Maxime Verhoeven

62 Chapter 4 College of Rheumatology (ACR) 12 (n=7, 8%) or the 2010 ACR/EULAR classification criteria (n=84, 92%), 13 and had a disease duration <12 months and active disease at baseline (DAS28≥2.6). Patients were randomly assigned to a treatment strategy with tocilizumab (TCZ) + placebo, MTX + placebo or their combination (TCZ+MTX) and treated to the target of sustained remission (i.e., a DAS28 <2.6 and swollen joint count of ≤4 joints of the 28 joints assessed, during ≥24 weeks). All 91 patients included were derived from the initial MTX + placebo strategy arm. The starting MTX dose was 10 mg/week orally and increased stepwise 5 mg every 4 weeks up to 30 mg/week until remission or the maximum tolerable dose. During the trial, GC use was not permitted. The tREACH (n=285) cohort was described earlier. 9 Importantly, in the tREACH the optimal MTX dose of 25 mg/week was reached within three weeks (combined with other conventional synthetic (cs) DMARDs and/or GCs) and therapy was targeted to low disease activity (DAS28 ≤3.2) at 3 months. If this failed, step-up treatment with additional csDMARDs (sulfasalazine and/or hydroxychloroquine) or bDMARDs (i.e., TNF-alpha inhibitor) was initiated. In both cohorts, folic acid (10 mg/week) was prescribed during MTX treatment. U-Act-Early was approved by the medical ethics committee of the University Medical Center Utrecht (ML22497), and tREACH by the medical ethics committee of Erasmus Medical Center (MEC-2006-252). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained for all patients. Outcome and clinical predictors The primary outcome was insufficient MTX response after 3 months of treatment start, defined as DAS28>3.2, where DAS28 was based on the erythrocyte sedimentation rate (DAS28-ESR). All predictors were dichotomized prior to analyses. Cut-off values were defined previously in the tREACH cohort as: DAS28 >5.1, HAQ >0.6, erythrocyte folate <750 nmol/L, current smoking, BMI >25kg/m 2 , ABCB1 rs1045642 (GG/GA vs AA) genotype, and ABCC3 rs4793665 (TC/CC vs TT) genotype. 9 As erythrocyte-folate levels were slightly higher in U-Act-Early, new cut-off points were examined for erythrocyte- folate (deciles) and BMI (>30kg/m 2 ) in U-Act-Early and tested for improvement of the model. The secondary outcome measure was non-response to MTX after 6 months of treatment, defined as DAS28-ESR>3.2. Erythrocyte folate and genetic variants In U-Act-Early erythrocyte folate and genetic variants included in the original prediction model 9 were determined from EDTA whole blood samples stored at -80°C, as described elsewhere. 14,15 DNA was obtained from whole blood using a MagNAPure Compact