Immune checkpoints in the tumor microenvironment

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Review

Immune checkpoints in the tumor microenvironment Salman M. Toora, Varun Sasidharan Naira, Julie Decocka, Eyad Elkorda,b,* a b

Cancer Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar Institute of Cancer Sciences, University of Manchester, Manchester, United Kingdom

ARTICLE INFO

ABSTRACT

Keywords: Immune checkpoints Immune checkpoint inhibitors Tumor microenvironment

Interactions between immune checkpoints (ICs) and their ligands negatively regulate T cell activation pathways involved in physiological immune responses against specific antigens. ICs and their ligands are frequently upregulated in the tumor microenvironment (TME) of various malignancies, and they represent significant barriers for induction of effective anti-tumor immune responses. Several IC inhibitors (ICIs) have been developed, with some currently in clinical trials and others have been approved for the treatment of different cancers. However, tumor cells are able to counteract the activity of ICIs and can commission additional inhibitory pathways via expression of other ICs/ligands within the TME. This review discusses the expression of various ICs/ligands in the TME and their impact on tumor immune evasion. Additionally, we discuss various regulatory mechanisms, including genetic and epigenetic, and other modulatory factors including hypoxia and the presence of immunosuppressive populations in the TME, which result in upregulation of ICs in various cancers. Moreover, we discuss the prognostic significance of ICs and their ligands, and the potential strategies to enhance treatment responses to ICIs. This review aims to advance our current knowledge on the role of ICs in the TME and the clinical benefits of targeting them.

1. Introduction Immune checkpoint (IC) pathways attenuate T cell responses to prevent autoimmunity and maintain immune homeostasis. However, tumors can hijack these immune-inhibitory pathways to attain immune resistance against tumor antigen-specific T cells. ICs and their ligands are frequently upregulated in the tumor microenvironment (TME) of various malignancies. These upregulations and their role in tumor immune evasion lead to the development of therapeutic agents to block interactions between ICs and their ligands to initiate effective antitumor responses. Several immune checkpoint inhibitors (ICIs) have been developed that have shown promising results in some cancers. However, studies have shown that the therapeutic efficacy of ICIs and clinical response are dependent on the nature of the tumor, as evident by low response rates and relapse in some cancer patients. These patients mainly present with ‘non-immunogenic’ tumors, characterized by lack of T cell activation and priming [1]. To date, blocking the interactions between cytotoxic T lymphocyte antigen 4 (CTLA-4) and CD80/86, and between programmed death 1 (PD-1) and PD-L1/2 are the most prominent and effective strategies for IC inhibition (ICI). Initial studies demonstrated that CTLA-4 blockade improved the overall survival in patients with metastatic melanoma

[2], while PD-1 blockade was associated with high objective response rates in renal cell cancer, small-cell lung cancer and melanoma patients [3]. ICIs have since then shown positive clinical responses in several malignances, including non-small cell lung cancer (NSCLC), colorectal cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, and hepatocellular carcinomas [4–9]. Fig. 1 shows an overview of the IC interactions with their ligands on tumor cells and lists the current US Food and Drug Administration (FDA) approved ICIs. Response to ICIs is highly reliant on the immune constituents of the TME due to dynamic interactions between immune cells and tumor cells. This review focuses on the expression of different ICs and their ligands in the TME, and the mechanisms of their regulation. Additionally, we discuss the prognostic significance of IC expression and their ligands in cancer patients and the potential strategies to enhance responses to ICIs for more effective anti-tumor immune responses. 2. Immune checkpoints in the tumor microenvironment Tumor growth is favored by upregulations of ICs in the TME, which suppress anti-tumor T cell responses. ICs are highly expressed on

⁎ Corresponding author at: Cancer Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar. E-mail addresses: [email protected], [email protected] (E. Elkord).

https://doi.org/10.1016/j.semcancer.2019.06.021 Received 30 May 2019; Received in revised form 15 June 2019; Accepted 28 June 2019 1044-579X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Salman M. Toor, et al., Seminars in Cancer Biology, https://doi.org/10.1016/j.semcancer.2019.06.021

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dysfunctional and exhausted CD8+ T cells characterized by defective effector function and reduced proliferation [10]. Some recent reports have shown significant accumulation of exhausted T cells, expressing multiple ICs including PD-1 and CTLA-4 in the TME of patients with breast and colorectal cancers [11,12]. Additionally, T regulatory cells (Tregs) in the TME upregulate multiple ICs including PD-1, lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) [13]; with FoxP3+ Tregs expressing TIM-3/LAG-3 and PD-1 being highly suppressive [14,15]. Bone marrow aspirates from acute myeloid leukemia patients showed an increase in Tregs and exhausted T cells indicated weakened immune responses [16]. Fig. 2 shows an example of accumulation of highly suppressive FoxP3+Helios+ Tregs, co-expressing PD-1 and CTLA-4, in the TME as compared to normal tissue. Key negative regulators of T cell activation include CTLA-4, PD-1, TIM-3, indoleamine 2,3-dioxygenase (IDO), V-domain Ig suppressor of T cell activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), TIGIT, B and T lymphocyte attenuator (BTLA) and LAG-3 [10,17]. In addition, recent studies have reported novel ICs that are associated with tumor immunity and may be targeted for effective antitumor immune responses, including the ectonucleotidases CD39 and CD73, carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), and the poliovirus receptor (PVR)-like receptors [18]. Specific glycan signatures found on tumor cells, such as N-glycans that stabilize PD-L1 by reducing its proteasomal degradation to enhance its immunosuppressive activity, are also proposed as novel ICs [19]. In this

Fig. 1. Overview of interactions between immune checkpoints and their ligands, and the therapeutic intervention through immune checkpoint inhibitors. T cells are activated through antigen presentation by MHC class II molecules on the surface of antigen-presenting cells (APCs) or MHC class I expressed on tumor cells, recognized by T cell receptor (TCR). Immune checkpoints such as, PD-1, CTLA-4, VISTA and TIGIT are expressed on activated or exhaustive T cells. Interactions of ICs with their ligands on tumor cells lead to T cell inactivation, which impede tumor cell killing by cytotoxic T cells. Monoclonal antibodies have been developed to inhibit these interactions to ensure sustained T cell activation and effective anti-tumor responses. These agents include ipilimumab targeting CTLA-4, nivolumab and pembrolizumab targeting PD-1 and atezolizumab, avelumab and durvalumab targeting PD-L1 on tumor cells.

Fig. 2. Immune checkpoint co-expression in the tumor microenvironment. Representative flow cytometric plots show the levels of CTLA-4+/− and PD-1+/− cells in CD3+CD4+FoxP3-Helios- non-Tregs and CD3+CD4+FoxP3+Helios+ Tregs isolated from colon normal tissue (A) and colon tumor tissue (B). FoxP3+Helios+ Treg are accumulated in the tumor microenvironment compared to colon normal tissue and the vast majority of these cells co-express CTLA-4 and PD-1. Additionally, the levels of FoxP3-Helios- non-Tregs that co-express CTLA-4 and PD-1 are also significantly higher in tumor tissue compared to normal tissue. These plots show that T cells in the tumor microenvironment co-express different immune checkpoints, highlighting the potential for combinational therapies to target them. 2

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section, we give a brief overview of the key ICs and their role in the TME.

Galectin-9 leads to dissociation of human leukocyte antigen B (HLA-B)associated transcript 3 (Bat3), which leads to decreased IFN-γ production and reduced T cell proliferation [48]. Moreover, these interactions trigger cell death in effector Th1 cells (Teff), thereby reducing tissue inflammation and leading to inhibition of autoimmune disease. In addition, interactions between CEACAM1 and TIM-3 are crucial for TIM-3 maturation, expression and functions on exhausted T cells [49]. Several functional anti-TIM-3 antibodies have been developed that are being tested to promote effective anti-tumor immunity in various in vitro and phase I clinical trials [50].

2.1. CTLA-4 CTLA-4 (CD152) is upregulated on T cells following engagement of the T cell receptor (TCR), and competes with the co-stimulatory molecule CD28 for binding to the B7 ligands (CD80 and CD86) on antigen presenting cells (APC) to effectively dampen T cell activation [20]. Its expression is associated with intracellular vesicles avidly trafficking to the surface immunologic synapse [21]. Upon binding to its ligands, CTLA-4 is phosphorylated to activate phosphoinositide 3-kinase (PI3K) pathways that lead to dephosphorylation of the CD3ζ chain, limiting the signaling potential of the TCR [22]. In addition, CTLA-4 inhibits T cell proliferation through the inhibition of IL-2 transcription [23]. CTLA-4 is constitutively expressed on Tregs and can affect Treg-mediated immune regulation [24,25] and studies have shown that CTLA-4blocking antibodies deplete Tregs in the TME [26–28]. Elevated CTLA-4 expression in the TME has been linked with poor prognosis in thymoma and Her2 negative breast cancer patients [29,30]. However, no correlations were found between CTLA-4 levels and overall survival of patients with different types of cancers [31]. In addition to treatment of melanomas and non-small cell lung carcinoma using ipilimumab (CTLA-4 inhibitor), combination therapy with a PD-1 blocker (nivolumab) improved the therapeutic efficacy in patients with microsatellite instability-high (MSI-H) or DNA mismatch repair deficient (dMMR) metastatic colorectal cancers [32,33] and lead to accelerated approval in treating such patients.

2.4. IDO IDO is a rate-limiting enzyme that has been shown to be highly expressed in the TME of various malignancies, and is associated with the conversion of the essential amino acid tryptophan (Trp) into catabolites known as kynurenines (Kyn) [51]. The IDO pathway has been proposed as an innate immune mechanism to defend against infections [52]. In the context of tumor immunity, studies have shown high IDO1 protein production by tumor cells and other immune cells in the TME. IDO1 assists immunosuppression via activation and expansion of Tregs and it also leads to increased levels of Kyn that abrogates Teff and NK cell functions [53]. Therefore, Kyn/Trp ratio may be used as a surrogate prognostic biomarker to monitor cancer growth and progression. Elevated Kyn/Trp ratios and elevated IDO1 activity have been associated with poor prognosis and low survival rates of cancer patients diagnosed with glioblastoma [54]. Currently, several IDO inhibitors, which primarily target the Kyn pathway are in clinical trials [55].

2.2. PD-1

2.5. VISTA

PD-1 signaling involves interactions between PD-1 and its ligands, PD-L1 and PD-L2, which counteract TCR signal transduction and costimulation [34,35]. PD-1 is primarily expressed on activated T cells upon TCR stimulation or exposure to the common gamma chain cytokines such as IL-2, IL-7, IL-15 and IL-21 [36]. Interactions with its ligands on APCs or tumor cells lead to impaired production of inflammatory cytokines, cell cycle arrest and diminished transcription of cell survival proteins such as Bcl-XL, dephosphorylation of ZAP70, and phosphorylation of PI3K by recruiting SHP1 and SHP2 phosphatases [37,38]. Tumors exploit these pathways via upregulation of PD-L1 to engage PD-1-expressing tumor-reactive T cells and assist in immunosuppression. Several studies have reported expansion of PD-1 expression within the TME of various malignancies [11,12,39–41]. Successful blockade of the PD-1/PD-L1 axes has shown positive responses in a variety of malignancies, which lead to FDA approvals of PD-1 blockers, such as pembrolizumab and nivolimumab as single or part of combined therapy regimes [42].

VISTA serves as a stimulatory ligand for APCs to favor immune activation but serves as a negative regulatory ligand for T cells, suppressing their activation, proliferation, and cytokine release [56]. It presents as both, ligand and receptor. VISTA is highly expressed within the TME on myeloid cells and also induces Foxp3 expression on CD4+ T cells [56]. In gastric cancer patients, VISTA expression was found at moderate levels on tumor cells compared to immune cells, the latter expressing VISTA in 83.6% of patients [57]. Moreover, VISTA expression correlated with PD-L1 expression, suggesting overlapping mechanisms with the PD-1 pathway but no correlation was reported with the disease outcome [57]. In addition, blocking VISTA effectively impaired the growth of established tumors, even though it was not detected at high levels in tumor-bearing mice [58]. VISTA was highly expressed in ovarian and endometrial tumor cells and suppressed T cell activity, while its inhibition significantly prolonged the survival of tumor-bearing mice [59]. VISTA is currently being explored as a candidate target for cancer immunotherapy.

2.3. TIM-3

2.6. KIR

TIM-3 (HAVCR2) is widely expressed on IFN-γ-producing T cells, FoxP3+ Tregs, dendritic cells (DCs), B cells, macrophages, natural killer (NK) cells, and mast cells [43]. Dysregulation of TIM-3 expression has been shown to be associated with onset of autoimmune diseases. Targeting TIM-3 leads to increased severity of experimental autoimmune encephalomyelitis [44]. Elevated TIM-3 expression correlates with suppression of T cell responses and T cell exhaustion, characterized by loss of T cell functions during chronic viral infections and during tumor development [45]. TIM-3 has been shown to be highly expressed within the TME, suggesting its potential role in tumor immunity. TIM-3 upregulation was associated with tumor antigen-specific exhausted CD8+ T cells in melanoma patients, which was effectively overturned by antiTIM-3 mAbs [46]. Mainly four putative ligands have been identified for TIM-3, Galectin-9, high mobility group protein B1 (HMGB1), phosphatidylserine (PtdSer) and CEACAM-1 [47]. Binding of TIM-3 with

KIR are a broad family of cell surface proteins found on natural killer (NK) cells. These molecules inhibit the killing function of NK cells by interacting with MHC I molecules, expressed on a variety of cellular populations, including tumor cells [60]. KIRs induce NK cell tolerance through a process called licensing, during which, KIR recognize a self MHC I molecule and prevent NK cell activation against self-tissue and auto-antigens [61]. Therefore, KIR ICIs can be used to treat cancers via unchecked cytotoxic ability of NK cells. Lirilumab is such ICI, currently in trials to treat leukemia and some solid tumors as monotherapy [62] and in combination with nivolumab and ipilimumab [63]. 2.7. TIGIT TIGIT is an immunosuppressive receptor expressed by NK and T cells. TIGIT exerts its immunosuppressive effects by competing with the 3

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immunoactivator receptor CD226 (DNAM-1) for binding to the ligands PVR (CD155) and PVRL2 (CD112) that modulate cytokine release by dendritic cells (DC), thereby indirectly inhibiting T cell activity [64]. TIGIT leads to the release of immunoregulatory cytokines, decrease in production of interferon gamma (IFN-γ) and IL-17, and prevents DC maturation [63]. In cancer, upregulation of TIGIT was reported in various malignancies and it has been shown to influence NK cellmeditated cytotoxic killing of tumor cells [65,66]. In addition, TIGIT blockade restored CD8+ T cell levels and activity, indicating that it affects anti-myeloma immune responses [67]. Phase I trials exploiting TIGIT as a novel target for ICI are currently underway [68].

and severe infection [84]. Furthermore, ICs can also be regulated through compensatory/mutual mechanisms. Reports showed that blockade of PD-1 can upregulate other checkpoints including TIGIT [85], TIM-3 [86], LAG-3 [87] and CTLA-4 [87] as compensatory mechanisms in many tumor settings. In murine HNSCC models, it was reported that PD-1 blockade upregulates TIM-3 in exhausted TILs as a compensatory mechanism [86]. In line with this, another pre-clinical ovarian study showed that triple blockade of PD-1/LAG-3/CTLA-4 resulted in 20% higher tumor-free survival in treated mice, compared with dual blockade of PD-1/CTLA-4 with increased infiltration of CD8+ T cells within the TME [87]. These data suggest that the virtual impact of ICs to anti-tumor immunity includes both canonical and compensatory mechanisms, which need to be considered in combinational therapy strategies. Notably, the compensatory upregulation of ICs also leads to an opportunity for selecting suitable alternative target(s) for combination therapy. Additionally, epigenetic modification play important roles in regulation of ICs expression as will be discussed below.

2.8. BTLA BTLA is an inhibitory receptor that is structurally and functionally associated with CTLA-4 and PD-1, and is expressed by the majority of lymphocytes. Ligation of BTLA by its ligand, herpes virus entry mediator (HVEM), blocks B and T cell activation, proliferation, and cytokine production [69]. Moreover, BTLA expressed on γδ T cells, negatively regulates the proliferation of potent effector Vγ9Vδ2T cells [70]. Unlike other ICs, BTLA-4 expression progressively decreases upon T cell activation, suggesting naïve T cells lose its expression during differentiation but its ligation constitutively inhibits T cell activation [71]. Increased BTLA and HVEM expressions have been shown to be associated with lymph node metastasis and poor prognosis of gastric cancer [72]. Tumor-specific CD8+ T cells in melanoma patients were inhibited by interaction of BTLA with HVEM on the tumor, even after progressive differentiation of the T cells [73]. Anti-BTLA monoclonal antibodies to serve as B cell receptor agonists and T cell differentiation antigen antagonists are currently in development in preclinical studies [74].

3.1. Epigenetic regulation of immune checkpoint expression in the tumor microenvironment Epigenetic modifications play a key role in the development and shaping of the primary tumor. The major epigenetic modifications include DNA methylation and post-translational histone modifications (PTMs), which can alter the structure of chromatin to regulate the gene expression without changing the existing nucleotide sequences [88]. Notably, it has been reported that epigenetic modulations occurring within the TME can favor tumor cells in immune resistance and evasion mechanisms [89]. Moreover, reversing these modifications can improve anti-tumor immune responses to restore immune surveillance and homeostasis [90]. Recent studies reported that DNA hypomethylation and decreased repressive histones, H3K9me3 and H3K27me3, on the promoter regions can upregulate the transcriptomic expression of ICs including PD-1 and CTLA-4, TIM-3 and LAG-3 in breast and colorectal TME [91–93]. Hence, advanced state-of-the-art technologies to screen genome-wide-epigenetic alterations of ICs in cancer could improve our understanding of IC regulation and pave the way for novel therapeutic methodologies.

2.9. LAG-3 LAG-3 (CD223) is expressed on activated T cells and NK cells [75]. MHC class II are recognized as putative ligands for LAG-3 [76]. Liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin) is also recognized as a ligand for LAG-3, these interactions can inhibit IFNγ release by Teff [77]. LAG-3 expressed on CD4+ T cells binds to MHC Class II on APCs, while LAG-3 expression on Teff and NK cells binds to LSECtin on tumor or liver cells [10]. LAG-3 pathway leads to a negative regulatory effect over T cell function, preventing tissue damage and autoimmunity. KIEELE motif, present on the cytoplasmic domain of LAG-3, is crucial for interaction of LAG-3 with downstream signaling molecules [78]. LAG-3 and PD-1 are frequently co-expressed and upregulated within the TME and lead to immune exhaustion and tumor growth [79]. LAG-3-mediated IC pathways impede cell cycle progression and its blockade improves anti-tumor immune responses [75,80]. Several LAG-3 modulating strategies are being investigated in pre-clinical and in clinical trials, such as BMS986016 in combination with nivolumab to treat melanoma patients [81].

3.1.1. Role of DNA methylation in immune checkpoint regulation DNA methylation of ICs and ligands is one of the most studied epigenetic alterations occurring within the TME of various malignancies. This modification is characteristically limited to cytosineguanine dinucleotide (CpG) islands [94]. Marked DNA hypomethylation of the promoter regions of ICs and ligands in various malignancies is associated with increased expression within the TME [92,93,95]. In melanoma, loss of global DNA methylation in the intergenic and repeat elements leads to constitutive expression of PD-L1, which inhibits the anti-tumor immune responses [95]. Another study on chronic lymphocytic leukemia showed that hypomethylation of three regions (intron, promoter and enhancer) within PD-1 is associated with its aberrant expression [96]. Furthermore, in non-small cell lung cancer both PD-1 and CTLA-4 were found to be hypomethylated, which positively correlated with their upregulation in tumor tissue, compared to matched normal tissue [97]. These data suggest that combination of targeted DNA hypermethylating agents together with immune checkpoint inhibitors can augment the anti-tumor responses; as the former may decrease their expression within the TME, which may augment the action of immune checkpoint inhibitor. The role of the promoter DNA demethylation in regulation of ICs/ligands in tumor-infiltrating T cells/ tumor cells is illustrated in Fig. 3.

3. Regulation of immune checkpoints There are limited information in the literature regarding the regulation of ICs expression in the TME. This could rely on multiple factors including hypoxia, tumor-associated macrophages/tumor-associated neutrophils (TAMs/TANs), exhausted T cells and compensatory mechanisms. It has been reported that under hypoxic conditions, the upregulation of CD73 and CD39 can induce extracellular adenosine level, which subsequently upregulates the expression of PD-1 and CTLA-4 [82]. TAMs and TANs also play key roles in the upregulation of both ICs and ligands in the TME. A study showed that immunosuppressive function of TAMs can upregulate both PD-1 and PD-L1 within the TME [83]. Additionally, T cell exhaustion occurs during chronic infections were reported to be associated with the co-expression of multiple inhibitory checkpoints, including PD-1 and LAG-3 for greater exhaustion

3.1.2. Role of histone modification in immune checkpoint regulation Histone modifications including acetylation, methylation, ubiquitination and phosphorylation are important players in the regulation of gene expression within the TME. It has been reported that histone acetylation 4

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3.1.3. Role of miRNA and lncRNA in immune checkpoint regulation Similar to coding proteins, miRNAs also have a large proportion of CpG islands, which are subjected to modifications in various malignancies [103,104]. Cortez et al. revealed a novel tumor immune evasion pathway mediated by p53/miR-34/PD-L1 that regulates the expression of PD-L1 in lung cancer [105]. Based on bioinformatic predictions, Fooladinezhad et al. suggested that expression of miR-3305p could inhibit the TIM-3 expression on AML cells [106]. Moreover, miRNAs including miR-146, miR-124, miR-138, miR-200, miR-424 (322), miR-9 and miR-29 regulate the expression of PD-1, CTLA-4, TIM3 and LAG-3 in various malignancies [107]. Reports showed that miRNAs can regulate the expression of ICs through direct or indirect interactions. Several miRNAs including miR-15/16, miR-138, miR-28 can directly interact with PD-1, CTLA-4, TIM-3 and LAG-3 and imitate the therapeutic effect of multiple ICI in various cancer models [107]. Similar to miRNAs, lncRNAs are also key players in the development and progression of tumor and are regulated by various epigenetic modifications [108,109]. It has been reported that co-expression of lncRNA AFAP1-AS1 and PD-1 was associated with poor prognosis in nasopharyngeal carcinoma [110]. These data suggest that epigenetic modifications play a key role in the expression of both miRNA/lncRNA and ICs/ligands in various malignancies. The above data suggest that the epigenetic modifications in miRNA/lncRNA positively correlate with the expression of ICs and ligands in various malignancies. 4. Immune checkpoint ligands in the tumor microenvironment Cancer cells overexpress inhibitory ligands due to oncogenic signaling and in response to inflammatory cytokines to enhance tumor tolerance and evade eradication by the immune system. Some studies have shown a strong correlation between IC ligand expression on tumor cells and response to IC blockade. Tumors expressing IC ligands respond better to ICIs, for example PD-L1+ tumors were more responsive to PD1/PD-L1 blockers compared to PD-L1− tumors [111]. CTLA-4 ligands (CD80/86) are abundantly present on tumor cells and antigen presenting cells in the TME [112]. Additionally, LSECtin, ligand for LAG-3, is expressed by liver and tumor cells [10], and galectin-9 is highly expressed on tumor cells [13]. Nectin-2 (CD112), ligand for TIGIT is also highly expressed on tumor cells [113]. Moreover, V-Set and immunoglobulin domain containing 3 (VSIG-3), ligand for VISTA, is elevated in different cancers and this interaction significantly affects T cell cytokine and chemokine secretion [114]. In addition to tumor cells, IC ligands are also expressed on immune cells such as myeloid cells. For instance, PD-L1 is expressed on monocytes within the TME and was found to inhibit T-cell proliferation and promote the induction of FoxP3+ Tregs [115]. Galectin-9 is also expressed on T cells and is shown to regulate immune responses by decreasing levels of Th17 cells and increasing the levels of FoxP3+ Tregs in vivo and in vitro [116]. Abundance of putative ligands for ICs in the TME may increase success of ICI since it shows that tumors are actively exploiting different IC pathways to inhibit T cell responses and therefore, provide additional opportunities/targets for ICI to block these pathways for restoration of effective anti-tumor responses. However, in some instances, patients with high IC ligand expression do not respond to IC blockade, while others with lower levels could respond. This may be due to the presence of other regulatory and compensatory mechanisms affecting tumor immune biology. Better understanding of individual patients’ TME may lead to improved ICI response by providing multiple targets for combination therapies, while better understanding of regulatory mechanisms of IC/ligand expression in the TME would assist in the success of these therapies.

Fig. 3. Effects of DNA methylation and histone modifications in the promoter region of ICs/ligands within the TME. The effects of DNA/histone epigenetic alterations in T cells (A) and cancer cells (B) are illustrated. The epigenetic modifications of ICs on T cells can lead to less responsive T cells, increased Treg levels, increased MDSC trafficking and impaired effector cytokine release. The epigenetic modifications in tumor cells can facilitate metastasis and immune evasion, and establish an immunosuppressive microenvironment.

enhances the cell surface expression of ICs and ligands, and inhibition of histone acetylase can favor immune checkpoint blockade by reducing the trafficking of myeloid-derived suppressor cells (MDSC) to the TME in mouse models [98]. Histone deacetylase (HDAC) inhibitor upregulated the expression of PD-L1 in melanoma, and improved response to PD-1 blockade [99]. Another in vitro study on breast cancer cell lines showed that histone acetyl transferase (HAT) is associated with the upregulation of PD-L1 and epithelial to mesenchymal transition (EMT) [100]. Moreover, Liu et al. found that tumor progression was controlled by limiting the suppressive function of Tregs upon treatment with one of the HAT inhibitors, EP300i, in immunocompetent mice [101]. Notably, preclinical studies in lung and melanoma showed that upregulation of T-cell chemokine expression upon HDAC inhibitor treatment could augment PD-1/ PD-L1 immuno-blockade therapy [99,102]. Furthermore, histone methylation is also associated with the upregulation of immune checkpoint expression within the TME. In the breast cancer TME, the abundance of repressive histones, H3K9me3 and H3K27me3, was found to be significantly reduced in PD-1, CTLA-4, TIM-3 and LAG3 promoter regions, compared with normal tissue [92]. Additionally, in the colorectal TME, binding of H3K27me3 in CTLA-4 and H3K9me3 in PD-1 and TIGIT promoter loci was significantly reduced in tumor tissue [93]. These reports rationalize that abundance of repressive histone methyl marks in the promoter region of ICs could be one of the significant causes of upregulation within the TME. The regulatory role of HAT and HDAC distribution in the promoter regions of ICs/ligands in TILs/tumor cells is summarized in Fig. 3. 5

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4.1. Regulatory mechanisms of PD-L1 expression in the tumor microenvironment

between IC expression in the TME and disease progression. In clear cell renal carcinoma, high IC expression in the absence of mature DC was associated with increased risk of disease progression, while low IC expression with localization of mature DC was associated with good prognosis [142]. A recent study reported that elevated LAG-3 expression correlates with better overall survival and progression free survival in esophageal squamous cell carcinoma patients [143]. Co-expression of LAG-3 with PD-1 on CD8+ TILs also has also gained attention as a prognostic marker in melanoma patients [144]. Moreover, in patients with nonsmall-cell lung cancer also, the presence of LAG-3+ TILs was associated with improved disease-specific survival [145]. TIM-3 expression in prostate cancer tissues is also proposed as an independent prognostic factor of poor prognosis, since low TIM-3 expression correlated with shorter overall survival in patients with metastatic prostate cancer [146]. In addition, it was recently reported that overexpression of TIGIT ligands, PVR and PVRL2, also correlates with poor survival in AML patients [147]. A study on 536 NSCLC patients presenting with stage I-IIIA showed diverging prognostic importance of CTLA-4 at late metastatic stages [148]. Furthermore, another study on thymoma patients showed that overexpression of CTLA-4 significantly correlated with poor survival [29]. In contrast, no significant correlation of increased intratumoral CTLA-4 expression with overall survival was reported in pooled analyses of patients with different cancers [31].

PD-L1 is generally not detectable in normal tissues but tumors upregulate PD-L1 in response to IFN-γ released by tumor-infiltrating lymphocytes to suppress local Teff function [111]. Tumor expression of PD-L1 is regulated by various mechanisms including genetic and epigenetic alterations, (post)transcriptional and (post)translational modifications. At genomic level, gene amplification and chromosomal translocation at 9p24.1, resulting in the use of an ectopic promoter, have been shown to increase the expression of PD-L1 in Hodgkin’s lymphoma [117]. Furthermore, an increasing number of transcription factors have been identified to regulate PD-L1 expression including IRF1, AP-1 components, NF-kB, STAT3, c-Myc, HIF1 and the transcription coactivators YAP1 and TAZ [118–120]. Emerging evidence suggests that the 3′UTR of PD-L1 is an important factor affecting the stability of the mRNA transcript. The Genomic structural variations resulting in a truncated 3′UTR are associated with increased expression of PD-L1 in multiple cancer types [121]. Furthermore, several miRNAs have been shown to directly target the 3′UTR of PD-L1. Numerous individuals’ miRNAs have been identified to suppress PD-L1 expression in different tumor types, including miR-17-5p in melanoma [122], miR217 in laryngeal cancer [123] and miR-424 in ovarian cancer [124]. At translational level, PD-L1 expression is controlled by activation of the PI3K/Akt/mTOR/S6K1 pathway through oncogenic and IFN-γ mediated signaling, or through PTEN loss in glioma and lung squamous cell carcinoma [125]. After translation, the stability of the PD-L1 protein can be altered by ubiquitination, de-ubiquitination, phosphorylation, glycosylation and interaction with PD-L1 binding partners [126,127]. In addition to posttranslational modifications of the PD-L1 protein, stability of the protein can also be regulated by its interaction with various binding partners, including CMTM6, Sigma1 and FKBP51 [128–130].

6. Targeting immune checkpoints Accumulating evidence suggests that blockade of key IC pathways can induce effective anti-tumor immunity. ICIs enable tumor-reactive T cells to overcome regulatory inhibitory mechanisms and mount effective anti-tumor responses. In addition to PD-1 and CTLA-4, several other ICs have emerged as potential targets. The current spectrum of FDA approved ICIs include an anti-CTLA4 antibody (ipilimumab), and anti-PD1 (pembrolizumab and Nivolumab) and anti-PD-L1 antibodies (atezolizumab, avelumab and durvalumab). Patients treated with ICI fall under three categories; responders, non-responders and those who showed initial response followed by disease progression [17,149]. Inadequate or defective generation of tumor-specific T cells and memory T cells are key factors that could be responsible for ICI failure [149]. However, the mechanisms of both, de novo and adaptive resistance to IC therapy are not well understood. Mechanisms involved in immune evasion contribute towards resistance to ICIs, hence, the immune profile of the TME primarily dictates the response to ICI therapy. Furthermore, the spatial distribution of infiltrating immune cells also determines the immune activation state of a tumor. In this respect, cancers have been classified into three main immune phenotypes based on the infiltration of immune cells; immune desert, immune excluded and inflamed [150]. Immune desert may be characterized by lack of T cell activation or priming, immune excluded may contain inhibitory signals for potent immune responses and immune inflamed tumors exhibit high immune cell infiltration [150]. Even though a tumor might be inflamed, anti-tumor immunity and immunotherapy response might be impaired. Lack of T cells with tumor antigen-specific TCRs, accumulation of immunosuppressive MDSC, TAMs and tumors with low Teff or low Teff to Treg ratios are all contributing factors to treatment resistance [1]. The remodeling of the tumor vasculature can alter proteins that control the homing and trafficking of immune cells [151]. Normalizing tumor vasculature through restoration of antiangiogenic factors stimulates immune activation. Khan et al. recently reviewed the clinical trials evaluating ICIs in combination with anti-angiogenic drugs to remodel tumor vasculature for effective infiltration of immune cells [151]. Additionally, alterations in pathways like IFN-γ signaling, the mitogen-activated protein kinase (MAPK) pathway and loss of PTEN expression lead to resistance to ICI [17,152].

5. Prognostic significance of immune checkpoints The prognostic significance of IC expression in the TME and periphery has been extensively explored across various cancers. Reports showed that the expression of ICs and ligands including PD-1, CTLA-4, TIGIT and PD-L1 is associated with poor clinical outcome in various cancers [3,131,132]. While some studies have reported significant correlations between IC expression and patient survival, others did not find any prognostic value for IC expression or even reported contradictory effects on prognosis, coupled with either improved or with worsened survival rates. Some studies have reported that elevated PDL1 expression has a negative impact on disease progression [133–135], while others did not find consistent effects of PD-L1 over-expression on survival rates [136,137]. Similarly, accumulation of ICs and their ligands do not always predict conclusive effects on survival rates. Elevated Galectin-9 expression was associated with improved overall survival in solid malignancies but no correlation was found with diseasefree survival or recurrence-free survival [138]. The composition of the TME is heterogeneous across patients and is a potential tool to predict malignant recurrence [139]. The immune profile of the TME influences the effects of IC expression on disease outcome, due to the dynamic crosstalk between immune cell populations to initiate potent immune responses. This is evident from studies that reported associations between high immune cell infiltration and high IC expression. The presence of high IC expression within the TME indicates high immune-cell infiltration, which is mostly associated with improved ICI response and disease regression. For instance, high PD-L1 expression in gastric cancer patients correlated with high penetration of TILs that corresponded with less risk of cancer progression [140]. In renal cell carcinoma patients, the response rate of PD-1 blockade positively correlated with PD-L1 expression on tumor cells [3,131,141]. However, the nature of tumor-infiltrates can also affect correlations 6

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6.1. Combination therapies of immune checkpoint inhibitors

using epigenetic modulators and ICIs to restore/improve immunosurveillance. It has been demonstrated that the synergetic effect of HDAC inhibitor, romidepsin, and anti-PD-1 mAb induced chemokine upregulation and improved the potentiality of T cells to restore immune homeostasis within the TME in a pre-clinical lung cancer model [102]. Some clinical trials investigating epigenetic and immune-oncology therapies are currently underway, which include combining azacytidine/guadecitabine with atezolizumab and entinostat/romidepsin/ vorinostat with pembrolizumab.

Although IC monotherapies have been able to extend the average life expectancy of cancer patients, only a fraction of patients benefit from ICIs [153]. This highlights the necessity for improvements in the therapeutic modalities. Numerous pre-clinical models to identify novel ICs and clinical trials evaluating the efficacy of ICIs as single or combined therapies to treat various malignancies are currently underway. These strategies include combinational therapies to target multiple ICs including PD-1, CTLA-4, TIM-3 and LAG-3 that are often co-expressed on immune cells [10]. Recent preliminary trials evaluating co-inhibitory blockade therapy with anti-PD-1/PD-L1 together with anti-CTLA-4 have recognized significant improvement in survival indices over monotherapy in melanoma and lung cancer patients [154]. Moreover, this combination treatment could expand T cell infiltration and reduce the suppressive function of Tregs and MDSC within the melanoma TME to restore antitumor immune responses [155]. Furthermore, the combined targeting of PD-1 with TIM-3 or LAG-3 could reverse T cell exhaustion and enhance their activation to improve anti-tumor immunity [79,156]. These reports indicate that dual IC blockade could improve the efficacy of immunotherapy. In addition, targeting KIR leads to NK cell activation [61] and synergistic ICI for T cell pathways provide a strong rationale for combination therapy to promote potent anti-tumor responses [63]. Some studies have shown that targeting single IC could lead to upregulation of other ICs within the TME, primarily as part of adaptive resistance exhibited in the TME. Anti-PD-1 therapy leads to TIM-3 upregulation in tumor-infiltrating lymphocytes in head and neck cancers [157]. Additionally, CTLA-4 blockade in prostate cancer patients leads to increased infiltration of immune cells and an increase in the levels of PD-L1 and VISTA expression in the TME [158]. Studies suggest that upregulation of other ICs following anti-PD-1 therapy provides new targets associated with resistance to PD-1 blockade [159]. This supports the rationale for combination therapies with different ICIs. Combining nivolumab with ipilimumab has been extensively investigated in melanoma patients. Treatment with nivolumab alone or combined with ipilimumab resulted in significantly improved objective response and prolonged progression-free survival than ipilimumab alone in melanoma patients [160–162]. Additionally, preclinical studies combining TIM-3 and PD-1 blockers improved survival in glioma models [163,164]. Dual blockade of PD-1 and LAG-3 also effectively restored functional anti-tumor immune responses and controlled tumor progression in a leukemic model [165]. Additionally, combining TSR-033, a novel potent anti-human LAG-3 therapeutic antibody, boosted the efficacy of PD-1 monotherapy in NSCLC model [166]. Sharma et al. listed the combination therapies being developed to overcome resistance to cancer immunotherapy [17]. Mutual regulation of ICs and their ligands could diminish the effect of specific ICI. For instance, PD-1 and TIGIT are often co-expressed on melanoma tumor infiltrating and tumor antigen-specific CD8+ T cells, and efficacy of PD-1 blockade can be increased by dual blockade with TIGIT inhibitors [85]. The exact mechanisms for this synergistic action remain largely unknown, although the co-stimulatory CD226/PVR signaling pathway might be involved since TIGIT can interfere with CD226 homodimerization and impede the binding of the ligand PVR as TIGIT has a higher affinity for PVR [167,168]. Thus, dual IC blockade could release the inhibitory signals from the PD-L1/PD-1 and possibly TIGIT/PVR pathway as well as the PD-1 and TIGIT-mediated negative regulation of the CD226 co-stimulatory receptor. In line with this, an imbalance in TIGIT/CD226 expression was found in CD8+ infiltrating cells in metastatic melanoma [85]. Combining epigenetic modulators with immunotherapeutics has shown significant advances in better clinical outcomes in some cancer patients. Recent studies have shown the indispensable role of epigenetics in the upregulation of various ICs in breast and colorectal cancer [91–93]. These reports unveil the importance for combinatorial therapy

6.2. Predictive biomarkers for immune checkpoint inhibition Currently, clinical decision making to treat patients with IC blockade is often overshadowed by the lack of response in the majority of patients and reports of immune-related adverse events (irAEs) [169]. This has spurred efforts to define predictive biomarkers of treatment response in order to enhance clinical benefit in relation to less adverse events. We have recently reviewed potential predictive biomarkers for ICI [153]. Herein, we briefly discuss the various approaches and indicators used as predictive biomarkers for successful ICI. Immune-inflamed tumors are more likely to respond to ICI compared to immune desert due to availability of surplus targets, provided by IC expression on infiltrating immune cells. In line with this, studies suggest that IC expression in the periphery also results in good response to ICI and peripheral blood analyses could be used as a non-invasive method to predict response to ICI. Indeed, peripheral absolute lymphocyte count [170], and neutrophil to lymphocyte ratio have been suggested as predictive biomarkers for ICI response [171]. Moreover, IC ligand expression on tumor cells, mainly PD-L1, is a robust predictive biomarker in addition to other potential predictive biomarkers such as mutational load and neoantigen burden [172]. Several trials have validated that PD-L1 expression correlates with an increased response to PD-1/PD-L1 blockade [173]. However, there are only few evidencebased FDA approvals of commercial IHC tests for PD-L1 expression that could be used as companion test [174]. Additionally, tumors with high mutational load express high levels of neoantigens, and are therefore predisposed to anti-tumor immune responses and effective ICI. Neoantigens arise from nonsynonymous mutations and are generally tumor-specific. Studies have shown that high neoantigen burden correlates with the cytolytic activity of intratumoral cytotoxic lymphocytes and NK cells [175]. Gene profiles and identification of specific neoantigens should lead to identification of novel predictive biomarkers for ICI. Certain limitations hinder the utility of predictive biomarkers for ICI. For instance, PD-L1 expression is frequently heterogeneous within tumors and can be inconsistent between primary tumor and distant metastases. Additionally, meta analyses of data from 4174 patients with advanced or metastatic cancers from eight different trials showed that both, PD-L1 positive and negative patients benefited from PD-1 and PDL1 inhibitors [173]. However, there was a 34% decrease in the risk of death in PD-L1 positive patients compared to 20% in PD-L1 negative patients [173]. Moreover, some reports fail to report significant correlations between mutational loads or neoantigen frequency with successful ICI, and lack of standardized protocols in assessing predictive biomarkers jeopardize the development of robust predictive biomarkers for ICI. Preexistence of autoimmune biomarkers in patients treated with ICIs can also have significant effects on the efficacy of ICIs and the occurrence of irAEs. A retrospective study conducted on 137 NCLC patients who underwent PD-1 IC therapy showed that preexisting antibodies were associated with clinical benefits and also, with the development of irAEs [176]. Therefore, risk to benefit ratio may also be assessed for individual patients by screening for preexisting autoimmune markers. 7

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6.3. Tumor macroenvironment factors modulating responses to immune checkpoint inhibitors

oncolytic virotherapy with ICI [193]. The T-Vec viral vector encoding GM-CSF was the first FDA approved oncolytic virus that in combination with anti-CTLA-4 (NCT01740297) or anti-PD-1 therapy (NCT 02263508) demonstrated a superior outcome over ICI alone in advanced stage melanoma [194,195]. Other viral vectors currently in phase I/II clinical trial are derived from viruses belonging to the families of herpes simplex viruses, adenoviruses, Coxsackie viruses, Vaccinia viruses, Reoviruses and Maraba viruses [193].

The efficacy of IC blockade could be modulated by many factors, including the mutual regulation of ICs, regulation by a wide range of immune cells, oncolytic viruses and gut microbiota. In accordance with the aforementioned concept of mutual IC regulation, Tregs with CTLA-4 and PD-1 expression associated with more potent immune suppression during chronic viral infection while Tregs with CTLA-4 and TIGIT expression more selectively inhibited Th1 and Th17 pro-inflammatory T cells [177,178]. Accumulating evidence suggests that MDSC might be involved in the regulation of Treg differentiation and expansion [179]. In addition, MDSCs could interfere with PD-1 blockade since PD-L1 expression can be upregulated in MDSCs via HIF-1α signaling or via transmission of tumor-derived exosomes [119,180]. Indeed, accumulation of MDSCs in metastatic 4T1 mouse tumors has been associated with suboptimal responses to antiPD-1 treatment with enhanced tumor eradication after MDSC depletion [98]. Likewise, tumor associated macrophages can express PD-1, which interferes with PD-1 blockade and disrupts T cell activation [83]. In recent years, it has become apparent that besides the TME, the gut microbiota can also modulate the efficiency of immune checkpoint inhibitors [181–184]. Studies reported better responses to anti-PD-1 immunotherapy in patients with a distinct gut microbiome, albeit with a different composition of bacterial strains [181,182,185]. These findings were corroborated by fecal material transfer experiments of human PD-1 responders into germ-free mice with similar tumor types, indicating that beneficial bacteria can inhibit tumor formation and progression. Further, fecal microbial transplantation from human CTLA-4 responders to germ-free mice with melanoma revealed the outgrowth of a single bacterial species in association with tumor regression [184]. Possible mechanisms underlying the microbiota-associated immune response include an enhanced tumor infiltration of Th1 T cells, reduced number of Tregs, increased secretion of the pro-inflammatory cytokine IL-12 by dendritic cells, cross-reactivity with microbial epitopes, immune-modulatory microbial metabolites and improved gut lining integrity; however; how gut bacteria can modulate these mechanisms remains unknown [186]. These encouraging findings formed the basis for two future human clinical trials using pills with purified single bacterial species isolated from feces of responding patients. Moreover, these findings suggest that the use of antibiotics during treatment with ICI could affect the efficiency of the treatment through gut microbial dysbiosis. Oncolytic virotherapy is a rapidly developing field that is gaining recognition as a novel immunotherapy approach. The rationale for using viruses for cancer treatment stems from the alterations observed systemically and in the local TME following viral infection. More specifically, viral infection has been shown to render immunologically ‘cold’ tumors into ‘hot’ tumors through the release of damage-associated molecular patterns by damaged and dying tumor cells, followed by cytokine secretion and enhanced tumor infiltration of T cells, NK cells and antigen presenting cells [187]. On the other hand, viral infections increase the expression of PD-L1 on cancer and immune cells; supporting the notion of a possible synergy of combinatorial treatment with checkpoint inhibitors [188–190]. Immunovirotherapy using a recombinant myxoid virus with anti-PD-1 therapy enhanced therapeutic outcome in a murine model compared to either monotherapy [191]. The authors further genetically altered the virus to secrete tumor-localized soluble PD-1 to counteract the inhibitory effect of tumor-derived PD-L1, resulting in enhanced efficacy compared to the unaltered virus combined with anti-PD-1 blockade [191]. Furthermore, treatment with attenuated measles virus engineered to secrete CTLA-4 and PD-L1 antibodies improved treatment efficacy in terms of prolonged overall survival and tumor remission in melanoma mouse models [192]. Following these encouraging preclinical studies, more than 15 clinical trials are ongoing investigating the safety and efficacy of combining

7. Future perspective on immune checkpoint inhibition Despite the remarkable success of some ICIs, to date the FDA has only approved six ICI therapies. Development of combination therapies to overcome the multi-faceted mechanisms of tumor-immune evasion and identification of novel ICIs and their therapeutic exploitation in combinational therapies are providing important preclinical results with plausible clinical benefits. However, some negative results to-date, in which no favorable clinical responses were recorded, would suggest that the complexity of response/resistance to ICIs, influenced by the TME and macroenvironment, would require a deeper understanding of the tumor-immune interaction on an individual patient basis. More importantly, pediatric application of ICI is a major challenge as irAEs in children can lead to severe complications. Although, the incidence of grade 3 or 4 irAES following ipilimumab therapy was 27% in children; pediatric toxicities were evident after a single dose [196]. Moreover, the immune landscape of pediatric tumors and the immature immune system in children render ICI risky and less effective. Currently, several clinical trials are underway testing the efficacy and toxicity of ICI in children. The use of predictive biomarkers with ICI combination therapies has great potential to improve response rates and induce tumor regression. Peripheral blood biomarkers for treatment response, deciphering complete immune landscape, encompassing various immune cell types and their functionality, interaction with tumor cells and T cells, and also considering the importance of microbiome for treatment response bear vital significance in development of successful ICI. Moreover, further investigations are required to establish correlations between IC/ligand expressions for the development of robust predictive biomarkers for ICI. Lastly, while sequencing technologies and analytical tools with standardized parameters are indispensable for successful ICI development, individual patient assessment of the TME composition remains very critical for all successful immunotherapeutic approaches. Acknowledgements This work was supported by a start-up grant [VR04] for Dr Eyad Elkord from Qatar Biomedical Research Institute, Qatar Foundation. References [1] R. Saleh, E. Elkord, Treg-mediated acquired resistance to immune checkpoint inhibitors, Cancer Lett. 457 (2019) 168–179. [2] F.S. Hodi, S.J. O’Day, D.F. McDermott, R.W. Weber, J.A. Sosman, J.B. Haanen, et al., Improved survival with ipilimumab in patients with metastatic melanoma, N. Engl. J. Med. 363 (8) (2010) 711–723. [3] S.L. Topalian, F.S. Hodi, J.R. Brahmer, S.N. Gettinger, D.C. Smith, D.F. McDermott, et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer, N. Engl. J. Med. 366 (26) (2012) 2443–2454. [4] P. Armand, M.A. Shipp, V. Ribrag, J.M. Michot, P.L. Zinzani, J. Kuruvilla, et al., Programmed death-1 blockade with pembrolizumab in patients with classical hodgkin lymphoma after brentuximab vedotin failure, J. Clin. Oncol. 34 (31) (2016) 3733–3739. [5] H. Borghaei, L. Paz-Ares, L. Horn, D.R. Spigel, M. Steins, N.E. Ready, et al., Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer, N. Engl. J. Med. 373 (17) (2015) 1627–1639. [6] A.B. El-Khoueiry, B. Sangro, T. Yau, T.S. Crocenzi, M. Kudo, C. Hsu, et al., Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial, Lancet 389 (10088) (2017) 2492–2502. [7] R.L. Ferris, G. Blumenschein Jr, J. Fayette, J. Guigay, A.D. Colevas, L. Licitra,

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