Validity of ultrasound as a surrogate marker of serum inflammatory cytokines after tocilizumab introduction in rheumatoid arthritis patients

doi:10.21203/rs.3.rs-3785334/v1

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Validity of ultrasound as a surrogate marker of serum inflammatory cytokines after tocilizumab introduction in rheumatoid arthritis patients

https://doi.org/10.21203/rs.3.rs-3785334/v1

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Background

Rheumatoid arthritis (RA) is characterized by persistent synovitis, which is evaluated using ultrasound. Ultrasound-based global assessment of the joints is correlated with serum cytokine levels. This correlation was validated in cross-sectional but not in prospective studies. We aimed to verify this validity in a prospective study of RA patients treated with the interleukin (IL)-6 receptor inhibitor tocilizumab.

Methods

Twenty-four consecutive RA patients started on tocilizumab treatment were prospectively enrolled. Serum levels of 27 cytokines/chemokines were measured using a multiplex immunoassay (Bio-Plex assay). Clinical examination and comprehensive ultrasound assessment of joints were conducted at baseline and 24 weeks.

Results

Clinical and ultrasound global scores showed significant improvements at 24 weeks (Disease Activity Score 28-joint count-erythrocyte sedimentation rate: p = 0.001, ultrasound power Doppler global score with 40 joints [PD40]: p = 0.028); however, there was no significant improvement in the inflammatory cytokine levels (IL-6: p = 0.93, vascular endothelial growth factor [VEGF]: p = 0.79, tumor necrosis factor [TNF]: p = 0.85). Before tocilizumab introduction, PD40 showed a significant correlation with multiple serum cytokines, including IL-6 (r = 0.584, p = 0.045), VEGF (r = 0.582, p = 0.047), TNF-α (r = 0.695, p = 0.012), and interferon gamma-induced protein-10 [IP-10] (r = 0.827, p = 0.001); this correlation was lost after 24 weeks. IL-6-associated cytokines (IL-8, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1a) and growth factors (VEGF, platelet-derived growth factor-bb, fibroblast growth factor-basic) altered > 50% of the correlated partner-cytokines after tocilizumab introduction.

Conclusions

The ultrasound global score is not a reliable surrogate marker of multiple inflammatory cytokine statuses during tocilizumab treatment. Thus, it should not be a rationale to determine tocilizumab discontinuation in RA patients.

rheumatoid arthritis

ultrasound

tocilizumab

A typical pathological feature of rheumatoid arthritis (RA) is persistent synovitis that results from the sustained recruitment of inflammatory cells into the joints [1]. At the site, T cells, together with B cells and other innate cells, form a complex network that regulates the production of pro-inflammatory cytokines [2, 3]. Tumor necrosis factor (TNF), interleukin (IL)-6, vascular endothelial growth factor (VEGF), and other cytokines play a central role in synovitis. These critical biomarkers contribute not only to RA disease severity and treatment response but also to complications and prognosis [4–6]. Targeting inflammatory cytokines with neutralizing antibodies is a strategy currently widely employed in the treatment of RA that can adequately suppress synovitis [7–9].

Ultrasound (US) has been increasingly used for the evaluation of synovitis in patients with inflammatory joint diseases. Synovial hypertrophy and effusion are detected by grayscale (GS) techniques and vascularization is detected by power Doppler (PD) techniques [10, 11]. US is a validated and reliable technology, and a comprehensive US assessment of joints is correlated with serum inflammatory cytokine levels [12–17]. Local assessment is associated with synovial pathophysiology and cytokines in the synovial fluid [18–20]. Moreover, the US global assessment using the sum of the US scores for multiple joints is as sensitive to change as the clinical composite measures used during treatment with biological medication targeting TNF and T cells [11, 21, 22].

Previous validation studies of the comprehensive US assessment of joints that showed a correlation with the serum cytokine levels were cross-sectional studies [12–17]. To date, no prospective studies have been conducted to assess the correlation between a global joint score and the serum cytokine levels. We believe that a prospective re-evaluation of the comprehensive US assessment of joints, focusing on the serum inflammatory cytokines as the gold standard, is essential for determining the utility of the US global joint scores in monitoring RA disease activity in the current controversial environment [23–26]. In addition, data are lacking regarding anti-IL-6 receptor antagonist treatment, which normalizes the C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR).

The present single-center prospective study aimed to examine whether a comprehensive US assessment of joints is correlated with serum inflammatory cytokines over time in RA patients treated with the IL-6 receptor inhibitor tocilizumab.

Patients and healthy donors

Twenty-four consecutive RA patients who were receiving tocilizumab were prospectively recruited from Juntendo University Hospital from January 2014 to December 2014. All patients fulfilled the American College of Rheumatology (ACR) 1987 revised criteria for the classification of RA [27] or the 2010 ACR/European League Against Rheumatism (EULAR) RA classification criteria [28]. The patients underwent an assessment of the number of swollen and tender joints they had and a global assessment on the Visual Analog Scale, and their blood samples were retrieved at baseline and at 24 weeks by blinded rheumatologists (MO) certified by the Japan College of Rheumatology. Clinical disease activity was estimated by the Disease Activity Score 28-joint count-ESR (DAS28-ESR) and the Clinical Disease Activity Index (CDAI). Nineteen age- and sex-matched healthy donors (HD) (the control group) were included in the study.

Ethics

This study was conducted in accordance with the tenets of the 1983 Declaration of Helsinki and received ethical approval from the institutional ethics committee of Juntendo University (no. 12–060). All study patients provided written informed consent to participate in the study.

US examination

A single rheumatologist (KM) experienced in musculoskeletal US, who was blinded to clinical and laboratory information, performed the US examination and the clinical assessment both at baseline and at week 24 using Noblus (Hitachi Medical Corporation, Tokyo, Japan) with an L64 linear probe (5–18 MHz). Systematic multiplanar GS and PD examination of 40 joints (GS40 and PD40) (shoulder, elbow, interphalangeal [IP], proximal interphalangeal, metacarpophalangeal [MCP], wrist, knee, ankle, and metatarsophalangeal; bilaterally) were performed in a standardized manner according to the EULAR guidelines [29]. Synovitis scores in GS and PD modes and combined scores were noted according to semi-quantitative scoring between 0 and 3 [30]. The US global score, GS40, PD40, and combined scores were the total scores for the 40 joints described above. The reduced joints’ global US7 [31], US12 [32], US18 [33], and US20 [34] scores (total scores of 7, 12, 18, and 20 joints, respectively) were also determined according to the referenced methods. PD variables were adjusted at the lowest permissible pulse repetition frequency to maximize sensitivity. Doppler gain was set just below the level at which Doppler noise appeared underlying the bone. The intraclass correlation coefficients of the semi-quantitative GS and PD scores were in good agreement (GS score, 0.87 and PD score, 0.92) [35]. All patients were scheduled to undergo US, clinical examination, and blood sample extraction on the same day just before tocilizumab administration.

Measurement of serum cytokines and chemokines

Serum cytokine and chemokine levels were measured using the Bio-Plex ProTM Human Cytokine 27-plex Assay (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions. Twenty-seven analytes were examined: IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, fibroblast growth factor (FGF)-basic, granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, IP-10, MCP-1, macrophage inflammatory protein (MIP)-1α, platelet-derived growth factor (PDGF)-bb, MIP-1β, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), VEGF, and TNF-α.

Correlation analysis

We conducted correlation analysis using the R corrplot package [36] for clinical measures, US global scores (7, 12, 18, 20, and 40 joints with GS, PD, and combined US), and cytokine levels in all combinations.

Cytokine network analysis

The cytokine network visualization of the Spearman’s rank correlation matrix between the cytokine levels was obtained using R’s qgraph package version 1.3 [37]. qgraph is a visualization tool and a tool for applying network analysis from data for estimating network structures, where the network can be weighted (correlations) and undirected. Here, each node represents a cytokine and each edge the correlation between the connecting nodes. For example, qgraph can draw a correlation network, not just as circular network but where strongly correlated nodes are drawn near each other.

Analysis for changes in correlated partner-cytokine levels

A pair of cytokines/chemokines showing significant correlation was called a partner-cytokine in this study. The cytokines that correlated at baseline but not at 24 weeks were defined as “lost” partner-cytokines, whereas those that did not correlate at baseline but significantly correlate at 24 weeks were defined as “gained” partner-cytokines. The rate of altered partner-cytokines (lost and gained partner-cytokines / total partner-cytokines) was determined.

Statistical analysis

Statistical analysis was performed using R software, version 3.5.3 [38]. Continuous data are presented as means with standard deviations or as medians with interquartile ranges. Data were compared using Fisher’s exact test or the Wilcoxon signed-rank test, as appropriate. The correlation was tested using Spearman’s method. P-values of < 0.05 were considered statistically significant.

Clinical examination and US determined efficacy of tocilizumab at 24 weeks

Studied patients’ backgrounds are described in Table 1. The DAS28-ESR (p = 0.001), CDAI (p = 0.037), CRP (p < 0.001), and ESR (p < 0.001) significantly improved at 24 weeks. PD40 (p = 0.028), combined US score (p = 0.025), US7 (p = 0.028), and US20 (p = 0.037) significantly decreased at 24 weeks. Although some of the determined cytokines showed changes, there were no cytokines that showed significant changes at 24 weeks (Fig. 1A).

Table 1

Patient background
Female, n (%)	21 (87.5)
Age, average (SD)	52.3 (11.6)
Bodyweight, average (SD)	55.3 (9.1)
Disease duration (years), median (IQR)	9.6 (7.2)
Total number of patients treated with biologics, n (%)	18 (75)
No. of patients treated with IFX, ETN, ADA, GLM, CZP, and ABT	5, 2, 4, 4, 1, 2
MTX used patients (%)	19 (79.2)
MTX dose, average (SD)	10.5 (3.4)
Concomitant use of corticosteroids, n (%)	12 (50)
Corticosteroid dose, average (SD)	6.4 (3.9)
DAS28-ESR, average (SD)	4.2 (1.5)
CDAI, average (SD)	15.5 (11.1)
MMP-3, average (SD)	131.7 (106.1)
Abbreviations: ABT: abatacept; ADA: adalimumab; CDAI: Clinical Disease Activity Index; CZP: certolizumab pegol; DAS28-ESR: Disease Activity Score 28-joint count-ESR; ETN: etanercept; GLM: golimumab; IFX: infliximab; IQR: interquartile range; MTX: methotrexate; MMP: matrix metalloproteinase; SD: standard deviation.
Patient background including sex, age, body weight, disease duration, treatment with biologics, type of biologics used in treatment, MTX use, MTX dose, concomitant use of corticosteroids, corticosteroid dose, DAS28-ESR score, CDAI score, and MMP-3 score are included in this table.

[Table 1 near here]

[Figure 1 near here]

Comparison of cytokine levels between RA patients and HD

At baseline, the cytokine levels in RA patients were significantly different from those in HD, except for MCP-1 and FGF-basic levels (Fig. 1B).

Correlation analysis with serum cytokine/chemokine levels

Among the 27 cytokines/chemokines evaluated, 24 (except for IL-2, IL-10, and IL-15) were within the detection limit in the serum of RA patients. At baseline (Fig. 2A), DAS28-ESR (r = 0.614, p = 0.034), CDAI (r = 0.707, p = 0.010), GS40 (r = 0.612, p = 0.034), and PD40 (r = 0.584, p = 0.045) were significantly correlated with IL-6. Moreover, CRP (r = 0.577, p = 0.05) and PD40 (r = 0.582, p = 0.047) were significantly correlated with VEGF. CRP (r = 0.736, p = 0.006), GS40 (r = 0.586, p = 0.045), PD40 (r = 0.827, p = 0.001), combined US score (r = 0.577, p = 0.05), and US7 (r = 0.662, p = 0.019) were significantly correlated with IP-10. However, at 24 weeks (Fig. 2B), there were no clinical or US scores showing significant correlations with cytokine levels. Only matrix metalloproteinase-3 (MMP-3) showed a significant inverse correlation with the chemokine RANTES (r = − 0.593, p = 0.042), and US20 showed a significant correlation with MIP-1a (r = 0.637, p = 0.026). The correlation matrices were considerably different before and after tocilizumab introduction.

[Figure 2 near here]

Cytokine network visualization of Spearman’s rank correlation matrix between the cytokine levels

Before tocilizumab introduction, growth factors, such as VEGF with IL-7 and IP-10; PDGF-bb with IL-6, IL-1b, IL-8, and MIP1-a; and FGF-basic with IL-17, showed several individual small-network blocks with strong correlations (expressed as thick lines in Fig. 3). After tocilizumab introduction in RA patients, VEGF, PDGF-bb, and FGF-basic lost strong correlations with other cytokines, and the entire network appeared as a one-network block consistent with the cytokine network in HD. TNF and GM-CSF were located outside of the network block in HD; however, they were located inside of the block in RA patients.

[Figure 3 near here]

Changes in correlated partner-cytokines: gained, lost, or conserved

Growth factors, such as VEGF, PDGF-bb, and FGF-basic, and cytokines, such as IL-13, IP-10, IL-8, MIP-1a, RANTES, IL-9, and IL-6, showed major changes (> 50%) in correlated partner-cytokines (Fig. 4, Table 2).

Table 2

List of cytokines and partner-cytokines
	Conserved correlates both at 0 week and 24 week	Gained correlates at 24 week	Lost correlates at 24 week
TNF-a	IL-1b, IL-1ra, IL-12, IFN-g, IL-4, IL-5, IL-6, MCP-1, Eotaxin, G-CSF, GM-CSF	IL-9, FGF-basic, IL-7	IP-10, IL-13, VEGF
IL-1b	TNF-a, IL-1ra, IFN-g, IL-4, IL-5, IL-6, MCP-1, Eotaxin, G-CSF, GM-CSF	IL-12, IL-9, FGF-basic, IL-7	IL-13, PDGF-bb
IL-1ra	TNF-a, IL-1b, IL-12, IFN-g, IL-4, IL-5, IL-6, MCP-1, Eotaxin, G-CSF, GM-CSF	IL-9, FGF-basic, IL-7	IP-10, IL-13, VEGF, PDGF-bb
IL-4	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-5, MCP-1, Eotaxin, G-CSF, GM-CSF	IL-9, IL-6, FGF-basic	IL-13, PDGF-bb
IL-5	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-4, MCP-1, Eotaxin, G-CSF, GM-CSF, PDGF-bb	IL-6	IL-13
IL-6	TNFa, IL-1b, IL-1ra, MCP-1, Eotaxin	IL-12, IFN-g, IL-4, IL-5, IL-9, FGF-basic, IL-7, G-CSF, GM-CSF	IL-8, MIP-1a, PDGF-bb
IL-9	IL-12	TNFa, IL-1b, IL-1ra, IFN-g, IL-4, IL-6, VEGF, FGF-basic, MCP-1, Eotaxin, IL-7, G-CSF, GM-CSF	None
IL-12	TNFa, IL-1ra, IL-12, IFN-g, IL-4, IL-5, IL-9, MCP-1, Eotaxin, GM-CSF	IL-1b, IL-6, VEGF, FGF-basic, IL-7, G-CSF	RANTES
IL-13	None	None	TNFa, IL-1b, IL-1ra, IFN-g, IL-4, IL-5, FGF-basic, MCP-1, Eotaxin, G-CSF, GM-CSF, PDGF-bb
IL-17	FGF-basic, MIP-1b, PDGF-bb	VEGF, GM-CSF	None
IFN-g	TNFa, IL-1b, IL-1ra, IL-12, IL-4, IL-5, MCP-1, Eotaxin, G-CSF, GM-CSF	IL-9, IL-6, FGF-basic, IL-7	IL-13, PDGF-bb
VEGF	FGF-basic	IL-12, IL-9, IL-17, GM-CSF, PDGF-bb	TNFa, IL-1ra, IP-10, MCP-1, Eotaxin
FGF basic	IL-17, VEGF, GM-CSF, PDGF-bb	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-4, IL-9, IL-6, MCP-1, Eotaxin, IL-7, G-CSF,	IL-13
PDGF-bb	IL-5, IL-17, FGF-basic,	VEGF, G-CSF, GM-CSF,	IL-1b, IL-1ra, IFN-g, IL-4, IL-13, IL-6, MCP-1,
G-CSF	TNFa, IL-1b, IL-1ra, IFN-g, IL-4, IL-5, MCP-1, Eotaxin, GM-CSF,	IL-12, IL-9, IL-6, FGF-basic,	IL-13, PDGF-bb
GM-CSF	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-4, IL-5, FGF-basic, MCP-1, Eotaxin, G-CSF,	IL-9, IL-6, IL-17, VEGF, IL-7,	IL-13, PDGF-bb
IL-7	VEGF, Eotaxin	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-9, IL-6, FGF-basic, MCP-1, GM-CSF	IP-10
IL-8	None	RANTES	IL-6, MIP-1a
IP-10	None	None	TNFa, IL-1ra, VEGF, MCP-1, Eotaxin, IL-7
MCP-1	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-4, IL-5, IL-6, Eotaxin, G-CSF, GM-CSF	IL-9, FGF-basic, IL-7	IP-10, IL-13, VEGF, PDGF-bb
MIP-1a	None	MIP-1b	IL-6, IL-8
MIP-1b	IL-17	MIP-1a	None
RANTES	None	IL-8	IL-12
Eotaxin	TNFa, IL-1b, IL-1ra, IL-12, IFN-g, IL-4, IL-5, IL-6, MCP-1, IL-7, G-CSF, GM-CSF	IL-9, FGF-basic,	IP-10, IL-13, VEGF,
Abbreviations: CSF: colony-stimulating factor; FGF: fibroblast growth factor; GM-CSF: granulocyte–macrophage colony-stimulating factor; IFN: interferon; IL: interleukin; IP: interferon gamma-induced protein; MCP: monocyte chemoattractant protein; G-CSF: granulocyte colony-stimulating factor; MIP: macrophage inflammatory protein; PDGF: platelet-derived growth factor; RANTES: regulated upon activation, normal T cell expressed, and presumably secreted; TNF: tumor necrosis factor; VEGF: vascular endothelial growth factor.
Measured cytokines and their significantly correlated cytokines at 0 and 24 weeks, as “partner-cytokines,” are listed in this table.
Cytokines that were correlated at baseline but not at 24 weeks are shown as lost partner-cytokines.
Cytokines that were not correlated at baseline but were significantly correlated at 24 weeks are shown as gained partner-cytokines.

[Table 2 near here]

[Figure 4 near here]

This prospective study investigated the validity of the US global score to estimate the serum inflammatory cytokine levels after tocilizumab introduction. The results showed that the correlation between the US global score and serum cytokine levels was lost after tocilizumab introduction.

Previous cross-sectional studies have shown the usefulness of the correlation between the US global score and serum cytokine levels. Contrary to our expectations, we found that the US global score did not correlate with serum cytokine levels when patients were treated with tocilizumab. Clinical disease activity and the US global score showed a significant decrease after tocilizumab treatment; however, the serum cytokine levels did not decrease significantly. This discrepancy could explain the reason for the loss of a significant correlation between the US global score and the serum cytokine levels. In the usual clinical settings, we should consider the interpretation of the US global scores in RA patients undergoing tocilizumab treatment because they may have underestimated serum inflammatory cytokine levels. This suggested that the US global score may not be an appropriate marker to determine whether tocilizumab should be discontinued after the achievement of clinical remission.

This study showed that the levels of multiple serum cytokines did not significantly decrease after tocilizumab introduction. This may be biologically plausible because tocilizumab is an IL-6 receptor antibody that works like a “cap” on the receptor, resulting in an IL-6 surge [39]. Tocilizumab suppresses only some of the downstream cytokines of the IL-6 receptor, such as IL-8, IP-10, MCP-1, and MIP-1a, for a certain duration in the initial treatment period. The reason for the significant improvement in clinical disease activity and the US global scores, despite the lack of significant changes in cytokine levels, could be explained by changes in the cytokine network configuration after tocilizumab introduction. For instance, growth factors, such as VEGF, PDGF-bb, and FGF-basic, which contribute to synovial hypertrophy or proliferation and angiogenesis [2–4, 26], showed a loss of significant correlation with their partner-cytokines after tocilizumab introduction. This suggested that the decreased synergistic effects [8, 26, 40, 41] of the growth factor without the support of partner-cytokines could be the more prominent and preceding factor in the reduction of clinical and US findings than the decrease in the growth factor level itself. Thus, the cytokine level and its network configuration should be a focus when assessing biological and clinical improvements.

Several limitations of this study should be acknowledged. First, the sample size was small, which might have caused the insignificant decrease observed in cytokine levels. Second, the observed duration was only 24 weeks. It would be better to follow-up for a longer duration—such as 1 or 2 years—to validate the US global score more accurately. To strengthen the concept of this study, multicenter studies with a larger sample size and longer duration are needed.

In summary, to the best of our knowledge, this study is the first to demonstrate that the US global score can underestimate serum inflammatory cytokine levels following the introduction of an IL-6 receptor inhibitor. The use of the US global score as collateral evidence of inflammatory cytokines could be inappropriate for RA patients treated with an IL-6 receptor antagonist. Thus, we should be cautious when interpreting and incorporating the US global score, particularly when judging the possibility of discontinuing IL-6 receptor antagonists in RA patients.

ACR: American College of Rheumatology

CDAI: Clinical Disease Activity Index

CRP: C-reactive protein

ESR: erythrocyte sedimentation rate

EULAR: European League Against Rheumatism

FGF: fibroblast growth factor

GM-CSF; granulocyte–macrophage colony-stimulating factor

GS: grayscale

HD: healthy donors

IL: interleukin

IP: interphalangeal

MCP: metacarpophalangeal

MIP: macrophage inflammatory protein

PD: power Doppler

PDGF: platelet-derived growth factor

RA: rheumatoid arthritis

RANTES: regulated upon activation, normal T cell expressed and presumably secreted

TNF: tumor necrosis factor

US: ultrasound

VEGF: vasucular endothelial growth factor

Ethical approval and consent to participate: This study was conducted in accordance with the tenets of the 1983 Declaration of Helsinki and received ethical approval from the institutional ethics committee of Juntendo University (no. 12-060). All study patients provided written informed consent to participate in the study.

Consent for publication: Not applicable.

Availability of data and materials: All data generated or analysed during this study are included in this published article.

Competing interests: The authors declare that they have no competing interests.

Funding: No funding was obtained for this study.

Authors' contributions: KM. collected the data, sample, performed US and assisted in preparing the manuscript. MO designed the study concept, analyzed the data and prepared the manuscript. All authors other than above critically reviewed the manuscript. MG measured cytokines. M-MY, KT, YK and NT supervised the study. All authors read and approved the final manuscript.

Acknowledgements: We would like to thank Editage (www.editage.com) for English language editing and publication support.

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No competing interests reported.

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Validity of ultrasound as a surrogate marker of serum inflammatory cytokines after tocilizumab introduction in rheumatoid arthritis patients

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Patients and healthy donors

Ethics

US examination

Measurement of serum cytokines and chemokines

Correlation analysis

Cytokine network analysis

Analysis for changes in correlated partner-cytokine levels

Statistical analysis

Results

Clinical examination and US determined efficacy of tocilizumab at 24 weeks

Comparison of cytokine levels between RA patients and HD

Correlation analysis with serum cytokine/chemokine levels

Cytokine network visualization of Spearman’s rank correlation matrix between the cytokine levels

Changes in correlated partner-cytokines: gained, lost, or conserved

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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