Chromatophoromas in Reptiles

Chromatophoromas are neoplasms that arise from pigment cells of reptiles, amphibians, and fish. They include melanophoromas (melanomas), iridophoromas, and xanthophoromas. Most chromatophoromas develop spontaneously, but genetic and environmental factors may also play a role in their oncogenesis. The diagnosis is typically through histologic examination. Immunohistochemistry and electron microscopy can be helpful for diagnosing poorly differentiated and/or poorly pigmented neoplasms. Aggressive surgical excision is the current treatment of choice. This review describes the clinical presentation, gross appearance, diagnostic applications, clinical behavior, and treatment of chromatophoromas in reptiles.


Background
The color of the skin of animals is determined by the amount of pigment within the epidermis and/or dermis that is produced by pigment cells. In mammals and birds, melanocytes are the sole type of pigment cell [1,2], whereas reptiles, amphibians, and fish possess a variety of pigment cells collectively termed chromatophores [2,3]. Chromatophores include melanophores (or melanocytes), iridiophores, leukophores, cyanophores, xanthophores, and erythrophores [1][2][3]. The latter two are often described collectively as xanthophores [4]. Melanophores, iridophores, and xanthophores have been described in reptiles [1][2][3]. Chromatophores in reptiles are typically localized in the dermis, which contrasts with mammalian epidermal melanocytes. Few reptile species are reported to also possess epidermal melanocytes [5]. Chromatophores or chromatophore-like cells (not melanomacrophages) are also commonly encountered in the coelom, especially in the parietal coelomic membrane of lizards, and scattered through the lung and liver [6]. The different types of pigment cells account for the wide variety of coloration observed in reptilian species and the ability, in some, to undergo rapid color changes [2,3]. These color adaptions aid in camouflage, communication, and sexual selection in these species [4].
Similar to mammalian melanocytes, chromatophores share a common stem cell of neural crest origin [6]. This stem cell migrates to the dermis during embryonic development and then further differentiates into the various chromatophores distributed throughout the dermis [6]. During differentiation, the membrane bound pigment organelles develop from the endoplasmic reticulum or vesicles from the Golgi apparatus that differ based on the respective types of chromatophores [6].
Chromatophores are divided into light-absorbing, pigment-producing chromatophores (melanophores and xanthophores) and structural or light-reflecting chromatophores (iridophores) with different genes involved in the development and differentiation of these two functionally different classes of chromatophores [7]. Melanophores contain melanincontaining melanosomes. Melanin, of the tyrosine-derived class of pigments, absorbs
There are a few reports of primary oral chromatophoromas [8,9,17], a single report of a primary pulmonary melanophoroma in a beaded lizard (Heloderma horridum exasperatum) [27], a single report of a primary small intestinal chromatophoroma in a green tree python (Chondropython viridis) [28], and a single report of a primary iris melanoma in a gecko (unspecified) [11] (Figure 1f, different animal).

Etiology of Chromatophoroma in Reptiles
The vast majority of reptilian chromatophoromas seem to develop spontaneously and no specific etiology has been identified. However, few mechanisms of oncogenesis have been investigated in some species.

Etiology of Chromatophoroma in Reptiles
The vast majority of reptilian chromatophoromas seem to develop spontaneously and no specific etiology has been identified. However, few mechanisms of oncogenesis have been investigated in some species.

UV Radiation
Artificial ultraviolet (UV) radiation, such as in solariums or tanning beds, has been associated with an increased incidence of cutaneous melanoma in humans [29]. In most reptiles, exposure to adequate natural or artificial UV light is essential for the synthesis of vitamin D3 and calcium metabolism [30,31]. Therefore, given that many captive reptiles are exposed to artificial UV light daily, it has been speculated that UV radiation may contribute to the development of cutaneous chromatophoromas [8,14]. This theory is supported by the increased incidence of cutaneous chromatophoromas in sun-loving, day active reptiles, such as bearded dragons (Pogona sp) and the paucity of tumors in nocturnal reptiles [8,9,13]. This theory may be further supported by a series of wild-caught elapid snakes in Australia that developed cutaneous chromatophoromas on the lateral and dorsal body surfaces [24]. However, in one retrospective study of artificial UV light exposure in various reptiles with cutaneous chromatophoromas, neoplasms were reported in several animals that did not receive artificial UV light as part of their husbandry [8]. Interestingly, a review of the archives at Northwest ZooPath (private veterinary diagnostic laboratory, Monroe, WA, USA) identified 87% (52/60 where the anatomical location was noted, appendages were not included) of tumors occurred on the dorsum or lateral aspects of the body, while only 14% (8/60) occurred on the ventrum, periocular region, or in the eye. Primary visceral chromatophoromas were rare and included tumors arising from one of each of the following tissues/organs: lung, kidney, epicardium, and colon.

Genetics
A genetic predisposition to developing cutaneous and oral iridophoromas has been postulated in the "lemon frost" color variant of the leopard gecko (Eublepharis macularius) [25,32]. This population is highly inbred due to selective breeding for unique color variations, and has a high incidence of iridophoromas [25,32] (Figure 2a-d). Guo et al., reported that 80% of the homozygous leopard geckos (from a large captive breeding colony) that had the lemon frost (lf) allele developed iridophoromas between 6 months to 5 years of age [25]. They localized the mutation to a single locus containing a strong candidate gene, serine peptidase inhibitor, Kunitz type 1 (SPINT1) [25]. SPINT1 is a tumor suppressor gene that has been implicated in the development of human cutaneous melanoma and unregulated proliferation of epithelial cells in mice and zebrafish [33,34]. It was concluded that a defect in SPINT1 could be leading to excessive proliferation and neoplastic transformation of iridophores within this inbred population of leopard geckos [25].

Mutations in Cancer Genes
Korabiowska et al. investigated the expression of DNA mismatch repair genes and growth arrest DNA damage genes in a non-specified cutaneous chromatophoroma in a jumping viper (Atropoides picadoi) and cutaneous melanoma in a bullsnake (Pituophis catenifer sayi) [35]. They found mutations in the growth arrest DNA damage gene GADD34 and mutations in the DNA mismatch repair gene MLH1 in both cases, and mutations in the DNA mismatch repair gene MSH2 in the melanoma [35]. It is unclear whether these mutations represent potential driver mutations of carcinogenesis.

Mutations in Cancer Genes
Korabiowska et al. investigated the expression of DNA mismatch repair genes and growth arrest DNA damage genes in a non-specified cutaneous chromatophoroma in a jumping viper (Atropoides picadoi) and cutaneous melanoma in a bullsnake (Pituophis catenifer sayi) [35]. They found mutations in the growth arrest DNA damage gene GADD34 and mutations in the DNA mismatch repair gene MLH1 in both cases, and mutations in the DNA mismatch repair gene MSH2 in the melanoma [35]. It is unclear whether these mutations represent potential driver mutations of carcinogenesis.
Cytologic examination can be challenging or misleading due to potential aspiration of normal chromatophores in the dermis or macrophages containing phagocytized granulocyte pigment, nuclear debris, or pigment granules from lysed chromatophores in an inflammatory lesion, which can have a similar cytologic appearance to chromatophoromas. Therefore, cytologic cases suspicious for chromatophoroma should always be confirmed by histologic examination. [20,23,27] (Figure 3a,b).
Cytologic examination can be challenging or misleading due to potential aspiration of normal chromatophores in the dermis or macrophages containing phagocytized granulocyte pigment, nuclear debris, or pigment granules from lysed chromatophores in an inflammatory lesion, which can have a similar cytologic appearance to chromatophoromas. Therefore, cytologic cases suspicious for chromatophoroma should always be confirmed by histologic examination.

Histology
Cutaneous chromatophoromas are primarily dermal, but can extend into the subcutis, underlying skeletal muscle, or vertebral column [9,12,13,24,38,39]. Malignant tumors tend to have an infiltrative growth pattern [9,12,13,32,40], but ultimate diagnosis is often based on metastatic spread. In two large case series of chromatophoromas in lizards and snakes (that included 26 and 42 cases, respectively), cases with substantial anaplasia (anisokaryosis and anisocytosis, nuclear atypia, and high mitotic counts) were associated with an increased likelihood of recurrence and/or metastasis in various species [9,12]. It should be noted that histologically benign appearing neoplasms (well-pigmented, mild anisokaryosis and anisocytosis, low mitotic counts) also were found to be metastatic in a few cases; so, presence, but not absence, of lymphatic invasion is a reliable indicator of more aggressive behavior [9,12]. Neoplastic cells are typically spindloid, less commonly epithelioid or polygonal, and with some frequency show a mixture of spindloid and polygonal cells [9,12,13]. Iridophoromas contain fine, light brown, or olive green cytoplasmic pigment that is birefringent under polarized light [9,12,13] (Figure 4a,b and Figure 5a,b). Xanthophoromas often contain red, brown, yellow, or orange cytoplasmic pigment [9,12] ( Figure 4c). Melanophoromas contain fine, brown, or black cytoplasmic pigment [9,12,13] (Figure 4d,e and Figure 5c-e). Mixed chromatophoromas are composed of a mixture of the previously described chromatophores [9,12,13]. Mixed melanophoroma-iridophoromas are most common [9,12,13], but an individual case of a mixed iridophoroma-xanthophoroma has been reported in a veiled chameleon (Chamaeleo calyptratus) [16]. The degree of pigmentation in chromatophoromas is usually high [9,12,13]. In most tumors, there is at least some degree of pigmentation [9,12,13]. Anisokaryosis and anisocytosis are often mild to moderate [9,12,13]. The mitotic count is typically low (0-2 mitotic figures in 10 high power fields) [9,12,13]. A subset of the bearded dragon (Pogona sp.) chromatophoromas and a single melanophoroma in a northern red-bellied cooter (Pseudemys rubriventris) were diagnosed as mucinous chromatophoromas based on the presence of abundant Periodic acid-Schiff positive, mucinous stroma within the neoplasm [9,13,19] (Figure 4e). Ulceration of the overlying surface is not uncommon in chromatophoromas (29 out of 208 cases, when reported in the literature) due to their raised cutaneous appearance [12,13,18,[22][23][24]36,41,42]. Intraepithelial neoplastic cells are only described in a small number of snake cases, which most likely represent epithelial invasion rather than junctional activity, as chromatophores do not reside in the epidermis in most reptiles [9,24,43]. Lymphatic invasion is uncommonly observed (7 out of 208 cases, when reported in the literature), even in cases that had widespread metastasis [9,12,20,21]. Neoplastic cells at metastatic sites typically have a similar histologic appearance as the primary neoplasm [9,12,17,19,37] (Figure 5a-e).   In reptiles, the term chromatophoroma is applied to a number of pigmented neoplasms, but it is a non-specific in regard to histogenesis and biological behavior. It is therefore important to further characterize the type of chromatophoroma (e.g., melanophoroma, iridophoroma, or xanthophoroma) based on the histologic features of the neoplastic cells when possible. This will help with elucidating potential clinical and behavior differences between the subtypes of chromatophoromas. The modifier "malignant" should be reserved for chromatophoromas (or their subtypes) that have evidence of lymphatic invasion or metastasis.

Immunohistochemistry
Most chromatophoromas have some degree of pigmentation within the mass that allows confirmation of chromatophore origin by histologic examination. Immunohistochemistry (IHC) can be helpful to diagnose amelanotic or non-pigmented chromatophoromas [13,17]. Reported IHC results conflict depending on the study, but overall, anti-

Family: Gekkonidae
An iris melanoma has been described in a generic gecko [11]. A search for "chromatophoromas, melanoma, melanophoromas, iridophoromas, and xanthophoromas" of the archives of Northwest ZooPath revealed chromatophoromas in these additional species: leaf tailed gecko (Uroplatus phantasticus) and tokay gecko (Gekko gecko).

Families: Diplodacylidae, Corytophanidae, Dactyloidae
No previously reported cases of chromatophoromas were found for Diplodacylidae, Corytophanidae, or Dactyloidae; however, a search for "chromatophoromas, melanoma, melanophoromas, iridophoromas, and xanthophoromas" of the archives of Northwest ZooPath revealed chromatophoromas in these additional species: giant new Caledonian gecko (Rhacodactylus leachianus), basilisk (Basiliscus basiliscus), and orient knight anole (Anolis equestris). The giant new Caledonian gecko was diagnosed with a dermal chromatophoroma with no report of metastasis. The basilisk was diagnosed with a malignant melanoma of the skin with metastasis to the liver and coelom. One orient knight anole was diagnosed with a colonic melanoma with no report of metastasis, and another was diagnosed with an amelanotic melanoma of the soft tissues of the leg with no report of metastasis.

Organs Affected by Metastases Citation
Great plains rat snake (Pantherophis emoryi)

Clinical Staging and Treatment
As in other species, clinical staging is helpful to assess the extent of neoplastic disease, predict prognosis, and form the best therapeutic plan for the patient. Additional clinical tests often include complete blood count (CBC) and blood chemistry, radiographs, ultrasound, computed tomography, or magnetic resonance imaging (MRI) [57]. The results of clinical staging were mentioned only in a small subset of reports of chromatophoromas in reptiles (7 cases out of 208) [21,23,32,36,45]. It was reported to be helpful to access the extent of neoplastic disease in these cases [21,23,32,36,45].
Surgical excision is the most common treatment for chromatophoromas in reptiles [9,12,13]. Complete surgical excision is curative in some cases, but the results of clinical staging can be helpful to determine if surgical excision is likely to be curative [9,12,13,32,36,45]. Complete surgical excision appeared to be curative in 63 out of 208 cases, though it should be noted that loss to follow up or lack of complete postmortem examination may falsely alter these numbers [9,12,13,32,36,45]. Complete surgical excision can be difficult to obtain in some cases due to the infiltrative growth pattern common to reptile chromatophoromas [9,12,13,32,40].
There are a few reported cases of reptile chromatophoromas treated with radiation with variable success. A malignant melanophoroma in a Hermann's tortoise (Testudo hermanmi) was treated with photon standing field irradiation (6 MV); three treatments of 9 Gy once per week using a linear accelerator (total dose: 27 Gy) after incomplete surgical excision [20]. The tortoise's condition declined 2 weeks after the last radiation treatment [20]. A melanoma in a common death adder (Acanthophis antarcticus) was treated with radioactive gold implants, but therapy was reported to be unsuccessful [41]. A melanin-producing malignant chromatophoroma in a yellow rat snake (Elaphe obsoleta quadrivittata) was treated with radiation therapy (6 MV electron beam); four equivalent fractions over a 15-day period (total 6000 cGy) after incomplete surgical excision [38]. The scales in the irradiated area became discolored and roughened, but the surgery site healed uneventfully [38]. The snake was irritable for several months and anorexic for 20 weeks following radiation therapy [38]. The snake died 10-months later and necropsy revealed a mass in the coelomic cavity with similar gross features to the previously removed cutaneous chromatophoroma [38]. No histologic examination was performed to confirm the identity of this mass [38].
In mammals, additional treatment options for melanomas include chemotherapy and melanoma vaccines [58]. Unfortunately, little is known about chemotherapy levels, dosages, and effects in reptiles for even the most common neoplasms, so this is still an area that needs investigation [57].

Conclusions
Chromatophoromas are a relatively common, primarily cutaneous neoplasm in reptiles, with an increasing number of reports in recent years [8,9,12,13]. The increased incidence and apparent species predispositions may be due in part to varying popularity of certain species as pets and exhibit animals, closer monitoring by caretakers, and advancements in veterinary medicine rather than true increased incidence. Some of the larger cases series originated from a small number of pathology groups, so overlapping and therefore over reporting of individual cases and species may have occurred.
Cutaneous chromatophoromas most commonly present as pigmented masses, but for clinicians it is also important to note that they can appear as flat, pigmented or nonpigmented scales [8,9,12,13]. Most chromatophoromas develop spontaneously, but UV (especially artificial UV) radiation may play a role in some cases, especially sun-loving reptiles such as bearded dragons [8]. A genetic component has also been described in an inbred population of leopard geckos [25]. Mutations in tumor suppressor genes most likely play an important role in chromatophoroma oncogenesis in reptiles, just like in mammals [25,35].
Cytologic examination can be helpful to diagnose chromatophoromas [19][20][21]23,27,36,37]. However, histologic examination is required for a definitive diagnosis and allows for assessment of malignant features and margin evaluation. While histologic examination is typically adequate to reach a diagnosis, IHC can be helpful for diagnosing poorly pigmented or amelanotic cases [13,17]. Reports of IHC effectiveness varies among studies and species, emphasizing the need for validation of IHC markers in reptilian species [9,12,13,16,17,[39][40][41][42]44,45]. Transmission electron microscopy can be used to definitely identify the chromatophore type based on visualization of pigment structures or platelets. This technique is typically not needed to achieve a definitive diagnosis, but can be used for poorly differentiated chromatophoromas [16]. Regardless, similar to amelanotic melanomas in mammals, ultrastructural identification of early-stage melanosomes can be challenging.
The primary therapy is complete surgical excision [9,12,13]. This is curative for some, but given the infiltrative growth pattern and metastatic potential of chromatophoromas in multiple reptile species, surgical excision may not be adequate to control neoplastic spread [9,12,13,32,40]. Cases with substantial anaplasia (anisokaryosis and anisocytosis, nuclear atypia, and high mitotic counts) have been associated with an increased likelihood of recurrence and/or metastasis in some species [9,12]. Absence of lymphatic invasion does not appear to be a reliable indicator of benign behavior [9,12,20,21]. Monitoring and clinical staging is especially important in these cases. Other treatment modalities have not been well studied in reptiles.