Multifocal hepatocellular carcinoma (mfHCC) arises via intrahepatic metastasis (IM) or multicentric occurrence (MO), each with distinct biological behavior and clinical implications, though mfHCC origin is rarely assessed in clinical practice. We aimed to characterize the clinicopathological and molecular features of IM-HCC, MO-HCC, and their surrounding non-tumor liver (NTL) tissues using multi-omics analyses.

We analyzed 76 tumor and 44 NTL biopsies from 22 patients, using whole-exome sequencing, RNA-sequencing, and proteomic/phosphoproteomic profiling. Patients were classified as IM, MO or mixed (IM + MO) according to their somatic mutations. A comparator cohort of 48 unifocal HCC and 15 normal livers was used. Clinicopathological parameters, pathway and transcription factor activities, immune infiltration, and targetable alterations were assessed.

Clonality analysis identified 10 IM, 9 MO, and 3 mixed patients. IM-HCCs showed more frequent macrovascular invasion and extrahepatic metastases, with upregulation of E2F/MYC-related cell cycle pathways, higher expression of metastasis-associated genes (e.g., TTK, BUB1, NUF2), higher CD8 + T-cell exhaustion, and shared actionable mutations (e.g. PTEN). MO-HCCs within patients displayed molecular dissimilarity comparable to tumors from different patients, though they also showed convergent kinase and pathway dysregulation. NTLs of IM-HCC patients had lower fibrosis, extracellular matrix signaling and pro-regenerative pathways (e.g., SOX2, TGFA) than those of MO-HCC.

The aggressive molecular features and immune exhaustion of IM-HCC support the need for combined therapies, while the convergence of kinase and pathway dysregulation of MO-HCC provides unified therapeutic opportunities. Key differences in fibrogenic and regenerative pathways may influence metastatic potential. Our findings provide insights into the biological behavior and therapeutic opportunities in mfHCC.