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Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide due to high recurrence rates after curative treatment and being frequently diagnosed at an advanced stage. Immune-checkpoint inhibition (ICPI) has yielded impressive clinical successes in a variety of solid cancers, however results in treatment of HCC have been modest. Vaccination could be a promising treatment to synergize with ICPI and enhance response rates. Cancer testis antigens (CTAs) were recently discovered to be widely expressed in HCC and expression in macroscopically tumor-free tissues correlated with recurrence, implying the presence of micro-satellites. To determine whether CTAs are immunogenic in HCC patients, we analyzed systemic T-cell and humoral responses against seven CTAs in 38 HCC patients using a multitude of techniques; flowcytometry, ELISA and whole antigen and peptide stimulation assays. CTA-specific T-cells were detected in all (25/25) analyzed patients, of which most had a memory phenotype but did not exhibit unequivocal signs of chronic stimulation or recent antigen encounter. Proliferative CD4+ and CD8+ T-cell responses against these CTAs were found in 14/16 analyzed HCC patients. CTA-peptide stimulation-induced granzyme B, IL2, and TNFa in 8/8 analyzed patients, including two MAGEA1 peptides included based on in silico prediction. Finally, IgG responses were observed in 13/32 patients, albeit with low titers. The presence of CD4+ and CD8+ T-cells and IgG responses shows the immunogenicity of these CTAs in HCC-patients. We hypothesize that vaccines based on these tumor-specific antigens may boost preexisting CTA-specific immunity and could enhance therapeutic efficacy of ICPI in advanced HCC.

Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC.

Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+ lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p < 0.001), Galectin-9 (p < 0.001) and HVEM (p < 0.001), and low CD8+TIL count (p = 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29; p