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Nottingham Digestive Diseases Centre, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UKNational Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
Nottingham Digestive Diseases Centre, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UKNational Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
Nottingham Digestive Diseases Centre, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UKNational Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
Dept of Gastroenterology and Hepatology, Royal Surrey NHS Foundation Trust, Surrey, UKDepartment of Clinical and Experimental Medicine, FHMS, University of Surrey, Surrey, UKInstitute of Liver Studies, Kings College Hospital NHS Foundation Trust. London, UK
Nottingham Digestive Diseases Centre, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UKNational Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
The reported risk of significant liver fibrosis from methotrexate has led to intensive monitoring strategies being recommended by multiple guidelines.
•
This large multicentre study using liver stiffness and enhanced liver fibrosis score (ELF) demonstrated that the risk of long-term methotrexate therapy on liver fibrosis in patients with rheumatoid arthritis or psoriasis is lower than previously estimated.
•
The findings add to the existing literature on the need to improve patients’ metabolic risk factors that are significantly associated with liver fibrosis.
•
In patients with rheumatoid arthritis, transient elastography would be a more reliable tool to screen for liver fibrosis because ELF may be confounded by the degree of inflammation in the joints.
Abstract
Background
The risk of significant liver fibrosis from prolonged methotrexate (MTX) exposure has been estimated in around 5% of patients, which has led to intensive monitoring strategies. However, the evidence is derived from retrospective studies that underreported risk factors of liver disease. We evaluated the risk of long-term MTX therapy on liver fibrosis in a longitudinal cohort study using two non-invasive markers.
Method
Between 2014-2021, adult patients diagnosed with Rheumatoid Arthritis (RA) or psoriasis for ≥2 years were recruited prospectively from six UK sites. MTX group included patients who received MTX for ≥6 months, whereas unexposed group included those who never received MTX. All patients underwent full liver profiling, enhanced liver fibrosis (ELF) markers, and transient elastography (TE).
Results
999 patients (mean age 60.8 ± 12 years, 62.3 % females) were included. Of 976 with valid TE values, 149 (15.3 %) had liver stiffness ≥7.9 kPa. Of 892 with valid ELF, 262 (29.4 %) had ELF ≥9.8. Age and BMI were independently associated with elevated liver stiffness and ELF. Neither MTX cumulative dose nor duration was associated with elevated liver stiffness. Diabetes was the most significant risk factor associated with liver stiffness ≥7.9 kPa (adjusted OR = 3.19, 95% CI 1.95 – 5.20, P <0.001). Regular use of non-steroidal anti-inflammatory drugs showed the strongest association with ELF ≥9.8 (OR = 1.76, 95% CI 1.20 – 2.56, P =0.003), suggesting the degree of joint inflammation in RA may confound ELF as a non-invasive marker of liver fibrosis
Conclusion
The risk of liver fibrosis attributed to MTX itself might have been previously overestimated; there is a need to consider modifying current MTX therapy monitoring guidelines.
The study has received funding from the National Institute of Health Research Nottingham Digestive Diseases Biomedical Research Unit and Nottingham Biomedical Research Centre [BRC-1215-20003] and Innovative Medicines Initiative (IMI) 2 of the European Union and the European Federation of Pharmaceutical Industries & Associations (EFPIA): TransBioLine. This work has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 821283. This Joint Undertaking receives the support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Disclosures:
GP Aithal has received consulting fees from Pfizer, GlaxoSmithKline, Clinicpace, Servier Pharmaceuticals, NuCANA Plc, AstraZeneca and BenevolentAI paid to the University of Nottingham. A Abhishek reports institutional research grants from AstraZeneca and Oxford Immunotec, personal author royalties from UpTodate and Springer, personal consulting fees from Inflazome and NGM Biopharmaceuticals, and personal payments for lectures from Menarini Pharmaceuticals and Cadilla Pharmaceuticals, in the past 36 months and unrelated to the current work. RJ Aspinall has received consulting fees and speaker honoraria from Intercept, Novartis UK, Falk Pharma and Norgine UK.
Authors contributions
EA: writing original draft, formal analysis, resources and visualisation; JIG: writing original draft, data curation, resources, project management and supervision; CC: formal analysis, review and editing; EB-T: resources, review and editing; AA: resources, review and editing, SM: review and editing, resources, project management and supervision; KJ: review and editing, resources, project management and supervision; AA: review and editing, resources, project management and supervision; DH: review and editing, resources, project management and supervision; RJA: review and editing, resources, project management and supervision; GPA: conceptualization, funding and writing original draft
Data Transparency Statement
The data that support the findings of this study are available on reasonable request to the corresponding author.
Current guidelines recommend intensive (2-3 monthly) monitoring strategies for long-term methotrexate therapy due to the potential risk of liver fibrosis.
•
Evaluation of the association using two validated non-invasive markers of liver fibrosis, liver stiffness and enhanced liver fibrosis score, in a large cohort of patients with rheumatoid arthritis or psoriasis shows that the reported risk has been previously overestimated.
•
The clinical focus should be to improve patients’ metabolic risk factors, diabetes and BMI, that are independently associated with liver stiffness.
•
There is a need to consider modifying current methotrexate therapy monitoring guidelines.
Introduction
Methotrexate (MTX) has been widely used as a disease-modifying drug in the treatment of rheumatoid arthritis and psoriasis with or without arthritis for several decades. It is recommended by the National Institute of Clinical Excellence (NICE) as the first-line treatment for both newly diagnosed rheumatoid arthritis (RA) and adult patients with moderate to severe psoriasis who need systemic therapy
Rheumatoid arthritis in adults: management. National Institute for Health and Care Excellence. NICE guideline 2020 October 2020; Available from: www.nice.org.uk/guidance/ng100.
Methotrexate for rheumatoid arthritis. Suggested guidelines for monitoring liver toxicity. American College of Rheumatology. Arthritis Rheum.1994; 37: 316-328
Low incidence of hepatotoxicity associated with long-term, low-dose oral methotrexate in treatment of refractory psoriasis, psoriatic arthritis, and rheumatoid arthritis. An acceptable risk/benefit ratio.
Aithal, G.P., Hepatotoxicity Related to Methotrexate, in Drug-Induced Liver Disease, D.L. Kaplowitz N, Editor. 2013, Elsevier: London, UK, Walthan, MA and San Diego, CA.
The main clinical concern arises due to the potential risk of significant liver fibrosis with prolonged methotrexate exposure, which has been estimated to occur in approximately 5% of patients (range: 3.5-7%), with some reports linking fibrosis to total cumulative dose
Aithal, G.P., Hepatotoxicity Related to Methotrexate, in Drug-Induced Liver Disease, D.L. Kaplowitz N, Editor. 2013, Elsevier: London, UK, Walthan, MA and San Diego, CA.
. Systematic reviews in patients with psoriasis and RA highlight the discrepancy in the available evidence regarding the risk of significant fibrosis from long-term MTX therapy
. Furthermore, most studies that assessed the association between MTX and hepatotoxicity were at high risk of selection bias and under-reported the main risk factors for liver disease e.g. obesity, diabetes, and alcohol use
. Due to the lack of specific biomarkers, it is difficult to distinguish whether the liver injury is due to MTX exposure or other underlying risk factors of liver disease that can cause chronic liver injury and lead to fibrosis.
The influence of the underlying disease itself on liver fibrosis and clinical outcome in MTX-exposed patients has been investigated in multiple studies. A recent population-based study in patients treated with MTX showed that cutaneous psoriasis or psoriatic arthritis were independently associated with liver disease events and cirrhosis compared to RA
. However, whether there was an effect of MTX on the liver disease, and to what degree, was not determined. Over a 24 year period, only 0.07% of adults with liver failure who had been listed for liver transplantation in the USA were attributed, wholly or partly, to MTX therapy
Although liver biopsy remains the gold standard test to quantify and stage liver fibrosis, it is an invasive procedure. It carries significant risks, including bleeding and hospitalisation, with an overall rate of bleeding up to 7 days after biopsy of 6.5 per 1000 biopsies (95% CI 5.8-7.1)
. Therefore, multiple non-invasive markers of liver fibrosis have emerged, including liver stiffness measurement through transient elastography (TE) and measurement of enhanced liver fibrosis blood biomarker panel (ELF)
. These non-invasive markers are used to select patients for further assessment by biopsy. TE for liver stiffness has a high-performance characteristic for detecting advanced fibrosis in non-alcoholic fatty liver disease (NAFLD)
. ELF score combines the quantitative measurements of three serological markers, procollagen type III N-terminal peptide (PIIINP), tissue inhibitor of matrix metalloproteinase 1 (TIMP1) and hyaluronic acid (HA), in an algorithm to produce an ELF score
. Latter has been validated in large cohorts of patients with chronic liver diseases and showed a high accuracy to predict mortality and liver-related clinical outcomes
Therefore, we aimed to establish the association between MTX exposure and liver fibrosis in a large cohort study of patients with RA or psoriasis using the two validated non-invasive surrogate measures of liver fibrosis, liver stiffness and ELF score.
Methods
Patients included and methods
From June 2014 to September 2021, eligible adult patients with RA and/or psoriasis were recruited from six different sites in the UK (Bradford, Brighton, Cornwall, Nottingham, Portsmouth, and Sussex). Each site independently elected to participate and enrol patients through the UK Clinical Research Network following adoption of the current study into the portfolio of the National Institute for Health and Care Research. Eligible patients were at least 18 years old and had established diagnoses of RA or psoriasis (with or without psoriatic arthritis) based on clinical, immunological and radiological changes for at least two years. All patients followed the standard of care pathway with weekly MTX, and folic acid supplementation as directed by their care team where appropriate. Patients were classified into two groups based on their exposure to MTX. MTX group included patients receiving MTX for more than six months prior recruitment. The unexposed group included patients who had never received MTX (no-MTX). Patients with other dermatological or rheumatological conditions or pre-existing liver disease, except for NAFLD or alcohol-related fatty liver disease, were excluded. The study was conducted according to the Declaration of Helsinki (Hong Kong Amendment) and Good Clinical Practice (European guidelines) with all participants providing written informed consent. The study protocol was approved by the East Midlands Health Research authority (REC Ref: 14/EM/0145) in April 2014. Clinical data, age, sex, weight, height, body mass index (BMI), waist circumference, diabetes, hyperlipidaemia, hypertension, alcohol consumption and detailed medication history were recorded at enrolment. The study did not include investigations to screen for hepatic steatosis; however, patients who are at risk of metabolic dysfunction-associated fatty liver disease (MAFLD) were identified using the international expert consensus criteria
In patients who were receiving MTX, dose and duration were recorded. Changes in dose over time were taken into account based on patients’ MTX monitoring charts and medication records and the total cumulative dose was calculated by the sum of all doses taken.
Liver investigations
On the day of recruitment, all patients had liver stiffness measurement through TE, and blood tests were taken for a full serological liver profile and ELF markers. The liver profile includes liver enzymes (aspartate transaminase level (AST), alanine transaminase level (ALT), gamma glutamyl-transpeptidase level (GGT), total bilirubin), and complete metabolic, virology and autoimmune serology (full blood count, urea and electrolytes, clotting profile, lipids, HbA1C, ferritin, alpha-1 antitrypsin, caeruloplasmin, HBsAg, Anti HCV, autoantibodies and immunoglobulins). ‘Elevated ALT’ was defined as above upper limit of normal (ULN), 45 IU/L.
Liver stiffness was estimated using TE (FibroScan®, Echosens, Paris, France) as previously described
. All patients had ten validated measures and IQR <30% of median liver stiffness. The cut off of 7.9 kPa was used to rule out advanced fibrosis, and 11.5 kPa to rule in cirrhosis, based on previous work in biopsy-proven NAFLD patients
. Assays of HA, PIIINP, and TIMP-1 were performed on an Immuno-1 autoanalyser at Nottingham University Hospitals using the manufacturer's reagents, and ELF score was calculated in accordance with the manufacturer's instructions (Siemens Healthineers). We used the manufacturer's thresholds, 9.8 to rule out advanced fibrosis and 11.3 to rule in cirrhosis, that have been shown to correlate with clinical outcomes in a large cohort of patients with mixed chronic liver disease with up to 7-year follow up
Demographic and clinical data were described using descriptive statistics, mean ± standard deviation (SD) for continuous measurements that are normally distributed, median and inter-quantile range (IQR) for non-normally distributed continuous variables and frequencies and percentiles for categorical data. Patients' pathological and clinical characteristics were compared using the Chi-square test for categorical variables or Fisher's exact test when one or more expected cell counts were less than 5. For continuous variables, Student's t-test was applied. For continuous outcome variables exhibiting a skewed distribution, they were transformed using the natural logarithms before t tests were conducted to satisfy the prerequisite assumptions of normality. P <0.05 was considered statistically significant. Correlation between liver fibrosis markers was done using Spearman's rank correlation. Multivariable logistic regression analysis was performed, including all variables that showed statistically significant association in the univariable analysis. We considered age, sex, diabetes, BMI and alcohol >14 units/week as priori confounders and were included in the final models regardless of their effect. Multivariable linear regression models were performed (fibrosis markers as continuous variables) in the exposed and unexposed groups using box-cox transformation of the dependent variables. Multivariable analyses were performed using MTX cumulative dose and MTX duration as independent variables in separate models. To study the independent influence of the diagnosis, we excluded patients with both RA and psoriasis from the multivariable analysis. Separate regression analyses were performed taking the risk of MAFLD as a single metabolic risk factor following its diagnostic criteria. All analyses were conducted using R programme version 4.0.3
The total number of patients recruited was 1024. Twenty-five patients (2.4%) were excluded from the analysis as they did not meet the inclusion criteria at the time of enrolment (11 patients in the unexposed group (no-MTX) previously received MTX and 14 patients in the MTX group had less than 6 months of exposure prior to recruitment). After exclusion, 999 patients were included in the analysis (876 exposed to MTX and 123 unexposed), as shown in Figure 1. Distribution of patients recruited across the sites is summarised in Supplementary Table 1.
The demographic and clinical characteristics of the 999 patients analysed are summarised in Table 1. A summary of medications taken in each group is shown in Supplementary Table 2. Patients who received MTX were older (P <0.001), predominantly females (P <0.01), and more often diagnosed with RA (P <0.001). In contrast, the unexposed group were more likely to drink alcohol > 14 units/week (P <0.001) and have received regular non-steroidal anti-inflammatory drugs (NSAIDs) (P = 0.01) and metformin (P = 0.02). There was no significant difference in ethnicity; most participants were white. The difference in the metabolic risk factors between groups (type 2 diabetes, dyslipidaemia, hypertension, BMI and MAFLD) was not statistically significant.
Table 1Demographics and clinical features of exposed (MTX) and unexposed (no-MTX) patients.
Characteristics
MTX (n = 876)
No-MTX (n = 123)
P
Age (years), mean (SD)
61.6 (11.6)
55.6 (13.5)
<0.001
Female, n (%)
560 (63.9)
62 (50.4)
0.004
Diagnosis, n (%)
<0.001*
o
RA
615 (70.2)
55 (44.7)
o
Psoriasis
241 (27.5)
67 (54.5)
o
Both
20 (2.3)
1 (0.8)
Ethnicity n (%)ro
0.09*
o
White
825 (94.2)
118 (95.9)
o
Black
6 (0.7)
0
o
Mixed Asian
0
1 (0.8)
o
South Asian
32 (3.7)
3 (2.5)
o
Asian
0
1(0.8)
o
Other
10 (1.1)
0
o
Unknown
3 (0.3)
0
Type 2 Diabetes n (%)
100 (11.5)
21 (17.1)
0.08
Unknown
5 (0.6)
0
Hyperlipidaemia n (%)
225 (25.9)
28 (22.8)
0.46
Unknown
7 (0.8)
0
Hypertension n (%)
296 (33.8)
36 (29.3)
0.32
BMI (kg/m2), mean (SD)
29.9 (6.7)
30.9 (7.5)
0.19
Waist circumference (cm), mean (SD)
99 (16.6)
103.1 (17.4)
0.01
MAFLD, n (%)
686 (78.3)
101 (82.1)
0.33
Alcohol > 14 units/week, n (%)
83 (9.5)
25 (20.3)
<0.001
oNot reported
5 (0.7)
0
MTX exposure, median (Q1, Q3)0
o
Dose (mg)
15 (12.5, 20)
NA
o
Duration (months)
72 (36, 132)
NA
o
Total cumulative dose (g)
4.8 (2.16, 7.95)
NA
Table 1. P values were derived from Pearson's Chi-squared for categorical variables and Student’s t-test for continuous variables. *Fisher's Exact Test was applied because one or more expected cell counts in the cross-tabulation were less than 5. Abbreviations: BMI: body mass index, MAFLD: metabolic dysfunction-associated fatty liver disease, MTX: methotrexate, RA: rheumatoid arthritis.
There was no significant difference in liver enzymes or AST/ALT ratio between the groups, as shown in Table 2. Distribution of ALT in exposed and unexposed patients is illustrated in Supplementary Fig. 1.
Table 2Liver enzymes in exposed (MTX) and unexposed (no-MTX) patients.
Table 2. P values were derived from Pearson's Chi-squared for categorical variables and Student’s t-test for the natural logarithms of continuous variables. Abbreviations: ALT: alanine transaminase, AST: aspartate aminotransferase, ULN: upper limit of normal (45 IU/L).
Out of 989 with ALT reported, 134 patients (13.5%) had elevated ALT, >45 IU/L (ULN). In MTX group, 112 out of 866 (12.9 %) had elevated ALT compared to 22 out of 101 in the unexposed group (17.9 %), P = 0.13. In the MTX group, patients with psoriatic arthritis (PsA) were more likely to have elevated ALT > 45 IU/L compared to RA (19 out of 99 PA (19.2%) compared to 65 out of 615 R A (10.6%), P = 0.01). However, there was no significant association between the type of arthritis and elevated ALT in multivariable analysis, Supplementary Table 3.
Non-invasive markers of liver fibrosis
Liver stiffness using TE
Liver stiffness from 23 patients (2.3%) could not be reliably obtained, so they were excluded from the analysis. Among the 976 patients with reliable liver stiffness, the median value of liver stiffness was 4.9 kPa (IQR 3.9 - 6.5), and 149 patients had liver stiffness ≥ 7.9 kPa (15.3%). Patients who were unexposed to MTX had higher median liver stiffness than those exposed (P = 0.049). Although a higher proportion of unexposed patients had liver stiffness ≥ 7.9 kPa, this difference did not reach statistical significance P = 0.08 (Table 3). Nonetheless, 14 unexposed (11.6%) met the cut off for cirrhosis compared to 47 exposed (5.5%), P = 0.01.
Table 3Liver stiffness in exposed (MTX) and unexposed (no-MTX) patients.
TE results
MTX (n = 855)
No-MTX (n = 121)
P
Liver stiffness (kPa), median, (Q1, Q3)
4.9 (3.9, 6.3)
5.3 (3.9, 6.8)
0.049
Liver stiffness groups, n (%)
0.08
o Low <7.9 kPa
731 (85.5)
96 (79.3)
o High ≥ 7.9 kPa
124 (14.5)
25 (20.7)
o Cirrhosis (≥ 11.5 kPa)
47 (5.5)
14 (11.6)
0.01
Table 3. P values were derived from Student’s t-test for the natural logarithm of liver stiffness and Pearson's Chi-squared for categorical variables.
In univariable analysis, factors that were significantly associated with elevated liver stiffness ≥ 7.9 kPa were male sex, psoriasis, BMI, diabetes, hyperlipidaemia and hypertension, with MTX duration showing a protective effect (Table 4, Supplementary Table 4). The use of metformin was not independently associated with elevated liver stiffness after adjusting for diabetes status. In multivariable analyses, neither MTX cumulative dose nor duration had a significant association (Table 4, Supplementary Table 4). Diabetes showed the strongest independent association with liver stiffness ≥ 7.9 kPa (adjusted OR = 3.19, 95% CI 1.95 – 5.20, P < 0.001). Other factors that showed significant association were age (P = 0.04), male sex (P = 0.02) and BMI (P < 0.001). When the risk of MAFLD was used as a single metabolic predictor in regression models, neither MTX cumulative dose nor duration were associated with elevated liver stiffness (Supplementary Tables 5 and 6). MAFLD showed the strongest association with elevated liver stiffness (adjusted OR = 2.73; 95% CI 1.58 - 5.08, P < 0.001). In this model, the association between psoriasis and elevated liver stiffness was statistically significant (adjusted OR = 1.76; 95% CI 1.19 – 2.60, P = 0.004).
Table 4Factors associated with elevated liver stiffness ≥ 7.9 kPa.
Factors
Unadjusted OR
P
Adjusted OR
95% CI
P
MTX cumulative dose
0.96
0.06
0.99
0.95 - 1.03
0.68
Age
1.003
0.63
1.02 *
1.00 - 1.04
0.04
Sex (Male)
1.56*
0.01
1.62 *
1.07 - 2.45
0.02
Psoriasis
1.74 **
0.003
1.51
0.98 - 2.32
0.06
BMI
1.13***
<0.001
1.13 ***
1.10 - 1.17
<0.001
Type 2 Diabetes
5.25***
<0.001
3.19 ***
1.95 - 5.20
<0.001
Hyperlipidaemia
1.97***
<0.001
1.23
0.77 - 1.94
0.37
Hypertension
2.33***
<0.001
1.34
0.87 - 2.06
0.18
Alcohol (>14 units/week)
0.76
0.37
0.68
0.33 - 1.32
0.28
* p<0.05 ** p<0.01 *** p<0.001
Table 4. Univariable and multivariable logistic regression model of liver stiffness ≥ 7.9 kPa in the whole population (MTX cumulative dose was used as the independent variable). Abbreviations: BMI: body mass index, CI: confidence interval, MTX: methotrexate, OR: odds ratio.
There was no statistically significant difference in procollagen type III N-terminal peptide (PIIINP), hyaluronic acid (HA) or ELF score between exposed and unexposed patients (Table 5). ELF score showed a weak correlation with liver stiffness (Spearman's rank correlation rho=0.22, 95% CI 0.16 – 0.29, P<0.001).
Table 5ELF scores in exposed (MTX) and unexposed (no-MTX) patients.
ELF fibrosis score
MTX (n = 876)
No-MTX (n = 123)
P
PIIINP (ug/L), mean (SD) (Values missing for 70 exposed and 14 unexposed)
8.42 (4.24)
8.74 (4.06)
0.44
HA (ug/L), median (Q1, Q3) (Values missing for 85 exposed and 18 unexposed)
51.89 (30.79, 89.92)
49.21 (26.11, 109.07)
0.76
ELF score, mean (SD) (Values missing for 89 exposed and 18 unexposed)
9.32 (0.98)
9.28 (0.96)
0.1
ELF groups, n (%)
0.16
o
Low < 9.8
562 (71.4)
68 (64.8)
o
High ≥ 9.8
225 (28.6)
37 (35.2)
o
Cirrhosis (≥11.3)
23 (2.9)
3 (2.9)
0.97
Table 5. P values were derived from Student’s t-test for PIIINP, HA and ELF scores, and the chi-square test for ELF groups. Abbreviations: ELF: enhanced liver fibrosis, HA: hyaluronic acid, PIIINP: procollagen type III N-terminal peptide.
Out of 892 patients with ELF score results, 28.6% of exposed patients had ELF ≥ 9.8 compared to 35.2% in the unexposed, and 2.9% of patients from each group had ELF ≥ 11.3 suggesting cirrhosis. However, there was no significant difference between groups (Table 5).
In the univariable analysis, factors that were associated with elevated ELF ≥9.8 were MTX cumulative dose, MTX duration, age, RA, hypertension and regular use of NSAIDs. In multivariable analysis, regular use of NSAIDs showed the strongest association with elevated ELF (P = 0.003), Table 6. When MTX duration was used as the independent variable, factors that were associated with elevated ELF were age, BMI and regular NSAIDs (Supplementary Table 7).
Table 6Factors associated with elevated ELF score ≥ 9.8
Factors
Unadjusted OR
P
Adjusted OR
95% CI
P
MTX cumulative dose
1.05***
<0.001
1.04 *
1.01 - 1.07
0.02
Age
1.06***
<0.001
1.07 ***
1.05 - 1.09
<0.001
Sex (Male)
1.17
0.30
1.15
0.83 - 1.60
0.39
Psoriasis
0.63**
0.007
0.87
0.60 - 1.26
0.47
BMI
1.003
0.72
1.03 *
1.01 - 1.06
0.01
Type 2 Diabetes
1.49
0.07
1.25
0.78 - 1.99
0.35
Hyperlipidaemia
1.27
0.14
Hypertension
1.66***
<0.001
1.09
0.77 - 1.54
0.62
Alcohol > 14 units
0.74
0.24
0.75
0.44 - 1.26
0.29
Regular NSAIDs
1.47*
0.02
1.76 **
1.20 - 2.56
0.003
* p<0.05 ** p<0.01 *** p<0.001
Table 6. Univariable and multivariable logistic regression model of ELF score ≥ 9.8 in the whole population (MTX cumulative dose as the independent variable). Abbreviations: BMI: body mass index, CI: confidence interval, MTX: methotrexate, NSAIDs: non-steroidal anti-inflammatory drugs, OR: odds ratio.
Enhanced liver fibrosis test in patients with psoriasis, psoriatic arthritis and rheumatoid arthritis: a cross-sectional comparison with procollagen-3 N-terminal peptide (P3NP).
British Journal of Dermatology.2017; 176: 1599-1606
, sensitivity analysis on patients with RA and psoriasis separately was performed. It showed that MTX cumulative dose, duration and regular NSAIDs were associated with elevated ELF >9.8 only in patients with RA which suggests that the association seen may be due to active arthritis rather than liver fibrosis (Supplementary Tables 8–11). When MAFLD was used as a single metabolic risk factor in regression models, it was not associated with elevated ELF whereas regular NSAIDs had the strongest association (Supplementary Tables 12 and 13).
Secondary analysis
We have performed a secondary analysis using linear regression models in each cohort to avoid potential selection bias that could have been generated due to an imbalance between the groups. Multivariable linear regression models in patients exposed to MTX showed results consistent with previous findings in all patients (Supplementary Tables 14–17). Age and BMI showed a significant linear relationship with liver stiffness and ELF in patients exposed to MTX. In the unexposed group, BMI and diabetes were significantly associated with liver stiffness but not with ELF (Supplementary Tables 18–19).
In patients with arthritis (RA or PsA) on prolonged MTX therapy, the type of inflammatory arthritis was not associated with elevated liver stiffness or ELF in multivariable logistic regression models (Supplementary Tables 20 and 21).
Liver biopsy
All recruited patients with elevated liver stiffness ≥7.9 kPa or ELF ≥9.8 were offered a liver biopsy to establish the histological fibrosis stage when suitable. However, most patients declined or were considered unsuitable for liver biopsy due to frailty. In addition, some patients underwent a liver biopsy as part of clinical care to investigate elevated liver enzymes. In total, liver biopsy was performed in 26 patients (22 exposed and 4 unexposed), as described in Table 7. In unexposed patients, the histology was in keeping with non-alcoholic steatohepatitis (NASH) in two patients, and autoimmune hepatitis and seronegative primary biliary cholangitis (PBC) in each of the others. Among the 22 patients exposed to MTX who had a liver biopsy, histology showed features of NASH in all patients. Out of these, 12 patients had at least fibrosis grade ≥F3 according to the Metavir score
In this multicentre large longitudinal cohort study involving about 1000 patients with psoriasis or rheumatoid arthritis, we have demonstrated that neither MTX cumulative dose (median 4.8 g) nor duration of exposure (median of 6 years) was associated with liver fibrosis using two non-invasive markers, liver stiffness and ELF score. Our results are consistent with two other studies that showed no association between MTX cumulative dose and elevated liver stiffness
Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study.
. Laharie's cohort study involved patients with a variety of inflammatory diseases, and their median liver stiffness was 4.6 kPa compared to 4.9 kPa in our study population
Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study.
. Furthermore, the latter study included 390 patients exposed to a median dose of only 1.3 g over 1.8 years and reported 6% of their patients had significant fibrosis, based on liver stiffness >7.9 kPa, compared to 14.7% in our study using the same threshold. However, our included patients were older and had significantly more risk factors for liver disease (BMI, type 2 diabetes, and higher alcohol intake) which might explain the higher proportion of patients with elevated liver stiffness. Diabetes was the most significant independent risk factor associated with elevated liver stiffness in our study, in addition to age, male sex, and BMI. In contrast, only BMI and alcohol consumption were associated with elevated liver stiffness in the Laharie et al.
Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study.
. The lack of association between MTX cumulative dose and liver fibrosis has been previously observed in retrospective studies involving patients with PS exposed to MTX using histology (n=71)
. Single centre studies involving patients with RA as well as inflammatory bowel disease (IBD) on MTX have described similar findings although all of these enrolled a very small number of patients (n=46-185)
Evaluation of liver fibrosis by transient elastography (Fibroscan®) in patients with inflammatory bowel disease treated with methotrexate: a multicentric trial.
Scandinavian Journal of Gastroenterology.2012; 47: 575-579
. The insufficient sample size of these previous studies and lack of unexposed group limited their power to identify independent risk factors associated with fibrosis through multivariable modelling.
PIIINP has been used for determining liver fibrosis in methotrexate therapy for many years
Aithal, G.P., Hepatotoxicity Related to Methotrexate, in Drug-Induced Liver Disease, D.L. Kaplowitz N, Editor. 2013, Elsevier: London, UK, Walthan, MA and San Diego, CA.
; this marker forms one of the three components of the ELF score. While ELF score has been recommended by the National Institute for Health and Care Excellence for the non-invasive detection of advanced fibrosis in NAFLD
Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease.
. Important limitations of PIIINP as a diagnostic test on its own include its lack of specificity to the liver and its association with arthritis and disease activity
. Our study showed no significant difference in PIIINP levels between patients exposed and unexposed to MTX and raises the question of its role in monitoring of liver fibrosis in these groups of patients and the cost-effectiveness of serial measures every three months.
In fact, a retrospective cohort study of patients with psoriasis treated with MTX of which 27 underwent liver biopsy showed that serial ELF score measurements had possibly superior diagnostic accuracy than serial PIIINP measures to detect fibrosis
. However, we found ELF score was similar among patients exposed and unexposed to MTX. In multivariable analyses, cumulative dose of MTX was associated with increase of ELF score. However, in sensitivity analysis, the association between MTX cumulative dose and ELF was only apparent in patients with RA. This is consistent with a recent study in RA patients, and a cross sectional study that showed the highest proportion of increased ELF score was seen in RA patients
Enhanced liver fibrosis test in patients with psoriasis, psoriatic arthritis and rheumatoid arthritis: a cross-sectional comparison with procollagen-3 N-terminal peptide (P3NP).
British Journal of Dermatology.2017; 176: 1599-1606
. Nonetheless, the association seen might reflect disease severity and inflammation at joints (rather than liver fibrosis) due to increase collagen turnover in inflammatory arthritis and hence, an increase of PIIINP. Although disease severity scores for psoriasis and RA were not captured consistently as part of our study, regular use of NSAIDs probably reflects disease activity in our study population. Regular use of NSAIDs was the most significant independent risk factor associated with elevated ELF ≥9.8, in addition to age and BMI. Severe disease activity has been shown to correlate with ELF (adjusted OR 5.850, 95% CI 1.740-19.673) in a cross-sectional study of psoriasis and RA patients with no significant difference between different medication subgroups, including MTX
Enhanced liver fibrosis test in patients with psoriasis, psoriatic arthritis and rheumatoid arthritis: a cross-sectional comparison with procollagen-3 N-terminal peptide (P3NP).
British Journal of Dermatology.2017; 176: 1599-1606
; however, the particular study did not evaluate liver fibrosis using liver stiffness. Similarly, a recent study in a RA cohort showed an association between cumulative dose of MTX and ELF, but not with liver stiffness. Furthermore, the Disease Activity Score for 28 joints (DAS-28) scale had the strongest correlation with ELF (Pearson correlation coefficient r = 0.51, P<0.001)
. Inflammation markers, such as high-sensitivity C-reactive protein (hs-CRP), were not priori markers included in this study; however, their association with PIIINP and ELF biomarkers could be investigated in future studies.
In multivariable analysis, using all risk factors, the type of disease was not significantly associated with liver fibrosis using both non-invasive fibrosis markers. Even in patients with arthritis exposed to MTX, the type of arthritis (RA compared to PsA) did not influence significant liver fibrosis using non-invasive markers. However, when metabolic risk factors were combined according to MAFLD criteria
, psoriasis was independently associated with elevated liver stiffness but not with ELF. This could be explained by merging all the metabolic risk factors in one variable, MAFLD status, it is assumed that they have similar effect on elevated liver stiffness. However, the degree of association between the different metabolic risk factors and liver stiffness varied, as shown in Table 4. A large population-based study showed that diagnosis differentially influence liver disease risk in the setting of methotrexate use independent of risk factors; patients with psoriasis were at high risk of cirrhosis and liver-related events compared to those with RA
. Nonetheless, the study did not adjust for BMI, which is a crucial risk factor and was associated with elevation of both liver fibrosis markers in our data.
The existing evidence indicates that prolonged MTX exposure does not influence worse clinical outcomes. In a meta-analysis of 32 randomised controlled trials of MTX versus comparator in adults with rheumatoid arthritis, psoriasis and inflammatory bowel disease showed that exposure to MTX was not associated with risk of liver failure, cirrhosis, or death (RR: 0.12, 95%CI 0.01–1.09)
. Moreover, in a population-based cohort of RA patients with chronic hepatitis B with more than six years of follow up, there was no increased risk of cirrhosis with long-term MTX use
Our study included the largest number of patients from multiple centres to date to assess the association between MTX exposure and liver fibrosis. In addition, our study has multiple strengths, including study design and a detailed characterisation of risk factors for liver disease that was lacking in previous studies. We used both liver stiffness and ELF score, two of the most validated non-invasive biomarkers of liver fibrosis accessible internationally, to investigate the association between MTX and liver fibrosis.
Despite its strengths, our study has a few limitations. The cut off points used for liver stiffness and ELF score are not validated specifically in patients with psoriasis/RA, but instead were extrapolated from the literature. Because the recruitment of patients in multiple centres was not consecutive, this might have generated selection bias, especially in the unexposed group which was smaller than the exposed group. Clinicians may have referred patients unexposed to MTX with risk factors of liver disease to obtain a liver fibrosis assessment (referral bias). We tried to correct for these potential biases by adjusting for risk factors of liver disease in the multivariable analysis and performing a secondary analysis on each group which demonstrated similar results.
Our study included a low number of liver biopsies. Biopsies are generally performed in only a selected patient sub-group when a non-invasive marker stratifies patients as being at high risk of having severe liver fibrosis. The use of a surrogate fibrosis marker such as liver stiffness can be considered a valuable alternative approach in a large population.
In conclusion, we found no association between MTX cumulative dose or duration and liver stiffness in patients with RA or psoriasis. This indicates that the risk of liver fibrosis due to MTX itself might have been previously overestimated in this population who is at higher risk of metabolic syndrome and NAFLD. Hence, this supports the current evidence on the need to improve patients’ metabolic risk factors that are associated with liver fibrosis. MTX cumulative dose and duration were associated ELF score in the RA subgroup which may reflect arthritis activity rather than liver fibrosis. The degree of inflammation, especially in those who have RA, may confound ELF as a marker to detect fibrosis; therefore, transient elastography would be a more reliable tool to screen for significant fibrosis in this group. Guidelines for monitoring patients on MTX should be revisited to compare non-invasive tests to the current reliance on liver enzymes and PIIINP, and an evaluation of the cost-effectiveness of regular assessments in this population should be considered in future studies.
Acknowledgements
The views expressed are those of the authors and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health. We thank all the research participants. We are grateful to David Simmons and Beth Robinson for study coordination, Davor Kresnik and Melanie Lingaya for technical assistance, Hrushikesh Divyateja (Nottingham University Hospitals NHS Trust) and Mark Pugh (Siemens) for assistance with ELF testing.
This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 821283. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Rheumatoid arthritis in adults: management. National Institute for Health and Care Excellence. NICE guideline 2020 October 2020; Available from: www.nice.org.uk/guidance/ng100.
Methotrexate for rheumatoid arthritis. Suggested guidelines for monitoring liver toxicity. American College of Rheumatology. Arthritis Rheum.1994; 37: 316-328
Low incidence of hepatotoxicity associated with long-term, low-dose oral methotrexate in treatment of refractory psoriasis, psoriatic arthritis, and rheumatoid arthritis. An acceptable risk/benefit ratio.
Aithal, G.P., Hepatotoxicity Related to Methotrexate, in Drug-Induced Liver Disease, D.L. Kaplowitz N, Editor. 2013, Elsevier: London, UK, Walthan, MA and San Diego, CA.
Enhanced liver fibrosis test in patients with psoriasis, psoriatic arthritis and rheumatoid arthritis: a cross-sectional comparison with procollagen-3 N-terminal peptide (P3NP).
British Journal of Dermatology.2017; 176: 1599-1606
Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study.
Evaluation of liver fibrosis by transient elastography (Fibroscan®) in patients with inflammatory bowel disease treated with methotrexate: a multicentric trial.
Scandinavian Journal of Gastroenterology.2012; 47: 575-579
Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease.
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