Objectives In rheumatoid arthritis, high levels of the cartilage turnover biomarker C-terminal cross-linking telopeptide of type II collagen (CTX-II) predict an increased risk of radiological progression. In very early inflammatory arthritis erosions are uncommon, therefore CTX-II requires validation against early markers of inflammatory arthritis such as power Doppler ultrasound (PDUS) synovitis and bone mineral density (BMD) loss.
Methods In 50 subjects with 12 weeks or less of inflammatory hand symptoms, urinary CTX-II and PDUS were performed at baseline and hand BMD at baseline and 12 months. CTX-II data were log transformed to a normal distribution. Associations between variables were examined using Pearson's r/Spearman's ρ correlations.
Results The mean 12- month change in BMD was −0.0068 g/cm2 and the geometric mean for baseline CTX-II/creatinine was 245.89 ng/mmol. Log-transformed baseline CTX-II showed a substantive negative association with change in average BMD over 12 months, controlling for baseline BMD and erythrocyte sedimentation rate (r=−0.359, p=0.044). The median total PDUS score was 3.0 and baseline CTX-II was significantly associated with baseline total PDUS (Spearman's ρ=0.482, p=0.002).
Conclusion Urinary CTX-II correlates with PDUS synovitis and hand BMD reduction very early in the course of inflammatory arthritis, suggesting that CTX-II has potential as a biomarker in very early inflammatory arthritis.
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Erosions and joint space narrowing on conventional radiography have historically been used as the clinically feasible measure of joint destruction in rheumatoid arthritis (RA). The development of erosions represents the endpoint in the inflammatory process that begins with synovitis1 and osteitis,2 and therefore erosions are rarely seen early on in the disease course. Early markers of disease activity are therefore particularly important. Dual energy x-ray absorptiometry (DEXA) can detect bone mineral density (BMD) reduction in the hands at the ‘prerheumatoid’ stage and this reduction has been shown to correlate with a diagnosis of RA at 12 months.3 In addition, BMD reduction in early RA has been shown to predict structural progression.4 Power Doppler ultrasound (PDUS) can identify synovitis in early undifferentiated arthritis5 and PDUS synovitis in RA low disease activity patients has been shown to predict structural progression.6
A biomarker that can accurately predict the earliest signs of structural progression (ie, initial cartilage loss) is therefore required. Type II collagen is the most abundant constituent of cartilage matrix.7 The C-terminal cross-linking telopeptide of type II collagen (CTX-II) can be measured in the urine using an immunoassay, providing a sensitive and specific measure of type II collagen breakdown.8 There is increasing published evidence for the validity of CTX-II as a biomarker in RA.9 CTX-II has been shown to be a responsive measure, closely following indicators such as inflammatory markers and disease activity scores,10 as well as being able to predict disease-modifying treatment effect.11 High baseline urinary levels of CTX-II have been shown to predict an increased risk of radiological progression in early RA, particularly in those without baseline radiographic damage.12
In the ‘prerheumatoid’ phase of inflammatory arthritis, however, the predictive validity of cartilage turnover biomarkers is uncertain. To assess CTX-II in the earliest stage of disease development, outcome measures that are known to correlate with disease activity and structural progression are desirable. The aim of this study was therefore to validate CTX-II against PDUS synovitis and hand BMD reduction in a cohort of very early inflammatory arthritis patients.
Fifty individuals were recruited into a longitudinal study of patients with very early inflammatory symptoms.13 Subjects with 12 weeks or less of inflammatory hand symptoms (defined as early morning stiffness ≥1 h in a minimum of one hand joint, with or without clinical synovitis) were consecutively recruited from an early arthritis clinic. Anti-cyclic citrullinated peptide (CCP) antibody was measured using a second-generation fluoroenzyme immunoassay (NR <7 U/ml) and rheumatoid factor (RF) status by nephelometry (normal range <20 IU/ml). Bilateral hand and feet conventional radiography was performed for reference at baseline and 12 months. All subjects were disease-modifying antirheumatic drug naive and received an intramuscular methylprednisolone injection following baseline assessment. Subsequent treatment was at the treating physician's discretion.
Urine samples (second void of the morning; non-fasting state) were obtained at baseline and frozen at −20°C. CTX-II was measured using a competitive ELISA (CartiLaps; IDS, Boldon, UK) based on a mouse monoclonal antibody raised against the EKGPDP sequence of human type II collagen C-telopeptide. The urinary CTX-II level (µg/l) was corrected by the urinary creatinine concentration (nanogram CTX-II/mmol creatinine). All measurements were performed in a central laboratory (Synarc, Lyon, France). No patients had marked liver and/or kidney function impairment that may have altered the urinary CTX-II levels.
BMD (g/cm2) of both hands was measured at baseline and 12 months using a Lunar Prodigy machine (GE Medical Systems, Belgium) and reported as an average (mean). Change over 12 months was then calculated. BMD measurements were repeated in a subset of 10 subjects from the original study cohort (n=50) to determine intrameasurement variability. These repeat measurements were performed by repositioning the subject's hands immediately after the baseline BMD measurements.
PDUS of bilateral metacarpophalangeal joints 1–5 and wrists (radiocarpal, intercarpal and ulnarcarpal joints) was performed by an experienced ultrasonographer (RJW) using a Philips HDI 5000 machine (Philips, Best, The Netherlands) employing a 15-7 MHz linear ‘hockey stick’ transducer and a medium wall filter. Joints were scored using a semiquantitative system1 (0–3 scale) and scores summated to produce a total score (maximum score 48) per patient.
As the CTX-II levels had a skewed distribution, the data were log transformed to a normal distribution before analysis. Descriptive statistics (mean and 95% CI for interval data (CTX-II, BMD), median and IQR for ordinal data (total PDUS score)) were calculated. Associations between variables were examined using Pearson's r correlation or Spearman's ρ as appropriate. Intraclass correlation coefficient (ICC) and smallest detectable change (SDC) (using the Bland–Altman coefficient of repeatability14) analyses were calculated for the 10 subjects with repeated BMD measurements. Analysis of covariance models that included baseline values as covariates were used to compare the change in average BMD over 12 months between patients who were RF/CCP positive or negative. All analyses were conducted using the SPSS 16.0.2 statistical package.
The characteristics of the 50 study subjects at baseline are shown in table 1. At 12 months, 38 subjects had developed persistent inflammatory arthritis, including RA and undifferentiated arthritis. One subject did not return for the follow-up visits, therefore their final diagnosis could not be recorded. The remaining subjects (N=11) had non-inflammatory arthritis. Five of 49 subjects (10%) had one or more erosions on conventional radiography at baseline and four of 40 (10%) at 12 months. At 12 months, one subject had become newly erosive and in two subjects erosions had apparently healed.
A total of 37 patients (32 inflammatory, five non-inflammatory) had both baseline urine specimens and baseline and 12-month hand DEXA data available.
In subjects with inflammatory arthritis the mean (95% CI) 12-month change in BMD was −0.0078 g/cm2 (−0.0135 to 0.0022) and the geometric mean (95% CI) baseline level of CTX-II/creatinine was 245.1 (191.7 to 313.4) ng/mmol. In subjects with non-inflammatory arthritis the mean (95% CI) 12-month change in BMD was −0.0001 (−0.0067 to 0.0065) and the geometric mean (95% CI) baseline level of CTX-II/creatinine was 250.85 (129.25 to 486.85). The means for the combined groups were −0.0068 (−0.0117 to −0.0018) and 245.89 (197.51 to 306.13), respectively. The ICC for BMD measurement showed very good agreement (n=10, ICC[1,1]=0.991 (0.967 to 0.998)). A Bland–Altman plot (data not shown) showed that there was no systematic bias and that the degree of error was not related to the magnitude of the measurement. The SDC was 0.0088. Thirteen of the 32 subjects with inflammatory arthritis showed 12-month BMD loss that exceeded this limit. None of those with non-inflammatory arthritis showed BMD loss exceeding the SDC.
Log-transformed baseline CTX-II/creatinine showed a substantive negative association with the change in average BMD over 12 months, controlling for BMD at baseline (n=37, Pearson's r=−0.439, p=0.007), as shown in figure 1. Baseline erythrocyte sedimentation rate (ESR) was available for 34 subjects; there was a substantive positive correlation between log-transformed baseline ESR and log-transformed baseline CTX-II/creatinine (r=0.399, p=0.019), and a substantive negative correlation between log-transformed baseline ESR and the change in average BMD, controlling for baseline BMD (r=−0.493, p=0.004).
The median (IQR) total PDUS score was 3.0 (0.0–12.5) for inflammatory arthritis patients and 0.0 (0.0–2.0) for non-inflammatory arthritis patients. Baseline CTX-II/creatinine was significantly associated with baseline total PDUS (Spearman's ρ=0.482, p=0.002; figure 2), as were baseline ESR (n=34, r=0.634, p<0.001) and C-reactive protein (n=37, r=0.529, p=0.001).
There were no substantive or significant associations between baseline PDUS and either DAS28–C-reactive protein (r=0.101, p=0.554) or DAS28–ESR (n=34, r=0.189, p=0.284). There was an association between baseline total PDUS and the change in average BMD over 12 months (r=−0.567, p<0.001). A supplementary data analysis is published online only.
This is the first study to address the role of the cartilage turnover biomarker CTX-II in patients with very early inflammatory disease. In this longitudinal study, we have shown that baseline CTX-II levels correlate with baseline PDUS synovitis and with deterioration in hand BMD over 12 months in patients with a very short inflammatory history. Using PDUS synovitis and BMD change as surrogate outcome measures for disease activity and structural progression, respectively, these results imply that CTX-II has concurrent and predictive validity as a biomarker in very early disease. We have also previously published work in this very early cohort showing that baseline PDUS synovitis correlates with persistent inflammatory arthritis at 12 months.13 This early stage of disease has not been extensively studied with respect to soluble biomarkers. Jensen et al15 have studied the relationship between soluble biomarkers of connective tissue metabolism and BMD in both unclassified polyarthritis and early RA, showing that matrix metalloproteinase 3 and pyridinoline were associated with BMD (measured by digital x-ray radiogrammetry). The study did not, however, investigate CTX-II. Haugeberg et al3 looked at only BMD in undifferentiated arthritis, showing that hand bone loss in undifferentiated arthritis is related to measures of disease activity and severity in those developing subsequent RA.
The main limitation of this study is the small number of study subjects. The principal reason for this was the difficulty capturing patients with 12 weeks or less of symptoms, as primary care physicians frequently refer patients with longer symptom durations. As only a small number of subjects had non-inflammatory diagnoses at 12 months, this study was not able to compare formally the CTX-II–BMD relationship between those with inflammatory and non-inflammatory diagnoses at 12 months. Similarly, it was not possible to assess any potential differences between those with RA and those with undifferentiated arthritis at 12 months.
Inflammatory hand symptomatology was the main inclusion criterion although other joint involvement (ie, tenderness with or without swelling) was not an exclusion. Involvement of other joints may have affected the biomarker level, although only one out of 37 (2.7%) subjects at baseline had evidence of other swollen joints than hands/wrists affected.
All subjects received an intramuscular methylprednisolone injection following baseline assessment so it was not possible to determine the effect of the steroid injection on bone loss in this cohort. However, one would surmise that such an effect would apply equally to non-inflammatory as to inflammatory patients and therefore not have a significant confounding effect on the results (although it cannot be ruled out that additional steroid therapy may have influenced the BMD change and with that the relations presented).
In summary, further research needs to be done to validate the results found in this study on a larger scale. This study suggests that CTX-II is a potentially useful biomarker in very early inflammatory patients, helping identify those patients who are at high risk of structural progression at the earliest possible stage allowing the timely instigation of appropriate therapy.
The authors would like to thank Sheena Stewart for performing the bone density scans and Karen Henshaw and Diane Corscadden for the initial processing of the urine samples.
Funding JEF is funded by a NIHR clinical lectureship. PE is an ARUK Professor of Rheumatology.
Conflicts of interest None.
Ethics approval This study was conducted with the approval of the Leeds Research Ethics Committee, Leeds, UK.
Provenance and peer review Not commissioned; externally peer reviewed.