Hepatitis B Virus Infection and Primary Hepatocellular Carcinoma"
2, 3
Edward Tabor," Robert J. GeretY,4 Charles L. Vogel,S Anne C. BayleY,6 Peter P. Anthony,7 Chao H. Chan,8 and Lewellys F. Barker 4. 9 ABSTRACT-Ninety-three patients with biopsy-proven primary hepatocellular carcinoma (PHC) from Uganda, Zambia, and the United States were examined for serologic evidence of hepatitis B virus (HBV) infection. Patients were tested for hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs), antibody to the hepatitis B core antigen (anti-HBc), hepatitis B e antigen (HBeAg), and its antibody (anti-HBe). Active HBV infection, as indicated by positive tests for HBsAg (with or without anti-HBs) and anti-HBc (without anti-HBs), was present in 62% of PHC patients (58 of 93), in contrast with 10% of African controls (9 of 90), and less than 1% of most United States adult populations reported in the literature. The presence of HBeAg or anti-HBe was rare among PHC patients and controls.-J Natl Cancer Inst 58: 1197-1200,1977.
Since the association between HBsAg and cases of PHC was first described by Sherlock et al. in 1970 (1), studies in many countries have found a higher prevalence of HBsAg or anti-HBs in the sera of patients with PHC compared to that in the sera of control groups. Most studies reported evidence of active HBV infection in fewer than 50% of patients with PHC. The full extent of the association between HB V and PHC may not have been demonstrated because of the relatively insensitive laboratory methods used for HBsAg detection in the past and because the tests for anti-HBs and anti-HBc were not readily available. In the present study, RIA to detect HBsAg and anti-HBs, CF and CEP assays to detect anti-HBc, and AGD to detect HBeAg and antiHBe were applied to the sera of biopsy-proven PHC patients from three countries, and the results were compared to those on sera from control groups or to the results obtained in extensive serologic studies reported in the literature. MATERIALS AND METHODS
Single serum samples from 47 PHC patients from Uganda, 19 PHC patients from Zambia, and 27 PHC patients from the United States were tested. The Ugandan patients were the subjects of an earlier study (2). Those from Zambia we're from the University Teaching Hospital, Lusaka. United States patients from different parts of the continental United States were provided by the Southeastern Cancer Study Group and by the Veterans Administration Hospital in Washington, D.C. Biopsy-proven PHC was present in all patients. Liver biopsies from patients from Uganda, Zambia, and the Southeastern Cancer Study Group were read by one pathologist (Dr. Peter Anthony). Biopsies from Washington, D.C. were read by staff pathologists at the Veterans Administration Hospital. Control sera included 50 hospital in-patients from Uganda with either melanoma or Kaposi's sarcoma and 40 healthy Zambian villagers. In both groups, controls were from the same geographic region as were the PHC patients. Controls from Uganda were 85% male, beVOL. 58, NO.5, MAY 1977
tween 24 and 89 years of age (compared to 69% male and an age range of 20-86 yr among PHC patients from Uganda). Controls from Zambia were 50% male and were between 2 and 81 years of age (compared to 89% male and an age range of 26-65 yr among PHC patients from Zambia). Since the prevalence of HBsAg and anti-HBs in the United States has been extensively studied, controls for the United States were chosen from three populations reported in the literature. These included 6,526 firsttime blood donors from 14 cities (3), 100 volunteer blood donors from Bethesda, Maryland (4), and 100 commercial blood donors from New Jersey (4). These populations were chosen to represent the range of HBV serologic findings in the United States. The 100 commercial blood donors were included to represent the greatest possible prevalence of serologic evidence of HBV exposure in the United States. Sera were tested for HBsAg by RIA (Ausria II; Abbott Laboratories, North Chicago, Ill.) (5). Anti-HBs was detected by RIA (Ausab; Abbott Laboratories) (6) and by passive hemagglutination (Virgo Reagents, Electro-Nucleonics, Bethesda, Md.) (7). Anti-HBc was detected by CF and CEP with HBcAg purified by ultracentrifugation from the liver of a chimpanzee experimentally infected with HBV (4, 8-10). The HBeAg and anti-HBe were tested by AGD (11) with the use of HBeAg from the serum of a chimpanzee experimentally infected with HBV and anti-HBe from two asymptomatic human HBsAg carriers (sera kindly provided by Mr. Roger Dodd, American National Red Cross). ABBREVIATIONS USED: HBsAg=hepatitis B surface antigen; PHC= primary hepatocellular carcinoma; anti-HBs=antibody to HBsAg; HBV=hepatitis B virus; anti-HBc=antibody to hepatitis B core antigen (HBcAg); RIA=radioimmunoassay; CF=complement fixation; CEP=counterelectrophoresis; AGD=agar gel diffusion; HBeAg= hepatitis B e antigen; anti-HBe=antibody to HBeAg. .
1197
Received May 18, 1976; accepted October 26, 1976. Supported in part by Public Health Service contracts NOI CM71343 and NOI CM43764 from the Division of Cancer Treatment, National Cancer Institute. 3 Presented in part at the Workshop on Hepatocellular Carcinoma, National Cancer Institute and Fogarty International Center, Bethesda, Md., October 31, 1975. 4 Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bldg. 29, 8800 Rockville Pike, Bethesda, Md. 20014. 5 Comprehensive Cancer Center of Florida, University of Miami Medical School, Miami, Fla. 33152 and The Southeastern Cancer Study Group. 6 Department of Surgery, University Teaching Hospital, Lusaka, Zambia. 7 Department of Pathology, Middlesex Hospital, London, England. 8 Medical Service, Veterans Administration Hospital, Washington, D.C. 20422. 9 We thank Mr. A. J. Shawver and Mr. D. Gilbert for their excellent technical assistance and Dr. Suresh Rastogi for statistical consultation. 1
2
J
NATL CANCER INST
1198
TABOR ET AL. EVIDENCE OF ACTIVE HBV INFECTION
RESULTS
One or more posiuve assays, indicating active HBV infection or recovery from prior HBV infection, were found in 83 of 93 PHC patients (89%). These included 44 of 47 PHC patients from Uganda [94%, compared to 38 of 50 controls (76%), P
2, 3
Edward Tabor," Robert J. GeretY,4 Charles L. Vogel,S Anne C. BayleY,6 Peter P. Anthony,7 Chao H. Chan,8 and Lewellys F. Barker 4. 9 ABSTRACT-Ninety-three patients with biopsy-proven primary hepatocellular carcinoma (PHC) from Uganda, Zambia, and the United States were examined for serologic evidence of hepatitis B virus (HBV) infection. Patients were tested for hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs), antibody to the hepatitis B core antigen (anti-HBc), hepatitis B e antigen (HBeAg), and its antibody (anti-HBe). Active HBV infection, as indicated by positive tests for HBsAg (with or without anti-HBs) and anti-HBc (without anti-HBs), was present in 62% of PHC patients (58 of 93), in contrast with 10% of African controls (9 of 90), and less than 1% of most United States adult populations reported in the literature. The presence of HBeAg or anti-HBe was rare among PHC patients and controls.-J Natl Cancer Inst 58: 1197-1200,1977.
Since the association between HBsAg and cases of PHC was first described by Sherlock et al. in 1970 (1), studies in many countries have found a higher prevalence of HBsAg or anti-HBs in the sera of patients with PHC compared to that in the sera of control groups. Most studies reported evidence of active HBV infection in fewer than 50% of patients with PHC. The full extent of the association between HB V and PHC may not have been demonstrated because of the relatively insensitive laboratory methods used for HBsAg detection in the past and because the tests for anti-HBs and anti-HBc were not readily available. In the present study, RIA to detect HBsAg and anti-HBs, CF and CEP assays to detect anti-HBc, and AGD to detect HBeAg and antiHBe were applied to the sera of biopsy-proven PHC patients from three countries, and the results were compared to those on sera from control groups or to the results obtained in extensive serologic studies reported in the literature. MATERIALS AND METHODS
Single serum samples from 47 PHC patients from Uganda, 19 PHC patients from Zambia, and 27 PHC patients from the United States were tested. The Ugandan patients were the subjects of an earlier study (2). Those from Zambia we're from the University Teaching Hospital, Lusaka. United States patients from different parts of the continental United States were provided by the Southeastern Cancer Study Group and by the Veterans Administration Hospital in Washington, D.C. Biopsy-proven PHC was present in all patients. Liver biopsies from patients from Uganda, Zambia, and the Southeastern Cancer Study Group were read by one pathologist (Dr. Peter Anthony). Biopsies from Washington, D.C. were read by staff pathologists at the Veterans Administration Hospital. Control sera included 50 hospital in-patients from Uganda with either melanoma or Kaposi's sarcoma and 40 healthy Zambian villagers. In both groups, controls were from the same geographic region as were the PHC patients. Controls from Uganda were 85% male, beVOL. 58, NO.5, MAY 1977
tween 24 and 89 years of age (compared to 69% male and an age range of 20-86 yr among PHC patients from Uganda). Controls from Zambia were 50% male and were between 2 and 81 years of age (compared to 89% male and an age range of 26-65 yr among PHC patients from Zambia). Since the prevalence of HBsAg and anti-HBs in the United States has been extensively studied, controls for the United States were chosen from three populations reported in the literature. These included 6,526 firsttime blood donors from 14 cities (3), 100 volunteer blood donors from Bethesda, Maryland (4), and 100 commercial blood donors from New Jersey (4). These populations were chosen to represent the range of HBV serologic findings in the United States. The 100 commercial blood donors were included to represent the greatest possible prevalence of serologic evidence of HBV exposure in the United States. Sera were tested for HBsAg by RIA (Ausria II; Abbott Laboratories, North Chicago, Ill.) (5). Anti-HBs was detected by RIA (Ausab; Abbott Laboratories) (6) and by passive hemagglutination (Virgo Reagents, Electro-Nucleonics, Bethesda, Md.) (7). Anti-HBc was detected by CF and CEP with HBcAg purified by ultracentrifugation from the liver of a chimpanzee experimentally infected with HBV (4, 8-10). The HBeAg and anti-HBe were tested by AGD (11) with the use of HBeAg from the serum of a chimpanzee experimentally infected with HBV and anti-HBe from two asymptomatic human HBsAg carriers (sera kindly provided by Mr. Roger Dodd, American National Red Cross). ABBREVIATIONS USED: HBsAg=hepatitis B surface antigen; PHC= primary hepatocellular carcinoma; anti-HBs=antibody to HBsAg; HBV=hepatitis B virus; anti-HBc=antibody to hepatitis B core antigen (HBcAg); RIA=radioimmunoassay; CF=complement fixation; CEP=counterelectrophoresis; AGD=agar gel diffusion; HBeAg= hepatitis B e antigen; anti-HBe=antibody to HBeAg. .
1197
Received May 18, 1976; accepted October 26, 1976. Supported in part by Public Health Service contracts NOI CM71343 and NOI CM43764 from the Division of Cancer Treatment, National Cancer Institute. 3 Presented in part at the Workshop on Hepatocellular Carcinoma, National Cancer Institute and Fogarty International Center, Bethesda, Md., October 31, 1975. 4 Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bldg. 29, 8800 Rockville Pike, Bethesda, Md. 20014. 5 Comprehensive Cancer Center of Florida, University of Miami Medical School, Miami, Fla. 33152 and The Southeastern Cancer Study Group. 6 Department of Surgery, University Teaching Hospital, Lusaka, Zambia. 7 Department of Pathology, Middlesex Hospital, London, England. 8 Medical Service, Veterans Administration Hospital, Washington, D.C. 20422. 9 We thank Mr. A. J. Shawver and Mr. D. Gilbert for their excellent technical assistance and Dr. Suresh Rastogi for statistical consultation. 1
2
J
NATL CANCER INST
1198
TABOR ET AL. EVIDENCE OF ACTIVE HBV INFECTION
RESULTS
One or more posiuve assays, indicating active HBV infection or recovery from prior HBV infection, were found in 83 of 93 PHC patients (89%). These included 44 of 47 PHC patients from Uganda [94%, compared to 38 of 50 controls (76%), P
