HEPATITIS VIRUSES AND LIVER CANCER

WILLIAM S. MASON, Ph.D., Senior Member; Research Associate Professor of Microbiology, University of Pennsylvania

Hepatitis B virus (HBV)chronically infects hepatocytes, the major cell type of the liver. This infection does not, by itself, appear to be cytopathic, and most individuals infected as adults clear the infection after a few months. However, about 10% of infected adults, and most people infected as children, will fail to clear the virus and will remain virus carriers throughout their lifetime. These individuals will experience chronic liver disease due to the immune response to viral antigens expressed on infected cells; as a consequence of the chronic liver damage, about 10% will eventually develop liver cancer, a generally fatal disease. The goals of the research in this laboratory are: to understand why some individuals fail to clear an infection, to devise protocols that will induce virus clearance, to determine whether the termination of a chronic infection reduces the risk of liver cancer, and to identify the sequence of changes in the liver that lead to hepatocellular carcinoma (HCC). Experiments towards these goals are being carried out in cell culture systems, as well as in animals chronically infected with duck HBV (DHBV) or woodchuck hepatitis virus (WHV).

Lamivudine ((-)-b-L-2'3'-dideoxy-3'-thiacytidine) is a nucleoside analog that reduces HBV titers in the sera of chronically infected patients by suppressing viral replication in the liver. Treatment is often associated with abatement in the severity of liver disease and, in some patients, with a complete loss of the virus. Unfortunately, in other patients treated for a year or more, the drug is no longer able to suppress virus replication due to the emergence of HBV variants that have a mutated DNA polymerase. In order to study how mutant virus emerges during lamivudine therapy and to learn more about the consequences of this therapy upon infection in the liver, we have characterized lamivudine treatment in woodchucks chronically infected with WHV. To this end, we have assessed the DNA polymerase genotype of virus in serum and liver throughout the course of lamivudine therapy beginning before treatment, extending through the period of virus suppression (usually about 9 to 12 months), and ending when virus titers had again risen to high levels.

Initial studies revealed that, as with HBV, a rise in virus titers during therapy was associated with the presence of viruses with mutations in the active site of the polymerase. Moreover, cell culture studies confirmed that these mutants were resistant to lamivudine. Our results also revealed, surprisingly, that emergence of mutant WHV was not always associated with an immediate rise in virus titers in the serum. In particular, one of the three types of mutant viruses that we identified became predominant in serum, though still at low titer, up to seven months before serum titers began to increase, a time when wild type virus was still predominant in the liver. A simple explanation for this result is that the increase in virus titers following emergence of drug resistant mutants can only occur as the pre-existing wild type virus is cleared from the hepatocyte population. Thus, sustained suppression of virus titers in the serum may be a measure of the stability of the infected state, rather than of a successful therapeutic outcome.

HBVs are strongly species-specific and a full replication cycle is only supported by primary hepatocyte cultures from the same species. Thus, most of our knowledge of the replication of HBV and WHV is derived from studies with the distantly related DHBV. In order to test the concepts developed with DHBV, we have developed procedures for culturing primary woodchuck hepatocytes that support a full cycle of WHV replication. Unfortunately, these cultures are not, by themselves, suitable for applying genetic approaches to studies of virus replication, as they are not readily transfected with viral DNAs. Moreover, the available liver cell lines do not support WHV production from transfected DNA. Delaney and Isom (Hepatology 28:1134, 1998) recently reported that baculoviruses could be used as vectors to deliver HBV DNA to human liver cell lines. We have evaluated this approach to deliver WHV DNA to primary woodchuck hepatocyte cultures. Recombinant baculoviruses were constructed, for expression of wild type WHV, WHV defective for surface antigen synthesis, and green fluorescent protein (GFP). All three recombinants were able to deliver target genes to woodchuck hepatocytes, as shown by subsequent replication of WHV DNA or expression of GFP. Thus, this approach is suitable for a genetic analysis of WHV reproduction, and a determination of the phenotype of envelope and X gene mutants is now being undertaken.

HCCs arise in nearly all woodchucks chronically infected with WHV. One or the other of two protooncogenes, Nmyc1 and Nmyc2, are expressed in most of these tumors, but not in normal liver. Expression of these genes is due to their transcriptional activation by nearby integration of WHV DNA. Several lines of research suggest that this aberrant expression plays, along with chronic liver injury, a key role in tumor outgrowth. However, it is not known if the integration of viral DNA plays a role in the outgrowth of preneoplastic lesions in infected woodchucks, or if it represents a late stage in tumorigenesis. The recent acquisition of a laser dissection microscope by Fox Chase provides the technology to assay for gene rearrangements in these preneoplastic cells. As a prelude to such an analysis, we have explored the use of PCR to detect viral DNA integrated near Nmyc1 or Nmyc2 in a series of uncharacterized woodchuck liver cancers harvested over the last several years. WHV DNA integration in the 3' untranslated region of Nmyc2 was detected in six of fourteen tumor samples. Using PCR, we were unable to detect a site of integration in the vicinity of either Nmyc1 or Nmyc2 in the remaining eight tumors. We are currently seeking to identify the integration sites in the remaining tumors, to determine why the PCR approach did not work for these, and if modifications to this approach could enhance the detection incidence. In any case, the current approach appears sufficient to initiate a search for WHV integrations near Nmyc2 in preneoplastic foci.

Chronic HBV infection in Senegal is seldom associated with a high level viremia past the age of 30. In contrast, about 20% of carriers in Haimin County, People's Republic of China, remain viremic for life. This difference suggests that there might be a stronger average immune response to infections in the viremic carriers in Senegal than in China, perhaps because infection begins at a greater age in Senegal. Deletion of putative T-cell epitopes of the HBV core gene has been reported in many studies. It is thought that the frequency of such deletion mutants of the core gene may be an indicator of the strength of the antiviral CTL response. Experiments were, therefore, initiated using PCR amplification of the viral core gene to determine the numbers of HBV carriers with a high incidence of core gene deletions in Senegal and in China. Among a set of 43 positive sera collected in China, all from patients with viremia in excess of 5x10⁶ per ml, only five contained a predominant PCR product that was derived from virus with a deletion in the core gene. In contrast, for 3 of 12 from Senegal, the predominant PCR product contained a core gene deletion. Moreover, while a unique DNA sequence ladder could be obtained by direct sequencing of PCR products from 88% of the Chinese samples, none (0/12) of the Senegalese samples yielded a distinct sequence ladder, apparently due to size and sequence heterogeneity. The sequence data suggest, again, a high level of core gene sequence heterogeneity in the Senegalese carriers, a conclusion supported by cloning and sequencing of the PCR products. Thus, our data are consistent with the hypothesis that the higher rate of seroconversion to a low viremic state in Senegal is associated with a greater immune response to the HBV core protein.

BENNETT, L.L., ALLAN, P.W., ARNETT, G., SHEALY, Y.F., SHEWACH, D.S., MASON, W.S., FOUREL, I., PARKER, W.B. Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase. Antimicrob. Agents Chemother. 42:1045-1051, 1998.

MASON, W.S., JILBERT, A.R. Hepatitis viruses. In Encyclopedia of Microbiology. Academic Press (in press).

MASON, W.S., ZHOU, T., NUNES, F., CONDREAY, L.D., LITWIN, S., SUMMERS, J. Antiviral therapy for chronic hepadnavirus infections. In Therapies for Viral Hepatitis, edited by R.F. Schinazi, J.-P. Sommadossi, H.C. Thomas. International Medical Press, London, pp. 177-184, 1998.

ZHOU, T., SAPUTELLI, J., ALDRICH, C.E., DESLAURIERS, CONDREAY, L.,MASON, W.S. Emergence of drug resistant populations of WHV in woodchucks treated with the antiviral nucleoside, lamivudine. Antimicrob. Agents Chemother. (in press).

Paper in press at time of previous report:

EVANS, A.A., O'CONNELL, A.P., PUGH, J.C., MASON, W.S., SHEN, F.-M., CHEN, G.-C., LIN, W.-Y., DIA, A., M'BOUP, S., DRAME, B., LONDON, W.T. Early resolution of viremia may be associated with a reduced risk of hepatocellular carcinoma. Cancer Epidemiol. Biomark. Prev. 7:559-565, 1998.

MASON, W.S., EVANS, A.A., LONDON, W.T. Hepatitis B virus replication, liver disease, and hepatocellular carcinoma. In Human Tumor Viruses, edited by D. McCance. ASM Press, Washington, DC., pp. 251-281, 1998.

MASON, W.S., CULLEN, J., MORALEDA, G., SAPUTELLI, J., ALDRICH, C.E., MILLER, D.S., TENNANT, B., FRICK, L., AVERETT, D., CONDREAY, L., JILBERT, A.R. Lamivudine therapy of WHV infected woodchucks. Virology 245:18-32, 1998.

MASON, W., MARION, P. Hepadnaviruses of avian hosts. In Encyclopedia of Virology. Academic Press (in press).

PUGH, J.C., GUO, J.-T., ALDRICH, C., RALL, G., TENNANT, B., ENGLAND, J.M., MASON, W.S. Aberrant expression of a cytokeratin in a subset of hepatocytes during chronic WHV infection. Virology 249: 68-79, 1998.

SEEGER, C., MASON, W.S. Chronic hepadnavirus infections of the woodchuck and duck. In Persistent Viral Infections, edited by R. Ahmed and I. Chen. John Wiley and Sons Ltd. pp 607-621, 1999.

SEEGER, C., MASON, W.S. Replication of the hepatitis B virus genome. In Hepatitis B virus: Molecular Mechanism in Disease and Novel Strategies for Therapy, edited by R. Koshy and W.H. Caselmann. Imperial College Press, London, pp. 1-20, 1998.

^§   Fox Chase researcher

^a   L. Condreay, M. Deslauriers: Glaxo Wellcome Corporation, Research Triangle Park, NC 27709

^b   F. Nunes, J. Wilson, K. Molnar-Kimber: University of Pennsylvania, Philadelphia, PA 19104

^c   Y. Qian: Present address-Yale University, New Haven, CN 06520

^d   F-M. Shen: Shanghai Medical University, Shanghai, PRC

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998