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NAFLD is a growing indication for liver transplantation
•
Cohort of 150 patients with at least one liver biopsy
•
Median follow-up after LT was 4.7 years
•
Recurrence of the initial disease is frequent and rapid after LT
•
Grade ≥2 steatosis at 1 year is highly predictive of recurrence of NASH and advanced fibrosis
Abstract
Background and Aims
Liver transplantation (LT) is the treatment of end-stage non-alcoholic fatty liver disease (NAFLD) (related decompensated cirrhosis and/or hepatocellular carcinoma). The aim of our study was to evaluate the risk of disease recurrence after LT and the factors influencing it.
Method
This retrospective multicenter study included adult transplanted for NAFLD cirrhosis between 2000 and 2019 in 20 participating French-speaking centers. Disease recurrence (steatosis, steatohepatitis and fibrosis) was diagnosed from liver graft biopsies.
Results
We analyzed 150 patients with at least one graft liver biopsy available ≥6 months, among 361 patients transplanted for NAFLD. The median age at LT was 61.3 years [54.4-64.6]. The median follow-up after LT was 4.7 years [2.8-8.1]. Steatosis and steatohepatitis cumulative recurrence rate at 5 years was 80.0% and 60.3%, respectively. Significant risk factors in multivariate analysis for steatohepatitis recurrence were a recipient age at LT < 65 years (OR: 4.214; p=0.044), an HDLc rate<1.15mmol/L after LT (OR: 3.463; p=0.013) and a grade ≥2 steatosis on the graft at 1 year (OR: 10.196; p=0.001). The cumulative incidence of advanced fibrosis (F3–F4) was 20.0% at 5 years and significant risk factors from multivariate analysis were metabolic syndrome before LT (OR: 8.550; p=0.038), long-term use of cyclosporine (OR: 11.388; p=0.031) and grade ≥2 steatosis at 1 year (OR: 10.720; p=0.049). No re-LT was performed for NAFLD-cirrhosis recurrence.
Conclusion
Our results strongly suggest that recurrence of initial disease after LT for NAFLD is ineluctable and progressive: the means to prevent it remain to be further evaluated.
Lay Summary
NAFLD is a growing indication for liver transplantation, but the analysis of disease recurrence, based on graft liver biopsies, has been poorly studied. Cumulative incidence at 5 years of steatosis, steatohepatitis and NAFLD-related significant fibrosis recurrence was 85.0%, 60.3% and 48.0%, respectively. Grade ≥2 steatosis on graft biopsy at 1 year (present in 25% of patients) is highly predictive of recurrence of steatohepatitis and advanced fibrosis: bariatric surgery should be discussed in these patients specifically.
Bourse d’appels offres à recherche 2018 – Agence de la Biomédecine.
Authors contributions:
Jérôme Dumortier had the idea of the project and participated in analysis and interpretation of data.
François Villeret collected the data.
François Villeret and Domitille Erard performed statistical analysis.
François Villeret and Jérôme Dumortier participated in writing of the manuscript.
François Villeret Sébastien Dharancy, Domitille Erard, Armand Abergel, Louise Barbier, Camille Besch, Olivier Boillot, Karim Boudjema, Audrey Coilly, Filomena Conti, Christophe Corpechot, Christophe Duvoux, François Faitot, Stéphanie Faure, Claire Francoz, Emiliano Giostra, Jean Gugenheim, Jean Hardwigsen, Marie-Noëlle Hilleret Jean-Baptiste Hiriart, Pauline Houssel-Debry, Nassim Kamar, Guillaume Lassailly, Marianne Latournerie, Georges-Philippe Pageaux, Didier Samuel, Claire Vanlemmens, Faouzi Saliba and Jérôme Dumortier were involved in medical care of the patients and approved the final version of the manuscript.
Introduction
Non-alcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in the world with a prevalence of nearly 24.1% in the USA adult population and 23.7% in Europe with many disparities: NAFLD affects an estimated 18.2% of the French population
. NAFLD is a spectrum of disease that ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) characterized by steatosis, inflammation, hepatocyte ballooning, and varying degrees of hepatic fibrosis which may progress to cirrhosis and end-stage liver disease
. Hepatocellular carcinoma (HCC) can develop on a cirrhotic or non-cirrhotic liver. Currently, no specific treatment is available for NAFLD and the only effective treatment is weight lost, for instance after bariatric surgery (BS)
. Liver transplantation (LT) may be indicated in case of decompensated NAFLD-related cirrhosis and/or HCC. LT for NAFLD is a growing indication worldwide: it is the second leading cause of LT in the USA but remains less frequent in Europe
. Patient survival at 5 years after LT for NAFLD without HCC has been reported to be 75.4% in Europe compared to 75% for alcohol-associated liver disease and 80% for Hepatitis B virus (HBV)-related disease
Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease.
Saeed N, Glass L, Sharma P, Shannon C, Sonnenday CJ, Tincopa MA. Incidence and Risks for Non Alcoholic Fatty Liver Disease and Steatohepatitis Post Liver Transplant: Systematic Review and Meta-analysis. Transplantation. 2019;
. The persistence of metabolic syndrome (MS) factors after LT, and even its aggravation because of immunosuppressive treatment, may suggest a recurrence of the initial disease on the graft. Recurrence rate of NAFLD is therefore estimated to be more than 80% at 5 years after LT. There is a need to better assess the prevalence of recurrence of NAFLD on the graft, its evolution and the associated risk factors.
The aim of the present study, based on a large retrospective cohort, was to describe the recurrence of initial disease (steatosis, NASH and fibrosis) on the graft after LT for NAFLD, based on liver biopsies, and to identify the factors influencing it.
Patients and methods
1 Study Population:
We included all adult patients transplanted in all French LT centers and in Geneva (Switzerland), based on the national database of the French Agence de la Biomédecine (ABM) and local databases (the “NASH” item did not exist in the ABM thesaurus before January the 1st 2018). We first selected all patients transplanted between January the 1st 2000 to 31 December 2019 for "other causes of cirrhosis", "cirrhosis of unknown cause", "metabolic disease" or "HCC" and “NASH” disease after January 2018 in the ABM database. All medical records were reviewed and patients were finally included after histopathological examination of available liver biopsy before the LT or the native liver compatible with a NAFLD cirrhosis, metabolic risks factors (diabetes, obesity or overweight, arterial hypertension) and the absence of other confounding etiology (alcohol consumption>10 g per week, autoimmune disease, viral hepatitis, Wilson's disease or hemochromatosis). The aim of this study was to investigate the recurrence of the initial disease based on graft biopsies performed ≥ 6 months after LT: the population is defined as all transplanted patients with a graft biopsy ≥ 6 months after LT.
This study was conducted in accordance with the Declaration of Helsinki. According to French law (Loi Jardé), retrospective studies do not require Institutional Review Board approval.
2 Clinical and biological characteristics at the time of listing
Cirrhosis characteristics at the LT registration time were specified including MELD score, CHILD-PUGH score and cirrhosis complications. Metabolic characteristics were recorded: dry weight at LT listing (without ascites or after paracentesis), height and higher body mass index (BMI) in their life were collected. BMI (kg/m2) was calculated from these values. Lipid profile, glycated hemoglobin (HbA1c) and diabetes therapy were collected. MS was defined according to the American Heart Association, replacing waist circumference with BMI over 30kg/m2
Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity.
. Cardiovascular (CV) events, CV check-up and pulmonary disease before LT were collected.
All patients received grafts from cadaveric or living donors. Donor’s characteristics (age, weight and BMI) were collected.
3 Follow-up after LT
Initial immunosuppressive regimen was based on a calcineurin-inhibitor (CNI): cyclosporine (CYA) or tacrolimus (TAC). Induction therapy by polyclonal antibodies or anti-interleukin-2 receptor antibodies was mainly administered in case of acute kidney injury. Starting on postoperative day 1, methylprednisolone was tapered to reach a maintenance dose of 0 to 5 mg/day at 6 months post-transplantation. Azathioprine (AZA), mycophenolate mofetil (MMF) or sirolimus/everolimus (mTOR-i: mTOR inhibitor) was either administered as part of an initial triple immunosuppressive regimen or introduced during follow-up as a maintenance immunosuppressive agent. Outpatient follow-up visits were usually conducted once a week during the first month after discharge from the hospital, twice a month during the second and third months, monthly for the rest of the first year, and every 3 or 12 months thereafter, regardless of the length of the observation period after LT. Additional visits were made when necessary. A complete laboratory investigation, including hematology, liver parameters, coagulation, electrolytes, total protein, renal parameters, fasting blood glucose, a lipid profile, and blood calcineurin inhibitor trough levels or mTOR-i levels, was conducted at each visit.
The prevalence of arterial hypertension, diabetes, dyslipidemia and specific associated drugs were recorded. CV events after LT were collected (defined by coronary heart disease, stroke or cardiac arrhythmia, and cardiorespiratory arrest). The presence of steatosis on non-invasive imaging examens (ultrasound or Computer Tomography scan, Magnetic Resonance Imaging, liver stiffness measurement) was specified.
Biological standards are considered abnormal if they are above or below the laboratory standard.
The end of follow-up corresponded to death, the last medical examination or date of loss to follow-up. All data were retrospectively collected until June 30th 2020.
4 Diagnosis of disease recurrence on liver graft
Histopathological data were collected from available liver graft biopsies. Depending on local center policy, protocol liver biopsy was performed after LT at 1, 2, 3, 5, 10 and 15 years in some. The liver pathology team at each LT center reviewed all biopsy samples. Steatosis was graded on a 0–3 semi-quantitative scale: (0) steatosis absent or in < 5% of hepatocytes;
steatosis in more than two-thirds of the hepatocytes. The grading of NASH (inflammation and hepatocyte ballooning) was performed according to the SAF (Steatosis, Activity and Fibrosis) scoring system
. The diagnosis of NASH was defined as a SAF activity score (presence of steatosis, ballooning and lobular inflammation according to the flip algorithm) ≥ 2
. Significant and advanced fibrosis were defined as a liver fibrosis stage ≥ F2 and ≥F3, respectively. For the analysis of fibrosis recurrence, we excluded possible factors that could aggravate fibrosis lesions such as cell rejection, or biliary obstacle. We excluded from the analysis of recurrence of initial disease on the graft patients with post-LT alcohol consumption >10 g per week.
5 Statistical analysis
All data were analyzed using SPSS software, version 23.0 (IBM, Armonk, NY, USA). Data were described in their totality using median with interquartile range [IQR] or mean with standard deviation (SD) for continuous variables and number (percentage) for categorical variables. Categorical variables were compared with the Chi-square or Fischer’s exact tests and quantitative variables were compared using the Student t-test or non-parametric tests (Mann-Whitney or Kruskall-Wallis tests) when appropriate.
The main event of interest was NAFLD recurrence. Steatosis, NASH and fibrosis recurrence rates were estimated using the Kaplan–Meier method. Determination of the risk factors for NASH recurrence was performed using Log Rank analysis or Mann-Whitney analysis (for quantitative variables). All significant variables in the univariate analysis with a level set at p < 0.1 were incorporated into multivariate models analyzed with binary logistic Cox regression. Logistic regression analyses (univariate and multivariate) were used to calculate the risk for liver steatosis, liver fibrosis and various parameters.
Results
1 Study population and metabolic characteristics at time of listing for LT
One hundred and fifty patients underwent liver biopsy ≥ 6 months after LT, among our global cohort of 361 patients transplanted from all 19 French LT centers and Geneva (Switzerland) (i.e. 20 French-speaking centers). These patients with available liver biopsy do not differ from (except for the duration of post-LT follow-up) patients without graft biopsy regarding pre and post-LT characteristics (Table 1). Patients were transplanted between January 2001 and June 2018. Median [IQR] follow-up after LT was 4.7 years [2.8-8.1] (range; 0.7-18.7 years). More than half of the patients had a MS when listed for LT. The median [IQR] BMI at time of LT time was 30.9 kg/m2 [26.7-33.9] but the median [IQR] higher BMI in their life was 35.0 kg/m2 [30.9-39.3]. In patients transplanted for HCC, the median [IQR] BMI at time of LT time was 30.3 kg/m2 [26.8 - 34.5] compared to 31.0 kg/m2 [27.3 – 33.3] in patients transplanted for ESLD (End-Stage Liver Disease) (not statistically significant; p=0.522). Thirty patients (20.0%) have at least one cardiovascular history before LT. Five patients (3.33%) had pre-LT angioplasty.
Table 1Characteristics of patients with liver biopsy before and after LT (on patient alive at 1-year n=286).
Comparison of patients with liver biopsy in whole cohort with patients with liver biopsy in centers performing protocol biopsies.
Gender (M/F)
98/52
102/34
63/40
16/3
0.076
0.010
0.499
Median age at LT (years) (IQR)
61.3 (54.4-64.6)
61.9 (57.9-65.7)
61.1 (57.5-65.5)
62.7 (57.7-65.8)
0.066
0.178
0.770
HCC
77 (51.3%)
75 (55.1%)
45 (43.7%)
12 (63.2%)
0.519
0.464
0.233
Median MELD score at LT listing (IQR)
13.1 (9.1 – 21.1)
14.6 (8.1-21.6)
14.3 (9.7-21.0)
12.6 (6.0-23.0)
0.912
0.583
0.848
Median CHILD-PUGH score at LT listing (IQR)
B9 (B7–C12)
B9 (A6-C12)
C10 (B8–C12)
C10 (B7–C12)
0.448
0.587
0.498
Median follow-up (years) (IQR)
4.9 (3.0-8.3)
3.5 (2.3-6.2)
4.5 (2.9-7.4)
3.4 (1.7-4.9)
0.001
0.001
0.0001
Characteristics before LT
BMI at LT time (kg/m2) (IQR)
30.9 (26.7-33.9)
31.1 (26.8-34.2)
31.0 (27.2-33.9)
34.0 (31.2-37.6)
0.608
0.183
0.721
Metabolic syndrome
77 (51.3%)
84 (61.8%)
52 (50.5%)
15 (78.9%)
0.076
0.143
0.213
Diabetes mellitus
112 (74.7%)
103 (75.7%)
78 (75.7%)
17 (89.5)
0.835
0.077
0.341
Median glycated hemoglobin level (%) (IQR)
5.8 (5.1-7.3)
5.7 (5.1-6.7)
5.9 (5.1-6.6)
6.4 (5.6-7.2)
0.423
0.727
0.278
Arterial Hypertension
122 (81.3%)
114 (83.8%)
77 (74.8%)
19 (100%)
0.664
0.067
0.146
Statin therapy
20 (13.3%)
16 (11.8%)
15 (14.6%)
4 (21.1%)
0.660
0.854
0.267
Fibrate therapy
4 (2.7%)
3 (2.2%)
3 (2.9%)
0 (0.0%)
0.768
0.220
0.214
Bariatric surgery
5 (3.3%)
3 (2.2%)
4 (3.9%)
0 (0.0%)
0.564
0.540
0.680
Cardiovascular history
30 (20.0%)
30 (22.1%)
20 (19.4%)
6 (31.6%)
0.147
0.549
0.212
Median serum creatinine level (μmol/L) (IQR)
80.5 (67.0-100.0)
85.0 (70.0-107.5)
80.0 (67.0-100.5)
77.0 (64.5-114.5)
0.146
0.434
0.487
Medium glomerular filtration rate (MDRD) (μmol/L) (IQR)
82.1 (60.3-106.7)
77.9 (58.7-100.8)
80.6 (59.8-106.5)
90.3 (60.2-104.7)
0.707
0.988
0.149
Characteristics after LT
BMI at 1 year after LT (kg/m2) (IQR)
29.5 (26.6-33.6)
29.4 (26.4-32.0)
29.5 (25.8-33.8)
32.0 (29.9-34.9)
0.519
0.079
0.478
Diabetes post-LT
111 (74.0%)
99 (72.8%)
78 (75.7%)
19 (100%)
0.669
0.038
0.352
Arterial hypertension post-LT
141 (94.0%)
111 (81.6%)
96 (93.2%)
18 (94.7%)
0.245
0.198
0.458
Dyslipidemia or lipid drugs
81 (54.0%)
93 (68.3%)
53 (51.5%)
6 (31.6%)
0.321
0.541
0.254
Metabolic syndrome post-LT
127 (84.7%)
118 (86.8%)
86 (83.5%)
18 (94.7%)
0.180
0.890
0.176
Donors characteristics
Median age (years) (IQR)
58.5 (43-68.3)
59.0 (47.0-69.0)
57.0 (44.0-69.5)
57.0 (53.0-65.0)
0.452
0.130
0.170
Gender (M/F)
73/48
70/36
45/24
7/4
0.691
0.120
0.240
Median BMI (kg/m2) (IQR)
24.8 (22.5-29.1)
24.8 (22.6-27.7)
24.9 (22.6-28.9)
24.3 (22.3-27.9)
0.555
0.257
0.360
Grade ≥1 steatosis
66 (44.0%)
42 (30.9%)
49 (47.6%)
9 (47.4%)
0.478
0.875
0.699
Grade ≥2 steatosis
13 (8.7%)
8 (5.9%)
9 (8.7%)
1 (5.3%)
0.247
0.743
0.547
Stage ≥1 fibrosis
36 (24.0%)
25 (18.4%)
16 (15.5%)
3 (15.8%)
0.364
0.788
0.272
Stage ≥2 Fibrosis
3 (2.0)
8 (5.9%)
2 (1.9%)
0 (0.0%)
0.785
0.890
0.450
BMI: body mass index; HCC: Hepatocellular Carcinoma; LT: Liver Transplantation; MELD: Model for End-Stage Liver Disease.
Univariate analysis was performed using Log Rank analysis or Mann-Whitney analysis (for quantitative variables). All significant variables in the univariate analysis with a level set at p < 0.1 were incorporated into multivariate models analyzed with binary logistic Cox regression. Values in bold are considered significant (<0.005).
∗ Comparison in whole cohort between patients with liver biopsy vs no liver biopsy.
∗∗ Comparison of patients in centers performing protocol biopsies (with and without biopsies).
∗∗∗ Comparison of patients with liver biopsy in whole cohort with patients with liver biopsy in centers performing protocol biopsies.
Univariate analysis was performed using Log Rank analysis or Mann-Whitney analysis (for quantitative variables). All significant variables in the univariate analysis with a level set at p < 0.1 were incorporated into multivariate models analyzed with binary logistic Cox regression. Values in bold are considered significant (<0.005).
∗ Because these variables are not independent, different multivariate analysis models were tested.
Because these variables are not independent, different multivariate analysis models were tested AZA: azathioprine; BMI: body mass index; CNI: Calcineurin Inhibitor; CST: corticosteroids; CYA: cyclosporine A; CV: Cardiovascular; HCC: Hepatocellular Carcinoma; HDL-c: High-Density Lipoproteins Cholesterol; LDL-c: Low-Density Lipoproteins; LT: Liver Transplantation; MMF: mycophenolate mofetil; mTOR-i: mTOR inhibitor; TG: triglycerides.
0.005
1.591 (0.001-2486)**
0.902**
≥ 35 kg/m2
0.289
0.688
HCC pre-LT
0.285
0.016
0.175 (0.016-1.896)
0.152
Pre-LT diabetes
0.378
0.413
Pre-LT HbA1c ≥ 7%
0.246
0.826
Pre-LT insulin therapy
0.600
0.646
Pre-LT arterial hypertension
0.733
0.187
Pre-LT metabolic syndrome
0.425
0.069
8.550 (1.125-64.983)
0.038
Active smoking before LT
0.677
0.447
Donors characteristics
Age of the donor (years)
0.074
0.552
Age ≥ 60 years
0.713
0.828
Age ≥ 70 years
0.648
0.868
Donors age + recipients age (years)
0.016
0.099
≥ 120 years
0.376
0.476
≥ 135 years
0.648
0.452
Donor BMI (kg/m2)
0.170
0.161
Graft steatosis (≥ 5%)
0.005
1.894 (0.915-3.923)**
0.086**
0.099
4.454 (1.257-103.209)
0.126
Grade ≥ 2 steatosis
0.028
2.837 (1.266-6.358)**
0.011**
0.605
Metabolic events after LT
BMI at 1 year after LT
0.121
0.019
≥ 30 kg/m2
0.007
1.198 (0.443-3.240)***
0.723***
0.001
3.578 (0.002-6102)
0.737
≥ 32 kg/m2
0.013
1.547 (0.679-3.525)***
0.299***
0.059
≥ 35 kg/m2
0.130
0.160
Weight difference 1-year post-LT (kg)
0.923
0.458
Diabetes post-LT
0.499
0.999
Arterial hypertension post-LT
0.609
0.634
TG ≥ 1.7mmol/L post-LT
0.353
0.363
LDLc ≥ 3.70mmol/L post-LT
0.244
0.890
HDLc < 1.15mmol/L
0.726
0.937
HbA1c ≥ 6.5%
0.127
0.324
HbA1c ≥ 7%
0.190
0.606
HbA1c ≥ 8%
0.249
0.109
Insulin therapy
0.901
0.420
Metabolic syndrome post-LT
0.641
0.739
Immunosuppressive regimen
Tacrolimus
0.370
0.756
MMF
0.738
0.971
CYA
0.990
0.005
11.388 (1.257-103.209)
0.031
mTOR-i
0.554
0.466
CST
0.277
0.934
AZA
0.681
0.873
CNI free therapy
0.370
0.756
Specifics therapies after LT
Statin therapy
0.290
0.840
Fibrate therapy
0.733
0.769
Insulin therapy
0.901
0.420
Dyslipidemia or lipid drugs
0.681
0.680
Complications after LT
OSAS
0.650
0.954
Acute rejection
0.973
0.656
Disease recurrence on the graft at 1 year
Grade ≥1 steatosis
0.0001
5.373 (1.593-18.121)****
0.007****
0.103
Grade ≥2 steatosis
0.0001
3.564 (1.605-7.917)****
0.002****
0.001
10.720 (1.006-114.259)
0.049
Grade 3 steatosis
0.001
4.596 (1.630-12.961)****
0.004****
0.109
NASH recurrence
0.0001
5.217 (2.013-13.519)****
0.001****
0.131
Each variable was tested in univariate analysis. All variables with a p value < 0.010 were retained for the multivariate model.
Univariate analysis was performed using Log Rank analysis or Mann-Whitney analysis (for quantitative variables). All significant variables in the univariate analysis with a level set at p < 0.1 were incorporated into multivariate models analyzed with binary logistic Cox regression. Values in bold are considered significant (<0.005).
∗ Because these variables are not independent, different multivariate analysis models were tested AZA: azathioprine; BMI: body mass index; CNI: Calcineurin Inhibitor; CST: corticosteroids; CYA: cyclosporine A; CV: Cardiovascular; HCC: Hepatocellular Carcinoma; HDL-c: High-Density Lipoproteins Cholesterol; LDL-c: Low-Density Lipoproteins; LT: Liver Transplantation; MMF: mycophenolate mofetil; mTOR-i: mTOR inhibitor; TG: triglycerides.
After LT, 94.0% of patients had hypertension, with 24 cases of de novo arterial hypertension (representing 85.7% of patients at risk). De novo diabetes developed in 28.9% of patients at risk after LT: 74.0% of patients had diabetes in the LT follow-up. After LT, 78.0% have dyslipidemia or were with anti-lipid therapy after LT. A de novo Obstructive Sleep Apnea syndrome (OSAS) after LT was diagnosed in 10.7% of patients.
The cumulative incidence of MS after LT at 1, 5 and 10 years was 73.5%, 86.2% and 92.5%, respectively (Figure 1); 73.1% of patients had a BMI over 25 kg/m2 and 59.3% were obese during follow-up. The median [IQR] BMI at 1 year and 5 years after LT was 29.5 kg/m2 [26.6-33.6] and 32.3 kg/m2 [28.2 – 35.5], respectively.
Figure 1Cumulative incidence of metabolic syndrome after LT (according to Kaplan-Meier estimates). The cumulative incidence of MS at 1, 5 and 10 years was 73.5%, 86.2% and 92.5%, respectively. There was no difference between patients with and without liver biopsy (p=0.896). (Kaplan–Meier analysis)
Five patients (3.3%) had history of BS: 3 before LT and 2 after LT. The median [IQR] time between the BS and the LT was 11.0 years [8.2-11.8] and the median [IQR] weight loss after BS was 20.0 kg [10.0-35.0]. No complication of BS was reported in these patients, including ESLD secondary to BS. Two (1.3%) patients underwent a BS after LT, with the median [IQR] time between LT and BS was 3.4 years [3.2-3.5]. Sleeve gastrectomy was the only type of BS after LT. A patient with NASH recurrence histologically proven at 1 year after LT underwent a sleeve gastrectomy at 3 years after LT allowing an improvement of the hepatic histology with complete regression of steatosis, NASH resolution and reduction of fibrosis stage from 3 to 2 on the liver biopsy 5-years after LT. A BS was under discussion for 10 (6.7%) patients at the end of follow-up.
Eight patients (5.3%) entered an intensive nutrition program (defined by specialized rehabilitation services) after LT.
3 NAFLD recurrence after LT and associated risk factors
One hundred and fifty patients underwent at least one liver graft biopsy after LT (222 liver biopsies in total, 94.3% performed as protocol biopsy). In centers performing protocol biopsies after LT, a liver graft biopsy was available at 1 year for 103 patients (out of 110 patients with ≥ 12 months follow-up, i.e 93.6%), 25 at 5 years (out of 28 patients with ≥ 5 years follow-up, i.e. 89.3%) and 12 at 10 years (out of 13 patients ≥ 10 years follow-up, i.e. 92.3%) (Supplementary Table 2). Cumulative incidence rates of steatosis, NASH and advanced fibrosis did not significantly differ when comparing the entire cohort and centers performing protocol biopsies (Figure 2).
Figure 2Grade ≥1 (A), grade ≥2 (B) and grade ≥3 (C) steatosis recurrence and NASH (D), NAFLD-related ≥ grade 2 fibrosis (E) and NAFLD-related ≥ grade 3 fibrosis (F) cumulative incidence after LT in whole cohort and centers with biopsy protocol. (Based on 150 liver biopsies) (According to Kaplan-Meier estimates). A: Steatosis ≥1 grade recurrence was 68.0% at 1 year and 85.0% at 5 years in whole cohort. P=0.381. B: Steatosis ≥2 grade recurrence was 27.8% at 1 year and 64.5% at 5 years in whole cohort. P=0.713. C: Steatosis ≥3 grade recurrence was 5.0% at 1 year and 30.3% at 5 years in whole cohort. P=0.378. D: NASH recurrence was 14.9% at 1 year and 60.3% at 5 years in whole cohort. P=0.823. E: NAFLD-related ≥ grade 2 fibrosis was 18.2% at 1 year and 48.0% at 5 years in whole cohort. P=0.333. F: NAFLD-related ≥ grade 3 fibrosis was 1.4% at 1 year and 20.0% at 5 years in whole cohort. P=0.745. (Kaplan–Meier analysis)
3.1 Steatosis recurrence on the graft (Figure 2-A/B/C and Table 2)
Steatosis (≥ 5% i.e., all stages) cumulative recurrence on the graft concerned the majority of patients (68.0%) at 1 year and 85.0% at 5 years after LT (Figure 2-A). The cumulative incidence of grade 2 steatosis recurrence on the graft at 1 and 5 years after LT was 27.8% and 64.5%, respectively; one third of recipients developed at least grade 2 steatosis (Figure 2-B). The cumulative incidence of grade 3 steatosis is shown in Figure 2-C. In multivariate analysis, only the BMI ≥ 31kg/m2 at time of LT (OR: 2.727; 95%CI: 1.048-7.098; p=0.040) was significantly associated with a risk of steatosis recurrence. There was a trend for patients with unmanaged diabetes (defined as HbA1c ≥ 8%). We found no relationship with weight gain or BMI at 1 year after LT. At 1 and 5 years after LT, in patients with grade ≥ 1 steatosis on biopsy, 33.6% and 75.0% respectively had steatosis on imaging US scan performed at the same time as the liver biopsy. At 1 and 5 years after LT, in patients with grade ≥ 2 steatosis on liver biopsy, 63.2% and 100% respectively had steatosis on imaging US scan performed at the same time as the liver biopsy. Seven patients (4.7%) had non-invasive assessment of steatosis by Controlled Attenuation Parameter (CAP) after LT.
NASH cumulative incidence recurrence rate was 14.9% at 1 year and 60.3% at 5 years post-LT (Figure 2-D). The median [IQR] delay between the LT and the NASH recurrence was 2.07 years [1.1-4.8]. On the 43 cases of NASH recurrence (28.7%), 33 patients (76.7%) had steatosis on US or CT scan.
The median [IQR] values of ALAT, ASAT and GGT at NASH recurrence diagnosis were respectively 35 IU/L [20-56], 32.0 IU/L [22 – 44] and 43 IU/L [28-87]. Twenty-six (60.5%) patients had GGT above the upper limit, 12 (27.9%) ALAT above the upper limit and 10 (23.3%) ASAT above the upper limit at the time of NASH recurrence diagnosis.
In multivariate analysis, risk factors of NASH recurrence were an age at the time of LT less than 65 years (OR: 4.214; 95%CI: 1.038-17.108; p=0.044), a post-LT HDL-c level less than 1.15 mmol/L (OR: 3.463; 95%Cl: 1.304-18.780; p=0.019), a grade 1 steatosis on the graft at 1 year after LT (OR: 10.521; 95%Cl: 2.127-52.046; p=0.004), or a grade 2 steatosis (OR: 10.196; 95%Cl: 3.553-29.257; p=0.001). Donor characteristics (age, BMI or steatosis on the graft) were not associated with NASH recurrence.
3.3 Recurrence of NAFLD-related fibrosis on the graft (Figure 2-E/F and Table 3)
Significant fibrosis (stage ≥ 2) cumulative incidence after LT was 18.2% at 1 year, 48.0% at 5 years and 65.0% at 10 years (Figure 2-E). There was no significant difference between the entire cohort and the protocol centers in which biopsies were not clinically directed (p=0.333). In multivariate analysis, risk factors to NAFLD-related significant fibrosis included a recipient age at time of LT < 60 years (OR: 2.368; 95%Cl: 1.125-4.986; p=0.023), an initial graft steatosis over grade 2 (OR: 2.837; 95%Cl: 1.266-6.358; p=0.011) and the presence at 1 year after LT of a grade ≥ 1 steatosis (OR: 5.373; 95%Cl: 1.593-18.121; p=0.007), grade ≥ 2 (OR: 3.564; 95%Cl:1.605-7.917; p=0.002), grade ≥3 (OR: 4.596; 95%Cl: 1.630-12.961; p=0.004) or NASH recurrence (OR: 5.217; 95%Cl: 2.013-13.519; p=0.001).
Cumulative incidence of advanced fibrosis (F3–F4) was 20.0% at 5 years and 48.0% at 10 years (Figure 2-F). Six patients (4.0%) had recurrent NAFLD cirrhosis after LT after a median delay of 7.1 years; one patient presented a decompensated cirrhosis 15 years after LT. Concerning NAFLD-related advanced fibrosis, risk factors identified in multivariate analysis were a MS before LT (OR: 8.550; 95%Cl: 1.125-64.983; p=0.038), a long-term use of CYA (OR: 11.388; 95%Cl: 1.257-103.209; p=0.031) and grade 2 steatosis at 1 year (OR: 10.720; 95%Cl: 1.006-114.259; p=0.049). Initial graft steatosis does not affect the occurrence of advanced fibrosis. The age of the recipient did not influence the occurrence of advanced fibrosis.
Non-invasive evaluation of fibrosis by liver stiffness measurement was performed in 9 patients (6.0%) during follow-up.
Figure 3 shows the cumulative incidence of advanced NAFLD-related fibrosis according to the presence of at least 1 risk factor: without any risk factor, the risk of advanced fibrosis was estimated at 5.3% at 5 years compared to 58.3% with at list one risk factor (p=0.001).
Figure 3Advanced fibrosis cumulative incidence according to the presence of risk factors. (Based on 150 liver biopsies) (According to Kaplan-Meier estimates). Risk factors considered for analysis: Pre-LT metabolic syndrome, grade ≥2 steatosis at 1 year after LT and long-term use of CYA. In group with risk factors, incidence of advanced fibrosis was 5.3% at 1 year after LT, 58.3% at 5 years and 77.7% at 10 years. In group without risk factors, incidence of advanced fibrosis was 0.0% at 1 year, 5.3% at 5 years and 48.4% at 10 years after LT. The difference is significant between the 2 groups of patients (p=0.001). (Kaplan–Meier analysis)
We report herein the largest available cohort studying recurrence of initial disease after LT for NAFLD, including more than 150 patients with available post-LT liver biopsy. Previous single center series reported only a few dozen of cases
Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease.
. We found that recurrence of NAFLD was observed in almost all patients 10 years after LT, that NASH recurrence occurred in more than half of the patients at 5 years, and that NAFLD-related significant and advanced fibrosis occurred in 48.0% and 20.0% at 5 years, respectively. The median follow-up of our patients was 4.7 years: this is because most LT were performed after 2015 and protocol graft biopsies are usually planed at 1, 5 and 10 years. Our results, despite a median follow-up time of less than 5 years, strongly support a highly frequent recurrence of the initial disease. We previously reported that the survival of the overall cohort was 79.8% at 5-years
Since most of our patients have a MS at time of LT, our results are not surprising. BMI at LT time of LT was high in our population: we tried to take as much as possible of the dry, non-ascites weights of these patients. The impact of ascites seems minimal as there is no difference between patients transplanted for CHC indication and those transplanted for ESLD. The relatively low median CHILD score may explain a low weight of ascites on body weight and BMI before LT. Estimation of MS in patients with ELSD may be underestimated due to possible disappearance of MS components in the advanced stages of liver disease. MS not only usually persists, but may worsen and occur de novo after LT. The aggravation of MS is mainly related to post-LT dyslipidemia: 62.6% of all patients developed de novo dyslipidemia after LT, probably favored by immunosuppressive drugs. In the context of LT (all etiologies of initial liver disease considered) MS incidence after LT is estimated to range 44-58%
. This high proportion of MS recurrence/occurrence after LT can be explained by the pre-existing MS before LT, associated with immunosuppressive treatment, including CNI, mTOR-i and corticosteroids, which promote diabetes, dyslipidaemia, arterial hypertension and weight gain
. The second explanation can be the very low proportion of patients who have undergone BS or specialized nutritional rehabilitation program in our cohort.
We strongly confirm here the impact of MS on the graft with recurrence of steatosis, NASH and fibrosis. Available studies come from the USA and only one European study investigated the recurrence of NAFLD on the graft, in 11 patients
. The largest available study included 34 patients, and disclosed that NAFLD and NASH recurrence occurred in 88% and 44% of patients, respectively, after a median follow-up of 47 months after LT
Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease.
. Our study is based on 150 patients with at least one liver biopsy performed after LT: recurrence of steatosis was frequent and early, since recurrence rate of NASH was 60.3% at 5 years after LT. Not all patients of the global cohort had a liver biopsy, and one of the limitations of our work could be a selection bias regarding the indication of liver biopsies (disturbed liver tests, steatosis on imaging or clinical arguments). Nevertheless, there was no difference between patients who had a biopsy and those who did not, except that the median follow-up was a little bit longer for patients with available biopsy (4.9 vs. 3.5 months). In addition, liver biopsies were in most cases (> 90%) protocol biopsies at 1, 5 and 10 years, according to different centers strategy: this high proportion of protocol biopsy allows to reduce as much as possible a possible selection bias. There was no difference in recurrence of steatosis, NASH and advanced fibrosis between the whole population with graft biopsy and patients with protocol biopsy after LT (figure 2 and supplementary table 2). Finally, recurrence of initial graft disease was diagnosed on liver biopsies interpreted at each of the LT centers. All the biopsies were not reviewed centrally, but histological lesions were graded (and reported) according to standard validated scores. In addition, less than ten patients underwent non-invasive investigation of steatosis by CAP and fibrosis by liver stiffness measurement or blood tests: these tests are not validated after LT
Performance of transient elastography and serum fibrosis biomarkers for non-invasive evaluation of recurrent fibrosis after liver transplantation: A meta-analysis.
and the small number of patients did not allow statistical analyses.
In available literature, studies on NAFLD recurrence need to be interpreted with caution: some studies included cryptogenic cirrhosis and/or assessment of fibrosis/steatosis on the graft by imaging methods, identifying therefore only steatosis (not NASH), with a significant lack of sensitivity. Our study finds a poor correlation between histologically proven steatosis and imaging, especially for grade 1 steatosis. Few studies (and only some patients included in each study) have analyzed results of liver graft biopsies
Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease.
. In a meta-analysis including 17 studies (2,378 patients), the cumulative incidence of recurrent NAFLD and NASH was 82% and 38%, respectively, more than 5 years after LT
Saeed N, Glass L, Sharma P, Shannon C, Sonnenday CJ, Tincopa MA. Incidence and Risks for Non Alcoholic Fatty Liver Disease and Steatohepatitis Post Liver Transplant: Systematic Review and Meta-analysis. Transplantation. 2019;
. Recurrence of steatosis and NASH has an impact on graft fibrosis. After elimination of other causes of fibrosis, the cumulative incidence of significant fibrosis was 48.0% at 5 years and 65.0% at 10 years in our study. In the study from Bathi et al., NALFD-significant fibrosis recurrence was estimated to be 42.2% at 47 months, and this is consistent with our results
. No re-transplantation for recurrence of the initial disease was performed in our cohort. This low rate of re-LT is consistent with the literature: in a study of 1,295 patients, only 1 re-LT was performed and none in the Bhati's study
. Recurrence of cirrhosis occurred with a median time of 7 years after LT in our study, and therefore probably occurs in elderly transplanted patients, over 70 years old, generally considered too old for a second LT.
Our study did not aim to compare NAFLD patients with a non-NAFLD population. However, in the literature, few studies have analyzed recurrence vs. development of de novo NAFLD on the graft (i.e. in patients transplanted for non-NAFLD cirrhosis)
. In a retrospective study, 80 patients, transplanted for non-NAFLD cirrhosis (Alcohol related-disease with no relapse after LT, HBV or HCV infection) and presenting a de novo NAFLD after LT, were compared to 11 patients with recurrent NAFLD. Advanced fibrosis (71.4% vs.12.5%) and NASH (71.4% vs.17.2%) were more frequent and faster in patients with recurrent NAFLD compared to de novo NAFLD. These data were confirmed by a meta-analysis: at 5 years after LT, NASH recurrence was estimated at 38% vs.17% for de novo NASH
Saeed N, Glass L, Sharma P, Shannon C, Sonnenday CJ, Tincopa MA. Incidence and Risks for Non Alcoholic Fatty Liver Disease and Steatohepatitis Post Liver Transplant: Systematic Review and Meta-analysis. Transplantation. 2019;
Since our study shows a rapid and frequent recurrence of the initial disease on the graft and an evolution towards advanced fibrosis, it is of great relevance to identify risk factors in order to limit disease recurrence and/or progression. Few studies, which evaluated disease recurrence of NAFLD, are summarized in Table 4: only some of them have identified risk factors for recurrence of steatosis or fibrosis. Factors associated with recurrence of the initial disease on the graft can be separated into 3 categories: components of the MS (BMI before and after LT, dyslipidemia and diabetes), factors related to immunosuppressive drugs (CYA or corticosteroids) and demographic factors (such as the age at transplantation). Patients’ factors are not modifiable, and it is therefore necessary to try to manage the other factors to decrease the risk of disease recurrence on the graft. Regarding MS, intensive nutritional management is required along with treatment of diabetes, dyslipidemia and hypertension. The benefit of BS has been demonstrated in non-transplanted patients: in a recent French study, at 5 years after BS, NASH had resolved in 84% of patients and an improvement of liver fibrosis in 76% of patients
. BS could probably be discussed in a majority of patients after LT. Current data suggest that sleeve gastrectomy could be performed 6 to 12 months after LT: the BS type allows access to the bile ducts and a good absorption of immunosuppressant. In our study, only 5 patients had history of BS, including only 2 patients after LT. According to the French Haute Autorité de Santé (HAS) criteria, BS is indicated in cases of a BMI greater than 40kg/m2, or 35kg/m2 with metabolic complications (NASH, hypertension, OSAS, type 2 diabetes, disabling osteoarticular diseases)
. Taking these criteria, 41 (27.3%) of our patients met these criteria at 1 year after LT (personal data): from whom 21 have grade 2 steatosis on liver biopsy at 12 months. One of the explanations for the low proportion of BS in our population is probably the relatively old age of the patients: the expected benefit of BS is validated in patients with an age below 65 years
Clinical Practice Guidelines for the Perioperative Nutritional, Metabolic, and Nonsurgical Support of the Bariatric Surgery Patient—2013 Update: Cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery.
. Nevertheless, several recent studies have suggested that BS could be performed in patients over 65 years of age, with follow-up of up to 6 years and good efficacy on weight, physical activity and biological parameters
. Another explanation may be reluctance on the part of the patient or surgeon to perform a BS after a LT. Nevertheless, current data, based on series of small numbers, show good results of BS after LT
. The second modifiable factor after LT is immunosuppressive treatments: all maintenance immunosuppressive drug classes (CNI, mTOR-i and corticosteroids) have deleterious metabolic profile except antimetabolites (MMF and azathioprine). MMF monotherapy is possible but only in selected patients with long delay since LT and can therefore not be generally recommended
. Avoiding steroids and minimizing CNI and/or mTORi is feasible but probably has a minimal impact. We believe it is essential to reduce the dosage of immunosuppressive drugs to a minimum, and to perform a liver biopsy at 12 months of LT to determine steatosis. Recurrence of grade 2 steatosis on the graft at 12 months seems to be strongly associated with both recurrence of NASH and advanced fibrosis: we suggest discussing BS in these patients, who represent about a quarter of NAFLD transplanted patients in our population.
Table 4Recurrence of Initial Graft Disease after Liver Transplantation for NAFLD Cirrhosis (Major studies with liver histological evaluation).
In conclusion, we report here the largest cohort based on liver biopsies of NAFLD recurrence after LT. The recurrence of steatosis and steatohepatitis on the graft was rapid and very frequent. Several associated factors have been identified and need to be confirmed in other large studies. The performance of a protocol biopsy at 12 months after LT appears to be determining to identify patients at risk of progression to NASH or advanced fibrosis. In these patients with grade 2 steatosis, representing about 25% of patients, BS should be discussed. The management of metabolic factors and the place of BS need to be evaluated to reduce the risk of recurrence of the initial disease in this exponentially growing population of LT patients.
Data availability statement:
Due to the sensitive nature of the questions asked in this study, survey respondents were assured raw data would remain confidential and would not be shared.
Acknowledgements
We thank all LT teams that participated in this study.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease.
Saeed N, Glass L, Sharma P, Shannon C, Sonnenday CJ, Tincopa MA. Incidence and Risks for Non Alcoholic Fatty Liver Disease and Steatohepatitis Post Liver Transplant: Systematic Review and Meta-analysis. Transplantation. 2019;
Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity.
Performance of transient elastography and serum fibrosis biomarkers for non-invasive evaluation of recurrent fibrosis after liver transplantation: A meta-analysis.
Clinical Practice Guidelines for the Perioperative Nutritional, Metabolic, and Nonsurgical Support of the Bariatric Surgery Patient—2013 Update: Cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery.
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