HCC EV ECG score: An extracellular vesicle‐based protein assay for detection of early‐stage hepatocellular carcinoma

Abstract

Background and Aims: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC.Approach and Results: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations (EpCAM + CD63 + , CD147 + CD63 + , and GPC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort (n = 106) and an independent validation cohort (n = 72). Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90--1.00; nonviral: 0.94; 95% CI, 0.88-0.99). Conclusion: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

HCC biomarkers – state of the old and outlook to future promising biomarkers and their potential in everyday clinical practice

“…Probably the closest study to a clinical application is the HCC EV ECG score, an extracellular vesicle-based protein assay for detecting early-stage hepatocellular carcinoma ( 228 ). Here, exosomes isolated from plasma are analyzed for surface expression of EpCAM, CD63, CD147, CD63, and GPC3.…”

Advances of multi-omics applications in hepatic precancerous lesions and hepatocellular carcinoma: The role of extracellular vesicles

“…Sun N et al developed and optimized an HCC-specific EV surface protein detection method (referred as HCC EV ECG score), based on three HCC EV subpopulations of EpCAM + CD63 + , CD147 + CD63 + , and GPC3 + CD63 + , by which EVs were isolated from peripheral blood, and HCC EV surface proteins were detected. This new established detection model was confirmed to have an accuracy of more than 93% (AUC >0.93) in distinguishing early HCC from cirrhosis with excellent detection performance ( Sun et al, 2022 ). LC3B (MAP1LC3B) is a widely used marker of autophagy.…”

Challenges and barriers in hepatocellular carcinoma (HCC) surveillance for patients with non-alcoholic fatty liver disease (NAFLD)

“…Extracellular vesicles (EVs) represent another type of "liquid biopsy" holding promise in HCC. As EVs contain DNA, RNA, proteins, metabolites and lipids from both normal and tumor cells and are present in circulation, several studies have investigated their role in detecting HCC among at-risk patients [42][43][44][45] . A recently published phase 2 biomarker study revealed a 91% sensitivity and 90% specificity for distinguishing early-stage HCC from cirrhosis, of which NAFLD represented 20% [46] .…”