Background: Bone turnover balance favors bone formation, especially mineralization, during the first 3 years of treatment with TNF-α inhibitors (TNFi). Our aim was to evaluate the course of serum bone turnover markers (BTM) and to investigate if facilitation of mineralization reflected by BTM BALP continues to increase during 6 years of TNFi treatment in patients with ankylosing spondylitis (AS) in daily clinical practice. Methods: Included were outpatients from the University Medical Center Groningen (UMCG) participating in the Groningen Leeuwarden Axial SpA (GLAS) cohort who were treated with TNFi for at least 6 years. Serum markers of collagen resorption, bone regulation, collagen formation and facilitator of bone mineralization (sCTX, OC, PINP and BALP, respectively) were measured at baseline, 3 and 6 months, 1, 2, 4 and 6 years. Z-scores were calculated to correct for age and gender. Results: 53 AS patients were eligible for analyses (66% male, mean age 39±11 years). Disease activity showed rapid and sustained improvement after start of TNFi. Evaluating BTM, sCTX did not significantly change during 6 years of treatment. OC was only significantly increased at 3 months compared to baseline, with median change in Z-score of +0.5. PINP significantly increased at 3 and 6 months and 2 years of treatment, with maximum median change in Z-score of +0.3. Interestingly, BALP was significantly increased at all time points up to and including 2 years of TNFi treatment, with maximum change in median Z-score of +1.2, and decreased thereafter. Conclusion: In AS patients receiving long-term TNFi, bone turnover balance favored collagen formation and facilitation of mineralization during the first 2 years of treatment. Thereafter, at 4 and 6 years of follow-up, BTM Z-scores returned to pre-treatment levels.
Ankylosing spondylitis (AS), a chronic auto-inflammatory disease of especially the sacroiliac joints (SI) and spinal column, is characterized by inflammation, osteoproliferation and excessive bone loss [[
In AS patients with persisting high disease activity despite conventional therapy, switch to biologicals and tumor necrosis factor-alpha (TNF-α) inhibitors (TNFi) to start as a first biological is advised [[
In a placebo-controlled trial of 201 axSpA patients treated with infliximab for 2 years, baseline levels of collagen resorption marker serum type I collagen C-telopeptide (sCTX), bone regulation marker osteocalcin (OC) and facilitator of bone mineralization marker bone-specific alkaline phosphatase (BALP) were positively correlated with an increase in spinal BMD at 24 and 102 weeks of follow-up [[
Therefore, our goal was to evaluate the course of serum bone turnover markers (BTM) and to investigate if the facilitation of mineralization reflected by BTM BALP still continues to increase during 6 years of TNFi treatment in patients with AS in daily clinical practice.
Included were 53 consecutive AS outpatients from the University Medical Center Groningen (UMCG) participating in the Groningen Leeuwarden Axial SpA (GLAS) cohort who were treated with TNFi for at least 6 years, with a maximum of 2 different TNFi. Patients were excluded from analyses when they used antiresorptive drugs to treat osteoporosis (e.g. bisphosphonates) at baseline or during follow-up. Data for a specific visit was coded as missing when patients either had experienced a fracture or received systemic corticosteroids within 1 year from that particular visit due to the possible effect on BTM. All patients were over 18 years of age, fulfilled the modified New York criteria for AS and/or the Assessments in Ankylosing Spondylitis (ASAS) criteria for AS including MRI and started TNFi because of active disease according to the ASAS consensus statement [[
Demographics characteristics and clinical assessments were obtained from the regular GLAS outpatient visits: age, gender, symptom duration, human leukocyte antigen-B27 (HLA-B27) status, smoking status, history of extra-skeletal manifestations (inflammatory bowel disease (IBD), uveitis or psoriasis), history of peripheral arthritis, and medication use. Disease activity was assessed with AS Disease Activity Score with CRP (ASDAS
BMD of the lumbar spine (anterior-posterior projection L1-L4) was measured at baseline, 2, 4 and 6 years using dual-energy X-ray absorptiometry (Hologic QDR Discovery, Waltman, MA, USA). Z-scores, the number of SD from the normal mean corrected for age and gender, were calculated using the NHANES reference database. Low BMD was defined as lumbar spine Z-score ≤1.
Available radiographs of the cervical and lumbar spine were scored at baseline, 2, 4 and 6 years in chronological time order by two trained readers blinded for patient characteristics using the modified Stoke AS Spine Score (mSASSS; range 0–72). Longitudinal radiographs of included patients treated with TNFi were randomized and scored together with longitudinal radiographs of axSpA patients not treated with TNFi in order to avoid potential reader bias concerning the effect of treatment on mSASSS. The average mSASSS total score of the two readers was used in the analysis. When there was discrepancy of >5 points in mSASSS total scores, a third independent reader scored the radiographs [[
Standardized follow-up visits were performed at baseline (before start of TNFi), 3 and 6 months, 1, 2, 4 and 6 years. Bone turnover was studied by assessment of serum markers of bone resorption (sCTX), bone regulation (OC), collagen formation (procollagen type 1 N-terminal peptide; PINP) and facilitator of bone mineralization (BALP). sCTX was measured by electro-chemiluminescence (ECLIA; Elecsys 2010 Roche Mannheim, Germany; inter-assay coefficient of variation (IE-CV) 10.8%), OC by immunoradiometric assay (IRMA; BioSource Europe South Africa; IE-CV 9.4%), PINP by RIA (Orion Diagnostica, Espoo, Finland; IE-CV 9.0%) and BALP by enzyme-linked immunosorbent assay (ELISA; Metra Biosystems, Mountain View, CA, USA; IE-CV 5.5%). Bone turnover markers were measured in a NEN-EN-ISO 9001:2008 certified and NEN-EN-ISO 15189:2012 accredited laboratory. Serum was acquired of non-fasting patients during study visits of the GLAS cohort taking place at fixed hours (same half-day) and were stored within one hour at -20°C until analysis. BTM Z-scores were used for analyses. Z-scores express the number of standard deviations (SD) from the normal mean corrected for age and gender, and therefore were used to correct for the normal influence that age and gender have on bone turnover. These BTM Z-scores were calculated using matched 10-year-cohorts of a Dutch reference group (200 men or 350 women), checked for serum 25OHvitD levels >50 nmol/liter as well as for the absence of osteoporosis (BMD T-score >-2.5) after 50 years of age: (BTM value of individual patient–mean BTM value of matched 10-year-cohort of reference group) / SD of matched reference cohort. Additionally, the net effect of collagen metabolism was explored by calculating: collagen formation marker PINP Z-score–collagen degradation marker sCTX Z-score.
Statistical analysis were performed with PASW Statistics 23 (SPSS, Chicago, IL, USA). Results were expressed as mean ± SD or median (interquartile range P25-P75) for normally distributed and non-normally distributed data, respectively. Generalized estimating equations (GEE) were used to analyze BTM, BMD, and clinical assessments over time within subjects. GEE is a longitudinal analysis technique which makes use of all available longitudinal data and allows unequal numbers of repeated measurements [[
In total, 53 AS patients started TNFi between October 2004 and December 2014 and had a follow-up of at least 6 years. Baseline characteristics showed that 66% were male, mean age was 38.5 ± 11.3 years, median symptom duration 15 years (IQR 9–25), 87% were HLA-B27+, mean ASDAS was 3.8 ± 1.0, mean BASDAI 5.7 ± 2.0 and median CRP 14 mg/L (IQR 7–27) (Table 1). At baseline, low BMD at the lumbar spine was present in 36% of the patients. mSASSS was available in 33 of 53 included AS patients, with median 2.7 (IQR 0.8–10.1) and mean 7.3 ± 10.3. Etanercept, infliximab or adalimumab was prescribed as first TNFi in 60%, 2% and 38% of patients, respectively. 26% (n = 14) of patients switched to a second TNF-α inhibitor during follow-up (Fig 1). Reasons for switching were infections (n = 6), secondary inefficacy in combination with infections (n = 2), cardio-vascular symptoms (n = 2), allergic skin reaction (n = 1), development of inflammatory bowel disease (n = 1) or miscellaneous (n = 2) (Fig 1).
Graph: Fig 1 Flowchart of TNFi use of the 53 AS patients on TNFi for ≥6 years and ≤ 2 different TNFi.
Graph
Table 1 Baseline characteristics of the AS study population treated with TNFi for 6 years (n = 53).
Age (yrs) 38.5 ± 11.3 Gender (male) (n, %) 35 (66) Duration of symptoms (yrs) 15 (9–25) HLA-B27+ (n, %) 46 (87) History of IBD (n, %) 4 (8) History of uveitis (n, %) 11 (22) History of psoriasis (n, %) 1 (2) History of peripheral arthritis (n, %) 15 (31) Current NSAID use (n, %) 46 (90) Current DMARD use (n, %) 13 (25) BASDAI (range 0–10) 5.7 ± 2.0 BASDAI ≥4 (n, %) 43 (84) ASDASCRP 3.8 ± 1.0 ASDASCRP ≥2.1 (n, %) 47 (94) CRP (mg/L) 14 (7–27) Increased CRP >5 (n, %) 43 (84) Occiput to wall distance (cm) 0.0 (0.0–8.0) Chest expansion (cm) 3.0 (2.0–4.0) Modified Schober test (cm) 3.1 ± 1.4 Lateral lumbar flexion mean (cm) 10.2 ± 5.6 25(OH)D (nmol/L) 60.9 ± 26.5 sCTX (pg/mL) 254.8 ± 111.1 sCTX Z-score 0.8 ± 1.0 Osteocalcin (ng/mL) 12.1 ± 4.3 Osteocalcin Z-score -0.5 ± 0.7 PINP (μg/L) 48.7 ± 18.4 PINP Z-score 0.3 ± 0.9 BALP (U/L) 21.3 ± 7.4 BALP Z-score 1.1 ± 1.9 BMD LS Z-score -0.3 (-1.4–0.7) BMD Z≤-1 (n, %) 16 (36) mSASSS 2.7 (0.8–10.1)
1 Values are mean ± SD or median (range) unless otherwise indicated. AS: Ankylosing Spondylitis; HLA-B27+: human leukocyte antigen B27 positive; IBD: inflammatory bowel disease; NSAID: non-steroidal anti-inflammatory drug; DMARD: disease-modifying antirheumatic drug; BASDAI: Bath Ankylosing Spondylitis Disease Activity Index; ASDAScrp: ankylosing spondylitis disease activity score with CRP; CRP: C-reactive protein; BALP: bone-specific-alkaline phosphatase; PINP: procollagen type 1 N-terminal peptide; sCTX: serum C-telopeptide of type I collagen; BMD: bone mineral density; LS: lumbar spine; mSASSS: modified Stoke ankylosing spondylitis spinal score.
After start of TNFi, all disease activity assessments showed a rapid and sustained improvement. Both ASDAScrp and BASDAI decreased significantly during TNFi at all follow-up visits compared to baseline (Fig 2). Mean change in ASDAScrp was 2.0 ± 1.0 at 6 years compared to baseline. Furthermore, 81% of patients were clinical responder based on improvement in ASDAScrp of >1.1 at 6 years. Levels of acute phase reactant CRP quickly decreased following therapy and were found to remain significantly lower compared to baseline. However, CRP levels remained elevated (>5mg/L; mean 16 mg/L) in approximately 10% of patients at every visit during follow-up. Serum levels of 25OHvitD were stable at group level during follow-up, although 8% and 28% of the patients had baseline 25OHvitD <25 or <50 nmol/L, respectively. BMD Z-score of the lumbar spine improved significantly during TNFi at all follow-up visits compared to baseline. Significant improvement compared to the previous time point was found up to and including 4 years, thereafter, flattening of improvement was observed. Mean increase in mSASSS was 1.6, 1.3 and 1.2 points for 0–2, 2–4 or 4–6 years, respectively (Fig 2).
Graph: Fig 2 Disease activity index score ASDAScrp (A), BASDAI (B), levels of acute phase reactant CRP (C), levels of vitamin D (D), BMD (E) and mSASSS progression (F) during 6 years of treatment with TNFi in patients with AS (n = 53). * indicate p-value of <0.05, ** indicate p-value of <0.01 and *** indicate p-value of <0.001 compared to baseline. Horizontal lines represent mean (Fig A, B and D) or median (C, E and F) and dots represent individual values.
At baseline, median BTM Z-scores were -0.08, -0.55, 0.14 and 0.83 for sCTX, OC, PINP and BALP, respectively. At group level, bone resorption marker sCTX did not significantly change over time after start of TNFi. Bone regulation marker OC was significantly increased after 3 months of TNFi compared to baseline, with a median change in Z-score of +0.5. Bone collagen formation marker PINP was significantly increased at 3 and 6 months and 2 years of TNFi compared to baseline, with a maximum median change in Z-score of +0.3. Facilitator of bone mineralization marker BALP was significantly increased at all time points up to and including 2 years compared to baseline, with a maximum median change in Z-score of +1.2. Thereafter, at 4 and 6 years, BALP returned to levels not significantly different from baseline (Fig 3A–3D). All results remained similar when correcting for serum levels of 25OHvitD over time. Exploring the net effect of collagen metabolism (collagen formation marker PINP Z-score–collagen degradation marker sCTX Z-score) confirmed that the balance favored collagen formation. However, a significant increase in the net effect of collagen formation was only observed between baseline and 6 months with a median PINP–sCTX Z-score of 0.25 and 0.71 at baseline and 6 months, respectively (Fig 4). The number of patients with a BALP Z-scores above +2 SD was highest after 1 and 2 of TNFi treatment; in 22%, 45%, 41%, 28% and 27% of the patients at baseline, 1, 2, 4 or 6 years, respectively.
Graph: Fig 3 The effect of 6 years of TNFi on markers of bone turnover sCTX (A), OC (B), PINP (C) and BALP (D) in patients with AS (n = 53). * indicate p-value of <0.05, ** indicate p-value of <0.01 and *** indicate p-value of <0.001 compared to baseline. Horizontal lines represent mean and dots represent individual values.
Graph: Fig 4 The net effect of collagen formation and degradation during 6 years of TNFi.* indicate p-value of <0.05 compared to baseline. Horizontal lines represent mean and dots represent individual values.
This prospective study within the observational GLAS cohort is the first study that evaluated the prolonged course of BTM during continuous TNFi treatment in AS patients in daily clinical practice. At group level, the collagen degradation and formation products indicated a balance towards bone formation, especially mineralization, in the first 2 years of TNFi treatment, which is in concordance with our previous study of AS patients with 3 years of TNFi treatment [[
Mineralization, of which the outcome is BMD, from a physiological point of view is the enlargement of hydroxyapatite crystals with displacement of water from the collagen fibrils and fibers. This is a slow process which takes months, in which BALP is an essential facilitator. Previous research in AS demonstrated that following TNFi BMD of the lumbar spine (AP projection) significantly improves within 24 weeks [[
The pro-inflammatory cytokine TNF-α plays an important role in the inflammatory process of AS and particularly impacts bone metabolism. In healthy individuals, the process of bone metabolism, i.e. osteoclastogenesis and osteoblastogenesis, are well balanced ensuring the continued remodeling and renewing of the skeleton [[
In the process of bone formation, there is consensus that TNF-α mainly acts as inhibitor in the differentiation from pre-osteoblast to osteoblast [[
The various roles of TNF-α on disease pathogenesis and its influence on bone formation can also be found in the process of mineralization. TNF-α plays a role in the mineralization process of bone by increasing alkaline phosphatase activity through inhibition of RUNX2 and PPARγ expression [[
A strong aspect of our study is the long-term standardized follow-up including BTM of AS patients with active disease treated with TNFi for 6 years. Following the start of TNFi, disease activity markers ASDAS
A potential limitation might be that serum samples were acquired of non-fasting patients (in accordance with daily clinical practice), which may have given more variation in serum levels of BTM. Study visits of the GLAS cohort took place at fixed consultation hour (same half-days), therefore we do not expect a large influence of diurnal variation in our study. Also, the absence of a (historical) reference cohort of untreated AS patients with high disease activity can be seen as limitation. Although short-term data is available from the placebo-controlled trial with infliximab [[
To conclude, after an initial balance favoring collagen formation and facilitation of mineralization, bone turnover markers return to pre-treatment levels during long-term TNF-α inhibition in AS patients. Future research may reveal if BTM can be used as prognostic markers of bone related outcome in AS.
The authors would like to thank all patients who participated in the GLAS cohort. Furthermore, the authors wish to acknowledge Mrs. W. Gerlofs, Mrs. S. Katerbarg, Mrs. S. Lange, and Mrs. B. Toonder for their contribution to clinical data collection and L. Wagenmakers and K. Koerts for their contribution to BTM assessments.
- 25OHvitD
- 25-hydroxyvitamin D
• AS
- ankylosing spondylitis
• ASAS
- assessments in ankylosing spondylitis
- ASDAS
crp - Ankylosing Spondylitis Disease Activity Score with CRP
• BALP
- bone-specific alkaline phosphatase
• BASDAI
- Bath Ankylosing Spondylitis Disease Activity Index
• BMD
- bone mineral density
• BTM
- bone turnover marker
• CRP
- C-reactive protein
• ECLIA
- electro-chemiluminescence
• ELISA
- enzyme-linked immunosorbent assay
• GEE
- generalized estimating equations
• GLAS
- Groningen-Leeuwarden Axial SpA
• HLA-B27
- human leukocyte antigen-B27
• IBD
- inflammatory bowel disease
• IE-CV
- inter-assay coefficient of variation
• IRMA
- immunoradiometric assay
• mSASSS
- modified Stoke ankylosing spondylitis spinal score: OC: osteocalcin
• PINP
- procollagen type 1 N-terminal peptide
• RIA
- radioimmunoassay
• sCTX
- serum type I collagen C-telopeptide
• SD
- standard deviation
• SI
- sacroiliac
- TNF-α
- tumor necrosis factor-alpha
• TNFi
- TNF-α inhibitor
• UMCG
- University Medical Center Groningen
By Mark Siderius; Anneke Spoorenberg; Frans G. M. Kroese; Eveline van der Veer and Suzanne Arends
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