Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis

Abstract

Introduction: Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.

Methods: Adult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30% post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.

Results: Ten subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71%) randomized to abatacept and one out of three patients (33%) randomized to placebo experienced ≥30% improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95% confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.

Conclusions: Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.

Trial registration: ClinicalTrials.gov NCT00442611. Registered 1 March 2007.

Figures

Fig. 1

Intrinsic subset assignment. a Purple identifiers designate samples with increased expression of inflammatory gene signature and green identifiers correspond to samples with increased expression of normal-like gene signature. b Expression patterns of 645 intrinsic genes from [11] across samples from the study. ‘Intrinsic subset’ row shows results of formal intrinsic subset assignment using Spearman correlation statistics (see Methods). Color bar here and on subsequent figures refers to median-centered log2 fold change. c Changes in inflammatory gene signature between baseline and post-treatment. Improvers – solid lines, non-improver – dashed line, placebos – dotted lines

Fig. 2

Gene and pathway signatures in abatacept improvers. a Blue identifiers designate baseline and black identifiers designate post-treatment samples; b 398 genes showed significant differential expression (p < 0.05) between baseline and post-treatment improver samples during the course of abatacept treatment; c 133 pathways were significantly differentially expressed in improvers (FDR <10 %). Color bar here and on subsequent figures represents single sample Gene Set Enrichment Analysis Normalized Enrichment Score (ssGSEA NES)

Fig. 3

CD28 pathway trends across abatacept improver and non-improver samples. a Expression of 19 genes contributing the most to the GSEA enrichment score (core enrichment group) is shown in improvers. Genes are ordered by the GSEA rank metric score with those contributing the most to the enrichment score at the top and those contributing the least at the bottom. Array tree is from Fig. 1a. b CD28 pathway trends across improver and non-improver baseline (base) and post-treatment (post) samples. Expression values are for centroid vectors generated by averaging expression data for each of 19 genes across all respective samples (e.g., all improver bases). p-values are for paired (base vs. post) and unpaired (base vs. base) t-test comparisons. Graphs show mean with SD scatter plots

Fig. 4

Gene and pathway signatures between abatacept and placebo groups at baseline. a Blue identifiers are improvers, black identifiers are placebos and orange identifier is non-improver; b 1,640 genes had significant differential expression at baseline between abatacept and placebo groups (p < 0.05); c 15 GSEA pathways were significantly differentially expressed at baseline between abatacept and placebo groups (FDR <10 %)

Fig. 5

Gene and pathway signatures between abatacept and placebo groups post-treatment. a Blue identifiers are improvers, black identifiers are placebos and orange identifier is non-improver; b 1,354 genes were significantly differentially expressed between abatacept and placebo groups post-treatment (p < 0.05); c 63 pathways had significant differential expression between abatacept and placebo groups post-treatment (FDR <10 %)

Fig. 6

CD28 pathway trends across abatacept and placebo post-treatment groups. a Comparison of expression centroids for the entire set of genes annotated to CD28 pathway. p-value is for unpaired t-test with Welch’s correction. Graph is Tukey’s box and whiskers plot. b Comparison of expression centroids for the core enrichment subset of CD28 pathway from Fig. 3. p-value is for unpaired t-test. Graph shows mean with SD scatter plot