J. Blagojevic

European multicentre study validates enhanced liver fibrosis test as biomarker of fibrosis in systemic sclerosis

ObjectivesnTo validate enhanced liver fibrosis (ELF) test and its components-amino-terminal propeptide of procollagen type III (PIIINP), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and HA-as biomarkers of fibrosis in SSc in an independent, international, multicentre cohort.nnnMethodsnTwo hundred and fifty-four SSc patients from six Rheumatology Centres were included. Sera were collected and stored according to EUSTAR biobanking recommendations and analysed through automated high throughput diagnostics. Statistical analysis was performed with SPSS software.nnnResultsnTwo hundred and forty-seven SSc patients (mean age 55.7 ± 13.9 years, 202 F) were analysed. ELF score, TIMP-1 and PIIINP levels were higher in males (P = 0.0197, P = 0.0107, P = 0.0108 respectively) and in dcSSc (P = 0.001, P = 0.0008, P < 0.0001 respectively). ELF score and the single markers significantly correlated with modified Rodnan skin score (r = 0.37, P < 0.0001), disease activity and severity (P < 0.0001 for all markers, except for HA P = 0.0001) and inversely with forced vital capacity, (FVC) % (TIMP-1, r = -0.21, P = 0.0012; PIIINP, r = -0.26, P = 0.0001), TLC% (ELF score, r = -0.20, P = 0.0036; TIMP-1, r = -0.32, P < 0.0001; PIIINP, r = -0.28, P < 0.0001), diffusion capacity of the lung for carbon monoxide (DLCO) % (P < 0.0001 for all markers, except for HA P = 0.0115). Multivariate analysis indicated that age (P < 0.001), modified Rodnan skin score (P < 0.001) and DLCO% (P = 0.005) were independently associated with ELF score.nnnConclusionnBetween the first and this validation studies, the value of the ELF score as independent marker of skin and lung involvement in SSc is confirmed in 457 patients. A longitudinal study is on-going to identify an SSc specific algorithm with predictive value for skin and lung progression.

SAT0229 A Novel Serum Test Based Algorithm To Aid in Very Early Diagnosis of Systemic Sclerosis (VEDOSS)

Background VEDOSS project relied on a specific set of inclusion criteria identifying patients in very early stage of systemic sclerosis (SSc) to be submitted to a tight follow up and unravel factors linked to the progression of the disease. The components of the ELF score (PIIINP, TIMP-1 and HA) have been shown to correlate with severity of skin and lung fibrosis in SSc. Objectives Our aim was to analyze the concentrations of ELF components in a subset of patients enrolled in the VEDOSS study and determine their potential diagnostic value. Methods Sera from 114 patients enrolled into the VEDOSS data base and 67 SSc controls fulfilling ACR/EULAR 2013 criteria (subsets: 33 diffuse; 34 limited) were obtained from 2 centres. Serum concentrations of ELF components were determined on Siemens Advia Centaur platform. Logistic regression analysis of the results was performed employing classification of SSc as state variable. Results Among the 114 patients enrolled from VEDOSS, 30 subjects had primary Raynauds phenomenon (PRP); 54 had Raynauds phenomenon (RP) and at least one of the following “red flags” (risk factors) for SSc: (Puffy fingers, ANA, SSc specific autoantibodies or SSc-specific capillaroscopic pattern) without fulfilling 2013 ACR/EULAR criteria (score <9). 30 VEDOSS patients fulfilled new ACR/EULAR criteria (score >9) despite having no evidence of skin involvement nor interstitial lung disease at lung high resolution CT or pulmonary arterial hypertension, left ventricular systolic impairment or renal involvement. All serum biomarkers concentrations correlated with age (p<0.05 for all). Logistic regression analysis using the three biomarkers as variables identified a specific model (SSc score) ranging from -3.92 to 5.77. The new score showed good ability to discriminate between patients with SSc (VEDOSS patients already classified as SSc and SSc controls) versus VEDOSS patients not yet classified as SSc (AOUC under ROC curve: 0.853, 95%CI 0.797–0.910). Within the VEDOSS database, SSc score showed a fair ability to discriminate between VEDOSS patients fulfilling SSc classification criteria, despite lack of skin internal organ involvement (lung, heart and kidneys), and those not fulfilling the criteria (AOUC =0.756, 95%CI 0.646–0.865). Indeed, patients fulfilling SSc classification criteria, had average score of 0.44 vs -0.86 of patients not fulfilling the criteria and the score remained significant even after correcting for age (p=0.026). Furthermore, SSc controls had significantly higher SSc score compared to VEDOSS patients already classified as SSc, confirmed after age correction (p<0.000). No difference in SSc score has been observed between PRP and VEDOSS patients not yet classified as SSc. Conclusions Our data indicate that the SSc score is a simple serum test based model that can be used in patients with RP to aid in the very early diagnosis of SSc. Furthermore, the identification of VEDOSS patients with a high SSc score test and already classified as SSc even in the absence of internal organ involvement can be used in intervention trials aimed to prevent further disease progression. Disclosure of Interest None declared

FRI0239 Complementary Value of ELF Test and NT-proBNP in Reflecting Fibrosis and Vasculopathy in Systemic Sclerosis

Background The ELF test and its components (PIIINP, TIMP-1 and HA) have been shown to correlate with skin, lung and overall fibrosis and not with any vascular manifestation of Systemic Sclerosis (SSc)1. On the contrary, NT-proBNP has been suggested to be useful for stratification of SSc patients, especially to identify those at risk of pulmonary hypertension2. Objectives Aims of this study were: a) to validate ELF score and its single analytes on an independent cohort of scleroderma patients; b) to evaluate whether NT-proBNP could provide additional value to the development of an SSc-specific algorithm. Methods 250 sera from SSc patients from a single UK centre were analysed employing a high-throughput in vitro diagnostic of a routine NHS pathology lab to measure ELF score, its analytes and NT-proBNP levels. All patients fulfilled 2013 ACR/EULAR classification criteria for SSc. Clinical, laboratory and instrumental data were collected at time of sampling. Statistical analysis was performed using SPSS. P<0.05 was considered statistically significant. Results Multivariate analysis of ELF score (including the variables found statistically significant in univariate analysis) identified age, mRSS and DLCO% as independently associated with ELF score (p<0.0001 for all), confirming results previously published on an independent Italian cohort. As previously shown, ELF score and single analytes were not associated with heart and vascular manifestations of the disease. On the contrary NT-proBNP significantly correlated with heart severity (p<0.0001) and peripheral vasculopathy (p=0.005). Its levels were higher in patients with current digital ulcers (p=0.001), digital pitting scars (p=0.01), telangiectasias (p=0.01), systemic arterial hypertension (p=0.004), pulmonary artery hypertension (PAH) (p=0.01), diastolic dysfuction (p=0.002), reduced ejection fraction (p=0.0002), arrhythmias (p<0.0001) and dyspnoea (p=0.003) compared to those without the manifestation. Multivariate analysis identified presence of arrhythmias (p<0.0001), age (p<0.0001), PAH (p<0.001) and DLCO% (p=0.006) as independently associated with NT-proBNP. All the biomarkers significantly correlated with total Medsgers severity scale and EScSGactivity index (p<0.0001). Conclusions Our findings validate the value of ELF score in a second independent cohort of 250 SSc sera and suggest that NT-proBNP has a complementary value correlating with other aspects of the disease such as PAH and heart severity. Longitudinal multicentres studies are warranted to determine the sensitivity to change and the predictive value of these biomarkers in SSc patients and to build up a new combined scleroderma specific algorithm including markers of fibrosis and vasculopathy. References Abignano G et al. Ann Rheum Dis 2014 Avouac J et al. Arthritis Care Res 2015 Disclosure of Interest None declared

SAT0198 The Desscipher Project in Systemic Sclerosis (SSC): Observational Data on Digital Ulcers (DU) Prevention from The Eustar Group

Background In SSc, the management of DU is a problem in clinical practice, therefore their prevention is highly warranted. Sildenafil, iloprost, calcium channel blockers (CCB), ACE inhibitors (ACEi) and bosentan, alone or in combination, are used for DU prevention without unanimous agreement. Objectives DeSScipher (“to decipher the optimal management of SSc”) is an EC-funded FP7 research project that consists of five observational trials (OT) addressing different aspects of SSc. The aim of OT1 was to evaluate the efficacy of vasoactive and vasodilating drugs or a combination therapy for the management of DU in SSc. Methods Longitudinal data were collected for up to 24 months by 28 DeSScipher centres. In SSc patients, the efficacy of sildenafil, bosentan, sildenafil+bosentan, iloprost or CCB /ACEi for the prevention of new DU was evaluated. Clinical features, ulcer type and other drugs were also recorded. Time without new DU, proportion of patients without new DU, risk of developing new DU and the mean number of new DU per patient were evaluated at 6 months follow-up. Results In OT1, 1394 patients were enrolled. The history of DU was a significant risk factor for developing new DU (OR=3.146; (95%CI:1.19–8.31), p=0.021), therefore analysis was focused on secondary prevention (prevention of new DU in patients with DU history). 473/1394 (33.9%) had a history of DU and 268/473 (56.7%) had follow up data (58.2% limited and 41.8% diffuse SSc subset). 47/268 (17.5%) were on Bosentan, 33/268 (12.3%) on Sildenafil, 40/268 (14.9%) on Iloprost, 31/268 (11.6%) on Sildenafil and Bosentan combination and 117/268 (43.7%) on CCB/ACEi alone. In patients with history of DU in the last 24 weeks, the treatment with CCB/ACEi alone was associated with 7 fold increased risk of developing new DU compared to all other treatment arms (OR =7.313; 95%CI:1.248–42.85, p=0.027). Patients on concomitant immunosuppressive treatment had a trend towards faster DU recurrence. Conclusions History of DU in the past 24 weeks should prompt a more aggressive preventive strategy other than therapy with CCB/ACEi alone due to its lower efficacy to prevent DU recurrence compared to the other treatment options. Acknowledgement The DeSScipher project was funded by the European Communitys Framework Programme 7 (FP7-HEALTH-2012.2.4.4–2 - Observational trials in rare diseases) under grant agreement N° 305495. We acknowledge the contribution of the following EUSTAR centers: Ancona (member N°34), Assiut (168), Bad Bramstedt (187), Belgrade (55), Bucharest (100), Cluj-Napoca (16), Erlangen (106), Frankfurt (124), Hamburg (64), Iasi (162), Istanbul (21), Istanbul (133), Köln (44), Monserrato (142), Moscow (78), Moscow (190), Padua (31), Rome (94), Salford/Manchester (80), Tübingen (56), Wuppertal (192). Disclosure of Interest None declared

AB0197 Soluble Semaphorin 3E (SEMA3E): A Biomarker for Dysregulation of Vascular Tone Control in Systemic Sclerosis (SSC)

Background SSc is a vasculopathy characterised by dysregulation of angiogenesis and vascular tone control leading to severe ischemia and loss of capillaries. In the complex system of interconnection between blood vessels and nerves there is an emerging evidence suggesting that proteins involved in transmitting axonal guidance cues, such as class III semaphorins, play a role in the regulation of vascular development. Sema3E has been shown to repel endothelial cells, modulating the cytoskeleton and focal adhesion structures leading to cellular apoptosis. Objectives To investigate if soluble Sema3E could be involved in the dysregulation of vascular tone control in primary Raynauds phenomenon (pRP) and in secondary RP investigating (Very Early Diagnosis of Systemic Sclerosis) VEDOSS and SSc patients. Methods Sema3E serum levels were measured by quantitative colorimetric sandwich ELISA in 102 pRP serum samples without autoantibodies positivity and without nailfold videocapillaroscopy (NVC) alterations, 100 SSc, 48 VEDOSS patients and 104 age- and sex-matched healthy controls. SSc and VEDOSS patients were evaluated for internal organ damage. Sema3E levels were expressed as median and range and compared by Mann-Whitney U test. p≤0.05 were considered statistically significant. Results Sema3E levels were significantly increased in pRP (median 1.00 ng/mL, range 0.00-5.84 ng/mL), VEDOSS (median 0.79 ng/mL, range 0.00-1.45 ng/mL) and SSc patients (median 0.85 ng/mL; range 0.00-18.01 ng/mL) versus healthy controls (median 0.55 ng/mL; range 0.00-1.47 ng/mL) (p<0.001). Sema3E was significantly higher in pRP than VEDOSS (p=0.025), while no significant differences were detected between pRP and SSc. In SSc with early NVC pattern, Sema3E levels were significantly increased (median 1.16 ng/mL range 0.63-9.53 ng/mL) in comparison to those detected in the late NVC pattern (median 0.75 ng/mL range 0.00-3.78 ng/mL, p=0.028). Moreover, Sema3E levels were significantly increased in SSc patients without ulcers (median 0.98 ng/mL; range 0.00-18.01 ng/mL) in comparison to those with digital ulcers (median 0.79 ng/mL; range 0.00-3.78 ng/mL, p=0.038). Sema3E was significantly higher in pRP compared to late SSc NVC pattern (p=0.04) and to SSc patients with digital ulcers (p=0.031). Conclusions Our data show that Sema3E sera levels are significantly increased in pRP, VEDOSS and SSc patients. Circulating Sema3E levels are higher in SSc patients without digital ulcers and with early NVC pattern. In particular, Sema3E may be involved in the early modifications of neuro-vascular mechanisms leading to dysregulation of vascular tone control. Disclosure of Interest None declared

FRI0515 Neuropilin-1 Contributes to Impaired Angiogenesis in Systemic Sclerosis and Correlates to Clinical Features

Background In systemic sclerosis (SSc) vascular involvement is a primary event characterized by vascular tone dysfunction and increased circulating levels of vascular endothelial growth factor (VEGF). Neuropilin-1 (NRP1) is a receptor for both class-3 semaphorin (sema) family of axon guidance molecules and VEGF. NRP1 is required for optimal VEGF/VEGFR-2 signaling, and NRP1-deficient mice exhibit vascular defects including disorganized blood vessels, lack of normal branching and missing capillary networks. Sema3a controls physiological and pathological angiogenesis. Objectives In the present study, we investigated the possible involvement of sema3a/NRP1 axis in SSc and its correlation to nailfold videocapillaroscopy (NVC) patterns and digital ulcers. Methods Soluble NRP1 (sNRP1) and sema3a levels were measured by quantitative colorimetric sandwich ELISA in serum samples from SSc patients (n=49) and age- and sex-matched healthy controls (n=39). Patients were classified according to NVC patterns (early, active and late). NRP1 and sema3a protein expression was evaluated by immunofluorescence and western blot in skin biopsies from SSc patients (n=10) and healthy controls (n=8). NRP1 expression was also evaluated in human dermal microvascular endothelial cells from SSc patients (SSc-MVECs) and healthy controls (H-MVECs) at basal level, and in H-MVECs after stimulation for 24 hours with recombinant human VEGF165 (10 ng/ml), SSc sera (n=3) and healthy sera (n=3). Results Circulating sNRP1 levels were significantly reduced in SSc patients (median 0.22 ng/ml) compared with healthy controls (median 0.69 ng/ml) (p=0.005). In particular, sNRP1 levels were significantly lower in either SSc patients with active or late NVC patterns than in controls (both p<0.05). Moreover, sNRP1 levels were significantly decreased in SSc patients with digital ulcers compared both with patients without digital ulcers (p=0.009) and controls (p=0.001). No significant differences in sema3a levels were detected between patients and controls. NRP1 expression was decreased in clinically affected skin biopsies from SSc patients compared with healthy skin, especially in dermal endothelial cells and stromal cells. H-MVECs showed higher NRP1 protein expression compared with SSc-MVECs. Stimulation with recombinant VEGF165 strongly upregulated NRP1 expression in H-MVECs. NRP1 expression in H-MVECs increased after treatment with healthy sera compared with basal condition, while it decreased after challenging with SSc sera (both p<0.005 vs basal H-MVECs). Sema3a expression was not different in skin biopsies from SSc patients compared with controls. Conclusions NRP1 expression is significantly decreased in SSc, and lower sNRP1 levels correlate with the severity of nailfold capillary modifications and presence of digital ulcers. NRP1 might play a role in the vascular damage and in the impairment of the angiogenic process in SSc. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3662

FRI0481 Microvascular Modifications in Very Early Systemic Sclerosis Patients

Background Great interest is devoted today to patients with very early diagnosis of systemic sclerosis (VEDOSS), characterized by the presence of Raynaud phenomenon (RP), puffy fingers, Antinuclear antibodies (ANA), plus the presence of disease related autoantibodies (anti-centromere – ACA – and antiTopoisomerase I – ATA) and/or nailfold videocapillaroscopy (NVC) scleroderma pattern [1,2]. In established SSc, NVC is today considered an outcome measure [3]. Objectives To describe microvascular modifications in VEDOSS patients and to retrospectively evaluate te effectiveness of NVC in monitoring the evolution of microvascular disease. Methods Patients with RP attending our outpatient clinics from March 2010 to October 2013 were enrolled. Autoantibodies were tested and NVC was performed at baseline and at follow-up an optical probe equipped with 200x lens. Giant capillaries, micro-haemorrhages, capillary loss and ramified/bushy capillaries were searched in NVC images and then clustered/scored according to the medium value of the alteration, as following: 0% present, score 0; from 0% to 33%, score 1; from 33% to 66%, score 2; over 66%, score 3 [4]. Scores given to each parameter were summed and compared between baseline and follow-up assessment: a decrease of the total score of at least 2 points was considered as improvement, an increase of at least 2 points as worsening, otherwise as stable. Results 167 patients were enrolled (18 males, mean age 51±15 years, 10±8 years since RP onset), with ANA positive in 96/167 (57.5%), ACA and ATA in 41/167 (24.5%) and 17/167 (10.2%) respectively. On NVC, giant capillaries were found in 62/167 (37.1%), haemorrhages in 72/167 (43.1%), loss of capillaries in 2/167 (1.2%) and ramified/bushy capillaries in 10/167 (5.9%). At baseline, giant capillaries and haemorrhages were associated with ANA plus ACA/ATA positivity (p<0.01), also confirmed for VEDOSS patients. Comparing baseline to follow up NVC evaluation (mean time 1.4±0.5 years), the presence of ANA plus ACA/ATA showed a trend to association (p=0.07) with worsening of microvascular involvement, which was statistically significant when VEDOSS criteria were satisfied (p<0.05); a trend toward improvement was seen in ANA negative patients (p=0.08). Conclusions NVC confirms its ability in detecting capillary modification in VEDOSS patients, as well as its effectiveness in monitoring microvascular disease evolution in time. References Avouac J, Fransen J, Walker UA et al. Preliminary criteria for the very early diagnosis of systemic sclerosis: results of a Delphi Consensus Study from EULAR Scleroderma Trials and Research Group, Ann Rheum Dis 2011Mar;70:476-81. Minier T, Guiducci S, Bellando-Randone S et al. Preliminary analysis of the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) EUSTAR multicentre study: evidence for puffy fingers as a pivotal sign for suspicion of systemic sclerosis. Ann Rheum Dis.2013 Aug 12. Cutolo M, Sulli A, Pizzorni C et al. Capillaroscopy as an Outcome Measure for Clinical Trials on the Peripheral Vasculopathy in SSc-Is It Useful? Int J Rheumatol 2010. Sulli A, Secchi ME, Pizzorni C, Cutolo M.Scoring the nailfold microvascular changes during the capillroscopic analysis in systemic sclerosis patients.Ann Rheum Dis.2008 Jun;67(6):885-7.Epub 2007 Nov 23 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2957