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Clearance of hepatitis B surface antigen (HBsAg) after surgical resection of hepatocellular carcinoma
Journal of Hepatology, 1987;4:45-51 Elsevier
45
HEP 00258
Clearance of hepatitis B surface antigen (HBsAg) after surgical resection of hepatocellular carcinoma Tsung-Teh Wu I , Hey-Chi Hsu l, Ding-Shinn Chen 2, Jin-Chuan Sheu 2, Ih-Jen Su I, Shiu-Ling Chen I and Sou-Ming Chuang 1 Departments of 1Pathology and 21nternal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (Republic of China) (Received 31 July, 1986) (Accepted 23 October, 1986)
Summary The serum HBsAg in 4 chronic HBsAg carrier patients with hepatocellular carcinoma (HCC) cleared within 4-38 months after surgical resection of their hepatic tumors. Two patients developed anti-HBs. During the follow-up period from 21 to 28 months after HBsAg clearance, none of the patients regained positive serum HBsAg. Two patients who had had tissue HBsAg present, exclusively in the tumor, showed quick HBsAg clearance after resection. The other 2 patients had a delayed HBsAg clearance. One had tissue HBsAg in both the tumor and nontumoral liver. Only 1 patient bad tissue FIBsAg in the liver, but not in the tumor. During the same period of observation of 323 chronic HBsAg carriers, who had a variety of histologically-verified chronic liver diseases and were followed for more than 6 months, only 1 cleared the antigen. The spontaneous HBsAg clearance in our HBsAg carriers (1/323) was significantly lower than that (4/64) of HBsAg-positive HCC patients with tumor resection, P < 0.004. The mechanisms of HBsAg clearance in HCC patients after surgical resection of tumors are discussed.
Introduction It is generally agreed that the hepatitis B surface antigen (HBsAg) carrier state is prolonged once established, although early studies have indicated that
it can terminate spontaneously. In a series of 210 blood donors followed for 4-20 months, 13 (6.2%) cleared HBsAg spontaneously [1]. In another series of 414 carriers followed for 2-42 months, 4 (1.0%) cleared HBsAg [2]. However, in neither group was
This work was partially supported by grants from the National Science Council of the Republic of China (NSC74-0610-B002-32, NSC75-B002-0412-17). Correspondence address: Professor Hey-Chi Hsu, Department of Pathology, National Taiwan University Hospital, Taipei. Taiwan, Republic of China. 0168-8278/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
T.-T. WU et al.
46 the carrier status defined by 6-month duration; all 4 cases of Helske's series [2] cleared the antigen within 6 months. According to the definition of the 6-month duration carrier state, only 1 case has cleared HBsAg among 227 cases followed for 6-76 months (mean: 44 months) by Sampliner et al. [3]. It is generally believed that clearance of HBsAg is an unusual event if the HBsAg has been documented as present for as long as 6 months [4]. The mechanisms of HBsAg clearance and its biologic significance are unknown, although antiviral therapy has been shown to clear HBsAg in some carriers with chronic hepatitis B [5]. It appears, however, that HBsAg clearance in HBsAg carriers, particularly after surgical resection of hepatocellular carcinoma (HCC), has not been previously reported. Patients with HCC are well known to follow a rapid fatal course when the tumor has reached its advanced stage. If the tumor is detected in the early stage and small in size, the clinical course can frequently be prolonged [6,7], and surgical resection has become possible in many patients [8]. These patients' longer survival makes it possible to investigate their possible HBsAg clearance after tumor resection. Among 64 patients of HCC followed for more than 6 months after tumor resection, 4 subsequently cleared serum HBsAg. This study reports their data.
Patients and Methods
Patients From May 1979 to December 1984, 131 HCC patients received surgical resection of the tumor at National Taiwan University Hospital. Sixty-eight were ~<5 cm in diameter (small HCC); 63 were larger (large HCC). Among these, 64 cases (49 small and 15 large HCCs) had serial follow-up serum HBsAg tests for more than 6 and up to 57 months (mean + SD: 32.5 + 12.9 months). The findings formed the basis of this study. During roughly the same period from January 1980 to December 1984, 323 chronic HBsAg carriers who had biopsy-proven chronic hepatitis or cirrhosis and had serial serologic follow-up of HBV markers for more than 6 and up to 63 months (mean
+ SD: 29.6 + 12.9 months) formed the control group.
Histologic examinations Paraffin sections of 3 - 4 Bm thickness were stained with hematoxylin and eosin (HE). Liver HBsAg and HBcAg in the liver and HCC tissues were demonstrated by both peroxidase-antiperoxidase (PAP) method, and/or avidin-biotin-peroxidase complex (ABC) techniques by using rabbit anti-HBs and antiHBc (Dako Corporation, Santa Barbara, CA, USA) [9,101. Serologic assays Serum HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc were measured by radioimmunoassays (Abbott Laboratories, North Chicago, IL, USA).
Results
From among 64 HCC patients who had been followed for more than 6 months after tumor resection, serum HBsAg cleared in 3 patients (3/49) with small HCC (Cases 1, 3 and 4) and 1 (1/15) with large HCC (Case 2). Two cases (Cases 2 and 3) seroconverted to anti-HBs. The other 2 patients remained negative for anti-HBs. In the control group of 323 chronic HBsAg carriers with biopsy-proven chronic hepatitis or cirrhosis, only one (0.3%) seroconverted to anti-HBs. The serum HBsAg of this patient was of low titer 21 months after biopsy and had completely disappeared with
TABLE 1 COMPARISON OF HBsAg CLEARANCE BETWEEN HEPATOCELLULAR CARCINOMA (HCC) PATIENTS AFTER TUMOR RESECTION AND CARRIERS WITH CHRONIC LIVER DISEASES HBsAg clearance HCC group Chronic carriers
Yes
No
4 (6.3%) I (0.113¢'~)
60 322 P < 0.004
HBsAG CLEARANCE AFTER RESECTION OF HEPATOCELLULAR CARCINOMA
47
c o n v e r s i o n to H B s A g - / a n t i - H B s + 18 m o n t h s later.
f r o m h e m a t e m e s i s and tarry stool due to r u p t u r e d
T h e a n t i - H B s had persisted for 24 m o n t h s up to the
e s o p h a g e a l varices, r e c e i v e d a p o r t o s y s t e m i c shunt
latest f o l l o w - u p in D e c e m b e r 1985.
and liver biopsy in 1977. L i v e r cirrhosis was con-
T h e H B s A g c l e a r a n c e rate was significantly h i g h e r
firmed, and both H B s A g and H B c A g w e r e d e t e c t e d
in the H C C g r o u p ( 6 . 3 % ) than in the c o n t r o l g r o u p
in liver cells. R e g u l a r a b d o m i n a l s o n o g r a p h i c follow-
( 0 . 3 % ) , P < 0.004 ( T a b l e 1). T h e annual c l e a r a n c e
up r e v e a l e d a small hepatic t u m o r in the right lobe in
rate of H B s A g was 2 . 6 % in patients with H C C , and
A u g u s t 1983; thus a w e d g e resection of the t u m o r
0 . 1 % in those with c h r o n i c hepatitis or cirrhosis.
was p e r f o r m e d in D e c e m b e r 1983. F o u r m o n t h s later, the first p o s t o p e r a t i v e
b l o o d sample
demon-
strated c l e a r a n c e of H B s A g and, up to the last folReport of Cases
low-up in M a r c h 1986, 27 m o n t h s after resection, the s e r u m H B s A g r e m a i n e d negative. A n t i - H B s was not
T h e b r i e f clinical c o u r s e and serologic profiles of
d e t e c t a b l e . T h e patient felt well and had no t u m o r re-
the 4 H C C patients with H B s A g c l e a r a n c e are sum-
c u r r e n c e . T h r o u g h o u t the course, s e r u m alpha-feto-
m a r i z e d in T a b l e 2; and the p a t h o l o g i c f e a t u r e s are
protein ( A F P ) was within n o r m a l limits.
shown in T a b l e 3.
T h e w e d g e - r e s e c t e d liver s p e c i m e n m e a s u r e d 2.8 x 2.5 x 1.5 cm and c o n t a i n e d a small hepatic t u m o r
Case 1
1.6 x 1.5 x 0,8 cm in size. T h e t u m o r was n o d u l a t e d
A 54-year-old man w h o , in 1976, had suffered
and e n c a p s u l a t e d ,
but with e x t r a c a p s u l a r invasion
TABLE 2 CLINICAL FEATURES OF 4 PATIENTS WITH HBsAg CLEARANCE FOLLOWING SURGICAL RESECTION OF HEPATOCELLULAR CARCINOMA Case No.
Age/ sex
HBeAg/ anti-HBe
HBsAg carriage
Anti-HBs
Duration
Clear/FU
Preop.
Postop.
1 2 3 4
54/M 66/M 66/M 70/M '~
-/+ -/+ -/+ -/+
6 yr 13 yr 7 yr ?
4 mo/21 mo 12 mo/27 mo 31 mo/28 mo 27 mo/27 mo
+, t -
+ + -
Abbreviations: Clear/FU = duration of HBsAg clearance after tumor resection/follow-up duration; preop. = pre-operatton; postop. = post-operation; t = low titer; + = positive;- = negative. An episode of non-A, non-B hepatitis was suspected 10 weeks after operation.
TABLE 3 PATHOLOGIC FEATURES OF THE 4 RESECTED HEPATOCELLULAR CARCINOMAS ACCOMPANIED BY HBsAg CLEARANCE Case No.
Tumor size/site
Liver status
HBsAg in liver/ HCC
HBcAg in liver/ HCC
Tumor recurrence;'
1 2
1.6/R 5.5/R (lst) 1.6/L (2nd) 4.4/R 4.8/L
C C
-/+ -/+ -/? +/+/+
-/+, f -/-/? -/-/-
No
3 4
PF, CPH C
Suspicious Yes No
Abbreviations: R = right lobe; L = left lobe; C = cirrhosis; PF = portal fibrosis; CPH = chronic persistent hepatitis; ? = unknown; f = focal. a After HBsAg clearance.
48
T.-T. WU et al.
and portal vein branch involvement. Multiple sections of the non-tumor liver failed to reveal tissue HBsAg, whereas the tumor portion had globular or inclusion type of cytoplasmic HBsAg in isolated tumor cells (Fig. 1). HBcAg was also demonstrated only in isolated tumor cells.
Case 2 A 66-year-old man who had had hepatomegaly and diarrhea in 1972 was found on biopsy to have fatty liver. He was regularly followed at the Government Employee Clinical Center. In 1980, AFP increased and HCC with cirrhosis was diagnosed. He received a right lobe segmentectomy in August 1981. HBsAg at that time was positive and anti-HBs was also positive in low titer. Tumor recurred in the left lobe, and a second operation was performed in December 1982. Seroconversion to anti-HBs occurred 12 months after the second operation, and anti-HBc was positive throughout follow-up. In his third operation, in April 1984, a tumor implant was resected from the omentum. Regular sonographic examination at the last follow-up in February 1986 revealed a suspected recurrent hepatic tumor in the left lobe near the prior operation site, but a biopsy was not performed. The first resected HCC from the right lobe measured 5.5 cm in diameter. It was well-encapsulated and with capsular invasion. Scattered globular-type
Fig. 1. Globular type cytoplasmicHBsAg is seen in a tumor cell (arrowhead) in the tumor portion of Case 1. PAP stain with mouse monoclonalanti-HBs, x600.
Fig. 2. Isolated globular cytoplasmic HBsAg is demonstrated in tumor cells (arrowheads) of Case 3. PAP stain with mouse monoclonal anti-HBs, x600.
cytoplasmic HBsAg was demonstrated in the tumor cells, but not in the nontumorous liver (Fig. 2). HBcAg was negative in both the tumor and the liver. The second resected specimen revealed a small tumor nodule about 1.5 cm in diameter. It was encapsulated but with extracapsular and liver invasion accompanied by a tumor thrombus in a small portal vein branch. Most of the resected tumor tissue was sent for virologic studies; the remaining tissue was inaclequate for tissue HBsAg and HBcAg study. The third operated specimen, in the omentum, was a 0.4 cm tumor nodule which was negative for HBsAg and HBcAg.
Case 3 A 66-year-old man who had been known to be a chronic HBsAg carrier since April 1978 developed elevated AFP in January 1980. Peritoneoscopy showed macronodular cirrhosis, and abdominal sonography revealed a mass in the left lobe of the liver. He received a segmentectomy in May 1981. Clearance of HBsAg occurred in October 1983, and antiHBs appeared 9 months later. Tumor recurrence was noticed in the left lobe on regular follow-up in May 1984, but the patient declined further surgery. The tumor had grown from 1.5 cm to 8.5 cm at the last follow-up in March 1986, 58 months after the tumor resection. The patient remained HBsAg-seronegative.
HBsAG CLEARANCE AFTER RESECTION OF HEPATOCELLULAR CARCINOMA The resected small HCC measured 4.4 × 4.4 x 4.2 cm in size, and was not encapsulated. Microscopically, there was liver, but no portal vein, invasion. The non-neoplastic liver parenchyma showed portal fibrosis and chronic persistent hepatitis. Tissue HBsAg was positive in the non-tumor, but not in the tumor, portion. Case 4
A 70-year-old man was found to have positive serum HBsAg and increased AFP in June 1981 during an annual physical check-up. Abdominal sonography and CT scan revealed a hepatic tumor about 5 cm in diameter in the left lobe. He received left lateral seg-
49
mentectomy in July. An episode of acute hepatitis probably due to superimposed post-transfusion nonA, non-B hepatitis, occurred 10 weeks after operation. Three months later, HBsAg was of low titer; it then disappeared 27 months after operation. At the last follow-up in March 1986, serum HBsAg remained negative as did anti-HBs. There was no local tumor recurrence despite elevated AFP. The resected small HCC measured 4.8 cm in diameter. It was well-encapsulated and showed no extracapsular or portal vein invasion. The non-neoplastic liver was cirrhotic. Tissue HBsAg was demonstrated both in the tumor and non-tumoral portions (Fig. 3), but not HBcAg.
Discussion
Fig. 3. A: Most of the tumor cells in the peripheral portion show invasion of the tumor capsule (c). B: Higher magnification shows cytoplasmic HBsAg of varying intensity in many tumor cells (arrowheads). PAP stain for HBsAg. A: x150; B: x600.
Delayed clearance of HBsAg is an unusual event. In a long-term follow-up observation Karen et ai. [11] reported HBsAg clearance with seroconversion to anti-HBs in 7 chronic HBsAg carriers, 5 with biopsy-proved chronic persistent hepatitis. Wallace et al. [12] observed a 6.0% loss of HBsAg among 115 HBsAg carriers, with an annual clearance rate of 1.0%, during follow-ups in a mean period of 6.1 years. The patients reported are healthy carriers or had Type B chronic hepatitis. HBsAg clearance in HCC patients has not been reported, probably because of the usual rapidly fatal course of symptomatic HCC. In Taiwan, chronic HBsAg carriers account for 15-17.8% of the general population [13-15], but the annual clearance rate of HBsAg as determined by the sensitive RIA test is not known. In the present series of 323 carriers with biopsy-proven chronic hepatitis or cirrhosis, spontaneous HBsAg clearance occurred rarely, accounting for only 0.3% (or 1/323) during a mean period of 2.5 years, or 0.1% per year. In contrast, a significantly higher annual clearance rate (2.6%) was demonstrated in HCC patients after surgical resection of the tumor. This information may provide some clues toward better understanding of the mechanisms of HBsAg clearance in HBsAg carriers. The mechanisms of the clearance of HBsAg car-
T.-T. WU et al.
50 rier status are not yet fully understood. Antiviral and interferon therapies have led to the clearance of HBsAg in some patients [16-19]. In contrast, none of the 15 patients of Omata et al. [20] nor the 10 patients of Smith et al. [21] showed any response. The reason for this discrepancy is not known. However, none of the present 4 patients were administered any antiviral therapy. The most remarkable and unusual findings were that H B s A g could be demonstrated in the tumor itself but not in the non-tumor portion of the livers of 2 patients (Cases 1 and 2) who had rapid clearance of serum HBsAg after tumor resection. These findings strongly suggest that the HBsAg-bearing H C C per se is responsible for the HBsAg antigenemia, and the removal of the tumor is responsible for the clearance of serum HBsAg. Previous studies have demonstrated the presence of H B s A g in the tumor cells in 15% of large H C C and 40% in small H C C [6], indicating a continued presence in the tumor cells of H B V genome which is capable of producing H B V antigens, as has also been suggested by H C C cell line culture and heterotransplant [22,23]. Several other possibilities can be raised to explain the mechanisms of H B s A g clearance in these patients. One is probably related to the reduction in HBsAg-producing cells either in the H C C and/or the non-tumorous liver, resulting in a lower serum H B s A g level undetectable with the present assay method [24]. These results are unable to prove or disprove this suggestion.
References
1 Szmuness W. Prince A. Brotman B, Hirsch RL. Hepatitis B antigen and antibody in blood donors: An epidemiologic study. J Infect Dis 1973; 127: 17-25. 2 Helske T. Carriers of hepatitis B antigen and transfusion hepatitis in Finland. Scand J Haematol 1974; 22 (Suppl): 1-65.
3 Sampliner RE, Carney E, Tabor E, Gerety R. In: W Szmuness. H.I Alter, JE Maynard (Eds.), Viral Hepatitis (1981 International Symposium). Franklin Institute Press, Philadelphia, PA, 1982: 722-723. 4 Sampliner RE. Follow-up and management of hepatitis B carriers. In: RJ Gerety (Ed.), Hepatitis B. Academic Press, Inc., Orlando, FL, 1985: 155-172. 5 Scullard GH, Pollard RB, Smith JL, Sacks S, Gregory PB,
However, the H B s A g clearance rate in the patients with large H C C (1/15) did not differ from those with small H C C (3/49), despite a much greater liver volume reduction after tumor resection in the large HCC. A n o t h e r possibility is the immunosuppressive effect of the H C C on the host's cellular immunity, which has been shown to improve after tumor resection [25]. The occurrence of anti-HBs after tumor resection in 2 of the 4 cases appears to support this speculation. The biologic significance of the H B s A g clearance in these patients is unknown, although H B s A g clearance has been suggested to imply a resolution of the underlying disease, elimination of the infectivity, and possibly elimination of the risk for H C C [4]. Two of these cases (Cases 1 and 4) had a relatively prolonged HCC-free period of 21-27 months after H B s A g clearance, and included 1 patient with extracapsular invasion and satellite nodules near the tumor. Because extracapsular invasion and satellite nodule formation in H C C have been shown to be associated with a high tumor recurrence rate [6], this finding seems to support the suggestion that H B s A g clearance may eliminate the risk of H C C [4]. However, the recurrence of tumor in the remaining 2 cases, despite clearance of H B s A g , contradicts this suggestion. Because of the limited number of cases, clarification of the biologic significance of the H B s A g clearance in H C C patients after tumor resection will require further study.
Robinson WS, Merigan TC. Antiviral treatment of chronic hepatitis B virus infection, Part 1 (Changes in viral markers with interferon combined with adenine arabinoside). J Infect Dis 1981; 143: 772-783. 6 Hsu HC, Sheu JC, Lin YH, et al. Prognostic histological features of resected small hepatocellular carcinoma (HCC) in Taiwan - - A comparison with resected large HCC. Cancer 1985; 56: 672-680. 7 Sheu JC, Sung JL, Chen DS, et al. Growth rate of asymptomatic hepatocellular carcinoma and its clinical implications. Gastroenterology 1985; 89: 259-266. 8 Chen DS, Sheu JC, Sung JL, et al. Small hepatocellular carcinoma: A clinicopathological study in thirteen patients. Gastroenterology 1982; 81 : 1109-1119. 9 Hsu HC, Lin WSJ, Tsai MJ, et al. Hepatitis-B surface antigen and hepatocellular carcinoma in Taiwan, with special
HBsAG CLEARANCE AFTER RESECTION OF HEPATOCELLULAR CARCINOMA reference to types and localization of HBsAg in the tumor cells. Cancer 1983; 52: 1825-1832. 10 Hsu SM. Raine L, Fanger H. Use of avidin-peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC and unlabeled antibody (PAP) procedure..I Histochem Cytochem 1981; 29: 577-580. 11 Karen LL, Allan GR, Mary A. Delayed HBsAg clearance in chronic hepatitis B viral infection. Hepatology 1981: 1: 586-589. 12 Wallace LMA, Brain JM, David BH, et al. The long-term serological course of asymptomatic hepatitis B virus carriers and the development of primary hepatocellular carcinoma. J Infect Dis 1985; 151: 604-609. 13 Sung JL, Chen DS. Hepatitis B surface antigen and antibody in liver disease in Taiwan (Proceedings 5th Asian Pacific Congress Gastroenterology, Singapore, 23-27 May). 1976: 265-269. 14 Beasley RP, Stevens CE. Epidemiology of hepatitis B infection in Taiwan (Proceedings International Symposium on Hepatitis in Taipei, 23-24 March). 1974: 1-10. 15 Tong MJ, Sun SC, Schaeffer BT, et al. Hepatitis-associated antigen and hepatocellular carcinoma in Taiwan, Ann Intern Med 1971; 75: 687-691. 16 Weller IV, Fowler M J, Monjardino J, Carreno V, Thomas HC, Sherlock S. Inhibition of hepatitis B viral replication by lymphoblastoid interferon. Phil Trans Roy Soc London 1982; 299: 128-130. 17 Lok ASF, Karayiannis P, Brown D, et al. A randomised study of the effect of adenine arabinoside monophosphate (4 or 8 week course) versus lymphoblastoid interferon on
18
19
20
21
22
23
24
25
51
hepatitis B virus (HBV) replication. Hepatology 1983; 3: 865. Lok ASF, Weller IVD, Karayinnis P, et al. Thrice weekly lymphoblastoid interferon is effective in inhibition hepatitis B virus replication. Liver 1984: 4: 45-49. Dooley JS, Davis GL, Waggoner JG, Hoofnagle JH. Low dose versus high dose recombinant leucocyte A interferon for chronic type B hepatitis. Hepatology 1983; 3: 853. Omata M, Yokosuka O, lmazeki F, Okuda K. In: GN Vyas, JL Dienstag, JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stratton, New York, NY, 1984: 670-676. Smith CI, Weissberg J, Bernhardt L, Merigan TC, Robinson WS. Acute Dane particle suppression with recombinant leucocyte A interferon in chronic hepatitis B virus infection. J Infect Dis 1983; 148: 907-913. Ziegler JL, Adamson RH, Barker LF. Fraumeni Jr JF~ Gerin J, Purcell RH. International workshop on hepatitis B and liver cancer: Special report. JNCI 1978: 60: 717. Bassendine MF, Arborgh BAM, Shipton U, et al. Hepatitis B surface antigen and alpha-fetoprotein secreting human primary liver cell cancer in athymic mice. Gastroenterology 1980; 79: 528-532. Hoofnagle JH, Seer LB, Bales ZB, Zimmerman HJ. Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. N Engl J Med 1978: 298: 1379-1383. Lee CS, Lee PH, Chang KJ, et al. Monitoring of T lymphocytes before and after surgical removal of hepatic tumors. Chin .1 Microbiol Immunol 1983; 16: 1-6.
45
HEP 00258
Clearance of hepatitis B surface antigen (HBsAg) after surgical resection of hepatocellular carcinoma Tsung-Teh Wu I , Hey-Chi Hsu l, Ding-Shinn Chen 2, Jin-Chuan Sheu 2, Ih-Jen Su I, Shiu-Ling Chen I and Sou-Ming Chuang 1 Departments of 1Pathology and 21nternal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (Republic of China) (Received 31 July, 1986) (Accepted 23 October, 1986)
Summary The serum HBsAg in 4 chronic HBsAg carrier patients with hepatocellular carcinoma (HCC) cleared within 4-38 months after surgical resection of their hepatic tumors. Two patients developed anti-HBs. During the follow-up period from 21 to 28 months after HBsAg clearance, none of the patients regained positive serum HBsAg. Two patients who had had tissue HBsAg present, exclusively in the tumor, showed quick HBsAg clearance after resection. The other 2 patients had a delayed HBsAg clearance. One had tissue HBsAg in both the tumor and nontumoral liver. Only 1 patient bad tissue FIBsAg in the liver, but not in the tumor. During the same period of observation of 323 chronic HBsAg carriers, who had a variety of histologically-verified chronic liver diseases and were followed for more than 6 months, only 1 cleared the antigen. The spontaneous HBsAg clearance in our HBsAg carriers (1/323) was significantly lower than that (4/64) of HBsAg-positive HCC patients with tumor resection, P < 0.004. The mechanisms of HBsAg clearance in HCC patients after surgical resection of tumors are discussed.
Introduction It is generally agreed that the hepatitis B surface antigen (HBsAg) carrier state is prolonged once established, although early studies have indicated that
it can terminate spontaneously. In a series of 210 blood donors followed for 4-20 months, 13 (6.2%) cleared HBsAg spontaneously [1]. In another series of 414 carriers followed for 2-42 months, 4 (1.0%) cleared HBsAg [2]. However, in neither group was
This work was partially supported by grants from the National Science Council of the Republic of China (NSC74-0610-B002-32, NSC75-B002-0412-17). Correspondence address: Professor Hey-Chi Hsu, Department of Pathology, National Taiwan University Hospital, Taipei. Taiwan, Republic of China. 0168-8278/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
T.-T. WU et al.
46 the carrier status defined by 6-month duration; all 4 cases of Helske's series [2] cleared the antigen within 6 months. According to the definition of the 6-month duration carrier state, only 1 case has cleared HBsAg among 227 cases followed for 6-76 months (mean: 44 months) by Sampliner et al. [3]. It is generally believed that clearance of HBsAg is an unusual event if the HBsAg has been documented as present for as long as 6 months [4]. The mechanisms of HBsAg clearance and its biologic significance are unknown, although antiviral therapy has been shown to clear HBsAg in some carriers with chronic hepatitis B [5]. It appears, however, that HBsAg clearance in HBsAg carriers, particularly after surgical resection of hepatocellular carcinoma (HCC), has not been previously reported. Patients with HCC are well known to follow a rapid fatal course when the tumor has reached its advanced stage. If the tumor is detected in the early stage and small in size, the clinical course can frequently be prolonged [6,7], and surgical resection has become possible in many patients [8]. These patients' longer survival makes it possible to investigate their possible HBsAg clearance after tumor resection. Among 64 patients of HCC followed for more than 6 months after tumor resection, 4 subsequently cleared serum HBsAg. This study reports their data.
Patients and Methods
Patients From May 1979 to December 1984, 131 HCC patients received surgical resection of the tumor at National Taiwan University Hospital. Sixty-eight were ~<5 cm in diameter (small HCC); 63 were larger (large HCC). Among these, 64 cases (49 small and 15 large HCCs) had serial follow-up serum HBsAg tests for more than 6 and up to 57 months (mean + SD: 32.5 + 12.9 months). The findings formed the basis of this study. During roughly the same period from January 1980 to December 1984, 323 chronic HBsAg carriers who had biopsy-proven chronic hepatitis or cirrhosis and had serial serologic follow-up of HBV markers for more than 6 and up to 63 months (mean
+ SD: 29.6 + 12.9 months) formed the control group.
Histologic examinations Paraffin sections of 3 - 4 Bm thickness were stained with hematoxylin and eosin (HE). Liver HBsAg and HBcAg in the liver and HCC tissues were demonstrated by both peroxidase-antiperoxidase (PAP) method, and/or avidin-biotin-peroxidase complex (ABC) techniques by using rabbit anti-HBs and antiHBc (Dako Corporation, Santa Barbara, CA, USA) [9,101. Serologic assays Serum HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc were measured by radioimmunoassays (Abbott Laboratories, North Chicago, IL, USA).
Results
From among 64 HCC patients who had been followed for more than 6 months after tumor resection, serum HBsAg cleared in 3 patients (3/49) with small HCC (Cases 1, 3 and 4) and 1 (1/15) with large HCC (Case 2). Two cases (Cases 2 and 3) seroconverted to anti-HBs. The other 2 patients remained negative for anti-HBs. In the control group of 323 chronic HBsAg carriers with biopsy-proven chronic hepatitis or cirrhosis, only one (0.3%) seroconverted to anti-HBs. The serum HBsAg of this patient was of low titer 21 months after biopsy and had completely disappeared with
TABLE 1 COMPARISON OF HBsAg CLEARANCE BETWEEN HEPATOCELLULAR CARCINOMA (HCC) PATIENTS AFTER TUMOR RESECTION AND CARRIERS WITH CHRONIC LIVER DISEASES HBsAg clearance HCC group Chronic carriers
Yes
No
4 (6.3%) I (0.113¢'~)
60 322 P < 0.004
HBsAG CLEARANCE AFTER RESECTION OF HEPATOCELLULAR CARCINOMA
47
c o n v e r s i o n to H B s A g - / a n t i - H B s + 18 m o n t h s later.
f r o m h e m a t e m e s i s and tarry stool due to r u p t u r e d
T h e a n t i - H B s had persisted for 24 m o n t h s up to the
e s o p h a g e a l varices, r e c e i v e d a p o r t o s y s t e m i c shunt
latest f o l l o w - u p in D e c e m b e r 1985.
and liver biopsy in 1977. L i v e r cirrhosis was con-
T h e H B s A g c l e a r a n c e rate was significantly h i g h e r
firmed, and both H B s A g and H B c A g w e r e d e t e c t e d
in the H C C g r o u p ( 6 . 3 % ) than in the c o n t r o l g r o u p
in liver cells. R e g u l a r a b d o m i n a l s o n o g r a p h i c follow-
( 0 . 3 % ) , P < 0.004 ( T a b l e 1). T h e annual c l e a r a n c e
up r e v e a l e d a small hepatic t u m o r in the right lobe in
rate of H B s A g was 2 . 6 % in patients with H C C , and
A u g u s t 1983; thus a w e d g e resection of the t u m o r
0 . 1 % in those with c h r o n i c hepatitis or cirrhosis.
was p e r f o r m e d in D e c e m b e r 1983. F o u r m o n t h s later, the first p o s t o p e r a t i v e
b l o o d sample
demon-
strated c l e a r a n c e of H B s A g and, up to the last folReport of Cases
low-up in M a r c h 1986, 27 m o n t h s after resection, the s e r u m H B s A g r e m a i n e d negative. A n t i - H B s was not
T h e b r i e f clinical c o u r s e and serologic profiles of
d e t e c t a b l e . T h e patient felt well and had no t u m o r re-
the 4 H C C patients with H B s A g c l e a r a n c e are sum-
c u r r e n c e . T h r o u g h o u t the course, s e r u m alpha-feto-
m a r i z e d in T a b l e 2; and the p a t h o l o g i c f e a t u r e s are
protein ( A F P ) was within n o r m a l limits.
shown in T a b l e 3.
T h e w e d g e - r e s e c t e d liver s p e c i m e n m e a s u r e d 2.8 x 2.5 x 1.5 cm and c o n t a i n e d a small hepatic t u m o r
Case 1
1.6 x 1.5 x 0,8 cm in size. T h e t u m o r was n o d u l a t e d
A 54-year-old man w h o , in 1976, had suffered
and e n c a p s u l a t e d ,
but with e x t r a c a p s u l a r invasion
TABLE 2 CLINICAL FEATURES OF 4 PATIENTS WITH HBsAg CLEARANCE FOLLOWING SURGICAL RESECTION OF HEPATOCELLULAR CARCINOMA Case No.
Age/ sex
HBeAg/ anti-HBe
HBsAg carriage
Anti-HBs
Duration
Clear/FU
Preop.
Postop.
1 2 3 4
54/M 66/M 66/M 70/M '~
-/+ -/+ -/+ -/+
6 yr 13 yr 7 yr ?
4 mo/21 mo 12 mo/27 mo 31 mo/28 mo 27 mo/27 mo
+, t -
+ + -
Abbreviations: Clear/FU = duration of HBsAg clearance after tumor resection/follow-up duration; preop. = pre-operatton; postop. = post-operation; t = low titer; + = positive;- = negative. An episode of non-A, non-B hepatitis was suspected 10 weeks after operation.
TABLE 3 PATHOLOGIC FEATURES OF THE 4 RESECTED HEPATOCELLULAR CARCINOMAS ACCOMPANIED BY HBsAg CLEARANCE Case No.
Tumor size/site
Liver status
HBsAg in liver/ HCC
HBcAg in liver/ HCC
Tumor recurrence;'
1 2
1.6/R 5.5/R (lst) 1.6/L (2nd) 4.4/R 4.8/L
C C
-/+ -/+ -/? +/+/+
-/+, f -/-/? -/-/-
No
3 4
PF, CPH C
Suspicious Yes No
Abbreviations: R = right lobe; L = left lobe; C = cirrhosis; PF = portal fibrosis; CPH = chronic persistent hepatitis; ? = unknown; f = focal. a After HBsAg clearance.
48
T.-T. WU et al.
and portal vein branch involvement. Multiple sections of the non-tumor liver failed to reveal tissue HBsAg, whereas the tumor portion had globular or inclusion type of cytoplasmic HBsAg in isolated tumor cells (Fig. 1). HBcAg was also demonstrated only in isolated tumor cells.
Case 2 A 66-year-old man who had had hepatomegaly and diarrhea in 1972 was found on biopsy to have fatty liver. He was regularly followed at the Government Employee Clinical Center. In 1980, AFP increased and HCC with cirrhosis was diagnosed. He received a right lobe segmentectomy in August 1981. HBsAg at that time was positive and anti-HBs was also positive in low titer. Tumor recurred in the left lobe, and a second operation was performed in December 1982. Seroconversion to anti-HBs occurred 12 months after the second operation, and anti-HBc was positive throughout follow-up. In his third operation, in April 1984, a tumor implant was resected from the omentum. Regular sonographic examination at the last follow-up in February 1986 revealed a suspected recurrent hepatic tumor in the left lobe near the prior operation site, but a biopsy was not performed. The first resected HCC from the right lobe measured 5.5 cm in diameter. It was well-encapsulated and with capsular invasion. Scattered globular-type
Fig. 1. Globular type cytoplasmicHBsAg is seen in a tumor cell (arrowhead) in the tumor portion of Case 1. PAP stain with mouse monoclonalanti-HBs, x600.
Fig. 2. Isolated globular cytoplasmic HBsAg is demonstrated in tumor cells (arrowheads) of Case 3. PAP stain with mouse monoclonal anti-HBs, x600.
cytoplasmic HBsAg was demonstrated in the tumor cells, but not in the nontumorous liver (Fig. 2). HBcAg was negative in both the tumor and the liver. The second resected specimen revealed a small tumor nodule about 1.5 cm in diameter. It was encapsulated but with extracapsular and liver invasion accompanied by a tumor thrombus in a small portal vein branch. Most of the resected tumor tissue was sent for virologic studies; the remaining tissue was inaclequate for tissue HBsAg and HBcAg study. The third operated specimen, in the omentum, was a 0.4 cm tumor nodule which was negative for HBsAg and HBcAg.
Case 3 A 66-year-old man who had been known to be a chronic HBsAg carrier since April 1978 developed elevated AFP in January 1980. Peritoneoscopy showed macronodular cirrhosis, and abdominal sonography revealed a mass in the left lobe of the liver. He received a segmentectomy in May 1981. Clearance of HBsAg occurred in October 1983, and antiHBs appeared 9 months later. Tumor recurrence was noticed in the left lobe on regular follow-up in May 1984, but the patient declined further surgery. The tumor had grown from 1.5 cm to 8.5 cm at the last follow-up in March 1986, 58 months after the tumor resection. The patient remained HBsAg-seronegative.
HBsAG CLEARANCE AFTER RESECTION OF HEPATOCELLULAR CARCINOMA The resected small HCC measured 4.4 × 4.4 x 4.2 cm in size, and was not encapsulated. Microscopically, there was liver, but no portal vein, invasion. The non-neoplastic liver parenchyma showed portal fibrosis and chronic persistent hepatitis. Tissue HBsAg was positive in the non-tumor, but not in the tumor, portion. Case 4
A 70-year-old man was found to have positive serum HBsAg and increased AFP in June 1981 during an annual physical check-up. Abdominal sonography and CT scan revealed a hepatic tumor about 5 cm in diameter in the left lobe. He received left lateral seg-
49
mentectomy in July. An episode of acute hepatitis probably due to superimposed post-transfusion nonA, non-B hepatitis, occurred 10 weeks after operation. Three months later, HBsAg was of low titer; it then disappeared 27 months after operation. At the last follow-up in March 1986, serum HBsAg remained negative as did anti-HBs. There was no local tumor recurrence despite elevated AFP. The resected small HCC measured 4.8 cm in diameter. It was well-encapsulated and showed no extracapsular or portal vein invasion. The non-neoplastic liver was cirrhotic. Tissue HBsAg was demonstrated both in the tumor and non-tumoral portions (Fig. 3), but not HBcAg.
Discussion
Fig. 3. A: Most of the tumor cells in the peripheral portion show invasion of the tumor capsule (c). B: Higher magnification shows cytoplasmic HBsAg of varying intensity in many tumor cells (arrowheads). PAP stain for HBsAg. A: x150; B: x600.
Delayed clearance of HBsAg is an unusual event. In a long-term follow-up observation Karen et ai. [11] reported HBsAg clearance with seroconversion to anti-HBs in 7 chronic HBsAg carriers, 5 with biopsy-proved chronic persistent hepatitis. Wallace et al. [12] observed a 6.0% loss of HBsAg among 115 HBsAg carriers, with an annual clearance rate of 1.0%, during follow-ups in a mean period of 6.1 years. The patients reported are healthy carriers or had Type B chronic hepatitis. HBsAg clearance in HCC patients has not been reported, probably because of the usual rapidly fatal course of symptomatic HCC. In Taiwan, chronic HBsAg carriers account for 15-17.8% of the general population [13-15], but the annual clearance rate of HBsAg as determined by the sensitive RIA test is not known. In the present series of 323 carriers with biopsy-proven chronic hepatitis or cirrhosis, spontaneous HBsAg clearance occurred rarely, accounting for only 0.3% (or 1/323) during a mean period of 2.5 years, or 0.1% per year. In contrast, a significantly higher annual clearance rate (2.6%) was demonstrated in HCC patients after surgical resection of the tumor. This information may provide some clues toward better understanding of the mechanisms of HBsAg clearance in HBsAg carriers. The mechanisms of the clearance of HBsAg car-
T.-T. WU et al.
50 rier status are not yet fully understood. Antiviral and interferon therapies have led to the clearance of HBsAg in some patients [16-19]. In contrast, none of the 15 patients of Omata et al. [20] nor the 10 patients of Smith et al. [21] showed any response. The reason for this discrepancy is not known. However, none of the present 4 patients were administered any antiviral therapy. The most remarkable and unusual findings were that H B s A g could be demonstrated in the tumor itself but not in the non-tumor portion of the livers of 2 patients (Cases 1 and 2) who had rapid clearance of serum HBsAg after tumor resection. These findings strongly suggest that the HBsAg-bearing H C C per se is responsible for the HBsAg antigenemia, and the removal of the tumor is responsible for the clearance of serum HBsAg. Previous studies have demonstrated the presence of H B s A g in the tumor cells in 15% of large H C C and 40% in small H C C [6], indicating a continued presence in the tumor cells of H B V genome which is capable of producing H B V antigens, as has also been suggested by H C C cell line culture and heterotransplant [22,23]. Several other possibilities can be raised to explain the mechanisms of H B s A g clearance in these patients. One is probably related to the reduction in HBsAg-producing cells either in the H C C and/or the non-tumorous liver, resulting in a lower serum H B s A g level undetectable with the present assay method [24]. These results are unable to prove or disprove this suggestion.
References
1 Szmuness W. Prince A. Brotman B, Hirsch RL. Hepatitis B antigen and antibody in blood donors: An epidemiologic study. J Infect Dis 1973; 127: 17-25. 2 Helske T. Carriers of hepatitis B antigen and transfusion hepatitis in Finland. Scand J Haematol 1974; 22 (Suppl): 1-65.
3 Sampliner RE, Carney E, Tabor E, Gerety R. In: W Szmuness. H.I Alter, JE Maynard (Eds.), Viral Hepatitis (1981 International Symposium). Franklin Institute Press, Philadelphia, PA, 1982: 722-723. 4 Sampliner RE. Follow-up and management of hepatitis B carriers. In: RJ Gerety (Ed.), Hepatitis B. Academic Press, Inc., Orlando, FL, 1985: 155-172. 5 Scullard GH, Pollard RB, Smith JL, Sacks S, Gregory PB,
However, the H B s A g clearance rate in the patients with large H C C (1/15) did not differ from those with small H C C (3/49), despite a much greater liver volume reduction after tumor resection in the large HCC. A n o t h e r possibility is the immunosuppressive effect of the H C C on the host's cellular immunity, which has been shown to improve after tumor resection [25]. The occurrence of anti-HBs after tumor resection in 2 of the 4 cases appears to support this speculation. The biologic significance of the H B s A g clearance in these patients is unknown, although H B s A g clearance has been suggested to imply a resolution of the underlying disease, elimination of the infectivity, and possibly elimination of the risk for H C C [4]. Two of these cases (Cases 1 and 4) had a relatively prolonged HCC-free period of 21-27 months after H B s A g clearance, and included 1 patient with extracapsular invasion and satellite nodules near the tumor. Because extracapsular invasion and satellite nodule formation in H C C have been shown to be associated with a high tumor recurrence rate [6], this finding seems to support the suggestion that H B s A g clearance may eliminate the risk of H C C [4]. However, the recurrence of tumor in the remaining 2 cases, despite clearance of H B s A g , contradicts this suggestion. Because of the limited number of cases, clarification of the biologic significance of the H B s A g clearance in H C C patients after tumor resection will require further study.
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hepatitis B virus (HBV) replication. Hepatology 1983; 3: 865. Lok ASF, Weller IVD, Karayinnis P, et al. Thrice weekly lymphoblastoid interferon is effective in inhibition hepatitis B virus replication. Liver 1984: 4: 45-49. Dooley JS, Davis GL, Waggoner JG, Hoofnagle JH. Low dose versus high dose recombinant leucocyte A interferon for chronic type B hepatitis. Hepatology 1983; 3: 853. Omata M, Yokosuka O, lmazeki F, Okuda K. In: GN Vyas, JL Dienstag, JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stratton, New York, NY, 1984: 670-676. Smith CI, Weissberg J, Bernhardt L, Merigan TC, Robinson WS. Acute Dane particle suppression with recombinant leucocyte A interferon in chronic hepatitis B virus infection. J Infect Dis 1983; 148: 907-913. Ziegler JL, Adamson RH, Barker LF. Fraumeni Jr JF~ Gerin J, Purcell RH. International workshop on hepatitis B and liver cancer: Special report. JNCI 1978: 60: 717. Bassendine MF, Arborgh BAM, Shipton U, et al. Hepatitis B surface antigen and alpha-fetoprotein secreting human primary liver cell cancer in athymic mice. Gastroenterology 1980; 79: 528-532. Hoofnagle JH, Seer LB, Bales ZB, Zimmerman HJ. Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. N Engl J Med 1978: 298: 1379-1383. Lee CS, Lee PH, Chang KJ, et al. Monitoring of T lymphocytes before and after surgical removal of hepatic tumors. Chin .1 Microbiol Immunol 1983; 16: 1-6.