Evaluation of necroptosis related genes RIPK1, RIPK3 and MLKL-p immunogenicity in hepatocellular carcinoma

Background & Objective: Necroptosis is a form of programmed necrosis. When necroptosis occurring in cancer, dispersed tumoural elements may contribute to boost the immune response. Receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and the mixed-lineage kinase domain-like protein (MLKL) are the main elements composing the subcellular pathway that realizes necroptosis. Aim of this study was to assess expression of RIPK1, RIPK3 and phosphorylated MLKL in a cohort of HCC patients and their correlation with infiltrating CD8+ T-cells and clinical follow-up data. Method: RIPK1, RIPK3 and MLKL-p expression was assessed with immunohistochemistry (IHC) in 83 FFPE samples of resected HCC patients. Expression was evaluated on a 4-tired scale. Tumoural and peritumoural infiltrating CD8+ T cells were automatically assessed on digitized sections. Co-localization of necroptotic factors was verified by multiplex imaging. Wilcoxon Rank Sum test and survival analysis were applied to: 1) compare the correlation between RIPK1, RIPK3, MLKL-p and their combination with T cell-infiltration; 2) evaluate the prognostic impact of these necroptotic kinases in HCC. Results were compared with those obtained from computational analysis of RNA-seq data in 373 HCC patients from TCGA. Results: RIPK1, RIPK3 and MLKL-p expression are significantly associated with tumoural but not peritumoural CD8+ T-cells infiltration (p-values < 6e-05 and > 0.4, respectively). By combining the IHC scores of the three kinases, the strength of the association with tumour infiltrating CD8+ T-cell increases (p-value 2.6e-09). Results are confirmed by TCGA RNAseq data. Conclusion: Necroptosis occurs in a subsets of HCC patients and it is correlated to the entity of infiltrating CD8+ T-cells.