Lupus RMD Open: first published as 10.1136/rmdopen-2020-001257 on 28 July 2020. Downloaded from Original research Urine and serum S100A8/A9 and S100A12 associate with active lupus nephritis and may predict response to rituximab treatment Jennifer C Davies,1 Angela Midgley,1 Emil Carlsson,1 Sean Donohue,1 Ian N Bruce,2 Michael W Beresford,1,3 Christian M Hedrich 1,3 To cite: Davies JC, Midgley A, ABSTRACT et al Key messages Carlsson E, . Urine and Background Approximately 30% of patients with the serum S100A8/A9 and S100A12 systemic autoimmune/inflammatory disorder systemic lupus associate with active lupus What is already known about this subject? erythematosus (SLE) develop lupus nephritis (LN) that affects nephritis and may predict ► Though ≈30% of patients with Systemic lupus treatment and prognosis. Easily accessible biomarkers do not response to rituximab treatment. erythematosus (SLE) develop lupus nephritis that exist to reliably predict renal disease. The Maximizing SLE RMD Open 2020;6:e001257. affects treatment and prognosis, easily accessible Therapeutic Potential by Application of Novel and Systemic doi:10.1136/rmdopen-2020- biomarkers do not exist to reliably predict renal Approaches and the Engineering Consortium aims to identify 001257 disease. indicators of treatment responses in SLE. This study tested ► Supplemental material is the applicability of calcium-binding S100 proteins in serum published online only. To view What does this study add? ► please visit the journal online and urine as biomarkers for disease activity and response to Serum S100A12, and serum and urine S100A8/A9 (http://dx.doi.org/10.1136/rmdo treatment with rituximab (RTX) in LN. (p<0.001) levels are elevated in SLE patients, and pen-2020-001257). Methods S100A8/A9 and S100A12 proteins were levels in urine and urine/serum ratios are elevated in quantified in the serum and urine of 243 patients with SLE patients with active LN. MWB and CH contributed from the British Isles Lupus Assessment Group Biologics ► Binary logistic regression and AUC analyses equally. Register (BILAG-BR) study and 48 controls matched for age suggests the combination of serum S100A8/A9 and ’ http://rmdopen.bmj.com/ Received 1 April 2020 using Meso Scale Discovery s technology to determine S100A12 can predict response to RTX treatment in Revised 27 May 2020 whether they perform as biomarkers for active LN and/or LN after 6 months. Accepted 7 June 2020 may be used to predict response to treatment with RTX. Renal disease activity and response to treatment was based How might this impact on clinical practice? on BILAG-BR scores and changes in response to treatment. ► While findings show promise for clinical application Results Serum S100A12 (p<0.001), and serum and urine of S100 proteins to predict active LN and response to S100A8/A9 (p<0.001) levels are elevated in patients with treatment with rituximab, they require to be SLE. While serum and urine S100 levels do not correlate confirmed in independent cohorts and longitudinal/ with global disease activity (SLE Disease Activity Index), prospective studies. levels in urine and urine/serum ratios are elevated in on September 30, 2021 by guest. Protected copyright. patients with active LN. S100 proteins perform better as biomarkers for active LN involvement in patients with SLE interactions of genetic factors, the environ- who tested positive for anti-double-stranded DNA ment and hormones contribute to disease 12 antibodies. Binary logistic regression and area under the expression. Tissue inflammation and organ curve analyses suggest the combination of serum S100A8/ damage are caused by immune cell dysregula- A9 and S100A12 can predict response to RTX treatment in tion including enhanced generation and acti- LN after 6 months. vation of effector T cells, altered cytokine Conclusions Findings from this study show promise for expression, B cell activation, autoantibody pro- clinical application of S100 proteins to predict active renal duction, immune complex deposition and disease in SLE and response to treatment with RTX. 34 © Author(s) (or their complement activation. employer(s)) 2020. Re-use Approximately 30% of adult-onset patients – permitted under CC BY. with SLE develop renal disease,5 7 and damage Published by BMJ. INTRODUCTION is strongly associated with morbidity and For numbered affiliations see Systemic lupus erythematosus (SLE) is mortality.89Diagnosing renal disease and mea- end of article. a systemic autoimmune/inflammatory disease suring its activity can be challenging, especially Correspondence to that can affect any organ of the human body. since frequently tested nephritis-associated pro- Christian Hedrich; christian. The molecular pathophysiology of SLE teinuria cannot reliably predict WHO nephritis [email protected] remains largely unknown, but complex class (that centrally influences treatment Davies JC, et al. RMD Open 2020;6:e001257. doi:10.1136/rmdopen-2020-001257 1 RMD Open RMD Open: first published as 10.1136/rmdopen-2020-001257 on 28 July 2020. Downloaded from decision) and/or inflammatory activity (since damage can PATIENTS AND METHODS result in renal protein loss). Thus, currently, renal biopsies Patients are used to reliably determine renal disease and/or the Serum and urine samples were collected from adult- form (WHO class) and extent of renal disease.10 onset patients with SLE (n=243) in the UK through Patients with SLE exhibit extraordinarily variable centres involved in the British Isles Lupus Assessment treatment responses, and we are currently unable to Group (BILAG) Biologics Register (BR). The BILAG- predict response to treatment options available. BR received ethical approval from the Health Research Though treatment recommendations for patients Authority dated 9 November 2009 (IRAS ref. 24407) with SLE-associated renal disease have been and written informed consent was obtained in accor- published,11 12 not all patients will respond to first- dance with the declaration of Helsinki. Serum (n=236) line agents and treatment optimisation not uncom- and urine (n=199) samples were collected at baseline in monly follows trial-and-error strategies. Thus, the patients who were being initiated on a change in immu- identification of pathomechanisms involved and the nosuppressive/immunomodulatory treatment. Samples establishment of predictive biomarkers are urgently were sent at ambient temperature in the overnight post needed to optimise treatment. Indeed, biologically for processing at a central laboratory at the Centre for informed individualised and target-directed Musculoskeletal Research, University of Manchester. approaches promise to be of benefit to both patients Clinical details including demographic data, concur- and healthcare systems by reducing disease burden, rent treatment, disease activity and treatment response time to remission, disease-associated damage and dis- were obtained from medical records through BILAG- ability, and treatment-associated cost.13 Depletion of BR. Patients were excluded from analysis if they were B lymphocytes with rituximab (RTX) can reduce SLE taking other biologic disease-modifying anti-rheumatic disease activity in a subset of patients with SLE, but drugs, and background doses of immunosuppressive only half of patients with SLE respond to RTX after drugs and corticosteroid were determined by the local 6months.14 15 However, biomarkers to predict treat- treating physician according to their usual clinical prac- ment responses and/or disease outcomes are currently tice ‘low-dose’ corticosteroids (table 1). All patients not available. included met the 1997 American College of Rheuma- Calcium-binding S100 proteins are mainly expressed tology classification criteria for SLE.23 Furthermore, in granulocytes and mononuclear blood cells such as serum and urine samples were obtained from 48 adult neutrophils and macrophages.16 17 More recently, healthy donors as controls (HC). Individual data points expression of these S100 proteins has been reported that required to be excluded from analysis are sum- in renal endothelial cells.18 Elevated serum concentra- marised in online supplementary table 1. tions of S100 proteins have been linked with systemic http://rmdopen.bmj.com/ inflammatory conditions in children and adults.19 Classification of disease activity Recently, elevated levels of pro-inflammatory The BILAG-2004 grade system scores disease activity as S100A12 and heterodimeric S100A8/A9 in serum follows: A (severe disease), B (moderate disease), and urine samples from patients with juvenile-onset C (mild or improving disease), D (inactive disease but SLE and in serum of adult-onset patients with SLE previous system disease) and E (system has never been – 24 were reported to correlate with renal disease.20 22 Pre- involved). For data interpretation, active disease of vious studies have not examined S100 proteins in the any system was defined as having grade A or B and inactive disease as D or E. Scores of C were not included serum and urine from the same patients and did not on September 30, 2021 by guest. Protected copyright. address the potential of combining S100 protein levels in the analysis to provide distinct disease states. To from serum and urine to determine systemic and/or generate global BILAG-BR scores, letters A, B, C, renal disease status. Furthermore, the predictive value D and E for each nine domains were converted to 25 of S100 expression regarding treatment responses has numerical scores, as previously described, and the not