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Non-alcoholic steatohepatitis-like pattern in liver biopsy of rheumatoid arthritis patients with persistent transaminitis during low-dose methotrexate treatment

  • Shunsuke Mori ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    moris@saisyunsou1.hosp.go.jp

    Affiliation Department of Rheumatology, Clinical Research Center for Rheumatic Diseases, NHO Kumamoto Saishunsou National Hospital, Kohshi, Kumamoto, Japan

  • Nobuyuki Arima,

    Roles Data curation, Formal analysis, Investigation, Resources, Writing – original draft, Writing – review & editing

    Affiliation Department of Pathology, Kumamoto Shinto General Hospital, Kumamoto, Japan

  • Masahiro Ito,

    Roles Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Pathology, Clinical Research Center, NHO Nagasaki Medical Center, Omura, Nagasaki, Japan

  • Shigetoshi Fujiyama,

    Roles Data curation, Formal analysis, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    Affiliation Department of Gastroenterology and Hepatology, Kumamoto Shinto General Hospital, Kumamoto, Japan

  • Yasuhiro Kamo,

    Roles Data curation, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Gastrointestinal Endoscopy Center, Sasebo Chuo Hospital, Sasebo, Nagasaki, Japan

  • Yukitaka Ueki

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Rheumatic and Collagen Disease Center, Sasebo Chuo Hospital, Sasebo, Nagasaki, Japan

Abstract

Objective

The mechanism of liver injury with low-dose methotrexate (MTX) is incompletely understood. This study was designed to evaluate the association between non-alcoholic fatty liver disease (NAFLD) and liver injury during MTX treatment for rheumatoid arthritis (RA).

Methods

Between October 2014 and May 2015, we enrolled all MTX users for RA and monitored participant serum hepatic transaminase levels for 1 year. All patients had normal transaminase levels before the first MTX prescription. Using diagnostic criteria for non-alcoholic steatohepatitis (NASH), we performed histological analyses for patients presenting persistent transaminitis, defined as elevations of hepatic transaminases in four of six determinations during the follow-up period. Possible risk factors for persistent transaminitis were also examined.

Results

We followed 846 RA patients with a mean cumulative MTX dose of 2.48 g and identified 51 patients presenting persistent transaminitis. According to multivariate logistic regression analysis, obesity (odds ratio [OR] 3.23, p < 0.001), type 2 diabetes (OR 3.52, p = 0.001), hypercholesterolemia (OR 2.56, p = 0.004), and hyperuricemia (OR 3.52, p = 0.019), which are recognized as risk factors for NAFLD, were independently associated with a risk of persistent transaminitis. Among patients with persistent transaminitis, 42 showed fatty liver at ultrasonography. These patients had no evidence of alcoholic fatty liver, chronic viral hepatitis, autoimmune liver diseases, or hereditary liver diseases. Biopsy specimens were obtained from 32 patients, and we found that a NASH-like pattern was the most prevalent histological abnormality. There was no significant impact of MTX dose and duration on the histological severity.

Conclusion

Risk factors and histological findings are similar between NAFLD/NASH and liver injury during low-dose MTX treatment for RA, which suggests a strong association between both entities. NAFLD/NASH may be an underlying condition causing persistent transaminitis in MTX-treated RA patients. The results of this study illustrate the need for monitoring liver injury in RA patients with NAFLD risk factors during MTX treatment.

Introduction

Over the last decade, the management of rheumatoid arthritis (RA) has progressed substantially through early and aggressive intervention with disease-modifying antirheumatic drugs (DMARDs) [1, 2]. Methotrexate (MTX) is currently used worldwide as the DMARD of first choice in the treatment of early and established RA, even after the advent of biological molecular-targeted agents [35]. With the continued use of MTX, however, hepatotoxicity has been an important safety concern in RA patients [6]. A recent systemic review of the medical literature showed that the incidence of hepatic transaminase elevations during the first 3 years of low-dose MTX use for RA was 13 per 100 patient-years, with a cumulative incidence rate of 31% [7]. Another systemic review also showed that 20% of RA patients had at least one episode of elevated liver enzymes during an average of 4.6 years of low-dose MTX treatment [8]. Nevertheless, the mode of action underlying liver injury during MTX treatment is incompletely understood, and it remains unclear whether low-dose MTX can independently contribute to liver injury in these patients.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide [911]. Although its prevalence has been reported variously depending on the population studied and the definition used, a recent meta-analysis study indicated that the global prevalence of imaging-diagnosed NAFLD is estimated to be 25% [12]. NAFLD encompasses a large spectrum of pathological changes, ranging from simple steatosis (nonalcoholic fatty liver [NAFL]) to the more progressive form of NAFLD, namely, nonalcoholic steatohepatitis (NASH). NASH is defined in histological terms, namely, the presence of hepatic steatosis and inflammation with hepatocellular injury (ballooning degeneration) and varying degrees of fibrosis, which can progress to cirrhosis, liver failure, and hepatocellular carcinoma [911]. NASH patients, in particular those with advanced stages of fibrosis, have a higher risk of liver-related mortality than non-NASH NAFLD patients [1216]. Obesity, type 2 diabetes, hyperlipidemia, hypertension, hyperuricemia, and metabolic syndrome have been recognized as risk factors for NAFLD [12, 1719]. With the growing epidemics of these conditions, the prevalence of NAFLD is expected to increase dramatically. It is of note that several risk factors for NAFLD are also associated with liver injury in psoriasis patients receiving MTX treatment [2023]. In addition, recent studies using liver biopsies have shown that the pathological features of liver injury during MTX treatment resembled those of NASH in psoriasis patients, suggesting that NASH can predispose or contribute to liver injury in these patients [21, 23]. Regarding the association between NASH and liver injury observed in RA patients during MTX treatment, however, there is little information available.

In this study, we performed a 1-year follow-up for MTX-treated RA patients and identified patients who developed persistent transaminitis during MTX treatment. For these patients, the grade and severity of hepatic steatosis, inflammation, cellular degeneration, and fibrosis were evaluated according to four pathological criteria for NASH diagnosis. We also examined the association between the development of persistent transaminitis and clinical features, such as RA-related factors, cumulative dose of MTX, and NAFLD risk factors.

Patients and methods

Patients and study design

Between October 2014 and May 2015, we consecutively enrolled all MTX users for RA at outpatient clinics for rheumatic diseases at NHO Kumamoto Saishunsou National Hospital and Sasebo Chuo Hospital in Japan. Eligible patients were required to have normal levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at the time of first starting MTX treatment. We confirmed that all candidate MTX users had exhibited normal levels of ALT and AST at the initiation of this drug. Patients were also required to fulfil the 1987 American College of Rheumatology (ACR) criteria or the 2010 ACR/European League Against Rheumatism (EULAR) criteria for diagnosis of RA [24, 25]. The exclusion criteria were being under 18 years of age, use of hepatotoxic drugs except RA medications, or having acute hepatic disease.

For each patient, demographic characteristics, RA-related factors, such as RA duration, Steinbrocker’s stage, clinical disease activity index (CDAI), health assessment questionnaire (HAQ), a level of serum C-reactive protein (CRP), positivity of anti-cyclic citrullinated peptide antibodies (anti-CCP Abs), the use of biological agents, steroids, nonsteroidal anti-inflammatory agents (NSAIDs), and acetaminophen, together with the presence of NAFLD risk factors and comorbidities, including hypertension, type 2 diabetes, chronic kidney disease (CKD), smoking history, body mass index (BMI), a level of serum low-density lipoprotein cholesterol (LDL-C), and level of serum uric acid, were examined at enrollment. The definitions of hypertension, diabetes, and CKD were described elsewhere [26].

To identify patients who developed persistent transaminitis, we monitored participant serum ALT and AST levels at each visit (i.e., every 4–8 weeks) for 1 year. Persistent transaminitis was defined as elevations in ALT and AST levels above the upper limit of normal (ULN) in four of six determinations during the 1-year follow-up period. If serum levels of transaminases were increased by greater than threefold the ULN, MTX was discontinued [27], but monitoring of the enzymes was continued to the end of the follow-up period. All patients who were found to have persistent transaminitis underwent abdominal ultrasonography (US), together with autoantibody tests (antinuclear antibody [ANA] and anti-mitochondrial antibody M2). ULN values for ALT and AST used in this study were 30 IU/l. Patients who presented abnormal findings suggesting fatty liver or chronic liver disease such as hepatic fibrosis and cirrhosis at US were scheduled to undergo liver biopsy. The exclusion criteria for liver biopsy in this study were the presence of secondary causes of hepatic fat accumulation, including chronic viral hepatitis (hepatitis B and hepatitis C), significant ethanol intake (> 30 g/day for males and > 20 g/day for females), autoimmune liver diseases (autoimmune hepatitis and primary biliary cholangitis), and hereditary liver diseases [911]. Patients who were seropositive for autoantibodies but had no pathological evidence to diagnose autoimmune liver disease were not excluded from liver biopsy because the presence of autoantibodies is reported in one-quarter of patients with NAFLD. Liver biopsy is therefore required to rule out autoimmune liver disease in NAFLD patients with positive autoantibodies [28, 29].

Abdominal US and HRCT

Abdominal US and HRCT were performed and viewed in random order and independently by two board-certified experts in hepatology (SF and YK). Final decisions were made by consensus in the event of a disagreement. Fatty liver was diagnosed when all of the following four abnormal findings were observed on US scan: bright liver, hepatorenal contrast, vascular blurring, and deep attenuation [30]. Chronic liver disease such as fibrosis and cirrhosis was identified based on the following morphological abnormalities: irregular or nodular liver surface, coarse or non-homogeneous liver parenchymal echotexture, and blunted or rounded liver edge, [31]. HRCT findings were used to identify fatty infiltration in the liver and determine its severity. For quantitative evaluations, the liver-to-spleen attenuation ratio (L/S ratio) was calculated. Hepatic and splenic attenuation values were measured on unenhanced HRCT scans using four circular region-of-interest (ROI) cursors in the liver (two in the right lobe and two in the left lobe) and two in the spleen. Average attenuation values of the liver and spleen were calculated and used to determine the L/S ratio. We interpreted an L/S ratio < 1.1 as indicative of fatty deposition > 30% [32].

Liver biopsy

For assessment of the type and severity of liver injury, we performed US-guided liver biopsy. Each biopsy specimen was evaluated using four pathological criteria, namely, the Matteoni criteria [33], the Brunt criteria for grading and staging [34], the NASH Clinical Research Network (NCRN) pathologic criteria (NAS: NAFLD Activity Score) [35], and the Younossi criteria [36]. First, we made a differential diagnosis between NASH and non-NASH NAFLD (NAFL) based on the following histological findings: steatosis, lobular inflammation, centrilobular ballooning degeneration of hepatocytes, Mallory-Denk bodies, centrilobular pericellular/perisinusoidal fibrosis, and bridging fibrosis. In the Matteoni criteria, types 1 and 2 were diagnosed with non-NASH NAFLD (NAFL), and types 3 and 4 were defined as NASH. Grading of disease activity and staging of fibrosis were made based on the NAS system and the Brunt criteria. In the NAS system, liver biopsy specimens were scored separately for steatosis (grades 0‒3), lobular inflammation (grades 0‒3), hepatocellular ballooning (grades 0‒2), and fibrosis (stages 0‒4). The sum of grades for steatosis, inflammation, and ballooning was used as the NAS. Fibrosis was scored as follows: stage 1, perisinusoidal or periportal fibrosis; stage 2, perisinusoidal and portal/periportal fibrosis; stage 3, bridging fibrosis; and stage 4, cirrhosis. Similarly, in the Brunt criteria, the grading of NASH activity was performed according to the degree of steatosis, ballooning degeneration, lobular and portal inflammation (grades 1‒3), and the staging of fibrosis was categorized as follows: stage 1, zone 3 perisinusoidal/pericellular fibrosis; stage 2, the above pattern plus periportal fibrosis; stage 3, the above two patterns plus bridging fibrosis; stage 4, cirrhosis. Patients’ liver biopsies were reviewed in random order and independently by two board-certified experts in liver pathology (NA and MI). Both observers were blinded to the patients’ clinical status. Final diagnosis was determined by consensus if there was a disagreement between their interpretations.

History of RA medications

Cumulative doses of MTX and steroids (prednisolone equivalent) at the time of enrollment as well as at the time of liver biopsy were calculated for each user. In the present study, 85% of patients were first diagnosed with RA in our institutions and started MTX treatment. Information on weekly MTX dose and duration of treatment was collected from each division’s computer database. The remaining 15% of patients started MTX treatment at other hospitals, and we scrutinized weekly MTX dose and duration from data-providing documents from each patient’s treating doctor in these hospitals. We confirmed that 5 mg/week of folic acid was prescribed concomitantly during MTX therapy.

Ethics approval

This study was conducted in accordance with the principles of the Declaration of Helsinki (2008). The protocol of this study also meets the requirements of the Ethical Guidelines for Medical and Health Research Involving Human Subjects, Japan (2014), and has been approved by the Human Research Ethics Committee of NHO Kumamoto Saishunsou National Hospital (No. 26–04). Informed written consent was obtained from all participants.

Statistical analysis

In univariate analyses for comparisons of categorical variables, differences between patient groups were analyzed using the chi-square test or Fisher’s exact probability test. Continuous variables were assessed by the independent-measures t-test for comparisons between two patient groups and analysis of variance (ANOVA) with a post hoc Tukey’s honest significant difference (HSD) test for comparisons between three patient groups. Multivariate logistic regression analysis was performed to evaluate the association between persistent transaminitis as a dependent variable and a set of independent variables considered to be significant risk factors in univariate analyses. A backward stepwise selection procedure was used to select significant independent variables. The strength of association between persistent transaminitis and these independent variables was estimated using odds ratios (ORs) and 95% confidence intervals (95% CIs). In addition, the receiver operating characteristic (ROC) curve and the corresponding area under the curve (AUC) value were calculated to provide an index of validity for the multivariate logistic regression model. For all tests, probability values (p values) < 0.05 were considered to indicate statistical significance. All calculations were performed using PASW Statistics version 22 (SPSS Japan Inc., Tokyo, Japan).

Results

Comparisons of clinical characteristics between RA patients with and without presenting persistent transaminitis during the 1-year follow-up period

A total of 846 MTX users for RA (mean cumulative dose of 2.48 g) were consecutively enrolled in this study and monitored their serum ALT and AST levels for 1 year. All patients had received MTX treatment for at least 1 month at the time of enrollment. Among these patients, persistent transaminitis was found in 51 patients. Nine of the 51 patients discontinued MTX treatment during the follow-up period because of an increase in ALT/AST levels > 3 times the ULN, but enzyme levels continued to be above the ULN. As shown in Table 1, age (59.0 vs. 63.8 years, p = 0.007), RA duration (8.3 vs. 11.3 years, p = 0.02), obesity (BMI ≥ 25, 45.1 vs. 19.7%, p < 0.001), type 2 diabetes (25.5 vs. 10.1%, p = 0.002), hypercholesterolemia (LDL-C levels ≥ 140 mg/dl, 33.3 vs. 17.1%, p = 0.007), and serum uric acid levels (5.1 vs. 4.6 mg/dl, p = 0.01) were significantly different between patients with persistent transaminitis and those without. Rates of CKD, hypertension, and current/ex-smokers were similar between both groups. In addition, there was no significant association of persistent transaminitis with sex or RA-related indexes, including RA stage, CDAI, HAQ, serum CRP levels, or anti-CCP positivity.

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Table 1. Baseline characteristics of MTX users for RA who did or did not develop persistent transaminitis during follow-up.

https://doi.org/10.1371/journal.pone.0203084.t001

In terms of RA treatment, the mean cumulative dose of MTX was not significantly different between patient groups. In addition to MTX, 43.1% of patients were receiving a biological agent. Steroids and NSAIDs were used in 21.2% and 21.0% of patients, respectively. There were no significant differences in the rates of prescription of these drugs between the two groups. In addition, there was no significant difference in the mean cumulative dose of steroids between both groups. Acetaminophen was not prescribed to any patients.

The clinical and therapeutic characteristics of nine patients who discontinued MTX due to an increase in ALT/AST > 3 times the ULN are summarized in Table 2. All patients except case 9 had at least one of the following conditions: obesity, type 2 diabetes, hyperlipidemia, and hyperuricemia. Cumulative MTX dose and treatment duration varied among patients.

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Table 2. Summary of details from MTX users for RA who exhibited ALT/AST levels > 3 times the ULN during a 1-year follow-up period.

https://doi.org/10.1371/journal.pone.0203084.t002

Factors associated with the development of persistent transaminitis during MTX treatment for RA

Advanced age (≥ 65 years), RA duration, obesity, type 2 diabetes, hypercholesterolemia, and hyperuricemia were included, as independent variables, in multivariate logistic regression analysis. As shown in Table 3, obesity (OR 3.23, p < 0.001), type 2 diabetes (OR 3.52, p = 0.001), hypercholesterolemia (OR 2.56, p = 0.004), and hyperuricemia (OR 3.52, p = 0.019) were significant factors associated with the development of persistent transaminitis during MTX treatment for RA. Advanced age was negatively associated with this abnormality (OR 0.29, p < 0.001). The final model yielded an AUC-ROC of 0.74 (p < 0.001).

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Table 3. Factors associated with the development of persistent transaminitis during MTX treatment for RA.

https://doi.org/10.1371/journal.pone.0203084.t003

Histological patterns in RA patients with persistent transaminitis

Fig 1 shows a flowchart for the evaluation of persistent transaminitis observed during the follow-up period. Of 51 patients with persistent transaminitis, 4 were diagnosed with viral infection (hepatitis B or C), and 2 did not show abnormal US findings. The remaining 45 patients showed fatty liver (42 patients) or hepatic fibrosis (3 patients) at US examinations, but they did not have any evidence of alcoholic fatty liver, chronic viral hepatitis, autoimmune liver diseases or hereditary liver diseases. In addition, none of the 45 patients showed symptoms or laboratory abnormalities that would lead us to suspect a diagnosis of endocrine disorders. Among these patients, 1 had a contraindication to liver biopsy and 12 refused to undergo this procedure. The mean L/S ratio (standard error) on the HRCT scans of the 13 patients was 0.99 (0.04), suggesting the presence of fatty liver with deposition > 30% in these patients.

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Fig 1. Flowchart for examinations of persistent transaminitis observed during MTX treatment for RA.

*Secondary causes of fatty liver include significant ethanol intake (> 30 g/day for male and > 20 g/day for female), viral hepatitis (hepatitis B and hepatitis C), autoimmune liver disease (autoimmune hepatitis and primary biliary cholangitis), and hereditary liver diseases. RA, rheumatoid arthritis; MTX, methotrexate; NASH, non-alcoholic steatohepatitis; NAFL, non-alcoholic fatty liver; IFH, interface hepatitis; US, ultrasonography; HRCT, high-resolution computed tomography.

https://doi.org/10.1371/journal.pone.0203084.g001

The remaining 32 patients underwent liver biopsy (Fig 1). Histological findings of these patients are shown in Table 4. Among them, 22 patients were found to have steatosis, centrilobular ballooning degeneration, and centrilobular perisinusoidal fibrosis. Based on these findings, they were classified as Matteoni criteria type 4. We diagnosed these patients as having a NASH-like pattern (NASH-like patients). Histological findings of the other 10 patients did not meet the Matteoni criteria for NASH. Seven patients had steatosis without lobular inflammation or centrilobular ballooning, and they were classified as Matteoni criteria type 1. These patients were diagnosed as having a NAFL-like pattern (NAFL-like patients). The histological diagnosis of the remaining 3 patients was interface hepatitis. Among the patients who underwent liver biopsy, advanced stage of fibrosis (stage ≥ 3) was found in 15 patients (bridging fibrosis), but cirrhosis was not detected in any cases. Among 9 patients whose serum transaminase levels were increased by greater than threefold the ULN, 8 had a NASH-like pattern and 1 had a NAFL-like pattern (Table 2).

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Table 4. Characteristics of RA patients who underwent liver biopsy for histological assessment of liver injury during MTX treatment.

https://doi.org/10.1371/journal.pone.0203084.t004

Characteristics of RA patients with NASH- or NAFL-like pattern

As shown in Table 4, mean L/S ratios on HRCT scans at the time of liver biopsy were significantly different between NASH-like patients and NAFL-like patients (0.96 vs. 1.19, p = 0.018), which showed that the former group had more severe fatty deposition in the liver compared with the latter group. Data on L/S ratios prior to starting MTX therapy were available in approximately half of the patients in both groups (0.97 in NASH-like patients and 1.18 in NAFL-like patients). The data suggested that in NASH-like patients, fatty liver with fatty deposition > 30% had already existed at the time of introducing MTX treatment. In the NAS system, the disease activity grades for steatosis, lobular inflammation, and centrilobular ballooning were higher in NASH-like patients compared with NAFL-like patients (NAS, 3.9 vs. 0.1). The fibrosis stage was also more advanced in the former patients than the latter patients (stage ≥ 3, 59.1% vs. 14.3%). Antinuclear antibody was present in 72.7% of NASH-like patients and 57.1% of NAFL-like patients. One NASH-like patient was positive for anti-mitochondrial antibody M2. They did not, however, fulfill the diagnostic criteria for autoimmune hepatitis [37] or primary biliary cholangitis [38]. Similar levels of transaminases were observed in both patient groups. In addition, there were no significant differences in age, RA duration, BMI, serum levels of LDL-C or uric acid, or rates of hypertension or type 2 diabetes between these groups.

Regarding MTX use at the time of liver biopsy, there were no significant differences in means of cumulative dose (2.34 vs. 2.39 g) or duration (46.5 vs. 47.3 months) between NASH-like patients and NAFL-like patients. In addition, there was no significant difference in rates of steroid users or mean cumulative dose of steroids between the two groups.

Discussion

In this study, the development of persistent transaminitis was observed in 51 out of 846 RA patients during MTX treatment. According to the multivariate regression analysis, obesity, type 2 diabetes, hypercholesterolemia, and hyperuricemia were significant factors associated with the development of persistent transaminitis. There was no significant difference in the mean cumulative dose of MTX. Among the 51 patients with persistent transaminitis, 42 showed fatty liver at US examinations. These patients had no evidence of alcoholic fatty liver, chronic viral hepatitis, autoimmune liver diseases, or hereditary liver diseases. Biopsy specimens were obtained from 32 patients, and the NASH-like pattern and NAFL-like pattern were found in 22 and 7 patients, respectively. There was no significant difference in means of exposure duration and cumulative dose of MTX between the two groups. Advanced stages of fibrosis were seen in 15 patients.

Obesity and type 2 diabetes have been reported to exert an influence on the development of liver injury during MTX treatment, especially fibrosis, in psoriasis patients [2023]. As for RA patients, Kent et al. identified obesity and hyperlipidemia as risk factors for the permanent discontinuation of MTX because of AST elevations [39]. Other studies showed that elevations of hepatic aminotransferases were more likely to occur in MTX users with obesity and hyperlipidemia [40, 41]. In the present study, we identified obesity, type 2 diabetes, hyperlipidemia, and hyperuricemia as possible risk factors for persistent transaminitis during low-dose MTX treatment for RA. These factors are well recognized as risk factors associated with NAFLD [12, 1719]. Dawwas and Aithal reported that compared with individuals with other liver diseases, patients with end-stage MTX-related liver diseases had a similar risk factor profile to those with NASH requiring liver transplantation [42]. In fact, we showed that more than 42 (82%) out of 51 patients of patients with persistent transaminitis had fatty liver at US examinations. We also found that the NASH-like pattern was the most prevalent histological pattern in liver biopsy samples. The similarity of risk factor profiles and histological findings between NAFLD/NASH and liver injury during MTX treatment may support the notion that these two entities share a common pathogenesis in RA patients.

Individuals susceptible to liver injury during MTX treatment may have underlying NAFLD/NASH. In the present study, all participants had normal ALT/AST levels prior to first starting MTX treatment, but the L/S ratios on HRCT scans at that time suggested the presence of underlying fatty liver in patients who developed NASH-like lesions during MTX treatment. Furthermore, the nine patients with severe transaminitis discontinued MTX during the follow-up period, but enzyme levels continued to be above the ULN. The exacerbation of preexisting NAFLD may be an important mechanism of liver injury observed in MTX-treated RA patients. Using data from a large cohort of RA and psoriatic arthritis patients initiating DMARDs, Curtis et al. indicated that transaminase elevations were 2.8-fold more likely in psoriatic arthritis patients than RA patients [43]. This may be explained by a higher incidence of NAFLD in psoriasis patients receiving systemic therapy compared with RA patients on such therapy [44].

It is important to evaluate histological changes during MTX treatment, especially the degree of hepatic fibrosis, because NAFLD patients with advanced stages of fibrosis is are at high risk for liver-related mortality [1216]. Through liver biopsies, we found fibrosis in 24 (2.8%) out of 846 RA patients with a mean cumulative MTX dose of 2.48 g. Advanced fibrosis (≥ stage 3 in the Brunt criteria and the NAS system) was present in 15 patients (1.8%). No patients showed cirrhosis in liver biopsies. In a case-control study with 1571 MTX-treated patients with inflammatory arthritis, Quintin et al. performed liver biopsy on 41 patients who had elevated liver enzymes (mean cumulative MTX dose of 1.3 g). Fibrosis was detected in 22 patients (1.4%), but in most cases, it was mild [45]. In a systemic literature review, Visser and van der Heijde showed that after 4 years of MTX use with a mean cumulative MTX dose of 2.4 g, mild fibrosis (grade IIIA in the Roenigk scale [46]), severe fibrosis (grade IIIB), and cirrhosis (grade IV) were present in 15.3%, 1.3%, and 0.5% of biopsy specimens from RA patients, respectively. Pre-MTX biopsies, however, showed that mild fibrosis, severe fibrosis, and cirrhosis were preexisting in 9%, 0.3%, and 0.3% of RA patients [7]. By performing histological analysis of liver biopsy specimens from 42 RA patients, Ros et al. showed that 14% presented mild fibrosis before treatment with low-dose MTX, but after 4 years, no histological progression was observed [47]. These findings suggested that the development of advanced or severe hepatic fibrosis is uncommon during low-dose MTX treatment for RA. Similar data were reported in psoriasis patients [22, 48].

In this study, we monitored liver injury during MTX treatment through serial measurement of serum ALT and AST levels according to major rheumatology guidelines for the use of MTX in daily clinical practice for RA [27]. Previous studies reported that serial abnormal AST tests are significantly correlated with histological staging of liver injury in RA patients receiving MTX [49, 50]. Recent studies with NAFLD patients have shown that the usage of ALT and AST levels is not appropriate to screen for the presence of hepatic fibrosis, however, because transaminase levels are often normal in patients with such histological changes [5154]. Therefore, we cannot exclude the possibility that some MTX-treated patients without persistent transaminitis in this study could have developed hepatic fibrosis during the treatment. Although liver biopsy is currently the most reliable approach for identifying the presence of hepatic fibrosis, routine surveillance biopsies are not recommended for RA patients receiving traditional doses of MTX because of cost, sampling errors, or procedure-related morbidity or mortality [6]. US evaluation of chronic liver disease such as hepatic fibrosis and cirrhosis is a reliable and effective alternative to pathological diagnosis [55, 56]. Conventional US is also the most common imaging technique to detect fatty liver. In NAFLD patients, however, this modality cannot grade hepatic fibrosis and cannot differentiate necro-inflammation and fibrosis (components of NASH) from simple steatosis [57, 58]. Considering the histological similarity between NAFLD/NASH and liver injury in RA patients receiving MTX treatment, conventional US may not be an appropriate tool for monitoring hepatic fibrosis during this treatment. Transient elastrography (TE), a recently developed non-invasive technique based on US monitoring of liver stiffness, has shown promising results for assessing the severity of hepatic fibrosis in non-obese patients with NAFLD [59, 60]. TE may be useful as a monitoring tool for hepatic fibrosis during low-dose MTX treatment of inflammatory arthritis such as RA [61].

Our findings are subject to several limitations. First, this study was not for patients who first began MTX treatment at enrollment. Therefore, we may have missed patients who discontinued MTX before the enrollment. Persistent transaminitis justifying liver biopsy is, however, considered a relatively rare event in the first year of MTX treatment for RA [41]. Thus, if we focused on the incident MTX cohort, we could not have obtained enough liver biopsies to evaluate the incidence of NASH-like lesions in liver injury occurring during MTX treatment for RA patients. Second, we did not examine biopsy specimens before the introduction of MTX or from patients without persistent transaminitis. These data would be useful to explore whether there may be a causative relationship between NASH and liver injury during MTX treatment. For ethical reasons, however, it was impossible to perform liver biopsy for patients without any signs or symptoms of liver disease. Finally, we did not include MTX-untreated RA patients as controls in this study. To examine the role that low-dose MTX can play in the development of persistent transaminitis in patients with NAFLD risk factors, it would be necessary to perform a prospective follow-up study including MTX-untreated controls. However, it was ethically unacceptable, because current guidelines recommend that MTX be part of the first treatment strategy in patients with active RA [1].

Conclusions

Persistent transaminitis during low-dose MTX treatment is more likely to occur in RA patients with NAFLD risk factors, such as obesity, type 2 diabetes, hypercholesterolemia, and hyperuricemia. The NASH-like pattern is the most prevalent histological abnormality in RA patients with persistent transaminitis. Cumulative dose and duration seem unlikely to influence the severity of pathological findings. These findings suggest the strong association between NAFLD/NASH and liver injury observed in MTX-treated RA patients. NAFLD/NASH may predispose to the development of persistent transaminitis in RA patients during MTX treatment. The prevalence of NAFLD risk factors is rising in the general population and probably in RA patients and, consequently, the risk of liver injury associated with MTX treatment may also increase. The results of this study demonstrate the need for monitoring liver injury in RA patients with NAFLD risk factors during low-dose MTX treatment.

References

  1. 1. Smolen JS, Landewe R, Breedveld FC, Buch M, Burmester G, Dougados M, et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update. Ann Rheum Dis 2014;73(3):492–509. pmid:24161836
  2. 2. Singh JA, Saag KG, Bridges SL Jr., Akl EA, Bannuru RR, Sullivan MC, et al. 2015 American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Rheumatol 2016;68(1):1–26. pmid:26545940
  3. 3. Pincus T, Yazici Y, Sokka T, Aletaha D, Smolen JS. Methotrexate as the "anchor drug" for the treatment of early rheumatoid arthritis. Clin Exp Rheumatol 2003;21(5 Suppl 31):S179–185.
  4. 4. Braun J. Methotrexate: optimizing the efficacy in rheumatoid arthritis. Ther Adv Musculoskelet Dis 2011;3(3):151–158. pmid:22870474
  5. 5. Jacobs JW. Lessons for the use of non-biologic anchor treatments for rheumatoid arthritis in the era of biologic therapies. Rheumatology (Oxford) 2012;51 Suppl 4:iv27–33.
  6. 6. Kremer JM, Alarcon GS, Lightfoot RW Jr., Willkens RF, Furst DE, Williams HJ, et al. Methotrexate for rheumatoid arthritis. Suggested guidelines for monitoring liver toxicity. American College of Rheumatology. Arthritis Rheum 1994;37(3):316–328. pmid:8129787
  7. 7. Visser K, van der Heijde DM. Risk and management of liver toxicity during methotrexate treatment in rheumatoid and psoriatic arthritis: a systematic review of the literature. Clin Exp Rheumatol 2009;27(6):1017–1025. pmid:20149325
  8. 8. Salliot C, van der Heijde D. Long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research. Ann Rheum Dis 2009;68(7):1100–1104. pmid:19060002
  9. 9. Watanabe S, Hashimoto E, Ikejima K, Uto H, Ono M, Sumida Y, et al. Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. J Gastroenterol 2015;50(4):364–377. pmid:25708290
  10. 10. European Association for the Study of Liver (EASL), European Association for the Study of Diabetes (EASD), European Association for the Study of Obesity (EASO). EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease. J Hepatol 2016;64(6):1388–1402. pmid:27062661
  11. 11. Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, et al. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67(1):328–357. pmid:28714183
  12. 12. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016;64(1):73–84. pmid:26707365
  13. 13. Rafiq N, Bai C, Fang Y, Srishord M, McCullough A, Gramlich T, et al. Long-term follow-up of patients with nonalcoholic fatty liver. Clin Gastroenterol Hepatol 2009;7(2):234–238. pmid:19049831
  14. 14. Stepanova M, Rafiq N, Makhlouf H, Agrawal R, Kaur I, Younoszai Z, et al. Predictors of all-cause mortality and liver-related mortality in patients with non-alcoholic fatty liver disease (NAFLD). Dig Dis Sci 2013;58(10):3017–3023. pmid:23775317
  15. 15. Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 2015;149(2):389–397 e310. pmid:25935633
  16. 16. Dulai PS, Singh S, Patel J, Soni M, Prokop LJ, Younossi Z, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis. Hepatology 2017;65(5):1557–1565. pmid:28130788
  17. 17. Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G, Bellentani S. Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos nutrition and liver study. Hepatology 2005;42(1):44–52. pmid:15895401
  18. 18. Hamaguchi M, Kojima T, Takeda N, Nakagawa T, Taniguchi H, Fujii K, et al. The metabolic syndrome as a predictor of nonalcoholic fatty liver disease. Ann Intern Med 2005;143(10):722–728. pmid:16287793
  19. 19. Li Y, Xu C, Yu C, Xu L, Miao M. Association of serum uric acid level with non-alcoholic fatty liver disease: a cross-sectional study. J Hepatol 2009;50(5):1029–1034. pmid:19299029
  20. 20. Malatjalian DA, Ross JB, Williams CN, Colwell SJ, Eastwood BJ. Methotrexate hepatotoxicity in psoriatics: report of 104 patients from Nova Scotia, with analysis of risks from obesity, diabetes and alcohol consumption during long term follow-up. Can J Gastroenterol 1996;10(6):369–375. pmid:9193771
  21. 21. Langman G, Hall PM, Todd G. Role of non-alcoholic steatohepatitis in methotrexate-induced liver injury. J Gastroenterol Hepatol 2001;16(12):1395–1401. pmid:11851839
  22. 22. Berends MA, Snoek J, de Jong EM, van de Kerkhof PC, van Oijen MG, van Krieken JH, et al. Liver injury in long-term methotrexate treatment in psoriasis is relatively infrequent. Aliment Pharmacol Ther 2006;24(5):805–811. pmid:16918884
  23. 23. Rosenberg P, Urwitz H, Johannesson A, Ros AM, Lindholm J, Kinnman N, et al. Psoriasis patients with diabetes type 2 are at high risk of developing liver fibrosis during methotrexate treatment. J Hepatol 2007;46(6):1111–1118. pmid:17399848
  24. 24. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31(3):315–324. pmid:3358796
  25. 25. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO 3rd, et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010;69(9):1580–1588. pmid:20699241
  26. 26. Mori S, Yoshitama T, Hirakata N, Ueki Y. Prevalence of and factors associated with renal dysfunction in rheumatoid arthritis patients: a cross-sectional study in community hospitals. Clin Rheumatol 2017;36(12):2673–2682. pmid:28884373
  27. 27. Visser K, Katchamart W, Loza E, Martinez-Lopez JA, Salliot C, Trudeau J, et al. Multinational evidence-based recommendations for the use of methotrexate in rheumatic disorders with a focus on rheumatoid arthritis: integrating systematic literature research and expert opinion of a broad international panel of rheumatologists in the 3E Initiative. Ann Rheum Dis 2009;68(7):1086–1093. pmid:19033291
  28. 28. Adams LA, Lindor KD, Angulo P. The prevalence of autoantibodies and autoimmune hepatitis in patients with nonalcoholic Fatty liver disease. Am J Gastroenterol 2004;99(7):1316–1320. pmid:15233671
  29. 29. Yatsuji S, Hashimoto E, Kaneda H, Taniai M, Tokushige K, Shiratori K. Diagnosing autoimmune hepatitis in nonalcoholic fatty liver disease: is the International Autoimmune Hepatitis Group scoring system useful? J Gastroenterol 2005;40(12):1130–1138. pmid:16378177
  30. 30. Yajima Y, Ohta K, Narui T, Abe R, Suzuki H, Ohtsuki M. Ultrasonographical diagnosis of fatty liver: significance of the liver-kidney contrast. Tohoku J Exp Med 1983;139(1):43–50. pmid:6220488
  31. 31. Kumada S, Tanaka S, Okaniwa S, Ogawa S, Kojima T, Nakajima M, et al. Abdominal ultrasonic diagnosis in medical examinations: guidlines from the Japan Society of Ningen Dock, the Japanese Society of Gastrointestinal Cancer Screening, and the Japan Society of Ultrasonics in Medicine. Jpn J Med Ultrasonics 2015;42 (2):201–224.
  32. 32. Iwasaki M, Takada Y, Hayashi M, Minamiguchi S, Haga H, Maetani Y, et al. Noninvasive evaluation of graft steatosis in living donor liver transplantation. Transplantation 2004;78(10):1501–1505. pmid:15599315
  33. 33. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116(6):1413–1419. pmid:10348825
  34. 34. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94(9):2467–2474. pmid:10484010
  35. 35. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41(6):1313–1321. pmid:15915461
  36. 36. Younossi ZM, Stepanova M, Rafiq N, Makhlouf H, Younoszai Z, Agrawal R, et al. Pathologic criteria for nonalcoholic steatohepatitis: interprotocol agreement and ability to predict liver-related mortality. Hepatology 2011;53(6):1874–1882. pmid:21360720
  37. 37. Lohse AW, Hennes E. Diagnostic criteria for autoimmune hepatitis. Hepatol Res 2007;37 Suppl 3:S509.
  38. 38. Lindor KD, Gershwin ME, Poupon R, Kaplan M, Bergasa NV, Heathcote EJ, et al. Primary biliary cirrhosis. Hepatology 2009;50(1):291–308. pmid:19554543
  39. 39. Kent PD, Luthra HS, Michet C Jr. Risk factors for methotrexate-induced abnormal laboratory monitoring results in patients with rheumatoid arthritis. J Rheumatol 2004;31(9):1727–1731. pmid:15338491
  40. 40. Suzuki Y, Uehara R, Tajima C, Noguchi A, Ide M, Ichikawa Y, et al. Elevation of serum hepatic aminotransferases during treatment of rheumatoid arthritis with low-dose methotrexate. Risk factors and response to folic acid. Scand J Rheumatol 1999;28(5):273–281. pmid:10568423
  41. 41. Schmajuk G, Miao Y, Yazdany J, Boscardin WJ, Daikh DI, Steinman MA. Identification of risk factors for elevated transaminases in methotrexate users through an electronic health record. Arthritis Care Res (Hoboken) 2014;66(8):1159–1166.
  42. 42. Dawwas MF, Aithal GP. End-stage methotrexate-related liver disease is rare and associated with features of the metabolic syndrome. Aliment Pharmacol Ther 2014;40(8):938–948. pmid:25185870
  43. 43. Curtis JR, Beukelman T, Onofrei A, Cassell S, Greenberg JD, Kavanaugh A, et al. Elevated liver enzyme tests among patients with rheumatoid arthritis or psoriatic arthritis treated with methotrexate and/or leflunomide. Ann Rheum Dis 2010;69(1):43–47. pmid:19147616
  44. 44. Ogdie A, Grewal SK, Noe MH, Shin DB, Takeshita J, Chiesa Fuxench ZC, et al. Risk of incident liver disease in patients with psoriasis, psoriatic arthritis, and rheumatoid arthritis: a population-based study. J Invest Dermatol 2018;138(4):760–767. pmid:29104161
  45. 45. Quintin E, Scoazec JY, Marotte H, Miossec P. Rare incidence of methotrexate-specific lesions in liver biopsy of patients with arthritis and elevated liver enzymes. Arthritis Res Ther 2010;12(4):R143. pmid:20637063
  46. 46. Roenigk HH Jr., Auerbach R, Maibach HI, Weinstein GD. Methotrexate guidelines—revised. J Am Acad Dermatol 1982;6(2):145–155. pmid:7037877
  47. 47. Ros S, Juanola X, Condom E, Canas C, Riera J, Guardiola J, et al. Light and electron microscopic analysis of liver biopsy samples from rheumatoid arthritis patients receiving long-term methotrexate therapy. Scand J Rheumatol 2002;31(6):330–336. pmid:12492247
  48. 48. Aithal GP, Haugk B, Das S, Card T, Burt AD, Record CO. Monitoring methotrexate-induced hepatic fibrosis in patients with psoriasis: are serial liver biopsies justified? Aliment Pharmacol Ther 2004;19(4):391–399. pmid:14871278
  49. 49. Kremer JM, Furst DE, Weinblatt ME, Blotner SD. Significant changes in serum AST across hepatic histological biopsy grades: prospective analysis of 3 cohorts receiving methotrexate therapy for rheumatoid arthritis. J Rheumatol 1996;23(3):459–461. pmid:8832983
  50. 50. Erickson AR, Reddy V, Vogelgesang SA, West SG. Usefulness of the American College of Rheumatology recommendations for liver biopsy in methotrexate-treated rheumatoid arthritis patients. Arthritis Rheum 1995;38(8):1115–1119. pmid:7639808
  51. 51. Mofrad P, Contos MJ, Haque M, Sargeant C, Fisher RA, Luketic VA, et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 2003;37(6):1286–1292. pmid:12774006
  52. 52. Fracanzani AL, Valenti L, Bugianesi E, Andreoletti M, Colli A, Vanni E, et al. Risk of severe liver disease in nonalcoholic fatty liver disease with normal aminotransferase levels: a role for insulin resistance and diabetes. Hepatology 2008;48(3):792–798. pmid:18752331
  53. 53. Uslusoy HS, Nak SG, Gulten M, Biyikli Z. Non-alcoholic steatohepatitis with normal aminotransferase values. World J Gastroenterol 2009;15(15):1863–1868. pmid:19370784
  54. 54. Yoneda M, Imajo K, Eguchi Y, Fujii H, Sumida Y, Hyogo H, et al. Noninvasive scoring systems in patients with nonalcoholic fatty liver disease with normal alanine aminotransferase levels. J Gastroenterol 2013;48(9):1051–1060. pmid:23184095
  55. 55. Nishiura T, Watanabe H, Ito M, Matsuoka Y, Yano K, Daikoku M, et al. Ultrasound evaluation of the fibrosis stage in chronic liver disease by the simultaneous use of low and high frequency probes. Br J Radiol 2005;78(927):189–197. pmid:15730982
  56. 56. Colli A, Fraquelli M, Andreoletti M, Marino B, Zuccoli E, Conte D. Severe liver fibrosis or cirrhosis: accuracy of US for detection—analysis of 300 cases. Radiology 2003;227(1):89–94. pmid:12601199
  57. 57. Saadeh S, Younossi ZM, Remer EM, Gramlich T, Ong JP, Hurley M, et al. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology 2002;123(3):745–750. pmid:12198701
  58. 58. Golabi P, Sayiner M, Fazel Y, Koenig A, Henry L, Younossi ZM. Current complications and challenges in nonalcoholic steatohepatitis screening and diagnosis. Expert Rev Gastroenterol Hepatol 2016;10(1):63–71. pmid:26469309
  59. 59. Wong VW, Vergniol J, Wong GL, Foucher J, Chan HL, Le Bail B, et al. Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology 2010;51(2):454–462. pmid:20101745
  60. 60. Yoneda M, Yoneda M, Mawatari H, Fujita K, Endo H, Iida H, et al. Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with nonalcoholic fatty liver disease (NAFLD). Dig Liver Dis 2008;40(5):371–378. pmid:18083083
  61. 61. Rouhi A, Hazlewood G, Shaheen AA, Swain MG, Barber CEH. Prevalence and risk factors for liver fibrosis detected by transient elastography or shear wave elastography in inflammatory arthritis: a systematic review. Clin Exp Rheumatol 2017; 35(6), 1029–1036. pmid:28598786