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1 Hepatitis B Viral Markers Hepatocellular Carcinoma in Patients With in Taiwan 1,2 Primary Mal-Ching Chien, M.D., 3 Myron J. Tong, Ph.D., M.D., 4 Kwang-Juel Lo, M.D., 5 Jaw-Kuang Lee, M.D., 5 David R. Milich, Ph.D., 6 Glrllh N. Vyal, Ph.D., 6 and 8ert L. Murphy, M.S. 7,8 ABSTRACT -The presence of hepatitis B viral markers in patients with primary hepatocellular carcinoma (PHC) was studied retrospectively at the Taiwan Veterans General Hospital in Taipei, Taiwan. Serum samples from 102 PHC patients and from 100 control individuals were tested for hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-hb s )' antibody to hepatitis B core antigen (anti-hbe), hepatitis Be antigen (HBeAg), and antibody to HBeAg (anti-hbe). Of the 102 PHC patients, 72 (71%) were positive for HBsAg. Nine (9%) additional patients were positive for anti-hbe alone in high titer, 19 (19%) had both anti HBe and anti-hb s ' and 9 (9%) had HBsAg, anti-hbe, and anti HBs. In the 100 controls, 12 (12%) were HBsAg-positive, whereas 22 (22%) had anti-hbe alone and 50 (50%) had both anti-hbe and anti-hb s ' Only 4 (4%) controls and no PHC patients had anti-hbs alone. Of the HBsAg-positive patients with PHC, 17 (29%) had HBeAg and 36 (61 %) had anti-hbe. The a-fetoprotein (AFP) levels above 400 ng/ml were found in 44% of the PHC patients. Values of AFP above 1X10 5 ng/ml were more frequently detected in PHC patients who were HBsAg-positive. Categorization of the geographic origins of the families whose members had PHC revealed that most families had originated from southern China. This study confirms that hepatitis B viral markers are frequently present in Chinese patients with PHC.-JNCI 1981; 66: In areas such as Asia and Africa HB,Ag is frequently present in patients with PHC (1-5). In Taiwan the relationship between HBV and PHC was first reported in 1971 (1). Recent studies have shown that anti-hb c was present in HBsAg-positive patients with PHC (6). At present it is presumed that anti-hb c, when detected in high titers in individuals who are HBsAg-negative, is indicative of ongoing HBV infection (7). HBeAg is considered to be an indicator of HBV infectivity and has been used to predict the clinical outcome in patients acutely infected with HBV (8, 9). However, HBeAg is of questionable value in the evaluation of the type or severity of chronic hepatitis B infection (10, 11). HBeAg has been detected in acute and chronic hepatitis B infections (12, 13), and in HBsAg-positive PHC patients (14). Anti-HBe has been reported only in a few patients with PHC (12). Another study described an association between anti-hbe and increased AFP levels and suggested that seroconversion from HBeAg to anti-hbe in HBsAg-positive cirrhotic patients may be indicative of early PHC (15). To gain further insight into the relationship between the HBV and PHC, we undertook the present study. We measured available serologic HBV markers in 102 PHC patients in Taiwan. MATERIALS AND METHODS Patients.-Sera of 102 Chinese patients with PHC were collected from the Department of Internal Medicine, Taiwan Veterans General Hospital. The patients consisted of 97 men and 5 women, and their average age was 52 years (range, yr). PHC was diagnosed histologically either by liver biopsy or autopsy in 36 patients. In the remaining patients, it was diagnosed by liver scan, increased AFP levels, peritoneoscopy, and celiac angiography. The control group consisted of 100 age- and sex-matched healthy individuals, who were seen at the outpatient clinic at the Taiwan Veterans General Hospital for their annual physical examinations. Laboratory tests.-hbsag and anti-hbs were measured by radioimmunoassay (Ausria II and AusAb tests; Abbott Laboratories, North Chicago, Ill.). The anti HBc was measured by radioimmunoassay by the method of Vyas and Roberts (16) and by the CorAb test (Abbott Laboratories). When the undiluted serum samples in the PHC patients were anti-hbc-positive alone, a 1:100 dilution of the sera was measured again for anti-hbc. When HB,Ag, anti-hb" and anti-hbc were ABBREVIATIONS USED: AFP=a-fetoprotein; anti-hb,=antibody to hepatitis B core antigen; anti-hb, = antibody to hepatitis B, antigen; anti-hb, = antibody to hepatitis B surface antigen; HB,Ag = hepatitis B, antigen; HB,Ag = hepatitis B surface antigen; HBV = hepatitis B virus; PHC= primary hepatocellular carcinoma(s). I Received May 16, 1980; accepted September 15, Supported in part by American Cancer Society Institutional Research Grant IN-21-R to the Los Angeles County/University of Southern California Comprehensive Cancer Center. 3 Liver Unit at the University of Southern California School of Medicine, Los Angeles, Calif Address reprint requests to Dr. TOQg at Liver Diagnostic Center, Huntington Memorial Hospital, 100 Congress St., Pasadena, Calif Division of Gastroenterology, Taiwan Veterans General Hospital, Taipei, Taiwan. 6 Department of Laboratory Medicine, University of California School of Medicine, San Francisco, Calif Center for Disease Control, Phoenix, Ariz We thank the personnel of the Department of Nuclear Medicine, Taiwan Veterans General Hospital. We also thank Dr. J. M. Weiner (University of Southern California School of Medicine) and Ms. J. H. Lin and Ms. C. M. Spengler (Liver Diagnostic Center, Huntington Memorial Hospital) for their valuable advice and technical assistance. 475

2 476 Chien, Tong, Lo, et at detected in the same individual, we performed an additional experiment to determine the specificity of the anti-hbs (Ling CM: Personal communication). We added 0.02 ml of strongly HBsAg-positive sera, subtype ad, to 0.2 ml of each serum sample in question, incubated the mixture at 25 C for 2 hours, and then tested the sera again for anti-hbs by AusAb. HBeAg and anti-hbe were measured by rheophoresis after fivefold concentration of the test sera with the use of polyacrylamide gel (Lyphogel; Gelman Instrument Co., Ann Arbor, Mich.) (10) and by radioimmunoassay (Abbott Laboratories). AFP was detected by radioimmunoassay (Alphafeto-125 kit; Dainabot Radioisotope Laboratories Ltd., Tokyo, Japan). When the AFP value was over 320 nglml, measurements were repeated with serial dilutions of the sera. Statistical analyses were performed by the chi-square test. RESULTS Viral markers.-of the 102 patients with PHC, 72 (71%) were positive for HBsAg (table I). Anti-HBc was detected in 100 of the 102 (98%) patients. Sixty-three (62%) of the 102 patients were positive for HBsAg and anti-hbc, 9 (9%) were positive in high titer for anti HBc alone, and 19 (19%) had both anti-hbc and anti HBs. Three viral markers, HBsAg, anti-hbs, and anti HBc, were detected in 9 (9%) PHC patients. In each instance, the presence of anti-hbs in an HBsAgpositive serum was confirmed by the neutralization test described in "Materials and Methods." Anti-HBs alone was not found in any PHC patient. In the 100 controls, 12 (12%) had circulating HBsAg and 84 (84%) had anti HBc. Among this last group, 22 (22%) were positive for anti-hbc alone and 50 (50%) had both anti-hbc and anti-hbs. Four (4%) controls and none of the PHC patients had anti-hbs alone. When measured by a radioimmunoassay method, the HBeAg was positive in 17 (29%) and anti-hbe was de- TABLE I.-Frequency of HB,Ag, anti-hb" anti-hbc in controls and in patients with PHC in Taiwan No. of HB,AgQ Anti-HB,Q Anti-HBo Q controls No. of patients with PHC 12 (12) 2 (2) + 4 (4) (12) 63 (62)b (50) 19 (19)b + 22 (22) 9(9)b (9) Total Positive for HB,Ag (I?) 72 (71) Positive for anti-hb, (54) 28 (27) Positive for anti-hb, (84) 100 (98) With no markers (12) 2 (2) Positive for at least 1 marker (88) 100 (98) Q - = negative; + = positive. b P<O.OOOI for all tests performed. TABLE 2.-Measurement of HB,Ag and anti-hb, by radioimmunoassay in controls and in HB,Ag-positive patients with PHC Parameter HB,Ag-positive 1 (9) Anti-HB,-positive 10 (91) HB,Ag-positive and anti-hb,-positive HB,Ag-negative and anti-hb,-negative o Total 11 No. of HB,Ag-positive Controls Patients with PHC 17 (29) 36 (61) 1 (2) 5 (8) 59 tected in 36 (61 %) of 59 HBsAg-positive patients with PHC (table 2). In the control group, I (9%) of II HB,Ag-positive persons had detectable HBeAg and 10 (91%) had anti-hbe. When sera from both groups were initially measured by rheophoresis, HBeAg was detected in 10% of the serum samples and anti-hbe in only 1% (table 3). Thus the radioimmunoassay technique is two times more sensitive for the detection of HBeAg and 60 times more sensitive for the measurement of anti-hb e AFP.-Seventy-six PHC patients had AFP levels above 20 nglml (table 4). Thirty-two patients had AFP values between 20 and 400 nglml with a mean of 152 ng/m!. Forty-four patients had AFP levels above 400 nglml, ranging between 737 and 1.5XI0 6 ng/ml. and having a mean value of 1.6XI0 5 ng/m!. Levels of AFP greater than IXI0 5 nglml were noted in 9 HBsAgpositive patients with PHC and in only I HBsAgnegative patient with PHC. Four (4%) of the controls had the following AFP levels above 20 ng/ml: 23, 35, 54, and 246 ng/m!. In these individuals, the serum alanine aminotransferase and aspartate aminotransferase levels were normal and the HB,Ag was not detected by radioimmunoassay. Of these 4 controls, 2 were anti HBc-positive only, and the remaining 2 were anti-hbspositive and anti-hbc-positive. Geographic distribution of families of PHC patients.-we attempted to categorize the geographic origins of the families of the patients with PHC and the controls (table 5). The area around the Yangtze River was considered as being in central China. Provinces to the north were considered to be in nothern China and those to the south of the central area were considered to be in southern China. The families of 66 TABLE 3.-Comparison of two methods of testing for HB,Ag and anti-hb, in HB,Ag-positive individuals Method of testing Q No. of individuals Radio- Parameter measured tested Rheophoresis immunoassay HB,Ag 70 7 (10) 18 (26) Anti-HB, 70 1 (1) 46 (66) Q Values are expressed as No. of serum samples positive.

3 Hepatitis B In Hepatocellular Carcinoma 477 TABLE 4.-AFP in 100 controls and in 100 patients with PHC AFP levels, No. of No. of PRC ng/ml a controls patients <20 96 (96) 24 (24) b (4) 32 (32) > (44) Total tested : AFP was considered positive when the value was >20 ng/ml. Th~ee sera were below 54 ng/ml (23, 35, and 54 ng/ml, respectively). One serum was 246 ngfml. (65%) of the 102 patients originated from southern China, whereas the families of 19 (19%) and 17 (17%) of the 102 patients originated from central and northern China, respectively. Also, all the families of the 12 HBsAg-positive controls originated from southern China. DISCUSSION PHC is a common malignant disease in areas such as Asia and Africa. In Taiwan the frequency of PHC among all cancers is reported to be as high as 22% and PHC is second only to nasopharyngeal carcinoma as the most common cancer in Chinese males (17). In Africa the frequency of PHC among all reported cancers was as high as 51 % in the Bantu (18). PHC is ~sua.lly f~und in patients with preexisting postnecrouc CIrrhOSIS. In a reported autopsy series, the association of PHC and cirrhosis was 60% in Bantu and between 79 and 91% in Taiwanese (18, 19). There have been numerous reports on the frequency of HBsAg in HBV-related diseases. The present study showed that the HBsAg positivity was 71 % in the PHC patients and 12% in the asymptomatic controls. Compared with the 15-20% frequencies of HBsAg previously reported (20, 21), the relatively lower frequency of HBsAg in our controls may be attributed to the older ~ndividuals who wer~ used as the age-matched group ill our study of pauents with PHC. Although the frequency of HBsAg-positive patients was similar to that of our previous communication (1), the measurement of HBV markers provided further information for categorization of the Chinese patients with PHC. In the present study we noted that all HBsAg-positive patients had circulating anti-hb c, and 5.-Frequency of HB,Ag-positive controls and patients wtth PHC according to geographic origins of their families TA~LE Territory No. of RB,Ag-positive Controls Patients with PRC All patients with PRC Northern China 0 10 (14) 17 (17) Central China 0 13 (18) 19 (19) Southern China 12 (100) 49 (68) 66 (65) Total (32%) of the 28 HB.Ag-negative patients had high titers of anti-hb c (in the absence of anti-hb.) in their sera. Recent reports indicated that the presence of anti HBc in high titer is indicative of ongoing HBV infection (22). If so, then 81 (79%) of 102 of our PHC patients were actively infected by HBV. This observation has been confirmed in other reports in which anti HBc was detected in 73-89% of patients with PHC (6, 23). However, to determine if the presence of anti-hbc in each instance truly represents current HBV infection, one must perform a confirmatory test such as staining liver tissue for the HBV antigens (24). Using the immunoperoxidase method, Omata et al. (25, 26) revealed that 75% of nonalcoholic liver disease patients with PHC, who were anti-hbc-positive, had hepatitis B core antigen in the nuclei of their hepatocytes. Anti HB. and anti-hb c together were detected in 19% of the patients with PHC and in the absence of HBsAg represent past infection by the HBV. Thus in our HB.Ag-negative PHC patients with both HBV antibodies, the relationship between HBV infection and ~HC. is unlikely and another etiologic source may be Imphcated. Three markers, HBsAg, anti-hbc, and anti HB., were detected in 9 of our PHC patients. To determine the specificity of the anti-hb. result, we performed an additional experiment: In each of these 9 sera, anti-hb. activity was neutralized by the addition of the HBsAg-positive sera. The presence of anti-hbs and HBsAg in the same sample may be due to the host's production of a small amount of anti-hbs that formed immune complexes and was detected by the sensitive radioimmunoassay tests (AusAb and Ausria II) presently in use. Alternatively, the HBsAg and anti HBs may be of different subtypes. HBcAg has been used as an indicator of viral infectivity and is found in HBsAg-positive patients with chronic persistent hepatitis, chronic active hepauus, and PHC (12-14). Recently, a significant correlation of HBsAg titers with HBeAg has been reported (27). Also, with advancing age, a decreasing prevalence of HBeAg and an increasing prevalence of anti-hbe in persisten.t HBsAg carriers were seen (28). In a study from TaIwan, HBeAg was found in 27% and anti-hbe in 2% of young HBsAg carriers, whereas in an older Chinese-American carrier population in the United States, HBeAg was found in 3.3% and anti-hbe was found in 24% (29,30). In Japan HBeAg was found in 22% and anti-hbe in 46% of asymptomatic HBsAg carriers (31). In the present paper, HBeAg was present in 9% of the. control HBsAg carriers and 91% were positive for anu-hbe. There are few reports on the frequencies of HBeAg and anti-hbe in patients with PHC. With the use of the less sensitive rheophoresis technique, HBeAg was detected in 2.3% and anti-hbe in 21 % of the PHC patients in Africa (15). In our PHC patients 29% were positive for HBeAg and 61 % had anti-hb e as revealed by a r~dioim~unoassay method. The higher frequency of anu-hbe ill PHC patients has been attributed to a stage of inactive liver disease (12) but may be due to the natural history of acquiring anti-hb e at a later age. JNC[, VOL. 66, NO.3, MARCH 1981

4 478 Chien, Tong, Lo, et al. As shown in our study, the frequency of anti-hbe was high in both the PHC patients and the controls, and the controls were age matched. Both HBeAg and anti HBe were detected in 1 PHC patient. This finding was thought to represent the host's transition stage from HBeAg to anti-hbe. The role of this antigen-antibody system in PHC is unclear. AFP is primarily produced by hepatic tumor tissue but may be produced by regenerating liver cells. Recently, AFP was detected in the cytoplasm of malignant cells (32). In Taiwan, with the use of a sensitive radioimmunoassay method, 97% of the healthy adults were found to have AFP levels below 20 ng/ml, and only 3 had AFP values between 21 and 40 ng/ml (33). In the present study that utilized the same radioimmunoassay method, 96% of the controls were found to have AFP levels below 20 ng/m!. However, 4 controls had AFP values of 23, 35, 54, and 246 ng/ml, respectively. All 4 had normal serum alanine aminotransferase and aspartate aminotransferase levels, and HB,Ag was not detected by radioimmunoassay. Two of these individuals had anti-hb c and anti-hb" and the remaining 2 had circulating anti-hb c only. The control who had an AFP level of 246 ng/ml had both anti-hb, and anti-hb c, which was attributed to convalescence from non-b viral hepatitis with regeneration of damaged liver tissue. The relationship between the AFP levels and circulating HB,Ag is still controversial (5, 34-36). AFP levels of greater than lx10 5 ng/ml were found in 9 of our HB,Ag-positive PHC patients but in only 1 of our HB,Ag-negative PHC patients. Chen and Sung (33) speculated that the association of HB,Ag and elevated AFP levels in patients with chronic active hepatitis and cirrhosis contributes to the higher risk of developing PHC. In addition, a long-term follow-up of chronic hepatitis patients at the Taiwan Veterans General Hospital showed that 92% of the patients with chronic active hepatitis were HB,Ag-positive (Lo K-J, Chien M-C, Tsai Y-T, et al.: Unpublished data), and PHC was detected in those- HB,Ag-positive chronic active hepatitis patients who developed AFP levels of above 400 ng/m!. In 1965 Tu and Lee (37) observed in Taiwan that PHC frequently occurred in Chinese patients whose origin was in the coastal provinces of China. Also, PHC has been frequently found in the Chinese residing overseas (38, 39). Most Chinese living outside China originated from the southern coastal provinces such as Kwangtung and Fukien. In the present study 68% of the families of PHC patients and all of the families of HB,Ag-positive controls originated from southern China. Dietary factors such as aflatoxin contamination and other environmental factors that may contribute to the high frequency of PHC in individuals originating from the south of China remain to be elucidated. This study showed that HBV markers are frequently detected in Chinese patients with PHC. Ninety-eight percent of PHC patients were positive for at least one HBV marker, and 79% had evidence of active HBV infection. Although a direct oncogenic property for the HBV has not yet been demonstrated, the high frequency of HBsAg positivity in PHC patients strongly suggests an initiating and a supporting role of the virus in the development of this cancer. REFERENCES (1) TONG MJ, SUN SC, SCHAEFFER BT, CHANG NK, Lo KJ, PETERS RL. Hepatitis-associated antigen and hepatocellular carcinoma in Taiwan. Ann Intern Med 1971; 75: (2) SIMONS MJ, YAP EH, SHANMUGARATNAM K. 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