JPC SYSTEMIC PATHOLOGY

INTEGUMENTARY SYSTEM

October 2022

I-N24 

 

SLIDE A

Signalment (JPC # 2785784):  Miniature schnauzer

 

HISTORY:  Firm, pedunculated dermal mass 

                                                

HISTOPATHOLOGIC DESCRIPTION:  Haired skin: Expanding the dermis and elevating the overlying hyperplastic and focally ulcerated epidermis is an unencapsulated, pedunculated, moderately cellular neoplasm composed of spindle cells arranged in short, interlacing streams and bundles separated by moderate amounts of collagenous matrix. Neoplastic cells have indistinct cell borders, moderate amounts of eosinophilic cytoplasm that occasionally contains brown, globular pigment (melanin) and an oval to elongate nucleus with finely stippled chromatin and 1 variably distinct nucleolus. Anisocytosis and anisokaryosis are mild and mitotic rate is 1 per 2.37 mm². Multifocally, there are individual or small nests of neoplastic cells within the epidermis (junctional activity). Multifocally within the dermis there are scattered macrophages with intracytoplasmic melanin. The epidermis is focally ulcerated with an overlying serocellular crust composed of cellular debris, degenerate neutrophils, hemorrhage, fibrin, and colonies of basophilic cocci. Subjacent to the crust within the superficial dermis there are increased numbers of small caliber blood vessels lined by reactive endothelium oriented perpendicular to the ulcer and perpendicular to abundant surrounding hypertrophied fibroblasts (granulation tissue). Rare apocrine glands are ectatic and surrounded by moderate numbers of lymphocytes, plasma cells, and fewer transmigrating neutrophils and macrophages (hidradenitis) and there is multifocal hemorrhage, fibrin and edema. The epidermis is hyperplastic, characterized by acanthosis, spongiosis, and rete ridge formation.

 

MORPHOLOGIC DIAGNOSIS:  Haired skin: Melanocytoma, miniature schnauzer, canine.

 

SLIDE B

Signalment (JPC # 2327682):  Age and breed unspecified dog.

 

HISTORY:  A mass on the digit

 

HISTOPATHOLOGIC DESCRIPTION:  Haired skin and bone, digit: Expanding the dermis, elevating a focally extensively ulcerated epidermis, and infiltrating the bone of the third phalanx (P3) is an unencapsulated, poorly demarcated, moderately cellular, infiltrative neoplasm composed of polygonal to spindle cells arranged in lobules, nests, and packets supported on a moderate fibrovascular stroma. Neoplastic cells have variably distinct cell borders, a scant to moderate amount of eosinophilic, granular cytoplasm that often contains brown pigment globules (melanin), and a round to oval nucleus with finely stippled chromatin and 1 to 2 prominent nucleoli. Anisocytosis and anisokaryosis are moderate and there are 2 to 3 mitotic figures per 2.37 mm². Multifocally, there are individual or small nests of neoplastic cells within the follicular epithelium (junctional activity). Within the neoplasm there is frequent single cell necrosis, moderate numbers of melanin-laden macrophages, and multifocal areas of lytic necrosis with replacement by hemorrhage, fibrin, edema, moderate numbers of viable and degenerate neutrophils, and fewer hemosiderin-laden macrophages. There is multifocal bone loss (osteolysis) with replacement by neoplastic cells. There are often scalloped trabeculae of immature woven bone extending from the remaining lamellar mature bone, basophilic reversal lines, and osteoclasts within Howship’s lacunae (bony remodeling).  

 

MORPHOLOGIC DIAGNOSIS:  Haired skin and bone, digit: Melanoma, breed unspecified, canine. 

 

GENERAL DISCUSSION:  

• Melanocytes: 

• Originate from neuroectoderm, and migrate to the skin and hair bulbs during fetal development
• Mature, dendritic, pigment-producing cells found between basal keratinocytes of the epidermis and hair bulb; contain E-cadherin cell surface markers which serve as adhesion mechanism between melanocytes and keratinocytes
• Produce melanin, which is stored within melanosomes, transferred to keratinocytes, and serve to protect the skin from UV radiation
• Melanoblasts which fail to reach the epidermis will reside in the dermis as intradermal melanocytes

• Melanocytic tumors are most common in dogs, horses, and some pig breeds

 

PATHOGENESIS:

• Unknown
• One recent study found that over 60% of oral malignant melanomas showed evidence of defective DNA mismatch repair proteins (Inanaga S et al., Vet Pathol 2021)
• A recent study evaluated the expression of checkpoint molecules (which suppress tumor-infiltrating lymphocytes [TILs] in tumors) in canine melanomas via quantification of mRNA. As in human melanomas, overexpression of three checkpoint molecules, PDCD1 (PD-1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) is a common feature of canine melanoma. The study found that the expression of these checkpoint molecules is modulated by interactions between CD3+ T cells and cancer cells (Stevenson VB, Vet Pathol 2021) 

 

TYPICAL CLINICAL FINDINGS:

• Mitotic index (MI) is a key prognostic indicator that predicts clinical behavior and should be determined in all melanocytic neoplasms; nuclear atypia, ulceration, and deep infiltration beyond the dermis are also associated with a poor outcome; see Smedley (2011) publication for detailed information 
• Degree of pigmentation and histologic pattern are not correlated with prognosis according to several studies
• Oral and lip melanocytic neoplasms:  Newer studies have shown a subpopulation of oral and lip melanocytic neoplasms with a favorable diagnosis, though historically, these locations were considered to be “almost invariably malignant”; oral and lip neoplasms composed of heavily pigmented, well-differentiated melanocytes with a low mitotic index (<4/10HPF) have a favorable prognosis
• Nailbed melanocytic neoplasms: often malignant
• Cutaneous melanocytic neoplasms:  often benign; canine melanocytic neoplasms with a MI of >3/10HPF are expected to have shorter survival times
• Malignant melanoma: 

• Often occur in older animals, though are reported in young animals of many species
• Metastasis most often occurs via lymphatics to regional lymph nodes and lungs, though has been reported in unusual locations such as the brain, heart and spleen
• Metastasis of an oral melanoma to the pituitary gland has been reported (Miller MA, Vet Pathol 2018)

 

TYPICAL GROSS FINDINGS:  

• Melanocytoma (benign melanoma, melanotic nevus):  Usually solitary, circumscribed, alopecic, gray to brown to black, cutaneous nodules that range from 1 to 4 cm in diameter; smooth or slightly papillated to pedunculated; ulceration is uncommon
• Malignant melanoma:  Sessile, variably circumscribed, unencapsulated; polypoid, dome-shape, or plaque-like; gray to brown to black; range from 1 to 10 cm in diameter; ulceration is common

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:  

• General features:

• Amount of pigmentation is variable in melanocytic neoplasms; a variety of cell shapes, from spindle cell, balloon cell (clear cell), epithelioid, and signet-ring cell, are possible
• Junctional activity (proliferation of neoplastic melanocytes in small nests within the epidermis at the epidermal/dermal or follicular/dermal junction) is a helpful diagnostic feature; see differentials for others tumors with junctional activity
• Breslow thickness:  A measurement from the top of the granular layer of the epidermis to the deepest invasive cell across the broad base of the tumor; prognostic factor used in humans; recent study evaluated prognostic value in dogs; dogs with tumor thickness >0.95 cm had a shorter survival time than those with tumor thickness < 0.95 cm; dogs with tumors > 0.75 cm had a shorter disease-free time (i.e., recurrence/metastasis was quicker) than those with tumors < 0.75cm (Silvestri S, Vet Path 2019)

• Melanocytoma (benign melanoma, melanotic nevus):

• Histological classifications:  Junctional (confined to the epidermis and dermoepidermal junction), compound (involving both dermis and epidermis), or dermal; compound and dermal are the most common in domestic animals
• Can be composed of any melanocytic cell type; most common are spindle cells (whorls or fingerprint patterns in the dermis), epithelioid cells (nests in the dermis or epidermis, and along the dermo-epidermal junction) or a combination
• Degree of pigmentation can vary; epithelioid cells are often darkly pigmented, and spindle cells are often lightly pigmented to amelanotic
• Mitotic figures are rare; MI <3 is reported to predict benign behavior
• Balloon cell melanocytoma variant:  dermal mass composed of large epithelioid to polygonal cells with abundant amphophilic to eosinophilic cytoplasm with a granular appearance; few neoplastic cells will contain fine melanin granules; nuclei are small, uniform and hyperchromatic
• Vasculogenic mimicry, a mechanism of tumor angiogenesis recognized in human melanocytomas, was reported in a palpebral melanocytoma in a dog; the neoplasm contained numerous lacunar and slit-like spaces filled by erythrocytes interspersed throughout the neoplastic melanocytes; spaces were lined by PLN-2 positive cells, but the cells were negative for factor VIII and CD31 (Nordio L, J Comp Path 2018)
• Recent case of melanocytoma effacing the lacrimal gland of a dog, blocking the drainage angle and causing buphthalmia and glaucoma (Mistui I, J Vet Diagn Invest 2022)

 

• Malignant melanoma: 

• Can be composed of a variety of melanocytic cell morphologies; most common are epithelioid cells, spindle cells or a combination
  • Epithelioid cells form clusters and/or nests
  • Spindle cells form sheets, bundles, and/or whorls
• Cells may be heavily pigmented or amelanotic; epithelioid and spindle cell form typically pigmented; balloon cell form and signet-ring cell form are usually poorly pigmented to amelanotic
• Cytoplasm is moderate to abundant
• Variable nuclear pleomorphism
• 3 or more mitotic figures per 2.37 mm²  (for cutaneous location), with mitotic atypia
• Varying degrees of junctional activity
• Variants:
  • Balloon cell malignant melanoma:  Dermal masses, sometimes multilobulated; junctional activity absent; large cells with large, vesicular nuclei, prominent nucleoli, low MI, abundant clear cytoplasm without visible melanin; rarely, few cells will contain pale, dust-like granules; rare multinucleated cells
  • Signet-ring malignant melanoma:  Round to polygonal cells with eccentric nuclei compressed by abundant pale eosinophilic or amphophilic cytoplasm without visible melanin; rarely, few cells contain pale granules; rare multinucleated cells 

 

ADDITIONAL DIAGNOSTIC TESTS:  

• Immunohistochemistry:  

• Melanocyte specific markers:
  • Melan-A (currently the most specific marker – melanocyte-differentiating protein recognized by cytotoxic T cells) useful in many species
  • PNL2, TRP-1 and TRP-2 used alone or as a cocktail with Melan-A is JKP recommendation
  • HMB-45:  Specific marker for melanoma cells
• Neural crest markers: S-100 and neuron specific enolase (NSE) are sensitive markers for melanocytes, but are not specific 
• Mesenchymal markers: Vimentin

• Prognostic markers:
  • FoxP3 (transcription factor forkhead box protein P3) and IDO (indoleamine 2,3-dioxygenase):  An increased risk of death due to canine melanoma is associated with a higher number of FoxP3⁺/HPF, higher percentage of CD3⁺ cells that were also FoxP3⁺ infiltrating and surround the tumor, and increased IDO⁺ cells/HPF (Porcellato I, Vet Pathol 2019)
  • FoxP3, CTLA-4, and IDO likely play a role in melanoma immunoescape. All show high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanoma compared to benign counterparts. FoxP3 expression >6.9 cells/HPF is an independent predictor of death; CTLA-4 expression associated with a worse prognosis; IDO expression associated with increased death hazard (Porcellato, I, Vet Pathol 2021)
  • MCAM/CD146 (melanoma cell adhesion molecule):  Overexpression of MCAM/CD146 was reported in canine oral melanomas, weak in cutaneous melanoma, and negative in ocular melanoma (Asa SA, J Comp Path 2017)
  • A recent study looked at tumor-infiltrating lymphocyte (TILs) presence and distribution in canine oral melanomas. Patients with higher TIL scores and an increased frequency of CD8+ T lymphocytes had higher survival rates, suggesting that TIL evaluation in canine oral melanoma is relevant to predicting tumor aggression and patient prognosis (Yasumaru, Vet Pathol 2021)
  • Canine oral melanomas were evaluated for galectin (Gal)-3, a prognostic marker for human neoplasms. Gal-3 cytoplasmic positivity was positively correlated with survival; Gal-3 nuclear positivity was negatively correlated with survival (Vargas THM, J Comp Path 2019)
  • A recent study found that E-cadherin expression as a prognostic factor differed depending on tumor origin. Cutaneous melanomas without E-cadherin frequently had more favorable clinical outcomes, whereas in amelanotic and oral melanomas, lower E-cadherin expression was correlated with more unfavorable outcomes (Silvestri S, Vet Pathol 2020)
  • Recent study found no apparent relationship between detected c-Kit mutations and KIT expression, indicating that, unlike in human oral melanoma, the use of tyrosine kinase inhibitor therapy to treat canine oral malignant melanoma is unlikely to be effective (Smedley, RC, Vet Pathol 2021)

• Histochemical stains:

• Fontana-Masson silver stain:  Highlights the small amounts of melanin in amelanotic melanomas
• Bleach:  Can be utilized to determine cytomorphologic characteristics in heavily pigmented melanocytes

• Cytologic findings:

• Benign and malignant neoplasms contain pleomorphic cells, ranging from epithelioid to spindle, or less often are discrete and round; well-differentiated cells contain abundant, fine, black-green cytoplasmic granules which may obscure the nucleus; benign forms contain small, uniform nuclei; malignant melanomas contain increased anisocytosis and anisokaryosis, coarse nuclear chromatin and prominent nucleoli
• Staging lymph nodes:  Consensus between routine cytology and histopathology for lymph node staging in patients with melanocytic neoplasms is poor and does not correlate with survival (Grimes, Vet Pathol 2017)

 

DIFFERENTIAL DIAGNOSIS: 

• Other melanocytic lesions:
• Melanocytoma-acanthoma (melanoacanthoma):  Rare, reported in dogs; circumscribed, unencapsulated, solitary, pigmented nodule, < 1 cm in diameter, combination of junctional melanocytoma with a benign epithelial tumor; epithelial cells forms nests and cords +/- cystic structures containing keratin, and melanocytic cells forms nests in the epidermis and sometimes within the epithelial cell cords, or may have a spindle shape and form whorls between epithelial cords
• Melanocytic hyperplasia:  In dogs, found on nipple; in cats, found on lips, eyelid, pinna, frequently in orange, cream or silver coat colors; hyperplasia of non-neoplastic melanocytes within the basal layer of the epidermis, with melanomacrophages in the superficial dermis

• Oculoderma melanocytosis (nevus of Ota) (case report): a dermal melanocytic hamartoma presenting as cutaneous facial hyperpigmentation that corresponds to the distribution of the ophthalmic and maxillary branches of the trigeminal nerve, often with ipsilateral ocular involvement; marked panuveal melanocytosis with extension into the sclera, bulbar conjunctiva, and connective tissue surrounding the optic nerve; melanocytes have a small nucleus with uniform nuclear morphology and absence of mitotic activity (Giannikaki, Vet Pathol 2019)
• Biphasic malignant melanoma-adenocarcinoma (case report):  recently reported in the digit of a dog; the melanocytic population was melan A positive and TRP2, PNL2, CKAE1/3, and CK7 negative; the epithelial population CKAE1/3 positive, and CK7, melan A, TRP2, and PNL2 negative (Needle, J Vet Diagn Invest 2018)

• Spindle cell neoplasms:

• Spindle cell melanoma:  Composed entirely of amelanotic spindle cells and impossible to distinguish from fibrosarcoma or neurofibrosarcoma without IHC stains
• Pigmented peripheral nerve sheath tumor (PNST):  May require IHC stains in some cases; PNST is GFAP positive and melanomas are rarely positive  
• Fibrosarcoma:  May require IHC stains in some cases; fibrosarcomas stain negative with  S100, melan-A, HMB-45, tyrosinase, and TRP-2

• Other neoplasms that display junctional activity:  Epitheliotrophic lymphoma, rarely cutaneous histiocytoma and mast cell tumors

 

COMPARATIVE PATHOLOGY:  

• Cats:  Melanomas are uncommon and are often amelanotic; greatest incidence in domestic shorthair cats; often occur on head
  1. Recently proposed grading scheme for feline nonocular melanomas evaluates tumor site, mitotic count, and intratumoral necrosis. Nonocular melanocytic neoplasms of the lips, oral or nasal mucosa, and nasal planum are high grade if they fulfill 1 or both of the following criteria: at least 4 mitoses in 10 HPF or the presence of intratumoral necrosis; those arising elsewhere are considered high grade if they fulfil both of the above criteria. (Pittaway R, Vet Pathol 2019)
• Horses:  Common in older gray horses, often on perineum, genital area and distal limbs; behavior is difficult to predict based on histological features, can have slow growth for years and then a sudden onset of malignant behavior; equine melanocytic tumors do not express Melan A, use PNL2 and S-100 instead; recent report of a primary sinonasal malignant melanoma with systemic metastasis in a 27 year old Anglo-Arabian (non-grey) gelding. (Hatai, H, J Vet Diagn Invest 2021)
• Pigs:  Sinclair miniature swine and melanoblastoma-bearing Libechov minipig (Hormel crosses, MeLiM strain, used in research) develop congenital melanoma; high incidence of spontaneous malignant melanoma, with reports of spontaneous regression  or malignant behavior with metastasis
• Guinea pigs: One recent report of subcutaneous S100, PNL-2, vimentin, and melan-A positive amelanotic malignant melanoma on the shoulder of a guinea pig.
• Gerbil: One of the most common cutaneous tumors in gerbils (in addition to SCC)
• Cattle:  
  1. Most are benign; may occur as a congenital lesion or at any age; Angus breeds overrepresented
  2. Case report of an amelanotic melanoma in a crossbred heifer calf with widespread metastases (Winslow CM, J Vet Diagn Invest 2017)
• Sheep and goats:  Uncommon and are generally pigmented; recent case report of a uveal melanoma in the anterior chamber that caused exophthalmos and glaucoma in an Aradi goat (El-Sahaey E, J Comp Pathol 2020)
• Avian species:  
  1. Penguins:  Malignant melanoma is relatively common in macaroni, Humboldt, and rockhopper penguins. Skin of the foot and hock, and subcutis and muscle near the beak and oral cavity are common sites. Histologically, neoplastic cells tend to be pleomorphic and heavily pigmented with prominent junctional activity. Surface ulceration correlates with systemic spread to liver, lung, adrenal gland, brain, and bone. Amelanotic melanomas not reported. PNL-2 can be used for IHC confirmation; S-100, Melan-A, and Ki67 cannot.
• Snakes: Chromatophoromas (I-N30) are neoplasms that arise from pigment-bearing cells (chromatophores) of the dermis; chromatophores include xanthophores, iridophores, and melanophores (containing melanosomes); melanocytic neoplasms are the most common; due to their dermal origin, there is neither junctional activity nor lentiginous spread; moderate to marked nuclear atypia appears consistently in neoplasms with a high risk of metastasis; San Francisco garter snake is overrepresented; S-100 + more reliable than PNL-2; sensitivity of melan A and HMB45 is extremely low in reptile melanocytic tumors; nuclear atypia is the most consistent feature associated with malignancy 
• Fallow deer:  Melanomas are common, often occurring on thin haired regions; periocular melanomas have an aggressive biologic behavior in white fallow deer (Hughes KL, J Vet Diagn Invest 2017)
• Siberian Tiger: Recent report of an elevated, ulcerated cutaneous mass on the flank that was removed and determined to be an amelanotic signet-ring melanoma. Surgical excision was curative with no recurrence four years later, suggesting that this variant is not as malignant as its counterpart in domestic cats (Hensel ME, J Comp Pathol 2021)

            

REFERENCES:  

 

1. Allnoch L, et al. Amelanotic malignant melanoma in a Himalayan Rex guinea pig (Cavia porcellus). J Comp Pathol. 2020; 181:13-17.
2. Asa SA. Immunohistochemical expression of MCAM/CD146 in canine melanoma. J Comp Path. 2017;157:27-33.
3. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th Ed. Ames, IA: Wiley Blackwell; 2016: 210.
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6. Giannikaki S, et al. Oculodermal melanocytosis: nevus of Ota in a dog. Vet Pathol. 2019;56(3):460-464.
7. Grimes JA, et al. Agreement between cytology and histopathology for regional lymph node metastasis in dogs with melanocytic neoplasms. Vet Pathol. 2017;54(4):579-587.
8. Hatai H, et al. Primary sinonasal malignant melanoma with systemic metastasis in a non-gray horse. J Vet Diagn Invest. 2021; 33(2):379-383.
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20. Porcellato, I, et al. FoxP3, CTLA-4, and IDO in canine melanocytic tumors. Vet Pathol, 2021; 58(1): 42-52.
21. Porcellato I, et al. FoxP3 and IDO in canine melanocytic tumors. Vet Pathol. 2019;56(2):189-199.
22. Raskin RE, Meyer DJ. Canine and Feline Cytology. A Color Atlas and Interpretation Guide. St. Louis, MO: Elsevier; 2016:73-74.
23. Silvestri S, et al. E-cadherin expression in canine melanocytic tumors: histological, immunohistochemical, and survival analysis. Vet Pathol. 2020;57(5):608-619.
24. Silvestri S, et al. Tumor thickness and modified Clark level in canine cutaneous melanocytic tumors. Vet Pathol. 2019;56(2):180-188.
25. Smedley RC, Thaiwong T, Deeth LE, Kiupel M. Correlation between KIT expression and c-kit mutations in 2 subtypes of canine oral melanocytic neoplasms. Vet Pathol. 2021;58(4):683-691.
26. Smedley RC, Spangler WL, Esplin DG, Kitchell BE, Bergman PJ, Ho HY, Bergin IL, Kiupel M. Prognostic markers for canine melanocytic neoplasms: A comparative review of the literature and goals for future investigation. Vet Pathol. 2011;48:54-72.
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