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 ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + 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.

FIGURE 1

A general workflow of developing HCC EV ECG score for distinguishing early-stage HCC from cirrhosis. Plasma samples from patients with HCC or liver cirrhosis were introduced to HCC EV surface protein assay that combined the use of covalent chemistry-mediated EV purification and duplex real-time immuno-PCR, to quantify eight subpopulations of HCC EVs, i.e., EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, GPC3⁺ CD63⁺ HCC EVs, ASGPR1⁺ CD63⁺ HCC EVs, EpCAM⁺ CD9⁺ HCC EVs, CD147⁺ CD9⁺ HCC EVs, GPC3⁺ CD9⁺ HCC EVs, and ASGPR1⁺ CD9⁺ HCC EVs. The resulting HCC EV surface protein signatures was then analyzed to generate HCC EV ECG score (accounted from EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, and GPC3⁺ CD63⁺ HCC EVs) for distinguishing early-stage HCC from at-risk liver cirrhosis. ASGPR1, Asialoglycoprotein receptor 1; EpCAM, epithelial cellular adhesion molecule; EV, extracellular vesicle; GPC3, Glypican 3 Protein; HCC, hepatocellular carcinoma; PCR, polymerase chain reaction.

FIGURE 2

Clinical study design flowchart depicting the recruitment and exclusions from the study cohort. Blood samples from 106 and 73 eligible participants were collected from UCLA (training cohort) and CSMC (independent validation cohort), respectively. In brief, HCC EV surface protein assay was utilized in the training cohort (UCLA cohort, n = 106) to identify the HCC EV subpopulations significantly associated with early-stage HCC over cirrhosis and establish the logistic regression model (i.e., HCC EV ECG score) for detecting early-stage HCC from cirrhosis. Model calibration was performed to evaluate the agreement between predicted probabilities from the model and observed event rates. Leave-one-out cross-validation was applied to estimate the performance of the established HCC EV ECG score in the training cohort. After one participant was excluded because of coexisting with other tumors, external validation of HCC EV ECG score was performed in the independent validation cohort (CSMC cohort, n = 72). CSMC, Cedars-Sinai Medical Center; ECG, EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, and GPC3⁺ CD63⁺ HCC EVs; EV, extracellular vesicle; GPC3, Glypican 3 Protein; HCC, hepatocellular carcinoma; UCLA, University of California, Los Angeles.

FIGURE 3

Validation of the four selected HCC-associated surface markers using TMA and HCC cells-derived EVs. (A) H&E staining and immunohistochemistry staining of the four selected HCC-associated surface protein markers, EpCAM, CD147, GPC3, and ASGPR1, on a TMA slide containing 708 HCC samples. Scale bar, 100 μm. (B) Percentage of tumors with negative (0), weak (1⁺), moderate (2⁺), strong (3⁺) staining for the four given surface protein markers. (C) The percentage of tumors staining for at least one of the four markers (combined) summarized in the bar chart. (D) Western blotting for the four surface protein markers in HepG2-derived EVs and Hep3B-derived EVs, with BSA as the negative control. ASGPR1, Asialoglycoprotein receptor 1; BSA, bovine serum albumin; EpCAM, epithelial cellular adhesion molecule; EV, extracellular vesicle; GPC3, Glypican 3 Protein; H&E, Hematoxylin and Eosin; HCC, hepatocellular carcinoma; TMA, tissue microarray.

FIGURE 4

HCC EV SPA for measuring subpopulations of HCC EVs and detecting early-stage HCC in the UCLA training cohort. (A) Heatmaps summarize relative duplex real-time immuno-PCR readouts of plasma samples from patients with early-stage HCC (BCLC Stage 0-A, n = 45) and patients with liver cirrhosis (n = 61). Significantly higher immuno-PCR signals of both (B) CD63⁺ and (C) CD9⁺ HCC EV subpopulations were observed in patients with HCC compared to those with cirrhosis. (D) ROC curve of HCC EV ECG score that was calculated by the signals from EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, and GPC3⁺ CD63⁺HCC EVs using HCC EV SPA, for detecting early-stage HCC from cirrhosis in the UCLA training cohort. (E) ROC curve of HCC EV ECG score after leave-one-out cross-validation for detecting early-stage HCC from cirrhosis in the UCLA training cohort. ASGPR1, Asialoglycoprotein receptor 1; AUROC, area under the receiver operating characteristic curve; BCLC, Barcelona Clinic Liver Cancer; EpCAM, epithelial cellular adhesion molecule; EV, extracellular vesicle; GPC3, Glypican 3 Protein; HCC, hepatocellular carcinoma; LOOCV, leave-one-out cross-validation; PCR, polymerase chain reaction; ROC, receiver operating characteristic; SPA, Surface Protein Assay; UCLA, University of California, Los Angeles.

FIGURE 5

HCC EV ECG score for detecting early-stage HCC in the CSMC independent validation cohort. (A) Heatmaps summarize relative duplex real-time immuno-PCR signals of plasma samples from patients with early-stage HCC (BCLC Stage 0-A, n = 35) and patients with liver cirrhosis (n = 37). Significantly higher immuno-PCR signals of (B) EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, and GPC3⁺ CD63⁺ HCC EVs were observed in patients with HCC compared to those with cirrhosis. (C) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis in the CSMC independent validation cohort. ASGPR1, Asialoglycoprotein receptor 1; AUROC, area under the receiver operating characteristic curve; BCLC, Barcelona Clinic Liver Cancer; CSMC, Cedars-Sinai Medical Center; EpCAM, epithelial cellular adhesion molecule; EV, extracellular vesicle; GPC3, Glypican 3 Protein; HCC, hepatocellular carcinoma; PCR, polymerase chain reaction; ROC, receiver operating characteristic.

FIGURE 6

Comparison between HCC EV ECG score and serum AFP for detecting early-stage HCC among all the participants and subgroup analyses. (A) Comparison of the performance of HCC EV ECG score and serum AFP for detecting early-stage HCC among all the participants (n = 172). (B) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among patients with viral etiology (n = 64). (C) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among patients with nonviral etiology (n = 114). (D) ROC curve of HCC EV ECG score for detecting early-stage HCC within Milan criteria (n = 68) from cirrhosis (n = 98). (E) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among patients with cirrhosis (n = 164). (F) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among patients with viral-related cirrhosis (n = 53). (G) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among patients with nonviral-related cirrhosis (n = 111). (H) ROC curve of HCC EV ECG score for detecting early-stage HCC from cirrhosis among a cohort with cirrhosis after frequency matching of the etiology (n = 117). AUROC with the sensitivity and specificity of the assays at the optimal cutoffs are listed for each graph. AFP, alpha-fetoprotein; AUROC, area under the receiver operating characteristic curve; ECG, EpCAM⁺ CD63⁺ HCC EVs, CD147⁺ CD63⁺ HCC EVs, and GPC3⁺ CD63⁺ HCC EVs; EV, extracellular vesicle; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic.