The Altered Serum Lipidome and its Diagnostic Potential for Non-Alcoholic Fatty Liver (NAFL)-Associated Hepatocellular Carcinoma

Abstract

Introduction: Non-alcoholic fatty liver disease (NAFLD) is affecting more people globally. Indeed, NAFLD is a spectrum of metabolic dysfunctions that can progress to hepatocellular carcinoma (NAFLD-HCC). This development can occur in a non-cirrhotic liver and thus, often lack clinical surveillance. The aim of this study was to develop non-invasive surveillance method for NAFLD-HCC. Methods: Using comprehensive ultra-high-performance liquid chromatography mass-spectrometry, we investigated 1,295 metabolites in serum from 249 patients. Area under the receiver operating characteristic curve was calculated for all detected metabolites and used to establish their diagnostic potential. Logistic regression analysis was used to establish the diagnostic score. Results: We show that NAFLD-HCC is characterized by a complete rearrangement of the serum lipidome, which distinguishes NAFLD-HCC from non-cancerous individuals and other HCC patients. We used machine learning to build a diagnostic model for NAFLD-HCC. We quantified predictive metabolites and developed the NAFLD-HCC Diagnostic Score, presenting superior diagnostic potential compared to alpha-fetoprotein (AFP). Patients metabolic landscapes show a progressive depletion in unsaturated fatty acids and acylcarnitines during transformation. Upregulation of fatty acid transporters in NAFLD-HCC tumors contributes to fatty acid depletion in the serum. Discussion: NAFLD-HCC patients can be efficiently distinguished by serum metabolic alterations from the healthy population and from HCC patients related to other etiologies (alcohol and viral hepatitis). Our model can be used for non-invasive surveillance of individuals with metabolic syndrome(s), allowing for early detection of NAFLD-HCC. Therefore, serum metabolomics may provide valuable insight to monitor patients at risk, including morbidly obese, diabetics, and NAFLD patients. Funding: The laboratory of JBA is supported by the Novo Nordisk Foundation (14040, 0058419), Danish Cancer Society (R98-A6446, R167-A10784, R278-A16638), and the Danish Medical Research Council (4183-00118A, 1030- 00070B). JMB is funded by the Spanish Ministry of Economy and Competitiveness and ’Instituto de Salud Carlos III’ grants (PI18/01075, Miguel Servet Programme CON14/00129 and CPII19/00008) co-financed by ’Fondo Europeo de Desarrollo Regional’ (FEDER); CIBERehd, Spain; IKERBASQUE, Basque foundation for Science, Spain; BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD); Department of Health of the Basque Country (2017111010), Euskadi RIS3 (2019222054, 2020333010); Department of Industry of the Basque Country (Elkartek: KK-2020/00008), AECC Scientific Foundation and European Commission Horizon 2020 program (ESCALON project no.: 825510). Declaration of Interest: Drs. Alonso and Arretxe are employed by OWL Metabolomics (One Way Liver, S.L). Dr. Banales is a member of the scientific advisory board of OWL Metabolomics. Remaining authors declare no conflict of interest. Ethical Approval: The study was performed following individual patient consent, local institutional review board (IRB), and approval from the Committees on Health and Research Ethics for the Capital Region of Denmark for use of archival material no. 17029679.