Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease globally [
However, to date, no study has investigated the impact if fibrosis progression per se is associated with an increased risk of future mortality. We aimed to confirm recent findings from other studies, evaluate risk factors for fibrosis progression, and examine if fibrosis progression per se is associated with increased mortality in NAFLD.
We retrospectively included all patients with NAFLD who had undergone two or more liver biopsies at the Karolinska University Hospital, Stockholm, Sweden in a cohort study. The methodology for the generation of the cohort and ascertainment of NAFLD has previously been described [
The cohort was stratified on progression or no progression of fibrosis between the two biopsies. Progression of fibrosis was defined as an increase of at least one stage of fibrosis. In patients who underwent more than two biopsies (
Data from the time of the baseline and follow-up biopsies were collected from patient charts as per Table
Clinical characteristics at baseline.
Parameter | Range | |
---|---|---|
Age (years) | 46 | 19–70 |
Sex, male, |
37 (62) | |
BMI (kg/m2) | 26.4 | 21.2–38.7 |
Smoking, ever, |
21 (37) | |
T2DM, |
10 (17) | |
Hypertension, |
14 (23) | |
Platelets (×10̄9/L) | 256 | 56–347 |
ALT (IU/L) | 88 | 10–307 |
AST (IU/L) | 46 | 14–289 |
Ferritin ( |
133 | 44–753 |
ALP (IU/L) | 200 | 47–876 |
GGT (IU/L) | 78 | 22–599 |
Bilirubin (mg/dL) | 0.58 | 0.18–2.22 |
PK-INR | 1 | 0.9–1.4 |
Albumin (g/dL) | 4.4 | 3.2–5.3 |
AST/ALT ratio | 0.7 | 0.4–4.5 |
CRP (mg/L) | 10 | 1–26 |
Cholesterol (mg/dL) | 221 | 77–402 |
Triglycerides (mg/dL) | 186 | 44–460 |
Glucose (mg/dL) | 95 | 67–305 |
NAS (0–8) | 4 | 1–8 |
NASH |
33 (61) | |
Fibrosis stage | ||
0 |
15 (25) | |
1 |
22 (37) | |
2 |
14 (23) | |
3 |
6 (10) | |
4 |
3 (5) |
Abbreviations: BMI: body mass index; DM2: diabetes mellitus type 2; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; GT: gamma glutamyltransferase; NAS: nonalcoholic fatty liver disease activity score.
Of the baseline biopsies, 54 were available, and for the follow-up biopsies, 41 were available for reevaluation including scoring of necroinflammatory changes and NASH. Available liver biopsies were reevaluated by two of the researchers (RH and HH), blinded to patient characteristics. The stage of fibrosis and the NAFLD activity score were determined according to Kleiner et al. [
Descriptive statistics for continuous variables are expressed as median (range), and categorical variables are presented as absolute numbers (percentages). Differences in continuous variables were analyzed using the Mann–Whitney
As NASH might be more important in early stages of fibrosis, we performed sensitivity analyses excluding cases with advanced fibrosis (stages 3-4). All analyses were performed using STATA v 13.0 (StataCorp, College Station, Texas, USA). A two-tailed
The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983, and approved by the Ethics Committee at Karolinska University Hospital, Stockholm, Sweden (Dnr: 2011/905-31/2 and 2015/1591-32).
Among the 510 patients with a histological diagnosis of NAFLD at baseline, 82 underwent a second biopsy. Of these, 17 patients were excluded due to missing clinical data from the time of the follow-up biopsy, and five patients were excluded because the second liver biopsy was performed within one year of the first, leaving 60 patients for analysis.
A flowchart for patient inclusion is presented in Figure
Flowchart for patient inclusion.
Clinical, histological, and laboratory features of the cohort are summarized in Table
Fibrosis staging at the baseline biopsy disclosed stage 0 in 15 (25.0%), stage 1 in 22 (36.7%), stage 2 in 14 (23.3%), stage 3 in 6 (10.0%), and stage 4 in 3 (5.0%) patients.
The median follow-up interval between the first and second liver biopsies was 8.4 years (range 1–34 years). Twenty-seven patients (45%) had liver biopsies more than ten years apart. A total of 26 (43%) patients had fibrosis progression while 34 (57%) patients had stable or regression of fibrosis. Time between the index and the follow-up biopsy was significantly longer in the group with fibrosis progression (median 16.2 versus 5.5 years,
Comparison of clinical characteristics at baseline and follow-up between patients with and without progression of fibrosis.
No fibrosis progression |
Range | Fibrosis progression |
Range | ||
---|---|---|---|---|---|
Sex, |
22 (65) | 15 (58) | 0.6 | ||
Age (years) | 42.5 | 19–70 | 50 | 20–66 | 0.28 |
BMI (kg/m2) | 26.7 | 23.5–38.7 | 26.6 | 21.2–32.2 | 0.29 |
T2DM, |
6 (18) | 4 (15) | 0.58 | ||
Hypertension, |
7 (21) | 7 (27) | 0.4 | ||
Platelets (×109/L) | 265 | 113–347 | 233 | 56–332 | 0.48 |
ALT (U/L) | 92 | 22–307 | 86 | 10–264 | 0.53 |
AST (U/L) | 49 | 29–135 | 46 | 14–288 | 0.64 |
Bilirubin (mg/dL) | 0.58 | 0.35–2.22 | 0.53 | 0.16–1.29 | 0.65 |
Albumin (g/dL) | 4.4 | 3.3–5.3 | 4.4 | 3.2–5.2 | 0.84 |
PK-INR | 1.0 | 0.9–1.3 | 1.0 | 1.0–1.2 | 0.45 |
Total cholesterol (mg/dL) | 232 | 77–402 | 189 | 127–340 | 0.09 |
NASH, yes (%) | 19/30 (63) | 14/24 (58) | 0.71 | ||
Years of follow-up | 5.5 | 1.1–27.2 | 16.2 | 1.7–33.7 | 0.01 |
Age (years) | 52 | 28–83 | 66 | 21–84 | <0.01 |
BMI (kg/m2) | 27.8 | 24.7–38.7 | 27.1 | 21–35 | 0.3 |
Delta BMI | 1.9 | −7.5 to 6.4 | 0.8 | −5.0 to 8.1 | 0.29 |
T2DM, |
18 (53) | 12 (46) | 0.4 | ||
Hypertension, |
13 (38) | 11 (42) | 0.48 | ||
Platelets (×109/L) | 238 | 61–465 | 215 | 92–505 | 0.25 |
ALT (U/l) | 59 | 11–343 | 64 | 17–604 | 0.62 |
AST (U/I) | 41 | 13–238 | 53 | 19–299 | 0.04 |
Bilirubin (mg/dL) | 0.58 | 0.23–1.87 | 0.79 | 0.23–5.73 | 0.22 |
Albumin (g/dL) | 4.2 | 1.7–5.2 | 3.7 | 2.0–4.8 | 0.007 |
PK-INR | 1.0 | 0.9–1.3 | 1.0 | 0.9–1.6 | 0.01 |
Total cholesterol (mg/dL) | 208 | 93–347 | 178 | 131–290 | 0.17 |
NASH, yes (%) | 10/24 (42) | 9/17 (53) | 0.54 |
Abbreviations: BMI: body mass index; DM2: diabetes mellitus type 2; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; GT: gamma glutamyltransferase; NAS: nonalcoholic fatty liver disease activity score.
The distribution of fibrosis stages at the baseline and follow-up biopsies is shown in Table
Distribution of the fibrosis stage at baseline and follow-up liver biopsies.
Baseline fibrosis stage | Follow-up fibrosis stage | |||||
---|---|---|---|---|---|---|
Stage 0 | Stage 1 | Stage 2 | Stage 3 | Stage 4 | Total | |
Stage 0 | 6 | 15 | ||||
Stage 1 | 2 | 13 | 22 | |||
Stage 2 | 1 | 3 | 3 | 14 | ||
Stage 3 | 0 | 1 | 0 | 2 | 6 | |
Stage 4 | 0 | 0 | 0 | 1 | 2 | 3 |
Total | 9 | 22 | 8 | 9 | 12 | 60 |
Cases with fibrosis progression are marked in bold.
Among the 41 patients with both baseline and follow-up biopsies available for scoring of steatosis, lobular inflammation and ballooning, 23 (62.1%) had NASH at baseline and 18 (48.7%) at follow-up. Of the 23 patients with baseline NASH, 10 (43.5%) experienced resolution of NASH, while 13 (56.5%) had NASH also at follow-up. Of the 14 patients without baseline NASH, five (35.7%) developed NASH while 9 (64.3%) did not.
The median rate of fibrosis progression was 0.15 stages/year (range 0.03–0.78) for patients with NASH at baseline and 0.11 stages/year (range 0.03–0.59) for patients with baseline NAFL (
The presence of NASH at baseline was not associated with fibrosis progression in the multivariate logistic regression analysis (aOR 1.04, 95% CI 0.30–3.64,
Steatohepatitis at baseline biopsy and association with fibrosis progression.
Parameter | OR | 95% CI | aOR1 | 95% CI | ||
---|---|---|---|---|---|---|
NASH | 0.81 | 0.27–2.43 | 0.71 | 0.97 | 0.25–3.69 | 0.96 |
NAS 5–8 | 0.88 | 0.30–2.56 | 0.81 | 0.74 | 0.21–2.62 | 0.65 |
1Adjusted for age at first biopsy, sex, BMI, type 2 diabetes, and time between biopsies. Abbreviations: OR: odds ratio; aOR: adjusted odds ratio; CI: confidence interval; NASH: nonalcoholic steatohepatitis; NAS: NAFLD activity score.
The respective association between NASH at baseline, high NAS at baseline, and fibrosis progression per se, with mortality using Cox regression.
Parameter | HR | 95% CI | aHR1 | 95% CI | ||
---|---|---|---|---|---|---|
NASH | 1.43 | 0.55–3.69 | 0.46 | 0.97 | 0.20–4.67 | 0.97 |
NAS 5–8 | 1.02 | 0.41–2.54 | 0.97 | 0.46 | 0.12–1.69 | 0.24 |
Fibrosis progression | 2.10 | 0.87–5.09 | 0.10 | 2.83 | 0.99–8.05 | 0.051 |
1Adjusted for sex, age, BMI, type 2 diabetes at first biopsy, and time between biopsies. Abbreviations: OR: odds ratio; aOR: adjusted odds ratio; CI: confidence interval; NASH: nonalcoholic steatohepatitis; NAS: NAFLD activity score.
Among the 26 patients with fibrosis progression on the follow-up biopsy, 24 had biopsies available for baseline biopsy scoring. Of these, 10 (42%) had NAFL and 14 (58%) had NASH at the baseline biopsy. There were no significant differences at baseline concerning steatosis, lobular inflammation, ballooning, NAS, or fibrosis stage between patients with and without fibrosis progression (Table
Histological disease activity at baseline and follow-up in patients with and without fibrosis progression. Fibrosis stage was determined in all biopsies.
Parameter | No fibrosis progression (mean) | SD | Fibrosis progression (mean) | SD | |
---|---|---|---|---|---|
Steatosis (1–3) | 1.73 | 0.94 | 1.79 | 0.88 | 0.88 |
Lobular inflammation (0–3) | 1.43 | 0.85 | 1.33 | 0.7 | 0.63 |
Ballooning (0–2) | 0.97 | 0.85 | 0.88 | 0.85 | 0.69 |
NAS (0–8) | 3.86 | 2.31 | 3.84 | 2.17 | 0.92 |
Fibrosis (0–4) | 1.47 | 1.16 | 1.15 | 1.04 | 0.28 |
NASH (number of patients, %) | 19/30 | 63% | 14/24 | 58% | 0.78 |
Steatosis (1–3) | 1.21 | 0.93 | 1.12 | 0.78 | 0.83 |
Lobular inflammation (0–3) | 1.17 | 0.92 | 1.29 | 0.69 | 0.52 |
Ballooning (0–2) | 0.54 | 0.78 | 0.94 | 0.83 | 0.11 |
NAS (0–8) | 2.92 | 1.98 | 3.35 | 1.73 | 0.43 |
Fibrosis (0–4) | 1.18 | 1.11 | 2.81 | 1.17 | <0.001 |
NASH (number of patients, %) | 10/24 | 42% | 9/17 | 53% | 0.54 |
∗54 biopsies were available for scoring of NASH at baseline and 41 at follow-up. Abbreviations: NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; SD: standard deviation.
After the follow-up biopsy, patients were followed for a median of 6.7 years (range 0.1–34.0). During this time, 21 patients (35%) died, eight (25%) in the group without fibrosis progression and thirteen (50%) in the group with fibrosis progression (
In the Cox regression model, only fibrosis progression was borderline associated with mortality (aHR 2.83, 95% CI 1.00–8.05,
No association between baseline NASH and fibrosis progression or mortality was found when excluding cases with fibrosis stages 3-4 from the analysis (data not shown).
In this retrospective cohort study of 60 NAFLD patients with sequential liver biopsies, a similar proportion of patients with NAFL and NASH had evidence of fibrosis progression. Thus, we confirm similar findings from other groups, which together strengthen the hypothesis that fibrosis progression also can occur in NAFLD patients without steatohepatitis at an initial baseline liver biopsy.
In addition, we found a trend towards increased mortality in patients with fibrosis progression, irrespective if NASH was present or not in the baseline liver biopsy and after adjustment for confounders. This finding is in line with results from previous studies implying that the fibrosis stage correlates with clinical outcomes and is the strongest predictor for overall and liver-related mortality [
The only significant risk factor for progression of fibrosis in the present study was the time between the two biopsies. In contrast, baseline T2DM and BMI were also associated with fibrosis progression in three recent studies with similar design as ours [
The major strength of this study was the long follow-up duration. The follow-up interval between the first and second liver biopsies is the longest hitherto documented. A long enough follow-up time is essential when studying fibrosis progression in patients with NAFLD, since this is a slow process. In a recent meta-analysis, the rate of fibrosis progression was estimated to one stage per seven years in subjects with NASH and per 14 years in those with NAFL [
The primary limitation of this study is a possible selection bias at the second biopsy, since cases with progressive fibrosis are probably more likely to undergo repeat examination than cases without. However, liver biopsies were historically performed more frequently compared to today, due to a lack of other modalities. Eighty-three percent of patients in this study were originally investigated before year 2000, thus reducing (but not excluding) the risk of selection bias. Since the biopsies were not planned per a specific protocol, the follow-up interval varies to a wide extent. Longer follow-up intervals may have an overrepresentation of older patients with more severe liver disease. As seen in Table
Other limitations include the small sample size, the lack of data on NASH at follow-up biopsy in 19 cases, and the absence of genetic status including
A major limitation of all paired biopsy studies in NAFLD, including ours, is the risk of residual confounding. Such confounding factors could comprise of lifestyle changes, including a change in alcohol consumption or medications during follow-up.
We did have access to data on development of BMI and type 2 diabetes, which were not associated with progression of fibrosis. Indeed, 35% of patients with fibrosis progression had a reduction in BMI during the follow-up period, suggesting that a reduction of BMI per se might not be enough to reduce the risk of fibrosis progression in some patients.
The clinical impact of this study together with the conclusions from previous studies [
The rate of fibrosis progression was quite slow in this study but comparable to what has been found in a recent meta-analysis [
Our study also demonstrates a trend towards higher mortality in patients with progressive fibrosis, which may have implications for selecting patients intended for more intense follow-up. This finding has to be confirmed in future studies on larger cohorts with a long enough follow-up time, which also should aim to find predictors for fibrosis progression. The present study could possibly contribute to future meta-analyses on this topic.
Both NAFL and NASH can lead to progressive fibrosis, with a mean fibrosis progression rate in the present study from 0.11 to 0.15 stages per year. Baseline steatohepatitis was not associated with an increased risk for fibrosis progression. Patients with progressive fibrosis appear to have a higher risk for mortality, regardless if NASH is present or not at baseline. Larger studies are needed to identify which patients with NAFL are at an increased risk for fibrosis progression.
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
All the authors declare that there is no conflict of interest regarding the publication of this paper.
Study conception and design were done by Hannes Hagström and Per Stål. Hannes Hagström, Olof Elfwén, and Rolf Hultcrantz were responsible for the acquisition of data. Statistical analysis was done by Hannes Hagström. Analysis and interpretation of data were done by Hannes Hagström, Olof Elfwén, Rolf Hultcrantz, Per Stål. Hannes Hagström, Olof Elfwén, and Per Stål did the drafting of the manuscript. Guarantors of the article are Hannes Hagström and Per Stål. All authors approved the final version of the article, including the authorship list. Hannes Hagström, Olof Elfwén, Rolf Hultcrantz, and Per Stål performed the critical revision of the manuscript.
Hannes Hagström was supported by grants from the Royal Swedish Academy of Sciences and the Swedish Gastroenterology Fund. Per Stål was supported by the Stiftelsen Tornspiran and the Stockholms Läns Landsting (ALF 20150403).