Introduction

Biliary tract cancers (BTC) are a class of highly heterogeneous malignancies that are mainly derived from biliary epithelial cells.[1] It pathologically involves adenocarcinoma, squamous cell carcinoma, and neuroendocrine tumors. Anatomically, BTC are commonly divided into four types, including gallbladder cancer (GBC), intrahepatic cholangiocarcinoma (ICC), and extrahepatic cholangiocarcinoma (ECC), in which ECC is further subclassified into perihilar and distal cholangiocarcinoma.[2] The incidence of BTC is ranked as the fifth carcinoma in the gastrointestinal malignancies in China and is still rising.[3,4] Of note, the high and increasing mortality accounts for the poor prognosis with 5-year survival rate <5%.[5] Radical resection is currently the only possible curative treatment; however, >70% of BTC patients are diagnosed at the advanced stage and miss the opportunity for surgery, mainly due to the atypical symptoms and complex anatomical location at the early stage, which is not easily detectable.[6] In the small population that meets surgical resection criteria, the local and distant recurrence rates remain as high as 50%.[1,7,8] In the advanced unresectable BTC, chemotherapy with other immunotherapy and/or targeted therapy emerge as the alternative regimens. At present, gemcitabine in combination with cisplatin is regarded as the first-line treatment for BTC.[9] Unfortunately, most BTC are not highly sensitive or rapidly develop resistance to these chemotherapy drugs. The overall objective response rate (ORR) is 26.1%, the median progression-free survival (PFS) is only 8.0 months, and the median overall survival (mOS) is 11.7 months,[9,10] demonstrating a malignant carcinoma.

Over the past decade, tumor immunotherapy has received significant attention in various cancer therapeutic fields, and recently it has been divided into two main categories: one is "PASSIVE" tumor immunotherapy, the approach that indirectly exerts a tumor killing function by activating the intrinsic immune system, such as immune checkpoint inhibitors (ICIs) therapy, tumor vaccines, cytokine therapy, and oncolytic virus immunotherapy; the other is "ACTIVE" tumor immunotherapy that directly eliminates tumor cells by infusing effector T cells into the body, such as adoptive cell therapy (ACT).[11,12] This approach has been proven to greatly improve the prognosis of various solid tumors and has yielded promising benefits to a number of chemo-resistant malignant tumors such as melanoma, renal cell carcinoma, and non-small cell lung cancer.[13,14] Indeed, the innovation of immunotherapy has notably advanced our knowledge of the conventional therapy and has been considered as the third revolution of cancer therapy following the traditional and targeted therapies [Figure 1].[15] To date, immense efforts in immunotherapy have been incorporated into multiple therapeutic regimens for cancer; however, so far as BTC is concerned, such studies have only just begun. In this review, we primarily focus on the cellular regulation of immune function in BTC and update the latest progress of clinical application of immunotherapy with current on-going multiple clinical trials.

Figure 1:

Classification of immunotherapy in BTC. BTC: Biliary tract cancers.

Cellular Immune Mechanism of BTC

The normal human immune system consists of innate immunity and adaptive immunity.[16] The innate immune system that is composed of macrophages, natural killer (NK) cells, and neutrophils functions as the first-line defense to rapidly eliminate exogenous pathogens within minutes to hours. The adaptive immune response involves T and B lymphocytes that invigorate specific cell and/or antibody-mediated immune reactions against tumor cells or other invaded organisms.[17] In the early phase of tumor development, both the innate and adaptive immune systems collaborate to suppress tumors by recognizing foreign antigens and releasing immune reactive factors. However, in the later stages of cancer, invasive tumor cells acquire the strong ability to escape from the immune surveillance, the so-called immune evasion.[18] One striking mechanism of this event is attributed to the altered tumor microenvironment in tumors including BTC. Sustaining interactions between tumor cells and adjacent varied stromal components (including immune cells, vascular cells, fibroblasts, cytokines, and extracellular matrix proteins, etc.) have (in) activated genetic and epigenetic changes in tumor cells, rendering tumor cells resistant to anti-tumor immunity derived from immune cells in BTC.[19] The Cancer Genome Atlas (TCGA) has shown that more than 70% of BTC tissues are enriched in immune cells and the degree of specific immune cell infiltration is closely related to the prognosis of cancer patients.[20] For example, one study indicates that the increased infiltration of cluster of differentiation 4 (CD4)+ and CD8+ T lymphocytes has significantly prolonged the OS of patients with BTC.[21] In contrast, the higher infiltration of tumor-associated neutrophils and regulatory T cells (Tregs) cells occurs in ECC, the worse the OS rate of patients.[22] Except these multiple immune lymphocytes involved in tumor immune escape, a number of inflammatory cells (e.g., macrophages) also participate in this process as these cells increase to release numerous cytokines and chemokines that inhibit the immune function of T cells.[23]

Recent Advances in BTC Immunotherapy ICIs

ICIs invigorate the immune system's ability to kill tumor cells by binding or blocking immune checkpoints.[24] The innovation of ICIs has opened a new therapeutic avenue to effectively treat cancer. Currently, the most commonly developed immune checkpoint blockers are specific antibodies against programed death-1 (PD-1), programed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte associated antigen 4 (CTLA4).[25]

In clinic, there are two main indicators available to evaluate patients' tolerance to these ICIs: one is tumor mutational burden (TMB) and the other is DNA mismatch repair (MMR) or microsatellite instability (MSI).[26] It is reported that tumors with high TMB and MMR deficient (dMMR) or MSI-high (MSI-H) may utilize abnormal proteins as neoantigens to activate anti-tumor responses and increase persistence. Interestingly, neither of these two indicators can account for a high proportion of BTC patients. Only 11% of BTC patients have high TMB of cancer-predisposing genes, such as BRCA1, BRCA2, RAD51D, MLH1, and MSH2.[27] The frequencies of DNA dMMR or MSI-H is 10% of ICC patients, 5–13% of ECC patients, and 5% of GBC patients.[28] Nevertheless, ICIs monotherapy or combined therapy may offer a promising benefit for these patients [Table 1].

Table 1 - Completed clinical trials assessing the use of immunotherapy for the treatment of BTC. Trial number Phase Sample size Treatment mPFS (months) mOS (months) ORR (%) ICIs monotherapy KEYNOTE-028 1b 24 Pembrolizumab 1.8 5.7 13.0 KEYNOTE-158 2 104 Pembrolizumab 2.0 7.4 5.8 NCT02829918 2 54 Nivolumab 3.7 14.2 11.0 JapicCTI-153098 1 30 Nivolumab 1.4 5.2 3.0 NCT01938612 1 42 Durvalumab 1.5 8.1 4.8 NCT03201458 2 39 Atezolizumab 1.9 – 2.8 Combination therapy CA209-538 2 39

Nivolumab

Ipilimumab

2.9 5.7 23.0 JapicCTI-153098 1 30

Nivolumab

Cisplatin/gemcitabine

4.2 15.4 36.7 NCT01938612 1 65

Durvalumab

Tremelimumab

1.6 10.1 10.8 NCT03875235 3 341 Durvalumab gemcitabine + cisplatin 7.2 12.8 – NCT03092895 2 92

Camrelizumab

FOLFOX4/GEMOX

5.3 12.4 16.3 NCT03201458 2 38

Atezolizumab

Cobimetinib

3.7 – 3.3 ChiCTR2100044476 2 38

Lenvatinib

Pembrolizumab/tislelizumab/sintilimab/camrelizumab

– 17.7 42.1 NCT01853618 2 20

Tremelimumab

Microwave ablation

3.4 6.0 12.5 Other strategy UMIN-000003207 1 9 HLA-A*2402 5.2 12.7 – UMIN-000005820 2 36

Surgery

DC vaccine + TILs

18.3 31.9 –

BTC: Biliary tract cancers; DC: Dendritic cell; FOLFOX4: 5-Fluorouracil, leucovorin, and oxaliplatin; GEMOX: Gemcitabine and oxaliplatin; HLA-A*2402: Lymphocyte antigen 6 complex locus K, TTK protein kinase, insulin-like growth factor-II mRNA-binding protein 3, and DEP domain containing 1; ICIs: Immune checkpoint inhibitors; mOS: Median overall survival; mPFS: Median progression-free survival; ORR: Objective response rate; TILs: Tumor-infiltrating lymphocytes; –: Not applicable.

ICIs monotherapy

Till date, several clinical trials have explored the efficacy of ICIs monotherapy in advanced BTC patients and have found moderate efficacy in unscreened patients.[29] PD-1 neutralizing antibodies (pembrolizumab, nivolumab, and camrelizumab) and PD-L1 neutralizing antibodies (durvalumab and atezolizumab) approved by Food and Drug Administration (FDA) are commonly engaged in a variety of human cancers.[30,31] A plethora of clinical evidence has reported the considerable benefits in cancers, which has been demonstrated in other review articles and will be not fully discussed here.

PD-1 and PD-L1 are reported to be expressed by aggressive BTC cells and inflammatory cells, and their upregulation is correlated with poor clinical prognosis.[32] There are two clinical trials, KEYNOTE-158 and KEYNOTE-028, which are settled to evaluate the efficacy of pembrolizumab in advanced BTC patients.[33] In KEYNOTE-158 (phase 2), all 104 American BTC patients at the advanced stage are enrolled with PD-1/PD-L1-positive and negative status and the results have shown that the ORR is only 5.8%. In KEYNOTE-028 (phase 1b), 24 patients with high PD-L1 expression have received pembrolizumab monotherapy and the ORR has increased to 13.0% (3/23; 95% CI, 2.8–33.6%). Therefore, regardless of PD-L1 expression, pembrolizumab offers durable anti-tumor activity in a small population of advanced BTC patients. The therapeutic potential of nivolumab monotherapy in 54 advanced BTC patients has also been evaluated in a multi-center phase II study.[34] The results reveal the ORR of examined patients is 22% (10/46) and the disease control rate (DCR) is 50%. Interestingly, while the study also suggests that there is a significant improvement of PFS in patients with high PD-L1 expression, the association between OS and high PD-L1 expression has not been proven. The role of the PD-L1 inhibitor atezolizumab has been demonstrated in a phase II trial that the anti-tumor activity of monotherapy is not effective in BTC.[35]

In addition to these neutralizing antibodies, researchers have developed a series of immune checkpoint small molecule inhibitors and are attempting to define effective one(s) from screening. The candidates are currently under preclinical studies or clinical phase I trial.[36] Meanwhile, multiple combining ICIs in advanced BTC patients are settled in some phase I and II trials (NCT01938612, CA209-538, NCT03668119, and NCT03704480), and the results are expected to offer guidance for future therapeutic strategies [Table 2].

Table 2 - On-going clinical trials of immunotherapy for the treatment of BTC. Trial number Phase Sample size Treatment Primary outcomes NCT03704480 2 10 Durvalumab + tremelimumab mOS NCT03668119 2 212 Nivolumab + ipilimumab ORR NCT05156788 2 40

Tislelizumab

Lenvatinib + GEMOX

mOS

mPFS

ORR

NCT03633773 1 9 MUC-1 CAR-T DCR NCT03801083 2 59 TILs ORR

BTC: Biliary tract cancers; CAR-T: Chimeric antigen receptor T-cell immunotherapy; DCR: Disease control rate; GEMOX: Gemcitabine and oxaliplatin; MUC-1: Mucin 1; mOS: Median overall survival; mPFS: Median progression-free survival; ORR: Objective response rate; TILs: Tumor-infiltrating lymphocytes.

Not limited to focusing on these specific ICIs, researchers have also added efforts to discover more immune checkpoints that may serve as alternative targets for therapy, such as lymphocyte activation gene 3 (LAG3) protein, T cell immunoglobulin domain and mucin domain-3 (TIM3), and T cell immunoreceptor with immunoglobulin (Ig) and ITIM domains protein (TIGIT).[37] In particular, TIGIT, known as the next "PD-1", has received great attention in clinical applications. TIGIT blockade has been found able to enhance anti-tumor responses of effector T-cells and NK-cells.[38]

Combination therapy

The efficacy of combined ICIs with other therapies has increasingly received attention and the more effective intervention emerges as the powerful approach to treat these lethal disorders. There are different combinations of therapies engaged in the clinic, such as ICIs with chemotherapy, targeted therapy, or microwave ablation.[39]

ICIs in combination with chemotherapy

Some preclinical studies have suggested that chemotherapy can increase HLA1 expression of tumor cells and promote cytokine release from tumor cells, thus invigorating anti-tumor immunity of T cells.[40] In the clinic, a number of studies demonstrate that ICIs in combination with chemotherapy significantly improve drug responses and prolong survival of advanced BTC patients. The monotherapy with nivolumab in BTC is fairly effective; however, when it is in combination with chemotherapy, the efficacy is notably improved. In a multi-center phase I study, the median OS of 30 BTC patients receiving nivolumab combined with chemotherapy is 15.4 months (90% CI, 11.8–not estimable) compared with 5.2 months (90% CI, 4.5–8.7) in 30 patients with monotherapy. The median PFS of the combination therapy is triple longer than that of nivolumab monotherapy (4.2 months vs. 1.4 months).[41] In China, a clinical trial with 77 BTC patients has evaluated the efficacy of a PD-1 inhibitor with chemotherapy in comparison with PD-1 inhibitor monotherapy and chemotherapy monotherapy.[42] The results uncover that the median OS and median PFS of a PD-1 inhibitor plus chemotherapy are 14.9 months (95% CI, 10.73–19.07) and 5.1 months (95% CI, 3.59–6.61), noticeably longer than the 4.1 months (95% CI, 2.79–5.42) and 2.2 months (95% CI, 1.10–3.30) of patients with PD-1 inhibitor monotherapy and the 6.0 months (95% CI, 3.66–8.34) and 2.4 months (95% CI, 1.12–3.68) of patients with chemotherapy alone. Last year, a multi-center phase II trial with 92 cases was set up in China to evaluate whether camrelizumab plus oxaliplatin-based chemotherapy could be the first-line therapy for advanced BTC.[43] The primary endpoint ORR was 16.3% (95% CI, 9.4–25.5) and DCR was 75.0% (95% CI, 64.9–83.4). Moreover, the median OS was 12.4 months (95% CI, 8.9–16.1) and the median PFS was 5.3 months (95% CI, 3.7–5.7). At the Chinese Society of Clinical Oncology (CSCO) annual meeting 2021, a study in China employing a small sample size demonstrated the efficacy of tislelizumab combined with lenvatinib and gemcitabine and oxaliplatin (GEMOX) regimen in the transformational treatment of potentially resectable, locally advanced BTCs, with an ORR as high as 80% (NCT05156788). TOPAZ-1 is the first international multi-center phase III clinical study in patients with advanced BTC (NCT03875235). The study aims to evaluate the efficacy and safety of durvalumab in combination with standard chemotherapy (gemcitabine/cisplatin) vs. chemotherapy alone as first-line treatment for advanced BTC. At the American Society of Clinical Oncology (ASCO) annual meeting 2022, the interim analysis of TOPAZ-1 showed that the median OS and the median PFS in the combination therapy group were 12.8 months and 7.2 months compared to 11.5 months and 5.7 months in the chemotherapy alone group. Therefore, the combination therapy of ICIs with chemotherapy drugs gives rise to benefits for advanced BTC patients.

ICIs in combination with targeted therapy

A monotherapy with atezolizumab and combined therapy of atezolizumab with cobimetinib, an mitogen-activated protein kinase kinase (MEK)-targeted drug, have been tested in a phase II trial on 77 patients with advanced BTC. The median PFS is 3.65 months in the combination therapy compared with 1.87 months in the monotherapy.[35] Lenvatinib, an oral multi-tyrosine kinase-targeted inhibitor, has been shown to enhance anti-tumor immune responses and increase the efficacy of PD-L1 inhibition.[44] In a phase II study conducted by Zhang et al[45], 38 patients with unresectable BTC have been treated with the combination of PD-1 inhibitors plus lenvatinib as the first-line treatment. Overall, the study reports that the ORR is 42.1% and DCR is 76.3%; a median event-free survival (EFS) of 8.0 months (95% CI, 4.6–11.4); and a median OS of 17.7 months (95% CI, not estimable). No treatment-related deaths are reported, while 34.2% of enrolled patients experience grade ≥3 treatment-related adverse events. At present, we have directed a clinical trial on GBC with erythroblastic leukemia viral oncogene homologue (ErbB) inhibitor sapitinib, based on our previous findings that GBC has high-frequency mutations in the ErbB signaling pathway and ERBB2/3 mutations that promote PD-L1-mediated immune escape.[46,47] The combination of sapitinib and atezolizumab could inhibit the progression of GBC, suggesting the feasibility of combining ICIs and targeted therapy. In addition, we further explore the additional targets that may participate in the ErbB signaling by single-cell transcriptome sequencing.[48] The study reveals that GBC with ErbB pathway mutations expresses high levels of secreted midkine (MDK) and promotes the differentiation of immunosuppressive macrophages, which in turn contribute to the immune escape of GBC by secreting C-X-C motif chemokine ligand 10 (CXCL10) resulting in Tregs activation. Thus, the additional key molecules involved in the ErbB mutation-mediated immune suppression may offer alternative targets having the potential for future therapeutic investigation.

ICIs in combination with microwave ablation

An anti-CTLA-4 monoclonal antibody tremelimumab can induce activation of cytotoxic T lymphocyte (CTL) by blocking the inhibitory signaling of CTLA-4. Thus, it has been used in combination with microwave ablation and chemotherapy to treat patients with advanced BTC. A clinical study has been conducted to investigate whether tremelimumab combined with microwave ablation could enhance the efficacy in patients with refractory BTC.[49] The results showed that the median PFS was 3.4 months (95% CI, 2.5–5.2 months) and OS was 6.0 months (95% CI, 3.8–8.8 months) in 16 cases, establishing the possibility of tremelimumab usage in combination with microwave ablation in refractory BTC.

Tumor vaccine

Given that tumor-derived antigens can stimulate T cells to activate immune responses, research scientists have developed tumor antigen-based vaccines by which patients build up the immune defense system specific for recognizing and lysing tumors.[50] Till date, tumor vaccines are mainly divided into polypeptide vaccines, dendritic cell (DC) vaccines, and nucleic acid vaccines, among which the polypeptide vaccines are the most extensively studied.

Polypeptide vaccines

Mutation of Wilms tumor protein 1 (WT1) and overexpression of mucin 1 (MUC1) are present in 80–90% of BTC.[51] Increased levels of both proteins are significantly correlated with poor prognosis of these patients. WT1 is a transcription factor with dual functions of activation and inhibition of targeted genes and it is highly expressed in leukemia and various solid tumors.[52] Although the molecular regulation related to its mutation form in BTC remains to be established, it is suggested to be related to the loss of zinc finger domains. Since 2005, MUC1 peptide vaccine has been developed for advanced cholangiocarcinoma, but the effectiveness is low.[53] Lately, WT1 peptide vaccine plus gemcitabine are engaged to treat patients with advanced BTC.[54] The research trial has enrolled 16 BTC patients, among whom eight patients have had stable diseases for 2 months. In addition to WT1 and MUC1, other types of peptide vaccines in BTC have also been created and therapeutic efficacy has been documented. A phase I clinical trial of a tetrapeptide vaccine in nine patients with advanced BTC has reported that 78% of patients are observed for specific T-cell responses. As a result, the median PFS and OS were 5.2 months and 12.7 months, respectively, demonstrating better prognosis.[55] Meanwhile, a phase I clinical trial with a class of tripeptide vaccines consisting of cell division cycle-related protein 1, cadherin 3, and kinesin family 20A for advanced BTC patients was conducted.[56] This study has revealed that the tumor vaccine is well tolerated and all patients develop peptide-specific T-cell immune responses in the body. In addition, a phase II clinical trial with a tripeptide vaccine harboring vascular endothelial growth factor receptors 1, 2 and kinesin family 20A has been recently publicized.[57] Of note, four out of six participants with advanced BTC in this study have displayed peptide-specific T lymphocyte responses. Although the substantial large cohorts with clinical trials warrant validating the benefit of polypeptide vaccines, the favorable outcomes of current studies are informative.

DC vaccines

Cell-based tumor vaccine is used as a powerful treatment option, in which the most common is DC vaccines that are loaded with cancer antigens on DC.[58] As professional antigen-presenting cells in the human body, immature DCs first enter tissues from blood to engulf antigens and present antigen-specific molecules to T cells that efficiently eliminate pathogens. DC precursors are loaded with tumor antigens in vitro and then infused back into the patients. Lysate-loaded DCs produced from treatment of a GBC cell line with heat shock in vitro induce activation of CD4+ T cells and CD8+ T cells in patients.[59]

Nucleic acid vaccines

In recent years, the innovation of nucleic acid tumor vaccines has emerged as a new effective regime in cancer immunotherapy. The success of genetic material-derived vaccines is largely ascribed to the rapid development of a series of high technology in biological applications including epigenetic modification of inserted nucleosides, development of new delivery materials, and refinement of messenger RNA (mRNA) transfer technology.[60] mRNA vaccine carrying plasmids that encode tumor-associated antigens can activate DCs and trigger specific immune response. For example, mRNA vaccines encoding three antigens cluster of differentiation 247 (CD247), Fc gamma receptor Ia (FCGR1A), and transformation/transcription domain associated protein (TRRAP) are expected to induce DC-mediated tumor immunity as "cold" cholangiocarcinoma mRNA vaccines, because co-expression of these three genes is associated with antigen-presenting cell infiltration and better prognosis of cholangiocarcinoma patients.[60]

Although a variety of tumor vaccines have been created and benefited BTC patients, multiple trials with the large samples and combination therapy with other drugs need to firmly establish the efficacy, ultimately improving the treatment.

ACT

ACT is a newly developed, promising cellular treatment in which, following expansion of patient-derived immune cells in vitro, these cells are administrated back into the patients to reinvigorate anti-tumor immunity.[61] A number of immunocompetent cells can be employed in this method, including tumor-infiltrating lymphocytes (TILs), T cell receptor chimeric T cells (TCR-T), chimeric antigen receptor T (CAR-T) cells, and cytokine-induced killer (CIK) cells. In BTC, to date, there are relatively limited clinical trials reported, which mainly focus on CAR-T, TIL, and CIK treatment.

CAR-T

CAR-T cells are patient-derived T cells genetically engineered with exogenous receptor(s) that specifically recognize tumor-expressing antigen. Technologies such as viral vectors-mediated gene therapy and CRISPR/Cas9 mediated genome editing make it possible to insert the recombinant DNA that codes CAR and eliminate the expression of TCR. Then CAR-T cells are administrated back to the patient to eliminate malignant cells. At present, a series of phase I and II clinical trials of CAR-T therapy for advanced BTC has been completed.

A phase I clinical trial of CAR-T cell therapy targeting human epidermal growth factor receptor 2 (HER2) in nine advanced BTC has demonstrated that one patient shows a partial response.[62] Three patients are in good physical condition, suggesting that CAR-T therapy is a potential approach to prolong survival for HER2-positive BTC patients. Likewise, another clinical study focuses on CAR-T therapy for patients with advanced BTC overexpressing epidermal growth factor receptor (EGFR). One of 17 patients has complete remission and 10 are well-controlled.[63] There is another ongoing study (NCT03633773) designed to evaluate the efficacy of MUC1-targeting CAR-T cells in patients with ICC. The results remain to be presented.

TIL

TIL is to isolate specific effector T cells from tumor tissue, expand the population in vitro, and then infuse them into the patients. Studies have shown that TIL may have a positive effect on patients with locally advanced or distant organ metastatic BTC.

A clinical trial using TIL in a patient with mutation in ERBB2-interacting protein has shown a remarkable inhibition of BTC.[64] Moreover, a clinical study in 2012 (UMIN000005820) with 62 cases of BTC demonstrated that the PFS of 36 ICC patients treated with surgery, DC vaccine, plus TILs was 18.3 months, in sharp comparison with only 7.7 months of PFS observed in 26 patients treated with surgery alone (P <0.05).[65] A phase II study on the efficacy and safety of TIL cell therapy in 59 patients with advanced BTC has also been initiated and the results will be published once it ends (NCT03801083).

CIK

CIK cell therapy involves culturing patients' peripheral blood lymphocytes in vitro and then infusing them back into the body for anti-tumor effects. In a clinical trial of CIK cell therapy plus DC vaccine in the treatment of patients with BTC, the result indicates that two of 85 patients have complete responses, 14 patients have partial responses, and 54 patients show good curative effects.[66]

In short, although the small clinical studies of ACT in BTC have begun and some have yielded encouraging benefits, multiple core-centers with longitudinal, case-control trials are essential to firmly demonstrate the efficacy. In addition, decreasing treatment cost should be also taken into account in order for this therapeutic approach to be affordable for a vast majority of cancer patients.

Cytokine therapy

Cytokines are involved in the regulation of the immune responses by binding to immune cell surface receptors that mediate a wide range of immune activities.[67] Therefore, cytokine therapy is to promote the growth and immune activity of immune cells. To date, there are a number of cytokines used to treat cancers, including interleukin-2 (IL-2), interferon-α (IFN-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

IL-2

IL-2 can stimulate T cells and NK cells to induce varied immunological activities. High doses of IL-2 have been appreciated to inhibit tumors, while low doses of IL-2 induce proliferation of Tregs that may act opposite anti-tumor function. As early as in the 1990s, high doses of IL-2 were used in the treatment of metastatic renal cell carcinoma and metastatic melanoma, but its multi-system toxicities have been taken into account.[68,69] Interestingly, the combination of IL-2 and 13-cis-retinoic acid (RA) can effectively reduce the toxicity of IL-2, thus achieving significant therapeutic effects on pancreatic and bile duct cancers.[70]

IFN-α

IFN-α mainly activates macrophages and NK cells to elicit anti-tumor immune responses.[71] For many years, IFN-α has been proved effective in a variety of malignancies, including melanoma, renal cell carcinoma, and chronic myelogenous leukemia (CML).[72] A recent study has reported that the combination of IFN-α and anti-PD-1-based immunotherapy significantly inhibits the proliferation and metastasis of cancer in patients with advanced liver cancer, making it possible to apply this technique to BTC in the future.[