Clonal state of human hepatocellular carcinoma and non-tumorous hepatocytes

We determined the clonal state in specimens of hepatocellular carcinoma (HCC) and non-tumorous hepatocytes from the integration mode of hepatitis B virus (HBV) DNA. The integration mode of HBV DNA in several parts of the tumors and non-tumorous regions of the same liver, as well as in metastatic tumors, was examined using the Southern blot analysis. In 13 of the 14 cases of HCC, the liver tumors, including metastatic tumors in lymph nodes and the lungs, were monoclonal. In one case, a different HCC clone was found in one part of the liver tumor. The integration of HBV DNA was also observed in non-tumorous tissues in 38 of the 78 cases (49%) of chronic hepatitis with and without HCC; in 16 cases of chronic hepatitis in which HBV DNA was integrated, several clones of the hepatocytes that had HBV DNA integrated into their chromosomal DNA and had proliferated clonally were found in non-tumorous tissues. These clones were different from the tumor clone of the same liver. Thus HCCs were usually monoclonal. The development of different tumor clones appeared to be unusual, but the nontumorous hepatocytes could have proliferated clonally from different multicentric clones before carcinogenesis. The clonal growth of the non-tumorous hepatocytes suggests that the integration of HBV DNA plays an important role in hepatocarcinogenesis.     

Influence of aflatoxin B1 intoxication on duck livers with duck hepatitis B virus infection

In an attempt to determine the effect of aflatoxin B1 (AFB) intoxication on livers with duck hepatitis B virus (DHBV) infection, domestic ducks were given 0.1 mg of AFB/kg body weight twice a week for a maximum period of 54 weeks employing various experimental designs. The ducks were infected with DHBV by i.v. inoculation of DHBV-positive sera within 24 h posthatch. The livers were examined histologically, immunohistochemically, and ultrastructurally, and the livers and sera were examined by molecular hybridization for DHBV DNA. AFB administration induced hepatocellular necrosis and marked biliary cell proliferation of the periportal areas, and finally liver cirrhosis. On short-term administration, the hepatocytes of DHBV-infected livers revealed a marked increase in incomplete particles of DHBV by immunostaining and electron microscopy, as compared to those without its administration. Long-term AFB administration provoked frequent nodular or cirrhotic changes. There was no significant increase in frequency of these changes in DHBV-positive ducks as compared to DHBV-negative ones. AFB administration induced hepatocellular carcinoma (HCC) in one DHBV-positive duck and in two DHBV-negative ducks. The HCC and cirrhotic livers revealed extrachromosomal but no integrated form of DHBV DNA by Southern blot hybridization analysis. Immunostaining demonstrated a heterogeneous distribution of DHBV from area to area in nodular and cirrhotic livers. Thus, AFB intoxication provoked various liver disorders independent of DHBV infection, and neither a cocarcinogenic effect of AFB and DHBV nor integration of viral DNA into the genome of neoplastic and nonneoplastic tissues was observed in the present experiments. Generally speaking, DHBV infection did not appear to accelerate hepatic disorders induced by AFB intoxication. However, AFB administration altered the DHBV in the liver in terms of its amount and distribution.     

Hit-and-run mechanism of HBV-mediated progression to hepatocellular carcinoma

AIM AND BACKGROUND: Hepatitis B virus is implicated in the development of hepatocellular caracinoma. No oncogenes have been identified within the viral genome. Furthermore, it frequently fragments after integration into the hepatocyte genome. Simultaneous investigations of hepatitis B virus integration patterns and genetic changes in precancerous tissues are important to understand the role played by hepatitis B virus integration in hepatocellular caracinoma. METHOD: We used a combination approach of dual characterization of highly polymorphic loci and the change in hepatitis B virus-DNA integration pattern. Large regenerative nodules were dissected from 6 explanted hepatitis B virus infected cirrhotic livers. Nodules within each liver segment were schematically mapped and histopathologically analyzed. Genomic DNA from each nodule was analyzed for hepatitis B virus integration and the genetic stability of 12 microsatellite loci including D3S2321, D8S1022, D17S1159, D4S2281, D5S1/2, D16S675, D16S685, D16S490, D16S526, D16S673, D16S677 and D16S690. RESULTS: Data from different liver segments revealed few viral integrations and average allele loss. The most exciting results came from a segment containing a set of clonally and spatially related nodules having similar histologic features, a progressive lineage of allele loss, HBV integration and loss of integration. CONCLUSIONS: This model portrait, a scenario of genetic events that precede tumor formation where the acquisition and loss of hepatitis B virus integrations in clonally related regenerative nodules, might explain how the virus acts as a hit-and-run mutagen.     

Integration of hepatitis B virus DNA in hepatocellular carcinoma

Integration of hepatitis B virus (HBV) DNA into genomic DNA was investigated in 34 livers bearing hepatocellular carcinoma (HCC) by Southern blot hybridization using 32P-labeled, cloned and purified HBV DNA as a probe. Rehybridization of nitrocellulose paper with a probe containing only the cloning vector was performed after dehybridization to avoid possible false-positive results. Integrated HBV DNA was detected in all 9 hepatitis B surface antigen (HBsAg)-seropositive cases and 3 out of 25 (12%) HBsAg seronegative cases. The hybridization patterns of viral DNA were the same among several cancer nodules in two HCC cases with multiple liver tumors, indicating unicentric hepatocarcinogenesis in these two cases. These results, obtained with avoidance of false-positive results, showed that only a minority of HBsAg-seronegative HCC cases in Japan had demonstrable HBV DNA in the tumors studied by the Southern blot hybridization technique.     

DUCK HEPATITIS B VIRUS DNA WITHIN HEPATIC MULTICENTRIC CANCER AND/OR METASTATIC CANCER

Duck hepatitis B vims (DHBV) DNA was detected in different tumorous nodules of ducks with hepatic multicentric cancer or intrahepatic metastasis by Southern blot technique. Among 7 ducks with hepatocellular carcinoma of multiple tumor nodules, the hybridization pattern of Integrated DHBV DNA In different tumorous nodules was identical in 3 cases and different in 2 cases. One case showed a similar hybridization pattern in two tumorous nodules and other one was negative tor DHBV DNA. Integrated DHBV DNA was also identified in a metastatic lung cancer of ducks with hepatocellular carcinoma. The hybridization pattern of metastasis of lungs was as the some as that in primary hepatocellular carcinoma. The same discrete hybridization bands In the different tumorous nodules indicate that these nodules might arise from one transformed cell. The different hybridization patterns In various tumorous nodules show that these tumorous nodules might arise from various transformed cells. The results suggest that the hyb     

Hepatitis B virus antigens in human primary hepatocellular carcinoma tissues.

The presence of hepatitis B virus (HBV) antigens was examined in specimens of liver tissue obtained at necropsy from black Senegalese patients suffering from primary hepatocellular carcinoma (PHC). The results were correlated with markers of hepatitis B infection in serum. Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) were sought for in 15 liver extracts. HBsAg was found in the liver in 10 of 12 cases with HBsAg-positive serum. HBcAg was detected in three livers. The HBsAg was detected in seven of eight livers by immunofluorescence and orcein staining. HBsAg-positive cells were mainly located in the peri-tumoral cirrhotic tissue, although positive hepatocytes were also found in 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 cytoplasm-nucleus pathway in the synthesis of the HBV 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 in swollen cisternae of the endoplasmic reticulum.     

Is mallory body formation a preneoplastic change? A study of 181 cases of liver bearing hepatocellular carcinoma and 82 cases of cirrhosis

The hypothesis that Mallory body formation by hepatocytes is a sign of preneoplasia was tested. This hypothesis was based on animal experiments but has not been tested in man. The authors studied the livers of 181 human autopsies in which hepatocellular carcinoma (HCC) was present and 82 cirrhotic livers from patients with alcoholism, HB viral infection, or cryptogenic cirrhosis. The frequency of Mallory bodies in nonneoplastic hepatocytes was 40% in the HCC-bearing livers with cirrhosis (LC). In HCC-bearing livers with pre-cirrhotic changes (PC), 25% showed Mallory body formation by nonneoplastic hepatocytes. In the cases of HCC, where there was no accompanying PC or LC, Mallory bodies were never found in the nonneoplastic hepatocytes. When the 82 cirrhotic livers without HCC and the 116 cirrhotic cases with HCC were combined, it was found that HCC was present in 70% of cirrhotic livers when the nonneoplastic liver cells contained Mallory bodies. When no Mallory bodies were found in the nonneoplastic liver cells, HCC was present in 53% of cases. The difference between the two groups was significant (P less than 0.05). The difference was significant for both HB viral hepatitis and cryptogenic cirrhosis but not for alcoholic cirrhosis. Likewise, when nonneoplastic hepatocytes formed Mallory bodies in cirrhotic livers, there was a statistically significant increase in the number of HCC cells that formed Mallory bodies (P less than 0.01). When nonneoplastic hepatocytes occurred in groups of Mallory body forming cells, the hepatocellular features were atypical and characteristic of dysplastic cells. The evidence indicates that when Mallory body formation was observed in HBsAg-positive and cryptogenic cirrhotic livers, they were associated with an increased frequency of HCC formation in man.     

Epidemiology, natural history and pathogenesis of hepatocellular carcinoma]

Hepatocellular carcinoma is one of the most prevalent tumors worldwide and its incidence is increasing due to hepatitis C virus infection. Other etiologic factors are hepatitis B virus infection, alcoholic liver disease and hemochromatosis. This tumor mainly develops in cirrhotic livers that are true precancerous states. Although mechanisms of hepatocarcinogenesis remain badly known, some signaling pathways are frequently deregulated: inactivation of the p53 tumor suppressor factor in 25% of HCC, activation of the Wnt signaling and the telomerase immortalization enzyme in most of tumors. Hepatitis viruses play a direct oncogenic role by interaction between viral proteins and cellular ones, which control cell homeostasis, or by integration of hepatitis B virus genome into the host genome. Furthermore, hepatitis viruses play an indirect oncogenic role by chronic inflammation and hepatocyte regeneration related to viral hepatopathy. In a near future, a better understanding of virus-specific oncogenic mechanisms should allow us to set up innovative preventive and curative therapeutic strategies.     

Liver iron stores and hepatitis B antigen status

Higher serum iron and ferritin levels noted in hepatitis B antigen (HBAg) carriers than in noncarriers suggests that virus might actively replicate in hepatocytes, stimulate ferritin synthesis, and result in increased liver iron stores. A comparative semiquantitative study of immunohistochemical ferritin (0-12) and hemosiderin (0-9) was performed on 54 normal, 13 cirrhotic, and 70 nonneoplastic livers from patients with hepatocellular carcinoma, in each group, comparing amounts in HBAg-positive and HBAg-negative patients. Mean scores for ferritin and hemosiderin were high in all three groups, normal livers averaging 8.3 and 6, respectively, cirrhotic livers, 8.5 and 7.4, respectively, and carcinoma livers, 5.6 and 6.1, respectively. In each group, there was no significant difference in ferritin and hemosiderin mean scores in HBAg-positive and HBAg-negative patients. The large liver iron stores do not appear to be a consequence of hepatitis B virus infection alone. Their role in the development of hepatocellular carcinoma is still to be elucidated.     

Dominance of functional androgen receptor allele with longer CAG repeat in hepatitis B virus-related female hepatocarcinogenesis

The CAG polymorphism in exon 1 of the androgen receptor (AR) gene has been shown associated with the development of human male hepatocellular carcinoma (HCC) with the shorter AR alleles conferring a higher risk. However, the significance of AR-CAG repeats in female hepatocarcinogenesis remains to be addressed. In this study, seventy-six pairs of female HCCs and corresponding nontumorous tissues were collected, and 180 cirrhotic nodules were microdissected from 7 cirrhotic livers. The clonality status, functional AR alleles, and CAG repeat number of each sample were determined by AR methylation analysis. In a total of 44 monoclonal HCCs, the mean of CAG repeats in the active alleles was significantly longer than that in the inactive alleles (22.0 +/- 2.8 versus 20.7 +/- 3.6; P = 0.047). When we divided HCCs into hepatitis B virus-positive [HBV(+)] and HBV(-) subgroups, the long AR allele dominance was found only in HBV(+) ones (P = 0.006 versus P = 0.923). Notably, the preference of long CAG repeat has also been found in the 100 monoclonal nodules (P = 0.013). For comparison of monoclonal nodules obtained from the same individual, a dominant long AR allele was found in 6 patients. The proportion of monoclonal cirrhotic nodules and HCCs expressing longer AR allele, 69 and 68%, are both significantly higher than 50%, the assumed value in normal liver (P < 0.001 for cirrhotic nodules and P = 0.005 for HCC). The dominance is again only prominent in HBV-infected HCCs [85% for HBV(+) HCC; P < 0.001 but 54% for HBV(-) HCC; P = 0.27]. The results indicated that in female hepatocarcinogenesis, hepatocytes expressing the longer AR allele seem to be favorably selected for autonomous growth and transformation, especially in synergy with HBV infection.     

Identify metastasis-associated genes in hepatocellular carcinoma through clonality delineation for multinodular tumor

Disease recurrence and metastasis are frequently observed in many successfullytreated localized cancers, including hepatocellular carcinoma in which intrahepatic and extrahepatic recurrence (metastasis) are frequently observed after curative resection. The present study aimed at identifying metastasis-associated genes through delineation of the clonality for multinodular liver cancer. The clonal relationship of 22 tumor foci from six patients was investigated by the genome-wide expression profile via cDNA microarray consisting of 23,000 genes. Tumor molecular properties including p53 protein overexpression and gene mutation, hepatitis B virus integration pattern, and genetic alteration examined by comparative genomic hybridization were compared. Results indicated that gene expression patterns could serve as the molecular fingerprint for clonality identification. Together with the molecular data from p53, hepatitis B virus integration and comparative genomic hybridization profiles, tumor nodules from five patients were confirmed with clonal relationship, and the expression profiles of the primary nodules were compared with their corresponding intrahepatic metastatic nodules. A total of 90 clones were found to be correlated with intrahepatic metastasis by Student's t test (P < 0.05). With reference to the primary tumor, 63 clones (39 known genes and 24 express sequence tags) were down-regulated whereas 27 clones (14 known genes and 13 express sequence tags) were up-regulated in the metastatic nodules. These metastasis-associated genes may provide clues to reveal patients with increased risk of developing metastasis, and to identify novel therapeutic targets for the treatment of metastasis.     

Detection of hepatitis B virus DNA sequences in bone marrow of children with leukemia

To investigate the possibility that hemopoietic cells may become infected with hepatitis B virus (HBV), viral DNA was studied by molecular hybridization in bone marrow aspirates of 51 children with leukemia. HBV-DNA was found in the bone marrow of eight children (15%) and Southern blot analysis revealed the presence of free, monomeric viral sequences. Only one of the eight children with HBV-DNA in bone marrow cells was HBsAg-positive in serum, whereas two additional patients were transiently HBsAg-positive in serum during follow-up, but were negative at the time HBV-DNA was found in bone marrow. Four other cases developed antibodies to HBV. Cases of myeloid leukemia were more frequently positive for HBV-DNA in bone marrow (55%), compared with cases of lymphoid leukemia (7%). These results indicate that hemopoietic cells are susceptible to infection with hepatitis B virus and stimulate new interest into the relation of HBV infection to the development of some forms of leukemia, as four of eight cases of myeloid leukemia were HBV-DNA positive in bone marrow aspirates at diagnosis, prior to receiving any transfusion therapy.     

Hepatic neoplasms in aflatoxin B1-treated, congenital duck hepatitis B virus-infected, and virus-free pekin ducks

To assess the effects of the combination of persistent hepadnavirus infection and chemical carcinogen exposure, aflatoxin B1 (AFB) was administered p.o. for 60 days to congenitally duck hepatitis B virus (DHBV)-infected and virus-free Pekin ducks, starting at 3 days of age, during a 28-month study. Hepatic neoplasia occurred only in AFB-dosed ducks. Hepatocellular carcinomas or biliary carcinomas occurred in 4 of 8 DHBV-infected and 3 of 4 DHBV-free ducks, and hepatocellular adenomas developed in 2 DHBV-infected AFB-dosed ducks that survived 20 months or longer. Altered foci of hepatocytes similar to those observed in chemical carcinogen-dosed rodents, characterized by enlarged eosinophilic hepatocytes or vacuolated cytoplasm, occurred in AFB-dosed ducks. Cells in foci or hepatic neoplasms did not contain histochemically detectable gamma-glutamyltranspeptidase but were distinguished from uninvolved parenchyma by altered glycogen content. Immunohistochemical staining indicated that DHBV core antigen persisted in liver, spleen, pancreas, and, to a lesser extent, kidney of most congenitally infected ducks up to 28 months of age. Hepatic neoplasms contained only patches of hepatocytes were detectable viral antigen. Southern blot analysis of restriction endonuclease-digested neoplastic and normal liver DNA revealed high molecular weight forms of DHBV DNA consistent with integration of viral DNA into the genome of hepatic neoplasms from 3 of 4 DHBV-infected ducks but not nontumorous liver. These findings indicate that AFB is a potent hepatic carcinogen in ducks and that persistent congenital DHBV infection did not contribute significantly to the emergence of hepatic neoplasia in ducks under these conditions.     

Hepatitis B virus-associated hepatocellular carcinoma in African patients

We have examined several tumors from South African patients with hepatitis B virus (HBV)-associated hepatocellular carcinoma for the presence of integrated viral DNA. In contrast with our findings in patients from Taiwan, few copies of the viral genome were integrated in each tumor. Furthermore, Southern hybridization showed similarities in integration patterns between different tumors. We are presently constructing genomic libraries from selected single-copy tumors in order to make a more detailed analysis of HBV DNA integration at the molecular level.     

Mono/oligoclonal pattern of Kaposi Sarcoma-associated herpesvirus (KSHV/HHV-8) episomes in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare lymphoma of B-cell origin, developed in serous cavities. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases co-infected with Epstein-Barr virus (EBV). In 15 primary PEL tumors including 10 EBV-positive cases, we analyzed the fused terminal repeat (TR) regions of KSHV episomes using pulsed-field gel electrophoresis and Southern blot. On the same genomic DNA samples, the cellular clonality was assessed by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IGH) VDJ gene rearrangements, associated in the EBV-infected cases, with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IGH gene rearrangements were detected in 13 tumors using Southern blot, in 11 cases using PCR, and in all cases considering both methods. EBV infection was monoclonal in all EBV-positive cases. However, only 5 PEL tumors were found to be monoclonally infected with KSHV. In the 10 other cases, we found a biclonal (2 bands; n = 4) or an oligoclonal pattern (3-6 bands; n = 6) of KSHV episomes. We hypothesized that the apparent discrepancy between viral and cellular clonalities in PEL might be due to several phenomena including complex mechanisms of genomic recircularization, insertion of duplicated sequences into the TR region and simultaneous infection of tumor cells with defective KSHV variants. KSHV infection of contaminating nontumoral cells, superinfection from lytically infected cells or viral integration events might also explain the oligoclonal pattern of KSHV infection. Several of these mechanisms, not mutually exclusive, might coexist in a single tumor.     

Integrated state of subgenomic fragments of hepatitis B virus DNA in hepatocellular carcinoma from mainland China

Hepatocellular carcinoma (HCC) samples from mainland China were examined for the presence and state of hepatitis B virus (HBV) DNA sequences. HBV DNA was detected by dot-blot hybridization in 13 of 17 cases of HCC from the Shanghai area and in three of six samples from Hangzhou. The HCC cases from Shanghai were then analyzed in more detail. Fifteen of the 17 patients had serologic evidence of past or present infection with HBV (with inadequate information available for the other two), and the 13 HCC samples positive for HBV DNA all came from serologically positive patients. Southern blot analysis showed that the HBV DNA sequences were always integrated in the HCC high-molecular-weight DNA; only one or two viral copies were present per tumor cell, and no common integration site was evident. Hybridization analyses using subgenomic probes of HBV DNA revealed that the tumors seldom retained an entire HBV genome. HBV S-region sequences were always present, X-region sequences were usually represented, and C-region sequences were rarely detectable in virus-positive tumors. A fragment within the HBV DNA X-region, between nucleotides 1441 and 1526, was found to hybridize nonspecifically with cellular DNA; reported sequence data indicated that this fragment would contain approximately 70% guanine + cytosine. Histologic sections were prepared from some of the frozen tissue specimens and stained by an indirect immunoperoxidase technique for hepatitis B surface antigen (HBsAg). Only 1 of 10 HBV DNA-positive samples contained HBsAg in the cytoplasm of tumor cells, although abundant HBsAg was present in adjacent normal cells in all 10 cases. There were no significant differences in histology between HCC that contained HBV DNA sequences and those that were virus negative. These data support the premise that HBV represents a major etiologic factor in the development of HCC in the Shanghai area of China, although the molecular basis of viral involvement remains obscure.     

Cirrhotic livers reveal genetic changes in the MDM2-P14ARF system of cell cycle regulators

The genesis of hepatocellular carcinoma is promoted by changes in the regulatory MDM2-P14ARF system. The incidence of such changes has to date not been analysed in non-tumourous livers showing regenerative proliferation. In the present study, 24 cirrhotic livers of alcohol-, autoimmue disorder- or HCV-caused genesis were screened for MDM2-P14ARF alterations at the level of protein, DNA and mRNA. Using confocal laser scanning microscopy, the absence of MDM2 and P14ARF expression was detected in all samples except three HCV-infected livers (four livers) which contained hepatocytes overexpressing MDM2 (P14ARF) protein. In two of the samples lacking P14ARF expression, laser microdissection and PCR demonstrated deletion of the P14ARF gene. The P14ARF gene amplified from other specimens did not carry mutations. MDM2 splicing variants were present in tissues from alcohol- and autoimmune disorder-induced cirrhoses. Sequencing of full-size mRNA revealed a MDM2 mis-sense mutation in an alcohol-induced cirrhosis. One sample contained regenerative nodules with genetic instability occurring at MDM2 locus D12S83 according to the data of automatic PCR fragment analysis. In summary, this study gives first evidence for different types of MDM2 and P14ARF alterations in cirrhotic livers. We suggest that the changes impair the regulatory MDM2-P14ARF system, thus possibly favouring regenerative proliferation and transformation.