Proteomic aptamer analysis reveals serum markers that characterize preclinical systemic sclerosis (SSc) patients at risk for progression toward definite SSc

Abstract

Background: The study of molecular mechanisms characterizing disease progression may be relevant to get insights into systemic sclerosis (SSc) pathogenesis and to intercept patients at very early stage. We aimed at investigating the proteomic profile of preclinical systemic sclerosis (PreSSc) via a discovery/validation two-step approach.

Methods: SOMAcan aptamer-based analysis was performed on a serum sample of 13 PreSSc (discovery cohort) according to 2001 LeRoy and Medsger criteria (characterized solely by Raynaud phenomenon plus a positive nailfold capillaroscopy and SSc-specific antibodies without any other sign of definite disease) and 8 healthy controls (HCs) age, gender, and ethnicity matched. Prospective data were available up to 4±0.6 years to determine the progression to definite SSc according to the EULAR/ACR 2013 classification criteria. In proteins with relative fluorescence units (RFU) > |1.5|-fold vs HCs values, univariate analysis was conducted via bootstrap aggregating models to determine the predicting accuracy (progression vs non-progression) of categorized baseline protein values. Gene Ontologies (GO terms) and Reactome terms of significant proteins at the adjusted 0.05 threshold were explored. Significant proteins from the discovery cohort were finally validated via ELISAs in an independent validation cohort of 50 PreSSc with clinical prospective data up to 5 years. Time-to-event analysis for interval-censored data was used to evaluate disease progression.

Results: In the discovery cohort, 286 out of 1306 proteins analyzed via SomaScan, were differentially expressed versus HCs. Ten proteins were significantly associated with disease progression; analysis through GO and Reactome showed differentially enriched pathways involving angiogenesis, endothelial cell chemotaxis, and endothelial cell chemotaxis to fibroblast growth factor (FGF). In the validation cohort, endostatin (HR=10.23, CI95=2.2-47.59, p=0.003) was strongly associated with disease progression, as well as bFGF (HR=0.84, CI95=0.709-0.996, p=0.045) and PAF-AHβ (HR=0.372, CI95=0.171-0.809, p=0.013) CONCLUSIONS: A distinct protein profile characterized PreSSc from HCs and proteins associated with hypoxia, vasculopathy, and fibrosis regulation are linked with the progression from preclinical to definite SSc. These proteins, in particular endostatin, can be regarded both as markers of severity and molecules with pathogenetic significance as well as therapeutic targets.

Keywords: Disease progression; Preclinical stage; Proteomic; Systemic sclerosis; Vasculopathy.

Fig. 1

Enrichment analysis in the discovery set. Enrichment analysis: genes related to the 10 proteins selected in the discovery cohort (see Table 1). A Gene Ontologies (GO) at the biological process level. B Reactome pathways

Fig. 2

Estimated time-to-evolution in the validation cohort. Survival estimates as calculated by the Turnbull’s method, in the validation cohort; T0 = blood draw

Fig. 3

Risk of evolution to definite systemic sclerosis in the validation cohort (replicated proteins). Survival estimates (Turnbull’s method) for dicothomized proteins (high/low serum levels) with prognostic significance in the validation cohort; time, years from blood draw. bFGF, basic fibroblast growth factor; PAF-AHβ, platelet-activating factor acetylhydrolase subunit beta

Fig. 4

Distribution of categorized proteins in the validation cohort. Distribution of cases with high or low validated protein levels in preclinical systemic sclerosis patients who did evolve (progressors) or who did not (non-progressors) into definite systemic sclerosis. Dicothomization was performed as described in the main text; for endostatin risk is associated with high serum levels; for the basic fibroblast growth factor (FGF) and platelet-activating factor acetylhydrolase subunit beta (PAFAH1B2) risk is associated with low serum levels; clusterization made on the basis of serum concentrations