Intratumor microbial sequencing of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often diagnosed at advanced stages, limiting curative options. Recent advances in conversion therapy have improved tumor resectability and patient survival. While intratumoral microbiota is known to influence the tumor microenvironment, its alterations following therapeutic interventions remain underexplored. This study (NCT06365034) included patients with initially unresectable HCC treated at the First Affiliated Hospital of Shandong First Medical University using conversion therapy with two or three modalities: immune checkpoint inhibitors (ICIs), tyrosine kinase inhibitors (TKIs), and locoregional therapies such as transarterial chemoembolization (TACE) or hepatic arterial infusion chemotherapy (HAIC). Primary endpoints included radical (R0) resection rate (100%), with secondary endpoints being objective response rate (19.7%), disease control rate (85.9%), and conversion rate (38.0%), disease-free survival (median: 10 months) and safety. Of the 27 patients achieving surgical resectability, 20 underwent successful resection, with fatigue as the most common adverse event. Intratumoral microbiome analysis excluded 5 patients who did not receive triple conversion therapy. Among the remaining patients, 11 showed high pathological response rates (residual tumor cells comprising less than 50% of the tumor bed), while 4 had low response rates (residual tumor cells comprising 50% or more than 50% of the tumor bed). We collected tumor tissues from 15 patients with uHCC who underwent surgical resection following triple conversion therapy. Additionally, tumor tissues and adjacent non-tumor tissues were obtained from 20 patients who underwent surgical resection at the time of initial diagnosis. All tissue samples were collected during surgery, processed into formalin-fixed paraffin-embedded (FFPE) blocks, labeled appropriately, and stored for subsequent use in microbiome analysis. 16S rRNA sequencing revealed that Escherichia_Shigella, Limosilactobacillus were more abundant in tumor tissues of patients with high pathological response rates, with functional predictions linking them to substance metabolism. This study highlights the prognostic potential of intratumoral microbiota in predicting conversion therapy efficacy in HCC.