Fig. 1

An HCC EV Digital Scoring Assay for HCC TR assessment. A The HCC EV Digital Scoring Assay is composed of a two-step workflow – Step 1: click chemistry-mediated enrichment of HCC EVs in 1.0-mL plasma by EV Click Beads in conjunction with the use of a cocktail of three TCO-grafted antibodies, and Step 2: absolute quantification of six HCC EV-specific genes by RT-dPCR. These six HCC EV-specific genes were selected and validated through i) a comprehensive data analysis pipeline based on our proprietary LiTA database, ii) RNAscope validation on HCC tissues and iii) a pilot study on early- or intermediate-stage HCC and liver cirrhosis patients. B A phase-2 retrospective case–control biomarker study was developed to evaluate the refined HCC EV Digital Scoring Assay for HCC TR assessment (i.e., distinguish post-Tx viable from nonviable HCC). Pre- and post-Tx plasma samples were collected from 100 patients with early- or intermediate-stage HCC, who received surgical resection, LT, local ablation, TARE, or TACE. The clinical status of these patients was classified as either post-Tx viable or nonviable in a 2:1 ratio. These patients were randomly divided into a training set (n = 49) and a validation set (n = 51). In the training set, all plasma samples were subjected to HCC EV Digital Scoring Assay to generate the respective pre-Tx and post-Tx HCC EV Digital Scores, and ∆ HCC EV Digital Scores (by subtracting the post-Tx with pre-Tx Scores). A logistic regression model was applied to integrate post-Tx HCC EV Digital Scores and ∆ HCC EV Digital Scores to establish HCC EV TR Scores, enabling effective differentiation of post-Tx viable and nonviable HCC. Subsequently, the study on the validation set reproduced the diagnostic efficacy of the HCC EV TR Scores for HCC TR assessment. ASGPR1, asialoglycoprotein receptor 1; CSMC, Cedars-Sinai Medical Center; EpCAM, epithelial cellular adhesion molecule; EV, extracellular vesicle; HCC, hepatocellular carcinoma; LiTA, Liver Transcriptome Atlas; LT, liver transplantation; mTz, methyltetrazine; RBC, red blood cell; RT-dPCR, reverse-transcription digital PCR; TACE, transarterial chemoembolization; TARE, transarterial radioembolization; TCO, trans-cyclooctene; TR, treatment response; Tx, treatment; UCLA, University of California, Los Angeles; WBC, white blood cell