PII-005 - ITPA ACTIVITY TO PREDICT REMISSION WITH METHOTREXATE IN JUVENILE IDIOPATHIC ARTHRITIS

S. Sindici Forgiarini¹, M. Lucafò¹, D. Curci², D. Selvestrel³, V. Moressa², S. Pagarin¹, B. Bellich², M. Franzin², A. Tommasini^2,4, M. Becker⁵, L. Ramsey⁶, S. Thompson^7,8, C. Langefeld⁹, E. Marrani¹⁰, G. Simonini¹⁰, G. Stocco¹¹, G. Decorti¹, A. Taddio^2,4, S. Pastore²; ¹University of Trieste, Trieste, Italy, ²IRCCS Burlo Garofolo, Trieste, Italy, ³International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy, ⁴University of Trieste, , , ⁵Duke University School of Medicine, Durham, NC, United States, ⁶Children's Mercy, Kansas City, MO, United States, ⁷Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, ⁸University of Cincinnati College of Medicine, , , ⁹Wake Forest School of Medicine, Winston-Salem, NC, United States, ¹⁰IRCCS Meyer Children's University Hospital, Firenze, Italy, ¹¹University of Trieste / Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy.

Background: Methotrexate (MTX) is the first-line therapy for juvenile idiopathic arthritis (JIA), but up to 40% of patients do not respond. Low ITPA activity in JIA patients has been linked to a reduced likelihood of clinical remission.

Methods: JIA patients from Trieste, Florence (Italy), and Kansas City (USA) receiving weekly MTX (10-15 mg/m2) for at least 12 months (199 patients, average age 9 ± 5 years, 73.9% females) were enrolled. Data were collected at baseline, 6 months, and every 3 months, with a follow-up of 12 months. ITPA activity was measured in erythrocyte lysates. In vitro, human hepatocytes (IHH) transfected either with an ITPA-containing (ITPA) or an empty (MOCK) plasmid were used for cytotoxicity (MTT) assay, ADORA2A expression (qPCR) and PKA expression (immunoblot). Remission, evaluated as a 6-month remission period beginning within one year of starting therapy, and ITPA activity association was assessed by logistic regression analysis and non-parametric tests, adjusting for clinical center by meta-analysis. Two-way ANOVA was used for in vitro tests.

Results: JIA patients reaching remission had higher ITPA activity than those who did not (all measurements: 210.9±91.3 vs 169.6±81.5 nmol IMP/h, p. meta=0.00018; onset: 230.3±96.3 vs 193.5±84.9 nmol IMP/h, p. meta=0.019). Predictive cutoffs of 164.7 and 222.6 nmol IMP/h were calculated for overall and onset measurements. IHH MOCK cells showed lower MTX cytotoxicity than IHH ITPA cells (IC50 MOCK±SEM: 273.8±155.14, IC50 ITPA±SEM: 13.33±10.82 nM, p< 0.0001). After 48h of 25 nM MTX treatment, ADORA2A expression was higher in MOCK cells (mean fold change difference (fc diff.)±SEM = 0.37±0.059, p< 0.0001). ITPA-overexpressing cells showed higher cAMP levels after 48h of treatment with both concentrations (25 nM: mean fc diff.±SEM = 0.82± 0.18, p< 0.01; 200 nM: mean fc diff.±SEM = 0.86±0.21, p< 0.01) and higher PKA expression after 72h (25 nM: mean fc diff.±SEM = 0.27± 0.077, p< 0.05; 200 nM: mean fc diff.±SEM = 0.44± 0.077, p< 0.001).

Conclusion: JIA patients with higher ITPA activity have increased probability of remission, both when measured during and at the onset of therapy. Differences observed in vitro suggest a different adenosine release, which may underlie the different response seen in patients. To be confirmed, adenosine will be quantified in IHH cells.