The influence of immune heterogeneity on the effectiveness of immune checkpoint inhibitors in multifocal hepatocellular carcinomas

Purpose Immune checkpoint inhibitor therapy is emerging as the promising option for patients with advanced hepatocellular carcinoma (HCC). We aimed to investigate the heterogeneity of different tumor nodules of the same patient with multifocal HCCs in response to immunotherapy and its molecular mechanisms. Experimental design We attained 45 surgical tumor samples including 33 small and 12 large nodules from 12 multifocal HCC patients and evaluated genomic and immune heterogeneity among tumors through whole genome sequencing (WGS) and RNA sequencing. Immunohistochemistry was performed to validate the expression of immune markers. The responses to anti-programed cell death protein-1 (PD-1) therapy in multifocal HCC patients were evaluated. Results The small and large tumors within the same patient presented with similar genomic characteristics, indicating their same genomic origin. We further found the small tumors had higher immune cell infiltration including more CD8+ T cells, M1 macrophage and monocytes as compared to large tumors. Besides, the expression of interferon signature predictive of response to anti-PD-1 therapy was significantly upregulated in the small tumors. Moreover, the immune pathways were more vigorous along with less active proliferation pathways in the small tumors. In keeping with this, we found that small nodules were more sensitive to anti-PD-1 therapy than large nodules in multifocal HCC patients. Conclusions The small tumors in multifocal HCC patients had higher immune cell infiltration and upregulation of immune pathways as compared to the large tumors, which can partially explain the different responses of small and large tumors in the same case to anti-PD-1 therapy.