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

Abstract

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.

Keywords: Biomarker; Fibrosis; Liver; MASLD; Proteomics.

Fig. 1

Differentially-abundant proteins identified in plasma from patients with suspected MASLD with early (F0-2) or advanced (F3-4) liver fibrosis. Plasma samples were collected from patients with suspected MASLD and early or advanced fibrosis, as determined from liver biopsies. Plasma proteins were submitted to MS-based label-free quantitative proteomics. For each protein, the Volcano plot displays the –log₁₀(Limma p-value) on the Y-axis; the X-axis corresponds to the log₂ fold-change between early and advanced fibrosis. The cut-off for statistical significance was set for a p-value providing an FDR of less than 5% according to the Benjamini–Hochberg procedure. Red and blue dots represent less abundant and more abundant proteins, respectively, when advanced stages are compared to early stages. Only the 10 most statistically significant proteins in each condition are annotated in this figure (for the complete list, see Supp. Table 1)

Fig. 2

Mean abundances of ALS and Gal-3BP in plasma samples correlate with fibrosis stages and phases. ALS (A) and Gal-3BP (B) levels based on MS abundance (discovery study, dark blue, left Y-axis, arbitrary units) and concentrations based on ELISA measurements (verification and validation studies, light blue and green, right Y-axis ng/mL)

Fig. 3

ALS and Gal-3BP abundances can distinguish between patients with early and advanced fibrosis. ALS abundance in discovery (MS analysis, arbitrary units) and verification (ELISA, concentration) studies – Grenoble cohort (A and B) and in validation study – Angers cohort (ELISA, concentration, C). Gal-3BP abundance results from discovery/verification tests – Grenoble cohort (D and E) and validation test – Angers cohort (F). Data are represented as boxplots. Statistical significance was verified using Mann–Whitney. Dots represent ALS and Gal-3BP abundance for individual patients

Fig. 4

Plasma concentration of ALS and Gal-3BP discriminate early (F0-2) from advanced (F3-4) fibrosis as well as the FibroTest panel; as a 2-protein panel they outperform FibroTest. ROC curves and AUROCs are shown with their respective 95% CIs for ALS/Gal-3BP quantified by ELISA, along with original FibroTest score (left). Combined concentrations of ALS and Gal-3BP compared to original FibroTest (right). Data presented correspond to the validation cohort. Details about CI calculation can be found in Supp. Mat. 6; the curves are displayed on two distinct plots for the sake of clarity only, to avoid confusion due to the extensive overlaps in CIs