Abatacept in individuals at high risk of rheumatoid arthritis (APIPPRA): a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial.

BACKGROUND: Individuals with serum antibodies to citrullinated protein antigens (ACPA), rheumatoid factor, and symptoms, such as inflammatory joint pain, are at high risk of developing rheumatoid arthritis. In the arthritis prevention in the pre-clinical phase of rheumatoid arthritis with abatacept (APIPPRA) trial, we aimed to evaluate the feasibility, efficacy, and acceptability of treating high risk individuals with the T-cell co-stimulation modulator abatacept. METHODS: The APIPPRA study was a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial done in 28 hospital-based early arthritis clinics in the UK and three in the Netherlands. Participants (aged ≥18 years) at risk of rheumatoid arthritis positive for ACPA and rheumatoid factor with inflammatory joint pain were recruited. Exclusion criteria included previous episodes of clinical synovitis and previous use of corticosteroids or disease-modifying antirheumatic drugs. Participants were randomly assigned (1:1) using a computer-generated permuted block randomisation (block sizes of 2 and 4) stratified by sex, smoking, and country, to 125 mg abatacept subcutaneous injections weekly or placebo for 12 months, and then followed up for 12 months. Masking was achieved by providing four kits (identical in appearance and packaging) with pre-filled syringes with coded labels of abatacept or placebo every 3 months. The primary endpoint was the time to development of clinical synovitis in three or more joints or rheumatoid arthritis according to American College of Rheumatology and European Alliance of Associations for Rheumatology 2010 criteria, whichever was met first. Synovitis was confirmed by ultrasonography. Follow-up was completed on Jan 13, 2021. All participants meeting the intention-to-treat principle were included in the analysis. This trial was registered with EudraCT (2013-003413-18). FINDINGS: Between Dec 22, 2014, and Jan 14, 2019, 280 individuals were evaluated for eligibility and, of 213 participants, 110 were randomly assigned to abatacept and 103 to placebo. During the treatment period, seven (6%) of 110 participants in the abatacept group and 30 (29%) of 103 participants in the placebo group met the primary endpoint. At 24 months, 27 (25%) of 110 participants in the abatacept group had progressed to rheumatoid arthritis, compared with 38 (37%) of 103 in the placebo group. The estimated proportion of participants remaining arthritis-free at 12 months was 92·8% (SE 2·6) in the abatacept group and 69·2% (4·7) in the placebo group. Kaplan-Meier arthritis-free survival plots over 24 months favoured abatacept (log-rank test p=0·044). The difference in restricted mean survival time between groups was 53 days (95% CI 28-78; p<0·0001) at 12 months and 99 days (95% CI 38-161; p=0·0016) at 24 months in favour of abatacept. During treatment, abatacept was associated with improvements in pain scores, functional wellbeing, and quality-of-life measurements, as well as low scores of subclinical synovitis by ultrasonography, compared with placebo. However, the effects were not sustained at 24 months. Seven serious adverse events occurred in the abatacept group and 11 in the placebo group, including one death in each group deemed unrelated to treatment. INTERPRETATION: Therapeutic intervention during the at-risk phase of rheumatoid arthritis is feasible, with acceptable safety profiles. T-cell co-stimulation modulation with abatacept for 12 months reduces progression to rheumatoid arthritis, with evidence of sustained efficacy beyond the treatment period, and with no new safety signals. FUNDING: Bristol Myers Squibb.

Monitoring immunE DysregulAtion foLLowing Immune checkpOint-inhibitioN (MEDALLION): protocol for an observational cancer immunotherapy cohort study.

BACKGROUND: Checkpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the form of immune-related adverse events (IrAEs), which may be sustained and life-altering. IrAEs may require high-dose and/or prolonged steroid use and represent a significant healthcare burden. They mimic immune-mediated inflammatory diseases (IMIDs) but understanding of their pathogenesis is limited. The MEDALLION project aims to determine targetable mechanisms of immune dysregulation in IrAE development, employing an immune monitoring approach to determine changes in circulating and tissue resident cells of CPI recipients who do/do not develop them and assessing the contribution of the microbiome in parallel. METHODS: MEDALLION is a non-randomised longitudinal cohort study aiming to recruit 66 cancer patient recipients of anti-PD1/PD-L1, anti-CTLA-4 or combination therapy. Eligible participants include those with malignant melanoma in the adjuvant or metastatic setting, mesothelioma and non-small cell lung carcinoma (NSCLC) treated in the metastatic setting. Comprehensive clinical evaluation is carried out alongside blood, skin swab and stool sampling at the time of CPI initiation (baseline) and during subsequent routine hospital visits on 6 occasions over a 10-month follow-up period. It is conservatively anticipated that one third of enrolled patients will experience a "significant IrAE" (SirAE), defined according to pre-determined criteria specific to the affected tissue/organ system. Those developing such toxicity may optionally undergo a biopsy of affected tissue where appropriate, otherwise being managed according to standard of care. Peripheral blood mononuclear cells will be analysed using multi-parameter flow cytometry to investigate immune subsets, their activation status and cytokine profiles. Stool samples and skin swabs will undergo DNA extraction for 16 S ribosomal RNA (rRNA) sequencing and internal transcribed spacer (ITS) gene sequencing to determine bacterial and fungal microbiome diversity, respectively, including species associated with toxicity. Stored tissue biopsies will be available for in situ and single-cell transcriptomic evaluation. Analysis will focus on the identification of biological predictors and precursors of SirAEs. DISCUSSION: The pathogenesis of IrAEs will be assessed through the MEDALLION cohort, with the potential to develop tools for their prediction and/or strategies for targeted prevention or treatment. TRIAL REGISTRATION: The study was registered on 18/09/2023 in the ISRCTN registry (43,419,676).

A Bayesian network approach incorporating imputation of missing data enables exploratory analysis of complex causal biological relationships.

Bayesian networks can be used to identify possible causal relationships between variables based on their conditional dependencies and independencies, which can be particularly useful in complex biological scenarios with many measured variables. Here we propose two improvements to an existing method for Bayesian network analysis, designed to increase the power to detect potential causal relationships between variables (including potentially a mixture of both discrete and continuous variables). Our first improvement relates to the treatment of missing data. When there is missing data, the standard approach is to remove every individual with any missing data before performing analysis. This can be wasteful and undesirable when there are many individuals with missing data, perhaps with only one or a few variables missing. This motivates the use of imputation. We present a new imputation method that uses a version of nearest neighbour imputation, whereby missing data from one individual is replaced with data from another individual, their nearest neighbour. For each individual with missing data, the subsets of variables to be used to select the nearest neighbour are chosen by sampling without replacement the complete data and estimating a best fit Bayesian network. We show that this approach leads to marked improvements in the recall and precision of directed edges in the final network identified, and we illustrate the approach through application to data from a recent study investigating the causal relationship between methylation and gene expression in early inflammatory arthritis patients. We also describe a second improvement in the form of a pseudo-Bayesian approach for upweighting certain network edges, which can be useful when there is prior evidence concerning their directions.

Characterization of age-associated B cells in early drug-naïve rheumatoid arthritis patients.

Age-associated B cells (ABCs) are an immune cell subset linked to autoimmunity, infection and ageing, and whose pathophysiological importance was recently highlighted using single cell synovial tissue profiling. To elucidate their pathophysiological relevance, peripheral blood (PB) ABCs from early rheumatoid arthritis (eRA) patients naïve to disease-modifying anti-rheumatic drugs (DMARDs) were compared with their synovial fluid (SF) counterparts, and to PB ABCs from psoriatic arthritis patients and healthy controls. PB and SF B-cell subsets were phenotyped by multi-parameter flow cytometry, sorted and subjected to gene expression profiling (NanoString nCounter® Immunology V2 Panel) and functional characterization (stimulated cytokine measurements by immunoassay). PB ABCs of eRA patients, which are transcriptionally distinct from those of control cohorts, express chemokine receptors and adhesion molecules, such as CXCR3, that favour homing to inflammatory sites over lymphoid tissue. These cells are an activated, class-switched B-cell subset expressing high levels of HLA-DR, co-stimulatory molecules and T-bet. Their secretion profile includes IL-12p70 and IL-23 but low levels of IL-10. High surface expression of FcRL family members, including FcRL3, furthermore suggests a role for these cells in autoimmunity. Finally, and unlike in the periphery where they are rare, ABCs are the predominant B-cell subsets in SF. These observations indicate the predilection of ABCs for inflammatory tissue in RA, where their propensity for antigen presentation and pro-inflammatory phenotype may support autoimmune pathology. Their potential as a therapeutic target therefore warrants further study.

Micro-RNA content of circulating extracellular vesicles in early rheumatoid arthritis as biomarkers and mediators of methotrexate efficacy.

OBJECTIVES: Extracellular vesicles (EVs) are abundant in body fluids, contributing to intercellular signalling by transferring cargo that includes microRNAs (miRs) - themselves implicated in pathobiology. For the first time we evaluated the potential of EV miRs to contribute diagnostic information in early RA, predict methotrexate (MTX) efficacy or shed light on the drug's mechanism of action. METHODS: 798 miRs isolated from serum-derived EVs of 46 patients with untreated RA, 23 with untreated polymyalgia rheumatica (PMR; inflammatory disease control group) and 12 in whom significant inflammatory disease had been excluded (non-inflammatory controls; NICs) were profiled (Nanostring); the same measurements were made for RA patients after 6 months' MTX treatment. Analyses took multiple testing into account. RESULTS: 28 EV miRs were robustly differentially expressed between early RA (but not PMR) patients and NICs after correction for age and sex, suggesting discriminatory value. Cross-validated partial least squared-discriminant analysis also indicated the predictive potential of a distinct baseline EV miR signature with respect to MTX-induced remission at 6 months. The change in expression of 13 miRs over the course of MTX treatment differed significantly between responders and non-responders, and four of those exhibiting increased relative abundance amongst responders have known roles in regulating the pathogenic potential of synovial fibroblasts, namely miR-212-3p, miR-338-5p, miR-410-3p, and miR-537. CONCLUSION: Our data highlight the potential of serum EV miRs as diagnostic and therapeutic biomarkers, highlighting a novel potential mechanism via which MTX may exert its therapeutic effect in early RA that warrants further investigation.

Number of days required to measure sedentary time and physical activity using accelerometery in rheumatoid arthritis: a reliability study.

This study aimed to determine the minimum number of days required to reliably estimate free-living sedentary time, light-intensity physical activity (LPA) and moderate-intensity physical activity (MPA) using accelerometer data in people with Rheumatoid Arthritis (RA), according to Disease Activity Score-28-C-reactive protein (DAS-28-CRP). Secondary analysis of two existing RA cohorts with controlled (cohort 1) and active (cohort 2) disease was undertaken. People with RA were classified as being in remission (DAS-28-CRP < 2.4, n = 9), or with low (DAS-28-CRP ≥ 2.4-≤ 3.2, n = 15), moderate (DAS-28-CRP > 3.2-≤ 5.1, n = 41) or high (DAS-28-CRP > 5.1, n = 16) disease activity. Participants wore an ActiGraph accelerometer on their right hip for 7 days during waking hours. Validated RA-specific cut-points were applied to accelerometer data to estimate free-living sedentary time, LPA and MPA (%/day). Single-day intraclass correlation coefficients (ICC) were calculated and used in the Spearman Brown prophecy formula to determine the number of monitoring days required to achieve measurement reliability (ICC ≥ 0.80) for each group. The remission group required ≥ 4 monitoring days to achieve an ICC ≥ 0.80 for sedentary time and LPA, with low, moderate and high disease activity groups requiring ≥ 3 monitoring days to reliably estimate these behaviours. The monitoring days required for MPA were more variable across disease activity groups (remission = ≥ 3 days; low = ≥ 2 days; moderate = ≥ 3 days; high = ≥ 5 days). We conclude at least 4 monitoring days will reliably estimate sedentary time and LPA in RA, across the whole spectrum of disease activity. However, to reliably estimate behaviours across the movement continuum (sedentary time, LPA, MPA), at least 5 monitoring days are required.

Interferon-α-mediated therapeutic resistance in early rheumatoid arthritis implicates epigenetic reprogramming.

OBJECTIVES: An interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to treatment. We wished to validate this effect and explore potential mechanisms of action. METHODS: In a multicentre inception cohort of eRA patients (n=191), we examined the whole blood IGS (MxA, IFI44L, OAS1, IFI6, ISG15) with reference to circulating IFN proteins, clinical outcomes and epigenetic influences on circulating CD19+ B and CD4+ T lymphocytes. RESULTS: We reproduced our previous findings demonstrating a raised baseline IGS. We additionally showed, for the first time, that the IGS in eRA reflects circulating IFN-α protein. Paired longitudinal analysis demonstrated a significant reduction between baseline and 6-month IGS and IFN-α levels (p<0.0001 for both). Despite this fall, a raised baseline IGS predicted worse 6-month clinical outcomes such as increased disease activity score (DAS-28, p=0.025) and lower likelihood of a good EULAR clinical response (p=0.034), which was independent of other conventional predictors of disease activity and clinical response. Molecular analysis of CD4+ T cells and CD19+ B cells demonstrated differentially methylated CPG sites and dysregulated expression of disease relevant genes, including PARP9, STAT1, and EPSTI1, associated with baseline IGS/IFNα levels. Differentially methylated CPG sites implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1α). CONCLUSIONS: Our data suggest that, in eRA, IFN-α can cause a sustained, epigenetically mediated, pathogenic increase in lymphocyte activation and proliferation, and that the IGS is, therefore, a robust prognostic biomarker. Its persistent harmful effects provide a rationale for the initial therapeutic targeting of IFN-α in selected patients with eRA.

The role of comorbidities alongside patient and disease characteristics in long-term disease activity in RA using UK inception cohort data.

OBJECTIVES: Control of disease activity in RA is a crucial part of its management to prevent long-term joint damage and disability. This study aimed to identify early predictors of poor disease activity at 5 and 10 years, focusing on comorbidities and clinical/sociodemographic factors at first presentation. METHODS: Patients from two UK-based RA cohorts were classified into two groups; low (<3.2) and moderate/high (≥3.2) DAS using 28 joint counts (DAS28) at 5/10 years. Clinical variables (e.g. rheumatoid nodules, erosions), sociodemographic factors (e.g. ethnicity, deprivation) and comorbidities were recorded at baseline and yearly thereafter. The Rheumatic Diseases Comorbidity Index quantified patient comorbidity burden. Binary logistic regression models (outcome low vs moderate/high DAS28) were fitted using multiple imputation. RESULTS: A total of 2701 patients living with RA were recruited (mean age 56.1 years, 66.9% female); 5-year data were available for 1718 (63.4%) patients and 10-year data for 820 (30.4%). Baseline Rheumatic Diseases Comorbidity Index was not associated with DAS28 at 5 [odds ratio (OR) 1.05, 95% CI 0.91, 1.22] or 10 years (OR 0.99, 95% CI 0.75, 1.31) in multivariable analyses. Sociodemographic factors (female gender, worse deprivation) and poorer baseline HAQ-Disability Index were associated with DAS28 ≥3.2 at both timepoints. Being seropositive was associated with 5-year DAS28 ≥3.2. CONCLUSION: This study demonstrates an association between sociodemographic and clinical factors and long-term RA disease activity, in models adjusting for comorbidity burden. The findings call for more holistic and targeted patient management in patients with RA and provide insights for more individualized management plans even on first presentation to rheumatology.

Predictors of poor function in RA based on two prospective UK inception cohorts. Do comorbidities matter?

OBJECTIVES: Evidence suggests that factors beyond disease activity associate with functional disability in RA. The primary study objective was to explore associations between comorbidities, sociodemographic factors and functional outcomes at five and 10 years. METHODS: RA patients from two UK prospective cohorts were grouped into low (<1.5) and high (≥1.5) five- and 10-year health assessment questionnaire (HAQ) score. Clinical variables (e.g. disease activity, rheumatoid nodules, erosions) and sociodemographic factors (e.g. ethnicity, deprivation) were recorded at baseline and yearly thereafter. Comorbidity was measured using the Rheumatic Diseases Comorbidity Index (RDCI). Binary logistic regression models were fitted using multiple imputation. RESULTS: In total, 2701 RA patients were recruited (mean age 56.1 years, 66.9% female). A total of 1718 (63.4%) had five-year and 820 (30.4%) 10-year follow-up data. In multivariable analysis, no association was found between RDCI and HAQ ≥ 1.5 at five or 10 years. Sociodemographic factors (increased age at disease onset, female gender, minority ethnicity) were associated with higher odds of HAQ ≥ 1.5 at five and 10 years, with worse deprivation additionally associated with HAQ ≥ 1.5 at 10 years (OR 0.79, 95% CI: 0.69, 0.90). CONCLUSION: Comorbidities at baseline have not been found to be associated with worse RA functional outcome in the long-term. On the other hand, sociodemographic factors, independently of disease measures, are associated with worse functional outcome in RA at five and 10 years, in models adjusting for comorbidity burden. Tailoring management interventions according to not only clinical disease parameters but also patient sociodemographic factors may improve long-term outcomes including functional disability.

Dendritic cell integrin expression patterns regulate inflammation in the rheumatoid arthritis joint.

OBJECTIVES: Immune dysregulation contributes to the development of RA. Altered surface expression patterns of integrin adhesion receptors by immune cells is one mechanism by which this may occur. We investigated the role of ß2 integrin subunits CD11a and CD11b in dendritic cell (DC) subsets of RA patients. METHODS: Total ß2 integrin subunit expression and its conformation ('active' vs 'inactive' state) were quantified in DC subsets from peripheral blood (PB) and SF of RA patients as well as PB from healthy controls. Ex vivo stimulation of PB DC subsets and in vitro-generated mature and tolerogenic monocyte-derived DCs (moDCs) were utilized to model the clinical findings. Integrin subunit contribution to DC function was tested by analysing clustering and adhesion, and in co-cultures to assess T cell activation. RESULTS: A significant reduction in total and active CD11a expression in DCs in RA SF compared with PB and, conversely, a significant increase in CD11b expression was found. These findings were modelled in vitro using moDCs: tolerogenic moDCs showed higher expression of active CD11a and reduced levels of active CD11b compared with mature moDCs. Finally, blockade of CD11b impaired T cell activation in DC-T cell co-cultures. CONCLUSION: For the first time in RA, we show opposing expression of CD11a and CD11b in DCs in environments of inflammation (CD11alow/CD11bhigh) and steady state/tolerance (CD11ahigh/CD11blow), as well as a T cell stimulatory role for CD11b. These findings highlight DC integrins as potential novel targets for intervention in RA.

Lymphocyte DNA methylation mediates genetic risk at shared immune-mediated disease loci.

BACKGROUND: Defining regulatory mechanisms through which noncoding risk variants influence the cell-mediated pathogenesis of immune-mediated disease (IMD) has emerged as a priority in the post-genome-wide association study era. OBJECTIVES: With a focus on rheumatoid arthritis, we sought new insight into genetic mechanisms of adaptive immune dysregulation to help prioritize molecular pathways for targeting in this and related immune pathologies. METHODS: Whole-genome methylation and transcriptional data from isolated CD4+ T cells and B cells of more than 100 genotyped and phenotyped patients with inflammatory arthritis, all of whom were naive to immunomodulatory treatments, were obtained. Analysis integrated these comprehensive data with genome-wide association study findings across IMDs and other publicly available resources. RESULTS: We provide strong evidence that disease-associated DNA variants regulate cis-CpG methylation in CD4+ T and/or B cells at 37% RA loci. Using paired, cell-specific transcriptomic data and causal inference testing, we identify examples where site-specific DNA methylation in turn mediates gene expression, including FCRL3 in both cell types and ORMDL3/GSDMB, IL6ST/ANKRD55, and JAZF1 in CD4+ T cells. A number of genes regulated in this way highlight mechanisms common to RA and other IMDs including multiple sclerosis and asthma, in turn distinguishing them from osteoarthritis, a primarily degenerative disease. Finally, we corroborate the observed effects experimentally. CONCLUSIONS: Our observations highlight important mechanisms of genetic risk in RA and the wider context of immune dysregulation. They confirm the utility of DNA methylation profiling as a tool for causal gene prioritization and, potentially, therapeutic targeting in complex IMD.

Targeting of tolerogenic dendritic cells to heat-shock proteins in inflammatory arthritis.

BACKGROUND: Autologous tolerogenic dendritic cells (tolDC) are a promising therapeutic strategy for inflammatory arthritis (IA) as they can regulate autoantigen-specific T cell responses. Here, we investigated two outstanding priorities for clinical development: (i) the suitability of using heat-shock proteins (HSP), abundant in inflamed synovia, as surrogate autoantigens to be presented by tolDC and (ii) identification of functional biomarkers that confirm tolDC regulatory activity. METHODS: Cell proliferation dye-labelled human peripheral blood mononuclear cells of IA (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) patients or healthy donors were cultured with HSP40-, HSP60- and HSP70-derived peptides or recall antigens (e.g. tuberculin purified protein derivative (PPD)) in the presence or absence of tolDC or control DC for 9 days. Functional characteristics of proliferated antigen-specific T-cells were measured using flow cytometry, gene expression profiling and cytokine secretion immunoassays. Repeated measures analysis of variance (ANOVA) with Bonferroni correction for comparisons between multiple groups and paired Student t test for comparisons between two groups were used to determine significance. RESULTS: All groups showed robust CD4+ T-cell responses towards one or more HSP-derived peptide(s) as assessed by a stimulation index > 2 (healthy donors: 78%, RA: 73%, PsA: 90%) and production of the cytokines IFNγ, IL-17A and GM-CSF. Addition of tolDC but not control DC induced a type 1 regulatory (Tr1) phenotype in the antigen-specific CD4+ T-cell population, as identified by high expression of LAG3, CD49b and secretion of IL-10. Furthermore, tolDC inhibited bystander natural killer (NK) cell activation in a TGFß dependent manner. CONCLUSIONS: HSP-specific CD4+ T-cells are detectable in the majority of RA and PsA patients and can be converted into Tr1 cells by tolDC. HSP-loaded tolDC may therefore be suitable for directing T regulatory responses to antigens in inflamed synovia of IA patients. Tr1 markers LAG3, CD49b and IL-10 are suitable biomarkers for future tolDC clinical trials.

Predicting drug-free remission in rheumatoid arthritis: A prospective interventional cohort study.

BACKGROUND: Many patients with rheumatoid arthritis (RA) achieve disease remission with modern treatment strategies. However, having achieved this state, there are no tests that predict when withdrawal of therapy will result in drug-free remission rather than flare. We aimed to identify predictors of drug-free remission in RA. METHODS: The Biomarkers of Remission in Rheumatoid Arthritis (BioRRA) Study was a unique, prospective, interventional cohort study of complete and abrupt cessation of conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs). Patients with RA of at least 12 months duration and in clinical and ultrasound remission discontinued DMARDs and were monitored for six months. The primary outcome was time-to-flare, defined as disease activity score in 28 joints with C-reactive protein (DAS28-CRP) ≥ 2.4. Baseline clinical and ultrasound measures, circulating inflammatory biomarkers, and peripheral CD4+ T cell gene expression were assessed for their ability to predict time-to-flare and flare/remission status by Cox regression and receiver-operating characteristic (ROC) analysis respectively. RESULTS: 23/44 (52%) eligible patients experienced an arthritis flare after a median (IQR) of 48 (31.5-86.5) days following DMARD cessation. A composite score incorporating five baseline variables (three transcripts [FAM102B, ENSG00000228010, ENSG00000227070], one cytokine [interleukin-27], one clinical [Boolean remission]) differentiated future flare from drug-free remission with an area under the ROC curve of 0.96 (95% CI 0.91-1.00), sensitivity 0.91 (0.78-1.00) and specificity 0.95 (0.84-1.00). CONCLUSION: We provide proof-of-concept evidence for predictors of drug-free remission in RA. If validated, these biomarkers could help to personalize immunosuppressant withdrawal: a therapy paradigm shift with ensuing patient and economic benefits.

Routine musculoskeletal ultrasound findings impact diagnostic decisions maximally in autoantibody-seronegative early arthritis patients.

OBJECTIVE: The diagnostic value added by musculoskeletal ultrasound (MSUS) over standard clinical and laboratory parameters has proved difficult to quantify. The additive contribution to diagnostic classification of a pragmatic, 15 min MSUS protocol was appraised in a large, unselected cohort of early arthritis clinic attendees. METHODS: Detailed baseline characteristics were recorded. Semi-quantitative MSUS scoring of the most symptomatic wrist, second/third MCPs and PIPs and second/fifth MTPs was recorded, along with the sonographer's scan impression (definitely inflammatory, possibly inflammatory or non-inflammatory). MSUS findings were available to rheumatologist diagnosticians during subsequent consultations. Persistent inflammatory arthritis (PIA) was classified only where patients were started on ≥1 DMARD. Multivariate and receiver operating characteristic (ROC) curve analyses were used to identify independent discriminators of PIA, and the added value of MSUS parameters. RESULTS: Eight hundred and thirty-one patients were enrolled, of whom 31.3% acquired a PIA diagnosis. Swollen joint count, CRP, age and ACPA status were non-redundant clinical/laboratory predictors of a PIA diagnosis by consulting rheumatologists, with good discriminatory utility (area under the ROC curve, AUROC, 0.88). While the additive contribution of summed parameters from the seven-joint MSUS protocol to this model was statistically significant (P = 0.004), it was numerically small (ΔAUROC 0.02). However, the additive contribution to diagnostic outcome of sonographer's scan impression over clinical parameters alone became substantial in the sub-cohort of ACPA-negative patients, increasing the AUROC by 9% from 0.81 to 0.90 (P < 0.0001). CONCLUSION: The clinical utility of a 15-min MSUS screen for diagnosing PIA requiring DMARDs is most evident among ACPA-negative patients attending an early arthritis clinic.

Expression of STAT3-regulated genes in circulating CD4+ T cells discriminates rheumatoid arthritis independently of clinical parameters in early arthritis.

OBJECTIVES: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. METHODS: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. RESULTS: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. CONCLUSION: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

Between a ROC and a hard place: Teaching prevalence plots to understand real world biomarker performance in the clinic.

The Receiver Operating Characteristic (ROC) curve and the Area Under the Curve (AUC) of the ROC curve are widely used in discovery to compare the performance of diagnostic and prognostic assays. The ROC curve has the advantage that it is independent of disease prevalence. However, in this note, we remind scientists and clinicians that the performance of an assay upon translation to the clinic is critically dependent upon that very same prevalence. Without an understanding of prevalence in the test population, even robust bioassays with excellent ROC characteristics may perform poorly in the clinic. While the exact prevalence in the target population is not always known, simple plots of candidate assay performance as a function of prevalence rate give a better understanding of the likely real-world performance and a greater understanding of the likely impact of variation in that prevalence on translation to the clinic.

What are the dominant cytokines in early rheumatoid arthritis?

PURPOSE OF REVIEW: Rheumatoid arthritis is a systemic disease of evolving immune dysregulation that culminates in joint destruction and disability. The principle by which pro-inflammatory cytokines may be therapeutically targeted to abrogate disease is well established, but has yet to translate into reliable cures for patients. Emerging insights into cytokine-mediated pathobiology during rheumatoid arthritis development are reviewed, and their implications for future treatment strategies considered. RECENT FINDINGS: Accumulating data highlight cytokine perturbations before the clinical onset of rheumatoid arthritis. Some of these have now been linked to the arthritogenic activation of autoantibodies and associated pain and bone destruction in affected joints. These observations suggest cytokines may trigger the transition from systemic immunity to arthritis. Cytokine exposure could furthermore 'prime' synovial stromal cells to perpetuate a dominant pro-inflammatory environment. By facilitating cross-talk between infiltrating immune cells and even sustaining ectopic lymphoid structure development in some cases, cytokine interplay ultimately underpins the failure of arthritis to resolve. SUMMARY: Successful therapeutic stratification will depend upon an increasingly sophisticated appreciation of how dominant players amongst cytokine networks vary across time and anatomical space during incipient rheumatoid arthritis. The prize of sustained remission for all patients justifies the considerable effort required to achieve this understanding.

IL-6-driven STAT signalling in circulating CD4+ lymphocytes is a marker for early anticitrullinated peptide antibody-negative rheumatoid arthritis.

OBJECTIVES: A previously identified signal transduction and activator of transcription-3 (STAT3) target-enriched gene signature in circulating CD4+ T cells of patients with early rheumatoid arthritis (RA) was prominent in autoantibody-negative individuals. Here, interleukin (IL)-6-mediated STAT signalling was investigated in circulating lymphocytes of an independent early arthritis patient cohort, seeking further insight into RA pathogenesis and biomarkers of potential clinical utility. METHODS: Constitutive and IL-6-induced expression of phosphorylated STAT1 (pSTAT1) and pSTAT3 was determined in T and B cells using Phosflow cytometric analysis in patients with RA and controls. Contemporaneous levels of serum cytokines were measured by immunoassay. Induced gene expression was measured in cultured CD4+T cells by quantitative real-time PCR. RESULTS: Among circulating lymphocytes of 187 patients with early arthritis, constitutive pSTAT3 correlated with serum IL-6 levels maximally in CD4+ T cells. Increased constitutive pSTAT3, but not pSTAT1, was observed in circulating CD4+ T cells of patients with early anticitrullinated peptide autoantibody (ACPA)-negative RA compared with disease controls, and these levels decreased alongside markers of disease activity with IL-6R-targeted treatment. Among patients presenting with seronegative undifferentiated arthritis (UA) the ratio of constitutive pSTAT3:pSTAT1 in CD4+ T cells contributed substantially to an algorithm for predicting progression to classifiable RA during a median of 20 months follow-up (area under receiver operator characteristic curve=0.84; p<0.001). CONCLUSIONS: Our findings support a particular role for IL-6-driven CD4+ T cell activation via STAT3 during the induction of RA, particularly as a feature of ACPA-negative disease. CD4+ T cell pSTAT measurements show promise as biomarkers of UA-RA progression and now require independent validation.

Components of treatment delay in rheumatoid arthritis differ according to autoantibody status: validation of a single-centre observation using national audit data.

OBJECTIVE: To determine whether time to treatment following symptom onset differs between RA patients according to autoantibody status. METHODS: A single-centre retrospective analysis of a UK early RA inception cohort was first undertaken to identify those components of the patient journey that differed by serological subtype. Data from a UK national audit of early inflammatory arthritis patients was accessed to replicate the key finding. RESULTS: A total of 173 RA patients were diagnosed over a 31-month period, of whom 80 (46%) were ACPA/RF double-seropositive (ACPA(+)/RF(+)), 53 (31%) ACPA(-)/RF(-), 17 (10%) ACPA(+)/RF(-) and 23 (13%) RF(+)/ACPA(-) Overall, ACPA(+)/RF(+) patients experienced significantly longer symptom duration before DMARD initiation. This was accounted for by delays in their presentation to primary care following symptom onset-a finding that was robustly confirmed in an independent dataset of 2192 UK early RA patients. In contrast, ACPA(-)/RF(-) patients were significantly more likely to experience delays in DMARD initiation after presenting to secondary care. CONCLUSION: Causes of treatment delays in early RA differ according to patients' autoantibody status. More insidious symptom onset and/or distinct health-seeking behaviours among ACPA(+)/RF(+) patients may contribute to late presentations in primary care, whereas ACPA(-)/RF(-) patients experience delayed diagnosis and treatment in secondary care. These observations inform the research agenda, potentially influencing the design of service delivery for early arthritis patients.