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Objectives: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. Methods: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. Results: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. Conclusions: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc., This work was supported by eU/eFPia/innovative Medicines initiative Joint Undertaking PrecisesaDs Grant no. 115 565.

We read with interest the recent report by Mathian et al 1 about 17 patients with systemic lupus erythematosus (SLE) and COVID-19, which has paved the way to a constellation of articles aiming to find clues on potential peculiarities in COVID-19 epidemiology and course among patients with SLE. An ongoing debate has also recently grown on the role of hydroxychloroquine (HCQ), with increasing data disconfirming a protective effect of this drug towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in general and specifically also in patients with SLE.2 3 We were thus prompted to summarise the evidence published so far on this latter topic to look for recurrent clinical or epidemiological patterns among different studies/cohorts. To this aim, we analysed all studies published in the English literature until June, 30th 2020 and identified 20 articles describing a total of 4059 patients with SLE, 255 of whom with a PCR-confirmed or presumptive (ie, based on symptoms or radiological findings) diagnosis of …

Background:the prognosis of Systemic lupus Erythematosus (SLE) patients has significantly improved over time, raising the need for more data about disease activity and damage accrual in the long term.Objectives:to investigate the risk of long term disease activity and to identify viable prognostic markers for disease flares in SLE patients with long standing diseaseMethods:data on SLE patients regularly followed at ASST PINI-CTO, Fondazione Ca’ Granda Policlinico and Ospedale San Raffaele, Milan (Milan Systemic Lupus Erythematosus Consortium, SMiLE, cohort) with disease duration ≥ 20 years, were retrospectively analyzed. Organ involvement as per the British Isles Lupus Assessment Group (BILAG) definitions was recorded along with achievement of clinical and complete remission (CR and CCR: clinical SLEDAI =0, PGA Results:data from 168 patients (table 1) were available for analysis. Remission (CR+CCR) and LLDAS were achieved in 22% and 61% at T15 and 25% and 71% at T20. LLDAS was not associated with a history of involvement in any BILAG domain, but it was inversely associated with treatment with mycophenolate at any time (50 vs 23% treated vs not treated; p=0.02). SDI>0 was found in 49% patients at T15 and in 71% at T20. LLDAS at T15 was associated with lower flare rates in the following five years (HR= 0.395, 95%, CI=0.239-0.653; Figure, left panel; pTable 1.Demographic, laboratory and clinical characteristics of patients with SLECharacteristics(n=168)Demographic characteristicsFemale, n (%)150 (89.3)Age at diagnosis years, median (IQR)24 (18-32)Clinical and serological features during 15 years follow up, n (%)Musculoskeletal140 (83)Mucocutaneous135 (80)Constitutional114 (68)Haematological103 (61)Nephritis81 (48)Cardiopulmonary49 (29)NPSLE31 (18)Positive anti-dsDNA136 (81)Hypocomplementemia136 (81)Positive antiphospholipid73 (44)CR / CCR at T1513 (8) / 23 (14)CR / CCR at T2017 (10) / 25 (15)Conclusion:LLDAS is common in SLE patients with long disease duration although up to 37% of patients with 15-year disease duration may experience a flare during the following 5 years. The flare risk increases with failure to attain LLDAS at T15 and with active serology. Late flares associate with damage accrual.References:[1]Aringer M, Ann Rheum Dis. 2019; Franklin K, Ann Rheum Dis 2019Table 2.therapy during the 20 year follow upTherapy (ever)%Prednisone83Hydroxychloroquine91Mycophenolate mofetil33Azathioprine50Methotrexate23Cyclophosphamide36Fig 1:risk of flare according to disease activityDisclosure of Interests:Maria Gerosa: None declared, Giuseppe Alvise Ramirez: None declared, Chiara Bellocchi: None declared, Lorenza Maria Argolini: None declared, Luca Moroni: None declared, Martina Cornalba: None declared, Nicola Farina: None declared, Lorenzo Dagna Grant/research support from: Abbvie, BMS, Celgene, Janssen, MSD, Mundipharma Pharmaceuticals, Novartis, Pfizer, Roche, SG, SOBI, Consultant of: Abbvie, Amgen, Biogen, BMS, Celltrion, Novartis, Pfizer, Roche, SG, and SOBI, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB, Enrica Bozzolo: None declared, Lorenzo Beretta Grant/research support from: Pfizer

Background:Myocarditis is an infrequent but potentially life-threatening inflammatory disorder and might be part of the spectrum of systemic lupus erythematosus (SLE). Little is known about the clinical and histologic features of myocarditis in SLE, especially compared to other forms of myocarditis.Objectives:to test for potential distinctive traits among myocarditis in SLE (MyoSLE), SLE without myocarditis (OnlySLE) and myocarditis without SLE (OnlyMyo)Methods:Patients with MyoSLE were identified from three centres and compared with 231 cross-sectionally enrolled patients with OnlySLE and 87 patients with OnlyMyo. MyoSLE patients were split into two groups based on myocarditis onset within (early onset) vs after (late onset) the first year from SLE diagnosis. OnlySLE patients were dichotomised in the same way based on disease duration at time of enrolment. Demographics and general clinical features were collected retrospectively. SLE disease activity index 2000 (SLEDAI-2K), SLE International Collaborating Clinics/American College of Rheumatology damage index (SDI), clinical and laboratory features were collected at time of myocarditis onset in MyoSLE and at enrolment in OnlySLE. Quantitative data are expressed as median [interquartile range].Results:Fourteen MyoSLE patients were identified, 50% with early onset. Women were equally frequent among MyoSLE (71%) and OnlySLE patients (87%) and less frequent in the OnlyMyo group (43%; pConclusion:Demographics of patients with MyoSLE are more similar to patients with OnlySLE than to OnlyMyo patients. MyoSLE might have distinct histological and pathogenic features compared to OnlyMyo. Patients with MyoSLE show similar patterns of disease activity and accrued damage at time of myocarditis onset compared to patients with OnlySLE with the same disease duration but might diverge later on in SLE course. aPL are frequent in MyoSLE and might both contribute to the pathogenesis of myocardial inflammation and account for the high prevalence of NPSLE and APS, especially in late onset cases.References:[1]Gartshteyn Y et al., Lupus, 2020[2]Thomas G et al., J Rheumatol, 2017[3]Peretto G et al., Int J Cardiol, 2019[4]McDonnell T et al., Blood Rev, 2019Disclosure of Interests:Giuseppe Alvise Ramirez: None declared, Maria Gerosa: None declared, Giacomo De Luca Speakers bureau: SOBI, Novartis, Celgene, Pfizer, MSD, Lorenzo Beretta Grant/research support from: Pfizer, Simone Sala: None declared, Giovanni Peretto: None declared, Luca Moroni: None declared, Francesca Mastropaolo: None declared, adriana cariddi: None declared, Silvia Sartorelli: None declared, Corrado Campochiaro Speakers bureau: Novartis, Pfizer, Roche, GSK, SOBI, Enrica Bozzolo: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB, Lorenzo Dagna Grant/research support from: The Unit of Immunology, Rheumatology, Allergy and Rare Diseases (UnIRAR) received unresctricted research/educational grants from Abbvie, Bristol-Myers Squibb, Celgene, Janssen, Merk Sharp & Dohme, Mundipharma Pharmaceuticals, Novartis, Pfizer, Roche, Sanofi-Genzyme, and SOBI., Consultant of: Prof Lorenzo Dagna received consultation honoraria from Abbvie, Amgen, Biogen, Bristol-Myers Squibb, Celltrion, Novartis, Pfizer, Roche, Sanofi-Genzyme, and SOBI.

Background:Genome-wide gene expression profiles and pathways analysis may help to discover deregulated processes underlying the pathogenesis of complex diseases or their phenotypic expression. Little or nothing is currently known about pathways associated with disease severity and damage in SSc.Objectives:To perform a whole blood transcriptome analysis and to characterize the individualized functional pathways associated with disease severity scores in SSc patients via a discovery and replication strategy.Methods:Whole blood samples were collected in RNA stabilizers from a discovery and a replication cohort of 67 and 34 patients, respectively. RNAseq data were generated by Illumina sequencing in two independent experiments pathways analysis was conducted according to the Functional Analysis of Individual Microarray Expression (FAIME) protocol (1). FAIME scores from Reactome pathways were correlated with the Scleroderma Clinical Trial Consortium Damage Index (SCTC-DI) total scores or with each of its two components (mortality and morbidity) as calculated from regression coefficient previously published (2). A non-parametric partial correlation analysis correcting the results for the use of steroids, immunosuppressants and disease duration was performed. Results independently associated with damage in both cohorts at the 0.1 level after 1000-fold permutation-testing were considered as significant and replicated.Results:A total of 1116 pathways were analyzed. None of them was associated in both cohorts with the total SCTC-DI; similarly, no association was found with the SCTC mortality component. On the contrary, 26 pathways showed an independent and replicated association with the SCTC morbidity component, including platelet degranulation, the transcriptional activity of SMAD2/SMAD3, Toll Like Receptor 2 (TLR2) cascade and related intracellular signaling events involving MyD88, IRAK and NF-kB, and the deregulation of selected transcription factors (TFAP2, E2F6).Conclusion:Selected molecular events involving the innate immune system, signaling and platelet metabolism are associated with the morbidity component of the SCTC-DI in SSc patients, reflecting an irreversible loss of function in several organs and apparatus. The sensitivity of these associations to individual change in accrual damage is to be determined.References:[1]PMID: 22291585; (2) PMID: 30928903Acknowledgments:This work was partially supported by EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant No. 115565Disclosure of Interests:Chiara Bellocchi: None declared, Marzia Rossato: None declared, Guillermo Barturen: None declared, Giulia Segatto: None declared, Barbara Vigone: None declared, Zuzanna Makowska Employee of: Bayer AG, Anne buttgereit Employee of: Bayer AG, Martin Kerick: None declared, Marta Alarcon-Riquelme: None declared, Javier Martin Ibanez: None declared, Lorenzo Beretta Grant/research support from: Pfizer

Background:The staging of interstitial lung disease (ILD) is important to monitor disease progression and for prognostication. A disease severity scale of Systemic Sclerosis (SSc)-related lung disease has long been proposed (i.e. Medsger’s severity scale). This scale was mostly developed by discussion and consensus and stage thresholds were not computed by a data-driven approach. Hidden Markov models (HMM) are methods to estimate population quantities for chronic diseases with a staged interpretation which are diagnosed by markers measured at irregular intervals.Objectives:To build a SSc-ILD specific disease severity scale with prognostic relevance via HMM modeling.Methods:A total of 358 SSc patients at risk for or with ILD were enrolled in a discovery (207 cases, Milan1) and in a validation (151 cases, Milan2, Pavia and Rome) cohort. Patients were included if satisfied the following criteria: 1) Diagnosis of SSc according to the EULAR/ACR 2013 criteria, 2) absence of anticentromere antibodies, 3) dcSSc subset or 4) other subsets with either 4a) ILD-related antibodies (Scl70, PmScl, Ku) or 4b) evidence of ILD on HRCT, 5) disease duration < 5 years at the time of the first pulmonary function test (PFT). Serial PFTs were retrieved and the time up to the last available visit -if the patient alive-, or to death due to pulmonary complications, was recorded. HMM were used to estimate the threshold of a 3-stage model (SL3SI, Scleroderma Lung 3-Stage Index) based on PFT functional values (normal/mild, moderate, severe involvement) in the discovery cohort. Survival estimates of the SL3SI model were compared to Medsger’s severity classes estimates and their predictive capability evaluated via the explained residual variation (R2) of prediction errors (the higher the better). One-hundred random replicates were generated to simulate the prediction effort in patients with different disease duration and lung severity.Results:Patients characteristics are summarized in the Table. Fifteen-years survival estimates for Mesdger’s classes in the discovery set were: normal=0.88, mild=0.86, moderate=0.84 and severe=0.71. The SL3SI was defined by the following thresholds: normal/mild, FVC and DLco >=75%; moderate FVC or DLco 74-55%; severe, FVC or DLco 2values at 15 yrs for the SL3SI compared to Medsger’s classes, providing evidence for a better predictive capability of the former (discovery: 0.31 vs 0.25; validation: 0.28 vs 0.19).Conclusion:The SL3SI, a simplified 3-stage functional model of SSc-ILD, yields better survival estimates and long-term prognostic information than Medsger’s classes. Its reproducibility and ease of use make it a useful tool for the functional and prognostic evaluation of SSc patients at risk for or with ILD.Table:VariablesDiscovery (n=207)Replication (n=151)DcSSc62 (30%)98 (64%)Age at first PFR48.6±1249.1±14.4Disease duration at first PFR1.7±1.61.3±2.4FVC90.5±18.191.1±20.2DLco70.7±19.861.3±20.1ILD on HRCT179 (86%)125 (80%)Scl70157 (76%)153 (78%)SSA63 (30%)32 (21%)n of visits38571473Follow-up time, yrs11±5.610.6±5.7Deaths27 (13%)23 (15%)Disclosure of Interests:Alessandro Santaniello: None declared, Chiara Bellocchi: None declared, Luca Bettolini: None declared, Marcello Cassavia: None declared, Gaia Montanelli: None declared, Adriana Severino: None declared, Monica Caronni: None declared, Corrado Campochiaro Speakers bureau: Novartis, Pfizer, Roche, GSK, SOBI, Enrico De Lorenzis: None declared, Gerlando Natalello: None declared, Paolo Delvino: None declared, Claudio Tirelli: None declared, Lorenzo Cavagna: None declared, Giacomo De Luca Speakers bureau: SOBI, Novartis, Celgene, Pfizer, MSD, Silvia Laura Bosello: None declared, Lorenzo Beretta Grant/research support from: Pfizer

Background Vasculopathy is one of the cardinal feature of systemic sclerosis (SSc) that can be observed in every patients from the earliest stages of the disease. Abnormalities in circulating angiogenetic factors and endothelial dysfunction markers in patients at different stages of the disease that include early or non-fibrotic (pre-fibrotic) subjects have not thoroughly described so far. Objectives To characterize vasculopathy in SSc patients through the determination of 7 selected vascular biomarkers in sera from a large cohort of patients with well defined and distinct clinical characteristics. Methods Sera from 224 subjects were obtained and concentrations of Angiopoietin 2, CXCL-16, E-Selectin, ICAM-1, IL-8, VCAM-1, VEGF were determined by a Luminex commercial assay. Subjects included 43 healthy controls (HC), 47 early systemic sclerosis patients (EaSSc) according to LeRoy and Medsger criteria (Raynaud9s phenomenon + abnormal capillaroscopy patterns and/or SSc-specific antibodies without other signs and symptoms of disease), 48 definitive SSc patients (defSSc) according to the 2013 ACR/EULAR criteria without skin fibrosis, 51 limited cutaneous (lcSSc) and 35 diffuse cutaneous (dcSSc) subjects. Results The four groups of patients showed well-distinct clinical and laboratory characteristics with a linear trend of decrease in FVC and DLco % predicted levels from EaSSc to defSSc to lcSSc and to dcSSc patients, and a linear trend of increase in CRP, ESR, gamamglobulin serum levels as well as the prevalence of lung fibrosis on the other way around. Serum concentrations of the studied analytes are reported in the table; for most vascular factors a characteristics linear trend (ANOVA polynomial test for trend Conclusions Our study demonstrate, for the first time, that circulating levels of vascular involvement start to increase in SSc patients from the earlies stages of the disease when clinical and laboratory findings of advanced disease cannot yet be detected. These abnormalities progress in parallel with the appraisal of the first sclerodermatous manifestation in defSSc and steadly increase with the onset of fibrotic manifestations. Acknowledgements The present work was financed by a grant from GILS (Gruppo Italiano per la Lotta alla Sclerodermia), theme “La diagnosi precoce della sclerodermia”. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is a chronic connective tissue disorder with the highest mortality of any autoimmune disease. SSc is a complex disease with a clear genetic component. This genetic susceptibility has been supported by a number of well-powered genetic association studies. In this regard, it is remarkable that several SSc genetic markers have been reported in the IL-12 pathway ( IL12A , STAT4 , IL12RB2 , IL12RB1 ). Furthermore, multiple clinical and experimental evidences have shown that this pathway is altered in SSc patients. TYK2 encodes the Tyrosine kinase 2 enzyme, which mediates the signaling of IL-12 receptor. Objectives We aimed to analyze the association of the TYK2 locus with SSc susceptibility. Methods The complete set of individuals included in this study reached 4,985 SSc patients and 11,621 healthy controls of European ancestry from Spain, Germany, The Netherlands, USA, Italy and United Kingdom. Initially, we analyzed all the polymorphisms located in the region that encompasses the TYK2 coding sequence and 20 kb up and downstream, in a previously published Immunochip-based dense genotyping study. We identified a common single nucleotide polymorphism (SNP), the V362F variant (rs2304256) and three rare variants: P1104A (rs34536443), A928V (rs35018800) and I684S (rs12720356), as the genetic markers that better explained the observed association in the region. Then, these variants were followed up in additional cohorts. Association and dependence relations were tested using logistic regression and conditional logistic regression, and pooled analyses were performed using the inverse variance method. Results Our pooled analysis showed that V362F variant reached the genome-wide significance level ( P =2.00x10 -10 , OR =0.84), while P1104A (rs34536443), A928V (rs35018800) and I684S (rs12720356) remained significant ( P =8.61x10 -3 , OR=0.79; P =1.95x10 -4 , OR=0.49; P random =0.016, OR=0.84, respectively). The analyses carried out for the main clinical features revealed that the observed association signals relied on the whole disease. Furthermore, our results revealed that the association of TYK2 with SSc was dependent on the interaction between the P1104A, A928V and I684S rare variants and that the previously observed association for the autoimmune related V362F variant, corresponded to a synthetic association dependent on the association of the three previously mentioned rare variants. Conclusions We report for the first time the association of TYK2 variants with SSc. Moreover, our data support that the highly significant association of a previously known autoimmune disease marker, the V362F variant (rs2304256), is dependent on the effect of three non-synonymous rare variants in this locus . Disclosure of Interest None declared

Jaccoud arthropathy (JA) is a non-erosive, deforming arthropathy of the metacarpophalangeal (MCP) and proximal interphalangeal joints, wrists and knees, first described as a rare complication of recurrent rheumatic fever1 and then extensively observed in systemic lupus erythaematosus (SLE) patients.2 3 Hand deformities present a typical picture of ulnar deviation and subluxation of the MCP, as observed in rheumatoid arthritis (RA). However, in JA, ligament laxity and muscle imbalance rather than the loss of bone and joint instability secondary to synovitis, determine the clinical picture.3 4 This hypothesis has been confirmed in an MRI study, as no signs of synovial …