A proteomics study of rheumatoid arthritis patients on etanercept identifies putative biomarkers associated with clinical outcome measures.

OBJECTIVES: Biologic DMARDs (bDMARDs) are widely used in patients with RA, but response to bDMARDs is heterogeneous. The objective of this work was to identify pretreatment proteomic biomarkers associated with RA clinical outcome measures in patients starting bDMARDs. METHODS: Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used to generate spectral maps of sera from patients with RA before and after 3 months of treatment with the bDMARD etanercept. Protein levels were regressed against RA clinical outcome measures, i.e. 28-joint DAS (DAS28) and its subcomponents and DAS28 <2.6 (i.e. remission). The proteins with the strongest evidence for association were analysed in an independent, replication dataset. Finally, subnetwork analysis was carried out using the Disease Module Detection algorithm and biological plausibility of identified proteins was assessed by enrichment analysis. RESULTS: A total of 180 patients with RA were included in the discovery dataset and 58 in the validation dataset from a UK-based prospective multicentre study. Ten individual proteins were found to be significantly associated with RA clinical outcome measures. The association of T-complex protein 1 subunit η with DAS28 remission was replicated in an independent cohort. Subnetwork analysis of the 10 proteins from the regression analysis identified the ontological theme, with the strongest associations being with acute phase and acute inflammatory responses. CONCLUSION: This longitudinal study of 180 patients with RA commencing etanercept has identified several putative protein biomarkers of treatment response to this drug, one of which was replicated in an independent cohort.

Pre-treatment calprotectin (MRP8/14) provides no added value to testing CRP alone in terms of predicting response to TNF inhibitors in rheumatoid arthritis in a post hoc analysis.

OBJECTIVES: The inflammatory protein calprotectin (MRP8/14) has been identified as a promising biomarker of treatment response in rheumatoid arthritis (RA). Our aim was to test MRP8/14 as a biomarker of response to tumour necrosis factor (TNF)-inhibitors in the largest RA cohort to date and to compare with C-reactive protein (CRP). METHODS: Serum MRP8/14 was measured in 470 patients with RA about to commence treatment with adalimumab (n=196) or etanercept (n=274). Additionally, MRP8/14 was measured in the 3-month sera of 179 adalimumab-treated patients. Response was determined using European League against Rheumatism (EULAR) response criteria calculated using the traditional 4-component (4C) DAS28-CRP and alternate validated versions using 3-component (3C) and 2-component (2C), clinical disease activity index (CDAI) improvement criteria and change in individual outcome measures. Logistic/linear regression models were fitted for response outcome. RESULTS: In the 3C and 2C models, patients with RA were 1.92 (CI: 1.04 to 3.54) and 2.03 (CI: 1.09 to 3.78) times more likely to be classified as EULAR responders if they had high (75th quartile) pre-treatment levels of MRP8/14 compared with low (25th quartile). No significant associations were observed for the 4C model. When only using CRP as a predictor, in the 3C and 2C analyses, patients above the 75th quartile were 3.79 (CI: 1.81 to 7.93) and 3.58 (CI: 1.74 to 7.35) times more likely to be EULAR responders and addition of MRP8/14 did not significantly improve model fit (p values=0.62 and 0.80, respectively). No significant associations were observed in the 4C analysis. Exclusion of CRP from the outcome measure (CDAI) did not result in any significant associations with MRP8/14 (OR 1.00 (CI: 0.99 to 1.01), suggesting that the associations were due to the correlation with CRP and that there is no additional utility of MRP8/14 beyond use of CRP in patients with RA starting TNFi therapy. CONCLUSION: Beyond correlation with CRP, we found no evidence to suggest that MRP8/14 explains additional variability in response to TNFi in patients with RA over and above CRP alone.

Non-trough adalimumab and certolizumab drug levels associated with a therapeutic EULAR response in adherent patients with rheumatoid arthritis.

OBJECTIVES: Interventions aimed at increasing TNF-α inhibitor serum drug levels (SDLs) may improve treatment response; however, previous studies suggesting SDL cut-offs have not accounted for treatment adherence. The aim of this study was to establish the relationship between adalimumab/certolizumab SDLs and EULAR good vs non-/moderate response and to define SDL cut-offs associated with good response in fully adherent patients. METHODS: In a prospective observational study, 475 patients with RA were treated with certolizumab (n = 192) or adalimumab (n = 283). At baseline and 3, 6 and 12 months, patients had 28-joint DAS, self-reported treatment adherence and SDLs measured. Fully adherent patients were analysed as a subgroup. Follow-up data at 3, 6 and 12 months were analysed separately. Median SDLs were compared in good vs non-/moderate response patients and receiver operating characteristics (ROC) curves were used to establish cut-off SDLs. RESULTS: Fully adherent good responders had significantly higher median adalimumab/certolizumab SDLs compared with non-/moderate responders (P = 0.04 and P = 0.0005, respectively). ROC analysis reported 3 month non-trough adalimumab SDLs discriminated good vs non-/moderate response with an area under the curve (AUC) of 0.63 (95% CI 0.52, 0.75), with a cut-off of 7.5 mg/l being 39.1% specific and 80.9% sensitive. Similarly, 3 month non-trough certolizumab SDLs discriminated good vs non-/moderate response with an AUC of 0.65 (95% CI 0.51, 0.78), with a cut-off of 26.0 mg/l being 43.9% specific and 77.8% sensitive. CONCLUSION: In fully adherent patients, higher SDLs are detected in good responders, suggesting that interventions to improve SDLs, such as encouraging adherence, could improve treatment response. The 3 month non-trough SDL cut-offs of 7.5 mg/l for adalimumab and 26.0 mg/l for certolizumab may be useful in clinical practice.

Pre-defined gene co-expression modules in rheumatoid arthritis transition towards molecular health following anti-TNF therapy.

BACKGROUND: No reliable biomarkers to predict response to TNF inhibitors (TNFi) in RA patients currently exist. The aims of this study were to replicate changes in gene co-expression modules that were previously reported in response to TNFi therapy in RA; to test if changes in module expression are specific to TNFi therapy; and to determine whether module expression transitions towards a disease-free state in responding patients. METHOD: Published transcriptomic data from the whole blood of disease-free controls (n = 10) and RA patients, treated with the TNFi adalimumab (n = 70) or methotrexate (n = 85), were studied. Treatment response was assessed using the EULAR response criteria following 3 or 6 months of treatment. Change in transcript expression between pre- and post-treatment was recorded for previously defined modules. Linear mixed models tested whether modular expression after treatment transitioned towards a disease-free state. RESULTS: For 25 of the 27 modules, change in expression between pre- and post-treatment in the adalimumab cohort replicated published findings. Of these 25 modules, six transitioned towards a disease-free state by 3 months (P < 0.05), irrespective of clinical response. One module (M3.2), related to inflammation and TNF biology, significantly correlated with response to adalimumab. Similar patterns of modular expression, with reduced magnitude, were observed in the methotrexate cohort. CONCLUSION: This study provides independent validation of changes in module expression in response to therapy in RA. However, these effects are not specific to TNFi. Further studies are required to determine whether specific modules could assist molecular classification of therapeutic response.

The Autoimmune Susceptibility Gene C5orf30 Regulates Macrophage-Mediated Resolution of Inflammation.

Genetic variants in C5orf30 have been associated with development of the autoimmune conditions primary biliary cirrhosis and rheumatoid arthritis. In rheumatoid arthritis, C5orf30 expression is cell-specific, with highest expression found in macrophages and synovial fibroblasts. C5orf30 is highly expressed in inflamed joints and is a negative regulator of tissue damage in a mouse model of inflammatory arthritis. Transcriptomic analysis from ultrasound-guided synovial biopsy of inflamed joints in a well characterized clinical cohort of newly diagnosed, disease-modifying antirheumatic drugs-naive rheumatoid arthritis patients was used to determine the clinical association of C5orf30 expression with disease activity. A combined molecular and computational biology approach was used to elucidate C5orf30 function in macrophages both in vitro and in vivo. Synovial expression of C5orf30 is inversely correlated with both clinical measures of rheumatoid arthritis disease activity and with synovial TNF mRNA expression. C5orf30 plays a role in regulating macrophage phenotype and is differentially turned over in inflammatory and anti-inflammatory macrophages. Inhibition of C5orf30 reduces wound healing/repair-associated functions of macrophages, reduces signaling required for resolution of inflammation, and decreases secretion of anti-inflammatory mediators. In an animal model of wound healing (zebrafish), C5orf30 inhibition increases the recruitment of macrophages to the wound site. Finally, we demonstrate that C5orf30 skews macrophage immunometabolism, demonstrating a mechanism for C5orf30-mediated immune regulation.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

Interleukin 1 receptor antagonist (IL1RN) gene variants predict radiographic severity of knee osteoarthritis and risk of incident disease.

OBJECTIVE: In these studies, we examined the association of single nucleotide polymorphisms (SNPs) of the IL1RN gene with radiographic severity of symptomatic knee osteoarthritis (SKOA) and the risk of incident OA. We also explored these genetic polymorphisms in patients with new onset rheumatoid arthritis (RA). METHODS: Over 1000 subjects who met American College of Rheumatology criteria for tibiofemoral OA were selected from three independent, National Institute of Health (NIH)-funded cohorts. CTA and TTG haplotypes formed from three SNPs of the IL1RN gene (rs419598, rs315952, rs9005) were assessed for association with radiographic severity, and risk for incident radiographic OA (rOA) in a nested case-control cohort. These IL1RN haplotypes were also assessed for association with disease activity (DAS28) and plasma inflammatory markers in patients with RA. RESULTS: Carriage of the IL1RN TTG haplotype was associated with increased odds of more severe rOA compared with age-matched, sex-matched and body mass index-matched individuals. Examination of the osteoarthritis initiative Incidence Subcohort demonstrated that carriage of the TTG haplotype was associated with 4.1-fold (p=0.001) increased odds of incident rOA. Plasma IL-1Ra levels were lower in TTG carriers, while chondrocytes from TTG carriers exhibited decreased secretion of IL-1Ra. In patients with RA, the TTG haplotype was associated with increased DAS28, decreased plasma IL-1Ra and elevations of plasma inflammatory markers (hsCRP, interleukin 6 (IL-6)). CONCLUSION: Carriage of the IL1RN TTG haplotype is associated with more severe rOA, increased risk for incident OA, and increased evidence of inflammation in RA. These data suggest that the IL1RN TTG risk haplotype, associated with decreased IL-1Ra plasma levels, impairs endogenous 'anti-inflammatory' mechanisms.

Differential DNA methylation correlates with response to methotrexate in rheumatoid arthritis.

OBJECTIVES: Identifying blood-based biomarkers that predict treatment response in RA is a clinical priority. We investigated differential DNA methylation as a candidate biomarker of response for the first-line drug used in RA, MTX. METHODS: DNA methylation was measured in DNA samples from individuals recruited to the Rheumatoid Arthritis Medication Study. Differentially methylated positions were compared between whole blood samples collected at baseline and at 4 weeks from patients who, by 6 months, had a good (n = 34) or poor response (n = 34) to MTX using linear modelling, adjusting for gender, age, cell composition, baseline 28-joint disease activity score (DAS28) and smoking status. Analyses also compared methylation with changes in DAS28 and changes in swollen joint count and tender joint count, and changes in CRP over the initial 6 months after MTX commencement. Differentially methylated positions showing significant differences with any response parameter were tested using pyrosequencing in an independent group of 100 patients from the Rheumatoid Arthritis Medication Study. RESULTS: In the discovery group, two CpG sites showed methylation changes at 4 weeks associated with clinical EULAR response by 6 months. Significant changes in methylation for three differentially methylated positions associated with change in tender joint counts, three with change in swollen joint count and a further four with change in CRP. Of the 12 CpGs, four showed replicated association in an independent dataset of samples from the Rheumatoid Arthritis Medication Study. CONCLUSION: These data represent an advance on current practice by contributing to a personalized medicine strategy allowing an escalation or change in therapy as early as 4 weeks.

Renegotiating dimensions of the self: A systematic review and qualitative evidence synthesis of the lived experience of self-managing rheumatoid arthritis.

BACKGROUND: As chronic illnesses, such as rheumatoid arthritis (RA), place an increased burden on health-care systems, the ability of individuals to self-manage these diseases is crucial. OBJECTIVE: To identify and synthesize the lived experience of self-management described by adults living with RA. DESIGN: A systematic search of five electronic databases (MEDLINE, CINAHL, EMBASE, PsycINFO and ASSIA) was undertaken to identify relevant studies. Data were extracted and quality-assessed using CASP guidelines. A meta-synthesis was conducted based on Thomas and Harden's thematic synthesis approach. RESULTS: The search identified 8423 publications. After removing duplicates, 6527 records remained of which 32 studies met the inclusion criteria. Quality of studies was moderate to high, yet a considerable lack of reflection on researcher bias was evident. Our analysis identified 28 dimensions of self-management RA across six domains: (a) cognitive-emotional, (b) behavioural, (c) social, (d) environmental, (e) physical and (f) technological. Cognitive-emotional experiences dominated the analysis. Renegotiating 'the self' (self-concept, self-esteem, self-efficacy) was a key focus of self-management among individuals with RA. CONCLUSION: Our findings highlight the focus of 'the self' as a central concern in the self-management of RA. Standardized self-management programmes may primarily focus on disease management and daily functioning. However, we suggest that personal biographies and circumstances should move to the fore of self-management support. REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews 2018: CRD42018100450. PATIENT OR PUBLIC CONTRIBUTION: Patient and public involvement was not explicit in this review. However, three authors provided a patient perspective on the self-management of arthritis and autoimmune disease.

Association of response to TNF inhibitors in rheumatoid arthritis with quantitative trait loci for CD40 and CD39.

OBJECTIVES: We sought to investigate whether genetic effects on response to TNF inhibitors (TNFi) in rheumatoid arthritis (RA) could be localised by considering known genetic susceptibility loci for relevant traits and to evaluate the usefulness of these genetic loci for stratifying drug response. METHODS: We studied the relation of TNFi response, quantified by change in swollen joint counts ( Δ SJC) and erythrocyte sedimentation rate ( Δ ESR) with locus-specific scores constructed from genome-wide assocation study summary statistics in 2938 genotyped individuals: 37 scores for RA; scores for 19 immune cell traits; scores for expression or methylation of 93 genes with previously reported associations between transcript level and drug response. Multivariate associations were evaluated in penalised regression models by cross-validation. RESULTS: We detected a statistically significant association between Δ SJC and the RA score at the CD40 locus (p=0.0004) and an inverse association between Δ SJC and the score for expression of CD39 on CD4 T cells (p=0.00005). A previously reported association between CD39 expression on regulatory T cells and response to methotrexate was in the opposite direction. In stratified analysis by concomitant methotrexate treatment, the inverse association was stronger in the combination therapy group and dissipated in the TNFi monotherapy group. Overall, ability to predict TNFi response from genotypic scores was limited, with models explaining less than 1% of phenotypic variance. CONCLUSIONS: The association with the CD39 trait is difficult to interpret because patients with RA are often prescribed TNFi after failing to respond to methotrexate. The CD39 and CD40 pathways could be relevant for targeting drug therapy.

Genome-wide association study of response to tumour necrosis factor inhibitor therapy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterised by chronic synovial joint inflammation. Treatment has been revolutionised by tumour necrosis factor alpha inhibitors (TNFi) but each available drug shows a significant non-response rate. We conducted a genome-wide association study of 1752 UK RA TNFi-treated patients to identify predictors of change in the Disease Activity Score 28 (DAS28) and subcomponents over 3-6 months. The rs7195994 variant at the FTO gene locus was associated with infliximab response when looking at a change in the swollen joint count (SJC28) subcomponent (p = 9.74 × 10-9). Capture Hi-C data show chromatin interactions in GM12878 cells between rs2540767, in high linkage disequilibrium with rs7195994 (R2 = 0.9) and IRX3, a neighbouring gene of FTO. IRX3 encodes a transcription factor involved in adipocyte remodelling and is regarded as the obesity gene at the FTO locus. Importantly, the rs7195994 association remained significantly associated following adjustment for BMI. In addition, using capture Hi-C data we showed interactions between TNFi-response associated variants and 16 RA susceptibility variants.

C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis.

The variant rs26232, in the first intron of the chromosome 5 open reading frame 30 (C5orf30) locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation, implying a central function in these organisms. Here, we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro, and gene profiling following C5orf30 inhibition confirmed up-regulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a previously unidentified negative regulator of tissue damage in RA, and this protein may act by modulating the autoaggressive phenotype that is characteristic of RASFs.

Histone deacetylase 1 regulates tissue destruction in rheumatoid arthritis.

Emerging evidence implicates epigenetic mechanisms in the pathogenesis of rheumatoid arthritis (RA). In this study, we have investigated the role of histone deacetylase (HDAC) enzymes in RA synovial fibroblasts (RASFs), a key cellular mediator of cartilage and bone destruction and determined effects of HDAC1 inhibition on both RASF phenotype in vitro, and joint inflammation and damage in the collagen-induced arthritis (CIA) model. Expression of HDACs 1-11 messenger ribonucleic acid (mRNA) was compared between RASFs and osteoarthritic synovial fibroblast (OASFs) using quantitative polymerase chain reaction. HDAC1 expression in RASFs was inhibited using small interfering RNA (siRNA) technology to assess effects on invasiveness, migration, proliferation and apoptosis. Effects of HDAC1 knockdown (KD) on the transcriptome were assessed using gene microarrays. The effects of siRNA-mediated HDAC(KD) on clinical scores, tissue inflammation and damage were assessed on CIA up to 47 days following immunization. Expression of HDAC1 was significantly higher in RASFs than OASFs. HDAC1(KD) resulted in reduced proliferation, invasion and migration in vitro and transcriptome profiling revealed effects on expression of genes regulating proliferation migration and inflammation. Furthermore, inhibition of HDAC1 in CIA resulted in reduced joint swelling, cartilage and bone damage and lower tumor necrosis factor in joint tissue. These results implicate HDAC1 as an important mediator of tissue damage in RA and support the potential therapeutic utility of inhibitors of this enzyme.

High frequency of antidrug antibodies and association of random drug levels with efficacy in certolizumab pegol-treated patients with rheumatoid arthritis: results from the BRAGGSS cohort.

OBJECTIVES: To evaluate (i) the association between random certolizumab drug levels, antidrug antibodies (ADAbs) and treatment response in patients with rheumatoid arthritis (RA); (ii) longitudinal factors associated with ADAbs and certolizumab drug levels. METHODS: This prospective cohort included 115 patients with RA treated with certolizumab. Serum samples were collected at 3, 6 and 12 months following treatment initiation. Drug levels and ADAbs were measured using ELISA and radioimmunoassay, respectively, at 3, 6 and 12 months. Disease Activity Score in 28 joints (DAS28) were measured at each visit and 12 months European League Against Rheumatism (EULAR) response was calculated. Patient self-reported adherence was collected longitudinally. Ordinal logistic regression and generalised estimating equation were used to test the association: (i) between drug levels, from serum sampled and treatment response; (ii) between ADAbs and drug levels; (iii) patient-centred factors and drug levels. RESULTS: ADAbs were detected in 37% (42/112 patients by 12 months). The presence of ADAbs were significantly associated with lower drug levels over 12 months (ß=-0.037, 95% CI -0.055 to 0.018, p<0.0001) but not independently with 12 months EULAR response (ß=0.0013 (95% CI -0.0032 to 0.00061), p=0.18). Drug level was associated with 12 months EULAR response (ß=0.032 (95% CI 0.0011 to 0.063), p=0.042). In the multivariate model, ADAb level and adherence were significantly associated with drug concentrations. CONCLUSIONS: This is the first study to demonstrate that higher certolizumab drug levels are associated with better 12 months EULAR response. ADAbs in certolizumab-treated patients with RA were detected at higher levels than previous studies and help determine the aetiology of a low drug level.

The predictive value of serum S100A9 and response to etanercept is not confirmed in a large UK rheumatoid arthritis cohort.

Objective: The aim was to correlate protein concentrations of S100A9 in pretreatment serum samples with response to the tumour-necrosis factor (TNF) inhibitor drugs etanercept in a large UK replication cohort. Methods: Pretreatment serum samples from patients with RA (n = 236) about to commence treatment with etanercept had S100A9 serum concentration measured using an ELISA. Following the experimental procedure, S100A9 concentrations were analysed with respect to EULAR response. Results: No evidence of association between S100A9 concentration and EULAR response to the TNF-inhibitor biologic drug etanercept was observed following multinomial logistic regression analysis (non-responder vs moderate responder, P = 0.957; and non-responder vs good responder, P = 0.316). Furthermore, no significant associations were observed when correlating pretreatment S100A9 concentrations with clinical parameters of disease activity (P > 0.05). Conclusion: In the largest replication cohort conducted to date, no evidence for association was observed to support the use of S100A9 as a clinical biomarker predictive of response to the TNF-inhibitor biologic drug etanercept.

Clinical utility of random anti-tumour necrosis factor drug testing and measurement of anti-drug antibodies on long-term treatment response in rheumatoid arthritis.

BACKGROUND: Up to 40% of patients with rheumatoid arthritis treated with anti-tumour necrosis factor (TNF) drugs do not respond because of primary inefficacy or loss of response. Although one explanation is that immunogenicity leads to the development of anti-drug antibodies and low drug concentrations, the clinical usefulness of pharmacological monitoring is debated. Our aim was to assess whether the presence of anti-drug antibodies and non-trough drug concentrations could predict treatment response in patients with rheumatoid arthritis treated with anti-TNF drugs. METHODS: 331 patients were selected from a multicentre prospective cohort (160 treated with adalimumab, 171 etanercept). Serum samples were collected at 3, 6, and 12 months after treatment initiation. Anti-drug antibodies were measured with RIA, drug concentrations with ELISAs, and Disease Activity Score in 28 joints (DAS28) at each timepoint. Linear and logistic regression, generalised estimating equation (GEE), and receiver operating characteristic curves were used to test the association and predictive value of anti-drug antibodies and non-trough drug concentrations on treatment response (ΔDAS28). FINDINGS: 835 serial samples were tested (414 adalimumab, 421 etanercept). Anti-adalimumab antibodies were detected in 31 (24·8%) of 125 patients who had completed 12 month follow-up and none of the etanercept patients. The presence of anti-drug antibodies was associated with lower adalimumab concentrations (Spearman r=-0·66, p=0·0041). At 3 months, anti-drug antibody formation and low adalimumab concentrations were significant predictors of poor treatment response at 12 months (area under curve [AUC] 0·68, 95% CI 0·54-0·81, and 0·66, 0·55-0·77, respectively; and both combined 0·71, 0·57-0·85). Adalimumab concentration was the most significant independent predictor of ΔDAS28 after adjustment for confounders (regression coefficient 0·12, 95% CI 0·06-0·18; p=0·003). High etanercept concentrations were associated with better treatment response (p=0·01), but low concentrations at 3 months were not a significant predictor of poor treatment response at 12 months (AUC 0·58, 95% CI 0·46-0·70). In the combined GEE model including adalimumab and etanercept, a body-mass index of 30 kg/m(2) or more was associated with low drug concentrations (regression coefficient 0·78, 95% CI 0·37-1·18; p<0·0001). INTERPRETATION: Pharmacological testing in anti-TNF initiated patients is clinically useful even in the absence of trough levels. At 3 months, presence of anti-drug antibodies and low adalimumab concentrations are a significant predictor for poor treatment response at 12 months. Strengths of this study include a large, prospective cohort and use of RIA to measure antibodies (less prone to drug interference). Although non-trough concentrations might have underestimated the frequency of antibodies, their presence still predicted response. FUNDING: MJ is a MRC Clinical Training Fellow supported by the North West England Medical Research Council Fellowship Scheme in Clinical Pharmacology and Therapeutics, which is funded by the UK Medical Research Council (grant number G1000417/94909), ICON, GlaxoSmithKline, AstraZeneca, and the Medical Evaluation Unit. Arthritis Research UK (grant ref 20385).

Detection of anti-drug antibodies using a bridging ELISA compared with radioimmunoassay in adalimumab-treated rheumatoid arthritis patients with random drug levels.

OBJECTIVE: To determine the concordance between RIA and bridging ELISA at detecting anti-drug antibodies (ADAbs) in the context of random adalimumab levels and investigate the additional clinical utility of detecting ADAbs in RA patients who test ADAb positive by RIA and negative by ELISA. METHODS: ADAb levels were determined using RIA and bridging ELISA in 63 adalimumab-treated RA patients (159 samples). Immunogenicity concordance was determined using receiver operating characteristic curves. To determine the additional clinical value provided by a positive RIA in the presence of negative ELISA, association between treatment response (ΔDAS28), adalimumab drug levels and ADAbs was evaluated longitudinally using generalized estimating equation. RESULTS: Of the 60 RIA+ samples (n = 31 patients), 19 (n = 10 patients) were also ELISA+, corresponding to 31.7% of samples. Area under the curve for detecting ADAbs using ELISA (compared with RIA) using receiver operating characteristic curves was 0.65 (95% CI: 0.59, 0.71); this increased to 0.91 (95% CI: 0.81, 0.99) if ADAbs were ⩾100 AU/ml using RIA. In RIA+/ELISA- patients, adalimumab levels were associated with ΔDAS28 over 12 months [regression coefficient: 0.098 (95% CI: 0.043, 0.15), P < 0.0001] and while ADAbs were significantly associated with drug level, they were not directly associated with ΔDAS28 over 12 months [ß coefficient: 0.00083 (95% CI: -0.0038 to 0.0054), P = 0.72]. CONCLUSION: ADAbs were detected using ELISA more frequently when present in high titres as measured by RIA. In RIA+/ELISA- patients, only drug levels were significantly associated with treatment response. Although ADAbs were not independently associated with treatment response, they may be helpful in determining the aetiology of low drug levels.

Predicting the risk of rheumatoid arthritis and its age of onset through modelling genetic risk variants with smoking.

The improved characterisation of risk factors for rheumatoid arthritis (RA) suggests they could be combined to identify individuals at increased disease risks in whom preventive strategies may be evaluated. We aimed to develop an RA prediction model capable of generating clinically relevant predictive data and to determine if it better predicted younger onset RA (YORA). Our novel modelling approach combined odds ratios for 15 four-digit/10 two-digit HLA-DRB1 alleles, 31 single nucleotide polymorphisms (SNPs) and ever-smoking status in males to determine risk using computer simulation and confidence interval based risk categorisation. Only males were evaluated in our models incorporating smoking as ever-smoking is a significant risk factor for RA in men but not women. We developed multiple models to evaluate each risk factor's impact on prediction. Each model's ability to discriminate anti-citrullinated protein antibody (ACPA)-positive RA from controls was evaluated in two cohorts: Wellcome Trust Case Control Consortium (WTCCC: 1,516 cases; 1,647 controls); UK RA Genetics Group Consortium (UKRAGG: 2,623 cases; 1,500 controls). HLA and smoking provided strongest prediction with good discrimination evidenced by an HLA-smoking model area under the curve (AUC) value of 0.813 in both WTCCC and UKRAGG. SNPs provided minimal prediction (AUC 0.660 WTCCC/0.617 UKRAGG). Whilst high individual risks were identified, with some cases having estimated lifetime risks of 86%, only a minority overall had substantially increased odds for RA. High risks from the HLA model were associated with YORA (P<0.0001); ever-smoking associated with older onset disease. This latter finding suggests smoking's impact on RA risk manifests later in life. Our modelling demonstrates that combining risk factors provides clinically informative RA prediction; additionally HLA and smoking status can be used to predict the risk of younger and older onset RA, respectively.

Genome-wide association study and gene expression analysis identifies CD84 as a predictor of response to etanercept therapy in rheumatoid arthritis.

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (ΔDAS) in the etanercept subset of patients (P = 8 × 10(-8)), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3' UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1 × 10(-11) in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better ΔDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry.