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A Comparative Review of Melanocytic Neoplasms

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

	Abstract

Top Abstract Introduction Pathogenesis Clinical Features Gross Pathology Histopathologic Variants Cytopathologic Variants Additional Diagnostic Techniques Future Diagnostics References

 
Melanoma is a devastating disease frequently encountered within both veterinary and human medicine. Molecular changes linked with neoplastic transformation of melanocytes include mutations in genes that encode proteins intrinsic to the regulatory pathways of two tumor suppressor proteins (retinoblastoma protein and p53), proto-oncogene mutation to oncogenes, altered expression of epithelial cadherin and CD44 adhesion molecules, and upregulation of angiogenic factors and other growth factors. Histologic evaluation of the primary mass is the most common means of diagnosis, with cytology used more frequently to document metastasis. Melanoma's highly variable histologic and cytologic patterns can make diagnosis by either method problematic. Adherent epithelioid morphology, including signet ring forms, and nonadherent round and spindle forms are recognized, with pigmentation an inconsistent finding. The site of the tumor, the thickness of the primary tumor or depth of invasion, and the number of mitotic figures per high-power field or per millimeter are used histologically to predict biologic behavior, whereas site and degree of pleomorphism are typically used for cytologic preparations. Diagnosis of amelanotic melanoma can be aided by ancillary diagnostic techniques. Tumor cells are usually positive for vimentin, S100, neuron-specific enolase, and Melan-A, and negative for cytokeratin. Melan-A as a positive marker is not as sensitive as the others are but is likely more specific. Monoclonal antibodies to human melanosome–specific antigens 1 and 5 cross-react with canine antigens for a combined sensitivity rate of 83%. Mouse monoclonal antibody IBF9 specifically recognizes canine melanoma antigen and also has good sensitivity. Serologic markers, including cytokines, cell adhesion molecules, and melanoma-inhibitory activity, are being investigated as potential sentinels of melanoma. Currently, there is no single diagnostic technique capable of differentiating benign from malignant melanocytic neoplasms or predicting survival time.

Key words: Animal; cytology; melanoma; mouth neoplasms; pathology; review; skin neoplasms.

	Introduction

Top Abstract Introduction Pathogenesis Clinical Features Gross Pathology Histopathologic Variants Cytopathologic Variants Additional Diagnostic Techniques Future Diagnostics References

 
A diagnosis of melanoma in domestic animals typically carries a grave prognosis. It is generally detected at a late stage when excision is rarely curative and metastasis is often already detectable within regional lymph nodes (LN). In dogs it is a common diagnosis, accounting for 7% of all malignant tumors.⁵⁰ It is the most common malignant neoplasm of the oral cavity and the second most frequent subungual neoplasm.¹⁴⁸ In cats it is not as common but carries the same poor prognosis. In gray horses melanoma is so common as to be considered almost inevitable.¹⁵⁰ In humans the cutaneous form of melanoma is expected to reach an incidence of 1 in 75 among individuals born in the year 2000.² Although oral melanoma is not common in humans, accounting for only 1–2% of all melanomas, as in domestic animals, it is generally very advanced when detected and has only a 5% 5-year survival rate.²⁰³

The purpose of this review is to provide a reference for diagnosticians and a resource for scientists in the field of melanoma research. Regardless of whether the neoplasm is initiated by chemical carcinogens or ultraviolet (UV) light, melanomas in all species share a similar biology in that they frequently recur and are predisposed to metastasis to regional LN. Animal melanomas are common and provide a useful model for a deadly human disease.¹⁴⁵

Terminology for this disease is not consistent in human and veterinary literature. To avoid confusion in terminology in this article and to seek a common ground for both groups of readers, we use the term "melanoma" for all malignant melanocytic tumors, whereas melanocytoma refers to benign forms.

	Pathogenesis

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Normal melanocytes are dendritic cells derived from neuroectodermal melanoblasts that have migrated during embryogenesis to the epidermis, dermis, and other sites. They are found within the basal layer of the epidermis interspersed between basal keratinocytes. They normally do not form attachments with or even touch each other but rather form adherent and regulatory junctions with five to eight neighboring keratinocytes via epithelial cadherin (E-cadherin) molecules.¹³⁸ Melanin is not retained within the normal melanocyte. It is packaged in melanosomes and transferred through their dendritic processes to keratinocytes. Conversion of normal melanocytes, that are nonpigmented and isolated from other melanocytes, to pigmented and clustered neoplastic melanocytes is a multistep process, with initiation as the first event, to be followed by promotion, transformation, and metastasis.^33,152,249

Virtually nothing is known about initiation of most animal melanomas, but initiation of as many as 65% of cutaneous melanomas in humans and in most of the melanomas diagnosed in Angora goats is thought to occur secondary to mutations generated by both UVA and UVB solar radiation.^96,180,218 This conclusion is based on epidemiologic linkage of melanoma with sun exposure by latitude and skin type (fair skin, freckling, and inability to tan). Furthermore, patients with the genetic disorder xeroderma pigmentosum, who have a defect in the enzymes required to repair light-induced DNA mutations (pyrimidine dimers), have a 1,000- to 2,000-fold increase in risk of developing skin cancer, including melanomas, squamous cell carcinomas (SCCs), and basal cell carcinomas.²⁴⁴ The initiators for all other cutaneous and noncutaneous melanomas are not known. Breed and familial clustering in domestic animals suggest that genetic susceptibility, possibly resulting in a greater frequency of spontaneously mutated cells, may be critical to initiation of many of these tumors.^{48,86,89,99,100}

In humans initiation within benign, pigmented precursor lesions contributes to a small percentage of melanoma cases.¹²⁷ The most important category of precursor lesion is the dysplastic nevus.¹²⁷ Multiple dysplastic nevi on an individual are associated with an increased risk of melanoma.¹⁹⁵ Other precursor lesions in humans include the giant congenital melanocytic nevus,¹²⁷ but only a single case of malignant transformation of a similar congenital lesion in a golden retriever has been reported.²⁴² Conroy⁴⁸ described two canine cases of melanoma that arose from junctional or dermal pigmented nevi (hamartomas), although in neither case was metastatic behavior observed. There is a single report of a primary melanoma in the skin of a dog that originated from a subcutaneous melanocytoma.¹⁶⁶ Melanoma has been reported to arise from primary acquired melanosis (PAM) in a dog's cornea and conjunctiva.⁵³ In humans approximately 46% of PAMs that demonstrate cellular atypia progress to melanoma.¹¹⁸ In general, however, with the exception of gray horses, malignant transformation of benign lesions is very uncommon in animals, and most melanomas are believed to arise de novo from melanocytes in the epidermis, dermis, ocular epithelium, and oral epithelium.⁴⁸

The next step in carcinogenesis requires a promoting factor(s). Promoters, possibly acting in an epigenetic fashion by disrupting gap-junctional intercellular communication, stimulate proliferation of the mutated cell, allowing for amplification of the cell population, persistence of the mutation, and opportunities for additional mutations.²³³ Promoters can include chronic trauma, chemical exposure, burns, hormones, infections, drugs, and other causes for reactive hyperplasia.^{33,38,119,233} Perhaps mucosal melanoma arises as a result of chronic injury (either mechanical or inflammatory) that results in reactive hyperplasia of the epithelium, disruption of normal keratinocyte-melanocyte interactions, and amplification of cells initiated spontaneously or by unidentified environmental factors.

Genetically or environmentally initiated DNA instability facilitates subsequent neoplastic transformation. Normal suppressors of cell proliferation and activators of apoptosis, e.g., retinoblastoma protein (Rb) and p53, are eventually superceded by unregulated growth factors or growth factor receptors (or both), and inhibitors of apoptosis, e.g., bcl-2.^160–162 For example, p53-triggered cell cycle arrest or apoptosis prevents maintenance of DNA mutations in any given cell line. Mutations in p53 (gene for a protein is italicized by convention) can prevent synthesis of p53 or result in abnormal p53 that cannot gain entry to the nucleus, resulting in perpetuation of a DNA mutation. Human melanomas with p53 expression are common, 20–40% among all cases, and may have a different pathogenesis compared with those without p53.²⁵¹ A canine case of multicentric melanocytoma was demonstrated to lack p53.¹⁹⁶ Conversely, overexpression of normal p53 was detected in metastatic melanomas in two horses, suggesting to those authors that p53 is not involved in equine melanoma tumorigenesis.²⁰²

Most melanoma cases in humans lack detectable p53 mutations. Mutations in INK4a, INK4b, and Waf-1 are more common.^160,231 INK4a and INK4b encode proteins p16^INK4a and p15^INK4b that are cyclin-dependent kinase inhibitors (CDKI) regulated by Rb. Entry into mitosis is triggered by cyclin-dependent kinases; therefore, INK4a and INK4b mutations ultimately remove the ability to inhibit mitosis and allow uncontrolled proliferation. INK4a also encodes p19^ARF, which is an activator of p53, via an alternative exon and reading frame. Waf-1 encodes the protein p21, another CDKI. Expression of Waf-1 is partially regulated by p53. The multicentric melanocytoma case described above lacked functional p21.¹⁹⁶ Thus, activities of two important tumor suppressors, p53 and Rb, are essentially sabotaged in the majority of melanoma by mutations in genes that encode proteins intrinsic to these regulatory pathways.²³¹

Expression of the apoptosis suppressor gene bcl-2 was detected in 16 of 18 human cutaneous melanomas examined by Morales-Ducret et al. 1995,¹⁶² but the bcl-2 gene product was also found in resting melanocytes in normal skin. On the other hand, atypical bcl-2 overexpression has been detected in human uveal melanoma.²⁸ Suppression of apoptosis complements disruption of the Rb and p53 pathways to favor uncontrolled proliferation.

Proto-oncogene mutation to oncogenes also favors proliferation and tumor development. Oncogenes detected in in vitro and in vivo melanoma studies include c-myc, c-erb-B-2, c-yes, c-kit, and ras.^12,142,160

Autonomous growth is an additional requirement for neoplastic progression. The chief autocrine growth factors linked with melanoma development are basic fibroblast growth factor (bFGF), melanoma growth stimulatory activity or growth regulated proteins, interleukin (IL)-8, IL-10, IL-18, platelet-derived growth factor-A, and -melanocyte stimulating hormone.^{115,134,151,245} Late-stage melanomas are also stimulated by factors such as IL-6 and tumor growth factor-ß that are normally inhibitory to melanocyte proliferation.¹³⁴

Neoplastic transformation, resulting from mutations as described above, occurs before metastasis. Metastasis itself is a multistep process starting with detachment from the primary mass, movement to and through endothelium, travel via blood or lymph (or both), adhesion and movement through endothelium, and finally attachment and proliferation within a secondary site. Neoplastic cells must downregulate and then upregulate various adhesion molecules, e.g., Ca²⁺-dependent adherent molecules (cadherins) and CD44. Many studies have demonstrated both in vitro and in vivo decreases in E-cadherin in melanoma cells, which is supportive of theories that diminished E-cadherin interactions with basal keratinocytes are critical to melanoma tumorigenesis.^113,152 But inconsistent decreases in membranous E-cadherin, concurrent with dramatic decreases in membranous placental cadherin and it's associated gamma catenin in late-stage primary and metastatic melanoma tumors have been reported by others.²⁰⁸

The adhesion molecule CD44 most likely plays a role in the facilitation of melanoma metastasis. CD44 is a widely expressed transmembrane glycoprotein that functions in T-cell activation, T-cell extravasation, LN homing, hyaluronate degradation, cell aggregation and migration, angiogenesis, and hematopoiesis.^91,219 CD44 interacts with hyaluronic acid to facilitate expansion of proliferating tissues. Reduced expression of standard CD44 and upregulation of CD44 variant number 5 (CD44v5) has been demonstrated to occur in melanoma metastatic to LN.^205,215 Melanoma metastasis, but not primary growth, was inhibited by antibody to CD44 in SCID mice injected with the human melanoma cell line SmelanomaU-2.¹⁰³

At both primary and metastatic locations, angiogenesis is critical to survival and growth of any neoplasm regardless of its derivation. Reports on microvascularity of melanoma and its relationship with tumor aggression are inconsistent but seem to favor the theory that angiogenesis is critical to the metastatic potential of melanoma and correlates with poor outcome.^139,165,172 One recent study examined serum concentrations of four angiogenic factors for a possible correlation with tumor progression and survival.²⁴⁰ Serum levels of angiogenin, vascular endothelial growth factor (VEGF), bFGF, and IL-8 were all significantly elevated in melanoma patients compared with healthy controls; however, only elevated levels of VEGF, bFGF, and IL-8, and not angiogenin, had a positive correlation with poor survival and tumor burden.

During this entire process tumor cells must escape immune surveillance, be physically able to survive and move within the circulatory or lymphatic system (or both), and be able to proliferate within a stromal environment different from their origin. One current theory suggests that avoidance of the immune system is facilitated by tumor production of IL-10.¹¹⁵

Other changes required of a melanocyte to become malignant are the subject of considerable research.²¹⁸ Evaluations of cultured melanoma lines, animal models, and clinical cases are helping in answering questions regarding both general tumor biology and melanoma pathogenesis.

	Clinical Features

Top Abstract Introduction Pathogenesis Clinical Features Gross Pathology Histopathologic Variants Cytopathologic Variants Additional Diagnostic Techniques Future Diagnostics References

 
Canine

Melanoma is relatively common in dogs, accounting for 3% of all neoplasms and up to 7% of all malignant tumors.⁵⁰ The most frequently affected sites are the oral cavity (56%), lip (23%), skin (11%), and digit (8%), with other sites, including the eye, accounting for only 2%.⁹⁰

Melanocytic neoplasms that arise in the oral cavity are virtually always considered malignant²⁶ and constitute the most common oral malignant neoplasm (Fig. 1).^26,232 They predominantly arise in the gingiva¹⁷⁵ and account for 33% of malignant neoplasms in this site,^{25,31,64,93,108} with fewer cases arising from the lingual, buccal, pharyngeal, tonsillar, and palatine epithelium. Oral melanomas grow rapidly, are invasive, and often recur after surgical resection. Presenting signs include dysphagia, halitosis, ptyalism, bleeding, and occasionally fracture of the mandible.^86,190 Seventy to ninety percent metastasize,^32,42,232 independent of location within the oral cavity.¹⁷ The most common metastatic sites are the regional LN, lung and viscera, including the heart (Figs. 2, 3).^48,232

View larger version (120K): [in this window] [in a new window]   Fig. 1. Oral melanoma. Canine. Nonpigmented, exophytic mass protruding from the hard palate. Formalin-fixed tissue.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Metastatic melanoma. Canine. Multiple, black, raised, smooth, shiny nodules disseminated throughout the thoracic viscera, effacing the lungs, pericardium, and mediastinal soft tissue. Fig. 3. Metastatic melanoma. Canine. Multiple, black, raised, smooth, shiny nodules disseminated throughout the liver.

Subungual melanoma is the second most common digital neoplasm after SCC.¹⁴⁸ Digits affected by subungual melanoma often have deformed nails, growth of a mass from the nailbed, or paronychia (Fig. 4). Subungual melanoma can result in destruction of phalangeal bone(s)⁷ but less commonly (5%) than in cases of subungual SCC (80%).¹⁴⁸ Marino et al.¹⁴⁸ reported radiographic evidence of metastasis in 58% of digital melanoma, either before or soon after surgery, and a median survival time of 12 months.

View larger version (159K): [in this window] [in a new window]   Fig. 4. Digital melanoma. Canine. Subgross appearance of malignant melanocytes forming an exophytic protrusion, which also effaces and invades underlying phalangeal bone. HE. Bar = 3.5 mm.

Melanoma incidence not only varies with site but also with breed. Cutaneous melanoma occurs more commonly in dogs with heavily pigmented skin, with the miniature schnauzer, standard schnauzer, and Scottish terrier at increased risk of developing neoplasm.^48,86,90 In addition to the above breeds, Irish setters and golden retrievers are at increased risk of developing subungual melanoma. The Irish Setter, Chihuahua, Golden Retriever, and Cocker Spaniel are at increased risk of developing melanoma of the lip.⁹⁰ German Shepherd Dogs and Boxers are more prone to developing oral melanoma.^175,232 The reason for breed predilection is not entirely clear, but it may reflect an underlying genetic risk or increased pigmentation (or both) in certain breeds. The latter is in contrast to humans where there is no proof of any difference in the relative frequency of oral melanoma in African-Americans and Africans, compared with white cohorts.⁹⁴ Furthermore, cutaneous melanoma is rare in Africans and other races with heavily pigmented skin.⁹⁴ Some authors report a predisposition to oral melanoma in Japanese, Indians, and Hispanics, although others have contradicted this observation.^24,203

Most dogs with melanoma are more than 10 years old with a range of 1–17 years.^7,86,232 An increased frequency has been described in male dogs.^64,90,232 This is similar to humans, where the male/female ratio of oral melanoma is 2–3:1.^13,203

Equine

Up to 15% of all equine skin tumors are melanocytic.^51,120 More than 90% of these tumors are benign at initial presentation, but approximately two-thirds are thought to progress to malignancy and are capable of widespread metastasis.^{92,120,124,130,198,212,213,224,241} The vast majority appear in gray or white horses, usually at or before the age of 5 years, corresponding to the time in their life when their coat color changes.^120,137,150 An early theory of equine melanocytic neoplasia suggested that dermal and visceral melanocytic tumors are a manifestation of a storage disease, rather than malignant neoplasia, and occur as a result of the accumulation of melanin in melanophages during the depigmentation process.^137,212 Melanocytic neoplasms also occur in non-gray or non-white horses, such as bays and chestnuts, and may be more likely to be malignant.¹²⁰ Arab breeds seem to have a predilection for cutaneous melanoma, but statistics may be skewed by the fact that most are gray.^120,136,198 A similar overrepresentation has been observed in Lippizaners and Percherons.⁶⁷

It has been more difficult to attribute any breed or color predisposition to ocular melanomas, which are rare, although such a correlation has been demonstrated in some cases.^14,67 The same is true for oral melanomas, which are uncommon, accounting for 17% of all oropharyngeal neoplasms in horses.⁶⁴

Melanomas are unusual in horses less than 6 years old. They increase in prevalence with age, with 80% of gray horses developing malignant forms by the time they are 15 years old.^92,150,241 There have been inconsistent reports regarding sex predilection. Sundberg et al. (1977)²²⁴ reported multiple dermal melanoma (otherwise called dermal melanomatosis) more frequently in males, whereas others suggest that there is no sex predilection.^120,224,241

Most equine melanomas arise in the skin of the perineal region, the ventral surface of the tail, and external genitalia.^8,92,150 More atypical sites have been reported, such as the coronary band,¹³⁰ foot,¹¹⁰ vertebral region,^124,198 and pelvic canal.¹²⁴ Rarely, the thoracic cavity,^167,225 nasal cavity,⁶² and eye^14,56,67,149 have been involved. Clinical signs range from interference with bridle, bits, and saddle to more serious obstructive lesions in the urogenital or gastrointestinal tract, progressing to life-threatening pulmonary or visceral metastasis.¹²⁰ Neurologic signs have been reported secondary to compression of the spinal cord by metastatic nodules.²¹² Other more unusual manifestations include Horner's syndrome and unilateral sweating.^157,167

Equine metanocytic neoplasms have traditionally been grouped according to one of the three growth patterns. Some grow slowly over many years without metastasizing, whereas others grow slowly initially, with a subsequent increase in the rate of growth after a few years. A third subset grows rapidly and is malignant from the beginning.^120,213 Valentine (1995) has suggested that there are actually at least four possible syndromes of equine melanoma, three of which have the potential for metastatic behavior.²⁴¹ Two of these three categories, dermal melanoma and dermal melanomatosis, are histologically very similar and can only be classified based on clinical features. Dermal melanomas are usually solitary, discrete lesions that are surgically excisable, occurring in a wide age-range of gray horses. Dermal melanomatosis denotes the presence of many lesions, often coalescing and usually occurring in typical locations, such as the genital or perineal region, of gray horses older than 15 years. These are not surgically curable and are much more likely to metastasize internally.^120,241 The third category refers to anaplastic melanoma in aged, non-gray horses. Although rare, it is the most aggressive, leading to death within months of diagnosis.²⁴¹ The fourth category is the melanocytic nevi, which are benign, pigmented lesions predominantly occurring in horses less than 6 years old.^120,241

Feline

Melanoma is uncommon in cats, accounting for less than 1% of all feline oral neoplasms^90,155,220 and approximately 0.5% of feline skin tumors.^32,64,86,182 The ocular and cutaneous forms are generally more common than melanoma of the oral cavity.^57,64,73,182 The most common cutaneous sites are head (Fig. 5), tail, distal extremities,¹⁴⁴ and lumbar area.^57,89,90,243 The prognosis is generally poor because of recurrence and regional metastasis in up to half the cases.^90,243 In a study of 23 cats with nonocular melanocytic tumors, approximately half were malignant, including all the three oral tumors included in the study.¹⁴⁴ In another study, four of the six reported cases of oral melanocytic tumors were malignant, with metastasis to viscera in one case and local invasion in three.¹⁸²

View larger version (147K): [in this window] [in a new window]   Fig. 5. Melanoma. Skin of dorsal skull between ears; feline. Exophytic, crusted, discrete mass elevating the epidermis.

The age-range is comparable in several surveys, affecting cats from 2 to 18 years of age but peaking at 8 to 12 years.^{57,84,86,144,182,243} No sex or breed predilection has been demonstrated.

Other species

Melanocytic neoplasms occurs in other domestic species, including cattle, sheep, alpaca, and swine^{106,178,192,252} as well as spontaneously in laboratory animals and in birds.^5,59 Of these species, swine are probably the most important for two reasons. Firstly, the Sinclair miniature and Duroc breeds have a genetic predisposition for melanomas.^178,229 Secondly, the Sinclair miniature pig has served as a model for spontaneous cutaneous melanoma in humans.^{111,156,178,221} The melanocytic tumors can be congenital and are often multiple.¹⁷⁸ In up to 90% of pigs, the tumors usually completely spontaneously regress, a feature that has made this such an attractive model, particularly for studying immunopathogenesis and its potential role in the therapy of melanoma.^111,179 On the other hand, 10–15% of affected pigs succumb to metastatic disease with involvement of regional LN, lungs and liver, as well as other viscera.^163,178 This model shares many features in common with human cutaneous melanomas, including spontaneous development of tumors, a wide spectrum of melanocytic tumors capable of malignant transformation, correlation between deeply invasive tumors and metastatic disease, and the pattern of metastatic spread and histopathology of tumor regression.¹¹¹ In humans the regression is usually partial and perhaps a poor prognostic indicator,¹⁸⁶ although complete remissions have been reported.²⁷ In both human and swine tumors, regression has been associated with a high number of tumor-infiltrative lymphocytes. These lymphocytes have been identified as CD8+ cytotoxic T cells¹⁶³ and display granulated lymphocyte morphology.¹⁹⁴ These are the lymphocytes that react to Melan-A/MART (melanoma antigen recognized by T cells), a cytoplasmic marker that is weaker or not evident in the population of melanoma cells remaining after partial remission of human melanomas.

Uveitis and vitiligo are associated with the spontaneous regression that occurs in these pigs. Cataracts and a band keratopathy, characterized by deposition of calcium salts beneath the basal corneal epithelial cells, have been less frequently observed.^74,135 Such observations have led to a profusion of studies aimed at elucidating the immunopathogenesis of regression. Peripheral blood lymphocytes in swine with regressing melanomas have significant lytic activity against cultured porcine melanocytes; lysis is age-dependent and does not occur in piglets less than 4 weeks old.¹⁹⁴ Histologic changes of regression are biphasic, consisting of tumor infiltration by what appear to be pigment-laden macrophages between 4 and 8 weeks of age, followed by a predominantly lymphocytic response over the next few months.⁹⁸ But the heavily pigmented cells may actually be terminally differentiated melanocytes and not monocyte-derived macrophages.⁹⁷ The end result is the destruction of normal as well as malignant melanocytes.¹³⁵ Circulating antibodies specific to pigment cells in pigs with regressing melanomas have been shown to increase with time and correlate with regression and vitiligo.⁵⁴ More recently, melanoma regression has been associated with loss of telomerase activity, leading eventually to DNA fragmentation and cell death.¹⁸¹ This is an important area of ongoing research, and a complete review of the swine melanoma model is beyond the scope of this article. Other potential animal models for the human disease include the laboratory mouse, the opossum, and the Xiphophorus fish.⁶³

In cattle, melanocytic tumors account for approximately 6% of all tumors, mostly arising in the skin and predominantly affecting cattle with red, gray, or black coats.¹⁵⁴ Reports of malignancy are very uncommon, but metastases can occur.²¹³ The majority of melanoma in cattle have been reported from the Indian subcontinent.^168,192,193 In contrast, in sheep and goats cutaneous melanocytic neoplasms metastasize frequently.²¹³ Angora goats are predisposed and have been proposed as a potential model of human melanoma because of the probable association with UV light.^96,180

Reports in wildlife and exotic species are much less common. There is a single case report of a poorly pigmented cutaneous melanocytoma in a ferret.²³⁶ Melanocytic neoplasms are very rare in birds, with only single case reports in the literature.^5,129

	Gross Pathology

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Melanoma can vary considerably in appearance, regardless of the site. Melanoma may be of any color, ranging from gray or brown to black, red, or even dark blue.^62,102,153 Pigmentation is not a specific feature because other neoplastic or nonneoplastic lesions can be phenotypically similar. Melanoma vary in size, but most fall within the range of 1–3 cm.^48,102 Cutaneous melanoma may be smooth domes, sessile nodules (Fig. 5), polypoid, plaquelike, or even lobulated masses;^86,102,166 larger ones are often ulcerated. In the horse they are often flat and firm, may be single or multiple, and can coalesce, creating a cobblestone appearance.¹²⁰ Alopecia and ulceration are variable features.

Lesions found at necropsy depend on how far the neoplasm has progressed and range from local primary tumor masses to widely disseminated metastases (Figs. 2, 3).^{57,86,198,254} Individual tumors vary from 1 mm to several centimeters diameter.²¹² Neoplastic masses can be compressive, although they are usually unencapsulated and infiltrative.¹⁸⁷ They can also be poorly defined, effacing normal structures rather than forming discrete masses.^110,130 Pigmentation is variable, but often the masses are black.

Regardless of site, melanomas can metastasize via the lymphatic or blood vessels, with regional LN being the usual first target.¹¹⁰ Any of the visceral organs can potentially be infiltrated, but the lungs are most commonly involved.^{17,21,92,130,158,182,198} Infrequently, extension and metastasis to serosal surfaces, such as pericardium, pleura and peritoneum, occur; associated effusions may be black.¹⁵⁷ Melanomas have the potential to invade bones and cause bony lysis in areas such as digit, maxilla, mandible, vertebrae, and ribs.^148,182,198

	Histopathologic Variants

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Histopathologic variants in animals

The term nevus, commonly used in describing pigmented melanocytic lesions of the epidermis and dermis in humans, is not used in veterinary dermatopathology.⁸⁸ When evaluating melanocytic neoplasms they may be compound, denoting both an epidermal-epithelial and dermal-submucosal component to the neoplasm, or dermal and submucosal with no identifiable epidermal or epithelial component (Fig. 6). Junctional refers to the proliferation of neoplastic melanocytes at the interface between the epidermis and dermis or epithelium and submucosa.⁸⁸ Pagetoid refers to the presence of either individual or small aggregates of neoplastic cells within the upper levels of the epidermis or epithelium⁹⁰ (Fig. 7) and was originally used to describe the carcinoma cells (Paget cells) that infiltrate the epidermis overlying mammary ductal carcinomas in situ.⁵²

View larger version (157K): [in this window] [in a new window]   Fig. 6. Melanoma. Skin; canine. The dermal neoplastic cells are fusiform and epithelioid. Melanocytic hyperplasia is evident in the epidermis, but it is nonneoplastic. HE. Bar = 40 µm. Fig. 7. Melanoma. Oral; canine. Pagetoid infiltration of oral mucosa by individual cells and nests of cells (arrows) and neurotization of submucosal neoplasm. HE. Bar = 40 µm.

View larger version (216K): [in this window] [in a new window]   Fig. 8. Melanoma. Ear; canine. Fig. 8a. Epithelioid cells arranged in packets. Cells are fairly uniform with a high nucleus to cytoplasmic ratio and sparse intracytoplasmic melanin. HE. Bar = 30 µm. Fig. 8b. Cytologic preparation of same tissue seen in Fig. 8a. Cells are round with moderately high nuclear to cytoplasmic ratios, round nuclei, fine lacy chromatin, multiple, distinct nucleoli, and a moderate amount of nonpigmented cytoplasm, stained light gray blue in the original image. A single mitotic figure is present in the center of the image. Wright-Giemsa. Bar = 10 µm. Fig. 9. Melanoma. Ear; feline. Fig. 9a. Pleomorphic round cells arranged in sheets. Many cells have abundant cytoplasm and sometimes two or more nuclei. HE. Bar = 30 µm. Fig. 9b. Cytologic preparation of same tissue seen in Fig. 9a. The arrow indicates a single giant nucleolus present in a giant neoplastic cell. Fine pigment granules are occasionally detectable in the neoplastic cells. Melanophages are identifiable by their coarse pigment granules. Wright-Giemsa. Bar = 20 µm. Fig. 10. Melanoma. Lip; canine. Fig. 10a. Mixed round and epithelioid cell proliferation. HE. Bar = 60 µm. Fig. 10b. Cytologic preparation of same tissue seen in Fig. 10a. The epithelioid neoplastic cells are found in adherent clusters. Nuclear to cytoplasmic ratios are high, with round nuclei, fine chromatin, distinct nucleoli, and a small amount of finely vacuolated cytoplasm. Pigmentation is not apparent. Wright-Giemsa. Bar = 10 µm.

View larger version (217K): [in this window] [in a new window]   Fig. 11. Melanoma. Head; feline. Fig. 11a. Mixed epithelioid and spindle cell pattern. Poorly pigmented epithelioid cells arranged in packets and nests with intervening more heavily pigmented spindle-shaped cells. HE. Bar = 30 µm. Fig. 11b. Cytologic preparation of same tissue seen in Fig. 11a. Many of the neoplastic cells were represented by only bare nuclei. Intact cells tended to be spindle form, with many fine pigment granules. Wright-Giemsa. Bar = 10 µm. Fig. 12. Metastatic (lungs) and primary (mouth) melanoma. Canine. Fig. 12a. Metastatic melanoma. Lungs; canine. Nonpigmented spindle-shaped cells arranged in tightly interweaving streams and bundles. The primary tumor was oral (see Fig. 12b). HE. Bar = 60 µm. Fig. 12b. Melanoma. oral; canine. Cytologic preparation of primary oral melanoma. The primary is pigmented and epithelioid, whereas the metastatic sites contain amelanotic spindle-shaped cells, seen in Fig. 12a. Wright-Giemsa. Bar = 10 µm. Fig. 13. Melanoma. Oropharynx; canine. Fig. 13a. Small groups of packeted epithelioid cells, with high nucleus to cytoplasmic ratios and prominent large nucleoli, intermingle with more spindle-shaped cells. HE. Bar = 30 µm. Fig. 13b. Cytologic preparation of same tissue seen in Fig. 13a. The round to oval neoplastic melanocytes contain 1–2 nuclei, with fine to slightly coarse chromatin, multiple, variably sized nucleoli, and moderately abundant fine to occasionally coarsely pigmented cytoplasm, which was stained very light gray blue in the original image. Nuclear clefting is visible in the cell in the lower left corner. Wright-Giemsa. Bar = 10 µm.

View larger version (171K): [in this window] [in a new window]   Fig. 14. Melanoma. Skin of foot; canine. A balloon cell variant. The cells have an abundant amount of pale eosinophilic but slightly dusty brown cytoplasm. There are fine fibrovascular trabeculae of connective tissue forming multiple small lobules. HE. Bar = 60 µm. Fig. 15. Melanoma. Oral cavity–maxilla; canine. There is a transition from cells that are round to more fusiform to cells with a basophilic cytoplasm, indicative of early chondroid differentiation.

Generally, cutaneous melanoma in domestic animals does not share the same degree of diversity described in the medical literature.¹⁷⁰ In veterinary pathology, because dysplastic nevi are not recognized as a specific entity, the pathologist is not required to differentiate between severely dysplastic nevi, melanoma in situ, and early invasive melanoma, all of which differ in their prognostic significance. Furthermore, most animal melanomas are advanced invasive masses when removed for histopathologic evaluation, corresponding to nodular melanoma in humans, whereas most melanomas in humans are the superficial spreading type, which are most often relatively small, thin tumors that are confined to the epidermis and dermis. Some direct histopathologic correlations can be made between human and canine melanoma in that both forms may be composed primarily of spindle cells, epithelioid or round cells, or a mixture of these cell types.⁷⁰ There are similarities between oral melanoma in dogs and humans. As in dogs, the prognosis for oral melanoma in humans is very poor regardless of morphology,⁷⁰ and no link has been found between survival rate and histologic type.⁷²

Cutaneous melanomas in humans are most commonly classified according to their growth pattern variants. The most common types are superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma.^36,222 This classification system is thought to represent subgroups with similar etiologies as well as biologic, clinical, and prognostic features,^183,222 although the validity of this theory has been questioned.²⁵⁰ All four have two growth phases of variable duration—radial growth with epidermal involvement only and vertical growth with dermal invasion.²²² Superficial spreading melanoma is the most common melanoma seen in Caucasians accounting for 70% of all melanomas.¹²⁷ This tumor is typically found on the trunk and extremities, with no predilection for chronically sun-damaged skin, and is composed of predominantly large, atypical epithelioid melanocytes. Nodular melanoma is predominantly dermal, with only a minor intraepidermal component. It is generally heavily pigmented, predominantly epithelioid, often ulcerated, and rapidly progressive, with essentially no clinically discernable radial growth phase (RGP).¹²⁷ It is the second most common melanoma, accounting for 15–30%,¹²⁷ with tumors generally found in skin that has been chronically sun damaged, e.g., the head, neck, and trunk. Lentigo maligna melanoma originates from lentigo maligna, which is typically seen on sun-exposed skin of elderly patients. The precursor lesion, lentigo maligna, is the early RGP (in situ) and can be present for years before the vertical growth phase (VGP) occurs, at which time the lesion is considered a melanoma. Spindle-shaped cells predominate over epithelioid cells in the VGP for this melanoma. Acral lentiginous melanoma occurs on the hands and feet, including the digits and subungual regions. This is the most common type of melanoma seen in Asians, African-Americans, and Hispanics.²²²

The variables listed below are used as prognostic indicators in humans with cutaneous superficial spreading and nodular melanomas to estimate 5- and 10-year survival with a 95% confidence level and to stage disease.^{2,11,36,45,146} Physicians are advised to use these variables for tumor node metastasis (TNM) staging of cutaneous melanomas with other growth patterns, e.g., lentigo maligna melanoma and acral lentiginous melanoma but with the caveat that these other groups may have different etiologies and different prognoses. Variables numbered 1–7 are those used by the Melanoma Staging Committee of the American Joint Committee on Cancer as the basis for the currently accepted staging system for cutaneous melanomas in people¹¹ and have been found to be independent predictors of survival. The remaining variables are not incorporated into the TNM system because they are difficult to objectively relate to survival curves, their status is controversial, or because they are not clearly independent of other factors, e.g., tumor thickness. Recording of these values for individual patients is recommended. It is interesting to note that morphologic atypia is not included in this list.

1) Modified Breslow²⁹ thickness of primary tumor: measured from stratum granulosum to deepest portion of neoplasm; considered the most significant.¹⁴⁶ Using the TNM staging protocol¹¹ assignments are as follows: T1 = <1.0 mm; T2 = 1.01–2.0 mm; T3 = 2.01–4.0 mm; T4 = >4.0 mm.

2) Ulceration: defined as microscopically detected absence of intact epidermis overlying a major portion of the primary melanoma that is not artifactual or secondary to trauma. Ulceration signifies locally advanced disease with higher risk of metastasis compared with nonulcerated tumors of like thickness.

3) Level of invasion: under new guidelines, Clark's levels⁴⁶ are now only used for thin melanomas (T1)¹¹

Level I—confined to the epidermis (melanoma in situ)

Level II—Extend from epidermis into papillary dermis, but papillary dermis not filled or expanded

Level III—Expand and fill the papillary dermis

Level IV—Infiltration of the reticular dermis

Level V—Infiltration of the subcutaneous fat

4) The number of metastatic LN and whether or not the metastasis is detected clinically or microscopically: although both indicate a poorer prognosis than no metastasis, macroscopic (clinical) detection is worse than microscopic detection (clinically occult).

5) Intralymphatic invasion: either satellite metastasis around the primary or in-transit metastasis between the primary and the regional LN; both sites have an equally poor prognostic significance that is about equivalent to detection of metastasis in multiple regional LN.

6) Site(s) of distant metastasis: melanomas metastatic to skin, subcutaneous tissue, and distant nodal sites have a better prognosis compared with lung metastasis, which is intermediate compared with other visceral sites, which have the poorest prognosis.

7) Elevated lactate dehydrogenase (LDH): elevated LDH is associated with diminished survival; a high LDH must be confirmed by two elevated LDH values determined more than 24 hours apart and not because of other factors, e.g., hemolysis or liver disease.

8) Number of metastatic sites: this variable was not incorporated into the TNM system because of the lack of standardization of diagnostic strategies used to search for metastatic sites.

9) Site: a prognostic advantage is seen in patients with nondistal appendicular tumors, as opposed to axial (trunk, head, neck), subungual, palms, or soles,^114,227 but this disappears when tumors are controlled for thickness, age, and sex.¹⁴⁶

10) Mitotic count: number per millimeter²: high counts are indicators of a worse prognosis.

11) Tumor infiltrating lymphocytes (TILs): brisk, nonbrisk, or absent; nonbrisk or lack of TILs is considered a high-risk attribute.

12) Age: poorer prognosis if > 60 years.

13) Growth phase: RGP versus VGP.²¹⁸

14) Sex: there is a slight survival advantage for women.

15) Regression: absent or present; regression being defined as evidence of loss of continuity of the lesion and fibrosis, which appears to correlate with high risk of metastasis, as opposed to actual regression of disease; surviving cells after regression represent a subpopulation that is more likely to migrate into lymphatics. The prognostic value of this trait is still uncertain.

Criteria of malignancy

Melanoma may be one of the few neoplasms in animals for which location is an important prognostic indicator in its own right. Melanocytic neoplasias involving the oral cavity, subungual region, and mucocutaneous junctions are considered malignant, regardless of any other single feature.⁷ Interestingly, this also seems to be the case with oral melanoma in humans. There is no evident relationship between histologic characteristics, including mitotic index and pigmentation, and survival rate.^70,190 The prognosis for human melanoma at mucocutaneous junctions and mucosal surfaces is also grave.²⁰³

In animal skin and eye melanocytic neoplasms, the most reliable histologic feature for distinguishing malignant from benign is the mitotic index.^{25,26,35,65,158,254} In the World Health Organization's Histologic Classification of Epithelial and Melanocytic Skin Tumors of Domestic Animals,⁸⁸ three or more mitotic figures per 10 high-power fields indicate malignancy. The identification of mitotic figures in sections must be undertaken with great care to ensure that only true mitoses are counted. Bleached sections should be used for pigmented neoplasms to avoid confusing mitoses with pyknotic nuclei and the small hyperchromatic nuclei of the spindle cells of the supporting stroma. The mitotic count will vary from area to area within the neoplasm, and the count should be performed in areas with the greatest concentration of mitotic figures. Where metastases have been confirmed, the mitotic rate in intraocular melanoma is usually greater than four per 10 high-power fields; less than two mitotic figures per 10 high-power fields is consistent with a melanocytoma.^35,254 In cats the value of mitotic activity to prediction of outcome is less clear, particularly with regard to ocular melanoma.^21,22,65 Some authors, however, report a link between high mitotic index and increased risk of metastatic disease.^57,68

Neoplastic cell morphology is also a useful discriminating feature.^25,69,88 Cytologic features of malignancy include the presence of a large nucleus, variation in nuclear size and shape, hyperchromasia, abnormal chromatin clumping, one or more nucleoli, and atypical mitotic figures.^65,86 Additional features favoring malignancy are the presence of neoplastic cells, individually or in nests, within the upper layers of the epidermis (Fig. 7),^65,86,88 although ulceration and necrosis may prevent evaluation of this feature. The presence or absence of junctional activity is not specific to melanoma and often occurs in melanocytomas.

The gold standard is, of course, lymphatic or vascular invasion.⁸⁸ Pathologists are often asked to evaluate mandibular LN for evidence of metastatic disease in dogs with oral melanoma. These nodes may be pigmented because of metastatic melanoma or the accumulation of melanophages within the medullary sinuses. The latter occurs when melanophages are transported to the regional LN after inflammation of the oral mucosa, particularly in dogs with pigmented oral epithelium. The cytologic features described above, when used to evaluate bleached sections, usually suffice to distinguish melanophages from melanoma.

In cats the following histologic features have been proposed to be of significance in identifying cutaneous melanoma: nuclear atypia (neoplasms with more extensive atypia are more likely to be malignant), mitotic activity (a trend of greater malignancy with increased numbers of mitoses), and tumor cell type (with epithelioid more likely to be malignant).⁸⁹ The relationship with cell type is not firmly established in the literature, in that two studies indicate a less precise relationship between cell type and malignant potential.^144,155 Furthermore, histologic determination of malignancy does not consistently correlate with clinical behavior.¹⁴⁴

The Callender system has been used historically to determine malignant potential in canine anterior uveal melanocytic neoplasms.^158,254 This system was designed to predict the behavior of ocular melanoma in humans,³⁷ which led to criticisms of its use in the veterinary literature for two main reasons. Firstly, it was designed for malignant tumors in humans, and most canine intraocular melanocytic tumors are benign.^234,254 Secondly, the morphology of the canine neoplastic melanocytes does not match the cell descriptions in the Callender classification.^35,158,254 Several authors favor a simpler system, dividing intraocular melanocytic tumors into benign and malignant using well-recognized cytologic features of malignancy as a basis for this classification.^158,254 Despite these controversies, some useful morphologic features can be used to ascribe malignancy, especially with canine anterior uveal melanoma. A greater risk of metastasis tends to occur in mixed and epithelioid cell types.²⁰⁶ Melanocytic neoplasms containing narrow spindle-shaped cells with small nuclei and no mitotic figures, or plump cells with large amounts of melanin and small round nuclei, are more likely to be benign.⁸¹ This is in contrast to the cat where cellular pleomorphism, degree of pigmentation, nuclear to cytoplasmic ratio, and number of nucleoli bear no relationship to the metastatic behavior of uveal melanocytic tumors.⁶⁸

Tumor size,^{26,80,105,200} degree of pigmentation, intensity of proliferating cell nuclear antigen (PCNA)–staining,²⁰⁰ the presence of necrosis, ulceration, or inflammation,^26,80,254 and p53 expression²⁰¹ are of limited prognostic value in animals. Depth of tumor invasion into the skin is an important criterion in human medicine but is felt to be too impractical in animals.⁶⁹ More sophisticated techniques, such as flow cytometry to detect chromosomal anomalies, offer no real advantage over histology in predicting tumor behavior.^25,112 The value of determination of a proliferative index using MIB-1, a monoclonal antibody to Ki-67, which identifies proliferating cells, has been investigated. One study examined 27 canine and feline melanocytic tumors, eight benign and 19 malignant, to correlate Ki-67 and PCNA activity with 6-month survival.²⁰⁰ Ki-67 activity correlated very well with poor survival but so did the presence of invasive growth and classification based on cytologic criteria. PCNA was significantly higher in malignant neoplasms, but levels did not demonstrate a strong correlation with poor survival. In a separate study, MIB-1 was applied to 68 cutaneous canine melanocytic neoplasms to determine if the level of immunoreactivity would correlate with 2-year survival. Eighteen of 68 tumors were classed as malignant histologically. The predictive value of the Ki-67 index for 2-year survival was 97%, which was only slightly higher than the predictive value associated with histologic evaluation (91%).¹³³

Differential diagnosis

Other tumors can look clinically very similar to melanoma, particularly those arising from the skin. These include melanocytoma as well as pigmented lesions of the epidermis and adnexa, e.g., feline basal cell tumors and carcinomas, trichoblastoma, trichoepithelioma, pilomatricoma, sebaceous, and apocrine neoplasms. Ceruminous cysts in the feline may be mistaken for multicentric melanoma of the pinna. Melanocytic hyperplasia (lentigo simplex) on the lips, eyelids, nose, and gingiva of orange, cream, and silver cats appear as pigmented macules at these sites. Hyperpigmented macular lesions clinically resembling melanoma may occur in the skin in dogs, primarily the abdomen and nipple. Similarly, epidermal hamartomas (pigmented epidermal nevi, canine seborrheic keratosis), and dermal hemangioma and hemangiosarcoma can appear as pigmented cutaneous tumors.^87,88

Nonmelanocytic neoplasms that commonly arise in the oral cavity and eye seldom present clinically as pigmented tumors; therefore, pigmentation is not a distraction.

	Cytopathologic Variants

Top Abstract Introduction Pathogenesis Clinical Features Gross Pathology Histopathologic Variants Cytopathologic Variants Additional Diagnostic Techniques Future Diagnostics References

 
Cytomorphology tends to reflect histologic findings, in that cells can be typed as epithelioid (Figs. 8b, 9b, 10b, 12b), round (Fig. 16), or spindle form (Figs. 11b, 17a–d),^{9,79,104,238,255} with agreement reported to be 78% in one study.⁴¹ Although, just as in histologic classification, cytomorphology is highly variable, a few generalizations can be made.^76,239 Nuclei tend to be central to eccentrically located, round to oval, with moderately dense to very fine chromatin and prominent round, oval, or angular nucleoli. Giant nucleoli are a common finding. Nuclei are usually solitary, but occasional binucleated and multinucleated forms are possible. Although not unique to melanoma, longitudinal folding of the nuclear membrane has been reported as a feature in oval nuclear forms stained with Papanicolaou.²⁰⁴ Folding has not been reported in veterinary literature; however, Papanicolaou staining is rarely used in veterinary clinical laboratories. Nuclear indentation or clefting can be seen. Nuclear to cytoplasmic ratios are moderately high to very high. Melanomas do not usually incite a vigorous acute inflammatory response, such as is seen with SCC, unless there is superficial ulceration of the tumor. It is more typical to detect chronic inflammation characterized by few to moderate numbers of melanophages. In cases with visceral metastasis, melanophages may also be observed in the peripheral blood and malignant effusions.²²⁸

View larger version (143K): [in this window] [in a new window]   Fig. 16. Cytology. Metastatic melanoma. Mandibular LN; canine. The nonadherent cells are round to very slightly spindle form, with very high nuclear to cytoplasmic ratios, round nuclei, multiple medium-sized nucleoli, and a small amount of light gray to gray-blue cytoplasm. Very rare fine blue-black pigment granules were occasionally detected. The determination of melanocytic derivation was helped by knowledge of the previous biopsy report of an oral melanoma and current physical examination findings, which revealed mandibular lymphadenopathy on same side. Wright-Giemsa. Bar = 20 µm.

View larger version (140K): [in this window] [in a new window]   Fig. 17a–d. Metastatic spindle cell melanoma. (a) Liver; canine and (b–d) jugular furrow; canine. The primary melanoma was oral. Fig. 17a. Histology. Liver–at necropsy. The pleomorphic metastatic cells are predominantly spindle shaped; however, the primary oral mass, as well as an earlier site of metastasis in the mandibular LN, was epithelioid (see Fig. 21). HE. Bar = 30 µm. Fig. 17b–d. Cytology. Jugular furrow–ante mortem. The cytologic interpretation was spindle cell tumor. Determination of melanocytic derivation was delayed until necropsy examination. There was no evidence of cytoplasmic pigmentation. Fig. 17c, d are enlarged details from Fig. 17b. Cells display marked anisokaryosis, with oval nuclei, fine chromatin, faint nucleoli, and occasional binucleation and nuclear clefting. Wright-Giemsa. Figure 17b: Bar = 50 µm. Figure 17c: Bar = 20 µm. Figure 17d: Bar = 10 µm.

Just as in histologic evaluations, distinguishing benign from malignant can be difficult. Using the number of mitotic figures per high-power field does not work well for cytologic specimens because mitotic figures may not preserve well enough to identify, and the number of cells per high-power field is not uniform. Identification of atypical mitotic figures is also difficult unless they are very bizarre because smearing can create chromosomal artifacts that mimic atypia. Generally, the standard cytologic criteria for malignancy is applied, i.e., marked anisocytosis and anisokaryosis, large to giant nucleoli, atypical nucleoli, and marked pleomorphism. Cytologically benign tumors should be assessed histologically to determine malignant potential, unless of course the cells are detected in an inappropriate location, e.g., LN.

Cytologic determination of specific melanocytic derivation can be problematic in amelanotic or poorly pigmented tumors because of the tendency to mimic other types of neoplasms. Cell size and general morphologic features of the round cell variant can be similar to b