Proteomic Analysis of Sera from Individuals with Diffuse Cutaneous Systemic Sclerosis Reveals a Multianalyte Signature Associated with Clinical Improvement during Imatinib Mesylate Treatment

Abstract

Objective: Imatinib has been investigated for the treatment of systemic sclerosis (SSc) because of its ability to inhibit the platelet-derived growth factor receptor and transforming growth factor-β signaling pathways, which have been implicated in SSc pathogenesis. In a 12-month open-label clinical trial assessing the safety and efficacy of imatinib in the treatment of diffuse cutaneous SSc (dcSSc), significant improvements in skin thickening were observed. Here, we report our analysis of sera collected during the clinical trial.

Methods: We measured the levels of 46 cytokines, chemokines, and growth factors in the sera of individuals with dcSSc using Luminex and ELISA. Autoantigen microarrays were used to measure immunoglobulin G reactivity to 28 autoantigens. Elastic net regularization was used to identify a signature that was predictive of clinical improvement (reduction in the modified Rodnan skin score ≥ 5) during treatment with imatinib. The signature was also tested using sera from a clinical trial of nilotinib, a tyrosine kinase inhibitor that is structurally related to imatinib, in dcSSc.

Results: The elastic net algorithm identified a signature, based on levels of CD40 ligand, chemokine (C-X-C motif) ligand 4 (CXCL4), and anti-PM/Scl-100, that was significantly higher in individuals who experienced clinical improvement than in those who did not (p = 0.0011). The signature was validated using samples from a clinical trial of nilotinib.

Conclusion: Identification of patients with SSc with the greatest probability of benefit from treatment with imatinib has the potential to guide individualized treatment. Validation of the signature will require testing in randomized, placebo-controlled studies. Clinicaltrials.gov NCT00555581 and NCT01166139.

Keywords: AUTOANTIBODIES; DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS; IMATINIB; NILOTINIB; PROTEIN MICROARRAYS.

Figures

Figure 1

Luminex identifies increased levels of multiple inflammatory proteins in the sera of individuals with dcSSc. The levels of 44 inflammatory proteins were measured in the sera of individuals with dcSSc at baseline (n = 26) and healthy controls (n = 8) by Luminex bead-based immunoassays. SAM was used to identify serum proteins at significantly different levels between groups (q 2). A hierarchically clustered (unsupervised, Euclidean distance) heatmap of the SAM-positive proteins is shown. The raw MFI of all proteins were log2 transformed prior to clustering. dcSSc: diffuse cutaneous systemic sclerosis; SAM: significance analysis of microarrays; MFI: mean fluorescence intensity; IL17: interleukin 17; MCP3: monocyte chemotactic protein 3; IL1RA: interleukin 1 receptor antagonist; GMCSF: granulocyte macrophage colony-stimulating factor; FGFB: fibroblast growth factor-β; VEGF: vascular endothelial growth factor; IFNG: interferon γ; TGFA: transforming growth factor-α; TNFA: tumor necrosis factor-α; GRO: growth-related oncogene (CXCL1); CRP: C-reactive protein; MDC: macrophage-derived chemokine (CCL22); IP10: interferon γ-induced protein 10.

Figure 2

Autoantigen microarray analysis of baseline sera from individuals with dcSSc. Sera from patients with dcSSc (n = 24) and healthy controls (n = 7) were used to probe microarrays with 28 known autoantigens. SAM was used to identify autoantigens with significantly different reactivity levels between groups (q 2). A hierarchically clustered (unsupervised, Euclidean distance) heatmap of the SAM-positive autoantigens is shown. All features were log2 transformed prior to clustering. dcSSc: diffuse cutaneous systemic sclerosis; SAM: significance analysis of microarrays; NPM1: nucleophosmin 1; MFI: mean fluorescence intensity.

Figure 3

Multianalyte signature predicts improvement in mRSS during treatment with imatinib and nilotinib. The elastic net algorithm was used to identify a signature, based on serum levels of CD40L, CXCL4, and PM/Scl-100 at baseline, that was predictive of clinical improvement during treatment with imatinib (defined as a decrease in mRSS ≥ 5 at 12 mos). (A) The signature was calculated for each individual patient in the imatinib trial at baseline, and was compared between clinical improvement groups. Bars represent group medians and the p values of Mann-Whitney U tests are shown. (B) ROC curve analysis was used to evaluate the accuracy of the baseline signature in predicting clinical response at 12 months in the imatinib trial. (C) Serum samples from a clinical trial of nilotinib for the treatment of dcSSc were used as an independent cohort to validate the signature. Baseline levels of CD40L, CXCL4, and PM/Scl-100 were used to calculate the signature for each individual patient, and ROC curve analysis was performed to assess its accuracy at predicting clinical improvement (reduction in mRSS ≥ 5) at 6 months. mRSS: modified Rodnan skin score; CXCL4: chemokine (C-X-C motif) ligand 4; ROC: receiver-operating characteristic; AUC: area under the curve.