Socioeconomic deprivation is associated with reduced response and lower treatment persistence with TNF inhibitors in rheumatoid arthritis.

OBJECTIVE: To investigate the association between socioeconomic deprivation and outcomes following TNF inhibitor (TNFi) treatment. METHODS: Individuals commencing their first TNFi in the British Society for Rheumatology Biologics Register for RA (BSRBR-RA) and Biologics in RA Genetics and Genomics Study Syndicate (BRAGGSS) cohort were included. Socioeconomic deprivation was proxied using the Index of Multiple Deprivation and categorized as 20% most deprived, middle 40% or 40% least deprived. DAS28-derived outcomes at 6 months (BSRBR-RA) and 3 months (BRAGGSS) were compared using regression models with the least deprived as referent. Risks of all-cause and cause-specific drug discontinuation were compared using Cox models in the BSRBR-RA. Additional analyses adjusted for lifestyle factors (e.g. smoking, BMI) as potential mediators. RESULTS: 16 085 individuals in the BSRBR-RA were included (mean age 56 years, 76% female), of whom 18%, 41% and 41% were in the most, middle and least deprived groups, respectively. Of 3459 included in BRAGGSS (mean age 57, 77% female), proportions were 22%, 36% and 41%, respectively. The most deprived group had 0.3-unit higher 6-month DAS28 (95% CI 0.22, 0.37) and were less likely to achieve low disease activity (odds ratio [OR] 0.76; 95% CI 0.68, 0.84) in unadjusted models. Results were similar for 3-month DAS28 (ß = 0.23; 95% CI 0.11, 0.36) and low disease activity (OR 0.77; 95% CI 0.63, 0.94). The most deprived were more likely to discontinue treatment (hazard ratio 1.18; 95% CI 1.12, 1.25), driven by ineffectiveness rather than adverse events. Adjusted estimates were generally attenuated. CONCLUSION: Socioeconomic deprivation is associated with reduced response to TNFi. Improvements in determinants of health other than lifestyle factors are needed to address socioeconomic inequities.

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.

High throughput screening of 0.5 million compounds against CRAF using Alpha CETSAⓇ.

The cellular thermal shift assay (CETSA®) has increasingly been used in early drug discovery to provide a measure of cellular target engagement. Traditionally, CETSA has been employed for bespoke questions with small to medium throughput and has predominantly been applied during hit validation rather than in hit identification. Using a CETSA screen versus the kinase CRAF, we assessed 3 key questions: (1) technical feasibility - could the CETSA methodology technically be applied at truly high throughput scale? (2) relevance - could hits suitable for further optimisation be identified? (3) reliability - would the approach identify known chemical equity. Here, we describe the first large scale AlphaLISA SureFire based CETSA (Alpha CETSA) approach allowing us to screen a large library of almost 0.5 million compounds. We discuss the issues overcome in automating and executing the screen and describe the resulting screen output.

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.

HLA-DRB1 haplotypes predict cardiovascular mortality in inflammatory polyarthritis independent of CRP and anti-CCP status.

BACKGROUND: Haplotypes defined by amino acids at HLA-DRB1 positions 11, 71 and 74 associated with susceptibility to rheumatoid arthritis (RA) are associated with radiological outcome, anti-TNF response and all cause-mortality in RA. RA is associated with cardiovascular (CV) morbidity and mortality, but the increased prevalence of risk factors of CV disease in RA only partially explains this association. The aim of this study was to investigate whether amino acids at positions 11, 71 and 74 of HLA-DRB1 are associated with cardiovascular (CV) mortality in inflammatory polyarthritis (IP). METHODS: The Norfolk Arthritis Register (NOAR) is an incidence register of IP: recruitment 1990-2007, final follow-up 2011. Two thousand five hundred fourteen patients had available genetic and mortality data. Amino acids at positions 11, 71 and 74 of HLA-DRB1 were determined. Univariate Cox proportional hazard models were applied to assess the association of genetic markers and both all-cause mortality and cardiovascular mortality. RESULTS: Among 2514 participants, 643 (25.6%) died during the study, and 343 (53.3%) of these deaths were attributed to CV causes. One thousand six hundred fifty (65.6%) participants were female, 709 (32.3%) were anti-CCP-positive and the median age of participants was 54. HLA-DRB1 haplotypes associated with susceptibility to rheumatoid arthritis (RA) consistently show the same magnitude and direction of association for overall and CV mortality in IP. For example, the SEA-haplotype, associated with the lowest susceptibility to RA, and the best radiographic outcome, was found to be associated with decreased CV mortality (HR 0.67, 95% CI 0.47, 0.91, p=0.023). Mediation analysis revealed associations were independent of anti-CCP status. CONCLUSIONS: HLA-DRB1 haplotypes associated with susceptibility to RA also predispose to increased risk of CV mortality in IP, independent of known CV risk factors. Associations were independent of anti-CCP status, which suggests in the future, genetic factors will add to the prediction of risk of cardiovascular mortality beyond serological markers.

Transcriptome-wide study of TNF-inhibitor therapy in rheumatoid arthritis reveals early signature of successful treatment.

BACKGROUND: Despite the success of TNF-inhibitor therapy in rheumatoid arthritis treatment, up to 40% of patients fail to respond adequately. This study aimed to identify transcriptome-based biomarkers of adalimumab response in rheumatoid arthritis (RA) to aid timely switching in non-responder patients and provide a better mechanistic understanding of the pathways involved in response/non-response. METHODS: The Affymetrix Human Transcriptome Array 2.0 (HTA) was used to measure the transcriptome in whole blood at pre-treatment and at 3 months in EULAR good- and non-responders to adalimumab therapy. Differential expression of transcripts was analysed at the transcript level using multiple linear regression. Differentially expressed genes were validated in independent samples using OpenArray™ RT-qPCR. RESULTS: In total, 813 transcripts were differentially expressed between pre-treatment and 3 months in adalimumab good-responders. No significant differential expression was observed between good- and non-responders at either time-point and no significant changes were observed in non-responders between time-points. OpenArray™ RT-qPCR was performed for 104 differentially expressed transcripts in good-responders, selected based on magnitude of effect or p value or based on prior association with RA or the immune system, validating differential expression for 17 transcripts. CONCLUSIONS: An early transcriptome signature of DAS28 response to adalimumab has been identified and replicated in independent datasets. Whilst treat-to-target approaches encourage early switching in non-responsive patients, registry evidence suggests that this does not always occur. The results herein could guide the development of a blood test to distinguish responders from non-responders at 3 months and support clinical decisions to switch non-responsive patients to an alternative therapy.

Pharmacogenetics of TNF inhibitor response in rheumatoid arthritis utilizing the two-component disease activity score.

Aim: TNF inhibitor drugs are a treatment option for rheumatoid arthritis, but response is not universal. Response is typically measured using the composite 4-component (4C) disease activity score 28 (DAS28) which contains more subjective measures. This study used a validated 2-component (2C) DAS28 score to determine whether SNPs associated with response were replicated in the UK population. Materials & methods: A literature review identified TNF inhibitor response SNPs. Linear regression was conducted to replicate associations with 4C or 2C-DAS28 response. Results: Eighteen independent SNPs were analyzed in 1828 patients. One and four associations with 4C and 2C-DAS28 response respectively were identified (p ≤ 0.05). Conclusion: Further genetic associations were replicated using the 2C-DAS28 which may reflect the objective nature of 2C-AS28.

Investigation of genetically regulated gene expression and response to treatment in rheumatoid arthritis highlights an association between IL18RAP expression and treatment response.

OBJECTIVES: In this study, we sought to investigate whether there was any association between genetically regulated gene expression (as predicted using various reference panels) and anti-tumour necrosis factor (anti-TNF) treatment response (change in erythrocyte sedimentation rate (ESR)) using 3158 European ancestry patients with rheumatoid arthritis. METHODS: The genetically regulated portion of gene expression was estimated in the full cohort of 3158 subjects (as well as within a subcohort consisting of 1575 UK patients) using the PrediXcan software package with three different reference panels. Estimated expression was tested for association with anti-TNF treatment response. As a replication/validation experiment, we also investigated the correlation between change in ESR with measured gene expression at the Interleukin 18 Receptor Accessory Protein (IL18RAP) gene in whole blood and synovial tissue, using an independent replication data set of patients receiving conventional synthetic disease modifying anti-rheumatic drugs, with directly measured (via RNA sequencing) gene expression. RESULTS: We found that predicted expression of IL18RAP showed a consistent signal of association with treatment response across the reference panels. In our independent replication data set, IL18RAP expression in whole blood showed correlation with the change in ESR between baseline and follow-up (r=-0.35, p=0.0091). Change in ESR was also correlated with the expression of IL18RAP in synovial tissue (r=-0.28, p=0.02). CONCLUSION: Our results suggest that IL18RAP expression is worthy of further investigation as a potential predictor of treatment response in rheumatoid arthritis that is not specific to a particular drug type.

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.

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.

Assessing the Role of DNA Methylation-Derived Neutrophil-to-Lymphocyte Ratio in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a disease of chronic systemic inflammation (SI). In the present study, we used four datasets to explore whether methylation-derived neutrophil-to-lymphocyte ratio (mdNLR) might be a marker of SI in new onset, untreated, and treated prevalent RA cases and/or a marker of treatment response to the tumour necrosis factor inhibitor (TNFi) etanercept. mdNLR was associated with increased odds of being a new onset RA case (OR = 2.32, 95% CI = 1.95-2.80, P < 2 × 10-16) and performed better in distinguishing new onset RA cases from controls compared to covariates: age, gender, and smoking status. In untreated preclinical RA cases and controls, mdNLR at baseline was associated with diagnosis of RA in later life after adjusting for batch (OR = 4.30, 95% CI = 1.52-21.71, P = 0.029) although no association was observed before batch correction. When prevalent RA cases were treated, there was no association with mdNLR in samples before and after batch correction (OR = 0.34, 95% CI = 0.05-1.82, P = 0.23), and mdNLR was not associated with treatment response to etanercept (OR = 1.10, 95% CI = 0.75-1.68, P = 0.64). Our results indicate that SI measured by DNA methylation data is indicative of the recent onset of RA. Although preclinical RA was associated with mdNLR, there was no difference in the mean mdNLR between preclinical RA cases and controls. mdNLR was not associated with RA case status if treatment for RA has commenced, and it is not associated with treatment response. In the future, mdNLR estimates may be used as a valuable research tool to reliably estimate SI in the absence of freshly collected blood samples.

Increased DNA methylation variability in rheumatoid arthritis-discordant monozygotic twins.

BACKGROUND: Rheumatoid arthritis is a common autoimmune disorder influenced by both genetic and environmental factors. Epigenome-wide association studies can identify environmentally mediated epigenetic changes such as altered DNA methylation, which may also be influenced by genetic factors. To investigate possible contributions of DNA methylation to the aetiology of rheumatoid arthritis with minimum confounding genetic heterogeneity, we investigated genome-wide DNA methylation in disease-discordant monozygotic twin pairs. METHODS: Genome-wide DNA methylation was assessed in 79 monozygotic twin pairs discordant for rheumatoid arthritis using the HumanMethylation450 BeadChip array (Illumina). Discordant twins were tested for both differential DNA methylation and methylation variability between rheumatoid arthritis and healthy twins. The methylation variability signature was then compared with methylation variants from studies of other autoimmune diseases and with an independent healthy population. RESULTS: We have identified a differentially variable DNA methylation signature that suggests multiple stress response pathways may be involved in the aetiology of the disease. This methylation variability signature also highlighted potential epigenetic disruption of multiple RUNX3 transcription factor binding sites as being associated with disease development. Comparison with previously performed epigenome-wide association studies of rheumatoid arthritis and type 1 diabetes identified shared pathways for autoimmune disorders, suggesting that epigenetics plays a role in autoimmunity and offering the possibility of identifying new targets for intervention. CONCLUSIONS: Through genome-wide analysis of DNA methylation in disease-discordant monozygotic twins, we have identified a differentially variable DNA methylation signature, in the absence of differential methylation in rheumatoid arthritis. This finding supports the importance of epigenetic variability as an emerging component in autoimmune disorders.

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.

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.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Previously reported PDE3A-SLCO1C1 genetic variant does not correlate with anti-TNF response in a large UK rheumatoid arthritis cohort.

AIM: A genetic variant has recently reached genome-wide significance for association with TNF-inhibitor response in rheumatoid arthritis patients. Here we undertake a replication study in a UK Caucasian population to test for association with TNF-inhibitor response. MATERIALS & METHODS: The genetic variant, rs3794271, located within the PDE3A-SLCO1C1 locus was analyzed for correlation with treatment response using both the EULAR classification criteria and absolute change in (Δ)DAS28 scores as outcome measures. RESULTS: Genotype data were available from 1750 TNF-inhibitor treated individuals. However, no evidence for association was observed (EULAR: p = 0.91 and ΔDAS28: p = 0.93). Furthermore, no significant associations were observed upon stratification by the anti-TNF received (p > 0.05). CONCLUSION: In the largest replication cohort conducted to date, no evidence for association was observed.

Differential Methylation as a Biomarker of Response to Etanercept in Patients With Rheumatoid Arthritis.

OBJECTIVE: Biologic drug therapies represent a huge advance in the treatment of rheumatoid arthritis (RA). However, very good disease control is achieved in only 30% of patients, making identification of biomarkers of response a research priority. We undertook this study to test our hypothesis that differential DNA methylation patterns may provide biomarkers predictive of response to tumor necrosis factor inhibitor (TNFi) therapy in patients with RA. METHODS: An epigenome-wide association study was performed on pretreatment whole blood DNA from patients with RA. Patients who displayed good response (n = 36) or no response (n = 36) to etanercept therapy at 3 months were selected. Differentially methylated positions were identified using linear regression. Variance of methylation at differentially methylated positions was assessed for correlation with cis-acting single-nucleotide polymorphisms (SNPs). A replication experiment for prioritized SNPs was performed in an independent cohort of 1,204 RA patients. RESULTS: Five positions that were differentially methylated between responder groups were identified, with a false discovery rate of <5%. The top 2 differentially methylated positions mapped to exon 7 of the LRPAP1 gene on chromosome 4 (cg04857395, P = 1.39 × 10(-8) and cg26401028, P = 1.69 × 10(-8) ). The A allele of the SNP rs3468 was correlated with higher levels of methylation for both of the top 2 differentially methylated positions (P = 2.63 × 10(-7) and P = 1.05 × 10(-6) , respectively). Furthermore, the A allele of rs3468 was correlated with European League Against Rheumatism nonresponse in the discovery cohort (P = 0.03; n = 56) and in the independent replication cohort (P = 0.003; n = 1,204). CONCLUSION: We identify DNA methylation as a potential biomarker of response to TNFi therapy, and we report the association between response and the LRPAP1 gene, which encodes a chaperone of low-density lipoprotein receptor-related protein 1. Additional replication experiments in independent sample collections are now needed.

Investigating CD11c expression as a potential genomic biomarker of response to TNF inhibitor biologics in whole blood rheumatoid arthritis samples.

INTRODUCTION: Gene expression profiling is rapidly becoming a useful and informative tool in a much needed area of research. Identifying patients as to whether they will respond or not to a given treatment before prescription is not only essential to optimise treatment outcome but also to lessen the economic burden that such drugs can have on healthcare resources. In rheumatoid arthritis (RA), there is of yet no genetic/genomic biomarker which can accurately predict response to TNF inhibitor biologics prior to treatment, despite much interest in this area. Multiple studies have reported findings on potential candidate genes; however, due to relatively small sample sizes or lack of sufficient validation, results have been disappointingly inconsistent. The aim of this research was to further explore the predictive value of a previously reported association between CD11c expression and response to the TNF inhibitor biologics, adalimumab and etanercept. METHODS: Real-time qPCR was performed using whole blood RNA samples obtained from seventy-five rheumatoid arthritis patients about to commence treatment with a TNF inhibitor biologic drug, whose response status was determined at 3-month follow-up using the EULAR classification criteria. Relative quantification of CD11c using the comparative CT method outputted differential expression between good-responders and non-responders as a fold-change. RESULTS: Relative expression of CD11c in patients receiving TNF inhibitor biologics yielded a decrease of 1.025 fold in good-responders as compared to non-responders (p-value = 0.36). Upon stratification of patients dependent upon the specific drug administered, adalimumab or etanercept, similar findings to the full cohort were observed, decreases of 1.015 (p-value = 0.33) and 1.032 fold (p-value = 0.13) in good-responders compared to non-responders, respectively. CONCLUSION: The results from this study reveal that CD11c expression does not correlate with response to TNF inhibitor biologics when tested for within pre-treatment whole blood samples of rheumatoid arthritis patients.