Poor association of GBV-C viremia with hepatocellular carcinoma

Journal of Heputology 1991; 21: 91-95 Printed in Denmark . AN rights reserved Munksgaard . Copenhagen

Copyright0 EuropeanAssociation for the Studyof the Liver1997 Journal of Hepatology ISSN 01684’278

Poor association of GBVC viremia with hepatocellular carcinoma Shoji Kubo’,

Shuhei Nishiguchi2, Tetsuo Kuroki2, Kazuhiro Hirohashi’, Hiromu Tanaka’, Tadashi Tsukamoto’, Taichi Shuto’ and Hiroaki Kinoshita’

‘Second Department of Surgery and 2Third Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan

Buckgrounai/Aims: The role of the hepatitis GB virus C in chronic liver disease is unclear. The clinical findings, results of laboratory tests, pathological findings, and outcome after liver resection for hepatocellular carcinoma in patients infected or not infected with GB virus C were compared. Met/zods: Serum samples from 213 patients were tested for hepatitis B virus, hepatitis C virus, and GB virus C. Of the 213 patients, 159 had hepatitis C viral RNA or antibodies (group l), seven had both hepatitis C viral RNA or antibodies and hepatitis B surface antigen (group 2), 25 had hepatitis B surface antigen (group 3), and 22 did not have hepatitis C viral RNA, hepatitis C antibodies, or hepatitis B surface antigen (group 4). Results: GB virus C RNA was detected in 12 of the

213 patients. Of the 12 patients, 11 were in group 1 and one was in group 4. There were no patients with hepatocellular carcinoma associated with GB virus C RNA alone. In groups 1 and 4, there were no differences in clinical findings, results of laboratory tests, pathological findings, or outcome after surgery in patients with and without GB virus C RNA. Conclusions: These results indicate a poor association of GB virus C viremia with hepatocellular carcinoma. We did not find clinically significant interaction between hepatitis C virus and GB virus C.

I

still controversial; some studies have suggested that hepatitis caused by GBV-C is usually mild (6,7). In this study, the role of GBV-C in hepatocellular carcinoma (HCC) was investigated.

N 1967, the serum of a 34-year-old surgeon whose initials were GB was found to contain a hepatitis agent not detected earlier (1). The host range and cross-challenge studies suggested that the “GB agent” was different from hepatitis A, B, C, D, and E viruses. Two viruses were identified in this agent, GB virus A (GBV-A) and GB virus B (GBV-B), and these organisms were suggested to be agents of non-A-E hepatitis (2,3). A similar virus, GB virus C (GBV-C), was isolated from human serum containing antibodies that recognize GBV-A and GBV-B recombinant proteins (4). Results of the polymerase chain reaction with reverse transcription (RT-PCR), which can be used to detect GBV-C, have suggested a role of GBV-C in hepatitis, especially fulminant hepatitis (5). However, whether GBV-C is involved in fulminant hepatitis is

Key words: GB virus C; Hepatitis B virus; Hepatitis C virus; Hepatitis G virus; Hepatocellular carcinoma; Liver resection.

Patients

and Methods

Patients

The subjects were all 213 patients who underwent liver resection at our institute for HCC between the beginning of 1991 and October 1995, and from whom serum samples taken earlier were still available. Blood was sampled before surgery and serum was immediately frozen at -80°C for assay later. Serum was tested for hepatitis B virus (HBV), hepatitis C virus (HCV), and GBV-C. Detection of virus markers

Received I1 September 1996: revised 27 January; accepted 4 February 1997

Correspondence: Shoji Kubo, M.D., Second Department of Surgery, Osaka City University Medical School, l-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan. Tel: 81-6-645-2151. Fax: 81-6-646-3071.

Hepatitis B surface antigen (HBsAg) was measured with an ELISA kit (International Reagents Corp., Kobe, Japan). Hepatitis B core antibodies (HBcAb) were measured by RIA (Dinabot, Tokyo, Japan). Samples were examined for hepatitis C virus antibodies 91

S. Kuho et ul.

(HCVAb) by second- or third-generation ELISA (Ortho Diagnostic Systems, Tokyo, Japan). Serum HCV RNA was detected by RT-PCR with primers derived from the highly conserved 5’-untranslated region (5’UTR) of the viral genome, as described previously (8). The patients with serum HCVAb or in whom HCV RNA was detected by RT-PCR were assumed to be infected with HCV Serum GBV-C RNA was extracted by the acid guanidinium-phenol-chloroform method from 150 ~1 of serum samples that had been stored frozen, converted to complementary DNA (cDNA) by RT, and amplified by nested PCR. We selected two sets of specific primers (outer, sense, 5’-GGT GAT GAC AGG GTT GGT AGG TC-3’; outer. antisense, 5’TTG CCC ATT CGC TCG ACA-3’; inner, sense, 5’GCA CGG TCC ACA GGT GTT G-3’: and inner, antisense, 5’-ACG CCT ATT GGT CAA GAG AGA CAT T-3’) based on the 5’-UTR of the entire HGV genome (GeneBank U44402 and U45966). The first PCR was done for 35 cycles with 1 min of denaturation at 94”C, 1 min of annealing at 5O”C, and 1 min of extension at 72°C. The second PCR was done for 30 cycles. PCR products were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide for detection of a band at the 146 basepair region. The specificity of the amplified products was checked by nucleotide sequence analysis. The samples were also examined by the Lcx GBV-C RNA assay system developed by Abbott Laboratories (North Chicago, IL, USA). Specimen preparation, as the first step of the Lcx GBV-C assay, was based on a column technique. The second step was RT-PCR in a single tube with a capture probe. The PCR primers and probe were specific for the 5’-UTR region of the GBV-C genome. After PCR amplification of the target DNA, the resulting hybrid of the PCR product and the capture probe could be detected by an Lcx analyzer based on microparticle enzyme immunoassay. Double-stranded DNA was sequenced by the dideoxy method with ddNTPs with fluorescent labels and inner sense or antisense primers. Data collection and analysis from gel electrophoresis during this step were done with the 373A DNA sequencing system (Perkin Elmer, Chiba, Japan). Informed consent was obtained from each patient. This study was done in accordance with the Helsinki Declaration of 1975 (revision of 1983) and was approved by the ethics committee of Osaka City University Medical School. Methods

We divided the 2 13 patients into groups, depending on which infective agents were detected. Group 1 con92

sisted of 159 patients

with HCV and without

HBsAg.

Group 2 consisted of seven patients with both HCV and HBsAg. Group 3 consisted of 25 patients without HCV and with HBsAg. Group 4 consisted of 22 patients without either HCV or HBsAg. In group 1, the patients were further divided into one subgroup of 53 patients without HBcAb (group la) and one subgroup of 106 patients with HBcAb (group lb). In group 4, the two subgroups were of eight patients without HBcAb (group 4a) and 14 patients with HBcAb (group 4b). We calculated the proportions of patients infected with GBV-C in each group and subgroup. We compared the clinical profiles in terms of mean age, sex ratio, proportion of alcoholics [intake, 86 g of ethanol per day for at least 10 years (9)]. proportion with a history of blood transfusion, results of laboratory tests [1.5-min indocyanine green retention test (ICGR,& aspartate aminotransferase (AST) activity, alanine aminotransferase (ALT) activity, total bilirubin, serum albumin. platelet count, and serum cx-fetoprotein (AFP)], pathological findings of the operative specimens, and outcome after surgery in the patients with and without GBV-C RNA. Statistics

Fisher’s exact test was used to evaluate the significance of differences in the sex ratio, proportion of alcoholics. proportion with a history of blood transfusion, proportion with chronic hepatitis or cirrhosis (diagnosed histologically), degree of differentiation of the tumor. and findings of intrahepatic metastasis or portal invasion. Student’s t-test was used to analyze differences in the mean age. The Mann-Whitney U-test was used to analyze differences in the results of laboratory tests. Tumor-free and cumulative survival rates after surgery were calculated by the Kaplan-Meier method. and the statistical significance of differences in the survival rates was evaluated by the log rank test. All p-values are two-tailed. A difference with a y-value less than 0.05 was considered to be significant.

Results In 12 (6%) patients of the 213 patients, GBV-C RNA was detected (Table 1). In 11 (7%) of the 159 patients in group 1, GBV-C RNA was detected. GBV-C RNA was detected in five (9%) of the 53 patients in group la and in six (6%) of the 106 patients in group 1b. In one of the six patients in group 1b. GBV-C RNA was detected by our method but was not detected by the method used by Abbott Laboratories. In one (5%) patient of the 22 patients in group 4, GBV-C RNA was detected. GBV-C RNA was not detected in any patient in group 2 or 3. In all patients in whom GBV-C RNA

Hepatitis GB virus and hepatoma TABLE

1

Viral markers

and numbers

of patients

Group (Subgroup)

1

with GBV-C

Viral markers HCV RNA

HBsAg

+

-

la lb 2 3 4

No. of patients

No. (%) of patients with GBV-C

159

11 (7)

HBcAb

_ + + -

+ +

-

_

53 106 I 25

0 0

22

1 (5)

4a

-

8

4b

+

14

Total

was detected by our method, analysis of the deduced amino acid sequence showed that the sequences of GBV-C were more than 97% identical to the sequence of GBV-C reported by Simons et al. (4). Clinical and pathological findings of the patients with and without GBV-C RNA in groups 1 and 4 are summarized in Table 2. In the 149 patients without GBV-C RNA in group 1, the mean age was 62 years (range, 43 to 70 years). The mean age of the 11 patients with GBV-C RNA in group 1 was 66 years (range, 60 to 72 years). There was no significant difference in the mean ages, sex ratios, proportions of alcoholic subjects, or proportions with a history of blood transfusion in the two groups. Nor were there significant differences in the ICGRis, serum AST, serum ALT, total bilirubin, albumin, platelet counts, or proportions of patients with abnormal levels of AFP (220 @ml). There was no significant difference in the proportions of patients with HCC of different degrees of differentiation, with associated cirrhosis, or with intrahepatic metastasis or portal invasion. We did not find any significant differences in clinical or pathological findings when we compared the findings between the patients with and without GBV-C RNA in each subgroup in group 1. The patient in group 4 with GBV-C RNA was 69 years old, which was older than the mean of the other patients in that group. Serum concentrations of AST, ALT, total bilirubin, and albumin and the platelet count were within the ranges of the other patients’ values. However, the result of the ICGRis test was higher than the mean for the other patients without GBV-C RNA in the same group. In all 12 patients with GBV-C RNA, the postoperative course was uneventful; no patients died. Abnormal changes in AST activity (to 100 II-l/l or more), ALT activity (100 IU/l or more), and total bilirubin (5 mg/dl

213

5 (9) 6 (6)

0 1 (7) 12 (6)

or more) did not occur during the perioperative period except for the first few days after surgery. Tumor-free survival rates at 3 years after surgery in patients without GBV-C in groups 1 and 4 were 51% and 48%, respectively. Cumulative survival rates at 3 years in these patients were 93% and 79%, respectively. In group 1, there was no significant difference in tumor-free and cumulative survival rates depending on GBV-C infection. The patient with GBV-C RNA in group 4 is alive with recurrence 2 years and 4 months after surgery.

Discussion HBV and HCV are risk factors for HCC. In Japan, about 70% of the patients with HCC have antibodies to HCV alone, another 10% have HBsAg, and another 10% have antibodies to both HCV and HBV (10). Recently, Yoshiba et al. (5) found GBV-C in Japanese patients, and suggested that it may be important in fulminant or subacute hepatitis. Masuko et al. (11) suggested, however, that infection with GBV-C does not cause inflammation of the liver because their 16 hemodialysis patients infected with GBV-C did not have elevated serum ALT activities. It is not known if GBV-C is a risk factor for HCC or if GBV-C affects the development of HCC when the patients have HCV or HBV Linnen et al. (12) found the prevalence of hepatitis G virus (HGV, which probably is GBV-C) in 769 volunteer blood donors in the United States to be 1.7%, and Jarvis et al. (13) found that of HGV in 125 donors in Edinburgh to be 3.2%; the prevalence of HGV is thus higher than that of HCV. Linnen et al. (12) reported detecting HGV in 18 (19%) of 96 patients with chronic HCV and in seven (10%) of 72 patients with chronic HBV. They also found two (7%) of 30 patients with HCC to have HGV, one patient had both HGV and HBV and another had both HGV and HCV

93

S. Kubo et UI TABLE

2

Clinical

findings,

results of laboratory

tests, and pathological Group

findings

of patients

with and without Group

1

Without

GBV-C

With GBV-C

Without

GBV-C 4 GBV-C

With GBV-C 1

148

II

13

Age, years, mean?SD Male:female Alcoholic Blood transfusion

621’6 124:25 49 (33%) 33 (22%)

6624 92 4 (36”%) 2 (18%)

5616 13:o 4 (31’%,) 1 ( 15%)

69 0:l 0 0

Results oj’hhorutory te,m* ICGR,5. ‘X, AST, IUil ALT, UI/l T-bil, mg/dl Albumin, mg/dl X IO4 Platelets. /mm3 AFP, 220 ng**

20 64 7s 0.8 3.6 12 73

19 78 71 0.9 3.6 14 6

10 41 34 0.7 3.4 19 6

42 51 52 1.0 3.4 12

No. of patients

Clinicalfindings

Putlzological,findings Diameter of tumor (cm)* Nontumorous tissue Fibrosis Chronic hepatitis Cirrhosis Differentiation of main tumor Well-differentiated Moderately differentiated Poorly differentiated Unknown Intrahepatic metastasis Portal invasion Operative

death

3-year survival rates Tumor-free (‘XI) Cumulative (%I)

(10, 34) (33, 111) (30, 130) (0.5. 1.4) (3.1, 4.1) (8. 20) (49’%>)

2.7 (1.7. 5.5) 7 (4%) 29 (ZO”/;,,l 1 13 (76%) 19 95 24 IO 30 30

(14%) (69%) (17%) (20%) (20’%,)

2 ( 1%) 51 93

(10. 26) (33. 81) (42. 112) (0.5. 1.3) (3.1. 4.0) (9, 20) (55%)

2.5 (1.5. 7.0)

94

3.0 (2.0. 18.9)

I 3.0

0 3 (27%) 8 (73%)

2 (IS%) 4 (3l’>h) 7 (54%)

0 0 1

1 (9%) IO (91%) 0 0 2 (18%) 3 (27%)

1 (8%) 7 (53”h)

4 (31’!
0 1 0 0 0 0

0

0

0

68 100

48 79

ICGR,,, 15-min test of indocyanine green retention; AST. aspartate aminotransferase; a-fetoprotein. * Median with 10 and 90 percentiles. ** Number of patients.

In this study of patients with HCC, GBV-C RNA was detected in 6% which is close to the prevalence reported by Linnen et al. (12) The prevalence of GBV-C was higher in our patients with HCC than in volunteer blood donors reported elsewhere [0.9% of 448 in Japan (1 l)]. However, the prevalence of GBV-C in our HCC patients seemed to be lower than in patients with HCV or HBV but without HCC (12). Details of HBV-HCV interactions are unclear, but these viruses do seem to interact in causing HCC, and an unexpectedly high incidence of double infections among HCC patients has been reported by several groups (14-16). In a Greek study, progression from chronic hepatitis to cirrhosis and to HCC seemed to be more likely when there was coinfection with HBV and HCV (16). Of our 12 HCC patients with GBV-C RNA, 11 patients were infected with HCV (six of the 11 patients had a history of infection with HBV) and one patient had a history of infection with HBV It

(5, 31) (28. 76) (20. 159) (0.5. 1.4) (2.9. 4.1) (8. 45) (46’!;,)

5 (38%) (I

ALT. alanine

_ _ aminotransferasc:

T-bil. total bilirubin;

AFP.

was difficult to decide whether GBV-C accelerates the development of WCC related to HBV or HCY because the number of HCC patients who had blood transfusions was too small for calculation of the time between blood transfusion and the detection of HCC, and because the time of infection with GBV-C, HCV, or HBV in the other patients could not be identified. However, in group 1, there was no significant difference in mean age between patients with and without GBVC RNA. In group 4, the one patient with GBV-C RNA was older than the mean age of the patients without GBV-C RNA. In our few patients infected with GBVC, the lack of a significant difference in the results of laboratory tests, pathological findings, perioperative course, or outcome between the different groups suggests but does not prove that there is no significant interaction between HCV and GBV-C. Previous studies showed that GBV-C can be transmitted by transfusion (1 l-1 3). In our study, only two

Hepatitis GB virus and hepatoma

of the 12 patients with GBV-C RNA had a history of blood transfusion. In Japan, although the recent increase in the number of patients with HCC related to HCV is thought to be due to certain medical practices no longer in use (the use of paid blood donors and the repeated use of needles), the routes of transmission of HCV are not yet fully understood (17,18). GBV-C may cause infection by the same routes as HCV In our study, we did not find any statistically significant effects of GBV-C on HCC. GBV-C may not be a risk factor for the development of HCC. However, GBV-C may have an undetected weak effect on the development of HCC, masked by HBV and HCV being strong risk factors. When a method to assay antibodies to GBV-C becomes available, it should be possible to decide if there is a weak effect or no effect.

Acknowledgements We thank Prof. S. Otani for valuable criticism and Ms. C. Latta for reading the manuscript. We also thank Abbott Laboratories for assaying serum samples for GBV-C. This work was supported by a grant from the Ministry of Education, Science, and Culture, Japan.

References 1. Deinhardt F, Holmes AW, Capps RB, Popper H. Studies on the transmission of human viral hepatitis to marmoset monkeys. I. Transmission of disease, serial passages, and description of liver lesions. J Exp Med 1967; 125: 673-88+plates 81-86. 2. Simons JN, Pilot-Matias TJ, Leary TP, Dawson GJ, Desai SM, Schlauder GG, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Nat1 Acad Sci USA 1995; 92: 3401-5. 3. Schlauder GG, Dawson GJ, Simons JN, Pilot-Matias TJ, Gutierrez RA, Heynen CA, et al. Molecular and serologic analysis in the transmission of the GB hepatitis agents. J Med Virol 1995; 46: 81-90. 4. Simons JN, Leary TP Dawson GJ, Pilot-Matias TJ, Muerhoff AS, Schlauder GG, et al. Isolation of novel virus-like

sequences associated with human hepatitis. Nature Med 1995; 1: 5649. 5. 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. 6. Kao J-H, Chen P-J, Chen D-S. GBV-C in the aetiology of fulminant hepatitis. Lancet 1996; 347: 120. 7. Kuroki T, Nishiguchi S, Tanaka M, Enomoto M, Kobayashi K. Does GBV-C cause fulminant hepatitis in Japan? Lancet 1996; 347: 908. 8. Nishiguchi S, Kuroki T, Ueda T, Fukuda K, Takeda T, Nakajima S, et al. Detection of hepatitis C virus antibody in the absence of viral RNA in patients with autoimmune hepatitis. Ann Intern Med 1992; 116: 21-5. 9. Liver Cancer Study Group of Japan. Primary liver cancer in Japan: clinicopathologic features and results of surgical treatment. Ann Surg 1990; 211: 277-87. 10. Shiratori Y, Shiina S, Imamura M, Kato N, Kanai F, Okudaira T, et al. Characteristic difference of hepatocellular carcinoma between hepatitis B- and C- [sic] viral infection in Japan. Hepatology 1995; 22: 1027-33. 11. Masuko K, Mitsui T, Iwano K, Yamazaki C, Okuda K, Meguro T, et al. Infection with hepatitis GB virus C in patients on maintenance hemodialysis. N Engl J Med 1996; 334: 1485-90. 12. Linnen J, Wages J Jr, Zhang-Keck Z-Y, Fry KE, Krawczynski KZ, Alter H, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996; 271: 505-8. 13. Jarvis LM, Davidson F, Hanley JP, Yap PL, Ludlam CA, Simmonds P Infection with hepatitis G virus among recipients of plasma products. Lancet 1996; 348: 1352-5. 14. Yu MC, Tong MJ, Coursaget P, Ross RK, Govindarajan S, Henderson BE. Prevalence of hepatitis B and C viral markers in black and white patients with hepatocellular carcinoma in the United States. J Nat1 Cancer Inst 1990; 82: 1038-41. 15. Kaklamani E, Trichopoulos D, Tzonou A, Zavitsanos X, Koumantaki Y, Hatzakis A, et al. Hepatitis B and C viruses and their interaction in the origin of hepatocellular carcinoma. JAMA 1991; 265: 19746. 16. Sherlock S. Viruses and hepatocellular carcinoma. Gut 1994; 35: 828-32. 17. Okuda K, Fujimoto I, Hanai A, Urano Y. Changing incidence of hepatocellular carcinoma in Japan. Cancer Res 1987; 47: 4967-72. 18. Kaneko S, Unoura M, Takeuchi M, Terasaki S, Ogino H, Matsushita E, et al. The role of hepatitis C virus in hepatocellular carcinoma in Japan. Intervirology 1994; 37: 108-13.

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