**2.1. History of immunotherapy**

**Treatment Cancer type Treating method Phase Study number**

Peptide vaccine EC IMF-001 I NCT01003808

VEGFR2, cisplatin, fluorouracil

EC URLC10, TTK, KOC1, VEGFR1,

Multiple cancer types (including

types (including

Multiple cancer types (including

Multiple cancer types (including

EC + GEJC Siewert I

EC + GEJC Siewert I

Solid tumors (including ESCC)

Solid tumors (including ESCC)

carcinoma; GEJC: gastroesophageal junction carcinoma.

EC Durvalumab

EC)

EC)

EC)

EC)

Cell vaccine Multiple cancer

EC CIK II NCT02490735

EC NY-ESO-1-TCR T cells II NCT01795976

EC LY6K, VEGFR1, VEGFR2 I NCT00561275

EC URLC10 I NCT00753844 EC, GC G17DT, cisplatin, fluorouracil III NCT00020787

EC + GEJC + GC Nivolumab/placebo III NCT02743494

EC Pembrolizumab + brachytherapy I NCT02642809 EC Nivolumab vs. paclitaxel/docetaxel III NCT02569242

EC + GC Pembrolizumab + trastuzumab II NCT02318901

(anti-PD-L1) + chemoradiotherapy

ACT: adoptive cell therapy; CIK: cytokine-induced killer cells; CTL: cytotoxic T-cells; VEGFR: vascular endothelial growth factor receptors; PD: programmed cell death receptor; EC: esophageal cancer; ESCC: esophageal squamous cell

**Table 1.** Recent or completed clinical trials of potential therapeutic approaches for immunotherapy of esophageal cancer.

LAG525 (anti-LAG3) + PD001

(anti-PD-1)

CTL I NCT00004178

Tumor cell vaccine I NCT01258868

H1299 lysate vaccine I/II NCT02054104

Allogeneic tumor vaccine I NCT01143545

Pembrolizumab vs. investigator's choice III NCT02564263

Pembrolizumab II NCT02971956

Nivolumab + ipilimumab I NCT02834013

I NCT00632333

I/II NCT02735239

I/II NCT02460224

**ACT**

**Tumor vaccine**

12 Esophageal Cancer and Beyond

**Immune checkpoints therapies**

> A few decades ago, several rare clinical regression of advanced cancer in response to immune stimulation aroused interests in the area of cancer treatment. A passionate handful of immunologists, oncologists and surgeons carried out forward-looking researches into the relations between tumor progress and body immune system. Their respectable efforts turned the seemingly insignificant clinical phenomenon into reproducible concrete success by revealing the hidden mechanisms of tumor response.

> At the earliest period of cancer immunotherapies, the exact mechanism remained unknown and the anti-tumor growth effect was limited yet providing impetus for in-depth study. The first effective immunotherapies aiming at directly modulating cell function using wellcharacterized recombinant cytokines including interleukin-2 (IL-2) and interferon alpha (IFNα), yet the safety was lowered associated with substantial toxicity. Other cytokines, including IFNγ, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-25, etc., failed to provide substantive benefit. The following breakthrough was the emerging conception of monoclonal antibodies (mAbs) targeting tumor cell surface receptor proteins (human epidermal growth factor receptor 2 (HER2)/Neu, epidermal growth factor receptor (EGFR), etc.) and were integrated into cancer care. Vaccination therapies using the modified peptide, whole tumor, recombinant proteins, dendritic cells (DCs), and adjuvants were only modestly successful. With

researching deeper into the cross-talk happening in the tumor microenvironment between tumor cells and immune cells, the modern era of immunotherapy launched with the extraordinary novel efficacy (and toxicities) of mAbs targeting immune checkpoints in patients with various cancer types in order to decrease the suppressive potential that would prevent immune cells from functioning. Current alternative approach to overcoming the suppressive tumor microenvironment was the administration of genetically modified autologous T cells targeting specific cancer-related antigens [5]. This could be done in the form of T cells in which modified specific T cell receptors (TCR) were inserted for a shared tumor antigen or a tumor-specific neoantigen accompanied by co-transduction of stimulatory cytokines such as IL-12 or could co-administrate with PD-1 pathway blockers to sustain the capability before being expanded in large numbers *in vitro* following lymphodepleting chemotherapy. These approaches have produced dramatic responses in a few patients with a variety of individual tumor types, especially hematological malignancies [6]. Further improvement of adoptive cell transfer therapy came from the modified T cells expressing chimeric antigen receptors (CAR) targeting tumor-specific binding domains. After several generations of amelioration, CAR-T therapy showed extraordinary effect in treating hematological malignancies yet treatment of patients with solid tumors using CAR-T cells had been less fruitful and had troubled clinicians much through the off-tumor/on-target toxicity. Given the virtually limitless possibility of *ex vivo* engineering of T cells, many of these potential hurdles will likely be resolved by the additional modification of T cell products prior to treatment.

Tumor microenvironment is constituted by various immune cells, endothelial cells, adipocytes, paravascular cells, nerve cells, fibroblasts, and extracellular matrix components around the cancer cells [10]. Some stromal cells in the tumor microenvironment have immunosuppressive potential to inhibit the function of immune effector cells and promote tumor progression. Thus inhibiting cancer cell apoptosis and promoting mechanisms such as proliferation, angiogenesis, drug resistance, and immune escape to promote tumorigenesis [11]. Chemokines CCL17 and CCL22 secreted by tumor cells or tumor-associated macrophages (TAMs) recruit CCR4+ regulatory T cells (Treg), which then inhibit immune effector cells by contacting directly or secretion of cytokines (IL-10 and IL-35). Th17 cells can be transformed into Treg cells stimulated by IL-6 and transforming growth factor-β (TGF-β). Inflammation and tumor-derived factors stimulate the activation of myeloid-derived suppressor cells (MDSCs). Activated MDSCs can directly inhibit the expression of CD8 + T cells. Activation and induction of Treg cells and other mechanisms contribute to immune escape of cancer cells. TAM cells and CAF cells promote the growth, invasion, metastasis, and angiogenesis of tumor cells through the secretion of cytokines, chemokines, and various growth factors. In addition, tumor cells and TAM cells can express pro-

Immunotherapy for Esophageal Cancer http://dx.doi.org/10.5772/intechopen.78644 15

grammed cell death ligand (PD)-L1/2 inhibiting T cell activation after binding to PD-1.

cells in esophageal cancer to explore potential therapeutic targets.

recurrence after radiotherapy and chemotherapy [20].

MDSCs have been shown to play an important role in promoting tumor immune escape, activating CAF cells and angiogenesis [12]. The presence of proinflammatory cytokines such as IL-1, IL-6 and prostaglandins in esophageal cancer microenvironment can activate MDSC [13]. MDSC inhibits activation of T cells by direct inhibition [14], cytotoxicity of natural killer cells (NK) [15], depletion of arginine and cysteine, induction of Treg cells, etc. to achieve immune escape [16]. Another group of immunosuppressive cells that exert similar functions are Treg cells. Under physiological conditions, Treg cells can regulate the activation and proliferation of T cells, B cells, and cytotoxicity of NK cells. But in tumor microenvironment, Treg cells can promote the occurrence and progression of tumor cells by secreting immunosuppressive related factors, secreting tumor-associated antigens (TAAs), and suppressing the cellular adverse reactions of immune effector cells and the release of granzymes [17]. Studies have shown that tumor cells and TAMs can recruit CCR4+ Treg cells to tumor sites by secreting CCL17 and CCL22 and other chemokines [18]. Treg cells are highly aggregated in tumor cites, promoting tumor invasion and metastasis, and are associated with disease severity, survival after chemotherapy, and prognosis [17]. Additionally, Th17 cells can secrete IL-17 and IL-22 and activate STAT3 related signaling pathways to promote angiogenesis and tumor growth [23]. However, the role of Th17 cells is still controversial. What factors affect the function of Th17 have not yet been well defined [18]. Therefore, we still have to know more about Th17

The tumorigenic mechanisms of TAMs are varied. Phenotype spectrum of macrophage ranges from M1 to M2: M1 macrophages represent the classical activated macrophages, with functions of cytokines secretion, antigen presentation, resistance to infection and anti-tumor ability, etc., while M2 macrophages secrete type II cytokines and induce activation of COX2/ prostaglandin E and other mechanisms that cause tumorigenesis [19]. The presence of cancer associated fibroblasts (CAFs) in patients with esophageal cancer is associated with microvessel density, and can also promote tumor progression and metastasis through epithelial mesenchymal transition (EMT). CAFs are also associated with 3-year survival rates and disease

Immunotherapy showed great potential in the treatment of esophageal cancer. Modern treatments of patients with esophageal cancer integrated immunotherapy with conventional surgical, chemotherapeutic, and radiation oncologic strategies. In spite of the seemingly gratifying results, the 5-year survival rate of patients with esophageal cancer in the middle and late stages is still lower than 15%, the 5-year survival rate of patients with locally advanced surgery alone is only 20–25% [7]. Postoperative chemotherapy or neoadjuvant chemoradiation only turns the 5-year survival rate up to only 30–35% [8]. One of the causes to blame of the rather poor prognosis of esophageal cancer is the rapid disease progression. More than 50% of patients already have visible metastases at the time of diagnosis [7]. Therefore, further investigation of esophageal cancer microenvironment and its impact on disease progression will lay a solid theoretical foundation for early diagnosis and treatment improvement of esophageal cancer.

In this chapter, we tried to converge concepts relevant to the complicated relationship between the host and the neoplastic tissue.

#### **2.2. Immune microenvironment and molecular correlations in esophageal cancer**

As early as 100 years ago, Paget [9] had put forward the hypothesis of "seeds and soil", which laid the foundation for the concept of tumor microenvironment. Numerous data indicate that many immune-related cells, factors and immune-related signaling pathways in the tumor microenvironment play an important role in the occurrence, metastasis, recurrence, angiogenesis, and drug resistance of tumors. The in-depth study of the tumor immune microenvironment is to search for molecular pathogenesis and new therapeutic models of esophageal cancer.

Tumor microenvironment is constituted by various immune cells, endothelial cells, adipocytes, paravascular cells, nerve cells, fibroblasts, and extracellular matrix components around the cancer cells [10]. Some stromal cells in the tumor microenvironment have immunosuppressive potential to inhibit the function of immune effector cells and promote tumor progression. Thus inhibiting cancer cell apoptosis and promoting mechanisms such as proliferation, angiogenesis, drug resistance, and immune escape to promote tumorigenesis [11]. Chemokines CCL17 and CCL22 secreted by tumor cells or tumor-associated macrophages (TAMs) recruit CCR4+ regulatory T cells (Treg), which then inhibit immune effector cells by contacting directly or secretion of cytokines (IL-10 and IL-35). Th17 cells can be transformed into Treg cells stimulated by IL-6 and transforming growth factor-β (TGF-β). Inflammation and tumor-derived factors stimulate the activation of myeloid-derived suppressor cells (MDSCs). Activated MDSCs can directly inhibit the expression of CD8 + T cells. Activation and induction of Treg cells and other mechanisms contribute to immune escape of cancer cells. TAM cells and CAF cells promote the growth, invasion, metastasis, and angiogenesis of tumor cells through the secretion of cytokines, chemokines, and various growth factors. In addition, tumor cells and TAM cells can express programmed cell death ligand (PD)-L1/2 inhibiting T cell activation after binding to PD-1.

researching deeper into the cross-talk happening in the tumor microenvironment between tumor cells and immune cells, the modern era of immunotherapy launched with the extraordinary novel efficacy (and toxicities) of mAbs targeting immune checkpoints in patients with various cancer types in order to decrease the suppressive potential that would prevent immune cells from functioning. Current alternative approach to overcoming the suppressive tumor microenvironment was the administration of genetically modified autologous T cells targeting specific cancer-related antigens [5]. This could be done in the form of T cells in which modified specific T cell receptors (TCR) were inserted for a shared tumor antigen or a tumor-specific neoantigen accompanied by co-transduction of stimulatory cytokines such as IL-12 or could co-administrate with PD-1 pathway blockers to sustain the capability before being expanded in large numbers *in vitro* following lymphodepleting chemotherapy. These approaches have produced dramatic responses in a few patients with a variety of individual tumor types, especially hematological malignancies [6]. Further improvement of adoptive cell transfer therapy came from the modified T cells expressing chimeric antigen receptors (CAR) targeting tumor-specific binding domains. After several generations of amelioration, CAR-T therapy showed extraordinary effect in treating hematological malignancies yet treatment of patients with solid tumors using CAR-T cells had been less fruitful and had troubled clinicians much through the off-tumor/on-target toxicity. Given the virtually limitless possibility of *ex vivo* engineering of T cells, many of these potential hurdles will likely be resolved by the

Immunotherapy showed great potential in the treatment of esophageal cancer. Modern treatments of patients with esophageal cancer integrated immunotherapy with conventional surgical, chemotherapeutic, and radiation oncologic strategies. In spite of the seemingly gratifying results, the 5-year survival rate of patients with esophageal cancer in the middle and late stages is still lower than 15%, the 5-year survival rate of patients with locally advanced surgery alone is only 20–25% [7]. Postoperative chemotherapy or neoadjuvant chemoradiation only turns the 5-year survival rate up to only 30–35% [8]. One of the causes to blame of the rather poor prognosis of esophageal cancer is the rapid disease progression. More than 50% of patients already have visible metastases at the time of diagnosis [7]. Therefore, further investigation of esophageal cancer microenvironment and its impact on disease progression will lay a solid theoretical foundation for early diagnosis and treatment improvement of

In this chapter, we tried to converge concepts relevant to the complicated relationship between

As early as 100 years ago, Paget [9] had put forward the hypothesis of "seeds and soil", which laid the foundation for the concept of tumor microenvironment. Numerous data indicate that many immune-related cells, factors and immune-related signaling pathways in the tumor microenvironment play an important role in the occurrence, metastasis, recurrence, angiogenesis, and drug resistance of tumors. The in-depth study of the tumor immune microenvironment is to search for molecular pathogenesis and new therapeutic models of esophageal

**2.2. Immune microenvironment and molecular correlations in esophageal cancer**

additional modification of T cell products prior to treatment.

esophageal cancer.

14 Esophageal Cancer and Beyond

cancer.

the host and the neoplastic tissue.

MDSCs have been shown to play an important role in promoting tumor immune escape, activating CAF cells and angiogenesis [12]. The presence of proinflammatory cytokines such as IL-1, IL-6 and prostaglandins in esophageal cancer microenvironment can activate MDSC [13]. MDSC inhibits activation of T cells by direct inhibition [14], cytotoxicity of natural killer cells (NK) [15], depletion of arginine and cysteine, induction of Treg cells, etc. to achieve immune escape [16]. Another group of immunosuppressive cells that exert similar functions are Treg cells. Under physiological conditions, Treg cells can regulate the activation and proliferation of T cells, B cells, and cytotoxicity of NK cells. But in tumor microenvironment, Treg cells can promote the occurrence and progression of tumor cells by secreting immunosuppressive related factors, secreting tumor-associated antigens (TAAs), and suppressing the cellular adverse reactions of immune effector cells and the release of granzymes [17]. Studies have shown that tumor cells and TAMs can recruit CCR4+ Treg cells to tumor sites by secreting CCL17 and CCL22 and other chemokines [18]. Treg cells are highly aggregated in tumor cites, promoting tumor invasion and metastasis, and are associated with disease severity, survival after chemotherapy, and prognosis [17]. Additionally, Th17 cells can secrete IL-17 and IL-22 and activate STAT3 related signaling pathways to promote angiogenesis and tumor growth [23]. However, the role of Th17 cells is still controversial. What factors affect the function of Th17 have not yet been well defined [18]. Therefore, we still have to know more about Th17 cells in esophageal cancer to explore potential therapeutic targets.

The tumorigenic mechanisms of TAMs are varied. Phenotype spectrum of macrophage ranges from M1 to M2: M1 macrophages represent the classical activated macrophages, with functions of cytokines secretion, antigen presentation, resistance to infection and anti-tumor ability, etc., while M2 macrophages secrete type II cytokines and induce activation of COX2/ prostaglandin E and other mechanisms that cause tumorigenesis [19]. The presence of cancer associated fibroblasts (CAFs) in patients with esophageal cancer is associated with microvessel density, and can also promote tumor progression and metastasis through epithelial mesenchymal transition (EMT). CAFs are also associated with 3-year survival rates and disease recurrence after radiotherapy and chemotherapy [20].

PD-1 is a member of the CD28 superfamily and is an important immunosuppressive molecule that inhibits the activation of T cells after binding to its ligand PD-L1/PD-L2 [4]. Multiple experiments confirmed that PD-L1 and PD-L2 are highly expressed in esophageal cancer [21], in which PD-L1 expression is closely related to tumor invasion depth and poor prognosis, whereas PD-L2 expression is associated with decreased CD8+ T cell infiltration [21]. The increasing PD-L2 expression can promote the secretion of Th2 cytokines such as IL-4/IL-13 [22]. These pieces of evidence suggest that blockers targeting PD-1 are of great significance in the treatment of esophageal cancer [23].

PD-1/PD-L1 blockers have achieved encouraging clinical results in treating melanoma, lung cancer and other cancer types [30, 31]. The potential role of PD-1 blockers in treatment of esophageal cancer can be predicted by genomic map of the tumor immune microenvironment. The expression of PD-L1 on MDSCs isolated from esophageal cancer tissue was higher obviously, and PD-1 expression was detected in approximately 60% of the tumor infiltrating lymphocytes (TILs) from esophageal cancer tissues [32]. Therefore, inhibition of the PD-1/

Immunotherapy for Esophageal Cancer http://dx.doi.org/10.5772/intechopen.78644 17

Preliminary results of ongoing studies indicated that PD-1 blocker Pembrolizumab had acceptable safety in PD-L1 positive esophageal cancer patients. The objective response rate for mid-term analysis was approximately 30% with a sustained response period of up to 40 weeks [33]. These results laid the foundation for the continued completion of checkpoint

CTLA-4, also known as CD152, belongs to the immunoglobulin superfamily and serves as an immunological checkpoint. When activated CD4+ helper T cells express CTLA-4, this kind of cells sends inhibitory signals to T cells [34]. High CTLA-4 expressing CD4+ Treg cells block T cells by reducing IL-2 secretion and downregulating IL-2 receptor expression then retard T cells in G1 phase of the cell cycle [35]. Ipilimumab and tremelimumab have two fully humanized monoclonal anti-CTLA-4 antibodies that have already received FDA approval for the treatment of melanoma and mesothelioma [36, 37]. A survey of tremelimumab has been completed in phase II clinical trials for treating advanced gastric and esophageal cancer (n = 18). Although only a 5% response rate was observed, four patients were controlled and one patient was observed with partial remission (25.4 Mo) after treatment and continued for several months [38]. The results of ongoing clinical trials are expected to further highlight the

Tumor vaccine treatment involves the administration of TAAs into patients thus triggering specific anti-tumor immune responses. Rosenberg et al. [39, 40] conducted a comprehensive review of 1306 cancer vaccine usage studies conducted before 2004 and found that the overall target response rate was only 3.3%. The explanation may be that these immune cells have low

For the treatment of esophageal cancer, some vaccine-based clinical trial reports have been published. A Phase I clinical trial of 10 patients with refractory stage III or IV esophageal squamous cell carcinoma treated with peptide vaccine found that 9 patients developed antigen-specific T cell immune response. One of the patients with liver metastases showed complete remission for 7 months, another had partial remission within all metastatic lung lesions, and 3 patients had progression-free survival lasting 2.5 months. The peptide vaccine used was derived from three HLA-A24-restricted cancer testis antigens (TTK protein kinase, lymphocyte antigen 6 complex locus K, and insulin-like growth factor-II mRNA binding protein 3) [41]. The multicenter, phase II clinical trial of the vaccine evaluated the OS, PFS, and immune response after vaccinations in patients with HLA-A\*2402 positive and negative esophageal squamous cell carcinoma, and immune response was observed in HLA-A\*2402

affinity or are inhibited by endogenous factors like the checkpoints mentioned above.

PD-L1 pathway for esophageal cancer treatment cannot be ignored.

inhibitor regimens pivotal study in esophageal cancer patients.

clinical value of monoclonal anti-CTLA-4 antibodies in esophageal cancer.

**2.4. Vaccine regimens**

In the early stages of esophageal cancer, TGF-β signaling suppresses tumor growth by downregulating the expression of Smad4 and c-Myc genes, while promoting growth and EMT in advanced esophageal cancer [24]. This "switching" effect is thought to be caused by the loss of adaptor proteins. For instance, an important adaptor protein, β2-spectrin, plays an important part in cell–cell interactions and maintenance of epithelial cell polarity. In esophageal adenocarcinoma, decreasing β2-spectrin in tumor cells results in increased expression of SOX9 and c-Myc, but it also reduces other TGF-β targets such as E-cadherin and cell cycle-regulated p21 and p27 genes [25]. In summary, these changes make TGF-β promote the progression and metastasis of the tumor by inducing EMT, especially in epithelial tumors like esophageal cancer.

In addition to growth factors, chemokines in the tumor microenvironment also play an important role in the development of tumors. Mainly, there is stromal cell derived factor-1 (CXCL12/SDF-1) secreted by fibroblasts [26], binding to its corresponding receptor CXCR4 or CXCR7 thus inducing tumor growth, promoting angiogenesis, stimulating tumor movement, invasion and metastasis [26]. SDF-1/CXCR4/CXCR7 axis is closely related to tumor invasion, metastasis and survival. However, the use of these separate components as indicators of prognostic analysis has yielded inconsistent results [27]. Nonetheless, CXCL12 has been shown to regulate the migration of CXCR4-positive tumor cells in esophageal adenocarcinoma *in vitro* and *in vivo*. Knockout of CXCR4 expression in KYSE-150 and TE-13 esophageal cancer cells by small interfering RNA can inhibit the proliferation, invasion and metastasis of tumor cells. Local CCL5 and CXCL10 in esophageal squamous cell carcinoma can recruit CD8+ T cells to the tumor site [28].

Further researches showed that remodeling body immune state by various means in esophageal cancer patients will be the main research direction of immunotherapy for esophageal cancer.

## **2.3. Checkpoint inhibitor regimens**

Immune system has sophisticated regulatory mechanisms. Several checkpoints are involved to maintain the balance between effective immune-responses fighting against infection or cancer state yet won't activate excessively to prevent damaging healthy cells. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1 are among the major inhibitory receptors expressed by Tregs which downregulate immune responses [29]. Inhibitory antibodies modulating these immune checkpoints have been most frequently used in immune-oncology trials in esophageal cancers.

PD-1/PD-L1 blockers have achieved encouraging clinical results in treating melanoma, lung cancer and other cancer types [30, 31]. The potential role of PD-1 blockers in treatment of esophageal cancer can be predicted by genomic map of the tumor immune microenvironment. The expression of PD-L1 on MDSCs isolated from esophageal cancer tissue was higher obviously, and PD-1 expression was detected in approximately 60% of the tumor infiltrating lymphocytes (TILs) from esophageal cancer tissues [32]. Therefore, inhibition of the PD-1/ PD-L1 pathway for esophageal cancer treatment cannot be ignored.

Preliminary results of ongoing studies indicated that PD-1 blocker Pembrolizumab had acceptable safety in PD-L1 positive esophageal cancer patients. The objective response rate for mid-term analysis was approximately 30% with a sustained response period of up to 40 weeks [33]. These results laid the foundation for the continued completion of checkpoint inhibitor regimens pivotal study in esophageal cancer patients.

CTLA-4, also known as CD152, belongs to the immunoglobulin superfamily and serves as an immunological checkpoint. When activated CD4+ helper T cells express CTLA-4, this kind of cells sends inhibitory signals to T cells [34]. High CTLA-4 expressing CD4+ Treg cells block T cells by reducing IL-2 secretion and downregulating IL-2 receptor expression then retard T cells in G1 phase of the cell cycle [35]. Ipilimumab and tremelimumab have two fully humanized monoclonal anti-CTLA-4 antibodies that have already received FDA approval for the treatment of melanoma and mesothelioma [36, 37]. A survey of tremelimumab has been completed in phase II clinical trials for treating advanced gastric and esophageal cancer (n = 18). Although only a 5% response rate was observed, four patients were controlled and one patient was observed with partial remission (25.4 Mo) after treatment and continued for several months [38]. The results of ongoing clinical trials are expected to further highlight the clinical value of monoclonal anti-CTLA-4 antibodies in esophageal cancer.

## **2.4. Vaccine regimens**

PD-1 is a member of the CD28 superfamily and is an important immunosuppressive molecule that inhibits the activation of T cells after binding to its ligand PD-L1/PD-L2 [4]. Multiple experiments confirmed that PD-L1 and PD-L2 are highly expressed in esophageal cancer [21], in which PD-L1 expression is closely related to tumor invasion depth and poor prognosis, whereas PD-L2 expression is associated with decreased CD8+ T cell infiltration [21]. The increasing PD-L2 expression can promote the secretion of Th2 cytokines such as IL-4/IL-13 [22]. These pieces of evidence suggest that blockers targeting PD-1 are of great significance in

In the early stages of esophageal cancer, TGF-β signaling suppresses tumor growth by downregulating the expression of Smad4 and c-Myc genes, while promoting growth and EMT in advanced esophageal cancer [24]. This "switching" effect is thought to be caused by the loss of adaptor proteins. For instance, an important adaptor protein, β2-spectrin, plays an important part in cell–cell interactions and maintenance of epithelial cell polarity. In esophageal adenocarcinoma, decreasing β2-spectrin in tumor cells results in increased expression of SOX9 and c-Myc, but it also reduces other TGF-β targets such as E-cadherin and cell cycle-regulated p21 and p27 genes [25]. In summary, these changes make TGF-β promote the progression and metastasis of the tumor by inducing EMT, especially in epithelial tumors like esophageal

In addition to growth factors, chemokines in the tumor microenvironment also play an important role in the development of tumors. Mainly, there is stromal cell derived factor-1 (CXCL12/SDF-1) secreted by fibroblasts [26], binding to its corresponding receptor CXCR4 or CXCR7 thus inducing tumor growth, promoting angiogenesis, stimulating tumor movement, invasion and metastasis [26]. SDF-1/CXCR4/CXCR7 axis is closely related to tumor invasion, metastasis and survival. However, the use of these separate components as indicators of prognostic analysis has yielded inconsistent results [27]. Nonetheless, CXCL12 has been shown to regulate the migration of CXCR4-positive tumor cells in esophageal adenocarcinoma *in vitro* and *in vivo*. Knockout of CXCR4 expression in KYSE-150 and TE-13 esophageal cancer cells by small interfering RNA can inhibit the proliferation, invasion and metastasis of tumor cells. Local CCL5 and CXCL10 in esophageal squamous cell carcinoma can recruit CD8+ T cells to

Further researches showed that remodeling body immune state by various means in esophageal cancer patients will be the main research direction of immunotherapy for esophageal

Immune system has sophisticated regulatory mechanisms. Several checkpoints are involved to maintain the balance between effective immune-responses fighting against infection or cancer state yet won't activate excessively to prevent damaging healthy cells. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1 are among the major inhibitory receptors expressed by Tregs which downregulate immune responses [29]. Inhibitory antibodies modulating these immune checkpoints have been most frequently used in immune-oncology trials in esopha-

the treatment of esophageal cancer [23].

16 Esophageal Cancer and Beyond

cancer.

the tumor site [28].

**2.3. Checkpoint inhibitor regimens**

cancer.

geal cancers.

Tumor vaccine treatment involves the administration of TAAs into patients thus triggering specific anti-tumor immune responses. Rosenberg et al. [39, 40] conducted a comprehensive review of 1306 cancer vaccine usage studies conducted before 2004 and found that the overall target response rate was only 3.3%. The explanation may be that these immune cells have low affinity or are inhibited by endogenous factors like the checkpoints mentioned above.

For the treatment of esophageal cancer, some vaccine-based clinical trial reports have been published. A Phase I clinical trial of 10 patients with refractory stage III or IV esophageal squamous cell carcinoma treated with peptide vaccine found that 9 patients developed antigen-specific T cell immune response. One of the patients with liver metastases showed complete remission for 7 months, another had partial remission within all metastatic lung lesions, and 3 patients had progression-free survival lasting 2.5 months. The peptide vaccine used was derived from three HLA-A24-restricted cancer testis antigens (TTK protein kinase, lymphocyte antigen 6 complex locus K, and insulin-like growth factor-II mRNA binding protein 3) [41]. The multicenter, phase II clinical trial of the vaccine evaluated the OS, PFS, and immune response after vaccinations in patients with HLA-A\*2402 positive and negative esophageal squamous cell carcinoma, and immune response was observed in HLA-A\*2402 positive patients (n = 35), but there was no statistical difference in OS compared to HLA-A\*2402-negative patients (n = 25) (4.6 mo vs. 2.6 mo, P > 0.05), yet there is a significant difference in PFS (P = 0.032) [42]. In a tumor vaccine trial hosted by Saito et al. [43] (n = 20), 4 patients with high levels of MAGE-A4 or MHC class I antigen in autologous tumor cells not only showed MAGE-A4 specific immune responses after vaccination, but compared with patients without using antibodies, their OS was also significantly prolonged. Wada et al. [44] used NY-ESO-1 as a cancer vaccine in 8 patients with esophageal cancer. The results showed that 7 patients had an immune response. Of the 6 patients evaluated for efficacy, 1 patient experienced partial remission, 2 patients continued to maintain progression-free status, and 2 patients had mixed clinical responses. Given the preliminary results of these peptide vaccines in clinical trials, safety inspections and the related researches combined with radiotherapy and chemotherapy are also being carried out gradually in treating multiple cancer types including esophageal cancer.

of genetically engineered T cells was carried out by Kageyama et al. [52], TCR-T cells were readopted to patients with MAGE-A4-positive recurrent esophageal cancer, and administered the MAGE-A4 peptide vaccine subsequently, the level of TCR-T cells in the peripheral blood of 10 subjects was monitored for 5 months, and 5 of them were able to detect specific T cells continuously. Seven subjects appeared tumor progress after 2 months of treatment, but

Immunotherapy for Esophageal Cancer http://dx.doi.org/10.5772/intechopen.78644 19

CAR-T is another type of genetically engineered T cells. CAR-T was obtained by translocating chimeric antigen receptor such as CARs into T cells. Gross et al. [53] successfully constructed the structure of CARs into T cells for the first time to exert their specific killing function. Up to now, tens of clinical trial data of CAR-T treatment on malignant hematological malignancies have been published. The Novartis' tisagenlecleucel, a synthetic bioimmune product of anti-CD19 CAR-T cells has been approved by FDA on treating relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in 2017 [54]. The early generation of CAR-T therapy for solid tumors did not appear ideal outcomes during clinical use [55]. The reasonable explanation might be lack of unique TAAs in solid tumors, less efficiency and persistency of T cell homing to tumor sites, and the intratumoral immunosuppressive environment strongly inhibited CAR-T cell function. Clinical trials targeting solid tumors using second or third-generation CAR techniques had been limited, but some of the more significant clinical trials are carrying out good results. Patients with metastatic or recurrent HER2-positive medulloblastoma treated with HER2-CAR-T had came out with stable conditions [56]. Feng et al. [57] showed the results of a clinical trial of an EGFR-CAR-T treatment for EGFR-positive relapsed/refractory NSCLC patients revealed partial remission in 2 of the 11 patients involved in the evaluation and 5 patients had a stable condition ranging from 2 to 8 months. There were no obvious adverse reactions in the entire clinical trial. In the treatment of esophageal cancer, no CAR-T therapy-related studies have been conducted yet. However, anti-tumor targets are now erupting in esophageal cancer. Points such as HER2 can also provide references for future steps in

Currently, comprehensive therapy shed light on tumor treatment to a large degree. Clinical practice has proven that it is difficult to achieve the best results with any single treatment method. Therefore, the principle of treatment for most tumors depends on comprehensive treatment. Recent research results showed that the combined usage of immunotherapy and chemotherapy in a variety of cancer treatment achieved better results than a single therapy, it can not only reverse the immunosuppression effects caused by the late stage of the tumor, increase the cross-presentation of tumor antigens, promote the proliferation of killer T cells and make it more easy to kill tumor cells, but can also reduce the incidence of adverse reactions from chemotherapy and reduce drug resistance of tumor cells to some extent. Combination of chemotherapy and immunotherapy has been a common available method in some cancer types. Neoadjuvant chemotherapy regimens together with HER2-targeted therapy achieved pathologic complete response in relapse-free survival among patients with breast cancer [58].

another 3 subjects survived more than 27 months.

the development of research.

**3. Combined treatment**
