Background and Aims: Lifestyle intervention aiming at weight loss (WL) is the cornerstone of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) management, but treatment response by non-invasive tests (NITs) is not yet established. Here, we aim to assess antifibrotic response to WL by collagen and non-collagen fibrogenesis markers.Methods: We included 146 patients with MASLD (median age 52 [46-61] years, 63% male) undergoing 6-month randomized dietary trial (2021-2023): Mediterranean Diet (n=49) or Low-Charb Diet (n=49) or nutritional advice (n=48). Inclusion criteria: MASLD and LSM (VCTE)<10 kPa, Body Mass Index (BMI) of 25-35 kg/m2, age<65 years. Exclusion criteria: HbA1c>9.5%, type 2 diabetes (T2D) treated with insulin of GLP1-RA. All parameters were assessed at baseline and 6 months. Serum non-collagen (TSP2, CD163 and IGFB7 - Mainz) and collagen (C4M, PRO-C3, PRO-C4, PRO-C6 and PRO-C8 - Nordic Bioscience) markers were measured through in-house standardized ELISA assay. Primary endpoint was changes in NIT levels between patients achieving <5% WL (group A n=91), compared to those achieving 5-10% WL (group B n=38) and ≥10% WL (group C n=17).Results: Median BMI was 30,4 [28,7–33,4] kg/m2, T2D and PNPLA3 GG were present in 31% and 21,8% of cases. PRO-C4 levels fell significantly in group B (p=0,041) and C (p=0,015), but not in group A (p=0,147). TSP2 levels fell substantially in group B (p=0,046) and in group C (p=0,004) but not in group A (p=0,714). FIB-4 and VCTE did not show clinically significant changes. Delta PRO-C4 showed a stepwise negative increase across the 3 groups: from mean -71,7 (SD 510,3) ng/ml to mean -228,7 (634,2) ng/ml to mean -695,5 (1735,8) ng/ml (ANOVA p=0,011). Delta TSP2 varied from mean -0,2 (13,5) ng/ml to mean -4,2 (14,5) ng/ml to mean -12,6 (15,8) ng/ml (ANOVA p=0,004). PRO-C4 and TSP2 showed significant association with delta weight changes: R2 0,05 (p=0,003) and 0,08 (p=0,0002). Delta TSP2> -1,96 ng/ml and delta PRO-C4> -703 ng/ml (both by Youden Index) were associated with WL >5% after adjustment for age, male sex, obesity, T2D and PNPLA3 GG: aOR 0,16 (0,04-0,63, p=0,008) and aOR 0,29 (0,29-0,96, p=0,043) respectively.Conclusion: In patients with MASLD without advanced fibrosis undergoing dietary intervention, serum TSP2 and PRO-C4 levels fell significantly in correlation to WL and were associated with WL>5%. This evidence suggests their clinical utility to monitor attenuation of hepatic fibrogenesis.

BACKGROUND & AIMS:Dietary interventions are a cornerstone of management for metabolic dysfunction-associated steatotic liver disease (MASLD). However, it remains unclear whether specific dietary patterns confer benefits beyond weight loss. We aimed to compare the effects of different dietary strategies on liver fat and metabolic health in patients with MASLD. METHODS:We conducted a 6-month, three-arm, parallel-group randomized controlled trial including patients with MASLD and overweight or obesity who were naïve to structured nutritional counselling. Participants were randomized to Mediterranean diet (MeD), low-carbohydrate, high-protein diet (LCHO), or standard nutritional recommendations. Liver fat content was assessed by controlled attenuation parameter (CAP). Secondary outcomes included changes in anthropometric and metabolic parameters, body composition, and ultra-processed food (UPF) consumption. Structural equation modelling was used to investigate pathways linking dietary allocation, weight loss, and UPF intake with changes in CAP. RESULTS:Of 173 enrolled participants, 148 (85.5%) completed the study. CAP decreased significantly within the LCHO and control groups (Δ -19.5 [-48.8 to 8.0], p = 0.004 and -11.0 [-32.0 to 12.0], p <0.001), with no significant between-group differences. All study arms achieved significant improvements in body weight, BMI, adiposity measures, metabolic profile, and dietary quality, including reductions in UPF intake. Structural equation modelling analyses showed no direct or total effect of dietary pattern on changes in CAP. In contrast, reductions in BMI and UPF consumption independently and significantly predicted CAP improvements: β = 14.34 (95% CI 10.55-18.13), and β = 0.64 (95% CI 0.25-1.03). CONCLUSIONS:In patients with MASLD, weight loss and reductions in UPF intake, rather than diet type, were the main drivers of improvements in liver fat and metabolic health. These findings support a patient-centered dietary approach in MASLD management. CLINICAL TRIAL NUMBER:NCT073043336. IMPACT AND IMPLICATIONS:This randomized controlled trial shows that weight loss and reductions in ultra-processed food intake, rather than the specific dietary pattern, are the primary factors associated with improvements in liver fat and metabolic health in MASLD. These findings challenge the emphasis on prescriptive dietary models and support a more flexible, patient-centered approach focused on sustainable weight reduction and improved dietary quality. Prioritizing reduction of ultra-processed foods may represent a practical and scalable strategy to enhance adherence and maximize long-term clinical benefit in routine care.

Background and Aims: Metabolic dysfunction associated steatotic liver disease (MASLD) is a rising cause of hepatocellular carcinoma (HCC), but reliable prognostic tools for risk stratification strategies are still under definition. Aim was to examine the incidence of HCC and the impact of acquired and genetic factors in a prospective multicenter cohort of patients with MASLD at increased risk.Methods: We included patients with MASLD or Metabolic and alcohol-associated liver disease (MetALD), aged 45-75 years (by allowing an alcohol intake limit <60/40 g/day Male/Female), with either fibrosis stage F3-F4, determined histologically or by noninvasive assessment (stiffness >7.9 kPa by Fibroscan and FIB-4 ≥ 1.3) or a family history of HCC in a first degree relative. Clinical parameters and genotyping for PNPLA3 rs738409, TM6SF2 rs58542926, GCKR rs1260326, MBOAT7 rs641738 and HSD17B13 rs72613567 were recorded and used to generate a polygenic risk score (PRS-5). Time to HCC progression was evaluated by Kaplan-Meier’s estimates of cumulative incidence rates. Cox regression analysis was used to identify predictors of HCC. Results: We enrolled 524 individuals (57% males) with a median follow-up of 44.5 months (IQR 15-57), 346 (65%) had cirrhosis, 280 (53%) diabetes and 100 (19%) MetALD. Twenty-nine (5.5%) patients developed HCC, of these 28 had cirrhosis (96.6%) and one advanced fibrosis (0.4%). The 5-years cumulative HCC incidence was 8.0% (95% CI: 5.2-11.8%), whereas annual incidence 1.5% (95% CI: 0.9-2.1%). Liver-related events were the main cause of mortality (37 deaths: 12 because of HCC, 10 liver decompensation, 7 cardiovascular events, 7 extra-hepatic cancer, and one infection). Kaplan-Meier analysis showed that progression to HCC was significantly associated with cirrhosis status (HR 15.98, 95% CI 2.17-117.6, p=0.00024) (Fig.1).For 426 patients (81%) complete genetic data were already available. PRS-5 values were significantly higher in patients with vs those without cirrhosis (0.47±0.25 vs. 0.39±0.26; p=0.0021). In patients with cirrhosis, incidence curve analysis did not show a significantly higher incidence of HCC in patients with high PRS-5 (≥0.495, 90th centile of reference population) despite nominally higher risk (HR 1.15, 95% CI 0.50-2.65, p=NS) (Fig.2). At Cox regression analysis, adjusted for age, sex, diabetes and BMI, higher FIB-4 was found to be a strong independent predictor of HCC progression (HR=1.34, 95% CI 1.20-1.49, p=1.5 × 10-7). The optimal FIB-4 cut-off for HCC prediction was 2.58 (Sn 89%, Sp 65%, AUC 0.82). Conclusion: In this multicenter prospective cohort of people with SLD, cirrhosis was confirmed as the strongest risk factor for HCC development. Genetic predisposition, as summarized by PRS-5, was associated with the presence of cirrhosis. FIB-4 score remained an independent predictor of progression to HCC. Additional analyses are ongoing to integrate genetic and clinical data for risk stratification improvement.

BACKGROUND:Non-invasive biomarkers with biological rationale are needed for identifying patients with progressive metabolic dysfunction-associated steatohepatitis (MASH). OBJECTIVE:To test the diagnostic performance of thrombospondin-2 (TSP2), insulin-like growth factor binding protein-7 (IGFBP7), growth differentiation factor (GDF15) and CD163, which were identified by liver transcriptomics as associated with the severity of MASLD. DESIGN:Retrospective study from three European centres in patients with liver biopsy for suspected MASLD. TSP2, IGFBP7, GDF15 and CD163 were measured by ELISAs. Liver stiffness (vibration controlled transient elastography, VCTE), FIB4, Agile3 and FAST scores served as comparators. Outcomes were at-risk MASH (ARM, steatohepatitis NAS>4 and fibrosis stages 2-4) and advanced fibrosis (AF, stages 3-4). Combinatorial scores included fibrosis-biomarkers and clinical/biological variables using multivariate logistic regression controlling for centres, age, body mass index (BMI) and gender. Training and validation sets were defined using a machine learning algorithm with a random split of the total cohort, for 100 iterations, and averaging of area under the receiver operating characteristic curve (AUROC) predictions. RESULTS:469 patients, 61% males, mean age 51.4 years, BMI 30.6 kg/m², 44% with type 2 diabetes, 54% with hypertension, 31% with AF and 46% with ARM were included. The TSP2 AUROC for AF was 0.812 (95% CI: 0.766 to 0.859) higher than for the other biomarkers (ranging from 0.680 to 0.769, p<0.001) and for the comparators (VCTE: 0.739, p<0.04; FIB4: 0.726, p<0.01 and Agile3: 0.755, p=0.20). The TSP2 AUROC for ARM was 0.812 (95% CI: 0.758 to 0.858) higher than for other biomarkers (ranging from 0.722 to 0.748, p<0.001) and comparators (FAST: 0.761, p=0.17). Combining TSP2 with clinical/biological variables (AST, age, platelets, albumin, GGT) increased AUROCs to 0.846 (95% CI: 0.807 to 0.885) for AF and 0.845 (95% CI: 0.806 to 0.880) for ARM and was significantly more performant than all other biomarkers. CONCLUSIONS:Serum TSP2, alone or with clinical/biological variables, potently discriminates AF and ARM in patients with MASLD.

IntroductionGenetic variants involved in lipid and glucose metabolism have been implicated in liver disease progression and hepatocellular carcinoma (HCC) development in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). However, their prognostic role in patients with established HCC remains unclear. We aimed to investigate the association between MASLD-related genetic variants and overall survival (OS) in patients with MASLD-related HCC.MethodsWe retrospectively evaluated 258 patients with MASLD-related HCC (median age: 73, 49–79 years; male sex: 86.8%); most patients had a diagnosis of early tumor (BCLC 0/A: n = 162; 62.8%). PNPLA3 rs738409, TM6SF2 rs58542926, MBOAT7 rs641738, GCKR rs780094, and HSD17B13 rs72613567 variants were genotyped. An independent cohort of viral-related HCC (n = 384) was analysed for exploratory comparison. Overall survival (OS) was evaluated using Kaplan-Meier analysis and multivariable Cox regression.ResultsAmong the investigated gene variants, only the MBOAT7 rs641738 TT genotype was associated with reduced OS in patients with MASLD-related HCC compared to CC/CT carriers (19.8 vs. 32.2 months; p = 0.006). At multivariable analysis, MBOAT7 rs641738 TT genotype retained a nominal association with poorer OS after adjustment for age, sex, tumor burden, and metabolic cofactors (aHR = 1.61, 95% CI 1.05–2.46; p = 0.028). Conversely, OS did not differ according to MBOAT7 rs641738 genotype in the exploratory viral-related HCC comparison cohort (p = 0.449).ConclusionsMBOAT7 rs641738 genotype was associated with poorer OS in patients with MASLD-related HCC. This finding was not observed in an exploratory viral-related HCC comparison cohort, but between-cohort differences limit etiological interpretation. These results should be considered hypothesis-generating and require external validation.

INTRODUCTION:Metabolic dysfunction-associated steatotic liver disease (MASLD) coexists with multiple comorbidities that contribute to impaired vascular function, worsening cognitive impairment, and quality of life. The aim of this study was to evaluate the impact of MASLD and related comorbidities on cognitive function and health-related quality of life (HRQoL). METHODS:A total of 601 overweight/obese patients with MASLD and/or type 2 diabetes mellitus were included. Liver stiffness (LS) measurement and steatosis were assessed by transient elastography and controlled attenuation parameter (CAP), respectively. Cognitive function and HRQoL were evaluated using the Repeatable Battery for the Assessment of Neuropsychological Status and Short Form (36) Health Survey (SF-36) questionnaires. RESULTS:MASLD-related comorbidities were found to significantly and clinically affect cognitive function and HRQoL. Patients with severe steatosis (CAP ≥ 300 dB/m, n = 378) exhibited median cognitive scores falling into the abnormal range ( P = 0.056), with statistically significant lower scores in physical functioning ( P < 0.001), vitality ( P = 0.011), general health ( P = 0.001), and immediate memory ( P = 0.034), as well as a trend toward lower visuospatial/construction scores ( P = 0.058) than CAP <300 dB/m. Among patients with significant or high LS (LS ≥ 8 kPa, n = 69), lower physical functioning ( P < 0.001), higher physical limitations ( P = 0.004), and worse general health ( P = 0.011) were observed compared with those with LS < 8 kPa. In the multivariate adjusted analyses, CAP ≥ 300 dB/m was significantly associated with cognitive impairment (odds ratio: 1.42, 95% confidence interval 1.0; 2.0, P = 0.045), whereas LS ≥ 8 kPa was associated with higher physical limitations (odds ratio: 1.9, 95% confidence interval 1.1; 3.2, P = 0.019). DISCUSSION:Our findings highlight the relationship between MASLD severity and impairment in cognitive function and HRQoL, underscoring its multifactorial nature. Specifically, severe hepatic steatosis may be a risk factor of cognitive decline, whereas significant or high LS seems to greatly affect HRQoL.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of adults globally. Despite efforts to develop non-invasive diagnostic tools, liver biopsy remains the gold standard for diagnosing metabolic dysfunction-associated steatohepatitis (MASH) and assessing fibrosis. This study investigated RNA aptamer panels, selected using APTASHAPE technology, for non-invasive MASLD diagnosis and fibrosis stratification. Aptamer panels were selected in a cohort of individuals with MASLD (development cohort, n = 77) and tested in separate cohorts: one with MASLD (test cohort, n = 57) and one assessed for bariatric surgery (bariatric cohort, n = 62). A panel distinguishing MASLD without steatohepatitis from MASH accurately stratified individuals in the developmentcohort (AUC = 0.83) but failed in the test and bariatric cohorts. It did, however, distinguish healthy controls from individuals with MASLD, achieving an AUC of 0.72 in the test cohort. A panel for fibrosis stratification differentiated F0 from F3–4 fibrosis in the development cohort (AUC = 0.68) but not in other cohorts. Mass spectrometry identified five plasma proteins as potential targets of the discriminative aptamers, with complement factor H suggested as a novel MASLD biomarker. In conclusion, APTASHAPE shows promise as a non-invasive tool for diagnosing and staging MASLD and identifying associated plasma biomarkers.

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) coexists with multiple comorbidities that contribute to impaired vascular function, worsening cognitive impairment, and quality of life. This study aimed to evaluate the impact of MASLD and related comorbidities on cognitive function and health-related quality of life (HRQoL). Methods: A total of 601 overweight/obese patients with MASLD and/or Type 2 Diabetes Mellitus were included. Liver stiffness (LS) measurement and steatosis were assessed by transient elastography and controlled attenuation parameter (CAP), respectively. Cognitive function and HRQoL were evaluated using the RBANS and SF-36 questionnaires. Results: MASLD-related comorbidities were found to significantly and clinically affect cognitive function and HRQoL. Patients with severe steatosis (CAP≥300 dB/m, n=378) exhibited median cognitive scores falling into the abnormal range ( p =0.056), with statistically significant lower scores in physical functioning ( p <0.001), vitality (p=0.011), general health ( p =0.001), and immediate memory (p=0.034), as well as a trend toward lower visuospatial/construction scores (p=0.058) than CAP<300 dB/m. Among patients with significant or high LS (LS≥8 kPa, n=69), lower physical functioning ( p <0.001), higher limitations physical ( p =0.004), and worse general health ( p= 0.011) were observed compared to those with LS<8 kPa. In the multivariate adjusted analyses, CAP≥300 dB/m was significantly associated with cognitive impairment (OR:1.42, 95% CI 1.0; 2.0, p =0.045), whereas LS≥8 kPa was associated with higher limitations physical (OR:1.9, 95% CI 1.1; 3.2, p =0.019). Conclusions: Our findings highlight the relationship between MASLD severity and impairment in cognitive function and HRQoL, underscoring its multifactorial nature. Specifically, severe hepatic steatosis may be a risk factor for cognitive decline, whereas significant or high liver stiffness appears to have a greater impact on HRQoL.