Kay Savage

HCV-associated hepatocellular carcinoma without cirrhosis

Abstract Background/Aims: Hepatocellular carcinoma is an aggressive malignancy and carriers a poor prognosis. Hepatitis B and C virus infection, cirrhosis and aflatoxin B 1 exposure are considered major risk factors. The role of hepatitis C virus in the causation of hepatocellular carcinoma has been debated. It is a positive, single-stranded RNA virus without a DNA intermediate in its replicative cycle, so that integration of hepatitis C virus nucleic acid sequences into the host genome seems unlikely. The most plausible explanation of hepatitis C virus-associated hepatocellular carcinoma so far is that the virus causes necroinflammatory hepatic disease with vigorous regeneration, fibrosis, and eventually cirrhosis. The aim of this study was to examine the relationship of hepatitis C, cirrhosis and hepatocellular carcinoma. Methods: Sixty-six consecutive patients with hepatocellular carcinoma undergoing resection or transplantation at the Royal Free Hospital were reviewed. A combination of serological data and polymerase chain reaction assay was used to assign hepatitis C virus and hepatitis B virus infection. Results: We found four HCV-RNA positive patietns with hepatocellular carcinoma without cirrhosis. All four cases were positive for HCV-RNA and negative for all markers of hepatitis B virus infection. Conclusions: These four cases show that hepatocellular carcinoma may develop in patients with hepatitis C virus without pre-existing cirrhosis. However, the precise role of hepatitis C virus in hepatocarcinogenesis, the carcinogenic potential of the different genotypes and whether this role is influenced by other risk factors still have to be clarified.

Expression and processing of gastrin in hepatocellular carcinoma, fibrolamellar carcinoma and cholangiocarcinoma.

BACKGROUND/AIMS Gastrin is a trophic factor within the normal gastrointestinal tract and is also a mitogen for a number of gastrointestinal and non-gastrointestinal tumours. Precursor forms of gastrin including progastrin (proG) and glycine-extended gastrin (G-gly) as well as the fully processed amidated gastrin (G-NH2) are expressed by tumours. There has been little study of the role of gastrin in either normal liver or liver tumours. The aim of this study was to identify the expression of CCK-B/gastrin receptor (CCK-BR), proG, G-gly and G-NH2 in normal liver and liver tumours. METHODS Tissue sections from patients with hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma as well as normal liver biopsies were assessed for expression of CCK-BR and gastrin isoforms. RESULTS Most liver tumours express CCK-BR and are able to process gastrin as far as proG and G-gly, although not as far as the amidated form. There appears to be little expression of the receptor and no expression of precursor forms of gastrin in normal liver. CONCLUSIONS Liver tumours express the CCK-BR and precursor forms of gastrin. This expression may be associated with tumour proliferation.

Orthotopic liver transplantation for chronic hepatitis in two patients with common variable immunodeficiency

Two patients with common variable immunodeficiency underwent orthotopic liver transplantation for chronic hepatitis, unequivocally caused by hepatitis C virus in one case. Although one patient had pneumonia 8 days after surgery and the other developed hepatic venular stenosis in the transplanted liver, both had a reasonably good quality of life for at least 15 months. However, both subsequently died of recurrent hepatitis C virus hepatitis or hemorrhage after splenectomy for hypersplenism. This shows that severe infection is not a major problem in patients with common variable immunodeficiency after liver transplantation provided they undergo prophylactic antimicrobial and immunoglobulin therapy. The longer term prognosis must be regarded as poor until more data are available following transplantation in similar patients.

Effect of Gastrin and Anti-Gastrin Antibodies on Proliferation of Hepatocyte Cell Lines

Gastrin (G-17) and its precursor glycine-extended gastrin (G-17-gly) have been shown to be trophic to some gastrointestinal tumors. This in vitro study assessed the effect of G-17, G-17-gly, anti-gastrin antibodies (anti-G-17), and the CCK-B receptor antagonist PD135,158 on three hepatoma cell lines (PLC/PRF/5, HepG2 and MCA-RH7777) and an embryonic liver cell line (WRL68). The pancreatic adenocarcinoma cell line AR42J was used as a positive control. G-17 and G-17-gly caused significant proliferation of AR42J and WRL68 cell lines. G-17-gly but not G-17 induced significant proliferation of the PLC/PRF/5 cell line. Anti-G-17 and PD135,158 significantly inhibited unstimulated AR42J and WRL68 cell lines. Anti-G-17 also inhibited the proliferative effects of G-17 and G-17-gly on AR42J, WRL68, and PLC/PRF/5 cell lines, whereas PD135,158 inhibited the proliferative effect of G-17 only. G-17 and G-17-gly as well as anti-G-17 and PD135,158 had no effect on HepG2 and MCA-RH77777 cell lines. It is concluded that G-17-stimulated proliferation is mediated via the CCK-B receptor and G-17-gly via a separate, as yet uncharacterized, receptor. There may therefore be a role for gastrin in embryonic hepatocellular proliferation and perhaps also in the proliferation of some hepatocellular tumors.

Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

Endocytosis of Anti-CCK-B/Gastrin Receptor Antibody and Effect on Hepatoma Cell Lines

Immunotherapy has considerable potential in the treatment of cancer. Here we report on the uptake of an antibody raised against the CCK-B/Gastrin receptor (CCK-BR) by liver embryonic and liver tumor cell lines. In all five cell lines studied, expression of CCK-BR and uptake of labeled anti-CCK-BR antibody was observed. The labeled anti-CCK-BR antibody was localized in both the cytoplasm and nucleus of cells. In addition, we found a coincidence between the uptake of the labeled antibody by cells and the occurrence of apoptosis (cell death). The results suggest that antibodies directed against CCK-BR have potential for targeting and possibly destroying tumor cells bearing the receptor.

Detection of HCV-RNA in paraffin-embedded liver biopsies from patients with autoimmune hepatitis

BACKGROUND/AIMS There may be a relationship between autoimmune hepatitis and viral infection. To examine this relationship, 19 patients with autoimmune hepatitis and/or chronic hepatitis C were studied. METHODS Patients were selected initially on the basis of having autoantibodies (anti-nuclear, anti-smooth muscle, or anti-liver-kidney microsomal) in serum. Formalin-fixed, paraffin-embedded liver biopsies from these patients were tested for HCV-RNA by polymerase chain reaction. The biopsies were examined histologically to detect features suggestive of chronic hepatitis C or autoimmune hepatitis. The results were correlated with serum anti-HCV and HCV-RNA, and with response to steroid therapy. RESULTS Five of the nineteen patients had detectable HCV-RNA in their liver biopsies. In two of three patients from whom serum was available, HCV-RNA was detectable. The remaining 14 patients were negative for HCV-RNA by tissue polymerase chain reaction. Serum was available from 11 of these patients, and serum HCV-RNA was negative in all. All of the three HCV-RNA-positive patients who were treated with steroids showed a partial response; tissue positivity for HCV-RNA was significantly higher in partial responders than in complete responders (60% vs 0%, p = 0.01). Severe portal and periportal inflammation with prominent plasma cells together with bridging parenchymal necrosis were seen more often in HCV-negative biopsies. Mild portal and periportal inflammation with portal lymphoid aggregates, apoptosis and spotty parenchymal necrosis were seen more in HCV-positive biopsies. CONCLUSIONS These results show that hepatitis C virus can be detected in some patients with circulating autoantibodies. The ability to detect HCV-RNA in paraffin-embedded archival material provides a valuable addition to the battery of available HCV tests.

Expression of CCK-B/gastrin receptor and endocytosis of anti-CCK-B/gastrin receptor antibody

Introduction: We have previously reported endocytosis of anti-CCK-BI gastrin receptor antibody (GR#l) by tumour cell lines. Aim: To demonstrate expression of CCK-B/gastrin receptor using RT-PCR, and confirm that uptake of anti-CCK-B/gastrin receptor antibody is dependent on expression of CCK-B/gastrin receptor. Materials and methods: GR#l is an antibody to the amino terminal end of the human CCK-BR. It was labelled with either Alexa-546 (red) or Alexa-488 (green) fluorescent marker. GR#l was added to live cells from the AR42J pancreatic adenocarcinoma cell line, as well as to fibroblast NIH3T3 cells transfected with human CCKB/gastrin receptor gene. GR#l was also added to non-transfected NIH3T3 cells as a negative control. Expression of CCK-B/gastrin receptor was demonstrated by RT-PCR. Total RNA was isolated from I . 3 X 106 cells using the Promega SV isolation system, according to the manufacturers instructions. RT-PCR was performed using l-2!Lg total RNA and the Access RT-PCR system (Promega). Specific primers were used for transcription and amplification of gastrin receptor and f3 actin as a positive control; a double round of PCR was used. Results were analysed using agarose gel electrophoresis. Results. Uptake of anti gastrin receptor antibody was seen in AR42J cells and in NIH3T3 cells transfected with gastrin receptor. No uptake was seen in non-transfected NIH3T3 cells. Using RT-PCR, mRNA for f3 actin was detected in all cell lines: mRNA for gastrin receptor was detected in AR42J cells and in transfected NIH3T3 cells, but not in non-transfected NIH3T3 cells. Conclusions. Anti gastrin receptor antibody was only taken up by cells expressing CCK-BR. This specific uptake confirms that this antibody may be a potential targeting agent in tumours expressing CCK-BR.

HCV-associated hepatocellular carcinoma -without cirrhosis

BACKGROUND/AIMS Hepatocellular carcinoma is an aggressive malignancy and carries a poor prognosis. Hepatitis B and C virus infection, cirrhosis and aflatoxin B1 exposure are considered major risk factors. The role of hepatitis C virus in the causation of hepatocellular carcinoma has been debated. It is a positive, single-stranded RNA virus without a DNA intermediate in its replicative cycle, so that integration of hepatitis C virus nucleic acid sequences into the host genome seems unlikely. The most plausible explanation of hepatitis C virus-associated hepatocellular carcinoma so far is that the virus causes necroinflammatory hepatic disease with vigorous regeneration, fibrosis, and eventually cirrhosis. The aim of this study was to examine the relationship of hepatitis C, cirrhosis and hepatocellular carcinoma. METHODS Sixty-six consecutive patients with hepatocellular carcinoma undergoing resection or transplantation at the Royal Free Hospital were reviewed. A combination of serological data and polymerase chain reaction assay was used to assign hepatitis C virus and hepatitis B virus infection. RESULTS We found four HCV-RNA positive patients with hepatocellular carcinoma without cirrhosis. All four cases were positive for HCV-RNA and negative for all markers of hepatitis B virus infection. CONCLUSIONS These four cases show that hepatocellular carcinoma may develop in patients with hepatitis C virus without pre-existing cirrhosis. However, the precise role of hepatitis C virus in hepatocarcinogenesis, the carcinogenic potential of the different genotypes and whether this role is influenced by other risk factors still have to be clarified.