Distinct Single-cell Immune Ecosystems Distinguish True and De Novo HBV-related Hepatocellular Carcinoma Recurrences

Summary Revealing differential tumor immune microenvironment (TIME) characteristics between true versus de novo hepatocellular carcinoma (HCC) recurrence could help optimal development and use of immunotherapies. Here, we studied the TIME of recurrent HBV-related HCCs by 5’and VDJ single-cell and bulk RNA-sequencing, flow cytometry, and multiplexed immunofluorescence. Analyses of mutational profiles, evolutionary trajectories, and clonal architecture using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The TIME of truly recurrent HCCs was characterized by an increased abundance in KLRB1 + CD8 + T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8 + T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed an upregulated GDF15 expression level on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited anti-tumor immunity in the TIME of truly recurrent lesions. In contrast, we found that myeloid cells’ crosstalk with T cells mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCC. In conclusion, our results support genomic diagnosis and immune profiling for guiding immunotherapy implementation based on the type of HCC recurrence and TIME. Highlights Truly recurrent lesions are seeded before primary tumor diagnosis, and that de novo cancer can occur earlier than the clinically used 2-year limit. ScRNA-seq unravels distinct immune ecosystems in true versus de novo HCC recurrences, highlighting the need for different immunotherapy strategies for two types of HCC recurrence. CD8 + T cells in de novo recurrence displayed cytotoxic and exhausted phenotypes while those in truly recurrent lesions showed a memory phenotype with weak cytotoxicity. HCC cells expressing the inhibitory molecule GDF15 were in the proximity of DCs only in truly recurrent lesions. High GDF15 expression level was associated with truly recurrent HCC and worse prognosis. Graphical abstract