Effectiveness of methotrexate and bridging glucocorticoids with or without early introduction of a 6-month course of etanercept in early RA: results of the 2-year, pragmatic, randomised CareRA2020 trial.

OBJECTIVES: To investigate if patients with early rheumatoid arthritis responding insufficiently to initial methotrexate (MTX) and bridging glucocorticoids (GCs) could benefit from early but temporary etanercept introduction as a second remission-induction attempt. METHODS: CareRA2020 (NCT03649061) was a 2-year, open-label, multicentre, pragmatic randomised controlled trial. Treatment-naïve patients started MTX and GC bridging (COBRA-Slim: CS). Within a time window from week (W) 8 until W32, early insufficient responders (28-joint Disease Activity Score - C-reactive Protein (DAS28-CRP) >3.2 between W8 and W32 or ≥2.6 at W32) were randomised to a Standard-CS strategy (adding leflunomide first) or Bio-induction-CS strategy (adding etanercept for 24 weeks). Additional treatment adaptations followed the treat-to-target principle. Longitudinal disease activity (DAS28-CRP) over 104 weeks (primary outcome), achievement of DAS28-CRP <2.6 28 weeks after randomisation, and biologic or targeted synthetic disease-modifying antirheumatic drug (b/tsDMARD) use at W104 were compared between randomisation groups. RESULTS: Following CS treatment, 142 patients were early responders; 55 early insufficient responders received Standard-CS and 55 Bio-induction-CS. Superiority of Bio-induction-CS over Standard-CS could not be demonstrated (ß=-0.204, (95% CI -0.486 to 0.078), p=0.157) for the primary outcome. More patients on Bio-induction-CS achieved DAS28-CRP <2.6 at 28 weeks after randomisation (59% (95% CI 44% to 72%) vs 44% (95% CI 31% to 59%) in Standard-CS) and they were treated less frequently with b/tsDMARDs at W104 (19/55, 35%) compared with Standard-CS (29/55, 53%). CONCLUSION: Half of the patients responded well to initial COBRA-Slim induction therapy. In early insufficient responders, adding etanercept for 6 months did not improve disease control over 104 weeks versus adding leflunomide first. However, temporary introduction of etanercept resulted in improved disease control early after randomisation and less patients on b/tsDMARDs at W104. TRIAL REGISTRATION NUMBER: NCT03649061. CTR PILOT APPROVAL BELGIUM: S59474, EudraCT number: 2017-004054-41.

Anti-citrullinated protein antibodies in the diagnosis of rheumatoid arthritis (RA): diagnostic performance of automated anti-CCP-2 and anti-CCP-3 antibodies assays.

This study compares the diagnostic performance of a second generation anti-cyclic citrullinated peptide antibody (CCP2) with a third generation anti-CCP antibodies assay (CCP3), as well as the combination of both tests. Serum samples of 127 patients were analyzed. IgG anti-CCP 2 and IgM rheumatoid factor were determined by EliA™ technique on a Phadia 250 instrument (Thermo Fisher Scientific), anti-CCP3 by the Quanta Flash™ anti-CCP3 IgG kit, BIO-FLASH Rapid Response Chemiluminscence Analyzer (INOVA Diagnostics). Diagnostic performance was compared using ROC-curves, sensitivity, specificity, likelihood ratios, and predictive values. Logistic regressions were used to investigate whether using both tests (anti-CCP2 and anti-CCP3) gives a better prediction of rheumatoid arthritis. At the manufacturer's cut-offs sensitivity and specificity were 79.4 and 61.0% for CCP3 and 80.9 and 69.5% for CCP2. No significant differences could be observed regarding the areas under the curve (AUC) of both ROC-curves. The optimal cut-off point for CCP2 was 10.5 U/ml (sensitivity of 75.0% and specificity of 80.0%) and 5.6 U/ml for CCP3 (sensitivity of 86.9% and specificity of 61.0%). Binary logistic regressions indicated that the likelihood of having rheumatoid arthritis (RA) is significantly higher when testing positive on both CCP2 and CCP3 compared to CCP2 or CCP3 alone. In our cohort, comparable performance was found between the two CCP assays. Positivity for both CCP2 and CCP3 resulted in the most specific identification of RA patients. In patients with joint complaints suspected of having RA and with a weakly positive CCP 2 (≥7 and ≤16 U/ml) CCP3 testing could be of additive value for diagnosing RA.

Indications for a genetic association of a VCP polymorphism with the pathogenesis of sporadic Paget's disease of bone, but not for TNFSF11 (RANKL) and IL-6 polymorphisms.

Paget's disease of bone (PDB) is, after osteoporosis, the second most common metabolic bone disorder in the elderly Caucasian population. Mutations in the sequestosome 1 gene (SQSTM1) are responsible for the etiology of PDB in a subset of patients, but the disease pathogenesis in the remaining PDB patients is still unknown. Therefore association studies investigating the relationship between genetic polymorphisms and sporadic PDB have been performed in order to find the susceptibility polymorphisms. In this paper, we sought to determine whether polymorphisms in 3 functional candidate genes play a role in the development of sporadic PDB: TNFSF11 (receptor activator of nuclear factor κB ligand, RANKL), VCP (valosin-containing protein) and IL-6 (interleukin 6). Analyzing 9 tag SNPs and 2 multi-marker tests (MMTs) in TNFSF11, 3 tag SNPs and 1 MMT in VCP and 8 tag SNPs in IL-6 in a population of 196 Belgian patients with sporadic PDB and 212 Belgian control individuals revealed that one VCP SNP (rs565070) turned out to be associated with PDB in this Belgian study population (p=5.5×10(-3)). None of the tag SNPs or MMTs selected for TNFSF11 or IL-6 was associated with PDB. Still, replication of our findings in the VCP gene in other populations is important to confirm our results. However, when combining data of VCP with those from other susceptible gene regions from previous association studies (i.e. TNFRSF11A, CSF1, OPTN and TM7SF4), independent effect of each gene region was found and the cumulative population attributable risk is 72.7%.

The majority of the genetic risk for Paget's disease of bone is explained by genetic variants close to the CSF1, OPTN, TM7SF4, and TNFRSF11A genes.

Paget's disease of bone (PDB) is one of the most frequent metabolic bone disorders (1-5%), next to osteoporosis, affecting individuals above age 55. Sequestosome1 mutations explain a part of the PDB patients, but still the disease pathogenesis in the remaining PDB patients is largely unknown. Therefore, association studies investigating the relationship between genetic polymorphisms and sporadic PDB have been performed to find the genetic risk variants. Previously such studies indicated a role of the OPG and RANK gene. The latter was recently confirmed in a genome-wide association study (GWAS) which also indicated the involvement of chromosomal regions harbouring the CSF1 and OPTN gene. In this study, we sought to replicate these findings in a Belgian and a Dutch population. Similar significant results were obtained for the single nucleotide polymorphisms and the haplotypes. The most significant results are found in the CSF1 gene region, followed by the OPTN and TNFRSF11A gene region (p values ranging from 1.3 × 10(-4) to 3.8 × 10(-8), OR = 1.523-1.858). We next obtained significant association with a polymorphism from the chromosomal region around the TM7SF4 gene (p = 2.7 × 10(-3), OR = 1.427), encoding DC-STAMP which did not reach genome-wide significance in the GWAS, but based on its function in osteoclasts it can be considered a strong candidate gene. After meta-analysis with the GWAS data, p values ranged between 2.6 × 10(-4) and 8.8 × 10(-32). The calculated cumulative population attributable risk of these four loci turned out to be about 67% in our two populations, indicating that most of the genetic risk for PDB is coming from genetic variants close to these four genes.

Genetic variation in the TNFRSF11A gene encoding RANK is associated with susceptibility to Paget's disease of bone.

RANK (receptor activator of nuclear factor-κB), encoded by TNFRSF11A, is a key protein in osteoclastogenesis. TNFRSF11A mutations cause Paget's disease of bone (PDB)-like diseases (ie, familial expansile osteolysis, expansile skeletal hyperphosphatasia, and early-onset PDB) and an osteoclast-poor form of osteopetrosis. However, no TNFRSF11A mutations have been found in classic PDB, neither in familial nor in isolated cases. To investigate the possible relationship between TNFRSF11A polymorphisms and sporadic PDB, we conducted an association study including 32 single-nucleotide polymorphisms (SNPs) in 196 Belgian sporadic PDB patients and 212 control individuals. Thirteen SNPs and 3 multimarker tests (MMTs) turned out to have a p value of between .036 and 3.17 × 10(-4) , with the major effect coming from females. Moreover, 6 SNPs and 1 MMT withstood the Bonferroni correction (p < .002). Replication studies were performed for 2 nonsynonymous SNPs (rs35211496 and rs1805034) in a Dutch and a British cohort. Interestingly, both SNPs resulted in p values ranging from .013 to 8.38 × 10(-5) in both populations. Meta-analysis over three populations resulted in p = .002 for rs35211496 and p = 1.27 × 10(-8) for rs1805034, again mainly coming from the female subgroups. In an attempt to identify the underlying causative SNP, we performed functional studies for the coding SNPs as well as resequencing efforts of a 31-kb region harboring a risk haplotype within the Belgian females. However, neither approach resulted in significant evidence for the causality of any of the tested genetic variants. Therefore, further studies are needed to identify the real cause of the increased risk to develop PDB shown to be present within TNFRSF11A.

Founder effect in different European countries for the recurrent P392L SQSTM1 mutation in Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is one of the most frequent metabolic bone diseases, affecting 1-5% of Western populations older than 55 years. Mutations in the sequestosome1 (SQSTM1) gene cause PDB in about one-third of familial PDB cases and in 2.4-9.3% of nonfamilial PDB cases, with the 1215C-->T (P392L) mutation being the most frequent one. We investigated whether a founder effect of the P392L SQSTM1 mutation was present in Belgian (n = 233), Dutch (n = 82), and Spanish (n = 64) patients without a PDB family history. First, direct sequencing analysis of exon 8 in these three populations showed that the P392L mutation occurred in 17 Belgian patients (7.3%), three Dutch patients without a family history (3.7%), and two Dutch patients with a family history. In the Spanish population, 15.6% of patients (n = 10) had the P392L mutation, including one homozygous mutant. This is by far the highest mutation frequency of all populations investigated so far. Next, we examined the genetic background of 33 mutated chromosomes by analyzing haplotypes. We genotyped four single-nucleotide polymorphisms (SNPs) in exon 6 and the 3'-untranslated region of SQSTM1 (rs4935C/T, rs4797G/A, rs10277T/C, and rs1065154G/T) and used software programs WHAP and PHASE to reconstruct haplotypes. Finally, allele-specific primers allowed us to assign the mutation to one of the two haplotypes from each individual. Sequencing results revealed that all 33 P392L mutations were on the CGTG (H2) haplotype. The chance to obtain this result due to 33 independent mutation events is 3.97 x 10(-14), providing strong evidence for a founder effect of the P392L SQSTM1 mutation in Belgian, Dutch, and Spanish patients with PDB.

Identification of sex-specific associations between polymorphisms of the osteoprotegerin gene, TNFRSF11B, and Paget's disease of bone.

UNLABELLED: We studied the role of TNFRSF11B polymorphisms on the risk to develop Paget's disease of bone in a Belgian study population. We observed no association in men, but a highly significant association was found in women, and this was confirmed in a population from the United Kingdom. INTRODUCTION: Juvenile Paget's disease has been shown to be caused by mutations in TNFRSF11B encoding osteoprotegerin. Although mutations in this gene have never been found in patients with typical Paget's disease of bone (PDB), there are indications that polymorphisms in TNFRSF11B might contribute to the risk of developing PDB. MATERIALS AND METHODS: We recruited a population of 131 Belgian patients with sporadic PDB and 171 Belgian controls. By means of the HapMap, we selected 17 SNPs that, in combination with four multimarker tests, contain most information on common genetic variation in TNFRSF11B. To replicate the findings observed in the Belgian study population, genotyping data of SNPs generated in a UK population were reanalyzed. RESULTS: In our Belgian study population, associations were found for two SNPs (rs11573871, rs1485286) and for one multimarker test involving rs1032129. When subsequently analyzing men and women separately, these associations turned out to be driven by women (56 cases, 78 controls). In addition, three other tagSNPs turned out to be associated in women only. These were rs2073617 (C950T), rs6415470, and rs11573869. Reanalysis of genotyping data from a UK study population indicated that the associations found for C950T and C1181G were also exclusively driven by women (146 cases, 216 controls). Meta-analysis provided evidence for risk increasing effects of the T allele of C950T and the G allele of C1181G in the female population (p = 0.002 and 0.003, respectively). The haplotypes formed by the SNPs associated in the Belgian population were also distributed differentially between female cases and controls. CONCLUSIONS: We showed for the first time that SNPs influencing the risk to develop PDB could be sex-specific. Further research is necessary to identify the causative variants in TNFRSF11B and to elucidate the molecular pathogenic mechanism.

Influence of bisphosphonates on the production of pro-inflammatory cytokines by activated human articular chondrocytes.

Bisphosphonates have anti-inflammatory effects in rheumatoid arthritis and chondroprotective effects in animal arthritis models but their influence on chondrocytes is not known. The aim of this study is to investigate whether bisphosphonates could influence the production of pro-inflammatory cytokines by activated chondrocytes. Therefore human articular cartilage explants were incubated at 48 h with clodronate, pamidronate or risedronate (10(-6) and 10(-8)mol/L), and dexamethasone (10(-8)mol/L). Subsequently, cultures were stimulated with IL-1, 10 ng/mL (n=6) or 1 ng/mL (n=10) for 48 h. Co-incubation was performed with or without bisphosphonates or dexamethasone. A flow cytometric microsphere-based immunoassay was used for the detection of the pro-inflammatory cytokines IL-6, IL-8, TNF-alpha, IL-1 and the regulatory cytokines IL-12p70 and IL-10 in the supernatants. Stimulation with IL-1 resulted in a dose dependent induction of IL-6 and IL-8, but no production of the other cytokines could be demonstrated. This production of IL-6 and IL-8 was neither inhibited nor enhanced by bisphosphonates. Only dexamethasone caused an inhibition of IL-6 production. In conclusion, there is no evidence on the level of articular cartilage cells that bisphosphonates would suppress or enhance IL-6 and IL-8 mediated joint destruction.