Immunology of Canine Melanoma

Abstract

Malignant melanoma is one of the most important tumors in dogs and is highly metastatic and aggressive disease. In recent years, molecular knowledge regarding canine melanoma has increased, and some chromosomal imbalances and tyrosine kinase pathways have been identified to be dysregulated. Mxoreover, canine melanoma is an immunogenic tumor that provides opportunities to administer immunotherapy to the patient. Podoplanin and chondroitin sulfate proteoglycan-4 (CSPG4) are markers against which monoclonal antibodies have been developed and tested in dogs in vivo with promising results. Owing to the importance of canine melanoma in the veterinary oncology field, this chapter reviews the most important aspects related to immunological involvement in the prognosis and treatment of canine melanoma.

1. Introduction

Canine melanoma is an aggressive tumor that originates from melanocytes in different sites in the animal body, including the oral cavity. Melanoma of the oral cavity is a highly aggressive disease and is the most prevalent cancer in the oral cavity [1, 2, 3, 4, 5, 6, 7, 8, 9]. According to the Oncology Pathology Working Group (OPWG) consensus, some of the most common histologic features of melanoma include intracytoplasmic melanin, variable cell morphology, junctional activity, pagetoid growth, presence of neoplastic cells at the mucosal–submucosal junction, and finely stippled to vesiculated nucleus with a prominent central nucleolus (“owl’s eye”) [9]. Distant metastasis, lymphatic invasion, nuclear atypia, mitotic index, tumor size/volume, and tumor score are also related to poor prognosis [6, 9, 10].

Canine melanoma is an immunogenic tumor, and investigations of different aspects associated with immune cells in tumor development and progression have been reported in the literature [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. Moreover, genomic and transcriptomic data from canine oral melanoma have revealed several pathways associated with inflammatory processes, including T-helper cell differentiation [1]. Thus, evaluation of the immune components of canine melanoma is pivotal for a better understanding of tumor biology and for developing new therapies. Therefore, this chapter reviews the association between the immune system and canine melanoma development, prognosis, and treatment.

2. Literature review

2.1 Tumor etiology

Canine oral melanoma is the most frequently diagnosed malignant tumor in dogs [2]. Oral melanoma is associated with a high local infiltration, metastatic rate, and poor prognosis. Biological behavior and presentation of melanoma vary remarkably and are influenced by anatomic site, stage, and histological features [1, 2]. Increasing age, with no relation to sex, is also a determinant for canine oral melanoma aggressiveness [2, 3, 6].

The etiology of canine oral melanoma is multifactorial, including environmental and genetic factors [6]. In cutaneous melanoma, hairy skin and sunlight exposure could be considered risk factors [2]. However, sunlight exposure cannot be considered a risk factor for oral melanoma. Chronic inflammation or trauma, deep bacterial infections, intralesional necrosis, chemical exposure, and burns are factors associated with canine melanoma development [6, 8].

Highly pigmented oral mucosa and purebred dogs, such as Airedale Terrier, Boston Terrier, Boxer, Chihuahua, Chow, Cocker Spaniel, Doberman Pinscher, English Springer Spaniel, Golden Retriever, Irish Setter, Miniature Schnauzer, Scottish Terrier, Poodles, Beauce Shepherds, Rottweilers, and Labrador Retrievers, are predisposed to melanocytic tumors, including oral melanoma [3, 4, 5]. This suggests that melanoma in dogs may have genetic factors [7, 8].

3. Melanoma clinical signs and diagnosis

Clinical signs of canine oral melanoma include halitosis, dysphagia, anorexia, weight loss, facial swelling or swelling of the lymph nodes, drooling, panting, loss of teeth, and oral and facial pain, which may induce the dog to avoid being touched. Dogs affected by oral melanoma can also be asymptomatic; in these cases, the owner or veterinarian may discover the oral mass only during routine examination. The owner may also notice blood in the water or food bowl or that oozing from the mouth [10, 11, 12, 13].

The diagnostic methods include biopsy of the tumor, followed by histopathological examination. Immunohistochemical tests can provide a reliable and definitive diagnosis [7]. The diagnosis may be confirmed by cytology of a fine-needle aspirate [10]. Immunofluorescence can also be used to distinguish melanomas from melanocytomas [14]. However, other tests, such as skull radiographs, chest X-rays, mandibular lymph node samples, abdominal ultrasound, serum biochemistry, and complete blood count, are crucial for a better patient health overview, metastasis detection, and safer treatment choice [15].

4. Prognostic factors for canine melanomas

A histological and epidemiological study including 384 cases of melanocytic tumors comprised 19% oral tumors, of which 59% were malignant. In contrast, analyses of melanocytic tumors of the skin identified only 12% of patients with malignant tumor [6]. Regarding the prognostic criteria for canine melanoma, mitotic index, nuclear atypia, tumor volume, the presence of metastasis, and the presence of deep inflammation or intralesional necrosis remain pivotal when determining patient outcome [6, 9].

A previous study of 67 oral melanoma samples suggested that free surgical margins and chemotherapy with carboplatin increased patient survival [16]. Another study indicated that melanocytic tumors were more common in middle-aged dogs with dark hair and undefined breeds. Histological analysis also revealed the prevalence of epithelioid cells [17]. Another study evaluated 338 canine oral melanoma cases, with an overrepresentation of breeds, such as Chow, Golden Retriever, and Pekingese/Poodle mix, but with no mention of hair color. Histological evaluation suggested the presence of polygonal and spindle cells [18]. However, the mismatched results suggested that epidemiological data for canine oral melanoma require detailed evaluation.

5. Immune system and cancer

The relationship between the immune system and the tumor’s ability to evade it is a hallmark of cancer [19]. An increasing number of studies have investigated the role of the immune system in tumor progression and its therapeutic potential for various tumors. The immune system comprises several cell types, and recognizes and eliminates biological, chemical, and physical dangers from the body via a series of humoral and cellular pathways and interactions [20]. In contrast, a neoplastic cell is an autologous cell that harbors suppression and overexpression of certain genes, mainly tumor suppressor genes and oncogenes, respectively. Neoplastic cells express proteins that are not recognized by the immune system; thus, receptors cannot indicate to the immune system that the cell is abnormal to the organism. Therefore, the immune system is an important factor in the body to prevent tumor progression. Taken together, it is necessary for the tumor to employ mechanisms to evade the antitumor immune response.

A neoplastic cell proliferates in an uncontrolled manner and each daughter cell may be the same as its mother cell; however, many cells accumulate new genetic defects, generating intratumor heterogeneity. This allows the neoplasm to adapt to different adversities for its progression, such as the antitumor action of the immune system. This process of interaction between neoplastic cells, immune cells, and the entire tumor microenvironment presents a complex cell-cell relationship. The most accepted “tumor immunoediting theory” that explains the events of this interaction is divided into the following three phases [21]:

1. Elimination: Cells of the immune system recognize and eliminate pre-neoplastic and neoplastic cells. In this phase, complete elimination of cells may occur, ending tumor initiation; however, some cells may escape surveillance of the immune system and proceed with carcinogenesis.

2. Balance: Tumor growth is equivalent to elimination of neoplastic cells by the immune system. In this process, tumor cells with lower immunogenicity, that is, those capable of evading the immune system, are selected.

3. Escape: Tumor growth occurs due to the reduction in/inability of the immune system to eliminate neoplastic cells and the rapid growth of neoplastic cells.

6. Tumor immune evasion mechanisms

The immune system can react with agents that are harmful to the body in several ways. Innate immunity, responsible for this first interaction, can corroborate the antitumor activity with a nonspecific reaction to the tumor [22], such as treatment with Bacillus de Calmette and Guerin of human melanomas [23], that initially induces a nonspecific response to this agent, but later affects tumor progression. In contrast, innate immunity, which is primarily responsible for the antitumor activity of the immune system, is triggered by recognition of tumor cells by CD8+ T cells. They eliminate tumor cells by various pathways, such as perforin/granzyme or by induction of apoptotic pathways [22].

The interaction between the immune system, neoplastic cells, and tumor microenvironment is extremely complex and orchestrated by numerous regulatory factors, whether intrinsic or extrinsic to neoplastic cells. Neoplastic cells employ several tactics for evading the immune system, and consequently, the permanence of the escape phase in the immunoediting process. Cells use several mechanisms to succeed in this process, ranging from the non-recognition of cells as non-self to the production of inhibitory factors and exhaustion of immune system cells, including the activation of bone marrow-derived suppressor cells, activation of regulatory T cells (Tregs), alteration of dendritic cell functions, production of cytokines, and non-recognition of cells due to non-expression of histocompatibility molecules [24].

7. Myeloid-derived suppressor cells (MDSCs)

An immune cell population responsible for suppression of the immune system in tumors is myeloid-derived suppressor cells (MDSCs). These cells are formed by populations of monocytes and immature granulocytes arising from the bone marrow during pathological conditions [25]. Numerous studies have indicated an increase in the number of cells of this type in cancer in humans and mice [26]; but, its role in dogs is unclear [27].

In humans, MDSCs are responsible for immunosuppression of the tumor site, allowing a more invasive and metastatic characteristic through the production of metalloproteinases [28, 29]. A study [25] on the immunosuppressive action of MDSCs indicated that MDSCs can suppress natural killer cells, dendritic cells, and T lymphocytes, in addition to potentiating the effects of Tregs by producing reactive oxygen species and inducible nitric oxide synthase (iNOs). MDSCs also produce immunoregulatory cytokines, such as TGF-β and IL-10 and decrease the expression of IL-12, which is responsible for the activation of T cells [30].

8. Induction of regulatory T cells by tumors

Tregs, in addition to natural killer cells, are CD4 T cells capable of directly suppressing CD4+ and CD8+ T cells. Thus, the presence of Tregs in the tumor microenvironment may be involved in a decrease in the immune response, and many studies have demonstrated that an increase in these cells is correlated with worse prognosis. Muir et al. [31] demonstrated the prognostic role of Tregs in canine lymphomas, with dogs with higher levels of Tregs showing worse prognoses. A study by Curiel et al. [32] also demonstrated that an increase in the number of these cells in ovarian carcinoma predisposes dogs to a lower survival rate.

9. Impaired dendritic cell activation and function

Dendritic cells are the main antigen-presenting cells (APC) that play an important role in the activation of CD8+ T cells and NK cells and are considered the main tumor surveillance cells. Neoplastic cells employ mechanisms to escape the immune system. The activation and alteration of dendritic cells, such as an increase in the production of IL-10 that antagonizes their antigen-presenting action, inactivates dendritic cells and reduces their ability to stimulate other cells. They induce anergy to specific antigens. Another study suggested that the release of IL-6 in the tumor environment keeps the dendritic cells immature, worsening the prognosis of patients with several tumors, as in the case of melanoma [33].

The importance of dendritic cells has been further explored in antitumor immunotherapy, including the use of dendritic cells in the production of antitumor vaccines [34].

10. Failure of tumor cells to activate immune system

Tumors can also prevent the activation of the immune system by decreasing the expression of type I and II histocompatibility molecules [27]. The expression of co-inhibitory molecules, such as CD73 and programmed death ligand-1 (PD-L1) allows tumors to evade the immune system. The programmed cell death protein-1/programmed death ligand-1 (PD-1/PD-L1) pathway promotes apoptosis of CD8+ T lymphocytes [35]. Whereas cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a co-inhibitor expressed by T cells that negatively regulates the activation of T cells. Therefore, the tumor can avoid the immune system because the immune cells are unable to recognize neoplastic cells either by apoptosis or exhaustion mechanisms. Many studies have focused on these pathways to inhibit tumor cells, and thus, restore the recognition and activation of the immune system.

11. Cytokines

Neoplastic cells produce different cytokines that alter the tumor microenvironment. The tumor microenvironment has an extremely delicate balance between antitumor effects and the origin of tumors arising from pro-inflammatory activity that is dependent on the mediator cells [36]. Therefore, the cytokines may have antitumor and protumor effects, depending on the interrelationship between the tumor and constituents of the tumor microenvironment [37]. Thus, the use of cytokines as therapeutic agents is quite complex, as their action is dependent on the status acquired in the tumor microenvironment. Lin and Karin [37] also showed that chronic inflammation leads to the production of several cytokines that allow tumor development, highlighting that the interaction between cells present in the tumor microenvironment determines the effects of the released cytokines. Catchpole et al. [38], for example, demonstrated an increase in the production of IL-10 and TGF-β and a decrease in that of IL-2, IL-4, and IFN-γ in the lymph nodes with melanoma metastasis. Each cytokine has different actions on different cells, and its effects can either help in carcinogenesis or confer antitumor activity (Table 1).

Main cytokines	Main activity
IL-2	Induces T-cell proliferation and differentiation into effector T cells; Increases cytotoxicity of “Natural killer” cells; Induces proliferation of B lymphocytes
IL-3	Promotes production/differentiation and proliferation of macrophages, monocytes, granulocytes, and dendritic cells
IL-4	Induces CD4+ T lymphocyte differentiation in cells with Th2 phenotype; Increases production of MHC-II; Induces growth and differentiation of B lymphocytes
IL-6	Pro-inflammatory and anti-apoptotic cytokine that may contribute to tumor development associated with chronic inflammation
IL-8	Chemotactic and activating factor of neutrophils and T lymphocytes
IL-10	Immunosuppressive cytokine produced by activated dendritic cells, macrophages, and T cells
IL-11	Stimulates proliferation of hematopoietic stem cells; Induces megakaryocyte maturation
IL-12	Stimulates synthesis of IFN-γ and TNF-α by T cells and natural killer cells decreasing angiogenesis
IFN-α, IFN-β	Induces apoptosis of tumor cells; Activation of natural killer cells; Inhibits tumor angiogenesis
IFN-γ	Promotes CD4+ T cell differentiation to Th1 phenotype; Activates macrophages; Modulates MHC I/II expression
TNF-α	Stimulates angiogenesis and metastasis of some tumors; Important pro-inflammatory cytokine
TGF-β	Immunosuppressive cytokine; Inhibits macrophage activation and B lymphocyte growth; High expression in various tumors

Table 1.

Main cytokines relevant to tumor immunotherapy.

Adapted from the small animal clinical oncology [27].

12. State-of-the-art of immunology in canine melanoma

Canine melanoma is an immunogenic tumor, and investigations of different aspects associated with immune cells in tumor development and progression have been reported in the literature [39, 40, 41]. Moreover, some studies have evaluated different cancer immunology aspects of canine melanoma, podoplanin [42], and chondroitin sulfate proteoglycan-4 (CSPG4) [40]. Podoplanin is a type I transmembrane protein that is expressed in different cells of the immune system, including lymphatic endothelial cells. Podoplanin overexpression has been investigated in several cancers, including canine melanomas. Since the development and application of a monoclonal antibody against podoplanin, these markers have been recognized as important for canine melanoma immunotherapy [43]. CSPG4 has become very important for canine melanoma owing to the number of vaccines produced against this protein [39, 44, 45]. Its expression has been reported in canine melanoma, and different clinical trials based on vaccines or electrogene therapy have been conducted [39, 44, 45]. A review of the PubMed database for the past ten years is provided in Table 2 summarizing clinical trials involving dogs with melanoma treated with immunotherapies.

Reference	Number of subjects and cancer type	Manuscript goal	Manuscript summary
Riccardo et al. [39]	80 oral melanomas	Evaluate the clinical efficacy of a vaccine targeting tumor antigen chondroitin sulfate proteoglycan (CSPG)4.	Authors developed a hybrid DNA vaccine against human/dog CSPG4 chimera, with results indicating a safe and immunogenic vaccine, prolonging the survival of melanoma-affected patients, and promising in clinical routine.
Saellstrom et al. [46]	32 cutaneous and oral melanomas	Evaluate the life-long follow-up of dogs affected by cutaneous and oral melanomas treated with episomal CD40L gene therapy.	Twenty out of thirty-two dogs experienced different degrees of side effects, including fever, local swollen lymph nodes, and increased live enzymes. The regular regimen administration adopted is three times a 1 mL injection.
Igase et al. [47]	30 cases with different cancers, including 23 cutaneous oral melanomas	Authors developed different PD-1 monoclonal antibodies (rat–dog chimeric and caninized anti-canine) and evaluated in vitro and in vivo the efficacy of antibodies.	Nineteen out of 30 patients experienced side effects, including fever and gastrointestinal symptoms. Antitumor responses are evaluated in 24 out of 30 cases and increased overall survival is achieved in vaccinated dogs than in historical control group.
Kamoto et al. [43]	Three oral melanomas	Evaluate a phase I/II clinical trial of an anti-podoplanin monoclonal antibody for canine oral melanoma treatment.	Study demonstrated the monoclonal antibody production and in vivo test in only three animals. No conclusion could be derived based on three subjects.
Maekawa et al. [48]	Nine cases are enrolled with seven having oral melanomas.	Evaluation of the toxicity and safety of a rat–dog chimeric anti-PD-L1 monoclonal antibody for canine cancer treatment.	The vaccine is safe and dogs experienced good antitumor response. However, it is a pilot study with low number of cases.
Piras et al. [45]	42 oral melanomas	Elucidate the disease-free and overall survival times after electro-vaccination with a plasmid encoding human CSPG4.	The protocol is safe and immunogenic, with clinical benefits for the patients.
Riccardo et al. [44]	33 oral melanomas	Evaluate the immunogenicity, safety, and therapeutic efficacy of a human CSPG4 DNA-based vaccine.	Authors provided evidence that anti-CSPG4 electro-vaccination prolongs overall survival of dogs with oral melanomas.
Westberg et al. [49]	19 oral and cutaneous melanomas	Evaluate safety and toxicity of a local adenovector CD40L (AdCD40L) immunogene treatment for dogs with oral and cutaneous melanoma.	AdCD40L therapy is safe and may provide benefit to patients.
Grosenbaugh et al. [50]	58 dogs with oral melanoma	Assess the safety and efficacy of a vaccine containing an insert encoding human tyrosinase gene as an adjuvant treatment for canine oral melanomas.	The vaccine is safe and may provide more benefits to the patients as an adjuvant treatment than historical control.

Table 2.

Summary of the articles published in the past ten years with clinical studies evaluating different immunotherapies for canine melanoma.

In addition to evaluating immunotargets for canine melanoma, several studies have investigated the association between immune markers and melanoma prognosis, including intratumoral infiltration of immune cells. The prognostic significance of CD3⁺ and CD20⁺ cell infiltration of canine oral melanoma has been investigated; and high infiltration of CD20⁺ cells is associated with metastasis, frequency of recurrence, shorter survival time, and high rate of tumor-related deaths, and disease-free interval [51]. Therefore, a high infiltration of CD20⁺ cells is associated with several negative prognostic factors. Yasumaru et al. [52, 53] evaluated the presence of CD8⁺ and CD4⁺ infiltrating T cells in oral melanoma using flow cytometry and concluded that tumor-infiltrating lymphocytes predict the aggressiveness and prognosis of patients with oral melanoma.

13. Conclusions

Canine melanoma is a complex disease, usually having poor prognosis when the tumor is located in the oral cavity. Canine melanoma is also a highly immunogenic tumor, with a close association with the immune system. Thus, a better understanding of its immunological components can help in the development of new immunotherapies.

Acknowledgments

We would like to thank the São Paulo Research Foundation (FAPESP) for the financial support of our research group (#2020/02255-0). We also would like to thank the National Council for Scientific and Technological Development (CNPq), grant number #302977/2021-0.

Conflict of interest

The authors declare no conflict of interest.