Download pdf - Hepatitis B x Antigen in Hepatitis B Virus Carrier Patients with Liver Cancer1

[CANCER RESEARCH 51, 4971-4977, September 15, 1991]

Hepatitis B x Antigen in Hepatitis B Virus Carrier Patients with Liver Cancer1

Wenliang Wang, W. Thomas London, and Mark A. Feitelson2-3

Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT

Formalin-fixed, paraffin-embedded specimens from 110 cases of primary hepatocellular carcinoma were stained for hepatitis B x antigen(HBxAg), hepatitis B surface antigen (HBsAg), and hepatitis B coreantigen (HBcAg). Eighty-four % of these patients were HBxAg positivein their tumor cells. Among the 110 cases studied, 80 had adjacentnontumorous tissue in the same block, and 65 of these nontumorous livertissues stained positive for HBxAg (81%). I IBsAy was positive in 19%of cases within tumor tissue and 61% in surrounding nontumorous tissue.HBcAg was positive in 11% of cases within tumor tissue and 26% insurrounding nontumorous tissue. These findings show that HBxAg is acommon marker in the liver of patients with hepatitis B virus (IIBV)-associated primary hepatocellular carcinoma and that it is closely associated with tumor cells in these individuals. In addition, the finding ofHBxAg in the absence of detectable HBsAg and HBcAg in the livertissues of many HBsAg carriers suggests that HBxAg could be expressedindependent of HBV replication and implies that the synthesis of thisantigen may be directed from integrated HBV DNA templates. Thefinding of HBxAg in the nucleus of hepatocytes from primary hepatocellular carcinoma patients with dysplasia, combined with the known trans-activating properties of HBxAg, implies that HBxAg plays one or moreimportant roles in hepatocarcinogenesis. The finding of HBxAg in bileduct epithelium and cholangiocarcinoma tissues is compatible with thehypothesis that HBV may contribute to this other primary tumor type inthe liver. Together, these results further implicate HBxAg in the patho-genesis of primary liver cancers.

INTRODUCTION

It is well established that the HBV chronic carrier state andthe presence of progressive liver disease are risk factors whichplay central roles in the development of PHC (1-5). HBVinfection may result in chronic hepatitis, followed by livercirrhosis (2, 3, 5, 6). Most PHC occurs in individuals withcirrhosis (80%) or CAH (10-15%) (3). PHC is closely associated with cirrhosis, particularly macronodular cirrhosis, whichis thought to play an important role in pathogenesis. However,the contribution of HBV to the pathogenesis of PHC remainsto be elucidated (1, 5-8).

The prevalence, distribution, and possible significance ofHBsAg and HBcAg in liver tissues from patients with chronicliver disease and PHC have been well documented (9-12).However, few studies focused on the characteristics of HBxAgin chronic infection have been reported. Evidence that HBxAgwith frans-activating properties could be produced from a viraltemplate integrated into the host genome during chronic infection (13), that HBxAg could trans-activate oncogenes such as

Received 4/3/91; accepted 7/8/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by USPHS Grants CA-40737, RR-05895, CA-06927, and CA-48656; Johnson & Johnson Focused Giving Grant 830702; andan appropriation from the Commonwealth of Pennsylvania.

2To whom requests for reprints should be addressed.3 Present address: Department of Pathology and Cell Biology, Thomas Jeffer

son University School of Medicine, 1020 Locust Street, Philadelphia, PA 19107.4The abbreviations used are: HBV, hepatitis B virus; PHC, primary hepato

cellular carcinoma; Anti-HBx, antibodies to the X gene products of hepatitis Band related viruses; CAH, chronic active hepatitis; ChC, cholangiocarcinoma;HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBxAg,hepatitis B X antigen; HChC, hepatocholangiocarcinoma.

c-myc(l4), and that HBxAg could "transform" nontumorigenic

cell lines into lines capable of growing as tumors in nude mice(14-16) support the hypothesis that HBxAg may play an important role in the pathogenesis of HBV-associated chronicliver disease, including PHC. Previous results from this laboratory, documenting the presence of HBxAg in dysplastic hepatocytes, combined with the predominantly nuclear distribution of HBxAg in patients with cirrhosis and dysplasia (17),also support this hypothesis. Hence, this study was carried outto test whether HBxAg was closely associated with tumor cellsin patients with PHC.

MATERIALS AND METHODS

One hundred ten cases of surgically resected specimens from HBVcarriers with PHC were obtained from the Pathology Laboratory of theFirst Teaching Hospital at the Fourth Military Medical University,Xi'an, People's Republic of China. For comparative studies, additional

HBV-infected patients with chronic liver diseases but no evidence ofPHC were also used. The characteristics of these latter populations ofpatients have been described earlier (17). Tissue staining for HBsAg,HBcAg, and HBxAg was carried out by a standard immunoperoxidaseprocedure (17) using poiyclonal anti-HBx raised from a syntheticpeptide antigen (spanning HBxAg residues 100-114) (18) or from arecombinant DNA-derived full length HBxAg polypeptide made froman expression vector in Escherichia coli (19). The recombinant HBxAgand corresponding antibody were kindly provided by Dr. X. Ma, Institute of Basic Medical Sciences, AMMS, Beijing, People's Republic of

China.Specificity controls were carried out as described (17). Fifteen unin-

fected human liver samples and several tissue types from other organs(spleen, lymph node, muscle, nerve, and gallbladder), were tested withimmune sera. Preimmune and normal rabbit sera were tested withpositive liver sections. The HBxAg synthetic peptide used to raiseimmune sera was tested for blocking in tissues staining positive withthe anti-HBx reagent. Recombinant DNA-derived HBxAg polypeptide(19) was used to block the reaction of antibodies raised against thisrecombinant polypeptide with positive tissues (kindly supplied by Dr.X. Ma). Liver powder made from uninfected human liver tissues (20)was used to absorb the primary antibodies prior to staining. Anti-HBxreagents were tested by Western blotting with E. coli lysate frombacteria expressing HBxAg polypeptide compared to a similar lysatefrom untransfected host cells. Hep G2.2.15 cells making HBV particleswere tested with anti-HBx reagents as were virus-negative Hep G2 cells.

Cell supernatants and extracts were also analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and Western blotting (18).Additional control tissues included liver containing unrelated met-astatic cancers.

Among the 110 carriers studied, there were three different tumortypes. One hundred two patients had PHC, 3 had HChC, and another5 had ChC. Eighty of these PHC patients had nontumorous liver in thesame histolÃ³gica! sections. The diagnosis of ChC and HChC wasconfirmed by Dr. Zachary Goodman, Hepatic Pathology Branch,Armed Forces Institute of Pathology. PHC was divided into threegrades of differentiation, based upon morphological criteria in eachcase, according to the guidelines suggested by the WHO (21) and others(22). Grade I represented differentiated PHC, grade II moderatelydifferentiated PHC, and grade III undifferentiated PHC.

The definitions and grades of CAH and liver cell dysplasia used inthese studies have been outlined earlier (17).

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HBxAg IN CARRIERS WITH LIVER CANCER

RESULTS

Frequency of HBxAg in Tumor and Surrounding NontumorTissues from PHC Patients. Among 110 HBsAg carrier caseswith liver tumors, 102 had PHC, 3 had HChC, and 5 had ChC.Eighty of these patients had surrounding nontumorous tissueon the same tissue section; cirrhosis was present in 54 casesand CAH was present in the remaining 26 (Table 1). Dysplasiawas present in 50 of 54 cases (93%) with liver cirrhosis and in12 of 26 cases (46%) with CAH. HBxAg was detected in 92 of110 patients with liver cancer (84%) within tumor cells usingpeptide antibodies, as described (17) (Table 1). Eighty-five of

these were seen in tumor cells of PHC, 3 in HChC, and 4 inChC. Representative results for PHC patients are presented inFig. 1. In addition to patients with PHC, 7 of 8 HBsAg carrierswith HChC or ChC were also HBxAg positive within tumorcells (Table 2; Fig. 2), suggesting that HBxAg may be a prevalent marker in these tumor types as well as in PHC. HBxAgwas also detected in the nonneoplastic tissues in 70 of the 80patients (88%) with these tissues available. Among these 70HBxAg-positive cases, 64 had PHC, 2 had HChC, and 4 hadChC. HBxAg was detectable in 45 of 54 PHC patients withcirrhosis (83%). Forty-three of these cirrhotic patients also haddysplasia. Among the remaining 20 PHC patients with nontumorous tissue, all had CAH, 10 had dysplasia, and 19 wereHBxAg positive (Table 1). HBxAg was also present in 2 ChCpatients with dysplasia (Table 2). As in earlier work (17), morethan 70% of patients with liver cell dysplasia were HBxAgpositive. These results show that HBxAg is often present innontumorous liver tissues adjacent to tumors. Among patientswith liver tumors, there were a handful who were also HBxAgpositive in bile duct epithelium (Fig. 2). In all cases, the stainingin bile ducts was generally weaker than in hepatocytes. Combined with the above results demonstrating the presence ofHBxAg in HChC and ChC sections, these results are consistentwith an association between HBV and bile duct associated and/or derived cells.

Many of the above sections were also stained with antibodiesraised against full-length HBxAg polypeptide made from arecombinant DNA expression vector in E. coli (19). The resultswere very similar to those obtained with the X peptide antibodies above, with the exception that the intensity of staining wassometimes lower using antibodies against the recombinant protein compared to the peptide antisera (data not shown). Specificity was demonstrated by showing that staining was absentfrom HBxAg-positive sections in which preimmune rabbitserum was used in place of anti-HBx as primary antibody (Fig.1) and by showing that staining could be completely blocked bypreincubation of the antisera with the immunizing antigen(HBxAg synthetic peptide or recombinant DNA-generatedHBxAg) prior to assay (Fig. 2). Controls outlined in "Materialsand Methods" have also been carried out with these antisera

(17) which confirm the specificity of antigen staining. Theseresults, then, support the conclusion that HBxAg is present inboth tumor and nontumorous liver cells from chronic carriers.

Cellular and Tissue Distribution of HBxAg. The percentageof HBxAg positive tumor cells varied among the different livercancer patients. Among the 80 patients with paired tumorousand nontumorous samples for evaluation, HBxAg was unde-tectable in tumor cells from 13 cases, was present in less than30% of tumor cells in 29 cases, and was detectable in morethan 30% of tumor cells in the remaining cases (Table 3). Inmost cases, the intensity of HBxAg staining increased with thepercentage of HBxAg-positive cells in liver sections, so that the

Table 1 Dsitribution of HBxAg in various types of liver cancer and surroundinghepatic tissues

Type ofPHCPHCWith

cirrhosisWith CAH

HChCWith CAH

ChCWith CAH

Total (%)TumorousCases10235110HBxAg

(%)85

(83)3(100)

4(80)

92 (84)NontumorousCases54"

20*2480HBxAg

(%)45

(83)19(95)2(100)4(100)

70 (88)Â°Fifty PHC patients with cirrhosis also had dysplasia. Forty-three PHC

patients with dysplasia were HBxAg positive; the other 2 HBxAg positive patientshad cirrhosis but no dysplasia.

h Ten PHC patients with CAH also had dysplasia. All patients in this category

with dysplasia were HBxAg positive.

patients with the largest percentage of HBxAg-positive tumorcells often had the most intense staining in those cells [usuallymoderate (++) to dark (+++) staining]. Similar analysis ofsurrounding nontumorous tissues in these patients showed 4cases without detectable HBxAg. In these cases, both the tumorous and nontumorous tissues were negative for HBsAg andHBcAg. HBxAg was present in less than 30% of nontumorouscells from an additional 7 patients and was present in morethan 30% of nontumorous liver cells from the remaining individuals (Table 3). Again, the intensity of staining correlatedwith the percentage of HBxAg-positive cells in these patients.When the frequency (percentage of HBxAg-positive cells) andintensity of HBxAg staining were compared in tumorous andnontumorous tissues among patients with CAH, cirrhosis, and/or dysplasia, both frequency and intensity of HBxAg stainingwas greater in nontumorous compared to tumor tissue. Hence,the proportion of patients with intense HBxAg staining wasgreater in the nontumorous compared to tumorous cells.

When the frequency and intensity of HBxAg staining wereexamined in patients without PHC and compared to the resultsof this study (Ref. 17; Table 4), it was observed that a higherproportion of PHC-negative patients with CAH or cirrhosiswere also HBxAg positive in more than 30% of their hepatocytes compared to patients with chronic persistent hepatitis andin tumor cells among patients with differentiated (grades I andII) or undifferentiated (grade III) PHC. An analogous observation was made when the parameter of HBxAg staining intensitywas evaluated (Table 4). For example, among patients withchronic persistent hepatitis and PHC, the intensity of HBxAgstaining in tissue was negative to mild (- to +). Combined with

the above finding that more than 30% of liver cells examinedin sections from most patients with CAH or cirrhosis werepositive for HBxAg and the staining in these cases was moderateto dark (++ to +++), these results suggest that the number ofcells expressing HBxAg and the intensity of staining increasesin carriers with progressive liver disease preceding the appearance of PHC.

The subcellular localization of HBxAg in the cells of tumorous and nontumorous tissues is summarized in Table 3. HBxAgwas localized mainly in the cytoplasm of the liver and tumorcells, and less frequently, on the cell membrane and/or in thenucleus. Both membranous and nuclear HBxAgs were observedin tumorous as well as nontumorous cells. A striking feature isthe relatively high frequency of HBxAg in the nuclei of hepatocytes in 18 of 50 cirrhotic patients with dyplasia (36%).Nuclear HBxAg was also observed in more than 50% of cirrhotic patients with dysplasia who had no evidence of PHC(Ref. 17; Table 4). In contrast, PHC patients with cirrhosis but

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D\ \ > /

\.ar;

"lâ€¢'*<?â€¢â€¢:

>~i

Fig. 1. Patterns of HBxAg expression in PHC tissues. A, localized distribution of HBxAg in a nodule of differentiated (grade I) PHC (upper right and middle)Staining is cytoplasmic. Cells in the upper left quarter are HBxAg negative. B, diffuse distribution of HBxAg in a region of undifferentiated PHC (grade III). Stainingis cytoplasmic and intense in many cells (arrows). C, HBxAg-positive PHC stained with preimmune rabbit serum from the same animal in which the anti-HBx peptideantiserum was later derived. D, membranous distribution of HBxAg in tumor cells from a patient with moderately differentiated PHC (grade II) (arrows). E, nucleardistribution of HBxAg in tumor cells from a patient with grade II PHC (arrows). Peptide antibodies were used for staining all panels. C, x 200; all other panels, x400.

no dysplasia did not have evidence of nuclear HBxAg, whilemore than 40% of PHC-negative patients with cirrhosis hadevidence of HBxAg (Tables 3 and 4). While these results areconsistent with the conclusion that the patterns of nuclearHBxAg are different among patients with CAH or cirrhosis,depending upon whether they also had PHC, the differencesmay be due to the fact that there are only 4 cirrhotic patientswith PHC compared to 33 without PHC (17) and that comparison of such small numbers is not enough to illustrate the truefrequencies. Hence, the finding of nuclear HBxAg in manypatients with dysplasia is consistent with the conclusion thatnuclear HBxAg plays an important role in pathogenesis ofchronic infection leading up to PHC.

Among PHC cases, membranous HBxAg was detected in10% of patients with cirrhosis and dysplasia, but in none of theindividuals with CAH (Table 3). In contrast, 30-40% of thepatients with chronic hepatitis and cirrhosis but no PHC werepositive for HBxAg on their hepatocyte membranes (Table 4).Hence, there is an apparent difference in the proportion of

patients with membranous HBxAg from those with and withoutPHC. These findings are also consistent with the conclusionthat membranous HBxAg may play a prominent role in theprocess leading up to PHC (7).

Both localized and diffuse staining was observed for HBxAgin PHC cells (Fig. 1; Table 3). Localized staining was thedominant pattern in the large majority of patients while diffusestaining was present in only 10-20% of PHC tissues. Sparsestaining was rare in PHC tissue, as it was in nontumorous liversamples (17). Further, the tissue distribution of HBxAg stainingdid not correlate with HBsAg or HBcAg, nor was it associatedwith any type of chronic liver disease. Similar patterns ofHBxAg were observed in tumorous and nontumorous tissuesfrom patients with HChC and ChC.

The patterns of HBxAg staining were also examined indifferentiated PHC (grades I and II) and compared to thepatterns in undifferentiated tumors (grade III) (Table 4). Seventy-five % of patients with differentiated PHC were HBxAgpositive in more than 30% of their cells, which was similar to

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that observed in the livers of chronic HBV carriers withoutPHC. In contrast, only 40% of patients with undifferentiatedPHC were HBxAg positive in more than 30% of their tumorcells. When the intensity of HBxAg staining is considered,moderate and intense staining was observed in most patientsindependent of the grade of PHC. Similar intensity profileswere also observed among HBV carriers without evidence ofPHC (Table 4). The cellular staining patterns for HBxAgshowed a dominant cytoplasmic distribution in nearly all patients, independent of the grade of PHC, and in patients withoutPHC. Membranous HBxAg was detected in 17% of patientswith differentiated PHC but was 30-40% in patients with

undifferentiated PHC and without PHC (Table 4). Membranous HBxAg was lowest among PHC patients with adjacentnontumorous tissue. Nuclear staining was lower in the tumorcells of PHC patients (24% for grades I and II; 19% for gradeIII) than in patients with cirrhosis (50%) or in cirrhotic patientswith dysplasia (54%). Finally, the distribution of HBxAg stain-

Table 2 HBV markers in liver sections from virus carriers withcholangiocarcinoma

Diagnosis inmmtu

Tumor moroustype tissue

HBV antigens in liver sections

SerumHBsAgstatus

Tumorous Nontumorous

HBsAg HBcAg HBxAg HBsAg HBcAg HBxAg

HChC CAHChC CAH"

HChC No tissueChC CAHHChC CAHChC No tissueChC CAHÂ°

ChC CAH" These patients have dysplasia in nontumorous liver tissue.

ing in tissue was mostly localized and, to a lesser extent, diffusein patients from all categories of patients (Table 4).

Patterns of HBsAg and HBcAg Expression in Patients withLiver Cancer in Relation to HBxAg. Fifteen % of patients withliver cancer were HBsAg positive in tumor cells. Seventeen of

D

* A 'f * '

Â«-

â€¢Â«â€¢c. .

Fig. 2. Detection of HBxAg in bile duct cells and primary liver tumors with ductular morphology. .â€¢(.HBxAg in bile duct epithelial cells from a HBV carrier withchronic hepatitis. B, control of same patient as in A in which peptide antibody was preincubated for l h at 37'C with an excess of immunizing synthetic peptide priorto staining. C, HBxAg in tumor cells from a patient with cholangiocarcinoma. Staining was carried out using antibodies raised against recombinant DNA-generatedHBxAg M, 17,000 polypeptide made in E. coli. HBxAg-positive cells are on the lower half. D, control of the same patient as in C, in which primary antibody waspreincubated for l h at 37*C with an excess of immunizing M, 17,000 HBxAg polypeptide prior to staining. /â€¢.',staining of a malignant fibrous histocytoma of the

liver with anti-HBx peptide antibodies from a patient without evidence of HBV infection.

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Table 3 Characteristics of HBxAg in tumor and surrounding nontumorous samples from Chinese HBV carriers with liver tumors"

Frequency incells*DiagnosisPHC/CAH

(n = 20)''

TumorousNontumorousNo.1719<30%

30-70%4

111 15HBxAg

positiveIntensity>70%

+ +++++2

31IS 1

I 12 6Distribution

incellsMc

CN4

17 50 18 1Distribution

intissueS0

0L

D14

315 4HBsAgpositive

(no.)112HBcAgpositive(no.)4

5

PHC/cirrhosis/Â±dysplasia (n = 54)

TumorousNontumorousHChC

(n =3)TumorousNontumorousChC

(n =5)TumorousNontumorous45513244195303122180213428000052001I292632331123000085'202045483244918Â«0000100000392032435310001133511115150100

" Samples presented here include those from patients in which both minorons and nontumorous samples were available.* The cutoff values for reporting the frequency of HBxAg positive cells were arbitrary.' C, cytoplasmic; D, diffuse; I., localized; M, membranous; N, nuclear; S, scattered.* Numbers in parentheses, number tested in each group.' All patients in these categories had dysplasia.

Table 4 Characteristics of HBxAg in liver samples from Chinese HBV carriers with different types of chronic liver disease

HBxAgpositiveDiagnosisCPH

CAHCirrhosis

Cirrhosis/dysplasia

PHC I, II*

NontumorousPHC III*

NontumorousNo.

tested28

8233656648

3626Frequency

incellsNo.18

1730655847

2725<30%7

143

1014

316230-70%8

40213837

231012>70%3

236

177

21111+3

4104

131Intensity++13

49164340

242016+++2

2413221422

48Distribution

incellsM"7

301021103

93C18

77306558

462624N2

8IS3514

1753Distribution

intissueS1

1122

000L13

5223374923

2513D4

246

267

252

12HBsAg

positive(no.)12

549

5614

313

16HBcAg

positive(no.)7

281

1412

1307

" M, membranous; C, cytoplasmic; N, nuclear; S, scattered; I . localized; D, diffuse; CPH, chronic persistent hepatitis; PHC I, II, (grade I) differentiated and (grade

II) moderately differentiated PHC; PHC III, (grade III) undifferentiated PHC.* Includes PHC patients with and without surrounding nontumorous tissue for evaluation.

19 HBsAg positive patients had PHC (3 grade I, 11 grade II,and 3 grade III) while 1 patient had HChC and another patienthad ChC (Tables 2-4). HBsAg was more prevalent in tumorcells from patients with differentiated liver cell carcinomas(grades I and II) compared to undifferentiated tumors (gradeIII). Among the 80 cases where hepatic tissue surroundingtumor nodules was available, 61% of the patients were HBsAg-positive in nontumorous tissue. Among the 47 HBsAg-positivecases with PHC, 34 had liver cirrhosis and 13 had chronichepatitis. The HBsAg-positive patient with HChC and the otherwith ChC both had CAH (Table 2). Eight patients who wereHBsAg positive in their tumor cells also had dysplasia. HBsAgstaining in tumorous and nontumorous tissues from the samecase was found in 14 cases (1 grade I, 10 grade II, and 3 gradeIII). The proportion of patients with HBsAg in surroundingnontumorous tissues was similar regardless of the tumor grade.The patterns of HBsAg staining could be described as thediffuse, membranous, and/or inclusion types in both tumorousand nontumorous tissues. The last type was observed to be themost frequent; multiple types of staining were observed in somecases. HBsAg was detected in 2 cases of PHC in the nuclei andnucleoli (data not shown).

Eleven % of patients with liver tumors were HBcAg positivein their tumor cells. Among patients with PHC, HBcAg wasobserved in the surrounding tissues from 20 cases; 15 with liver

cirrhosis (including 14 with dysplasia), and 5 with chronichepatitis (including 2 with dysplasia). A single patient with ChCwas also HBcAg positive in surrounding nontumorous tissue(Table 3). As with HBsAg, HBcAg was more prevalent in tumorcells from patients with differentiated liver cell carcinomascompared to undifferentiated tumors (Table 4). HBcAg-positivecells appeared in both tumorous and nontumorous tissues in 5PHC patients. HBcAg was demonstrated in the nuclei andnucleoli, in the cytoplasm, and/or on the cell membrane (datanot shown). Cytoplasmic HBcAg staining was observed intumor cells, while nuclear HBcAg staining was dominant innontumorous tissue. As with HBsAg, the proportion of patientswith HBsAg in surrounding nontumorous tissues was similarregardless of the tumor grade.

Among the 92 HBxAg positive tumors, 23% were associatedwith HBsAg and/or HBcAg in tumor cells; 6 of these caseswere associated with both antigens, 15 with only HBsAg, and6 with only HBcAg (Tables 3 and 4). Among the 72 HBxAgpositive cases of nontumorous liver tissue in patients with PHC48 were also positive for HBsAg and/or HBcAg. Fifteen ofthese samples were positive for both HBsAg and HBcAg, 29for HBsAg without HBcAg, and 5 with HBcAg but no HBsAg.Five of 7 HBxAg-positive cases of HChC and ChC had neitherHBsAg nor HBcAg in tumor tissue (Table 2). Two of thepatients in this group who were HBsAg positive in HChC or

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ChC were also HBsAg positive in surrounding liver tissue. Oneof the HBsAg-positive patients was also HBcAg positive insurrounding liver tissue (Table 2). Hence, HBxAg was expressed in tumor tissue more frequently than HBsAg or HBcAgin this population.

DISCUSSION

The patterns of HBxAg gene expression were determined inliver sections from 110 HBV carrier patients with liver cancerand were compared to liver HBsAg and HBcAg staining in 80of these patients where tumor and surrounding nontumoroussamples were available. HBxAg was found in both tumorousand surrounding nontumorous cells from more than 80% ofthese patients (Table 1), which is consistent with some observations (23), but not others (24). The differences may be due tothe antibody probes used for staining and/or the populationsused in the various studies. HBxAg was also found in manytumor tissue samples from HBsAg-positive carriers who hadundetectable levels of HBsAg and HBcAg in tumor (Tables 2-4). Again, similar observations have been made in some (23)but not all (24) studies. The presence of HBxAg as the onlyhepatitis B antigen marker in both tumor and many nontumorous samples implies that the production of HBxAg may occurindependent of viral replication. The discordance betweenHBxAg and HBcAg in these studies is consistent with theconclusion that HBxAg may be generated mainly from one ormore integrated HBV DNA templates, which are commonlyfound in PHC nodules (13, 25, 26) and in some cases of chronichepatitis (27).

The results from this study demonstrate a similar discordanceof HBxAg with HBsAg and HBcAg in nontumorous tissues. Incontrast, other studies show an apparent concordance of Xantigen in liver with classical virus markers, and especially withcore antigen (23, 28, 29) suggesting that X antigen expressionis related to patterns of virus replication. The latter concept isfurther supported by the reported findings of X antigen associated with core particles in the liver (30, 31), and with Xantigen associated with both hepatitis B e antigen and viralDNA in the serum (18). The differences in interpretation maybe due to the differences in the template producing X antigenand the characteristics of the X antigen polypeptides made.During viral replication, studies in this laboratory have shownthat X antigen polypeptides are made as X/core fusion components (30, 31), probably from supercoiled viral DNA, whichbecome closely associated with cytoplasmic core particles. Perhaps this is why several studies have shown a close associationbetween X antigen and viral replication (23, 28, 29). Alternatively, many patients with chronic hepatitis, and most withcirrhosis and/or liver cancer are HBcAg negative in the liverand have undetectable levels of virus replication. With little orno replicative forms of virus DNA in the liver, X antigenpolypeptide(s) may be made from integrated templates as components unfused to core antigen. Under the latter circumstances, the production of X antigen polypeptides would beexpected to occur independently of core particles and virusreplication. The latter circumstances appear to be the case inthe patients within this study, most of whom have little evidenceof virus replication and probably have only integrated HBVDNA.

The presence of HBxAg in the tumor cells from the majorityof patients strengthens a close association between chronicHBV infection and the appearance of liver cancer. The fact that

HBxAg has also been observed in a human hepatoblastoma cellline producing HBV particles (32), that X region viral DNAhas been detected in most liver tumor tissues examined (26),and that functional X polypeptide(s) have been produced fromsuch integrated species (13, 27) not only supports the idea ofHBxAg as a common antigen in patients with chronic liverdisease, including liver cancer, but also implies that it has oneor more roles in the initiation and/or progression of PHC. Thefinding of several HBxAg-negative tumors, even in patientswho were positive for HBxAg in surrounding nontumorouscells, suggests that the continued expression of HBxAg maynot be required for the persistence of liver cancer. Observationsthat liver tumor nodules from some HBV-infected individualsdo not have detectable HBV DNA (8) also suggest that tumorpersistence is not HBxAg dependent. However, recent reportsthat HBxAg expression is associated with the transformationof nontumorigenic to tumorigenic cell lines (14-16) and thatthe expression of HBxAg is associated with the appearance ofliver tumors in transgenic mice from one laboratory (33) suggestthat HBxAg may participate in one or more critical stepsimportant to the establishment of liver cancer.

The finding of HBxAg in bile duct cells from HBV carriers(Fig. 2) suggests that liver cells other than hepatocytes may beinfected with HBV. Infection of bile duct cells may be importantbecause HBxAg staining was also observed in several bile ductcell-derived tumors, namely, HChC and ChC (Fig. 2). These

results raise the possibility that HBV may be the etiologicalagent responsible for these other liver neoplasms in chronicHBV carriers. Further support for this possibility derives fromthe recent finding that the related duck hepatitis B virus wasdetected by immunochemical staining in bile duct epithelialcells and that bile duct proliferation is a feature of duck hepatitisB virus infection (34, 35). These observations are consistentwith the conclusion that hepadnaviruses may infect bile ductepithelial cells and that such infections could result in proliferation which may be an important step in the pathogenesis ofcholangiocarcinoma.

Although the mechanism by which HBxAg contributes to thepathogenesis of liver cancer remains to be determined, thewidely documented irons-activating activities of HBxAg (36,37) may play an important role. As noted previously (17) andhere (Tables 3 and 4), the relatively high frequency of HBxAgstaining in the nuclei of patients with dysplasia compared topatients with chronic hepatitis and PHC suggests that if alterations in host gene expression take place, these changes aremost likely to occur in cirrhotic patients (with dysplasia) justprior to the appearance of liver cancer. If so, then such /rani-activation may result in triggering the expression of cellulargenes critical for the onset of liver cancer. Alternatively, or inaddition, the putative protein kinase activity associated withHBxAg (38) may be important in altering the function of cellcycle-dependent or other growth-regulatory molecules impor

tant to hepatocellular homeostasis. These outcomes could occurindependent of the continued expression of HBxAg in tumorcells once the appropriate alterations in the behavior of cellshave been achieved. Hence, HBxAg may be more important tothe onset rather than the persistence of liver cancer.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Zachary Goodman, Hepatic Pathology Branch, Armed Forces Institute of Pathology, for confirming thehistological diagnosis of cholangiocarcinoma and hepatocholangiocar-

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cinoma in selected PHC patients. The authors would also like to thankthe experimental pathology laboratory at the Fox Chase Cancer Center,under the direction of Dr. AndrÃ©sKlein-Szanto, for preparation of theunstained slides used for the immunohistochemical staining in thesestudies.

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2. London, W. T. Primary hepalocellular carcinoma: etiology, pathogenesis andprevention. Hum. Pathol., 12: 1085-1087, 1981.

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1991;51:4971-4977. Cancer Res Wenliang Wang, W. Thomas London and Mark A. Feitelson Liver CancerHepatitis B x Antigen in Hepatitis B Virus Carrier Patients with

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