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The yin and yang of evasion and immune activation in HCC

  • Oxana V. Makarova-Rusher
    Affiliations
    Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
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  • José Medina-Echeverz
    Affiliations
    Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
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  • Austin G. Duffy
    Affiliations
    Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
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  • Tim F. Greten
    Correspondence
    Corresponding author. Address: NIH/NCI/CCR Building 10 Rm 12N226, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel.: +1 (301) 451 4723; fax: +1 (301) 480 8780.
    Affiliations
    Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
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Open AccessPublished:February 28, 2015DOI:https://doi.org/10.1016/j.jhep.2015.02.038

      Summary

      Current systemic treatment options for patients with hepatocellular carcinoma (HCC) are limited to sorafenib. With the recent FDA approval of the second PD1-PD-L1 pathway inhibitor, immunotherapy has gained even more interest as a potential novel treatment option for patients with HCC. This is due not only because of the failure of other treatment approaches in the past, but also because immunological mechanisms have been shown to play an important role during tumor development, growth, and treatment. Here we present a review of immunological mechanisms in the liver relevant for tumor progression and treatment. We summarize our current knowledge on immune activating and immune suppressing mechanisms during tumor initiation, development, and treatment. We try to explain the paradox of how inflammatory responses in a setting of chronic infection promote tumor development, while the primary aim of immunotherapy is to activate immunity. Finally we summarize recent advances in addition to providing an outlook for the immunotherapy of HCC.

      Keywords

      Introduction

      Primary liver cancer, a disease of etiologic and geographic diversity, is now the second most common cause of cancer related death worldwide, with hepatocellular carcinoma (HCC) accounting for the majority of these cases [
      • Ferlay J.
      • Soerjomataram I.
      • Dikshit R.
      • et al.
      Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.
      ]. Globally, the number of deaths from HCC is close to the number of new patients diagnosed. The majority of all HCC incidence is a consequence of chronic viral infection with hepatitis B (HBV) and C (HCV) [
      • Shlomai A.
      • de Jong Y.P.
      • Rice C.M.
      Virus associated malignancies: the role of viral hepatitis in hepatocellular carcinoma.
      ] and develops in the setting of persistent immune modulation by chronic viral inflammation. The HBV vaccine, which is the first adaptive immunity intervention to prevent cancer development, was introduced worldwide in the 1980s. Since that time, a decreased incidence of HCC has been reported in Asia, where HBV-associated HCC is endemic [
      • Park N.H.
      • Chung Y.H.
      • Lee H.S.
      Impacts of vaccination on hepatitis B viral infections in Korea over a 25-year period.
      ]. In recent years, diabetes and obesity (and consecutive non-alcoholic steatohepatitis (NASH)) have been receiving growing attention as risk factors for tumor development in the US [
      • Welzel T.M.
      • Graubard B.I.
      • Quraishi S.
      • et al.
      Population-attributable fractions of risk factors for hepatocellular carcinoma in the United States.
      ]. Surgery and local ablative therapies remain the only curative treatment option while intra-arterial treatments (transarterial chemoembolization – TACE- and selective intra-arterial radiotherapy-SIRT-) and systemic chemotherapy are used in the palliative setting in patients with more advanced disease [
      • Ulahannan S.V.
      • Duffy A.G.
      • McNeel T.S.
      • et al.
      Earlier presentation and application of curative treatments in hepatocellular carcinoma.
      ]. Currently, the only FDA approved systemic treatment for HCC is sorafenib, a multikinase inhibitor. Sorafenib was established as the standard of care in advanced HCC after a phase III randomized, placebo controlled clinical trial showed a small improvement in overall survival (OS) from 7.9 months to 10.7 months [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ]. Thus there is a clear and urgent need for new therapies for this deadly disease.
      Chronic hepatic inflammatory responses are the number one risk factor for liver tumor development. At the same time, immune based approaches aimed at enhancing tumor-specific immune responses are currently under investigation as therapeutic strategies in HCC cancer research. A better understanding of how the immune system promotes tumor growth will hopefully lead to the development of more effective treatment options and will be the focus of this review (Fig. 1). In addition we will discuss currently ongoing and recent clinical trials aiming to enhance anti-tumor immunity either by inducing antigen-specific T cells or by blocking immune inhibitory mechanisms.
      Figure thumbnail gr1
      Fig. 1The counteracting forces regulate the balance between tumor progression and tumor elimination. The forces mediating tumor progression include liver tolerance to antigens, immunosuppression of chronic inflammation and HCC dependent immune tolerance. On the opposite side of the equilibrium is anti-tumor immune response, which can be enhanced by immunotherapy such as immune checkpoint blockade.

      Tolerance inducing mechanisms in the liver

      Under physiological conditions, the liver has to perform multiple tasks, including the uptake of blood borne pathogens, the excretion of toxic waste substances, and the filtration of environmental or bacterial agents from the gastrointestinal tract. These functions result in enormous antigen exposure to the liver [
      • Jacob A.I.
      • Goldberg P.K.
      • Bloom N.
      • Degenshein G.A.
      • Kozinn P.J.
      Endotoxin and bacteria in portal blood.
      ,
      • Crispe I.N.
      Hepatic T cells and liver tolerance.
      ]. To prevent organ autoimmune damage from ongoing immune stimulation by a myriad of antigens, liver evolution resulted in the development of intrinsic liver tolerogenic mechanisms in the innate and adaptive immune response [
      • Crispe I.N.
      Hepatic T cells and liver tolerance.
      ,
      • Sriwatanawongsa V.
      • Davies H.S.
      • Calne R.Y.
      The essential roles of parenchymal tissues and passenger leukocytes in the tolerance induced by liver grafting in rats.
      ,
      • Knolle P.
      • Schlaak J.
      • Uhrig A.
      • Kempf P.
      • Meyer zum Buschenfelde K.H.
      • Gerken G.
      Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge.
      ,
      • Knolle P.A.
      • Germann T.
      • Treichel U.
      • et al.
      Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells.
      ,
      • Protzer U.
      • Maini M.K.
      • Knolle P.A.
      Living in the liver: hepatic infections.
      ] (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Under physiological conditions the liver has the capacity to induce tolerance against antigens delivered from the intestine. The identified immune responses promoting liver tolerance to antigens include constitutive inhibitory cytokine release (interleukin 10 (IL-10) by Kupffer cells (KC) and tumor growth factor beta (TGF-β) by KC or liver sinusoidal endothelial cells (LSEC)), and upregulation of immune checkpoints (programmed death-ligand 1 or 2 (PD-L1 or PD-L2) on hepatocytes (HC), hepatic stellate cells (HSC), KC, LSEC and intrahepatic leucocytes (LC), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on T regulatory cells (Treg)). All of these immune responses protect the liver from autoimmune damage by blocking activation of effector T cells in the liver microenvironment. While liver immune tolerance is beneficial in case of harmless antigens in cancer-free liver, it may be detrimental with respect to HCC antigen immune escape in cancer-bearing host.
      Under physiological conditions, cells of the innate immune system such as Kupffer cells, which are resident macrophages of the liver, release interleukin (IL)-10 [
      • Knolle P.
      • Schlaak J.
      • Uhrig A.
      • Kempf P.
      • Meyer zum Buschenfelde K.H.
      • Gerken G.
      Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge.
      ] and, consequently, inhibit immune responses. Furthermore, constitutive release of another active inhibitory cytokine, transforming growth factor (TGF)-β, by endothelial liver cells and Kupffer cells has been identified [
      • Bissell D.M.
      • Wang S.S.
      • Jarnagin W.R.
      • Roll F.J.
      Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation.
      ] as a contributor to local immunosuppression. Although liver sinusoidal endothelial cells (LSECs) can promote differentiation of T cells into memory-like T cells, which give rise to effector T cells after reactivation by antigen presenting cells (APCs) [
      • Bottcher J.P.
      • Schanz O.
      • Wohlleber D.
      • et al.
      Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.
      ] and induces a rapid generation of cytotoxic T cells [
      • Bottcher J.P.
      • Schanz O.
      • Garbers C.
      • et al.
      IL-6 trans-signaling-dependent rapid development of cytotoxic CD8+ T cell function.
      ], tolerogenic liver priming by endotoxins leads to defective antigen processing by LSECs, diminishing antigen-specific immune surveillance [
      • Knolle P.A.
      • Germann T.
      • Treichel U.
      • et al.
      Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells.
      ]. Moreover, a decrease in surface expression of immune co-stimulatory molecules, B7-1 (also known as CD80) and B7-2 (a.k.a. CD86) [
      • Knolle P.A.
      • Germann T.
      • Treichel U.
      • et al.
      Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells.
      ], on LSECs limits the ability of these APCs to activate CD4+ T cells, further decreasing adaptive immune system surveillance in liver tissues.
      The B7-1 and B7-2 molecules are expressed on various types of APCs and are essential components of the B7-CD28/CTLA-4 immune checkpoint pathways. Interactions of B7-1 and B7-2 transmit co-stimulatory signals to antigen-primed T cells when they are ligated with the CD28 receptor on T cells or co-inhibitory signals when they interact with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the inhibitory checkpoint receptor on T cells, therefore enhancing or downregulating T cell activation. With respect to the immunosuppressive role of CTLA-4 in the liver, CTLA-4 expression by Foxp3+CD25+CD4+ T regulatory cells (Tregs) has been linked to the induction of host immune tolerance after liver transplantation [
      • Li W.
      • Kuhr C.S.
      • Zheng X.X.
      • et al.
      New insights into mechanisms of spontaneous liver transplant tolerance: the role of Foxp3-expressing CD25+CD4+ regulatory T cells.
      ] and, thus represents a potential organ-specific mechanism to regulate immune activation. In addition to the B7-CD28/CTLA-4 pathway, another immune checkpoint pathway, PD-L1/PD-L2/PD-1 (programmed death- ligand 1 or 2/programmed cell death 1 receptor), inhibits immune activation in the liver [
      • Muhlbauer M.
      • Fleck M.
      • Schutz C.
      • et al.
      PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis.
      ,
      • Yu M.C.
      • Chen C.H.
      • Liang X.
      • et al.
      Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice.
      ,
      • Diehl L.
      • Schurich A.
      • Grochtmann R.
      • Hegenbarth S.
      • Chen L.
      • Knolle P.A.
      Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance.
      ,
      • Kassel R.
      • Cruise M.W.
      • Iezzoni J.C.
      • Taylor N.A.
      • Pruett T.L.
      • Hahn Y.S.
      Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
      ,
      • Gu X.
      • Wang Y.
      • Xiang J.
      • et al.
      Interferon- gamma triggers hepatic stellate cell-mediated immune regulation through MEK/ERK signaling pathway.
      ]. PD-L1, expressed on hepatocytes [
      • Muhlbauer M.
      • Fleck M.
      • Schutz C.
      • et al.
      PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis.
      ], hepatic stellate cells (HSCs) [
      • Yu M.C.
      • Chen C.H.
      • Liang X.
      • et al.
      Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice.
      ], LSECs [
      • Diehl L.
      • Schurich A.
      • Grochtmann R.
      • Hegenbarth S.
      • Chen L.
      • Knolle P.A.
      Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance.
      ], and Kupffer cells [
      • Kassel R.
      • Cruise M.W.
      • Iezzoni J.C.
      • Taylor N.A.
      • Pruett T.L.
      • Hahn Y.S.
      Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
      ], contributes to mechanisms of liver immune tolerance via induction of T cell apoptosis [
      • Muhlbauer M.
      • Fleck M.
      • Schutz C.
      • et al.
      PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis.
      ,
      • Yu M.C.
      • Chen C.H.
      • Liang X.
      • et al.
      Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice.
      ,
      • Gu X.
      • Wang Y.
      • Xiang J.
      • et al.
      Interferon- gamma triggers hepatic stellate cell-mediated immune regulation through MEK/ERK signaling pathway.
      ] or T cell dysfunction [
      • Diehl L.
      • Schurich A.
      • Grochtmann R.
      • Hegenbarth S.
      • Chen L.
      • Knolle P.A.
      Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance.
      ]. Furthermore, the physiologic expression of PD-L1 as well as PD-L2 and PD-1 can be increased in chronically inflamed livers [
      • Kassel R.
      • Cruise M.W.
      • Iezzoni J.C.
      • Taylor N.A.
      • Pruett T.L.
      • Hahn Y.S.
      Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
      ]. All of these tolerogenic responses can be protective with respect to harmless antigens, yet detrimental in the case of immune tolerance to tumor-associated antigens (TAAs) and HCC progression.

      Immunosuppression of chronic inflammation

      Chronic inflammation, characterized by the continued expression of different cytokines [
      • Calabrese F.
      • Valente M.
      • Giacometti C.
      • et al.
      Parenchymal transforming growth factor beta-1: its type II receptor and Smad signaling pathway correlate with inflammation and fibrosis in chronic liver disease of viral etiology.
      ,
      • Huang A.
      • Zhang B.
      • Yan W.
      • et al.
      Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis b virus infection through PD-1-induced IL-10.
      ] and recruitment of immune cells [
      • Kassel R.
      • Cruise M.W.
      • Iezzoni J.C.
      • Taylor N.A.
      • Pruett T.L.
      • Hahn Y.S.
      Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
      ,
      • Xu D.
      • Fu J.
      • Jin L.
      • et al.
      Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B.
      ,
      • Zhang H.H.
      • Mei M.H.
      • Fei R.
      • et al.
      Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.
      ] to the liver, contributes to HCC carcinogenesis and disease progression by enhancing immune suppression and thus allowing the growth of cancer cells [
      • Zhang H.H.
      • Mei M.H.
      • Fei R.
      • et al.
      Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.
      ,
      • Matsuzaki K.
      • Murata M.
      • Yoshida K.
      • et al.
      Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.
      ]. The inhibition of antigen-specific immune surveillance in the chronic inflammatory state is mediated by changes in expression of immune checkpoints [
      • Huang A.
      • Zhang B.
      • Yan W.
      • et al.
      Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis b virus infection through PD-1-induced IL-10.
      ,
      • Nebbia G.
      • Peppa D.
      • Schurich A.
      • et al.
      Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.
      ,
      • Peng G.
      • Li S.
      • Wu W.
      • Tan X.
      • Chen Y.
      • Chen Z.
      PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients.
      ,
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ], alterations in function of dendritic cells (DCs) [
      • Murakami H.
      • Akbar S.M.
      • Matsui H.
      • Horiike N.
      • Onji M.
      Decreased interferon-alpha production and impaired T helper 1 polarization by dendritic cells from patients with chronic hepatitis C.
      ,
      • Bain C.
      • Fatmi A.
      • Zoulim F.
      • Zarski J.P.
      • Trepo C.
      • Inchauspe G.
      Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.
      ,
      • Martinet J.
      • Dufeu-Duchesne T.
      • Bruder Costa J.
      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      ], increases in the frequency of Tregs [
      • Zhang H.H.
      • Mei M.H.
      • Fei R.
      • et al.
      Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.
      ,
      • Cabrera R.
      • Tu Z.
      • Xu Y.
      • et al.
      An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.
      ], and release of cytokines with immune suppressive functions such as IL-10 and TGF-β [
      • Huang A.
      • Zhang B.
      • Yan W.
      • et al.
      Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis b virus infection through PD-1-induced IL-10.
      ,
      • Cabrera R.
      • Tu Z.
      • Xu Y.
      • et al.
      An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.
      ,
      • Liu B.S.
      • Groothuismink Z.M.
      • Janssen H.L.
      • Boonstra A.
      Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections.
      ].
      In recent years, immune inhibitory checkpoints have been increasingly recognized as important players in immunosuppression of chronic inflammation. In chronic viral and autoimmune hepatitis, upregulation of PD-1 expression on – intrahepatic lymphocytes and its ligands PD-L1 (a.k.a. B7-H1) and PD-L2 (a.k.a. B7-H2), on Kupffer cells, LSECs, and leukocytes – have been positively correlated with the degree of liver inflammation [
      • Kassel R.
      • Cruise M.W.
      • Iezzoni J.C.
      • Taylor N.A.
      • Pruett T.L.
      • Hahn Y.S.
      Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
      ]. Furthermore, the increased frequency of circulating CD8+ T cells expressing PD-1 has been associated with progression of HBV-related cirrhosis to HCC [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ]. In addition to the PD-1 [
      • Peng G.
      • Li S.
      • Wu W.
      • Tan X.
      • Chen Y.
      • Chen Z.
      PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients.
      ,
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ,
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ] upregulation, other negative T cell regulatory checkpoint receptors such as CTLA-4 [
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ] and T cell immunoglobulin domain and mucin domain-3 (Tim-3) [
      • Nebbia G.
      • Peppa D.
      • Schurich A.
      • et al.
      Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.
      ,
      • Moorman J.P.
      • Wang J.M.
      • Zhang Y.
      • et al.
      Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.
      ], have been linked to reduced T cell effector function in chronic viral hepatitis. Collectively, these data suggest that immune inhibitory checkpoints contribute to immunosuppression in the chronic inflammatory state, allowing HCC progression.
      Interestingly, studies of patients with chronic HCV infection showed that blocking PD-1/PD-L1 or B7-CD28/CTLA-4 signaling restores the capacity for expansion and function of circulating HCV-specific CD8+ T cells, but not for intrahepatic HCV-specific CD8+ T cells [
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ,
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ]. The refractoriness of intrahepatic CD8+ T cells to PD-1/PD-L1 blockage can be explained by Treg-mediated immunosuppression, upregulation of CTLA-4 in HCV-specific hepatic CD8+ PD-1high T cells [
      • Zhang H.H.
      • Mei M.H.
      • Fei R.
      • et al.
      Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.
      ] and reduced expression of co-stimulatory molecules CD28 and CD127 (IL-7R alpha) by these cells [
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ,
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ]. The exhaustion of intrahepatic CD8+ T cells has been reversed by using combined PD-1 and CTLA-4 inhibition [
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      ]. Therefore, this finding suggests that a combination of immune targeting treatments might be necessary for restoration of antigen-specific CD8+ T cell functions in chronically inflamed livers.

      HCC immune tolerance

      In tumor-bearing hosts, the strategies promoting tolerance to tumor antigens include a decrease in recognition of malignant cells and suppression of immune system function in the tumor microenvironment [
      • Schreiber R.D.
      • Old L.J.
      • Smyth M.J.
      Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion.
      ], both of which can lead to cancer progression. Although the pathways involved in cancer-induced immune tolerance have not been fully identified, available data define multiple immune responses promoting HCC progression and thus indicate promising targets for therapy aimed at restoring anti-tumor immunity. These responses include changes in the number or function of immune cells, cytokine level and immune receptor or ligand expression.
      On a cellular level, failure of HCC-associated antigen presentation by APCs as a consequence of decreased expression of HLA class-I molecules [
      • Matsui M.
      • Machida S.
      • Itani-Yohda T.
      • Akatsuka T.
      Downregulation of the proteasome subunits, transporter, and antigen presentation in hepatocellular carcinoma, and their restoration by interferon-gamma.
      ] and ineffective tumor antigen processing [
      • Kurokohchi K.
      • Carrington M.
      • Mann D.L.
      • et al.
      Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma.
      ], contributes to the inability of the immune system to recognize liver cancer. Other immune responses contributing to HCC tolerance include increases in Tregs [
      • Ormandy L.A.
      • Hillemann T.
      • Wedemeyer H.
      • Manns M.P.
      • Greten T.F.
      • Korangy F.
      Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma.
      ,
      • Unitt E.
      • Rushbrook S.M.
      • Marshall A.
      • et al.
      Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.
      ], invariant natural killer T cells (iNKT) [
      • Cariani E.
      • Pilli M.
      • Zerbini A.
      • et al.
      Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.
      ], CD14+HLA-DR−/low monocytic-like myeloid-derived suppressor cells (MDSC) [
      • Hoechst B.
      • Ormandy L.A.
      • Ballmaier M.
      • et al.
      A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
      ], and tumor-associated macrophages (TAMs) [
      • Zhu X.D.
      • Zhang J.B.
      • Zhuang P.Y.
      • et al.
      High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.
      ,
      • Yeung O.W.
      • Lo C.
      • Ling C.
      • et al.
      Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma.
      ,
      • Ding T.
      • Xu J.
      • Wang F.
      • et al.
      High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection.
      ], as well as diminished CD4+ T helper cells [
      • Behboudi S.
      • Alisa A.
      • Boswell S.
      • Anastassiou J.
      • Pathan A.A.
      • Williams R.
      Expansion of anti-AFP Th1 and Tc1 responses in hepatocellular carcinoma occur in different stages of disease.
      ]. With respect to Tregs, an increase in CD4+CD25+ Tregs within tumor infiltrating lymphocytes (TILs) has been described in association with a decrease in number and function of CD8+ T cells [
      • Ormandy L.A.
      • Hillemann T.
      • Wedemeyer H.
      • Manns M.P.
      • Greten T.F.
      • Korangy F.
      Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma.
      ,
      • Unitt E.
      • Rushbrook S.M.
      • Marshall A.
      • et al.
      Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.
      ]. This finding suggests the use of immune treatments aimed at Treg depletion as a possible therapeutic strategy for restoration of anti-tumor immune responses. Furthermore, an increase in Treg numbers either in peripheral circulation or within TILs has been associated with decreased OS [
      • Gao Q.
      • Qiu S.J.
      • Fan J.
      • et al.
      Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.
      ,
      • Fu J.
      • Xu D.
      • Liu Z.
      • et al.
      Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients.
      ], and thus can be further explored as an immune prognostic marker in biomarker development.
      Different cytokines in association with a unique innate immunity signature within the tumor microenvironment have been reported to promote the development of HCC metastases. This poor prognostic signature includes an increase in immunosuppressive cytokines (IL-4, IL-5, IL-8, and IL-10) accompanied by suppression of immune activating cytokines (IL-1, tumor necrosis factor (TNF), and interferon gamma (IFN-γ)) [
      • Budhu A.
      • Forgues M.
      • Ye Q.H.
      • et al.
      Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment.
      ]. Furthermore, increased IL-10 levels in patients with advanced HCC have been associated with shorter OS and immune dysfunction, such as diminished activity of lymphokine-activated killer (LAK) and natural killer (NK) cells [
      • Hattori E.
      • Okumoto K.
      • Adachi T.
      • et al.
      Possible contribution of circulating interleukin-10 (IL-10) to anti-tumor immunity and prognosis in patients with unresectable hepatocellular carcinoma.
      ].
      On a receptor and ligand level, HCC immune tolerance is mediated by immunosuppression via decreased co-stimulatory or increased inhibitory checkpoint signaling. With respect to co-stimulatory molecules, significant reductions of B7-1 and B7-2 (immune co-stimulatory ligands) expression have been identified on HCC cells [
      • Fujiwara K.
      • Higashi T.
      • Nouso K.
      • et al.
      Decreased expression of B7 costimulatory molecules and major histocompatibility complex class-I in human hepatocellular carcinoma.
      ,
      • Tatsumi T.
      • Takehara T.
      • Katayama K.
      • et al.
      Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma.
      ] resulting in a decrease of B7/CD28 mediated activation of effector T cells. Furthermore, several other immune co-stimulatory molecules such as CD244 (2B4) [
      • Wu Y.
      • Kuang D.M.
      • Pan W.D.
      • et al.
      Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.
      ], CD28 [
      • Hsu P.N.
      • Yang T.C.
      • Kao J.T.
      • et al.
      Increased PD-1 and decreased CD28 expression in chronic hepatitis B patients with advanced hepatocellular carcinoma.
      ], CD40 [
      • Sugimoto K.
      • Shiraki K.
      • Ito T.
      • et al.
      Expression of functional CD40 in human hepatocellular carcinoma.
      ], CD137 (4-1BB) [
      • Morales-Kastresana A.
      • Sanmamed M.F.
      • Rodriguez I.
      • et al.
      Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
      ], and OX-40 [
      • Morales-Kastresana A.
      • Sanmamed M.F.
      • Rodriguez I.
      • et al.
      Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
      ] have been studied in liver cancer and will serve as targets for immune agonist antibodies in further clinical development.
      Immune inhibitory receptors and ligands also play a major role in induction and maintenance of HCC immune tolerance [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ,
      • Wu K.
      • Kryczek I.
      • Chen L.
      • Zou W.
      • Welling T.H.
      Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
      ,
      • Okazaki T.
      • Honjo T.
      The PD-1-PD-L pathway in immunological tolerance.
      ,
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      ,
      • Han Y.
      • Chen Z.
      • Yang Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      ,
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.
      ,
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ,
      • Li F.J.
      • Zhang Y.
      • Jin G.X.
      • Yao L.
      • Wu D.Q.
      Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.
      ,
      • Li H.
      • Wu K.
      • Tao K.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      ]. Several inhibitory checkpoints have been associated with immune dysfunction in HCC including CTLA-4 [
      • Han Y.
      • Chen Z.
      • Yang Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      ,
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.
      ,
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ], PD-1 and its ligand (PD-L1) [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ,
      • Wu K.
      • Kryczek I.
      • Chen L.
      • Zou W.
      • Welling T.H.
      Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
      ,
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      ], lymphocyte-activation gene 3 (LAG-3) [
      • Li F.J.
      • Zhang Y.
      • Jin G.X.
      • Yao L.
      • Wu D.Q.
      Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.
      ], Tim-3 and its ligand (galectin-9) [
      • Li H.
      • Wu K.
      • Tao K.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      ], and adenosine A2a receptor (A2aR) [
      • Stemmer S.M.
      • Benjaminov O.
      • Medalia G.
      • et al.
      CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.
      ]. In HCC patients, high CTLA-4 expression on Tregs in peripheral blood has been reported in association with a decrease in cytolytic granzyme B production by CD8+ T cells [
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.
      ]. In addition, CTLA-4 expression on CD14+DCs was associated with IL-10 and indoleamine-2,3-dioxygenase (IDO)-mediated inhibition of T cell proliferation and induction of T cell apoptosis [
      • Han Y.
      • Chen Z.
      • Yang Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      ].
      Another mechanism of tumor-induced immune tolerance is mediated by changes in the PD-L1/PD-1 immune checkpoint pathway. The increased expression of PD-1 has been reported on CD8+ T cells in patients with HCC [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ,
      • Wu K.
      • Kryczek I.
      • Chen L.
      • Zou W.
      • Welling T.H.
      Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
      ], as well as an increase in tumor infiltrating and circulating PD-1+CD8+ T cells associated with disease progression after curative hepatic resection [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ]. In addition to the upregulation of PD-1 on T cells, its ligand, PD-L1, is highly expressed on peritumoral stroma cells (Kupffer cells, LSECs, and monocytes) as well as cancer cells, promoting a PD-L1/PD-1 pathway-driven inhibition of anti-tumor T cell responses [
      • Wu K.
      • Kryczek I.
      • Chen L.
      • Zou W.
      • Welling T.H.
      Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
      ,
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      ,
      • Wang B.J.
      • Bao J.J.
      • Wang J.Z.
      • et al.
      Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma.
      ,
      • Gao Q.
      • Wang X.Y.
      • Qiu S.J.
      • et al.
      Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma.
      ]. This new understanding of increased PD-1/PD-L1 expression provides the rationale for the use of PD-1 and PD-L1 immune checkpoint blocking antibodies in HCC treatment.
      However, because HCC-induced immune tolerance in the setting of a tolerogenic liver environment and chronic inflammation is associated with multiple immunosuppressive mechanisms, dual or triple combinations of immune targeting agents along with inhibitory checkpoint blockage as a backbone of therapy are the most promising strategies for clinical development.

      Spontaneous tumor-specific immune responses

      Despite multiple immunosuppressive alterations, the majority of liver cancer patients mount detectable adaptive immune responses against tumor antigens [
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ,
      • Thimme R.
      • Neagu M.
      • Boettler T.
      • et al.
      Comprehensive analysis of the alpha-fetoprotein-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
      ]. The detected TAAs against which HCC-specific CD8+ T cells have been identified include alpha-fetoprotein (AFP) [
      • Thimme R.
      • Neagu M.
      • Boettler T.
      • et al.
      Comprehensive analysis of the alpha-fetoprotein-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
      ,
      • Butterfield L.H.
      • Ribas A.
      • Meng W.S.
      • et al.
      T-cell responses to HLA-A∗0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.
      ], highly immunogenic cancer-testis antigen NY-ESO-136 [
      • Bricard G.
      • Bouzourene H.
      • Martinet O.
      • et al.
      Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
      ,
      • Gehring A.J.
      • Ho Z.Z.
      • Tan A.T.
      • et al.
      Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.
      ,
      • Korangy F.
      • Ormandy L.A.
      • Bleck J.S.
      • et al.
      Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-1 in hepatocellular carcinoma.
      ], other cancer-testis antigens such as synovial sarcoma X breakpoint 2 (SSX-2) [
      • Bricard G.
      • Bouzourene H.
      • Martinet O.
      • et al.
      Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
      ,
      • Gehring A.J.
      • Ho Z.Z.
      • Tan A.T.
      • et al.
      Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.
      ], catalytic enzyme telomerase reverse transcriptase (TERT) [
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ,
      • Mizukoshi E.
      • Nakamoto Y.
      • Marukawa Y.
      • et al.
      Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma.
      ], cyclophilin B40 [
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ], melanoma antigen gene-A (MAGE-A) [
      • Bricard G.
      • Bouzourene H.
      • Martinet O.
      • et al.
      Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
      ], and fetal oncoprotein glypican-3 (GPC3) [
      • Hiroishi K.
      • Eguchi J.
      • Baba T.
      • et al.
      Strong CD8(+) T-cell responses against tumor-associated antigens prolong the recurrence-free interval after tumor treatment in patients with hepatocellular carcinoma.
      ,
      • Komori H.
      • Nakatsura T.
      • Senju S.
      • et al.
      Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma.
      ]. Generally, in vitro antigen-specific stimulation is required to detect these T cells indicating that T cells only exist at very low frequencies. As a promising treatment strategy, enhancement of TAA-directed immune responses by vaccines and immune checkpoint combinations may result in HCC growth control and tumor regression. In addition to immune responses against classical TAAs, spontaneous immune responses against neo-antigens which are specific for individual tumors and important for immunotherapy have been identified in patients with melanoma [
      • van Rooij N.
      • van Buuren M.M.
      • Philips D.
      • et al.
      Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma.
      ,
      • Robbins P.F.
      • Lu Y.C.
      • El-Gamil M.
      • et al.
      Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
      ]. In future, the identification of tumor-specific mutated epitopes inducing immune responses in patients with HCC might provide additional targets for immunotherapy.
      Other immune responses, which can serve as targets for enhancement by immunotherapy, include an increase in TILs and cytotoxic NKs. The presence of CD4+ and CD8+ TILs has been associated with a decrease in HCC recurrence after liver transplantation or hepatic resection [
      • Cariani E.
      • Pilli M.
      • Zerbini A.
      • et al.
      Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.
      ,
      • Unitt E.
      • Marshall A.
      • Gelson W.
      • et al.
      Tumour lymphocytic infiltrate and recurrence of hepatocellular carcinoma following liver transplantation.
      ]. In addition, after hepatic resection or radiofrequency ablation (RFA), the detection of cytotoxic (CD56dim) or activating killer immunoglobulin-like receptor (KIR2DS5) NK cell phenotypes has been correlated with improved OS [
      • Cariani E.
      • Pilli M.
      • Zerbini A.
      • et al.
      HLA and killer immunoglobulin-like receptor genes as outcome predictors of hepatitis C virus-related hepatocellular carcinoma.
      ]. Therefore, therapies aimed at increasing TILs or cytotoxic NK cells could improve outcomes after potentially curative surgery.
      Collectively, all of these facts suggest that an HCC-bearing host harbors multiple immune regulatory changes that support tumor tolerance and disease progression rather than anti-tumor immunity and cancer elimination. The identification of immune responses that affect the equilibrium between primary liver cancer progression and tumor elimination provides the targets for HCC immunotherapy development (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Immune status of an HCC-bearing host is characterized by different immune responses leading to immunity or immune tolerance and thereby promoting tumor cell death or growth, respectively. Unfortunately, multiple immune alterations associated with carcinogenesis and disease progression shift the immune response towards tumor immune tolerance and disease progression. Treatment approaches such as immune checkpoint blockers can bring the balance back towards immunity and cancer cell death.

      Clinical trials

      Multiple immune targeted therapeutic strategies aimed at inducing or enhancing anti-tumor immunity against HCC have been evaluated in clinical trials. Most of the agents have been investigated as single agent therapies and are in early phases of clinical trial development (Table 1).
      Table 1Clinical trials of immune targeted therapies in hepatocellular carcinoma.
      IFN-interferon (randomized clinical trials with more than 50 patients enrolled), RBV-ribavirin, Ad.IL-12-adenoviral vector encoding human interleukin-12 genes, GI-gastrointestinal, TβRI inhibitor-transforming growth factor-β receptor I inhibitor, TACE-transarterial chemoembolization, RFA-radiofrequency ablation, AdV-AFP-expressing replication-deficient adenovirus, A(3)AR agonist-A3 adenosine receptor agonist, RR-recurrence rate, RFS-recurrence free survival, PR-partial response, SD stable disease, OS-overall survival, ds-days, ws-weeks, ms-months, yr-year, QW-every week, QM-every month, no difference no statistically significant difference, N/A-not available, G3-CTL-glypican-3-specifc cytotoxic T lymphocytes.

      Vaccines

      For more than a decade, vaccines targeting HCC TAAs have been investigated in early clinical trials with a mixed history of success and failure [
      • Lee W.C.
      • Wang H.C.
      • Hung C.F.
      • Huang P.F.
      • Lia C.R.
      • Chen M.F.
      Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial.
      ,
      • Butterfield L.H.
      • Ribas A.
      • Meng W.S.
      • et al.
      T-cell responses to HLA-A∗0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.
      ,
      • Tada F.
      • Abe M.
      • Hirooka M.
      • et al.
      Phase I/II study of immunotherapy using tumor antigen-pulsed dendritic cells in patients with hepatocellular carcinoma.
      ,
      • Greten T.F.
      • Forner A.
      • Korangy F.
      • et al.
      A phase II open label trial evaluating safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma.
      ,
      • Palmer D.H.
      • Midgley R.S.
      • Mirza N.
      • et al.
      A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma.
      ]. Approximately two thirds of patients with HCC have been reported to have detectable immune responses to one or more TAAs [
      • Mizukoshi E.
      • Nakamoto Y.
      • Arai K.
      • et al.
      Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
      ]. These findings support vaccine treatments as an attractive strategy for therapeutic development. AFP was the first TAA to be targeted in clinic for HCC treatment. AFP is a self-antigen normally present in fetal development, therefore AFP is well-tolerated by the host immune system. Different modalities have been used to overcome AFP immune tolerance and induce immune responses in clinical vaccination settings. In 2003, the first HCC vaccine clinical trial reported detectable T cell responses to AFP after HLA-A∗0201 patients with AFP-positive HCC were immunized with a vaccine to four HLA-A∗0201-restricted AFP peptides [
      • Butterfield L.H.
      • Ribas A.
      • Meng W.S.
      • et al.
      T-cell responses to HLA-A∗0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.
      ]. Subsequently, a phase I/II clinical trial was completed with only transient immunologic responses detected, possibly due to the limited number of antigens used or deficient CD4+ helper T cell support [
      • Butterfield L.H.
      • Ribas A.
      • Meng W.S.
      • et al.
      T-cell responses to HLA-A∗0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.
      ]. In an attempt to overcome these limitations, a phase II clinical trial used DCs pulsed with lysates of a hepatoblastoma cell line containing multiple antigens. Of 25 patients receiving 3 or more DC infusions, disease control rate was 28% (1 partial response, 6 stable disease) and disease stabilization time 6–16 months. However, a short median survival of only 168 days was reported. Notably, detectable antigen-specific T cell immune responses were associated with clinical responses and a fall in AFP expression [
      • Palmer D.H.
      • Midgley R.S.
      • Mirza N.
      • et al.
      A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma.
      ]. Better clinical outcomes were observed in another HCC vaccine trial, whereby DCs pulsed with autologous tumor lysates were administered in two different treatment strategies: 5 courses of DCs weekly or 5 induction treatments followed by monthly boost vaccination maintenance. Reported outcomes among 31 treated patients were: 12.9% partial response, 54.8% stable disease and overall 1 year survival rate of 10.7% (±9.4) vs. 63.3% (±12.0) favoring the maintenance treatment schedule [
      • Lee W.C.
      • Wang H.C.
      • Hung C.F.
      • Huang P.F.
      • Lia C.R.
      • Chen M.F.
      Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial.
      ]. A cell-free vaccine platform was tested in the adjuvant settings when two patients were vaccinated with plasmid DNA followed by AFP-expressing replication-deficient adenovirus injection. AFP-specific T cell responses were observed [
      • Butterfield L.H.
      • Economou J.S.
      • Gamblin T.C.
      • Geller D.A.
      Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients.
      ], but further studies are clearly needed to verify the clinical efficacy of this approach. Lastly, telomerase reverse transcriptase peptide vaccines were tested in patients with advanced HCC. In a phase II trial of GV1001, the immunomodulatory effects of low-dose cyclophosphamide and GM-CSF were used for immune sensitization. In 40 patients treated with this combination, no radiological responses or detectable GV1001-specific immune responses were reported, although an immune response such as a decrease in the number of CD4+CD25+Foxp3+ Tregs was detected [
      • Greten T.F.
      • Forner A.
      • Korangy F.
      • et al.
      A phase II open label trial evaluating safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma.
      ]. In another trial HLA-A∗24:02-restricted GPC3298–306 or HLA-A∗02:01-restricted GPC3144–152 peptides were tested and demonstrated a promising outcome: detectable GPC3-specific CTL response in 30 out of 33 patients and a positive correlation of GPC3-specific CTL with survival [
      • Sawada Y.
      • Yoshikawa T.
      • Nobuoka D.
      • et al.
      Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival.
      ].
      These results showed the potential of HCC vaccines to elicit immunological and clinical anti-tumor responses. New and emerging preclinical and clinical evidence can guide the design of effective treatment strategies with the aim of augmenting these responses and translating the serologic anti-tumor immune activation seen into significant clinical benefit. In the recent study, detection of T cell responses against multiple TAA was associated with superior tumor growth control [
      • Flecken T.
      • Schmidt N.
      • Hild S.
      • et al.
      Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma.
      ]. Therefore, the development of multi-antigen vaccines may have the potential to improve the modest clinical efficacy of vaccination observed in clinical trials evaluating single-antigen vaccines. Importantly, the B7-CD28/CLTA-4 immune checkpoint pathway was described as inhibiting CD4+ T cell proliferation induced by DCs in response to a specific antigen [
      • Laurent S.
      • Carrega P.
      • Saverino D.
      • et al.
      CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.
      ]. Furthermore, B7-CD28/CTLA-4 ligation increases IL-10 production by DCs [
      • Laurent S.
      • Carrega P.
      • Saverino D.
      • et al.
      CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.
      ], which is the inhibitory cytokine capable of strongly downregulating the antigen presenting function of human monocytes [
      • de Waal Malefyt R.
      • Haanen J.
      • Spits H.
      • et al.
      Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.
      ]. These antigen-masking capacities of immune checkpoints provide a preclinical rational for vaccine and immune checkpoint inhibitor combinations to achieve enhanced TAA-specific immune activation. Among other future strategies, computer-guided HCC epitope-optimization [
      • Hong Y.
      • Peng Y.
      • Guo Z.S.
      • et al.
      Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.
      ], which increases immunogenicity of known TAAs by biomedical engineering, can be used to enhance anti-tumor immune responses. To further maximize clinical benefit, epitope-optimized vaccines can be combined with immune checkpoints targeting antibodies. These combinations have the potential to achieve a synergistic immunostimulatory effect thereby providing more meaningful clinical benefits of vaccine therapies.

      Adoptive cell transfer

      Adoptive cell transfer (ACT) is an autologous infusion of ex-vivo selected, activated and expanded TILs, which are extracted from patient’s tumor or from peripheral blood. ACT has been investigated in adjuvant settings for HCC treatment. In a randomized clinical trial, 76 out of 150 patients who had undergone curative resection received adjuvant activated autologous lymphocyte infusions. The untreated cohort of 74 patients was considered as the control group. Although an improvement in recurrence-free outcomes was observed, no significant difference in OS was reported between treated and control groups [
      • Takayama T.
      • Sekine T.
      • Makuuchi M.
      • et al.
      Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial.
      ]. Different strategies have been applied to augment ACT treatment clinical benefits such as systemic administration of cytokines [
      • Rosenberg S.A.
      • Lotze M.T.
      • Muul L.M.
      • et al.
      Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer.
      ] or vaccination [
      • Shimizu K.
      • Kotera Y.
      • Aruga A.
      • et al.
      Postoperative dendritic cell vaccine plus activated T-cell transfer improves the survival of patients with invasive hepatocellular carcinoma.
      ]. In a recently published clinical trial, patients with HCC after curative resection were treated with an autologous tumor lysate-pulsed DC vaccine and activated T cell transfer (ATVAC) combination. Patient outcomes after surgery alone (52 patients) were compared with combination treatment (42 patients) and an impressive difference was reported in OS, 41.0 months vs. 97.7 months, respectively [
      • Shimizu K.
      • Kotera Y.
      • Aruga A.
      • et al.
      Postoperative dendritic cell vaccine plus activated T-cell transfer improves the survival of patients with invasive hepatocellular carcinoma.
      ]. This significant clinical benefit provided by a dual immunotherapy combination supports a combined modality approach in the development of new adaptive immunity targeted therapies.

      Cytokines

      Innate immunity treatments with cytokines have been extensively studied for treatment of HCC. Interferon (IFN), which is used in the treatment of hepatitis C, was a logical choice for HCC treatment development for its possible dual antiviral and anti-tumor action. Multiple randomized clinical trials of different schedules and classes of IFN have been completed with mixed results reported with regard to recurrence-free and survival outcomes [
      • Kubo S.
      • Nishiguchi S.
      • Hirohashi K.
      • et al.
      Effects of long-term postoperative interferon-alpha therapy on intrahepatic recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. A randomized, controlled trial.
      ,
      • Llovet J.M.
      • Sala M.
      • Castells L.
      • et al.
      Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma.
      ,
      • Shiratori Y.
      • Shiina S.
      • Teratani T.
      • et al.
      Interferon therapy after tumor ablation improves prognosis in patients with hepatocellular carcinoma associated with hepatitis C virus.
      ,
      • Mazzaferro V.
      • Romito R.
      • Schiavo M.
      • et al.
      Prevention of hepatocellular carcinoma recurrence with alpha-interferon after liver resection in HCV cirrhosis.
      ,
      • Sun H.C.
      • Tang Z.Y.
      • Wang L.
      • et al.
      Postoperative interferon alpha treatment postponed recurrence and improved overall survival in patients after curative resection of HBV-related hepatocellular carcinoma: a randomized clinical trial.
      ,
      • Lo C.M.
      • Liu C.L.
      • Chan S.C.
      • et al.
      A randomized, controlled trial of postoperative adjuvant interferon therapy after resection of hepatocellular carcinoma.
      ,
      • Chen L.T.
      • Chen M.F.
      • Li L.A.
      • et al.
      Long-term results of a randomized, observation-controlled, phase III trial of adjuvant interferon Alfa-2b in hepatocellular carcinoma after curative resection.
      ,
      • Ishikawa T.
      • Higuchi K.
      • Kubota T.
      • et al.
      Combination PEG-IFN a-2b/ribavirin therapy following treatment of hepatitis C virus-associated hepatocellular carcinoma is capable of improving hepatic functional reserve and survival.
      ]. Although IFN treatment has the potential to benefit selected patients with HCC, it comes at the expense of a poor side effect profile such as flu-like symptoms (chills, fever, headache, myalgia, malaise), myelosuppression, depression, and hepatotoxicity. One possibility to overcome these effects may be an intratumoral application, which has been achieved using an adenovirus-based approach or transduced dendritic cells [
      • Sangro B.
      • Mazzolini G.
      • Ruiz J.
      • et al.
      Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors.
      ,
      • Mazzolini G.
      • Alfaro C.
      • Sangro B.
      • et al.
      Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas.
      ].
      Among new promising cytokine targeting approaches, a novel small molecule inhibitor of TGF-β receptor I, LY2157299, is under active investigation for HCC. Preliminary results of LY2157299 therapy were reported in a phase II clinical trial, where 109 were enrolled and a median OS of 36 weeks (93.1 weeks in AFP responders vs. 29.6 weeks in non-AFP responders) was observed [
      • Faivre S.J.
      • Santoro A.
      • Kelley R.K.
      • et al.
      A phase 2 study of a novel transforming growth factor-beta (TGF-β1) receptor I kinase inhibitor, LY2157299 monohydrate (LY), in patients with advanced hepatocellular carcinoma (HCC).
      ]. Currently, LY2157299 is being evaluated in a phase II, non-randomized trial as a single agent or in combination with sorafenib (NCT01246986).

      Immune checkpoint inhibitors

      To date, all immune checkpoint targeted therapies consist of monoclonal antibodies developed for a specific immune target. Although several immune checkpoint blocking agents were described in preclinical models, the majority of clinically developed therapies are antibodies that target PD-1, PD-L1 and CTLA-4 molecules. Among those, ipilimumab (anti-CTLA-4) in 2011 as well as pembrolizumab and nivolumab (anti-PD-1) in 2014 were approved by the FDA for the treatment of melanoma. Among CTLA-4 targeted therapies, only tremelimumab, a fully human IgG2 monoclonal antibody, has been clinically evaluated in HCC. A phase II, non-controlled, multicenter tremelimumab clinical trial enrolled patients with HCC and chronic HCV who were not eligible for surgery or locoregional therapy. In this trial, 21 patients with advanced HCC and Child-Pugh scores A (57.1%) or B (42.9%) were enrolled. Each treated patient received 15 mg/kg tremelimumab every 90 days as a single agent therapy. The trial showed a partial response rate of 17.6%, disease control rate of 76.4% and median OS of 8.2 months. Although 45% of patients experienced above grade 3 transaminase toxicity, the transaminitis was transient, only being observed with the first tremelimumab dose and without the need for systemic steroids. Notably, tremelimumab induced a progressive decrease in viral load in most of the patients and a complete viral response in 3 patients [
      • Sangro B.
      • Gomez-Martin C.
      • de la Mata M.
      • et al.
      A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C.
      ]. These results indicate a dual effect of anti-tumor and antiviral activity of tremelimumab, suggesting that immune checkpoint treatment can be particularly beneficial in patients with a viral etiology such as HBV or HCV-related HCC. Currently, we are testing tremelimumab in combination with local therapies (TACE or RFA) in a pilot phase I/II clinical trial [
      • Duffy A.G.
      • Ulahannan S.V.
      • Fioravanti S.
      • et al.
      A pilot study of tremelimumab, a monoclonal antibody against CTLA-4, in combination with either transcatheter arterial chemoembolization (TACE) or radiofrequency ablation (RFA) in patients with hepatocellular carcinoma (HCC).
      ].
      Among PD-1/PD-L1 targeted treatments, nivolumab (BMS-936558), which is a fully human IgG4 monoclonal antibody targeting PD-1 receptor, is under active investigation in a phase I dose escalation clinical trial [
      • Sangro B.
      • Crocenzi T.S.
      • Welling T.H.
      • et al.
      Phase I dose escalation study of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in patients (pts) with advanced hepatocellular carcinoma (HCC) with or without chronic viral hepatitis.
      ]. This trial was designed to evaluate safety and preliminary activity of nivolumab in patients with HCC with or without HBV or HCV infection [
      • Sangro B.
      • Crocenzi T.S.
      • Welling T.H.
      • et al.
      Phase I dose escalation study of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in patients (pts) with advanced hepatocellular carcinoma (HCC) with or without chronic viral hepatitis.
      ]. Additionally, another anti-PD-1 monoclonal antibody pidilizumab (CT-011) was evaluated in phase I clinical trial (NCT00966251). Unfortunately, the trial was terminated early because of slow accrual without reporting any collected results. Although other immune checkpoint antibodies including LAG3, TIM-3 and NK-inhibitory receptors were described to have antineoplastic activity in preclinical models, their clinical efficacy in patients with HCC has not yet been reported [
      • Li F.J.
      • Zhang Y.
      • Jin G.X.
      • Yao L.
      • Wu D.Q.
      Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.
      ,
      • Li H.
      • Wu K.
      • Tao K.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      ]. Furthermore, preclinical studies of other immune modulatory molecules such as CD244 (2B4) [
      • Wu Y.
      • Kuang D.M.
      • Pan W.D.
      • et al.
      Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.
      ], CD137 (4-1BB) [
      • Morales-Kastresana A.
      • Sanmamed M.F.
      • Rodriguez I.
      • et al.
      Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
      ], and OX-40 [
      • Morales-Kastresana A.
      • Sanmamed M.F.
      • Rodriguez I.
      • et al.
      Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
      ] identified another area for further clinical development of immune agonist treatments.

      Other immune based approaches

      Several other immune targeted agents have been evaluated in phase I/II clinical trials. Treatment with lenalidomide, an immune modulator, was investigated in patients with advanced HCC. A phase II trial testing the efficacy of lenalidomide after sorafenib failure reported a partial response of 15% and OS 7.6 months in patients with advanced HCC [
      • Safran H.
      • Charpentier K.P.
      • Kaubisch A.
      • et al.
      Lenalidomide for second-line treatment of advanced hepatocellular cancer: a Brown University Oncology Group Phase II Study.
      ]. Another phase II trial evaluated the efficacy of a monoclonal antibody against glypican 3, which is an oncofetal protein expressed in liver cancer cells. In this trial, patients with advanced HCC were randomized in a 2:1 ratio to anti-glypican antibody (GC33) vs. placebo. A total of 185 patients were treated with no difference in progression free survival or OS observed between the two groups. Notably, analysis of drug exposure revealed that high GC33 exposure had a favorable OS relative to placebo, 9.7 vs. 6.7. Therefore, the investigators concluded that a suboptimal dose of GC33 was potentially responsible for the lack of treatment benefit [
      • Yen C.J.
      • Daniel B.
      • Kudo M.
      Randomized phase II trial of intravenous RO5137382/GC33 at 1600 mg every other week and placebo in previously treated patients with unresectable hepatocellular carcinoma (HCC; NCT01507168).
      ]. In addition, safety data has been reported for several immune targeted therapies in HCC treatment, namely mapatumumab, an agonist to tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) [
      • Sun W.
      • Nelson D.
      • Alberts S.R.
      • et al.
      Phase Ib study of mapatumumab in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC) and chronic viral hepatitis.
      ], tigatuzumab (CS-1008), a death receptor 5 agonist [
      • Saleh M.N.
      • Percent I.
      • Wood T.E.
      • et al.
      A phase I study of CS-1008 (humanized monoclonal antibody targeting death receptor 5 or DR5), administered weekly to patients with advanced solid tumors or lymphomas.
      ], and CF102, an A(3) adenosine receptor agonist [
      • Stemmer S.M.
      • Benjaminov O.
      • Medalia G.
      • et al.
      CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.
      ]. These early investigations reported good tolerability justifying their further development.

      Future perspectives: biomarkers, combinations and outcome assessment

      Novel opportunities have opened for HCC therapy development after the reported significant clinical benefits of immune checkpoint blockade not only in traditionally highly immunogenic cancers such as melanoma, but also in other solid tumors. As part of effective HCC immune therapeutic strategies, biomarker identification, combination of immune checkpoint targeting treatment with known or new immune modulating therapies, and an effective outcome assessment criteria warrant further investigations.
      Detection of biomarkers to guide treatment selection can improve patient care by predicting potential responders to particular immunotherapies. The use of biomarkers offers a promise of personalized immune treatment combinations by defining which immune pathway is the driver in each particular patient and, thus, by identifying the optimal therapeutic target. However, the dynamic expression and heterogeneous geographic distribution of immune molecules present challenges for biomarker development. These characteristics can lead to errors in patient selection if patients are excluded from potentially beneficial treatments based on immune receptors or ligand presence in a specific time and place. Therefore, the identification of immune molecules which are more static in expression or use alterations in specific immune components such as immunomodulatory cytokines (IL-4, IL-10, TGF-β), immunomodulatory cells (TILs, circulating Treg, TAA-specific CD8+ effector T cells) as surrogate biomarkers could be crucial in successful immune biomarker development.
      Although immune-directed treatments such as cytokines, vaccines, and activated immune cell infusions have been investigated for HCC treatment, low clinical responses were reported after single immune agent therapy, most likely because multiple immune pathways are involved in HCC progression in the setting of an immune tolerogenic liver microenvironment. Typically, HCC-bearing host immune status is characterized by maintenance of tumor immune tolerance in a setting of chronic inflammation with a major input from several immune inhibitory checkpoints (PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3) [
      • Shi F.
      • Shi M.
      • Zeng Z.
      • et al.
      PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
      ,
      • Wu K.
      • Kryczek I.
      • Chen L.
      • Zou W.
      • Welling T.H.
      Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
      ,
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      ,
      • Han Y.
      • Chen Z.
      • Yang Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      ,
      • Li F.J.
      • Zhang Y.
      • Jin G.X.
      • Yao L.
      • Wu D.Q.
      Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.
      ,
      • Li H.
      • Wu K.
      • Tao K.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      ,
      • Wang B.J.
      • Bao J.J.
      • Wang J.Z.
      • et al.
      Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma.
      ]. In addition, physiologic liver immune tolerance to antigens, which among other immune alternations is characterized by the upregulation of immune checkpoints, plays a contributory role in HCC progression and resistance to immunotherapy.
      To overcome these dual liver and tumor immune tolerance phenomena, the use of combination immunotherapies with inhibitory checkpoint blockage offers the greatest promise. Immune checkpoint pathway-targeting agents combined with the previously investigated immune therapies may provide an additional boost to immune system activation and give a “second chance” to already known immune targeted treatments. Furthermore, the development of immunotherapy in adjuvant settings, i.e. after TACE procedure, can augment conventional treatment benefits. Additionally, double or triple immune checkpoint combinations with new immune targets can be used for anti-tumor immune response induction or enhancement. In future investigations, simultaneous treatments with most clinically developed immune checkpoint targeting therapies (anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies), or combinations of checkpoint blockage with antibodies to co-stimulatory (CD-137, OX-40) or co-inhibitory signals (TIM-3, LAG-3, NK-inhibitory receptors), new epitope-optimized vaccines [
      • Hong Y.
      • Peng Y.
      • Guo Z.S.
      • et al.
      Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.
      ], or novel cytokine therapies [
      • Serova M.
      • Tijeras-Raballand A.
      • Dos Santos C.
      • et al.
      Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen.
      ] are potential strategies for successful treatment development. Another strategy to potentiate immune responses is the combination of immunostimulatory drugs with RFA. This rationale is supported by data showing RFA-induced increase in TAAs-specific T cell responses [
      • Mizukoshi E.
      • Yamashita T.
      • Arai K.
      • et al.
      Enhancement of tumor-associated antigen-specific T cell responses by radiofrequency ablation of hepatocellular carcinoma.
      ]. In contrast to RFA, combining immunotherapy with sorafenib is complicated by the notion that sorafenib has been associated with immunosuppressive effects such as inhibition of NK function [
      • Krusch M.
      • Salih J.
      • Schlicke M.
      • et al.
      The kinase inhibitors sunitinib and sorafenib differentially affect NK cell antitumor reactivity in vitro.
      ], increase in MDCS [
      • Chen Y.
      • Huang Y.
      • Reiberger T.
      • et al.
      Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice.
      ], intratumoral accumulation of Tregs and M2-types macrophages, and upregulation of PD-L1 expression [
      • Chen Y.
      • Ramjiawan R.R.
      • Reiberger T.
      • et al.
      CXCR4 inhibition in tumor microenvironment facilitates anti-PD-1 immunotherapy in sorafenib-treated HCC in mice.
      ].
      Finally, a unique feature of the immune tumor response that brings additional challenges in immune treatment development is the potential to induce a local immune inflammatory reaction. This reaction can appear as an initial increase in size or enhancement of HCC lesions on magnetic resonance imaging or computed tomography before tumor size regression or resolution of enhancement. This phenomenon dictates usage of the Immune-Related Response Criteria (irRC), which is a criteria based on the total tumor volume changes and confirmatory imaging at least 4 weeks apart prior to declaring disease progression. To further improve the outcome assessment, the irRC can be used in conjunction with HCC lesion measurements to incorporate the tumor viability features as an additional metric for treatment response in radiologic assessment.

      Financial support

      This work was supported by the Intramural Research Program of the NCI , NIH .

      Conflict of interest

      The authors declare no conflicts of interest in relation to this manuscript.

      References

        • Ferlay J.
        • Soerjomataram I.
        • Dikshit R.
        • et al.
        Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.
        Int J Cancer. 2014;
        • Shlomai A.
        • de Jong Y.P.
        • Rice C.M.
        Virus associated malignancies: the role of viral hepatitis in hepatocellular carcinoma.
        Semin Cancer Biol. 2014; 26: 78-88
        • Park N.H.
        • Chung Y.H.
        • Lee H.S.
        Impacts of vaccination on hepatitis B viral infections in Korea over a 25-year period.
        Intervirology. 2010; 53: 20-28
        • Welzel T.M.
        • Graubard B.I.
        • Quraishi S.
        • et al.
        Population-attributable fractions of risk factors for hepatocellular carcinoma in the United States.
        Am J Gastroenterol. 2013; 108: 1314-1321
        • Ulahannan S.V.
        • Duffy A.G.
        • McNeel T.S.
        • et al.
        Earlier presentation and application of curative treatments in hepatocellular carcinoma.
        Hepatology. 2014;
        • Llovet J.M.
        • Ricci S.
        • Mazzaferro V.
        • et al.
        Sorafenib in advanced hepatocellular carcinoma.
        N Engl J Med. 2008; 359: 378-390
        • Jacob A.I.
        • Goldberg P.K.
        • Bloom N.
        • Degenshein G.A.
        • Kozinn P.J.
        Endotoxin and bacteria in portal blood.
        Gastroenterology. 1977; 72: 1268-1270
        • Crispe I.N.
        Hepatic T cells and liver tolerance.
        Nat Rev Immunol. 2003; 3: 51-62
        • Sriwatanawongsa V.
        • Davies H.S.
        • Calne R.Y.
        The essential roles of parenchymal tissues and passenger leukocytes in the tolerance induced by liver grafting in rats.
        Nat Med. 1995; 1: 428-432
        • Knolle P.
        • Schlaak J.
        • Uhrig A.
        • Kempf P.
        • Meyer zum Buschenfelde K.H.
        • Gerken G.
        Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge.
        J Hepatol. 1995; 22: 226-229
        • Knolle P.A.
        • Germann T.
        • Treichel U.
        • et al.
        Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells.
        J Immunol. 1999; 162: 1401-1407
        • Protzer U.
        • Maini M.K.
        • Knolle P.A.
        Living in the liver: hepatic infections.
        Nat Rev Immunol. 2012; 12: 201-213
        • Bissell D.M.
        • Wang S.S.
        • Jarnagin W.R.
        • Roll F.J.
        Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation.
        J Clin Invest. 1995; 96: 447-455
        • Bottcher J.P.
        • Schanz O.
        • Wohlleber D.
        • et al.
        Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.
        Cell Rep. 2013; 3: 779-795
        • Bottcher J.P.
        • Schanz O.
        • Garbers C.
        • et al.
        IL-6 trans-signaling-dependent rapid development of cytotoxic CD8+ T cell function.
        Cell Rep. 2014; 8: 1318-1327
        • Li W.
        • Kuhr C.S.
        • Zheng X.X.
        • et al.
        New insights into mechanisms of spontaneous liver transplant tolerance: the role of Foxp3-expressing CD25+CD4+ regulatory T cells.
        Am J Transplant. 2008; 8: 1639-1651
        • Muhlbauer M.
        • Fleck M.
        • Schutz C.
        • et al.
        PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis.
        J Hepatol. 2006; 45: 520-528
        • Yu M.C.
        • Chen C.H.
        • Liang X.
        • et al.
        Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice.
        Hepatology. 2004; 40: 1312-1321
        • Diehl L.
        • Schurich A.
        • Grochtmann R.
        • Hegenbarth S.
        • Chen L.
        • Knolle P.A.
        Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance.
        Hepatology. 2008; 47: 296-305
        • Kassel R.
        • Cruise M.W.
        • Iezzoni J.C.
        • Taylor N.A.
        • Pruett T.L.
        • Hahn Y.S.
        Chronically inflamed livers up-regulate expression of inhibitory B7 family members.
        Hepatology. 2009; 50: 1625-1637
        • Gu X.
        • Wang Y.
        • Xiang J.
        • et al.
        Interferon- gamma triggers hepatic stellate cell-mediated immune regulation through MEK/ERK signaling pathway.
        Clin Dev Immunol. 2013; 2013: 389807
        • Calabrese F.
        • Valente M.
        • Giacometti C.
        • et al.
        Parenchymal transforming growth factor beta-1: its type II receptor and Smad signaling pathway correlate with inflammation and fibrosis in chronic liver disease of viral etiology.
        J Gastroenterol Hepatol. 2003; 18: 1302-1308
        • Huang A.
        • Zhang B.
        • Yan W.
        • et al.
        Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis b virus infection through PD-1-induced IL-10.
        J Immunol. 2014; 193: 5461-5469
        • Xu D.
        • Fu J.
        • Jin L.
        • et al.
        Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B.
        J Immunol. 2006; 177: 739-747
        • Zhang H.H.
        • Mei M.H.
        • Fei R.
        • et al.
        Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.
        J Viral Hepatitis. 2010; 17: 34-43
        • Matsuzaki K.
        • Murata M.
        • Yoshida K.
        • et al.
        Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.
        Hepatology. 2007; 46: 48-57
        • Nebbia G.
        • Peppa D.
        • Schurich A.
        • et al.
        Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.
        PLoS One. 2012; 7: e47648
        • Peng G.
        • Li S.
        • Wu W.
        • Tan X.
        • Chen Y.
        • Chen Z.
        PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients.
        Mol Immunol. 2008; 45: 963-970
        • Nakamoto N.
        • Cho H.
        • Shaked A.
        • et al.
        Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
        PLoS Pathog. 2009; 5: e1000313
        • Murakami H.
        • Akbar S.M.
        • Matsui H.
        • Horiike N.
        • Onji M.
        Decreased interferon-alpha production and impaired T helper 1 polarization by dendritic cells from patients with chronic hepatitis C.
        Clin Exp Immunol. 2004; 137: 559-565
        • Bain C.
        • Fatmi A.
        • Zoulim F.
        • Zarski J.P.
        • Trepo C.
        • Inchauspe G.
        Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.
        Gastroenterology. 2001; 120: 512-524
        • Martinet J.
        • Dufeu-Duchesne T.
        • Bruder Costa J.
        • et al.
        Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
        Gastroenterology. 2012; 143: e8
        • Cabrera R.
        • Tu Z.
        • Xu Y.
        • et al.
        An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.
        Hepatology. 2004; 40: 1062-1071
        • Liu B.S.
        • Groothuismink Z.M.
        • Janssen H.L.
        • Boonstra A.
        Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections.
        J Leukoc Biol. 2011; 89: 981-988
        • Shi F.
        • Shi M.
        • Zeng Z.
        • et al.
        PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients.
        Int J Cancer. 2011; 128: 887-896
        • Nakamoto N.
        • Kaplan D.E.
        • Coleclough J.
        • et al.
        Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
        Gastroenterology. 2008; 134 (37 e1-2): 1927-1937
        • Moorman J.P.
        • Wang J.M.
        • Zhang Y.
        • et al.
        Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.
        J Immunol. 2012; 189: 755-766
        • Schreiber R.D.
        • Old L.J.
        • Smyth M.J.
        Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion.
        Science. 2011; 331: 1565-1570
        • Matsui M.
        • Machida S.
        • Itani-Yohda T.
        • Akatsuka T.
        Downregulation of the proteasome subunits, transporter, and antigen presentation in hepatocellular carcinoma, and their restoration by interferon-gamma.
        J Gastroenterol Hepatol. 2002; 17: 897-907
        • Kurokohchi K.
        • Carrington M.
        • Mann D.L.
        • et al.
        Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma.
        Hepatology. 1996; 23: 1181-1188
        • Ormandy L.A.
        • Hillemann T.
        • Wedemeyer H.
        • Manns M.P.
        • Greten T.F.
        • Korangy F.
        Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma.
        Cancer Res. 2005; 65: 2457-2464
        • Unitt E.
        • Rushbrook S.M.
        • Marshall A.
        • et al.
        Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.
        Hepatology. 2005; 41: 722-730
        • Cariani E.
        • Pilli M.
        • Zerbini A.
        • et al.
        Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.
        PLoS One. 2012; 7: e32493
        • Hoechst B.
        • Ormandy L.A.
        • Ballmaier M.
        • et al.
        A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
        Gastroenterology. 2008; 135: 234-243
        • Zhu X.D.
        • Zhang J.B.
        • Zhuang P.Y.
        • et al.
        High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.
        J Clin Oncol. 2008; 26: 2707-2716
        • Yeung O.W.
        • Lo C.
        • Ling C.
        • et al.
        Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma.
        J Hepatol. 2014;
        • Ding T.
        • Xu J.
        • Wang F.
        • et al.
        High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection.
        Hum Pathol. 2009; 40: 381-389
        • Behboudi S.
        • Alisa A.
        • Boswell S.
        • Anastassiou J.
        • Pathan A.A.
        • Williams R.
        Expansion of anti-AFP Th1 and Tc1 responses in hepatocellular carcinoma occur in different stages of disease.
        Br J Cancer. 2010; 102: 748-753
        • Gao Q.
        • Qiu S.J.
        • Fan J.
        • et al.
        Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.
        J Clin Oncol. 2007; 25: 2586-2593
        • Fu J.
        • Xu D.
        • Liu Z.
        • et al.
        Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients.
        Gastroenterology. 2007; 132: 2328-2339
        • Budhu A.
        • Forgues M.
        • Ye Q.H.
        • et al.
        Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment.
        Cancer Cell. 2006; 10: 99-111
        • Hattori E.
        • Okumoto K.
        • Adachi T.
        • et al.
        Possible contribution of circulating interleukin-10 (IL-10) to anti-tumor immunity and prognosis in patients with unresectable hepatocellular carcinoma.
        Hepatol Res. 2003; 27: 309-314
        • Fujiwara K.
        • Higashi T.
        • Nouso K.
        • et al.
        Decreased expression of B7 costimulatory molecules and major histocompatibility complex class-I in human hepatocellular carcinoma.
        J Gastroenterol Hepatol. 2004; 19: 1121-1127
        • Tatsumi T.
        • Takehara T.
        • Katayama K.
        • et al.
        Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma.
        Hepatology. 1997; 25: 1108-1114
        • Wu Y.
        • Kuang D.M.
        • Pan W.D.
        • et al.
        Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.
        Hepatology. 2013; 57: 1107-1116
        • Hsu P.N.
        • Yang T.C.
        • Kao J.T.
        • et al.
        Increased PD-1 and decreased CD28 expression in chronic hepatitis B patients with advanced hepatocellular carcinoma.
        Liver Int. 2010; 30: 1379-1386
        • Sugimoto K.
        • Shiraki K.
        • Ito T.
        • et al.
        Expression of functional CD40 in human hepatocellular carcinoma.
        Hepatology. 1999; 30: 920-926
        • Morales-Kastresana A.
        • Sanmamed M.F.
        • Rodriguez I.
        • et al.
        Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
        Clin Cancer Res. 2013; 19: 6151-6162
        • Wu K.
        • Kryczek I.
        • Chen L.
        • Zou W.
        • Welling T.H.
        Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7–H1/programmed death-1 interactions.
        Cancer Res. 2009; 69: 8067-8075
        • Okazaki T.
        • Honjo T.
        The PD-1-PD-L pathway in immunological tolerance.
        Trends Immunol. 2006; 27: 195-201
        • Kuang D.M.
        • Zhao Q.
        • Peng C.
        • et al.
        Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
        J Exp Med. 2009; 206: 1327-1337
        • Han Y.
        • Chen Z.
        • Yang Y.
        • et al.
        Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
        Hepatology. 2014; 59: 567-579
        • Kalathil S.
        • Lugade A.A.
        • Miller A.
        • Iyer R.
        • Thanavala Y.
        Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.
        Cancer Res. 2013; 73: 2435-2444
        • Mizukoshi E.
        • Nakamoto Y.
        • Arai K.
        • et al.
        Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma.
        Hepatology. 2011; 53: 1206-1216
        • Li F.J.
        • Zhang Y.
        • Jin G.X.
        • Yao L.
        • Wu D.Q.
        Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.
        Immunol Lett. 2013; 150: 116-122
        • Li H.
        • Wu K.
        • Tao K.
        • et al.
        Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
        Hepatology. 2012; 56: 1342-1351
        • Stemmer S.M.
        • Benjaminov O.
        • Medalia G.
        • et al.
        CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.
        Oncologist. 2013; 18: 25-26
        • Wang B.J.
        • Bao J.J.
        • Wang J.Z.
        • et al.
        Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma.
        World J Gastroenterol. 2011; 17: 3322-3329
        • Gao Q.
        • Wang X.Y.
        • Qiu S.J.
        • et al.
        Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma.
        Clin Cancer Res. 2009; 15: 971-979
        • Thimme R.
        • Neagu M.
        • Boettler T.
        • et al.
        Comprehensive analysis of the alpha-fetoprotein-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
        Hepatology. 2008; 48: 1821-1833
        • Butterfield L.H.
        • Ribas A.
        • Meng W.S.
        • et al.
        T-cell responses to HLA-A∗0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.
        Clin Cancer Res. 2003; 9: 5902-5908
        • Bricard G.
        • Bouzourene H.
        • Martinet O.
        • et al.
        Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.
        J Immunol. 2005; 174: 1709-1716
        • Gehring A.J.
        • Ho Z.Z.
        • Tan A.T.
        • et al.
        Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.
        Gastroenterology. 2009; 137: 682-690
        • Korangy F.
        • Ormandy L.A.
        • Bleck J.S.
        • et al.
        Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-1 in hepatocellular carcinoma.
        Clin Cancer Res. 2004; 10: 4332-4341
        • Mizukoshi E.
        • Nakamoto Y.
        • Marukawa Y.
        • et al.
        Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma.
        Hepatology. 2006; 43: 1284-1294
        • Hiroishi K.
        • Eguchi J.
        • Baba T.
        • et al.
        Strong CD8(+) T-cell responses against tumor-associated antigens prolong the recurrence-free interval after tumor treatment in patients with hepatocellular carcinoma.
        J Gastroenterol. 2010; 45: 451-458
        • Komori H.
        • Nakatsura T.
        • Senju S.
        • et al.
        Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma.
        Clin Cancer Res. 2006; 12: 2689-2697
        • van Rooij N.
        • van Buuren M.M.
        • Philips D.
        • et al.
        Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma.
        J Clin Oncol. 2013; 31: e439-e442
        • Robbins P.F.
        • Lu Y.C.
        • El-Gamil M.
        • et al.
        Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
        Nat Med. 2013; 19: 747-752
        • Unitt E.
        • Marshall A.
        • Gelson W.
        • et al.
        Tumour lymphocytic infiltrate and recurrence of hepatocellular carcinoma following liver transplantation.
        J Hepatol. 2006; 45: 246-253
        • Cariani E.
        • Pilli M.
        • Zerbini A.
        • et al.
        HLA and killer immunoglobulin-like receptor genes as outcome predictors of hepatitis C virus-related hepatocellular carcinoma.
        Clin Cancer Res. 2013; 19: 5465-5473
        • Lee W.C.
        • Wang H.C.
        • Hung C.F.
        • Huang P.F.
        • Lia C.R.
        • Chen M.F.
        Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial.
        J Immunother. 2005; 28: 496-504
        • Tada F.
        • Abe M.
        • Hirooka M.
        • et al.
        Phase I/II study of immunotherapy using tumor antigen-pulsed dendritic cells in patients with hepatocellular carcinoma.
        Int J Oncol. 2012; 41: 1601-1609
        • Greten T.F.
        • Forner A.
        • Korangy F.
        • et al.
        A phase II open label trial evaluating safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma.
        BMC Cancer. 2010; 10: 209
        • Palmer D.H.
        • Midgley R.S.
        • Mirza N.
        • et al.
        A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma.
        Hepatology. 2009; 49: 124-132
        • Butterfield L.H.
        • Economou J.S.
        • Gamblin T.C.
        • Geller D.A.
        Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients.
        J Transl Med. 2014; 12: 86
        • Sawada Y.
        • Yoshikawa T.
        • Nobuoka D.
        • et al.
        Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival.
        Clin Cancer Res. 2012; 18: 3686-3696
        • Flecken T.
        • Schmidt N.
        • Hild S.
        • et al.
        Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma.
        Hepatology. 2014; 59: 1415-1426
        • Laurent S.
        • Carrega P.
        • Saverino D.
        • et al.
        CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.
        Hum Immunol. 2010; 71: 934-941
        • de Waal Malefyt R.
        • Haanen J.
        • Spits H.
        • et al.
        Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.
        J Exp Med. 1991; 174: 915-924
        • Hong Y.
        • Peng Y.
        • Guo Z.S.
        • et al.
        Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.
        Hepatology. 2014; 59: 1448-1458
        • Takayama T.
        • Sekine T.
        • Makuuchi M.
        • et al.
        Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial.
        Lancet. 2000; 356: 802-807
        • Rosenberg S.A.
        • Lotze M.T.
        • Muul L.M.
        • et al.
        Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer.
        N Engl J Med. 1985; 313: 1485-1492
        • Shimizu K.
        • Kotera Y.
        • Aruga A.
        • et al.
        Postoperative dendritic cell vaccine plus activated T-cell transfer improves the survival of patients with invasive hepatocellular carcinoma.
        Hum Vaccin Immunother. 2014; 10: 970-976
        • Kubo S.
        • Nishiguchi S.
        • Hirohashi K.
        • et al.
        Effects of long-term postoperative interferon-alpha therapy on intrahepatic recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. A randomized, controlled trial.
        Ann Intern Med. 2001; 134: 963-967
        • Llovet J.M.
        • Sala M.
        • Castells L.
        • et al.
        Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma.
        Hepatology. 2000; 31: 54-58
        • Shiratori Y.
        • Shiina S.
        • Teratani T.
        • et al.
        Interferon therapy after tumor ablation improves prognosis in patients with hepatocellular carcinoma associated with hepatitis C virus.
        Ann Intern Med. 2003; 138: 299-306
        • Mazzaferro V.
        • Romito R.
        • Schiavo M.
        • et al.
        Prevention of hepatocellular carcinoma recurrence with alpha-interferon after liver resection in HCV cirrhosis.
        Hepatology. 2006; 44: 1543-1554
        • Sun H.C.
        • Tang Z.Y.
        • Wang L.
        • et al.
        Postoperative interferon alpha treatment postponed recurrence and improved overall survival in patients after curative resection of HBV-related hepatocellular carcinoma: a randomized clinical trial.
        J Cancer Res Clin Oncol. 2006; 132: 458-465
        • Lo C.M.
        • Liu C.L.
        • Chan S.C.
        • et al.
        A randomized, controlled trial of postoperative adjuvant interferon therapy after resection of hepatocellular carcinoma.
        Ann Surg. 2007; 245: 831-842
        • Chen L.T.
        • Chen M.F.
        • Li L.A.
        • et al.
        Long-term results of a randomized, observation-controlled, phase III trial of adjuvant interferon Alfa-2b in hepatocellular carcinoma after curative resection.
        Ann Surg. 2012; 255: 8-17
        • Ishikawa T.
        • Higuchi K.
        • Kubota T.
        • et al.
        Combination PEG-IFN a-2b/ribavirin therapy following treatment of hepatitis C virus-associated hepatocellular carcinoma is capable of improving hepatic functional reserve and survival.
        Hepatogastroenterology. 2012; 59: 529-532
        • Sangro B.
        • Mazzolini G.
        • Ruiz J.
        • et al.
        Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors.
        J Clin Oncol. 2004; 22: 1389-1397
        • Mazzolini G.
        • Alfaro C.
        • Sangro B.
        • et al.
        Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas.
        J Clin Oncol. 2005; 23: 999-1010
        • Faivre S.J.
        • Santoro A.
        • Kelley R.K.
        • et al.
        A phase 2 study of a novel transforming growth factor-beta (TGF-β1) receptor I kinase inhibitor, LY2157299 monohydrate (LY), in patients with advanced hepatocellular carcinoma (HCC).
        J Clin Oncol. 2014; 32 (abstr LBA173)
        • Sangro B.
        • Gomez-Martin C.
        • de la Mata M.
        • et al.
        A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C.
        J Hepatol. 2013; 59: 81-88
        • Duffy A.G.
        • Ulahannan S.V.
        • Fioravanti S.
        • et al.
        A pilot study of tremelimumab, a monoclonal antibody against CTLA-4, in combination with either transcatheter arterial chemoembolization (TACE) or radiofrequency ablation (RFA) in patients with hepatocellular carcinoma (HCC).
        J Clin Oncol. 2014; 32 (abstr e15133)
        • Sangro B.
        • Crocenzi T.S.
        • Welling T.H.
        • et al.
        Phase I dose escalation study of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in patients (pts) with advanced hepatocellular carcinoma (HCC) with or without chronic viral hepatitis.
        J Clin Oncol. 2013; 31 (abstr TPS3111)
        • Safran H.
        • Charpentier K.P.
        • Kaubisch A.
        • et al.
        Lenalidomide for second-line treatment of advanced hepatocellular cancer: a Brown University Oncology Group Phase II Study.
        Am J Clin Oncol. 2013;
        • Yen C.J.
        • Daniel B.
        • Kudo M.
        Randomized phase II trial of intravenous RO5137382/GC33 at 1600 mg every other week and placebo in previously treated patients with unresectable hepatocellular carcinoma (HCC; NCT01507168).
        J Clin Oncol. 2014; 32 (5c, abstr 4102)
        • Sun W.
        • Nelson D.
        • Alberts S.R.
        • et al.
        Phase Ib study of mapatumumab in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC) and chronic viral hepatitis.
        in: J Clin Oncol. 29. 2011 (abstr 261)
        • Saleh M.N.
        • Percent I.
        • Wood T.E.
        • et al.
        A phase I study of CS-1008 (humanized monoclonal antibody targeting death receptor 5 or DR5), administered weekly to patients with advanced solid tumors or lymphomas.
        J Clin Oncol. 2008; : 3537
        • Serova M.
        • Tijeras-Raballand A.
        • Dos Santos C.
        • et al.
        Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen.
        Cancer Res. 2013; 73: 2094
        • Mizukoshi E.
        • Yamashita T.
        • Arai K.
        • et al.
        Enhancement of tumor-associated antigen-specific T cell responses by radiofrequency ablation of hepatocellular carcinoma.
        Hepatology. 2013; 57: 1448-1457
        • Krusch M.
        • Salih J.
        • Schlicke M.
        • et al.
        The kinase inhibitors sunitinib and sorafenib differentially affect NK cell antitumor reactivity in vitro.
        J Immunol. 2009; 183: 8286-8294
        • Chen Y.
        • Huang Y.
        • Reiberger T.
        • et al.
        Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice.
        Hepatology. 2014; 59: 1435-1447
        • Chen Y.
        • Ramjiawan R.R.
        • Reiberger T.
        • et al.
        CXCR4 inhibition in tumor microenvironment facilitates anti-PD-1 immunotherapy in sorafenib-treated HCC in mice.
        Hepatology. 2014;