Han et al. Arthritis Research & Therapy (2016) 18:93 DOI 10.1186/s13075-016-0995-0 RESEARCH ARTICLE Open Access Baseline CXCL10 and CXCL13 levels are predictive biomarkers for tumor necrosis factor inhibitor therapy in patients with moderate to severe rheumatoid arthritis: a pilot, prospective study Bobby Kwanghoon Han1*, Igor Kuzin2, John P. Gaughan3, Nancy J. Olsen4 and Andrea Bottaro2 Abstract Background: TNF inhibitors have been used as a treatment for moderate to severe RA patients. However, reliable biomarkers that predict therapeutic response to TNF inhibitors are lacking. In this study, we investigated whether chemokines may represent useful biomarkers to predict the response to TNF inhibitor therapy in RA. Methods: RA patients (n = 29) who were initiating adalimumab or etanercept were recruited from the rheumatology clinics at Cooper University Hospital. RA patients were evaluated at baseline and 14 weeks after TNF inhibitor therapy, and serum levels of CXCL10, CXCL13, and CCL20 were measured by ELISA. Responders (n = 16) were defined as patients who had good or moderate response at week 14 by EULAR response criteria, and nonresponders (n = 13) were defined as having no response. Results: Responders had higher levels of baseline CXCL10 and CXCL13 compared to nonresponders (p =0.03 and 0.002 respectively). There was no difference in CCL20 levels. CXCL10 and CXCL13 were highly correlated with each other, and were higher in seropositive RA patients. CXCL10 and CXCL13 levels were decreased after TNF inhibitor therapy in responders. Baseline additive levels of CXCL10 + 13 were correlated with changes in DAS score at 14 weeks after TNF inhibitor therapy (r = 0.42, p = 0.03), and ROC curve analyses for predictive ability of CXCL10 + 13 showed an AUC of 0.83. Conclusions: Elevated baseline levels of CXCL10 and CXCL13 wereassociatedwithfavorableresponsetoTNF inhibitor therapy in RA. Subjects with high CXCL10 and high CXCL13 may represent a subset of RA patients whose inflammatory reactions are primarily driven by TNF. Keywords: CXCL10, CXCL13, TNF inhibitor, Rheumatoid arthritis Background for moderate to severe RA patients who have inad- Rheumatoidarthritis(RA)isadiseasethatischarac- equate responses to conventional disease-modifying an- terized by synovial inflammation, cartilage and bone tirheumatic drugs (DMARDs) including methotrexate. destruction, and systemic features [1, 2]. Advances in However, reliable predictive biomarkers of therapeutic understanding the pathogenesis of the disease have response for TNF inhibitor therapy are lacking [3]. fostered the development of new therapeutics. Tumor Several studies have been conducted to discover pre- necrosis factor (TNF) inhibitors are used as a treatment dictive biomarkers for RA therapies. It has been reported that type I interferon (IFN) signature is associated with * Correspondence: [email protected] the therapeutic response to TNF inhibitors and rituximab 1Division of Rheumatology, Cooper Medical School of Rowan University, [4–7]. C-X-C motif chemokine 10 (CXCL10) is induced Camden, NJ 08103, USA by type I and II IFNs [8, 9]. Recruitment and activation of Full list of author information is available at the end of the article © 2016 Han et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Han et al. Arthritis Research & Therapy (2016) 18:93 Page 2 of 7 C-X-C chemokine receptor type 3 (CXCR3)-positive T etanercept treatment). The research protocol was ap- helper 1 (Th1) lymphocytes and monocytes by CXCL10 proved by the Institutional Review Board of Cooper may lead to TNF production in RA [10, 11]. In other University Hospital and all patients provided written in- studies, C-X-C motif chemokine 13 (CXCL13) has been formed consent for participation in the study. reported to be a predictive biomarker for RA therapies [12, 13]. CXCL13, a chemokine that attracts B lympho- Measurement of chemokine levels cytes and follicular helper T lymphocytes (TFH) by bind- Freshly isolated serum samples were aliquoted and stored ing to C-X-C chemokine receptor type 5 (CXCR5), is in a −80 °C freezer until use. Commercial enzyme-linked upregulated in a subset of T cells by TNF or interleukin 6 immunosorbent assay (ELISA) kits were used for serum (IL-6) in RA [14, 15] as well as in follicular dendritic cells measurements of CXCL10 (R&D, Minneapolis, MN, USA), in the germinal center of lymphoid tissues [16]. Lastly, CXCL13 (Sigma-Aldrich, St Louis, MO, USA), and CCL20 baseline interleukin 17 (IL-17) levels are inversely corre- (Sigma-Aldrich, St Louis, MO, USA). lated with response to TNF inhibitor therapy in RA [17]. C-C motif chemokine 20 (CCL20) attracts T helper 17 (Th17) lymphocytes expressing C-C chemokine receptor Statistical analyses type 6 (CCR6), and induced by interleukin 1β (IL-1β), Statistical analyses were performed using SAS v9.4 (SAS IL-17, and TNF [18, 19]. Institute, Cary, NC, USA), and graphs were generated In this study, we investigated whether these three using GraphPad Prism 6.0 (GraphPad Software, La Jolla, chemokines may represent useful predictive biomarkers CA, USA). Continuous variables were compared using for TNF inhibitor therapy in RA patients. We measured Wilcoxon ranked sum test, and dichotomous variables ’ CXCL10, CXCL13, and CCL20 in RA patients who were were compared using Fisher s exact test. Correlations initiating TNF inhibitor therapy and correlated each between pairs of continuous variables were performed chemokine level with the therapeutic response. using Spearman correlation coefficient. Differences be- tween pretreatment and posttreatment chemokine levels Methods were compared using analysis of variance (ANOVA) Patients and assessment for repeated measures. Receiver operating characteris- Patients with RA who met the inclusion and exclusion tic (ROC) curve analysis was performed to assess the criteria were recruited during routine care in the rheuma- predictive ability of cytokine variables. In all the tests, p tology clinics at the Cooper University Hospital (Camden, atwo-sided value <0.05 was considered significant. NJ, USA). The inclusion criteria for this study were: (1) diagnosis of RA by American College of Rheuma- Results tology (ACR) criteria; (2) active RA defined by disease Baseline serum CXCL10 and CXCL13 levels are higher in activity score (DAS) >4.4; (3) inadequate response to responders to TNF inhibitor therapy methotrexate; (4) clinical indication for initiating adalimu- Twenty-nine RA patients who were about to start either mab or etanercept treatment. The exclusion criteria were: adalimumab or etanercept after having an inadequate (1) diagnosis of other connective tissue diseases including response to methotrexate and other DMARDs were systemic lupus erythematosus, systemic sclerosis, or derm- recruited. Five patients had been treated with a TNF atomyositis, or interstitial lung disease; (2) diagnosis of inhibitor previously, and their last treatment was at least chronic infection including viral hepatitis or human 3 months ago. After 14 weeks of TNF inhibitor therapy, immunodeficiency virus; (3) history of malignancy. All using EULAR response criteria, the patients were classi- 29 patients except one were continued on baseline fied into 16 good and moderate responders (collectively methotrexate and other DMARDs while taking adali- termed hereafter as ‘responders’) and 13 nonresponders. mumab (22 patients) or etanercept (7 patients). The pa- Their baseline characteristics, summarized in Table 1, tients were assessed and peripheral blood samples were showed no significant differences between responders obtained at baseline and 14 weeks after TNF inhibitor and nonresponders. therapy. The results of rheumatoid factor (RF), anti- Baseline chemokine levels were measured by ELISA be- cyclic citrullinated peptide antibody (anti-CCP), and fore starting TNF inhibitor therapy and compared between erythrocyte sedimentation rate (ESR) tests were ob- responders and nonresponders (Fig. 1a). Responders had tained as part of patient care. Responders were defined significantly higher serum levels of CXCL10 (606 ± 581 as patients who had good to moderate response at week vs 283 ± 265 pg/ml, p = 0.03) and CXCL13 (383 ± 644 vs 14 by European League Against Rheumatism (EULAR) 27 ± 24 pg/ml, p = 0.002) compared to nonresponders. response criteria (14 with adalimumab and 2 with eta- There was no difference in CCL20 levels between re- nercept treatment), and nonresponders were defined as sponders and nonresponders (14 ± 13 vs 19 ± 31 pg/ml, having no response (8 with adalimumab and 5 with p = 0.78). Han et al. Arthritis Research & Therapy (2016) 18:93 Page 3 of 7 Table 1 Baseline characteristics of RA patients Chemokine levels were compared between seropositive Responders Nonresponders p value and seronegative patients. Baseline CXCL10 and CXCL13 (n = 16) (n = 13) levels were higher in anti-CCP-positive patients than in Age (years) 51.6 ± 12.7 50.7 ± 8.1 0.80a anti-CCP-negative patients (p =0.02 and p = 0.005 re- Gender (female %) 69 (11/16) 77 (10/13) 0.70b spectively). Only baseline CXCL13 levels were higher in p Duration (years) 6.5 ± 4.9 7.4 ± 7.5 0.66a RF-positive patients than in RF-negative patients ( = 0.02) (Table 2). There were no significant differences in RF or anti-CCP positive (%) 75 (12/16) 54 (7/13) 0.27b a posttreatment CXCL10 and CXCL13 levels between RF- DAS28 ESR 6.2 ± 1.1 6.7 ± 0.6 0.15 positive and RF-negative patients (p =0.57 and p =0.72 a ESR (mm) 37 ± 31 30 ± 22 0.79 respectively), and anti-CCP-positive and anti-CCP-negative Values are presented as mean with standard deviation.