Current progress in immunotherapy of nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma is one of the highly prevalent malignant tumors and the most common type of ear, nose, and throat cancer. The exact cause of various cancer is still unclear; however, a diversity of risk factors for the development of nasopharyngeal cancer have been reported, including genetic changes, viral infection, and environmental factors, among which, Epstein-Barr-Virus plays an important role in the oncogenesis of nasopharyngeal carcinoma. Nasopharyngeal carcinoma is highly malignant and prone to metastasis, while most of them are moderately sensitive to radiation therapy; hence, radiation therapy combined with chemotherapy is currently the standard treatment for nasopharyngeal carcinoma. However, this combination therapy tends to cause more complications and tumor recurrence, which not only increases the financial burden to patients, but also adversely affects their physical and mental health. In recent years, immunotherapy has been emerging as a new strategy to treat malignant tumors including nasopharyngeal carcinoma and has achieved favorable results. The immunotherapy for nasopharyngeal carcinoma can control or even eliminate tumor cells by inducing and enhancing the collective immune function. Currently, the main immunotherapeutic approaches for nasopharyngeal carcinoma include successive immune cell therapy, immune checkpoint inhibitors, and tumor vaccines. However, the efficacy of immunotherapy in nasopharyngeal carcinoma still require improvement. In this review, we summarized the current progress and efficacy of immunotherapy, particularly by targeting EVB, in the treatment of nasopharyngeal carcinoma.

Introduction

Nasopharyngeal cancer occurs in the nasopharynx behind the nose and above the back of throat. It is one of the highly prevalent malignant tumors and the most common type of ear, nose, and throat cancer [1-3]. The specific pathogenesis of this cancer is still unclear but multiple risk factors have been reported, including genetic changes, viral infection, and environmental factors [4-7]. The main clinical manifestations of nasopharyngeal carcinoma include snotty blood or nasal bleeding and headache. Advanced nasopharyngeal carcinoma can lead to cachexia or even death.

The traditional treatment methods for nasopharyngeal cancer include surgery and radiotherapy, but due to the limitation of anatomical location, surgery is difficult to implement. Since nasopharyngeal cancer is more sensitive to radiation therapy, radiation therapy combined with chemotherapy is currently the preferred treatment option for nasopharyngeal cancer [8-10]. Although the efficacy of the combination treatment regimen was better than monotherapy in overall survival rate and recurrence survival rate, the adverse side effects from the combination treatment therapy were much severe, especially the acute adverse effects were significantly increased. In addition, the combination treatment therapy does not significantly reduce the incidence of distant metastasis. For example, Sun et al. retrospectively analyzed the clinical data of 123 patients with nasopharyngeal carcinoma and found that the prognosis of patients in the concurrent radiotherapy group was not significantly better than that of the radiotherapy alone group, while the incidence of neutropenia, hemoglobin reduction, and radiation mucosal reaction was significantly increased [11].

Epstein-Barr-Virus (EBV) plays an important role in the development of nasopharyngeal carcinoma [12-14]. Almost all nasopharyngeal carcinoma subtypes, including basal-like, keratinized, and non-keratinized types, are associated with EBV [15,16]. Depending on the cell type, EBV-infected cells undergo different latent cycles and express different viral proteins. Because viral proteins are almost exclusively expressed in malignant cells, EBVs present as ideal targets for immunotherapy to treat nasopharyngeal carcinoma [13,17-19]. Since the blockade of CTLA-4 and PD-1 as well as CAR T-cell therapies have produced significant therapeutic responses in cancer patients, immunotherapy has revolutionized the current cancer treatment regimens. Moreover, immunotherapy can not only persistently remove the small amount of disseminated tumor cells but also improve the body immune function damaged by radiotherapy and chemotherapy, demonstrating the complementary advantages in combination with other conventional therapies [13,20,21].

Adoptive cell transfer therapy

Adoptive cell transfer therapy, also known as cellular immunotherapy, involves several different immunotherapy methods. Basically, in this type of therapy, the biologically active immune effector cells from patients are isolated and cultured in vitro to expand their number by thousands of times or are manipulated to enhance their cytotoxic function or to induce them to differentiate into immune effector cells with anti-tumor activity. Then, these cells are infused them back into patients to eliminate pathogens, cancer cells, and the mutated cells in blood and tissues to exert their anti-tumor effects [22].

Tumor infiltrating lymphocyte (TIL) therapy

In the early stages of cancer, the immune system plays defensive roles by attacking the tumor via mobilizing special immune cells of lymphocytes. These lymphocytes, also known as TILs, discovered by Rosenberg et al., can recognize and attack tumor and penetrate the tumor mass [23]. However, the tumor microenvironment and the expression of PD-1 can inhibit the activity of TILs, as a result, TILs are unable to effectively kill the tumor cells in tumor tissue. Therefore, growing efforts have been made to enrich certain type of lymphocytes in tumor tissues by in vitro culture methods, which are then infused back to patients to play an anti-tumor role. Moreover, combining this enrichment with PD-1 can further improve the therapeutic efficacy [24]. In a phase I clinical trial conducted by Li et al. [25], 20 patients with nasopharyngeal carcinoma were infused with in vitro-expanded autologous TILs after synchronous chemoradiotherapy, and 19 out of 20 patients exhibited complete remission with only mild adverse effects. In addition, after infusion of TILs, increased expressions of LMP-1, LMP-2 and EBNA-1-specific T cells could be detected in the blood of 65% of patients, while plasma EBV load was significantly reduced in 85% of patients. Hence, the findings from this clinical trial confirmed that adoptive cell transfer therapy with TILs produced sustained antitumor activity and anti-EBV immune response in patients with nasopharyngeal carcinoma.

Engineered T-cell receptor (TCR) therapy

Nevertheless, not all patients have T cells that can recognize the tumor. For these patients, an engineered TCR therapy can be applied. This approach starts with obtaining T cells from the patient first. Then, in vitro manipulation of these cells will activate and expand these anti-tumor T cells while equipping them with new T cell receptors that enable them to recognize specific cancer antigens. TCR-based adoptive therapy allows the modification of the optimal target for each patient’s tumor and different types of T cells. This personalized therapy offers patients higher probability of remission. At present, the application of TCR-T in patients with nasopharyngeal carcinoma is still under clinical trials, and the specific treatment plan and efficacy need to be investigated in subsequent studies [26].

Chimeric antigen receptor (CAR) T-cell therapy

The basic principle of CAR T-cell therapy is to genetically modify the patient’s own T-lymphocytes, which are then processed and cultivated to meet the requirements for therapeutic purposes before these cells are infused back to the patients [27]. These modified T-lymphocytes can specifically recognize tumor cells that express antigenic targets, thereby activating T-lymphocytes to effectively kill tumor cells.

One example of this approach was reported by Zhao et al. [12], in which using LMP2A as the target, the authors successfully prepared LMP2A CAR T cells and confirmed by in vitro and in vivo experiments that compared with CD19 CAR T cells and T cells, LMP2A CAR T cells had a stronger cytotoxic effect on nasopharyngeal carcinoma cells, particularly, a significant targeting toxicity on LMP2A-positive nasopharyngeal carcinoma cells.

Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTLs) has been proven clinically effective, but it has never been evaluated in combination with chemotherapy in the first-line treatment. One follow-up study evaluated the efficacy of adding 5-fluorouracil (5-FU) as maintenance chemotherapy to the induction chemotherapy with Paclitaxel-Gemcitabine-Carboplatin (PGC) in a cohort of nasopharyngeal carcinoma patients. While this regimen was effective, it was also highly toxic, with almost 80% of patients experiencing grade 3 to 4 neutropenia [28]. However, when using the combination of EBV-CTLs, the chemotherapy efficacy is improved with tolerable side effects. Chia et al. found that the median survival was increased to 29.9 months in patients with recurrent and/or metastatic nasopharyngeal carcinoma receiving GC-CTL regimen (gemcitabine and carboplatin (GC) followed by up to six doses of EBV-CTL), while it was only 17.7 months in those receiving PGC chemotherapy and 21.4 months in those receiving PGC-5-FU chemotherapy [22]. The two-year overall survival rates for patients receiving GC-CTL, PGC-5-FU, and PGC regimens were 62.9%, 42.9% and 29.5%, respectively, and the three-year overall survival rates were 37.1%, 25% and 16.4%, respectively, demonstrating the benefit of using CTL combination therapy. Their study also found a positive correlation between LMP2-specific T cells and survival.

Natural killer (NK) cell therapy

NK cells present in the human bloodstream as the first responders. They directly recognize the abnormal cells and release cytotoxic granule contents, e.g., perforin and granzyme, which are recognized by target cells before the T cells are deployed, triggering the self-destruction of cancer cells. They can also eliminate circulating cancer stem cells to prevent metastasis. Growing studies are exploring the potential of integrating these NK cells with cancer-targeting CARs [29-31]. As an example, Mei et al. studied the cytotoxic effect of the modified NK cells on human nasopharyngeal carcinoma cells and found that the modified NK cells could eliminate the nasopharyngeal carcinoma cells, and IL-2 and IL-15 treatments could up-regulate the expression of NKG2D in the modified NK cells and restore their cytotoxicity to CNE2 cells [30].

Immune checkpoint inhibitors (ICIs)

It has been well known that ICIs block the function of immune checkpoint proteins to enhance the immune response or to relieve immune suppression. Programmed death 1 (PD-1, also known as CD279) belongs to the immunoglobulin superfamily expressed on the surface of T cells and Pro-B cells and inhibits T-cell activation through binding to its ligands PD-L1 or PD-L2, whereas PD-1 pathway inhibitors, e.g., PD-L1 and PD-L2 inhibitors, inhibit PD-1 binding to ligands, thereby enhancing T-cell activity, which are particularly effective in the late phase of the immune response [32].

A study on the regulatory mechanisms of PD-L1 in EBV-positive nasopharyngeal carcinoma showed that interferon (IFN)-γ, IFN-β and EBV latent membrane protein 1 (LMP1) induced cellular PD-L1 expression [33]. In addition, PD-1 inhibitory monoclonal antibodies can enhance the cytotoxic effects of NK cells in nasopharyngeal carcinoma by mediating the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), leading to antitumor effects [34]. A study by Hsu et al. revealed that anti-PD-1 antibody Pembrolizumab showed favorable antitumor activity and a manageable safety profile in patients with recurrent or metastatic nasopharyngeal carcinoma [35]. In addition, Ma et al. found that anti-PD-1 antibody Nivolumab had better anti-tumor activity without unexpected toxicity, resulting a better 1-year overall survival of patients than that previously reported from a similar population [36]. Furthermore, a study by Lee et al. showed that PD-L1 was substantially expressed in nasopharyngeal carcinoma cells, and high PD-L1 expression (IHC2+) was associated with better local progression-free survival and PFS [37]. However, in contrast to this study, a study by Chan et al. showed that no prognostic value of PD-L1 expression levels was observed in patients with nasopharyngeal carcinoma [38]. The possible explanation for these different results could be different PD-L1 antibodies or scoring algorithms used in those studies and different baseline characteristics of enrolled patients.

Tumor vaccine therapy

Tumor vaccine therapy is tumor-specific active immunotherapy which includes tumor cell vaccine, tumor nucleic acid vaccine, tumor peptide vaccine, anti-idiotype antibody tumor vaccine, tumor genetic engineering vaccine, and dendritic cell vaccine. Anti-idiotype antibody (Anti-ID Abs) is a specific antibody against the antigenic determinant (idiotype) in the variable region of an antibody, which can be used as an important reference for immunogenicity analysis [39,40]. There has been some progress in tumor vaccine therapy, mostly in basic research and clinical trial stage. The Newcastle disease virus (NDV)-modified autologous tumor cell vaccine fNDV ATV developed by the German Cancer Center has achieved promising results in a multicenter, multitumor clinical trial. NDV is an avian paramyxovirus with a multi-tissue affinity for immunostimulatory characteristics. More importantly, NDV can replicate selectively in the cytoplasm of tumor cells independent of host cell proliferation, and it is a non-pathogenic virus that can effectively and safely infect tumor cells. Currently, fNDV.ATV is usually prepared in a PBS environment, where every 10 million cells are warmed with 32HU NDV ulster for one hour, and the cellular vaccine is inactivated by 200 Gy r-radiation or MMC treatment before it can be used for intradermal injection in patients with nasopharyngeal carcinoma. T cells are accumulated locally via NDV-induced chemokines, and some T cells express T cell receptors (TCRs) which interact with TAAs peptides present as MHC-TAAs complexes on the surface of tumor cells to mediate first signal generation. Tumor cells that lack co-stimulatory molecules are not able to transmit additional co-stimulatory signals and therefore fail to activate tumor-specific T lymphocytes [41-43]. Yue et al. prepared a live vaccine with the NDV La Sota strain and inoculated the vaccine in rabbits by the spray aspiration. After being proved to be safe and effective, the vaccine was then inoculated by the same way in nasopharyngeal carcinoma patients who have undergone radiation therapy. The results showed that the 3-year recurrence rate of the treated group was significantly lower than that of the control group (4.00% vs. 26.0%).

Dendritic cell vaccine is another antitumor immunotherapy that has been intensively studied in recent years and has shown promising therapeutic effects in multiple preliminary clinical trials of various tumors. Lin et al. loaded dendritic cells of nasopharyngeal carcinoma patients with a restricted epitope polypeptide of latent membrane antigen 2 (LMP2) and then infused the cells back into the patient [44]. Among the 16 patients tested, 9 patients showed strong cytotoxic T lymphocytes (CTLs) activity against LMP2 peptide. Also, the tumor volume decreased in 2 patients 3 months after treatment. In vitro experiments showed that CTLs activated by DC-loaded LMP2A1 1 peptide binding epitopes could effectively inhibit the growth of poorly differentiated nasopharyngeal carcinoma cell lines.

In another study by Wang et al., the bispecific and multivalent antibodies with more antigen binding would improve the immunogenicity of the vaccine. The bispecific anti-idiotype antibody vaccine G22-I50, produced through the genetic engineering approach to enhance its immunogenicity, induced a strong humoral and cell-mediated immune response, suggesting its potential application in the prevention and treatment of nasopharyngeal carcinoma [45]. The ability of bispecific antibodies to improve vaccine immunogenicity is because they can enhance the killing effect on target cells by recognizing and binding two different antigens to attach immune cells, viral molecules, and other factors to tumor cells. They can also bind different antigens on the same tumor cells to enhance their binding specificity, thus reducing side effects such as off-target toxicity. On the other hand, anti-idiotype is a strong stimulator of CD4 response. Chen Luo et al. found that three injections of PCDNA3.1-G22, an anti-idiotype antibody, induced CD4+ T helper cell activity and CD8+ cytolytic activity, thereby protecting mice against tumor cell challenge. This might explain why anti-idiotypic antibodies could elicit strong immune responses [40]. In addition, Taylor et al. conducted a dose-escalating phase IA trial in British to determine whether the MVA recombinant cowpox vaccine was broadly applicable, as it was constructed from a Chinese-derived EBV strain [46]. Their results revealed that vaccination increased EBNA-1 (EBV nuclear antigen 1) in 7 of 14 (50%) patients and increased LMP2 reactivity in 6 of 14 (43%) patients by more than 2-fold. The vaccination also increased the proportion of epitope specific CD4+ and CD8+ T cells to 120% and 420%, respectively. Most importantly, EBV strain variation and HLA diversity did not alter the immunogenicity of the vaccine, demonstrating the feasibility of using this approach in Southeast Asia, where nasopharyngeal carcinoma has the highest incidence rate.

Conclusion and prospective

Currently, immunotherapy has been widely used in the treatment of various tumors, including nasopharyngeal carcinoma (Table 1), and has achieved promising therapeutic effect in improving the survival and the quality of life of cancer patients [47,48]. However, immunotherapy still has some limitations. The efficacy of immune monotherapy is relatively low, which needs to be combined with chemotherapy and radiotherapy. In addition, drug resistance is the major challenge when using immune checkpoint inhibitors, as studies have shown that resistance is observed in up to approximately 60% of patients receiving anti-PD-1 therapy [49-51].

Table 1.

Clinical studies of immunotherapy in patients with nasopharyngeal carcinoma

Authors	Immunotherapy regimen	Therapeutic effect	Safety
Li et al. [14]	Treatment with CCRT and ACT using expanded auto-TIL	The short-term CR was 69.5% in the 23 enrolled patients at the completion of CCRT and 83% (19/23, DFS >9) at 3 months after ACT when using auto-TILs.	18 exhibited DFS for longer than 12 months. Only one patient presented a residual neck lymph node 3 month after ACT.
Chia et al. [13]	GC-CTL regimen/PGC-5-FU and PGC regimens*	The median survival was 29.9 months in patients receiving GC-CTL regimen, 17.7 months in those receiving PGC chemotherapy, and 21.4 months in those receiving PGC-5-FU chemotherapy.	Our results include additional follow-up data collected in 2007 that contained 11 more deaths as compared with 15 deaths reported in the original publication.
Hsu et al. [18]	Nivolumab	Partial response and stable disease were observed in 7 and 14 patients, respectively, for an ORR of 25.9% (95% CI, 11.1 to 46.3) over a median follow-up of 20 months.	Patients had better activity, no unexpected toxicity, and better 1-year overall survival than previous data from a similar population.
Ma et al. [19]	Nivolumab	The overall ORR was 20.5%. The 1-year overall survival rate was 59% (95% CI, 44.3% to 78.5%) and 1-year progression-free survival (PFS) rate was 19.3% (95% CI, 10.1% to 37.2%).	There was no association between survival and PD-L1 expression or plasma Epstein-Barr virus DNA clearance. There was no unexpected toxicity to nivolumab.
Taylor et al. [24]	MVA recombinant cowpox vaccine	Vaccination increased EBNA-1 (EBV nuclear antigen 1) in 7 of 14 (50%) patients and increased LMP2 reactivity in 6 of 14 (43%) patients by more than 2-fold, and also increased the mass and frequency of epitope-specific CD4+ and CD8+ T cells to 120% and 420%.	EBV strain variation and HLA diversity did not alter the immunogenicity of the vaccine.

^*GC-CTL: Gemcitabine and Carboplatin (GC) followed by EBV-specific Cytotoxic T Lymphocytes; PGC-5-FU: Paclitaxel-Gemcitabine-Carboplatin (PGC)-5-Fluorouracil (5-FU).

Therefore, overcoming the acquired resistance against immune checkpoint inhibitors is an urgent unmet medical need. Furthermore, the safety of immunotherapy is also a concern. Currently, there is no definitive standard for assessing the safety of immunotherapy, which needs to be addressed.

Disclosure of conflict of interest

None.