Continuously elevated serum matrix metalloproteinase-3 for 3 ~ 6 months predict one-year radiographic progression in rheumatoid arthritis: a prospective cohort study

Patients with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR) [10] or 2010 ACR/EULAR classification criteria for RA [11] were recruited from the Department of Rheumatology of Sun Yat-sen Memorial Hospital, Guangzhou, People’s Republic of China. Inclusion criteria also included: patients with active disease activity, defined as simplified disease activity index (SDAI) > 3.3 [12] and poor prognosis of one or more of the following features: functional limitation (Health Assessment Questionnaire score ≥ 1 [13]), positive rheumatoid factor (RF) or anti-cyclic citrullinated peptide antibody (anti-CCP), extraarticular disease (e.g., presence of rheumatoid nodules, secondary Sjogren’s syndrome, RA vasculitis, Felty’s syndrome, and RA lung disease) and bony erosions by radiography [14]. Exclusion criteria included: relevant concurrent liver disease (aspartate aminotransferase > 100 IU/L or alkalinephosphatase > 100 IU/L), renal disease (serum creatinine > 1.5 mg/dl), hematological disease (total white blood cell count < 4 × 10⁹/L, platelet count < 100 × 10⁹/L), or severe respiratory disease, malignancy, pregnancy or plans to become pregnant, and psychological problems that would make adherence to the study protocol impossible. This study was conducted in compliance with the Helsinki Declaration. The Medical Ethics Committee of Sun Yat-sen Memorial Hospital approved the protocol. All patients agreed to participate in this study and gave written informed consent.

All patients were treated according to the 2008 ACR recommendations for the management of RA [14]. Therapy was decided according to the disease duration, disease activity, presence of poor prognosis features and patients’ willingness. Disease duration was divided into three categories: < 6 months (short), 6–24 months (intermediate), and > 24 months (long) [14]. Disease activity defined by SDAI was divided into four categories: > 26.0 [high disease activity (HDA)], > 11.0 and ≤ 26.0 [moderate disease activity (MDA)], > 3.3 and ≤ 11.0 [low disease activity (LDA)], and ≤ 3.3 (remission) [12]. Adjustment of therapy was based on the T2T strategy adherence to the disease activity measured by SDAI. The therapeutic target was defined as remission (primary target) or LDA (alternative target) measured by SDAI and patients were divided into a T2T-achieving group (defined as SDAI remission + LDA at the 12th month) and a T2T-non-achieving group (MDA + HDA at the 12th month).

All patients were followed up at regular intervals including the same predefined assessment points (0, 1st, 3rd, 6th, and 12th month) and the following indicators were assessed: 28-joint tender and swollen joint count (28TJC and 28SJC, both 0–28), Patient global assessment of disease activity (PtGA, 0–10 cm, 10 = worst status), Provider global assessment of disease activity (PrGA, 0–10 cm, 10 = worst status), Pain visual analogue scale (Pain VAS, 0–10 cm, 10 = most pain), Chinese language version of the Stanford health assessment questionnaire (HAQ, 0–3, 3 = most functional disability) [15], erythrocyte sedimentation rate [(ESR) mm/h, normal range: 0–20 mm/h (female), 0–15 mm/h (male)], C-reactive protein [(CRP), mg/dl, normal range: 0–0.5 mg/dl], RF (mg/L, determined by nephelometry, Siemens Healthcare Diagnostics, Munich, Germany, normal range: 0–20 mg/L), and anti-CCP [U/ml, measured by enzyme-linked immunosorbent assay (ELISA), Aesku Diagnostics, Wendelsheim, Germany, normal range: 0–18 U/ml]. Disease activity was assessed with SDAI, clinical disease activity index (CDAI) and disease activity score in 28 joints (DAS28) with four variables including CRP [DAS28 (4)-CRP].

Conventional radiographs of bilateral hands and wrists (anteroposterior view) were performed at baseline and the 12th month visit. All radiographs were scored according to the Sharp/van der Heijde score of hands by two experienced observers (MJD from rheumatology and ZX from radiology), who were not aware of the patients’ clinical findings [16]. Sixteen areas for erosion and fifteen for joint space narrowing of hands were assessed in each hand/wrist. The maximum score per single joint for erosions is 5, and for joint space narrowing is 4, with the sum of the erosion (0 ~ 160) and joint space narrowing (0 ~ 120) subscores constituting the total Sharp score of hands (0 ~ 280). Reliability and agreement were assessed using an intra-class correlation coefficient (ICC): the mean ICC for inter-observer agreement was 0.997. Radiographic progression was defined as a change of total Sharp score more than 0.5 units [17], and rapid radiographic progression (RRP) was defined as a change of total Sharp score more than 5 units from baseline to one year [18].

Serum samples were collected from all the RA patients after overnight fasting and stored at −80 °C until analysis. Serum levels of soluble MMP-3 were measured with a human MMP-3 detection kit (AESKU Diagnostics, Wendelsheim, Germany) according to the manufacturer’s instructions. This kit detects total MMP-3 (pro- and active MMP-3) in human serum. Measurements were done in duplicate. Serum samples were placed in designated microwells. In addition, calibrators, negative and positive controls were added to the designated microwells to construct a standard curve. The plates were then incubated for 30 min at 26 °C and washed with wash buffer three times. Then 100 μl TMB substrate was added to each well and incubated for 30 min at 26 °C, protected from intense light. Then 100 μl of stop solution was added to each well, using the same order as for the substrate, and incubated for a minimum of 5 min. The absorbance of each well was read at 450 nm (optionally 450/620 nm) within 30 min. The normal ranges of serum MMP-3 concentrations were 18 ng/ml - 60 ng/ml (female) or 24 ng/ml – 120 ng/ml (male). The assays were performed blindly, without knowledge of the patient’s clinical data.

Statistical analyses were performed with SPSS for Windows 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Data are presented as frequencies and percentages for categorical variables and median and interquartile range (IQR) for continuous variables. The Mann–Whitney or Kruskal-Wallis rank-sum test was used to compare the differences of continuous variables between two or three groups. The Wilcoxon matched-pairs signed ranks sum test was used to compare the differences of continuous variables between indicators at baseline and each time point. The Chi-square test or Fisher exact test was used for categorical variables in different groups. Univariate and multivariate logistic regression analyses were used to identify predictors of radiological progression and control confounding factors. Variables were included in the model if P < 0.05 or removed if P > 0.10 according to the forward selection technique. The one-way analysis of variance (ANOVA) for repeated measures analysis with Bonferroni correction for multiple comparisons tests was used to compare the difference of dynamic disease activity indicators between two groups. Survival curves were used to show the ratio of patients with abnormal disease activity indicators during the therapies. Log-rank tests were used to compare survival curves of disease activity indicators between two groups. The abilities of disease activity indicators to predict one-year radiographic progression were evaluated by positive and negative predictive values (PPV and NPV, respectively). The predictive accuracy was assessed by receiver operating characteristic (ROC) curve analysis with area under the curve (AUC) and the cutoff point was determined by the Youden index. All significance tests were two-tailed and were conducted at the 5 % significance level.