Dual alcohol and metabolic‐related liver disease: Results from a population of liver transplant patients

If alcohol‐related liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are now the two main indications for liver transplantation (LT), it has been recognized that both conditions can coexist in varying degrees and the concept of dual‐aetiology fatty liver disease (DAFLD) has been proposed. This retrospective study aimed to evaluate, in a cohort of patients transplanted for ALD and NAFLD, the prevalence of DAFLD before LT and the impact on liver graft outcome.


| INTRODUC TI ON
The three major causes of chronic liver disease worldwide are viral hepatitis (B and C), alcohol, and metabolic syndrome, with some significant geographical disparities. 1 Alcohol-related liver disease (ALD) remains the most common cause of liver cirrhosis in Europe. 1 From 2012 to 2017, age-standardized death rate for nonalcoholic fatty liver disease (NAFLD) and ALD liver cirrhosis increased annually by 1.4% and .5% respectively, whereas there was no increase for viral hepatitis liver cirrhosis, probably because of hepatitis B virus (HBV) vaccination programs and direct antiviral treatments for hepatitis C virus (HCV). 1 During the past two decades, NAFLD prevalence increased, reaching 25%. 1 This evolution is strongly related to the epidemic of worldwide obesity, type 2 diabetes, hyperlipidaemia, and hypertension as components of the metabolic syndrome. 2r reasons of simplification, especially in epidemiological analyses, liver disease patients are usually classified into a single aetiology group.It must be pointed out that multiple factors can be synergistic for liver disease progression in individuals.The most described association in the past was excessive alcohol consumption and HCV infection in addicted patients. 3Similarly, to evaluate the risk of severe liver disease, it has been shown that the interaction between alcohol consumption and metabolic syndrome has to be addressed. 4,5D and NAFLD are distinguished by average alcohol intake, since they share the same histological features. 6It seems too simple to categorize patients either in ALD or NAFLD according to their alcohol consumption since there is often a clinical overlap between both factors.4][5] It is therefore clear that both conditions (alcohol use and metabolic syndrome) can coexist in varying degrees in a significant proportions of patients and the concept of Dual-Aetiology Fatty Liver Disease (DAFLD) has been proposed. 7th the decline of HCV, ALD is the leading indication for liver transplantation (LT) worldwide and represent more than one-third of all LT in Europe. 8,9In 2013, in the United States, NAFLD has become the second cause of chronic liver disease among patients awaiting LT with a rapid increase over the last decade. 10,11In Europe, the same trend was observed and LT for NAFLD increased significantly from 1.2% in 2002 to 8.4% in 2016. 8ctors associated with the development of liver disease before LT can persist or emerge after LT as persistent or new risk factors for graft injury.The prevalence of metabolic syndrome before LT remains low except for NAFLD patients.Whatever the initial indication for LT, metabolic syndrome has been reported in up to 40%-60% of patients after LT and especially in patients with initial ALD. 12Concerning alcohol relapse, even if most patients remain abstinent or occasional drinkers after LT, up to 20% of the patients will experience severe alcohol relapse. 13After LT, alcohol relapse, metabolic syndrome, or both are determinants for graft injury. 14,15aft injury after LT described as recurrent or de novo fatty liver disease according to the indication of LT is a common finding. 16As in immunocompetent patients, fatty liver graft disease can progress to steatohepatitis, significant and advanced fibrosis, and finally cirrhosis. 15,17,18The present retrospective study aimed to evaluate, in a large cohort of patients transplanted for ALD, the prevalence of DAFLD before LT and the impact on liver graft after LT.We hypothesized that among the so-called ALD patients, patients with metabolic syndrome (DAFLD) or without metabolic syndrome (ALD), prior LT might not have the same outcome after LT.The aims of the present study were to (1) evaluate DAFLD's prevalence in a population of socalled ALD LT patients, (2) describe the characteristics of DAFLD patients before and after LT, and (3) analyse the impact on the graft and evaluate if DAFLD patients have different determinants of post-LT fatty liver graft disease.

| Patient selection and study design
From 1990 to 2010, all patients who underwent a first LT for socalled ALD at Hospital Edouard Herriot, Lyon, France and who survived more than 1 year were included in the study population.We excluded patients awaiting LT for ALD associated with recognized other causes of liver disease except metabolic syndrome (mainly HBV or HCV infection, or autoimmune hepatitis).Before LT, ALD was confirmed by a multidisciplinary transplant team (surgeon, nurses and hepatologist) and histologically by a pre-transplant biopsy or by the histological analysis of the explanted liver.
In this population of patients transplanted for so-called ALD, we identified patients for whom initial liver disease could have been DAFLD.Before LT, DAFLD was defined as patients with a history of excessive alcohol consumption and a history or at listing: obesity (BMI ≥ 30 kg/m 2 ) associated with either diabetes or hypertension.
Since the evaluation of obesity in patients with end-stage liver disease and ascites is a challenge, and waist circumference does not seem to be an accurate indicator, we used a BMI ≥ 30 kg/m 2 without ascites or after paracentesis (dry-weight) as the best indicator for obesity in our population. 19In addition, the interpretation of lipid profile in the context of end-stage liver disease is not valid with very low serum levels of lipids and lipoproteins; therefore, we excluded the results of the lipid profile to define properly a DAFLD patient. 20ring the same inclusion period, patients transplanted for NAFLD

Key Points
Dual alcohol and metabolic-related liver disease has recently emerged as an indication of liver transplantation.In the future, this particular population needs to be identified as a specific entity since post-liver transplantation outcome on the graft is different from alcohol-related liver disease and more similar to nonalcoholic fatty liver disease patients.
were included as a control group of "pure metabolic" patients with regard to mean alcohol consumption less than 3 drinks per day in men and 2 drinks per day in women.In summary, before LT, patients were separated in three groups: LT for ALD, DAFLD, and NAFLD.

| Liver transplantation and follow-up
All patients received blood group compatible grafts from cadaveric or living donors.Outpatient follow-up visits were on average scheduled once a week for the first month after hospital discharge, twice a month for the second and third months, monthly during the first year, and every 3-12 months thereafter.Additional visits were also scheduled when necessary for specific issues.A complete laboratory investigation, including haematology, liver parameters, coagulation, electrolytes, total protein, renal parameters, fast blood glucose, and lipid profile was performed at each visit.An initial liver biopsy sample was taken at the end of the transplantation procedure.Patients then underwent liver graft biopsy using a standardized protocol at 1, 5, 10, 15, and 20 years as part of the routine management in our centre and when clinically indicated.Informed consent was obtained from the patient before liver biopsy in all cases.
Clinical data were retrospectively collected in patient's charts at two different points during follow-up: at LT registration and last follow-up.At registration, severity of liver disease was assessed using the MELD and Child-Pugh score.The following data were collected: age, gender, BMI, hypertension, immunosuppression regimen, treatments, hepatocellular carcinoma.Data on the donor, such as age, BMI, gender were reported as well as the type of graft.Patients were followed until death or last follow-up.February 1, 2020 was the endpoint of the study.

| Metabolic syndrome after LT
Metabolic syndrome was defined according to the 2009 joint interim statement of the International Diabetes Federation and the National Heart, Lung and Blood institute. 21We chose to use this recent definition since it was designed in collaboration with multiple scientific associations.In the past, the principal difference between all definitions of metabolic syndrome was related to waist circumference.
Waist circumference recommendations were then made according to geographical origins.The European threshold for abdominal waist circumference is equivalent to a BMI of approximately 30 kg/m 2 .
Metabolic syndrome after LT was diagnosed when at least 3 of the Patients with drug treatments for elevated triglycerides, for reduced HDL-c, for elevated glucose and antihypertensive drug treatment were considered to be respectively alternate indicators to fulfil the criteria of metabolic syndrome.

| Alcohol/tobacco consumption
All patients with a history of alcohol consumption were advised to remain abstinent after LT.Alcohol relapse after LT was evaluated at each follow-up visit and was based on the patient's or his/her relative's or by the primary care physician report.Any alcohol use after LT was considered to be a relapse.Severe alcohol relapse was defined as a declared mean alcohol use of more than 20 g/day (women) or 30 g/day (men) for at least 6 months.Patients were divided into three groups: no alcohol relapse, occasional and severe alcohol relapse.In case of alcohol relapse, support was offered by a psychiatric and addiction specialist to control and withdraw any alcohol use.
Date of relapse was reported and the cumulative years of excessive drinking were defined as the total years of follow-up after exclusion of periods of abstinence.Smoking (tobacco), before and after LT, was categorized according to: never smoking, past, and active smoking.

| Histological data
Liver biopsy was fixed in formalin, processed according to conventional procedures, and cut into 4-μm-thick sections stained with haematoxylin-eosin-safran, Sirius red, and Perls' coloration.For each biopsy, three section levels were analysed, and the included biopsies had to measure more than 10 mm with at least 10 hepatic portal spaces.All biopsy samples were reviewed by experienced pathologists.Evaluation and reporting of histopathological features were performed according to the steatosis, activity and fibrosis (SAF) scoring scale.Steatosis was assessed by the percentage of hepatocytes containing large or medium-sized lipid droplets from 0 to 3 grades: S0: <5%; S1: 5%-33%, S2: 34%-66%, S3 > 67%.Lobular inflammation was defined as a focus of 2 or more inflammatory cells within the lobule organized either as microgranulomas or located within the sinusoids: grade 0: none; 1: 2 foci per lobule.Ballooning of hepatocytes was graded from 0 to 2 (0: normal hepatocytes; 1: presence of clusters of hepatocytes with a rounded shape and pale cytoplasm, but normal size; 2: as for grade 1, but with at least one enlarged ballooned hepatocyte).All liver biopsies were reassessed according to the FLIP algorithm.The FLIP algorithm uses steatosis as the entry criteria into the algorithm, then weighted by hepatocellular ballooning and lobular inflammation.All cases with <5% steatosis were labelled as no fatty liver disease whereas all cases presenting with at least grade 1 of each of the three features (steatosis, ballooning and lobular inflammation) were classified as steatohepatitis.The remaining cases were diagnosed as fatty liver disease with bland steatosis.Stage of fibrosis was assessed using the NASH Clinical Research Network fibrosis score as follows: F0: none, F1: perisinusoidal zone 3 or peri-portal fibrosis stage, F2: perisinusoidal and peri-portal fibrosis without bridging stage, F3: bridging stage and F4: cirrhosis.Fibrosis ≥ F2 was considered to be significant fibrosis and F3-F4 fibrosis to be advanced fibrosis. 21The same algorithm and scoring system were used regardless of the cause of liver graft injury (alcohol relapse, metabolic syndrome or both).Post-LT fatty liver disease and steatohepatitis were related to post-LT clinical phenotype: metabolic-related, severe alcohol-related or both.

| Statistical Analysis
Categorical data are presented as proportions and percentage and continuous variables are presented as median and range.
Comparisons between qualitative and quantitative variables were respectively performed using the chi-square test and non-parametric Kruskal-Wallis test or one-way ANOVA.A p-value < .05 was considered to be significant.All analyses were performed using SPSS software, version 21 (IBM SPSS Statistics for Windows, Version 21.0.Armonk, NY, USA).

| What was the prevalence of DALFD in the population of so-called ALD LT patients?
From 1990 to 2010, 366 patients were transplanted in our centre for so-called ALD as the main LT indication out of 907 LT recipients and survived more than 1 year.According to our definition, 333 patients (90.9%) were classified as ALD and 33 patients (9.1%) as DAFLD.
These two groups (ALD and DAFLD), were also compared with the group of 24 patients transplanted for NAFLD during the same period.LT for DAFLD and NAFLD emerged recently and represented 3.7% and .7% of all LT from 2000 to 2004 and 6.4% and 8.2% from 2005 to 2010, respectively (Figure 1).

| What were the characteristics of DAFLD patients before LT?
The characteristics of patients transplanted for ALD, DAFLD, and NAFLD before LT are described in Table 1.Patients transplanted for DAFLD compared with ALD patients were significantly older, had more features of metabolic syndrome including BMI ≥ 30, diabetes, and hypertension.In contrast, DAFLD were not different from NAFLD patients in terms of age and metabolic syndrome features, such as diabetes, hypertension except for a BMI ≥ 30.Alcohol abstinence and tobacco consumption was not different in patients transplanted for ALD or DAFLD.The severity of liver disease according to MELD and Child-Pugh score at registration was not different between ALD and DAFLD patients but ALD patients had more ascites.
Patients transplanted for DAFLD and NAFLD had significantly more HCC than ALD patients.At the time of LT, donor gender, donor age, and the type of graft was not different in ALD and DAFLD patients.
Patients transplanted for DAFLD received graft from donors with significantly higher BMI compared with ALD patients (Table 1).

| What were the characteristics of DAFLD patients after LT?
Patient's follow-up was not different between ALD and DAFLD patients, but was significantly longer compared with NAFLD patients  1).ALD patients experienced significantly more severe alcohol relapse than DAFLD patients, but there was no difference for smoking habits.After LT, metabolic syndrome was significantly more prevalent in patients with DAFLD compared with ALD patients, but there was no difference in patients with DAFLD compared with NAFLD patients.
The main differences of metabolic syndrome features between DAFLD and ALD were BMI ≥ 30 kg/m 2 , diabetes, and hypertension.There was no difference of lipid profile or treatment.DAFLD were similar to NAFLD for all the features of the metabolic syndrome (including all the stages of obesity) except for HDL-C cholesterol or statins treatment (Table 1).Figure 2 summarizes alcohol and metabolic trajectories after LT according to pre-LT group.Compared with ALD patients, DAFLD patients had twice more post-LT metabolic syndrome.At the last follow-up, ALD and DALFD patients had less tacrolimus-based immunosuppression than NAFLD patients but compared with ALD patients, DAFLD patients received more everolimus (Table 1).

| What are the determinants of fatty liver graft disease?
Histological examination of liver graft biopsies at reperfusion showed no difference between all groups for graft steatosis or

| DISCUSS ION
In the past two decades, due to the epidemic of obesity, the prevalence of metabolic syndrome increased in the general population and in patients awaiting LT. [22][23][24] ALD is the main indication for LT in Europe, but NAFLD has emerged as an indication for LT in the past 10 years. 8,16The present study is the first one to report, in a large population of LT patients, that patients transplanted for so-called LT, de novo metabolic syndrome is common and especially in ALD patients. 25The prevalence of metabolic syndrome in the general population is around 25%-30%, but it is much higher in LT population around 40%-60%, and it can reach 65% for NAFLD transplanted patients. 12,26,27In our cohort, DAFLD patients had higher rates (66.7%) of metabolic syndrome.This is probably related to the presence of pre-LT metabolic features that persisted after LT: 100% had a BMI ≥ 30 kg/m 2 , 75.8% had hypertension, and 60.6% had diabetes before LT.In a 2018 meta-analysis in- to liver failure and LT.Recurrent NAFLD has been reported from 24.0% to 88.2% and steatohepatitis in 30%-40% of LT patients for NAFLD. 18,31In a meta-analysis of 2166 patients, pooled prevalence of de novo fatty liver graft disease was globally of 26% and of 37% in patients transplanted for ALD. 32In two recent studies with longer follow-up, the prevalence of de novo fatty liver graft disease was higher of 56.4 and 53.0%. 14,33Authors admitted that these high rates might be explained by a low threshold of alcohol consumption to define ALD and some patients could have been probably transplanted for NAFLD or DAFLD rather than ALD as in our study.We previously compared de novo NAFLD (n = 80) and recurrent NAFLD (n = 11) and suggested that they were different entities and that recurrent NAFLD appeared to be earlier and was more severe. 34In our cohort, NAFLD patients had less biopsies and were followed for a shorter time, that could explain slightly less fatty liver disease in NAFLD patients compared with DAFLD patients.Nevertheless, in a study comparing patients transplanted for ALD and NAFLD, there was no difference in recurrent and de novo fatty liver disease and steatohepatitis, but information on metabolic syndrome before and after LT and alcohol relapse was lacking. 33In our cohort, prevalence of fatty liver graft disease was higher than that in all the previous studies and especially for DAFLD patients.A proportion of recipients with fatty liver graft disease will progress towards steatohepatitis.De novo steatohepatitis (2%-10.4%)appears to be much less frequent than recurrent steatohepatitis (30%-40%) but a recent report by Galvin et al. in   2019 reported in patients with de novo fatty liver disease, 51.7% of steatohepatitis. 14,18,32,35Here again in our cohort, we report high prevalence of steatohepatitis in patients with fatty liver graft disease.These high rates of fatty liver disease and steatohepatitis could be explained by the long follow-up of DAFLD patients and to the fact that patients had protocolized biopsies which is the best way to estimate liver graft injury.DAFLD patients also emerged in the 2000's concomitantly with the epidemic of obesity and metabolic syndrome.The era of transplantation studied probably plays a role in the prevalence of each type of fatty liver graft disease.Galvin et al. also noticed that liver graft injury increased with the increased prevalence of diabetes and median BMI in their population. 35ver graft fibrosis is the most relevant histological lesion since it can lead to recurrent cirrhosis and liver decompensation, which directly impacts patients and grafts survival.Most studies reported a very low rate of significant fibrosis induced by de novo fatty liver graft disease ranging from 0 to 17.7% compared with 20%-71% in recurrent NAFLD. 14,34,36,37In our study, significant fibrosis in patients with fatty liver graft disease was also higher than previously described probably due to a longer follow-up and there was no significant difference among the three groups.
In ).We previously reported that alcohol relapse is known to induce rapid and severe liver graft injury that can lead to recurrent cirrhosis, with no significant impact of metabolic syndrome. 25,38Indeed, in the present analysis, ALD patients were the only group with liver graft injury related only to alcohol relapse.DAFLD and NAFLD had more liver graft injury related to metabolic syndrome.Our study is also the first one to describe liver injury related to both metabolic syndrome and alcohol relapse which is equally prevalent in ALD and DAFLD group.Post-LT management of liver graft injury needs to be therefore adjusted to causal factors.
The main limitation of our study is that it is particularly difficult to diagnose metabolic syndrome in patients awaiting LT, since cirrhotic patients with end-stage liver disease are often malnourished, with ascites and hypotension.Due to liver failure and impaired glycidic and lipidic metabolism, lipids profile, and lipoproteins are also not interpretable.Since the definition of metabolic syndrome cannot be applied to a population of chronic liver disease patients awaiting LT, we had to build our own definition.We chose obesity (BMI ≥ 30 kg/ m 2 ) as a corner stone of our definition associated with hypertension and diabetes.The definition we chose to define DAFLD might be in a way too selective (BMI ≥ 30 kg/m 2 ) but it allows to select only patients with "real" metabolic syndrome.The retrospective nature of our study, could have also missed patients who progressively lost metabolic features (impaired lipidic and glucidic metabolism) and developed sarcopenia with liver disease progression.Nevertheless, a history of obesity, diabetes and hypertension reported in medical charts allowed us to identify patients with a history of metabolic syndrome before liver failure consequences.Unfortunately, we also did not have access to genetic polymorphisms.
In conclusion, our results strongly support that DAFLD is a recent and emerging indication of LT.DAFLD patients were at high risks of post-LT metabolic syndrome and increased liver graft injury related to metabolic features more than alcohol relapse compared with ALD patients.In the future, this particular population needs to be individualized since graft injury is different from ALD

significant fibrosis F ≥ 2 .
A total of 926 post-LT biopsies was available: 800 biopsies in the ALD group, 81 in the DAFLD group and 45 in the NAFLD group.In the ALD group, the patients had a biopsy in median every 4.8 years (range: 4.1-6.7 years); in the DALFD group in median, every 4.3 years (range: 3.45-4.9years), and in the NAFLD group in median, every 3.7 years (range: 2.9-4.2) (p = .0001).The diagnosis rate of fatty liver graft disease after LT was of 68.6% in the ALD group, 84.9% in the DAFLD group and 62.5% in the NAFLD group (Figure3).After LT, DAFLD patients had significantly more fatty liver graft disease due to metabolic syndrome features than ALD patients.NAFLD patients had no alcohol-related fatty liver graft disease.Compared with ALD patients, DAFLD patients had no fatty liver disease only related to alcohol relapse.Compared with NAFLD, DAFLD patients had more fatty liver graft disease.In patients with fatty liver graft disease, steatohepatitis was diagnosed in 35.5% in the ALD group, in 51.5% in the DAFLD group and in 50.0% in the NAFLD group.In NAFLD's patients, significant and advanced fibrosis, in patients with fatty liver graft disease, was related to metabolic features while it was partly due to alcohol relapse in DAFLD and in ALD patients (Figure3).Women with post-LT steatohepatitis have developed more post-LT significant fibrosis (F ≥ 2) (50.7% vs. men 24.5% (p < .001))and more advanced fibrosis (F3-F4) (25.3% vs. men 10.8% (p = .003)).
ALD, could have been transplanted for DAFLD.DAFLD as an indication for LT has emerged in the 2000s alongside with NAFLD.NAFLD patients compared to ALD patients had more metabolic comorbidities, were older and indication of LT was more frequently HCC rather than liver failure.Half of DAFLD patients were also transplanted for HCC similar to NAFLD patients.This is in accordance with the US study from Wong et al. also who reporting high prevalence of HCC in NAFLD (21.0%) compared with ALD (7.5%) patients. 10Before and after LT, DAFLD patients seem to have the same behaviour as NAFLD patients rather than ALD patients in terms of metabolic syndrome, with two times less alcohol relapse.Fatty liver graft disease and steatohepatitis was increased in DAFLD compared with ALD patients and was more related to metabolic features like in NAFLD patients.Nevertheless, DAFLD patients had no more significant and advanced fibrosis than ALD or NAFLD patients.LT is probably the best human model to study the impact of factors associated with the development of liver disease since all patients receive a new functional liver graft and are followed closely (and histologically) after LT.Factors associated with the development of liver disease before LT can persist or emerge after LT, as persistent or new risk factors for graft injury.After cluding studies from 2007 to 2015 and 3539 LT recipients, pre-LT BMI and diabetes were the two risks factors for post-LT metabolic syndrome with in this study a prevalence of 35%.12 Similar to recurrent metabolic syndrome, patients can also experience alcohol relapse (occasional or severe) after LT and Kodali et al. reported in a 2018 meta-analysis an alcohol relapse rate of 26.3% (18.0%-36.7%).In our cohort, alcohol relapse was in the same range for ALD patients, but two times lower in DAFLD patients, reflecting that DAFLD patients could be a different population in between NAFLD and ALD patients.After LT, the association of metabolic syndrome and alcohol relapse (10.2% in ALD and 6.1% in DAFLD patients) has to the best of our knowledge never been described before.The synergistic deleterious effect of this association has been studied outside LT context and severe alcohol consumption has been associated with increased risk of metabolic syndrome but there are no available data in LT field.28In 2020, Eslam et al. proposed a new definition for the diagnosis of metabolic-associated fatty liver disease (MAFLD) with positive criteria, simple and independent of other liver disease. 29This new definition is based on liver steatosis in addition to overweight, type 2 diabetes or evidence of metabolic dysregulation independently from average alcohol intake.Thus, patients with MAFLD represent a very heterogeneous group of patients in terms of severity of alcohol consumption and metabolic syndrome.We chose not to use this definition for our present work since post-LT natural history of patients transplanted for ALD seems different than patient transplanted for NAFLD or DAFLD.We also wanted to study precisely the impact of the association of alcohol and metabolic F I G U R E 3 Determinants of liver graft injury according to post LT metabolic features and alcohol relapse.syndrome (DAFLD) compared with alcohol only and metabolic only.In 2023, a group of expert proposed a new term for NAFLD to be MASLD (metabolic-associated steatotic liver disease) and MetALD for patients with MASLD and increased alcohol intake which could partly represent our DAFLD patient group even if we were more stringent regarding metabolic features with the definition of DAFLD. 30However, ALD patients with few elements of metabolic syndrome were classified as ALD in the present study, even if they would be classified now as MetALD alcohol predominant in the new nomenclature.Alcohol and metabolic syndrome are frequently implicated in liver graft injury since they initially led
Evolution of LT indications from 1990 to 2010.TA B L E 1 ALD n TA B L E 1 (Continued) (Continues) (Table addition to pre-LT comorbidities, new risk factors of liver graft injury can emerge after LT, especially de novo metabolic syndrome.A very interesting and original point herein was to study the determinants of fatty liver graft disease according to post-LT alcohol relapse and post-LT metabolic features, since they are different according to LT indication.Most studies take into account dichotomizing alcohol relapse vs. metabolic syndrome and this may explain the important heterogeneity of reported graft injury.Hejlova et al. found that the major risk factors for the development of graft steatosis were: LT for ALD (OR 3.62, IC 95% 2.04-6.42),BMI increase (OR 1.27, IC 95% 1.17-1.38)and post-LT alcohol consumption (OR 9.46, IC 95%: 2.32-38.6