Preliminary experience in radionuclide therapy of hepatocellular carcinoma using hepatic intra-arterial radio-conjugates.

INTRODUCTION: In a minority of the cases, resection of hepatocellular carcinoma (HCC) is potentially curative but local recurrence is common. Adjuvant intra-arterial radio-conjugate therapy could potentially reduce the rate of local recurrence and increase disease-free and overall survival. However, in the majority of cases, treatment of HCC is largely palliative. A wide range of palliative treatment options are available and these include external radiation, hepatic intra-arterial chemo-embolisation, systemic chemotherapy and percutaneous ethanol injection. The long-term survival rate is poor. Hepatic intra-arterial radio-conjugate therapy provides a new and promising means of palliation. MATERIALS AND METHODS: We share our initial experience in the treatment of patients with HCC. A total of 32 patients were recruited between October 1999 and June 2001. Group 1 comprised 15 patients who had potentially curative resection of HCC who were treated with Iodine-131 (I131) lipiodol as a form of adjuvant therapy. Group 2 comprised 17 patients with unresectable HCC, 12 of whom were treated with Yttrium-90 (Y90) microspheres and 5 with Rhenium-188 (Re188) lipiodol. The radio-conjugates were administered via the intra-arterial route. RESULTS: Thirteen of the 15 patients in group 1 who were treated with I131 lipiodol following curative resection of HCC were free of disease, 1 patient died and 1 patient who developed recurrence was retreated with Re188 lipiodol and was subsequently free of disease. The 6-month disease-free survival rate was 100% and the 12-month disease-free and overall survival rates were 72% and 85%, respectively. Of the 12 patients in group 2 who were treated with Y90 microspheres for unresectable HCC, 6 had stable disease, 2 showed tumour regression and 4 died. The 6-month and 12-month survival rates were 75% and 66%, respectively. Of the 5 patients in group 2 who were treated with Re188 lipiodol for unresectable HCC, 4 had stable disease and 1 had regression of the right lobe tumour but progression of the left lobe tumour. CONCLUSION: Our results in the adjuvant treatment of patients with I131 lipiodol following curative resection of early HCC and in the palliative treatment of unresectable HCC using Y90 microspheres and Re188 lipiodol are preliminary and not fully conclusive. These preliminary results have to be confirmed in larger groups of patients and by prospective, randomised, controlled trials. This study highlights the preliminary experience in radionuclide therapy of HCC using hepatic intra-arterial radio-conjugates in a local context.

Hepatitis B virus X protein in the proteasome of mammalian cells: defining the targeting domain.

The hepatitis B virus X (HBX) protein has been implicated in both hepatitis B virus-related pathogenesis and also in diverse cellular processes. The diversity of its activities may be mediated through its interaction with cellular organelles. However no clearly defined subcellular localization of HBX is available. We report here the localization of HBX in the proteasome complexes using green fluorescent protein tag. A new proteasome-targeting domain has also been defined in HBX by deletion study. Furthermore, a functional role of HBX in the cellular processes mediated by the proteasome complexes has been suggested by its cell cycle-independent localization in the proteasome. Further analysis of the functional role of HBX in the proteasome complexes should provide more information on the underlying mechanism of HBX ativities.

Intra-familial evidence of horizontal transmission of hepatitis B virus surface antigen mutant G145R.

OBJECTIVES: To provide intra-familial evidence on the horizontal transmission of hepatitis B virus surface antigen (HBsAg) mutant G145R. METHODS: Serum samples from family members of 10 vaccinated infants who carried this G145R mutant were collected. The presence of the mutant was analysed by polymerase chain reaction (PCR) and sequencing. RESULTS: The G145R mutant was identified in family members of three of the 10 infants. In family 1, the mutant found initially in child 1 was identified in another child and the father. In families 2 and 3, the G145R mutant detected previously in child 1 was detected in the father. Additional mutations in HBsAg were identified in at least two members in family 1 and 2, suggesting horizontal transmission of the mutant among them. The G145R mutant was found in samples with high levels of neutralizing antibody against HBV (anti-HBs). In addition, liver damage was seen in one G145R carrier infant. CONCLUSIONS: The G145R mutant could be transmitted horizontally among family members, and this could occur in the presence of high levels of anti-HBs. Improvement of detection system for the G145R and other HBsAg mutant will be needed for their effective control.

Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance.

Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HBX variants from HBV-related human hepatocellular carcinoma (HCC). These HBX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HBX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HBX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HBX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HBX.

Mutations and deletions in core promoter and precore stop codon in relation to viral replication and liver damage in Singaporean hepatitis B virus carriers.

BACKGROUND: The core promoter of hepatitis B virus (HBV) is crucial for the viral replication and mutations may lead to the establishment of chronic infection and development of liver diseases. We analysed this region in Singaporean HBV carriers and assessed their association with viral replication and liver damage. MATERIALS AND METHODS: Thirty-three Singaporean HBV carriers were selected. Serological markers for HBV infection and indicators for liver functions were analysed using commercial kits. Among these patients, 17 were chronic carriers, 10 had cirrhotic livers and 6 others had hepatocellular carcinoma (HCC). The region on the HBV genome covering the entire core promoter and core gene was amplified for each patient by polymerase chain reaction. The amplified DNA fragments were sequenced and analysed. RESULTS: The incidence of mutations in the core promoter or the precore gene product (stop codon at amino acid 28) was not significantly higher compared with the wild type sequences in patients with liver damage. Most mutations in either the core promoter or precore gene significantly reduced the viral replication, as indicated by HBV DNA levels. High levels of HBV DNA were found in three mutants with deletion in the same region, presumably the binding site of liver enriched factor, within the core promoter. CONCLUSION: Our findings revealed a different mutation pattern in the core promoter in Singaporean HBV carriers. While most mutations may not be directly associated with the development of liver diseases, deletions in the core promoter could contribute to enhanced viral replication and should be studied further.

Detection of hepatitis B virus surface antigen mutants in paraffin-embedded hepatocellular carcinoma tissues.

The antigenic "a" determinant of the human hepatitis B virus surface antigen (HBsAg) is highly conserved and involved in inducing neutralizing antibody. Mutations on this determinant have been associated with the hepatitis B virus (HBV) escape from vaccination but are also found in chronic HBV carriers, but their involvement in liver diseases including hepatocellular carcinoma (HCC) is unclear. To investigate the possible liver disease-associated role of HBsAg mutants, their incidence was analyzed in 11 paraffin embedded HCC tissues using an improved DNA extraction method. Mutations on the "a" determinant (Thr126Ser, Gly145Arg, a double mutant Thr126Ser/Gln129Asn, Met 133Leu and Thr140Ile) were identified in 5 samples while the wild type sequence was found in 2 others. Future characterization of these HCC-associated HBsAg mutants should provide new insights on their role in the pathogenesis of HCC.

Mutation "hot spot" in HLA class I-restricted T cell epitope on hepatitis B surface antigen in chronic carriers and hepatocellular carcinoma.

Mutations in the human hepatitis B virus (HBV) genome contribute to its escape from host immune surveillance and result in persistent infection. We report the identification of frequent mutations encompassing residues 29 to 53 of the HBV surface antigen in chronic HBV carriers as well as in patients with hepatocellular carcinoma. The location of these mutations, not found in patients with acute hepatitis and vaccinated infants, coincides with a human leukocyte antigen class I-restricted cytotoxic T lymphocyte epitope. Significantly, mutations occur at a higher frequency (83%) compared with those identified on the immunogenic "a" determinant (25%) of the corresponding patients. Our findings therefore suggest the potential importance of this novel mutation "hot spot" in the establishment of chronic HBV infection.

Human hepatitis B virus mutants: significance of molecular changes.

Human hepatitis B virus, the leading pathogen for hepatitis B, is a compact DNA virus with viral genes that largely overlap. An increasing number of mutations have emerged following human interventions such as vaccination and anti-viral therapy. While vaccine escape mutants are characterized by mutations on the antigenic hepatitis B surface antigen, those carrying mutations in other viral proteins are either resistant to anti-viral therapy or implicated in acute liver diseases. Molecular identification of these various mutants should shed new lights on the underlying mechanism of hepatitis B virus viral escape and resistance and provide helpful information on their effective eradication.

Hepatitis B virus variants with lamivudine-related mutations in the DNA polymerase and the 'a' epitope of the surface antigen are sensitive to ganciclovir.

Lamivudine is a new antiviral agent effective against hepatitis B viral (HBV) infections but can result in virus-drug resistance associated with mutations in the conserved 'YM552DD' motif of the HBV DNA polymerase. Due to their overlapping coding regions in the HBV genome, mutations in the DNA polymerase may result in substitutions in the hepatitis B surface antigen (HBsAg), albeit outside the antigenic 'a' epitope. Here we report the identification of a novel type of lamivudine-related mutations located in both the polymerase (YM552DD-->Y1552DD) and the 'a' epitope of HBsAg (Gly130-->Asp130). The same virus carried a HBsAg Gly145-->Arg145 mutation prior to therapy. Both the wild type HBV and lamivudine-related mutants with the Gly145-->Arg145 HBsAg mutation were suppressed following ganciclovir treatment, indicating a beneficial additive effect of both drugs against different forms of HBV mutants.

Detection of hepatitis B surface antigen mutants and their integration in human hepatocellular carcinoma.

Vaccine escape hepatitis B virus surface antigen (HBsAg) mutants are capable of independent replication and have been implicated in acute hepatitis. We now report the detection of these mutants with changes at various positions of the antigenic 'a' determinant in human hepatocellular carcinoma (HCC). Southern blot analysis indicated that the HBsAg mutant with the Glycine to Arginine change at position 145 was integrated in HCC, whereas those with a Threonine at position 133 instead of a Methionine were identified in the serum of aggressive HCC. Further studies on HBsAg mutants in HCC should provide new insights on their involvement in the hepatocarcinogenesis.

Current aspects of hepatitis B surface antigen mutants in Singapore.

Mutations occurring on the major antigenic 'a' determinant of hepatitis B surface antigen (HBsAg) have been detected in immunized Singapore children, despite immunoprophylaxis with hepatitis B immune globulin and HBV vaccine. These vaccine-escape HBsAg mutants display a predominance of the Gly145-to-Arg145 mutation in the antigenic 'a' determinant. Our latest follow-up studies indicate the stability of this as well as other vaccine-escape HBsAg mutants over time. We have also identified HBsAg mutants in immunized children with amino acid substitutions outside the 'a' determinant. Transmission of various vaccine-escape HBsAg mutants have been shown in our epidemiological studies. Detection of HBsAg mutants, carrying amino acid changes at various position of the 'a' determinant in random population, points to their emergence in Singapore before the implementation of the vaccination programme. Of significant interest is the recent identification of vaccine-escape HBsAg mutants in hepatocellular carcinoma.

Significance of the minor i and t determinants of hepatitis B virus in hepatocellular carcinoma.

Stored sera from asymptomatic hepatitis B virus (HBV) carriers and hepatitis B virus surface antigen-positive hepatocellular carcinoma (HCC) patients were tested for HBV subtypes, such as subtype determinants d, y, w, r and also antigenic determinants isoleucine (i) and threonine (t) by direct S gene nucleotide sequencing. Significant changes in minor i and t determinants in hepatocellular carcinoma patients with adr hepatitis B carriers were seen. The adr subtype with t determinant was present in 14/25 (56%) of HCC patients compared with only two of 28 (7%) in asymptomatic hepatitis B carriers (P<0.001). However, the adr subtype with i determinant was present in nine of 25 (36%) of the HCC patients and also present in 24/28 (86%) of asymptomatic carriers (P<0.001). No significant changes were seen with the adw subtypes. These results show that i and t minor determinant changes are more common with adr subtypes associated with HCC than with the adw subtype. Whether these subtle changes are pathologically relevant or only a polymorphism of hepatitis B genotypes will depend on subsequent follow-up studies.

Acute viral hepatitis E: clinical and serologic studies in Singapore.

Seroprevalence of hepatitis E is now documented in many countries around the world, but studies of its clinical manifestations and serologic course have been confined to endemic areas. We have prospectively evaluated the occurrence, evolution, and outcome of acute hepatitis E in our patients. Fifteen patients (11 men, 4 women; median age: 41 years) were diagnosed to have acute, sporadic hepatitis E between July 1993 and January 1995; 10 of the 15 were followed up. Sera anti-hepatitis E virus (HEV) immunoglobulin (Ig)G and IgM antibodies and HEV ribonucleic acid in the blood and stool were tested at weeks 1 and 2; serial tests for hepatitis E antibodies and liver function were carried out at months 1, 3, 6, 9, 12, and 18. Coinfection with hepatitis A and superinfection on chronic hepatitis B were found in 3 and 2 patients, respectively. One patient had transient passage of virus in the stool, but none was viremic. Eighty-seven percent of patients lost their IgM antibodies within 3 months, but anti-HEV IgG, once present, persisted throughout follow-up. All patients but one had complete recovery. A higher than reported level of alanine transaminase (mean: 28.5 times normal) and the lack of viremia during acute infection in our patients may be due to increased immune-mediated viral clearance.

Hepatitis E in Singapore--a seroprevalence study.

The seroprevalence of anti-HEV IgG was determined in a hospital-based population in a general medical unit. Patients who were otherwise well but admitted for acute, non-hepatological conditions represent the "healthy" general population, and those admitted primarily with liver disorders were studied. The seroprevalence of anti-HEV IgG was found to be 10.5% in the "healthy" population and 14.7% amongst those with liver diseases. The lack of travel history and past history of jaundice suggests presence of local cases and subclinical manifestation in some of the infected patients. There is an association between seroprevalence of hepatitis A and E, suggesting common predisposing factors for both infections. Anti-HAV IgG has a higher seroprevalence. Retesting of anti-HEV IgG in those who were initially positive found persistence of antibodies beyond twelve months. Both anti-HAV IgG and anti-HEV IgG were found more commonly in the older age groups.