Diagnostic non-invasif des stéatohépatites non alcooliques : évaluation de nouveaux candidats biomarqueurs plasmatiques

NAFLD has become a Public Health problem in developed countries. Liver biopsy remains the controversial gold standard examination. Alternatives lack specificity. There is a clear need for circulating biomarkers. Our main objective was to evaluate some biomarker candidates in human plasma for assessing the severity of NAFLD, in particular discriminating NASH from steatosis. We have selected 4 biomarker candidates (BHMT1, ADH1B, ADH4, CK18) from omics databases, literature searches and previous investigations. Concentrations of the candidates have been determined in 53 plasmas from biopsy-proven NAFLD patients thanks to immunoassays. Two subgroups were defined according to SAF score. Orthogonal analyses thanks to targeted proteomics are about to be finished. Quantification of CK18 with ELISA confirmed literature data, with higher plasmatic concentrations among significant disease group compared to mild disease group, for total as well as caspase-cleaved CK18 (respectively p=0.0001; p=0.0004). Both CK18 are correlated with necroinflammatory parameters, steatosis and fibrosis. BHMT1 was not quantifiable by immunoassay. No difference has been observed between 2 subgroups in terms of ADH1B and ADH4 plasma levels. Results from LC-MS/SRM are only preliminary. Total CK18 and CK18 fragments are serious candidate biomarkers for assessing NAFLD severity, including discriminate NASH from NAFL. Regarding BHMT1, ADH1B and ADH4, results from targeted proteomics are long awaited. Larger-scale studies are necessary to confirm these preliminary results.Les stéatopathies dysmétaboliques, ou NAFLD, sont devenues un véritable problème de santé publique. La biopsie hépatique reste à ce jour l’examen de référence, cependant reste un mauvais gold standard. Les alternatives moins invasives manquent de spécificité. Il existe donc un réel besoin de biomarqueurs circulants capables de limiter le recours à la biopsie. L’objectif principal de cette étude est d’évaluer une sélection de candidats biomarqueurs plasmatiques pour la sévérité des NAFLD. Quatre candidats protéiques on été sélectionnés (BHMT1, ADH1B, ADH4, CK18), par le biais de recherches bibliographiques exhaustives, de données “omiques”, et d’investigations préalables en protéomique. Les concentrations de chaque candidat ont été obtenues pour 53 plasmas issus de patients porteurs d’une NAFLD prouvée histologiquement. Deux sous-groupes ont été définis selon le score SAF. La combinaison de tests immunologiques et d’analyses protéomique ciblée (mode LC-MS/SRM) fait la force de ce travail. Les taux plasmatiques étaient plus élevés pour CK18 dans le groupe “maladie significativement active”, comparativement au groupe “maladie modérément active”, pour la forme complète de CK18 M65 et son fragment M30 (respectivement p=0.0001; p=0.0004). Une corrélation était notée avec les paramètres histologiques de necroinflammation, stéatose et fibrose. BHMT1 n’a pas pu être quantifié par test ELISA. Aucune différence n’a été observée pour ADH1B et ADH4. CK18 reste une protéine très intéressante pour l’appréciation de la sévérité globale des NAFLD. Concernant BHMT1, ADH1B et ADH4, les résultats d’analyses en protéomique ciblée manquent incontestablement pour se prononcer

Comprehensive and comparative exploration of the Atp7b(-/-) mouse plasma proteome

International audienceWilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b-/- mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b-/- mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b-/- mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b-/- mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b-/- mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts

Validation of the new 2021 EASL algorithm for the noninvasive diagnosis of advanced fibrosis in NAFLD

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Impact of Type 2 Diabetes on the Accuracy of Noninvasive Tests of Liver Fibrosis With Resulting Clinical Implications

International audienceBackground & aims: Noninvasive tests (NITs) of liver fibrosis have been suggested to be less accurate in type 2 diabetes mellitus (T2DM). We aimed to compare the accuracy of 6 NITs between patients with or without T2DM, explain any differences, and adapt diagnostic algorithms for clinical practice accordingly.Methods: We included 1051 patients with nonalcoholic fatty liver disease with liver biopsy, blood fibrosis tests (Nonalcoholic Fatty Liver Disease Fibrosis Score, FIB4, Fibrotest, FibroMeter), vibration-controlled transient elastography (VCTE), and the combinatory elasto-blood test FibroMeterVCTE. The study endpoint was advanced fibrosis on liver biopsy.Results: NIT areas under the receiver operating characteristic curve were significantly lower in patients with T2DM, mostly because of a decrease in specificity. For FIB4, this decrease in specificity was only related to the higher age of patients with T2DM enrolled. For Fibrotest, FibroMeter, and FibroMeterVCTE, the decrease in specificity was related to age but also to higher alpha2-macroglobulin level, which is known to increase in T2DM. Sensitivity was unaffected by T2DM, but it masked a doubled raw number of false negatives because of the 2-fold higher prevalence of advanced fibrosis in that setting. The sequential algorithm FIB4-vibration-controlled transient elastography had 90.3% accuracy in patients without T2DM vs 79.0% in those with (P < .001). Algorithms using first-line specialized tests maintained a low rate of false negatives and false positives in T2DM.Conclusions: The decrease in NIT accuracy observed in T2DM is partly biased by the different characteristics of the groups studied, but also linked to T2DM itself through modification of the levels of some NIT biomarkers. Specialized tests should be used first-line to diagnose advanced liver fibrosis in T2DM

Impact of Type 2 Diabetes on the Accuracy of Noninvasive Tests of Liver Fibrosis With Resulting Clinical Implications

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Plasma ALS and Gal-3BP differentiate early from advanced liver fibrosis in MASLD patients

International audienceBackground: Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect 30% of the world’s population, and its prevalence is increasing in line with obesity. Liver fibrosis is closely related to mortality, making it the most important clinical parameter for MASLD. It is currently assessed by liver biopsy – an invasive procedure that has some limitations. There is thus an urgent need for a reliable non-invasive means to diagnose earlier MASLD stages. Methods: A discovery study was performed on 158 plasma samples from histologically-characterised MASLD patients using mass spectrometry (MS)-based quantitative proteomics. Differentially abundant proteins were selected for verification by ELISA in the same cohort. They were subsequently validated in an independent MASLD cohort ( n = 200). Results: From the 72 proteins differentially abundant between patients with early (F0-2) and advanced fibrosis (F3-4), we selected Insulin-like growth factor-binding protein complex acid labile subunit (ALS) and Galectin-3-binding protein (Gal-3BP) for further study. In our validation cohort, AUROCs with 95% CIs of 0.744 [0.673 – 0.816] and 0.735 [0.661 – 0.81] were obtained for ALS and Gal-3BP, respectively. Combining ALS and Gal-3BP improved the assessment of advanced liver fibrosis, giving an AUROC of 0.796 [0.731. 0.862]. The {ALS; Gal-3BP} model surpassed classic fibrosis panels in predicting advanced liver fibrosis. Conclusions: Further investigations with complementary cohorts will be needed to confirm the usefulness of ALS and Gal-3BP individually and in combination with other biomarkers for diagnosis of liver fibrosis. With the availability of ELISA assays, these findings could be rapidly clinically translated, providing direct benefits for patients

Mass Spectrometry-Based Proteomics Reveal Alcohol Dehydrogenase 1B as a Blood Biomarker Candidate to Monitor Acetaminophen-Induced Liver Injury

International audienceAcute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select from among these candidates those that were specifically detected in the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease, and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement in the international normalized ratio (INR) within 10\textendash 48 h, and was associated with favorable outcomes. In conclusion, the combination of omics data exploration and proteomics revealed ADH1B as a new blood biomarker candidate that could be useful for the monitoring of acetaminophen-induced ALI

New sequential combinations of non-invasive fibrosis tests provide an accurate diagnosis of advanced fibrosis in NAFLD

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