melanophoromas and iridophoromas in reptiles - · pdf filemelanophoromas and iridophoromas in...

J. Comp . Path. 2011, Vol. 8, 1e 11

www.elsevier.com/locate/jcpa

DISEASE IN WILDLIFE OR EXOTIC SPECIES

Melanophoromas and Iridophoromas in Reptiles

Cor

002

doi

Pl

do

K. O. Heckers*, H. Aupperle*, V. Schmidt† and M. Pees†

*LABOKLIN, Labor f€ur klinische Diagnostik, Steubenstr. 4, 97688 Bad Kissingen and †Klinik f€ur V€ogel und Reptilien,Veterin€armedizinische Fakult€at, An den Tierkliniken 17, 04103 Leipzig, Germany

resp

1-99

:10.1

ease

i:10

Summary

Chromatophoromas are tumours of pigment-producing cells of the skin and are rarely reported in reptiles.These tumours are subclassified on the basis of the type of pigment. The present study characterizes chroma-tophoromas arising in 26 reptiles, including six snakes, 19 lizards and a tortoise. These include the first reportsof melanophoromas in a yellow anaconda (Eunectes notaeus), pigmy rattlesnake (Sistrurus spp.), southern watersnake (Nerodia fasciata), veiled chameleon (Chamaeleo calyptratus) and leopard gecko (Eublepharis macularius); thefirst reports of benign iridophoromas in a savannah monitor (Varanus exanthematicus), veiled chameleon andbearded dragon (Pogona vitticeps); and the first description of a malignant iridophoroma in a bearded dragon.Additionally, in three bearded dragons a ‘mucinous’ type of melanophoroma is described for the first time.Chromatophoromas generally arose from the skin of the body and head and ranged in size from 0.2 to2.0 cm in diameter. In six cases the animals were humanely destroyed immediately after diagnosis. Three fur-ther animals were humanely destroyed following recurrence of their tumour. Six of these nine reptiles had vis-ceral metastases. Grossly, melanophoromas (n¼ 20) were grey or black, while iridophoromas (n¼ 6) werewhite in colour. Microscopically, most of the tumours were composed of spindle cells with varying pigmenta-tion and 0e2 mitoses per 10 high power fields. Six of the 20 melanophoromas were classified as malignant dueto the presence of intravascular tumour cells, visceral metastases, high pleomorphism and/or mitotic figures.Five of the six iridophoromas were classified as benign and the one malignant tumour was defined by the pres-ence of intravascular tumour cells and visceral metastases. Immunohistochemically, melan A and S100 werecoexpressed by all of the chromatophoromas.

� 2011 Elsevier Ltd. All rights reserved.

Keywords: chromatophoroma; iridophoroma; melanophoroma; reptile

Introduction

Three types of pigment cells (chromatophores) occurwithin the integument of reptiles.

Tumours of pigment cells are generally referred toas chromatophoromas and are classified according totheir specific chromatophores as: (1) melanophoro-mas or melanomas (melanin-producing cells), (2)xanthophoromas (carotenoid or pteridine-producingcells) or (3) iridophoromas (crystalline purine-producing cells containing reflecting granules of gua-nine, adenine, hypoxanthine or uric acid) (Bagnara,1966; Bagnara and Hadley, 1973; Rohrlich, 1974;Bagnara et al., 1979; Gopalakrishnakone, 1986;Morrison, 1995).

ondence to: K. O. Heckers (e-mail: [email protected]).

75/$ - see front matter

016/j.jcpa.2011.07.003

cite this article in press as: Heckers KO, et al., Melanophoromas and

.1016/j.jcpa.2011.07.003

In general, melanophoromas are considered raretumours of reptiles (Korabiowska et al., 1997, 1998).In a study of 1,941 reptiles, Sinn (2004) found one mel-anophoroma in a snake, while Ippen and Schr€oder(1977) found no chromatophoromas in 5,000 reptilepost-mortem examinations. Most reports of chromato-phoromas have involved snakes such as colubrids, par-ticularly the San Francisco garter snake (Thamnophis

sirtalis tetrataenia), and in animals of the families Crotali-dae, Boidae and Viperidae (Schlumberger and Lucke,1948; Frye et al., 1975; Ryan et al., 1981; Ramsay andFowler, 1992; Gregory et al., 1997; Garner et al., 2004;Suedmeyer et al., 2007). Metastases have beenreported in several cases of melanophoromas (Ball,1946; Elkan, 1974; Garner et al., 2004). Few cases ofmelanophoromas are described in lizards (Mikaelianet al., 2000; Johnson, 2003; Garner et al., 2004;

� 2011 Elsevier Ltd. All rights reserved.

Iridophoromas in Reptiles, Journal of Comparative Pathology (2011),

mailto:[email protected]

http://dx.doi.org/10.1016/j.jcpa.2011.07.003



http://www.elsevier.com/locate/jcpa

Table 1

Frequency of chromatophoromas in the archive of

reptilian tumours

Species Number of

tumours

Chromatophoromas Melanophoromas Iridophoromas

Lizards 105 20.0% 13.7% 6.3%

Bearded

dragons

51 29.4% 21.6% 7.8%

Snakes 61 9.8% 9.8% eTortoises and

turtles

13 7.6% 7.6% e

Total number 179 14.5% 11.2% 3.3%

2 K.O. Heckers et al.

Irizarry-Rovira et al., 2006; Simpson, 2008). In tortoisesthere is one report of a malignant melanophoroma ofthe carapace in a Hermann’s tortoise (Testudo

hermanni) (Heckers et al., 2011). Ametastatic iridophor-oma is described in a pine snake (Pituophis melanoleucus;Jacobson et al., 1989) and one malignant iridophoromais reported in a boa constrictor (Boa constrictor; AFIP,2000). One publication reports a xanthophoroma ina canebrake rattlesnake (Crotalus horridus atricaudatus;Gregory et al., 1997).

The diagnosis of amelanotic melanomas in mam-mals is often based on immunohistochemical detec-tion of melan A and S100 protein (Glage, 2001;Ramos-Vara et al., 2002). Melan A is expressed inmelanocytes and in melanoma cells (Glage, 2001),while S100 protein is expressed in numerous cell typesincluding glial cells and ependymal cells of the centralnervous system, glial cells of peripheral nerves(Schwann cells), melanocytes and melanocytictumour cells (Orchard and Wilson, 1994). Immuno-histochemical characterization of reptilian chromato-phoromas has rarely been reported. Irizarry-Roviraet al. (2006) were the first to report detection of melanA in reptiles in amelanophoroma from a green iguana(Iguana iguana). Reptilian melanophoromas do not al-ways show expression of S100 (Gregory et al., 1997;Korabiowska et al., 1997; Kusewitt et al., 1997).

Although several case reports of chromatophoro-mas in reptiles are available, comparative pathologi-cal studies of a larger number of cases do not exist.Therefore, the aim of the present study was to charac-terize and compare the clinical findings and the gross,microscopical and immunohistochemical features ofchromatophoromas in 26 reptiles.

Materials and Methods

Animals

Twenty-six chromatophoromas were selected for anal-ysis from an archive of 179 reptilian tumours collectedover 9 years. The chromatophoromas arose in a num-ber of different species of varying age (Table 1). Ineach case the owners of the animals had observedskinmasses that had been present for a period of severalweeks.

In bearded dragons the chromatophoromas werelocated in the skin of the shoulder/thorax (n¼ 4), thehead (n¼ 3), tail (n¼ 2), leg (n¼ 1) and in the oralcavity (n¼ 2). The origin was not documented inthree cases. In other reptiles (snakes, chameleonsand a savannah monitor) the chromatophoromaswere located in the skin of the thorax (n¼ 6), abdomen(n¼ 3) and head (n¼ 1) or in the oral cavity (n¼ 1).

Most chromatophoromas were excised surgically(n¼ 23), fixed in 4%buffered formalin and submitted

Please cite this article in press as: Heckers KO, et al., Melanophoromas and

doi:10.1016/j.jcpa.2011.07.003

for histopathological investigation. Six animals werehumanely destroyed at various times (1e635 days)after histological diagnosis and submitted for nec-ropsy examination. These included a yellow ana-conda (Eunectes notaeus), a bearded dragon (Pogonavitticeps), a veiled chameleon (Chamaeleo calyptratus),a leopard gecko (Eublepharis macularius), a Hermann’stortoise and a pigmy rattlesnake (Sistrurus spp.).Three other reptiles were submitted for necropsyexamination without previous histological investiga-tion. These included a garter snake with small pig-mented dermal tumours near to the eye and on thedorsum, a bearded dragon with a largenon-excisable grey mass with some dark regions onthe ventral abdomen and a bearded dragon witha non-excisable, ulcerated white mass ventral to thebase of the tail (Table 2). During necropsy examina-tion the animals were inspected in detail and repre-sentative samples were fixed in formalin for furtherexamination.

Histopathology

The histological examinations were performed by twoveterinary pathologists according to a standard pro-tocol. Samples were embedded in paraffin wax andstained with haematoxylin and eosin (HE). PeriodicacideSchiff (PAS) staining was also performed forthe mucinous tumours. If necessary, sections ofheavily pigmented melanophoromas were bleachedin H2O2 10%. Polarized light was used to visualizebirefringence, which is characteristic of iridophores(Jacobson et al., 1989; Irizarry-Rovira et al., 2006)and this examination was used to differentiatebetween melanophoromas and iridophoromas.

Mitotic figures and nucleoli were counted in 10high power fields (�40 microscope objective) in ran-domly selected areas and the mean numbers were cal-culated. The degree of pigmentation was gradedaccording to a modification of the system describedby Smedley et al. (2011) as amelanotic (lack of pig-ment granules or only single cells containing very


Table 2

Details of reptiles included in the study

Species Number of animals Sex (n¼) Age (years) Type of chromatophoroma (n¼)

Bearded dragon (Pogona vitticeps) 15 Female (6)

Male (3)Unknown (6)

Mean 3.7

(range 2e7)

Melanophoroma (11)

Iridophoroma (4)

Veiled chameleon (Chamaeleo calyptratus) 2 Male (2) 3

1

Melanophoroma

Iridophoroma

Leopard gecko (Eublepharis macularius) 1 Female 10 MelanophoromaSavannah monitor (Varanus exanthematicus) 1 Unknown 5 Iridophoroma

Garter snake (Thamnophis sirtalis) 2 Female

Unknown

Unknown

7

Melanophoroma

Yellow anaconda (Eunectes notaeus) 1 Female 17 Melanophoroma

Pigmy rattlesnake (Sistrurus spp.) 1 Female 3 Melanophoroma

Boa constrictor (Boa constrictor) 1 Female 13 Melanophoroma

Southern water snake (Nerodia fasciata) 1 Male 11 MelanophoromaHermann’s tortoise (Testudo hermanni) 1 Female 13 Melanophoroma

Melanophoromas and Iridophoromas in Reptiles 3

few granules), mild (some or most cells with mild pig-mentation), moderate (most cells are moderately pig-mented), marked (most cells are intensely pigmented)or dispersed.

Index number LI Value

1 none 0

2 weak 1

3 moderate 2

4 strong 3

Immunohistochemistry

The expression of melan A and S100 protein wasdetected by the streptavidinebiotin immunoperoxi-dase technique. Intensively pigmented melanophoro-mas were bleached in order to evaluate theimmunohistochemistry (IHC). To avoid reductionof labelling intensity through the bleaching process,mildly pigmented melanophoromas were examinedwith and without bleaching. As a control for themelan A and S100 markers the expression of thesemolecules in three fibrosarcomas, a myxoma, a myxo-sarcoma and a squamous cell carcinoma from differ-ent reptile species was investigated.

Citrate buffer (pH6.0) pretreatment (30 min, 96�C)was required for the detection of both antigens. An ini-tial blocking stepwith 20%goat serum inTris-bufferedsaline (TBS; 50 mM, pH 7.6) for 20 min at room tem-perature was performed. All antisera were diluted inTBS. Mouse monoclonal anti-human melan A (1 in50; DAKO GmbH, Hamburg, Germany) was incu-bated with the sections for 24 h at room temperature.For this reaction the secondary antibody was ratanti-mouse IgG (1 in 250;Vector Laboratories, Burlin-game, California,USA) incubatedwith the sections for30 min at room temperature. Rabbit anti-S100 (1 in400; DAKO GmbH) was incubated with the sectionsfor 24 h at room temperature. The secondary antibodyfor this reaction was goat anti-rabbit IgG (1 in 800;DAKO Cytomation, Glostrup, Denmark) incubatedwith the sections for 30 min at room temperature. Avi-dinebiotineperoxidase complex (horseradish peroxi-


doi:10.1016/j.jcpa.2011.07.003

dase streptavidin; 1 in 250; Vector Laboratories) andAEC-substrate (Zytomed Systems, Berlin, Germany)were added subsequently to ‘visualize’ labelling. Slideswere counterstained with Mayer’s haematoxylin (BioOptica, Milan, Italy).

Negative controls were performed by substitutingmouse (DAKO GmbH) or rabbit (DAKO GmbH)control serum for the primary antibodies. A caninemelanoma served as a positive control, as did thelabelling of chromatophores in normal skin adjacentto the tumours.

The labelling intensity was evaluated semiquantita-tively and graded as follows: 0, negative; 1, weak (palered); 2, moderate (red); 3, strong (deep red). Theimmunoreactive score (IRS) (Remmele and Stegner,1987) was calculated according to the following for-mula, which was modified by €Ozgen et al. (1997):

IRS ¼ 1=100X

fPPn �max½4� ðLIn � 1Þ; 1�g4n¼2

where n is the index, PP is the percentage of positivecells, 4¼ 5 is the weight and LI is the labelling inten-sity. The assignment of labelling intensity and LIvalue is:

Statistics

Statistical analyses included the ShapiroeWilk-test,Levene t-test, Fisher’s exact test (Median test) andPearson and Spearman correlation tests. Results were



considered statistically significant where P# 0.05. Allanalyses were performed using SPSS software, version16.0 (SSPS Inc., Chicago, Illinois, USA).

Fig. 1. Dermal melanophoroma in the skin of the thorax of a2-year-old bearded dragon. The mass is 2.0� 1.5� 1.0 cmin size. Photograph courtesy Dr. J. Wiechert.

Results

Twenty of the 26 tumours were diagnosed as melano-phoromas and six tumours with birefringent cytoplas-mic granules were identified as iridophoromas. Six ofthe melanophoromas were classified as malignant dueto the presence of intravascular neoplastic cells, vis-ceral metastases, high pleomorphism and/or the pres-ence of mitotic figures. Most iridophoromas wereclassified as benign (n¼ 5), with only one being con-sideredmalignant due to the presence of intravascularneoplastic cells and neoplastic cells in the viscera.

The age of the affected animals ranged between 1and 17 years (median 5 years; Table 2).

When all 179 reptilian tumours were considered,chromatophoromas (n¼ 26) were the most frequent,followed by adenocarcinomas of different organs(n¼ 23), squamous cell carcinomas (n¼ 18), fibrosar-comas (n¼ 18), lymphomas (n¼ 11), papillomas(n¼ 11), osteochondrosarcomas (n¼ 9) and lipomas(n¼ 9). In lizards, chromatophoromas (n¼ 19) werethe most frequent tumours followed by squamous cellcarcinomas (n¼ 14), fibrosarcomas (n¼ 10), adenocar-cinomas of different organs (n¼ 9), lymphomas (n¼ 6)and papillomas (n¼ 5). The most common tumours ofsnakes were adenocarcinomas of different organs(n¼ 13), fibrosarcomas (n¼ 8), chromatophoromas(n¼ 6), lymphomas (n¼ 6) and osteochondrosarcomas(n¼ 5). In the groupof tortoises and turtles themost fre-quent tumours were papillomas (n¼ 3) and squamouscell carcinomas (n¼ 3).

Fig. 2. Dermal melanophoroma measuring 4.0� 3.0� 2.5 cmfrom a 7-year-old bearded dragon after excision and fixa-tion. This is a mucinous type of melanophoroma showingthe cut surface with colouration from white to brown toblack.

Gross Features of Melanophoromas

Melanophoromas occurred in 11 bearded dragons andnine other reptiles. The melanophoromas ranged be-tween 0.2 and 2.0 cm in diameter with the exceptionof one tumour that measured 4.0� 3.0� 2.5 cm. Thecut surface of themelanophoromas in bearded dragonsafter fixation was grey (n¼ 4), white (n¼ 3), black(n¼ 2) or mixed white and black (n¼ 2). In the otherreptile species the cut surface was black (n¼ 6), grey(n¼ 2) or mixed white and black (n¼ 1) (Fig. 1). Ingeneral, the melanophoromas appeared significantlydarker than the iridophoromas (P¼ 0.004). The con-sistency of the tumours was firm in 17 of 20 cases. Inthree bearded dragons a ‘mucinous’ type of melano-phoroma was found. These had a gelatinous consis-tency and released jelly-like material into theformalin (Fig. 2). In six of the nine necropsy examina-tions visceral metastases were found (Table 3).


doi:10.1016/j.jcpa.2011.07.003

Histopathological Findings of Melanophoromas in Bearded

Dragons

Most melanophoromas (9/11) in bearded dragonswere located in the dermis and infiltrated the deeperdermis and musculature. Infiltration of the epidermiswas not found. Epidermal ulceration was present intwo cases. In two cases, in which melanophoromaswere located in the oral cavity, the tumour cellswere locally invasive. The melanophoromas werecomposed of spindle-shaped cells, which were par-tially arranged in bundles and streams and separatedby fine fibrous septa with a few capillaries.

In seven cases the oval or round nuclei had dis-persed chromatin or were hyperchromatic with mild


Table 3

Summary of microscopical findings of melanophoromas

Species Shape of cells Pleomorphism Mitotic figures/HPF Degree of pigmentation Metastases

Bearded dragon

n¼ 11

Spindle shaped, n¼ 11 Mild, n¼ 4

Moderate, n¼ 6Marked, n ¼1

0, n¼ 4

1, n¼ 62, n¼ 1

Amelanotic, n¼ 2

Mild, n¼ 3Marked, n¼ 1

Dispersed, n¼ 5

Yes

Veiled chameleon Spindle shaped Marked 3 Dispersed Yes

Leopard gecko Epithelioid Marked 11 Dispersed YesGarter snake 1

Garter snake no. 2

Spindle shaped, n¼ 2 Moderate, n¼ 2 0, n¼ 1

1, n¼ 1

Dispersed, n¼ 2 No

Yellow anaconda Spindle shaped Moderate 2 Dispersed YesPigmy rattlesnake Spindle shaped Mild 0 Marked No

Boa constrictor Spindle shaped Moderate 0 Dispersed No

Southern water snake Spindle shaped Mild 0 Marked No

Hermann’s tortoise Spindle shaped Moderate 2 Dispersed Yes


or moderate anisokaryosis. The number of nucleoliwas 0e2. The mean number of mitoses per HPFwas 0e2. Atypical mitotic figures were present infive cases. Neoplastic cells had a moderate amountof cytoplasm, but the degree of pigmentation washighly variable. In most cases amelanotic cells wereinterspersed with nests of markedly pigmented cells(Fig. 3; Table 3).

In one adult bearded dragon with a markedly pig-mented melanophoroma of the face, marked cellularpleomorphism was present. The cells were spindleshaped, heavily pigmented and had hyperchromaticround to oval nuclei with <1 mitosis per HPF.Tumour cell emboli were found in lymphatic vessels,but no necropsy examination was performed in orderto determine if visceral metastases were present.

In the three cases of mucinous tumours, all frombearded dragons, the defining feature was a large

Fig. 3. Dermal melanophoroma from a 5-year-old beardeddragon. The tumour consists of sheets of epithelioid tospindle-shaped cells with large hyperchromatic nuclei,a prominent nucleolus and a variable degree of pigmenta-tion. HE. Bar, 10 mm.


doi:10.1016/j.jcpa.2011.07.003

amount of basophilic, PAS-positive, mucinous inter-stitial matrix. The cells were arranged in interlacingbundles and loose whorls. The cells were mostly ame-lanotic with dispersed foci of moderate to marked pig-mentation. The nuclei had a mildly dispersedchromatin pattern. The mean number of mitoseswas 0e1 per HPF. The growth was invasive into thedermis and musculature in all cases, but tumour cellemboli in lymphatic or blood vessels and metastaseswere not found (Fig. 4).

Birefringence was not exhibited by any of the mel-anophoromas. Mild inflammatory infiltration wasseen in seven tumours. Heterophils were the domi-nant inflammatory cells, but some lymphocytes,plasma cells and histiocytes were also present.

Metastatic melanophoromas had a significantlygreater pleomorphism (P¼ 0.047) than those tu-mours without metastases. The number of nucleoli

Fig. 4. Mucinous type of dermal melanophoroma from a 7-year-old bearded dragon. Loosely-arranged tumour cells liewithin large amounts of basophilic mucinous stroma. HE.Bar, 10 mm.



(P¼ 0.041) and mitoses (P¼ 0.001) was higher thanin the cases without visceral metastases. The degree ofpigmentation was not associated with malignancy(P¼ 0.274).

Histopathological Findings of Melanophoromas in other

Reptile Species

The histomorphological findings in melanophoromasin the other reptile species (n¼ 9)were not significantlydifferent compared with the findings in the beardeddragons. The tumours were located in the dermis (8/9) and infiltrated the deeper dermis and musculature.One melanophoroma was located in the oral cavity(leopard gecko) and showed invasive growth into theinterstitial tissue. Neoplastic infiltration of the epider-mis was found in the yellow anaconda and the pigmyrattlesnake. Epidermal ulceration was present in theboa constrictor and the yellow anaconda. The mor-phology of the neoplastic cells was predominately spin-dle shaped (8/9 cases), but epithelioid in the leopardgecko (Table 3). Tumour cell emboli in lymphaticand/or blood vessels were found in the biopsy of the sec-ond recurrence in the yellow anaconda and in theHer-mann’s tortoise. These tumours were characterized byspindle-shaped cells with hyperchromatic round tooval nuclei. Cells were mostly amelanotic with dis-persed foci of moderate (yellow anaconda) or markedpigmentation (Hermann’s tortoise) and there were1e2 mitoses per HPF. Five melanophoromas showedmild infiltration of heterophils. Additionally, in threecases some lymphocytes, plasma cells and histiocytes,and in the tumour of the yellow anaconda some multi-nucleated giant cells, were observed.

Visceral Metastases of Melanophoromas

Melanophoromas were present in the viscera in addi-tion to the skin in five cases, including three lizards,

Table

Characteristics of metasta

Species Primary location Metas

Bearded dragon Skin

Melanophoroma

Lung, gut, liver, k

Bearded dragon Skin

Iridophoroma

Skin, muscle, heart,

liver, pancreas, k

parietal serosa of

Veiled chameleon 1 SkinMelanophoroma

Heart, lung, tongue,spleen, kidneys, bon

serosa of the coel

Leopard gecko Oral cavityMelanophoroma

Lun

Yellow anaconda Skin

Melanophoroma

Heart, stomach, gut,

uterus, ovar

Hermann’s tortoise CarapaceMelanophoroma

Lung, liver, spleendeferens, adrenal g


doi:10.1016/j.jcpa.2011.07.003

one snake and one tortoise (Table 4) and these meta-static lesions affected 18 different tissues. Neoplasticfoci occurred predominately in the kidney, liver,lung, intestine and abdominal fat bodies as well asin the stomach and heart. In four of five cases multipleorgans were affected. In two cases the dermal tumoursshowed intravascular tumour cells and during nec-ropsy examination, visceral metastases were seen. Inthree cases no tumour cell emboli were found in der-mal neoplasms, but visceral metastases were located.

In general, the morphological features of the meta-static tumours corresponded to that described for thedermal neoplasms, but some differences are notable.In the veiled chameleon and the leopard gecko theprimary tumours showed a higher number of mitoticfigures and a higher degree of cellular atypia than thevisceral metastases. The primary oral tumour of theleopard gecko was amelanotic, while the neoplasticcells in the lungs were moderately pigmented. In theyellow anaconda with recurring tumours the cellulardifferentiation changed from epithelioid (first dermallesion) to spindle shaped (first recurrence in skin, sec-ond recurrence in skin and visceral metastases). Atfirst, the nuclei showed a homogenous chromatin pat-tern that changed to a mildly dispersed and finally toa vacuolated pattern.

Clinical Outcome of Reptiles with Melanophoromas

In five cases there was no recurrence of the cutaneoustumour after surgical removal. The animals weremonitored by the referring veterinarian over a periodof 7 months (last cases) to 7 years. Recurrences wereobserved in three cases: one directly after surgeryand, in the other two, within a period of 7 monthsafter surgery. The animals were subsequentlyhumanely destroyed. In the case of the yellow ana-condawith a dermalmelanophoroma two recurrences

4

tic chromatophoromas

tasis Gross findings in viscera

idneys, fat bodies Dark grey blotches, 0.2e0.4 cm

lung, stomach, gut,

idneys, fat bodies,

the coelomic cavity

Firm, 0.2e1.0 cm white spots and masses

stomach, gut, liver,e, fat bodies, parietal

omic cavity, brain

Black spots 0.2e0.4 cm and diffuse blackcolour of the viscera

g One grey spot, <0.1 cm

pancreas, kidneys,

ies, fat body

Firm, 1.0� 1.0� 0.4 cm mass in the heart,

black spots 0.3e0.6 cm in other organs

, kidneys, ductuslands, thyroid gland

Mild to moderate diffuse swelling of liverand kidneys with black spots, 0.2 cm



occurred within a period of 21 months. After removalof the second recurrent tumour, the snake was hu-manely destroyed due to the poor prognosis. Eightreptiles were humanely destroyed directly after diag-nosis because of poor prognosis and/or poor state ofhealth. Another three reptiles died a few weeks toa few months after diagnosis, one with visceralmetastases (Hermann’s tortoise) and two of secondarydiseases (bearded dragon and pigmy rattlesnake). Infive cases visceral metastases occurred; four of thesepatients were humanely destroyed and one died. Infive cases no follow-up information was available.

Gross Findings in Iridophoromas

Iridophoroma occurred in four bearded dragons, oneveiled chameleon and one savannahmonitor. The iri-dophoromas appeared as single white intradermalnodules. The savannah monitor had one white mass(1.0� 0.5� 0.5 cm) on its neck (Fig. 5). Twobearded dragons had solitary pale nodules measuring0.2 cm and 0.4 cm in diameter on the head andthroat, respectively. One bearded dragon developeda solitary white mass measuring 3.0� 2.0� 2.0 cmon the thorax. Another bearded dragon hada 1.5e2.0 cm ulcerated mass on the ventral side ofits tail and further masses in the viscera. In the veiledchameleon the iridophoromas appeared as multifocalwhite blotches of the skin (0.1e0.3 cm) near the yel-low stripes of the thorax and abdomen.

Histopathological Findings in Iridophoromas

In HE-stained slides iridophores contained moderateto large amounts of fine to coarse golden-brown toolive-green pigment granules that were larger thanthe black pigment granules found in melanophores

Fig. 5. Iridophoroma on a savannahmonitor with focal, irregular,white thickening of the dorsal scales. The mass is1.3� 1.3� 0.7 cm in size. Photograph courtesy ofDr. J. Wiechert.


doi:10.1016/j.jcpa.2011.07.003

(Fig. 6). Diagnosis of iridophoromas (n¼ 6) wasbased on the detection of numerous birefringent,strongly anisotropic granules within the neoplasticcells when examined under polarized light (Fig. 7).Further histomorphological findings in iridophoro-mas were not significantly different from the melano-phoromas (Table 5). All iridophoromas showedinfiltrative growth and inflammation was not found.

Visceral Metastases of Iridophoromas

The female bearded dragon with a tumour at the baseof the tail had numerous iridophoromas in severalorgans (Table 4). The spindle-shaped cell morphol-ogy, invasive growth, marked pigmentation andmitotic index (1 per HPF) were similar in all sites.The lymphatic and blood vessels associated with thetumour of the tail contained numerous tumour cellemboli. Visceral metastases affected 11 differentorgans in this case.

Clinical Outcome of Reptiles with Iridophoromas

In three cases (bearded dragon, savannah monitorand veiled chameleon) surgical removal was curative

Fig. 6. Dermal iridophoroma from a 5-year-old bearded dragon.The tumour is composed of pigmented cells containingamber-coloured granules (iridophores). Single blackmela-nophores are located in the dermis. HE. Bar, 15 mm.


Fig. 7. Dermal iridophoroma from a 3-year-old bearded dragon.Under polarized light there are variably pigmentedspindle-shaped cells containing marked amounts of aniso-tropic crystalline material. HE. Bar, 10 mm.


and there was no recurrence of the tumours overa 2-year period. One bearded dragon was humanelydestroyed directly after diagnosis because of thepoor prognosis of an inoperable tumour and the ani-mal’s poor state of health. In two cases no clinicalfollow-up information was available.

Immunohistochemistry

IHC was performed in 24 of the 26 cases. In four casesIHC was necessary for final diagnosis because the tu-mours were unpigmented. Treatment with H2O2 didnot affect the IHC. Melan A labelling was not seen inany of the control tumours, while S100 protein was ex-pressed in the fibrosarcomas, the myxoma and themyxosarcoma, but not in the squamous cell carcinoma.

In all chromatophoromas melan A and S100 pro-tein were coexpressed in varying intensity (Figs. 8and 9). The median expression intensity (IRS) of 18melanophoromas was 2.3 (0.2e9.1) for melan Aand 4.9 (1.9e9.5) for S100 protein. In the sixiridophoromas it was 1.4 (0.2e2.6) for melan A and4.3 (3.2e5.7) for S100 protein.

Discussion

Chromatophoromas appear to be relatively commontumours in reptiles. In bearded dragons melanophor-

Table

Summary of microscopical fi

Species Shape of cells Pleomorphism

Bearded dragon

n¼ 4

Spindle shaped,

n¼ 4

Mild, n¼ 2

Moderate, n¼ 2

Veiled chameleon 2 Spindle shaped ModerateSavannah monitor Spindle shaped Mild


doi:10.1016/j.jcpa.2011.07.003

omas represented 21.6% of all tumours in the studyand represented 85%of the chromatophoromas foundin lizards. To the authors’ knowledge there are no pre-vious reports of melanophoromas in anacondas,pigmy rattlesnakes, southernwater snakes, veiled cha-meleons, leopard geckos or savannah monitors.

In eight cases surgical removal of the tumour wascurative. Recurrence was observed in three casesand visceral metastases occurred in six cases. In tencases the animals were humanely destroyed and threeother reptiles died a short time after diagnosis.

The microscopical appearance of the melanophor-omas was similar in all species of reptiles (infiltrativegrowth, spindle-shaped cells, mild to moderate aniso-karyosis, 1e2 nucleoli, few mitotic figures and vari-able pigmentation). These findings are consistentwith previous reports (Garner et al., 2004; Irizarry-Rovira et al., 2006; Simpson 2008). Threemelanophoromas with an atypical mucinousappearance were identified in bearded dragons.This type of melanocytic neoplasm has not beenpreviously described in reptiles or mammals. Thetumours were characterized by the presence ofa proteoglycan-rich, PAS-positive mucinous intersti-tial matrix, which was released into the formalin dur-ing fixation. Myxosarcoma was considered asa differential diagnosis, but was excluded due to thepresence of pigmentation, the cellular morphologyand the expression of melan A. This type of melano-phoroma has invasive growth and mild cellular aty-pia, but does not metastasize to the viscera.

Lymphatic and/or vascular invasion is generallyregarded as the best indicator of malignancy(Garner et al., 2004); however, in cases with visceralmetastases a significantly higher cellular pleomor-phism and increased numbers of nucleoli and mitosesin dermal melanophoromas were also detected. Thesesigns of malignancy were not as pronounced in the vis-ceral sites as in the primary dermal tumours. Cellularatypia is a common feature of malignant tumours inthe dog (Spangler and Kass, 2006), but in the presentstudy some well-differentiated dermal chromatophor-omas had widespread visceral metastasis, so this fea-ture of malignancy may not necessarily apply toreptilian tumours. Similarly, mitotic activity is animportant means of distinguishing between benign

5

ndings of iridophoromas

Mitotic figures/HPF Degree of pigmentation Intravascular growth

0, n¼ 2

1, n¼ 2

Marked, n¼ 4 Yes

0 Marked No0 Marked No


Fig. 8. Dermal melanophoroma from a 2-year-old beardeddragon. Moderate melan A expression is seen in most ofthe tumour cells. IHC. Bar, 15 mm.


and malignant melanocytic tumours in mammals. Indogs, but not in cats, >3 mitoses per HPF are usuallyindicative of malignancy (Goldschmidt et al., 1998).In the present study a mitotic activity of $2 perHPF appeared to be associated with malignancy;however, melanophoromas with a mitotic activity of1 per HPF also showed lymphatic invasion and vis-ceral metastases. The location of melanophoroma inthe skin (oral or digital) does not seem to be a prognos-tic factor in reptiles, as described in dogs (Spanglerand Kass, 2006).

In mammals, cellular morphology is not of prog-nostic significance in malignant melanoma(Goldschmidt et al., 1998). In reptiles the cellularmorphology was predominantly spindle shaped; how-ever, epithelioid differentiation was seen in two of the

Fig. 9. Dermal melanophoroma from an adult bearded dragon.MarkedS-100 expression is seen inmost of the tumour cells.IHC. Bar, 10 mm.


doi:10.1016/j.jcpa.2011.07.003

five cases with multiple melanophoromas. Infiltrativegrowth was present in all solitary and multiple mela-nophoromas, so this feature does not appear to bea good criterion of malignancy in reptiles. Further-more, the size of the tumour, the degree of pigmenta-tion and the presence of tumour-associatedinflammation were not predictive of the nature ofthe tumour. Infiltration of single cells or nests ofneoplastic melanocytes in the upper epidermis, whichis an indicator of malignancy in mammals(Goldschmidt et al., 1998), was not observed intumours in the present study. In summary, lymphaticand/or vascular invasion and metastases are the bestindicators of malignancy in reptiles, followed by a mi-totic activity of $2 per HPF, greater cellular pleo-morphism and increased numbers of nucleoli.

In reptiles, iridophoromas have been reported onlyin two species of snakes (Jacobson et al., 1989; AFIP,2000). In the present study, iridophoromas wereidentified only in lizards, particularly in beardeddragons, with a frequency of 7.8% of totalneoplasms. This is also the first report of benigniridophoromas in the savannah monitor, the veiledchameleon and the bearded dragon, as well as ofa malignant iridophoroma in a bearded dragon.These tumours were characterized by a white colourafter fixation. Microscopically, the ambercolouration of the granules in the iridophores wasnoticeably different from the black melanin granulesof melanophores. Furthermore, birefringence wasclearly seen in these tumours confirming thediagnosis.

Similar criteria to those described above may beused to distinguish between benign andmalignant iri-dophoromas. According to Gopalakrishnakone(1986) iridophores were normally present in smallnumbers in the internal viscera of a Chinese softshellturtle (Trionyx sinensis); however, we investigated theinternal viscera of 10 bearded dragons by polarizedlight microscopy and iridophores were not found (un-published data). This confirms the assumption thatthe numerous iridophoromas found in the viscera ofone of the bearded dragons were visceral metastases.

Melan A is one of the most commonly used melano-cytic differentiation markers for human and caninemelanomas (Jungbluth, 2008; Smedley et al., 2011).Its specificity in canine melanoma is high (89e92%for melanomas; Ramos-Vara et al., 2000; Ramos-Vara and Miller, 2011). S100 protein is a widely usedmelanocytic marker with high sensitivity for humanmelanomas (97e100%) and with slightly lowersensitivity (76%) for canine melanomas (Ramos-Vara et al., 2000; Ohsie et al., 2008). Both, melan Aand S100 protein were detected by IHC in allchromatophoromas in the present study. The



specificity of melan A and the sensitivity of S100protein in melanophoromas and iridophoromasreported in other species were confirmed in thereptile chromatophoromas in this study. Theexpression pattern and intensity did not varysignificantly between melanophoromas andiridophoromas. The detection of S100 protein inmelanophoromas was in contrast to the results ofother studies (Kusewitt et al., 1997, Irizarry-Roviraet al., 2006) where S100 protein was not detected.This is probably due to the fact that differentantibodies and pretreatments were used. Kusewittet al. (1997) used the same antibodies, but theseauthors examined a death adder (Acanthophis antarcti-cus), a species of snakenot included in the present study.

Immunohistochemical studies of S100 protein andmelan A expression in iridophoromas are notreported. The mild labelling of melan A in iridophor-omas is probably caused by the similar origin of bothcell lines; dermal chromatophores, including melano-phores, xanthophores and iridophores, all derive fromthe neural crest (Bagnara et al., 1979).

In summary, chromatophoromas in reptiles, espe-cially in bearded dragons, but also in other lizards,occurredmore often than assumed by the low numberof case reports in the literature and their potential ma-lignancy should be considered clinically. Furtherstudies are necessary to understand the biologicalbehaviour of these tumours in more detail. Melan Ais useful for differentiating reptilian amelanotic mela-nophoromas from other sarcomas.

References

AFIPWednesday Slide Conference e No. 29, 3 May 2000,Case I e S-49-98 (AFIP 2681629) http://vp4.afip.org/wsc/wsc99/99wsc29.htm.

Bagnara JT (1966) Cytology and cytophysiology of nonme-lanophore pigment cells. International Review of Cytology,20, 173e205.

Bagnara JT, Hadley ME (1973) Chromatophores and Color

Change. Prentice-Hall, Englewood-Cliffs, p. 202.Bagnara JT, Matsumoto J, Ferris W, Frost SK, Turner

et al. (1979) Common origin of pigment cells. Science,203, 410e415.

Ball HA (1946) Melanosarcoma and rhabdomyoma in twopine snakes (Pituophis melanoleucus). Cancer Research Jour-nal, 6, 134e138.

Elkan E (1974) Malignant melanoma in a snake. Journal ofComparative Pathology, 84, 51e57.

Frye FL, Carney J, Harshbarger J, Zeigel R (1975) Malig-nant chromatophoroma in a western terrestrial gartersnake. Journal of the American Veterinary Medical Associa-

tion, 167, 557e558.Garner MM, Hernandez-Divers SM, Raymond JT (2004)

Reptile neoplasms: a retrospective study of case submis-


doi:10.1016/j.jcpa.2011.07.003

sions to a specialty diagnostic service. Veterinary Clinics ofNorth America: Exotic Animal Practice, 7, 653e671.

Glage S (2001) Immunhistochemische Diagnostik von intraokul€a-ren Melanomen bei Hund und Katze. Inaugural dissertationTier€arztliche Hochschule Hannover, pp. 1e129.

Goldschmidt MH, Dunstan RW, Stannard AA, vonTscharner C, Walder EJ et al. (1998) Melanocytic tu-mours and tumour like lesions. In: Histological Classifica-

tion of Epithelial and Melanocytic Tumours of the Skin of

Domestic Animals, Armed Forces Institute of Pathologyin cooperation with the American Registry of Pathologyand theWorldHealthOrganization Collaborating Cen-ter forWorldwideReference onComparativeOncology,Washington DC, pp. 38e40.

Gopalakrishnakone P (1986) The structure of the pigmentcells in the turtle Trionyx sinensis. Archivum histologicum

Japonicum, 49, 421e435.Gregory CR, Harmon BG, Latimer KS, Hafner S,

Campagnoli RP et al. (1997) Malignant chromatophor-oma in a canebrake rattlesnake (Crotalus horridus atricau-datus). Journal of Zoo and Wildlife Medicine, 28, 198e203.

Heckers KO, Schmidt V, Krastel D, Hildebrandt G,Kiefer I et al. (2011) Malignant melanophoroma ina Hermann’s tortoise (Testudo hermanni) e a case report.Tier€arztliche Praxis, 39 (K), 45e50.

Ippen R, Schr€oder HD (1977) Zu den Erkrankungen derReptilien. In: Erkrankungen der Zootiere 19th International

Symposium, Poznan, pp. 15e29.Irizarry-Rovira AR,Wolf A, Ramos-Vara JA (2006) Cuta-

neous melanophoroma in a green iguana (Iguana iguana).Veterinary Clinical Pathology, 35, 101e105.

Jacobson ER, Ferris W, Bagnara JT, Iverson WO (1989)Chromatophoromas in a pine snake. Pigment Cell andMelanoma Research, 2, 26e33.

Johnson JD (2003) Laser removal of a palpebral melanomain a green iguana. Proceedings of the 10th Annual Meeting of

the Association of Reptile and Amphibian Veterinarians, Min-neapolis, pp. 41e42.

JungbluthAA (2008) Serological reagents for the immunohis-tochemical analysis of melanoma metastases in sentinellymph nodes. Seminars in Diagnostic Pathology, 25, 120e125.

Korabiowska M, Brinck U, Frye FL, Harshbarger JC,Droese M et al. (1998) Expression of growth arrest andDNA damage genes and DNA mismatch repair genesin snake melanomas. In Vivo, 12, 539e542.

Korabiowska M, Brink U, Frye FF, Harshbarger JC,Schauer A et al. (1997) Immunohistochemical and pho-tometric analysis of snake melanomas. In Vivo, 11,415e420.

Kusewitt DF, Reece RL, Miska KB (1997) S-100 immuno-reactivity in melanomas of two marsupials, a bird, anda reptile. Veterinary Pathology, 34, 615e618.

Mikaelian I, Lynch S, Harshbarger JC, Reavill DR (2000)Malignant chromatophoroma in a day gecko. Exotic PetPractice, 5, 73e74.

Morrison RL (1995) A transmission electron microscopic(TEM) method for determining structural colours re-flected by lizard iridophores. Pigment Cell and Melanoma

Research, 8, 28e36.


http://vp4.afip.org/wsc/wsc99/99wsc29.htm

http://vp4.afip.org/wsc/wsc99/99wsc29.htm


Ohsie SJ, Sarantopoulus GP, Cochran AJ, Binder SW(2008) Immunohistochemical characteristics of mela-noma. Journal of Cutaneous Pathology, 35, 433e444.

OrchardG,Wilson JE (1994) Immunocytochemistry in thediagnosis of malignant melanoma. British Journal of Bio-medical Science, 51, 44e56.

€Ozgen S, RaschK,KroppG, SchoonHA, Aupperle H et al.(1997) Aetiopathogenesis and therapy of equine hydro-mukometra: preliminary data. Pferdeheilkunde, 13,533e536.

Ramos-Vara JA, Beissenherz ME, Miller MA,Johnson GC, Pace LW et al. (2000) Retrospective studyof 338 canine oral melanomas with clinical, histologic,and immunohistochemical review of 129 cases. VeterinaryPathology, 37, 597e608.

Ramos-Vara JA, Miller MA (2011) Immunohistochemicalidentification of canine melanocytic neoplasms with an-tibodies to melanocytic antigen PNL2 and tyrosinase:comparison with Melan A. Veterinary Pathology, 48,443e450.

Ramos-Vara JA, Miller MA, Johnson GC, Turnquist SE,Kreeger JM et al. (2002) Melan A and S 100 protein im-munohistochemistry in feline melanomas: 48 cases. Vet-erinary Pathology, 39, 127e132.

Ramsay EC, Fowler M (1992) Reptile neoplasms at theSacramento Zoo, 1981e1991. In: Proceedings of the JointConference of the American Association of Zoo Veterinarians

and the American Association of Wildlife Veterinarians, Oak-land, pp. 153e155.

RemmeleW, Stegner HE (1987) Recommendation for uni-form definition of an immunoreactive score (IRS) forimmunohistochemical estrogen receptor detection(ER-ICA) in breast cancer tissue. Der Pathologe, 8,138e140.


doi:10.1016/j.jcpa.2011.07.003

Rohrlich ST (1974) Fine structural demonstration ofordered arrays of cytoplasmic filaments in vertebrate iri-dophorese a comparative survey. Journal of Cell Biology,62, 295e304.

Ryan MJ, Hill DL, Whitney GD (1981) Malignant chro-matophoroma in a gopher snake. Veterinary Pathology,18, 827e829.

Schlumberger HG, Lucke B (1948) Tumors of fishes, am-phibians, and reptiles. Cancer Research, 8, 657e754.

Simpson S (2008) Pigmented cutaneous neoplasm in a cen-tral beardeddragon. In:Proceedings of theUnusual andExotic

Pet Veterinarians Annual Conference, Brisbane, pp. 285e288.Sinn AD (2004) Pathologie bei Reptilien e eine retrospektive

Studie.M€unchen,Universit€atM€unchen, Veterin€armedi-zinische Fakult€at, Dissertation.

Smedley RC, Spangler WL, Esplin DG, Kitchell BE,Bergman PJ et al. (2011) Prognostic markers for caninemelanocytic neoplasms: a comparative review of the lit-erature and goals for future investigation. Veterinary Pa-thology, 48, 54e72.

SpanglerWL, Kass PH (2006) The histologic and epidemi-ologic bases for prognostic considerations in canine mel-anocytic neoplasms. Veterinary Pathology, 43, 136e149.

Suedmeyer WK, Bryan JN, Johnson G, Freeman A (2007)Diagnosis and clinical management of multiple chroma-tophoromas in an eastern yellowbelly racer (Coluber con-strictor flaviventris). Journal of Zoo andWildlifeMedicine, 38,127e130.

Iridophoromas

½ R

A

in

eceived, January 14th, 2011

ccepted, July 5th, 2011

Reptiles, Journal of Comparativ

�

e Pathology (2011),

melanophoromas and iridophoromas in reptiles - · pdf filemelanophoromas and iridophoromas in...

Documents