OBJECTIVE:Stratification of therapeutic responses may help identify efficacious therapies for osteoarthritis (OA). In the PROMOTE randomised trial, participants with elevated baseline high-sensitivity C-reactive protein (hs-CRP) showed greater pain reduction after methotrexate treatment. We set out to interrogate a broader panel of serum/plasma inflammatory response markers relevant to methotrexate actions as potential biomarkers of therapeutic effect. Our objectives were to: (i) characterize changes in these systemic markers during methotrexate treatment; determine whether (ii) baseline levels or (iii) changes in any marker during treatment were associated with treatment response; and (iv) compare these findings with the more established clinical inflammatory marker, hs-CRP. DESIGN:Plasma/serum samples from participants in PROMOTE's biological substudy were analysed for 35 inflammatory markers at baseline (pre-treatment) and at 6-months (post-treatment), by MesoScale V-plex multiplex assay. Those with paired biological and clinical data at both baseline and 6-months were included in the substudy analysis set. Relationships between markers and overall data structure were assessed by Pearson correlation and Principal Component analysis. Associations between markers (baseline levels or change over time) and change in average knee pain severity in past week (numerical rating scale, NRS) were evaluated by univariable linear regression, adjusting for baseline age, sex, and body mass index. Least Absolute Shrinkage and Selection Operator (LASSO) regression with bootstrap resampling enabled marker selection. Benjamini-Hochberg correction adjusted for multiple testing (Padj). RESULTS:87 participants with paired blood marker and clinical data were eligible for substudy analysis. 18/35 markers were quantifiable and analysed. Systemic IL-8 and TNF-α levels decreased (Padj=0.015, 0.048 respectively) while IL-15 increased (Padj=0.033) with methotrexate treatment over 6-months. Analysing within this active treatment randomised arm, higher baseline IFN-γ was associated with greater reduction in NRS pain change (0.66 [0.01, 1.31], P=0.047), as was decreasing TNF-α over 6-months (2.25 [0.00, 4.5], P=0.049). LASSO identified higher IFN-γ, lower plasma IL-15 and IL-16, and younger age as the most important baseline predictors of pain improvement. hs-CRP was highly selected by LASSO for treatment response in both arms. In a secondary univariate treatment arm-by-biomarker interaction analysis, of the 19 markers, only hs-CRP showed consistent effects in adjusted models (at baseline, coeffic. 2.34 [0.53, 4.15], P=0.001; change over 6-months, (0.36 [0.06, 0.66], P=0.018). CONCLUSIONS:Blood measurement of IFN-γ, TNF-α, IL-15 and IL-16 as well as hs-CRP could act as potential markers to stratify the treatment response by average knee pain to methotrexate in knee osteoarthritis.

Despite being commonly reported by patients, the occurrence of episodic ‘flare’ and factors affecting it have not previously been studied in hand osteoarthritis. We investigated the associations between short-term fluctuations in hand pain and clinical/constitutional factors (including female hormonal and reproductive exposures) in individuals with hand osteoarthritis. HOPE-Cohort was a remote, 6-month prospective study. Males and females were recruited UK-wide from the community, primary and secondary care, with clinically-diagnosed hand osteoarthritis (≥ 2 joints involved), who were not in flare at baseline. Baseline data collected included age, sex, BMI; clinical involvement of hand joints; psychological status (including pain self-efficacy, anxiety); and in females, age of menopause, history of oophorectomy/hysterectomy, Hormone Replacement Therapy (HRT) use and menopausal symptoms score. Hand pain severity was recorded daily for 6-months by participant smartphone (REDCap weblink within daily SMS) using a 0-10 numerical rating scale (NRS). Participants self-reported onset of flare, triggering an additional flare survey to further characterise flare episodes. These data sources yielded the following outcomes: (i) average hand pain severity (NRS) at baseline; (ii) hand pain variability across 6-months, by intra-individual standard deviation (IISD) of daily pain NRS; and (iii) count of participant-reported flares. In sex-stratified analyses, associations between baseline variables and these outcomes were assessed by univariable and multivariable regression (linear or Poisson, depending on the outcome) using STATA IC 18.0. 132 participants (median age 64) were recruited, of whom 121 (94%) provided pain scores on ≥ 90 of 180 possible days. 114 (86%) were females of whom 87% were post-menopausal, with history of hysterectomy and oophorectomy in 18% and 9% respectively. 87% (105/121) experienced ≥1 hand osteoarthritis flare over 6-months. Amongst females, median flare frequency was 2.5 (IQR 1,4, range 0,11). During a flare, daily pain NRS increased by median 1.5 (IQR 0.9,2.5). Median flare duration was 5 days. In females, adverse scores on all psychological measures, prior hysterectomy and higher menopausal symptoms score were all associated with greater baseline hand pain severity. Greater baseline pain NRS was associated with greater within-participant pain variability (increase in IISD β 0.09 [95%CI:0.06,0.13]; R2=0.22). After baseline pain adjustment, lower pain self-efficacy, higher BMI and absence of current HRT were all associated with greater hand pain variability. Greater baseline pain was also associated with greater frequency of participant-reported flares, IRR 1.17 (95%CI:1.07,1.27). After adjusting for baseline hand pain, several factors remained associated with increased flare frequency, including history of hysterectomy, oophorectomy, plus menopause factors (lower age; increasing interval since). This study provides novel data on the varying pain experience in hand osteoarthritis and evidence for flare episodes being a recognisable feature for many. In post-menopausal females, underlying hormonal factors may be relevant in explaining the course and severity of hand pain. M. Gulati: Grants/research support; MG has a Clinical Research Fellowship from Versus Arthritis [Grant number 21604]. MG is in receipt of a Clarendon Scholarship [Oxford University Press]. G. Brewer: None. T.A. Perry: None. B. Marsden: None. M. Schlussel: None. N.K. Arden: None. G.S. Collins: None. K. Vincent: Consultancies; K.V. has had consultancy fees paid to her institution from Gedeon Richter, Reckitts, AbbVie, Bayer Healthcare and Gesynta. Honoraria; K.V. has received honoraria for lectures. T.L. Vincent: Consultancies; T.V. is a current advisory board member for Zoetis. Grants/research support; T.V. has received research funding in the past 5 years from Pfizer, UCB, Novartis, Fidia, Biosplice. A. Silman: None. F.E. Watt: Consultancies; F.W. has received consultancy fees from Pfizer in the past 5 years. Grants/research support; FW has a fellowship from UK Research & Innovation (UKRI) [Grant numbers MR/S016538/1, MR/S016538/2 and MR/Y003470/1).

Hand pain and hand osteoarthritis are common, particularly among peri-/post-menopausal women. Despite this, there are few studies on long-term outcomes and their determinants in this population. We examined associations between demographic and clinical factors including female reproductive and hormonal exposures on hand pain in women over 20 years. The “Chingford 1000 Women Study” is a community-based cohort of females aged 45-64 years representing a high-risk population for painful hand osteoarthritis. Participants were recruited from general practice and followed for over 20 years. Those with other painful hand conditions (e.g. inflammatory arthritis) were excluded. Baseline variables analysed included age, BMI, hand joint bony swelling (binary), hand radiographic osteoarthritis (ROA, binary), previous hysterectomy, bilateral oophorectomy and (in post-menopausal females) use/duration of hormone replacement therapy (HRT). ‘Symptomatic established hand osteoarthritis’ (seHOA) was defined as hand pain plus bony swelling and/or ROA ≥2 hand joints. Our outcomes included presence/absence of hand pain in either hand at i) Year 10 and ii) Year 20 respectively (binary), and iii) number of visits at which hand pain was reported in ‘complete cases’ (with Years-3,5,10,15&20 data). Associations between exposures and outcomes were assessed fitting a logistic regression adjusting for age+BMI alone or in a multivariable model, using STATA IC 18.0. Of 834 eligible participants, 249 (30%) reported hand pain at baseline, of whom 174 (70%) had seHOA. By Year 20, cohort retention was 435 (52%), of whom 367 (84%) provided hand pain information at all necessary visits for the complete case analysis. At Years 10 and 20 respectively, prevalence of hand pain was 85 (13%) and 74 (17%), representing 32 (25%) and 19 (25%) of those with seHOA at baseline. Persistent hand pain (at all visits after pain initially reported) was infrequent: 18 (5%) of all complete cases and 6 (10%) of those with baseline seHOA. Baseline hand joint bony swelling and/or ROA were associated with future reporting of hand pain (at both Years 10 and 20) in those with baseline hand pain. In the absence of baseline pain, there were no such associations. In age+BMI-adjusted models, bilateral oophorectomy and prior HRT use (≥2 years) were each associated with hand pain at Year-10: relative risk (RR) 2.5(95% CI: 1.5,4.3) and 2.1(95% CI: 1.2,3.7) respectively. Only the association of bilateral oophorectomy with hand pain persisted in the multivariable model. In age+BMI-adjusted models, hysterectomy, bilateral oophorectomy and baseline HRT use were all associated with greater odds of more visits with reported hand pain. We describe long-term outcomes for females experiencing hand pain and osteoarthritis. They add to evidence that bilateral oophorectomy and hysterectomy, themselves associated with either abrupt loss of oestrogen/other sex hormones and/or their dysregulation, appear to be important risk factors for long-term hand pain. M. Gulati: Grants/research support; M.G. has a Clinical Research Fellowship from Versus Arthritis [Grant number 21604]. M.G. is in receipt of a Clarendon Scholarship [Oxford University Press]. T.A. Perry: None. D.J. Hart: None. T.D. Spector: None. M. Schlussel: None. G.S. Collins: None. K. Vincent: Consultancies; K.V. has had consultancy fees paid to her institution from Gedeon Richter, Reckitts, AbbVie, Bayer Healthcare and Gesynta. Honoraria; K.V. has received honoraria for lectures. T.L. Vincent: Consultancies; T.V. is a current advisory board member for Zoetis. Grants/research support; T.V. has received research funding in the past 5 years from Pfizer, UCB, Novartis, Fidia, Biosplice. N.K. Arden: None. A. Silman: None. F.E. Watt: Consultancies; F.W. has received consultancy fees from Pfizer in the past 5 years. Grants/research support; F.W has a fellowship from UK Research & Innovation (UKRI) [Grant numbers MR/S016538/1, MR/S016538/2 and MR/Y003470/1).

Objective The ArmeD SerVices TrAuma RehabilitatioN OutComE (ADVANCE) study is investigating long-term combat-injury outcomes; this sub-study aims to understand the association of osteoarthritis (OA) biomarkers with knee radiographic OA (rOA), pain and function in this high-risk population for post-traumatic OA. Design ADVANCE compares combat-injured participants with age, rank, deployment and job-role frequency-matched uninjured participants. Post-injury immunoassay-measured serum biomarkers, knee radiographs, Knee Injury and Osteoarthritis Outcome Scale, and six-minute walk tests are reported. The primary analysis, adjusted for age, body mass, socioeconomic status, and ethnicity, was to determine any differences in biomarkers between those with/without combat injury, rOA and pain. Secondary analyses were performed to compare post-traumatic/idiopathic OA, painful/painfree rOA and injury patterns. Results A total of 1145 male participants were recruited, aged 34.1 ± 5.4, 8.9 ± 2.2 years post-injury (n = 579 trauma-exposed, of which, traumatic-amputation n = 161) or deployment (n = 566 matched). Cartilage oligomeric matrix protein (COMP) was significantly higher in the combat-injured group compared to uninjured (p = 0.01). Notably, COMP was significantly lower in the traumatic-amputation group compared to non-amputees (p < 0.001), decreasing relative to number of amputations (p < 0.001). Leptin was higher (p = 0.005) and adiponectin lower (p = 0.017) in those with v without knee pain, associated with an increased risk of 22% and 17% for pain, and 46% and 34% for painful rOA, respectively. There were no significant differences between trauma-exposed and unexposed participants with rOA. Conclusions The most notable findings of this large, unique study are the similarities between those with rOA regardless of trauma-exposure, the injury-pattern and traumatic-amputation-associated differences in COMP, and the relationship between adipokines and pain.

Purpose (the aim of the study): Osteoarthritis (OA) exerts a profound impact on quality of life. In the absence of disease-modifying OA drugs (DMOADs), there is a critical need to unravel the molecular drivers of the disease and discern potential variations among individuals. The advent of large-scale, high-throughput omics-related biotechnologies enable us to address this need at an unprecedented scale. The STEpUP OA (Synovial Fluid To Define Molecular Endotypes by Unbiased Proteomics in Osteoarthritis) consortium was established to answer a primary question of whether distinct molecular OA endotypes exist using large-scale proteomics of knee OA synovial fluid (SF). We hypothesised that molecular endotypes may delineate specific patient groups, thereby influencing diagnosis, predicting disease course, and guiding treatment response.

Aims:To explore key stakeholder views around feasibility and acceptability of trials seeking to prevent post-traumatic osteoarthritis (PTOA) following knee injury, and provide guidance for next steps in PTOA trial design. Methods:Healthcare professionals, clinicians, and/or researchers (HCP/Rs) were surveyed, and the data were presented at a congress workshop. A second and related survey was then developed for people with joint damage caused by knee injury and/or osteoarthritis (PJDs), who were approached by a UK Charity newsletter or Oxford involvement registry. Anonymized data were collected and analyzed in Qualtrics. Results:Survey responses (n = 19 HCP/Rs, 39 PJDs) supported studies testing pharmacological agents preventing PTOA. All HCP/Rs and 30/31 (97%) PJDs supported the development of new treatments that improved or delayed knee symptoms and damage to knee structure. PJDs thought that improving structural knee damage was more important than knee symptoms. Both groups found studies more acceptable as expected future benefit and risk of PTOA increased. All drug delivery routes were acceptable. Workshop participants (around n = 60) reflected survey views. Discussions suggested that stratifying using molecular testing for likely drug response appeared to be more acceptable than using characteristics such as sex, age, and BMI. Conclusion:Our findings supported PTOA drug intervention studies, including situations where there is low risk of disease, no expected benefit of treatment, and frequent treatment administration. PJDs appeared less risk-averse than HCP/Rs. This work reinforces the benefits of consensus and involvement work in the co-creation of PTOA drug trial design. Involvement of key stakeholders, such as PJDs with different risks of OA and regulatory representatives, are critical for trial design success.

BACKGROUND:Treatments for osteoarthritis (OA) are limited. Previous small studies suggest that the antirheumatic drug methotrexate may be a potential treatment for OA pain. OBJECTIVE:To assess symptomatic benefits of methotrexate in knee OA (KOA). DESIGN:A multicenter, randomized, double-blind, placebo-controlled trial done between 13 June 2014 and 13 October 2017. (ISRCTN77854383; EudraCT: 2013-001689-41). SETTING:15 secondary care musculoskeletal clinics in the United Kingdom. PARTICIPANTS:A total of 207 participants with symptomatic, radiographic KOA and knee pain (severity ≥4 out of 10) on most days in the past 3 months with inadequate response to current medication were approached for inclusion. INTERVENTION:Participants were randomly assigned 1:1 to oral methotrexate once weekly (6-week escalation 10 to 25 mg) or matched placebo over 12 months and continued usual analgesia. MEASUREMENTS:The primary end point was average knee pain (numerical rating scale [NRS] 0 to 10) at 6 months, with 12-month follow-up to assess longer-term response. Secondary end points included knee stiffness and function outcomes and adverse events (AEs). RESULTS:A total of 155 participants (64% women; mean age, 60.9 years; 50% Kellgren-Lawrence grade 3 to 4) were randomly assigned to methotrexate (n = 77) or placebo (n = 78). Follow-up was 86% (n = 134; methotrexate: 66, placebo: 68) at 6 months. Mean knee pain decreased from 6.4 (SD, 1.80) at baseline to 5.1 (SD, 2.32) at 6 months in the methotrexate group and from 6.8 (SD, 1.62) to 6.2 (SD, 2.30) in the placebo group. The primary intention-to-treat analysis showed a statistically significant pain reduction of 0.79 NRS points in favor of methotrexate (95% CI, 0.08 to 1.51; P = 0.030). There were also statistically significant treatment group differences in favor of methotrexate at 6 months for Western Ontario and McMaster Universities Osteoarthritis Index stiffness (0.60 points [CI, 0.01 to 1.18]; P = 0.045) and function (5.01 points [CI, 1.29 to 8.74]; P = 0.008). Treatment adherence analysis supported a dose-response effect. Four unrelated serious AEs were reported (methotrexate: 2, placebo: 2). LIMITATION:Not permitting oral methotrexate to be changed to subcutaneous delivery for intolerance. CONCLUSION:Oral methotrexate added to usual medications demonstrated statistically significant reduction in KOA pain, stiffness, and function at 6 months. PRIMARY FUNDING SOURCE:Versus Arthritis.

Background: Despite an acute knee injury being a major risk factor for osteoarthritis, the factors that initiate and maintain this risk of longer-term knee symptoms are poorly understood. Bioactive lipids derived from omega-3 and -6 polyunsaturated fatty acids have key roles in the regulation of the inflammatory response and have been linked to joint damage and osteoarthritis pain in translational models.Hypothesis: There would be associations between systemic levels of bioactive lipids and knee symptoms longitudinally after an acute knee injury and related knee surgery.Study Design: Controlled laboratory study.Methods: This study analyzed a subset of young, active adults who had sustained an acute knee injury (recruited via a surgical care pathway) and healthy age- and sex-matched controls. Surgery, if performed, was conducted after the baseline serum sample was taken and before the 3-month and 2-year visits. Liquid chromatography-tandem mass spectrometry of 41 bioactive lipids was carried out in sera of (1) 47 injured participants (median age, 28 years) collected at baseline (median, 24 days after injury), 3 months, and 2 years, along with the Knee injury and Osteoarthritis Outcome Score, and (2) age- and sex-matched controls.Results: Levels of the omega-3 polyunsaturated fatty acids eicosapentaenoic acid (P <= .0001) and docosahexaenoic acid (P <= .0001) and the pro-resolving lipid mediators 17- and 14-hydroxydocosahexaenoic acid, and 18-hydroxyeicosapentaenoic acid were all significantly greater at baseline in injured participants compared with the later time points and also higher than in healthy controls (P = .0019 and P <= .0001, respectively). Levels of pro-inflammatory prostaglandins E2 and D2, leukotriene B4, and thromboxane B2 were significantly lower at baseline compared with the later time points. Higher levels of 8,9-, 11,12-, and 14,15-dihydroxyeicosatrienoic acid (DHET) were cross-sectionally associated with more severe knee pain/symptoms according to the Knee injury and Osteoarthritis Outcome Score at 2 years (P = .0004, R2 = 0.251; P = .0002, R2 = 0.278; and P = .0012, R2 = 0.214, respectively).Conclusion: The profile of pro-resolving versus pro-inflammatory lipids at baseline suggests an initial activation of pro-resolution pathways, followed by the later activation of pro-inflammatory pathways.Clinical Relevance: In this largely surgically managed cohort, the association of soluble epoxide hydrolase metabolites, the DHETs, with more severe knee symptoms at 2 years provides a rationale for further investigation into the role of this pathway in persisting knee symptoms in this population, including potential therapeutic strategies.

Purpose (the aim of the study): A major challenge for osteoarthritis (OA) research and clinical care is selecting the most appropriate outcome measure for different populations. These often involve a blend of subjective, patient-reported measures, such as the Knee injury and OA Outcome Score (KOOS), and objective, clinician-reported measures, such as functional tasks like the 6-minute walk test (6MWT). The UK-based prospective ADVANCE cohort study was established to assess the long-term outcomes following combat trauma sustained during the Afghanistan war.

Purpose (the aim of the study): Approximately 1 in 2 people with a history of significant knee injury develop symptomatic radiographic osteoarthritis (OA), so called post-traumatic osteoarthritis (PTOA) of the knee. The impacts of PTOA are well-described, but little is known about the genetic risk of developing OA after knee injury and whether this differs from idiopathic OA (iOA). Knowledge of genetic variants associated with PTOA would improve our understanding of this subgroup of disease. Our aim is to identify genome-wide significant signals associated with PTOA of the knee.

Objective: The global impact of osteoarthritis is growing. Currently no disease modifying osteoarthritis drugs/therapies exist, increasing the need for preventative strategies. Knee injuries have a high prevalence, distinct onset, and strong independent association with post-traumatic osteoarthritis (PTOA). Numerous groups are embarking upon research that will culminate in clinical trials to assess the effect of interventions to prevent knee PTOA despite challenges and lack of consensus about trial design in this population. Our objectives were to improve awareness of knee PTOA prevention trial design and discuss state-of-the art methods to address the unique opportunities and challenges of these studies. Design: An international interdisciplinary group developed a workshop, hosted at the 2023 Osteoarthritis Research Society International Congress. Here we summarize the workshop content and outputs, with the goal of moving the field of PTOA prevention trial design forward. Results: Workshop highlights included discussions about target population (considering risk, homogeneity, and possibility of modifying osteoarthritis outcome); target treatment (considering delivery, timing, feasibility and effectiveness); comparators (usual care, placebo), and primary symptomatic outcomes considering surrogates and the importance of knee function and symptoms other than pain to this population. Conclusions: Opportunities to test multimodal PTOA prevention interventions across preclinical models and clinical trials exist. As improving symptomatic outcomes aligns with patient and regulator priorities, co-primary symptomatic (single or aggregate/multidimensional outcome considering function and symptoms beyond pain) and structural/physiological outcomes may be appropriate for these trials. To ensure PTOA prevention trials are relevant and acceptable to all stakeholders, future research should address critical knowledge gaps and challenges.

Purpose (the aim of the study): The molecular mechanisms of osteoarthritis (OA) remain largely unexplained. STEpUP OA (Synovial Fluid To Define Molecular Endotypes by Unbiased Proteomics in Osteoarthritis) is an international consortium which has used SomaLogic SomaScan technology to measure over 7000 proteins in the synovial fluid (SF) from >1700 individuals with OA or following joint injury. This has generated an unprecedented molecular and clinical data resource with which to interrogate molecules and pathways associated with disease. As part of this work, we set out to compare the SF proteomic profiles of individuals with advanced and non-advanced radiographic knee OA.

Background: Professional football is a risk factor for knee OA, but it is unclear whether it is also a risk factor for foot/ankle OA. Objectives: To compare the prevalence of foot/ankle outcomes (pain, GP-diagnosed OA, radiographic OA (ROA), and surgery) between male retired professional footballers and general population control men. Methods: A postal questionnaire was sent to 878 footballers and 1060 controls who participated in our previous knee OA study. Pain was defined as “current” (in the past month) and as “ever” (lasting ≥3 months in the past). GP-diagnosed OA and foot/ankle surgery were self-reported. Socioeconomic status in deciles was obtained according to post code. Foot and ankle radiographs were undertaken in those who indicated willingness to have radiographs. ROA was assessed using the La Trobe atlas and defined as osteophyte (OP) ≥2 or joint space narrowing (JSN) ≥2. The adjusted relative risk (aRR) with 95% confidence intervals (CI) were calculated using Poisson regression model on self-reported outcomes and foot/ankle ROA prevalence between groups. Results: 468 footballers (53%) and 619 controls (58%) completed the questionnaire. Of these, 113 footballers and 319 controls completed the radiographic assessment. Footballers were 4 years younger and had higher socioeconomic status than controls (Table 1). Compared to controls, footballers were more likely to have current foot/ankle pain (aRR 1.32, 95% CI 1.03-1.68), GP-diagnosed foot/ankle OA (aRR 1.79, 95%CI 1.18-2.71), and forefoot/ankle surgery (aRR 2.82, 95% CI 1.94-4.09), but not ever foot/ankle pain (Table 2). Prevalence of foot/ankle ROA did not differ between groups as defined but was significantly different when both OP and JSN ≥2 (i.e., definite OP plus definite JSN) was used to define ROA. The ankle was the most common joint affected by OA in footballers over controls. Both foot/ankle injury and injection were reported more commonly in footballers than controls. Injuries and injections correlated with each other, and the most commoninjection was corticosteroid. Conclusion: Professional footballers were more likely to have foot/ankle OA than controls in their post-retirement. Injury and injection were both associated with an increased risk of foot/ankle OA. Further research on preventing injury and reducing injections is needed. Keywords: osteoarthritis; foot/ankle; footballers. REFERENCES: NIL. Acknowledgements: The Football Association (FA), Professional Footballers’ Association (PFA) and Versus Arthritis provided financial support for the study. We are grateful to the footballers and control men in the East- Midlands who participated in the study. Disclosure of Interests: None declared.Table 1Characteristics of study population.FootballersControlsp-valueQuestionnaires, n468619Age (years), mean (SD)63.27 (10.4)68.42 (9.2)<0.001a*Body mass index (kg/m2), mean (SD)27.16 (3.2)26.81 (4.1)0.124aSocioeconomic status, mean (SD)7.50 (2.4)6.59 (3.0)<0.001a*Charlson Comorbidity Index, mean (SD)1.07 (2.0)1.33 (2.2)0.048a*SF-36 components summary, mean (SD)Physical components72.02 (21.6)69.25 (23.9)0.046a*Mental components77.98 (19.2)77.27 (20.3)0.559aFoot/ankle injury, n (%)190/425 (44.7)112/598 (18.7)<0.001b*Foot/ankle injections, n (%)221/416 (53.1)36/576 (6.2)<0.001b*Gout, n (%)56/486 (12.0)70/619 (11.3)0.737bPattern 3 finger ratio (2D<4D), n (%)300/452 (66.3)373/598 (62.3)0.181bHallux valgus, n (%): Current118/454 (25.9)159/607 (26.1)0.941bConstitutional (aged 20 years)27/428 (6.3)39/568 (6.9)0.710bFlat foot, n (%)39/443 (8.8)55/600 (9.1)0.840bBody pain, n (%)103/468 (22.0)177/619 (29.0)0.014b*a = Independent sample T-test. b= Chi-Square test.SD: Standard deviation. Table 2Prevalence of foot/ankle OA between footballers and controls.Prevalence, n (%)FootballersControlsaRR1 (95% CI)Self-reported Outcomes, n468619Current foot/ankle pain150/429 (35.0)150/574 (26.0)1.32 (1.03 - 1.68)*Ever foot/ankle pain ≥3 months133/438 (30.0)160/590 (27.0)1.15 (0.89 - 1.48)GP-diagnosed foot/ankle OA55/424 (13.0)47/581 (8.0)1.79 (1.18 - 2.71)*Surgery forefoot/ ankle103/426 (24.1)47/595 (7.9)2.82 (1.94 - 4.09)**Major risk factorsInjury190/425 (44.7)112/598 (18.7)2.27 (1.77 - 2.93)**Injection221/416 (53.1)36/576 (6.2)8.13 (5.68 - 11.63)*** p<0.05. ** p<0.001.1 aRR = Adjusted Rlative Risk: for age, socioeconomic status, and Charlson Comorbidity Index.

Background Osteoarthritis (OA) has a lifetime risk of over 40%, imposing a huge societal burden. Clinical variability suggests that it could be more than one disease. Synovial fluid To detect Endotypes by Unbiased Proteomics in OA (STEpUP OA) was established to test the hypothesis that there are detectable distinct molecular endotypes in knee OA. Methods OA knee synovial fluid (SF) samples (N=1361) were from pre-existing OA cohorts with cross-sectional clinical (radiographic and pain) data. Samples were divided into Discovery (N = 708) and Replication (N=653) datasets. Proteomic analysis was performed using SomaScan V4.1 assay (6596 proteins). Unsupervised clustering was performed using k-means, assessed using the f(k) metric, with and without adjustments for potential confounders. Regression analyses were used to assess protein associations with radiographic (Kellgren and Lawrence) and knee pain (WOMAC pain), with and without stratification by body mass index (BMI) or biological sex. Adjustments were made for cohort (random intercept) or intracellular protein, using an intracellular protein score (IPS). Analyses were carried out in R according to a pre-published plan. Results No distinct SF molecular endotypes were identified in OA but two indistinct clusters were defined in non-IPS regressed data which were stable across subgroup analyses. Clustering was lost after IPS regression adjustment. Strong, replicable protein associations were observed with radiographic disease severity, which were retained after adjustment for cohort or IPS. Pathway analysis identified a strong epithelial to mesenchymal transition (EMT) pathway, and weaker associations with angiogenesis, complement and coagulation. The latter were variably lost after adjustment for BMI or biological sex. Associations with patient reported pain were weaker. Conclusion These data support knee OA as a biologically continuous disease in which disease severity is associated with a strong, robust, tissue remodelling signature. Subtle differences were found in pathways after stratification by BMI or sex.### Competing Interest StatementTAP, YD, PH, SL, AS, NKA, AJP, DF, MK, BM, AMV and SK declare no conflicts of interest. FW has received consultancy fees from Pfizer. LSL has received consultancy fees from Arthro Therapeutics AB, and is an advisory board member of AstraZeneca. LJD has received consultancy fees from Nightingale Health PLC. TLV has no conflicts to declare with the exception of grant income for STEpUP OA from industry partners (see above). RAM is a shareholder of AstraZeneca. SB and JM are employees and shareholders of Novartis. CTA has received consultancy fees from Novartis, and has received honoraria for educational purposes also from Novartis. TJW is a shareholder of Chondropeptix BV. DAW has received consultancy fees from GlaxoSmithKline plc, AKL Research & Development Limited, Pfizer Ltd, Eli Lilly and Company, Contura International, and AbbVie Inc, has received honoraria for educational purposes from Pfizer Ltd and AbbVie Inc, is a board member (Director) of UKRI and Versus Arthritis Advanced Pain Discovery Platform. ### Funding StatementThe study was supported by Kennedy Trust for Rheumatology Research (grant number: 171806), Versus Arthritis (grant number: 22473), Centre for Osteoarthritis Pathogenesis Versus Arthritis (grant numbers: 21621, 20205), Galapagos, Biosplice, Novartis, Fidia, UCB, Pfizer (non-consortium member) and Somalogic (in kind contributions). The funders Kennedy Trust for Rheumatology Research, Versus Arthritis and Pfizer had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The funders Galapagos, Biosplice, Novartis, Fidia, UCB and SomaLogic were all active consortium members, attending consortium meetings. As such they made contributions to the study design and support of data collection, decision to publish and review and commenting on the manuscript. In addition, SomaLogic, UCB and Novartis were members of the Data Analysis Group.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:University of Oxford Medical Sciences Central University Research Ethics Committee (CUREC) granted ethical approval for the processing, storage and use of samples and linked data for this project on 1st November 2019 (R67029/RE001).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesThe minimal datasets upon which this data relies and all R code, including the html vignette, are available at https://github.com/ndorms-tperry/STEpUP-OA-Primary-Manuscript. The full STEpUP OA dataset may be made available by application to the Data Access and Publication Group of STEpUP OA (stepupoa@kennedy.ox.ac.uk) once the primary analysis manuscript is published, in accordance with what is stipulated in our Consortium Agreement. This may attract an access fee to cover administrative processing. Neither the minimal dataset nor the full STEpUP OA dataset include patient identifiable data.

Objectives To develop a protocol for largescale analysis of synovial fluid proteins, for the identification of biological networks associated with subtypes of osteoarthritis. Methods Synovial Fluid To detect molecular Endotypes by Unbiased Proteomics in Osteoarthritis (STEpUP OA) is an international consortium utilising clinical data (capturing pain, radiographic severity and demographic features) and knee synovial fluid from 17 participating cohorts. 1746 samples from 1650 individuals comprising OA, joint injury, healthy and inflammatory arthritis controls, divided into discovery (n = 1045) and replication (n = 701) datasets, were analysed by SomaScan Discovery Plex V4.1 (>7000 SOMAmers/proteins). An optimised approach to standardisation was developed. Technical confounders and batch-effects were identified and adjusted for. Poorly performing SOMAmers and samples were excluded. Variance in the data was determined by principal component (PC) analysis. Results A synovial fluid standardised protocol was optimised that had good reliability (<20% co-efficient of variation for >80% of SOMAmers in pooled samples) and overall good correlation with immunoassay. 1720 samples and >6290 SOMAmers met inclusion criteria. 48% of data variance (PC1) was strongly correlated with individual SOMAmer signal intensities, particularly with low abundance proteins (median correlation coefficient 0.70), and was enriched for nuclear and non-secreted proteins. We concluded that this component was predominantly intracellular proteins, and could be adjusted for using an ‘intracellular protein score’ (IPS). PC2 (7% variance) was attributable to processing batch and was batch-corrected by ComBat. Lesser effects were attributed to other technical confounders. Data visualisation revealed clustering of injury and OA cases in overlapping but distinguishable areas of high-dimensional proteomic space. Conclusions We have developed a robust method for analysing synovial fluid protein, creating a molecular and clinical dataset of unprecedented scale to explore potential patient subtypes and the molecular pathogenesis of OA. Such methodology underpins the development of new approaches to tackle this disease which remains a huge societal challenge.

IntroductionHand osteoarthritis is more common in women, and its risk increases around the time of the menopause. We set out to describe the timing between menopause and the onset of symptomatic hand osteoarthritis (OA), and associations with the use of hormone replacement therapy (HRT) or its discontinuation, describing any identifiable subgroups of women.MethodsRetrospective healthcare-records study of sequential women referred to a specialist hand OA clinic, 2007–2015. Confirmation of hand OA diagnosis was by clinican, by accepted criteria. Demographics and clinical variables were from healthcare-records, recorded by standardised proforma. Outcomes of interest were reported age of onset of hand symptoms, reported age at final menstrual period (FMP), time from FMP to reported onset of hand symptoms and time from cessation of HRT to reported onset of hand symptoms. Exposure categories for systemic HRT use were never users, current users, previous users. Analysis of Variance compared groups; linear regression analysed associations of exposure with outcome.Results82/92(89%) of eligible women were post-menopausal, mean age at FMP 49.9 years (SD5.4). In these post-menopausal women, median time from FMP to hand symptom onset was 3 years. 48/82 (59%) developed hand symptoms within the defined peri-menopausal period (FMP ± 4 years), whilst some women developed their symptoms before or after (range −25, 30 years). In women who discontinued HRT prior to symptom onset, the median time from HRT cessation to onset of hand symptoms was 6 months. Past HRT users were older at hand symptom onset than women who had not taken HRT [coeff.4.7 years (0.92, 8.39); P = 0.015].ConclusionsThis study adds to evidence associating the menopause/sex hormone deficiency with hand OA symptom onset in a sizeable subgroup of women (but not all). HRT use/cessation appears to influence the timing of onset of hand OA symptoms. It is not possible to interpret from this type of study whether sex hormone deficiency is causative of disease or modulates its symptoms. It is also not possible to judge whether painful hand osteoarthritis in post-menopausal women is a subtype of disease. Further investigation is indicated of sex-specific subtypes and potential for personalised medicine for post-menopausal women with hand osteoarthritis, as a clearly definable high-risk subgroup.