POS0185\u2005IDENTIFICATION AND VALIDATION OF TWO NOVEL SERUM BIOMARKERS ASSOCIATED WITH THE SEROLOGICAL STATUS OF RHEUMATOID ARTHRITIS PATIENTS.

Abstract

Despite the diagnostic value of Rheumatoid Factor (RF) and Anti-Citrullinated Protein Antibodies (ACPA), more serological markers are needed in order to improve early diagnosis and treatment response of the Rheumatoid Arthritis (RA) patients. Increased knowledge about how these two major autoreactivities arise is crucial for understanding how RA develops and what mechanisms drive pathogenesis.We aimed to investigate, using a proteomic strategy, novel protein biomarkers associated with RF and/or ACPA that might be useful to stratify seropositive and seronegative RA patients.A shotgun proteomic analysis was performed on 80 sera from the RA cohort of the Rheumatology Unit of the University Hospital of Santiago de Compostela (CHUS). Sera were classified as seropositive or seronegative according to their RF and ACPA values, and were then analyzed employing the iTRAQ labelling technique (Sciex) followed by LC-MALDI-MS/MS analysis (MALDI-TOF). A Multiple Reaction Monitoring (MRM) method was subsequently developed using the Skyline Software for the simultaneous quantification of 26 peptides belonging to ten putative protein biomarkers. The quantitative targeted analysis was performed using peptides with isotope labelled amino acids as internal standards. Serum levels of orosomucoid 1 (ORM1) and haptoglobin (HPT) were measured using commercially available ELISA Kits in the whole RA cohort (n=260) from the Rheumatology Unit of the University Hospital of A Coruña (HUAC).For the initial screening, eighty sera were grouped according to the ACPA/RF status in 4 pools (20 patients/pool). Using an iTRAQ technology-based quantitative proteomic approach, the abundance of eleven proteins was altered in the sera from ACPApos/RFpos, 13 proteins in ACPAneg/RFpos and 12 proteins in ACPApos/RFneg, compared to ACPAneg/RFneg. Vitamin D binding protein (VTDB) was the unique protein that resulted increased in all the comparisons. For the biomarker verification phase, all the samples from the CHUS cohort were analyzed individually (n=80). Using the MRM technology, 26 peptides belonging to ten putative protein biomarkers associated with double positivity were simultaneously quantified. The statistical analysis showed a significant modulation of 9 peptides (belonging to 4 different proteins) in ACPApos/RFpos, 7 peptides (5 proteins) in ACPAneg/RFpos, and 9 peptides (6 proteins) in ACPApos/RFneg compared to ACPAneg/RFneg (p<0.05). Two acute phase reactants (ORM1 and HPT) displayed the same modulation in both screening and verification phases, thus confirming their association with the double positivity. Finally, in the biomarker validation phase, a total of 260 patients from CHUAC were included (Table 1). RA patients were classified as follows: (1) 112 patients (43.1%) were ACPApos/RFpos; (2) 73 patients (28.1%) were ACPAneg/RFneg; (3) 51 patients (19.6%) were ACPAneg/RFpos; and (4) 24 patients (9.2%) were ACPApos/RFneg. Serum levels of ORM1 and HPT (Figure 1), measured by commercial immunoassays, confirmed their increased values in double seropositive patients (p=0,0053 ORM1; p=0,0026 HPT). Finally, the increased level of ORM1 resulted associated with RF rather than ACPA status (p=0,0008 ACPAneg/RFpos); whereas HPT was associated with ACPA rather than RF status (p=0,0112 ACPApos/RFneg).Table 1.The different phases of RA biomarker development followed in this study.DISCOVERYPHASEVERIFICATIONPHASEVALIDATIONPHASESource centerCHUSCHUSCHUACN° ofsamplesn= 4n= 80n= 260ACPA+RF+Pool 1ACPA+RF+20ACPA+RF+112ACPA-RF-Pool 2ACPA-RF-20ACPA-RF-73ACPA-RF+Pool 3ACPA-RF+20ACPA-RF+51ACPA+RF-Pool 4ACPA+RF-20ACPA+RF-24N° ofbiomarkersORM1, ORM2, HPT, A2GL, AACT, RBP4, PLMN, IC1, VDBP, APOBORM1, HPT, A2GL, AACTORM1, HPTFigure 1.The determination of ORM1 and HPT in sera provides novel information useful for patient stratification, which might improve diagnostic and prognostic approaches and facilitate the development of personalized medicine strategies in RA.This work is supported by grants from Fondo de Investigación Sanitaria (RD16/0012/0002, PT17/0019/0014) integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”.None declared