INTRA-HEPATIC MICROBIAL HETEROGENEITY AMONG MULTIFOCAL TUMORS IN HCC PATIENTS AND ITS ASSOCIATION WITH HOST GENOMIC AND TRANSCRIPTOMIC ALTERATIONS

Background & Aim: 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 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.
Method: We collected 290 HCC nodule tissue samples (3 to 7 nodules per case) 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. IM or MO nodule was evaluated by the proportion of shared somatic mutations, the common clusters of clone and genomic distance. Gene-taxa correlation was conducted by LASSO regression.
Results: Compared to adjacent non-tumor tissues, inter-tumor microbial communities in mHCC were heterogeneous. Streptococcus (50/58), Acinetobacter (48/58), Stenotrophomonas (46/58), and Corynebacterium (46/58) 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 132 IM nodules and 15 MO nodules were identified. Transcriptome sequencing analysis showed pathways of epithelial-mesenchymal transition, angiogenesis, inflammation and cell cycle were upregulated in IM nodules, implying high metastatic activity in the IM nodules. We observed alpha diversity was significantly decreased in IM nodules (P=0.005) compared to MO nodules. The bacterial community architecture was significantly distinct between IM and MO nodules (P=0.01). Enterococcus, Streptococcus minor, Helicobacter pylori and Fusobacterium were consistently enriched in the IM nodules, while Corynebacterium,Stenotrophomonas and Bacteroides were depleted. In particular, abundances of 13 bacteria biomarkers could distinguish IM nodules from MO nodules with AUC of 0.85. Finally, integrative microbiome profile and host transcriptomic analysis revealed that the IM nodules-enriched bacteria taxa were positively associated with host metastasis-related gene expression, including FGFR1, MMP1 and CDH2, suggesting hepatic bacteria taxa might contribute to HCC intrahepatic metastasis.
Conclusions: 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 on the contribution of intrahepatic microbiota to multifocal HCC heterogeneity.