IDDF2024-ABS-0141 Intra-hepatic microbial heterogeneity among multifocal tumors in hepatocellular carcinoma patients and its association with host genomic and transcriptomic alterations

<h3>Background</h3> About half of patients with hepatocellular carcinoma (HCC) are initially diagnosed with multi-nodule, which developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO). The signature of the intrahepatic microbiome in multifocal HCC (mHCC) and its association with HCC genomic alterations remain elusive. We aim to identify the microbial signature in mHCC and its contribution to HCC multi-nodule heterogeneity. <h3>Methods</h3> We collected 300 HCC nodule tissue samples and 58 matched adjacent non-tumor liver tissues from 58 mHCC patients who underwent partial hepatectomy. Microbial profiling, host gene mutation, and gene expression were performed using 16S ribosomal RNA gene sequencing, whole-exome sequencing, and bulk RNA sequencing. The presence of intrahepatic microbiome was determined by immunohistochemistry staining, electron microscopy, and fluorescence <i>in situ</i> hybridization. <h3>Results</h3> Compared to adjacent non-tumor tissues, inter-tumor microbial communities in mHCC were heterogeneous. <i>Streptococcus</i>, <i>Stenotrophomonas</i>, and <i>Lactobacillus</i> were the most prevalent bacterial genera in mHCC, whose abundance varied highly across nodules in mHCC patients. The host inter-tumor genomic alteration presented considerable heterogeneity, of which 156 IM nodules and 15 MO nodules were identified. We observed alpha diversity significantly decreased in IM compared to MO nodules (<i>P</i>=0.002). The bacterial community architecture was also significantly distinct between IM and MO nodules (<i>P</i>=0.012). <i>Enterococcus</i>, <i>Streptococcus minor</i>, and <i>Fusobacterium</i> were consistently enriched in the IM nodules, while <i>Stenotrophomonas</i>, <i>Corynebacterium</i>, and <i>Bacteroides</i> were depleted. In particular, an abundance of 12 bacterial biomarkers could distinguish IM from MO nodules with an AUROC of 0.87. Transcriptomic analysis showed epithelial-mesenchymal transition, cell cycle, and angiogenesis pathways were upregulated in IM nodules. Integrative analysis revealed that the IM nodules-enriched bacteria taxa were positively associated with host metastasis-related gene expression, including FGFR, HGF, and MMP1. Finally, we validated the presence of intrahepatic bacteria in mHCC tissues. <h3>Conclusions</h3> We comprehensively demonstrated that inter-tumor microbiota of multi-HCC nodules is heterogeneous. The bacteria community architecture was different between IM and MO nodules. IM nodules-enriched bacteria were positively associated with host metastasis-related gene expression. Thus, our findings provide new insights into the contribution of intrahepatic microbiota to multifocal HCC heterogeneity.