P162 Biosamples from VEDOSS patients show classic pathological signs of Scleroderma:Opportunity for a biological diagnosis of disease
Authors: Ross Rebecca L , Clarke Emily , Merchant Will , Georgiou Ioanna , Mulipa Panji , Carriero Antonio , Abignano Giuseppina , Herrick Ariane , Denton Christopher , Riobo-Del Galdo Natalia , Del Galdo Francesco 
Published: April 2023

Abstract

Background/Aims
The VEDOSS study has recently indicated that more than 50% of patients affected by Raynaud’s phenomenon (RP) and specific SSc anti-nuclear antibodies (ANA) and/or nailfold videocapillaroscopy changes (NVC) (sRP) satisfied ACR/EULAR 2013 criteria within 5 years, indicating a window of opportunity for early detection of SSc. Here we aimed to determine whether sera, skin or dermal fibroblasts cultured from VEDOSS patients showed any biological hallmark of SSc.

Methods
VEDOSS patients were enrolled in the national inception cohort for patients at risk of Scleroderma (Kennedy Cohort, n = 64) . Sera were tested for IFN-inducible chemokines and biomarker of extracellular matrix turnover (ELF test) and compared to Healthy control (HC) and SSc sera (n = 56, 64, respectively). 3mm skin punch biopsies were taken from the forearms of 12 VEDOSS patients, and 5 healthy controls and 6 SSc patients. Biopsies were analysed for haematoxylin and eosin (H&E), masson trichrome staining (MT) and immunohistological (IHC) staining for CD45. A dermatopathologist blinded to clinical features undertook the analysis. A probabilistic image analysis model was used to detect areas of immunopositivity (CD45) and collagen staining (MT) within the samples, using HeteroGenius Medical Imaging Manager colour analysis. In addition, 7 biopsies along with 2 healthy control and 3 SSc, were used to explant fibroblasts cultures. mRNA and protein were isolated from primary fibroblasts and processed for RT-qPCR and western blotting analyses.

Results
Skin biopsies from VEDOSS patients showed evidence of fibrosis and increased collagen bundles within the dermis evidenced in H&E and MT staining, as usually seen in SSc, compared to HC. IHC showed increased number of CD45+ cells in VEDOSS. Semiquantitative analysis of histopathological samples indicated a significant correlation between MT and CD45 staining for VEDOSS (R = 0.8828 P = 0.0016). In vitro, fibroblasts from VEDOSS patients showed an average 5-fold higher collagen mRNA levels compared to HC fibroblasts, at a similar range to that seen in SSc fibroblasts, confirmed at protein level via western blotting. Sera from VEDOSS patients showed significantly higher IFN-inducible chemokines compared to HC and an intermediate levels compared to SSc [Mean (STDV) HC = 4.69(0.29) vs. VEDOSS= 4.93 (0.39); SSc= 5.30 (0.54)]. ELF score was within normal range for most patients with SRP and showed no statistical difference to HC.

Conclusion
Although pilot in nature, this study suggests that patients with no clinical signs of skin fibrosis in their forearms already show biomarker signs of SSc both in their sera, skin biopsies and fibroblast level. Our data indicate that skin thickening is a late manifestation of SSc pathogenesis and early window of opportunity of patients with VEDOSS could be targeted for immune intervention and antifibrotic intervention.
Disclosure
R.L. ross: None. E. Clarke: None. W. Merchant: None. I. Georgiou: None. P. Mulipa: None. A. Carriero: None. G. Abignano: None. A. herrick: None. C. Denton: None. N. Riobo-Del Galdo: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.