Abstract: Sjögren’s syndrome (SS) is a systemic autoimmune disease whose main characteristic is involvement of the exocrine glandular system. Thus, its most common clinical manifestation is eye and mouth dryness which, alongside fatigue and pain, contributes to poor health-related quality of life (HRQoL). A growing body of evidence recognises the adipose tissue as an active endocrine organ secreting bioactive mediators involved in metabolic and inflammatory disorders. A relationship between obesity and symptoms in SS has not yet been elucidated. Objectives: To explore potential associations between obesity-related immune-metabolic biomarkers and clinical symptoms in SS and sicca patients. Methods: Proteomics analysis of 184 cardio-immuno-metabolic proteins was assessed on sera from 53 SS (50 females (F), 3 males (M); mean age 54 years) and 60 sicca (56 F, 4 M; mean age 57 years) patients. Participants were enrolled in the Birmingham Optimising Assessment in Sjögren`s Syndrome (OASIS) cohort and examinations included the EULAR SS Patient Reported Index (ESSPRI), Schirmer’s test, unstimulated whole saliva, minor labial salivary gland biopsy, EuroQoL-5 dimension (EQ-5D) tool, immunological parameters, body max index (BMI) and Hb1Ac. Participants with SS fulfilled 2016 ACR/EULAR criteria. Non-SS sicca patients were anti-Ro antibody negative, displayed objective oral and/or ocular dryness, and did not have a physician diagnosis for SS. Linear regression analysis and univariate models were performed to identify the key predictors of symptoms. Results: HRQoL as measured as EQ-5D utility values, symptoms as assessed by ESSPRI, and BMI did not differ between the SS and sicca group. However, strong correlations between BMI (or fat or fat-free mass) and EQ-5D and ESSPRI scores were found in the sicca but not in the SS group. Among several proteins investigated, ADM, TNFRSF13B, FGF23, IL10RB, CD5, CD40, IL1RA, TNRSFN9, TNFRSF10A, TNFRSF11A, TRAILR2, GAL9, SPON2, ACE2, LEP, CD4, IL12B, SLAMF1, PD-L1 positively correlated with symptoms in the sicca group (R ≥ 0.3; Holm’s adjusted P ≤ 0.05). Adrenomedullin (ADM) showed the strongest correlation with ESSPRI (R = 0.56; P 〈 0.0001); age (R = 0.45; P = 0.0003); BMI (R = 0.43, P = 0.0008); Ocular Surface Disease Index (R = 0.32, P = 0.03); EQ-5D utility value (R = -0.45, P = 0.001) and VAS patient (R = -0.41, P = 0.008). There was no association between ADM and gender, Schirmer`s test, disease and symptom duration (P 〉 0.05). ADM was independently associated with ESSPRI scores in non-SS sicca patients when corrected for BMI, age, HbA1c, depression and anxiety scores. None of the above mentioned proteins correlated with clinical symptoms in the SS group. Conclusion: The study suggests that obesity-related immune-metabolic factors may play a role in regulating the symptoms in non-SS sicca patients. ADM appears to be a strong independent predictor of symptoms in these patients but not in SS. Disclosure of Interests: Valentina Pucino: None declared, Jason D. Turner: None declared, Florian Kollert Employee of: Novartis, Saaeha Rauz: None declared, Andrea Richard: None declared, Jon Higham: None declared, Ana Poveda-Gallego: None declared, Simon J. Bowman Consultant of: Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio, Glapagos, Speakers bureau: Novartis, Francesca Barone: None declared, Benjamin Fisher: None declared

Abstract: The travel distance from home to a treatment centre, which may impact the stage at diagnosis, has not been investigated for retinoblastoma, the most common childhood eye cancer. We aimed to investigate the travel burden and its impact on clinical presentation in a large sample of patients with retinoblastoma from Africa and Europe. Methods A cross-sectional analysis including 518 treatment-naïve patients with retinoblastoma residing in 40 European countries and 1024 treatment-naïve patients with retinoblastoma residing in 43 African countries. Results Capture rate was 42.2% of expected patients from Africa and 108.8% from Europe. African patients were older (95% CI −12.4 to −5.4, p 〈 0.001), had fewer cases of familial retinoblastoma (95% CI 2.0 to 5.3, p 〈 0.001) and presented with more advanced disease (95% CI 6.0 to 9.8, p 〈 0.001); 43.4% and 15.4% of Africans had extraocular retinoblastoma and distant metastasis at the time of diagnosis, respectively, compared to 2.9% and 1.0% of the Europeans. To reach a retinoblastoma centre, European patients travelled 421.8 km compared to Africans who travelled 185.7 km (p 〈 0.001). On regression analysis, lower-national income level, African residence and older age (p 〈 0.001), but not travel distance (p=0.19), were risk factors for advanced disease. Conclusions Fewer than half the expected number of patients with retinoblastoma presented to African referral centres in 2017, suggesting poor awareness or other barriers to access. Despite the relatively shorter distance travelled by African patients, they presented with later-stage disease. Health education about retinoblastoma is needed for carers and health workers in Africa in order to increase capture rate and promote early referral.

Abstract: Sjögren’s syndrome (SS) is a complex autoimmune disease with exocrine gland dysfunction leading to substantial morbidity. There are 10 published genetic susceptibility loci. Objectives: Our genome-wide association study (GWAS) aimed to identify additional risk loci of genome-wide significance (GWS; p 〈 5E-08) in European-derived primary SS. Methods: A total of 3232 cases and 17481 controls genotyped on GWAS arrays and 619 cases and 6171 controls genotyped on ImmunoChip (IC) arrays were imputed after quality control. Logistic regression was calculated adjusting for ancestry using the first 4 principal components to identify SS-associated SNPs. GWAS and IC results were meta-analyzed using weighted Z-scores. Bayesian statistics were used to assign posterior probabilities and define credible SNP sets for each locus. Bioinformatic analyses were used to predict functionality. Results: Seven novel loci exceeded GWS in the GWAS analysis: NAB1 , MIR146A-PTTG1 , XKR6 , MAPT-CRHR1 , RPTOR-CHMP6-BAIAP2 , TYK2 and SYNGR1 . Meta-analysis with IC data identified three more novel loci exceeding GWS: CD247 , PRDM1-ATG5 and TNFAIP3 . Several additional loci with suggestive association (p 〈 1E-05) were also identified: ADAMTSL2 , CGNL1 and PHRF1 . Several identified loci have reported functional implications in immune regulation and autoimmune disease. In lupus, rs2431697 correlated with rs2431098, which was shown to alter MIR146A expression, resulting in type I interferon pathway imbalance. Similarly, TYK2 risk association reportedly drives interferon, IL10 and RET signaling pathways. PRDM1 encodes Blimp-1, a master regulator of immune cell differentiation. CD247 encodes the zeta subunit of the T cell receptor complex. XKR6 is implicated in apoptotic cell ingestion. ATG5 is also involved in apoptosis, as well as autophagy and antigen presentation. Additional bioinformatics analyses (Haploreg, Regulome DB, ENCODE, etc.) revealed immune-relevant functional implications for each risk locus. The SS-associated credible set included variants downstream of TNFAIP3 in a region reported to abolish looping between an enhancer and the TNFAIP3 promoter in lupus and a coding variant that has been shown to alter NF-kB activity and neutrophil extra-cellular traps. The rs2293765 in the 5’ UTR of NAB1 showed evidence of enhancer/promoter activities. The rs2069235 in the SYNGR1 locus showed enhancer and transcription start site activities in B and T cells. The rs7210219 in the MAPT-CRHR1 locus showed enhancer/promotor activities in various tissues. Conclusion: We have identified ten novel genetic susceptibility loci associated with SS pathology. Our finding increases the current number of GWS regions in SS patients of European origin, from 10 to 20. Future work is needed to identify and characterize the functional variants in each region. Disclosure of Interests: Bhuwan Khatri: None declared, Tove Ragna Reksten: None declared, Kandice L Tessneer: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, R Hal Scofield Grant/research support from: Pfizer, Simon J. Bowman Consultant of: Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio, Glapagos, Speakers bureau: Novartis, Joel Guthridge Grant/research support from: Xencor, Bristol Myers Squibb, DXterity, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, Lars Ronnblom Grant/research support from: AZ, Speakers bureau: AZ, Blake M Warner: None declared, Xavier Mariette: None declared, Roald Omdal: None declared, Javier Martin Ibanez: None declared, Maria Teruel: None declared, Janicke Liaaen Jensen: None declared, Lara A Aqrawi: None declared, Øyvind Palm: None declared, Marie Wahren-Herlenius: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, A Darise Farris Speakers bureau: Biogen, Marta Alarcon-Riquelme: None declared, Wan-fai Ng: None declared, Kathy L Sivils: None declared, Gunnel Nordmark: None declared, Christopher Lessard: None declared

Abstract: Today, there are still no DMARDs licensed for primary Sjögren Syndrome (pSS) patients. Among the explanations, are the limitations of current outcome measures used as primary endpoints: e.g; high placebo response rate, evaluation of either symptoms or systemic activity, and important features not being assessed. The NECESSITY consortium ( https://www.necessity-h2020.eu/ ), including pSS experts from academia, pharmaceutical industry and patient groups formed to develop a new composite responder index, the Sjögren’s Tool for Assessing Response (STAR) that solve the issues of current outcome measures in pSS and is intended for use in clinical trials as an efficacy endpoint. Objectives To develop a composite responder index in primary Sjögren’s syndrome (pSS): the STAR. Methods To develop the STAR, the NECESSITY consortium used data-driven methods, based on 9 randomized controlled trials (RCTs), and consensus techniques, involving 78 experts and 20 patients. Based on reanalysis of rituximab trials (TRACTISS and TEARS) and literature review, the Delphi panel identified a core set of domains to include in the STAR, with their respective outcome measures. STAR options combining these domains were designed and proposed to the panel to select and improve them. For each STAR option, sensitivity to change was estimated by the C-index (derived from Effect size) in all 9 RCTs. Delphi rounds were run for selecting STAR among these options. The Delphi panel also voted to classify trials as positive, negative or “in between” in regards to primary but also key secondary endpoints. For the options remaining before the final vote, meta-analyses of the RCTs were performed separately for positive and “in between” trials together, and for negative trials. Results The Delphi panel identified 5 core domains (systemic activity, patient symptoms, lachrymal gland function, salivary gland function and biological parameters), and 227 STAR options, combining these domains, were selected to be tested for sensitivity to change. After two Delphi rounds, meta-analyses of the 20 remaining options were performed. The candidate STAR was selected by a final vote based on metrological properties and clinical relevance. In positive/in between trials, candidate STAR detected a difference between arms (OR 3.29, 95%-CI [2.07;5.22], whereas it did not in negative trials (OR 1.53, 95%-CI [0.81;2.91] ). Conclusion The candidate STAR is a composite responder index, including in a single tool all main disease features, and is designed for use as a primary endpoint in pSS RCTs. Its rigorous and consensual development process ensures its face and content validity. The candidate STAR showed good sensitivity and specificity to change. The candidate STAR will be prospectively validated in a dedicated three arms RCT of the NECESSITY consortium that will evaluate combination of synthetic DMARDs (hydroxychloroquine + lefunomide or hydroxychloroquine + mycophenolate vs placebo). We encourage the use of STAR in any ongoing and future trials. Table 1. Candidate STAR Domain Point Definition of response Systemic activity 3 Decrease of clinESSDAI ≥ 3 Patient reported outcome 3 Decrease of ESSPRI ≥ 1 point or ≥ 15% Lachrymal gland function 1 Schirmer: If abnormal score at baseline: increase ≥ 5 mm from baseline If normal score at baseline: no change to abnormal Or Ocular Staining Score: If abnormal score at baseline: decrease ≥ 2 points from baseline If normal score at baseline: no change to abnormal Salivary gland function 1 Unstimulated Whole Salivary Flow: If score 〉 0 at baseline: increase ≥ 25% from baseline If score is 0 at baseline: any increase from baseline or Ultrasound: Decrease ≥ 25% in total Hocevar score from baseline Biological 1 Serum IgG levels: decrease ≥ 10% or Rheumatoid Factor levels: decrease ≥ 25% Candidate STAR responder ≥ 5 points ESSDAI: EULAR Sjögren syndrome disease activity index; ESSPRI: EULAR Sjögren syndrome patient reported index; IgG: Immunoglobulin G; Acknowledgements NECESSITY WP5 STAR development participants: Suzanne Arends (University Medical Center Groningen, Department of Rheumatology and Clinical Immunology, Groningen 9700 RB, Netherlands), Francesca Barone (Centre for Translational Inflammation Research, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK), Albin Björk (Division of Rheumatology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden), Coralie Bouillot (Association Française du Gougerot Sjögren et des Syndromes Secs, France), Guillermo Carvajal Alegria (University of Brest, Inserm, CHU de Brest, LBAI, UMR1227, Brest, France; Service de Rhumatologie, Centre de Référence Maladies Autoimmunes Rares CERAINO, CHU Cavale Blanche, Brest, France), Wen-Hung Chen (GlaxoSmithKline, Research Triangle Park, North Carolina, USA), Kenneth Clark (GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom), Konstantina Delli (Department of Oral and Maxillofacial Surgery, University Medical Center Groningen (UMCG), University of Groningen, The Netherlands), Salvatore de Vita (Rheumatology Clinic, University Hospital of Udine, Italy), Liseth de Wolff (University Medical Center Groningen, Department of Rheumatology and Clinical Immunology, Groningen 9700 RB, Netherlands), Jennifer Evans (Novartis Pharmaceuticals corporation USA), Stéphanie Galtier (Institut de Recherches Internationales Servier (IRIS), Suresnes Cedex, France), Saviana Gandolfo (Rheumatology Clinic, Department of Medical area, University of Udine, ASUFC, 33100 Udine, Italy), Mickael Guedj (Institut de Recherches Internationales Servier (IRIS), Suresnes Cedex, France), Dewi Guellec (CHU de Brest, Service de Rhumatologie, Inserm, CIC 1412, Brest, France), Safae Hamkour (Center of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht 3584 GA, Netherlands), Dominik Hartl (Novartis Institutes for BioMedical Research, Basel, Switzerland), Malin Jonsson (Section for Oral and Maxillofacial Radiology, Department of Clinical Dentistry, Faculty of Medicine and Dentistry, University of Bergen, Norway), Roland Jonsson (Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Department of Rheumatology, Haukeland University Hospital, Bergen, Norway), Frans Kroese (University Medical Center Groningen, Department of Rheumatology and Clinical Immunology, Groningen 9700 RB, Netherlands), Aike Albert Kruize (University Medical Center Utrecht, Department Rheumatology and Clinical Immunology, Utrecht, Netherlands), Laurence Laigle (Institut de Recherches Internationales Servier (IRIS), Suresnes Cedex, France), Véronique Le Guern (AP-HP, Hôpital Cochin, Centre de référence maladies auto-immunes et systémiques rares, service de médecine interne, Paris, France), Wen-Lin Luo (Department of Biometrics and Statistical Science, Novartis Pharmaceuticals, East Hanover, New Jersey), Esther Mossel (University Medical Center Groningen, Department of Rheumatology and Clinical Immunology, Groningen 9700 RB, Netherlands), Wan-Fai Ng (Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK), Gaëtane Nocturne (Department of Rheumatology, Université Paris-Saclay, INSERM U1184: Centre for Immunology of Viral Infections and Autoimmune Diseases, Assistance Publique-Hôpitaux de Paris, Hôpital Bicêtre, Le Kremlin Bicêtre, Paris, France), Marleen Nys (Global Biometric Sciences, Bristol Myers Squibb, Braine L’Alleud, Belgium), Roald Omdal (Clinical Immunology Unit, Department of Internal Medicine, Stavanger University Hospital, PO Box 8100, 4068, Stavanger, Norway), Jacques-Olivier Pers (LBAI, UMR1227, University of Brest, Inserm, Brest, France and CHU de Brest, Brest, France), Maggy Pincemin (Association Française du Gougerot Sjögren et des Syndromes Secs, France), Manel Ramos-Casals (Department of Autoimmune Diseases, Hospital Clinic de Barcelona Institut Clinic de Medicinai Dermatologia, Barcelona, Catalunya, Spain), Philippe Ravaud (Centre d’Epidémiologie Clinique, Hôpital Hôtel-Dieu, Assistance Publique-Hôpitaux de Paris, Paris, France), Neelanjana Ray (Global Drug Development - Immunology, Bristol Myers Squibb Company, Princeton, New Jersey, USA), Alain Saraux (HU de Brest, Service de Rhumatologie, Univ Brest, Inserm, UMR1227, Lymphocytes B et Autoimmunité, Univ Brest, Inserm, LabEx IGO, Brest, France), Athanasios Tzioufas (Rheumatology Clinic, Department of Medical area, University of Udine, ASUFC, 33100 Udine, Italy), Gwenny Verstappen (University Medical Center Groningen, Department of Rheumatology and Clinical Immunology, Groningen 9700 RB, Netherlands), Arjan Vissink, Marie Wahren-Herlenius (Division of Rheumatology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden). We thank the following experts: Esen Karamursel Akpek, Alan Baer, Chiara Baldini, Elena Bartoloni, Marí-Alfonso Begona, Johan Brun, Vatinee Bunya, Laurent Chiche, Troy Daniels, Paul Emery, Robert Fox, Roberto Giacomelli, John Gonzales, John Greenspan, Robert Moots, Susumu Nishiyama, Elizabeth Price, Christophe Richez, Caroline Shiboski, Roser Solans Laque, Muthiah Srinivasan, Peter Olsson, Tsutomu Takeuchi, Frederick Vivino, Paraskevi Voulgari, Daniel Wallace, Ava Wu, Wen Zhang. We thank the anonymous patients from the NECESSITY Patient Advisory Group and the Sjögren Foundation for their valuable contribution to the Delphi process. We thank EW StClair and AN Baer who generated the baminercept data and made them publicly available. Disclosure of Interests Raphaèle Seror Consultant of: GlaxoSmithKline, Boehringer, Janssen and Novartis, Grant/research support from: GlaxoSmithKline and Amgen, Gabriel Baron: None declared, Marine Camus: None declared, Divi Cornec Consultant of: GlaxoSmithKline, Bristol Myers Squibb, Janssen, Amgen, Pfizer and Roche, Elodie Perrodeau: None declared, Simon J. Bowman Consultant of: Abbvie, Astra Zeneca, Galapagos and Novartis Pharmaceuticals, Michele Bombardieri Consultant of: UCB, Amgen/Medimmune, Janssen, and GlaxoSmithKline, Grant/research support from: Amgen/Medimmune, Janssen, and GlaxoSmithKline, Hendrika Bootsma: None declared, Jacques-Eric Gottenberg Consultant of: AbbVie, Bristol Myers Squibb, Eli Lilly, Galapagos, Gilead, Pfizer, Roche, Sanofi, Novartis, MSD, CSL-Behring and Genzyme, Grant/research support from: Bristol Myers Squibb, Benjamin Fisher Speakers bureau: Bristol Myers Squibb and Novartis, Consultant of: Novartis, Bristol Myers Squibb, Janssen and Servier, Grant/research support from: Servier, Galapagos and Janssen, Wolfgang Hueber Shareholder of: Novartis Pharma, Employee of: Novartis Pharma, Joel van Roon: None declared, Valerie Devauchelle-Pensec: None declared, Peter Gergely Shareholder of: Novartis Pharma, Employee of: Novartis Pharma, Xavier Mariette Consultant of: Bristol Myers Squibb, Galapagos, GlaxoSmithKline, Janssen, Novartis, Pfizer and UCB, Grant/research support from: Ose Pharmaceuticals, Raphaël Porcher: None declared

Abstract: Sjogren’s disease (SjD) is an autoimmune disease characterized by reduced function of exocrine glands (i.e., salivary and lacrimal glands). Epithelial cell damage resulting from lymphocytic infiltration has been implicated in SjD etiology [1]. How genetic and epigenetic changes influence epithelial-immune cell interactions in SjD pathogenesis remain understudied. Objectives Evaluate the role of SjD risk loci in salivary gland tissue to gain insights into the potential genes involved in salivary gland dysfunction. Methods SNPs from 16 regions with SNP-SjD associations (P 〈 5x10-8) in our GWAS study (3232 SjD cases) and meta-analysis of ImmunoChip data (619 SjD cases) [2] were interrogated for eQTLs using Genotype-Tissue Expression (GTEx) minor salivary gland data. Subsequent analysis identified genes that were both eQTLs in the minor salivary gland and significantly expressed in RNA-seq and ATAC-seq data from the submaxillary salivary gland epithelial cell line, A253. Pathway enrichment analysis was performed using gProfiler on the genes where coalescence of e QTL, RNA-seq, and ATAC-seq data was observed. To further validate the results, we performed transcriptome-wide association study (TWAS) analysis using GWAS summary statistics and minor salivary gland eQTL GTEx data. Results In total, 5884 genome-wide significant SNPs from 16 SjD risk loci were identified as potential minor salivary gland eQTLs using two discovery thresholds: p(FDR) 〈 0.05 provided by eQTL study (3566 SNPs) and p(FDR) 〉 0.05 and p 〈 0.05 in eQTL study (2318 SNPs). Further analysis revealed 10 SjD risk loci with SNPs that were minor salivary gland eQTLs for a total of 155 unique genes that had a coalescence of RNA- and ATAC-seq data in A253 cells. Many SNPs altered the expression of the nearest gene to the risk allele (i.e., index gene), such as IRF5 and TNPO3 on chromosome 7 at 128Mb; however, this locus had 12 additional genes that were eQTLs in minor salivary gland. In contrast, other loci had no reported eQTLs for the index gene, but several reported eQTLs for other genes, such TYK2 on chromosome 19 at 10Mb that showed no change in TYK2 expression but eQTLs for 8 distant genes, including ICAM1 . Pathway enrichment analysis revealed an enrichment in Butyrophilin (BTN) family interactions (R-HSA-8851) (PAdj=1.564x10-5), including the BTN2A1 , BTN2A2 , BTN3A1 , BTN3A2 and BTN3A3 gene cluster in the MHC region. In further support, TWAS of the minor salivary gland and the SjD GWAS summary statistics (after Bonferroni correction) showed association between SjD and BTN3A2 (p=1.24x10-42), as well as many other loci in the MHC region. In addition, several long non-coding (lnc) RNAs on chromosome 17 were significant, peaking at RP11-259G18.1 (p=4.43x10-10). Conclusion This study shows that SjD-associated risk alleles influence disease by altering gene expression in immune cells and minor salivary glands. Further, our analysis suggests that altered gene expression in the minor salivary gland expands beyond effects on the index gene to several genes on each locus. Interestingly, we observed minor salivary gland eQTLs for several BTN family genes, which act as cell-surface binding partners to regulate cell-cell interactions, including interactions between epithelial cells and activated T cells [3]. Future work will assess chromatin-chromatin-interactions within the 10 SjD risk loci in salivary gland cells and tissues to map local chromatin regulatory networks that regulate gene expression. Additional transcriptional studies of SjD minor salivary gland tissues will provide further insights into how altered gene expression in the salivary gland influences SjD pathology. References [1]Verstappen. Nat Rev Rheumatol 2021;17(6):333-348. [2]Khatri, et al. Annals of Rheumatic Diseases 2020;79:30-31. [3]Arnett HA, Viney JL. Nature Reviews Immunology 2014;14:559-569. Disclosure of Interests Farhang Aghakhanian: None declared, Mandi M Wiley: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Astrid Rasmussen: None declared, Simon J. Bowman Consultant of: Abbvie, Galapagos, and Novartis in 2020-2021., Lida Radfar: None declared, Roald Omdal: None declared, Marie Wahren-Herlenius: None declared, Blake M Warner: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, Patrick M Gaffney: None declared, Judith A. James: None declared, Lars Ronnblom: None declared, R Hal Scofield: None declared, Xavier Mariette: None declared, Marta Alarcon-Riquelme: None declared, Wan Fai Ng: None declared, Kathy Sivils Employee of: Current employee of Janssen, Gunnel Nordmark: None declared, Umesh Deshmukh: None declared, A Darise Farris: None declared, Christopher Lessard: None declared

Abstract: Given the similarity in symptoms between primary Sjogren’s syndrome (SjS) and non-SjS sicca syndrome (sicca), we sought to characterise clinical and proteomic predictors of symptoms in both groups in order to better understand disease mechanisms and help guide development of immunomodulatory treatments. These have not, to date, unequivocally improved symptoms in SjS clinical trials. Methods Serum proteomics was performed using O-link inflammation and cardiovascular II panels. SjS (n=53) fulfilled 2016 ACR/European Alliance of Associations for Rheumatology (EULAR) criteria whereas sicca (n=60) were anti-Ro negative, displayed objective or subjective dryness, and either had a negative salivary gland biopsy or, in the absence of a biopsy, it was considered that a biopsy result would not change classification status. Linear regression analysis was performed to identify the key predictors of symptoms. Cluster analysis was completed using protein expression values. Results EULAR-Sjögren’s-Syndrome-Patient-Reported-Index (ESSPRI), EuroQoL-5 Dimension utility values, and anxiety and depression did not differ between SjS and sicca. Correlations between body mass index (BMI) and ESSPRI were found in sicca and to a lesser extent in SjS. Twenty proteins positively associated with symptoms in sicca but none in SjS. We identified two proteomically defined subgroups in sicca and two in SjS that differed in symptom burden. Within hierarchical clustering of the SjS and sicca pool, the highest symptom burden groups were the least distinct. Levels of adrenomedullin (ADM), soluble CD40 (CD40) and spondin 2 (SPON2) together explained 51% of symptom variability in sicca. ADM was strongly correlated with ESSPRI (spearman’s r=0.62; p 〈 0.0001), even in a multivariate model corrected for BMI, age, objective dryness, depression and anxiety scores. Conclusions Obesity-related metabolic factors may regulate symptoms in sicca. Further work should explore non-inflammatory drivers of high symptom burden in SjS to improve clinical trial outcomes.

Abstract: The TRial for Anti-B-Cell Therapy In patients with pSS (TRACTISS) is the largest multi-centre, placebo-controlled, phase-III trial with the administration of 2 cycles of Rituximab (RTX) or placebo at week 0 and 24, with trial clinical endpoints at week 48. Despite the primary endpoints (30% reduction in fatigue or oral dryness) were not met, RTX treated patients showed an improvement in secondary endpoints, such as unstimulated whole salivary flow (UWSF), and salivary gland (SG) total ultrasound score 1,2 . Additionally, recent post-hoc analysis of TRACTISS using novel CRESS composite endpoints 3 , highlighted a significantly increased response rate in the RTX vs placebo arm. Objectives To perform the first longitudinal analysis of matched transcriptomic and histological data of SG biopsies of pSS patients treated with RTX vs placebo at 3 time points, over 48 weeks, from the TRACTISS cohort, in order to identify mechanisms of response/resistance to B cell depletion. Methods 29 pSS patients randomised to RTX or placebo arm consented for labial SG biopsies at week 0, 16 and 48. Patients received two 1000mg cycles of RTX or placebo at week 0 and 24. SG focus score, inflammatory aggregate area fraction, B-cells (CD20+), T-cells (CD3+), follicular dendritic cells (FDCs) (CD21+) and plasma cells (CD138+) density were assessed using quantitative digital image analysis. RNA sequencing with deconvolution and pathway analysis was performed to identify genes signatures and consensus gene modules as biomarkers of disease evolution and response/resistance to therapy. Results Placebo-treated SGs showed worsening of SG inflammation highlighted by the increment of aggregate size, B-cell density, development of new FDC networks, and a higher ectopic GC prevalence over 48 weeks, compared to RTX-treated patients. No difference in focus score, total T-cell and plasma cell infiltration was observed. RTX downregulated genes involved in immune cell recruitment and inflammatory aggregate organisation (e.g. CXCL13, CCR7 and PDCD1). Gene signature-based analysis of 35 immune cell types using XCell highlighted how RTX blocked class-switched and memory-B-cells accumulation in SGs over 48 weeks. Pathway analyses confirmed the downregulation of leukocyte migration, MHC-II antigen presentation, and T-cell co-stimulation immunological pathways, such as the CD40 receptor complex pathway. Among RTX-treated patients, only CRESS-responders demonstrated prevention of worsening B cell-driven molecular pathology signatures over time and a significant improvement in UWSF, in parallel with the upregulation of molecular pathways associated to SG restoration of the glandular epithelium. None of the above effects were observed at week 16 after the first RTX cycle. Conclusion Two RTX infusions repeated at week 24 exerted beneficial effects on labial SG inflammatory infiltration in pSS by downregulating genes involved in immune cell recruitment, activation and organisation in ectopic GCs. Conversely, all the above parameters showed significant evolution in placebo treated patients over 48 weeks demonstrating progression of SG immunopathology. Clinical responders to RTX based on CRESS response criteria were characterised by preservation of exocrine function which appear driven by SG epithelial restoration. References [1]Fisher, B. A. et al. Effect of rituximab on a salivary gland ultrasound score in primary Sjögren’s syndrome: results of the TRACTISS randomised double-blind multicentre substudy. Ann. Rheum. Dis. 77 , 412–416 (2018). [2]Bowman, S. J. et al. Randomized Controlled Trial of Rituximab and Cost-Effectiveness Analysis in Treating Fatigue and Oral Dryness in Primary Sjögren’s Syndrome. Arthritis Rheumatol. 69 , 1440–1450 (2017). [3]Arends, S. et al. Composite of Relevant Endpoints for Sjögren’s Syndrome (CRESS): development and validation of a novel outcome measure. Lancet Rheumatol. 3 , e553–e562 (2021). Disclosure of Interests None declared

Abstract: The pathogenic role of B-cells in primary Sjögren’s Syndrome (pSS) is well established and B cell abnormalities. Because of the substantial role of B-cells, rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, has been considered as a potential biologic disease modifying drug to reduce disease activity in pSS. To date, the TRial for Anti-B-Cell Therapy In patients with pSS (TRACTISS) is the largest multi-centre, placebo-controlled trial with RTX. Despite the unmet primary endpoints (30% reduction in fatigue or oral dryness, measured by visual analogue scale), RTX treated patients showed an improvement in unstimulated whole salivary flow (Bowman et al. Arthritis Rheumatol 2017;69:1440–1450). Objectives: To provide the first longitudinal transcriptomic and histological analysis at 3 time points over 48 weeks of labial SGs of pSS patients treated with RTX, in comparison to placebo, from the TRACTISS cohort. Methods: 26 pSS patients randomised to RTX or placebo arm consented for labial SG biopsies at baseline, weeks 16 and 48. Patients received two 1000mg cycles of RTX or placebo at baseline and week 24. SG focus score, inflammatory aggregate area fraction, B-cells (CD20+), T-cells (CD3+), follicular dendritic cells (FDCs) (CD21+) and plasma cells (CD138+) density were assessed by H & E and immunofluorescence staining. The histological analysis was performed by digital imaging using QuPath software. RNA was extracted from matched labial SG lobules and sequenced with Illumina platform. A Principal Component Analysis (PCA) and features driving the PCA were investigated along with the most influential gene loadings. The limma-voom R pipeline was used to extract Differential Expressed Genes (DEGs) between placebo and RTX group at week 48, and gene ontology (GO) enrichment analysis performed through EnrichR to derive GO terms and pathways associated with DEGs. Results: Placebo-treated labial SGs showed a worsening of inflammation highlighted by the increment of B-cell density, development of new FDC networks, and a higher ectopic GC prevalence at week 48, compared to RTX-treated patients. No difference in total T-cells and plasma cell infiltration was observed. RTX downregulated genes involved in immune cell recruitment and inflammatory aggregate organisation (e.g. CCR7, CCL19, CD52, and PDCD1) and gene signature-based analysis of 64 immune cell types highlighted how RTX preferentially blocked class-switched- and memory-B-cells infiltration in SGs at week 48. Pathway analyses confirmed the downregulation of leukocyte migration, MHC class II antigen presentation, and T-cell co-stimulation immunological pathways, such as the CD40 receptor complex pathway. The analysis of placebo SGs transcriptomic at week 48 showed a higher expression of genes linked to ectopic GC organisation, such as CXCL13, CCL19, LTβ, in female compared to male subjects. Gender was confirmed as a key co-variate responsible for most of the variation in the PCA, together with the SG focus score and the foci area fraction. Conclusion: Treatment with RTX showed beneficial effects on labial SG inflammatory infiltration in pSS, by downregulating genes involved in immune cell recruitment, activation and organisation in ectopic GCs. Class-switched-B-cells, memory-B-cells and FDC network development were primarily affected appearing to be responsible for the lack of progression in SG B cell infiltration in the RTX compared to the placebo arm in which clear worsening of SG immunopathology over 48 weeks was detected in female patients. Although a clear association with the clinical improvement in unstimulated salivary flow observed at week 48 in RTX-treated patients could not be established given the low number of patients consenting to 3 longitudinal biopsies it is conceivable that RTX is responsible for preserving exocrine function. Acknowledgements: SJB receives a salary contribution from the NIHR Birmingham Biomedical Research Centre. Disclosure of Interests: Elena Pontarini: None declared, Farzana Chowdhury: None declared, Elisabetta Sciacca: None declared, Sofia Grigoriadou: None declared, Felice Rivellese: None declared, Davide Lucchesi: None declared, Katriona Goldmann: None declared, Liliane Fossati-Jimack: None declared, Paul Emery: None declared, Wan Fai Ng: None declared, Nurhan Sutcliffe: None declared, Colin Everett: None declared, Catherine Fernandez: None declared, Anwar Tappuni: None declared, Myles Lewis: None declared, Costantino Pitzalis: None declared, Simon J. Bowman Consultant of: SJB In 2020 I have received consultancy fees from Novartis, Abbvie and Galapagos., Michele Bombardieri: None declared