Rheumatoid arthritis (RA) is characterized by synovial inflammation in multiple joints and irreversible joint destruction in the absence of adequate treatment 12. Among various disease-modifying anti-rheumatic drugs (DMARDs), methotrexate (MTX) has been the most widely used of these for RA. Moreover, new biological agents, such as TNF-α blockade and anti-CD20 monoclonal antibody, need a combination treatment with MTX to reduce the formation of neutralizing antibodies that can diminish the therapeutic efficacy 3.

Thus, unless patients have systemic conditions that make MTX treatment unfeasible, such as liver or interstitial lung disease, MTX is usually administered to most RA patients, either alone or with other DMARDs 45. Despite their potent efficiency, long-term use of MTX can induce serious adverse events such as hepatitis 6, although the development of liver cirrhosis due to MTX use has rarely been reported 7. Other DMARDs or non-steroidal anti-inflammatory drugs (NSAIDs) may not be free from concerns over these adverse effects. Particularly, leflunomide, which is newly developed and is often used in combination with MTX, has been reported to significantly increase the risk of liver toxicity potentially elevating liver enzyme levels, or inducing other serious diseases such as liver fibrosis 89.

From a clinical point of view, physicians may stop DMARDs or reduce their doses, when the levels of liver enzymes are highly elevated. However, drug-induced liver fibrosis can sometimes progress without changes of enzyme levels, or of structure on ultrasonography. In these cases, only liver biopsy can detect silent liver fibrosis, but it cannot be performed in all patients taking DMARDs, because it is invasive and unethical.

Recently, non-invasive liver stiffness measurement (LSM) using transient elastography (FibroScan^®, EchoSens, Paris, France) was introduced to assess the severity of liver fibrosis, and to screen the normal population for identifying people potentially at risk of underlying chronic liver disease 101112. So far, a few studies using transient elastography have shown a relationship between MTX use and liver fibrosis, but the association still remains controversial 131415. Furthermore, the combined effect of MTX and other DMARDs on liver fibrosis has not been described. Hence, in the present study, we assessed the correlation of the dose of MTX and silent liver fibrosis and investigated medication that correlated with abnormal liver stiffness measurement (LSM) using transient elastography in RA patients receiving MTX.

In clinical practice, if the results of liver-related laboratory tests are abnormal, the potential liver damage and progression of fibrosis can easily be spotted by physicians, and the drug regimen or treatment strategy can be altered. However, when patients with RA have no evidence of underlying chronic liver disease and have persistently normal liver-related laboratory results, the silent progression of liver fibrosis can be missed. Indeed, data are scarce on the prevalence of silent liver fibrosis, and on how to monitor or detect this adverse outcome due to long-term use of DMARDs by patients with RA and normal liver function. Furthermore, no significant relationship between Roenigk grading of liver fibrosis and quantitative results of liver-related laboratory tests was identified in a previous study of liver biopsies performed in 16 patients with RA and long-term use of MTX 18. Thus, in this cross-sectional study, we focused only on patients with RA who were not suspected of having underlying chronic liver disease, and found that leflunomide combined with MTX had a significant correlation with silent liver fibrosis.

So far, in addition to age, alcohol consumption, duration of RA, serum albumin level, obesity, and pre-existing pulmonary fibrosis, the cumulative dose and duration of MTX use have been reported as risk factors for histological fibrosis or cirrhosis in patients with RA receiving MTX 192021. Therefore, concerns related to hepatotoxicity of MTX, including elevated liver enzymes or progression of fibrosis, has limited physicians' use of MTX in RA patients with viral hepatitis or liver cirrhosis 22. Despite these reports, in this study, we found that the cumulative dose of MTX did not significantly correlate with LSM values, and was statistically equivalent between patients with normal and abnormal LSM values, similar to the results of previous studies 1314. Further, when we stratified our study population into two groups based on a previously proposed cutoff MTX cumulative dose of 4,000 mg 13, the proportion of patients with abnormal LSM values was equivalent between the groups (P = 0.572). Also, when we reanalyzed 52 RA patients receiving MTX but not leflunomide, we found that 9 patients (17.3%) had abnormal LSM values and there were no significant differences between patients with and without abnormal LSM values. However, these results did not suggest that the cumulative dose of MTX may not be related to silent liver fibrosis in RA patients. Our study design, based on the study of patients with normal liver function, without underlying chronic liver disease, and who had been exposed to MTX for more than 24 weeks, might have reduced the extent of the effect of MTX. To clarify this, a further study will be necessary, including RA patients regardless of the administration of MTX.

Meanwhile, leflunomide has been reported to increase the frequency of abnormal liver enzyme up to 19% 23. The currently recommended monitoring guidelines suggest leflunomide dose reduction or discontinuation when ALT levels are more than two to three times the normal level 24. However, no reports are available that propose practical guidelines for monitoring leflunomide hepatotoxicity in RA patients with normal liver function. In our study, we found that the cumulative dose of leflunomide correlated closely with LSM values, and could be used as an independent predictor for abnormal LSM values. We selected 53 patients (51%) who received both leflunomide and MTX, and obtained the cutoff for the leflunomide cumulative dose of 19,170 mg (5.3 years with 10 mg tablets or 2.6 years with 20 mg tablets), because in Korea, leflunomide is usually administered with MTX 25. Patients with a cumulative dose of leflunomide over 19,170 mg had a significantly higher risk of having an abnormal LSM value than those with less than 19,170 mg; the hazard ratio was 12.75. On the other hand, when we divided patients into two groups according to the presence of leflunomide and compared LSM values between the two groups, patients receiving MTX plus leflunomide (n = 53) had higher LSM values than those receiving MTX only (n = 52) (5.0 ± 2.2 vs 4.3 ± 0.9, P = 0.035). However, we found no significant difference in the frequency of abnormal LSM values according to the presence of leflunomide (P = 0.134). Thus, in RA patients receiving MTX and a cumulative dose of leflunomide over 19,170 mg (rather than the fact of its administration), we suggest that transient elastography be performed to for check silent liver fibrosis, even if the patient has normal liver function.

In our study, silent liver fibrosis was assessed using noninvasive transient elastography, instead of invasive liver biopsy. We defined 5.3 kPa as the cutoff for abnormal LSM values, which was adopted from a previous study that investigated the normal range of LSM values in healthy living liver and kidney donors in South Korea (5^th - 95^th percentiles for LSM 3.9 to 5.3 kPa) 10. Although our patients had normal liver function and were without chronic liver disease, their range of LSM values seemed slightly higher (5^th to 95^th percentiles 3.2 to 6.7 kPa) than healthy Koreans, potentially because of long-term use of DMARDs. A value of 5.3 kPa in our study seems relatively low to predict the presence of clinically significant liver fibrosis. However, this strict cutoff may draw physicians' attention to silent liver fibrosis in patients with RA receiving MTX and leflunomide, and encourage the adjustment of hepatotoxic medication doses to prevent irreversible liver fibrosis.

Our study has several issues. First, the lack of histological data is the main limitation, especially in patients with abnormal LSM values. Second, the cutoff LSM value of 5.3 kPa is not high enough to analyze the prevalence of clinically significant fibrosis 26. Further study with higher prevalence of high LSM values can overcome this issue. Third, because this study was cross-sectional, baseline LSM values prior to the initiation of DMARDs were not available. Finally, serial measurements of LSM for monitoring changes in the fibrotic burden were not available, in spite of its noninvasiveness. If future studies can serially measure LSM, they might reveal a dynamic correlation between LSM and differing doses of DMARDs, including MTX and leflunomide.

In this study, the cumulative dose of leflunomide correlated closely with the presence of silent liver fibrosis, reflected by abnormal LSM values, and it was the only independent predictor of abnormal LSM in patients with RA, who had received MTX over 24 weeks. However, further studies are required to investigate whether treatment regimens or strategies should be modified when abnormal LSM values are identified.

ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; AUROC: area under the receiver operator characteristic curve; BMI: body mass index; DMARD: disease-modifying anti-rheumatic drug; GGT: gamma-glutamyltranspeptidase; HDL: high density lipoprotein; INR: international normalized ratio; kPa: kilopascals; LSM: liver stiffness measurement; MTX: methotrexate; NSAID: non-steroidal anti-inflammatory drug; OR: odds ratio; RA: rheumatoid arthritis; TNF: tumor necrosis factor.

The authors declare that they have no competing interests.

All authors contributed to the study concept, design, and acquisition and interpretation of data. SWL, HP, SK performed the statistical analysis. SWL, SKL, SK and YB drafted and revised the manuscript. All authors have read and approved the manuscript for publication.