Introduction

Rheumatoid arthritis (RA) is a pathologically heterogeneous disease, with variability between patients in the number of affected joints, antibody titers, serum cytokine levels, and severity of joint damage. Histological and molecular heterogeneity in synovial tissue of patients with RA has also been demonstrated [1,2,3]. Genome-wide expression analysis of synovial tissues from a large RA cohort demonstrated distinct molecular and cellular phenotypes, which reflect the heterogeneity present in the RA synovium [4]. Some of the clinical and treatment response heterogeneity may be explained by myeloid-, lymphoid-, and fibroid-dominant synovial subtypes of RA [5]. Although a wide range of treatment options have shown clinical benefit in patients with RA, it is unknown which patients will respond to specific treatments. Synovial tissue is often unavailable before the initiation of treatment; therefore, the use of serum biomarkers to predict which patients will respond to a specific treatment is an area of interest for rheumatologists.

The biomarker C-X-C motif chemokine ligand 13 (CXCL13) is a B-cell chemoattractant that is expressed by follicular dendritic cells in secondary lymphoid tissue and ectopic germinal centers [6]. Studies have shown a correlation between synovial tissue expression of CXCL13 and CXCL13 serum levels [7] and suggested that CXCL13 has a role in RA pathogenesis related to accumulation of B cells in inflamed synovium [8]. CXCL13 has been shown to be associated with the lymphoid phenotype of patients with RA [4]. Soluble intercellular adhesion molecule-1 (sICAM-1) is an adhesion molecule that is upregulated in a variety of cell types in response to TNFα signaling and has been shown to be associated with the myeloid synovial phenotype in patients with RA [4]. CXCL13 (lymphoid) and sICAM-1 (myeloid) were shown to differentially predict response in patients who received the anti–IL-6 receptor antibody tocilizumab (TCZ) as monotherapy and had prior inadequate response to methotrexate (MTX) [4]. In clinical practice, patients vary in terms of duration of RA and previous therapy. Studies are needed to analyze the association of biomarkers with disease activity and treatment response in varied populations of patients who receive TCZ either as monotherapy or in combination with MTX for the treatment of RA. This study aimed to determine whether there is an association of CXCL13 and sICAM-1 with disease activity and response to TCZ in patients with early RA and those with inadequate response to disease-modifying antirheumatic drugs (DMARD-IR).

Materials and methods

Patient inclusion and exclusion criteria and methods for the FUNCTION and LITHE trials were described previously [9, 10]. Patients in FUNCTION had moderate to severe RA of ≤ 2 years’ duration (early RA) and had not previously received MTX or biologic agents [9]. Patients in LITHE had moderate to severe RA for ≥ 6 months with an inadequate response to MTX (DMARD-IR) [10]. The FUNCTION and LITHE study protocols were approved by an ethics committee or institutional review board at each participating center before the start of the study. All patients provided written informed consent in accordance with the Declaration of Helsinki.

For the present study, patients from FUNCTION and LITHE placebo (PBO) + MTX arms, TCZ 8 mg/kg monotherapy arm (FUNCTION only), and TCZ 8 mg/kg + MTX arms were selected based on baseline and week 24 serum sample availability. Patients from the TCZ 4 mg/kg arms were not selected. Serum CXCL13 and sICAM-1 levels were measured using a commercial ELLA (automated enzyme-linked immunosorbent assay) and performed in accordance with the relevant national and international regulations and guidance by Microcoat Biotechnologie GmbH (Bernried am Starnberger See, Germany). The CXCL13 assay (ProteinSimple, San Jose, CA, USA) was conducted using 25 μL of serum samples, which were diluted two-fold. The lower limit of quantification as calculated for undiluted serum ranged from 1.14 to 1.58 pg/mL depending on the kit lot of the assay and the upper limit of quantification was 12,220 pg/mL for all kit lots of the assay. The precision, as determined from the analysis of quality control samples, was satisfactory throughout the analytical study and ranged from 0.124 to 10.211% for low quality control (LQC) and from 0.384 to 10.153% for high quality control (HQC). The mean concentration of LQC was 20.387 pg/mL with a coefficient of variation (CV) of 7.4%. The mean concentration of HQC was 974.169 pg/mL with a CV of 5.5%.

The sICAM-1 assay (ProteinSimple, San Jose, CA, USA) was conducted using 5 μL of serum samples, which were diluted 100-fold. The lower limit of quantification as calculated for undiluted serum was 410 pg/mL and the upper limit of quantification as calculated for undiluted serum was 1,563,000 pg/mL. The precision, as determined from the analysis of quality control samples, was satisfactory throughout the analytical study and ranged from 0.064 to 17.640% for LQC and from 0.160 to 11.660% for HQC. The mean concentration of LQC was 114.531 pg/mL with a CV of 9.7%. The mean concentration of HQC was 5240.573 pg/mL with a CV of 6.8%.

Statistical analysis

Baseline clinical characteristics were compared between the FUNCTION biomarker population and the LITHE biomarker population using the Welch t test on transformed data; P values <0.05 were considered statistically significant. Correlations between CXCL13 or sICAM-1 levels and DAS28-ESR scores were evaluated according to the Pearson correlation coefficient. Changes in CXCL13 and sICAM-1 from baseline to week 24 were compared between treatment arms using the Welch t test. The effect of treatment and baseline DAS28-ESR, CXCL13, and sICAM-1 on the likelihood of DAS28-ESR remission and 50% improvement per the American College of Rheumatology (ACR50) response at week 24 was estimated via logistic regression. Log transformation was used for the biomarker values to ensure a more normal distribution of the data. Results were not adjusted for multiple testing.

Results

Overall, 458 of 869 patients from FUNCTION (TCZ 8 mg/kg monotherapy, n = 157; TCZ 8 mg/kg + MTX, n = 160; PBO + MTX, n = 141) and 287 of 791 patients from LITHE (TCZ 8 mg/kg + MTX, n = 137; PBO + MTX, n = 150) were included. Mean disease duration in FUNCTION (early RA) was significantly shorter than in LITHE (DMARD-IR; 0.45 vs 8.65 years; P < 0.0001); mean ESR at baseline was significantly higher in patients in FUNCTION than in those in LITHE (Table 1). At baseline, CXCL13 levels correlated moderately with DAS28-ESR in the early-RA population (r = 0.36; P < 0.0001) and weakly in the DMARD-IR population (r = 0.21; P = 0.0003) (Fig. 1). Correlation between sICAM-1 levels and DAS28-ESR was low in both the early-RA (r = 0.14; P = 0.0029) and DMARD-IR populations (r = 0.17; P = 0.0040).

Table 1 Baseline Clinical Characteristics
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Fig. 1
figure 1

Correlation Between Serum CXCL13 and sICAM-1 Levels and DAS28-ESR at Baseline. a Patients in the FUNCTION trial (early RA). b Patients in the LITHE trial (DMARD-IR). CXCL13, C-X-C motif chemokine ligand 13; DAS28-ESR, Disease Activity Score in 28 joints calculated with erythrocyte sedimentation rate; DMARD-IR, inadequate response to disease-modifying antirheumatic drugs; RA, rheumatoid arthritis; sICAM-1, soluble intercellular adhesion molecule-1

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In the early-RA population, baseline CXCL13 correlated weakly with all 4 components of DAS28-ESR (tender joint count, r = 0.2; swollen joint count [SJC], r = 0.24; ESR, r = 0.34; Patient Global Assessment [PGA], r = 0.24; P < 0.000031 for all), whereas sICAM-1 only correlated weakly with SJC (r = 0.15; P < 0.0031) and ESR (r = 0.17; P < 0.00031). In the DMARD-IR population, baseline CXCL13 correlated weakly with ESR (r = 0.20; P < 0.0031) and PGA (r = 0.22; P < 0.00031), and sICAM-1 did not significantly correlate with any of the DAS28-ESR components.

The proportion of patients who achieved ACR50 response and DAS28-ESR remission by study population and treatment arm are shown in Fig. 2. Response to treatment was similar between patients in the present biomarker analysis and the intent-to-treat (ITT) populations in the FUNCTION and LITHE trials (data not shown). CXCL13 decreased significantly at week 24 in all treatment arms in both the early-RA and DMARD-IR populations, with the greatest reductions observed in the TCZ + MTX and TCZ monotherapy arms (Fig. 3a). In the early-RA population, the effect of TCZ monotherapy on CXCL13 was similar to that of TCZ + MTX. sICAM-1 decreased significantly at week 24 in the TCZ monotherapy arm in patients with early RA and the TCZ + MTX arms in both the early-RA and DMARD-IR populations but not in the PBO + MTX arms (Fig. 3b).

Fig. 2
figure 2

Proportion of Patients Who Achieved ACR50 Response and DAS28-ESR Remission at Week 24 by Study and Treatment Arm. ACR50, 50% improvement per the American College of Rheumatology; DAS28-ESR, Disease Activity Score in 28 joints calculated with erythrocyte sedimentation rate; DMARD-IR, inadequate response to disease-modifying antirheumatic drugs; mono, monotherapy; MTX, methotrexate; PBO, placebo; RA, rheumatoid arthritis; TCZ, tocilizumab

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Fig. 3
figure 3

Change in CXCL13 and sICAM-1 From Baseline to Week 24 by Study and Treatment Arm. a CXCL13. b sICAM-1. BL, baseline; CXCL13, C-X-C motif chemokine ligand 13; DAS28-ESR, Disease Activity Score in 28 joints calculated with erythrocyte sedimentation rate; DMARD-IR, inadequate response to disease-modifying antirheumatic drugs; MTX, methotrexate; NS, not significant; PBO, placebo; RA, rheumatoid arthritis; sICAM-1, soluble intercellular adhesion molecule-1; TCZ, tocilizumab. * P < 0.01. ** P < 0.001. *** P < 0.0001

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Change in CXCL13 correlated moderately with change in DAS28-ESR at week 24 in both the early-RA and DMARD-IR populations (r = 0.33; P < 0.0001 for both) (Fig. 4). Change in sICAM-1 correlated moderately with change in DAS28-ESR at week 24 in the DMARD-IR population (r = 0.26; P = 0.0002) but weakly in the early-RA population (r = 0.16; P = 0.0005). Although the treatment arm had a significant effect on the likelihood of DAS28-ESR remission and achievement of ACR50, the effects of baseline levels of CXCL13 and sICAM-1 were not significant (Additional file 1: Table S1). Furthermore, biomarker categories based on high vs low baseline CXCL13 and sICAM-1 levels did not predict response to TCZ or MTX in the early-RA or DMARD-IR populations (data not shown).

Fig. 4
figure 4

Correlation Between Change in Serum Levels of CXCL13 and sICAM-1 and Change in DAS28-ESR at Week 24. CXCL13, C-X-C motif chemokine ligand 13; DAS28-ESR, Disease Activity Score in 28 joints calculated with erythrocyte sedimentation rate; DMARD-IR, inadequate response to disease-modifying antirheumatic drugs; MTX, methotrexate; PBO, placebo; RA, rheumatoid arthritis; sICAM-1, soluble intercellular adhesion molecule-1; TCZ, tocilizumab

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We assessed differences between extreme responders and nonresponders in the FUNCTION and LITHE trials by defining highest DAS28 response as those patients who achieve DAS28 remission status at 24 weeks and defining lowest DAS28 response as those patients who failed to achieve a decrease in DAS28 of at least 0.6. In the FUNCTION trial, patients receiving TCZ who achieved remission had a mean (SD) sICAM1 level of 398.89 (99.62) ng/ml and CXCL13 level of 162.64 (109.69) pg/ml, whereas patients who failed to achieve a DAS28 response had a sICAM1 level of 397.34 (117.33) ng/ml and CXCL13 level of 102.63 (33.75) pg/ml. Similarly in the LITHE trial, patients receiving TCZ who achieved remission had a mean (SD) sICAM1 level of 421.28 (139.49) ng/ml and CXCL13 level of 165.09 (104.84) pg/ml, whereas patients who failed to achieve a DAS28 response had a sICAM1 level of 426.94 (150.03) ng/ml and CXCL13 level of 176.13 (153.22) pg/ml. Therefore, even in patients with extremes of DAS28 response, baseline levels of CXCL13 and sICAM1 did not differ significantly.

A higher proportion of patients in the ITT population of the LITHE trial were receiving glucocorticoids at baseline (PBO + MTX, 70%; TCZ + MTX, 62%) [10] than patients in the ITT population in the FUNCTION trial (PBO + MTX, 38%; TCZ + MTX, 33%; TCZ mono, 40%) [9]. Inclusion criteria related to glucocorticoids were similar between studies [9, 10].

Discussion

In the present study, the association of CXCL13 with disease activity in RA was stronger in early-RA patients than in DMARD-IR patients. Prior exposure to DMARDs may have affected baseline CXCL13 levels, despite patients having active RA. Furthermore, a study of DMARD-naïve patients with early RA (< 3 months) proposed that high levels of CXCL13 possibly indicated recent inflammation [11]. Changes in CXCL13 and, to a lesser extent, sICAM-1 correlated significantly with changes in DAS28-ESR at week 24. Notably, in early-RA patients, the effect of TCZ monotherapy on changes in CXCL13 was similar to that observed with TCZ + MTX. The decrease in CXCL13 with disease activity was consistent with a previous study analyzing CXCL13 in patients treated with rituximab [7].

In the DMARD-IR patients, CXCL13 levels decreased significantly in patients in the PBO + MTX group. We suspect that this decrease in CXCL13 level is due to the anti-inflammatory effect of methotrexate treatment in these patients, despite their previously reported DMARD-IR status. This effect on the levels of an inflammatory biomarker could reflect better MTX treatment adherence due to the closer medical monitoring while enrolled in a clinical trial.

The results of the present study differed from those of a study of patients with RA (MTX-IR) from the ADACTA trial, which demonstrated that patients with high baseline CXCL13 levels were more likely to respond to TCZ monotherapy than patients with high sICAM-1 levels [4]. The effect of MTX on CXCL13 and sICAM-1 may have contributed to the discordant study results. More patients in the present study received TCZ + MTX (n = 297) than TCZ monotherapy (n = 157). However, a study of patients with inadequate response to tumor necrosis factor inhibitors who received sarilumab + DMARDs was also unable to fully replicate the biomarker results from ADACTA [12].

Although this study is among the first to evaluate the serum biomarkers CXCL13 and sICAM-1 in early-RA and DMARD-IR patients who received TCZ, it does have limitations. This study used available samples only, which may have resulted in a biased population. However, DAS28 and ACR50 results were similar between patients in the present biomarker study and the ITT populations in the FUNCTION and LITHE trials. In addition, Clinical Disease Activity Index values were not calculated, although they may have been informative because changes in ESR may explain some of the association between decreased CXCL13 and sICAM-1 and decreased disease activity.

Conclusions

In summary, baseline CXCL13 and sICAM-1 levels did not predict response to TCZ at week 24, suggesting that although CXCL13 and sICAM-1 were modestly associated with RA disease activity, they do not predict response to TCZ in all RA populations.