1090. Unique Full Length Hepatitis B Virus Genomes Isolated from Children With Hepatocellular Carcinoma

M. Chang; H. Huang; H. Wang; L. Chen; D. Tsuei; Y. Ni

 

Background and Aims

The cause of early oncogenesis in hepatitis B virus (HBV)-related childhood hepatocellular carcinoma (HCC) remains unclear. This study is aimed at investigating whether the existence of possible unique HBV strains contributes to the early oncogenesis of childhood HCC.

 

Methods

We investigated the full-length genomic sequences of HBV in the serum of 7 children with HCC and another 21 children with chronic HBV infection. HBV full-length sequences from 48 Asian adults with HCC and 16 Asian adults with chronic HBV infection, which were retrieved from GenBank, were used for comparison. Using phylogenetic tree construction and direct sequence comparison, we analyzed the distinct features of HBV genomes from children with HCC.

 

Results

Phylogenetic analysis indicated that the majority (71.4%) of HBV from children with HCC contained recombinant sequences of both genotypes B and C and clustered in two very closely related clans that were clearly distinguishable from HBV genomes of patients (children and adults) with chronic HBV infection and adults with HCC. Sequence analysis identified a consensus 12-bp pre-S2 deletion in 57.1% of HBV genomes of children with HCC. Other characteristic mutations were also found throughout HBV genome.

 

Conclusion

The majority of full-length sequences of HBV isolated from children with HCC belonged to recombinant strains of genotype B and C , which are clearly distinguishable from HBV genomes of patients with chronic HBV infection and adults with HCC. Those HBV strains from HCC children contained unique mutations, particularly a short pre-S2 deletion, which may be associated with early HCC development.

 


1093.Treatment of Hepatitis B Using Intron A vs. Peginterferon in Combination with Lamivudine in Children

P. A. Harren; K. Ventura; M. Martinez; D. Jan; R. S. Brown; S. J. Lobritto

 

Aim

To assess the safety, tolerability and efficacy of 2 combination therapy regimens using interferon (IFN) and lamivudine (LAM) to treat E antigen (Ag) positive pediatric hepatitis B (HBV) infected patients.

 

Methods:

34 patients (age 2.5-6 years) at a single center were assessed over a 5 years. Baseline labs included liver profile (LFT), complete blood count (CBC), thyroid function test (TFT), HBV E Ag, HBV E antibody (Ab) and HBV DNA quantitative PCR. All patients had a liver biopsy with the intent to treat to grade and stage disease. Exclusion criteria included HIV co-infection, stage 4 disease, severe thrombocytopenia, thyroid disease, uncontrolled depression and history of non-compliance. Two treatment regimens were used. Group A: 16/34 patients received Intron A, 5 million unit/m2 SQ daily (max dose 10 million units) for 5 months with LAM (staggered by one month) 4mg/kg/day orally (max dose 100mg) until E Ag seroconversion or resistance. Group B: 6/34 patients received of Peginterferon Alfa 2b (Peg-IFN), 1.5mcg/kg/week for 24 weeks with LAM as above. Monthly assessments included vital signs, weights, LFT, CBC, TFT, HBV DNA, HBV E Ag, and HBV E Ab.

 

Results

Twenty-four males and 10 females were assessed. Liver biopsies demonstrated Stage 0-3, Grade 0-3 disease. Group A demographics: 4 Asian, 2 Hispanic, 6 African American, 4 Caucasian with vertically transmitted disease in 13/16. At screening all had HBV DNA > 200 million copies, 6/16 had normal LFT. On therapy15/16 had undetectable HBV DNA with 8/16 achieving E Ag seroconversion at mean of 44 weeks (range 22-69). Safety data: none developed thyroid dysfunction, 2/16 patients had neutropenia (1 treated with IFN dose reduction, 1 treated with Neupogen), 1 patient required an antidepressant, 1 patient stopped IFN due to seizure, and 2/16 required nutritional supplements for weight loss. One subject developed LAM resistance. Group B demographics: 3 Asian, 2 Hispanic, 1 African American all with vertical transmission. At screening all had HBV DNA > 200 million copies and normal LFT. No patient cleared DNA on therapy and no patient achieved HBeAg seroconversion. Two of 6 required nutritional supplements for weight loss and no other adverse effects occurred.

 

Conclusion

Combination therapy with IFN and LAM in a staggered manner is safe and effective in pediatric patients with chronic HBV infection. Combination therapy with Peg-IFN and LAM at the dosing assessed is not equally efficacious and should not be used in pediatric patients until further clarification of dosage and frequency.

 


1094. HBeAg Seroconversion Correlates with a High Number of Core Gene Mutations and with Genotypes B and D in Pediatric Patients

I. Carey; M. Mytilinaiou; A. Giannattasio; S. Bansal; P. Cheeseman; D. Cebecauerova; G. Mieli-Vergani; D. Vergani

 

Background

The outcome of hepatitis B virus (HBV) infection results from the interaction between virus and host immune response. While effective immune recognition usually controls viral replication, escape mutations may arise from the immune system selective pressure, with consequent persistent infection.

 

Aim

To investigate whether there is a relationship between presence of mutations within HBV core gene, HBV genotypes, HBV DNA viral load and HBeAg seroconversion in five different HBV infection settings.

 

Patients:

One hundred paediatric patients (median age 13.1 years, 54 males) were divided into 5 groups according to their HBeAg/HBsAg status and aminotransferases activity (Table 1).

 

Methods

HBV DNA was isolated from serum samples. The presence of point mutations within HBV pre-core and core promoter regions and amino acid substitutions within immunodominant epitopes of HBV core gene was analysed by direct sequencing (mutations/patient). HBV genotypes were determined by direct sequencing. HBV DNA viral load was quantified by real-time PCR (log10 copies/ml).

 

Results

A higher number of mutations within the HBV pre-core region and HBV core gene was detected in HBeAg negative patients than in those HBeAg positive (pre-core: 4.50.2 vs.2.60.2, p=0.001 and core: 5.50.4 vs. 4.10.3, p=0.04). Genotypes B and D were more prevalent in HBeAg negative children than in those HBeAg positive (80% vs. 58%, p<0.001). HBV-DNA viral load was significantly higher in groups A (mean SEM, 7.90.2), B (6.80.3), and D (7.51.2) compared to C (3.40.3) and E (1.00.4) (p=0.001). Among HBeAg negative children, those with genotypes B and D had a higher number of pre-core mutations and tended to have a lower number of core mutations than those with genotypes A, C and E (pre-core: 5.50.4 vs. 4.10.2, p=0.04; 4.70.3 vs. 5.70.3, p=0.1, respectively).

 

Conclusions

Irrespective of genotype, emergence of HBV core gene mutations is associated with HBeAg seroconversion. In patients with genotypes B and D, who more frequently lose HBeAg, seroconversion is associated also to a high number of pre-core mutations. To what extent these mutations result from the selective pressure of the immune system or are virally determined remains to be clarified.

 

Group

HBeAg

HBsAg

ALT

N

Stage

A

Positive

Positive

Normal

46

Immunotolerant

B

Positive

Positive

Elevated

24

Immunoactive

C

Negative

Positive

Normal

22

Low replication

D

Negative

Positive

Elevated

2

HBeAg negative hepatitis

E

Negative

Negative

Normal

6

Self-limited

 


1095. Graft Histology Up to 10 Years after Pediatric Liver Transplantation: High Incidence of Severe Fibrosis, Correlated with Transplant-related Factors

R. Scheenstra; P. M. Peeters; H. J. Verkade; A. S. Gouw

 

Introduction

Previously we showed that 31% of liver grafts have portal fibrosis at 1 year after pediatric liver transplantation (LTx; Transplantation 2000; 6:236). The presence of fibrosis correlated positively with transplant related factors, including cold ischemia time (CIT) and biliary complications.

 

Aim

To assess progress and development of fibrosis in liver grafts in the long term after pediatric LTx and to identify possible risk factors.

 

Methods

We reviewed protocol biopsies obtained at 5 and 10 years after LTx from the originally included 77 patients/grafts. We analyzed the relationships between histology and parameters of transplantation and follow up. Severity of fibrosis was evaluated using a three point scale: 0 = no fibrosis, 1 = portal fibrosis without bridging (mild fibrosis), 2 = portal fibrosis with occasional bridging (moderate fibrosis), 3 = diffuse portal bridging (severe fibrosis/cirrhosis).

 

Results

At 5 and 10 years after LTx, 66/77 (85%) and 55/77 (71%) biopsies were available, resp. Missing biopsies at 10 yrs after LTx included 7 deaths and 6 retransplants. Between 1 year and 5 years after LTx, the prevalence of fibrosis increased from 31% to 67%, but remained stable thereafter (10 yrs: 65%). However, the percentage of patients with severe fibrosis continued to increase, from 10% (5 yrs) to 27% (10 yrs). The presence of fibrosis at 1 year tended to correlate with re-transplantation (p=0.054). The incidence of severe fibrosis after 10 years was significantly higher in patients who had fibrosis at 1 year after LTx, compared with those without fibrosis at 1 year (p= 0.004). At 5 years, ASAT and γGT values were significantly higher in patients with fibrosis than in those without fibrosis (ALAT 23 13 vs. 36 28 U/l; GGT 30 52 vs. 112 149 U/l; resp., each p <0.05). At 10 years after LTx, corresponding values were not significantly different. Sixty four percent (64%) of patients without fibrosis at 1 yr after LTx had developed some degree of fibrosis at 10 years (25/39). The development of fibrosis starting after 1 year was strongly related to CIT, donor/recipient ratio and type of preservation fluid (these factors were also strongly interrelated). The CIT of the grafts that developed late fibrosis was longer than that of grafts without fibrosis at 10 yr (11.4 3.9 h vs. 7.2 3.8 h; p = 0.004).

 

Conclusion

At 5 and 10 years after pediatric LTx, the majority of grafts have fibrosis in protocol biopsies. At least in 25% of the grafts, fibrosis progresses to cirrhosis in 10 years after pediatric LTx. Even fibrosis that only develops after the first year post LTx is strongly correlated with transplant related factors.

 


899. Replication of Hepatitis B Virus (HBV) Starts after Birth

M. J. Quasdorff; D. Nierhoff; G. Holz; T. Goeser; U. Protzer

 

Vertical transmission of Hepatitis B Virus (HBV) is thought to occur during birth, but there are only very limited data on the replicative potential of HBV in case of an earlier infection time point. Since HBV replication is discussed to depend on hepatocyte differentiation, the purpose of this study was to define the starting point and analyze the changes in HBV replication along with hepatocyte maturation during fetal and postnatal liver development in an HBV-transgenic mouse model.

 

Methods

We analyzed different stages of fetal and postnatal liver development, starting at embryonic day (ED) 12.5 in HBV 1.3 xfs transgenic mice. Breeding pairs were selected based on high HBeAg serum levels, indicating efficient HBV replication. To assess HBV replication in fetal, postnatal and adult livers, we quantified expression of HBV pregenomic RNA (pgRNA) and HBV DNA by real-time PCR and HBV core and envelope proteins by Western blot analysis. Additionally, expression levels of hepatocyte nuclear factors (HNF) and peroxisome proliferator-activated receptor gamma coactivator - 1 alpha (PGC-1 alpha) as main regulators of hepatocyte differentiation and HBV gene expression were quantified by Western blot analysis relative to beta-actin. Single-cell suspensions of fetal livers and adult hepatocytes were fixed on cytospins and costained for HBV core protein and epithelial-specific pancytokeratin.

 

Results

Low levels of pgRNA were detected at all analyzed stages of liver development, but expression of pgRNA significantly increased compared to ED 12.5: 7-fold on ED 15.5, 20.8-fold on ED 18.5, 29.0-fold on 2 days post-natally and 190.0-fold in adult liver (AL). Using Western blot or immunocytochemistry, we did neither detect HBV core nor large envelope proteins before birth. Correlating with these results, HNF4alpha, PGC-1alpha and HNF1 were detected at ED 15.5 at the protein level and markedly increased until birth: 30.9-, 32.5- and 61.6-fold, 58.9-, 363- and 444.5-fold and 43.3-, 110.0- and 114.7-fold on ED 18.5, 2 days postnatally and in AL, respectively. HBV DNA increased continuously from ED 18.5 onwards.

 

Conclusion

HBV replication starts around birth, although the transcription template is present before as a transgene. Hepatocyte differentiation and expression levels of HNF4alpha, PGC-1alpha and HNF1alpha contribute to the efficiency of HBV pgRNA transcription and protein production and determine the starting point of HBV replication.