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

BACKGROUND: 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.

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

BACKGROUND AND AIMS: The European Association for the Study of the Liver (EASL) has recently proposed an algorithm for the diagnosis of advanced liver fibrosis. We aimed to evaluate the diagnostic accuracy of this algorithm in nonalcoholic fatty liver disease (NAFLD). APPROACH AND RESULTS: One thousand fifty-one patients with NAFLD, liver biopsy, and four noninvasive tests (NITs; Fibrosis-4 [FIB4], vibration controlled transient elastography [VCTE], FibroMeter, Fibrotest) were included. The enhanced liver fibrosis (ELF) score was available in 396 patients. A cohort of 230 patients from primary care/diabetes clinics had FIB4, VCTE, and ELF. Compared with the performance of single NITs, agreement between two NITs (FIB4 and VCTE, VCTE and patented serum tests) increased specificity and positive predictive value by 20%, thus justifying the sequential use proposed in the EASL algorithm. The FIB4/VCTE/FibroMeter and FIB4/VCTE/Fibrotest algorithms performed similarly, providing 85% diagnostic accuracy and a liver biopsy requirement rate of only 10%. The FIB4/VCTE/ELF algorithm performed similarly in the subgroup where ELF was available. Simulations of algorithm accuracies at different prevalence showed that positive predictive values rapidly increased, reaching a plateau above 75% starting at 15% prevalence. Negative predictive values remained higher than 90% up to 25% prevalence. The rate of liver biopsy requirement remained stable, increasing by only 5% between low and high prevalence settings. When the EASL algorithm was applied in the primary care/diabetes clinic cohort, liver biopsy requirement was only 3%, and the agreement among the three steps provided 75% positive predictive value. CONCLUSIONS: Our study validates the algorithm proposed by the EASL in its latest 2021 guidelines for the diagnosis of advanced fibrosis in the setting of NAFLD.

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

BACKGROUND & 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.

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

Acute 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 10-48 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.

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

Wilson'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.

The prognostic impact of cirrhosis on patients receiving maintenance haemodialysis.

BACKGROUND: Further study is needed on the prognostic impact of cirrhosis on haemodialysis patients. AIM: To evaluate cirrhosis' impact according to severity on survival and to provide therapeutic guidelines for haemodialysis cirrhotic patients. METHODS: Patients with end-stage renal failure treated with haemodialysis were included retrospectively from 01/01/2000 to 31/12/2004 and prospectively from 01/01/2005 to 31/12/2014 in our French Region. Clinical data, presence of cirrhosis and its severity were recorded at the beginning of haemodialysis. The primary endpoint was 2-year survival. RESULTS: Seven thousand three hundred and fifty-four patients (96%) without cirrhosis and 304 patients (4%) with cirrhosis were included. Two-year survival in noncirrhotic patients was higher than in cirrhotic patients (71.7% vs 54.4%, P < 0.0001). Patients with decompensated cirrhosis had a worse 2-year outcome (44.1%) as compared to compensated cirrhotic (62.8%, P = 0.002) and noncirrhotic patients (71.7%, P < 0.0001). Compensated and decompensated cirrhosis were independent predictive factors of 2-year mortality. In sensitivity analysis restricted to cirrhotic patients, 2-year survival of Child-Pugh A patients was higher than in Child-Pugh B and C patients (65.5% vs 27.7% vs 0%, P < 0.0001). Development of predictive models based either on severity scores (MELD and Child-Pugh) and extrahepatic comorbidities allowed correct classification of around 70% of patients in terms of mortality and may help to better stratify mortality risk in this population. CONCLUSIONS: Cirrhosis is independently associated with mortality in haemodialysis patients. Patients with severe cirrhosis have a poor 2-year outcome. Severity of cirrhosis and presence of extrahepatic comorbidities should be considered when deciding to initiate renal replacement therapy.

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

BACKGROUND & AIMS: Advanced liver fibrosis is an important diagnostic target in non-alcoholic fatty liver disease (NAFLD) as it defines the subgroup of patients with impaired prognosis. The non-invasive diagnosis of advanced fibrosis is currently limited by the suboptimal positive predictive value and the grey zone (representing indeterminate diagnosis) of fibrosis tests. Here, we aimed to determine the best combination of non-invasive tests for the diagnosis of advanced fibrosis in NAFLD. METHODS: A total of 938 patients with biopsy-proven NAFLD were randomized 2:1 into derivation and validation sets. All patients underwent liver stiffness measurement with vibration controlled transient elastography (VCTE) and blood fibrosis tests (NAFLD fibrosis score, Fibrosis-4 [FIB4], Fibrotest, Hepascore, FibroMeter). FibroMeterVCTE, which combines VCTE results and FibroMeter markers in a single test, was also calculated in all patients. RESULTS: For the diagnosis of advanced fibrosis, VCTE was significantly more accurate than the blood tests (area under the receiver operating characteristic curve [AUROC]: 0.840 ±â€¯0.013, p ≤0.005). FibroMeter was the most accurate blood test (AUROC: 0.793 ±â€¯0.015, p ≤0.017). The combinatory test FibroMeterVCTE outperformed VCTE and blood tests (AUROC: 0.866 ±â€¯0.012, p ≤0.005). The sequential combination of FIB4 then FibroMeterVCTE (FIB4-FMVCTE algorithm) or VCTE then FibroMeterVCTE (VCTE-FMVCTE algorithm) provided an excellent diagnostic accuracy of 90% for advanced fibrosis, with liver biopsy only required to confirm the diagnosis in 20% of cases. The FIB4-FMVCTE and VCTE-FMVCTE algorithms were significantly more accurate than the pragmatic algorithms currently proposed. CONCLUSION: The sequential combination of fibrosis tests in the FIB4-FMVCTE and VCTE-FMVCTE algorithms provides a highly accurate solution for the diagnosis of advanced fibrosis in NAFLD. These algorithms should now be validated for the diagnosis of advanced liver fibrosis in diabetology or primary care settings. LAY SUMMARY: The evaluation of liver fibrosis is mandatory in non-alcoholic fatty liver disease (NAFLD), as advanced fibrosis identifies the subgroup of patients with impaired prognosis. FibroMeterVCTE is a new fibrosis test combining blood markers and the result of vibration controlled transient elastography (VCTE) into a single diagnostic test. Our results show that FibroMeterVCTE outperforms other blood fibrosis tests and VCTE alone for the diagnosis of advanced fibrosis in a large multi-centric cohort of 938 patients with biopsy-proven NAFLD. Sequential algorithms using a simple blood test or VCTE as a first-line procedure, then FibroMeterVCTE as a second-line test accurately classified 90% of patients.