Definite and indeterminate nonalcoholic steatohepatitis share similar clinical features and prognosis: A longitudinal study of 1893 biopsy-proven nonalcoholic fatty liver disease subjects

2076 | Liver International. 2021;41:2076–2086.wileyonlinelibrary.com/journal/liv Received: 20 January 2021 | Revised: 1 April 2021 | Accepted: 6 April 2021 DOI: 10.1111/liv.14898 ORIGINAL ARTICLE Definite and indeterminate nonalcoholic steatohepatitis share similar clinical features and prognosis: A longitudinal study of 1893 biopsyproven nonalcoholic fatty liver disease subjects Javier Ampuero1,2,3 | Rocío Aller4 | Rocío Gallego- Durán2,3 | Javier Crespo5 | Javier Abad6 | Águeda González- Rodríguez7 | Judith Gómez- Camarero8 | Joan Caballería3,9 | Oreste Lo Iacono10 | Luis Ibañez3,11 | Javier García- Samaniego3,12 | Rosa Martín- Mateos3,13 | Rubén Francés3,14 | Conrado Fernández- Rodríguez15 | Moisés Diago16 | Germán Soriano3,17 | Raúl J. Andrade3,18 | Raquel Latorre19 | Francisco Jorquera3,20 | Rosa M. Morillas3,21 | Desam Escudero22 | Pamela Estévez23 | Manuel Hernández- Guerra24 | Salvador Augustín25 | María Jesús Pareja- Megia26 | Jesús M. Banales3,27 | Patricia Aspichueta28,3 | Salvador Benlloch3,29 | José Miguel Rosales30 | Javier Salmerón31 | Juan Turnes32 | Manuel Romero- Gómez1,2,3 | on behalf of HEPAmet Registry 1Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Seville, Spain 2SeLiver Group, Instituto de Biomedicina de Sevilla, Seville, Spain 3CIBERehd, Madrid, Spain 4Centro de Investigación de Endocrinología y Nutrición, Hospital Clínico Universitario de Valladolid, Universidad de Valladolid, Valladolid, Spain 5Digestive Department, Hospital Universitario Marqués de Valdecilla, Santander, Spain 6Digestive Department, Hospital Universitario Puerta de Hierro, Madrid, Spain 7Liver Research Unit, Hospital Universitario Santa Cristina Instituto de Investigación Sanitaria Princesa, Madrid, Spain 8Digestive Department, Hospital Universitario de Burgos, Burgos, Spain 9Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBPAS), Barcelona, Spain 10Digestive Department, Hospital Universitario Tajo, Aranjuez, Spain 11Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain 12IdiPAZ, Hospital Universitario La Paz, Madrid, Spain 13Digestive Department, Hospital Universitario Ramón y Cajal, Madrid, Spain 14Hospital General Universitario de Alicante, Universidad Miguel Hernández, Elche, Spain 15Hospital Universitario Fundación de Alcorcón, Universidad Rey Juan Carlos, Móstoles, Spain 16Digestive Department, Hospital General Universitario de Valencia, Valencia, Spain 17Digestive Department, Hospital de la Santa Creu i San Pau, Barcelona, Spain 18Unidad de Gestión Clínica de Enfermedades Digestivas, Instituto de Investigación Biomédica de Málaga- IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga, Spain 19Digestive Department, Hospital Universitari Son Llátzer, Mallorca, Spain 20Servicio de Aparato Digestivo, Complejo Asistencial Universitario de León, IBIOMED, León, Spain 21Digestive Department, Hospital Germans Trias i Pujol, Badalona, Spain Abbreviations: AHT, arterial hypertension; BMI, body mass index; MAFLD, metabolicassociated fatty liver disease; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; T2DM, type 2 diabetes mellitus. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Liver International published by John Wiley & Sons Ltd. | 2077 AMPUERO Et Al 22Hospital Clínico Universitario de Valencia, Universitat de València, Valencia, Spain 23Digestive Department, Complejo Hospitalario Universitario de Vigo, Vigo, Spain 24Digestive Department, Hospital Universitario de Canarias, Santa Cruz de Tenerife, Spain 25Digestive Department, Hospital Vall d'Hebrón, Barcelona, Spain 26Pathology Department, Hospital Universitario Virgen de Valme, Sevilla, Spain 27Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute— Donostia University Hospital, University of the Basque Country (UPV/ EHU), Ikerbasque, San Sebastian, Spain 28Department of Physiology, Faculty of Medicine and Nursing, Biocruces Research Institute, University of Basque Country UPV/EHU, Leioa, Spain 29Digestive Department, Hospital Universitari i Politecnic La Fe, Valencia, Spain 30Digestive Department, Agencia Sanitaria Costa del Sol, Marbella, Spain 31Digestive Department, Hospital Universitario San Cecilio, Granada, Spain 32Digestive Department, Complejo Hospitalario de Pontevedra, Pontevedra, Spain Correspondence Javier Ampuero, Digestive Disease Department and CIBERehd, Virgen del Rocio University Hospital, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain. Email: jamp[email protected] Funding information This project has been partially funded by the ‘Consejería de Salud de la Junta de Andalucía’ (PI- 0075- 2014) and the ‘Spanish Ministry of Economy, Innovation and Competition, Instituto de Salud Carlos III’ (PI19/01404, PI16/01842, PI17/00535 and GLD19/00100). The funders have not had any role in the design, analysis, writing or interpretation of this project. Handling Editor: Salvatore Petta Abstract Background and Aim: Histological score systems may not fully capture the essential nonalcoholic steatohepatitis (NASH) features, which is one of the leading causes of screening failure in clinical trials. We assessed the NASH distribution and its components across the fibrosis stages and their impact on the prognosis and their relationship with the concept of metabolicassociated fatty liver disease (MAFLD). Methods: Spanish multicenter study including 1893 biopsyproven nonalcoholic fatty liver disease (NAFLD) patients from HEPAmet registry. NASH was diagnosed by NAS score ≥4 (including steatosis, ballooning and lobular inflammation) and fibrosis by Kleiner score. The presence of MAFLD was determined. Progression to cirrhosis, first episode of decompensated cirrhosis and death were collected during the followup (4.7 ± 3.8 years). Results: Fibrosis was F0 34.3% (649/1893), F1 27% (511/1893), F2 16.5% (312/1893), F3 15% (284/1893) and F4 7.2% (137/1893). NASH diagnosis 51.9% (982/1893), and its individual components (severe steatosis, ballooning and lobular inflammation), increased from F0 (33.6%) to F2 (68.6%), and decreased significantly in F4 patients (51.8%) (P = .0001). More than 70% of non- NASH patients showed some inflammatory activity (ballooning or lobular inflammation), showing a similar MAFLD rate than NASH (96.2% [945/982] vs. 95.2% [535/562]) and significantly higher than nonalcoholic fatty liver (NAFL) subjects (89.1% [311/349]) (P < .0001). Progression to cirrhosis was similar between NASH (9.5% [51/539]) and indeterminate NASH (7.9% [25/316]), and higher than steatosis (5% [14/263]) (logRank 8.417; P = .015). Death and decompensated cirrhosis were similar between these. Conclusions: The prevalence of steatohepatitis decreased in advanced liver disease. However, most of these patients showed some inflammatory activity histologically and had metabolic disturbances. These findings should be considered in clinical trials whose main aim is to prevent cirrhosis progression and complications, liver transplant and death. KEYWORDS ballooning, fatty liver disease, inflammation, metabolicassociated fatty liver disease, natural coursesteatohepatitis, steatosis 2078 | AMPUERO Et Al 1 | INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of histological features that range from simple steatosis to nonalcoholic steatohepatitis (NASH) and, finally, cirrhosis.1 Splitting NAFLD into three successive stages (steatosis, noncirrhotic NASH and cirrhotic NASH) has provided a convenient conceptual framework for improving the diagnostic methods and identifying areas of potential future drug development.2 However, the diagnosis of NAFLD is challenging sometimes, so we need to consider a more pragmatic approach to targeting these individuals. In this scenario, the term “metabolicassociated fatty liver disease” (MAFLD) has been recently proposed.3 The diagnosis of MAFLD is based on recognizing underlying alterations in metabolism,4 beyond the histological classification of NAFLD. MAFLD is defined by the presence of steatosis (by histology or imaging) and overweight or at least two metabolic risk factors. Notably, steatosis could be absent in the case of advanced liver disease.5 Subjects enrolled in NASH clinical trials are usually required to have a NAS ≥ 4 and a fibrosis stage from F1 to cirrhosis by liver biopsy. However, up to half of the screened individuals fail to meet these eligibility criteria.6 We should consider that NAS score was developed as a tool to measure NAFLD changes during therapeutic trials instead of as a surrogate for the histologic diagnosis of NASH.7 Notably, previous studies have reported that 20%– 40% of the NAFLD population does not display a definite NASH histological diagnosis,8,9 making these individuals ineligible for enrollment. Consequently, the large number of suboptimal biopsies adds significantly to the cost and duration of clinical trials. However, liver fibrosis is considered the strongest predictor of adverse clinical outcomes.10 Besides, treatment goals for patients with advanced liver disease are to halt or slow fibrosis progression, prevent clinical decompensation, reduce the need for liver transplantation and improve survival.11 The liver biopsy must keep being essential in the management of NAFLD,12 but we should avoid converting this tool into a barrier to diagnose, treat or identify atrisk patients. For instance, the development of noninvasive tests and imagingbased methods has allowed a wide expansion of liver fibrosis assessment in atrisk patients.1 3 - 1 6 Thus, the liver biopsy should be relocated to maximize its advantages in selected patients. In a Spanish large cohort of individuals with biopsyproven NAFLD followedup during 4.7 years, we aimed to assess the prevalence of NASH and its components (steatosis, ballooning and lobular inflammation) across the fibrosis stages and link them with prognostic outcomes to determine if NAS score ≥4 reflects a reliable scenario in clinical practice as inclusion criterium in NAFLD clinical trials. 2 | METHODS 2.1 | Selection of patients This is an observational study of 1893 patients with biopsyconfirmed NAFLD who had been enrolled and prospectively followed up from the Spanish HEPAmet Registry. This registry is governed by the Spanish Association for the Study of the Liver and the Network of Biomedical Research Centre for the Study of the Liver and Digestive Diseases (CIBERehd). Data monitoring is a fundamental element of the registry, ensuring data procurement accuracy and minimization of bias. Patients underwent a liver biopsy according to the routine decisions in the clinical practice (eg, presence of fatty liver by imaging, increased aminotransferase levels, based on noninvasive tests and suspected advanced liver disease by imaging or laboratory tests) or at the time of bariatric surgery. The inclusion criterion was biopsyproven NAFLD, irrespective of the existence of NASH or fibrosis stage. Exclusion criteria were significant alcohol intake (≥30 g daily for men and ≥20 g daily for women) and evidence of concomitant liver disease (ie, viral or autoimmune hepatitis, HIV, druginduced fatty liver, hemochromatosis or Wilson's disease). The study was performed in agreement with the Declaration of Helsinki and approved by the Ethics and Clinical Research Committee of every centre. All patients were informed of the nature of the study and gave their written consent to participate. 2.2 | Clinical assessment Demographic characteristics, anthropometric measurements and laboratory tests (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), triglycerides, cholesterol, HDL- c, LDL- c, fasting glucose, HbA1c, insulin, creatinine and albumin) were recorded at the same time of liver biopsy. An overnight (12- h) fasting blood sample was taken for routine biochemical analyses. Homeostatic model assessment (HOMA) Key points • The prevalence of definite NASH histological criteria decreases in advanced liver disease, precluding these subjects from receiving new therapeutic options. • Patients with NASH and not- NASH specific inflammatory activity share similar clinical features and prognosis, and they are different from those with simple steatosis. • Most of the patients with significant fibrosis and cirrhosis without a welldefined NASH show metabolic disturbances. • This is the first study in clinical practice reinforcing the renaming of MAFLD. | 2079 AMPUERO Et Al was calculated based on insulin and glucose (fasting insulin (mIU/ ml) × fasting glucose (mg/ml)/405). We defined the following metabolic risk factors at baseline17: (a) type 2 diabetes mellitus (T2DM), defined by fasting blood glucose ≥126 mg/dl, or Hb1Ac >6.5%, or use of blood glucoselowering agents; (b) arterial hypertension (AHT), determined by blood pressure ≥130/85 mm Hg or use of blood pressurelowering agents; (c) low HDL, defined by HDL- c < 40 mg/dl in men or <50 mg/dl in women or use of lipidlowering agents; (d) hypertriglyceridemia, characterized by triglyceride levels ≥150 mg/dl or use of lipidlowering agents; (e) overweight and obesity, determined by a body mass index (BMI) >25 and 30 kg/m2, respectively; (f) insulin resistance, defined by HOMA > 2.5 in absence of T2DM. Metabolic syndrome was considered when at least three single components were present, while MAFLD was defined according to the recent consensus.3 Followup was defined, depending on the endpoint, as the time from the liver biopsy to the progression to cirrhosis (for noncirrhotic patients), the first event of cirrhosis complication (ascites, hepatic encephalopathy and variceal bleeding) or death. They were collected whenever the investigator received notice about the event. In the case of no event, patients were censored at 10 years of followup or at the end of the study period (May 2020). In noncirrhotic patients at baseline, the progression to cirrhosis was considered when typical findings of cirrhosis were observed during the followup in a second liver biopsy (3/90), abdominal ultrasound (38/90) or transient elastography (>16 kPa) (36/90), or when an episode of decompensated cirrhosis occurred (13/90) (TARGETNASH criteria18). 2.3 | Histological assessment The diagnosis of NAFLD was based on histological criteria. All liver biopsies were assessed by experienced hepatopathologists (leaded by MJPM), associated with the LITMUS histopathologists group,19 who were blinded regarding the patient's evaluation and clinical data. Samples of <15- mm length or <10 portal tracts were considered not suitable for diagnosis and were excluded. Several histological aspects were measured. Steatosis, lobular inflammation and hepatocyte balloon degeneration were systematically assessed according to the NASH CRN Scoring System: (a) Steatosis was rated as Grade 0 (<5%), Grade 1 (5%– 33%), Grade 2 (33%– 66%) and Grade 3 (>66%); (b) hepatocyte ballooning was considered as 0 (none), 1 (mildfew) and 2 (moderatemarked); (c) lobular inflammation was rated as 0 (none), 1 (<2 foci/20 optical field), 2 (2- 4 foci/20 optical field) and 3 (>4 foci/20 optical field). Although NASH CRN does not define exactly NASH by using the NAS score,20 we determined NASH according to NAS score ≥4 (with at least 1 point each in inflammation and ballooning).7 This threshold was based on the inclusion criteria of most Food and Drug Administration (FDA)- approved clinical trials to develop NAFLD therapeutic drugs. The severity of fibrosis was staged from 0 to 4. 2.4 | Objectives We assessed if NAS score ≥4 as inclusion criterion in NAFLD clinical trials reflects a reliable scenario in clinical practice, due to some cases with higher NAS score could not have findings of definite NASH and other cases of lower NAS score do.20 To achieve this goal, secondary aims were (a) to analyze the presence and the distribution of NAS score ≥4 and of its single components (steatosis, inflammation and ballooning) across the fibrosis stages; (b) to analyze the histological features of patients with apparent lack of NASH (NAS < 4), who are usually excluded from clinical trials; (c) to perform an exploratory analysis to determine the prognosis of three histological patterns based on NAS score and liver inflammation (NASH vs. indeterminate NASH vs. NAFL) in terms of progression to cirrhosis, the appearance of the first episode of decompensated cirrhosis and death. 2.5 | Statistical analyses Data were reported as the mean ± standard deviation for normal and median (interquartile range) for nonnormal continuous variables, while frequency was used for discrete variables. In the univariate comparisons, we used the Student's t test and ANOVA with Bonferroni adjustments for continuous samples and chisquare test or Fisher's exact test for the qualitative ones. Nonparametric alternatives (Mann– Whitney U and Kruskal– Wallis tests) were used for nonnormal distributions. Survival analysis was assessed by the Kaplan– Meyer method, and differences between patient subgroups were evaluated by the logrank test. Values were supposed to be statistically significant when P < .05. On the other hand, the annual rate of progression to cirrhosis and the appearance of the first episode of decompensated cirrhosis and death were computed by dividing the number of patients with the defined event by the number of person years of which patients were followed. We multiply rates by 100 to transform in cases per 100 person years. The method used for missing data was completecase analysis because statistical packages excluded individuals with any missing value. STATA (12.0, STATA Corporation, College Station, TX, USA) statistical package was used in all analyses and GraphPad Prism (version 6.0; GraphPad Software, Inc, La Jolla, CA) for graphics. 3 | RESULTS 3.1 | Baseline features of the study population The baseline features of the study cohort are stated in Tables 1 and S1. Briefly, steatohepatitis (NAS ≥ 4) was present in 51.9% (982/1893) of the overall population. According to liver fibrosis: F0 34.3% (649/1893), F1 27% (511/1893), F2 16.5% (312/1893), F3 15% (284/1893) and F4 7.2% (137/1893). The 41.6% (788/1893), 36.8% (696/1893) and 21.6% (409/1893) showed Grades 1, 2 and 2080 | AMPUERO Et Al 3 of steatosis, respectively. On the other hand, lobular inflammation was seen in 73.5% (1391/1893) of patients (50.5% had mild and 23% moderate- to- severe inflammation), while ballooning was present in 67% (1269/1893) of subjects (47.6% showed mildfew and 19.4% moderatemarked). The histological activity in patients, stratified by fibrosis stage, is shown in Table 2. 3.2 | Histological features depending on the fibrosis stage We analyzed the distribution of severe steatosis (Grade 3), ballooning and lobular inflammation according to the fibrosis stage (Figure 1A). All these three single components of the histological definition of steatohepatitis were less frequent in patients with F0. The prevalence of these features, according to the fibrosis stage, showed an inverted U- shaped curve. Particularly, severe steatosis, ballooning and lobular inflammation increased from F1 to F2, while their percentage was similar in F3 and decreased in F4 patients. According to the NAS score, the same trend was observed for the distribution of NASH- specific inflammation, which was significantly lower in cirrhotic than in F2 and F3 patients (Figure 1B). Besides, we analyzed the distribution of the following histological patterns by fibrosis stage: steatohepatitis (NAS ≥ 4) versus indeterminate NASH (at least, presence of ballooning or lobular inflammation) versus NAFL (lack of ballooning and lobular inflammation); in the overall cohort (Figure 2A) and depending on the presence of diabetes mellitus (Figure 2B), obesity (BMI > 30) (Figure 2C), the indication of the liver biopsy (Figure 2D) and the sex (Figure 2E). On the other hand, the distribution of steatohepatitis, as well as their individual components, is shown in Table S2, according to the presence of MAFLD clinical criteria. Briefly, the presence of metabolic derangement influenced a higher inflammatory activity in the liver only in patients with F0: ballooning 51.6% versus 22.5%, lobular inflammation 57.5% versus 32.5% and NASH 35% versus 12.5% (P < .05 for all comparisons). TABLE 1 Baseline features of the overall population Characteristic Overall cohort (n = 1893) Male sex 48% (909/1893) Age; years ± SD 51.8 ± 12.3 BMI ± SD (kg/m2)35.3 ± 8.8 Obesity (BMI > 30 kg/m2)61.5% (1164/1893) Arterial hypertension 48.7% (922/1893) Type 2 diabetes mellitus 36.7% (695/1893) Glucose ± SD (mg/dl) 118 ± 43 HOMA- IR ± SD 5.5 ± 5.2 HOMA > 2.5 (in absence of T2DM) 14.4% (273/1893) Total cholesterol ± SD (mg/dl) 194 ± 45 H D L - c ± SD (mg/dl) 49 ± 15 LDL- c ± SD (mg/dl) 117 ± 38 Triglycerides ± SD (mg/dl) 166 ± 116 Hypertriglyceridemia 48.1% (910/1893) AST ± SD (IU/L) 43 ± 38 ALT ± SD (IU/L) 60 ± 53 GGT ± SD (IU/L) 102 ± 57 Phosphatase alkaline ±SD (IU/L) 95 ± 53 Bilirubin ± SD (mg/dl) 0.68 ± 0.4 Albumin ± SD (g/dl) 4.39 ± 0.4 Creatinine ± SD (mg/dl) 0.83 ± 0.2 Platelet count ± SD (×109/L) 234 ± 71 INR ± SD 1.03 ± 0.1 Abbreviations: BMI, body mass index; SD, standard deviation. TABLE 2 Histological activity, including steatohepatitis, steatosis, ballooning and lobular inflammation, stratified by fibrosis stage Fibrosis stage F0F1F2F3F4 NASH (NAS > 4) 33.6% (218/649) 56.2% (287/511) 68.6% (214/312) 67.6% (192/284) 51.8% (71/137) Steatosis Grade 1 50.5% (328/649) 37.4% (191/511) 34.3% (107/312) 34.9% (99/284) 46% (63/137) Grade 2 34.4% (191/649) 38.7% (198/511) 36.2% (113/312) 39.8% (113/284) 35.8% (49/137) Grade 3 15.1% (98/649) 23.9% (122/511) 29.5% (92/312) 25.4% (72/284) 18.2% (25/137) Ballooning 050.2% (326/649) 25.2% (129/511) 18.6% (58/312) 23.2% (66/284) 32.8% (45/137) 139% (253/649) 54% (276/511) 51% (159/312) 52.5% (149/284) 46.7% (64/137) 210.8% (73/649) 20.7% (106/511) 30.4% (95/312) 24.3% (69/284) 20.4% (28/137) Lobular inflammation 044.1% (286/649) 20.9% (107/511) 11.9% (37/312) 15.1% (43/284) 21.2% (29/137) 143.8% (284/649) 56.2% (287/511) 53.5% (167/312) 51.4% (146/284) 52.6% (72/137) 210.3% (67/649) 18.6% (95/511) 28.8% (90/312) 30.3% (86/284) 22.6% (31/137) 31.8% (12/649) 4.3% (22/511) 5.8% (18/312) 3.2% (9/284) 3.6% (5/137) Abbreviation: NASH, nonalcoholic steatohepatitis. | 2081 AMPUERO Et Al FIGURE 1 Histological features depending on the fibrosis stage. A, Prevalence of severe steatosis (Grade 3)*, ballooning (>1)**, and lobular inflammation (>1)***. B, NAS score. *P values for steatosis. F0 versus F4 0.356; F1 versus F4 0.162; F2 versus F4 0.012; F3 versus F4 0.104. **P values for ballooning. F0 versus F4 0.0002; F1 versus F4 0.074; F2 versus F4 0.0009; F3 versus F4 0.036. ***P values for inflammation. F0 versus F4 <0.0001; F1 versus F4 0.953; F2 versus F4 0.010; F3 versus F4 0.036 FIGURE 2 Distribution of definite nonalcoholic steatohepatitis (NASH), indeterminate NASH, and nonalcoholic fatty liver (NAFL). A, Overall cohort. B, Depending on the presence of type 2 diabetes mellitus (T2DM). C, Depending on the presence of obesity. D, Indication of the liver biopsy. E, Comparison between men and women (A) (B) (C) (D) (E) 2082 | AMPUERO Et Al 3.3 | Histological features in patients without NASH Despite 31.4% of patients showing F2 had no NASH, 83.7% of these patients showed some inflammatory component (ballooning or lobular inflammation) in the liver biopsy (Figure 3A). Also, a third of patients with advanced fibrosis were not diagnosed with NASH. However, up to 71.7% of them showed some inflammatory degree in the liver (Figure 3B). In cirrhotic patients, the percentage of patients without steatohepatitis was 48.2%, although up to 68.2% of them showed at least ballooning or lobular inflammation (Figure 3C). 3.4 | Baseline features according to the histological pattern According to the histological pattern, the baseline features of the overall population are shown in Table 3. Briefly, both NASH and indeterminate NASH groups showed similar rates for metabolic factors such as obesity, diabetes mellitus and arterial hypertension, as well as they were not significantly different for liver tests like bilirubin, albumin or platelet levels. Instead, NASH patients showed significantly higher AST and ALT levels. Also, MAFLD criteria were present in 96.2% (945/982) of NASH patients and 95.2% (535/562) in indeterminate NASH, while it was 89.1% (311/349) in patients with NAFL (P < .0001). On the other hand, the distribution of MAFLD by histological pattern and fibrosis stage is shown in Figure S1. 3.5 | Prognosis according to the histological pattern After the exclusion of patients who underwent liver biopsy during bariatric surgery (n = 539) and were enrolled in clinical trials (n = 120), 1234 subjects (65.2% of the overall cohort) were included in the survival analysis. During the followup (mean time 4.7 ± 3.8 years), 2.8% (34/1234) of patients died, 1.9% (24/1234) suffered from a first decompensation (10.3% (12/116) among baseline cirrhotic patients), 8.1% (90/1118) of noncirrhotics suffered progression to cirrhosis and 1.1% (13/1234) developed hepatocellular carcinoma. Fibrosis stage was the main histological determinant on the prognosis of the overall cohort (mortality was F0 1.8% [7/392] vs. F1 1.6% [5/315] vs. F2 3.1% [6/196] vs. F3 2.8% [6/215] vs. F4 9.6% [10/116]; logRank 22.369, P = .0001). On the other hand, despite having more patients with advanced liver disease, NASH patients had not a worse survival (2.7% [16/598]) than those with indeterminate NASH (1.4% [5/355]) or NAFL (4.6% [13/281]) (logRank 3.175; P = .204). Also, the three groups suffered a similar rate of first cirrhosis decompensation during the study (2% [12/598] vs. 1.7% [6/355] vs. 2.1% [6/281]; P = .806). Nevertheless, the incidence of progression to cirrhosis was significantly higher for both NASH patients (9.5% [51/539]; HR 2.35 [95% CI 1.30- 4.26]) and those with indeterminate NASH (7.9% [25/316]; HR 1.90 [95% CI 1.01- 3.66] [p = ns between them]) compared to subjects with NAFL (5% [14/263]; HR 1.00 [reference]) (logRank 8.417; P = .015) (Figure 4A). Consequently, the annual incidence rate of FIGURE 3 Histological features in patients without a definite nonalcoholic steatohepatitis (NASH) diagnosis. A, Significant fibrosis. B, Advanced fibrosis. C, Cirrhosis 31.4% 68.6% Steatosis and lobular inflammation Steatosis and ballooning Simple steatosis 0 100 50 90 80 70 60 40 30 20 10 Steatosis and lobular inflammation and ballooning 32.4%67.6% Steatosis and lobular inflammation Steatosis and ballooning Simple steatosis 0 100 50 90 80 70 60 40 30 20 10 Steatosis and lobular inflammation and ballooning 51.8% 48.2% 0 100 50 90 80 70 60 40 30 20 10 (A) (B) (C) Steatosis and lobular inflammation Steatosis and ballooning Simple steatosis Steatosis and lobular inflammation and ballooning | 2083 AMPUERO Et Al progression to cirrhosis was higher for patients with NASH and indeterminate NASH than those with NAFL (Figure 4B). On the other hand, the impact of the histological pattern on both mortality and decompensated cirrhosis was not influenced by MAFLD despite showing higher percentages of these outcomes in dysmetabolic patients (P > .05). According to progression to cirrhosis, MAFLD also did not modify the impact of patients with NASH or indeterminate NASH. By contrast, patients showing NAFL and MAFLD showed higher progression to cirrhosis than those metabolically healthy patients (6.1% [14/231] vs. 0% [0/32]; logRank 3.677, P = .049) (Figure 4C). 4 | DISCUSSION Our study observed that the prevalence of NASH, defined by NAS ≥ 4 (with at least 1 point each in inflammation and ballooning), decreased in advanced liver disease, particularly in cirrhotic patients. Also, more than 70% of patients with NAS < 4 showed some inflammatory degree in the liver biopsy. Both NASH and indeterminate NASH patients shared a similar rate of metabolic risk factors (including obesity, diabetes, arterial hypertension and dyslipidemia) and liver tests (albumin, bilirubin and platelets), although those with NAS ≥ 4 presented higher levels of AST and ALT. Besides, these subgroups showed a significantly higher proportion of MAFLD criterion than simple steatosis, focusing this term on patients with a higher likelihood of some hepatic inflammatory grade. Interestingly, both NASH and indeterminate NASH showed similar progression rates to cirrhosis, decompensated cirrhosis and mortality. Therefore, our findings cast doubt on current selection criteria for clinical trials because many subjects, who would benefit from an experimental treatment to halt the progression of the liver disease, could be excluded from clinical trials due to the absence of one single component of definite NASH. A relatively high proportion of our patients did not show findings related to definite NASH (notably, half of the cirrhotic patients and a third of those with advanced fibrosis could not be diagnosed from NASH). A recent Italian study demonstrated that up to 33% of patients with NAFLD- related significant fibrosis did not show NASH (up to 10% had no inflammation), although the authors did not assess TABLE 3 Baseline features of the overall cohort according to the histological pattern Characteristic NASH Indeterminate NASH NAFL P value for the trend Male sex 44.6% (438/982)a,b 51.6% (290/562) 51.9% (181/349) .008 Age; years ± SD 51.9 ± 12.4 52.1 ± 12.4 50.8 ± 11.9 .243 BMI ± SD (kg/m2)35.9 ± 8.7a 35.2 ± 8.7 34 ± 9 .005 Obesity (BMI ≥30 kg/m2) 70.9% (640/903)a 67.5% (349/517)c 59.7% (175/293) .002 Arterial hypertension 51.8% (515/982)a 48.2% (271/562)c 40.7% (142/349) .002 Type 2 diabetes mellitus 40.4% (397/982)a 37.4% (210/562)c 25.2% (88/349) .0001 Glucose ± SD (mg/dl) 121 ± 44a 117 ± 46 112 ± 37 .005 HbA1c (%) 6.5 ± 1.5a 6.4 ± 1.4c 6 ± 1.3 .017 Total cholesterol ± SD (mg/dl) 194 ± 47 193 ± 44 194 ± 43 .913 H D L - c ± SD (mg/dl) 48 ± 15b 51 ± 17 49 ± 13 .003 LDL- c ± SD (mg/dl) 116 ± 39 118 ± 37 120 ± 36 .346 Triglycerides ± SD (mg/dl) 177 ± 120a,b 160 ± 125 147 ± 75 .0001 Hypertriglyceridemia 53% (520/982)a,b 46.3% (260/562)c 37.2% (130/349) .0001 AST ± SD (IU/L) 48 ± 46a,b 38 ± 25 36 ± 28 .0001 ALT ± SD (IU/L) 67 ± 61a,b 53 ± 40 54 ± 46 .0001 Bilirubin ± SD (mg/dl) 0.67 ± 0.4 0.70 ± 0.4 0.70 ± 0.4 .252 Albumin ± SD (g/dl) 4.39 ± 0.4 4.37 ± 0.5 4.41 ± 0.4 .520 Creatinine ± SD (mg/dl) 0.82 ± 0.3 0.83 ± 0.2 0.83 ± 0.2 .682 Platelet count ± SD (×109/L) 233 ± 70 231 ± 71 240 ± 74 .209 INR ± SD 1.03 ± 0.1 1.04 ± 0.2 1.03 ± 0.2 .830 MAFLD criteria 96.2% (945/982)a 95.2% (535/562)c 89.1% (311/349) .0001 Cirrhosis 7.2% (71/982) 8% (45/562) 6% (21/349) .530 Abbreviations: BMI, body mass index; MAFLD, metabolicassociated fatty liver disease; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis; SD, standard deviation. ap<0.05 for comparison between NASH and indetermined NASH. bp<0.05 for comparison between NASH and NAFL. cp<0.05 for comparison between indetermined NASH and NAFL. 2084 | AMPUERO Et Al the distribution of NAS single components across the different fibrosis stages.21 Another French study defined histologically severe disease as a SAF activity score ≥3 or advanced fibrosis (not requiring steatohepatitis), assuming that patients with NAFLD- related advanced liver disease could not have some definite elements of NASH.22 Besides, steatofibrosis demonstrated to have higher overall mortality than NASH in NAFLD patients.23 Interestingly, despite the lack of a definite NASH diagnosis, most of the patients included in our study showed some inflammatory degree in the liver biopsy, irrespective of the fibrosis stage. Indeterminate NASH, a subclassification recognized by the NASH CRN system, is characterized by steatosis with inflammation and hepatocyte injury but atypical of definite steatohepatitis, and it can be associated with varying amounts of fibrosis.5 It has been documented that fibrosis can progress even in patients without baseline steatohepatitis features, due to ballooning and lobular inflammation predicted fibrosis progression.24 Consequently, these patients would more likely develop complications and should be cautiously monitored. In this scenario, we observed that both patients with NASH and indeterminate NASH showed similar FIGURE 4 A, Kaplan– Meier curve to show the impact of the histological pattern on the progression to cirrhosis. B, Annual incidence rate of progression to cirrhosis depending on histological pattern and fibrosis stage (cases per 100 personyears). C, Kaplan– Meier curve to show the impact of metabolicassociated fatty liver disease (MAFLD) on the progression to cirrhosis in patients with nonalcoholic fatty liver (NAFL) (A) (B) (C)