- Email: [email protected]
A novel phenotypic assay for monitoring hepatitis B virus (HBV) drug resistance during antiviral treatment
Parallel
Session
2: Basic
abnormalities. After 21 mo of median follow-up, 24 pts (60%) suffered recurrence (19 in the same nodule; 4 in other segments; 1 seeding along the subcutaneous needle-track). Tumor size < 4 cm and alphafetoprotein < 100 nglml were significantly associated with complete response, while previous treatment, Milan Criteria and Child stage were not. Mild complications were observed in 20% of pts. Recurrences were treated mainly with 2nd ablation (13 pts: 68%). Ultimately, 6 pts received OLT and in 4 vital residual HCC was found. At 21 months overall patients and disease free survival were 68% and 21%. Conclusions: RFA + HA1 appears to control HCC < 4 cm. In larger lesions such a combination cannot be considered a definitive treatment. Risk of seeding was confirmed.
I 14
A PHASE I CLINICAL OF ADENOVIRUS
TRIAL OF INTRATUMORAL
ENCODING
WITH HEPATOCELLULAR ADVANCED
INTERLEUKIN-12
CARCINOMA
GASTROINTESTINAL
INJECTION IN PATIENTS
AND OTHER
TUMORS
G.D. Mazzolini, B. Sanero, .I. Ruiz, M. Herraiz, J.I. Herrero, .I. Quiroga, A. Benito, .I. Larrache, J. Pueyo, J.C. Subtil, J. Sola, B. Sadaba, l? Sarobe, I. MeleroC. Qian, J. Prieto. Liver Unit And Depts. Of Radiology, Farmacology And Pathology. Clinica Universitmia, Pamplona, Spain Introduction: very few treatments are effective for liver metastasis from gastrointestinal tumors, including hepatocellular carcinoma (HCC). AFIL12 (an adenoviral vector encoding human IL-12 genes) has shown a potent antitumor effect in pre-clinical setting. Aim: to evaluate the feasibility and safety of the intratumoral injection of AF-IL12 into patients with advanced primary or secondary liver tumors. Secondary end-points were the evaluation of biological activity and tumor response. Patients and Methods: AF-IL12 was administered in doses ranging from 2.5 x lOel0 to 3 x lOe12 vp to 7 cohorts of 3 patients. Injection was repeated after 30 days in case of stable or responding disease (up to 3 injections per patient). A thorough clinical and analytical follow-up was performed. Results: 21 patients (primary: 9; non-primary: 12) have received 44 injections. Intratumoral administration was successfully achieved in 100% of patients, mainly with US-guide. Frequent although transient adverse reactions included fever, malaise, sweating and lymphopenia. No dose-limiting toxicity has been reached. A dose-dependant viral shedding was observed and viral DNA could be detected in blood as far as day 5. No toxic serum K-12 and II+-y levels were found. We observed a non-dose dependent rise in the levels of II+-y short after AFII-12 administration presumably due to viral infection. In contrast, we noted a dose-dependent K-12 production (up to 520 pg/ml). Three patients demonstrated a 25% decrease in serum tumor markers. One partial response and 6 stabilizations were observed. Conclusions: Intratumoral injection of AF-IL12 in our population was feasible and safe. Some indicators of biological activity were also observed. Further studies are necessary to establish the clinical effectiveness of this promising strategy.
Parallel Session 2: Basic Viral Hepatitis
I15
CO-CHAPERONE REPLICATION
CHIP INDUCES
STIMULATION
OF HBV
IN VITRO
F! Hoffmann’, S. Alberti2, M.A. Gonzalez-Carmona’, J. Hoehfeld2, W.H. Caselmann’. ‘Department Of Internal Medicine, University Of Bonn; 2Department Of Cell Biology, University Of Bonn, Bonn, Get-many
Introduction: To initiate HBV replication HBV polymerase bonucleincomplex with HBV-RNA and chaperones Hsp70
forms a riand Hsp90.
Viral Hepatitis
7
Hsp70 and Hsp90 are controlled by co-chaperons named Hip and Hop, which encourage the chaperon activity, and CHIP and Bag-l, which mediate chaperon - interaction with the ubiquitin/proteasome-system. CHIP is one of the most important co-chaperons, because it acts as a Ubiquitinligase and tags chaperon-bound proteins for proteasomal degradation. Our objective is to elucidate the influence of CHIP on HBV replication in tissue culture. Methods: To show the association of HBV polymerase with Hsp70/Hsp90 we translated the HBV polymerase in vitro and tagged it radioactively. After addition of HSP70/Hsp90 the formed complex was immunoprecipitated by anti-chaperon-antibodies and the polymerase was detected by autoradiography. After addition of CHIP, the conjugation enzyme UbcHSb and the ubiquitin activating enzyme El we examined whether the polymerase is a suitable substrate for CHIP Next we infected HBV secreting HepG2.2.15 cells with CHIP-recombinant adenoviruses and detected CHIP overexpression by western blot. The influence of CHIP on HBV replication was assayed by a Southern blot using polymerase-DNA. The detection of viral particles in the medium was done by capture assay. Results: Hsp90/Hsp70 bound HBV polymerase can be ubiquitinated through a complex of the ubiquitin-ligase CHIP and UbcHSb in vitro. After infection of HepG2.2.15 cells, in which HBV replicates, with CHIP recombinant adenoviruses the CHIP overexpression lead to a strong stimulation of viral replication and sezemation of viral particles into the medium. Conclusions: Our results show that CHIP is an important regulator of HBV-replication. This seems to be due to the CHIP-mediated ubiquitination of the HBV-polymerase. In our next experiments we plan to examine the effects of this ubiquitination on activity and stability of the polymerase.
I
16
A NOVEL PHENOTYPIC
ASSAY FOR MONITORING
B VIRUS (HBV) DRUG RESISTANCE
HEPATITIS
DURING ANTIVIRAL
TREATMENT
D. Durantel, S. Durantel, C. Pichoud, B. Werle, M.-N. Brunelle, C. Trepo, F. Zoulim. INSERM Unit 271, Lyon, France HBV drug resistance is a major problem during antiviral therapy. Therefore our aim was to design new and fast cloning strategies to study the evolution of the replication capacity and the drug susceptibility of HBV genomes isolated from patients undergoing lamivudine and/or adefovir treatment. Methods: Viral DNA was extracted from sera, PCR amplified, then cloned in more-than-full-length configuration (1.1 or 1.3 genome unit) into two different types of vectors that enable, after Huh7 cell transfection, the initiation of the intracellular HBV cycle. Northern and Southern blots analysis were performed to characterise the expression of viral RNAs and DNA replicative intermediates for HBV clinical isolates and compare their replication capacity with that of wild type strains. Results: A detailed study regarding the biological properties and drug susceptibility, i.e. variation of ICSOs and 9Os, of naturally occurring HBV mutants has been performed. Throughout the corn--se of therapy, HBV genomes from patients were cloned and evaluated for their replication competence in vitro. For each replication-competent clone obtained, the level of replication and sensitivity to antivirals were related to the genotypes and the pattern of mutations. Multiclonal analysis enabled to evaluate the sensitivity of viral quasi-species to different drugs by determining IC5OslIC9Os. Conclusions: Our phenotypic assay enables the characterisation of the sensitivity to different antivirals of a viral population in less than 4 weeks. This assay will enable a better monitoring and adaptation of antiviral therapy, and will provide a new tool for the study of the molecular biology of HBV clinical isolates. Objective:
Session
2: Basic
abnormalities. After 21 mo of median follow-up, 24 pts (60%) suffered recurrence (19 in the same nodule; 4 in other segments; 1 seeding along the subcutaneous needle-track). Tumor size < 4 cm and alphafetoprotein < 100 nglml were significantly associated with complete response, while previous treatment, Milan Criteria and Child stage were not. Mild complications were observed in 20% of pts. Recurrences were treated mainly with 2nd ablation (13 pts: 68%). Ultimately, 6 pts received OLT and in 4 vital residual HCC was found. At 21 months overall patients and disease free survival were 68% and 21%. Conclusions: RFA + HA1 appears to control HCC < 4 cm. In larger lesions such a combination cannot be considered a definitive treatment. Risk of seeding was confirmed.
I 14
A PHASE I CLINICAL OF ADENOVIRUS
TRIAL OF INTRATUMORAL
ENCODING
WITH HEPATOCELLULAR ADVANCED
INTERLEUKIN-12
CARCINOMA
GASTROINTESTINAL
INJECTION IN PATIENTS
AND OTHER
TUMORS
G.D. Mazzolini, B. Sanero, .I. Ruiz, M. Herraiz, J.I. Herrero, .I. Quiroga, A. Benito, .I. Larrache, J. Pueyo, J.C. Subtil, J. Sola, B. Sadaba, l? Sarobe, I. MeleroC. Qian, J. Prieto. Liver Unit And Depts. Of Radiology, Farmacology And Pathology. Clinica Universitmia, Pamplona, Spain Introduction: very few treatments are effective for liver metastasis from gastrointestinal tumors, including hepatocellular carcinoma (HCC). AFIL12 (an adenoviral vector encoding human IL-12 genes) has shown a potent antitumor effect in pre-clinical setting. Aim: to evaluate the feasibility and safety of the intratumoral injection of AF-IL12 into patients with advanced primary or secondary liver tumors. Secondary end-points were the evaluation of biological activity and tumor response. Patients and Methods: AF-IL12 was administered in doses ranging from 2.5 x lOel0 to 3 x lOe12 vp to 7 cohorts of 3 patients. Injection was repeated after 30 days in case of stable or responding disease (up to 3 injections per patient). A thorough clinical and analytical follow-up was performed. Results: 21 patients (primary: 9; non-primary: 12) have received 44 injections. Intratumoral administration was successfully achieved in 100% of patients, mainly with US-guide. Frequent although transient adverse reactions included fever, malaise, sweating and lymphopenia. No dose-limiting toxicity has been reached. A dose-dependant viral shedding was observed and viral DNA could be detected in blood as far as day 5. No toxic serum K-12 and II+-y levels were found. We observed a non-dose dependent rise in the levels of II+-y short after AFII-12 administration presumably due to viral infection. In contrast, we noted a dose-dependent K-12 production (up to 520 pg/ml). Three patients demonstrated a 25% decrease in serum tumor markers. One partial response and 6 stabilizations were observed. Conclusions: Intratumoral injection of AF-IL12 in our population was feasible and safe. Some indicators of biological activity were also observed. Further studies are necessary to establish the clinical effectiveness of this promising strategy.
Parallel Session 2: Basic Viral Hepatitis
I15
CO-CHAPERONE REPLICATION
CHIP INDUCES
STIMULATION
OF HBV
IN VITRO
F! Hoffmann’, S. Alberti2, M.A. Gonzalez-Carmona’, J. Hoehfeld2, W.H. Caselmann’. ‘Department Of Internal Medicine, University Of Bonn; 2Department Of Cell Biology, University Of Bonn, Bonn, Get-many
Introduction: To initiate HBV replication HBV polymerase bonucleincomplex with HBV-RNA and chaperones Hsp70
forms a riand Hsp90.
Viral Hepatitis
7
Hsp70 and Hsp90 are controlled by co-chaperons named Hip and Hop, which encourage the chaperon activity, and CHIP and Bag-l, which mediate chaperon - interaction with the ubiquitin/proteasome-system. CHIP is one of the most important co-chaperons, because it acts as a Ubiquitinligase and tags chaperon-bound proteins for proteasomal degradation. Our objective is to elucidate the influence of CHIP on HBV replication in tissue culture. Methods: To show the association of HBV polymerase with Hsp70/Hsp90 we translated the HBV polymerase in vitro and tagged it radioactively. After addition of HSP70/Hsp90 the formed complex was immunoprecipitated by anti-chaperon-antibodies and the polymerase was detected by autoradiography. After addition of CHIP, the conjugation enzyme UbcHSb and the ubiquitin activating enzyme El we examined whether the polymerase is a suitable substrate for CHIP Next we infected HBV secreting HepG2.2.15 cells with CHIP-recombinant adenoviruses and detected CHIP overexpression by western blot. The influence of CHIP on HBV replication was assayed by a Southern blot using polymerase-DNA. The detection of viral particles in the medium was done by capture assay. Results: Hsp90/Hsp70 bound HBV polymerase can be ubiquitinated through a complex of the ubiquitin-ligase CHIP and UbcHSb in vitro. After infection of HepG2.2.15 cells, in which HBV replicates, with CHIP recombinant adenoviruses the CHIP overexpression lead to a strong stimulation of viral replication and sezemation of viral particles into the medium. Conclusions: Our results show that CHIP is an important regulator of HBV-replication. This seems to be due to the CHIP-mediated ubiquitination of the HBV-polymerase. In our next experiments we plan to examine the effects of this ubiquitination on activity and stability of the polymerase.
I
16
A NOVEL PHENOTYPIC
ASSAY FOR MONITORING
B VIRUS (HBV) DRUG RESISTANCE
HEPATITIS
DURING ANTIVIRAL
TREATMENT
D. Durantel, S. Durantel, C. Pichoud, B. Werle, M.-N. Brunelle, C. Trepo, F. Zoulim. INSERM Unit 271, Lyon, France HBV drug resistance is a major problem during antiviral therapy. Therefore our aim was to design new and fast cloning strategies to study the evolution of the replication capacity and the drug susceptibility of HBV genomes isolated from patients undergoing lamivudine and/or adefovir treatment. Methods: Viral DNA was extracted from sera, PCR amplified, then cloned in more-than-full-length configuration (1.1 or 1.3 genome unit) into two different types of vectors that enable, after Huh7 cell transfection, the initiation of the intracellular HBV cycle. Northern and Southern blots analysis were performed to characterise the expression of viral RNAs and DNA replicative intermediates for HBV clinical isolates and compare their replication capacity with that of wild type strains. Results: A detailed study regarding the biological properties and drug susceptibility, i.e. variation of ICSOs and 9Os, of naturally occurring HBV mutants has been performed. Throughout the corn--se of therapy, HBV genomes from patients were cloned and evaluated for their replication competence in vitro. For each replication-competent clone obtained, the level of replication and sensitivity to antivirals were related to the genotypes and the pattern of mutations. Multiclonal analysis enabled to evaluate the sensitivity of viral quasi-species to different drugs by determining IC5OslIC9Os. Conclusions: Our phenotypic assay enables the characterisation of the sensitivity to different antivirals of a viral population in less than 4 weeks. This assay will enable a better monitoring and adaptation of antiviral therapy, and will provide a new tool for the study of the molecular biology of HBV clinical isolates. Objective: