Immunogenic role of Necrosis-inducing complex RIPK1-RIPK3-MLKL-P in Hepatocellular Carcinoma

Objectives. Necroptosis (NCP) is a form of programmed cell death characterized by the lysis of cellular elements and it involves the formation of a necrosis-inducing complex consisting of the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and the mixed-lineage kinase domain-like protein (MLKL) (1). NCP is involved in promoting inflammation under pathological conditions and recent studies on NCP have shown that some cancer cells may show a preferential reduction in the expression of the key mediators of NCP, suggesting that it may negatively regulate tumorigenesis. When NCP occurs in cancer cells, dispersed elements in the tumor microenvironment may contribute to cancer development by influencing local immune response, which can be assessed by the quantification of T cell infiltration in cancer. This can be done by using a reliable and reproducible method as the immunoscore (2), that may be applied also to hepatocellular carcinoma (HCC) (3). Aim of this study was to assess expression of RIPK1, RIPK3 and phosphorylated MLKL in cohort HCC patients and their correlation with T cell infiltration and clinical follow-up data. Methods. RIPK1, RIPK3 and MLKL-p expressions were assessed by immunohistochemistry in 76 FFPE samples of resected HCC patients. Expression was evaluated on a 4-tired scale based on the percentage of positivity stain in tumor cells, where 0 indicates absent staining, 1+ indicates less than 5%, 2+ indicates positive staining between 6% to 50%, and 3+ indicates positive staining >50%. Both overall and disease-free survival analyses were performed on the patients stratified by high and low expression of the necrosis-inducing complex. Specifically, patients showing 3+ staining in at least two genes of the necrosis-inducing complex were grouped separately from the other cases. Tumor and peritumoral infiltrating CD3 and CD8 T assessment on fully digitized HCC sections and molecular quantification of RIPK1, RIPK3 and MLKL-p are in progress. Results. RIPK1 showed 0+ in 18/76 cases; 1+ in 27/76 cases; 2+ in 20/76 cases and 3+ in 11/76 with cytoplasmic localization or in the membrane. RIPK3 immunoprofile resulted in 13/76, 10/76, 11/76 and 42/76 positive cases for 0+,1+,2+ and 3+ respectively; with cytoplasmic localization. MLKL-p resulted in 35/76, 24/76, 13/76 and 4/76 positive cases respectively 0+, 1+, 2+ and 3+; with cytoplasmic localization or in the membrane (Fig. 1). Both overall and disease-free survival analyses showed significant correlation between poor prognosis and down-regulation of the necrosis-inducing complex, with log- rank test p-values 0.048 and 0.012, respectively. Conclusions. Considering our preliminary data NCP occurs in a subset of HCC patients and it could represent an easy and reliable parameter for the evaluation of HCC immunogenicity and for the evaluation of tumoral aggressiveness. These results will be compared to those obtained in larger cohorts from publicly available datasets (e.g. data from TCGA and ICGC consortia) to validate the role of NCP in the hepatocellular carcinoma.