Distinct single-cell immune ecosystems distinguish true and de novo HBV-related hepatocellular carcinoma recurrences

ObjectiveRevealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies. DesignWe performed 5'and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts. ResultsAnalyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised 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 elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells' cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients. ConclusionTrue and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.