PROJECT SUMMARY/ABSTRACT Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Local regional therapies, such as local ablation and transarterial chemoembolization (TACE), are the most commonly used treatment modality for HCC. Following treatment, patients undergo radiologic imaging for evaluation of treatment response. Assessing treatment response to local regional therapies is often challenging due to treatment-related nonspecific changes and definitive assessment typically requires repeated cross-sectional imaging. Similarly, there is poor radiologic-histological correlation of post-treatment tumor viability, with discordant results seen in 38% of HCC patients after local regional therapies. While serum alpha-fetoprotein (AFP) can aid to assess treatment response, less than half of all patients with HCC have an elevated AFP before treatment. This limits its utility for broader implementation. Extracellular vesicles (EVs) are phospholipid bilayer-enclosed particles released by both normal and tumor cells. Compared to other types of liquid biopsy, EVs have advantages in their higher frequency in circulation and the simplicity of sample preservation. One major challenge of this approach, however, is that tumor-derived EVs typically co-exist with those from non-tumor sources, resulting in complex background signals. To overcome this technical barrier for selective purification of tumor-derived EVs, our joint research team developed a novel nanotechnology platform named EV Click Chip with the support of NIH/NCI R01 funding (R01 CA253651). When used in combination with reverse-transcription droplet digital PCR (RT-ddPCR), this HCC EV Digital Scoring Assay (EV Click Chip + RT-ddPCR) is able to purify HCC EVs and quantify HCC-specific mRNA signatures from the purified HCC EVs. We recently showed this assay can distinguish early-stage HCC from liver cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.93. Given the potential of HCC EV Digital Scoring Assay for reflecting the presence of HCC and tumor burden, we hypothesized it could be used for evaluating treatment response in HCC patients receiving locoregional therapies. The study aims of this two-year R01 revision application are to investigate the performance of HCC EV Digital Scoring Assay (EV Click Chip + RT-ddPCR) for evaluation of HCC treatment response to local ablation or TACE.