h t . J . Cancer: 24, 421-429 (1979)
HEPATITIS B VIRUS ANTIGENS IN HUMAN PRIMARY HEPATOCELLULAR CARCINOMA TISSUES A. GOUDEAU Ph. MAUPAS l, P. COURSACET l , J. DRUCKER ', J . P. CHIRON2, F. DENISz, and I. DIOPMAR^ Institut de Virologie, FacultCs de Mkdecine et de Pharmacie, 2 bis, Boulevard Tonnelle, 37000 Tours, France; and Services de Microbiologie et de Pathologie Infectieuse, Faculti de Mkdecine de Dakar, Dakar, SCnkgal 133,
The presence of hepatitis B virus (HBV) antigens was examined i n specimens of liver tissue obtained a t necropsy f r o m black Senegalese patients suffering from primary hepatocellular carcinoma (PHC). The results were correlated w i t h markers of hepat i t i s B infection i n serum. Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) were sought for i n 15 liver extracts. HBsAg was found i n the liver in 10 of 12 cases with HBsAg-positive serum. HBcAg was detected in three livers. The HBsAg was detected i n seven of eight livers by immunofluorescence and orcein staining. HBsAG-positive cells were mainly located i n the peri-tumoral cirrhotic tissue, although positive hepatocytes were also found i n tumour nodules in liver from one of the patients. HBcAg was found in five of seven cases by immunofluorescence in hepatocytes of the cirrhotic areas. HBcAg fluorescence was primarily nuclear but, in some lobules, a patchy cytoplasmic fluorescence was observed. This suggests a cytoplasmnucleus pathway i n the synthesis o f the H B V core antigen. Electron microscopy was performed on two HBsAg- and HBcAg-positive cases. Fibrillar and crystalline cytoplasmic inclusions were observed in tumour cells. In the same cells, 20-25 nm virus-like particles were present i n swollen cisternae of the endoplasmic reticulum.
There is a growing body of evidence supporting a close association between hepatitis B virus (HBV) infection and primary hepatocellular carcinoma (PHC). Extensive serologic studies have reported a high frequency of HBV markers in PHC patients compared to matched control groups (Maupas et al., 1975, 1977; Prince et al., 1975; Rlumberg et al., 1975; LarouzC et al., 1976; Tabor el al., 1977; Trichopoulos et al., 1978). The correlation between HBV and PHC, found in several parts of the world, is more significant that any other association between PHC and hepatocarcinogens, such as aflatoxin or nitrosamine, known to induce liver tumours in animals. More recently, the presence of HBV antigens in PHC was demonstrated in frozen sections or paraffin-embedded liver tissues. HBsAg was localized by orcein staining (Kostich and rngham, 1977; Tan et al., 1977; Cohen et al., 1978); inimunofluorescence (Bodin et al., 1974; Akeyama e / al., 1974; Nazarewicz et al., 1977); and immunoperoxidase techniques (Nayak et a/., 1977). Immunospecific stains were also used to localize HBcAg on frozen sections of PHC tissues (Nayak e t a / . , 1977). In the present study, HBV antigens were sought for in liver tissues obtained at necropsy from 15 black Senegalese patients who died of primary hepatocellular carcinoma. HBsAg and HBcAg were
assayed in liver extracts and the results correlated with serologic markers of HBV infection. Eight specimens were studied using the orcein stain on paraffin-embedded tissues and/or immunofluorescence on snap-frozen sections to detect the presence of HBsAg. Immunofluorescence was also used to detect HBcAg in seven samples. Two HBsAg- and HBcAg-positive specimens were selected and studied by electron microscopy for cellular abnormalities and HBV particles in either cytoplasm or nuclei of infected cells. MATERIAL A N D METHODS
Liver tissues and sera Specimens of liver tissue were obtained at necropsy from 15 Senegalese patients who died of primary hepatocellular carcinoma at Le Dantec hospital, Dakar (Internal Medicine Department, Pr. Sankale). All were adult males (age: 19-58 years) (Table I). A serum sample was obtained in each case within the week preceding death and tested for HBsAg by radioimmunoassay (RIA) (Ausria 11-125, Abbott) and for anti-HBs by RIA (Ausab, Abbott). Anti-HBc was detected by counterelectrophoresis (CEP) and complement fixation using purified HBcAg antigen (Coursaget et al., 1976). HBeAg and anti-HBe were sought for by agar gel diffusion (AGD) (Coursaget et al., 1978). Alpha-foetoprotein was tested by CEP (anti-alfa-foetoprotein, Institut Pasteur). Liver extracts were prepared by homogenizing tissue for 15 min at 45,000 rpm (Homogenizor H451, Equipement Industriel) in Tris-buffered saline 0.02 M PH 7.4, at a concentration of 25% w/v. Homogenates were sonicated three times for 1 rnin at 4" C, the sonicator (MSE Ultrasonicator) being set at 8 pm peak to peak and 28 K cycles. Cell debris was removed by two low-speed centrifugations: 30 min at 1,900 g and 15 rnin at 12,000 g (Beckman TJ6 and L5-65). Pellets from a subsequent ultra-centrifugation for 90 min at 400,000 g (Beckman L5-65) were resuspended in phosphate-buffered saline (PBS) and tested for HBsAg by RIA and for HBcAg by CEP using an anti-HBc positive serum from an HBsAg carrier. This serum was tested for anti-HBc by RIA (Corab, Abbott) and found positive at a dilution. It was negative for antiHBs when tested by RIA and negative for anti-HBe To whom reprint requests should be addressed. Received: March 9, 1979, and in revised form July 18, 1979.
GOUDEAU ET AL. TABLE I CLINICAL A N D BIOLOGICAL DATA FOR PHC PATIENTS STUDIED
+ + ++
+ + + ++ + t
~Multimicronodular R and L lobes atrophic fibrosis R and L lobes Massive tumor R and L lobes L lobe Multimacronodular R and L lobes, L lobe important necrosis Massive tumor R, R and L lobes micronodules L Massive tumor R, R and L lobes, multimicronodules inipoI tarlt necrosis R and L lobes ~. Massive tumor L lobe R and L lobes Two massive tumors L lobe Several nodules in Multimicronodular R and L lobes R and L lungs R and L lobes Multimicronodular R and L lobes R and L lobes Multimacronodular R and L lobes, important necrosis R and L lobes Multirnacronodular R and L lobes L lobe One tumor in Multimacronodular R and L lobes omentum major R and L lobes Multimacronodular L lobe only L lobe Massive tumor L, R and L lobes nodules R lobe Massive tumor R, R and L lobes ~. nodules L lobe ~
' B, Bassari: W, Woloff; T, Toucouleur; P, Peuhl: D, Diola; R, right: L, left
by AGD. It was non-reactive when tested with homogenate of normal human liver tissue. Each liver specimen was obtained a t necropsy within 8 to 12 h of death. Samples were taken from the tumour and non-tumour areas of both lobes. Metastases were taken whenever present. Blocks of 1 cm3 were wrapped in aluminum foil and immediately frozen a t -20" C . I n the case of eight liver samples, blocks were cut from the different areas, fixed in 20 % buffered formalin and embedded in paraffin. Serial paraffin sections of 4 ,mi thickness were stained with haematoxylin and eosin, Massontrichrome and orcein according to the method of Shikata et a!. (1974). Liver samples from the same eight patients were thawed and snap-frozen sections (nitrogen, isopentane) of 4 pm were cut, fixed in acetone for 2 min a t room temperature and tested by irnmunofluorescence for HBV core and envelope antigens. Immunofluorescence Immunofluorescence was carried out by the indirect method using antisera of human origin. Anti-HBs serum was obtained from a nurse vaccinated against hepatitis B virus infection (Maupas e f al., 1976, 1978). This antiserum contained more than 2.5 IU/ml when tested in reference to the HBIG International Reference Standard (Dr. Cou-
rouce, CNTS, Paris). The anti-HBs antiserum was anti-HBc-negative by RIA and anti-HBe-negative by AGD. HBcAg was detected using the human anti-HBc described previously and a preparation of purified anti-HBc antibody with similar results. Anti-HBc immunoglobulins were purified from a pool of sera from three HBsAg carriers. Sera were positive for anti-HRc by R I A at a to dilution. Immunoglobulin fractions were prepared by two steps of 30 % ammonium sulphate precipitations and subsequent ion exchange chromatography on D E 52 (Whatman, Maidstone, Kent, England). Anti-HBc immunoglobulins were anti-HBc-positive by RIA at a dilution, anti-HBs-negative by RIA, and antiHBe-negative by AGD. Anti-HBs and anti-HBc antisera were controlled by immunofluorescence for the presence of autoimmune antibody with preparations of human lung, breast, bowel, and uterus tissue as well as normal human liver tissue. Two drops of antiserum diluted 1 :5 were layered on the slide a n d incubated for 30 min at room temperature in a moist chamber. After I5 niin washing in PBS, PH 7.4, fluorescein isothiocyanateconjugated anti-human imniunoglobuiin (goat antiserum, anti-IgA, IgG, I g M ; Behringwerke Paris, France) was added and incubated for 30 min. Three washings in PBS were necessary to remove the
HEPATITIS B MARKERS IN LIVER CARCINOMA
FIGURE 1 - Nodule of well-differentiated liver cell carcinoma. The surrounding parenchyma is invaded by fibrosis and regenerative niacronodules. ( x 100).
rzmaining dye. The slides were observed under buffered glycerine with a fluorescent microscope (Leitz, Wetzlar. W. Germany). Elcctron microscopy
Two liver specimens were studied by electron microscopy after they had shown positive fluorescence for HBsAg and HBcAg. Sections were fixed in 2 % glutaraldehyde in PBS then washed in PRS. Post-fixation was carried out for 1 h in 2% OsO, in PBS. Pieces were embedded in araldite and cut with a Reichert OMU3 ultramicrotome. Ultra-thin sections were stained by uranyl acctate and lead citrate according to the method of Reynolds (1 963). Observations were made with a Jeol JEM 100 B electron microscope. RESULTS
Sera and liver extrncts Results of serologic tests on sera and liver extracts are presented in Table 11. In I0 of 12 cases where HBsAg was detected in the serum, HBsAg was also present in corresponding liver preparation by RIA. HBcAg was found by CEP in three liver extracts from patients in whom HBsAg was positive in the serum. In one case (No. 13), where anti-HBs was detectable in the serum, the liver was negative for HBsAg and HBcAg. Two patients were positive for
FIGURE2 -Scattered orcein-positi~~ cells in cirrhosig nodule (patient No. 5 ) ( ~ 2 0 0 ) .
GOUDEAU ET AL. TABLE I1 HBV SERlC MARKERS AND ALPHA-FCETOPROTEIN IN PHC PATIENTS. HBsAG A N D HBcAG IN CORRESPONDING LIVER EXTRACTS.
1' 2 3' 4' 5'
6 7 8 9 10 ' ll 12 13 14 15 '
+ + t + + + + ++ +
12 (80%) 3 (20%)
+-k t + + 4+ -t+ +-
+-t -1 + -1 ++ + + + ++ +13 (87%)
++ + +
' Also studied by orcein staining and/or immunofluorescence. HBsAg and anti-HBs simultaneously: the liver sample from patient No. I 1 was HBsAg-positive, and from patient No. 12 HbcAg-positive. In one patient (No. 15) with no serologic markers of prior infection by HBV, the liver was negative for both HBV antigens. HbeAg was negative in all cases. Anti-HBe was positive in three cases with HBsAg-
positive serum. Alpha-foetoprotein was positive in 11 cases. Histopathologic examination revealed that P H C was associated with cirrhosis in all cases. Multilobular cirrhosis of the post-necrotic type occupied thc liver pa-
FIGURE 3 ~- Peri-tuniour cluster of orcein-positive cells [patient No. 5). The same specimen showed rare orcein-positive cells in tumour nodules ( x 200).
FIGURE 4 .. -~ Orcein staining of HBsAg in hepatocytes in cirrhotic area (patient No. 5 ) . HBsAg has the appearance of a dense introcytoplasmic inclusion ( ?I 600).
HEPATITIS B MARKtKS IN LIVER CARCINOMA TABLE 111 HBsAC A N D HBcAG IN LIVER TISSUES AS SHOWN BY ORCEIN STAINING A N D IMMUNOFLUORESCENCE
I 3 4 5 10
I1 12 15
f ’ l i ? -1t ii-
+ + ~
i NT i -t-
L i-, scattered orcein-positive cells. positive cells. - Not tested.
, clusters of orcein
orcein staining. Positive cells were either scattered throughout the cirrhotic parenchyma or gathered in dense clusters, especially in the peri-tumour areas (Fig. 2, 3). In orcein-positive cells the entire cytoplasm was stained or, more often, HBsAg had the appearance of a voluminous cytoplasmic inclusion (Fig. 4).The nucleus and membrane were not stained. 1mmitnoflitoresceric.e
Indirect inmunofluorescence (IF) for HBsAg was positive in four of seven cases (57”/,), all of these patients having HbsAg in their serum. HBsAg was detected in the cytoplasm of numerous hepatocytes of peri-tumour cirrhotic tissue (Fig. 5 ) , in a pattern very similar to that found with orcein staining. A diffuse cytoplasmic fluorescence was observed; cell membrane and nucleus were negative. In one case (patient No. 5) HBsAg was also found in the cytoplasm of a few cells in the tumour. In both orcein and HBsAg IF, the antigen was not uniformly distributed and five fields of five blocks were studied whenever available before any definite conclusion was drawn. Fluorescence for HBcAg was observed in five liver samples (71 %) associated with positive HBsAg fluorescence in fivz cases and with orcein-positive staining in five. HBcAg fluorescence of the nuclei was homogeneous while patchy staining was observed in the cytoplasm of some cells (Fig. 6).
FIGURE 5 - lndirect immunofluorescence. HBsAg in non-tuniour hepatocytes (patient No. 4 ) ( 200).
renchyma. A dense fibrosis and voluminous regenerative nodules, two or mori: portal triads, were observed in every case except patient No. 9 where the cirrhosis was of the micronodular type. Nodules of well-differentiated trabecular hepatocellular carcinoma were scattered over this cirrhotic background (Fig. 1). The architecture of tumour tissue was often destroyed by necrosis, thus limiting further interpretation, No ground-glass hepatocytes or “sanded” nuclei were seen with conventional histologic stains. Shikata’s orcein stain revealed large brown-reddish inclusions of HBsAg in the cytoplasm of hepatocytes of the cirrhotic nodules in six of seven HBsAg positive cases (86%) (Table Ill). The one HBVnegative PHC (No. 15) was also negative after
FIGURE6 Indirect inllllunOflL10TescenCe. HBcAg in n~iclei ( a ) and cytoplasm (a and b ) of cirrhosis cells (patient N o 4) ((I >,600x 1.5, b \I 600 ~ 2 . 5 ) . ~
GOUDEAU ET AL.
FIGURE 7 - Tumour hepatocyte. Arrows indicate para-crystalline structures in amorphic inclusion body of the cytoplasm (bar:100 nm).
Liver sections from patient No. 15, negative for HBV serologic markers, were negative by all IF assays.
cells. These particles were detected in vacuoles issuing from the endoplasmic reticulum (Fig. 8).
Electron microscopy Liver speciniens from patients No. 4 and 5, positive for HBsAg and HBcAg by IF, were studied by electron microscopy. Tumour cells were greatly altered with an enlarged and irregularly-shaped nucleus and a prominent nucleolus. The cytoplasm was poor in endoplasmic reticulum and glycogen granules when compared to normal hepatocytes ; mitochondria were swollen and their cristae were reduced in number. In the cytoplasm of tuniour cells, unusual inclusions were present. These inclusions were ovoid, two or three times the size of a mitochondrion, and not surrounded by a limiting membrane. There were two or three inclusions per cell in nearly half of the cells observed. In the inner part of the inclusions 100- to 200-nm long fibrils, with diameters of 5 to 10 nm, were arranged in crystalline structures (Fig. 7). Fibrils were surrounded by an amorphous matrix which was not limited by a czllular membrane. N o similar structures were seen in cells from the cirrhotic tissue. I n liver from patient No. 5, several clusters of 20- to 25-nm virus-like particles were observed in the cytoplasm of inclusion-containing
HBsAg was found in liver extracts from 10 of the 12 patients with HBsAg-positive serum. Liver tissue of patients with anti-HBsAg only (No. 13) o r antiHBc only (No. 14) or with no serum markers (No. 15) were HBsAg-negative. HBcAg was detected in liver extracts from only three HBsAg-positive patients but results may have been altered by the presence of circulating anti-HBc in all but two of the studied cases or by limited sensitivity of the CEP. Anti-HBe was detzcted in the serum of three of the 12 HbsAg-positive P H C (25%) and HBeAg in none of the patients. These results are similar to those reported by Werner at a / . (1976) and argue against the hypothesis that seroconversion to anti-HBe is a sign of resolution of the liver disease. Orcein-positive cells werz found in six or seven H BsAg-positive cases (86 %), a prevalence similar to that reported by Cohen e t a / . (1978). The overall percentage of orcein-positive livers was 75 Od, almost identical to 74% reported by Tan et a / . in Singapore (1977). After orcein staining, most of the HBsAgpositive cells were present in the cirrhotic areas and confined to one lobule while adjacent lobules were
HEPATITIS B MARKERS IN LIVER CARCINOMA
FIGURE 8 - Tumour hepatocyte; 25-nm virus-like particles (arrow) in cisterna of endoplasmic reticulum (bar: SO
negative as observed by Tan et a / . (1977) and Cohen et a/. (1978). Inside a positive lobule a diffuse distribution of HBsAg was occasionally found. More often, though, HBsAg-positive cells were clustered in a segment of the lobule especially at the margin of tuniour nodules, as reported by Portmann e t a l . (1975). In one case (patient No. 5 ) a few HBsAg cells were observed in well-differentiated PHC nodules. According to this irregular distribution of HBsAg-producing cells, it seems that replication of HBV may take place in scattered lobules of the cirrhotic parenchyma. This has to be taken into account upon examination of small fragments of liver tissue or samples containing mainly neoplasic tissue (Tan etal., 1977). For instance, sample No. 12 was found initially negative by orcein and IF and then positive by IF when additional sections from slightly different areas were examined. HBsAg was detected by indirect imniunofluorescence in the cytoplasm of liver cells, mainly in peritumour cirrhotic areas. In one case (patient No. 5 ) scanty HBsAg fluorescence was noted in liver cells of rare tumour nodules as reported previously from other laboratories (Kostich and Ingham, 1977; Nazarewicz e l a/., 1977; Nayak et a/., 1977). This suggests that HBV replication, though diminishing, still persists in the neoplastic cell. However, our data
indicate that most of the HBV antigen production occurs in the cells of the cirrhotic nodules. HBcAg was not confined to the nuclei of hepatocytes but involved both cytoplasm and nucleus. However, nuclei of positive cells were stained homogeneously while only patchy cytoplasmic fluorescznce was observed. This confirms recent data suggesting that synthesis of HBcAg, like the capsid of other D N A viruses, occurs in the cytoplasm and probably in the endoplasmic reticulum. HBcAg would then migrate to the nucleus where assembly of the corc particles would take place (Huang, 1977; Gudat and Bianchi, 1977). IF failed to detect HBcAg in tumour cells, even in preparation where HBsAg was positive. In two of 15 cases HBsAg and anti-HBs were present simultaneously in the serum. It was not possible to determine the anti-HBs subtype by agar gel diffusion because of the insufficient antibody titre. However, it may be speculated that these patients had recovered from infection by one HBV subtype and had been subsequently infected by another subtype (Tabor et al., 1977; Trichopoulos et a/., 1978). Ongoing HBV infection was revealed in these two cases by the detection of HBsAg (No. 11) and HBcAg (No. 12) in the liver tissues. Electron microscopy of two liver specimens (patients No. 4 and No. 5 ) revealed fibrillar and
GOUDEAU ET AL.
crystalline inclusions in the cytoplasm of some tumour cells. These inclusions seemed more “organized” than similar fibrillar bodies described by Smetana et a/. (1972) in hepatoma of unknown HBV marker content or by Bodin et af. (1974) in tumour cells of HBsAg-positive hepatoma. These inclusions were clearly distinct from alcoholic hyaline bodies. Moreover, our patients, being Moslem, did not consume alcohol. It is not clear whether these inclusions represented some form of cell degeneration or were related to HBV infection. Virus-like particles measuring 20-25 nni were observed in swollen cisternae of the endoplasmic reticulum of neoplastic cells, mainly in those containing fibrillar inclusions. Similar findings have been reported by Campion el a/. (1972) and related by direct immunofluorescence to HBsAg material. Careful examination of the preparation failed to detect intracisternal tubular particles, described by Stein et a / . (1972), and intranuclear spherical particles (Huang 1975), which are thought to be respectively HBsAg and HBcAg materials in infected cells of HBsAg carriers. The lower frequency of HBsAg-positive cases among patients with cirrhosis than among those with PHC, and the discrepancy between tumour and non-tuniour areas in regard to H B V replication, argue against the hypothesis that HBV induces the malignant transformation, by integration into the
genome of the host cell, as has been shown for other DNA viruses. However, it was recently reported that HBsAg has been detected by IF and orcein in neoplastic cells of a bone metastasis of PHC (Doury P I a/., 1977). Moreover, hepatitis R virus D N A has been found, though not in integrated form, in HBVrelated hepatoma (Summers et a/., 1978; London, 1978). Finally, an HBsAg-producing cell line has been successfully established from P H C tissue (Alexander et a/., 1976). A variety of models involving HBV as an etiologic agent may be advanced to explain these somewhat discordant data. HBV infection could lead successively to chronic hepatitis followed in most cases by postnecrotic cirrhosis. Ultimately, P H C would evolve from the post-necrotic regenerative nodules with H BV replication becoming increasingly defective as in papovavirus-induced tumours (Mellors, 1960). Other carcinogens might play a synergistic role in this process (Sun er a/., 1971). ACKNOWLEDGEMENTS
We gratefully acknowledge the help of Dr. Th. Planiol (Medical Biophysics) and Drs. P. Jobard and A. Benatre (Pathology) for generously allowing us to use their departments. We thank Mr. P.Y. Sizaret for the electron niicroscopic study and Ms. Ch. Bourderioux for skillful technical assistance.
A N T I G E N E S D U VIRUS DE L’HEPATITE B DANS DES TISSUS D E CANCER P R I M I T I F DU FOlE HUMAIN La presence des antigenes constitutifs d u virus de I’hepatite B (HBV) a kt6 recherchee dans des echantillons d e tissu hepatique preleves B I’autopsie de malades senegalais atteints de cancer primitif d u foie. Les resultats de l’examen des tissus ont ete compares avec la presence de marqueurs seriques d’une infection par le virus HB. Les antigenes de I’enveloppe (AgHBs) et de la capside virale (AgHBc) ont ete recherches dans 15 extraits de foie. L’antigene HBs a ete retrouve dans 10 preparations de foie sur 12 malades ayant une serologie HBs positive, l’antigene HBc daris 3. L’antigene HBs a ete detecte dans 7 foies sur 8 etudies par la coloration a l’orceine et par imrnunofluorescence indirecte. Les cellules AgHBs positives etaient principalement localisees dans le parenchyme cirrhotique. Chez un patient, toutefois, quelques hepatocytes AgHBs positifs ont ete observes dans les nodules tumoraux. L’antigene HBc a ete retrouve dans 5 cas sur 7 etudies en immunofluorescence. Les cellules positives etaient situees dans le tissu cirrhotique. La fluorescence HBc etait principalement nucleaire mais, dans quelques lobules, une fluorescence cytoplasmique granulaire a ete observee. Ce resultat suggere qu’il existe un courant cytoplasme-noyau au cours de la synthese de la capside du virus de I’hepatite B. Une etude en microscopie electronique a ete realisees dans deux cas ou I’AgHBs et I’AgHBc etaient presents dans les tissus. Des inclusions fibrillaires et cristallines ont ete observees dans les cellules tumorales. Dans les m&mescellules, des particules d’aspect viral de 20-25 nm etaient presentes dans des citernes du reticulum endoplasniique.
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HEPATITIS B MARKERS IN LIVER CARCINOMA
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