Hepatic gene expression during treatment with peginterferon and ribavirin: Identifying molecular pathways for treatment response

Abstract

The reasons for hepatitis C treatment failure remain unknown but may be related to different host responses to therapy. In this study, we compared hepatic gene expression in patients prior to and during peginterferon and ribavirin therapy. In the on-treatment group, patients received either ribavirin for 72 hours prior to peginterferon alpha-2a injection or peginterferon alpha-2a for 24 hours, prior to biopsy. The patients were grouped into rapid responders (RRs) with a greater than 2-log drop and slow responders (SRs) with a less than 2-log drop in hepatitis C virus RNA by week 4. Pretreatment biopsy specimens were obtained from a matched control group. The pretreatment patients were grouped as RRs or SRs on the basis of the subsequent treatment response. Gene expression profiling was performed with Affymetrix microarray technology. Known interferon-stimulated genes (ISGs) were induced in treated patients. In the pretreatment group, future SRs had higher pretreatment ISG expression than RRs. On treatment, RRs and SRs had similar absolute ISG expression, but when it was corrected for the baseline expression with the pretreatment group, RRs showed a greater fold change in ISGs, whereas SRs showed a greater change in interferon (IFN)-inhibitory pathways. The patients pretreated with ribavirin had heightened induction of IFN-related genes and down-regulation of genes involved in IFN inhibition and hepatic stellate cell activation.

Conclusion: These data suggest that ISG inducibility is important for the treatment response and that ribavirin may improve outcomes by enhancing hepatic gene responses to peginterferon. Collectively, these mechanisms may provide a molecular basis for the improved efficacy of combination therapy.

Fig. 1

Study design. The patients in the on-treatment group were given either 180 μg of peginterferon-alpha 2a subcutaneously 24 hours prior to liver biopsy or 1000/1200 mg of ribavirin daily for 72 hours plus 180 μg of peginterferon-alpha 2a subcutaneously 24 hours prior to liver biopsy. After the biopsy, the patients were continued on the therapy for 48 weeks.

Fig. 2

Heat map showing distinct gene expression patterns in pretreatment and on-treatment groups. The map is based on the 364 genes with a greater than 1.5-fold difference in the expression with P <0.01. Red indicates increased gene expression, and blue indicates decreased gene expression. The white line across and down the heat map provides a statistically significant separation of the 2 groups and up-regulated or down-regulated genes. Within the 2 groups, the treatment response is indicated as follows: N, naive treatment; RR, rapid responder; and SR, slow responder.

Fig. 3

Gene pathways with significant differential gene expression between pretreatment and on-treatment patients. For each known pathway, the number of genes with differing expression between the groups was calculated, and a P value was determined on the basis of the likelihood of finding the given number of genes by chance alone. The inverse logarithm of the P value is shown on the x axis. Pathways with a cutoff of 2 (equivalent to a P value of 0.01) are shown. In addition to interferon signaling, pathways involved in immune responses and apoptosis were most affected by the interferon treatment.

Fig. 4

Analyses of the gene expression by a quantitative polymerase chain reaction. Selected genes from the microarray analysis were quantified by a TaqMan real-time polymerase chain reaction. (A) On-treatment versus pretreatment. (B) Future RRs versus SRs (pretreatment group). (C) RRs versus SRs (on-treatment group by the fold change). Gene expression in the on-treatment group was normalized for the baseline expression with the mean expression level in the pretreatment group. The fold change was calculated as on-treatment RRs/pretreatment RRs or on-treatment SRs/pretreatment SRs. (D) Peginterferon and ribavirin versus peginterferon. RR indicates rapid responder; and SR, slow responder.

Fig. 5

Supervised hierarchical clustering by a gene list. The patients are categorized on the basis of gene expression profiles. The number of branch points between patients reflects the degree of similarity in the expression pattern. (A) On-treatment group versus the pre-treatment group. (B) Future slow responders versus rapid responders in the pretreatment group. (C) Rapid responders versus slow responders in the on-treatment group divided by the pretreatment group. (D) Peginterferon plus ribavirin versus peginterferon alone.

Fig. 6

Postulated scheme of the importance of gene expression before and during peginterferon and ribavirin treatment and its effect on treatment outcome. The strength of the adaptive immune response and early endogenous IFN production likely determine whether the initial HCV infection is cleared or becomes chronic. High ISG expression, presumably from increased endogenous IFN production, is accompanied by up-regulation of IFN-inhibitory pathways. This leads to reduced ISG induction with IFN treatment. Ribavirin enhances the efficacy of IFN directly through up-regulation of the IFN receptor and indirectly through effects on apoptosis and possibly HSCs. Ultimately, the degree of ISG induction with therapy may determine treatment outcome. HCV indicates hepatitis C virus; HSC, hepatic stellate cell; IFN, interferon; ISG, interferon-stimulated gene; RR, rapid responder; and SR, slow responder.