Prospective direct comparison of non-invasive liver tests in outpatients with type 2 diabetes using intention-to-diagnose analysis.

BACKGROUND: No prospective diagnostic studies have directly compared widespread non-invasive liver tests in patients with type 2 diabetes (T2D) using the intention-to-diagnose method for each of the three main histological features of metabolic dysfunction associated steatotic liver disease - namely fibrosis, metabolic dysfunction-associated steatohepatitis (MASH), and steatosis. AIMS: To compare the performance of nine tests using the intention-to-diagnose rather than the standard method, which would exclude non-evaluable participants METHODS: Biopsy was used as the reference with predetermined cut-offs, advanced fibrosis being the main endpoint. The Nash-FibroTest panel including FibroTest-T2D, SteatoTest-T2D and MashTest-T2D was optimised for type 2 diabetes. FibroTest-T2D was compared to vibration-controlled transient elastography stiffness (VCTE), two-dimensional shear-wave elastography stiffness (TD-SWE), and Fibrosis-4 blood test. NashTest-T2D was compared to aspartate aminotransferase. SteatoTest-T2D was compared to the controlled attenuation parameter and the hepatorenal gradient. RESULTS: Among 402 cases, non-evaluable tests were 6.7% for VCTE, 4.0% for hepatorenal gradient, 3.2% for controlled attenuation parameter, 1.5% for TD-SWE, 1.2% for NashTest-T2D, and 0.02% for Fibrosis-4, aspartate aminotransferase and SteatoTest-T2D. The VCTE AUROC for advanced fibrosis was over-estimated by 6% (0.83 [95% CI: 0.78-0.87]) by standard analysis compared to intention-to-diagnose (0.77 [0.72-0.81] p = 0.008). The AUROCs for advanced fibrosis did not differ significantly in intention-to-diagnose between FibroTest-T2D (0.77; 95% CI: 0.73-0.82), VCTE (0.77; 95% CI: 0.72-0.81) and TD-SWE(0.78; 0.74-0.83) but were all higher than the Fibrosis-4 score (0.70; 95% CI all differences ≥7%; p ≤ 0.03). For MASH, MashTest-T2D had a higher AUROC (0.76; 95% CI: 0.70-0.80) than aspartate aminotransferase (0.72; 95% CI: 0.66-0.77; p = 0.035). For steatosis, AUROCs did not differ significantly between SteatoTest-T2D, controlled attenuation parameter and hepatorenal gradient. CONCLUSIONS: In intention-to-diagnose analysis, FibroTest-T2D, TD-SWE and VCTE performed similarly for staging fibrosis, and out-performed Fibrosis-4 in outpatients with type 2 diabetes. The standard analysis over-estimated VCTE performance. CLINICALTRIAL: gov: NCT03634098.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Clinical Interest of Serum Alpha-2 Macroglobulin, Apolipoprotein A1, and Haptoglobin in Patients with Non-Alcoholic Fatty Liver Disease, with and without Type 2 Diabetes, before or during COVID-19.

In patients with non-alcoholic fatty liver disease (NAFLD) with or without type 2 diabetes mellitus (T2DM), alpha-2 macroglobulin (A2M), apolipoprotein A1 (ApoA1), and haptoglobin are associated with the risk of liver fibrosis, inflammation (NASH), and COVID-19. We assessed if these associations were worsened by T2DM after adjustment by age, sex, obesity, and COVID-19. Three datasets were used: the "Control Population", which enabled standardization of protein serum levels according to age and sex (N = 27,382); the "NAFLD-Biopsy" cohort for associations with liver features (N = 926); and the USA "NAFLD-Serum" cohort for protein kinetics before and during COVID-19 (N = 421,021). The impact of T2DM was assessed by comparing regression curves adjusted by age, sex, and obesity for the liver features in "NAFLD-Biopsy", and before and during COVID-19 pandemic peaks in "NAFLD-Serum". Patients with NAFLD without T2DM, compared with the values of controls, had increased A2M, decreased ApoA1, and increased haptoglobin serum levels. In patients with both NAFLD and T2DM, these significant mean differences were magnified, and even more during the COVID-19 pandemic in comparison with the year 2019 (all p < 0.001), with a maximum ApoA1 decrease of 0.21 g/L in women, and a maximum haptoglobin increase of 0.17 g/L in men. In conclusion, T2DM is associated with abnormal levels of A2M, ApoA1, and haptoglobin independently of NAFLD, age, sex, obesity, and COVID-19.

Prospective external validation of a new non-invasive test for the diagnosis of non-alcoholic steatohepatitis in patients with type 2 diabetes.

BACKGROUND: One of the unmet needs in patients with type 2 diabetes mellitus (T2DM) is the prediction of non-alcoholic liver disease by non-invasive blood tests, for each of the three main histological features, fibrosis, non-alcoholic steatohepatitis (NASH) and steatosis. AIMS: To validate externally the performances of a recent panel, Nash-FibroTest, for the assessment of the severity of fibrosis stages, NASH grades and steatosis grades. METHODS: We prospectively analysed 272 patients with T2DM. Standard definitions of stages and grades were used, and analyses were centralised and blinded. The performances of the FibroTest, NashTest-2 and SteatoTest-2 were assessed using the Obuchowski measure (OM), the main outcome recommended as a summary measure of accuracy includeing all pairwise stages and grades comparisons, which is not provided par the extensively used binary area under the ROC curve. RESULTS: The diagnostic performance of each component of the panel was significant. OM (SE; significance) of the FibroTest, the NashTest-2 and the SteatoTest-2 was 0.862 (0.012; P < 0.001), 0.827 (0.015; P < 0.001) and 0.794 (0.020; P < 0.01), respectively. For ballooning and lobular inflammation, OM was 0.794 (0.021; P < 0.001) and 0.821 (0.017; P < 0.001), respectively. In a post hoc analysis the FibroTest outperformed VCTE by 4.1% (2.5-6.5; P < 0.001) for reliability, with a non-significant difference for OM for fibrosis staging, 0.859 (0.012) for FibroTest vs 0.870 (0.009) for VCTE. CONCLUSIONS: From a single blood sample, the panel provides non-invasive diagnosis of the stages of fibrosis, and the grades of NASH and steatosis in patients with T2DM. TRIAL REGISTRATION NUMBER: NCT03634098.