Pharmacogenomics of interferon-beta therapy in multiple sclerosis: baseline IFN signature determines pharmacological differences between patients

Abstract

Background: Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNbeta in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNbeta therapy.

Methodology: Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNbeta therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNbeta. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation.

Principal findings: Pharmacogenomics revealed a marked variation in the pharmacological response to IFNbeta between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNbeta therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R = -0.7208; p = 0.0016). The negative correlation was maintained after three (R = -0.7363; p = 0.0027) and six (R = -0.8154; p = 0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n = 30; R = -0.4719; p = 0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNbeta indicating that differential responsiveness to IFNbeta is an intrinsic feature of peripheral blood cells at baseline.

Conclusion: These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNbeta.

Figure 1. A. Biological response to IFNß therapy in MS patients

Two-way hierarchical cluster analyses using gene expression ratio's (biological response). This diagram contains genes that were at least two-fold up- or downregulated after IFNß therapy in at least seven patients. Upregulated genes after therapy are indicated by a red colour, downregulated by a green colour and genes that show no differences in expression after therapy are indicated in black. B. Cluster of IFN-induced genes Selection of genes clustering together based on similar biological response profiles within the patient group. The genes clustered together with a correlation of 0.925 and are known to be induced by IFN. The mean expression ratio of all genes in this IFN cluster is referred to as the biological IFN response.

Figure 2. Correlation between baseline and biological response to IFNß therapy.

Biological responses were calculated, using a set of IFN-induced genes (A and B) or a single IFN-induced gene (C and D) and correlated with baseline levels, resulting in a significant negative correlation. In C and D the expression levels of RSAD2 is measured by quantitative realtime PCR and normalized to the expression levels of GAPDH. A. IFN cluster as described in Figure 1B; B. Selection of 15 genes; C. Biological response after three months, using RSAD2 gene expression levels; D Biological response after six months using RSAD2 gene expression levels.

Figure 3. Comparative analysis between different treatment regimens.

Comparison of biological response of Avonex treated patients and Betaferon or Rebif treated patients. A. Average biological response using the set of 15 IFN-induced genes in the test group of 16 RRMS patients; B. Biological response using PCR based gene expression levels for RSAD2 in the second independent validation group of 30 RRMS patients.