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Infection with GB virus C in the patients with primary hepatocellular carcinoma in Japan
Hepatology Research 8 (1997) 37 – 43
Infection with GB virus C in the patients with primary hepatocellular carcinoma in Japan Takao Shibayama a, Seishuu Hayashi a, Shunichi Saeki a, Takeshi Tanaka a, Hiroo Ohtake a, Satoshi Tanaka a, Naoto Sawada b, Hiroaki Okamoto c,* a
Department of Internal Medicine, Tokyo Metropolitan Komagome Hospital, Tokyo 113, Japan b Institute of Immunology, Tokyo 112, Japan c Immunology Di6ision, Jichi Medical School, Minamikawachi-Machi Tochigi-Ken 329 -04, Japan Received 24 February 1997; received in revised form 12 May 1997; accepted 20 May 1997
Abstract RNA of recently reported, putative non-A to E hepatitis virus designated GB virus C (GBV-C) was determined by reverse-transcription polymerase chain reaction in sera from 231 Japanese patients with primary hepatocellular carcinoma. GBV-C RNA was detected in 21 patients (9% of the total), including two of the 23 patients (8%) with markers of hepatitis B virus infection, 11 of the 114 patients (10%) with markers of hepatitis C virus infection, seven of the 86 patients (8%) with markers of both hepatitis B and C virus infections, and one of the eight patients (13%) without such markers. These results indicate that the contribution of GBV-C infection to the development of hepatocellular carcinoma would be small either by itself or in cooperation with hepatitis B and C viruses. © 1997 Elsevier Science Ireland Ltd. Keywords: Hepatitis viruses; Hepatitis B virus; Hepatitis C viruses; Hepatocellular carcinoma; Chronic hepatitis; Liver cirrhosis
* Corresponding author. Tel.: + 81 285 44 2111, ext. 3334; fax: + 81 285 44 1557. 1386-6346/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 0 4 9 - 1
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T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
1. Introduction Infection with hepatitis viruses is closely associated with primary hepatocellular carcinoma (HCC). In particular, hepatitis B virus (HBV) and hepatitis C virus (HCV) are common in patients with HCC [1,2]. There are patients with HCC, however, who do not have evidence of infection with HBV or HCV, or ingested known carcinogenic agents such as aflatoxin. The proportion of patients with HCC of unknown etiology varies widely in different districts. It is higher in places where HBV and HCV infection is uncommon while lower in areas where these viruses are endemic. There are patients with acute or chronic liver disease who are without markers of HCV or HBV infection, or non-viral etiologies, and in whom infection with unknown hepatitis virus is suspected [3]. Their disease is classified as non-A to E hepatitis, because the infection with hepatitis A, D or E virus infection is excluded from them also. Recently, a viral agent possibly responsible for a part of non-A to E hepatitis has been discovered independently by two groups of investigators, and named GB virus C (GBV-C) [4,5] or hepatitis G virus [6]. They both are a single-stranded, positive RNA virus with a genomic length of 9400 nucleotides, resemble fla6i6iridae and distantly related to HCV. Because they share 86% of nucleotides and 96% of amino acid sequence, they are considered to be different isolates of the same virus, and will be referred to as GBV-C collectively. GBV-C transmits by transfusions [7] and injection-drug abuse [6,8], and persists indefinitely longer than 16 years in hemodialysis patients [9]. The exact hepatitis-inducing capacity of GBV-C is yet to be determined. GBV-C has been implicated in some patients with fulminant hepatitis of unknown etiology [10]. RNA of GBV-C has been reported in seven of 17 patients (36%) with chronic hepatitis of non-A to E etiology in Italy where it occurs only in 1% of normal controls [11]. In Japan, GBV-C RNA has been reported in one of 12 patients (8%) with cryptogenic hepatitis without markers of HCV or HBV infection [12]. In as much as liver cirrhosis as a sequel of chronic hepatitis is proposed as a precancerous state, hepatitis viruses other than HCV and HBV may be associated with the development of HCC. We tested for GBV-C RNA in sera from 231 patients with HCC and evaluated its association with the development of HCC either alone or in concert with HCV and HBV infections.
2. Materials and methods
2.1. Patients During 4.5 years from March, 1990 to September, 1994, 231 consecutive patients with HCC received care at the Department of Internal Medicine of Tokyo Metropolitan Komagome Hospital. They included 155 men and 76 women with the mean 9SD age of 639 8 years. The diagnosis of HCC was based on angiography or histology of liver tissues obtained by echo-guided biopsy. The patients with
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
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antibody to HCV (anti-HCV), HCV RNA or both were classified into type C; those with at least one of hepatitis B surface antigen (HBsAg), HBV DNA, antibody to HBsAg (anti-HBs) and antibody to HBV core (anti-HBc) into type B; those with both markers of HCV and HBV infections into B plus C type; and the remaining patients into non-B, non-C type. Patients with daily intake of \ 80 g alcohol for longer than 5 years were categorized as drinkers. Patients were asked if they had a history of transfusions or family members with liver disease. Sera were obtained from them and tested for GBV-C RNA. The study was approved by the ethics committee of the institution, and an informed consent was obtained from every patient.
2.2. Determination of GBV-C RNA GBV-C RNA was determined by reverse-transcription (RT)-polymerase chain reaction (PCR) with nested primers deduced from conserved areas in the 5% untranslated region by the method described previously [13]. Briefly, RNAs were extracted from 100 ml of serum, reverse-transcribed to cDNA and amplified by a two-stage PCR. The first round was performed with sense cG75 and antisense cG58 primers for 35 cycles and the second round was carried out with nested primers, sense cG134 and antisense c G131, for 25 cycles. Expected sizes of amplicons in the first and second rounds of PCR were 242 and 208 base pairs, respectively, and they assured a proper amplification of GBV-C sequences. Care was taken to avoid cross-contamination with 1 positive and 2 negative controls inserted in every 20 samples.
2.3. Determination of markers of HCV and HBV infections HBsAg and anti-HBs were determined by passive hemagglutination using commercial kits (MyCell, Institute of Immunology Co., Ltd., Tokyo, Japan) and anti-HBc was determined by hemagglutination inhibition by the method described previously [14]. Sera were serially diluted two-fold, and hemagglutination with 22 or higher dilutions was considered reactive for HBsAg and anti-HBs, and hemagglutination inhibition with 23 or higher dilutions was recorded positive for anti-HBc. All sera without detectable HBsAg were tested for HBV DNA by PCR with primers deduced from the S gene [14]. Anti-HCV was determined by a third-generation enzyme-linked immunosorbent assay (Ortho ELISAIII, Ortho Diagnostic Systems, Tokyo, Japan) with an absorbance at 492 nm exceeding 0.65 considered reactive. Nucleic acids extracted from 100 ml of serum were tested for HCV RNA by RT-PCR with primers deduced from the 5% untranslated region.
2.4. Statistical analyses Frequency between groups was compared using the Fisher’s exact test and x 2 test. Group means were compared by the Student’s t-test.
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
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3. Results
3.1. GBV-C RNA in patients with HCC of 6arious etiologies Overall, GBV-C RNA was detected in 21 of the 231 patients (9%) with HCC. Table 1 compares the detection of GBV-C RNA in patients with HCC who were classified into four categories by the presence or absence of markers of HCV or HBV infection. GBV-C RNA was detected in two of the 23 patients (9%) with HCC of type B, 11 of the 114 patients (10%) with HCC of type C, and seven of the 86 patients (8%) with HCC of dual types (B and C). GBV-C RNA was detected in one of the eight patients (13%) who were without serological or molecular biological markers of HCV or HBV infection and classified into nonB, non-C type. The patient accounted for 0.4% of the 231 patients with HCC studied. There were no appreciable differences in the proportion of male patients, mean age, the frequency of accompanying cirrhosis or history of transfusion among the four categories of patients with HCC. No differences were observed in the frequency of GBV-C RNA between male and female patients (14/155 or 9% vs. 7/76 or 9%). Alcohol intake was reported by five of the eight patients (63%) with HCC of non-B, non-C type somewhat more frequently than by 65 of the 223 patients (29%) of type B, type C or B and C type.
3.2. Comparison of the patients with HCC who had or did not ha6e GBV-C RNA There were no appreciable differences in demographic, virological and biochemical features, as well as history of transfusion, alcohol intake and family history of liver disease, between the 21 patients with GBV-C RNA and the 210 patients without GBV-C RNA in serum. Restricted to the 200 patients with HCV infection (Table 2), however, the 18 with GBV-C RNA reported alcohol intake significantly more frequently than the remaining 182 without GBV-C RNA (6% vs. 32%, P B0.05).
Table 1 GBV-C RNA, liver cirrhosis and a history of transfusion in patients with hepatocellular carcinoma of various types Type of liver disease
N
Male (%)
Age
GBV-C RNA %
Liver cirrhosis%
Transfusion%
B C B and C Non-B, non-C Total
23 114 86 8 231
17 70 61 7 155
59912 6597 63 97 64 95 63 9 8
2 11 7 1 21
16 97 75 6 194
3 45 31 1 80
(74) (61) (71) (88) (67)
(9) (10) (8) (13) (9)
(70) (85) (87) (75) (84)
(13) (39) (36) (13) (35)
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
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Table 2 Comparison of the patients with HCV-associated hepatocellular carcinoma who were with or without GBV-C RNA in serum Features
N Male (%) Age Liver cirrhosis HCV RNA HBsAg/HBV DNAa ALT (IU/l) AST (IU/l) High AFP (]7.7 ng/ml) Total protein (g/100 ml) g-globulin g-GTP (IU/l) Platelets (×1010/l) Transfusion Alcohol intake Liver disease in family members
GBV-C RNA
Differences
(+)
(−)
18 11 (61%) 65 98 13 (72%) 18 (100%) 1 (6%) 85 932 86 921 17 (94%) 7.29 0.5 1.8 90.6 99 977 10.4 9 3.9 6 (33%) 1 (6%) 2 (11%)
182 120 (66%) 64 97 159 (87%) 176 (97%) 10 (5%) 91 952 106 9 87 160 (88%) 7.1 90.7 1.8 9 0.6 104 9163 10.0 9 4.6 70 (38%) 58 (32%) 35 (19%)
NSb NS NS NS NS NS NS NS NS NS NS NS NS NS PB0.05 NS
Abbreviations and normal values: alanine aminotransferase (ALT), 4 – 43 IU/L; aspartate aminotransferase (AST), 11–32 IU/l; alpha-fetoprotein (AFP), B7.7 ng/ml; total protein, 6.4 – 8.1 g/100 ml; g-globulin, 1.4–2.4 g/100 ml; g-glutamyl transpeptidase (g-GTP), 6 – 70 IU/l; platelets, 10 – 40×1010/l. a Positive for HBsAg, HBV DNA or both. b Not significant.
3.3. The patient with HCC of non-B, non-C type who was infected with GBV-C The patient was a 60-year-old male and did not have any serological or molecular biological evidence of HCV or HBV infection. He received transfusion due to hematoemesis and melena from esophageal varices in August, 1990. A mass lesion of 10 cm in diameter representing HCC was identified in his liver which obstructed portal vein partially and compressed the right atrium. He died of peritoneal bleeding due to the rupture of tumor and liver insufficiency in March, 1991. He was diagnosed to have liver cirrhosis at autopsy. However, the extent of piecemeal necrosis and infiltration with inflammatory cells were minimal, which could be ascribed to a high dose of alcohol taken by him.
4. Discussion Of the 231 consecutive patients with HCC in the present study conducted in Japan, 23 (10%) had evidence of HBV infection, 114 (49%) possessed that of HCV infection, and 86 (37%) had both. The remaining eight patients (3%) were without any serological or molecular biological markers of HCV or HBV infection, and their HCC was classified into a non-B, non-C type.
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T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
Excessive alcohol intake was reported more frequently by the eight patients without markers of HBV or HCV infection (non-B, non-C) than by the remaining 223 patients of type B, type C or B and C type (63% vs. 29%); the difference fell short of being significant, however. This might represent factors other than HBV or HCV infection promoting the development of HCC in them. Restricted to the 200 patients with HCV infection, also, the 18 with GBV-C RNA reported alcohol intake significantly more frequently than the remaining 182 without GBV-C RNA (6% vs. 32%, P B0.05). Except that, there were no differences in demographic, clinical and viral variables between the patients with and without GBV-C coinfection. We have reported that GBV-C infection do not influence HCV RNA titer or is associated with particular HCV genotypes [9,15]. Should GBV-C turn out to be a non-A to E hepatitis virus, it would be responsible for a part of HCC cases not ascribable to HBV or HCV infection. The present results did not support a role of GBV-C in inducing HCC. GBV-C RNA was detected in only one of the eight patients (13%) with HCC of a non-B, non-C etiology. However, it was detected in 9% and 10% of patients with HCC of type B and type C, respectively, and in 8% of patients with HCC of B plus C etiology. Even in the single patient with HCC of non-B, non-C type, an exclusive causal role was not assigned to GBV-C. He had received a transfusion in the past which might have transmitted GBV-C to the patient after the development of HCC. Overall, GBV-C RNA was detected in 21 of the 231 patients with HCC (9%), at a prevalence much higher than that in Japanese blood donors reported at 0.9% [9]. However, 20 of them (95%) were coinfected with HBV, HCV or both. The coinfection of GBV-C with HBV and HCV has been reported [4,6,8,9], which would be attributable to common routes for these blood-borne viruses to penetrate into the community. Despite close association of hepatitis virus infection with the development of HCC, the mechanism of carcinogenesis is not understood even for HBV which can integrate into host DNA [16]. The integration is rarely clonal and mostly random with various franking host DNA sequences. Activation of oncogenic genes and tumor-repressing gene has been proposed, and the transactivating function of the X protein speculated in view of frequent integration of the X gene in tumor tissues. None of these theories can explain the majority of HCC cases of type B. However, an early occurrence of HCC in some children infected with HBV and the development of HCC unaccompanied by cirrhosis in HBV carriers leave a possibility for a carcinogenic capacity of this virus. A causal relationship is much less clear for HCV, which does not integrate into host DNA. Regeneration of hepatocytes in repairing necroinflammatory damage by HCV during a long period is implicated in the carcinogenesis. Since both HCV and GBV-C are classified in the fla6i6iridae family, a carcinogenic capacity of GBV-C, if any, would be similar to that of HCV and indirect. However, there have been no definitive lines of evidence to support a role of GBV-C in the induction of chronic liver disease. Post-transfusion or community-acquired non-A to E hepatitis is reported to be mild, and rarely, if ever, becomes chronic [17,18]. Taken together, GBV-C would
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hardly induce liver cirrhosis to bring the patients into a precancerous state for the development of HCC. GBV-C might work synergistically with HBV and HCV toward the development of HCC. However, such a mechanism is not clearly established even for the patients coinfected with HBV and HCV. Combined with the present survey of GBV-C RNA in patients with HCC, the role of GBV-C in inducing HCC would be minimal.
References [1] Popper H, Shafritz DA, Hoofnagle JH. Relation of the hepatitis B virus carrier state to hepatocellular carcinoma. Hepatology 1987;7:764 – 72. [2] Di Bisceglie AM. Hepatitis C and hepatocellular carcinoma. Semin Liver Dis 1995;15:64 – 9. [3] Alter HJ, Bradley DW. Non-A, non-B hepatitis unrelated to the hepatitis C virus (non-ABC). Semin Liver Dis 1995;15:110–20. [4] Simons JN, Leary TP, Dawson GJ, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1995;1:564 – 9. [5] Leary TP, Muerhoff AS, Simons JN, et al. Sequence and genomic organization of GBV-C: a novel member of the Flaviviridae associated with human non-A-E hepatitis. J Med Virol 1996;48:60 – 7. [6] 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. [7] Wang JT, Tsai FC, Lee CZ, et al. A prospective study of transfusiontransmitted GB virus C infection: similar frequency but different clinical presentation compared with hepatitis C virus. Blood 1996;8:1881–6. [8] Aikawa T, Sugai Y, Okamoto H. Hepatitis G infection in drug abusers with chronic hepatitis C (correspondence). New Engl J Med 1996;334:195 – 6. [9] 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. [10] 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. [11] Fiordalisi G, Zanella I, Mantero G, et al. High prevalence of GB virus C infection in a group of Italian patients with hepatitis of unkonwn etiology. J Infect Dis 1996;176:181 – 3. [12] 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. [13] Shimizu M, Osada K, Okamoto H. Transfusion-transmitted hepatitis G virus following open heart surgery (letter). Transfusion 1996;36:937. [14] Iizuka H, Ohmura K, Ishijima A, et al. Correlation between anti-HBc titers and HBV DNA in blood units without detectable HBsAg. Vox Sang 1992;63:107 – 11. [15] Nagayama R., Miyake K., Okamoto H. Effect of interferon on GB virus C and hepatitis C virus in hepatitis patients with the co-infection. J Med Virol 1997;52:156 – 60. [16] Buendia MA. Hepatitis B viruses and hepatocellular carcinoma. Adv Cancer Res 1992;59:167 – 226. [17] 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. [18] Alter MJ, Gallagher M, Morris TT, et al. Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. New Engl J Med 1997;336:741 – 6.
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Infection with GB virus C in the patients with primary hepatocellular carcinoma in Japan Takao Shibayama a, Seishuu Hayashi a, Shunichi Saeki a, Takeshi Tanaka a, Hiroo Ohtake a, Satoshi Tanaka a, Naoto Sawada b, Hiroaki Okamoto c,* a
Department of Internal Medicine, Tokyo Metropolitan Komagome Hospital, Tokyo 113, Japan b Institute of Immunology, Tokyo 112, Japan c Immunology Di6ision, Jichi Medical School, Minamikawachi-Machi Tochigi-Ken 329 -04, Japan Received 24 February 1997; received in revised form 12 May 1997; accepted 20 May 1997
Abstract RNA of recently reported, putative non-A to E hepatitis virus designated GB virus C (GBV-C) was determined by reverse-transcription polymerase chain reaction in sera from 231 Japanese patients with primary hepatocellular carcinoma. GBV-C RNA was detected in 21 patients (9% of the total), including two of the 23 patients (8%) with markers of hepatitis B virus infection, 11 of the 114 patients (10%) with markers of hepatitis C virus infection, seven of the 86 patients (8%) with markers of both hepatitis B and C virus infections, and one of the eight patients (13%) without such markers. These results indicate that the contribution of GBV-C infection to the development of hepatocellular carcinoma would be small either by itself or in cooperation with hepatitis B and C viruses. © 1997 Elsevier Science Ireland Ltd. Keywords: Hepatitis viruses; Hepatitis B virus; Hepatitis C viruses; Hepatocellular carcinoma; Chronic hepatitis; Liver cirrhosis
* Corresponding author. Tel.: + 81 285 44 2111, ext. 3334; fax: + 81 285 44 1557. 1386-6346/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 0 4 9 - 1
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T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
1. Introduction Infection with hepatitis viruses is closely associated with primary hepatocellular carcinoma (HCC). In particular, hepatitis B virus (HBV) and hepatitis C virus (HCV) are common in patients with HCC [1,2]. There are patients with HCC, however, who do not have evidence of infection with HBV or HCV, or ingested known carcinogenic agents such as aflatoxin. The proportion of patients with HCC of unknown etiology varies widely in different districts. It is higher in places where HBV and HCV infection is uncommon while lower in areas where these viruses are endemic. There are patients with acute or chronic liver disease who are without markers of HCV or HBV infection, or non-viral etiologies, and in whom infection with unknown hepatitis virus is suspected [3]. Their disease is classified as non-A to E hepatitis, because the infection with hepatitis A, D or E virus infection is excluded from them also. Recently, a viral agent possibly responsible for a part of non-A to E hepatitis has been discovered independently by two groups of investigators, and named GB virus C (GBV-C) [4,5] or hepatitis G virus [6]. They both are a single-stranded, positive RNA virus with a genomic length of 9400 nucleotides, resemble fla6i6iridae and distantly related to HCV. Because they share 86% of nucleotides and 96% of amino acid sequence, they are considered to be different isolates of the same virus, and will be referred to as GBV-C collectively. GBV-C transmits by transfusions [7] and injection-drug abuse [6,8], and persists indefinitely longer than 16 years in hemodialysis patients [9]. The exact hepatitis-inducing capacity of GBV-C is yet to be determined. GBV-C has been implicated in some patients with fulminant hepatitis of unknown etiology [10]. RNA of GBV-C has been reported in seven of 17 patients (36%) with chronic hepatitis of non-A to E etiology in Italy where it occurs only in 1% of normal controls [11]. In Japan, GBV-C RNA has been reported in one of 12 patients (8%) with cryptogenic hepatitis without markers of HCV or HBV infection [12]. In as much as liver cirrhosis as a sequel of chronic hepatitis is proposed as a precancerous state, hepatitis viruses other than HCV and HBV may be associated with the development of HCC. We tested for GBV-C RNA in sera from 231 patients with HCC and evaluated its association with the development of HCC either alone or in concert with HCV and HBV infections.
2. Materials and methods
2.1. Patients During 4.5 years from March, 1990 to September, 1994, 231 consecutive patients with HCC received care at the Department of Internal Medicine of Tokyo Metropolitan Komagome Hospital. They included 155 men and 76 women with the mean 9SD age of 639 8 years. The diagnosis of HCC was based on angiography or histology of liver tissues obtained by echo-guided biopsy. The patients with
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
39
antibody to HCV (anti-HCV), HCV RNA or both were classified into type C; those with at least one of hepatitis B surface antigen (HBsAg), HBV DNA, antibody to HBsAg (anti-HBs) and antibody to HBV core (anti-HBc) into type B; those with both markers of HCV and HBV infections into B plus C type; and the remaining patients into non-B, non-C type. Patients with daily intake of \ 80 g alcohol for longer than 5 years were categorized as drinkers. Patients were asked if they had a history of transfusions or family members with liver disease. Sera were obtained from them and tested for GBV-C RNA. The study was approved by the ethics committee of the institution, and an informed consent was obtained from every patient.
2.2. Determination of GBV-C RNA GBV-C RNA was determined by reverse-transcription (RT)-polymerase chain reaction (PCR) with nested primers deduced from conserved areas in the 5% untranslated region by the method described previously [13]. Briefly, RNAs were extracted from 100 ml of serum, reverse-transcribed to cDNA and amplified by a two-stage PCR. The first round was performed with sense cG75 and antisense cG58 primers for 35 cycles and the second round was carried out with nested primers, sense cG134 and antisense c G131, for 25 cycles. Expected sizes of amplicons in the first and second rounds of PCR were 242 and 208 base pairs, respectively, and they assured a proper amplification of GBV-C sequences. Care was taken to avoid cross-contamination with 1 positive and 2 negative controls inserted in every 20 samples.
2.3. Determination of markers of HCV and HBV infections HBsAg and anti-HBs were determined by passive hemagglutination using commercial kits (MyCell, Institute of Immunology Co., Ltd., Tokyo, Japan) and anti-HBc was determined by hemagglutination inhibition by the method described previously [14]. Sera were serially diluted two-fold, and hemagglutination with 22 or higher dilutions was considered reactive for HBsAg and anti-HBs, and hemagglutination inhibition with 23 or higher dilutions was recorded positive for anti-HBc. All sera without detectable HBsAg were tested for HBV DNA by PCR with primers deduced from the S gene [14]. Anti-HCV was determined by a third-generation enzyme-linked immunosorbent assay (Ortho ELISAIII, Ortho Diagnostic Systems, Tokyo, Japan) with an absorbance at 492 nm exceeding 0.65 considered reactive. Nucleic acids extracted from 100 ml of serum were tested for HCV RNA by RT-PCR with primers deduced from the 5% untranslated region.
2.4. Statistical analyses Frequency between groups was compared using the Fisher’s exact test and x 2 test. Group means were compared by the Student’s t-test.
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
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3. Results
3.1. GBV-C RNA in patients with HCC of 6arious etiologies Overall, GBV-C RNA was detected in 21 of the 231 patients (9%) with HCC. Table 1 compares the detection of GBV-C RNA in patients with HCC who were classified into four categories by the presence or absence of markers of HCV or HBV infection. GBV-C RNA was detected in two of the 23 patients (9%) with HCC of type B, 11 of the 114 patients (10%) with HCC of type C, and seven of the 86 patients (8%) with HCC of dual types (B and C). GBV-C RNA was detected in one of the eight patients (13%) who were without serological or molecular biological markers of HCV or HBV infection and classified into nonB, non-C type. The patient accounted for 0.4% of the 231 patients with HCC studied. There were no appreciable differences in the proportion of male patients, mean age, the frequency of accompanying cirrhosis or history of transfusion among the four categories of patients with HCC. No differences were observed in the frequency of GBV-C RNA between male and female patients (14/155 or 9% vs. 7/76 or 9%). Alcohol intake was reported by five of the eight patients (63%) with HCC of non-B, non-C type somewhat more frequently than by 65 of the 223 patients (29%) of type B, type C or B and C type.
3.2. Comparison of the patients with HCC who had or did not ha6e GBV-C RNA There were no appreciable differences in demographic, virological and biochemical features, as well as history of transfusion, alcohol intake and family history of liver disease, between the 21 patients with GBV-C RNA and the 210 patients without GBV-C RNA in serum. Restricted to the 200 patients with HCV infection (Table 2), however, the 18 with GBV-C RNA reported alcohol intake significantly more frequently than the remaining 182 without GBV-C RNA (6% vs. 32%, P B0.05).
Table 1 GBV-C RNA, liver cirrhosis and a history of transfusion in patients with hepatocellular carcinoma of various types Type of liver disease
N
Male (%)
Age
GBV-C RNA %
Liver cirrhosis%
Transfusion%
B C B and C Non-B, non-C Total
23 114 86 8 231
17 70 61 7 155
59912 6597 63 97 64 95 63 9 8
2 11 7 1 21
16 97 75 6 194
3 45 31 1 80
(74) (61) (71) (88) (67)
(9) (10) (8) (13) (9)
(70) (85) (87) (75) (84)
(13) (39) (36) (13) (35)
T. Shibayama et al. / Hepatology Research 8 (1997) 37–43
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Table 2 Comparison of the patients with HCV-associated hepatocellular carcinoma who were with or without GBV-C RNA in serum Features
N Male (%) Age Liver cirrhosis HCV RNA HBsAg/HBV DNAa ALT (IU/l) AST (IU/l) High AFP (]7.7 ng/ml) Total protein (g/100 ml) g-globulin g-GTP (IU/l) Platelets (×1010/l) Transfusion Alcohol intake Liver disease in family members
GBV-C RNA
Differences
(+)
(−)
18 11 (61%) 65 98 13 (72%) 18 (100%) 1 (6%) 85 932 86 921 17 (94%) 7.29 0.5 1.8 90.6 99 977 10.4 9 3.9 6 (33%) 1 (6%) 2 (11%)
182 120 (66%) 64 97 159 (87%) 176 (97%) 10 (5%) 91 952 106 9 87 160 (88%) 7.1 90.7 1.8 9 0.6 104 9163 10.0 9 4.6 70 (38%) 58 (32%) 35 (19%)
NSb NS NS NS NS NS NS NS NS NS NS NS NS NS PB0.05 NS
Abbreviations and normal values: alanine aminotransferase (ALT), 4 – 43 IU/L; aspartate aminotransferase (AST), 11–32 IU/l; alpha-fetoprotein (AFP), B7.7 ng/ml; total protein, 6.4 – 8.1 g/100 ml; g-globulin, 1.4–2.4 g/100 ml; g-glutamyl transpeptidase (g-GTP), 6 – 70 IU/l; platelets, 10 – 40×1010/l. a Positive for HBsAg, HBV DNA or both. b Not significant.
3.3. The patient with HCC of non-B, non-C type who was infected with GBV-C The patient was a 60-year-old male and did not have any serological or molecular biological evidence of HCV or HBV infection. He received transfusion due to hematoemesis and melena from esophageal varices in August, 1990. A mass lesion of 10 cm in diameter representing HCC was identified in his liver which obstructed portal vein partially and compressed the right atrium. He died of peritoneal bleeding due to the rupture of tumor and liver insufficiency in March, 1991. He was diagnosed to have liver cirrhosis at autopsy. However, the extent of piecemeal necrosis and infiltration with inflammatory cells were minimal, which could be ascribed to a high dose of alcohol taken by him.
4. Discussion Of the 231 consecutive patients with HCC in the present study conducted in Japan, 23 (10%) had evidence of HBV infection, 114 (49%) possessed that of HCV infection, and 86 (37%) had both. The remaining eight patients (3%) were without any serological or molecular biological markers of HCV or HBV infection, and their HCC was classified into a non-B, non-C type.
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Excessive alcohol intake was reported more frequently by the eight patients without markers of HBV or HCV infection (non-B, non-C) than by the remaining 223 patients of type B, type C or B and C type (63% vs. 29%); the difference fell short of being significant, however. This might represent factors other than HBV or HCV infection promoting the development of HCC in them. Restricted to the 200 patients with HCV infection, also, the 18 with GBV-C RNA reported alcohol intake significantly more frequently than the remaining 182 without GBV-C RNA (6% vs. 32%, P B0.05). Except that, there were no differences in demographic, clinical and viral variables between the patients with and without GBV-C coinfection. We have reported that GBV-C infection do not influence HCV RNA titer or is associated with particular HCV genotypes [9,15]. Should GBV-C turn out to be a non-A to E hepatitis virus, it would be responsible for a part of HCC cases not ascribable to HBV or HCV infection. The present results did not support a role of GBV-C in inducing HCC. GBV-C RNA was detected in only one of the eight patients (13%) with HCC of a non-B, non-C etiology. However, it was detected in 9% and 10% of patients with HCC of type B and type C, respectively, and in 8% of patients with HCC of B plus C etiology. Even in the single patient with HCC of non-B, non-C type, an exclusive causal role was not assigned to GBV-C. He had received a transfusion in the past which might have transmitted GBV-C to the patient after the development of HCC. Overall, GBV-C RNA was detected in 21 of the 231 patients with HCC (9%), at a prevalence much higher than that in Japanese blood donors reported at 0.9% [9]. However, 20 of them (95%) were coinfected with HBV, HCV or both. The coinfection of GBV-C with HBV and HCV has been reported [4,6,8,9], which would be attributable to common routes for these blood-borne viruses to penetrate into the community. Despite close association of hepatitis virus infection with the development of HCC, the mechanism of carcinogenesis is not understood even for HBV which can integrate into host DNA [16]. The integration is rarely clonal and mostly random with various franking host DNA sequences. Activation of oncogenic genes and tumor-repressing gene has been proposed, and the transactivating function of the X protein speculated in view of frequent integration of the X gene in tumor tissues. None of these theories can explain the majority of HCC cases of type B. However, an early occurrence of HCC in some children infected with HBV and the development of HCC unaccompanied by cirrhosis in HBV carriers leave a possibility for a carcinogenic capacity of this virus. A causal relationship is much less clear for HCV, which does not integrate into host DNA. Regeneration of hepatocytes in repairing necroinflammatory damage by HCV during a long period is implicated in the carcinogenesis. Since both HCV and GBV-C are classified in the fla6i6iridae family, a carcinogenic capacity of GBV-C, if any, would be similar to that of HCV and indirect. However, there have been no definitive lines of evidence to support a role of GBV-C in the induction of chronic liver disease. Post-transfusion or community-acquired non-A to E hepatitis is reported to be mild, and rarely, if ever, becomes chronic [17,18]. Taken together, GBV-C would
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hardly induce liver cirrhosis to bring the patients into a precancerous state for the development of HCC. GBV-C might work synergistically with HBV and HCV toward the development of HCC. However, such a mechanism is not clearly established even for the patients coinfected with HBV and HCV. Combined with the present survey of GBV-C RNA in patients with HCC, the role of GBV-C in inducing HCC would be minimal.
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