TRANSCRIPTOMIC AND PROTEOMIC PROFILING OF PRECLINICAL SYSTEMIC SCLEROSIS

Background: Systemic sclerosis (SSc) is a rare systemic autoimmune disease with a high morbidity and mortality. In the preclinical phase of the disease, patients present exclusively Raynaud phenomenon (RP) with a nailfold video-capillaroscopy positive for scleroderma pattern and/or the presence of specific autoantibodies. Research investigations on preclinical systemic sclerosis (Pre-SSc) patients are rare due to the exceptionality and the difficulty to intercept this specific subgroup of subjects that in five years will progress into a definite SSc diagnosis in about 50% of cases. Objectives: to perform a comprehensive investigation of preclinical SSc at gene expression, proteomic and clinical level with the aim to identify specific early-stage disease biomarkers as well as progression biomarkers. Methods: gene expression analysis through RNAseq technique was performed on whole blood samples of 35 Pre-SSc and 16 matched healthy controls (HC) collected at baseline and after four years. Gene expression module changes analysis was performed comparing evolving Pre-SSc, stable Pre-SSc and HC at baseline and follow-up. A proteomic analysis through SOMAscan technology was assessed on serum at baseline of a subgroup of 16 Pre-SSc (8 evolving Pre-SSc vs 8 stable Pre-SSc) and 8 HC selected on a clinical basis, matching for age, gender and autoantibody profile. Finally, proteins emerged from SOMAscan analaysis to be predictive of progression were validated on a validation cohort of 50 subjects with preclinical features whose serum aliquots were available at baseline. Results: out of 35 Pre-SSc, 15 (42.9%) progressed toward a definite SSc after four years. The presence of gastro-esophageal reflux and a shorter time of RP were associated with a shorter time of progression. RNA expression change analysis of evolving Pre-SSc vs stable Pre-SSc identified 73 genes with a corrected p value ≤0.05. At baseline, Pre-SSc had type I IFN gene expression modules (M 3.4, M 5.12, M 1.2) increased in comparison to HC. NK gene expression modules were decreased in evolving Pre-SSc over time. Out of 286 proteins assessed by SOMAscan, 10 proteins were able to predict at baseline progressors from not progressors. Evolving Pre-SSc showed increased levels of NKp30, Endostatin, bFGF, ECM1, FGF18, Fibronectin 1.3, PAFAH1B2, FABP and decreased levels of PHI and Ubiquitin1. High levels of endostatin and reduced serum levels of PAFAH1B2 were confirmed with ELISAs in the validation cohort to correlate with a shorter time to progression. Conclusion: a type I IFN signature distinguished preclinical SSc from HC and a reduced NK signature was associated to SSc progression. Proteins linked to pathways of fibrosis, extracellular matrix organization, positive regulation of cell proliferation, angiogenesis, signal transduction were discovered to predict disease progression. Moreover, endostatin emerged as a biomarker worthy of future mechanistic investigations.