Fig. 5

A pilot study to refine the 6 HCC EV-specific genes from the 12 HCC EV-specific gene candidates and calculate HCC EV Digital Score. A To refine the 6 HCC EV-specific genes from the 12 HCC EV-specific gene candidates, a pilot study of HCC EV Digital Scoring Assay was conducted to differentiate early- or intermediate-stage Tx-naïve HCC (BCLC 0-B, n = 35) from liver cirrhosis (n = 35), where the disease window of the HCC cases matched that of the HCC TR assessment cohort. B The 6 differentially expressed genes ALB, APOH, FGB, FGG, H2 AX, and TF, capable of better differentiating early- or intermediate-stage HCC from liver cirrhosis were identified. C Heatmaps summarizing relative intensities of the 6 HCC EV-specific genes across the early- or intermediate-stage HCC and liver cirrhosis patients. Scale: log₂(transcript + 1). D A weighted Z-score formula and the respective weights of the six genes were developed to integrate the 6-gene readouts into HCC EV Digital Scores. In this formula, i represents each gene, with weights assigned based on the relative signal-to-noise ratio in the average gene copies (log₂ transformed) of HCC patients relative to those of cirrhosis patients. E Boxplot summarizing the HCC EV Digital Scores calculated for the early- or intermediate-stage HCC and liver cirrhosis patients. F Receiver operating characteristic (ROC) curves of HCC EV Digital Score for differentiating early- or intermediate-stage HCC and liver cirrhosis patients (AUROC = 0.85, 95% CI: 0.76—0.94). AUROC, area under the receiver operating characteristic curve; EV, extracellular vesicle; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic; RT-dPCR, reverse-transcription digital PCR