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T2001 Quasispecies Changes Detected in Hypervariable Region 1 of Hepatitis C Genome With High-Resolution DNA Melting Analysis in Treated and Untreated Chronic Hepatitis C Patients
T2001 Quasispecies Changes Detected in Hypervariable Region 1 of Hepatitis C Genome With High-Resolution DNA Melting Analysis in Treated and Untreated Chronic Hepatitis C Patients Irena Hrstic, Blazenka Grahovac, Branko Kolaric, Rajko Ostojic, Boris Vucelic BACKGROUND AND AIMS: Hepatitis C virus exists in an individual as pool of closely related but genetically distinct variants. Development of resistant variants can be the reason of treatment failure. Aim of this study was to determine the changes in HCV quasispecies in untreated and treated group of patients in region susceptible for mutations. METHODS: Quasispecies were detected by high-resolution DNA melting analysis in hypervariable region1 at two points: 1. forty untreated patients: at baseline and after 6 months; 2. sixty-nine treated patients: at baseline and at the end of treatment. Second sample was collected for 40 untreated and for 24 treated patients (45 had ETVR). For each sample result was shown as melting curve with 4 parameters: Tm (melting temperature) specifies the temperature where half of initial DNA is denaturated; H (high) specifies the amount of melted DNA at Tm; W (wide) is range from minimal and maximal needed temperature and A (area) is area under the curve and depends of all 3 parameters. RESULTS: In untreated group mean age was 40,0 y and the most common genotype was 1 (57,5%) while in treated group mean age was 43,4 y with majority of patients with genotype 1 infection (83,3%). Obtained baseline Tm was statistically significantly correlated with genotype obtained with commercial kits (p= 0,05) while no significance was found between W, H, A and genotype. No statistical significances have been found in Tm, H, W and A for untreated and treated patients between paired samples but medians have changed in both groups (table 1 and 2). Trend of changes differed between groups. In untreated patients W and H raised after 6 months while in treated patients W and H declined. CONCLUSIONS: Without treatment, quasispecies become more heterogeneous while therapy makes them more homogeneous, towards resistant variants. Since no significant changes have been found in Tm it might be concluded that all observed changes developed between same genotype at baseline. Untreated patients
T1999
AASLD Abstracts
Gene Expression Changes in Hepatitis C Infected Liver as Measured by Encode Tiling Array Analysis of Both 5' CAP and 3' poly(A) RNA Milan E. Folkers, Don Delker, Christopher I. Maxwell, Cassie A. Nelson, David A. Nix, Curt H. Hagedorn Aims: To perform a more comprehensive analysis of gene expression changes in HCV infected cirrhotic (HCV) liver including both annotated and unannotated genomic regions. We hypothesized that standard RNA isolation techniques may under-represent some gene expression differences in HCV liver and that RNAs from previously undefined genes are differentially regulated in HCV as compared to control liver. Background: To date, no studies have used tiling arrays to assess previously unannotated regions in HCV liver for novel genes altered during chronic HCV infection. Although array studies of annotated genes have been done in HCV liver, they have primary focused on samples of poly(A)+ RNA. Recent data has suggested that many mammalian mRNAs lack or have short 3' polyadenylated ends and are likely to be underrepresented or absent in such studies. Methods: Chronic hepatitis C cirrhotic (HCV) liver and normal human liver were prepared using a high-affinity eIF4E variant which binds 5' capped (Pol II) RNAs with a ten-fold higher affinity than the wildtype protein; poly(A)+ RNA was isolated with oligo(dT). Each sample had cDNA synthesized and probes prepared for Agilent ENCODE tiling array (representing 1% of the genome and tiling both annotated and unannotated regions). Real-time PCR analysis of selected transcripts was done with a total of 7 HCV and 7 control liver specimens. RACE and sequencing was performed on RNA from unannotated regions to better define their 5' and 3' ends. Results: Tiling array analysis of 5' capped RNA samples identified 69 differentially expressed annotated genes (fold change >1.5 and Bonferroni adjusted p-value <0.05) in HCV liver compared to control liver. Analysis of poly(A)+ RNA identified 52 annotated genes that were differentially expressed. Multiple immune response transcripts, including five leukocyte immunoglobulinlike receptor transcripts, interferon regulatory factor 1 (IRF1) and interferon alpha receptor (IFNAR1), were identified exclusively in 5' cap RNA. This analysis also identified three candidate novel Pol II RNAs that were increased in HCV liver. A novel transcript from chromosome 14 was also expressed in Huh7.5 cells following acute HCV infection. This novel RNA may be part of the host antiviral defense response. Conclusion: This study provides evidence that analysis of 5' capped RNA adds information to gene expression studies of HCV liver biospecimens. In addition, this approach to gene expression analysis identified putative novel Pol II genes that were upregulated in HCV liver - one of these was also increased in acutely HCV infected liver cells. The function of this RNA is presently being investigated.
Treated patients
T2002 Improved Sustained Virologic Response (SVR) in “Difficult-to-Cure” Patients Treated With Telaprevir (T) in Combination With Peginterferon Alfa-2a (P) and Ribavirin (R): An Analysis From the PROVE3 Study Andrew J. Muir, Adrian M. Di Bisceglie, Nathalie Adda, Michael P. Manns, Norah Terrault, Leif Bengtsson, Shelley George, John McHutchison Background and Aims: PROVE3 is a Phase 2b randomized controlled study that evaluated the safety and efficacy of telaprevir-based regimens in patients with genotype 1 hepatitis C virus (HCV) who did not achieve an SVR with prior PR therapy. In this subgroup analysis we assessed whether T/PR improved SVR rates in patients with factors associated with lower virologic response rates to PR. Methods: The two T/PR treatment arms, T12/PR24 (n=115) and T24/PR48 (n=113), evaluating 12 weeks or 24 weeks of T with 24 or 48 weeks of PR, respectively, were compared with the control arm PR48 (n=114) for each factor. The statistical evaluation of SVR rate was performed using a multivariate logistic regression model including the potential predictive factors: baseline HCV RNA level (<800,000 IU/mL versus ≥800,000 IU/mL), age (≤50 years versus >50 years), race (black versus white), sex, body mass index (<25, 25-<30 and ≥30 kg/m2), liver fibrosis stage (cirrhosis/bridging fibrosis versus any other), and prior response (non-response versus relapse). Results: The analysis of each variable indicated that RVR and SVR rates were higher in the T12/PR24 and T24/PR48 groups than in the PR48 group (see Table). A multivariate logistic regression analysis performed on all subjects indicated that treatment (P<0.001), baseline HCV RNA level (P= 0.017), and prior response (P<0.001) were predictors of SVR. The analyses based on each treatment group indicated that prior non-response was a negative predictor of response among telaprevir-treated patients (P=0.01 for T12/PR24 and P=0.001 for T24/PR48) and high baseline HCV RNA level was a negative predictor of response in the PR48 arm (P<0.001). Conclusions: Telaprevir-based therapy improved RVR and SVR rates in patients with factors associated with lower SVR rates with PR therapy, including baseline viral load, sex, age, BMI, race, and fibrosis stage. RVR and SVR rates
T2000 Liver Gene Expression Signature to Predict Response to Pegylated Interferon Plus Ribavirin in Chronic Hepatitis C A. Ducès, Ivan Bièche, Benedicte Jardin-Watelet, M. Lapalus, S. De Muynck, Eve Dupas, Isabelle Molina, N. Lambert, E. Nicolas, B. Sallenave, E. Estrabaud, Nathalie Jullian, M. Martinot-Peignoux, Olivier Lada, V. Paradis, Dominique Valla, Pierre Bedossa, Daniel Laune, M. Vidaud, Patrick Marcellin, T. Asselah BACKGROUND AND AIMS The standard of care treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. METHODS Pretreatment liver biopsies from 140 patients treated with pegylated interferon plus ribavirin were studied. Among these patients 68 (49%) had sustained virological response (SVRs), 51 (36%) non response (NR) and 21 relapse (RR) (15%). Patients were mainly infected with genotype 1, 3, 4 and 2. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of 54 genes from the literature involved in various cellular and molecular mechanisms associated with liver gene expression during hepatitis C infection. RESULTS The expression profiles of responders differ significantly from those of NRs. The most notable changes in gene expression were mainly observed in the IFN-stimulated genes, and in growth factors/cytokines genes. Interestingly, the basal liver levels of expression of IFN-stimulated genes were higher in NRs in comparison with RR and SVRs. In NRs, the failure to respond to exogenous PEG-IFN could indicate a blunted response to IFN. This raises the possibility that, in NRs, the IFN-stimulated genes are already maximally induced. CONCLUSION NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a molecular gene signature. The genes included in the signature encode molecules secreted in the serum and provide a logical functional approach for the development of serum markers to predict response to treatment.
AASLD Abstracts
S-846
T1999
AASLD Abstracts
Gene Expression Changes in Hepatitis C Infected Liver as Measured by Encode Tiling Array Analysis of Both 5' CAP and 3' poly(A) RNA Milan E. Folkers, Don Delker, Christopher I. Maxwell, Cassie A. Nelson, David A. Nix, Curt H. Hagedorn Aims: To perform a more comprehensive analysis of gene expression changes in HCV infected cirrhotic (HCV) liver including both annotated and unannotated genomic regions. We hypothesized that standard RNA isolation techniques may under-represent some gene expression differences in HCV liver and that RNAs from previously undefined genes are differentially regulated in HCV as compared to control liver. Background: To date, no studies have used tiling arrays to assess previously unannotated regions in HCV liver for novel genes altered during chronic HCV infection. Although array studies of annotated genes have been done in HCV liver, they have primary focused on samples of poly(A)+ RNA. Recent data has suggested that many mammalian mRNAs lack or have short 3' polyadenylated ends and are likely to be underrepresented or absent in such studies. Methods: Chronic hepatitis C cirrhotic (HCV) liver and normal human liver were prepared using a high-affinity eIF4E variant which binds 5' capped (Pol II) RNAs with a ten-fold higher affinity than the wildtype protein; poly(A)+ RNA was isolated with oligo(dT). Each sample had cDNA synthesized and probes prepared for Agilent ENCODE tiling array (representing 1% of the genome and tiling both annotated and unannotated regions). Real-time PCR analysis of selected transcripts was done with a total of 7 HCV and 7 control liver specimens. RACE and sequencing was performed on RNA from unannotated regions to better define their 5' and 3' ends. Results: Tiling array analysis of 5' capped RNA samples identified 69 differentially expressed annotated genes (fold change >1.5 and Bonferroni adjusted p-value <0.05) in HCV liver compared to control liver. Analysis of poly(A)+ RNA identified 52 annotated genes that were differentially expressed. Multiple immune response transcripts, including five leukocyte immunoglobulinlike receptor transcripts, interferon regulatory factor 1 (IRF1) and interferon alpha receptor (IFNAR1), were identified exclusively in 5' cap RNA. This analysis also identified three candidate novel Pol II RNAs that were increased in HCV liver. A novel transcript from chromosome 14 was also expressed in Huh7.5 cells following acute HCV infection. This novel RNA may be part of the host antiviral defense response. Conclusion: This study provides evidence that analysis of 5' capped RNA adds information to gene expression studies of HCV liver biospecimens. In addition, this approach to gene expression analysis identified putative novel Pol II genes that were upregulated in HCV liver - one of these was also increased in acutely HCV infected liver cells. The function of this RNA is presently being investigated.
Treated patients
T2002 Improved Sustained Virologic Response (SVR) in “Difficult-to-Cure” Patients Treated With Telaprevir (T) in Combination With Peginterferon Alfa-2a (P) and Ribavirin (R): An Analysis From the PROVE3 Study Andrew J. Muir, Adrian M. Di Bisceglie, Nathalie Adda, Michael P. Manns, Norah Terrault, Leif Bengtsson, Shelley George, John McHutchison Background and Aims: PROVE3 is a Phase 2b randomized controlled study that evaluated the safety and efficacy of telaprevir-based regimens in patients with genotype 1 hepatitis C virus (HCV) who did not achieve an SVR with prior PR therapy. In this subgroup analysis we assessed whether T/PR improved SVR rates in patients with factors associated with lower virologic response rates to PR. Methods: The two T/PR treatment arms, T12/PR24 (n=115) and T24/PR48 (n=113), evaluating 12 weeks or 24 weeks of T with 24 or 48 weeks of PR, respectively, were compared with the control arm PR48 (n=114) for each factor. The statistical evaluation of SVR rate was performed using a multivariate logistic regression model including the potential predictive factors: baseline HCV RNA level (<800,000 IU/mL versus ≥800,000 IU/mL), age (≤50 years versus >50 years), race (black versus white), sex, body mass index (<25, 25-<30 and ≥30 kg/m2), liver fibrosis stage (cirrhosis/bridging fibrosis versus any other), and prior response (non-response versus relapse). Results: The analysis of each variable indicated that RVR and SVR rates were higher in the T12/PR24 and T24/PR48 groups than in the PR48 group (see Table). A multivariate logistic regression analysis performed on all subjects indicated that treatment (P<0.001), baseline HCV RNA level (P= 0.017), and prior response (P<0.001) were predictors of SVR. The analyses based on each treatment group indicated that prior non-response was a negative predictor of response among telaprevir-treated patients (P=0.01 for T12/PR24 and P=0.001 for T24/PR48) and high baseline HCV RNA level was a negative predictor of response in the PR48 arm (P<0.001). Conclusions: Telaprevir-based therapy improved RVR and SVR rates in patients with factors associated with lower SVR rates with PR therapy, including baseline viral load, sex, age, BMI, race, and fibrosis stage. RVR and SVR rates
T2000 Liver Gene Expression Signature to Predict Response to Pegylated Interferon Plus Ribavirin in Chronic Hepatitis C A. Ducès, Ivan Bièche, Benedicte Jardin-Watelet, M. Lapalus, S. De Muynck, Eve Dupas, Isabelle Molina, N. Lambert, E. Nicolas, B. Sallenave, E. Estrabaud, Nathalie Jullian, M. Martinot-Peignoux, Olivier Lada, V. Paradis, Dominique Valla, Pierre Bedossa, Daniel Laune, M. Vidaud, Patrick Marcellin, T. Asselah BACKGROUND AND AIMS The standard of care treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. METHODS Pretreatment liver biopsies from 140 patients treated with pegylated interferon plus ribavirin were studied. Among these patients 68 (49%) had sustained virological response (SVRs), 51 (36%) non response (NR) and 21 relapse (RR) (15%). Patients were mainly infected with genotype 1, 3, 4 and 2. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of 54 genes from the literature involved in various cellular and molecular mechanisms associated with liver gene expression during hepatitis C infection. RESULTS The expression profiles of responders differ significantly from those of NRs. The most notable changes in gene expression were mainly observed in the IFN-stimulated genes, and in growth factors/cytokines genes. Interestingly, the basal liver levels of expression of IFN-stimulated genes were higher in NRs in comparison with RR and SVRs. In NRs, the failure to respond to exogenous PEG-IFN could indicate a blunted response to IFN. This raises the possibility that, in NRs, the IFN-stimulated genes are already maximally induced. CONCLUSION NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a molecular gene signature. The genes included in the signature encode molecules secreted in the serum and provide a logical functional approach for the development of serum markers to predict response to treatment.
AASLD Abstracts
S-846