Phenotypic Characteristics of the Tumour Microenvironment in Primary and Secondary Hepatocellular Carcinoma

Abstract

(1) Background: The intra-tumoural heterogeneity (ITH) of hepatocellular carcinoma (HCC) and its microenvironment (TME) across primary and secondary disease is poorly characterised. (2) Methods: Intra-tumoural (IT) and peri-tumoural (PT) staining of matched primary and secondary samples was conducted to evaluate the distribution of CD4+/FOXP3+ and CD8+/PD1+ T-cells. Samples underwent PD-L1/2 immunostaining, tumour mutational burden (TMB) evaluation, and high-resolution T-cell receptor (TCR) sequencing to derive T-cell clonality and targeted transcriptomics. (3) Results: We analysed 24 samples from matched primary (<i>n</i> = 11) and secondary (<i>n</i> = 13; 5 synchronous, 6 metachronous) deposits, 11 being extrahepatic (84.6%). IT CD8+ density was lower than PT in both primary (<i>p</i> = 0.005) and secondary deposits (<i>p</i> = 0.01), consistent with immune exclusion. PD-L1+ tumours displayed higher IT and PT CD8+/PD1+ cell density compared to PD-L1- (<i>p</i> < 0.05), and primary IT infiltrate was enriched in CD4+/FOXP3+ cells, compared to PT regions (<i>p</i> = 0.004). TCR-sequencing demonstrated enrichment of the top T-cell clonotype in secondary versus primary HCC (<i>p</i> = 0.02), without differences in overall productive clonality (<i>p</i> = 0.35). TMB was similar across primary versus secondary HCC (<i>p</i> = 0.95). While directed gene set analysis demonstrated the uniformity of transcriptional signatures of individual immune cell types, secondary deposits demonstrated higher <i>COLEC12</i> (<i>p</i> = 0.004), <i>CCL26</i> (<i>p</i> = 0.02), <i>CD1E</i> (<i>p</i> = 0.02) and <i>CD36</i> (<i>p</i> = 0.03) expression with downregulation of <i>CXCL1</i> (<i>p</i> = 0.03), suggesting differential regulation of innate immunity. (4) Conclusion: Immune exclusion is a defining feature of the HCC TME. Despite evidence of homogeneity in somatic TMB, secondary HCC is characterised by the expansion of a distinct T-cell clonotype and differential regulation of innate immune pathways.