Supplementary Table S3: Metabolite Set Enrichment Analysis in HCC Cases Compared to Controls with Cirrhosis

Supplementary Table S1: Baseline characteristics of the derivation cohort comprised of participants with HCC, matched controls without cirrhosis, and unmatched controls with cirrhosis who underwent plasma metabolomics profiling

Supplementary Table S4: Random Forest Classifier Analysis for Importance of Individual Plasma Metabolites and Clinical Covariates for Prediction of Hepatocellular Carcinoma in HCC Cases compared to Controls with Cirrhosis

OBJECTIVES:We have shown that semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), is a highly effective therapy for metabolic dysfunction-associated steatotic liver disease (MASLD) in people with HIV (PWH). The OWLiver® metabolomics algorithms allow non-invasive MASLD severity staging without liver biopsy, but the use of metabolomic data for disease monitoring with GLP-1RA therapy has not been reported. METHODS:SLIM LIVER (ACTG A5371) was an open-label, phase 2b, single-arm trial of semaglutide in PWH and MASLD (2021-2022). Adult participants who showed clinical response (>2.27 kg weight loss) to semaglutide during the study (N = 36) had their serum analysed for metabolomic profiling by the OWLiver® panel. RESULTS:Twenty-four weeks of low-dose semaglutide led to significant improvement in MASLD disease severity by serum metabolomic profiling across all MASLD disease severity categories, suggesting histologic improvements. CONCLUSIONS:The OWLiver® metabolomics algorithms can be used to monitor MASLD severity during GLP-1RA therapy.

Supplementary Table S8: Random Forest classifier analysis of plasma metabolite profiles: HCC cases compared to all controls (derivation cohort)

Supplementary Table S6: Associations of Individual Plasma Metabolites with Risk of Hepatocellular Carcinoma in Multivariable-Adjusted Unconditional Logistic Regression Models in HCC Cases versus All Controls

Supplementary Table S5: Baseline characteristics of the validation cohort comprised of controls with cirrhosis matched to cases with cirrhosis and incident hepatocellular carcinoma

Supplementary Figure S1: Metabolite set enrichment analysis displaying the top 25 metabolic pathways enriched in HCC cases relative to all controls

Supplementary Table S2: Subgroup Analyses: Associations of Individual Plasma Metabolites with Risk of Hepatocellular Carcinoma in Multivariable-Adjusted Unconditional Logistic Regression Models in HCC Cases vs. Controls with Cirrhosis

Background:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of liver-related morbidity and mortality among people with human immunodeficiency virus (PWH). We aimed to determine the prevalence of MASLD and clinically significant fibrosis among PWH and investigate if racial and ethnic differences exist after adjustment for clinical risk factors and PNPLA3 genotype. Methods:This cross-sectional analysis included adult PWH prospectively enrolled in 2 US multicenter studies from 2018 to 2023. MASLD was defined as a controlled attenuation parameter (CAP) score ≥263 dB/m with at least 1 cardiometabolic risk factor and clinically significant fibrosis as a liver stiffness measurement ≥8 kPa upon vibration-controlled transient elastography. Multivariable logistic regression analysis was performed to examine the association of race/ethnicity with the presence of MASLD and clinically significant fibrosis, adjusting for clinical risk factors and PNPLA3 genotype. Results:Of the 996 participants, the mean age was 53.9 years (standard deviation, 12 years) and 72% were male. The prevalence of MASLD and clinically significant fibrosis were 49% and 14%, respectively. Non-Hispanic Black (NHB) persons had the lowest prevalence of MASLD (43%) compared to non-Hispanic White (NHW) (53%) and Hispanic individuals (60%) (P < .001). Similarly, NHB persons had the lowest prevalence of clinically significant fibrosis (12%) compared to NHW (20%) and Hispanic individuals (13%) (P = .005). After adjusting for clinical risk factors and PNPLA3 genotype, NHB had a lower odds of MASLD (odds ratio [OR], 0.39 [95% confidence interval {CI}, .23-.66]) and clinically significant fibrosis (OR, 0.38 [95% CI, .20-.71]) compared to NHW persons. Conclusions:NHB PWH have a lower risk of MASLD and clinically significant fibrosis compared to NHW and Hispanic individuals, even after controlling for clinical risk factors and PNPLA3 genotype.

BACKGROUND & AIMS:Hepatic steatosis has been associated with major adverse cardiovascular events independently of other cardiovascular risk factors and the severity of coronary artery disease. Nevertheless, the relationship between hepatic steatosis, coronary plaque composition, and major adverse cardiovascular events remains unclear. METHODS:A central core laboratory analyzed Prospective Multicenter Imaging Study for Evaluation of Chest Pain (PROMISE) participants randomized to the computed tomography arm. Hepatic steatosis was assessed on noncontrast computed tomography using standard hepatic and splenic attenuation methods. Coronary computed tomography angiography was used to quantify total, calcified, noncalcified, and low-attenuation plaque volume and burden (% vessel volume). Multivariable regression and mediation analyses assessed relationships between hepatic steatosis, plaque components, and major adverse cardiovascular events (death, myocardial infarction, unstable angina hospitalization). The median follow-up was 25 months (interquartile range, 18-33 months). RESULTS:Among 3637 patients (age, 60.6 ± 8.2 years; 51.4% female), 25.5% had hepatic steatosis and were slightly younger, more often male, had more cardiovascular risk factors, and had a higher rate of major adverse cardiovascular events (4.1% vs 2.5%) (all P < .05). After adjustment for clinical risk factors, hepatic steatosis was associated with greater noncalcified plaque burden (β, 0.15%; 95% confidence interval, 0.04-0.27; P = .008). Hepatic steatosis was associated with increased risk of major adverse cardiovascular events independent of atherosclerotic cardiovascular disease risk score, obesity, obstructive stenosis, and noncalcified plaque burden (adjusted hazard ratio, 1.69; 95% confidence interval, 1.12-2.54; P = .012), whereas noncalcified plaque burden mediated 11% of the association between hepatic steatosis and major adverse cardiovascular events. CONCLUSIONS:Hepatic steatosis was associated with greater noncalcified plaque burden and increased risk of major adverse cardiovascular events, with noncalcified plaque burden accounting for a portion of this association, suggesting a link between hepatic steatosis and vulnerable coronary atherosclerosis. These findings support integrated cardiometabolic risk assessment in patients undergoing coronary computed tomography angiography. CLINICALTRIALS:gov number, NCT01174550.

Background & Aims: The current prevalence of fatty liver disease (FLD) due to alcohol-associated (AFLD) and nonalcoholic (NAFLD) origins in US persons with HIV (PWH) is not well defined. We prospectively evaluated the burden of FLD and hepatic fibrosis in a diverse cohort of PWH. Approach & Results: Consenting participants in outpatient HIV clinics in 3 centers in the US underwent detailed phenotyping, including liver ultrasound and vibration-controlled transient elastography for controlled attenuation parameter and liver stiffness measurement. The prevalence of AFLD, NAFLD, and clinically significant and advanced fibrosis was determined. Univariate and multivariate logistic regression models were used to evaluate factors associated with the risk of NAFLD. Of 342 participants, 95.6% were on antiretroviral therapy, 93.9% had adequate viral suppression, 48.7% (95% CI 43%–54%) had steatosis by ultrasound, and 50.6% (95% CI 45%–56%) had steatosis by controlled attenuation parameter ≥263 dB/m. NAFLD accounted for 90% of FLD. In multivariable analysis, old age, higher body mass index, diabetes, and higher alanine aminotransferase, but not antiretroviral therapy or CD4+ cell count, were independently associated with increased NAFLD risk. In all PWH with fatty liver, the frequency of liver stiffness measurement 8–12 kPa was 13.9% (95% CI 9%–20%) and ≥12 kPa 6.4% (95% CI 3%–11%), with a similar frequency of these liver stiffness measurement cutoffs in NAFLD. Conclusions: Nearly half of the virally-suppressed PWH have FLD, 90% of which is due to NAFLD. A fifth of the PWH with FLD has clinically significant fibrosis, and 6% have advanced fibrosis. These data lend support to systematic screening for high-risk NAFLD in PWH.

Supplementary Table S7: Metabolite Set Enrichment Analysis in HCC Cases Compared to All Controls

Supplementary Figure S2: Performance of Random Forest-derived pre-diagnostic plasma metabolite signatures and covariate-based signatures in predicting HCC among HCC cases and all controls

Despite increasing incidence of hepatocellular carcinoma (HCC) in vulnerable populations, accurate early detection tools are lacking. We aimed to investigate the associations between prediagnostic plasma metabolites and incident HCC in a diverse population. In a prospective, nested case-control study within the Southern Community Cohort Study, we conducted prediagnostic LC/MS metabolomic profiling in 150 incident HCC cases (median time to diagnosis, 7.9 years) and 100 controls with cirrhosis. Logistic regression assessed metabolite associations with HCC risk. Metabolite set enrichment analysis identified enriched pathways, and a random forest classifier was used for risk classification models. Candidate metabolites were validated in the UK Biobank (N = 12 incident HCC cases and 24 cirrhosis controls). In logistic regression analysis, seven metabolites were associated with incident HCC (MeffP < 0.0004), including N-acetylmethionine (OR = 0.46; 95% confidence interval, 0.31-0.66). Multiple pathways were enriched in HCC, including histidine and CoA metabolism (FDR P < 0.001). The random forest classifier identified 10 metabolites for inclusion in HCC risk classification models, which improved HCC risk classification compared with clinical covariates alone (AUC = 0.66 for covariates vs. 0.88 for 10 metabolites plus covariates; P < 0.0001). Findings were consistent in the UK Biobank (AUC = 0.72 for covariates vs. 0.88 for four analogous metabolites plus covariates; P = 0.04), assessed via nuclear magnetic resonance spectroscopy. Prediagnostic metabolites, primarily amino acid and sphingolipid derivatives, are associated with HCC risk and improve HCC risk classification beyond clinical covariates. These metabolite profiles, detectable years before diagnosis, could serve as early biomarkers for HCC detection and risk stratification if validated in larger studies. Prevention Relevance: Our findings support the need for larger prospective studies examining the role of prediagnostic plasma metabolomics for the preventive management of HCC in diverse patients across multiple etiologies of liver disease. This approach could improve HCC care by identifying metabolic changes years before diagnosis, potentially enhancing screening and early detection practices.

Disclosure: N. Chamorro-Pareja: None. K.E. Corey: Intercept. M.S. Haines: BioAge. C.C. Dobbie: None. J. Lin: None. M.L. Chicote: None. J.S. Husseini: None. M.A. Bredella: None. L.E. Dichtel: Perspectum Diagnostics, Lumos Pharma, Recordati, Novo Nordisk, Third Rock Ventures, Marea Therapeutics, Merida Biosciences, Flare Therapeutics. Background: Daily, subcutaneous growth hormone (GH) administration has been demonstrated to reduce hepatic steatosis in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD). Ibutamoren is an investigational oral, small molecule GH secretagogue. We hypothesized that GH augmentation via ibutamoren would reduce intrahepatic lipid content (IHL) and improve body composition in a pilot study of individuals with MASLD. Methods: Thirteen adults with BMI ≥25 kg/m2 and MASLD without diabetes were enrolled in an open-label pilot study of daily oral ibutamoren 25 mg for 6 months (NCT05364684). The primary endpoint was absolute change in percent IHL by proton magnetic resonance spectroscopy. Secondary endpoints included absolute change in ALT, HbA1c and body composition by dual-energy x-ray absorptiometry. Data are reported as mean ± SD. The open-label pilot cohort was compared to a historical, prospective, placebo group studied under a similar protocol (NCT02217345). Results: Seven subjects completed the 6-month ibutamoren study (IBU) and were compared to 10 historical placebo controls (PBO) with similar mean weight change (IBU 1.7±2.8% vs PBO 2.9±1.9%, p=0.3). Baseline mean age (46±12 y), BMI (33±4 kg/m2), sex distribution (35% female) and IHL (17.1±13.8%) did not differ between IBU and PBO. IGF-1 Z-score was significantly augmented in IBU vs PBO, as expected (delta IGF-1 Z-score 0.9±0.5 vs -0.3±0.6, p<0.001). Three IBU subjects dropped out for treatment-emergent adverse events (n=2 hyperglycemia, n=1 brain fog). In an intent-to-treat analysis, there was no difference in absolute change in IHL in IBU vs PBO (3.9±6.7% vs 6.7±6.1%, p=0.4). Changes in serum ALT (8±18 IU/L vs 4±7 IU/L, p=0.6), appendicular lean mass (0.7±0.9 kg vs 1.0±0.6 kg, p=0.6) and visceral adipose tissue (-1.8±11.9 cm2 vs 5.3±13.0 cm2, p=0.3) did not differ in IBU vs PBO, respectively. HbA1c increased modestly within in the non-diabetic range in IBU vs PBO (5.3±0.2% to 5.7±0.4% vs 5.4±0.2% to 5.4±0.2%, p=0.008 for delta). In an as-treated secondary analysis excluding one IBU subject with suspected non-adherence to ibutamoren administration (based on lack of change in IGF-1 Z-score at 6 months), there was a beneficial effect on IHL in IBU vs PBO (1.5±2.3% vs 6.6±6.1%, p=0.03). All other findings were similar to the intent-to-treat analysis. Conclusion: In this pilot study, we demonstrated a possible beneficial signal of ibutamoren on IHL in an as-treated secondary analysis but were unable to demonstrate this effect in our primary analysis or an effect on body composition in either analysis. Future larger studies could investigate oral ibutamoren co-administered with weight loss medications such as GLP-1 receptor agonists, a combination which may offset the negative glycemic effect of ibutamoren while harnessing the potential for GH augmentation to further reduce liver fat and preserve muscle mass. Presentation: Saturday, July 12, 2025

Background: Clinical guidelines emphasise identifying patients at risk of chronic liver disease progression. To avoid biopsy drawbacks, noninvasive imaging tests (NITs) have become part of standard-of-care. We assessed the real-world clinical profile, referral trends, and use of magnetic resonance imaging (MRI)-based tests, multiparametric MRI (mpMRI) and magnetic resonance elastography (MRE), as part of chronic liver disease management. Methods: Patients referred for abdominal imaging as part of standard-of-care were eligible for inclusion irrespective of liver aetiology or referral pathway. Liver fibrosis was assessed using MRE and disease severity using mpMRI (disease activity [iron-corrected T1, cT1], liver fat content [LFC] and iron). T-tests were used for group comparisons; Kaplan-Meier analyses for disease progression and area under the receiver operating characteristic (AUC) for diagnostic accuracy. Results: Over 18 months, 256 patients (53 years, 51% female, 48% with BMI > 30 kg/m(2)) were referred for liver imaging. The majority (66%) had steatotic liver disease (SLD). Of those with low MRE (73%) and low FIB-4 (42%), 36% had elevated cT1 (> 875 ms). Those with MRE > 5 kPa had cT1 > 875 ms. During follow-up, those with low MRE (< 3.14 kPa) but elevated cT1 (> 800 ms) had significant disease worsening (HR: 3.1, p = 0.0035) compared to all others. In the SLD group, cT1 (AUC: 0.71) outperformed LFC (AUC: 0.64) and MRE (AUC: 0.53) in predicting disease progression. Conclusion: Regardless of aetiology, patients with low fibrosis risk (MRE) but high disease activity (cT1) face a three-times higher risk of progression. Integrating both biomarkers into standard care, especially for SLD, can guide management adjustments.

Disclosure: N. Chahal: None. A.K. Arpante: None. J.E. Johnson: None. C.A. Dash: None. M.W. Ockene: None. I. Zheng: None. A. Boutin: None. H. Lee: None. K.E. Corey: None. T.L. Stanley: None. S.K. Grinspoon: None. L.T. Fourman: None. Background: Over 1 million HIV-negative individuals are born to mothers with HIV annually. While mounting evidence indicates an increased risk of metabolic abnormalities among this population in early life, long-term sequelae of perinatal HIV exposure without infection (PHEU) into adulthood remain largely unknown. This study examined the prevalence and prenatal correlates of obesity and metabolic comorbidities in the oldest known cohort of young adults with PHEU versus matched unexposed controls. Methods: In this prospective study, we evaluated 41 young adults with PHEU and 41 controls matched 1:1 on ≥4 of the following factors: age, sex, race/ethnicity, maternal education, maternal substance use disorder, and breastfeeding. Obesity was defined as BMI ≥30 kg/m2 with elevated waist circumference (≥88 cm in females, ≥102 cm in males), or BMI >40 kg/m2. Metabolic comorbidities included impaired glucose tolerance (HbA1c ≥5.7%, fasting glucose ≥100 mg/dL, and/or 2-hour glucose ≥140 mg/dL on oral glucose tolerance test), insulin resistance (HOMA-IR ≥3), hypertension (clinical diagnosis, SBP ≥140 mmHg, and/or DBP ≥90 mmHg), dyslipidemia (total cholesterol ≥200 mg/dL, HDL-C <40 mg/dL, and/or triglycerides ≥150 mg/dL), and hepatic steatosis (controlled attenuation parameter ≥263 dB/m on elastography). A composite metabolic comorbidity score was derived with 1 point assigned to each of the 5 selected outcomes. Framingham 30-year risk of cardiovascular disease (CVD) was calculated. Prenatal history was ascertained via maternal interview and medical records. Results: Young adults with PHEU (24±5 y, 46% male) had higher rates of obesity (51% vs. 29%, P=0.04), impaired glucose tolerance (21% vs. 3%, P=0.01), insulin resistance (24% vs. 2%, P=0.004), hypertension (20% vs. 0%, P=0.003), dyslipidemia (46% vs. 15%, P=0.002), and hepatic steatosis (44% vs. 15%, P=0.006) compared to matched controls. These differences conferred a higher metabolic comorbidity score (1.5±1.4 vs. 0.3±0.7, P<0.001) and 30-year CVD risk (9.7% vs. 5.8%, P=0.02). Relationships of PHEU with BMI, comorbidity score, and CVD risk persisted upon adjusting for young adult (e.g., adverse childhood experiences) and maternal factors (e.g., BMI). PHEU was associated with a higher rate of ≥1 metabolic comorbidity in young adults with (100% vs. 58%, P=0.001) and without obesity (42% vs. 15%, P=0.04). In the PHEU group, lower maternal prenatal CD4 count was associated with higher adult CVD risk (ρ=-0.42, P=0.03). Conclusions: Young adults with PHEU bear a greater burden of obesity and metabolic comorbidities versus unexposed peers. Maternal HIV-related immune dysregulation may contribute to these outcomes. These findings highlight the critical role of the intrauterine environment in shaping adult cardiometabolic disease risk and may have broader implications for other groups including offspring of mothers with obesity. Presentation: Monday, July 14, 2025

Acute inflammation is an essential response that our bodies use to combat infections1. However, in the absence of infections, chronic inflammation can have a pivotal role in the onset and progression of chronic diseases, such as arthritis, cancer, autoimmune disorders, metabolic-dysfunction-associated steatohepatitis (MASH), and most ageing-associated pathologies2,3. The underlying mechanisms that distinguish chronic inflammation from its acute counterpart remain unclear, posing challenges to the development of targeted therapies for these major diseases. Here we identify a mechanism that separates the two responses: during chronic but not acute inflammation, chromatin remodelling is influenced by nuclear autophagy, in which the WSTF protein of the ISWI chromatin-remodelling complex interacts with the ATG8 autophagy protein family in the nucleus. This interaction leads to WSTF nuclear export and subsequent degradation by autophagosomes and lysosomes in the cytoplasm. Loss of WSTF leads to chromatin opening over inflammatory genes, amplifying inflammation. Cell-penetrating peptides that block the WSTF-ATG8 interaction do not affect acute inflammation but suppress chronic inflammation in senescence as well as in MASH and osteoarthritis in mouse models and patient samples. The ability to specifically target chronic inflammation without blunting acute inflammation offers an approach for treating common chronic inflammatory diseases.