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Primary hepatocellular carcinoma with hepatitis B virus-DNA integration in a 4-year-old boy
PRIMARY HEPATOCELLULAR CARCINOMA WITH HEPATITIS B VIRUS-DNA INTEGRATION IN A 4-YEAR-OLD BOY T o M o v u g l TANAKA, MD,* HIROYUKI MIYAMOTO, MD,* OKIO HINO, MD,* TOMOYUKI KITAGAWA,MD,* MICHIO KOIKE, MD,* TADASHI hZUKA, MD,* HARUttlKO SAKAMOTO,MD,~ AND AKIRA OOSHIMA, MD w
The autopsy findings in a case of hepatocelMar carcinoma with hepatitis B virus (HB~9-DNA integration in a Japanese boy, 4 years and 10 month of age, are reported. The boy was an H B V cadier born in a typical familial cluster of H B V infection in Japan. He had been asymptomatic until the sudden manifestation of the liver tumor. Histopathologic examination revealed a well. differentiated, adult-type hepatocelhdar carcinoma without hepatic cirrhosis. H B V - D N A sequences were detected in the tumor cell DNA by the Southern blot hyb~4dization method. This is the youngest patient with hepatocelhdar carcinoma with H B V - D N A integration reported to date, suggesting that HBV may manifest its oncogenic properties after a shorter incubation period than generally believed. HUM PATnOL 1 7:202--204, 1986. T h e association o f hepatitis B virus (HBV) infection with primary hepatocellular carcinoma (HCC) has been demonstrated by man)' seroepidemiologic studies) T h e incidence o f HCC is markedly higher in areas in which HBV infection is endenfic than in other areas. These seroepidemiologic studies indicate that HBV nfight play a principal causal role in the generation o f h u m a n HCC. It was demonstrated recently by the transfer hydridization technique o f Southern z that HBV is integrated at very high frequencies in the DNA o f HCC tumor ceils in carrier patients, 3.a providing t i m b e r evidence of an oncogenic role for HBV in relation to HCC. T h e present report describes the autopsy findings in a case of HCC with H B V - D N A integration that developed in a 4-year-old boy with HBV antigenenfia belonging to a typical familial HBV cluster in Japan. To our knowledge, this is the youngest reported patient with HCC in whom H B V - D N A was proved to be imegrated in the tumor cell genome. This case may lead to better understanding of the phase o f viral integration in host cells and its significance in h u m a n hepatocarcinogenesis. ,
patient died four months after admission due to an extremely distended liver tumor and cachexia. T h e familial cluster o f HBV infection was as follows: T h e patient's older brother was both HBsAg- and HBeAgpositive but had no symptoms of liver disease. His younger brother had received prophylactic treatment for HBV infection by our protocol, inchlding the administration of two doses o f anti-HBs hyperimmune gammaglobtflin and three doses o f HBV vaccine, and was anti-HBs-positive at the time of this stud)'. T h e patient's mother was both anti-HBeand HBsAg-positive, but detailed serologic findings were not recorded at tile birth o f the subject o f tiffs study. T h e patient's father was auti-HBs-positive, but it is not clear when his seroconversion occurred.
Pathologic Findings Autopsy perntission was granted for the liver only. T h e postmorten examination o f the liver was perfornted within two hours after the patient's death. T h e liver weighed 2,240 g. On gross exantination, widespread, yellowish white multinodular unencapsulated lesions, with a maximal size o f approximately 5 x 6 x 9 cm, were found in the right lobe o f the liver. T h e centers o f some of these lesions were necrotic. Scattered intrahepatic metastatic lesions were observed in the bilateral lobes. T h e tumor had invaded the right portal vein, forming tumor tissue emboli. In solid regions of the tumor, hard, white, fibrous connective tissue was predonfinant. A small noncancerous region was observed in the left lobe of the liver. Light microscopy revealed an adult-type primary hepatocellular carcinoma. T u m o r cells showed trabecular (fig. la) and tubular (fig. Ib) patterns; t h e ) u m o r cells Were focall)' pleomorphic, and multinucleated giant cells were present (fig. lc). Bile formation was recognized witlfin the regions composed o f tubtdar and pleomorphic tumor:cells~ T h e noncancerous portion o f the liver showed condensed portal fibrosis with chronic inflanmmtory infihration. However, obvious regenerative nodules were not fotlnd. HBsAg was detected in the cytoplasm of noncancerous hepatocytes (fig. ld) and in a small n u m b e r of tumor cells by immunoperoxidase staining. HBcAg was not detectable in the nuclei o f either normal hepatocytes or tumor cells by the application of immunoperoxidase or immunofluorescence techniques to paraffin-embedded sectionsP DNA was extracted from tile autopsy tumor specime n,
i
R E P O R T OF A CASE A Japanese boy, 4 years and 10 months of age, previously in good health and with no history o f blood transfusion, was admitted to Wakayama Medical College Hospital on September 17, 1983, complaining of a cough and abdominal stiffness. T h e liver was palpable 11 cm below the midline o f the right costal margin. Laboratory data f r o m liver f u n c t i o n tests p e r f o r m e d on admission are shown in table 1. A plain C T scan disclosed a large, lowdensity area in the center of the right lobe o f the liver, with e n h a n c e m e n t on contrast c o m p u t e d t o m o g r a p h y . T h e chest C T scan showed several small sbadows in the right lung, which were interpreted as metastases. Irradiation with 60Co and chemotherapy were unsuccessful, and the
Table t.
Received July 3, 1985; revision accepted for publication August 28, 1985. * Department of Microbiology, Wakayama Medical College, Wakayama, Japan. t Department of Pathology, Cancer Institute, Tokyo, Japan. Department of Pediatrids, Wakayama Medical College, Wakayama, Japan. w Department of Pathology, Wakayanm Medical College, Wak~iyama, Japan. Address correspondence and reprint requests to Dr. ltino: Department of Pathology, Cancer hlstitute, Kami-lkebukuro 1-371, Toshinm-ku, Tokyo 170, Japan.
202
Liver Function Test Results on Admission
Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Lactate dehydrogenase Total cholesterol Total bilirubin cc-fetoprotein ttBsAg Subtype Anti-ttBsAg Anti-HBcAg HBeAg : Anti-ttBeAg
101 U/I 46 U/I 250 U/I 308 U/I 693 mg/I 1.1 mg/l " 210 x 10 t ng/l x 320 (R-PHA) adr (MO) Negative (PHA) x 2048 (IAttA) Negative (MO) Positive (MO)
ABBREVIATIONS: ,XIO, Micro-Ouchterlon)" method; PHA, Passive hemagglutination test; R-PHA, reversed passive liemaggltttination test; IAtlA, innnune adherence hemagglutination test.
FIGURE t. Hepatocellular carcinoma showing trabecular (a], tubular (b], and pleomorphic patterns with multinucleated giant cells (c]. HBsAg-positive hepatocytes are present in a noncancerous region [d]. [a, b, and c, Hematoxylin-eosin stain, x 125. d, lmmunoperoxidase method and hematoxylin stain, x 250.)
203
HUMAN PATHOLOGY
UND
HIN
Volume IL No. 2 [February 1986]
DISCUSSION
Barn
--23.5Kb
--
9.TKb
--
6.6Kb
---
&.3Kb
--
2.2Kb
--
2. t K b
FIGURE2. Representative autoradiographs of Southern blot anal,/sis of hepatoceIlular carcinoma. UND indicates undigested; HIN, Hind Ill-digested; Barn, Barn Hl-digested. Molecular weight markers of known length are shown on the right.
a n d S o u t h e r n blot analysis was p e r f o r m e d with c l o n e d H B V - D N A probes, as described previously. 4 Briefly, total celhdar D N A was extracted by t r e a t i n g the tissue o v e r n i g h t with 2 p e r cent SDS/500 Isg/ml proteinase K, followed by r e p e a t e d p h e n o l extraction. A p o r t i o n o f the purified D N A (10 ~g) was digested with a restriction e n z y m e ( H i n d III o r Barn HI). T h e digested a n d u n d i g e s t e d samples were separated by electrophoresis in agarose slab gel (1 p e r cent). T h e D N A in the gel was blotted o n t o nitrocellulose filters a n d hybridized with cloned 3 z P - H B V - D N A covering the e n t i r e viral g e n o m e . -t A f t e r h y b r i d i z a t i o n , the filter was washed several times with 0.1 X SSC at 65~ a n d exposed for o n e to five days o n Kodac X - o m a t XRP-1 film for aut o r a d i o g r a p h y at - 7 0 ~ With the D N A that was not digested by restriction enzymes, a s m e a r o f hybridization was s e e n in t h e l f i g h - m o l e c u l a r - w e i g h t r e g i o n o f t h e gel, w h e r e a s a f t e r t r e a t m e n t with H i n d I I I o r Barn H I o n e m a j o r b a n d or two discrete bands, respectively, were .demo n s t r a t e d (fig. 2). N o t h i n g was d e m o n s t r a t e d in the same gel after rehybridization with a p u r e pBR322 probe after waslfing. Since it is k n o w n that H i n d I I I does not cleave viral D N A a n d that Barn H I cleaves viral D N A at o n e site, in the a d r subtype o f HBV, 6 /his restriction p a t t e r n indicates i n t e g r a t i o n o f H B V - D N A in at least o n e site o f the host DNA. D N A extracted f r o m several d i f f e r e n t portions o f the t u m o r gave rise to exactly the same hybridization pattern, indicating the clonality o f t m n o r cells with respect to H B V - D N A integration.
Ill H B V carriers, liver diseases a n d H C C usually develop in late a d n h h o o d . Occasionally, however, H C C develops in childhood, a n d the y o u n g e s t patient in a case o f H C C associated with H B V a n t i g e n e m i a r e p o r t e d previously was a 6-year-old boy. 7 T i l e patient o f tile p r e s e n t case was m u c h y o u n g e r a n d m o r e significant titan tltat o f the previously r e p o r t e d case because clonal H B V - D N A integration was proved. A h h o u g h the diagnosis o f H C C was m a d e w h e n this patient was 4 years a n d 10 m o n t h s o f age, the establishment o f the c a n c e r cell should lmve occurred m u c h earlier, p r e s u m a b l y within the first two )ears after birth: the r e p o r t e d v o l u m e - d o u b l i n g time for some rapidly g r o w i n g i n f a n t i l e m a l i g n a n t t u m o r s is about 20 days, a n d , theoretically, 30 d o n b l i n g times are r e q u i r e d for a single c a n c e r cell, I0 p.m in d i a m e t e r , to attain a clinically detectable size, i.e., larger t h a n 1 c m in diameter. 8 It has not b e e n clarified w h e n a n d how f r e q u e n t l y H B V - D N A is integrated into the D N A o f hepatocytes in infected patients. A c c o r d i n g to Scotto et al., 9 viral integration occurs in infantile life, as earl)' as two m o n t h s after infection, as d e m o n s t r a t e d by clonal integration patterns, i.e., discrete b a n d s in the lfigh-molecular-weight r e g i o n o n S o u t h e r n blot analysis after H i n d I I I digestion. However, analyses by restriction p a t t e r n a n d molecular c l o n i n g o f H C C w i t h v i r a l i n t e g r a t i o n h a v e to d a t e r e v e a l e d n o c o m m o n site for i n t e g r a t i o n in host DNA. I n t e g r a t i o n in host hepatocytes may occur at r a n d o m within the DNA, a n d a clonal i n t e g r a t i o n p a t t e r n f r o m infected n o n n e o plastic liver tissue, therefore, could not be expected. Indeed, only r a n d o m i n t e g r a t i o n patterns were revealed in the study o f infected n o n n e o p l a s t i c livers o f Kam et al. l~ T h e relevance o f the data o f Scotto et al. is questionable, therefore, unless clonal, almost neoplastic, growth o f particular hepatocytes with viral i n t e g r a t i o n occurred. T h e p r e s e n t data are i m p o r t a n t because they clearly d e m o n s t r a t e that 1) the H B V - D N A integration in bepatocytes m a y occur earl)" in the lives o f carrier patients a n d 2) in this case the viral i n t e g r a t i o n probably played a m o r e direct causal role in the carcinogenesis t h a n it would in adult HCC, in which it is possible that HBV infection, with i n t e g r a t i o n into DNA, may act merely as a co-carcinogenic factor. 4 Molecular analysis o f the m o d e o f integration o f the viral g e n o m e into the host D N A in the p r e s e n t case is now in progress. REFERENCES 1. Beasle)" RP, ttwang LY, Lin CC, et al: ttepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan. Lancet 2: 1129, 1981 2. Southern EM: Detection of specific sequences anaong DNA fragments separated by gel electrophoresis. J Mol Biol 98:503, 1975 3. Shafritz D, Shouval D, Sherumn HI, et ah Integration of hepatitis B virus DNA into geuome of liver cells in chronic liver disease and hepatocellular carcinoum. N EnglJ Med 305:1067, 1981 4. Hino O, Kitagawa T, Sugano th Relationship between serum and histochenfical markers for hepatitis B virus and rate of x'iralintegration in hepatocellular carcinoma in Japan. Int J Cancer 35:5, 1985 5. Huang Stl, Minassian tl, More JD: Application of immunofluorescent staining on paraffin section improved by trypsin digestion. Lab Invest 35:383, 1976 6. Ono Y, Onda H, Sakada R, et ah The complete nucleotide sequences of the clonal hepatitis B virus DNA: subtype adr and adw. Nucleic Acids Res 11:1747, 1983 7. Shimoda T, Uchida T, Miyata It, et al: A 6-year-old boy having hepatocellular carcinonm associated with hepatitis B surface antigenenfia. AmJ Clin Pathol 74:827, 1980 8. Steel GG: Growth Kinetics of Tumors. Oxford, Clarendon Press, 1977 9. ScottJ, ttadchooel M, Hery C, et ah Hepatitis B virus DNA in children's liver diseases: detection by blot hybridisation in liver and serum. Gut 2-1:618, 1983 10. Kam W, Rail LB, Smuckler EA, et al: tlepatitis B viral DNA in liver and serum of asymptomatic carriers, l'roc Natl Acad Sci USA 79:7522, 1982
204
The autopsy findings in a case of hepatocelMar carcinoma with hepatitis B virus (HB~9-DNA integration in a Japanese boy, 4 years and 10 month of age, are reported. The boy was an H B V cadier born in a typical familial cluster of H B V infection in Japan. He had been asymptomatic until the sudden manifestation of the liver tumor. Histopathologic examination revealed a well. differentiated, adult-type hepatocelhdar carcinoma without hepatic cirrhosis. H B V - D N A sequences were detected in the tumor cell DNA by the Southern blot hyb~4dization method. This is the youngest patient with hepatocelhdar carcinoma with H B V - D N A integration reported to date, suggesting that HBV may manifest its oncogenic properties after a shorter incubation period than generally believed. HUM PATnOL 1 7:202--204, 1986. T h e association o f hepatitis B virus (HBV) infection with primary hepatocellular carcinoma (HCC) has been demonstrated by man)' seroepidemiologic studies) T h e incidence o f HCC is markedly higher in areas in which HBV infection is endenfic than in other areas. These seroepidemiologic studies indicate that HBV nfight play a principal causal role in the generation o f h u m a n HCC. It was demonstrated recently by the transfer hydridization technique o f Southern z that HBV is integrated at very high frequencies in the DNA o f HCC tumor ceils in carrier patients, 3.a providing t i m b e r evidence of an oncogenic role for HBV in relation to HCC. T h e present report describes the autopsy findings in a case of HCC with H B V - D N A integration that developed in a 4-year-old boy with HBV antigenenfia belonging to a typical familial HBV cluster in Japan. To our knowledge, this is the youngest reported patient with HCC in whom H B V - D N A was proved to be imegrated in the tumor cell genome. This case may lead to better understanding of the phase o f viral integration in host cells and its significance in h u m a n hepatocarcinogenesis. ,
patient died four months after admission due to an extremely distended liver tumor and cachexia. T h e familial cluster o f HBV infection was as follows: T h e patient's older brother was both HBsAg- and HBeAgpositive but had no symptoms of liver disease. His younger brother had received prophylactic treatment for HBV infection by our protocol, inchlding the administration of two doses o f anti-HBs hyperimmune gammaglobtflin and three doses o f HBV vaccine, and was anti-HBs-positive at the time of this stud)'. T h e patient's mother was both anti-HBeand HBsAg-positive, but detailed serologic findings were not recorded at tile birth o f the subject o f tiffs study. T h e patient's father was auti-HBs-positive, but it is not clear when his seroconversion occurred.
Pathologic Findings Autopsy perntission was granted for the liver only. T h e postmorten examination o f the liver was perfornted within two hours after the patient's death. T h e liver weighed 2,240 g. On gross exantination, widespread, yellowish white multinodular unencapsulated lesions, with a maximal size o f approximately 5 x 6 x 9 cm, were found in the right lobe o f the liver. T h e centers o f some of these lesions were necrotic. Scattered intrahepatic metastatic lesions were observed in the bilateral lobes. T h e tumor had invaded the right portal vein, forming tumor tissue emboli. In solid regions of the tumor, hard, white, fibrous connective tissue was predonfinant. A small noncancerous region was observed in the left lobe of the liver. Light microscopy revealed an adult-type primary hepatocellular carcinoma. T u m o r cells showed trabecular (fig. la) and tubular (fig. Ib) patterns; t h e ) u m o r cells Were focall)' pleomorphic, and multinucleated giant cells were present (fig. lc). Bile formation was recognized witlfin the regions composed o f tubtdar and pleomorphic tumor:cells~ T h e noncancerous portion o f the liver showed condensed portal fibrosis with chronic inflanmmtory infihration. However, obvious regenerative nodules were not fotlnd. HBsAg was detected in the cytoplasm of noncancerous hepatocytes (fig. ld) and in a small n u m b e r of tumor cells by immunoperoxidase staining. HBcAg was not detectable in the nuclei o f either normal hepatocytes or tumor cells by the application of immunoperoxidase or immunofluorescence techniques to paraffin-embedded sectionsP DNA was extracted from tile autopsy tumor specime n,
i
R E P O R T OF A CASE A Japanese boy, 4 years and 10 months of age, previously in good health and with no history o f blood transfusion, was admitted to Wakayama Medical College Hospital on September 17, 1983, complaining of a cough and abdominal stiffness. T h e liver was palpable 11 cm below the midline o f the right costal margin. Laboratory data f r o m liver f u n c t i o n tests p e r f o r m e d on admission are shown in table 1. A plain C T scan disclosed a large, lowdensity area in the center of the right lobe o f the liver, with e n h a n c e m e n t on contrast c o m p u t e d t o m o g r a p h y . T h e chest C T scan showed several small sbadows in the right lung, which were interpreted as metastases. Irradiation with 60Co and chemotherapy were unsuccessful, and the
Table t.
Received July 3, 1985; revision accepted for publication August 28, 1985. * Department of Microbiology, Wakayama Medical College, Wakayama, Japan. t Department of Pathology, Cancer Institute, Tokyo, Japan. Department of Pediatrids, Wakayama Medical College, Wakayama, Japan. w Department of Pathology, Wakayanm Medical College, Wak~iyama, Japan. Address correspondence and reprint requests to Dr. ltino: Department of Pathology, Cancer hlstitute, Kami-lkebukuro 1-371, Toshinm-ku, Tokyo 170, Japan.
202
Liver Function Test Results on Admission
Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Lactate dehydrogenase Total cholesterol Total bilirubin cc-fetoprotein ttBsAg Subtype Anti-ttBsAg Anti-HBcAg HBeAg : Anti-ttBeAg
101 U/I 46 U/I 250 U/I 308 U/I 693 mg/I 1.1 mg/l " 210 x 10 t ng/l x 320 (R-PHA) adr (MO) Negative (PHA) x 2048 (IAttA) Negative (MO) Positive (MO)
ABBREVIATIONS: ,XIO, Micro-Ouchterlon)" method; PHA, Passive hemagglutination test; R-PHA, reversed passive liemaggltttination test; IAtlA, innnune adherence hemagglutination test.
FIGURE t. Hepatocellular carcinoma showing trabecular (a], tubular (b], and pleomorphic patterns with multinucleated giant cells (c]. HBsAg-positive hepatocytes are present in a noncancerous region [d]. [a, b, and c, Hematoxylin-eosin stain, x 125. d, lmmunoperoxidase method and hematoxylin stain, x 250.)
203
HUMAN PATHOLOGY
UND
HIN
Volume IL No. 2 [February 1986]
DISCUSSION
Barn
--23.5Kb
--
9.TKb
--
6.6Kb
---
&.3Kb
--
2.2Kb
--
2. t K b
FIGURE2. Representative autoradiographs of Southern blot anal,/sis of hepatoceIlular carcinoma. UND indicates undigested; HIN, Hind Ill-digested; Barn, Barn Hl-digested. Molecular weight markers of known length are shown on the right.
a n d S o u t h e r n blot analysis was p e r f o r m e d with c l o n e d H B V - D N A probes, as described previously. 4 Briefly, total celhdar D N A was extracted by t r e a t i n g the tissue o v e r n i g h t with 2 p e r cent SDS/500 Isg/ml proteinase K, followed by r e p e a t e d p h e n o l extraction. A p o r t i o n o f the purified D N A (10 ~g) was digested with a restriction e n z y m e ( H i n d III o r Barn HI). T h e digested a n d u n d i g e s t e d samples were separated by electrophoresis in agarose slab gel (1 p e r cent). T h e D N A in the gel was blotted o n t o nitrocellulose filters a n d hybridized with cloned 3 z P - H B V - D N A covering the e n t i r e viral g e n o m e . -t A f t e r h y b r i d i z a t i o n , the filter was washed several times with 0.1 X SSC at 65~ a n d exposed for o n e to five days o n Kodac X - o m a t XRP-1 film for aut o r a d i o g r a p h y at - 7 0 ~ With the D N A that was not digested by restriction enzymes, a s m e a r o f hybridization was s e e n in t h e l f i g h - m o l e c u l a r - w e i g h t r e g i o n o f t h e gel, w h e r e a s a f t e r t r e a t m e n t with H i n d I I I o r Barn H I o n e m a j o r b a n d or two discrete bands, respectively, were .demo n s t r a t e d (fig. 2). N o t h i n g was d e m o n s t r a t e d in the same gel after rehybridization with a p u r e pBR322 probe after waslfing. Since it is k n o w n that H i n d I I I does not cleave viral D N A a n d that Barn H I cleaves viral D N A at o n e site, in the a d r subtype o f HBV, 6 /his restriction p a t t e r n indicates i n t e g r a t i o n o f H B V - D N A in at least o n e site o f the host DNA. D N A extracted f r o m several d i f f e r e n t portions o f the t u m o r gave rise to exactly the same hybridization pattern, indicating the clonality o f t m n o r cells with respect to H B V - D N A integration.
Ill H B V carriers, liver diseases a n d H C C usually develop in late a d n h h o o d . Occasionally, however, H C C develops in childhood, a n d the y o u n g e s t patient in a case o f H C C associated with H B V a n t i g e n e m i a r e p o r t e d previously was a 6-year-old boy. 7 T i l e patient o f tile p r e s e n t case was m u c h y o u n g e r a n d m o r e significant titan tltat o f the previously r e p o r t e d case because clonal H B V - D N A integration was proved. A h h o u g h the diagnosis o f H C C was m a d e w h e n this patient was 4 years a n d 10 m o n t h s o f age, the establishment o f the c a n c e r cell should lmve occurred m u c h earlier, p r e s u m a b l y within the first two )ears after birth: the r e p o r t e d v o l u m e - d o u b l i n g time for some rapidly g r o w i n g i n f a n t i l e m a l i g n a n t t u m o r s is about 20 days, a n d , theoretically, 30 d o n b l i n g times are r e q u i r e d for a single c a n c e r cell, I0 p.m in d i a m e t e r , to attain a clinically detectable size, i.e., larger t h a n 1 c m in diameter. 8 It has not b e e n clarified w h e n a n d how f r e q u e n t l y H B V - D N A is integrated into the D N A o f hepatocytes in infected patients. A c c o r d i n g to Scotto et al., 9 viral integration occurs in infantile life, as earl)' as two m o n t h s after infection, as d e m o n s t r a t e d by clonal integration patterns, i.e., discrete b a n d s in the lfigh-molecular-weight r e g i o n o n S o u t h e r n blot analysis after H i n d I I I digestion. However, analyses by restriction p a t t e r n a n d molecular c l o n i n g o f H C C w i t h v i r a l i n t e g r a t i o n h a v e to d a t e r e v e a l e d n o c o m m o n site for i n t e g r a t i o n in host DNA. I n t e g r a t i o n in host hepatocytes may occur at r a n d o m within the DNA, a n d a clonal i n t e g r a t i o n p a t t e r n f r o m infected n o n n e o plastic liver tissue, therefore, could not be expected. Indeed, only r a n d o m i n t e g r a t i o n patterns were revealed in the study o f infected n o n n e o p l a s t i c livers o f Kam et al. l~ T h e relevance o f the data o f Scotto et al. is questionable, therefore, unless clonal, almost neoplastic, growth o f particular hepatocytes with viral i n t e g r a t i o n occurred. T h e p r e s e n t data are i m p o r t a n t because they clearly d e m o n s t r a t e that 1) the H B V - D N A integration in bepatocytes m a y occur earl)" in the lives o f carrier patients a n d 2) in this case the viral i n t e g r a t i o n probably played a m o r e direct causal role in the carcinogenesis t h a n it would in adult HCC, in which it is possible that HBV infection, with i n t e g r a t i o n into DNA, may act merely as a co-carcinogenic factor. 4 Molecular analysis o f the m o d e o f integration o f the viral g e n o m e into the host D N A in the p r e s e n t case is now in progress. REFERENCES 1. Beasle)" RP, ttwang LY, Lin CC, et al: ttepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan. Lancet 2: 1129, 1981 2. Southern EM: Detection of specific sequences anaong DNA fragments separated by gel electrophoresis. J Mol Biol 98:503, 1975 3. Shafritz D, Shouval D, Sherumn HI, et ah Integration of hepatitis B virus DNA into geuome of liver cells in chronic liver disease and hepatocellular carcinoum. N EnglJ Med 305:1067, 1981 4. Hino O, Kitagawa T, Sugano th Relationship between serum and histochenfical markers for hepatitis B virus and rate of x'iralintegration in hepatocellular carcinoma in Japan. Int J Cancer 35:5, 1985 5. Huang Stl, Minassian tl, More JD: Application of immunofluorescent staining on paraffin section improved by trypsin digestion. Lab Invest 35:383, 1976 6. Ono Y, Onda H, Sakada R, et ah The complete nucleotide sequences of the clonal hepatitis B virus DNA: subtype adr and adw. Nucleic Acids Res 11:1747, 1983 7. Shimoda T, Uchida T, Miyata It, et al: A 6-year-old boy having hepatocellular carcinonm associated with hepatitis B surface antigenenfia. AmJ Clin Pathol 74:827, 1980 8. Steel GG: Growth Kinetics of Tumors. Oxford, Clarendon Press, 1977 9. ScottJ, ttadchooel M, Hery C, et ah Hepatitis B virus DNA in children's liver diseases: detection by blot hybridisation in liver and serum. Gut 2-1:618, 1983 10. Kam W, Rail LB, Smuckler EA, et al: tlepatitis B viral DNA in liver and serum of asymptomatic carriers, l'roc Natl Acad Sci USA 79:7522, 1982
204