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

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 metabolic‐associated fatty liver disease (MAFLD).


| INTRODUC TI ON
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, non-cirrhotic 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 at-risk patients. For instance, the development of non-invasive tests and imaging-based methods has allowed a wide expansion of liver fibrosis assessment in at-risk patients. [13][14][15][16] Thus, the liver biopsy should be relocated to maximize its advantages in selected patients. In a Spanish large cohort of individuals with biopsy-proven NAFLD followed-up 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.

| Selection of patients
This is an observational study of 1893 patients with biopsy-confirmed 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 non-invasive 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, drug-induced 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.

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 well-defined NASH show metabolic disturbances.
• This is the first study in clinical practice reinforcing the renaming of MAFLD.
We defined the following metabolic risk factors at baseline 17 : (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 pressure-lowering agents; (c) low HDL, defined by HDL-c < 40 mg/dl in men or

| Histological assessment
The diagnosis of NAFLD was based on histological criteria. All liver biopsies were assessed by experienced hepato-pathologists (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 (mild-few) and 2 (moderate-marked); (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.

| 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.

| Statistical analyses
Data were reported as the mean ± standard deviation for normal and median (interquartile range) for non-normal continuous variables, while frequency was used for discrete variables. In the uni-

| Baseline features of the study population
The baseline features of the study cohort are stated in Tables 1   and S1

| 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  Figure 2D) and the sex ( Figure 2E).

| 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).

| Baseline features according to the histological pattern
According to the histological pattern, the baseline features of the overall population are shown in Table 3 Figure S1.

| 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),  Figure 4A). Consequently, the annual incidence rate of

| D ISCUSS I ON
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 inflam- about how the disease appeared and how they have to proceed. 28,29 For instance, primary biliary cirrhosis has been renamed to cholangitis owing to these reasons. 30 The study has some limitations usually observed when realworld data are evaluated. Ideally, the biopsies of all patients should be assessed by one single pathologist to avoid introducing some interobserver variability. Besides, the sampling variability of the biopsies might explain part of the findings. Despite these concerns, the results were consistent and robust after analyzing the data centre by centre (in fact, almost half of the cohort was reviewed by a single central pathologist -MJPM-). Probably, the fact of having a very selected group of expert hepato-pathologists who review all the biopsies after an initial discussion to homogenizes the interpretation of histologic criteria 19 and that patients whose samples are not suitable for diagnosis are excluded from Hepamet Registry could mitigate the worries. Also, a mean follow-up time of 4.7 years could not be enough to observe a high number of prognostic outcomes (eg, hepatocellular carcinoma). However, we performed an exploratory analysis in which we found statistically significant differences for cirrhosis progression, probably because there were fast progressors, including the use of annual incidence rate that considers for subjects the actual time at risk and does not require to complete the total follow-up time.
In summary, steatohepatitis features seem to be absent in half of the patients with significant fibrosis and cirrhosis, which could increase the screening failure rates and preclude to receive new therapeutic options in a subset of patients at high risk of progression and extrahepatic and/or liver-related outcomes. Future studies should individualize the most appropriate criteria for diagnosing and including patients in clinical trials according to the integration of clinical and histological features due to the NAS score limitation for such purpose.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
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