Deconvoluting synovial fluid molecular endotypes in knee osteoarthritis: primary results from the STEpUP OA Consortium

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 Statement TAP, 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 Statement The 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 Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The 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. Yes I 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). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The 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.