Objectives To investigate the relation between ankylosing spondylitis disease activity score (ASDAS), Bath ankylosing spondylitis disease activity index (BASDAI) and treatment response and biomarkers of inflammation (C-reactive protein (CRP), interleukin-6 (IL-6), YKL-40), angiogenesis (vascular endothelial growth factor (VEGF)), cartilage (C-terminal crosslinking telopeptide of type II collagen (CTX-II), matrix metalloproteinase-3 (MMP-3), total aggrecan, cartilage oligomeric matrix protein) and bone (C-terminal crosslinking telopeptide of type I collagen, osteocalcin) turnover in 60 patients with axial spondyloarthritis initiating tumour necrosis factor alpha (TNFα) inhibitor therapy.
Methods ASDAS (CRP-based), BASDAI and biomarkers were determined before and seven times during 46 weeks of TNFα inhibitor therapy.
Results Very high ASDAS were associated with high levels of inflammatory biomarkers, while high BASDAI were not related to any biomarkers. Mixed modeling demonstrated significant longitudinal associations between ASDAS and IL-6, VEGF, MMP-3 and osteocalcin and between BASDAI and CRP, IL-6 and VEGF. Major improvement in ASDAS was associated with larger percentage decreases in biomarkers of inflammation, angiogenesis, MMP-3 and increases in aggrecan and osteocalcin. BASDAI response was associated with larger decreases in CRP and IL-6. Biomarkers with moderate/high differences in responsiveness for major versus no/clinically important improvement in ASDAS were CRP, IL-6, VEGF, aggrecan and osteocalcin, and VEGF and CTX-II for BASDAI response versus non-response.
Conclusion Levels and changes of 10 biomarkers in patients with axial spondyloarthritis during anti-TNFα therapy were documented. Construct validity and responsiveness of IL-6, VEGF, MMP-3, total aggrecan and osteocalcin were demonstrated. ASDAS was more associated with these biomarkers than BASDAI, and may better reflect the inflammatory disease processes.
ClinicalTrials.gov identifier NCT00133315.
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Axial spondyloarthritis is characterised by inflammation in the sacroiliac joints and spine. In more advanced disease, erosions, syndesmophytes and ankylosis develop. The pathogenesis of spondyloarthritis is not known in detail. The various biological disease processes in spondyloarthritis may be reflected in levels of and changes in different circulating biochemical markers of inflammation,1,–,4 angiogenesis4,–,6 and cartilage4 5 7,–,10 and bone turnover.11,–,13 The relationship between such biomarkers and the new composite measure of disease activity for patients with ankylosing spondylitis (AS), the ankylosing spondylitis disease activity score (ASDAS),14 15 have not been investigated. Correspondingly, it has not been explored if the ASDAS better reflects the biological disease processes than the Bath ankylosing spondylitis disease activity index (BASDAI).16
In this study we investigated circulating biomarkers of inflammation (C-reactive protein (CRP), interleukin 6 (IL-6), YKL-40), angiogenesis (vascular endothelial growth factor (VEGF)), cartilage (C-terminal crosslinking telopeptide of type II collagen (CTX-II), total aggrecan, matrix metalloproteinase 3 (MMP-3), cartilage oligomeric matrix protein (COMP)) and bone turnover (C-terminal crosslinking telopeptide of type I collagen (CTX-I), total osteocalcin) in patients with axial spondyloarthritis initiating tumour necrosis factor alpha (TNFα) inhibitor therapy, in order to explore the relation between these biomarkers, ASDAS, BASDAI and ASDAS and BASDAI response.
Patients and methods
Patients and study design
The prospective Biomarkers in Spondyloarthritis (BIOSPA) study17 comprised 60 patients with: (1) spondyloarthritis according to the European Spondyloarthropathy Study Group criteria;18 (2) sacroiliitis on x-rays19 or on MRI;20 (3) BASDAI greater than 30 mm; and (4) clinical indication for anti-TNFα therapy. Patient characteristics are given in table 1.
Clinical and biochemical assessments (table 1) were performed at baseline and seven times during the study. At week 22, 35 (58%) patients fullfilled the primary response criterion (BASDAI response, ie, a decrease in BASDAI ≤50% or 20 mm)21 and 18 (30%) were BASDAI non-responders. ASDAS including BASDAI questions 2, 3 and 6, patient's global assessment and serum CRP were calculated.14,15 The cut-offs for inactive ASDAS disease activity (<1.3) (n=0; 0%), moderate (≥1.3; <2.1) (n=2; 3%), high (≥2.1; ≤3.5) (n=19; 32%) and very high (>3.5) (n=35; 58%) disease activity and no improvement (ΔASDAS <1.1) (n=14; 23%), clinically important improvement (≥1.1; <2.00) (n=10; 17%) and major improvement (≥2.0) (n=23; 38%) in ASDAS were defined according to ASAS (Assessment of SpondyloArthritis international Society).22 We defined moderate (>30; <50 mm) (n=24; 40%) and high (≥50 mm) (n=36; 60%) BASDAI disease activity. Seven (12%) and six (10%) patients were excluded before weeks 22 and 46, respectively, due to adverse events (n=9), lack of efficacy (n=2) or non-compliance (n=2). ASDAS could not be calculated in four (7%) patients at baseline and in five (8%) patients at week 22 (in a total of five (10%) patients) because of missing values. Patients excluded did not differ from patients included in the analyses.
The study was approved by the ethical committee (H-KF-02-050/04) and conducted in accordance with the Helsinki II Declaration.
Blood samples were centrifuged at 3000g for 10 min at room temperature 30–120 min after sampling. Serum and plasma samples were stored at 80°C and second morning spot urine at 20°C until analysis. All samples from a patient were analysed in duplicate in the same assay. No patients had increased liver enzymes above twice the upper limit of normal or increased serum creatinine. If a patient had an infection (n=1) at the study visit or had been treated with glucocorticoids less than 6 weeks before (n=2) the biomarker results from this visit they were excluded from the biomarker analyses.
CRP was analysed with turbidimetry (DakoCytomation, Glostrup, Denmark). A highly sensitive CRP method was used for levels from 0 to 90 mg/l. The following commercial ELISA assays were used: highly sensitive IL-6, VEGF and MMP-3 (Quantikine, R&D Systems, Abingdon, UK), YKL-40 (Quidel, CA, USA), COMP (Wieslab, Malmö, Sweden), total aggrecan (IDS Nordic, Herlev, Denmark) and CTX-I, CTX-II and total osteocalcin (Nordic Biosciences, Herlev, Denmark). Urinary CTX-II was corrected for urinary creatinine concentration. The biomarker analyses were performed according to the manufacturer's instructions. Biomarker levels in healthy subjects are shown in table 1.
Biomarker levels were compared with the reference levels of healthy subjects. Patients were stratified according to baseline ASDAS (≤3.5 vs >3.5), BASDAI (>30 and <50 vs ≥50), swollen joint count (0 vs ≥1) and CRP (≤8 mg/l vs >8 mg/l), and according to improvement in ASDAS (ΔASDAS <2.0 vs ≥2.0; and <1.1 vs ≥2.0) and BASDAI response at weeks 22 and 46, and compared regarding pretreatment biomarker levels and changes. The cut-off of BASDAI was chosen to obtain the same frequency of patients with high versus low disease activity as in the ASDAS analyses. From a clinical perspective, the definitions of moderate and high BASDAI disease activity seemed reasonable. Pooling patients with no and clinically important improvement is, from a clinical point of view, problematical. We therefore also performed the analyses for patients with no versus major improvement in ASDAS, although this reduced the number of patients in the lower response group. Comparisons were performed using the χ2 test, Fisher's exact test, the Mann–Whitney test and Wilcoxon sign rank test. Correlations were examined with Spearman rank correlation coefficients. The longitudinal associations between ASDAS, BASDAI, CRP and biomarker levels were assessed by the use of multivariate linear mixed modelling including data from baseline, weeks 2, 6 14 and 22. ASDAS, BASDAI and CRP were the dependent variables, whereas time, biomarker levels, gender, age, diagnosis (axial spondyloarthritis associated with vs without psoriasis) and anti-TNFα therapy were explanatory fixed effects and each patient a random effect. The analyses were performed for each biomarker separately, except for CRP in the analyses of ASDAS, which includes CRP. Before the analyses ASDAS, BASDAI and the biomarker levels at all time points were logarithmically transformed due to skewed distribution of data. Graphically, the assumption of linearity, variance homogeneity and normal distribution was fullfilled for all variables except for time, which was analysed as a non-ordinal parameter. No interactions were found between the dependent and explanatory variables or between the biomarker levels and gender, age, diagnosis and anti-TNF therapy. The standardised response mean (SRM) was calculated as the difference between the mean baseline scores and follow-up scores at week 22 divided by the SD of the change scores.23 Values less than 0.20, 0.20 or more to less than 0.50, 0.50 or more to less than 0.80 and 0.80 or more were considered to represent no, small, moderate and high correlation and responsiveness,23 respectively. Analyses were made using SPSS version 12.0 and SAS version 9.2. p Values less than 0.01 in the Spearman correlation analyses and less than 0.05 in all other analyses were considered statistically significant.
Compared with healthy subjects, the 60 spondyloarthritis patients had significantly higher baseline levels of CRP, IL-6, VEGF, YKL-40, MMP-3 and CTX-II, lower levels of aggrecan and levels within the normal range of COMP, CTX-I and osteocalcin (table 1).
ASDAS correlated weakly/moderately with BASDAI, CRP, VEGF and IL-6. Only weak and non-significant correlations were seen between BASDAI and the biomarkers (table 2). Swollen joint count correlated with COMP and MMP-3; CRP with IL-6, VEGF and aggrecan (inverse).
Very high ASDAS were more frequent in patients with high BASDAI (35 (86%) vs 2 (10%), p<0.0001) and elevated CRP (32 (91%) vs 11 (52%), p=0.002) than in patients with moderate BASDAI or normal CRP. The frequency of peripheral arthritis in patients with very high versus moderate/high ASDAS (9 (26%) vs 2 (11%)) and in patients with high versus moderate BASDAI (10 (28%) vs 1 (4%)) did not differ.
At baseline, patients with very high ASDAS had higher levels of CRP, IL-6 and VEGF compared with patients with moderate/high ASDAS, while no differences were seen between patients with moderate versus high BASDAI (table 3).
Patients with peripheral arthritis had higher MMP-3 and COMP than patients without peripheral arthritis, and patients with elevated CRP had higher IL-6, VEGF and lower aggrecan compared with patients with normal CRP (see supplementary table 1, available online only).
Patients with major improvement in ASDAS at week 22 had higher baseline CRP, IL-6, CTX-II and MMP-3 and lower YKL-40 compared with patients without major improvement (table 4), and they had higher VEGF but not CTX-II compared with patients with no improvement in ASDAS. BASDAI responders had higher CRP and VEGF, and lower YKL-40 than BASDAI non-responders. Similar results were found when patients were stratified according to treatment response at week 46.
After the initiation of anti-TNFα therapy ASDAS, BASDAI, biomarkers of inflammation, angiogenesis, CTX-II and MMP-3 decreased in patients, whereas aggrecan, COMP and osteocalcin increased, while CTX-I stayed unchanged (see supplementary table 2, available online only).
From baseline to week 22 the percentage change in ASDAS correlated weakly/moderately with percentage changes in BASDAI, CRP, IL-6, MMP-3 and osteocalcin (inverse) (table 2). BASDAI correlated with CRP. The percentage change in CRP correlated with percentage changes in IL-6, VEGF and osteocalcin (inverse).
The longitudinal analyses demonstrated an association between ASDAS and IL-6, VEGF, MMP-3 and osteocalcin (inverse) during the first 22 weeks of the study (table 5). BASDAI was associated with CRP, IL-6 and VEGF. CRP was associated with IL-6, VEGF, YKL-40 and MMP-3 and inversely with aggrecan, COMP and osteocalcin. ASDAS was associated with age in all models, while BASDAI only was associated with age in the models comprising YKL-40, CTX-II, aggrecan, MMP-3 and COMP (data not shown). In the models including MMP-3, gender was also independently associated with ASDAS and CRP.
Patients with very high ASDAS, high BASDAI, major improvement in ASDAS and BASDAI responders differed from patients with moderate/high ASDAS, moderate BASDAI, no/clinically important improvement and BASDAI non-responders, respectively, in their significant decreases in VEGF and MMP-3 from week 2 and onwards and increase in osteocalcin in the first part of the study. No such changes were seen in the latter groups (see supplementary table 2, available online only). CTX-II decreased in the second part of the study in BASDAI.
Patients with very high baseline ASDAS had larger percentage decreases in CRP, IL-6, VEGF and MMP3 and higher increases in aggrecan at week 22 (table 4), and they had larger percentage decreases in CRP and YKL-40 at week 46 compared with patients with moderate/high ASDAS. Patients with high BASDAI had larger percentage decreases in MMP-3 at week 22 and in YKL-40 at week 46 compared with patients with moderate BASDAI.
Patients with major improvement in ASDAS and BASDAI responders had larger percentage decreases in CRP and IL-6 at week 22 (table 4). Patients with major improvement in ASDAS had larger decreases in MMP-3, and increases in aggrecan and osteocalcin compared with patients with no/clinically important improvement. Similar results were obtained when patients with major improvement in ASDAS were compared with patients with no improvement, except that larger decreases were also seen for VEGF in the latter group. At week 46 patients with major improvement in ASDAS had larger decreases in CRP, IL-6 and MMP-3 compared with patients with no/clinically important improvement and BASDAI non-responders in YKL-40 compared with BASDAI non-responders.
Moderate/high responsiveness (SRM ≥0.5) was seen in CRP, IL-6, VEGF, CTX-II and aggrecan in patients with major improvement in ASDAS and BASDAI responders (table 6) from baseline to week 22, and also in MMP-3 and osteocalcin in patients with major improvement in ASDAS and YKL-40 in BASDAI responders. All 10 biomarkers demonstrated low responsiveness in patients with a lower treatment response, except for IL-6 in BASDAI non-responders.
Biomarkers with moderate/high ability to demonstrate difference in responsiveness (ΔSRM ≥0.5) between patients with major versus no/clinically important improvement in ASDAS were CRP (0.60), IL-6 (0.80), VEGF (0.66), aggrecan (0.54) and osteocalcin (1.31) (table 6). Similar results were observed in difference in responsiveness when patients with major versus no improvement in ASDAS were compared. VEGF (0.53) and CTX-II (0.59) showed the highest difference in responsiveness between BASDAI responders versus non-responders.
In this study, baseline levels and changes in 10 biomarkers of inflammation, angiogenesis, cartilage and bone turnover were investigated in patients with axial spondyloarthritis during anti-TNFα therapy, and their relation with ASDAS and BASDAI disease activity and treatment response were studied.
In the univariate analyses baseline levels of the inflammatory biomarkers (CRP, IL-6 and VEGF) showed weak to moderate associations with ASDAS and no or weak associations with BASDAI (except for VEGF). However, ASDAS and BASDAI were both longitudinally associated with biomarkers of inflammation and angiogenesis, and ASDAS also with MMP-3 and osteocalcin after adjustment for possible confounders. This suggests that ASDAS and BASDAI both reflect systemic inflammatory disease activity, and ASDAS also osteoblast activity. ASDAS comprises CRP and to what extent the association between ASDAS and biomarkers of inflammation was caused by this was not analysed. The biomarkers of inflammation demonstrated moderate/high difference in responsiveness between patients with major versus no/clinically important improvement in ASDAS. In contrast, this was only observed for VEGF in BASDAI responders versus non-responders.
No previous studies have examined the relation between ASDAS and circulating biomarkers, and only a few studies have investigated the relation between biomarkers, BASDAI and treatment response. In accordance with others we found significant decreases in CRP,4 9 24 IL-61 4 5 and VEGF4,–,6 in patients with AS or axial spondyloarthritis treated with TNFα inhibitors. Like others we found no or weak correlations at baseline between BASDAI and CRP,1 4 9 IL-64 and VEGF,1 5 12 and between changes in BASDAI and CRP4 9 and IL-6.4 As a result of the low number of patients we did not perform regression analyses of biomarker levels for the prediction of treatment response. However, higher baseline levels of and changes in CRP were associated with a positive clinical response regardless of response criteria. Previous studies have reported that high baseline CRP was associated with decreases in BASDAI scores,4 and normal CRP was associated with not achieving a BASDAI50 response25 to anti-TNFα therapy.
ASDAS and BASDAI showed weak associations with the cartilage biomarkers COMP and aggrecan, while ASDAS was longitudinally associated with MMP-3. Major improvements in ASDAS and BASDAI response were related to MMP-3 and the latter two also to CTX-II. A moderate/high difference in responsiveness was demonstrated for CTX-II between BASDAI responders versus non-responders.
The presence of peripheral arthritis was associated with higher MMP-3 and COMP and percentage changes in these biomarkers. In our and other studies9 10 of axial spondyloarthritis patients, patients with peripheral arthritis had higher serum MMP-3 than patients without peripheral arthritis. MMP-3 are produced by chondrocytes and by macrophages and fibroblasts in the synovium.26 MMP-3 levels in synovial fluid are 1000 times higher than in paired serum samples, and MMP-3 from inflamed peripheral joints contributes to the serum level.10 In patients with peripheral spondyloarthritis synovial sublining MMP-3 protein expression was best to differentiate responders from non-responders to treatment with TNFα inhibitor (SRM 1.11) and active compared with inactive treatment (1.59).8 However, the difference in responsiveness of systemic MMP-3 was low in our study, which may be explained by other sources of MMP-3 from outside the joints and spine.
One study has investigated urinary CTX-II in patients with AS during anti-TNFα therapy,9 and found that changes in BASDAI correlated weakly with CTX-II, but not at baseline. CTX-II decreased in patients treated with adalimumab but not in patients treated with placebo, which was shown after 12 and 24 weeks of treatment.9 We found no correlations or longitudinal associations between ASDAS, BASDAI and CTX-II from baseline to week 22. However, CTX-II decreased first after 22 weeks of anti-TNFα therapy. An explanation for this may be that 50% of our patients were treated with infliximab 3 mg/kg during the first 22 weeks, and after evaluation of treatment response at week 22, non-responders and incomplete responders had more efficient treatment.
The aggrecan ELISA assay detects intact aggrecan and fragments containing the G1 and/or G2 domains released by ADAMTs enzymes.27 MMP-3 and aggrecan did not correlate. Aggrecan increased rapidly and persistently, indicating that this biomarker of aggrecan turnover is sensitive to changes in disease activity during anti-TNFα therapy. This was also found for aggrecan epitope 846 in patients treated with etanercept but not with placebo,3 suggesting that the inflammatory processes of spondyloarthritis suppress normal aggrecan turnover reversibly.
Weak associations were observed between ASDAS and BASDAI and biomarkers of bone turnover, except for an inverse correlation and longitudinal association between ASDAS and osteocalcin. Osteocalcin levels increased in patients with major improvement in ASDAS and decreased in patients with no/clinically important improvement. Moderate/high differences in responsiveness of osteocalcin were observed between patients with major versus no/clinically important improvement in ASDAS and major versus no improvement in ASDAS. Trends towards greater percentage increases in osteocalcin from baseline to week 24 in patients treated with infliximab but not with placebo are reported12 and increases in osteocalcin after 12 weeks of anti-TNFα therapy.13 In our study, osteocalcin increased transiently from weeks 2 to 22. In accordance with others, we observed no treatment effects on biomarkers of bone resorption.9 12 13 The increase in osteocalcin may reflect osteblast activation because of new bone formation due to the development of new syndesmophytes/ankylosis or an increase in systemic bone formation resulting from the decrease in systemic inflammation. However, only baseline osteocalcin has been associated with increased bone mineral density in the spine and hip in patients with AS.12
Limitations of our study include the low sample size and the large number of tests performed, which increases the probability of having significant results by chance. The use of three different TNFα inhibitors and the use of infliximab in a dose of 3 mg/kg make it more difficult to interpret the results of the clinical non-responders.
In conclusion, this, the first study to compare ASDAS with biomarkers of inflammation, angiogenesis and cartilage and bone turnover, demonstrated that ASDAS and BASDAI were both associated with biomarkers of inflammation in patients with axial spondyloarthritis initiating anti-TNFα therapy. Furthermore, ASDAS was associated with MMP-3 and osteocalcin and changes in ASDAS with changes in CTX-II, MMP-3 and osteocalcin, while BASDAI response was not related to biomarkers of cartilage and bone turnover. This indicates that ASDAS may better reflect the inflammatory disease processes in spondyloarthritis than BASDAI. The levels of CRP, IL-6, VEGF, CTX-II, aggrecan and osteocalcin changed during anti-TNFα therapy and in parallel with conventional measures of disease activity and with moderate to high differences in SRM. Therefore, the construct validity and responsiveness of these parameters as biomarkers of disease activity have been documented.
The authors would like to thank technician Teresa Rozenfeld, Glostrup Hospital, for analysing the biomarkers, and the staff at the Department of Clinical Immunology at Nykøbing Falster County Hospital for providing blood samples from healthy volunteer blood donors. The authors also wish to thank statistician Ib Jarle Christensen, Finsen Laboratory, Copenhagen Biocenter, Copenhagen, Denmark, for calculating the longitudinal associations between biomarker levels and ASDAS, BASDAI and CRP. Abbott Denmark printed and distributed the case report forms used in the study.
Funding SJP's salary during her position as a PhD student was financed by the Faculty of Health Sciences, University of Copenhagen, Denmark. Furthermore, the authors are grateful for the grant support received for the biomarker analyses: the Danish Rheumatism Association; the Danish Psoriasis Research Foundation; the Toyota Foundation in Denmark; the Research Council of Herlev Hospital; the Bjarne Jensen Foundation; the A.P. Møller Foundation for the Advancement of Medical Science; the Foundation of 1870; the Scandinavian Journal of Rheumatology's grant; Oldermand Slagtermester Peter Ryholts Grant. The University of Copenhagen and the above-mentioned associations and foundations all provided non-profit funding and played no role in the design and conduct of the study; or in the collection, analysis, and interpretation of the data; or in the preparation, review and approval of the manuscript.
Competing interests None.
Ethics approval This study was conducted with the approval of the local ethics commitee in Copenhagen.
Provenance and peer review Not commissioned; externally peer reviewed.