Serum levels of the soluble urokinase plasminogen activator receptor (suPAR) correlates with disease activity in early rheumatoid arthritis and reflects joint damage over time

Soluble urokinase plasminogen activator receptor (suPAR) is intensively studied as a biomarker of inflammation and disease outcome in various diseases. In rheumatoid arthritis (RA), suPAR have shown an association with inflammation and swollen joints, but data on suPAR in relation to early disease course and disease progression are lacking. This study investigates the potential of suPAR to predict or reflect disease outcome in early RA. Serum suPAR was measured by enzyme-linked immunosorbent assay at disease onset and after 3 and 36 months in 252 patients from a Swedish prospective observational early RA cohort. Levels and changes of suPAR were analyzed in relation to the 28-joint disease activity score (DAS28) and joint damage according to the Larsen score at inclusion and during follow-up. 100 healthy blood donors served as controls. Circulating levels of suPAR were higher in RA patients at all time points as compared to healthy controls. Baseline suPAR was significantly associated with baseline disease activity whereas suPAR levels at 36 months were associated with joint damage at 36 months. No predictive value of suPAR levels or changes in suPAR levels over time were found. In conclusion, suPAR levels associate with disease activity in early untreated RA and reflects joint damage at later stages. Increased suPAR in established RA could indicate patients in need of frequent monitoring of joint status, irrespective of disease activity. In the view of suPAR as a rapidly emerging biomarker, it is important to be aware of its ability to reflect both inflammation and subsequent damage. Soluble urokinase plasminogen activator receptor (suPAR) is intensively studied as a biomarker of inflammation and disease outcome in various diseases. In rheumatoid arthritis (RA), suPAR have shown an association with inflammation and swollen joints, but data on suPAR in relation to early disease course and disease progression are lacking. This study investigates the potential of suPAR to predict or reflect disease outcome in early RA. Serum suPAR was measured by enzyme-linked immunosorbent assay at disease onset and after 3 and 36 months in 252 patients from a Swedish prospective observational early RA cohort. Levels and changes of suPAR were analyzed in relation to the 28-joint disease activity score (DAS28) and joint damage according to the Larsen score at inclusion and during follow-up. 100 healthy blood donors served as controls. Circulating levels of suPAR were higher in RA patients at all time points as compared to healthy controls. Baseline suPAR was significantly associated with baseline disease activity whereas suPAR levels at 36 months were associated with joint damage at 36 months. No predictive value of suPAR levels or changes in suPAR levels over time were found. In conclusion, suPAR levels associate with disease activity in early untreated RA and reflects joint damage at later stages. Increased suPAR in established RA could indicate patients in need of frequent monitoring of joint status, irrespective of disease activity. In the view of suPAR as a rapidly emerging biomarker, it is important to be aware of its ability to reflect both inflammation and subsequent damage. AT A GLANCE COMMENTARYEnocsson H, et al.BackgroundSoluble urokinase plasminogen activator receptor (suPAR) is intensively studied as a biomarker of inflammation and outcome in various diseases. Herein, suPAR in serum was evaluated over time in recent-onset rheumatoid arthritis (RA).Translational SignificancesuPAR correlated with disease activity at baseline, whereas follow-up levels associated with structural joint damage. Thus, increased suPAR in patients with established RA may reflect joint destruction. Due to the emerging interest in suPAR as a prognostic biomarker, it is important to acknowledge that suPAR may reflect different aspects of disease depending on disease duration.IntroductionThe membrane-bound urokinase plasminogen activator receptor (uPAR; CD87) is a multi-ligand receptor encoded by the PLAUR gene and expressed on endothelial cells, smooth muscle cells, fibroblasts and immune cells such as neutrophils and monocytes.1Thuno M Macho B Eugen-Olsen J. suPAR: the molecular crystal ball.Disease Markers. 2009; 27: 157-172https://doi.org/10.3233/DMA-2009-0657Crossref PubMed Scopus (370) Google Scholar The receptor and its soluble form suPAR, is involved in processes like proteolysis, tissue remodeling, cell migration and inflammation via interaction with its ligand urokinase plasminogen activator (uPA; also known as urokinase), and lateral interactions with other receptors such as integrins, vitronectin and formyl peptide receptors.1Thuno M Macho B Eugen-Olsen J. suPAR: the molecular crystal ball.Disease Markers. 2009; 27: 157-172https://doi.org/10.3233/DMA-2009-0657Crossref PubMed Scopus (370) Google Scholar, 2de Paulis A Montuori N Prevete N et al.Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2.J Immunol. 2004; 173: 5739-5748Crossref PubMed Scopus (100) Google Scholar, 3Kanse SM Kost C Wilhelm OG Andreasen PA Preissner KT. The urokinase receptor is a major vitronectin-binding protein on endothelial cells.Exp Cell Res. 1996; 224: 344-353https://doi.org/10.1006/excr.1996.0144Crossref PubMed Scopus (232) Google ScholarIncreased uPAR expression and circulating suPAR levels were initially described as prognostic factors in various forms of malignancies and has later been described as a marker of immune activation, disease severity and prognosis in multiple inflammatory and infectious diseases.1Thuno M Macho B Eugen-Olsen J. suPAR: the molecular crystal ball.Disease Markers. 2009; 27: 157-172https://doi.org/10.3233/DMA-2009-0657Crossref PubMed Scopus (370) Google Scholar,4Enocsson H Wetterö J Skogh T Sjöwall C. Soluble urokinase plasminogen activator receptor levels reflect organ damage in systemic lupus erythematosus.Transl Res. 2013; 162: 287-296https://doi.org/10.1016/j.trsl.2013.07.003Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 5Sjöwall C Martinsson K Cardell K Ekstedt M Kechagias S. Soluble urokinase plasminogen activator receptor levels are associated with severity of fibrosis in nonalcoholic fatty liver disease.Transl Res. 2015; 165: 658-666https://doi.org/10.1016/j.trsl.2014.09.007Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Andersen O Eugen-Olsen J Kofoed K Iversen J Haugaard SB. Soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in HIV-infected patients receiving highly active antiretroviral therapy.J Med Virol. 2008; 80: 209-216https://doi.org/10.1002/jmv.21114Crossref PubMed Scopus (105) Google Scholar, 7Ostrowski SR Ullum H Goka BQ et al.Plasma concentrations of soluble urokinase-type plasminogen activator receptor are increased in patients with malaria and are associated with a poor clinical or a fatal outcome.J Infect Dis. 2005; 191: 1331-1341https://doi.org/10.1086/428854Crossref PubMed Scopus (67) Google Scholar, 8Enocsson H Wirestam L Dahle C et al.Soluble urokinase plasminogen activator receptor (suPAR) levels predict damage accrual in patients with recent-onset systemic lupus erythematosus.J Autoimmun. 2020; 106102340https://doi.org/10.1016/j.jaut.2019.102340Crossref PubMed Scopus (20) Google ScholarRheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and subsequent damage to bone and cartilage, but also systemic inflammation, fatigue, increased risk of cardiovascular disease and other extra-articular manifestations.9Scott DL Wolfe F Huizinga TW. Rheumatoid arthritis.Lancet. 2010; 376: 1094-1108https://doi.org/10.1016/S0140-6736(10)60826-4Abstract Full Text Full Text PDF PubMed Scopus (2311) Google Scholar The measurement of anti‐citrullinated protein antibodies (ACPA) is a diagnostic tool and their presence associates with an increased risk of early erosions and extra-articular manifestations.10Willemze A Trouw LA Toes RE Huizinga TW. The influence of ACPA status and characteristics on the course of RA.Nat Rev. 2012; 8: 144-152https://doi.org/10.1038/nrrheum.2011.204Crossref Scopus (149) Google Scholar Other than presence of ACPA, only a limited number of biomarkers have shown potential to prognosticate disease progression in RA.11Mahler M Martinez-Prat L Sparks JA Deane KD. Precision medicine in the care of rheumatoid arthritis: Focus on prediction and prevention of future clinically-apparent disease.Autoimmun Rev. 2020; 19102506https://doi.org/10.1016/j.autrev.2020.102506Crossref PubMed Scopus (24) Google ScholarA few previous studies have investigated circulating suPAR in rheumatoid arthritis.12Slot O Brunner N Locht H Oxholm P Stephens RW. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis.Ann Rheum Dis. 1999; 58: 488-492https://doi.org/10.1136/ard.58.8.488Crossref PubMed Scopus (115) Google Scholar, 13Toldi G Beko G Kadar G et al.Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.Clin Chem Lab Med. 2013; 51: 327-332https://doi.org/10.1515/cclm-2012-0221Crossref PubMed Scopus (27) Google Scholar, 14Koga T Okada A Kawashiri S et al.Soluble urokinase plasminogen activator receptor as a useful biomarker to predict the response to adalimumab in patients with rheumatoid arthritis in a Japanese population.Clin Exp Rheumatol. 2011; 29: 811-815PubMed Google Scholar In these studies, circulating levels of suPAR were increased in patients compared to healthy controls,12Slot O Brunner N Locht H Oxholm P Stephens RW. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis.Ann Rheum Dis. 1999; 58: 488-492https://doi.org/10.1136/ard.58.8.488Crossref PubMed Scopus (115) Google Scholar, 13Toldi G Beko G Kadar G et al.Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.Clin Chem Lab Med. 2013; 51: 327-332https://doi.org/10.1515/cclm-2012-0221Crossref PubMed Scopus (27) Google Scholar, 14Koga T Okada A Kawashiri S et al.Soluble urokinase plasminogen activator receptor as a useful biomarker to predict the response to adalimumab in patients with rheumatoid arthritis in a Japanese population.Clin Exp Rheumatol. 2011; 29: 811-815PubMed Google Scholar and correlated with the number of swollen joints12Slot O Brunner N Locht H Oxholm P Stephens RW. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis.Ann Rheum Dis. 1999; 58: 488-492https://doi.org/10.1136/ard.58.8.488Crossref PubMed Scopus (115) Google Scholar,13Toldi G Beko G Kadar G et al.Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.Clin Chem Lab Med. 2013; 51: 327-332https://doi.org/10.1515/cclm-2012-0221Crossref PubMed Scopus (27) Google Scholar also among patients with limited disease activity.13Toldi G Beko G Kadar G et al.Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.Clin Chem Lab Med. 2013; 51: 327-332https://doi.org/10.1515/cclm-2012-0221Crossref PubMed Scopus (27) Google Scholar However, none of the previously published studies addressed early RA, i.e. a phase of particular importance to tailor antirheumatic treatment in order to prevent future joint damage and disability.15Smolen JS Landewe RBM Bijlsma JWJ et al.EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update.Ann Rheum Dis. 2020; 79: 685-699https://doi.org/10.1136/annrheumdis-2019-216655Crossref PubMed Scopus (1058) Google Scholar To fill this knowledge gap, we aimed to evaluate the potential of serum suPAR as a biomarker of disease progression in recent-onset RA.Material and MethodsPatients and controlsPatients were included in a Swedish prospective observational early RA cohort denoted TIRA-2, which has been described in detail previously.16Martinsson K Johansson A Kastbom A Skogh T. Immunoglobulin (Ig)G1 and IgG4 anti-cyclic citrullinated peptide (CCP) associate with shared epitope, whereas IgG2 anti-CCP associates with smoking in patients with recent-onset rheumatoid arthritis (the Swedish TIRA project).Clin Exp Immunol. 2017; 188: 53-62https://doi.org/10.1111/cei.12901Crossref PubMed Scopus (10) Google Scholar In short, patients with < 12 months symptom duration fulfilling either the American College of Rheumatology 1987 (ACR87) criteria (96%) or a clinical RA diagnosis based on symmetrical small joint arthritis and >60min morning stiffness (4%) were enrolled and followed prospectively.16Martinsson K Johansson A Kastbom A Skogh T. Immunoglobulin (Ig)G1 and IgG4 anti-cyclic citrullinated peptide (CCP) associate with shared epitope, whereas IgG2 anti-CCP associates with smoking in patients with recent-onset rheumatoid arthritis (the Swedish TIRA project).Clin Exp Immunol. 2017; 188: 53-62https://doi.org/10.1111/cei.12901Crossref PubMed Scopus (10) Google Scholar Serum samples and clinical variables were collected at inclusion and at 3, 6, 12, 24 and 36 months of follow-up. Larsen score was recorded at inclusion and at the 36 months follow-up visit. In this study, 252 patients were analyzed for serum suPAR at three time points (0, 3 and 36 months). Inclusion samples and 3 months samples were chosen to represent the dynamic first phase of the disease where changes in disease activity are most likely to appear. Long term effects were studied at 36 months.No patient had received disease-modifying anti-rheumatic drugs prior to the initial blood sampling at the first visit (baseline). Baseline characteristics are shown in Table I.Table IBaseline characteristics of the 252 early rheumatoid arthritis patientsMean (range) or number (%)Age58 (19-85)Female179 (71)*Available for 238 patients.RF positive141 (56)ACPA positive173 (69)†Available for 149 patients. The patients were classified as current smokers if smoking cigarettes regularly within one year of inclusion.Smoking Never42 (28) Current38 (26) Previous59 (40) Irregular8 (5.4) Non-cigarette smokers2 (1.3)‡Available for 250 patients.Shared epitope carriage (≥1 copies)187 (75)§Available for 231 patients.Larsen score2.5 (0-23)║Available for 236 patients.DAS285.1 (2.0-8.1)Methotrexate initiated227 (90)Oral corticosteroids initiated159 (62)Abbreviations: ACPA, anti-citrullinated protein antibodies; DAS28, 28-disease activity score; RF, rheumatoid factor. Available for 238 patients.† Available for 149 patients. The patients were classified as current smokers if smoking cigarettes regularly within one year of inclusion.‡ Available for 250 patients.§ Available for 231 patients.║ Available for 236 patients. Open table in a new tab ACPA was analyzed by fluorescence enzyme immunoassay on the Phadia 250 instrument with 2nd generation cyclic citrullinated peptides (CCP) as antigen (EliA; Thermo Fisher AB, Uppsala, Sweden). Rheumatoid factor (RF) assessments were performed locally, by agglutination test, according to clinical routine at the respective participating rheumatology unit. Details about shared epitope genetics are described elsewhere.17Roos K Martinsson K Ziegelasch M et al.Circulating secretory IgA antibodies against cyclic citrullinated peptides in early rheumatoid arthritis associate with inflammatory activity and smoking.Arthritis Res Ther. 2016; 18: 119https://doi.org/10.1186/s13075-016-1014-1Crossref PubMed Scopus (31) Google ScholarOne hundred healthy blood donors (49 men, 51 women; mean age 43.6 years; range, 21-71 years) served as control subjects.Peripheral venous blood was drawn from each individual at every visit. Serum was prepared and stored at -70 ˚C until analysis.EthicsThis study was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects. The TIRA-2 study was approved by the regional ethical review board in Linköping, Sweden (Decision No. M168-05/2006).Clinical variablesThe patients were prospectively followed with regular assessments of 28-joint Disease Activity Score (DAS28),18Prevoo ML van 't Hof MA Kuper HH van Leeuwen MA van de Putte LB van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis.Arthritis Rheum. 1995; 38: 44-48https://doi.org/10.1002/art.1780380107Crossref PubMed Scopus (5009) Google Scholar erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), radiographic joint damage according to the Larsen method,19Larsen A. How to apply Larsen score in evaluating radiographs of rheumatoid arthritis in long-term studies.J Rheumatol. 1995; 22: 1974-1975PubMed Google Scholar and information regarding ongoing medication. A 3-year radiographic progression score was calculated by subtracting the Larsen score at baseline from the Larsen score at the 36 months follow-up visit.suPAR measurementssuPAR was measured using the human uPAR duoset enzyme-linked immunosorbent assay (ELISA) (R&D systems, Abingdon, United Kingdom). This ELISA contains a monoclonal capture antibody generated against an amino acid sequence found on each of the suPAR subunits (D1, D2 and D3) which is combined with a polyclonal detection antibody. Considering the potential interference of autoantibodies directed towards the Fc-part of IgG (i.e. rheumatoid factors; RF) with suPAR detection in the ELISA, suPAR spiking was performed in two RF positive and two RF negative samples. Recombinant suPAR (standard from the uPAR duoset ELISA) was added in 2 different concentrations to the respective serum sample, and the sera was thereafter analyzed by the ELISA assay. The recovery was comparable between RF+ (range 80-113%; mean 98%) and RF- sera (range 85-93%; mean 88%) (Supplementary Table 1).Sera from patients and controls were diluted 1:10, 1:15 or 1:20 and analyzed as duplicates according to manufacturer guidelines (R&D systems). For every patient, samples taken at 0 and 3 months were assayed on the same plate to avoid interassay variation. Samples from the 36 months visit were analyzed separately from months 0 and 3. However, values from all samples were normalized according to an internal control that was assayed on every plate. If coefficient of variation (CV) values of assay duplicates differed more than 20%, samples were rerun. Details and raw data from the ELISA are found in Supplementary data 1. Absorbance was measured at 450 nm using Sunrise microplate reader (Tecan Austria GmbH, Grödig, Austria) and data were processed in Magellan software version 7.1 (Tecan).StatisticsAge, suPAR and DAS28 had a Gaussian distribution at all three timepoints whereas CRP, ESR, Larsen score, number of swollen joints, number of tender joints and 3-year radiographic progression were generally not normally distributed. Therefore, students-t-test or One-way ANOVA was used to compare suPAR levels, age and DAS28 in two and three groups, respectively. The repeated measures ANOVA was performed to compare suPAR levels among the patients at the three different time points. When this ANOVA was statistically significant, the Tukey-Kramer post hoc test was performed, comparing all groups with each other. For comparisons between patients and controls we used ANOVA with Dunnett's post hoc test. Comparisons regarding CRP, ESR, Larsen score or 3-year radiographic progression were performed with Mann-Whitney U test. All correlations were evaluated using Spearman's correlation for comparability. Multiple linear regressions were validated by controlling the normality of residuals, linearity of the model, absence of autocorrelation (Durbin-Watson; value between 1.8 and 2.2), collinearity (VIF<2 and Tolerance>0.5) and by detection of outliers (scatter dot of Leverage vs DfFit).A two-tailed P value lower than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA) or SPSS Statistics 19-20 (IBM, Armonk, NY, USA) software.ResultssuPAR levels and subject characteristics over timeMean suPAR levels among patients differed significantly between all time-points analyzed (Fig 1A), with the highest mean suPAR levels found 3 months after inclusion (8.47 ng/mL) and the lowest at 36 months (7.14 ng/mL) (Fig 1A). Mean suPAR levels were significantly higher in RA patients at all three visits (P < 0.001) compared to healthy controls (Fig 1A). ESR, CRP and DAS28 decreased significantly between inclusion and three months (P < 0.001). Changes of suPAR, DAS28 and Larsen score over time are shown in Fig 1B.There were no significant differences in suPAR levels between men and women among patients at any timepoint. Among the controls, women had higher suPAR levels (mean 4.20 ng/mL) compared to men (mean 3.02 ng/mL; P = 0.005). A significant correlation between age and baseline suPAR was found for the patients (rho = 0.227; P < 0.001) but not among controls.Mean baseline levels of suPAR were lower in ACPA positive patients compared with ACPA negative patients (7.44 vs 8.86 ng/mL; P = 0.001) and levels of ACPA showed a weak negative correlation with baseline suPAR (rho = -0.167, P = 0.008). No statistical significance in suPAR levels was found between RF-positive and RF-negative patients.No difference in baseline suPAR was found dependent on cigarette smoking. However, current smokers had significantly higher suPAR levels at 3 months (mean = 9.38;) compared with the remaining patients where smoking status was available (mean = 8.00 ng/mL; P = 0.016) as well as compared with never smokers (mean = 7.93; P = 0.048). Levels of suPAR were also increased at 36 months among current smokers (mean = 8.25 ng/mL) compared with the remaining patients (mean = 6.65 ng/mL; P < 0.001) or never smokers (mean = 6.60; P = 0.001). Patients carrying shared epitope (one or two alleles) did not differ significantly regarding mean suPAR levels at any timepoint compared to patients without shared epitope.Associations of suPAR and Clinical Outcome in RA PatientsCorrelation analyses were performed between serum suPAR levels at all three timepoints versus clinical variables (CRP, ESR, DAS28, Larsen score and radiographic progression over 3 year) at the same timepoints (Supplementary Table 2). Correlations were seen between suPAR and DAS28 at baseline (P < 0.001, rho = 0.246), as well as between suPAR and Larsen score at 36 months (P = 0.001, rho = 0.240). We also performed correlation analysis between baseline suPAR and baseline DAS28 among the patients with 36-months radiographic data available (n = 176), and this revealed a similar correlation (P = 0.001, rho = 0.263) as for the whole group of patients (Supplementary Table 2).Furthermore, changes in suPAR level between 0 and 3 months, 0 and 36 months as well as 3 and 36 months were correlated with clinical outcome variables, but no significances accompanied with a rho>0.2 were found (data not shown). Furthermore, baseline suPAR or 3 months suPAR did not correlate with 36 months Larsen score or radiographic progression.We then divided patients into three groups (as equal sized as possible) according to DAS28 and Larsen score, respectively, at baseline and 36 months (Fig 2). The group with the highest baseline DAS28 had significantly higher suPAR levels at baseline. In addition, patients with Larsen score > 5 at 36 months had higher levels of suPAR at 36 months compared to patients with Larsen score <2. No significant differences in suPAR were seen based on Larsen score at baseline or DAS28 at 36 months.Fig 2suPAR levels in patient groups stratified according to disease activity and radiographic damage. (A) Baseline (M0) suPAR and its association with baseline 28-joint disease activity score (DAS28). (B) Baseline suPAR at different baseline radiographic damage (Larsen score). (C) 36 months (M36) suPAR levels at different M36 disease activity. (D) M36 suPAR and its association with Larsen score. Bars and error bars indicate mean and 95% CI.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The associations between suPAR and clinical outcomes are independent of age and ACPAThe association between suPAR and DAS28 at baseline was tested in linear regression models. suPAR levels at baseline differed according to ACPA status, thus an adjusted model with suPAR and ACPA was created revealing that suPAR was associated with DAS28 independent of ACPA status (Table II). Next, variables with potential effect on DAS28 was tested one by one in a linear regression (age, sex, shared epitope, ACPA and smoking status) with DAS28 as the dependent variable. Except for the association between suPAR and DAS28, only age was significantly associated with DAS28 among the tested variables. Based on this, an optimized model was created with age and suPAR as independent variables revealing a significant impact of both age and suPAR on DAS28 (Table II).Table IILinear regression analysesDependent variable*One outlier (high suPAR) was found when validating these models. Removal of the outlier resulted in a lower p-value for suPAR in all three models.ModelIndependent variablesStandardized BetaP valueModel R2DAS28 M0Only suPARsuPAR M00.210.0010.04(n = 236)DAS28 M0AdjustedsuPAR M00.190.0030.05(n = 236)ACPA positive M0NSDAS28 M0OptimizedsuPAR M00.170.0090.082(n = 236)Age0.200.002Abbreviations: ACPA, anti‐citrullinated protein antibodies; DAS28, 28-joint disease activity score; M0, 0 months (inclusion); suPAR, soluble urokinase plasminogen activator receptor. One outlier (high suPAR) was found when validating these models. Removal of the outlier resulted in a lower p-value for suPAR in all three models. Open table in a new tab At 36 months, suPAR was associated with Larsen score at 36 months. Larsen score was categorized into a binary variable (Larsen score 0-5 vs >5) prior to logistic regression analysis (Table III). An adjusted model with current cigarette smoking (baseline) and suPAR (36 months) as independent variables did not reach statistical significance, but data on both smoking and Larsen was only available for 111 patients. Next, variables potentially associated with Larsen score at 36 months were added one by one to a logistic regression. Shared epitope, smoking status and sex did not reach statistical significance, whereas age and ACPA positivity were positively associated with Larsen score >5. An optimized model with suPAR (36 months), age and ACPA-positivity (baseline) as independent variables (n = 176) did not remove the statistical significance for suPAR (P = 0.023) although ACPA positivity (P = 0.007) and age (P = 0.012) also had a significant impact on Larsen score at 36 months.Table IIILogistic regression analysesDependent variableModelIndependent variablesP valueNagelkerke R2Larsen M36Only suPARsuPAR M360.0110.06(cut-off 5)(n = 176)Larsen M36*Smoking data were available for 149 patients of which 111 also had Larsen score data at 36 months.AdjustedsuPAR M36NSN/A(cut-off 5)(n = 111)Current smoker M0NSLarsen M36OptimizedsuPAR M360.0230.16(cut-off 5)(n = 176)ACPA positive M00.007Age0.012Abbreviations: ACPA, anti‐citrullinated protein antibodies; M0, 0 months (inclusion); M36, 36 months; suPAR, soluble urokinase plasminogen activator receptor. Smoking data were available for 149 patients of which 111 also had Larsen score data at 36 months. Open table in a new tab DiscussionThis is the first study to address suPAR as a prognostic biomarker in recent-onset RA. We found elevated suPAR levels in untreated recent-onset RA in association with disease activity, and further increasing levels during 3 months of early disease. The association with disease activity at baseline is in line with previous reports on established disease,12Slot O Brunner N Locht H Oxholm P Stephens RW. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis.Ann Rheum Dis. 1999; 58: 488-492https://doi.org/10.1136/ard.58.8.488Crossref PubMed Scopus (115) Google Scholar,13Toldi G Beko G Kadar G et al.Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.Clin Chem Lab Med. 2013; 51: 327-332https://doi.org/10.1515/cclm-2012-0221Crossref PubMed Scopus (27) Google Scholar but increasing levels during the initial 3 months is surprising, as disease activity robustly declines during the same time-period. In a study by Koga et al, baseline suPAR levels were related to anti-TNF treatment outcome among patients with established RA. Interestingly, patients classified as non-responders had higher pre-treatment suPAR levels in comparison with good responders, despite similar pre-treatment disease activity.14Koga T Okada A Kawashiri S et al.Soluble urokinase plasminogen activator receptor as a useful biomarker to predict the response to adalimumab in patients with rheumatoid arthritis in a Japanese population.Clin Exp Rheumatol. 2011; 29: 811-815PubMed Google Scholar This may indicate a multifactor