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Tumor immune microenvironment and immune checkpoint inhibitors in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is the main prevalent histological type of esophageal cancer, predominantly constituting 90% of cases worldwide. Despite the development of multidisciplinary therapeutic approaches, its prognosis remains unfavorable. Recently, the development of monoclonal antibodies inhibiting programmed death 1 (PD‐1) or programmed death‐ligand 1 (PD‐L1) has led to marked therapeutic responses among multiple malignancies including ESCC. However, only a few patients achieved clinical benefits due to resistance. Therefore, precise and accurate predictive biomarkers should be identified for personalized immunotherapy in clinical settings. Because the tumor immune microenvironment can potentially influence the patient's response to immune checkpoint inhibitors, tumor immunity, such as PD‐L1 expression on tumors, tumor‐infiltrating lymphocytes, tumor‐associated macrophages, and myeloid‐derived suppressor cells, in ESCC should be further investigated. In this review, accumulated evidence regarding the tumor immune microenvironment and immune checkpoint inhibitors in ESCC are summarized.


| IMMUNE CHECK P OINT INHIB ITOR S IN E SCC
An escape or evasion of the immune system is now established as one of the hallmarks of cancer. 14 Malignant cells can escape immune destruction by developing mechanisms regularly employed by the immune system to regulate itself. PD-L1 engages PD-1 receptor and induces PD-1 signaling, resulting in T-cell-mediated immune response suppression. 15 Tumor cells can co-opt the PD-1 pathway to evade immune responses by expressing PD-1 ligands on the cell surface and engaging immune effector cells with PD-1 receptor expression. Therefore, the PD-1/PD-L1 pathway has attracted much attention for its roles in tumor immunology and as immune-based therapeutic targets. To date, many clinical trials have focused on immune checkpoint inhibitors including PD-1/PD-L1 inhibitors in ESCC (Table 1). 16 Nivolumab is a high-affinity, humanized IgG4 monoclonal PD-1 antibody. 17 The ATTRACTION-01 trial is a multicenter phase II study assessing the safety and antitumor efficacy of nivolumab in esophageal cancer. 8 This study included 65 patients with advanced ESCC who were refractory or could not tolerate standard chemotherapy.
Treatment-related death was not observed, and only 17 (26%) patients had grade 3-4 adverse events (AEs). The objective response rate was 17%: 8 patients with partial response (PR) and 3 with complete response. The 1-and 2-y overall survival (OS) rates were 45% and 17%, respectively. Currently, results of phase III clinical trial (ATTRACTION-03) comparing nivolumab with docetaxel or paclitaxel in patients with ESCC refractory to fluoropyrimidine and platinum have been released. 18 At a minimum follow-up of 18 mo, the median OS improved from 8 mo in patients randomized to chemotherapy to 11 mo in those randomized to nivolumab, corresponding to a significant 23% reduction in the risk of death (hazard ratio [HR], 0.77; P = .019). Collectively, nivolumab might be considered as a new standard 2nd-line treatment strategy to address the high unmet needs of patients with advanced ESCC.
Pembrolizumab is a potent, highly selective, fully humanized IgG4-k monoclonal antibody against PD-1. 17 The KEYNOTE-028 trial is a multicohort phase IB study, including 83 patients with esophageal cancer with standard chemotherapy failure who were PD-L1 positive, and 78% of these patients had ESCC. With a median follow-up of 7 mo, the overall response (OR) rate was 30%, and that in the subgroup of ESCC patients was 28%. 19  In the ESCC subgroup, the median OS was 8 mo with pembrolizumab and 7 mo with chemotherapy (HR = 0.78, P = .0095). 21 These trials might indicate that pembrolizumab can be considered as a new 2ndline treatment for patients with ESCC.
Clinical trials of other anti-PD-L1 antibodies (eg, durvalumab) for esophageal cancer are ongoing. In a phase I trial of durvalumab monotherapy for advanced solid tumors, 7 of 22 patients experienced grade 2 treatment-related AEs, and 1 patient experienced grade 3 AE. One patient had a PR, and the disease control rate at 12 weeks was 36%. 22 Currently, several trials investigated the efficacy of durvalumab as a monotherapy for esophageal cancer, or in combination with chemotherapy, chemoradiotherapy, or immunotherapy. 23 In recent years, dual immune checkpoint inhibition and immunotherapy combined with cytotoxic agents are investigated to increase the therapeutic response to immune checkpoint inhibitors for patients with ESCC. 24 A randomized phase III study (CheckMate 648) F I G U R E 1 Cancer resistance to immune checkpoint inhibitors can be driven both by tumor cell-intrinsic and tumor cell-extrinsic factors of nivolumab plus ipilimumab (anti-CTLA-4 antibody) or nivolumab combined with fluorouracil plus cisplatin vs. fluorouracil plus cisplatin in patients with advanced or metastatic ESCC is ongoing. 25 In addition, a randomized phase III trial (KEYNOTE-590) compared fluorouracil plus cisplatin plus pembrolizumab with fluorouracil plus cisplatin as the 1st-line treatment for patients with locally advanced/ metastatic esophageal cancer. 26 Currently, only anti-PD-1 inhibitors (pembrolizumab and nivolumab) can be used for patients with ESCC in a clinical setting in Japan. Pembrolizumab was granted approval in December 2018 for the treatment of patients with unresectable/metastatic solid tumors harboring high microsatellite instability (MSI) or DNA mismatch repair (MMR) gene deficiency. Nivolumab has been approved in February 2020 for the treatment of patients with unresectable advanced or recurrent esophageal cancer that has progressed following chemotherapy.

| TIME
Although immune checkpoint inhibitors have manifested dramatic clinical effectiveness in human malignancies, the majority of patients still showed de novo or adaptive resistance. 27 Therefore, appropriate biomarkers to select patients who are likely to respond to these drugs as well as combination therapy to overcome resistance should be found. Cancer resistance to immune checkpoint inhibitors can be driven both by tumor cell-intrinsic (eg, PD-L1 expression, tumor mutation load, microsatellite instability-high status) and extrinsic factors (eg, TILs, TAMs, MDSCs) that contribute to immune evasion. 10 Thus, the concept of TIME has attracted increasing interests for developing and optimizing immunotherapeutic approaches, identifying predictive biomarkers, and selecting the most appropriate treatment approach for a given ESCC patient ( Table 2). The schematic view of TIME is shown in Figure 2. Recently, as will be described later, 4 different types of TIME have been proposed based on the presence or absence of TILs and PD-L1 expression on tumor cells. 28 This stratification may shed light on novel therapeutic approaches for rationally designing idealized combination therapies based on tumor immunology. 29

| PD -L1 E XPRE SS I ON ON C AN CER CELL S
PD-L1 expression on tumor cells is one of the most reasonable predictive biomarkers for the therapeutic efficacy of immune checkpoint inhibitors. 30 In fact, the predictive role of PD-L1 expression for PD-1/PD-L1 inhibitors has been reported in many types of cancers. 31 Regarding esophageal cancer, in the KEYNOTE-180 trial, PD-L1 expression was evaluated using the combined positive score   In majority of these studies, PD-L1 overexpression was associated with poor clinical outcomes. 32 However, 2 other studies demonstrated that PD-L1 overexpression was related to favorable prognosis. 33,34 In a previous study, using a non-biased database of 305 curatively resected esophageal cancers, PD-L1 expression was found to be associated with an unfavorable clinical outcome in esophageal cancer, supporting its role as a prognostic biomarker. 32 status has been reported to be a significant predictor of progression-free survival with pembrolizumab independently of PD-L1. 36 We have recently reported that the expression of PD-L2 as well as PD-L1 is associated with unfavorable clinical outcomes in esophageal cancer. 37 We have also found that PD-L2 and PD-L1 exhibit   Figure 3B). This proposed classification of TIME types may be important in designing optimal immunotherapeutic strategies. 28,29 Type I tumors are most likely to benefit from single-agent PD-1/

| TAMS
Macrophages have a crucial role in phagocytosis, antigen presentation, and cytokine and/or growth factor production and are promising effectors of cancer immunotherapy. In response to microenvironmental stimulations, macrophages polarize into anti-tumorigenic M1 or pro-tumorigenic M2 phenotype. TAMs are defined as macrophages located in, or at the close vicinity of, the tumor. As TAMs can produce various pro-tumorigenic factors, including growth factors, cytokines, and proteases, they can be attractive targets for recalibrating the immune response within TIME.

| MDSC s
MDSCs have been extensively investigated as one of the most crucial immunosuppressive cells in TIME. 53 They may drive tumor progression through cytokine and chemokine secretion with protumorigenic functions, depending on TIME. High infiltration of MDSCs has been reportedly associated with poor prognosis in patients with esophageal cancer. 54 In ESCC, IL-6 or other signaling pathways mediated by aldehyde dehydrogenase can regulate the activation of MDSCs. 55 A previous study showed that MDSCs were heterogeneous, and CD38 could serve as a marker for MDSCs with increased immunosuppressive ability in esophageal cancer. 56 Various therapeutic approaches are designed targeting MDSCs in an attempt to eradicate cancer cells. 57 Some of these approaches are currently undergoing clinical trials to estimate the efficacy and safety of their application in cancer patients.

| DENDRITI C CELL S
Dendritic cells (DCs) play a crucial role in a forefront of an immune response due to their advanced ability to recognize foreign antigens and mobilize naive T cells to effectors. Thus, DCs are recognized a promising target to activate the immune system in immunotherapeutic strategies against malignant cells. In the last decade, many researchers have tried to develop immunotherapeutic strategies against human cancers through vaccination. A cluster of LAMP3 + DCs appeared to be the mature form of conventional DCs and express diverse immune-relevant ligands and potentially regulate multiple subtypes of lymphocytes. 35 In ESCC, mature LAMP3 + DCs has been reportedly associated with increasing tumor-infiltrating CD8 + T cells. 58 Recently, promising findings of a phase I study on LV305, an engineered harmless virus targeting DCs, have been released. 59

| MI CROSATELLITE IN S TAB ILIT Y IN E SCC
MSI is a strong mutator phenotype due to genetic alterations caused by genetic and epigenetic inactivation of DNA MMR genes. 64

| CON CLUS ION
Over the last decade, our understanding on mechanisms underlying TIME in ESCC has rapidly improved, allowing the phenomenal development of cancer immunotherapy. Challenges in moving forward are to put much effort into the immunologic and biologic exploration in ESCC setting to, more precisely, tailor various available or emerging immunotherapeutic approaches. In the near future, large prospective trials should be designed and conducted to validate reliable predictive biomarkers, allowing the selection of patients with ESCC with the highest chance of benefiting from immunotherapy. Ongoing intensive efforts to establish biomarkers for immunotherapy response prediction hold great promise to maximize patient benefits from an immunotherapeutic approach including immune checkpoint inhibitor.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.