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

• Main Authors: Petros Fessas, Paolo Spina, Renzo L. Boldorini, Mario Pirisi, Rosalba Minisini, Francesco A. Mauri, Fraser Simpson, Paola Olivieri, Alessandra Gennari, Ching Ngar Wong, Abdul Siddique, Robert D. Goldin, Ayse U. Akarca, Teresa Marafioti, David J. Pinato
• Format: Article
• Language: English
• Published: MDPI AG 2021-04-01
• Series: Cancers
• Subjects: hepatocellular carcinoma; intra-tumoural heterogeneity; tumour microenvironment
• Online Access: https://www.mdpi.com/2072-6694/13/9/2137

(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.