Genomic and the tumor microenvironment heterogeneity in multifocal hepatocellular carcinoma

Background and Aims: Ambiguous understanding of tumors and tumor microenvironments (TMEs) hinders accurate diagnosis and available treatment for multifocal hepatocellular carcinoma (HCC) covering intrahepatic metastasis (IM) and multicentric occurrence (MO). Here, we characterized the diverse TMEs of IM and MO identified by whole-exome sequencing at single-cell resolution. Approach and Results: We performed parallel whole-exome sequencing and scRNA-seq on 23 samples from 7 patients to profile their TMEs when major results were validated by immunohistochemistry in the additional cohort. Integrative analysis of whole-exome sequencing and single-cell RNA sequencing found that malignant cells in IM showed higher intratumor heterogeneity, stemness, and more activated metabolism than those in MO. Tumors from IM shared similar TMEs while distinct TMEs were noticed in those from MO. Furthermore, CD20+ B cells, plasma cells, and conventional type II dendritic cells (cDC2s) were decreased in IM relative to MO while T cells in IM exhibited a more terminally exhausted capacity with a higher proportion of proliferative/exhausted T cells than that in MO. Both CD20 and CD1C correlated with better prognosis in multifocal HCC. Additionally, MMP9+ tumor-associated macrophages were enriched across IM and MO, which formed cellular niches with regulatory T cells and proliferative/exhausted T cells. Conclusions: Our findings deeply decipher the heterogeneous TMEs between IM and MO, which provide a comprehensive landscape of multifocal HCC.