BackgroundAutoantibodies to citrullinated protein (ACPA; measured as anti-CCP; aCCP) and rheumatoid factor (RF) appear years before clinical onset of RA and are essential tools in today’s classification criteria for RA. In animal models, antibodies to joint specific proteins (JP) can induce arthritis, and they are also present at onset of RA [1]. As there is a need for increased precision for early diagnosis of RA as well as identification of different subtypes of the disease, we aim to assess whether autoantibodies to native or modified JP can be used for early and precise diagnosis of RA.ObjectivesTo study whether antibodies to JP, alone or in combination with ACPA/RF, could increase the diagnostic sensitivity and specificity in untreated early (ue)RA patients.MethodsAntibodies to JP were analysed in serum from patients in three independent ueRA cohorts as well as from population controls without rheumatic diseases (WINGA, Gothenburg and MFM-ÅUS, Malmö n=1062). ERAp (n=66), the smallest and most recent cohort was chosen for screening, and BARFOT and TIRA-2 (n=1939) for validation. We have developed a bead-based multianalyte flow immunoassay [2] and screened approx. 350 peptides derived from JPs of interest. We included monoclonal antibodies as assay calibrators and determined limit of detection (LoD). To assess positivity for autoantibodies to JP of interest above LoD, we used 5MAD (median absolute deviation) of the control populations as the cut-off.ResultsIn the ERAp cohort, 5 autoantibodies discriminated RA patients from controls with 81% sensitivity and 100% specificity (Table 1). The same autoantibodies had 68% sensitivity and 98% specificity in the combined BARFOT and TIRA-2 cohorts. Together with RF and aCCP, only 2 of the 5 autoantibodies added statistically significant diagnostic value, increasing the sensitivity from 48% to 61% with 99% specificity. In aCCP- and RF-negative ueRA patients (n=536), the novel biomarkers identified 22.5% of the patients with 99% specificity compared to controls.Table 1.Diagnostic capacity of the joint-specific antibodiesTest panelPerformanceGroup of patientsaCCP+RF+JP+SensitivitySpecificityAUC(ROC)ERApAll patients (n=66)--X81%100%89%RF and aCCP-neg patients (n=7)1------BARFOT and TIRA-2, combined dataAll patients (N=1939)--X68%98%86%All patients (N=1939)X--58%99%78%All patients (N=1939)2XX-48%100%84%All patients (N=1939)2, 3XXX61%99%86%RF and/or aCCP-pos patients (N=1403)--X84%99%93%RF and aCCP-neg patients (N=536)--X22%99%67%RA, literature valuesAnti-CCP testXN/AN/A53–71%95–96%N/A1Not analysed due to lack of power2This patient population is both aCCP+ and RF+3Only 2 of the 5 autoantibodies added statistically significant to the diagnostic valueAUC, Area under the curve; ROC, receiver operating characteristic curve; N/A, not applicable. Controls without rheumatic diseases: N=935 for BARFOT / TIRA-2 and N=27 for ERAp.ConclusionAutoantibodies to JP discriminate ueRA patients better then aCCP and RF alone and add an increased diagnostic value in particular for seronegative patients.References[1]Holmdahl, R., V. Malmstrom, and H. Burkhardt, Autoimmune priming, tissue attack and chronic inflammation - the three stages of rheumatoid arthritis. Eur J Immunol, 2014. 44(6): p. 1593-9.[2]Viljanen, J., et al., Synthesis of an Array of Triple-Helical Peptides from Type II Collagen for Multiplex Analysis of Autoantibodies in Rheumatoid Arthritis. ACS Chem Biol, 2020. 15(9): p. 2605-2615. Correction: ACS Chem Biol, 2020. 15(11): p. 3072AcknowledgementsBARFOT study group.Disclosure of InterestsErik Lönnblom: None declared, Monica Leu Agelii: None declared, Outi Sareila Employee of: Part time employee in Vacara AB, Ingiäld Hafström: None declared, Maria Andersson: None declared, Lei Cheng: None declared, Göran Bergström: None declared, Anna-Karin H Ekwall: None declared, Anna Rudin: None declared, Alf Kastbom: None declared, Christopher Sjowall: None declared, Bingze Xu: None declared, Lennart T.H. Jacobsson: None declared, Johan Viljanen: None declared, Jan Kihlberg: None declared, Inger Gjertsson: None declared, Rikard Holmdahl Shareholder of: Rikard Holmdahl the founder of Vacara AB.
Background:The optimal first-line treatment of patients (pts) with early rheumatoid arthritis (RA) is yet to be established.Objectives:The primary aim was to assess and compare the proportion of pts who achieved remission with active conventional therapy (ACT) and with three different biologic therapies after 24 wks. Secondary aims were to assess and compare other efficacy measures.Methods:The investigator-initiated NORD-STAR trial (NCT01491815) was conducted in the Nordic countries and Netherlands. In this multicenter, randomized, open-label, blinded-assessor study pts with treatment-naïve, early RA with DAS28>3.2, and positive RF or ACPA, or CRP >10mg/L were randomized 1:1:1:1. Methotrexate (25 mg/week after one month) was combined with: 1) (ACT): oral prednisolone (tapered quickly);or: sulphasalazine, hydroxychloroquine and mandatory intra-articular (IA) glucocorticoid (GC) injections in swollen joints <wk 20; 2) certolizumab 200 mg EOW SC (CZP); 3) abatacept 125 mg/wk SC (ABA); tocilizumab 162 mg/wk SC (TCZ). IA GC was allowed in all arms <wk 20. Primary outcome was clinical disease activity index remission (CDAI≤2.8) at wk 24. Secondary outcomes included CDAI remission over time and other remission criteria. Dichotomous outcomes were analyzed by adjusted logistic regression with non-responder imputation (NRI). Non-inferiority analyses had a pre-specified margin of 15%.Results:812 pts were randomized. Age was 54.3±14.7 yrs (mean±SD), 31.2% were male, DAS28 5.0±1.1, 74.9% were RF and 81.9% ACPA positive. Fig 1 shows the adjusted CDAI remission rates over time with 95% CI. Table shows crude remission and response rates and absolute differences in adjusted remission and response rates (superiority analysis). Differences in remission and response rates with CZP and TCZ, but not with ABA, remained within the pre-defined non-inferiority margin versus ACT, Fig 2.Figure 1.CDAI remission over time (adj. estimates with 95% CI)Figure 2.Non-inferiority analysis of protocol population. Estimated differences in CDAI remission rates between Arm 1 (active conventional therapy) and Arms 2, 3, and 4 (biologic arms) as reference with 95% confidence intervals, adjusted for gender, ACPA status, country, age, body-mass index and baseline DAS28-CRP. ABA, abatacept; CZP, certolizumab-pegol; MTX, methotrexate; TCZ, tocilizumab.Conclusion:High remission rates were found across all four treatment arms at 24 wks. Higher CDAI remission rate was observed for ABA versus ACT (+9%) and for CZP (+4%), but not for TCZ (-1%). With the predefined 15% margin, ACT was non-inferior to CZP and TCZ, but not to ABA. This underscores the efficacy of active conventional therapy based on MTX combined with glucocorticoids and may guide future treatment strategies for early RA.Table.Primary and key secondary outcomes at 24 weeks (ITT)Active conventional therapy (ACT)Certolizumab+MTXAbatacept+MTXTocilizumab+MTXNo of pts (ITT)200203204188§Crude remission and response ratesCDAI remission42.0%47.8%52.5%41.0%ACR/EULAR Boolean remission34.0%38.4%37.3%31.4%DAS28 remission63.5%68.5%69.6%63.3%SDAI remission41.5%49.8%51.5%42.6%EULAR good response71.5%76.9%79.9%71.3%Difference (95% CI) in rates with Arm 1 as reference (adjusted)CDAI remissionRef4% (-5 to 13%)9% (0.1 to 19%)-1% (-10 to 9%)ACR/EULAR Boolean remissionRef4% (-6 to 13%)5% (-5 to 14%)-4% (-13 to 6%)DAS28 remissionRef3% (-6 to 11%)5% (-4 to 13%)-1% (-10 to 8%)SDAI remissionRef6% (-3 to 18%)9% (-0.3 to 18%)1% (-8 to 11%)EULAR good responseRef4% (-4 to 14%)8% (-2 to 18%)0.4% (-10 to 11%)§17 patients allocated to Tocilizumab did not receive it due to its unavailability and were excluded from ITT.Acknowledgments:Manufacturers provided CZP and ABA.Disclosure of Interests:Merete L. Hetland Grant/research support from: BMS, MSD, AbbVie, Roche, Novartis, Biogen and Pfizer, Consultant of: Eli Lilly, Speakers bureau: Orion Pharma, Biogen, Pfizer, CellTrion, Merck and Samsung Bioepis, Espen A Haavardsholm Grant/research support from: AbbVie, UCB Pharma, Pfizer Inc, MSD Norway, Roche Norway, Consultant of: Pfizer, AbbVie, Janssen-Cilag, Gilead, UCB Pharma, Celgene, Lilly, Paid instructor for: UCB Pharma, Speakers bureau: Pfizer, AbbVie, UCB Pharma, Celgene, Lilly, Roche, MSD, Anna Rudin Consultant of: Astra/Zeneca, Dan Nordström Consultant of: Abbvie, Celgene, Lilly, Novartis, Pfizer, Roche and UCB., Speakers bureau: Abbvie, Celgene, Lilly, Novartis, Pfizer, Roche and UCB., Michael Nurmohamed Grant/research support from: Not related to this research, Consultant of: Not related to this research, Speakers bureau: Not related to this research, Björn Gudbjornsson Speakers bureau: Novartis and Amgen, Jon Lampa Speakers bureau: Pfizer, Janssen, Novartis, Kim Hørslev-Petersen: None declared, Till Uhlig Consultant of: Lilly, Pfizer, Speakers bureau: Grünenthal, Novartis, Gerdur Gröndal: None declared, Mikkel Ǿstergaard Grant/research support from: AbbVie, Bristol-Myers Squibb, Celgene, Merck, and Novartis, Consultant of: AbbVie, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Eli Lilly, Hospira, Janssen, Merck, Novartis, Novo Nordisk, Orion, Pfizer, Regeneron, Roche, Sandoz, Sanofi, and UCB, Speakers bureau: AbbVie, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Eli Lilly, Hospira, Janssen, Merck, Novartis, Novo Nordisk, Orion, Pfizer, Regeneron, Roche, Sandoz, Sanofi, and UCB, Marte Heiberg: None declared, Jos Twisk: None declared, Simon Krabbe: None declared, Kristina Lend: None declared, Inge Olsen: None declared, Joakim Lindqvist: None declared, Anna-Karin H Ekwall Consultant of: AbbVie, Pfizer, Kathrine L. Grøn Grant/research support from: BMS, Meliha C Kapetanovic: None declared, Francesca Faustini: None declared, Riitta Tuompo: None declared, Tove Lorenzen: None declared, Giovanni Cagnotto: None declared, Eva Baecklund: None declared, Oliver Hendricks Grant/research support from: Pfizer, MSD, Daisy Vedder: None declared, Tuulikki Sokka-Isler: None declared, Tomas Husmark: None declared, Maud-Kristine A Ljosa: None declared, Eli Brodin: None declared, Torkell Ellingsen: None declared, Annika Soderbergh: None declared, Milad Rizk Speakers bureau: AbbVie, Åsa Reckner: None declared, Per Larsson: None declared, Line Uhrenholt Speakers bureau: Abbvie, Eli Lilly and Novartis (not related to the submitted work), Søren Andreas Just: None declared, David Stevens: None declared, Trine Bay Laurberg Consultant of: UCB Pharma (Advisory Board), Gunnstein Bakland Consultant of: Novartis, UCB, Ronald van Vollenhoven Grant/research support from: BMS, GSK, Lilly, UCB, Pfizer, Roche, Consultant of: AbbVie, AstraZeneca, Biogen, Biotest, Celgene, Gilead, Janssen, Pfizer, Servier, UCB, Speakers bureau: AbbVie, Pfizer, UCB
BackgroundActivated fibroblast-like synoviocytes (FLS) are key effector cells in the joint in rheumatoid arthritis (RA). Local FLS proliferation is responsible for synovial hyperplasia, a key feature of the RA synovium correlating with disease activity. PDGF and IL-1b are known FLS mitogens. LBH is a transcription regulator and tumor suppressor, recently identified as a RA risk gene. We have demonstrated that LBH regulates FLS proliferation and that LBH expression is regulated by growth factors and by epigenetic mechanisms[1]. Methotrexate (MTX) is still the first-line treatment of RA but the target cells and mechanism of action of the low dose used in rheumatic diseases is largely unknown. Increased expression of cell cycle checkpoint genes[2] and modified DNA methylation[3] in immune cells have recently been described.ObjectivesThe aim of this study was to investigate the effects of MTX on PDGF and IL1b-induced FLS proliferation in vitro and in particular on the expression of LBH, cell cycle genes (CDKN1A/p21 and CCND1/cyclinD1) and on genes regulating DNA methylation (DNMTs) in order to further understand the pharmacodynamics of this drug in RA and to identify novel markers for drug response.MethodsPrimary FLS from RA patients and from patients with osteoarthritis (OA) were plated on day 0 in DMEM complete, pre-treated 24 hours with MTX or control medium day 1, and stimulated with 20ng/ml PDGF+2 ng/ml IL-1b with or without 1 uM MTX in DMEM with 1% FBS for 24–48 hours starting day 2. Cells were then harvested for qPCR for gene expression and flowcytometry for cell cycle analysis.ResultsStimulating RA-FLS cultures (n=3) with PDGF+IL-1b for 24 hours, pushed 24,5±3,5% cells into G2/M phase compared to 3,4±0,8% in unstimulated controls. Interestingly, treating PDGF+IL1b stimulated FLS with MTX, significantly inhibited cell cycle progression (4,6±1,9% in G2/M phase, p=0,02). PDGF+IL-1b stimulation of FLS for 24 hours reduced LBH mRNA expression. However, in the presence of 1uM MTX the LBH mRNA expression was significantly higher in RA-FLS (3,2±0,5 fold, p=0.002, n=5) and in OA-FLS (2,2±0,5 fold, p=0,02, n=5) after PDGF+IL-1b stimulation compared to untreated controls. In addition, MTX treatment strikingly increased the CDKN1A expression 14,3±4,4 fold (p=0,006) of treated vs untreated stimulated FLS. Furthermore, we found that 1 uM MTX restored and increased a lowered DNMT1 mRNA expression to 144±12% after PDGF+IL1b stimulation. There were no significant effects of MTX on CCND1 or DNMT3a expression at investigated time points.ConclusionsTherapeutic doses of MTX reduce mitogen induced FLS proliferation and significantly revert mitogen-induced reduction of LBH and p21 expression in RA FLS. MTX restores expression of DNMT1 suggesting that MTX might regulate gene expression and proliferation by affecting the epigenome.References Ekwall, A.K., et al., The Rheumatoid Arthritis Risk Gene LBH Regulates Growth in Fibroblast-like Synoviocytes. Arthritis Rheumatol, 2015. 67(5): p. 1193–202.Spurlock, C.F., 3rd, et al., Methotrexa...
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