Molecular signatures in skin associated with clinical improvement during mycophenolate treatment in systemic sclerosis

Abstract

Heterogeneity in systemic sclerosis (SSc) confounds clinical trials. We previously identified "intrinsic" gene expression subsets by analysis of SSc skin. Here we test the hypotheses that skin gene expression signatures including intrinsic subset are associated with modified Rodnan skin score (MRSS) improvement during mycophenolate mofetil (MMF) treatment. Gene expression and intrinsic subset assignment were measured in 12 SSc patients' biopsies and 10 controls at baseline, and from serial biopsies of 1 cyclophosphamide-treated patient and 9 MMF-treated patients. Gene expression changes during treatment were determined using paired t-tests corrected for multiple hypothesis testing. MRSS improved in four of seven MMF-treated patients classified as the inflammatory intrinsic subset. Three patients without MRSS improvement were classified as normal-like or fibroproliferative intrinsic subsets. A total of 321 genes (false discovery rate (FDR)<5%) were differentially expressed at baseline between patients with and without MRSS improvement during treatment. The expression of 571 genes (FDR<10%) changed between pre- and post-MMF treatment biopsies for patients showing MRSS improvement. Gene expression changes in skin are only seen in patients with MRSS improvement. Baseline gene expression in skin, including intrinsic subset assignment, may identify SSc patients whose MRSS will improve during MMF treatment, suggesting that gene expression in skin may allow targeted treatment in SSc.

Figure 1. Changing pathological factors in the skin during treatment

Hematoxylin and eosin stained skin biopsies. a) Arm biopsy from a healthy control subject (scale bar=20μm), and biopsy pairs (pre- and post-treatment as indicated) from three non-improvers (upper panel) and four improvers (lower panel) during MMF, representative photomicrographs. Total and arm modified Rodnan skin score (MRSS) as well as fibrosis score are listed below. b) COMP immunofluorescence for pre- and post-treatment biopsies for a healthy volunteer (N1000) and four improvers (SSc3, 5, 6 and 10) and three non-improvers (SSc8, 12, 16) with quantification below. Scale bar=50μm.

Figure 2. Improvers cluster within the inflammatory intrinsic subset

We selected 2775 intrinsic genes with a False Discovery Rate of 3%. Genes and microarray samples were clustered hierarchically. The sample dendrogram (a) shows the statistically significant intrinsic groups. Branch points above each * are significant at p ≤ 0.005. The dendrogram branches are colored to reflect the major intrinsic subsets of normal-like (green), inflammatory (purple) and diffuse-proliferation (red). Patient identifiers indicate systemic sclerosis samples (SSc) and normal healthy controls (Norm); those in the MMF study are colored to reflect improvers (blue) and non-improvers (orange). b) Overview of the gene expression profiles. c and d) Inflammatory clusters, e) mitotic fibroproliferative cluster, f) DNA replication proliferation cluster.

Figure 3. Comparison of baseline gene expression between improvers and non-improvers

Baseline gene expression in arm and back samples between improvers (imp) and non-improvers (non-imp) was compared. a) Blue identifiers indicate improvers and gold indicates non-improvers. b) There were 321 genes identified (FDR<5%) with significant differential expression between improvers and non-improvers during MMF.

Figure 4. Gene expression changes during MMF treatment between improvers and non-improvers

571 genes showed changes in expression during MMF treatment (FDR<10%). Patients that were classified as non-improvers show low levels expression of these genes, which either do not change expression or show increased expression.

Figure 5. Validation of biologically relevant microarray findings using quantitative reverse transcription polymerase chain reactions immunofluorescence

Results are the relative expression values normalized to the mean expression in arm samples of control subjects, *p<0.05.