AB0338 EVALUATION OF THE ASSOCIATION OF ALLELES OF INTOLERANCE RISK TO METOTREXATE IN RHEUMATOID ARTHRITIS PATIENTS UNDER TREATMENT WITH BIOLOGIC THERAPIES

Background:Metotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA) both in monotherapy and in combination with biologic disease-modifying antirheumatic drugs (bDMARDs), it usually well tolerated but AEs may appear that causing toxicity that requires suspension of the treatmentObjectives:Determine the prevalence of certain polymorphisms among patients that receive bDMARD in monotherapy or in combination with MTX to confirm its relevance as biomarkers of intolerance. Evaluate the influence of certain polymorphisms in the effectiveness of monotherapy or combined treatment in patients, through“Disease Activity Score 28”(DAS28), SDAI Simple disease activity index (SDAI), Clinical disease activity index (CDAI) and each one of its componentsMethods:Retrospective observational multicentric study (University Hospital Complex, Granada, Carlos de Haya Hospital, Malaga and University Hospital Reina Sofia, Cordoba), of cases-control of 227 patients with RA (criteria ACR/EULAR), of which 120 received MTX and bDMARD combined therapy (cases) and other with only bDMARD (controls). All of them had been or were currently treated with MTX, remained with stable doses of bDMARD, and had a DNA sample stored before the inclusion in the studyDNA was isolated from total peripheral blood and by fluorescent probe HybProbe and/ or Taqman, 10 polymorphisms of 10 protein coding genes were determined involved in the metabolism and toxicity of MTX according to current evidenceBesides the type of polymorphism, data on the activity of the disease were analysed (DAS28VSG, DAS28PCR, SDAI, CDAI, at the start of the MTX income, of the BT, and in the inclusion visitA descriptive and comparative study was carried out on all that and afterwards an assessment was made through a multiple logistic regression analysis (MLR) on the risk of intolerance to MTXResults:An analysis was carried out on 227 patients (120 cases and 107 controls) with an average age of 60 (12,1) being women 78,4%, with a time of evolution since diagnosis of 14,84 (7,78) years48,9% registered adverse events (AE) MTX related, mainly gastrointestinal, hepatobiliary and skin-subcutaneous tissue. The percentage of AE appearance was superior in the monotherapy group than in the group with combined therapyThe most prevalent polymorphism (84,6% (IC95%: 84,09%-85,11%) and in cases (86,0% (IC95%: 79,43%-92,57%) washomozygous CC (ITPase-c94a); in controlshomozygous GG (GGH-T401C) (87,5% (IC95%:81,58%-93,42%)There were no significant differences in the parameters of activity between groups, in both, patients were best basally controlled than at the start of the MTX income and/or bDMARDBeinghomozygous-AA for the DHFR genewas significantly associated (p<0.05) with the appearance of AE (none of the 4 homozygous AA patients for that gene had AE)In MLR,homozygous GG(ref. heterozygous AG) in polymorphismGGH-T401C,beinghomozygous CC(ref. heterozygous TC) in polymorphismABCC2-C24TandPCR (mg/dL) at the start of bDMARDresulted independent predictive factors of MTX intoleranceConclusion:Polymorphisms T401C for the GGH gene and C24T for the ABCC2 gene and PCR at the start of the bDMARD resulted independent predictive factors of MTX intolerance. Polymorphismhomozygous AAforDHFR genewas related to significant protection against appearance of AEDisclosure of Interests:Alejandro Escudero Contreras: None declared, Rafaela Ortega Castro: None declared, Jerusalem Calvo Gutierrez: None declared, Natalia Mena-Vázquez: None declared, Rafael Cáliz Cáliz: None declared, Eduardo Collantes Estevez Grant/research support from: ROCHE and Pfizer, Speakers bureau: ROCHE, Lilly, Bristol and Celgene, Antonio Fernandez-Nebro: None declared, Maria del Carmen Abalos-Aguilera: None declared, Chary Lopez-Pedrera Grant/research support from: ROCHE and Pfizer., Mª Teresa Ruiz Jimenez Employee of: Roche Farma, SPAIN, Font Ugalde Pilar: None declared

DHFR silencing effects on biological behaviour of ovarian carcinoma cells in vitro

Background: Dyhidrofolate reductase (DHFR) is involved in the DNA synthesis and is expressed highly in platinum resistant ovarian carcinoma tissue . The relationship between DHFR gene and platinum drug resistance in ovarian carcinoma (OC) is still unclear. Methods: To design targeting hairpin siRNA of DHFR gene, and constructed lentiviral vector carrying DHFR gene, then though flow cytometry、high performance liquid chromatography and electron microscope, DHFR gene expression was down-regulating to investigate the biological behaviour of DHFR in OC. Results: DHFR mRNA expression in ovarian carcinoma was higher than normal ovarian tissues, in patients with omentum metastasis was lower than that in patients without omentum metastasis, in patients with CR for treatment was lower than that of patients with SD or PD or PR, cisplatin-sensitive patients was lower than that in cisplatin-multidrug resistant cases. The median survival period of patients with DHFR mRNA expression lower than 0.331(Youden index) was 16.4 months, but of that higher than 0.331 was 44.5 months. COX multivariate analysis showed that DHFR mRNA expression was a dependent prognostic factor. The apoptosis rate of DHFR-pGCSIL-SKOV3 was obvious higher than pGCSIL-SKOV3 and SKOV3 cells at 24h and 48h(P＜0.05). GO/G1 stage rate of three groups at 24h、48h and 72h were decreasing but the G2/M and S stage rates were raising. The cells in the cisplatin concentration (2.5、5.0μg/mL)at 24h and 48h,the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell were significantly higher than pGCSIL-SKOV3 and SKOV3 cells. The microfilament increased and gathered together, mitochondrial structure also change obviously under no drug. But there were rarely microfilament in three group cells induced by IC50 cisplatin concentration(4.4、5.5、4.9µg/ml)at 24 and 48 h, while at 72 h, there are obvious increase and inordinate microfilament. Conclusion: DHFR silencing inhibits cell growth and cisplatin resistance, there were certain contact between resistance increases with microfilament gathered and the change of the mitochondria.The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.