BACKGROUND: Reliable biomarkers are warranted to identify patients with metabolic dysfunction-associated steatotic liver disease (MASLD), including metabolic dysfunction-associated steatohepatitis (MASH), at risk for developing hepatocellular carcinoma (HCC). RESEARCH AND METHODS: We evaluated whether circulating histones can predict this risk. Plasma histones were measured using imaging flow cytometry in patients with MASLD (n = 25), MASH (n = 25), HCC (n = 40), and 30 healthy controls. RESULTS: We detected (p < 0.05), compared to controls: 1) elevated levels of H2A, H3, H2A/H2B/H3/H4, macroH2A1.1, macroH2A1.2 in MASLD/MASH and HCC; 2) decreased levels of macroH2A1.2/H2B/H3/H4 in MASLD/MASH and increased in HCC; 3) reduced H4 levels discriminating between MASH and non-MASH. Machine-learning analysis showed that being diabetic/dyslipidemic, having decreased H2A (p = 0.002) and H4 (p = 0.0156) levels favor MASH. CONCLUSIONS: Our data indicate plasma histones H2A and H4 as new biomarkers of liver disease progression. The identification of histone-based biomarkers using imaging flow cytometry could provide a rapid approach to discriminate between non-MASH and MASH, and to predict the risk of HCC development.

Patients with fatty liver disease linked to metabolism (MASLD or its more severe inflammatory form MASH) need better ways to spot who might later develop liver cancer (HCC). In this study, we measured different types of histones (proteins that bind our DNA and then float in the blood if cells die) using a special imaging technique in 25 people with MASLD, 25 with MASH, 40 with HCC, and 30 healthy people. We found clear differences: several histones and histone complexes were higher in all patient groups than in healthy controls. Some histone levels were lower in MASLD/MASH but rose again in HCC, while histone H4 was particularly low in MASH and helped tell MASH apart from milder disease. Using machine learning, the strongest signals for having MASH (rather than a simpler fatty liver) were diabetes or high blood fats combined with lower circulating histones H2A and H4. Overall, blood levels of histones H2A and H4 look like promising new markers to track how serious the liver disease is, distinguish MASH from less severe forms, and possibly predict who will go on to develop liver cancer. This histone test is fast and could be useful in everyday practice.

CEMAD Digestive Diseases Center Fondazione Policlinico Universitario A Gemelli IRCCS Università Cattolica del Sacro Cuore Rome Italy

Computational Biology and Bioinformatics Unit Fondazione Policlinico Universitario A Gemelli IRCCS Roma Italy

Department of Adaptive Biotechnologies Global Change Research Institute CAS Brno Czech Republic

Department of Medical Genetics Medical University of Varna Varna Bulgaria

Department of Stem Cell Biology and Transplantology Research Institute of the Medical University of Varna Varna Bulgaria

Research Unit of Genetics of Complex Phenotypes Bambino Gesù Children's Hospital IRCCS Rome Italy

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