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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

Objective

To investigate whether baseline concentrations of one-carbon metabolism biomarkers are associated with treatment nonresponse and adverse events in rheumatoid arthritis (RA) patients receiving methotrexate (MTX).

Methods

A prospective derivation cohort (n = 285) and validation cohort (n = 102) of RA patients receiving MTX were studied. Concentrations of plasma homocysteine, serum vitamin B12, serum folate, erythrocyte vitamin B6, and erythrocyte folate were determined at baseline and after 3 months of treatment. Nonresponse after 3 months was assessed using the Disease Activity Score in 28 joints (DAS28) and the European League Against Rheumatism (EULAR) response criteria. Adverse events at 3 months were assessed using biochemical parameters and health status questionnaires. Analyses were corrected for baseline DAS28, age, sex, MTX dose, comedications, and presence of the methylenetetrahydrofolate reductase 677TT genotype.

Results

In the derivation cohort, the mean DAS28 scores at baseline and 3 months were 4.94 and 3.12, respectively, and 78% of patients experienced adverse events. This was similar between the 2 cohorts, despite a lower MTX dose in the validation cohort. Patients with lower levels of erythrocyte folate at baseline had a higher DAS28 at 3 months in both the derivation cohort (β = −0.15, P = 0.037) and the validation cohort (β = −0.20, P = 0.048). In line with these results, lower baseline erythrocyte folate levels were linearly associated with a 3-month DAS28 of >3.2 in both cohorts (derivation cohort, P = 0.049; validation cohort, P = 0.021) and with nonresponse according to the EULAR criteria (derivation cohort, P = 0.066; validation cohort, P = 0.027). None of the other biomarkers (levels at baseline or changes over 3 months) were associated with the DAS28 or treatment nonresponse. Baseline levels of the biomarkers and changes in levels after 3 months were not associated with incidence of adverse events.

Conclusion

A low baseline concentration of erythrocyte folate is associated with high disease activity and nonresponse at 3 months after the start of MTX treatment and could be used in prediction models for MTX outcome. None of the investigated one-carbon metabolism biomarkers were associated with incidence of adverse events at 3 months.

Methotrexate (MTX) is the cornerstone disease-modifying antirheumatic drug (DMARD) in the treatment of rheumatoid arthritis (RA). In significant numbers of patients, MTX fails to achieve adequate suppression of disease activity and induces adverse events, which impacts the ability to increase or even continue the therapeutic dose ([1]). Patients who do not respond to MTX or develop severe adverse events within 3 months after the start of MTX treatment are frequently treated with biologic agents, alone or in combination with MTX ([2]). The ability to predict MTX nonresponse and MTX-induced adverse events before the initiation of this DMARD treatment is paramount, since the first months following diagnosis represent a window of opportunity during which outcomes can be more effectively modulated by therapy ([3]). To ensure that only those patients who are nonresponsive to MTX receive early additional treatment with biologic agents, and those who are responsive to MTX are spared from treatment with costly biologic agents, it is necessary to identify nonresponders and patients prone to experience adverse events at baseline. In order to predict MTX nonresponse and occurrence of adverse events, risk factors for these outcomes should be identified ([4, 5]).

Earlier studies have examined clinical and genetic risk factors for MTX nonresponse in RA patients ([1, 6]). Besides clinical and genetic determinants, phenotypic markers (metabolites/proteins) could also be potential predictors of MTX nonresponse. MTX is a folate antagonist that uses the same transport mechanisms as folate ([7]). MTX, as well as food-derived folate or supplemented folates, are taken up intracellularly via solute carrier 19A1 and incorporated into the folate pathway (one-carbon metabolism). Inside cells, MTX inhibits key enzymes involved in one-carbon metabolism, and this mechanism is responsible for the therapeutic effects of MTX, as well as its adverse event profile. Important phenotypic markers of one-carbon metabolism, such as concentrations of plasma homocysteine, serum vitamin B12, serum folate, erythrocyte vitamin B6, and erythrocyte folate, may determine the extent of MTX nonresponse and MTX-related adverse events. Nevertheless, these biomarkers have rarely been studied as risk factors of MTX outcome ([8, 9]).

We therefore investigated whether these one-carbon metabolism biomarkers, measured at baseline, could be associated with MTX nonresponse and incidence of adverse events over 3 months of followup in a prospective cohort study of RA patients receiving MTX. We also validated our findings in an independent validation cohort of RA patients.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

Patients

Data from 2 prospective cohorts of RA patients, all of whom were white, were collected. The derivation cohort consisted of patients who were enrolled in the Treatment in the Rotterdam Early Arthritis Cohort (tREACH) study, which is a multicenter, stratified, single-blind clinical trial of patients with early RA, as previously described ([10]). The validation cohort consisted of patients with RA from the Methotrexate in Rotterdam, Netherlands (MTX-R) cohort. These latter patients were started on MTX treatment between January 2006 and March 2011 in the Department of Rheumatology of Erasmus University Medical Center (UMC) (Rotterdam, The Netherlands). The medical ethics committee of Erasmus UMC approved both studies, and patients gave their written informed consent before inclusion.

The derivation cohort included patients receiving MTX who fulfilled the American College of Rheumatology/European League Against Rheumatism (EULAR) 2010 criteria for RA ([11]). Patients in the validation cohort were included when diagnosed as having RA by the physician. Patients from the derivation cohort were started on an MTX dosage of 25 mg/week. The patients in this cohort were randomized to receive either MTX alone or cotreatment with sulfasalazine, hydroxychloroquine, and corticosteroids ([10]), whereas in the validation cohort, the dosage of MTX and comedications were chosen by the physician. In both cohorts, patients received folic acid (10 mg/week) during MTX treatment. All patients were assessed at baseline and after 3 months of treatment.

Biomarkers

Three research tubes of blood samples were obtained during every study visit, in addition to routine blood samples for determination of the erythrocyte sedimentation rate (ESR) and levels of C-reactive protein (CRP), alanine aminotransferase (ALT), leukocytes, and thrombocytes. One serum tube was centrifuged for 10 minutes at 1,700g at a temperature of 4°C, and the serum was divided into aliquots and stored at −80°C. One EDTA tube was immediately put on ice after collection and centrifuged for 10 minutes at 1,700g at a temperature of 4°C, and plasma and aliquots of cell pellets were stored at −80°C. One EDTA tube was kept at room temperature, and the whole blood was divided into aliquots and stored at −80°C.

The concentration of homocysteine was determined in EDTA–plasma using isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS; Waters Acquity UPLC Quattro Premier XE), by an adapted method ([12]). For chromatographic separation, a Waters Symmetry C8 column (2.1 × 100 mm) with a precolumn (Waters) was used. Concentrations of vitamin B12 and folate in the serum were measured using an electrochemiluminescence immunoassay (Modular E170; Roche). The concentration of vitamin B6 was measured in whole blood with an isotope-dilution LC-MS/MS assay, as described previously ([13]). For the erythrocyte folate assay, 100 μl whole blood was diluted with 1,600 μl of a 10 gm/liter ascorbic acid solution (pH 4) and incubated for 3 hours at room temperature. Tubes were centrifuged at 2,000g and analyzed with an electrochemiluminescence immunoassay for folate (Modular E170; Roche). The concentration of erythrocyte folate was measured in whole blood from the EDTA tube at room temperature within 24 hours after sample collection. The sustained stability of erythrocyte folate at room temperature for up to 24 hours has been proven in a previous study ([14]). The erythrocyte folate levels were corrected for those of serum folate and hematocrit.

Routine hematology parameters were measured using a Sysmex XE-2100 instrument, and the ESR was measured using a Sysmex InteRRliner. Routine chemistry parameters were measured on a Roche Modular P analyzer. Isolation of DNA and genotyping for the methylenetetrahydrofolate reductase (MTHFR) 677T allele was done using a previously described method ([5]).

Assessment of treatment nonresponse and adverse events

The primary outcome assessed was the Disease Activity Score in 28 joints (DAS28) with ESR (DAS28-ESR) ([15]), which was assessed at baseline and after 3 months of followup. In rheumatology practice, physicians assess disease activity levels using a DAS28 cutoff value of >3.2 to define active disease, and assess treatment response according to whether changes in disease activity meet the EULAR response criteria for RA ([16]); both of these are used as the decision points for continuing or stopping medication. Therefore, MTX nonresponse was defined as a DAS28 score of >3.2 and failure to meet the EULAR response criteria.

Specifically, the EULAR response criteria are based on an attained level of improvement in the DAS28 as well as extent of change in the DAS28 over a defined followup period. Patients are classified as either nonresponders, moderate responders, or good responders. In this study, we dichotomized the EULAR criteria into nonresponse versus moderate/good response. Of note, the EULAR response criteria allow assessment of only those patients whose DAS28 at baseline is ≥3.3.

Adverse events were assessed with biochemical and self-reported measures. Gastrointestinal symptoms, malaise, psychological disorders, MTX-related hepatotoxicity, MTX-related depression of bone marrow, and “other” adverse events were counted as an adverse event. Dichotomized categories of ≥1 adverse event (versus none) and ≥3 adverse events (versus ≤2) were also analyzed as outcome variables. The adverse event categories of gastrointestinal symptoms, malaise, psychological disorders, and “other” adverse events were all accumulations of different symptoms that were reported on the patient health status questionnaires. Gastrointestinal symptoms comprised diarrhea, vomiting, sickness, and abdominal pain. Malaise comprised fatigue, dizziness, headache, sleeplessness, and not feeling well. Psychological disorders involved depression and personality changes. “Other” adverse events comprised dyspnea, alopecia, infection, mucositis, epistaxis, and skin-related disorders. If a patient reported experiencing none of these symptoms over the followup and also did not meet the criteria for hepatotoxicity or bone marrow depression, the patient was scored as having no adverse event. Hepatotoxicity was defined as an ALT level >3 times the upper limit of normal. Bone marrow depression was defined as a leukocyte count of <3.0 × 109/liter or thrombocyte count of <100 × 109/liter.

We did not determine the percentage of patients with adverse events at baseline. The category of ≥3 adverse events was assigned to patients who experienced more than one symptom over the followup. For example, if a patient reported having the symptoms of diarrhea, vomiting, and sickness, he or she was scored as having gastrointestinal symptoms and as having ≥3 adverse events.

Statistical analysis

Statistical comparisons were made using Student's t-test, chi-square test, or Mann-Whitney U test, as appropriate. Multivariate regression analysis was used to examine the associations between biomarker levels at baseline and the change in biomarker levels and the DAS28 at 3 months. Results are expressed as the standardized beta coefficient. Logistic regression analysis was used for the dichotomous outcome measures of DAS28 >3.2 (versus DAS28 ≤3.2), nonresponse (versus moderate/good response) according to the EULAR criteria, and presence (versus absence) of adverse events. Results are expressed as the odds ratio (OR) with 95% confidence interval (95% CI).

If necessary, biomarker levels were normalized by transformation to their natural logarithm, to improve plots of the residual analyses. To examine whether there was a linear or nonlinear association, biomarkers were analyzed continuously, and concentrations were analyzed in quintiles according to ranges of values (from lowest levels in the first quintile to highest levels in the fifth quintile). To test whether there was a significant (P < 0.05) effect modification, interaction terms, defined as, for example, biomarker × comedication, were included in all multivariate models. If an interaction term was significant, analyses were stratified. Analyses were corrected for confounders, including age, sex, baseline DAS28, MTX dose, presence of the MTHFR 677TT genotype, use of other DMARDs, and use of corticosteroids ([6, 17]).

In addition, we investigated whether the results based on the DAS28-ESR, the routine outcome measure used in our studies, would be comparable to results assessed with the DAS28-CRP in the pooled cohort. We also examined the potential effect modification of the MTHFR 677 T allele genotype, by stratifying the significant biomarker–outcome associations by genotype in the pooled cohort. The potential effect modification was explored by defining the interaction term as significant biomarker × presence of MTHFR 677 T allele variants.

All statistical analyses were performed using the SPSS statistical package, version 20.0.0.1.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

Characteristics of the patients

Flow charts for the derivation cohort and validation cohort, with numbers of patients assessed for eligibility and reasons for dropping out, are given in Figure 1. The 3-month followup data from the derivation cohort were reported in an earlier study ([18]). For the present study, 285 patients from the derivation cohort were included at baseline, and 270 were still participating after 3 months. For the validation cohort, 102 patients were included at baseline, and 84 were still participating after 3 months. Table 1 shows the baseline characteristics of both cohorts. The mean MTX dosage was higher in the derivation cohort compared to the validation cohort. In the validation cohort, the DAS28 was lower, more patients were taking nonsteroidal antiinflammatory drugs, fewer patients were taking corticosteroids, and more patients had received MTX via subcutaneous injection when compared to patients in the derivation cohort.

image

Figure 1. Flow chart of the distribution of rheumatoid arthritis (RA) patients in the derivation and validation cohorts at baseline and followup. MTX = methotrexate; ACR = American College of Rheumatology; tREACH = Treatment in the Rotterdam Early Arthritis Cohort.

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Table 1. Baseline characteristics of the cohorts*
 Derivation cohort (n = 285)Validation cohort (n = 102)P
  1. IQR = interquartile range; ESR = erythrocyte sedimentation rate; CRP = C-reactive protein; MTHFR = methylenetetrahydrofolate reductase; VAS = visual analog scale; DAS28 = Disease Activity Score in 28 joints; MTX = methotrexate; NSAIDs = nonsteroidal antiinflammatory drugs; DMARDs = disease-modifying antirheumatic drugs.

Laboratory parameter   
Plasma homocysteine, median (IQR) μmoles/liter11 (10–14)12 (10–16)0.264
Serum vitamin B12, median (IQR) pmoles/liter290 (231–404)286 (230–376)0.588
Serum folate, median (IQR) nmoles/liter17 (13–24)17 (13–23)0.742
Erythrocyte vitamin B6, median (IQR) nmoles/liter80 (64–97)74 (64–102)0.485
Erythrocyte folate, median (IQR) nmoles/liter844 (662–1,165)1,079 (868–1,326)<0.001
Rheumatoid factor positive, %6641<0.001
Anti–cyclic citrullinated peptide antibody positive, %7041<0.001
ESR, median (IQR) mm/hour23 (13–40)19 (9–33)0.011
CRP, median (IQR) mg/liter8 (4–23)7 (3–14)0.444
MTHFR 677T allele, %53420.107
Clinical parameter   
Sex, % male30290.991
Age, mean ± SD years54 ± 1452 ± 160.299
Patient global assessment of general health on 100-mm VAS, mean ± SD53 ± 2254 ± 260.704
28–tender joint count, median (IQR)6 (3–10)4 (1–8)<0.001
28–swollen joint count, median (IQR)6 (3–10)3 (1–7)<0.001
DAS28, mean ± SD4.94 ± 1.154.26 ± 1.43<0.001
Medication   
MTX dosage, mean ± SD mg/week25 ± 115 ± 2<0.001
NSAIDs, %1436<0.001
Other DMARDs, %62570.408
Oral corticosteroids, %6211<0.001
Parenteral corticosteroids, %323<0.001
Subcutaneous MTX injections, %06<0.001

Biomarker concentrations

Baseline levels of the one-carbon metabolism biomarkers were comparable in both cohorts, with the exception of the baseline erythrocyte folate level, which was lower in the derivation cohort (median 844 nmoles/liter versus 1,079 nmoles/liter in the validation cohort; P < 0.001) (Table 1). Figure 2 shows all biomarker concentrations at baseline and at 3 months in both cohorts. The majority of one-carbon metabolism biomarker concentrations remained stable over time, apart from serum folate, whose concentration increased in both cohorts (derivation cohort, median 17 nmoles/liter at baseline versus 31 nmoles/liter at 3 months; validation cohort, median 17 nmoles/liter at baseline versus 26 nmoles/liter at 3 months) (each P < 0.001). The erythrocyte folate level increased only in the derivation cohort (median 844 nmoles/liter at baseline versus 940 nmoles/liter at 3 months; P = 0.023) (Figure 2).

image

Figure 2. Concentrations of one-carbon metabolism biomarkers at baseline (circles) and after 3 months of methotrexate treatment (triangles) in the 2 cohorts. Results are shown as the median with interquartile range.

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Treatment response

In both cohorts, disease activity decreased over time. In the derivation cohort, the mean ± SD DAS28 was 4.94 ± 1.15 at baseline and decreased to 3.12 ± 1.19 after 3 months. In the validation cohort, the DAS28 decreased from 4.26 ± 1.43 at baseline to 2.92 ± 1.23 at 3 months. When treatment response was determined according to the EULAR response criteria, 46% of patients were good responders, 38% were moderate responders, and 16% were nonresponders after 3 months of MTX treatment in the derivation cohort. In the validation cohort, 43% were good responders, 39% were moderate responders, and 18% were nonresponders. In comparing the derivation cohort with the validation cohort, the DAS28 (mean 3.12 versus 2.92; P = 0.174) and EULAR nonresponse rate (16% versus 18%; P = 0.879) were comparable after 3 months.

A lower baseline level of erythrocyte folate was associated with a higher DAS28 after 3 months (derivation cohort, β = −0.15, P = 0.037; validation cohort, β = −0.20, P = 0.048) (Table 2). In line with these results, the results of logistic regression analyses, in which the erythrocyte folate level (stratified into quintiles) was the independent variable and a DAS28 >3.2 was the dependent variable, showed a linear trend toward association in both the derivation cohort (P = 0.049) and the validation cohort (P = 0.021) (Figure 3). In summary, a low concentration of erythrocyte folate at baseline was associated with MTX nonresponse (a higher DAS28) at 3 months of treatment. There were no significant interaction terms.

Table 2. Linear regression analysis of associations between folate biomarker levels and the Disease Activity Score in 28 joints after 3 months of methotrexate treatment in both cohorts*
BiomarkerDerivation cohortValidation cohort
Standardized βPStandardized βP
  1. Analyses were corrected for age, sex, baseline Disease Activity Score in 28 joints, methotrexate dose, use of other disease-modifying antirheumatic drugs, use of corticosteroids, and presence of the methylenetetrahydrofolate reductase 677TT genotype.

Baseline    
Plasma homocysteine−0.070.3100.160.106
Serum vitamin B120.080.243−0.040.728
Serum folate−0.060.362−0.120.223
Erythrocyte vitamin B60.010.861−0.160.083
Erythrocyte folate−0.150.037−0.200.048
Change from baseline to 3 months    
Plasma homocysteine−0.010.8420.000.973
Serum vitamin B12−0.040.5410.080.430
Serum folate−0.010.924−0.060.520
Erythrocyte vitamin B6−0.040.5830.000.998
Erythrocyte folate0.020.7480.090.333
image

Figure 3. Logistic regression analyses of associations between biomarker concentrations (stratified as second to fifth quintiles, from lowest to highest levels) and a Disease Activity Score in 28 joints (DAS28) of >3.2 after 3 months of methotrexate (MTX) treatment in the 2 cohorts. Results are shown as the odds ratio (OR) with 95% confidence interval, relative to the first quintile (set as an OR of 1), corrected for age, sex, baseline DAS28, MTX dose, use of other disease-modifying antirheumatic drugs, use of corticosteroids, and presence of the methylenetetrahydrofolate reductase 677TT genotype.

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In the derivation cohort, there was a trend toward a linear association of the baseline erythrocyte folate level with nonresponse to treatment according to the EULAR criteria (DAS28 >3.2) at 3 months (P = 0.066), and in the validation cohort, this association was significant (P = 0.027). There were no associations between the baseline levels of homocysteine, vitamin B12, serum folate, or vitamin B6 and a EULAR-defined nonresponse to MTX at 3 months.

Except for erythrocyte folate, none of the other one-carbon metabolism biomarkers, measured at baseline, were associated with the DAS28 at 3 months (Table 2). Moreover, changes in the levels of any of the one-carbon metabolism biomarkers over time (from baseline to 3 months) showed no association with either the DAS28 or the treatment nonresponse at 3 months. We also performed the analyses of the association between erythrocyte folate levels and MTX response using 2 different versions of the DAS28, the DAS28-ESR and the DAS28-CRP, in the pooled cohort. Both results were comparable (DAS28-ESR, β = −0.15, P = 0.009; DAS28-CRP, β = −0.13, P = 0.038).

In the pooled cohort, the concentration of erythrocyte folate was associated with the DAS28 (β = −0.15). When the pooled cohort was stratified according to genotype, the association of erythrocyte folate levels with the DAS28 was similar between patients with the MTHFR 677CC genotype and those with the MTHFR CT genotype (β = −0.16 and β = −0.15, respectively). In the MTHFR TT stratum, the effect was smaller (β = −0.11). However, the interaction term (MTHFR 677CC versus MTHFR CT versus MTHFR TT × baseline erythrocyte folate) was not significant (P = 0.45). The interaction terms with the MTHFR 677 genotype, when divided into 2 categories (CC/CT versus TT and CC versus CT/TT × baseline erythrocyte folate), were also not significant (P = 0.31 and P = 0.26, respectively). Thus, there was no evidence of an effect modification of the MTHFR 677 genotype on baseline erythrocyte folate levels in this study.

Adverse events

Figure 4 shows the percentage of patients with adverse events in both cohorts. The percentage of patients with any adverse event over 3 months was comparable in both cohorts (78% in the derivation cohort versus 80% in the validation cohort). Only the percentage of patients with malaise after 3 months of MTX treatment was significantly higher in the validation cohort compared to the derivation cohort (49% versus 32%; P = 0.004). No significant associations between baseline levels of the one-carbon metabolism biomarkers and incidence of adverse events were found after 3 months. Furthermore, changes in the biomarkers between baseline and 3 months were also not associated with the occurrence of any adverse events.

image

Figure 4. Percentage of patients who reported experiencing no adverse events, ≥3 adverse events, or specific categories of adverse events over 3 months of followup in the 2 cohorts. Percentage values are shown over the bars.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

In RA patients, low baseline levels of erythrocyte folate were linearly associated with nonresponse to short-term MTX treatment in 2 independent cohorts. None of the one-carbon metabolism biomarkers were associated with incidence of adverse events over 3 months.

Our study is the first to demonstrate that a low level of erythrocyte folate at baseline is associated with nonresponse in 2 independent prospective cohorts of RA patients receiving treatment with MTX. We showed that the effect was similar between the derivation cohort and the validation cohort and was independent of the response criteria used. Erythrocyte folate levels have been associated before with MTX outcome in 2 cross-sectional studies in RA patients ([8, 9]). In these studies, a higher erythrocyte folate level was associated with higher disease activity. However, those patients were being treated with MTX and folic acid at the time of blood collection. Therefore, these associations could not be used for prediction of MTX outcome, since erythrocyte folate concentrations may be influenced by MTX competition and folate supplementation. In our cohorts, patients were not receiving folic acid or MTX at baseline. Studies on the effects of folic acid supplementation on the MTX response have reported either no effects ([19]) or a negative association ([20]). Taken together, these results suggest that lower concentrations of folate during MTX treatment facilitate higher effectiveness of MTX in the competition with folate for transporter proteins, polyglutamylation proteins, and target enzymes for MTX.

In contrast, we found that a lower baseline erythrocyte folate concentration was associated with MTX nonresponse in the 2 independent cohorts. A possible explanation for this finding may be that in individuals with lower concentrations of folate, the absorption, transportation, cellular uptake, and retention of folates may be less effective. Since MTX is structurally similar to folate and uses the same means of transportation and metabolism, patients with low baseline levels of intracellular folate may less easily accumulate MTX intracellularly during therapy. In this sense, measurement of the baseline erythrocyte folate level is a sort of functional assay for the body's capacity to accumulate and retain cellular folate, and thereby predicts how much MTX will be taken up and accumulated during therapy.

This hypothesis is supported in different ways. First, to test this hypothesis, we measured the total concentration of MTX in the erythrocytes of all patients in our 2 cohorts, using a recently described isotope-dilution LC-MS/MS assay ([21]). The median total MTX concentration after 3 months of MTX treatment was 130 nmoles/liter packed erythrocytes (interquartile range [IQR] 92–167) in the derivation cohort and 117 nmoles/liter packed erythrocytes (IQR 78–157) in the validation cohort. Patients with lower baseline erythrocyte folate concentrations achieved lower total erythrocyte MTX concentrations after 3 months of MTX therapy, and vice versa (β = 0.17, P = 0.011), when analyses were corrected for sex, age, MTX dose, MTX administration route, and cohort. In line with this result, others have also shown that erythrocyte folate levels were positively associated with erythrocyte MTX levels ([22]).

Second, in a recent study of patients with juvenile arthritis, we observed that a genetic polymorphism in the influx transporter solute carrier 19A1 was associated with a diminished response to MTX treatment, and efflux transporter polymorphisms were associated with an improved response ([23]). This finding underscores the need for effective uptake and cellular retention of MTX.

Levels of homocysteine and vitamin B12 were not associated with MTX outcome in this study. An earlier study also reported no association of the baseline homocysteine concentration or the 3-month change in homocysteine concentration with MTX response or toxicity in RA ([24]). An increased homocysteine concentration after MTX initiation was observed in earlier studies ([24-27]). A decrease in homocysteine levels was observed in groups of patients who received supplementation with folic or folinic acid ([24, 26]). In the present study, we observed no significant change in the homocysteine concentration between baseline and 3 months of treatment. This may be explained by the fact that all patients in our study received folic acid and MTX. The baseline levels of folate in the serum were not associated with MTX nonresponse in our study. We did find a significant increase in serum folate levels after 3 months. This can be explained by folic acid supplementation. The skewed distribution is probably a result of the combination of the short-elimination half-life of folic acid and the variation in time span between folic acid intake and sample collection. The increase in the erythrocyte folate levels in the derivation cohort could not be replicated in the smaller validation cohort. Erythrocytes live ∼3 months, and therefore the 3-month data could be diluted.

We observed no association between a lower erythrocyte vitamin B6 concentration and MTX nonresponse. However, earlier research showed that vitamin B6 levels are inversely associated with systemic markers of inflammation ([28]). In addition, mild vitamin B6 deficiency characterizes a subclinical at-risk condition in inflammation-related diseases ([29]). Patients with lower vitamin B6 concentrations could thus have higher levels of inflammatory disease and, accordingly, a higher chance of being a nonresponder. However, baseline vitamin B6 levels were not related to the baseline CRP level (P = 0.319) or baseline DAS28 (P = 0.755) in our study, and there was no significant association between the baseline vitamin B6 level and the DAS28 after 3 months.

The derivation cohort had lower baseline erythrocyte folate concentrations compared to the validation cohort. Higher disease activity will cause higher activity of the immune system, and this could have caused higher usage of folate and may also have influenced the body's capacity to accumulate cellular folate. This might explain the lower baseline erythrocyte folate level in the cohort with higher baseline disease activity. Erythrocyte folate levels increased after 3 months of therapy only in the derivation cohort. Patients in both cohorts received 10 mg/week folic acid. The patients in the derivation cohort had a lower erythrocyte folate concentration compared to the patients in the validation cohort. It is plausible that the erythrocyte folate levels in the patients in the derivation cohort increased to within the same range as those in the validation cohort after 3 months of treatment with 10 mg/week folic acid.

In a systematic review that included 3,463 RA patients who had received long-term treatment with MTX ([30]), the authors reported that 72.9% of patients experienced any adverse event, 30.8% had gastrointestinal adverse events, 18.5% developed liver toxicity, 5.5% developed central nervous system toxicity, and 5.2% had cytopenia. In our derivation and validation cohorts after 3 months of MTX treatment, the percentages of patients with any adverse event (78% and 80%, respectively), gastrointestinal symptoms (43% and 42%, respectively), psychological disorders (9% and 13%, respectively), malaise (32% and 49%, respectively), and other adverse events (35% and 24%, respectively) were slightly higher than the values reported in the systematic review. In contrast, the percentage of patients who developed hepatotoxicity (1% and 2%, respectively) and bone marrow depression (0% and 1%, respectively) after MTX treatment appeared to be lower in the present study.

There are some differences between the systematic review and our study. First, the adverse events reported herein were measured after 3 months of treatment, whereas in the systematic review, the adverse events were measured after a mean of 36.5 months (range 27–132 months). When we determined the incidence of these events after 9 months of treatment in our derivation and validation cohorts, we found that 14% and 31% of the patients, respectively, had experienced gastrointestinal symptoms and 4% in each cohort had experienced psychological disorders (results not shown), indicating that fewer patients had developed adverse events after long-term treatment.

Second, there were differences in dosages used. The mean MTX dosage in the systematic review was 8.8 mg/week, whereas in our cohorts, the mean dosages were 15 mg/week and 25 mg/week.

Third, the conditions reported as gastrointestinal symptoms in our cohorts (diarrhea, vomiting, sickness, and abdominal pain) differed from those in the systematic review (stomatitis, ulcer, abdominal pain, gastrointestinal bleed, dyspepsia, nausea, vomiting, diarrhea, weight loss, and appetite loss).

Fourth, the low incidence of hepatotoxicity and bone marrow depression in our cohorts could be attributed to our use of a strict definition of these toxicities. We used the definitions recommended by the Dutch Association of Rheumatologists, in which hepatotoxicity is defined as an ALT level 3 times the upper limit of normal, and bone marrow depression is defined as a leukocyte count of <3.0 × 109/liter or thrombocyte count of <100 × 109/liter. In the systematic review, liver toxicity was defined as an increase in the aspartate aminotransferase and/or ALT level above the upper limit of normal, and presence of cytopenia (defined as a decrease of >2 gm/dl in the hemoglobin level, or a platelet count of <150 × 109/liter, or a white blood cell count of <3.5 × 109/liter).

Most of the studies in the systematic review did not report or insufficiently reported the use of folic acid. However, a total coverage of folic acid use would probably only have resulted in fewer adverse events being reported in the systematic review, because another systematic review reported a 79% reduction in mucosal and gastrointestinal side effects with the use of folic acid (OR 0.21, 95% CI 0.10–0.44) ([19]). In summary, we acknowledge that there are some differences in the incidence of adverse events between the literature and our cohorts, but these could be attributed to the above-mentioned differences in definitions and population characteristics.

None of the investigated baseline biomarker levels were associated with occurrence of adverse events after 3 months of treatment. In contrast to this finding, 12 patients with juvenile arthritis who had a history of intolerance to MTX treatment were shown to have significantly lower cellular folate concentrations when compared to 81 patients who had never been treated with MTX ([31]). In addition, low-to-normal initial levels of plasma folate and red blood cell folate have been associated with the future toxicity of MTX in RA patients ([32]). In our cohorts, all patients were treated with folic acid. This treatment has been proven to reduce MTX-related adverse events in RA patients ([19]). This could have diluted the relationship between the investigated biomarker concentrations and adverse events in our study.

The percentages of patients with adverse events in the 2 cohorts were similar when the groups of patients were compared according to their different dosing schemes. A significantly higher percentage of patients experienced malaise in the validation cohort, although the MTX-dosing scheme was lower (15 mg/week) than in the derivation cohort (25 mg/week). Malaise is a subjective parameter that is collected in patient self-report questionnaires. The questionnaires in the derivation cohort had closed questions, such as “Were you tired last week?” In contrast, the questionnaires in the validation cohort had a more open character, such as “Write down all adverse events from last week.” With closed questions, one would suspect that the scores would be higher, because patients would be more aware of possible adverse events. However, this difference in questionnaire designs probably did not have an influence, since we observed a lower percentage of patients with malaise only in the group who responded to closed questions on the health status questionnaires. Taken together, these results indicate that a higher MTX-dosing scheme did not lead to more adverse events in our study.

There are some limitations to the present study. First, the hypothesis was based on the MTX working mechanism, and therefore MTX monotherapy would be ideal. However, more than one-half of the patients in both cohorts received other DMARDs in addition to MTX. These drugs can also cause a response in terms of modulating disease activity, and can produce similar side effects as those related to MTX. Therefore, we corrected all of the analyses for the use of other DMARDs. The corrected results were not significantly different from the uncorrected results.

Second, the difference in MTX dosage between the 2 cohorts was considerable (25 mg/week versus 15 mg/week; P < 0.001), but all of our analyses were done in the 2 cohorts separately. Nevertheless, the difference in MTX dosage within each cohort was minimal, with standard deviations of 1 mg/week in the derivation cohort (median 25 mg/week, range 10–25) and 2 mg/week in the validation cohort (median 15 mg/week, range 5–25). We corrected for the MTX dosage in all of our analyses.

Third, the levels of erythrocyte folate at baseline were not linearly associated with treatment nonresponse according to the EULAR criteria at 3 months (P = 0.066) in the derivation cohort, although there was a trend toward association. This might be explained by the smaller sample size of the derivation cohort, due to the restriction that only patients with a baseline DAS28 of ≥3.3 could be assessed when applying the EULAR response criteria.

Fourth, unfortunately, we did not register information on the time relationship between folate supplementation and administration of the MTX dose and blood sample withdrawal. Patients in both cohorts were advised to take folate supplementation 2 days after receiving the MTX dose. However, it would be extremely difficult to monitor in what way patients followed this instruction.

Moreover, the time between blood sample withdrawal and MTX dose or folate supplementation was not registered. The timing of folate supplementation and blood sample withdrawal could have an impact on the 3-month serum folate concentrations, but would have less impact on the erythrocyte folate concentrations at 3 months. We are most interested in predicting the clinical response at the start of treatment (baseline), prior to administration of any medication. Our results showed that the baseline erythrocyte folate level was predictive of the clinical response to MTX after 3 months of treatment. If the time between folate supplementation and blood sample withdrawal were to be standardized, it could be that there might be an effect of the 3-month serum folate concentration on disease activity. In contrast, the 3-month erythrocyte folate concentration did not show an effect on the DAS28 after 3 months (derivation cohort, β = −0.08, P = 0.283; validation cohort, β = −0.12, P = 0.260).

In conclusion, our study is the first prospective study to show that a lower baseline erythrocyte folate level was associated with nonresponse to MTX after 3 months of treatment, as measured according to the DAS28, in 2 independent cohorts. Thus, the baseline erythrocyte folate level may be a promising new biomarker for prediction models of MTX nonresponse. In contrast, baseline levels of plasma homocysteine, serum vitamin B12, serum folate, and erythrocyte vitamin B6 were unrelated to MTX nonresponse in RA. None of the investigated folate biomarkers were associated with occurrence of adverse events after 3 months.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. de Rotte had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. de Rotte, Pluijm, van Zeben, Lindemans, Hazes, de Jonge.

Acquisition of data. de Rotte, de Jong, Barendregt, van der Lubbe, de Sonnaville, Hazes, de Jonge.

Analysis and interpretation of data. de Rotte, de Jong, Pluijm, Ćalasan, Hazes, de Jonge.

Acknowledgments

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES

The authors thank all patients who are enrolled in the tREACH and MTX-R cohorts. Without their active cooperation, this study would not be possible. The tREACH trial involves the following rheumatology centers in The Netherlands: Erasmus UMC (Rotterdam), Sint Franciscus Hospital (Rotterdam), Maasstad Hospital (Rotterdam), Vlietland Hospital (Schiedam), Admiraal de Ruyter Hospital (Goes and Vlissingen), Zorgsaam Hospital (Terneuzen), and Albert Schweitzer Hospital (Dordrecht). The authors thank the following people from these centers for their contributions to the tREACH and MTX-R studies: R. Aartsen, C. Alfenaar, C. Alves, R. Arendse, M. Baak-Dijkstra, J. Bal-overzier, N. Basoski, S. Beer, E. den Boer, F. Bonte, R. Brouwer, H. Buijs, N. Buijs, E. Colin, R. Dolhain, C. Fleming, F. Fodili, A. Gerards, J. van Gorp, P. Griffioen, B. Grillet, B. Hamelink, K. Han, S. Heil, L. van Hove, M. Huisman, M. de Jager, S. Joziasse, P. Krijger, M. van Krugten, C. van Leeuwen, J. Luime, J. Nijs, B. Schaeybroeck, W. Schilleman, S. Schrauwen, T. Sutter, W. Verbree, A. van der Voordt, M. de Vroed, M. de Waart, M. Walter, A. Weel, H. Wintjes, B. van Zelst, and L. Zwang.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgments
  8. REFERENCES
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