Melanoma of the dog and cat: consensus and guidelines

Melanoma of the dog and cat poses a clinical challenge to veterinary practitioners across the globe. As knowledge evolves, so too do clinical practices. However, there remain uncertainties and controversies. There is value for the veterinary community at large in the generation of a contemporary wide-ranging guideline document. The aim of this project was therefore to assimilate the available published knowledge into a single accessible referenced resource and to provide expert clinical guidance to support professional colleagues as they navigate current melanoma challenges and controversies. Melanocytic tumors are common in dogs but rare in cats. The history and clinical signs relate to the anatomic site of the melanoma. Oral and subungual malignant melanomas are the most common malignant types in dogs. While many melanocytic tumors are heavily pigmented, making diagnosis relatively straightforward, melanin pigmentation is variable. A validated clinical stage scheme has been defined for canine oral melanoma. For all other locations and for feline melanoma, TNM-based staging applies. Certain histological characteristics have been shown to bear prognostic significance and can thus prove instructive in clinical decision making. Surgical resection using wide margins is currently the mainstay of therapy for the local control of melanomas, regardless of primary location. Radiotherapy forms an integral part of the management of canine oral melanomas, both as a primary and an adjuvant therapy. Adjuvant immunotherapy or chemotherapy is offered to patients at high risk of developing distant metastasis. Location is the major prognostic factor, although it is not completely predictive of local invasiveness and metastatic potential. There are no specific guidelines regarding referral considerations for dogs with melanoma, as this is likely based on a multitude of factors. The ultimate goal is to provide the best options for patients to extend quality of life and survival, either within the primary care or referral hospital setting.


Introduction
Tumors of melanin-producing cells in dogs and cats pose a significant challenge to all practitioners, regardless of experience and facilities.There is a broad spectrum of malignancy and, while patterns of behavior exist, tumors comply with these rules with variable veracity.The following basic rule always applies: advanced clinical stage and evidence of elevated proliferation rate correlate with poorer outcomes.No single treatment is consistently best, and all treatment modalities have a potential application.The purpose of this guideline document is to provide a succinct yet comprehensive overview of melanoma management in dogs and cats.

History and clinical signs of melanoma in dogs
The history and clinical signs relate to the anatomic site of the melanoma.
Melanocytic tumors of the haired skin typically manifest as raised pigmented mass lesions; signs of inflammation are usually absent.Small brown or black masses are the most common; however, the lesions can be large, flat, or wrinkled (2,19) [LOE 2c-5, OEG B].Lesions can be multiple.They may be present for a long time.Cutaneous melanomas are commonly phenotypically benign (1,2,19,20) [LOE 2c-5, OEG B].Whereas melanocytomas are usually solitary, small, pigmented, firm, and freely moveable over deeper structures, malignant melanomas tend to be fast-growing tumors, often ulcerated and pigmented (21) [LOE 4b, OEG C].Certain sites may be associated with malignancy: for example, the digit, footpad, and scrotum (2,21) Subungual lesions usually present as a swollen painful distal phalanx or a non-healing lesion near the claw.Patients present with lameness or excessive licking of the site (22) [LOE 4c, OEG C]; subungual melanomas frequently metastasize (5,22,23) Clinical signs of ocular melanoma relate to the structure affected and may include a mass lesion, glaucoma, hyphema, anterior uveitis, epiphora, conjunctival vascular injection, mucopurulent ocular discharge, and/or protrusion of the third eyelid, depending on the site of the lesion (7) Incidence, prevalence, history and clinical signs.Recommendations: 1 Multiple expressions are used to describe tumors of melanocytic origin.It is recommended that clear and transparent terms are used to ensure a common language and comparability between studies.We would like to reinforce the meanings of the following expressions: Melanoma and Malignant melanoma have the same meaning and refer to malignant tumors derived from melanocytes; Melanocytoma refers to a benign tumors derived from melanocytes; Melanocytic tumors refers to tumors derived from melanocytes, regardless of whether they are benign or malignant.2 Incidence and prevalence: in dogs, there are known breed predispositions to melanoma.Whether prognoses with melanoma differ according to breed is not known.Given the significance of genotype in human melanoma subtypes, this may be an interesting area of future study.3 In cats, peak age incidence differs between melanomas of the pinna and melanomas of other sites.Although it is reasonable to presume an aetiological difference between melanomas occurring in sun-exposed and non-exposed sites, there is no evidence to suggest a difference in clinical progression or prognosis.

Biological aspects and genetics
There are two important differences between human and canine melanomas: 1. canine melanoma is not induced by ultraviolet (UV) Melanomas in the oral cavity are similar to human mucosal melanomas in that benign and malignant forms cannot be discriminated by visual examination (3,(24)(25)(26) [LOE 2a-5, OEG D].Pigmentation can vary within a single tumor (Figure 1).Tumor burden and pigmentation are inconsistent indicators of malignant potential.
While many melanocytic tumors are heavily pigmented, making diagnosis relatively straightforward, melanin pigmentation is variable (35,36) [LOE 5, OEG D].The cytoplasmic melanin pigment granules may be fine and dust-like, needle-shaped, or coarse and granular.Small amounts of intracellular gray/black/green pigment may be seen (36) [LOE 5, OEG D].If the neoplastic cells are disrupted during sample collection or preparation, melanin pigment may be evident in the background of the smear.Infrequently, melanomas may be devoid of melanin pigmentation, rendering cytological diagnosis challenging or impossible (6,34) [LOE 5, OEG D].
Fine needle aspirates from lymph nodes can be extremely helpful for staging dogs with melanoma, particularly in patients with gross lymphadenomegaly (17) [LOE 4a, OEG A] (Figure 1).However, diagnosis of lymph node metastasis can be challenging due to the varied appearance of metastatic melanocytes and the presence of melanin-containing macrophages (melanophages) (37) [LOE 4b, OEG A].Dermal melanin is taken up by macrophages, which then traffic to the lymph nodes.Differentiation between melanocytes and melanophages may require histology, immunohistochemistry, or other histochemical staining techniques.

Biopsy
The identification of neoplastic cells containing melanin pigment allows for a cytologic diagnosis of melanoma in many cases (2,38) [LOE 5, OEG D].However, poorly pigmented or amelanotic melanomas are more challenging to diagnose cytologically, and histopathology with or without immunohistochemistry is required to confirm a diagnosis (38) [LOE 5, OEG D].
Differentials for oral melanoma include squamous cell carcinoma, fibrosarcoma, lymphoma, and odontogenic tumors.Cutaneous melanoma can resemble any neoplastic and non-neoplastic skin mass, and for subungual melanoma, SCC and nail-bed infections are differentials (39) [LOE 5, OEG D].Since oral tumor surfaces, especially those of the large melanocytic tumors, are often ulcerated and/or necrotic, large incisional biopsies, such as deep wedge or core punch biopsies are frequently required to make a definitive diagnosis.For oral tumors, biopsies should only be obtained through the mucosa and not the skin to avoid tumor seeding (40) [LOE 5, OEG D].Tru-cut biopsy needles can be used, but samples obtained using this method are generally small, making melanin granule detection challenging and potentially limiting a diagnosis to "sarcoma" or "malignant tumor" on histopathology.If Tru-cut biopsies are used, sampling several different areas should be performed.Electrocautery should only be used for hemostasis once the biopsy tissue is removed since biopsy tissues could be damaged by the heat (41) [LOE 5, OEG D].Finally, all resected tissues should be submitted and properly prepared with an optimized fixation ratio of 1-part tissue to 9-parts 10% neutral buffered formalin (41) [LOE 5, OEG D].
On histopathology, a proliferation of neoplastic cells at dermoepidermal junctions referred to as "junctional activity, " and neoplastic cells found in intraepithelial nests can aid in the diagnosis of oral melanoma (35,38) [LOE 5, OEG D].These features are often observed because of the preservation of epithelium over melanocytic tumors.Thus, if one suspects a poorly pigmented or amelanotic melanoma, a biopsy should be taken, preserving part of the epithelium and improving diagnostic accuracy.

Histopathology
Specific histochemical staining techniques are employed to demonstrate the presence of melanin: Fontana-Masson (black) and Schmorl's (blue-green).Bleaching helps visualization of intracellular structures by removing melanin from the tissues.Prussian blue stain aids differentiation of melanin granules from hemosiderin by identifying the presence of iron.
The proliferation marker Ki-67 is useful to distinguish benign and malignant forms, and has prognostic value (26,44,45) [LOE 4a-4b, OEG A].A higher percentage of cells express c-kit in melanocytomas than in malignant types (46) [LOE 4b, OEG A], but c-kit expression did not correlate with prognosis in malignant melanomas (46,47) [LOE 4b, OEG A].A further summary of prognostically-relevant histological features follows in section: Consideration of Prognostic Indicators and in Table 1.

Biomarkers
In human studies, diagnostic biomarkers currently used to assist in the diagnosis of melanoma are usually specific only for melanocytic neoplasms and not necessarily for their ability to metastasize (55) [LOE 5, OEG D].In dogs, the high molecular weight melanomaassociated antigen chondroitin sulfate proteoglycan-4 (CSPG4) was found to be a biomarker for malignant melanoma.However, there was no association between CSPG4 staining and clinical stage (56)

Diagnosis. Recommendations:
• A large and/or deep incisional/core biopsy, avoiding ulcerated or necrotic areas, is recommended to make an ACCURATE diagnosis of a melanocytic tumor.This is particularly pertinent in amelanotic or poorly pigmented melanoma.• Oral melanoma must be biopsied via the mucosal surface, not the skin, to avoid the risk of preventing future curative surgery by iatrogenic tumor seeding.
• If a definitive diagnosis of a suspected melanoma is not made on routine histology, immunohistochemistry is indicated.

Diagnosis. Opinion:
• In the coming years, it is likely that genomic testing will be able to yield a definitive melanoma diagnosis in cases in which histology and immunohistochemical evaluations have proved non-diagnostic.

Clinical staging of canine melanomas
Clinical staging of canine oral melanomas is straightforward.The goals are to determine the clinical stage according to the World Health Organization (61) [LOE 5, OEG D] (Box 1), to provide prognostic criteria and to guide therapeutic decision-making.Clinical staging evaluates three classical segments: the primary tumor (size and local extension, T segment), the locoregional lymph nodes (N segment), and the presence of distant metastasis (M segment), mainly in the lungs.A validated clinical stage scheme has been defined for canine oral melanoma.For all other locations and for feline melanoma, TNM-based staging applies.Tumor evaluation (segment T) The size of the local tumor is the first criterion to evaluate.Primary tumor size is of prognostic significance and corresponds to the fundamentals of the TNM classification as follows: Stage 1: a tumor less than 2 cm in diameter without any metastasis; Stage 2: a tumor greater than 2 and less than 4 cm in diameter without any metastasis; and Stage 3: a tumor greater than 4 cm in diameter and/or with confirmed lymph node involvement.
The tumor size can be clinically measured with a caliper or by cross-sectional imaging with computed tomography (CT).The longest diameter should be used for the classification.Although depth and nature of tissue invasion can be presumed to be prognostically significant, they have not been shown to supersede tumor size and are not included in the recognized clinical stage scheme.

Locoregional lymph node evaluation (segment N)
Lymph node evaluation is an important step in the clinical staging of dogs with melanoma (2,4,22,25,49) [LOE 3a-4a, OEG B].In a study with 100 dogs with oral melanomas, cytological or histopathological evidence of mandibular lymph node metastasis could be demonstrated in 53% of the cases, including in nodes of normal size (17) [LOE 4a, OEG C].Likewise, subungual melanoma is highly malignant, with 19 to 30% of the dogs reportedly having regional lymph node metastasis at time of diagnosis (22,62) Lymph node size is an unreliable predictor of metastasis (17) [LOE 4a, OEG C].Similarly, CT (63) [LOE 4c, OEG C] and cytology (37) [LOE 4b, OEG C] have been shown to carry low sensitivity and accuracy for assessment of cervical lymph node metastasis in dogs with oral melanoma.Thus, when lymph node status is uncertain, histopathology should remain the gold standard for the assessment of lymph nodes for metastatic disease with, in some instances, the need for immunohistochemistry (Melan-A) to aid differentiation between melanocytes and melanophages.
In oropharyngeal melanoma, bilateral or contralateral lymph node metastasis is not uncommon.Determination and selective biopsy of the sentinel lymph node is regarded by some to be a more advanced method of staging (37,(64)(65)(66) [LOE 4b-4c, OEG C].However, when an oral tumor is present, the local lymphatic drainage may be abnormal.Metastasis to contralateral lymph nodes and to the retropharyngeal lymph nodes without mandibular lymph node involvement have been described (64,67,68) [LOE 4b, OEG B].Currently, bilateral excisional biopsy of mandibular and retropharyngeal nodes for staging is advocated (62); removal via a single incision has been described (69) [LOE 4b, OEG D].The parotid lymph node can also be included in the clinical staging.Furthermore, there is not only 1 lymph node per lymphocenter (e.g., dogs can have between 2 and 5 mandibular lymph nodes), and each could be metastatic.

Distant metastasis (segment M)
Detection of lung metastasis is fundamental in clinical staging of dogs with melanoma.It can be performed by radiography with 3-view thoracic radiographs (70) [LOE 4a, OEG B].However, CT is more sensitive than radiography for the detection of small pulmonary nodules, especially in large dogs (71-73) [LOE 4b-4c, OEG C].The lower size threshold for detection is approximately 1 mm by CT, whereas it is between 7 and 9 mm by radiography.In canine cancer generally, only 9% of pulmonary nodules that can be detected by CT are visible on radiographs.In patients with thoracic radiographs without pulmonary nodules, 13 to 39% show nodules on a CT scan (71) [LOE 4c, OEG C].
Abdominal metastases are rare in dogs with melanoma, but have been described in the abdominal lymph nodes, liver, adrenal glands, and other sites (2,74,75) [LOE 4a-5, OEG B].The use of abdominal ultrasonography or CT should be considered.Skeletal metastases are also recognized; clinically occult lesions would not be expected to be identified except by CT.

Clinical stage. Recommendations:
• Clinical stage determination is fundamental to therapeutic decision-making for canine and feline melanoma.• We recommend that the size and site of the primary tumor are recorded prior to further intervention to improve communication between colleagues in the event that patient care might be shared.• Thoracic imaging can be performed by radiography (3-view radiography is preferred), but CT is more sensitive.• Abdominal metastases are rare but have been described so abdominal imaging would be required to achieve thorough clinical stage determination.

Surgical treatment
Surgical resection using wide margins is currently the mainstay of therapy for the local control of melanomas, regardless of primary location.

Oral melanoma
In canine oral melanoma, surgical planning using thin-slice contrast CT with multiplanar reconstruction is regarded as the gold standard, especially in all maxillary and caudal mandibular locations.Because resection of melanomas affecting the jaw usually involves a segmental mandibulectomy or maxillectomy, resection width in the bone is based on the detectable bony changes on CT (76) [LOE 4b, OEG B].For the surgical techniques of jaw resection, please refer to the respective surgical textbooks or monographs.
A minimal surgical margin for melanomas has not been established but, in our experience, a minimum of 1 to 2 cm of healthylooking bone is usually sufficient to attain clean margins.On the soft tissue side (e.g., lip, sublingual floor of the mouth, soft palate), the surgeon should strive for wider margins (i.e., a minimum of 2 cm).Histopathologic confirmation of completeness of excision is important; all bony and soft tissue margins should be histologically evaluated.There is no consensus among veterinary pathologists regarding surgical margin assessment.Therefore, the veterinarian should discuss with their pathology lab how completeness of resection is assessed and how assessment can be optimized (77,78) [LOE 3a, OEG D].Helpful techniques to improve margin assessment include applying surgical ink to the cut surfaces before fixation in formalin or taking resection margin biopsies from bony and soft tissue margins, which are submitted in a separate container.With the latter technique, the surgeon can define which and how many sites are examined histologically.In 70 dogs treated with curative-intent jaw resections (49) [LOE 4a, OEG C], resection of 2 to 3 cm bone margins and 1 cm soft tissue margins resulted in tumor-free margins in almost 73% of cases.Only 17% developed local tumor recurrence.The progressionfree interval in this study was 508 days, with a median survival time (MST) of 723 days.In a separate study (79) [LOE 4a, OEG C], almost 80% of resections (73/92) were considered complete after "wide" excision.Recurrence rate was 8.3% (6/73) and the MST was 354 days.
In cats, oral melanoma is rare.A case series of 8 cats treated with radical mandibulectomy reported 6/8 adequately prehending and swallowing food by 3 months after surgery (80) [LOE 4c, OEG C].In essence, the same resection guidelines apply in cats and dogs, but in cats, less is known about the resection margins necessary to obtain tumor-free margins.Resection width in cats is limited by the much smaller anatomical size of the oral structures.This makes both critical assessment of the resectability of the tumor by appropriate imaging, as well as examination of surgical bony and soft tissue margins, even more important.

Subungual melanomas
For subungual melanomas, amputation of the affected digit at the metacarpophalangeal or metatarsophalangeal joint is the treatment of choice and usually results in tumor-free margins (81) [LOE 4c, OEG C].Amputation at the level of the interphalangeal joints is contraindicated because tumor-free soft tissue margins are frequently not achieved.In large tumors, taking resection margin biopsies of the adjacent skin and soft tissue may be indicated to assess completeness of resection.

Melanoma of the foot pad
Melanoma of the foot pad can be addressed by partial or complete full-thickness resection of the affected foot pad, including the entire fat cushion layer.Assessment of the lateral and deep margins is important, and the use of surgical ink or tumor bed biopsies is recommended.Reconstruction of a weight-bearing foot pad surface can be achieved by transposition of one or both of the second and fifth digital pads using a phalangeal fillet technique (82,83) [LOE 4c-5, OEG C].

Melanoma of the haired skin
In non-metastatic melanoma of the haired skin, wide surgical resection is the treatment of choice, whenever anatomically possible.Again, evidence-based recommendations for the resection width have not been established, but most authors recommend a 2 to 3 cm lateral margin and a subfascial resection for the deep extension.Assessment of completeness of resection, using techniques already described, is recommended.

Ocular melanoma
In ocular melanoma, the location of the tumor on or in the eye is of importance in choosing surgical technique, and outlined as follows (7,84)

Nasal planum melanoma
In nasal planum melanoma in cats, a wide to radical resection (nosectomy) is recommended for both benign and malignant melanoma.Benign feline nasal planum melanoma may progress to malignancy, and radiation therapy does not result in durable remissions (13) [LOE 4c, OEG C].
Surgical excision of tributary lymph nodes in oral, subungual, and cutaneous melanomas adds important information regarding the clinical stage of the disease and is therefore frequently recommended.However, the therapeutic value of lymph node dissection in canine melanoma has not been studied systematically.Based on the evidence available, lymph node biopsy is more likely a diagnostic rather than a therapeutic procedure and may not offer a survival advantage.

Radiotherapy
Radiotherapy forms an integral part of the management of canine oral melanomas, both as a primary and an adjuvant therapy (88-95) [LOE 4a-4c, OEG B].It is also described in the context of feline oral melanomas and in canine non-oral melanoma treatment; although the indications are fewer, responses appear broadly comparable (94, [96][97][98] [LOE 4a-4c, OEG C].
Although the reason remains controversial, melanoma exhibits sensitivity to coarsely fractionated radiation protocols, with attendant benefits in terms of risk of acute toxicity, costs, and time.This does not mean that melanomas are insensitive to other, more fractionated courses of therapy.An optimal protocol remains undefined.In the context of oral melanoma, it is noteworthy that the canine mandible is a bone that is sensitive to hypofractionation, increasing the risk of osteoradionecrosis (99,100) [LOE 4c, OEG C].Lower dose per fraction protocols can be used to reduce late effect incidence in dogs with better prognoses (99 Treatment protocols reported range from daily 3Gy fractions for 4 weeks (57Gy) through to weekly 10Gy fractions for 3 weeks (30Gy).Most published studies are retrospective (88-90, 95, 96, 101, 102) [LOE 4a-4c, OEG B].This and the variety of fractionation protocols, equipment used, concomitant therapies, and clinical stage of cancer treated make comparisons between protocols challenging.Outcomes appear to differ between cases that undergo radiotherapy in the macroscopic and the microscopic (post-surgery) settings (89, 93,94,101) [LOE 4a-4c, OEG B].
Macroscopic tumors are expected to shrink or disappear in more than 80% of cases; responses become evident within 2 to 3 weeks of initiating therapy.Reported average remission durations range from 3 to 9 months (89, 93,94,101)  Multiple small studies describe the impact of radiotherapy and chemotherapy in combination.There is an absence of evidence that combining the treatments, with or without surgery, leads to a consistent improvement in outcome (88-90, 101, 102) [LOE 4a-4c, OEG C].Most studies have failed to demonstrate a positive survival impact of the addition of chemotherapy.However, the small number of cases reported, and a lack of uniformity of tumors and treatments, may mask a genuine difference in outcome.Although statistically significant differences in outcomes may not exist, data from small studies suggested that time to local recurrence was longer in oral melanoma cases receiving adjuvant radiotherapy and chemotherapy in combination compared with adjuvant radiotherapy alone (101) [LOE 4c, OEG C], and time to progression was improved in dogs receiving radiotherapy for melanomas at any site when temozolomide was added to treatment (94 There is a general acceptance that technological advances to improve accuracy of radiation targeting will enable greater differentiation between target and non-target tissues, delivery of greater doses of radiation to cancer tissues, and a superior outcome for the patient (103, 104) [LOE 5, OEG D].Data do not yet exist to substantiate this hypothesis in the field of canine oral melanoma.The complex anatomy of the oral cavity and the infiltrative nature of malignant melanoma apply limits to the improvements that can be gained, but it appears reasonable to believe that further improvements in locoregional melanoma control are possible.

General principles
While locoregional tumor control can be achieved by means of resection and/or irradiation of the primary tumor and regional lymph nodes, development of metastatic disease very often leads to death.Adjuvant systemic therapy is offered to patients without evidence of macroscopic metastases that are at high risk of developing microscopic disease, and therefore distant metastasis.

Adjuvant immunotherapy
A therapeutic vaccine containing xenogeneic plasmid DNA with an insert encoding human tyrosinase (ONCEPT ® Canine Melanoma Vaccine) has been evaluated in a prospective, multicenter clinical trial in dogs with locally controlled (surgically removed primary tumor, regional lymph nodes, and irradiation for incompletely resected tumors) stage 2 and 3 oral malignant melanoma (105) [LOE 2c, OEG B].The vaccine was delivered transdermally by use of a needle-free intramuscular vaccination device in 4 doses every 2 weeks, followed by booster injections at 6-month intervals.Statistically significant improvement in survival time was observed (MST not reached; >50% of dogs survived longer than 437 days) versus the classic local-controlonly treatment protocol received by a historical control group (MST of 324 days).Another group of vaccinated dogs with stage 1 to 3 oral malignant melanoma reached an MST of 445 days (106)  The canine ONCEPT ® vaccine can be safely administered to cats with oral, ocular/periorbital, dermal, mucocutaneous, lip, and subcutaneous melanomas.There are no data regarding its efficacy (108) [LOE 4b, OEG C].

Adjuvant chemotherapy
For canine oral, digital, and cutaneous melanoma, there is no evidence that addition of cytotoxic drugs (carboplatin, cisplatin, melphalan) to surgical treatment or radiotherapy leads to a significant increase in survival time (79,89,95,101,102,109)

Treatment of macroscopic disease
Overall response rates for treatment in dogs with measurable disease are disappointing and have not exceeded 18% for dogs treated with cisplatin and piroxicam (110) [LOE 4c, OEG C] or 28% for dogs treated with carboplatin (111) [LOE 4b, OEG C].Furthermore, complete responses are rare and short in duration.The ONCEPT ® vaccine is not indicated for macroscopic disease, but there is information about its use in this setting with an MST of 179 days (106) [LOE 4c, OEG C].Treatment.Recommendations:

Other modalities of treatment
• Surgical resection using wide margins is currently the mainstay of therapy for the local control of melanomas, regardless of primary location.CT imaging is the optimal strategy for surgical planning of jaw-invasive melanoma.Treatment.Opinions: • An increased array of immunotherapy options is predicted to become available for treatment of melanoma.As a result, radiotherapy is likely to assume greater therapeutic significance because it increases epitope expression.• Immunotherapy appears to be the optimal method of treating microscopic disease, provided that macroscopic disease is effectively controlled.
• The ONCEPT ® Canine Melanoma Vaccine has an evidence base for treatment of stage 2 and 3 malignant melanoma with adequate local tumor control.• There is limited scientific literature to support the use of other medical therapies for melanoma.A pivotal study of gilvetmab in canine stage 3 melanoma is ongoing at the time of writing.

Consideration of prognostic indicators
The definition and identification of existing or new prognostic factors in canine melanoma should follow the regimented, systematic approach already described in the literature to clearly identify and consider these factors as truly valuable to the clinician (25,118) [LOE 2a-3a, OEG C].Location is the major prognostic factor, although it is not completely predictive of local invasiveness and metastatic potential.The oral/mucosal  Oral melanoma decision tree. 10.3389/fvets.2024.1359426 Frontiers in Veterinary Science 11 frontiersin.orgsurgical excision (48) [LOE 4a, OEG C]; however, mitotic index of 3/10hpf or higher, high Ki-67 index, and survivin expression seem to be related to metastatic potential and reduced overall survival (26,52,54,119) [LOE 4a-4b, OEG C].Ulceration might be a negative prognostic factor in cutaneous tumors (26,52,81) [LOE 4a-4b, OEG C].Melanoma in the digits is generally malignant (86%), and all subungual melanocytic tumors are malignant (5) [LOE 2c, OEG B].They have a high metastatic propensity, with regional or distant pulmonary metastasis evident at the time of diagnosis in 30 to 40% of cases, and subsequent development of regional or distant metastasis in most of the remainder (22,62) 1).Following a modified World Health Organization staging system from I to IV, based on tumor size, lymph node involvement, and metastases in dogs with oral melanomas, has been found to be extremely prognostic (49)  In cats, tumor location, mitotic index and presence of intratumoral necrosis have been attributed with prognostic significance.A two-tier grading scheme has been proposed, with tumors of the nose, lip and oral cavity considered high-grade if mitotic index is greater than or equal to four OR if there are confluent aggregates of necrotic neoplastic cells.Other non-ocular tumors are considered high-grade if they exhibited BOTH a high mitotic index and intratumoral necrosis (121) [LOE 4a, OEG C].With so-defined high-grade tumors, median survival time was 90 days whereas only 19% of low-grade tumors adversely impacted survival.

Melanoma referral considerations
There are no specific guidelines regarding referral considerations for dogs with melanoma, as this is likely based on a multitude of factors.Clinician experience and comfort level, access to diagnostic imaging, and proximity to referral have an impact.Tumor location impacts decision-making, as mandibular and maxillary tumors are more likely to be referred due to the higher level of expertise needed for surgical excision.Access to ONCEPT ® Canine Melanoma Vaccine will also affect decision-making as it is limited to selected veterinarians in some geographies and is not available at all in others.
If a referral is to be sought, tumor size should be recorded before excision/referral, as this is deemed a strong prognostic factor.If a biopsy or excision is performed prior to referral, pre-operative digital photographs of the lesion can greatly assist patient assessment and treatment planning.Similarly, staging (imaging and lymph node assessment), when feasible, is paramount before referral, as this may impact the therapy and prognosis.It is also important to note that incomplete excision is associated with poorer outcomes so, if complete excision may not be possible in primary care, referral should be considered.
The ultimate goal is to provide the best options for patients to extend quality of life and survival, either within the primary care or referral hospital setting.

1
FIGURE 1 Oral melanoma.(A-D) Heterogenous appearance of oral melanoma in a cocker spaniel.(A) Outward appearance.(B) Visualization of the intraoral aspect illustrates the importance of oral examination in forming the clinical suspicion of melanoma.(C,D) The intraoperative and excision specimen images reveal the absence of an appreciable surgical margin.This is acceptable when the goal of surgery is palliation of the consequences of the presence of a large mass or as a precursor to definitive radiotherapy.(D) Heterogenous appearance of oral melanoma: the tumor grossly exhibits melanotic and amelanotic parts.(E) Malignant melanoma cytology from mandibular gingiva.Cellular borders are usually distinct, though sometimes faint.Cytoplasm is basophilic or light basophilic and granulated; occasionally, small vacuoles or light black to deep green small cytoplasmic granules are seen.Nuclei are eccentrically located and may be irregular; they often show large, dark, prominent nucleoli.(F-I) Oral malignant melanoma with lymph node metastasis in a dachshund.(F) A small primary tumor was located medial to a left upper molar tooth.(G) The patient was presented with left submandibular lymph node enlargement.(H,I).Tumor cells are spindle-shaped and contain large amounts of melanin pigment.The melanoma cells are large and elongated, with length 4-5x and width 2-3x accompanying red blood cells.The cytoplasmic margins are indistinct, the cytoplasm is vacuolated and black/green cytoplasmic granules are apparent.The nuclei are round to oval with stippled, coarse chromatin; nucleoli are distinct and vary in size and number.(J) Spindle-shaped form of malignant melanoma cytologically resembles a sarcoma, though melanin granules are also evident in this sample.(K,L) Oral melanoma with lymph node metastasis; melanophages versus malignant melanoma.(K) A macrophage (melanophage) with intracytoplasmic melanin pigment (arrow) is seen among reactive lymphoid population.Free melanin granules are also visible.Melanophages in themselves do not indicate tumor metastasis, as they can be seen in reactive or normal lymph nodes from dogs without melanocytic tumors.(L) Intracytoplasmic melanin pigment is evident in a large epithelioid melanoma cell (arrow) among a population of reactive lymphoid cells.(E,I,J-L) Original magnification x 1,000.(H) Original magnification x 800.
[LOE 4a-c, OEG B].Cytotoxic drugs (cisplatin 10-30 mg/m 2 intravenously or carboplatin 90 mg/m 2 intravenously) may have a role as a radiation sensitizer given once weekly approximately 1 h before radiotherapy in the management of dogs with incompletely excised oral melanomas (90) [LOE 4b, OEG C].The median time to metastasis was 10.2 months, the MST was 11.9 months, and the local recurrence rate was 15% (90) [LOE 4b, OEG C].As previously mentioned, adjuvant temozolomide after radiotherapy prolonged time to progression without a significant influence on survival time (94) [LOE 4c, OEG C].
Multiple studies have demonstrated a correlation between the expression of COX-2 in canine melanoma and/or melanoma cell lines and proliferation and survival of cells(112, 113)  [LOE 2b, OEG B], but no clinical trials investigating nonsteroidal anti-inflammatory drug (NSAID) effectiveness in canine and feline melanoma cases have been published.Local treatment modalities, such as intralesional cisplatin implants or electrochemotherapy with intralesional bleomycin, have been reported for a limited number of patients(114, 115)  [LOE 4a-b, OEG C].Cytotoxic drugs in a metronomic setting could have some influence for immune cells in the microenvironment of a tumor, but the clinical significance of this remains unknown(79) [LOE 4c, OEG C].Scientific information concerning the treatment of malignant melanoma with tyrosine kinase inhibitors is anecdotal.Masitinib used for stage 3 and 4 malignant melanoma showed only mild effectiveness(116) [LOE 4c, OEG C].Checkpoint inhibitors successfully used for human melanomas are not currently available for canine and feline patients though the first caninized anti-PD-1 monoclonal antibody has recently been awarded conditional licensure in the United States for treatment of canine melanoma.Gilvetmab was given to 25 dogs with stage 2 or 3 melanoma.Complete response was noted in 2/25, partial response in 3/25 and stable disease in 10/25 cases (117) [LOE 4b, OEG D].
10.3389/fvets.2024.1359426Frontiers in Veterinary Science 10 frontiersin.orgsubtype carries the worst prognosis, with a high degree of local invasiveness and high metastatic potential, and reported MST between 3 and 24 months, according to the stage.Oral benign melanomas might exist (50) [LOE 4a, OEG C]; however, caution is recommended because of their unpredictable behavior (2) [LOE 5, OEG D].Lip and tongue locations might have a better prognosis compared with other locations within the mouth; welldifferentiated mucosal forms have been described (18) [LOE 4a, OEG C].Melanomas involving the haired skin are often cured with a clean-margin
(18,d 2. whereas ocular and cutaneous truncal melanomas are most common malignant forms in humans, in dogs these anatomic forms are typically benign.Instead, oral and subungual malignant melanomas are the most common malignant types in dogs; these are rare but also aggressive in humans(18, 23)[LOE 2b-5, OEG B]. light

TABLE 1
Prognostic factors for canine melanoma.
Oral: number of positive nuclei per grid Other sites: % of positive nuclei when 500 cells counted Oral: <19.5, favorable Other sites: <15%, favorable (2, 25, 26, 45, 52) [LOE 3a-4b, OEG B] Survivin Nuclear expression prognostic for cutaneous melanoma only Poor (26, 54) [LOE 4b, OEG C] [LOE 4a,  OEG C].Two retrospective clinical studies did not reveal any benefit from the use of the vaccine but, in both studies, local control of the • Surgical excision of tributary lymph nodes in oral, subungual, footpad and cutaneous melanoma is recommended.It adds important information regarding the clinical stage of the disease and it improves local tumor control.• When adequate local control cannot be achieved surgically, radiotherapy should be considered.• Explicit recommendations for treatment of canine oral melanoma are presented in Figure 2.