Rheumatoid arthritis (RA) is a prototypical autoimmune arthritis affecting nearly 1% of the world population and is a significant cause of worldwide disability. Though prior studies have demonstrated the appearance of RA-related autoantibodies years before the onset of clinical RA, the pattern of immunologic events preceding the development of RA remains unclear. To characterize the evolution of the autoantibody response in the preclinical phase of RA, we used a novel multiplex autoantigen array to evaluate development of the anti-citrullinated protein antibodies (ACPA) and to determine if epitope spread correlates with rise in serum cytokines and imminent onset of clinical RA. To do so, we utilized a cohort of 81 patients with clinical RA for whom stored serum was available from 1–12 years prior to disease onset. We evaluated the accumulation of ACPA subtypes over time and correlated this accumulation with elevations in serum cytokines. We then used logistic regression to identify a profile of biomarkers which predicts the imminent onset of clinical RA (defined as within 2 years of testing). We observed a time-dependent expansion of ACPA specificity with the number of ACPA subtypes. At the earliest timepoints, we found autoantibodies targeting several innate immune ligands including citrullinated histones, fibrinogen, and biglycan, thus providing insights into the earliest autoantigen targets and potential mechanisms underlying the onset and development of autoimmunity in RA. Additionally, expansion of the ACPA response strongly predicted elevations in many inflammatory cytokines including TNF-α, IL-6, IL-12p70, and IFN-γ. Thus, we observe that the preclinical phase of RA is characterized by an accumulation of multiple autoantibody specificities reflecting the process of epitope spread. Epitope expansion is closely correlated with the appearance of preclinical inflammation, and we identify a biomarker profile including autoantibodies and cytokines which predicts the imminent onset of clinical arthritis.

Objective. To evaluate levels of biomarkers in preclinical rheumatoid arthritis (RA) and to use elevated biomarkers to develop a model for the prediction of time to future diagnosis of seropositive RA.Methods. Stored samples obtained from 73 military cases with seropositive RA prior to RA diagnosis and from controls (mean 2.9 samples per case; samples collected a mean of 6.6 years prior to diagnosis) were tested for rheumatoid factor (RF) isotypes, anti-cyclic citrullinated peptide (anti-CCP) antibodies, 14 cytokines and chemokines (by bead-based assay), and C-reactive protein (CRP).Results. Preclinical positivity for anti-CCP and/or >2 RF isotypes was >96% specific for future RA. In preclinical RA, levels of the following were positive in a significantly greater proportion of RA cases versus controls: interleukin-1␣ (IL-1␣), IL-1␤, IL-6, IL-10, IL-12p40, IL-12p70, IL-15, fibroblast growth factor 2, flt-3 ligand, tumor necrosis factor ␣, interferon-␥-inducible 10-kd protein, granulocyte-macrophage colony-stimulating factor, and CRP. Also, increasing numbers of elevated cytokines/chemokines were present in cases nearer to the time of diagnosis. RA patients who were >40 years old at diagnosis had a higher proportion of samples positive for cytokines/chemokines 5-10 years prior to diagnosis than did patients who were <40 years old at diagnosis (P < 0.01). In regression modeling using only case samples positive for autoantibodies highly specific for future RA, increasing numbers of cytokines/chemokines were predictive of decreased time to diagnosis, and the predicted time to diagnosis based on cytokines/chemokines was longer in older compared with younger cases.Conclusion. Levels of autoantibodies, cytokines/ chemokines, and CRP are elevated in the preclinical period of RA development. In preclinical autoantibodypositive cases, the number of elevated cytokines/ chemokines is predictive of the time of diagnosis of future RA in an age-dependent manner.Multiple studies have demonstrated that levels of disease-related biomarkers may be elevated prior to the onset of symptomatic rheumatoid arthritis (RA). These biomarkers include rheumatoid factor (RF) and antibodies to citrullinated protein antigens, as well as secre-

Corresponding author: V. Michael Holers, MD, Division of Rheumatology, University of Colorado at Denver and Health Sciences Center, Barbara Davis Center, 1775 North Ursula Street, Aurora, Colorado 80045, Phone: (303) Fax: (303) 724-7581, Michael.Holers@uchsc.edu. Address for reprint requests: Same as above * These two authors contributed equally to the study. COMPETING INTERESTSThe authors declare that they have no competing interests. AUTHORS' CONTRIBUTIONSDM participated in the design and analysis of this study and the writing of this manuscript. KD participated in the analysis of data and preparation of manuscript. LP participated in the analysis of samples and data and preparation of manuscript. AB and AL contributed statistical expertise in the analysis of data for this manuscript. CW participated in subject evaluation and determination of eligibility for this study MR participated in the design of this study and procurement of the serum samples for analysis WG participated in the design of this study and procurement of the serum samples for analysis, as well as subject evaluation and determination of eligibility for this study. JN participated in the design and analysis of this study and preparation of manuscript. VMH participated in the design and analysis of this study, antibody testing, and manuscript preparation. DISCLAIMERThe views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or U.S. Government.The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article to be published in Annals of Rheumatic Diseases editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our license http://ard.bmjjournals.com/ifora/licence.pdf. Objectives-This study investigated factors that may influence the prevalence and timing of appearance of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies during the pre-clinical phase of rheumatoid arthritis (RA) development. NIH Public AccessMethods-243 serial pre-diagnosis serum samples from 83 subjects with RA were examined for the presence of RF and anti-CCP antibodies.Results-57% and 61% of subjects had at least one pre-diagnosis sample positive for RF or anti-CCP, respectively. Gender and race were not significantly associated with the prevalence or timing of pre-clinical antibody appearance. Pre-clinical anti-CCP positivity was strongly associated with the development of erosive RA (OR 4.64; 95% CI 1.71-12.63; p=0.003), but RF was not (p=0.11). Additionally, as age at the time of diagnosis of RA increased the duration of pre-diagnosis antibody positivity for RF and anti-CCP increased, with the longest duration of preclinical antibody positivity seen in patients diagnosed with RA over the age of 40. In ...

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Objective Anti-carbamylated protein (anti-CarP) antibodies could further elucidate early RA pathogenesis and predict clinical disease. We compared diagnostic accuracy of anti-CarP antibodies for future RA to other RA-related antibodies in military personnel. Methods Stored pre-RA diagnosis serum samples from 76 RA cases were tested for anti-CarP Fetal Calf Serum (FCS), anti-CarP Fibrinogen (Fib), anti-CCP2, RF-Neph, and RF-isotypes (IgM, IgG, and IgA). Positivity for all antibodies was determined as ≥2SD of log-transformed means from controls. Relationships between autoantibodies and future RA were assessed in prediagnosis serum for all RA cases compared to controls using sensitivity, specificity, and logistic regression. Differences in diagnostic accuracy between antibody combinations were assessed using comparisons of area under the curves (AUCs). Results Anti-CarP-FCS was 26% sensitive and 95% specific for future RA, where anti-CarP-Fib was 16% sensitive and 95% specific for future RA. Anti-CarP-FCS positivity was associated with future RA, while anti-CarP-Fib trended towards association. The antibody combination of anti-CCP2 and/or ≥2 RFs (RF-Neph and/or RF-isotypes) resulted in an AUC of 0.72 for future RA, where the AUC was 0.71 with the addition of anti-CarP-FCS to this prior combination. Conclusion Adding anti-CarP-FCS to antibody combinations did not improve AUC. However, anti-CarP-FCS was associated with future onset of RA, and was present in prediagnosis serum in ~10% of RA cases negative for anti-CCP2, but positive for RF.