MMP-3 as a predictor for structural remission in RA patients treated with MTX monotherapy

Background The study was undertaken to assess the efficacy of methotrexate (MTX) monotherapy on the radiographic progression of individual rheumatoid arthritis (RA) patients, each of whom had received MTX monotherapy for 3 years with an option to change to biological disease-modifying anti-rheumatic drugs (bDMARDs). We also looked for predictors of radiographic non-progression in these patients. Methods Rheumatoid patients (n = 161) were prospectively followed for 3 years while receiving low-dose MTX monotherapy unless disease was otherwise active and/or adverse events appeared. Their disease activity and radiographic progression were evaluated with reference to disease activity score 28 (DAS28), modified health assessment of questionnaire (mHAQ) and other indices. The change in van der Heijde-modified total Sharp score per year (∆TSS) was assessed using probability plots, in which the patients were classified into the subgroups showing structural remission (REM; ∆TSS ≤0.5), radiographic progression (∆TSS >3) or rapid radiographic progression (RRP; ∆TSS >5). Results MTX monotherapy, continued until disease became active and/or adverse event appeared, was associated with a significant improvement (p <0.0001) in the DAS28-ESR (3) scores, % DAS28 remission, and mHAQ scores each year, from baseline to 3 years. The mHAQ remission rate (∆mHAQ <0.5) and Boolean remission were also improved from 16 to 60 % and 0.8 to 24.0 %, respectively. We found that the ratio of patients classified as REM increased yearly from 62/161 (38.5 %) to 69/137 (50.4 %), while those classified as ∆TSS >3 decreased from 55/161 (34.2 %) to 28/137 (20.4 %) and those in RRP decreased from 35/161 (21.7 %) to 15/137 (10.9 %). Receiver operating characteristic (ROC) curve analyses showed that serum matrix metalloproteinase-3 (MMP-3) <103.7 ng/ml at outset predicts a patient subgroup that exhibits no radiographic progression. Conclusions Half of rheumatoid patients treated with MTX monotherapy for 3 years exhibited structural remission, and this outcome can be predicted at the outset by lower serum MMP-3. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0948-7) contains supplementary material, which is available to authorized users.

Recently, O'Dell et al. have shown that treating patients with MTX monotherapy initially, while later providing an option to step up to combination therapy produces outcomes similar to those seen with combination therapies consisting of cDMARDS and/or bDMARDs that are provided from the outset [19]. In their study, approximately 30 % of the patients treated by MTX monotherapy did not require subsequent combination therapy. However, this subgroup was reported to be clinically and radiographically indistinguishable from those who required it. For MTX monotherapy to be more effectively employed as a first-line drug to halt radiographic progression. it would be useful at outset to ascertain which patients would benefit most from MTX monotherapy and which would require combination therapy including biologic agents. The purpose of the present study was to assess the efficacy of low-dose MTX monotherapy, a regimen that is commonly prescribed in Japan, and to potentially identify a subgroup of patients on MTX monotherapy, without radiographic evidence of disease progression. In the present study, 161 patients with rheumatoid arthritis were followed unless disease was otherwise active or significant adverse events appeared. Disease progression was scored as the change in the modified total Sharp score per year (ΔTSS), starting at baseline and continuing for 3 years. Patients were classified into subgroups exhibiting structural remission (REM; ΔTSS ≤0.5), radiographic evidence of progression (ΔTSS >3) or radiographic evidence of rapid progression (RRP; ΔTSS >5) [20].

Patients and the study design
Patients were included who had RA (n = 161) as determined by the 1987 American College of Rheumatology (ACR) criteria [21] and who had started MTX monotherapy for the first time between January 2005 and August 2010 (Table 1). Patient consent was in accordance with protocols approved by the respective institutional ethical committees of Kohnan Hospital (protocol H16.11.9) and Kyushu University (protocol 875). Only patients who underwent MTX monotherapy for at least 7 months were included, and they were prospectively followed for 3 years while receiving low-dose MTX monotherapy, unless disease was otherwise active and/or adverse events appeared. The dose of MTX used was allowed to vary according to disease activity. Patients who had already been receiving low-dose prednisolone (mean 5, median 5, range 2-10) per day were included and allowed to continue this dosing (Table 1), whereas patients who were just starting prednisolone were excluded. Exclusion criteria were disease activity score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR) >4.2 and clinical disease activity index (CDAI) >22, and/or the emergence of a significant adverse event: in these cases, additional therapies, including bDMARDs, were initiated. Patients were also assessed using the 2010 ACR/European League Against Rheumatism (EULAR) criteria [22].

Assessment
Disease activity was evaluated by joint counts, visual analog scale (VAS), morning stiffness, grip strength, Creactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), matrix metalloproteinase-3 (MMP-3), anti-citrullinated protein antibody (ACPA), disease activity score in 28 joints (DAS28), and the modified health assessment questionnaire (mHAQ). Patients were followed prospectively for 3 years until cessation of MTX monotherapy. Incomplete data from patients who had dropped out because of our exclusion criteria (n = 25) were assessed in two ways: (1) clinical endpoints were imputed as the last observation carried forward (LOCF) and (2) clinical endpoints were assessed as observed. We obtained similar results with both methods. We also followed the sequelae of patients who had dropped out, and observations that were applicable were described in the results. Radiographic evidence of progression was assessed in the hands and wrists and scored chronologically as recommended by Bruynesteyn et al. [23] using the TSS as previously described [24]. The change in TSS per year was evaluated from the baseline and annual evaluation of patients, and recorded as the TSS year-progression (ΔTSS). From these data, patients were classified into three groups: those with structural remission (REM) (ΔTSS ≤0.5), those with radiographic evidence of progression (ΔTSS >3), and those with radiographic evidence of rapid progression (RRP) (ΔTSS >5) [22]. Baseline and annual data were assessed in relation to radiographic evidence of progression to identify a factor that could predict which subgroup of patients may respond to treatment, i.e., patients who showed no radiographic evidence of progression,

Effect of low-dose MTX monotherapy on radiographic evidence of progression
Radiographic evidence of progression as determined by mean ΔTSS significantly decreased over the 3-year study period. Respective measurements (mean ± SD (n), median, statistical significance) were as follows: ΔTSS 0-1 year, 3.3 ± 5.1 (n = 161), 1.0, p <0.0001; ΔTSS 1-2 years, 2.7 ± 5.2 (n = 145), 1.0, p <0.0001; and ΔTSS 2-3 years, 2.2 ± 4.4 (n = 137), 0.5, p <0.0001. Cumulative probability plots showed that after 1 year of MTX monotherapy, 62/ 161 of the patients (38.5 %) were classified into the REM category, 55/161 (34.2 %) into ΔTSS >3, and 35/161 (21.7 %) into RRP (Fig. 2, upper). Among the 62 patients in the REM category, 2 (3.2 %) were clinically stable and moved to another hospital: subsequent follow up showed that these patients had inactive disease for the remaining 2 years. One other patient (1.6 %) with clinically active disease had MTX monotherapy stopped and infliximab (IFX) therapy initiated because the patient had a DAS28-ESR (3) >3.2. Among the 20 patients in the ΔTSS >3 but not RRP category, 3 patients (15.0 %) had active disease and thus IFX therapy was added. Two patients (10.0 %) were stable and moved to another hospital: subsequent follow up showed that they had inactive disease for the remaining 2 years. Among the 35 patients in the RRP category, 8 patients (22.9 %) had active disease and biologic agents were added to their regimen. One of these patients developed mandibular myelitis and MTX therapy was stopped for 4 months and then resumed.
During the second year of MTX monotherapy (years 1-2), 60/145 patients (41.4 %) were classified as REM, 37/145 (25.5 %) as ΔTSS >3, and 23/145 (15.9 %) as RRP (Fig. 2, middle). Among the 60 patients classified as REM, one patient (0.017 %) showed signs of liver injury and thus MTX therapy was stopped and tocilizumab Fig. 2 Cumulative probability plots for the change in van der Heijde modified total Sharp score per year (ΔTSS). Patient subgroups corresponding to structural remission (REM), radiographic evidence of progression (ΔTSS >3), or radiographic evidence of rapid progression (RRP) are indicated. Bio indicates a patient who began new treatment with a biologic agent. Ch indicates a patient who moved to another hospital because of inactive disease but continued methotrexate (MTX) treatment. Inf indicates a patient who developed infection, mandibular myelitis, and thus discontinued MTX treatment. Liv indicates a patient who had abnormal liver tests and thus discontinued MTX treatment. IP indicates a patient who developed interstitial pneumonitis and thus discontinued MTX treatment. St indicates a patient who developed stomatitis and thus discontinued MTX treatment (TCZ) was initiated. IFX was initiated in one other patient (0.017 %) due to the patient's personal preference, although the disease was inactive. Among the 14 patients classified as ΔTSS >3 but not RRP, disease became clinically active in one patient (7.1 %) and thus biologic agents were added. Another patient (with ΔTSS ≤3) developed interstitial pneumonitis and thus MTX was stopped and tacrolimus was initiated. Among the 23 patients with RRP, disease became clinically active in 3 (11.3 %) and thus biologic agents were added. One other patient (0.04 %) developed stomatitis and thus MTX therapy was stopped.
In summary, during the 3 years of MTX monotherapy, the percentage of patients classified into the REM group increased from 38.5 to 50.4 % (p = 0.0466), those in ΔTSS >3 decreased from 34.2 to 20.4 % (p = 0.0095), and those in RRP decreased from 21.7 to 10.9 % (p = 0.0190) (Fig. 2).
Search for a factor that predicts which patients will show no radiographic evidence of progression with MTX monotherapy We searched for a factor that might identify a subgroup of patients who did not have radiographic evidence of progression as assessed by ROC curve analysis. We found that patients with serum MMP-3 levels <103.7 ng/ml at baseline came to be classified in neither the ΔTSS >3 nor the RRP groups, with negative predictive values (NPV) of 88.7 and 96.8 %, respectively (Fig. 3). This cutoff level of 103.7 ng/ml was valid when male and female patients were studied separately, with an NPV of 88.1 % for ΔTSS >3 and 87.8 % for RRP, Fig. 3 Receiver operating characteristics curve analysis of a radiographic evidence of progression (van der Heijde modified total Sharp score year-progression (ΔTSS) >3)) and b radiographic evidence of rapid progression (RRP), showing that serum matrix metalloproteinase-3 (MMP-3) levels measured at the outset of methotrexate monotherapy can predict radiographic evidence of non-progression of disease. AUC area under the curve respectively (Additional file 1: Figure S2). We also noted that as the clinical course of therapy proceeded, lower serum MMP-3 levels predicted a subgroup that exhibited no radiographic evidence of progression: 98.0 ng/ml as measured after 1 year of MTX monotherapy with an NPV of 86.8 % for this 1-2 year period, and 68.8 ng/ml after 2 years of MTX monotherapy, with NPV of 93.2 % for the 2-3 year period (Fig. 4).

Discussion
Initial application of MTX monotherapy followed by combination therapy with bDMARDs, if necessary, is a reasonable therapeutic strategy for treating patients with early RA [25]. In the present study, we found that a substantial proportion of patients with RA fared well with just MTX monotherapy: they did not exhibit radiographic evidence of progression and did not require combination therapy. This result was in part due to the careful monitoring of patients and to the availability of other treatment options to individuals who exhibited active disease and/or adverse events. Nevertheless, it was somewhat surprising to find that approximately half of the patients who would have been considered to have a poor prognosis as assessed by the EULAR/ACR criteria [22] nevertheless had no radiographic evidence of progression for 3 years while being treated only with lowdose MTX monotherapy. The dose of MTX used was significantly lower than those used in other countries, due to national healthcare regulations unique to Japan at that time [26]. We considered the possibility that genetic factors unique to the Japanese population might underlie the high efficacy of this low-dose MTX monotherapy. However, our findings are consistent with those of O'Dell et al. [19] who reported that approximately 30 % of Caucasian patients fared well with MTX monotherapy and did not require combination therapy. The patients in our studies may be more typical of the patients encountered by clinicians in daily practice compared to those in the O'Dell study, as most patients had a significantly longer history of disease prior to entering the study (average 4.4 years). Many of these patients had previous exposure to other therapies including sulfasalazine (36 %) or bucillamine (38 %). Our observations may more closely represent the outcomes to be expected with low-dose MTX monotherapy in a typical real-life patient population, and indicates that MTX monotherapy may As to which patients might benefit most from MTX monotherapy and which would require additional therapy with biologic agents or others to halt progression as seen on radiography, we found that low serum MMP-3 measured at the outset of MTX monotherapy, was a good predictor of radiographic non-progression. We further noted that lower serum MMP-3 measured during the course of MTX monotherapy is also a good predictor. The results are in line with the finding of Ma et al., who found that continuously elevated serum MMP-3 predicts radiographic evidence of progression for 1 year in the patients treated with various cDMARDs in a treat-to-target (T2T) protocol [27]. Previous studies have shown that joint destruction is halted if all of the joint-destroying MMPs including MMP-3, MMP-9 and MMP-13 are normalized [28,29]. Although serum MMP-3 levels alone are a crude reflection of disease progression [30], they presumably reflect the levels of other joint-destroying MMPs as well, as for example shown in the collagen-induced arthritis model in mice [31]. These may explain why serum MMP-3, as determined by ROC curve analysis, is negatively correlated with later radiographic evidence of progression in the present study.
We also noted that clinically active disease emerged primarily in patients in the RRP group. Patients classified as REM or ΔTSS ≤3 seldom developed clinically active disease during the course of MTX monotherapy, and this was especially true among patients who remained in REM for more than 1 year of MTX monotherapy. These results support the rationale for the use of MTX monotherapy as a first choice to halt radiographic evidence of progression, unless or until disease becomes active and/or adverse events appear. This rationale is further supported by recent studies that suggest that MTX may even extend the life expectancy of patients with rheumatoid disease [32,33].

Conclusions
In conclusion, we found that structural remission and radiographic evidence of non-progression were respectively induced in 50.4 and 79.6 % of patients with rheumatoid disease treated continuously with MTX monotherapy, with an option to change to bDMARDs for 3 years. We also found that low serum MMP-3 measured at the outset of MTX monotherapy can be a good predictor of radiographic evidence of non-progression.