Advances and clinical applications of immune checkpoint inhibitors in hematological malignancies

Abstract

Immune checkpoints are differentially expressed on various immune cells to regulate immune responses in tumor microenvironment. Tumor cells can activate the immune checkpoint pathway to establish an immunosuppressive tumor microenvironment and inhibit the anti-tumor immune response, which may lead to tumor progression by evading immune surveillance. Interrupting co-inhibitory signaling pathways with immune checkpoint inhibitors (ICIs) could reinvigorate the anti-tumor immune response and promote immune-mediated eradication of tumor cells. As a milestone in tumor treatment, ICIs have been firstly used in solid tumors and subsequently expanded to hematological malignancies, which are in their infancy. Currently, immune checkpoints have been investigated as promising biomarkers and therapeutic targets in hematological malignancies, and novel immune checkpoints, such as signal regulatory protein α (SIRPα) and tumor necrosis factor-alpha-inducible protein 8-like 2 (TIPE2), are constantly being discovered. Numerous ICIs have received clinical approval for clinical application in the treatment of hematological malignancies, especially when used in combination with other strategies, including oncolytic viruses (OVs), neoantigen vaccines, bispecific antibodies (bsAb), bio-nanomaterials, tumor vaccines, and cytokine-induced killer (CIK) cells. Moreover, the proportion of individuals with hematological malignancies benefiting from ICIs remains lower than expected due to multiple mechanisms of drug resistance and immune-related adverse events (irAEs). Close monitoring and appropriate intervention are needed to mitigate irAEs while using ICIs. This review provided a comprehensive overview of immune checkpoints on different immune cells, the latest advances of ICIs and highlighted the clinical applications of immune checkpoints in hematological malignancies, including biomarkers, targets, combination of ICIs with other therapies, mechanisms of resistance to ICIs, and irAEs, which can provide novel insight into the future exploration of ICIs in tumor treatment.

Keywords: Immune checkpoint, hematological malignancies, biomarkers, therapeutic targets, drug resistance

1. Background

Immune homeostasis can be influenced by immune checkpoint molecules that are expressed on immune cells and tumor cells, which regulate the immune system, and targeting immune checkpoints could affect immune homeostasis [1]. Ligand-receptor pairs that exert inhibitory effects on immune responses are referred to as immune checkpoint molecules. Additionally, the inhibitory pathways may uphold self-tolerance and counteract the activation procedure to prevent excessive harm, which also promotes tumor cells to evade immune destruction, also called immune escape. In recent years, new advances in the mechanisms of tumor promotion by immune checkpoints have emerged continuously. For example, a new study demonstrates that programmed cell death protein 1 (PD-1) signaling inhibits T-cell tumors by restricting the production of glycolytic energy and acetyl coenzyme A (CoA) in a mouse model of T-cell non-Hodgkin lymphoma (T-NHL) and tumor cells from patients with T-NHL [2]. Monoclonal antibodies (mAbs) have been recognized as a means of immune checkpoint blockade (ICB), which could relieve the immune cells from suppression and enable them to identify and eliminate tumor cells [3].
Promising tumor immunotherapies known as immune checkpoint inhibitors (ICIs) have been recognized for their ability to enhance anti-tumor immune responses by targeting immune checkpoints present on both immune cells and tumor cells [4]. A variety of ICIs targeting specific immune checkpoints are currently available in the clinic. In 2011, ipilimumab, the first block antibody against immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), was authorized for treating melanoma [5]. In 2014, the first PD-1 targeting agent, pembrolizumab, was approved by U.S. Food and Drug Administration (FDA) for melanoma treatment [6, 7]. In 2016, nivolumab, the first PD-1 inhibitor approved to treat hematological malignancies, was approved for treating individuals with relapsed classic Hodgkin lymphoma (c-HL) after autologous hematopoietic stem cell transplantation (auto-HCT). In 2017, approval for the treatment of relapsed/refractory c-HL (r/r c-HL) was granted to pembrolizumab in 2017, after undergoing more previous treatment regimens [8]. At the present time, ICIs have become the most widely used anti-tumor therapy [4], which have enhanced the clinical management of aggressive tumors, including improving patient survival, changing the way to assess efficacy and manage adverse effects, especially in metastatic melanoma [7]. Additionally, promising progress has been made in the application and research of ICIs independently and in combination with other drugs in hematological malignancies [9, 10].
As numerous studies have verified the clinical significance and prognostic value of ICIs, the expressions of immune checkpoints may have the potential to become significant biomarkers in forecasting prognosis and responsiveness to ICIs. In addition to classical immune checkpoints like PD-1 [11] and CTLA-4 [12], emerging immune checkpoints have been gradually revealed to be prognostic biomarkers for tumors, including V-domain Ig suppressor of T cell activation (VISTA) in extranodal natural killer/T-cell lymphoma [13], lymphocyte activation gene 3 (LAG-3) [14] in diffuse large B-cell lymphoma (DLBCL).
Despite the promising future of ICIs, a considerable proportion of tumor patients fail to show a positive response to ICIs therapy or respond briefly before developing resistance, with significant variation among different types of tumors [15]. Perturbations of any steps of anti-tumor immunity can contribute to ICIs resistance, including the recruitment and stimulation of T cells, the induction of T cell effector activities, and the formation of effector memory T cells [16]. The approaches to overcome drug resistance include focusing on other checkpoint molecules, enhancing T cell exposure to antigens, or combining ICIs with other therapeutic modalities, including cytokine therapies. ICIs treatment can lead to a range of immune-related adverse events (irAEs) that collectively manifest as various side effects. The future application of ICIs in tumor treatment may involve predictive models that rely on the theory of integrated biomarker determination. Combination therapy presents new opportunities for the use of ICIs in tumor therapy, including oncolytic viruses (OVs) [17], neoantigen vaccines [18], bsAb [19], bio-nanomaterials [20], tumor vaccines [21], and cytokine-induced killer (CIK) cells [22].
Given the emerging research of immune checkpoints in the field of hematological malignancies in recent years, we focused on the current advances and potential clinical applications of ICIs in hematological malignancies, including the identification and utilization of biomarkers, the clinical studies of ICIs as standalone treatments or in combination, and the possible obstacles.

2. Immune Checkpoints in Differenct Immune Cells

Immune checkpoints, which have both similarities and differences on the surface of immune cells, antigen-presenting cells (APCs), or tumor cells, have been extensively studied in the last decade [23-25]. Their types and distribution, which will be described separately in the following sections, are shown in Figure 1.

Figure 1: The interaction between immune cells and tumor cells through immune checkpoints. The immune component in the tumor microenvironment consists of different types of immune cells, which are highly associated with the anti-tumor immunological state. The expression of immune checkpoint proteins by tumor cells dysregulates the anti-tumor immunity, suppresses the immune function of T cells, and favors the growth and expansion of cancer tumor cells. Abbreviation: APC, antigen-presenting cells; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; FGL-1, fibrinogen-like protein 1; Gal-9, galectin-9; HVEM, herpes virus entry mediator; LAG-3, lymphocyte activation gene 3; MHC, major histocompatibility complex; NK cells, natural killer cells; PD-1, programmed cell death protein 1; PD-L1/2, programmed death-ligand 1/2; Treg, regulatory T cells. TCR, T cell receptor; TIM-3, T cell immunoglobulin domain and mucin domain 3; TIGIT, T cell Ig and ITIM domain; VISTA, V-domain Ig suppressor of T cell activation; VISTAL, VISTA ligand.

2.1 Natural killer cells (NK cells)

NK cells, whose chief effector functions are cell killing and the releasement of pro-inflammatory cytokine, can respond to virally infected cells [26, 27].
Additional inhibitory immune checkpoints related to NK cells, apart from the inhibitory receptors linked to the major histocompatibility complex (MHC) class I, have been discovered, including the well-known checkpoints like CTLA-4, PD-1, LAG-3, and T cell immunoglobulin domain and mucin domain 3 (TIM-3) [28]. Recent reports have identified B7- cluster of differentiation (CD) 28 family members, including B7-H3, B7-H7, and VISTA, as potential candidates for inhibiting NK cells. NK cell-based immune checkpoint targets, including siglec-7 and -9, CD200, and CD47, have recently been discovered within the siglec family receptors [28]. CD200 is regarded as a marker of tumor progression due to its elevated expression in different types of tumors in both non-hematological [29] and hematological malignancies, including multiple myeloma (MM) [30] and acute myeloid leukemia (AML) [31]. Evidence suggests that the CD200-CD200 receptor (CD200R) inhibitory pathway directly contributes to suppressing NK cells. The overexpression of CD200 in AML patients suppressed the anti-tumor responses of NK cells, consequently elevating the likelihood of relapse in these individuals. These findings clearly demonstrated that the inhibition of NK cell cytotoxicity could be achieved by targeting cells that express CD200 [32], leading to immune escape and tumor progression.
Recently, a novel immune checkpoint signal regulatory protein α (SIRPα) was reported in NK cells, which could interact with target CD47 and counter other signals, such as interleukin 2 (IL-2) and CD16. Overexpression of CD47 in the NK-sensitive erythroleukemia cell line K562 significantly attenuated the killing of K562 by NK cells. Moreover, SIRPα deficiency or blockage increased the cytotoxic ability of rhesus monkey NK cells, which suggested that the disruption of the SIRPα-CD47 immune checkpoint could enhance the immune response of NK cells against tumors and inhibit NK cell-mediated tumor-killing effects [23].
The formation of tumors has been linked to the existence of malfunctioning NK cells, including aberrant activation of inhibitory immune checkpoints [28]. Hence, ICIs that can restore the anti-tumor activity of NK cells may serve as a viable choice for immunotherapy against tumors. In addition, combining anti-PD-1 or anti-PD-L1 inhibitors and NK cell-specific checkpoint inhibitors, such as anti-KIR or anti-NKG2A inhibitors, can be used for combination immunotherapy in hematological malignancies based on checkpoint inhibition. With the development of novel checkpoints, combining these checkpoints for synergistic anti-tumor responses is a future direction to fully utilize the tumor-killing role of NK cells.

2.2 Regulatory T cells (Tregs)

Tregs have crucial functions in balancing immune homeostasis and suppressing autoimmune responses [33] because they can regulate T cells, B cells, NK cells, dendritic cells (DCs), and macrophages through both humoral and direct cell interactions [34]. However, Tregs impede immune surveillance against tumors in healthy patients and hinder the formation of potent immune responses against tumors in individuals with tumors by inhibiting the growth of cytotoxic CD8+ T cells [33, 35]. Treg-mediated suppression mechanisms encompass a range of molecules, including CTLA-4, LAG-3, IL-10, IL-35, IL-2, forkhead box protein P3 (Foxp3), transforming growth factor-β (TGF-β), and others [36].
Previous studies have unveiled the impact of ICIs on T effector cells, but there is limited understanding regarding their influence on Tregs [33]. In a novel study, Bauer et al. [24] treated transgenic mice that spontaneously developed B cell lymphomas due to restricted overexpression of the proto-oncogene c-myelocytomatosis viral oncogene homolog (c-MYC) in B cells with anti-PD-1 (clone J43) and anti-CTLA-4 (clone UC10-4B9), resulting in reduced upregulation of Foxp3, CD25, and IL-10 in Tregs, as well as a decrease in their inhibitory ability. During B cell lymphoma progression, intratumoral Tregs showed elevated expression of Foxp3, CD25, CTLA-4, and IL-10 compared to Tregs from healthy mice, which exhibited a positive association with heightened immunosuppressive capabilities. This function could be attributed to the change towards a pro-inflammatory environment promoted by ICIs [24]. It has been reported that in c-HL and NHL, such as DLBCL, ICIs therapy reduced the infiltration of immunosuppressive CD4+/CD25+/Foxp3+ Tregs [37, 38]. As discussed above, targeting immune checkpoints on Tregs may offer a hopeful strategy for effective tumor immunotherapy.

2.3 DCs

DCs, B cells, and macrophages are typically regarded as the three significant groups of APCs. However, DCs can convey tumor antigens to the draining lymph nodes, thereby triggering the activation of T cells, an essential step for the development of T cell-mediated immunity [39]. Conventional DCs (cDCs) especially excel in presenting exogenous and endogenous antigens to T cells and regulating the proliferation, survival, and effectiveness of T cells [40]. Antigens from tumor cells could be captured by cDCs and presented to T cells within the tumor microenvironment or when cDCs moved to lymph nodes connected to the tumor [41].
Although the response to PD-1/programmed cell death 1 ligand 1 (PD-L1) blocking can be promoted by anti-TIM-3 antibodies through reducing T cell exhaustion, the effectiveness of TIM-3 blockade may extend to patients with tumors lacking significant T cell infiltration [42]. In co-stimulatory molecules, CD80 and CD86 expressed by DCs controlled activation or suppression of T cells through the interaction between CD28 or CTLA4 [43]. Peng et al. [25] found that DCs infiltrating the expressed a high level of PD-L1, which played a crucial role in limiting anti-tumor immune responses. PD-L1 expression on DCs was increased during antigen presentation in order to shield DCs from the cytotoxic effects of activated T cells. A high density of DCs has been correlated with favorable prognosis in c-HL [44] and cutaneous T cell lymphoma (CTCL) [45]. Plasmacytoid DCs (pDCs), which are closely related to the AML and chronic myeloid leukemia (CML) progression, are hematopoietic cells, mainly developed from a myeloid branch including the macrophage DC progenitor with monocytes, cDCs and pDCs differentiation potential [46]. Therefore, although the mechanism behind immune checkpoints in DCs remains unclear, DCs still have the potential to act as target cells for ICIs to improve efficacy in tumor treatment.

2.4 Macrophages

Macrophages are an important cell type in the innate immune response, with CD47 serving as the main regulator for macrophages. Blocking CD47 allows macrophages to phagocytose leukemia cells for therapeutic purposes [47]. Since first confirmed as a tumor antigen in human ovarian tumors [48], CD47 has been progressively shown to be overexpressed in a wide range of hematological malignancies, such as acute lymphoblastic leukemia (ALL) [49], AML [50] and CML [51], which appears to be a universal indication for tumor cells to avoid phagocytosis by the innate immune system, particularly macrophages [52]. In addition, higher CD47 expression levels were negatively associated with good treatment response and prognosis in patients with acute myeloid leukemia and CML [51]. Previous studies have confirmed that CD47 messenger RNA (mRNA) and protein levels are higher in leukemic stem cells of AML patients than in normal healthy stem cells, and elevated CD47 was highly correlated with poor prognosis [49].
The involvement of CD47 in the tumor-mediated evasion of phagocytosis was initially reported in cases of AML. Compared to normal cell counterparts, both mouse and human AML cells exhibited increased expression of CD47, which was directly linked to disease pathogenesis through the evasion of macrophages [53]. Consequently, clinical studies of CD47-targeted agents have been underway for AML and myelodysplastic syndromes (MDS), either as monotherapy or in combination therapy. Macrophages exhibited strong phagocytosis of primary AML patient leukemic cells when exposed to the anti-CD47 blocking antibody (clone B6H12), whereas the immunoglobulin G (IgG) control or a non-blocking anti-CD47 antibody did not show the same effect. Within 14 days of treatment, anti-CD47 antibody treatment eliminated tumor cells of peripheral blood (PB) and bone marrow (BM) in primary AML patient-derived xenografted mice in vivo. Furthermore, an anti-CD47 antibody (magrolimab) that has been humanized for clinical purposes exhibited comparable elimination of leukemia and prolonged survival in vivo [54]. In addition to AML, similar pre-clinical observations were noted in MDS patients [52].
Recently, new immune checkpoints have been identified in macrophages, including the cell surface glycoprotein CD137, also called 4-1BB, which is a member of the tumor necrosis factor (TNF) receptor superfamily [55, 56]. Stoll et al. [57] discovered that CD137 was expressed on circulating monocytes of healthy individuals and at even greater levels on cells derived from tumor patients. Monocytes that exhibit elevated levels of CD137 demonstrate enhanced ability to engulf MM and lymphoma cells treated with anti-CD38 or anti-CD20 mAbs, respectively, due to their heightened phagocytic capacity for antibody-dependent phagocytosis [57]. Therefore, CD137 was identified as a new potential immune checkpoint on human macrophages, suggesting possible therapeutic benefits in the treatment of tumors.
In summary, tumor cells mediate immune escape through various immune checkpoints located on the surface of immune cells, and ICIs targeting these immune checkpoints may have better efficacy. In addition, cellular immunotherapies are evolving in hematological malignancies as novel therapies, mainly including chimeric antigen receptor T-cell (CAR-T) therapy, NK-cell-based immunotherapy (CAR-NK therapy), and allogeneic hematopoietic stem cell transplantation (allo-HSCT). DC vaccines are also being experimented in murine T cell lymphoma models [58] and AML patients [59]. Therefore, both immune cells themselves and ICIs targeting immune checkpoints of immune cells have great therapeutic potential in hematological malignancies. Meanwhile, combining ICIs with targeted immune cell chemotherapeutic agents is a promising strategy to improve the treatment response rate of hematological malignancies.

ICIs have been rapidly developed in solid tumors while are less effective in hematological malignancies, which has been rapid progress in recent years. However, numerous unsatisfactory clinical problems still need to be solved in the application of ICIs due to immature technology and other reasons, such as resistance and adverse effects. In recent years, there has been a growing focus on researching immune checkpoints, including biomarkers, combination therapies with ICIs, resistance, and toxicities.

3.1 Prognostic biomarkers

The lack of specific biomarkers for prognostic stratification and accurate diagnosis makes hematological malignancies the most challenging type of tumor to diagnose. It is worth noting that researchers have proved that immune checkpoints could be used as promising biomarkers for diagnosis and prognosis prediction in hematological malignancies.

3.1.1 PD-1/PD-L1

According to recent research, higher levels of immune checkpoints have been linked to poor prognosis and worse treatment efficacy in hematological malignancies. For example, PD-1 and PD-L1 expression were poor prognostic indicators in patients with aggressive adult T-cell leukemia-lymphoma (ATLL) [12]. Cuccaro et al. [11] found that increased PD-L1 expression in PB was associated with advanced disease, systemic symptoms, and inferior progression-free survival (PFS) in HL patients, which proved that PD-L1 expression in PB might be a potential indicator for prognosis in HL. High PD-1/PD-L1 expression was associated with poor prognosis in aggressive acquired immunodeficiency syndrome (AIDS)-associated non-Hodgkin's lymphoma (NHL) [60]. The high level of CD4+ PD1+ and CD8+ PD1+ T lymphocytes were both prognostic factors of AML patients and ALL patients [61]. Elevated levels of soluble PD-L1 (sPD-L1) were associated with poor prognosis in MM [62].
In addition to prognostic markers, the researchers explored the potential of PD-1 and PD-L1 as biomarkers in predicting treatment response, testing safety, and detecting disease progression [63-65]. In research for cutaneous T cell lymphoma, PD-1+ T cells were involved in the formation of spatial biomarkers that could be strongly associated with response to pembrolizumab treatment [65]. In another study, by quantifying PD-1 in patients with FL and those who converted to DLBCL, researchers found that high levels of PD-1 in the follicles were associated with a significantly shorter time to transformation-free survival, indicating that PD-1 expression in follicular lymphoma (FL) tumor tissues prior to treatment could be used as a risk-predictive biomarker for transformation to DLBCL [63]. Through analysis of metabolic markers on immune cells from lymphoma patients undergoing autologous transplantation, including DLBCL, FL, and T-NHL, other researchers found that lymphoma patients with a sustained increase in PD-1 expression on T cells had a shorter median survival after autologous transplantation, suggesting that PD-1 expression on T cells could be used as an unfavorable biomarker for lymphoma patients undergoing autologous transplantation [64].
All of the above studies suggested that PD-1 and PD-L1 may serve as potential biomarkers in hematological malignancies. Based on these results, a large number of studies have come to explore the potential of targeting PD-1 and PD-L1 for the treatment of hematologic malignancies. However, since the therapeutic effect of anti-PD-1/PD-L1 therapy varies considerably in hematological malignancies with high heterogeneity, it is necessary to detect the expression level of PD-1 and PD-L1 to decide whether to use ICIs targeting PD-1 and PD-L1, as well as to predict their therapeutic responses. Furthermore, larger sample size experiments need to be used to validate their potential as prognostic markers for hematological malignancies.

3.1.2 CTLA-4

CTLA-4 has been reported to be a poor prognostic indicator in patients with aggressive ATLL [12]. Previous analysis indicated that unsuitable manifestation of CTLA-4 on CD4+ T cells in active MM was linked to unfavorable clinical outcomes. MM patients with decreased CTLA-4 levels expression may be prone to experiencing early relapse [66]. In a research on MDS patients, soluble CTLA-4 (sCTLA-4) levels were higher in MDS patients compared to controls, and sCTLA-4 levels were significantly higher in patients with high-risk MDS compared to the intermediate-risk group [67]. The higher the patient's CTLA-4 levels, the higher the risk of transformation to AML and the higher the mortality rate after follow-up, suggesting that elevated sCTLA-4 levels in MDS patients are an indicator of poor prognosis in MDS [67]. In another study of AML patients, the mRNA expression of CTLA-4 was significantly upregulated in AML patients compared to healthy controls. In addition, CTLA-4 expression was found to be associated with poor prognosis, and regression analysis revealed that CTLA-4 expression level was an independent predictor of prognosis in AML patients [68].
Based on these results, a large number of studies have begun to explore the potential of targeting CTLA-4 for the treatment of hematological malignancies, with promising results. However, similar to the results of anti-PD-1/PD-L1 therapy, anti-CTLA-4 therapy in hematological malignancies has shown markedly variable results. For example, in a phase 1 clinical study of ipilimumab in B-cell non-Hodgkin lymphoma (B-NHL) patients, the complete response rate (CRR) was only 5.6% [69]. However, in another phase 1 clinical study of ipilimumab in allo-HSCT patients, the CRR could reach 23% [70]. Therefore, in order to confirm the potential of CTLA-4 as a prognostic biomarker in hematological malignancies, it is necessary to conduct studies with a larger sample size.

3.1.3 TIM-3

In addition, microenvironmental expressions of TIM-3 were strongly correlated with better overall survival, which were important prognostic factors in patients with ATLL [71]. NK cells play a crucial role in immune responses against AML, and the expression of TIM-3 is significantly high in NK cells derived from AML individuals, which is associated with enhanced functional authorization and superior capabilities as effectors. Racova et al. [72] constructed prognosis-related biomarkers of active immunity against AML by NK cell frequency and TIM-3 expression levels. Similarly, Tim-3+ Foxp3+ Treg cells were highly enriched in the tumor microenvironment (TME) of DLBCL patients, which were correlated with the poor prognosis [73]. In addition to prognostic biomarkers, TIM-3 has potential as a biomarker for predicting chemotherapy efficacy. In a study of DLBCL patients, TIM-3 expression was increased in CD3+ T cells from DLBCL patients compared to healthy controls, and the level of TIM-3 expression was decreased after four courses of standard chemotherapy. Patients with low TIM-3 expression had a higher treatment efficacy than patients with high TIM-3 expression, indicating that TIM-3 may serve as a potential indicator of chemotherapy efficacy in DLBCL patients [74]. Another study also demonstrated that, after three years of follow-up, the rate of Tim-3 positive expression was higher in treatment-effective DLBCL patients than in treatment-ineffective patients, and Tim-3 positivity was an independent risk factor for the prognosis of DLBCL [75].
Collectively, TIM-3 has the potential to serve as a potential biomarker for hematological malignancies. Furthermore, we need more research with larger sample sizes to demonstrate the ability of TIM-3 as a biomarker and the reliability of predicting treatment efficacy for different hematological malignancies.

3.1.4 Others

In addition to the above familiar immune checkpoints, novel immune checkpoints are constantly being identified as potential biomarkers in hematological malignancies. For example, microenvironmental expressions of tumor necrosis factor receptor superfamily member 4 ligand (OX40L) were strongly correlated with better overall survival, which were important prognostic factors in patients with ATLL [71]. Moreover, AML patients with high levels of OX40L expression on tumor cells had significantly worse survival than patients with low OX40 expression, suggesting OX40 was a novel prognostic marker for AML patients [76].
Several studies have demonstrated that high expression of LAG-3 was correlated with worse outcomes and functioned as an independent prognostic indicator in DLBCL and MDS patients [14, 77, 78]. Moreover, VISTA was an independent prognostic factor for patients with extra-nodal natural killer/T cell lymphoma (ENKTCL), providing that VISTA could be a promising immune biomarker to perform prognostic stratification or diagnosis for ENKTCL [13].
Another study in AML patients showed an imbalance in the distribution of TIGIT and CD226 (the competitive co-stimulatory receptor for TIGIT) on γδ T cells, with a decrease in CD226+ γδ T cells and an increase in TIGIT+ γδ T cells in patients with de novo AML, whereas TIGIT-CD226+ γδ T cells were restored in patients with AML who reached complete response after chemotherapy [79]. In addition, non-M3 AML patients with higher TIGIT+ CD226− γδ T cells had lower overall survival [79].
Tumor necrosis factor-alpha-inducible protein 8-like 2 (TIPE2) is a newly identified negative regulator of anti-tumor immunity that plays a crucial function in preserving immune homeostasis. It has been shown in pan-cancer studies that TIPE2 might be a promising immune checkpoint biomarker in different hematological malignancy types, including AML, and might serve as a promising target for immunotherapy [80]. The high expression of the novel immune checkpoint molecule, siglec-15, on peritumoral macrophage predicted the positive outcome in primary central nervous system lymphoma (PCNSL), indicating that siglec-15 might represent an independent prognostic factor [81].
Taken together, immune checkpoints have been demonstrated to be prognostically relevant in hematological malignancies, particularly in lymphoma and leukemia, which potentially improve the prognosis and stratification accuracy. In addition, a large amount of prognosis-related statistics could help researchers make useful references for the development of new ICIs and select the immune targets and directions for research and development. Immune checkpoints are no longer to be used merely as prognostic biomarkers for hematological malignancies, and new studies have expanded to explore their potential as biomarkers for safety and clinical outcomes. There are individualized differences in the efficacy of ICIs among patients with hematological malignancy, so more experiments with larger sample sizes are needed to repeatedly validate the ability of immune checkpoints to be clinical biomarkers. Meanwhile, two or more immune checkpoints have been found to be concurrently associated with the prognosis in hematological malignancies and may have a synergistic effect, which also provides a mechanistic basis for the subsequent combined application of ICIs targeting different immune checkpoints.

3.2 The advances of ICIs in hematological malignancies

A variety of ICIs targeting specific immune checkpoints have been currently available in the clinic, including anti-CTLA-4 and anti-PD-1 [82]. Recent clinical studies on ICIs in hematological malignancies are shown in Table 1.

4. Barriers to Application of Immune Checkpint Inhibitors in Hematolocical Malignancies

4.1 Drug resistance of ICIs in hematological malignancies

Drug resistance, which is primarily associated with biological processes related to tumor immunity, is an important factor affecting the effectiveness of ICIs. The speculated mechanisms of resistance to ICIs can be broadly categorized into several groups, including inadequate tumor antigenicity, tumor-intrinsic IFN-γ signaling, loss of MHC, and disordered regulation of oncogenic signaling [135]. The mechanism of tumor resistance to ICIs is systematically illustrated in Figure 2.

Figure 2: The molecular mechanisms of acquired resistance to ICIs in tumor cells. (A)The disruption and downregulation of antigen presentation machinery: the mutations and expression loss of MHC-I or B2M lead to the inhibition of tumor antigen presentation and the decrease of TCR engagement. (B)The loss of IFN-γ sensitivity: the mutations and expression loss of IFN-γ-R or JAK1/2 lead to the insensitivity to IFN-γ in tumor microenvironment and the resistance to anti-PD-1/anti-PD-L1 treatment mediated by T cell response. (C)Tumor-mediated immunosuppression and exclusion: activated WNT signaling leads to the augment of β-catenin or the mutations and loss of PTEN, which can ultimately promote the production of immune-suppressive cytokines that reduce the infiltration and function of CD8+ T cells in tumor microenvironment. (D)Additional inhibitory checkpoints: the upregulation of additional immune checkpoints such as TIM-3, LAG-3, and VISTA can be found at the time of acquired resistance to PD-1 inhibitors. Abbreviation: B2M, Beta-2-microglobulin; IFN-γ, interferon-γ; IFN-γ-R, interferon-γ receptor; IRF1, interferon regulatory factor; JAK1, Janus kinase 1; JAK2, Janus kinase 2; LAG-3, lymphocyte activation gene 3; MHC-I, major histocompatibility complex I; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PTEN, phosphatase and tensin homolog; STAT1, signal transducer and activator of transcription 1; STAT2, signal transducer and activator of transcription 2; TIM-3, T cell immunoglobulin domain and mucin domain 3; VISTA, V-domain Ig suppressor of T cell activation.

Firstly, following treatment with ICIs, loss of function mutations in Beta-2-microglobulin (B2M) could lead to MHC I loss and represent a molecular route of immune escape [136]. Recently, truncation changes in B2M have been repeatedly found in acquired resistance to ICIs [137-139]. The absence of MHC I and II expression may also be due to the loss-of-function mutations in Janus kinase (JAK) 1/2 and B2M genes. It has been reported that the expression of MHC molecules can be induced by several drugs, such as Toll-like receptor agonists, histone deacetylase (HDAC) inhibitors, etc. [140]. Cellular therapies, such as CD40 agonists or CAR-T, can also be effective in tumors with impaired expression of such MHC molecules [141].
Secondly, the essential initial process in the JAK-STAT pathway, which triggered the apoptosis of tumor cells, was the activation of receptor-associated kinases JAK1 and JAK2 binding to the IFN-γ-receptor 1/ receptor 2 (R1/R2) [15]. Several cases of inactivating mutations in JAK1 or JAK2 suggested that mutations in this pathway may lead to the progression of ICIs resistance [137, 142, 143]. A study in NK cell/T-cell lymphoma found that the HDAC inhibitor chidamide, recently approved for the treatment of patients with r/r peripheral T-cell lymphoma (PTCL), was associated with an ORR and CRR of 39% and 18%, respectively [144]. In-vitro studies have shown that overactive JAK-STAT signaling in NKTL cell lines is associated with resistance to chidamide [144]. Another study in cutaneous T-cell lymphoma showed that n-(4-ethoxycarbophenyl) retinamide (ECPIRM), the 13-cis retinoic acid derivative, inhibited the expression of the JAK/STAT pathway, thereby inhibiting cell proliferation and promoting apoptosis, which may also address the resistance to ICIs triggered by the JAK/STAT pathway [145]. Therefore, inhibition of JAK-STAT activity could reprogram chromatin from a drug-resistant to a sensitive state, overcome drug resistance to ICIs and produce synergistic anti-tumor effects in vitro and in vivo.
Thirdly, the absence of the tumor suppressor phosphatase and tensin homolog (PTEN), which regulated phosphatidylinositol 3-kinase (PI3K) activity, had also been observed in cases of acquired resistance to ICIs [146-148]. PTEN deficiency in lymphoid malignancies has been associated with advanced disease, chemotherapy resistance, and poor survival [149]. The combination of PI3K inhibitors and ICIs could be a potential strategy to improve the drug resistance to ICIs in hematological malignancies, but a large number of clinical trials are still needed to further validate the feasibility.
Lastly, several studies have reported the increased expression of other immune checkpoints at the time of acquired resistance, including TIM3 [150], VISTA [151], and LAG-3 [139]. As mechanisms of resistance were inferred from circumstantial data in some reports, the exact mechanism of resistance to ICIs remains uncertain. One study included 19 AML patients treated with azacitidine and avelumab, and the findings demonstrated that PD-L2 expression was increased during treatment in both BM and PB [152]. Therefore, high expression of PD-L2 in BM may be an essential mechanism for the resistance to anti-PD-L1 therapy in AML patients [152]. In general, unremitting research on tumor resistance to ICIs will expand the spectrum of patients who can benefit from ICIs. Though the mechanism of ICIs resistance in hematological malignancies is still unclear, there are still feasible solutions, such as the combination of ICIs with chemotherapeutic agents, antiangiogenic agents, or radiotherapy. Combination application is the current research hotspot for resisting and reversing immune resistance, which still needs further clinical research. In view of the possible increased risk of toxicities, the combination of multiple ICIs requires careful assessments of benefits and risks before determining the treatment regimen.

4.2 Toxicity of ICIs in hematological malignancies

So far, an unavoidable problem with ICIs is irAEs [153, 154]. The mechanisms of irAEs related to ICIs are complex and not fully comprehended but are currently known to be associated with aberrant T cell activity [133, 155]. Shared antigens between tumor and normal tissue have been thought to activate nascent T cell response [133]. Nascent alterations in the peripheral B cell pool can also be used to explain the mechanisms of irAEs related to ICIs [156]. IrAEs related to ICIs can manifest in different human systems due to the disruption of the body's immune balance by ICIs [133]. IrAEs related to ICIs consist of dozens of different conditions that affect almost every organ system, including the skin, endocrine system, digestive system, etc. [157, 158]. ICIs-related irAEs of hematological malignancies are shown systematically in Figure 3. IrAEs related to ICIs can be severe and even lethal in certain instances, especially in patients with underlying diseases [159].

Figure 3: ICIs-related IrAEs in a variety of systems in hematological malignancies. Several systems can be affected by ICI-related irAEs in hematological malignancies, including the skin, digestive system, respiratory system, endocrine system, cardiovascular system, urinary system, and central nervous system. In comparison, cutaneous toxicity and gastrointestinal toxicity are more common but less severe, while pulmonary toxicity, cardiotoxicity, neurological toxicity, and nephrotoxicity are rare but more severe, even fatal. Abbreviation: ICIs, immune checkpoint inhibitors; irAEs, immune-related adverse events.

Skin toxicity is the most common type of irAE related to ICIs in hematological malignancies, especially MM [160]. The usual and typical manifestations are maculopapular rash, pruritus, and exfoliative dermatitis [161, 162], but rare skin necrosis [163] and Stevens-Johnson syndrome [164] may also occur. Rash and pruritus occurred in 0%-17% of patients receiving ipilimumab monotherapy and were mostly mild, usually defined as body surface area (BSA) grade 1-2. Another PD-1 inhibitor, pembrolizumab, can also cause skin toxicity [165]. Grade 1 (affecting less than 10% of BSA) and grade 2 (10%–30% BSA) skin irAEs are generally treated symptomatically and usually do not affect the continued use of ICIs. In a word, skin toxicity of hematological malignancies is diverse and variable in severity, with a high degree of individual variability. Skin toxicity is not as severe compared to other tissue toxicities, but it can have an impact on a patient's quality of life.
Endocrine organs, such as the thyroid, pituitary, adrenal, and pancreas, are frequently affected in patients who receive ICIs, leading to the development of irAEs [166, 167]. Osteomalacia, thyroid dysfunction, insulin-deficient diabetes mellitus, and primary adrenal insufficiency are among the reported ICIs-related irAEs. Hypophysitis was associated with anti-CTLA-4 therapy [69], while thyroid dysfunction was associated with anti-PD-1 therapy [168]. Diabetes and adrenal insufficiency are comparatively rare but can be fatal if left untreated [167]. The use of ipilimumab, either alone or in combination with other therapies, was linked to the occurrence of thyroid dysfunction (0%-6%) in cases of hematological malignancies such as CLL [162] and AML [70] after allo-HSCT. There was a comparable occurrence of thyroid toxicity in cases where nivolumab and pembrolizumab were administered. Hypothyroidism was reported in 0%-29% and 0%-17% [169] of cases, respectively, while hyperthyroidism occurred in 0%-13%[168] and 0%-17% [170]of cases. Both of these two agents above also showed the occurrence of adrenal insufficiency with a maximum incidence of 6% [86, 161]. Thyroid diseases generally do not require ICIs discontinuation. Cortisol levels should be tested to prevent adrenal crisis when TSH is decreasing [160]. Overall, endocrine toxicities are diverse, highly variable, and potentially fatal, requiring electrolyte and hormone examinations to detect this kind of irAEs.
Frequent occurrences of gastrointestinal (GI) irAEs to ICIs have been found particularly during anti-CTLA-4 therapy [166, 171]. The clinical manifestations are diversified, including diarrhea, abdominal pain, hematuria, and even some extraintestinal manifestations. Meanwhile, upper gastrointestinal symptoms such as nausea and vomiting are less common [153, 171]. Mild colitis is usually treated symptomatically with fluid and electrolyte repletion [153]. If symptoms get worse, ICIs need to be stopped immediately, and steroids should be given orally or intravenously as appropriate [172].
In general, pulmonary symptoms were more noticeable with anti-PD-1 or anti-PD-L1 monoclonal antibodies than with anti-CTLA-4 inhibitors. Pulmonary toxicity in ICIs is uncommon, but when present, it has the potential to worsen rapidly or even be fatal [166]. Pneumonia is one of the most common causes of ICIs discontinuation and is the primary cause of treatment-related mortality in hematological malignancies. Clinical symptoms include dyspnea, cough, and chest pain, usually appearing about 10-12 weeks after ICIs treatment [160]. The incidence of pulmonary toxicity for ipilimumab monotherapy was 0%-11%. Nivolumab and pembrolizumab caused pneumonitis or upper respiratory tract infection in 0%-24% [69] and 0%-13% [161] of patients.
Cardiotoxicity in hematological malignancies occurs occasionally. The incidence rate of myocarditis was predicted to be 1.14%, with a median time to onset of 34 days [173]. Cases of myocarditis were observed in hematological malignancy patients such as r/r MM receiving ICIs therapy, and two cases were fatal and were caused by the combination of pembrolizumab and dexamethasone [174-176]. Pericarditis with pericardial pain, myocarditis with difficulty breathing caused by fluid accumulation in the lungs or arrhythmias with heart palpitations and fainting may indicate cardiotoxicity [177, 178]. Therefore, the incidence of cardiac irAEs is low, but the mortality rate is high [179]. European Society for Medical Oncology (ESMO) Clinical Practice Guidelines recommend electrocardiography and troponin for all individuals [153]. When myocarditis is confirmed, it is necessary to discontinue ICIs and administer high-dose corticosteroids to patients [172].
Other rare irAEs to hematological malignancies include nephrotoxicity and rheumatologic toxicity [180, 181]. Arthralgia and myalgia are the most commonly reported rheumatic irAEs, while arthritis, myositis and vasculitis were also observed in trials of hematological malignancies treated with ICIs, such as CLL [90], r/r HL [182], AML [90], MDS [90], CML [90], r/r primary mediastinal B-cell lymphoma [183], r/r peripheral T cell lymphoma [180]. These adverse events are more likely to occur in anti-PD-1 ICIs and may occur later than other irAEs [166]. The most common manifestation of nephrotoxicity is acute kidney injury (AKI) due to acute tubulointerstitial nephritis [166, 181].
In general, irAEs to hematological malignancies can occur in all systems, among which cardiotoxicity and pulmonary toxicity, as well as endocrine toxicity, are more dangerous and require close monitoring of patients. The current strategy for dealing with irAEs is based on timely monitoring, early detection, and effective intervention [133]. While searching for alternatives to high-dose corticosteroids to develop unique strategies for the treatment of irAEs [155, 184], more scientific prevention and monitoring measures are also necessary. So, is it necessary to resume the use of ICIs after control of irAEs? There is still controversy over this question. The general recommendation is based on the grade determination of irAEs. For patients with grade 2 irAEs, ICIs can be reintroduced after the adverse effects have resolved to grade 1, and for grade 3 irAEs, it is generally recommended that ICIs should be discontinued. Importantly, restarting ICIs after the interruption of ICIs due to irAEs should be done in consultation with a specialist physician. If severe or life-threatening irAEs have occurred, treatment with these ICIs must be permanently discontinued, and restarting ICIs should be done by choosing a different type of ICIs as much as possible. Besides, restarting ICIs should be done by monitoring the recurrence of previous irAEs and considering permanently discontinuing the treatment with these ICIs if irAEs are present again. It is more important to individualize the decision based on patient outcomes and whether irAEs are controlled.

Declarations

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Competing Interests

The authors declare that they have no competing interests.

Authors' Contributions

Wenyue Sun wrote this manuscript and created figures and tables. Xin Wang and Shunfeng Hu revised the manuscript. Xin Wang provided guidance throughout the preparation of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was funded by the National Natural Science Foundation (No.82270200, No.82070203 and No.81770210); Key Research and Development Program of Shandong Province (No.2018CXGC1213); Taishan Scholars Program of Shandong Province (No.tspd20230610, NO.tsqnz20231251); Translational Research Grant of NCRCH (No.2021WWB02, No.2020ZKMB01); Shandong Provincial Engineering Research Center of Lymphoma; Academic Promotion Programme of Shandong First Medical University (No. 2019QL018); China Postdoctoral Science Foundation (No. 2023M741506); Shandong Provincial Natural Science Foundation (No. ZR2023QH193).

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