Dottorato in TECNOLOGIE AVANZATE IN CHIRURGIA

Titolo della tesi: Novel liquid biopsy tests for highly accurate diagnosis of NASH and liver fibrosis: a multicenter prospective cohort trial

Background: Approval of drugs for non-alcoholic steato-hepatitis (NASH) requires evidence of significant effects on histological endpoints, yet liver biopsy is an invasive procedure. Therefore, novel non-invasive biomarkers of NASH and/or liver fibrosis are needed. Methods: Proteomics was performed with iTRAQ and nano-LC-MS/MS, and flow-cytometry assay used to measure PLIN2 and RAB14 in peripheral blood CD14+CD16− monocytes. Caucasian patients with clinical risk factors for NASH and/or fibrosis without secondary causes, underwent percutaneous liver biopsy; 50 healthy subjects underwent needle liver biopsy during elective laparoscopic cholecystectomy (Training-cohort: 100 subjects; Validation-cohort: 150 subjects). Results: Monocyte PLIN2 and RAB14 were highly and scarcely expressed in NASH and liver fibrosis, respectively. Biomarker performance was compared with histology. Algorithm for NASH used PLIN2 mean-florescence-intensity (MFI) which, combined with triglyceride, ALT plasma levels and presence/absence of diabetes as covariates, showed an AUROC of 96% (CI: 93-100%) in the training and 98% (CI: 96-100%) in the validation cohort, with Accuracy: 91%, Sensitivity: 98%, Specificity: 85% in training and 96%, 94%, and 98% in validation cohort. Prediction of fibrosis (RAB14-MFI) used BMI, HDL-cholesterol, plasma glucose, and ALT levels as covariates, and yielded an AUROC of 95% (CI: 88-100%), with Accuracy: 95%, Sensitivity: 98% and Specificity: 87% in training and 97% (CI: 90-100%), 91%, 90%, and 93% in validation cohort. Novel biomarkers performed substantially better compared to the most widely used biomarkers (FIB4, NAFLD Fibrosis-Score, and AST-to-Platelet-ratio). Conclusions: Novel liquid biopsy test is highly accurate in diagnosing NASH and/or liver fibrosis, and is markedly more reliable than the most commonly used biomarkers, and on par with gold standard liver biopsy-based histology.