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BackgroundIn up to 20% of patients with systemic sclerosis (SSc) none of the established SSc-specific autoantibodies are present [1]. Notwithstanding, in many of these patients high-titer autoantibodies can be detected on the HEp-2 indirect immunofluorescence assay (HEp-2 IIFA) which suggests the presence of an autoantibody to an intracellular protein expressed by the HEp-2 cell line. Immunoprecipitation of unlabeled cell extract followed by gel-free liquid chromatography tandem mass-spectrometry analysis has the potential to identify new autoantigens in an unbiased manner.ObjectivesTo identify new autoantigens through immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP + LC-MS/MS) in HEp-2 IIFA-positive patients with SSc in whom none of the established SSc-specific autoantibody specificities are present.MethodsForty-nine patients from the University Hospitals Leuven that fulfilled the EULAR-ACR 2013 classification criteria for systemic sclerosis or LeRoy and Medsger’s criteria for early systemic sclerosis and who were negative on the EliA CTD Screen (Thermo Fisher Scientific, United States), which includes centromere protein B, topoisomerase I, RNA polymerase III, fibrillarin, PM-Scl and U1 ribonucleoprotein, were identified. Immunoprecipitation was performed by incubation of sera of these patients (1/30 in 300 µl Tris-buffered saline) with Pierce A/G magnetic beads, subsequent cross-linking with bissulfosuccinimidyl suberate (BS3) followed by incubation with nuclear extract of HeLa cells (100-150 µg) overnight at 4°C. The eluted protein was analyzed through liquid chromatography with tandem mass spectrometry. Mass spectrometry data were matched against the Uniprot Homo Sapiens database with the Mascot search engine. Candidate autoantigens were confirmed through immunoprecipitation followed by western blot of the eluate with target-specific polyclonal rabbit antibodies or western blot of recombinant protein incubated with sera of the index patients.ResultsWe identified multiple new autoantigens, including the THO complex subunit 1 (THOC1) and other subunits of the THO complex in 3 patients, nuclear valosin-containing protein like-2 (NVL) in 2 patients, nucleolar and coiled-body phosphoprotein 1 (NOLC1) and multiple interacting proteins in 1 patient, probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2) in 1 patient, telomeric repeat-binding factor 2 (TERF2) and TERF2-interacting protein (TERF2IP) in 1 patient and regulator of chromosome condensation 1 (RCC1) in 1 patient. The new targets were confirmed through immunoprecipitation-western blot or western blot of recombinant protein incubated with sera (Figure 1). Furthermore, in 10 patients known SSc-associated autoantigens were strongly immunoprecipitated including multiple Th/To subunits in 5 patients, RuvBL1/2 in 2 patients, multiple PM-Scl subunits in 2 patients (who both were negative on the EliA CTD Screen), and fibrillarin in 1 patient (who was also negative on the EliA CTD .).Figure 1.Immunoprecipitation-western blot with target-specific rabbit polyclonal antibody (1/500-2000 dilution), numbers corresponding to order of description of patients, HC healthy control. NE nuclear extract. RP recombinant protein WB-RP western blot of recombinant protein incubated with patient’s sera.ConclusionMultiple new autoantigens were identified and confirmed in patients with SSc without previously identified autoantibody specificity. Further evaluation of reactivity against the newly identified autoantigens in patients with SSc with known autoantibody specificities and other cohorts is required. IP + LC-MS/MS can identify new and established autoantigens in patients with SSc.References[1]Meier FMP, Frommer KW, Dinser R, et al. Update on the profile of the EUSTAR cohort: an analysis of the EULAR Scleroderma Trials and Research group database. Ann Rheum Dis 2012;71:1355–60. doi:10.1136/annrheumdis-2011-200742Disclosure of InterestsJean-Baptiste Vulsteke: None declared, Daniel Blockmans: None declared, Petra De Haes: None declared, Steven Vanderschueren: None declared, Patrick Verschueren: None declared, Kristl G Claeys: None declared, Wim Wuyts Grant/research support from: Boehringer-Ingelheim, Galapagos, Roche, Jan Leo Lenaerts: None declared, Ellen De Langhe: None declared, Xavier Bossuyt Consultant of: Inova Diagnostics, Thermo Fisher Scientific.

BackgroundAnti-transcriptional intermediary factor 1-gamma (TIF1-gamma) autoantibodies are strongly linked to cancer-associated dermatomyositis (DM). In addition to anti-TIF1-gamma autoantibodies, autoantibodies to TIF1-alpha and TIF1-beta have been described, often coexisting with TIF1-gamma or other known autoantibodies [1,2]. Thus far, anti-TIF1-beta autoantibodies without other known autoantibodies have been identified in only 3 patients with dermatomyositis, of which none had cancer [1,2].ObjectivesTo report on a patient with cancer-associated dermatomyositis and isolated anti-TIF1-beta autoantibodies.MethodsSerum of a patient with cancer-associated dermatomyositis without known autoantibody specificity was evaluated by immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP + LC-MS/MS). Mass spectrometry data were matched against the Uniprot Homo Sapiens database with the Mascot search engine using Proteome Discoverer. Additional immunoprecipitation of radiolabeled cell extract followed by autoradiography were performed. Clinical and laboratory data were retrieved from the electronic health record.ResultsTIF1-beta was identified in the immunoprecipitate of the serum of the patient by IP + LC-MS/MS, but not TIF1-gamma or TIF1-alpha. These results were confirmed by immunoprecipitation of radiolabeled cell extract (Figure 1A). The HEp-2 indirect immunofluorescence assay showed a nuclear fine speckled pattern (AC-4, maximum titre > 1/1280, Figure 1B), which corresponds to the subcellular localization of TIF1-beta [3]. The patient, a 64-year old male, presented with dermatomyositis with classical cutaneous and muscular involvement (max. serum creatine kinase level 370 U/l). A renal cell carcinoma was found during the diagnostic work-up for which a partial nephrectomy was performed. There was a good response of both cutaneous and muscular disease activity to treatment with hydroxychloroquine and methylprednisolone.Figure 1.A Immunoprecipitation with 35S-methionine labeled K562 cells with subsequent radiography. C Positive control with anti-TIF1-beta and anti-U1-RNP autoantibodies, 1 anti-TIF1-beta-positive patient (patient here described), 2 negative patient, 3 anti-TIF1-gamma/alpha-positive patient B Nuclear fine speckled pattern on HEp-2 indirect immunofluorescence assay, 40X magnificationConclusionIsolated anti-TIF1-beta autoantibodies should be considered in patients with dermatomyositis without known myositis-specific autoantibodies and can be associated with cancer.References[1]Fujimoto M, Hamaguchi Y, Kaji K, et al. Myositis-specific anti-155/140 autoantibodies target transcription intermediary factor 1 family proteins. Arthritis Rheum 2012;64:513–22. doi:10.1002/art.33403[2]Satoh M, Chan JYF, Ross SJ, et al. Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis. Arthritis Res Ther 2012;14:1–8. doi:10.1186/AR3802/FIGURES/2[3]Thul PJ, Åkesson L, Wiking M, et al. A subcellular map of the human proteome. Science 2017;356. doi:10.1126/science.aal3321Disclosure of InterestsJean-Baptiste Vulsteke: None declared, Petra De Haes: None declared, Minoru Satoh: None declared, Ellen De Langhe: None declared, Xavier Bossuyt Consultant of: Inova Diagnostics, Thermo Fisher Scientific

Background:RISE-SSc (NCT02283762) was a multicenter, double-blind, Phase IIb study of riociguat in early dcSSc. Primary endpoint was change in mRSS from baseline to Wk 52.Objectives:Exploratory, descriptive analyses of riociguat target engagement and effects on disease biomarkers in RISE-SSc and their relationship with effects on the primary endpoint. All biomarker p-values are for information only.Methods:Pts with dcSSc (duration ≤18 mo; modified Rodnan skin score [mRSS] 10–22 units) were randomized to riociguat 0.5−2.5 mg tid (n=60) or placebo (n=61). Biomarkers of target engagement (cGMP), inflammation and/or vascular/endothelial function (e.g. high-sensitivity C-reactive protein [hsCRP], soluble platelet endothelial cell adhesion molecule 1 [sPECAM-1], soluble E-selectin, chemokine ligand 4 [CXCL-4]), and fibrosis (e.g. alpha-smooth muscle cell actin [alphaSMA], pro-collagen mRNA expression) were measured in plasma, serum, and skin biopsies at baseline and Wk 14.Results:Mean±SD change from baseline in mRSS was –2.09±5.66 (n=57) with riociguat and –0.77±8.24 (n=52) with placebo (p=0.08). From baseline to Wk 14, plasma cGMP rose by mean (SD) 94% (78%) (n=52) with riociguat and 10% (39%) (n=52) with placebo (nominal pConclusion:Primary study endpoint (change in mRSS) was not met. Plasma cGMP rose with riociguat, confirming engagement with the NO-sGC-cGMP pathway. Serum sPECAM-1 (marker of endothelial activation) and CXCL-4 (marker of progressive SSc) fell with riociguat; hsCRP and E-selectin did not. Some serum and skin biomarkers of higher disease activity at baseline were associated with a greater effect of riociguat on skin fibrosis.Acknowledgments:RISE-SSc was jointly funded by Bayer AG and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.Disclosure of Interests:Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Frank Kramer Employee of: Bayer AG, Josef Höfler Employee of: Josef Höfler is an employee of Staburo GmbH, Munich, Germany, contracted by Bayer AG to perform the biomarker analyses, Mercedeh Ghadessi Employee of: Bayer AG, Peter Sandner Employee of: Bayer AG, Yannick Allanore Grant/research support from: BMS, Inventiva, Roche, Sanofi, Consultant of: Actelion, Bayer AG, BMS, BI, Christopher Denton Grant/research support from: GlaxoSmithKline, CSL Behring, and Inventiva, Consultant of: Medscape, Roche-Genentech, Actelion, GlaxoSmithKline, Sanofi Aventis, Inventiva, CSL Behring, Boehringer Ingelheim, Corbus Pharmaceuticals, Acceleron, Curzion and Bayer, Masataka Kuwana Grant/research support from: Acetelion, Consultant of: Acetelion, Bayer, Chugai, Corbus Pharmaceuticals, CSL Behring and Reata Pharmaceuticals. He was a member of the SENSCIS trial Steering Committee (Boehringer Ingelheim), Marco Matucci-Cerinic Grant/research support from: Actelion, MSD, Bristol-Myers Squibb, Speakers bureau: Acetelion, Lilly, Boehringer Ingelheim, Janet Pope Grant/research support from: AbbVie, Bristol-Myers Squibb, Eli Lilly & Company, Merck, Roche, Seattle Genetics, UCB, Consultant of: AbbVie, Actelion, Amgen, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eicos Sciences, Eli Lilly & Company, Emerald, Gilead Sciences, Inc., Janssen, Merck, Novartis, Pfizer, Roche, Sandoz, Sanofi, UCB, Speakers bureau: UCB, Tatsuya Atsumi Grant/research support from: Eli Lily Japan K.K., Alexion Pharmaceuticals, Inc., Bristol-Myers Squibb Co., AbbVie Inc., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Astellas Pharma Inc., Consultant of: Gilead Sciences, Inc., Eli Lilly Japan K.K., UCB Japan Co. Ltd., AbbVie Inc., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Speakers bureau: Eli Lilly Japan K.K., UCB Japan Co. Ltd., Bristol-Myers Squibb Co., AbbVie Inc., Eisai Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Takeda Pharmaceutical Co., Ltd., Astellas Pharma Inc., Radim Bečvář Consultant of: Actelion, Roche, László Czirják Consultant of: Actelion, BI, Roche-Genentech, Lilly, Medac, Novartis, Pfizer, Bayer AG, Ellen De Langhe Consultant of: member of advisory board for Boehringer, Eric Hachlla: None declared, Tomonori Ishii: None declared, Osamu Ishikawa: None declared, Sindhu Johnson Grant/research support from: Boehringer Ingelheim, Corbus Pharmaceuticals, GlaxoSmithKline, Roche, Merck, Bayer, Consultant of: Boehringer Ingelheim, Ikaria, Kaisa Laapas Employee of: Partly in-sourced to Bayer, Valeria Riccieri: None declared, Elena Schiopu: None declared, Richard Silver: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Chiara Stagnaro: None declared, Viginia Steen Grant/research support from: The associated affiliation has received grants/research from Boehringer Ingelheim, Corbus Pharmaceuticals, CSL Behring, Eicos, Galapagos, Immune Tolerance Network, Reata, Consultant of: Virginia Steen has acted as a consultant for Boehringer Ingelheim, Corbus, CSL Behring, Eicos, Forbius, Wendy Stevens: None declared, Gabriella Szücs: None declared, Marie-Elise Truchetet: None declared, Melanie Wosnitza Employee of: Bayer AG, Dinesh Khanna Shareholder of: Eicos Sciences, Inc./Civi Biopharma, Inc., Grant/research support from: Dr Khanna was supported by NIH/NIAMS K24AR063120, Consultant of: Acceleron, Actelion, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Corbus Pharmaceuticals, Horizon Therapeutic, Galapagos, Roche/Genentech, GlaxoSmithKline, Mitsubishi Tanabe, Sanofi-Aventis/Genzyme, UCB

Background:The presence of myositis specific anti-melanoma differentiation associated protein 5 (MDA5) autoantibodies is associated with mucocutaneous ulcerations, rapidly progressing interstitial lung disease (RPILD), arthritis and mild muscle involvement in patients. RPILD is the major cause of mortality. At present it is unknown which domain of the MDA5 protein is the main elicitor of an immunogenic response.Objectives:The aim of this study is to delineate the domains in the MDA5 protein that are the target of autoantibodies.Methods:Anti-MDA5 IgG were isolated from MDA5(+) patient plasma (7 UPMC, 1 KI and 1 KULeuven) by affinity chromatography using an in-house affinity column as described earlier in Ossipova et al, 2014(1). 8 constructs covering different regions of the MDA5 protein were recombinantly produced in E.coli (Uniprot ID Q9BYX4, Figure 1). An in-house ELISA was developed to identify the domains with the main epitope(s) by measuring the reactivity of the plasma samples and purified autoantibodies against these MDA5 protein constructs, similar to what was reported by Fernandes-Cerqueira et al, 2018(2). The biotinylated MDA5 proteins were immobilized on streptavidin coated plates and subsequently incubated with primary antibodies (purified autoantibodies(2) or original plasma) and a HRP-conjugated secondary antibody. The ELISA was developed by the addition of TMB substrate and the optical density (OD) was measured at 450 nm.Figure 1.Graphical presentation of the constructs representing different (combinations of) domains of the MDA5 protein.Results:The preliminary data suggest the main reactivity of the plasma samples and the corresponding purified autoantibodies is directed towards the helicase domains and that there is variability between the patients in the reactivity towards domains located at the end of the protein.Conclusion:The study aims to resolve the main immunogenic domain of the MDA5 protein, which will lead to more insight in the disease mechanisms. The preliminary results suggest this domain is in the center of the MDA5 protein, but further experiments are necessary. We will use this set up to study differences in reactivity between patients (from different cohorts) and assess if differences in antibody reactivity could be linked to clinical features such as RPILD. Such correlations might be beneficial to predict the disease progression and to apply personal treatment approaches.References:[1]Ossipova E, Cerqueira CF, Reed E, Kharlamova N, Israelsson L, et al. Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther. 2014;16(4):R167.[2]Fernandes-Cerqueira C, Renard N, Notarnicola A, Wigren E, Gräslund S, et al. Patients with anti-Jo1 antibodies display a characteristic IgG Fc-glycan profile which is further enhanced in anti-Jo1 autoantibodies. Scientific reports. 2018;8(1):17958.Disclosure of Interests:Eveline Van Gompel: None declared, Catia Cerqueira: None declared, Edvard Wigren: None declared, Susanne Gräslund: None declared, Karine Chemin: None declared, Begum Horuluoglu: None declared, Ellen De Langhe: None declared, Olivier Benveniste: None declared, Ingrid E. Lundberg Consultant of: Consulting fees from Corbus Pharmaceuticals, Inc, Grant/research support from: Research grants from Bristol Myers Squibb and AstraZeneca.

Background:Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the main cause of death in SSc and accounts for up to 30-35% of SSc-mortality (1-2). All SSc cases, irrespective of the extent of the skin disease, should be evaluated for ILD (3). The epidemiology of SSc-ILD in Belgium is unknown. In literature, the prevalence of ILD in SSc varies between 19% and 52%. However, different criteria were used to diagnose ILD (4). In 2008, Goh et al. proposed a flow diagram to diagnose SSc-ILD based on chest high-resolution CT-scan (HRCT) and pulmonary function tests (PFTs). Their categorization into limited or extensive ILD has prognostic value (5).Objectives:To determine the prevalence and incidence rate of SSc-ILD in Flanders.Methods:Up to 12-year follow-up data of consecutive SSc patients were obtained by 2 Flemish expert centres (University Hospitals Ghent and Leuven). Patients fulfilling the LeRoy and/or ACR-EULAR classification criteria were included consecutively in the prospective cohort (6). Patients received HRCT at baseline and on indication thereafter, as well as yearly PFT. All HRCTs were centrally analyzed (Ghent) and patients were categorized according to the Goh criteria as without ILD, with limited ILD (limILD) or with extensive ILD (extILD) (5).Results:Between 2006 and 2018, 797 SSc patients (557 Ghent/240 Leuven; 22% limited SSc (LSSc)/59% limited cutaneous SSc (LcSSc)/19% diffuse cutaneous SSc (DcSSc)) had baseline HRCT and PFT. The baseline characteristics are depicted in the table. The mean age (SD) was 53 +/-15 years and the majority of patients was female (76%).272 SSc patients had ILD at baseline, implicating a baseline prevalence of 34% (272/797). The baseline prevalences were 35% and 55% for the LcSSc and DcSSc subgroups respectively. During a median follow-up of 39 months (IQR: 11-79 months), 44 patients were diagnosed with incidental SSc-ILD, resulting in an incidence rate of 21,0/1000 person-years (PY), 95% CI:15,2-28,1. The incidence rates were 21,7/1000 PY, 95%CI: 14,3-31,6 and 43.9/1000PY, 95%CI: 22.7-76.8 for the LcSSc and DcSSc subgroups respectively.Table.Baseline characteristicsSSc (n=797)LcSSc (n=470)DcSSc (n=149)age (years) °53+/-1554+/-1554+/-14♂/♀ *193(24%)/604(76%)109(23%)/361(77%)58(39%)/91(61%)Disease Duration (months) #for 718: 22 (5-72)for 443: 25 (5-80)for 145: 16 (7-52)LSSc/LcSSc/DcSSc *178(22%)/470(59%)/149(19%)follow-up (months) #39 (11-79)38.5 (9.75-81)44 (17.5-78)Anti-centromere antibodies§252/538 (47%)163/317 (51%)19/108 (18%)Anti-topoisomeraseI antibodies§119/519 (23%)66/297 (22%)45/112 (40%)ILD at baseline, *272 (34%)163 (35%)82 (55%)LimILD, *230 (29%)139 (30%)67 (45%)ExtILD, *42 (5%)24 (5%)15 (10%)New ILD during follow-up, §44/52527/30712/67°: mean +/- standard deviation, *: number of patients (percent), #: median (interquartile range), §= number of patients/total number of patients with available data (%)Conclusion:In an unselected cohort of SSc patients, a third of the patients has ILD at baseline which is in line with previous prevalence reports. Importantly, this is the first study reporting incidence rates of SSc-ILD.References:[1]Steen VD and Medsger TA, Ann Rheum Dis 2007;66:940-4[2]Elhai M et al. Ann Rheum Dis 2017;76:1897-1905[3]Smith V et al. RMD Open 2019;4:e000782. doi:10.1136/rmdopen-2018-000782[4]Bergamasco A et al. Clinical Epidemiology 2019;11:257-73[5]Goh N et al. Am J Respir Crit Care Med 2008;177:1248-54[6]van den Hoogen et al. Arthritis Rheumatol 2013;65:2737-47Disclosure of Interests:Els Vandecasteele Grant/research support from: my institution has received a research grant from the Research Foundation Flanders FWO), Speakers bureau: my institution has received speaker fees from Actelion, Karin Melsens: None declared, Daniel Blockmans Consultant of: yes, Speakers bureau: yes, Charlotte Carton: None declared, Filip De Keyser: None declared, Ellen De Langhe Consultant of: member of advisory board for Boehringer, Bernard Lauwerys: None declared, Yves Piette: None declared, Amber Vanhaecke: None declared, Koen Verbeke: None declared, Wim Wuyts Grant/research support from: my institution has received a grant from Boehringer Ingelheim and Roche, Consultant of: my institution has received payments for consultancy from Boehringer Ingelheim and Roche, Speakers bureau: my institution has received speaker fees from Boehringer Ingelheim and Roche, Guy Brusselle: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl

Background:RISE-SSc (NCT02283762) was a multicenter Phase IIb trial of riociguat in pts with early (duration ≤18 months) dcSSc and modified Rodnan skin score (mRSS) 10−22 units. Pts were randomized double-blind to placebo or riociguat 0.5–2.5 mg t.i.d. for 52 weeks. The primary endpoint, mRSS change from baseline to Week (Wk) 52, did not reach statistical significance (p=0.08, riociguat vs placebo), but there were favorable trends in some other outcomes.Objectives:To present open-label long-term extension (LTE) results of RISE-SSc.Methods:Pts who completed Wk 52 of double-blind therapy could enter LTE on riociguat. Endpoints included mRSS, adverse events (AEs), and serious AEs (SAEs).Results:Of 60 pts randomized to riociguat and 61 to placebo, 42 (riociguat−riociguat group) and 45 (former placebo group), respectively, entered LTE. At LTE start, mean±SD mRSS was 16.4±3.2 and 16.3±4.2 units, and mean disease duration was 8.9±7.8 and 8.9±5.8 months, in the riociguat−riociguat and former placebo groups, respectively. Other demographics/disease characteristics were also comparable. Median duration of riociguat treatment was 1092 d in riociguat−riociguat pts and 649 d in former placebo pts. Throughout the study, mRSS decreased in both groups (Figure 1). From Wk 52 to last visit, mRSS fell by −3.02±5.51 in riociguat−riociguat patients and −3.96±5.43 in former placebo pts. Rates of mRSS regression (decrease by >5 units and ≥25% from Wk 52 to last visit) and of % declines in mRSS were similar in the two groups (Figure 2). mRSS progression (increase by >5 units and ≥25% from Wk 52 to last visit) occurred in 1 pt (2%) in each group. During the entire study, rescue therapy agents were used in 15 (36%) riociguat−riociguat pts and 17 (38%) former placebo pts. AEs were reported from Wk 52 to last visit in 82 pts (94%): 40 (95%) riociguat−riociguat and 42 (93%) former placebo. Most common AEs overall: nasopharyngitis (24%), gastroesophageal reflux disease (17%), diarrhea (15%), and hypotension (14%). AEs of special interest (dizziness, postural dizziness, or hypotension) occurred in 5 riociguat−riociguat pts (12%) and 4 former placebo pts (9%). SAEs were reported in 21 (24%) pts: 10 (24%) riociguat−riociguat pts and 11 (24%) former placebo pts, with no SAE reported in >1 patient, no SAEs of special interest, and no deaths.Conclusion:During LTE riociguat treatment, mRSS decreased in both groups from Wk 52 onwards and mRSS progression was uncommon. Riociguat had acceptable safety, similar to the main study, with no new safety signal.Acknowledgments:RISE-SSc was jointly funded by Bayer AG and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.Disclosure of Interests:Dinesh Khanna Shareholder of: Eicos, Grant/research support from: NIH NIAID, NIH NIAMS, Consultant of: Acceleron, Actelion, Bayer, BMS, Boehringer-Ingelheim, Corbus, Galapagos, Genentech/Roche, GSK, Mitsubishi Tanabi, Sanofi-Aventis/Genzyme, UCB Pharma, Janet Pope Grant/research support from: AbbVie, Bristol-Myers Squibb, Eli Lilly & Company, Merck, Roche, Seattle Genetics, UCB, Consultant of: AbbVie, Actelion, Amgen, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eicos Sciences, Eli Lilly & Company, Emerald, Gilead Sciences, Inc., Janssen, Merck, Novartis, Pfizer, Roche, Sandoz, Sanofi, UCB, Speakers bureau: UCB, Marco Matucci-Cerinic Grant/research support from: Actelion, MSD, Bristol-Myers Squibb, Speakers bureau: Acetelion, Lilly, Boehringer Ingelheim, Masataka Kuwana Grant/research support from: Acetelion, Consultant of: Acetelion, Bayer, Chugai, Corbus Pharmaceuticals, CSL Behring and Reata Pharmaceuticals. He was a member of the SENSCIS trial Steering Committee (Boehringer Ingelheim), Christopher Denton Grant/research support from: GlaxoSmithKline, CSL Behring, and Inventiva, Consultant of: Medscape, Roche-Genentech, Actelion, GlaxoSmithKline, Sanofi Aventis, Inventiva, CSL Behring, Boehringer Ingelheim, Corbus Pharmaceuticals, Acceleron, Curzion and Bayer, Yannick Allanore Grant/research support from: BMS, Inventiva, Roche, Sanofi, Consultant of: Actelion, Bayer AG, BMS, BI, Melanie Wosnitza Employee of: Bayer AG, Marie-Elise Truchetet: None declared, Gabriella Szücs: None declared, Wendy Stevens: None declared, Viginia Steen Grant/research support from: The associated affiliation has received grants/research from Boehringer Ingelheim, Corbus Pharmaceuticals, CSL Behring, Eicos, Galapagos, Immune Tolerance Network, Reata, Consultant of: Virginia Steen has acted as a consultant for Boehringer Ingelheim, Corbus, CSL Behring, Eicos, Forbius, Chiara Stagnaro: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Richard Silver: None declared, Elena Schiopu: None declared, Valeria Riccieri: None declared, Frank Kramer Employee of: Bayer AG, Sindhu Johnson Grant/research support from: Boehringer Ingelheim, Corbus Pharmaceuticals, GlaxoSmithKline, Roche, Merck, Bayer, Consultant of: Boehringer Ingelheim, Ikaria, Osamu Ishikawa: None declared, Tomonori Ishii: None declared, Eric Hachlla: None declared, Ellen De Langhe Consultant of: member of advisory board for Boehringer, László Czirják Consultant of: Actelion, BI, Roche-Genentech, Lilly, Medac, Novartis, Pfizer, Bayer AG, Radim Bečvář Consultant of: Actelion, Roche, Tatsuya Atsumi Grant/research support from: Eli Lily Japan K.K., Alexion Pharmaceuticals, Inc., Bristol-Myers Squibb Co., AbbVie Inc., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Astellas Pharma Inc., Consultant of: Gilead Sciences, Inc., Eli Lilly Japan K.K., UCB Japan Co. Ltd., AbbVie Inc., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Speakers bureau: Eli Lilly Japan K.K., UCB Japan Co. Ltd., Bristol-Myers Squibb Co., AbbVie Inc., Eisai Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Pfizer Inc., Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Takeda Pharmaceutical Co., Ltd., Astellas Pharma Inc., Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche

Background and objectives Fibrosis is the formation of excess fibrous connective tissue. It disturbs cell, tissue and organ homeostasis, leading to severe disease manifestations in various disorders. Transcriptional regulation of genes and pathways with pro- or anti-fibrotic effects is therefore a therapeutic target. DOT1-like histone H3 methyltransferase (DOT1L) is the only known H3K79 methyltransferase. It functions as a positive mediator of gene transcription, of DNA repair, as a regulator of the cell cycle, and has been associated with Wnt signalling. Our aim is to study the role of DOT1L in downstream events associated with pro-fibrotic cascades, hypothesising that it may act as an epigenetic masterswitch in the fibrotic process. Materials and methods MRC5 cells (a human fetal lung fibroblast cell line) were cultured for up to 22 days with 0 and 3µM of EPZ04777 (DOT1L inhibitor). During that period, the cells were stimulated at different time-ponts, with 0 to 5 ng/mL TGFβ 1 for 24 h. We performed mRNA and protein expression analysis. Total RNA was extracted from cell lysates and reverse-transcribed, and gene expression levels were quantified with real-time quantitative PCR (qPCR). Expression was normalised to Hprt and subsequently normalised to the vehicle condition using the comparative cycle threshold method (ΔΔCT). Total cell lysates were homogenised in Cell Extraction Buffer and proteins were analysed by immunoblot, including assessment of the H3K79 methylation status. Results Long-term treatment progressively decreases the methylation status of H3K79. Upon stimulation with TGFβ1, MRC5 cells show upregulation of type I Collagen mRNA expression and collagen deposition. Moreover, αSMA mRNA expression is also increased, indicating a fibroblast to myofibroblast differentiation. TGFβ1 stimulation also increased DOT1L expression and activity in the absence of EPZ04777, as shown by qPCR and H3K79 methylation status. Upon treatment with EPZ04777, we also observed an increase in collagen and αSMA expression, suggesting that loss of DOT1L activity results in pro-fibrotic changes in lung fibroblasts. Conclusions DOT1L has homeostatic effects in the healthy lung fibroblast, since its inhibition triggers a pro-fibrotic transcriptional response. Its upregulation by TGFβ1 could represent a regulatory feedback loop that requires further investigation.

Background Pulmonary fibrosis, either idiopathic or secondary to diseases such as systemic sclerosis, is a devastating and life threatening disorder for which effective treatment is still lacking. The bleomycin-induced pulmonary fibrosis model is well-characterized and the most widely used mouse model. The resulting fibrosis is routinely quantified by labor-intensive end-stage histological assessments, requiring many animals and lack the ability to follow-up on disease progression and potential therapeutic effects in the individual animal. At present, imaging tools for the evaluation of lung disease with good temporal and spatial resolution in vivo are limited. Objectives To optimize and evaluate lung MRI protocols to visualize disease onset and progression in the bleomycin-induced model of lung fibrosis. We compared prospectively and retrospectively gated MRI sequences and validated our results with established CT imaging of lung fibrosis and histochemical techniques. Methods Animal Model: Male C57Bl/6 mice were intratracheally instilled with bleomycin (0.05U in 50 µl of PBS) or sham. The mice were scanned with MRI and CT at baseline and weekly until 3 weeks after instillation. After the last imaging time point, mice were sacrificed, ex vivo CT data were acquired and the lungs were isolated for histological analysis and quantification as described before 3. MRI images were acquired at 9.4T (Bruker Biospin, 20 cm) in combination with a 7.5cm quadrature coil, using the following sequences: (1) a respiratory triggered RARE sequence (TR 6000ms TEeff=15.9ms, 50slices of 0.5mm thick), (2) a respiratory triggered ultra short echo (UTE) sequence (FID mode, TR= 20ms, TE=0.4ms, 8 slices, 0.6mm slice thickness) and (3) a retrospectively gated FLASH sequence IntraGate ( TR/TE = 30/1.26 ms, 17 deg flip angle, 5 slices covering the lung, slice thickness 1 mm). For reconstruction, 70% of the respiration and ECG period was used (Paravision 5.1, Bruker)). MRI data were quantified using ImageJ. CT methods: retrospectively gated CT images were acquired on a small animal µCT scanner (SkyScan 1076, Bruker microCT) and quantified. Results The prospectively gated UTE and RARE protocols as well as retrospectively gated IntraGate-FLASH imaging were able to visualize an increase of hyperintense focal spots over time, corresponding to progression of lung fibrosis as corroborated by lung CT images. Quantification of the mean lung signal intensity shows an increase over time, which was confirmed by the decrease in aerated lung volume quantified from the CT data and by histology. UTE, RARE and IntraGate-FLASH images of control animals confirmed the absence of contrast without fibrosis induction. Conclusions The evaluated MRI protocols were all able to non-invasively visualize and quantify lung disease progression. Moreover, the IntraGate-FLASH protocol does not need setup of respiratory triggering for lung imaging, making it an easy to use and efficient alternative to more conventional sequences. Where CT provides poor soft tissue contrast, MRI has the potential to provide contrast differences between vasculature, fibrotic areas and inflammation, without concerns for radiotoxicity when scanning the same animal repeatedly. Disclosure of Interest None Declared