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Background: Type I interferons (IFN-Is) play a role in a broad range of rheumatic and musculoskeletal diseases (RMDs), and compelling evidence suggests that their measurement could have clinical value, although testing has not progressed into clinical settings. Objective: To develop evidence-based points to consider (PtC) for the measurement and reporting of IFN-I assays in clinical research and to determine their potential clinical utility. Methods: EULAR standardised operating procedures were followed. A task force including rheumatologists, immunologists, translational scientists and a patient partner was formed. Two systematic reviews were conducted to address methodological and clinical questions. PtC were formulated based on the retrieved evidence and expert opinion. Level of evidence and agreement was determined. Results: Two overarching principles and 11 PtC were defined. The first set (PtC 1-4) concerned terminology, assay characteristics and reporting practices to enable more consistent reporting and facilitate translation and collaborations. The second set (PtC 5-11) addressed clinical applications for diagnosis and outcome assessments, including disease activity, prognosis and prediction of treatment response. The mean level of agreement was generally high, mainly in the first PtC set and for clinical applications in systemic lupus erythematosus. Harmonisation of assay methodology and clinical validation were key points for the research agenda. Conclusions: IFN-I assays have a high potential for implementation in the clinical management of RMDs. Uptake of these PtC will facilitate the progress of IFN-I assays into clinical practice and may be also of interest beyond rheumatology.

ObjectivesTo systematically review the literature for assay methods that aim to evaluate type I interferon (IFN-I) pathway activation and to harmonise-related terminology.MethodsThree databases were searched for reports of IFN-I and rheumatic musculoskeletal diseases. Information about the performance metrics of assays measuring IFN-I and measures of truth were extracted and summarised. A EULAR task force panel assessed feasibility and developed consensus terminology.ResultsOf 10 037 abstracts, 276 fulfilled eligibility criteria for data extraction. Some reported more than one technique to measure IFN-I pathway activation. Hence, 276 papers generated data on 412 methods. IFN-I pathway activation was measured using: qPCR (n=121), immunoassays (n=101), microarray (n=69), reporter cell assay (n=38), DNA methylation (n=14), flow cytometry (n=14), cytopathic effect assay (n=11), RNA sequencing (n=9), plaque reduction assay (n=8), Nanostring (n=5), bisulphite sequencing (n=3). Principles of each assay are summarised for content validity. Concurrent validity (correlation with other IFN assays) was presented for n=150/412 assays. Reliability data were variable and provided for 13 assays. Gene expression and immunoassays were considered most feasible. Consensus terminology to define different aspects of IFN-I research and practice was produced.ConclusionsDiverse methods have been reported as IFN-I assays and these differ in what elements or aspects of IFN-I pathway activation they measure and how. No 'gold standard' represents the entirety of the IFN pathway, some may not be specific for IFN-I. Data on reliability or comparing assays were limited, and feasibility is a challenge for many assays. Consensus terminology should improve consistency of reporting.

BackgroundType I interferons (IFN-I) contribute to a broad range of rheumatic and musculoskeletal diseases (RMDs). Compelling evidence suggests that the measurement of IFN-I pathway activation may have clinical value. Although several IFN-I pathway assays have been proposed, the exact clinical applications are unclear. We summarise the evidence on the potential clinical utility of assays measuring IFN-I pathway activation.MethodsA systematic literature review was conducted across three databases to evaluate the use of IFN-I assays in diagnosis and monitor disease activity, prognosis, response to treatment and responsiveness to change in several RMDs.ResultsOf 366 screened, 276 studies were selected that reported the use of assays reflecting IFN-I pathway activation for disease diagnosis (n=188), assessment of disease activity (n=122), prognosis (n=20), response to treatment (n=23) and assay responsiveness (n=59). Immunoassays, quantitative PCR (qPCR) and microarrays were reported most frequently, while systemic lupus erythematosus (SLE), rheumatoid arthritis, myositis, systemic sclerosis and primary Sjogren's syndrome were the most studied RMDs. The literature demonstrated significant heterogeneity in techniques, analytical conditions, risk of bias and application in diseases. Inadequate study designs and technical heterogeneity were the main limitations. IFN-I pathway activation was associated with disease activity and flare occurrence in SLE, but their incremental value was uncertain. IFN-I pathway activation may predict response to IFN-I targeting therapies and may predict response to different treatments.ConclusionsEvidence indicates potential clinical value of assays measuring IFN-I pathway activation in several RMDs, but assay harmonisation and clinical validation are urged. This review informs the EULAR points to consider for the measurement and reporting of IFN-I pathway assays.

OBJECTIVES: To assess to what extent leflunomide (LEF) and hydroxychloroquine (HCQ) therapy in patients with primary Sjögren's syndrome (RepurpSS-I) targets type I IFN-associated responses and to study the potential of several interferon associated RNA-based and protein-based biomarkers to predict and monitor treatment. METHODS: In 21 patients treated with LEF/HCQ and 8 patients treated with placebo, blood was drawn at baseline, 8, 16 and 24 weeks. IFN-signatures based on RNA expression of five IFN-associated genes were quantified in circulating mononuclear cells and in whole blood. MxA protein levels were measured in whole blood, and protein levels of CXCL10 and Galectin-9 were quantified in serum. Differences between responders and non-responders were assessed and receiver operating characteristic analysis was used to determine the capacity of baseline expression and early changes (after 8 weeks of treatment) in biomarkers to predict treatment response at the clinical endpoint. RESULTS: IFN-signatures in peripheral blood mononuclear cell and whole blood decreased after 24 weeks of LEF/HCQ treatment, however, changes in IFN signatures only poorly correlated with changes in disease activity. In contrast to baseline IFN signatures, baseline protein concentrations of galectin-9 and decreases in circulating MxA and Galectin-9 were robustly associated with clinical response. Early changes in serum Galectin-9 best predicted clinical response at 24 weeks (area under the curve 0.90). CONCLUSIONS: LEF/HCQ combination therapy targets type-I IFN-associated proteins that are associated with strongly decreased B cell hyperactivity and disease activity. IFN-associated Galectin-9 is a promising biomarker for treatment prediction and monitoring in pSS patients treated with LEF/HCQ.

Objective To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters. Methods Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets. Results Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters. Conclusion Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.

INTRODUCTION: Activated B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of pSS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to investigate if the eMZL clonotype is detectable in prediagnostic blood and tissue biopsies of pSS patients.METHODS/RESULTS: We studied prediagnostic tissue biopsies of three pSS patients diagnosed with eMZL and four pSS controls through immunoglobulin (IG) gene repertoire sequencing. In all three cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. Among controls, we observed transient elevation of clonotypes in two pSS patients. To evaluate if eMZL clonotypes may also be detected in the circulation, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and two years prior to eMZL relapse in two pSS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis in both cases. Next, we selected three pSS patients who developed eMZL lymphoma and five additional pSS patients who remained lymphoma-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of three years before eMZL diagnosis. In two out of three eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed consisted of stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in pSS.DISCUSSION: In conclusion, our results indicate that eMZL clonotypes in pSS patients are detectable prior to overt eMZL diagnosis in both tissue biopsies and peripheral blood through immunogenetic s

Background Type I interferons (IFN-I) contribute to a broad range of rheumatic and musculoskeletal diseases (RMDs). Compelling evidence suggests that the measurement of IFN-I pathway activation may have clinical value. Although several IFN-I pathway assays have been proposed, the exact clinical applications are unclear. We summarise the evidence on the potential clinical utility of assays measuring IFN-I pathway activation. Methods A systematic literature review was conducted across three databases to evaluate the use of IFN-I assays in diagnosis and monitor disease activity, prognosis, response to treatment and responsiveness to change in several RMDs. Results Of 366 screened, 276 studies were selected that reported the use of assays reflecting IFN-I pathway activation for disease diagnosis (n=188), assessment of disease activity (n=122), prognosis (n=20), response to treatment (n=23) and assay responsiveness (n=59). Immunoassays, quantitative PCR (qPCR) and microarrays were reported most frequently, while systemic lupus erythematosus (SLE), rheumatoid arthritis, myositis, systemic sclerosis and primary Sjögren's syndrome were the most studied RMDs. The literature demonstrated significant heterogeneity in techniques, analytical conditions, risk of bias and application in diseases. Inadequate study designs and technical heterogeneity were the main limitations. IFN-I pathway activation was associated with disease activity and flare occurrence in SLE, but their incremental value was uncertain. IFN-I pathway activation may predict response to IFN-I targeting therapies and may predict response to different treatments. Conclusions Evidence indicates potential clinical value of assays measuring IFN-I pathway activation in several RMDs, but assay harmonisation and clinical validation are urged. This review informs the EULAR points to consider for the measurement and reporting of IFN-I pathway assays.

Objectives To systematically review the literature for assay methods that aim to evaluate type I interferon (IFN-I) pathway activation and to harmonise-related terminology. Methods Three databases were searched for reports of IFN-I and rheumatic musculoskeletal diseases. Information about the performance metrics of assays measuring IFN-I and measures of truth were extracted and summarised. A EULAR task force panel assessed feasibility and developed consensus terminology. Results Of 10 037 abstracts, 276 fulfilled eligibility criteria for data extraction. Some reported more than one technique to measure IFN-I pathway activation. Hence, 276 papers generated data on 412 methods. IFN-I pathway activation was measured using: qPCR (n=121), immunoassays (n=101), microarray (n=69), reporter cell assay (n=38), DNA methylation (n=14), flow cytometry (n=14), cytopathic effect assay (n=11), RNA sequencing (n=9), plaque reduction assay (n=8), Nanostring (n=5), bisulphite sequencing (n=3). Principles of each assay are summarised for content validity. Concurrent validity (correlation with other IFN assays) was presented for n=150/412 assays. Reliability data were variable and provided for 13 assays. Gene expression and immunoassays were considered most feasible. Consensus terminology to define different aspects of IFN-I research and practice was produced. Conclusions Diverse methods have been reported as IFN-I assays and these differ in what elements or aspects of IFN-I pathway activation they measure and how. No â € gold standard' represents the entirety of the IFN pathway, some may not be specific for IFN-I. Data on reliability or comparing assays were limited, and feasibility is a challenge for many assays. Consensus terminology should improve consistency of reporting.

Background/Purpose: Primary Sjögren’s Syndrome (SS) is characterized by B lymphocyte hyperactivity with B cell activating factor (BAFF) acting as an important regulator. Single Nucleotide Polymorphisms (SNPs) of the BAFF gene have been implicated in the pathogenesis of several autoimmune diseases characterized by heightened fatigue levels, including primary SS. We aimed to explore potential associations between BAFF SNPs and fatigue status of primary SS patients. Methods: Fatigue status was assessed in 199 consecutive primary SS patients (Greek cohort) using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) scale. Clinical, histological, laboratory, psychometric and personality data were also collected. DNA extracted from peripheral blood of all patients underwent evaluation for the presence of five BAFF SNPs (rs9514827, rs1041569, rs9514828, rs1224141, rs12583006) by PCR. To confirm our findings, an independent replicative cohort of 62 primary SS patients (Dutch cohort) was implemented. Finally, 52 multiple sclerosis (MS) patients were served as disease controls (MS cohort). Analysis of BAFF SNPs in association with fatigue levels was performed by the online platforms SNPStats and SHEsis and the SPSS 26 and Graph Pad Prism 8.00 software. Results: TT genotype of the rs9514828 BAFF polymorphism was significantly less frequent in the fatigued primary SS patients of the Greek cohort compared to the non-fatigued (14.1% vs 33.3%). The corresponding ORs [95%CI] in the dominant and overdominant models were 0.33 [0.15-0.72], p=0.003 and 0.42 [0.23-0.78], p=0.005 respectively. The association remained significant after adjustment for the variables contributing to fatigue in the univariate analysis (OR [95% CI]: 0.3 [0.1-0.9], p=0.026). Accordingly, in the Dutch cohort, there was a trend of lower mental fatigue among patients carrying the TT rs9514828 BAFF genotype compared to their CC counterparts (4.1 ± 2.4 vs 6.0 ± 2.2 respectively, p=0.06). The rs9

Introduction: Myxovirus resistance protein 1 (MxA) is a biomarker that is elevated in patients with viral infections. The goal of this study was to evaluate the diagnostic value of MxA in diagnosing COVID-19 infections in the emergency department (ED) patients. Methods: This was a single-center prospective observational cohort study including patients with a suspected COVID-19 infection. The primary outcome of this study was a confirmed COVID-19 infection by RT-PCR test. MxA was assessed using an enzyme immunoassay on whole blood and receiver operating chart and area under the curve (AUC) analysis was conducted. Sensitivity, specificity, negative predictive value, and positive predictive value of MxA on diagnosing COVID-19 at the optimal cut-off of MxA was determined. Results: In 2021, 100 patients were included. Of these patients, 77 patients had COVID-19 infection and 23 were non-COVID-19. Median MxA level was significantly higher (p <.001) in COVID-19 patients compared to non-COVID-19 patients, respectively 1933 and 0.1 ng/ml. The AUC of MxA on a confirmed COVID-19 infection was 0.941 (95% CI: 0.867–1.000). The optimal cut-off point of MxA was 252 ng/ml. At this cut-off point, the sensitivity of MxA on a confirmed COVID-19 infection was 94% (95% CI: 85%–98%) and the specificity was 91% (95% CI: 72%–99%). Conclusion: MxA accurately distinguishes COVID-19 infections from bacterial infections and noninfectious diagnoses in the ED in patients with a suspected COVID-19 infection. If the results can be validated, MxA could improve the diagnostic workup and patient flow in the ED.

OBJECTIVES: Clinical phenotyping and predicting treatment responses in SLE patients is challenging. Extensive blood transcriptional profiling has identified various gene modules that are promising for stratification of SLE patients. We aimed to translate existing transcriptomic data into simpler gene signatures suitable for daily clinical practice. METHODS: Real-time PCR of multiple genes from the IFN M1.2, IFN M5.12, neutrophil (NPh) and plasma cell (PLC) modules, followed by a principle component analysis, was used to identify indicator genes per gene signature. Gene signatures were measured in longitudinal samples from two childhood-onset SLE cohorts (n = 101 and n = 34, respectively), and associations with clinical features were assessed. Disease activity was measured using Safety of Estrogen in Lupus National Assessment (SELENA)-SLEDAI. Cluster analysis subdivided patients into three mutually exclusive fingerprint-groups termed (1) all-signatures-low, (2) only IFN high (M1.2 and/or M5.12) and (3) high NPh and/or PLC. RESULTS: All gene signatures were significantly associated with disease activity in cross-sectionally collected samples. The PLC-signature showed the highest association with disease activity. Interestingly, in longitudinally collected samples, the PLC-signature was associated with disease activity and showed a decrease over time. When patients were divided into fingerprints, the highest disease activity was observed in the high NPh and/or PLC group. The lowest disease activity was observed in the all-signatures-low group. The same distribution was reproduced in samples from an independent SLE cohort. CONCLUSIONS: The identified gene signatures were associated with disease activity and were indicated to be suitable tools for stratifying SLE patients into groups with similar activated immune pathways that may guide future treatment choices.