Methodological development of molecular endotype discovery from synovial fluid of individuals with knee osteoarthritis: the STEpUP OA Consortium

Objectives To develop and validate a pipeline for quality controlled (QC) protein data for largescale analysis of synovial fluid (SF), using SomaLogic technology. Design Knee SF and associated clinical data were from partner cohorts. SF samples were centrifuged, supernatants stored at −80 °C, then analysed by SomaScan Discovery Plex V4.1 (>7000 SOMAmers/proteins). Setting An international consortium of 9 academic and 8 commercial partners (STEpUP OA). Participants 1746 SF samples from 1650 individuals comprising OA, joint injury, healthy controls and inflammatory arthritis controls, divided into discovery (n=1045) and replication (n=701) datasets. Primary and secondary outcome measures An optimised approach to standardisation was developed iteratively, monitoring reliability and precision (comparing coefficient of variation [%CV] of ‘pooled’ SF samples between plates and correlation with prior immunoassay for 9 analytes). Pre-defined technical confounders were adjusted for (by Limma) and batch correction was by ComBat. Poorly performing SOMAmers and samples were filtered. Variance in the data was determined by principal component (PC) analysis. Data were visualised by Uniform Manifold Approximation and Projection (UMAP). Results Optimal SF standardisation aligned with that used for plasma, but without median normalisation. There was good reliability (<20 %CV for >80% of SOMAmers in pooled samples) and overall good correlation with immunoassay. PC1 accounted for 48% of variance and strongly correlated with individual SOMAmer signal intensities (median correlation coefficient 0.70). These could be adjusted using an ‘intracellular protein score’. PC2 (7% variance) was attributable to processing batch and was batch-corrected by ComBat. Lesser effects were attributed to other technical confounders. Data visualisation by UMAP revealed clustering of injury and OA cases in overlapping but distinguishable areas of high-dimensional proteomic space. Conclusions We define a standardised approach for SF analysis using the SOMAscan platform and identify likely ‘intracellular’ protein as being a major driver of variance in the data. Strengths and limitations ### Competing Interest Statement YD, TAP, PH, SL, AS, NKA, DF, BM, AMV, SK, VB, JMA and VK declare no conflicts of interest. FW has received consultancy fees from Pfizer, and has a leadership role at the Medical Research Council (panel member) and Osteoarthritis and Cartilage (Associate Editor). LSL has received consultancy fees from Arthro Therapeutics AB, and is an advisory board member of AstraZeneca (non consortium member). 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 (consortium members). MK has received support for attending the Gordon Research Conference, OARSI meeting, International Cartilage Repair Society, Munster University, is a board member of the Dutch Arthritis Society (Chair of Visitation Board), and has a leadership role at Osteoarthritis Research Society International (Board of Directors Member). 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 of UKRI (Director) and Versus Arthritis Advanced Pain Discovery Platform. TLV directs the Centre for OA pathogenesis (grant numbers 21612 and 20205) and has additional grant support from Versus Arthritis, the European Research Council, the Medical Research Council and FOREUM. LJD is supported by a Wellcome trust fellowship grant 208750/Z/17/Z and Kennedy Trust for Rheumatology Research for the present manuscript. LJD is also supported by grants from the MRC and the Helmsley Charitable Trust. FEW is supported by a UKRI Future Leaders Fellowship (MRC number: MR/S016538/1 and MR/S016538/2). FW, NKA and SK are members of the Centre for Sport, Exercise and Osteoarthritis Research Versus Arthritis (grant number 21595). MK is supported by grants from CIHR, NSERC, The Arthritis Society Canada, Krembil Foundation, CFI, Canada Research Chairs program, and has received support from the University Health Network Foundation, Toronto for the present manuscript. TJW is supported by grants from NWO-TTW Perspectief (#P15-23), Stichting de Weijerhorst and ReumaNederland (LLP14) for the present manuscript, and is a shareholder of Chondropeptix BV. BDMT is supported through the United Kingdom Medical Research Council programme (grant MC UU 00002/2). For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising. LB is supported by grants from Kennedy Trust for Rheumatology Research (grant number 171806) and UK Medical Research Council (grant MC UU 00002/2). DAW is supported by grants from Pfizer Ltd, UCB Pharma, Orion Corporation, GlaxoSmithKline Research and Development, and Eli Lilly and Company, Versus Arthritis, UKRI, Nuffield Foundation. ### Funding Statement The study was supported by Kennedy Trust for Rheumatology Research (grant number: 171806), Versus Arthritis (grant number: 22473), Centre for OA Pathogenesis Versus Arthritis (grant numbers: 21621, 20205), Galapagos, Biosplice, Novartis, Fidia, UCB, Pfizer (non consortium member) and Somalogic (in kind contributions). YD, TAP, PH, SL, AS, NKA, DF, BM, AMV, SK, VB, JMA and VK declare no conflicts of interest. FW has received consultancy fees from Pfizer, and has a leadership role at the Medical Research Council (panel member) and Osteoarthritis and Cartilage (Associate Editor). LSL has received consultancy fees from Arthro Therapeutics AB, and is an advisory board member of AstraZeneca (non consortium member). 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 (consortium members). MK has received support for attending the Gordon Research Conference, OARSI meeting, International Cartilage Repair Society, Munster University, is a board member of the Dutch Arthritis Society (Chair of Visitation Board), and has a leadership role at Osteoarthritis Research Society International (Board of Directors Member). 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 of UKRI (Director) and Versus Arthritis Advanced Pain Discovery Platform. TLV directs the Centre for OA pathogenesis (grant numbers 21612 and 20205) and has additional grant support from Versus Arthritis, the European Research Council, the Medical Research Council and FOREUM. LJD is supported by a Wellcome trust fellowship grant 208750/Z/17/Z and Kennedy Trust for Rheumatology Research for the present manuscript. LJD is also supported by grants from the MRC and the Helmsley Charitable Trust. FEW is supported by a UKRI Future Leaders Fellowship (MRC number: MR/S016538/1 and MR/S016538/2). FW, NKA and SK are members of the Centre for Sport, Exercise and Osteoarthritis Research Versus Arthritis (grant number 21595). MK is supported by grants from CIHR, NSERC, The Arthritis Society Canada, Krembil Foundation, CFI, Canada Research Chairs program, and has received support from the University Health Network Foundation, Toronto for the present manuscript. TJW is supported by grants from NWO-TTW Perspectief (#P15-23), Stichting de Weijerhorst and ReumaNederland (LLP14) for the present manuscript, and is a shareholder of Chondropeptix BV. BDMT is supported through the United Kingdom Medical Research Council programme (grant MC UU 00002/2). For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising. LB is supported by grants from Kennedy Trust for Rheumatology Research (grant number 171806) and UK Medical Research Council (grant MC UU 00002/2). DAW is supported by grants from Pfizer Ltd, UCB Pharma, Orion Corporation, GlaxoSmithKline Research and Development, and Eli Lilly and Company, Versus Arthritis, UKRI, Nuffield Foundation. ### 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). Each site ensured compliance with local ethical and data protection policies and appropriate written informed consent from each participant via existing approvals which included collaborative use. All signed a material transfer agreement prior to movement of samples/data. 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 In accordance with the STEpUP OA Consortium Agreement and the Data Access and Publication Group, protein and clinical data will be available for bone fide research relating to osteoarthritis through an application to the STEpUP OA Data Access and Publication group once the discovery and replication analyses are in press and if it does not infringe patent position. This may be subject to an access fee (to be confirmed).