Malignant melanoma occurs in various parts of the human body, but the most common sites are the sole and toes, which account for 40% of cases, followed by the head and neck, trunk, and fingers. According to the classic Clark classification, the four types of melanomas are lentigo lentiginous melanoma (ALM), nodular melanoma (NM), superficial spreading melanoma (SSM), and lentigo malignant melanoma (LMM), while those that occur in the mucosa are classified as mucosal melanoma (MCM). Some cannot be classified into any of these categories. In the United States, SSM accounts for 70%, ALM is < 5%, and MCM is very rare [1, 2]. However, in Japan, ALM (40%) and MCM (10%) account for half of the cases [3].
In humans, oral melanoma is aggressive malignancy and has a worse prognosis than cutaneous melanoma [4]. Oral melanoma accounts for 0.2–8% of all melanomas [5] and 1–2% of all oral malignancies [6]. Currently, melanoma treatment in humans includes surgical excision, radiation therapy, chemotherapy, melanoma vaccines, immune therapy, and targeted therapy [7]. However, advanced-stage melanoma and metastasis are difficult to treat. Therefore, it is necessary to find a therapy to increase the survival rate and quality of life for patients with advanced-stage disease.
In a report comparing postoperative adjuvant therapy with recurrence-free survival (RFS) rates performed for one year, the RFS rates in the COMBI-AD (dabrafenib plus trametinib), CheckMate 238 (nivolumab), and EORTC 1325 (pembrolizumab) were 88%, 70%, and 75%, respectively. Dabrafenib plus trametinib, a molecularly targeted therapy, is more effective. However, at the end of treatment, this difference disappears, with 3-year RFS rates of 58%, 58%, and 63.7%, respectively [8]. These results indicate that the tendency to relapse from the moment of discontinuation of its administration is a common problem with molecularly targeted drugs. In malignant melanoma, the number of genetic mutations (TMB) varies by disease type and is high in SSM but very low in ALM and MCM [9]. For this reason, some have suggested that immune checkpoint inhibitors (ICIs) may be more effective in Europe and the United States, where SSM is more common, but in Japan, where ALM and MCM account for half of all patients, ICI may be less effective. Among ICI, the frequency of immune-related adverse events (irAE) does not change much with increasing doses of anti-PD-1 antibody as a single agent, but the frequency of adverse events (AEs) (e.g., gastrointestinal disorders) increases with the dose of anti-CTLA-4 antibody, both as a single agent and in combination [10]. Furthermore, when anti-PD-1 and anti-CTLA-4 antibodies are combined, various irAEs occur simultaneously, and gastrointestinal symptoms, skin and liver damage are more likely to occur when they are combined simultaneously [11].
Considering the above, we investigate the use of oncolytic viruses as adjuvant therapy in patients with melanoma. In this study, we propose that the use of dogs is useful as a clinical model for melanoma therapy with oncolytic viruses because metastases from primary tumors occur frequently and in a relatively short period (within a year) after surgery, similar to those in humans.
Melanoma is common cancer in canines. The most common location was hairy skin. However, primary melanomas can occur in the oral cavity, gastrointestinal tract, footpad, eye, nail bed, nasal cavity, anal sac, or mucocutaneous junction [12]. Oral cancer accounts for approximately 6% of canine cancer [12]. Malignant melanoma is the most common type of canine oral tumor and is reported to be aggressive cancer with high local invasiveness and a high rate of metastasis [13]. Melanomas can occur in the oral cavities of dogs along the gingiva, lips, tongue, and hard palate [14]. Currently, treatment options for malignant melanoma include surgery, radiation therapy, and immunotherapy in the veterinary field. Surgical excision is the first choice treatment. However, the available treatment options for canines with advanced-stage disease are limited and the prognosis is very poor. Therefore, the identification of a promising new strategy for cancer therapy is important. One promising therapeutic approach is oncolytic virotherapy. Several oncolytic viruses have been used for canine cancer therapy [15]. In recent years, immunotherapy, including targeted antibodies, checkpoint inhibitors, cytokines, and adjuvants, have been applied as monotherapy or in combination with current treatments for human and dog cancer. Melanomas encountered in dogs are primary tumors and present metastases that recur postoperatively in a relatively short period compared to humans. Due to the relative ease with which these melanomas can be obtained, it is beneficial to use canine clinical samples to study human treatment models.
Oncolytic virotherapy has recently been approved as a novel therapeutic agent for cancer treatment. An oncolytic virus selectively infects, replicates, and kills cancer cells without harming normal cells [16]. In humans, the benefits of oncolytic viruses for the treatment of cancer have been studied [17]. Oncolytic viruses are being developed clinically as treatment options in humans. Herpes simplex virus (HSV) has been used to treat patients with cutaneous or subcutaneous metastases of the breast, epithelial cancer of the head and neck, gastrointestinal adenocarcinoma, and malignant melanoma [18]. Patients with metastatic melanoma are currently being treated with HSV in a phase II study [19]. An adenovirus formulation has been reported to be a treatment option for bladder cancer [20]. Reoviruses have been used for the treatment of prostate cancer [21], metastatic colorectal cancer [22] and malignant glioma [23]. Several oncolytic viruses have been studied in the veterinary field; however, only a few clinical trials have reported the use of oncolytic viruses for the treatment of canine cancer [15]. For example, strains of canine adenovirus, canine distemper virus, and vaccinia virus have been used for canine cancer therapy in preclinical studies. Further, adenovirus has been used for the treatment of canine osteosarcoma and canine mammary carcinoma [24]. The measles virus (MV) belongs to the genus Morbillivirus of the Paramyxoviridae family and has been reported to treat canine mammary cancer [25].
The Newcastle Disease Virus (NDV) is an avian paramyxovirus with a negative single-stranded RNA genome. It is characterized by selective viral replication in tumor cells [26]. NDV is an avian pathogen that causes respiratory diseases. However, this virus is an oncolytic virus that replicates tumors and is selective in mammals. NDV-infected tumor cells have been reported to produce type I interferons (IFNs), induce the expression of class I major histocompatibility complex (MHC) and cell adhesion molecules, for example, hemagglutinin-neuraminidase (HN) protein on the surface of tumor cells increases the expression of class I MHC [27]. NDV has also been proposed for the treatment of human carcinoma [28]. Phase I and II clinical trials are progressing to evaluate NDV for treatment in humans [29]. A recombinant NDV-green fluorescent protein (GFP) has also been developed and has been reported to exert anti-tumor activity and anti-tumor immunity-inducing effects using tumors derived from mice [30, 31]. Previously, we generated a canine IFNγ (cIFNγ) gene expressing recombinant NDV (rNDV) using reverse genetics. Virus-infected cells induced cell death without producing any infectious virus particles because the amino acid sequence at the viral F protein cleavage site was transformed (G-R-Q-G/S-R; L) [32]. Recombinant NDV (rNDV) is safer than wild-type NDV for patient inoculation. NDV-infected tumor cells have been reported to induce apoptosis through caspase activation [33]. The indirect oncolytic mechanism is an immune-mediated response associated with both innate and adaptive immune responses [27, 34].
Interferon-γ (IFNγ) is the only member of the type II interferon family, which is generated following several innate and adaptive immune cell responses. IFNγ is a cytokine with antitumor activities, involved in the control of tumor initiation and progression [35]. IFNγ is useful for adjuvant immunotherapy in different types of cancer [36]. Interleukin-2 (IL-2) is a T cell growth factor. It is mainly secreted by CD4 + and CD8 + T lymphocytes and can stimulate proliferation of cytolytic activity and cytokine secretion by T lymphocytes and natural killer cells [37]. In humans, antitumor efficacy has been reported in patients with metastatic melanoma [38]. Interferon regulatory factor 1 (IRF-1) is a tumor suppressor gene with antiproliferative and pro-apoptotic effects on cancer cells [39]. Previous descriptions of IRF-1 expression have been associated with caspase activation [40].
The purpose of this study was to develop a novel therapy for malignant oral melanoma using the recombinant Newcastle disease virus. In this study, we applied the oncolytic recombinant NDV (rNDV) to induce cell-mediated immunity in treated patients and succeeded to achieve the oncolytic activity together with the production of canine IFNγ by rNDV-cIFNγ infection.