Digestive Diseases and Sc&nces, Vol. 37, No. 1 (January 1992), pp. 65-72
Hepatitis B Virus Markers and Antibodies to Hepatitis C Virus in Japanese Patients with Hepatocellular Carcinoma NOBUKAZU YUKI, MD, NORIO HAYASHI, MD, AKINORI KASAHARA, MD, HIDEKI HAGIWARA, MD, KAZUHIRO KATAYAMA, MD, HIDEYUKI FUSAMOTO, MD, and TAKENOBU KAMADA, MD
Sera from Japanese patients with chronic liver disease were tested for hepatitis B virus (HBV) markers and antibodies to hepatitis C virus (anti-HCV), and the results were correlated to the presence o f hepatocellular Carcinoma. In chronic non-A, non-B liver disease, anti-HCV prevalence was high both in patients with hepatocellular carcinoma (78/89, 88%) and without it (66/84, 79%), while previous HBV infection was more common in patients with hepatocellular carcinoma (65/89, 73%) than in those without it (46/84, 55%) (P < 0.05). Coexistence o f anti-HCV and antibodies to H B V was observed frequently in patients with hepatocellular carcinoma (56/89, 63%) compared with patients without it (39/84, 46%) (P < 0.05). In chronic H B V carriers, anti-HCV was more common in patients with hepatOcellular carcinoma (12/38, 32%) than in those without it (3/62, 5%) (P < 0.01). These results suggest that infection with the two viruses may be a risk factor for more serious liver disease. KEY WORDS: chronic liver disease; hepatitis B virus; hepatitis C virus; hepatocellular carcinoma.
A close relationship between the development of hepatocellular carcinoma (HCC) and persistent hepatitis B virus (HBV) infection has been stressed in various areas (1-7). Recent findings suggest that integrated HBV sequences in cellular DNA could contribute to liver carcinogenesis by activation of cellular genes (8-12). To date, there also have been a few reports suggesting a putative association of HCC and the agent(s) of non-A, non-B viral hepatitis (13, 14). However, this has not been well
Manuscript received December 14, 1990; revised manuscript received May 6, 1991; accepted May 6, 1991. From the First Department of Medicine, Osaka University Medical School, Osaka, Japan. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. Address for reprint requests: Dr. Norio Hayashi, First Department of Medicine, Osaka University Medical School, Fukushima 1-1-50, Fukushima-ku, Osaka 553, Japan.
defined because of the lack of serological markers for this agent(s). Recently, Choo et al cloned the genome of a non-A, non-B hepatitis virus, termed hepatitis C virus (HCV) (15), Part of the nonstructural region of the HCV genome was expressed as a fusion polypeptide with human superoxide dismutase in yeast (C100-3 antigen), and a specific assay for the detection of antibodies to this antigen (anti-C100-3) was established (16). Using this system, epidemiological studies on the relationship between HCV infection and the development of HCC can be how performed more accurately. Several reports have demonstrated the potential etiologic,role of HCV in the development of HCC (17-20). In this Study, we determined the prevalence of anti-C100-3 and HBV markers in Japanese patients with chronic liver disease. The results were corre-
Digestive Diseases and Sciences, Vol. 37, No. 1 (January 1992)
0163-2116/92/0100-0065506.50/09 1992Plei~uinPublishingCorporation
65
YUKI ET AL TABLE 1. CLINICAL CHARACTERISTICS AND PREVALENCE OF ANTI-C100-3 IN PATIENTS WITH HEPATOCELLULAR CARCINOMA
Patients*
N
Non-A, non-B HCC Alcoholic HCC HBsAg-positive HCC
89 21 38
Aget (years) 63 -+ 7 59 --- 9 54 --- 10:~,w
Sex (M/F)
Past history of blood transfusion
Anti-ClO0-3 prevalence
72/17~ 21/0 33/5
16/89 (18%) 3/21 (14%) 2/38 (5%)
78/89 (88%) 13/21 (62%)w 12/38 (32%)~:,w
*HCC, hepatocellular carcinoma. tValues are mean - SD. :~P < 0.05 vs alcoholic HCC. w < 0.01 vs non-A, non-B HCC. lated to the p r e s e n c e of H C C to investigate the implication of the two viruses in the d e v e l o p m e n t of HCC. M A T E R I A L S AND M E T H O D S
Serum Samples. Sera were obtained from 148 Japanese patients with HCC. In each case, the tumor was revealed by imaging procedures (ultrasonography, computed tomography plus angiography). The tumor occupied one segment of the liver in 32 cases, two segments in 29, and more than two segments in 87. Of these, 119 patients had undergOne transcatheter arterial embolization before blood collection, and the other 29 patients had received no treatment. In 18 patients, HCC and preexisting cirrhosis were proved pathologically. The other patients also had preexisting chronic liver disease and showed clinical features of cirrhosis, although liver histology was not available. There were 126 men and 22 women (mean age 60 -- 9 years). Thirty-eight patients had hepatitis B surface antigen (HBsAg), and 21 were alcoholics with the average daily consumption of ethanol exceeding 135 g for more than 10 years. The other 89 patients were negative for HBsAg and showed no evidence of alcoholic, autoimmune, or drug-induced liver disease. Sera also were obtained from 146 patients with chronic liver disease who had no ultrasonographic and clinical evidence of HCC (92 men, 54 women; mean age 50 - 13 years). Of these, 84 patients had chronic non,A, non-B liver disease, and liver histology showed the features of chronic persistent hepatitis (CPH) in 10 cases, of chronic active hepatitis (CAH) in 43, and of liver cirrhosis in 31. The other 62 patients had chronic HBV infection and were known to be carriers of HBsAg for more than 12 months. Fourteen were asymptomatic HBsAg carriers with normal liver function tests, and 48 were HBsAg carriers with chronic liver disease. The liver histology of these 48 showed the features of CPH in 17 cases, of CAH in 15, and of liver cirrhosis in 16. All samples were collected for this study over three years and Stored in screw-cap vials at - 8 0 ~ C. When tested, each sample was amber, not turbid or viscous, and was used without heat inactivation. Serological Testing. HBsAg , hepatitis B e antigen (HBeAg), antibody to HB~Ag (anti-HB~) and antibody to hepatitis B core antigen (anti-HBc) were determined using commercially available radioimmunoassays (Dainabot Co., Ltd., Tokyo, Japan). Anti-C100-3 was tested with an ELISA system (Ortho Diagnostic Systems Co., Ltd.,
66
Tokyo, Japan). The assay was performed according to the manufacturer's instructions. Microtiter plates, which had been coated with a recombinant C100-3 antigen, were incubated with test sera at 37~ C for 1 hr, followed by incubation with per0xidase-conjugated monoclonal antihuman IgG antibody for another 1 hr. Then a solution containing o-phenylene diamine and H202 was added, and the resulting optical density at 492 nm (OD492) was measured using a photo-enzyme-linked immunosorbent assay colorimeter (Titertek Multiscan MCC/340, Flow Laboratories, McLean, VA). The mean value of OD492 for three negative controls of the kits plus 0.400 OD units was taken as the cutoff value, which ranged from 0.423 to 0.430 (mean 0.427). The positive controls of the kits always gave OD492 of >1.0. All assays were done in duplicate. To compare ELISA results of different sampies, the ratio of the mean OD reading of the duplicate results to the cutoff value (cutoff index) was calculated. In preliminary experiments, we examined the relationship between the ELISA cutoff index and anti-Cl00-3 titer expressed by the highest twofold dilution of the test serum showing positive results. The ELISA cutoff index was found to reach the ceiling in proportion to an increase in anti-C 100-3 titer and did not exceed 7.0. Based on these observations, we adopted the anti-C100-3 titer expressed by the highest twofold dilution as well as the ELISA cutoff index for quantitative estimation of anti-C 100-3. A total of 294 serum samples were tested for anti-C100-3, and 21 samples were positive for anti-C100-3 with cutoff index of
Hepatitis B Virus Markers and Antibodies to Hepatitis C Virus in Japanese Patients with Hepatocellular Carcinoma NOBUKAZU YUKI, MD, NORIO HAYASHI, MD, AKINORI KASAHARA, MD, HIDEKI HAGIWARA, MD, KAZUHIRO KATAYAMA, MD, HIDEYUKI FUSAMOTO, MD, and TAKENOBU KAMADA, MD
Sera from Japanese patients with chronic liver disease were tested for hepatitis B virus (HBV) markers and antibodies to hepatitis C virus (anti-HCV), and the results were correlated to the presence o f hepatocellular Carcinoma. In chronic non-A, non-B liver disease, anti-HCV prevalence was high both in patients with hepatocellular carcinoma (78/89, 88%) and without it (66/84, 79%), while previous HBV infection was more common in patients with hepatocellular carcinoma (65/89, 73%) than in those without it (46/84, 55%) (P < 0.05). Coexistence o f anti-HCV and antibodies to H B V was observed frequently in patients with hepatocellular carcinoma (56/89, 63%) compared with patients without it (39/84, 46%) (P < 0.05). In chronic H B V carriers, anti-HCV was more common in patients with hepatOcellular carcinoma (12/38, 32%) than in those without it (3/62, 5%) (P < 0.01). These results suggest that infection with the two viruses may be a risk factor for more serious liver disease. KEY WORDS: chronic liver disease; hepatitis B virus; hepatitis C virus; hepatocellular carcinoma.
A close relationship between the development of hepatocellular carcinoma (HCC) and persistent hepatitis B virus (HBV) infection has been stressed in various areas (1-7). Recent findings suggest that integrated HBV sequences in cellular DNA could contribute to liver carcinogenesis by activation of cellular genes (8-12). To date, there also have been a few reports suggesting a putative association of HCC and the agent(s) of non-A, non-B viral hepatitis (13, 14). However, this has not been well
Manuscript received December 14, 1990; revised manuscript received May 6, 1991; accepted May 6, 1991. From the First Department of Medicine, Osaka University Medical School, Osaka, Japan. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. Address for reprint requests: Dr. Norio Hayashi, First Department of Medicine, Osaka University Medical School, Fukushima 1-1-50, Fukushima-ku, Osaka 553, Japan.
defined because of the lack of serological markers for this agent(s). Recently, Choo et al cloned the genome of a non-A, non-B hepatitis virus, termed hepatitis C virus (HCV) (15), Part of the nonstructural region of the HCV genome was expressed as a fusion polypeptide with human superoxide dismutase in yeast (C100-3 antigen), and a specific assay for the detection of antibodies to this antigen (anti-C100-3) was established (16). Using this system, epidemiological studies on the relationship between HCV infection and the development of HCC can be how performed more accurately. Several reports have demonstrated the potential etiologic,role of HCV in the development of HCC (17-20). In this Study, we determined the prevalence of anti-C100-3 and HBV markers in Japanese patients with chronic liver disease. The results were corre-
Digestive Diseases and Sciences, Vol. 37, No. 1 (January 1992)
0163-2116/92/0100-0065506.50/09 1992Plei~uinPublishingCorporation
65
YUKI ET AL TABLE 1. CLINICAL CHARACTERISTICS AND PREVALENCE OF ANTI-C100-3 IN PATIENTS WITH HEPATOCELLULAR CARCINOMA
Patients*
N
Non-A, non-B HCC Alcoholic HCC HBsAg-positive HCC
89 21 38
Aget (years) 63 -+ 7 59 --- 9 54 --- 10:~,w
Sex (M/F)
Past history of blood transfusion
Anti-ClO0-3 prevalence
72/17~ 21/0 33/5
16/89 (18%) 3/21 (14%) 2/38 (5%)
78/89 (88%) 13/21 (62%)w 12/38 (32%)~:,w
*HCC, hepatocellular carcinoma. tValues are mean - SD. :~P < 0.05 vs alcoholic HCC. w < 0.01 vs non-A, non-B HCC. lated to the p r e s e n c e of H C C to investigate the implication of the two viruses in the d e v e l o p m e n t of HCC. M A T E R I A L S AND M E T H O D S
Serum Samples. Sera were obtained from 148 Japanese patients with HCC. In each case, the tumor was revealed by imaging procedures (ultrasonography, computed tomography plus angiography). The tumor occupied one segment of the liver in 32 cases, two segments in 29, and more than two segments in 87. Of these, 119 patients had undergOne transcatheter arterial embolization before blood collection, and the other 29 patients had received no treatment. In 18 patients, HCC and preexisting cirrhosis were proved pathologically. The other patients also had preexisting chronic liver disease and showed clinical features of cirrhosis, although liver histology was not available. There were 126 men and 22 women (mean age 60 -- 9 years). Thirty-eight patients had hepatitis B surface antigen (HBsAg), and 21 were alcoholics with the average daily consumption of ethanol exceeding 135 g for more than 10 years. The other 89 patients were negative for HBsAg and showed no evidence of alcoholic, autoimmune, or drug-induced liver disease. Sera also were obtained from 146 patients with chronic liver disease who had no ultrasonographic and clinical evidence of HCC (92 men, 54 women; mean age 50 - 13 years). Of these, 84 patients had chronic non,A, non-B liver disease, and liver histology showed the features of chronic persistent hepatitis (CPH) in 10 cases, of chronic active hepatitis (CAH) in 43, and of liver cirrhosis in 31. The other 62 patients had chronic HBV infection and were known to be carriers of HBsAg for more than 12 months. Fourteen were asymptomatic HBsAg carriers with normal liver function tests, and 48 were HBsAg carriers with chronic liver disease. The liver histology of these 48 showed the features of CPH in 17 cases, of CAH in 15, and of liver cirrhosis in 16. All samples were collected for this study over three years and Stored in screw-cap vials at - 8 0 ~ C. When tested, each sample was amber, not turbid or viscous, and was used without heat inactivation. Serological Testing. HBsAg , hepatitis B e antigen (HBeAg), antibody to HB~Ag (anti-HB~) and antibody to hepatitis B core antigen (anti-HBc) were determined using commercially available radioimmunoassays (Dainabot Co., Ltd., Tokyo, Japan). Anti-C100-3 was tested with an ELISA system (Ortho Diagnostic Systems Co., Ltd.,
66
Tokyo, Japan). The assay was performed according to the manufacturer's instructions. Microtiter plates, which had been coated with a recombinant C100-3 antigen, were incubated with test sera at 37~ C for 1 hr, followed by incubation with per0xidase-conjugated monoclonal antihuman IgG antibody for another 1 hr. Then a solution containing o-phenylene diamine and H202 was added, and the resulting optical density at 492 nm (OD492) was measured using a photo-enzyme-linked immunosorbent assay colorimeter (Titertek Multiscan MCC/340, Flow Laboratories, McLean, VA). The mean value of OD492 for three negative controls of the kits plus 0.400 OD units was taken as the cutoff value, which ranged from 0.423 to 0.430 (mean 0.427). The positive controls of the kits always gave OD492 of >1.0. All assays were done in duplicate. To compare ELISA results of different sampies, the ratio of the mean OD reading of the duplicate results to the cutoff value (cutoff index) was calculated. In preliminary experiments, we examined the relationship between the ELISA cutoff index and anti-Cl00-3 titer expressed by the highest twofold dilution of the test serum showing positive results. The ELISA cutoff index was found to reach the ceiling in proportion to an increase in anti-C 100-3 titer and did not exceed 7.0. Based on these observations, we adopted the anti-C100-3 titer expressed by the highest twofold dilution as well as the ELISA cutoff index for quantitative estimation of anti-C 100-3. A total of 294 serum samples were tested for anti-C100-3, and 21 samples were positive for anti-C100-3 with cutoff index of
