Maxime Verhoeven

63 Validation of a prediction model for insufficient response to MTX (Roche Life Science, Almere, The Netherlands) and genotypes were determined for ABCB1 rs1045642 and ABCC3 rs4793665 using real-time PCR with Taqman, as described previously. 15 Samples were tested for deviation from the Hardy-Weinberg equilibrium (HWE). SNPs were determined in the same lab and according to the same protocols as the tREACH study. 9 Importantly, there were methodological differences in the measurement of folate between the tREACH and U-Act-Early cohorts. The Elecsys® Folate III assay (Ref 7027290190; Roche Diagnostics) has been re-standardized since 2017 in accordance with the WHO International Standard NIBSC Code 03/178. This resulted in 10% lower erythrocyte folate levels (U-Act-Early) compared to those quantified using the previous assay (tREACH). Furthermore, serum folate levels, required for folate correction in whole blood, were not available in U-Act-Early. As serum folate levels take up only a small part of the total folate concentrations, whole blood folate levels were corrected for the average serum folate concentration in the tREACH (25 nmol/L). Statistical analysis Clinical and laboratory parameters for the tREACH (derivation) and U-Act-Early (validation) cohorts were compared. The difference in DAS28 at 3 months (compared to baseline) was expressed as a mean with standard deviation (±SD) and assessed using a paired sample t-test. Differences between cohort variables were tested using an independent two-group t-test, if the assumptions of normal distribution (visual inspection) and equal variances (tested using the Levene’s test) were met. If these assumptions were not met, non-parametric Mann Whitney U-test was performed. Differences in proportions were tested using a two-sample proportion test. Due to missing informed consents at the start of this validation study, eight subjects from the tREACH (derivation) cohort were excluded from analyses in this study. That is why the model as described previously was first re-analyzed on the tREACH data excluding these eight subjects, resulting in negligible differences in effect sizes compared to previous study. 9 Next, to validate the prediction model in the external validation cohort (U-Act- Early), the predictors, DAS28 >5.1, HAQ >0.6, erythrocyte folate <750 nmol/L, current smoking, BMI >25kg/m 2 , ABCB1 rs1045642 genotype and ABCC3 rs4793665 genotype were entered into a multivariable logistic regression and the probability for insufficient response was calculated using the pROC package in R according to the following formula: , where b0 represents the constant, b, b2 and bn represent the regression coefficients for each of the predictors x1, x2, xn. Subsequently, an ROC curve with AUC was constructed using the predicted probabilities and compared with results in the tREACH. The previously developed prediction model in tREACH (derivation) was considered successfully validated in the U-Act-Early (validation) if the AUC-ROC was not significantly 4