Immune checkpoints include stimulatory and inhibitory checkpoint molecules. In recent years, inhibitory checkpoints, including cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), and programmed cell death ligand 1 (PD-L1), have been identified to suppress anti-tumor immune responses in solid tumors. Novel drugs targeting immune checkpoints have succeeded in cancer treatment. Specific PD-1 blockades were approved for treatment of melanoma in 2014 and for treatment of non-small-cell lung cancer in 2015 in the United States, European Union, and Japan. Preclinical and clinical studies show immune checkpoint therapy provides survival benefit for greater numbers of patients with liver cancer, including hepatocellular carcinoma and cholangiocarcinoma, two main primary liver cancers. The combination of anti-PD-1/PD-L1 with anti- CTLA-4 antibodies is being evaluated in phase 1, 2 or 3 trials, and the results suggest that an anti-PD-1 antibody combined with locoregional therapy or other molecular targeted agents is an effective treatment strategy for HCC. In addition, studies on activating co-stimulatory receptors to enhance anti-tumor immune responses have increased our understanding regarding this immunotherapy in liver cancer. Epigenetic modulations of checkpoints for improving the tumor microenvironment also expand our knowledge of potential therapeutic targets in improving the tumor microenvironment and restoring immune recognition and immunogenicity. In this review, we summarize current knowledge and recent developments in immune checkpoint-based therapies for the treatment of hepatocellular carcinoma and cholangiocarcinoma and attempt to clarify the mechanisms underlying its effects. Keywords: Immune checkpoint, Hepatocellular carcinoma, Cholangiocarcinoma, Immunotherapy, Epigenetics Background Immunotherapy has emerged as a promising therapy Globally, primary liver cancer accounts for 6% of all can- and is being investigated in various tumors including cers and 9% of all death from cancer. It is the sixth most liver cancer . Emerging evidence supports that the common cancer and the second leading cause of cancer blockade of immune checkpoints is among the most death. The important primary liver cancers include he- promising approaches in cancer immunotherapy [4–6]. patocellular carcinoma (HCC), accounting for approxi- The activity of the immune system is mostly regulated mately 75%, and cholangiocarcinoma, accounting for by immune cells called T cells. In the tumor microenvir- approximately 6%. Although either surgical resection or onment, T cells can recognize tumor antigens, which are liver transplant can be used for the treatment of liver presented to T cell receptors by antigen-presenting cells cancer, limitations are caused by high recurrence rates (APCs). Besides signal via T cell receptors, T cell re- after resection and low-ratio eligibility for surgery and sponse is fine-tuned by a group of cell surface molecules, transplant because this cancer is often detected at a late named immune checkpoints. They can be either stimu- stage [1, 2]. In the tumor microenvironment, cancer cells latory or inhibitory, and participate in various stages of and host immune responses interact to promote or in- T cell response (Fig. 1)[6–11]. Many cancers are able to hibit the pathologic progression of cancer. The immune evade the immune system, mainly by overexpressing in- system can identify cancer cells, and mobilizing the im- hibitory ligands to damp T cell attack. As a result, fewer, mune response is able to eliminate cancer . and damaged T cells were found in patients with HCC, which contributed to the progression of this cancer . * Correspondence: email@example.com; firstname.lastname@example.org Recently, in vitro and in vivo results show histone dea- Department of Surgery, University of Colorado Anschutz Medical Campus, cetylase inhibitors (HDACi) and DNA methyltransferase RC1-North Building, P18-8116, Aurora, CO 80045, USA inhibitors (DNMTi), two important epigenetic drugs, Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital affiliated to China Medical University, Shenyang 110004, Liaoning, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 2 of 12 Fig. 1 Illustration of stimulatory and inhibitory immune checkpoints between T-cells, APCs, and cancer cells. Blockade of inhibitory immune checkpoints can positively regulate T-cell activation and prevent immune escape of cancer cells within the tumor microenvironment. Activation of stimulatory immune check points can augment the effect of immune checkpoint inhibitors in cancer therapeutics. Red, inhibitory immune checkpoints; blue, stimulatory immune checkpoints can up-regulate expression of inhibitory immune check- CTLA-4 inhibits T cell response by directly delivering an points in either immune or cancer cells [13–15]. Epigen- inhibitory signal to T cell, and interfering with the bind- etic modifiers function importantly in priming and ing between B7 and CD28 . In 31 HCC patients, it enhancing the therapeutic effect of the host immune sys- was found the addition of anti-CTLA-4 antibody re- tem on cancer [14, 15]. The purpose of this review is to sulted in an increase in the frequency of give a brief overview of the role for immune checkpoints tumor-associated antigens (TAA)-specific cytotoxic T related to liver cancer progression. It also provides new cells in 60% of HCC patients, accompanied with en- insights into the epigenetic mechanism in checkpoint hanced antitumor effect of tumor-specific T cells . In immunotherapy and checkpoint blocking – based thera- addition, CTLA-4 is shown to be important for regula- peutic approaches for treatment of liver cancer. tory T cell (Treg) function. Tregs control functions of the effector T cells, and thus crucially maintain periph- Immune checkpoints and hepatocellular carcinoma eral tolerance . Unlike effector T cells, Tregs consti- The most ex vivo studied and clinically relevant check- tutively express CTLA-4 to exert their immune point proteins are CTLA-4, PD-1, and PD-L1 (Tables 1 suppression [21, 22]. Treg-specific CTLA-4 deficiency and 2). The expression of inhibitory immune checkpoints was shown to affect in vivo Treg suppressive function can be dysregulated in a tumor microenvironment, which and promote tumor immunity [21, 22]. In a rat liver can lead to improvement of T cell-mediated immune re- transplantation model with tumor recurrence, hepatic sponse through cancer immunotherapy . The PD-1 expressions of CTLA-4, TGF-β and PD-L1 were in- pathway is found to suppress T cell activation mainly creased in the tumor tissues from small-for-size liver within peripheral tissues at the later phase, whereas the graft group compared to whole graft group. The results CTLA-4 pathways are involved in regulation of T suggested that up-regulation of CTLA-4 may mediate cell-mediated immune responses primarily in lymph the mobilization of Tregs by small-for-size graft injury, nodes at the priming phase . contributing to HCC recurrence after liver transplant- ation . HCC-derived Tregs down-regulated CD80/86 CTLA-4 expression on splenic DCs in a CTLA-4 dependent CTLA-4 is a CD28 homolog and primarily located in manner, and inhibition of CTLA-4 could prevent the intracellular compartments in resting naive T cells. Treg-mediated suppression in anti-tumor immune Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 3 of 12 Table 1 Immune checkpoints expression in liver cancers Cancer type Number TNM Stage Tumor differentiation Tumor size (cm) Immune Cellular expression Year Reference (I + II / III + IV) (I + II / III + IV) checkpoints Human HCC 217 Operable, 101 7.26 (1.0–2.5) PD-L1/PD-1 neoplastic and 2016  (tumor samples) resected (46%)/ 116 (53%) inflammatory cells Human HCC 176 97/52 112/64 5.3 (PD-L1 )/ PD-L1 CD68+ macrophages 2016  low 4.9 (PD-L1 ) high Human HCC 90 Operable, resected 73/17 4.2 (1.3–15) PD-L1 peritumoral 2017  hepatocytes Human HCC 294 59/87 140/6 110(<5) /36(≥5) PD-L1/PD-1 tumor infiltrating 2017  and CTLA-4 Human HCC 69 35/34 50/19 7/21(Tim-3 low)/ Tim-3 CD14+ monocytes 2015  17/24 (Tim-3 high) Human HCC 171 100/71 NR 98/73 PD-1 neoplastic and 2016  and Tim-3 inflammatory cells Human ICC 31 9/22 13/18 20 (<5) / 11 (>5) PD-L1 neoplastic and 2009  and PD-1 inflammatory cells Human ICC 27 16/11 19/8 NR PD-L1 ICC cells 2016  HCC hepatocellular carcinoma, ICC Intrahepatic cholangiocarcinoma, NR not reported responses . Thus, CTLA-4 could not only enhance PD-L1+ Kupffer cells interact with PD-1 + CD8+ T cells the antitumor effect of effector T cells but also maintain and contribute to dysfunction of effector T cells in HCC. self-tolerance and the suppressive function of Tregs in Elevated PD-L1 expression in HCC is indeed associated liver cancer immunity. with poorer prognosis in HCC patients . In 217 HCCs, PD-L1 was expressed by both neoplastic and PD-1/PD-L1 intra-tumoral inflammatory cells, which are related to PD-L1 is the main ligand for PD-1, which is crucial for tumor aggressiveness. It also suggests that the PD-L1/ tumor immunity. In addition, PD-L1 also interacts with PD-1 immune checkpoint could be targeted in the treat- B7-1 to inhibit T cell immunity, and the role of this ment of particular HCC variants . More recently, 90 interaction in cancer immunity is still unclear . Bind- HCC patients with PD-L1 expression in peritumoral he- ing of PD-L1 to its receptor can suppress T cell migra- patocytes were demonstrated to have a significantly tion, proliferation, and secretion of cytotoxic mediators, higher risk of cancer recurrence or metastasis and and thus blocks the “cancer immunity cycle” . In the cancer-related death . Immunohistochemistry data in HCC tumor microenvironment, PD-L1 expression is 294 HCC tissue samples showed PD-1 and PD-L1 ex- mainly expressed in Kupffer cells but is slightly pression was significantly related to high CD8+ expressed on other APCs or HCC tumor cells . CD8 tumor-infiltrating lymphocytes (TILs). Only high + T cells and Kupffer cells in human HCC tumor tissues Edmondson–Steiner grade was markedly related to high expressed high levels of PD-1 and PD-L1, respectively. PD-1 expression. High PD-L1 expression was Table 2 Pre-clinical studies with immune checkpoints in therapy of liver cancers Cancer type Number TNM Stage Tumor differentiation Tumor Immune Therapy Target cells Year Reference (I + II / III + IV) (I + II / III + IV) size (cm) checkpoints Human HCC 71 57 / 14 58 / 13 36 (≤5) / PD-L1 PD-L1and PD-1 Kupffer cells 2009  35 (>5) and PD-1 antibodies and CD8 T cells Human HCC NR NR NR NR PD-L1 Specific shRNA for HCC cell lines 2017  PD-L1 and DNMT1 Human HCC 31 22/9 21/10 9(≤5) / CTLA-4 CTLA-4 antibodies Tumor-Associated 2011  22(>5) Antigen-Specific T Cells Mice HCC NR NR NR NR CTLA-4 CTLA-4 antibodies Regulatory T cells 2017  Human HCC 59 54 / 4 unknown, NR NR LAG3, PD-1, Blocking antibodies tumor-infiltrating 2017  n =1 Tim3 and to LAG3, PD-1, TIM3 T cells CTLA4 or CTLA4 Human HCC 21 8/13 NR NR GITR GITR ligand tumor-infiltrating 2013  Tregs HCC hepatocellular carcinoma, ICC Intrahepatic cholangiocarcinoma, NR not reported Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 4 of 12 demonstrated as an independent poor prognostic factor antibody with PD-L1 blockade further augmented TIL re- for disease-free survival in the high CD8+ TILs group. sponses to polyclonal stimuli and TAA . This suggests Further, combined high expression of PD-L1 and CD8+ that LAG-3 plays an important role in T-cell suppression TIL is an important prognostic factor related to the im- in the HCC microenvironment and might be a promising mune checkpoint pathway in HCC. Also, this result immunotherapeutic target for HCC. Further clinical trials would be helpful in evaluating the applicable group of about Tim-3, Lag-3 or TIGIT blockers should be per- PD-1/PD-L1 blocking agent for HCC patients . formed in liver cancer treatment. PD-L1 expression was significantly increased in tumors with a high number of tumor-infiltrating lymphocytes Co-stimulatory immune checkpoints (ρ = 0.533, p < 0.001). High PD-L1 expression was asso- The best characterized co-stimulatory ligands that have ciated with significantly shorter overall survival . been investigated in hepatocellular carcinoma are B7-1 These clinic data further support that PD-L1 is an im- and B7-2. These two important immune checkpoints are portant mediator in the progression and an important mainly expressed on professional antigen-presenting target in the anti-tumor therapy for liver cancer. cells. B7-1 and B7-2 can bind to both CD28 and CTLA-4, and thus regulate T cell activation via selective Other inhibitory checkpoints interacting with either CD28 or CTLA-4 . Expres- Several other inhibitory receptors, including T-cell im- sion of costimulatory molecules, including B7-1 and munoglobulin- and mucin-domain-containing molecule-3 B7-2, have been found to be down-regulated in HCC (Tim-3) and LAG-3, are also upregulated on TAA-specific cells . This down-regulation may lead to suppression CD8+ T-cells in various cancer types, and are also in- of activation of effector T-cells mediated by B7/CD28. volved in progression of liver cancer. Tim-3 is strongly The glucocorticoid-induced tumor necrosis factor recep- expressed on CD4+ and CD8+ T-cells obtained from tor (GITR) and the inducible T-cell co-stimulator (ICOS) HCC lesions in contrast to the surrounding liver tissue. are co-stimulatory checkpoints and regulate the im- Tim-3 is expressed on tumor-associated macrophages munosuppressive Tregs function. Importantly, GITR and (TAM), which contributes to HCC growth . Intri- ICOS are up-regulated in Tregs infiltrating HCC and guingly, a high number of Tim3+ tumor infiltrating cells may function as potential targets for immunotherapeutic and Tim3+ TAM in HCC lesions are associated with a interventions for antitumor therapy . poor prognosis . In 171 patients with hepatitis B virus (HBV)-related HCC, both PD-1 and Tim-3 expressions in Immune checkpoints and cholangiocarcinoma liver infiltrating lymphocytes were significantly high in Intrahepatic cholangiocarcinoma (ICC) represents the tumor tissues compared to tumor adjacent tissues. The second most common primary liver malignancy, ac- up-regulation of PD-1 and Tim-3 were related to higher counting for 10–20% of all primary liver cancers . tumor grades . There is a significant positive intercor- Although ICC is traditionally viewed as a rare cancer, its relation between the levels of PD-1 and Tim-3 expression incidence has been steadily rising, with recent reports in tumor tissues and tumor adjacent tissues. The expres- showing the incidence of ICC in the USA has increased sions of PD-1 and Tim-3 in tumor tissues and tumor adja- from 0.44 to 1.18 cases/100,000 over the past three de- cent tissues were significantly associated with PD-1 and cades . The prognosis for ICC continues to be poor, Tim-3 polymorphisms, with genotype AA of PD-1 with surgery as the only definitive option for cure. rs10204525 and genotypes GT + TT of Tim-3 rs10053538 Median survival rate is low because most patients are respectively . LAG-3 is another important inhibi- not eligible for curative resection. As such, there is an tory immune check point and exerts synergistic effects increasing need for the development of novel adjuvant with PD-1/PD-L1 on T cell activation in the tumor therapies for patients with ICC. microenvironment. In HCC-vaccine-immunized mice, STAT3-blocked HCC vaccine downregulated expres- PD-1/PD-L1 sion of PD-1, TIGIT, and LAG-3, which could prevent In contrast to HCC, immunotherapy in cholangiocarci- cancer-induced dysfunction of CD8+ T and natural noma has been limited and mostly ineffective . How- killer cells . Recently, expression of LAG3 was ever, a high frequency of tumor-infiltrating lymphocytes found to be significantly higher on tumor-associated and PD-L1 expression suggest that checkpoint inhibition antigen (TAA)-specific CD8+ tumor-infiltrating T may prove effective . Expression of PD-L1 was found helper cells and CD8+ cytotoxic T cells in tumors than both in tumor-associated macrophages and in the tumor those in tumor-free liver tissues and blood of HCC pa- front. Patients with tumors exhibiting PD-L1 expression tients . Interestingly, blocking LAG-3 increased ex around the tumor front had a lower overall survival than vivo proliferation of CD4+ and CD8+ TIL and effector tumor front-positive patients . In 31 surgically cytokine production. Combination of LAG-3 blocking resected ICC samples from Asian patients, PD-L1 Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 5 of 12 expression was significantly higher in tumor tissue than HCC . Importantly, HCC DNA methylation is highly that in adjacent tissue . High levels of PD-L1 expres- enriched in immune function-related gene PD-1 . sion were also found in Western patients with ICC, Interestingly, Liu et al. found highly upregulated DNA which resulted in tumor poor differentiation, higher ma- methyltransferase 1 (DNMT1) is positively correlated lignant tumor stage and higher levels of apoptotic CD8+ with PD-L1 overexpression in sorafenib-resistant HCC TILs, and therefore led to lower chance of survival . cells. PD-L1 further induced DNMT1-dependent DNA More recently, in occupational cholangiocarcinoma, hypomethylation and restored the expression of PD-L1 expression was found in biliary intraepithelial methylation-silenced Cadherin 1, a metastasis suppres- neoplasia and intraductal papillary neoplasm. Cholangio- sor in HCC . carcinoma cells expressed PD-L1 in a low number of Accumulating evidence also shows histone deacetyla- cases of occupational cholangiocarcinoma, while tion regulates immune checkpoint expression and plays carcinoma cells expressed PD-L1 in all cases. Moreover, an important role in cancer progression. HDAC is have PD-L1 and PD-1 were also expressed in been shown to sensitize cancer cells to immune check- tumor-associated macrophages and tumor-infiltrating T point therapy by upregulating the immune checkpoints cells expressed. The number of PD-L1-positive mono- CTLA-4, PD-1, PD-L1, and PD-L2 on tumor cells and nuclear cells, PD-1-positive lymphocytes, and TILs . For example, inhibition of the class I HDAC1, CD8-positive lymphocytes infiltrating within the tumor HDAC2 and/or HDAC3 led to acetylation of the PD-L1 was markedly high in occupational cholangiocarcinoma. and PD-L2 promotors, which augmented up-regulation Immunostaining with mAbs detected human leukocyte of PD-L1/L2 protein and RNA transcription in melan- antigens (HLA) class I defects in 60% of ICC tumors oma patients, in melanoma cell lines and in a syngeneic and PD-L1 expression in 30%. Patients bearing tumors mouse model of melanoma . Interestingly, Lienlaf et with HLA class I defects and PD-L1 expression had a al.  found HDAC6i (ACY-241) reduced PD-L1 pro- significantly reduced survival rate. The results suggested duction and increased co-stimulatory checkpoint (CD28) PD-L1 up-regulation mediates immune escape in chol- levels, and thus suppressed tumor growth in vivo. In the angiocarcinoma and could be potential biomarker of re- WM164 HDAC6KD cells, the expression of PD-L2, sponse to anti-PD-1/PDL1 immunotherapy . The B7-H4 and TRAIL-R1 were largely diminished, while role of other immune checkpoints for cholangiocarci- B7-H3, Galectin-9 and TRAIL-R2 were moderately de- noma is still not well established. creased. In breast cancer cells, CD137, a co-stimulatory checkpoint, was found to be up-regulated by HDACi Epigenetic mechanism in checkpoint (SAHA) treatment . Therefore, inhibitory and immunotherapy co-stimulatory checkpoints can be up-regulated or In cancer, two important epigenetic mechanisms include down-regulated by different HDAC isoforms in different hypermethylation, which is mediated by DNMTs, and tumor types. To date, the immune modulatory activity histone deacetylation, which is mediated by HDACs. of HDAC inhibitors on tumor-specific immunity includ- Epigenetic dysregulation is a crucial mechanism under- ing immune checkpoints has not been well demon- lying the progression of cancer [46–49]. Some epigenetic strated or characterized in HCC. regulators can act negatively and positively in immune Recent evidence suggests that noncoding RNAs, such responses and lead to immune evasion , which pro- as microRNAs (miRNAs) and long noncoding RNAs vides a novel mechanism in immune checkpoint therapy (lncRNAs), may also have direct epigenetic functions by for treatment of cancers. recruiting specific protein complexes to genomic DNA, Recently, epigenetic modifications of the key immune and specifically to some promoters modulating the ex- checkpoints including PD-1, PD-L1, and CTLA-4 were pression of the corresponding genes. MiRNAs and analyzed in non-small cell lung cancer tissues from 39 lncRNAs play important roles in regulating expression patients . It was shown that CTLA-4 and PD-1, but of immune checkpoints in various tumors . In hu- not PD-L1, are hypomethylated in human lung tumors. man malignant pleural mesothelioma, the levels of This hypomethylation also led to increased expression of miR-15b, miR-16, miR-193a-3p, miR-195, and miR-200c these two genes as shown by transcriptome analysis . were significantly lower in the immune checkpoint In a phase 2 trial, hypomethylating agents such as vori- PD-L1-positive samples. Likewise, PD-L1 and nostat and azacitidine upregulated mRNA expression of miR-138-5p levels were inversely correlated in human PD-L1, PD-L2, PD-1 and CTLA-4 in 61 patients with colorectal cancer tumors, and miR-138-5p inhibited acute myeloid leukemia . More recently, profiling PD-L1 expression in tumor models in vivo . In lung DNA methylation in peripheral blood mononuclear cells cancer, it was demonstrated that the p53/miR-34/PD-L1 and T cells from HCC patients show that a broad signa- and miR-200/ZEB1/PD-L1 axis are novel mechanisms in ture of DNA methylation intensifies with progression of tumor immune evasion [61, 62]. Moreover, it is recently Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 6 of 12 demonstrated that transfection of human CD4+ T cells treatment in HCC patients, nivolumab showed a man- with miR-138 suppressed expression of CTLA-4, PD-1, ageable safety profile, including acceptable tolerability. and Foxp3 in glioma preclinical models . Whether The objective response rate was 20% (95% CI 15–26) in the association between miRNA expression and immune patients treated with nivolumab 3 mg/kg in the checkpoint levels in tumors can be translated into a pre- dose-expansion phase and 15% (95% CI 6–28) in the dictive marker of checkpoint inhibitor therapy in liver dose-escalation phase . Early data from the biliary cancer requires further investigation. Interactions among tract cohort of Keynote-028 reported an objective re- three kinds of RNAs were revealed in the ‘lncRNA-miR- sponse rate of 17% and a further 17% achieved stable NA-mRNA’ competing endogenous RNA network. Sev- disease in PD-L1 positive pretreated advanced cholan- eral biomarkers were identified for diagnosis of diabetic giocarcinoma . pancreatic cancer, such as lncRNAs (HOTAIR, CECR7 Immunotherapy is promising for HCC and cholangio- and UCA1), hsa-miR-214, hsa-miR-429, CCDC33 and carcinoma. However, even for those patients who respond CTLA-4. Notably, interactions of ‘CECR7-hsa-- to the single agent immunotherapy, combinational ther- miR-429-CTLA4’ were highlighted in the endogenous apy may be more potent and lead to more durable re- RNA network, which is very important in enhancing the sponse. At the 2016 ASCO meeting, an ongoing phase I progression of pancreatic cancer . Some miRNAs trial showed trans catheter arterial chemoembolization. and lncRNAs might be involved in the “cancer immunity Radiofrequency, or cryoablation induced a peripheral im- cycle” regulated by immune checkpoints such as mune response which may enhance the effect of CTLA-4 and PD-L1-PD-1 and could be the subject of anti-CTLA-4 treatment. This combination is safe and future investigations in liver cancer. leads to the accumulation of intratumoral CD8+ T cells Taken together, a wave of translational research high- and activation of T cells in peripheral blood in responding lights the mechanistic and functional link between epi- patients. Encouraging clinical activity was seen with ob- genetic regulation and immune checkpoints in the jective confirmed responses and a PFS of 5.7 months development and progression of primary tumors includ- (NCT01853618) . Another pilot study for the com- ing liver cancer. bined effect of immune checkpoint blocking and ablative therapies has been initiated in patients with advanced liver Checkpoint-blocking based therapeutic approaches cancer (NCT02821754). Chemotherapy such as cisplatin Over the last decade, there has been significant progress can reduce PD-L2 expression on tumor cells [69, 70]. Both in our understanding of the immune system which has these studies show that chemotherapy can enhance antitu- led to development of numerous immune checkpoints mor immunity and thus may combine and augment blockades that have altered the management and prog- immune checkpoint therapy for treatment of liver cancer. nosis in some cancers including liver cancer (Table 2). As previously discussed, epigenetic modulators enhance As more such drugs are developed, we will have multiple cell surface expression of immune checkpoints. Several additional options and indications for these inhibitors in studies provided evidence to support increased expression the near future. Among these pathways, the PD-1/ of checkpoint inhibitors on tumor cells following epigen- PD-L1 and the B7-1/B7-2/CTLA-4 have been identified etic treatment, which enhances responses to immune as clinically available inhibitors. checkpoint therapy [56, 71]. Recently, the role of HDACi These immune checkpoint drugs such as nivolumab, and histone methyltransferases in tumor immunity and pembrolizumab, and ipilimumab have already been FDA cancer therapy has been investigated. In approved in non-small cell lung cancer, renal cell carcin- melanoma-bearing mice, HDACi upregulated expression oma, melanoma, Hodgkin lymphoma, and urothelial of PD-L1 and PD-L2 through increased histone acetyl- bladder cancer . Trials investigating immune check- ation. Further, combination of HDACi and PD-1 blockade point blockades in HCC and cholangiocarcinoma are in led to higher efficiency in slowing tumor progression and progress and early signals of efficacy have recently been improving survival rate than single agent therapy . reported (Table 3). Encouraging clinical outcomes were 3-Deazaneplanocin A and 5-aza-2′deoxycytidine, two im- reported from an ongoing phase I/II trial of the portant DNMTi, enhanced the therapeutic efficacy of anti-PD-1 antibody nivolumab at the 2015 American PD-L1 blockade in reducing tumor volume, increasing Society of Clinical Oncology (ASCO) Annual Meeting tumor infiltrating CD8+ T cells and Th1-type chemokine held in Chicago . Waterfall plots showed that the expression in ovarian cancer in C57/BL6 mice . tumor size decreased to some extent in all cohorts in- Chiappinelli et al. demonstrated that 5-azacytidine, sensi- cluding uninfected, HBV-infected, and hepatitis C tized tumors to anti-CTLA-4 immune checkpoint therapy virus-infected HCC patients. It was significant and stable compared to 5-azacytidine or anti-CTLA-4 alone in a in the response to the treatment of nivolumab in HCC mouse model of melanoma . Enhancer of zeste homo- patients. In another recent ongoing trial of nivolumab log 2 blockade led to reduced PD-L1 mRNA levels and a Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 7 of 12 Table 3 Clinical trials with immune checkpoints therapy in liver cancers Cancer type Number Study arms Stage Status Design Primary Estimated Trial NCT outcome completion HCC 35 Nivolumab (anti PD-1 Ab) + LRT Phase 1 Recruiting Single Group July 2019 July 2019 NCT02837029 (Yttrium 90Y glass microspheres) Assignment HCC 154 PDR001 (anti PD-1 Ab) Phase 1 Recruiting Non-Randomized January 12, 2020 January 12, 2020 NCT02947165 + NIS793 (anti TGF-b Ab) HCC 114 Durvalumab (anti PD-1L Ab) Phase 1 Recruiting Non-Randomized March 2018 September 2018 NCT02572687 + ramucirumab (anti-VEGF-R2 Ab) HCC 51 Durvalumab (anti PD-1 L Ab) Phase 1 Recruiting Non-Randomized November 9, 2017 November 9, 2017 NCT02740985 + AZD4635 HCC 61 Tremelimumab (anti CTLA-4 Ab) Phase 1 Active, Non-Randomized December 31, 2017 December 31, 2018 NCT01853618 not recruiting Liver cancer 60 Ipilimumab (anti CTLA-4 Ab) Phase 1 Recruiting Non-Randomized May 2019 May 2019 NCT02668770 + MGN1703 (Toll-like receptor agonist) HCC 120 Ipilimumab (anti CTLA-4 Ab) Phase 1 Recruiting Randomized August 2019 August 2019 NCT02239900 + stereotactic body radiation HCC 75 Nivolumab (anti PD-1 Ab) Phase 1/2 Recruiting Non-Randomized April 2018 March 2019 NCT02423343 + galunisertib (TGF-b inhibitor) HCC 620 Nivolumab (anti PD-1 Ab) Phase 1/2 Recruiting Non-Randomized July 22, 2018 July 9, 2019 NCT01658878 + ipilimumab (anti CTLA-4 Ab) HCC 108 PDR001 (anti PD-1 Ab) Phase 1/2 Recruiting Non-Randomized December 24, 2018 December 24, 2018 NCT02795429 + INC280 (c-Met inhibitor) HCC 50 Prembrolizumab (anti PD-1 Ab) Phase 1/2 Recruiting Single Group September 2019 October 2019 NCT02886897 + dendritic cells, Assignment cytokine-induced killer cells HCC 15 Prembrolizumab (anti PD-1 Ab) Phase 1/2 Recruiting Single Group December 2019 December 2019 NCT02940496 Assignment HCC 50 Nivolumab (anti PD-1 Ab) Phase 1/2 Recruiting Single Group June 23, 2020 June 23, 2020 NCT02859324 + CC-122 Assignment (immunostimulatory pathway modifier) HCC 90 Durvalumab (anti PD-1 L Ab), Phase 1/2 Recruiting Non-Randomized April 30, 2020 April 30, 2021 NCT02821754 Tremelimumab (anti CTLA-4 Ab) + LRT HCC 620 Nivolumab (anti PD-1 Ab), Phase 1/2 Recruiting Non- Randomized September 4, 2018 July 9, 2019 NCT01658878 Nivolumab + Ipilimumab, Nivolumab + cabozantinib, Nivolumab + Ipilimumab + cabozantinib HCC 28 Pembrolizumab (Keytruda) Phase 2 Recruiting Single Group April 2018 April 2019 NCT02658019 (anti PD-1 Ab) Assignment HCC 440 Durvalumab (anti PD-1 L Ab) Phase 2 Recruiting Randomized March 20, 2020 March 20, 2020 NCT02519348 + Tremelimumab (anti CTLA-4 Ab) HCC 726 Nivolumab (anti PD-1 Ab) Phase 3 Recruiting Randomized October 1, 2018 June 22, 2019 NCT02576509 HCC 408 Prembrolizumab (anti PD-1 Ab) Phase 3 Active, not Randomized February 1, 2019 February 1, 2019 NCT02702401 recruiting HCC 1200 Durvalumab (anti PD-1 L Ab) Phase 3 Not yet Randomized February 27, 2020 March 29, 2021 NCT03298451 + tremelimumab recruiting (anti CTLA-4 Ab) decrease in PD-L1+ Pax3+ in melanoma cells, which was Combination therapy with immunotherapy and maintained during concomitant IL-2cx or anti-CTLA-4 chemotherapy or radiation therapy are being studied and immunotherapy . Taken together, these discoveries reported to be synergistic through multiple mechanisms. create a highly promising basis for combination studies As more data of these combinations is available, it will using epigenetic and immune checkpoint therapy in pa- likely improve outcomes for patients with this rare ag- tients with various cancers including liver cancer gressive group of cancers, and we will also be able to de- (Table 4). velop further trials to upgrade our understanding of Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 8 of 12 Table 4 Ongoing clinical trials combining epigenetic drugs and immune checkpoint blockade therapy in cancers Cancer type Number Immune checkpoint inhibitors Epigenetic drugs Stage Status Design Trial NCT HCC 90 Durvalumab Guadecitabine Phase 1 Recruiting Single Group NCT03257761 Assignment Unresectable NSCLC 41 Nivolumab ACY-241 Phase 1 Recruiting Single Group NCT02635061 and ipilimumab Assignment Metastatic unresectable 45 Pembrolizumab Entinostat Phase 1 Recruiting Single Group NCT02453620 HER2-negative Assignment breast cancer Advanced solid tumors 30 Pembrolizumab Entinostat Phase 1 Recruiting Randomized NCT02909452 Unresectable stage III/IV 17 Ipilimumab Panobinostat Phase 1 Recruiting Single Group NCT02032810 melanoma Assignment Advanced CRC 30 Pembrolizumab Romidepsin Phase 1 Recruiting Randomized NCT02512172 and/or 5-AZA MSS advanced CRC 30 Pembrolizumab Romidepsin Phase 1 Recruiting Randomized NCT02512172 and/or 5-AZA MDS following 27 Pembrolizumab Entionstat Phase 1 Recruiting Single Group NCT02936752 DNMTi-failed therapy Assignment Advanced solid tumors 45 Nivolumab RRx-001 Phase 1 Active, not Single Group NCT02518958 or lymphomas recruiting Assignment MM 19 Ipilimumab SGI-110 Phase 1 Recruiting Single Group NCT02608437 Assignment MDS 73 Durvalumab with or Azacytidine Phase 1 Recruiting Non-Randomized NCT02117219 without tremelimumab Advanced cell 62 Atezolizumab Entinostat Phase 1/2 Recruiting Single Group NCT03024437 carcinoma Assignment Breast cancer 88 Atezolizumab Entinostat Phase 1/2 Recruiting Randomized NCT02708680 DLBCL 5 Rituximab Belinostat Phase 2 Active, not Single Group NCT01686165 recruiting Assignment Metastatic uveal 29 Pembrolizumab Entinostat Phase 2 Recruiting Single Group NCT02697630 melanoma Assignment DLBCL 42 Rituximab Panobinostat Phase 2 Active, not Randomized NCT01238692 recruiting Advanced solid tumors 119 Durvalumab Mocetinostat Phase 1/2 Recruiting Single Group NCT02805660 and NSCLC Assignment NSCLC and melanoma 202 Pembrolizumab Entinostat Phase 1/2 Recruiting Non-Randomized NCT02437136 HNSCC and SGC 49 Pembrolizumab Vorinostat Phase 1/2 Active, not Single Group NCT02538510 recruiting Assignment Stage IV NSCLC 100 Pembrolizumab Vorinostat Phase 1/2 Recruiting Randomized NCT02638090 DLBCL 83 Rituximab Vorinostat Phase 1/2 Active, not Single Group NCT00972478 recruiting Assignment Lymphoma/leukaemia 40 Rituximab Vorinostat Phase 1/2 Active, not Single Group NCT00918723 recruiting Assignment Advanced renal or 42 Pembrolizumab Vorinostat Phase 2 Recruiting Non-Randomized NCT02619253 urothelial cell carcinoma Hormone therapy-resistant 87 Pembrolizumab Vorinostat Phase 2 Recruiting Randomized NCT02395627 breast cancer AML 182 Nivolumab 5-AZA Phase 2 Recruiting Non-Randomized NCT02397720 Metastatic CRC 31 Nivolumab 5-AZA Phase 2 Active, not Single Group NCT02260440 recruiting Assignment Advanced/metastatic 100 Nivolumab 5-AZA Phase 2 Active, not Randomized NCT02546986 NSCLC recruiting MDS 120 Nivolumab and/or 5-AZA Phase 2 Recruiting Non-Randomized NCT02530463 ipilimumab Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 9 of 12 Table 4 Ongoing clinical trials combining epigenetic drugs and immune checkpoint blockade therapy in cancers (Continued) Cancer type Number Immune checkpoint inhibitors Epigenetic drugs Stage Status Design Trial NCT Refractory/relapsed AML 37 Lirilumab 5-AZA Phase 2 Active, not Single Group NCT02399917 recruiting Assignment MDS 12 Lirilumab and nivolumab 5-AZA Phase 2 Active, not Non-Randomized NCT02599649 recruiting Metastatic 71 Pembrolizumab 5-AZA Phase 2 Recruiting Non-Randomized NCT02816021 melanoma NSCLC 120 Nivolumab 5-AZA and/or Phase 2 Recruiting Randomized NCT01928576 entinostat NSCLC 60 Nivolumab 5-AZA- CdR/ Phase 2 Recruiting Randomized NCT02795923 tetrahydrouridine Advanced solid 60 Durvalumab 5-AZA Phase 2 Recruiting Single Group NCT02811497 tumors Assignment Advanced/metastatic 100 Pembrolizumab Oral azacytidine Phase 2 Active, not Randomized NCT02546986 NSCLC recruiting PR recurrent OC 38 Pembrolizumab Guadecitabine Phase 2 Recruiting Single Group NCT02901899 Assignment PR recurrent OC 20 Pembrolizumab Oral azacytidine Phase 2 Recruiting Randomized NCT02900560 MDS 120 Durvalumab Oral azacytidine Phase 2 Recruiting Randomized NCT02281084 MDS, AML 213 Durvalumab Azacytidine Phase 2 Active, not Randomized NCT02775903 recruiting Refractory/recurrent 138 Avelumab Entinostat Phase 2 Recruiting Randomized NCT02915523 epithelial OC DLBCL 304 Rituximab 5-AZA Phase 3 Recruiting Randomized NCT02951156 HCC hepatocellular carcinoma, NSCLC Non-small cell lung cancer, HER2 human epidermal growth factor receptor 2, CRC colorectal cancer, 5-AZA Azacitydine, MSS Microsatellite stable, MDS Myelodysplastic syndromes, DNMTi DNA methyltransferase inhibitor, MM Multiple myeloma, DLBCL Diffuse large B cell lymphoma, HNSCC head and neck squamous cell carcinoma, SGC salivary gland cancer, AML Acute myeloid leukaemia, OC ovarian cancer therapies targeting liver cancers. Therefore, immuno- may enable re-expression of immune related thera- therapy offers hope to liver cancer patients with a dismal peutic genes, especially in combination of immunother- prognosis that has not seen significant changes in ther- apy [79, 80]. They can also increase expression of apy for a long time. immune checkpoints to synergize with immune check- point blockade therapy, leading to improving Limitations and perspectives of immune anti-tumor responses . checkpoint therapy Resistance to immune checkpoint blockades is still com- Conclusions monly observed in most cancer patients . Failure of Most liver cancers are diagnosed at an advanced stage, immune checkpoint inhibitors therapy can result from while the therapy is limited. Immune checkpoint therapy three categories: (1) mutations of the immunogenicity of provides survival benefit for liver cancer treatment. Epi- cancer itself. The mutations influence expression of genetic regulation mechanistically and functionally links components of antigen-processing and presentation ma- with immune checkpoints. Epigenetic mechanisms of chinery (e.g., transporter associated with antigen pro- checkpoint blocking prove to be promising in treat- cessing, HLA class molecules, and β2 microglobulin), ing liver cancers and determining patient prognosis. Fur- novel tumor-associated antigens (e.g., cancer-testis anti- ther investigations are required to explore the clinical gens, neoantigens), and cytokines; (2) expression of al- potential in combination with epigenetic and immune ternative immune checkpoint ligands on tumor cells checkpoint therapy for liver cancer treatment. (and/or immune cells). Expression of alternative co-inhibitory immune checkpoints (e.g., CTLA-4, Abbreviations APC: Antigen presenting cell; ASCO: American Society of Clinical Oncology; TIM-3, LAG-3, and VISTA) has been associated with re- BTLA: B- and T-lymphocyte attenuator; CTLA-4: Cytotoxic T lymphocyte– sistance to PD-1 blockade [76, 77]; or (3) defects in T associated antigen 4; DNMT1: DNA methyltransferase 1; DNMTi: DNA cell infiltration. Diminished infiltration of T cells led to methyltransferase inhibitors; GITR: Glucocorticoid-induced tumor necrosis factor receptor-related gene; HBV: Hepatitis B virus; HCC: Hepatocellular resistance to PD-1 blockade in melanoma patients . carcinoma; HDACi: Histone deacetylase inhibitors; HLA: Human leukocyte However, epigenetic modifying agents including antigens; HVEM: Herpesvirus entry mediator; ICC: Intrahepatic demethylating agents and histone deacetylase inhibitors cholangiocarcinoma; IDO: Indoleamine 2,3-dioxygenase; KIRs: Killer cell Xu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:110 Page 10 of 12 immunoglobulin-like receptors; LAG-3: Anti-lymphocyte activation gene-3; 15. Chiappinelli KB, Zahnow CA, Ahuja N, Baylin SB. Combining epigenetic and lncRNAs: long noncoding RNAs; miRNAs: microRNAs; PD-1: Programmed cell immunotherapy to combat cancer. Cancer Res. 2016;76:1683–9. death protein-1; PD-L1: Programmed cell death ligand 1; 16. Philips GK, Atkins M. Therapeutic uses of anti-PD-1 and anti-PD-L1 TAA: Tumor-associated antigens; TAM: Tumor-associated macrophages; antibodies. Int Immunol. 2015;27:39–46. TILs: Tumor-infiltrating lymphocytes; Tim-3: T-cell immunoglobulin- and 17. Buchbinder EI, Desai A. CTLA-4 and PD-1 pathways: similarities, differences, mucin-domain-containing molecule-3; Tregs: Regulatory T cells; and implications of their inhibition. Am J Clin Oncol. 2016;39:98–106. VISTA: V-domain Ig suppressor of T-cell activation 18. Ramagopal UA, Liu W, Garrett-Thomson SC, Bonanno JB, Yan Q, Srinivasan M, Wong SC, Bell A, Mankikar S, Rangan VS, Deshpande S, Korman AJ, Almo SC. Acknowledgements Structural basis for cancer immunotherapy by the first-in-class checkpoint The authors acknowledge the contribution of all investigators at the inhibitor ipilimumab. Proc Natl Acad Sci U S A. 2017;114:E4223–32. participating study sites. 19. Mizukoshi E, Nakamoto Y, Arai K, Yamashita T, Sakai A, Sakai Y, Kagaya T, Yamashita T, Honda M, Kaneko S. Comparative analysis of various tumor- Funding associated antigen-specific t-cell responses in patients with hepatocellular This work was supported by Shenyang Science and Technology Project carcinoma. Hepatology. 2011;53:1206–16. (No. 17-230-9-16). 20. Duggleby R, Danby RD, Madrigal JA, Saudemont A. Clinical grade regulatory CD4(+) T cells (Tregs): moving toward cellular-based immunomodulatory Authors’ contributions therapies. Front Immunol. 2018;9:252. 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Journal of Experimental & Clinical Cancer Research – Springer Journals
Published: May 29, 2018
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