an animal model for human melanoma

Photochemistry and Photobiology, 1996, 64(3): 577-580

An Animal Model for Human Melanoma

Adele Green*', Rachel Neale', Roger Kelly2, Ian Smith2, Effie Ablett3, Barry Meyersl and Peter Parsons3 'Epidemiology and Population Health Unit and 3Cancer Unit, Queensland Institute of Medical Research and 'School of Veterinary Science, University of Queensland, Queensland, Australia

Received 8 February 1996; accepted 9 May 1996

ABSTRACT

Experimental animal models that are directly relevant to human melanoma are lacking. We propose the Angora goat as a potentially useful field model with experimental potential and to this end have examined the prevalence and site distribution of all skin cancers in 28 Angora goat herds in Queensland, Australia. The prevalence of benign melanocytic lesions (lentigines) and their experimental induction by sunlight were also investigated. Among 1731 goats over 2 years of age, 139 malignant skin tumors were excised from 95 affected animals. The prevalence of squamous cell carcinoma (SCC) was 3.8% and of melanoma, 2.2%. Main site of occurrence of melanoma (83%) was the dorsal surface of the ear; in contrast SCC occurred mostly (84%) on the perineum. Lentigines were darker and more prevalent on the exposed compared with the unexposed surface of the ear in Angoras, analogous to the higher prevalence of nevi on the exposed compared with the less exposed inner surface of the arm in humans. Lentigines, which were also found on the perineum though lighter in color than on the dorsal ear, were absent in young animals under 3 months but were numerous in 1-3 year olds. Furthermore in an experimental substudy eight goats, having one flank repeatedly shorn and the contralateral flank left unshorn, revealed consistently more solar lentigines on the shorn flank (P < 0.05) when both sides were examined after 9 months. Histopathological examination of paired skin biopsies from five of these goats also showed more abundant pigmentation in skin from the exposed, as compared with the unexposed flank. These findings indicate that sunlight induces tumors and lentigines in goats in a highly site- specific manner. The Angora goat model may suggest paradigms for explaining the site differences observed for human melanoma and may also be useful in the fu- ture clarification of molecular changes following carcinogenic levels of sun exposure.

*To whom correspondence should be addressed at: Queensland In- stitute of Medical Research, PO Royal Brisbane Hospital, Queens- land 4029, Australia. Fax: 6 1-7-3362 0 I I 1 ; e-mail: [email protected].

0 1996 American Society for Photobiology 003 I -8655/96 $5.00+0.00

INTRODUCTION

There is sufficient evidence that solar radiation causes cutaneous malignant melanoma in humans and that UVA and UVB radiation are carcinogenic in experimental animals (1 ) . Despite extensive investigation however, the steps involved in the development of melanoma after sun exposure are not fully understood. Experimental animal models of skin neo- plasia are mostly relevant to squamous cell carcinoma (SCC)?; the few experimental animal models available for studies of melanoma induction offer little evidence that solar UV alone is responsible. Melanocytic tumors occur sponta- neously in gray horses in old age and are probably the man- ifestation of a melanin storage disease (2). Congenital melanoma in miniature pigs (3) and fish have given insights into the role of oncogenes but are not appropriate for the study of etiology given that a carcinogen does not appear to be involved. Indeed melanoma can be routinely induced in few animals tested except the opossum (4.5) and hybrid fish (6). though the direct relevance to humans of the opossum model is diminished because melanomas arise from hair follicle melanocytes and they lack epidermal melanocytes, which are the presumed target cells in human melanoma. Also the relevance to humans of a photoreactivating repair enzyme in the opossum is uncertain, though there is in vifro evidence that cyclobutyl pyrimidine dimer photolyase is present in human cells (7).

Another potential animal model for human melanoma is the Angora goat, given the similarity to humans of the histopathological features (8) and course of the disease, com- mencing in the skin, followed by local invasion and metastasis to lymph nodes (9). Squamous cell carcinomas are also prevalent among Angora goats. The Angora is a brown-eyed, white-skinned goat covered with dense white mohair except for the ears, nose and perineum. There are few data available regarding the prevalence and incidence of caprine melanoma, but peak age of occurrence appears to be around 4-5 years (9); in Australian Angora goats the average lifespan is 8-1 2 years. Consistent with the hypothesis that caprine melanoma is caused at least in part by exposure to solar UVB radiation, the cases reported have occurred in countries at low latitude and thus with high solar UV (northern Africa, India), and within Australia, prevalence rates in goats are higher in northern subtropical compared with southern

?Abbreviation: SCC, squamous cell carcinoma.

577

578 Adele Green et a/.

Table 1. 1731 Angora goats in Queensland

Site distribution of prevalent melanoma and SCC among

Melanoma SCC

Site No. (%) No. (%)

regions (I. Smith, unpublished data). Furthermore the com- mon sites of occurrence have little protection from the sun, namely ear, face and perineum; and pale-colored and white goats appear to be at high risk of melanoma. Moreover the histopathology of pigmented lesions from exposed sites in goats resembles that of lentigo simplex or lentigo rnaligna in humans (8). Besides sun exposure, other predisposing fac- tors in goats including inherent genetic susceptibility and intense exposure to certain antihelmintic chemicals ( e .g hal- ogenated salicylanilides, benzimidazoles) have been sug- gested (I. Smith, unpublished data). The photolyase activity in cells from goats is not known.

Site of occurrence of melanoma potentially provides clues to its carcinogenesis because of site-specific differences in intensity of solar dose received. The pattern observed in hu-

Figure 1. Example of primary malignant melanoma on dorsal surface of ear of female Angora goat.

mans is enigmatic, however, given present limited knowl- edge of pathogenesis of melanoma. Angora goat herds in Queensland, Australia, were examined for skin cancers with the aim of exploring site-specific differences in detail. As

recent sun exposure on Proliferative melanocYtic lesions in previously unexposed skin.

numbers of pigmented lesions (clinically, Solar lentigines) on the shorn and unshorn sides were compared. Skin biopsies ( 1 cm di- ameter) from the mid-flank areas of both sides of five goats were collected, fixed for 24-36 h in neutral buffered formalin, embedded

tions for histopathological examination. Action spectroscopy exper- iments were not performed in view of the logistic difficulties of conducting such studies in goats.

the goat was to the effect Of in paraffin and stained with hematoxylin and eosin to prepare set-

MATERIALS AND METHODS Animals. Twenty-eight Angora goat herds located in central and southeastern Queensland between 21" and 28" south latitude were ascertained for study. Herd size ranged from 16 to 232 animals over 2 years old with an average of 62 goats per herd. Although purebred animals now comprise a very small proportion of the Angora goat population in Australia (due to the repeated backcrossing of feral females to Angora males over the last three decades), the animals used in the present study were 90-95% Angora in genetic consti- tution. All goats were systematically examined for the presence of skin lesions by one of two registered veterinarians (R.N., IS.) during 1993. Suspected malignancies were biopsied or excised using standard surgical techniques, with the exception of two tumors that were clinically diagnosed as large, classical SCC. All surgical specimens and sections of normal skin from perineum and ear were examined by a veterinary pathologist (R.K.).

Separate counts of nonmalignant melanocytic lesions were made on the dorsal and ventral surfaces of the ears and on the perineum in 19 randomly selected goats in one of the herds in the study. The goats ranged in age from I month to 8 years, and all were females with the exception of two goats (aged 1 and 10 months respectively). In addition, skin taken at autopsy was fixed in formalin, embedded in paraffin and sections subjected to standard immunohistochemistry using S-100 antibody.

Experimenral sun exposure. In an experimental substudy. 12 bucks from a herd in southeast Queensland (28" latitude) were shorn, 6 on their left sides and 6 on their right, once a fortnight for some 9 months during which time they grazed outdoors as usual, in the sun with only a few trees providing shade. After 9 months both sides of the eight goats still available for study were shorn and the

RESULTS The majority (approximately 95%) of the goats examined were female. Among the 1731 goats over 2 years old examined, a total of 139 malignant skin tumors were excised from 95 affected animals. The majority (67%) were SCC and the remainder were melanomas (Table I). Of the 28 herds examined, 16 herds (57%) had at least one goat affected by melanoma; 15 herds (54%) had at least one goat affected by SCC; goats from 1 I herds (39%) were affected by both types of tumor and goats from 8 herds (29%) had no prevalent malignant skin tumors. The prevalence of goats affected by SCC was 3.8% and b? melanoma, 2.2%. Be- tween herds, prevalence of goats affected by melanoma ranged from 0% to 12% and prevalence of goats affected by SCC from 0% to 16%. Sites of occurrence of melanoma were the dorsal surface of the ear (Fig. 1) (83%), the perineum (15%) and the eyelids (2%). In contrast SCC occurred mostly on the perineum (84%). and less on the ear (lo%), eyelids (5%) and nose ( 1 % ) (Table 1).

The distributions of lentigines were examined on the 19 goats of various ages. None were observed before the age of 3 months and maximum counts were reached by 2 years of age. Among goats aged over 2 years, there were on average about eight-fold more lentigines on the dorsal than on

Photochemistry and Photobiology, 1996, 64(3) 579

Figure 2. Typical appearance of dorsal and ventral surfaces of shaved right and left ears of an Angora goat showing higher number of lentigines on the dorsal (exposed) surface.

the ventral surface of the ear ( P < 0.001) (Figs. 2 and 3). Macroscopically, lentigines on the dorsal surface were darker than on the ventral surface (Fig. 2); those on the perineum were of intermediate color. Crude average counts on the perineum were comparable to those of the ventral surface of the ear, but after adjustment for the difference in surface area (greater on either surface of the ear by a factor of 2.4), the lentigo count on the dorsal ear per unit surface area of skin was almost three times the count on the perineum, though the difference was not significant (P < 0.2) (Fig. 3). Density of lentigines was about five times greater on the perineum than on the ventral ear, however ( P < 0.005). Nu- merous melanocytes were revealed in the basal layer by re- action with S-100 antibody.

Histology of non-neoplastic skin from the sites of interest showed more intense melanocytic activity from both dorsal and ventral surfaces of the ear than from the perineum. Dif- ferences could not be quantified however, because of the extreme variability in melanocyte number according to whether or not lentigines had been captured in the sections of normal skin.

Experimental sun exposure

Consistently more solar lentigines occurred on the goats’ shorn compared with unshorn flanks (P < 0.05) (Table 2). Pathological examination of the five paired skin biopsies (performed blind to fleece status) showed greater melanocytic activity or more abundant pigmentation in skin from the exposed compared with the unexposed flanks of three of the five (B, D, E: Table 2). The intensity of melanocytic activity in the skin sections again varied in accordance with the extent to which lentigines had been captured in the plane of section. Severe hyperkeratosis was also observed in the sun-exposed skin of one animal (C).

DISCUSSION We have shown previously that caprine melanoma may provide a unique animal model for human melanoma, based on its natural history and on histopathology (8). We have now extended the potential heuristic value of the model to exploring the reasons for the observed site distribution of mel-

- dorsal ear

+ventral ear li *perineum /

1 11 21 31 41 51 61 71 81 91

age (months)

Figure 3. Distribution of lentigines on the dorsal and ventral surfaces of the ears and the perineum (adjusted for surface area) in 19 Angora goats according to age.

anoma in humans. There is a clear predilection of melanomas in Angoras to occur on the chronically exposed dorsal surface of the ear, despite the apparently similar sun exposure received by other less-haired sites such as the nose and perineum. Site variation of caprine melanoma is thus analogous to that of melanoma occurring in humans where there is relative sparing of certain sun-exposed sites. For example in the Queensland population the dorsum of the hand and the forearm receive similar sun exposure, yet there is a relative deficit of melanomas occurring on the dorsum of the hand compared with the forearm. Rates of melanoma on the forearm are 4 times higher than those on the back of the hand in men and 1 1 times higher in women (10).

There are several possible explanations for these obser- vations in Angora goats, which have been derived from strains introduced into Australia more than 100 years ago (the study herds have not been influenced by the recent introduction of animals from the United States and South Af- rica). There may be greater net solar UV received at the surface of the ear than on the nose or perineum when the animal is grazing outdoors, which is not readily appreciated

Table 2. ocytic activity among Angora bucks

Experimental induction of pigmented lesions and melan-

Histology No. lentigines Animal Shorn Unshorn Shorn vs unshorn

A 12 2 B >20 9

C 14 3 D 4 2

E 8 2 F 7 1 G 3 0 H 0 0

Total 68 19

No difference Melanocytic activity and pigment

No difference Slightly greater demoepidermal

Pigmentation more abundant Not biopsied Not biopsied Not biopsied

production much greater

pigmentation

580 Adele Green et a/.

by casual observation. The ears of the Angora are generally held so that the dorsal surface of the ear is only slightly angled to the ground and thus receives maximal incident solar radiation, in contrast to the other exposed sites such as the perineum (exposed because Angoras usually hold their tails erect). The dorsal surface of the ear has short sparse hair compared with the glabrous skin of nose and perineum and thus the hair follicle melanocytes may provide many more target cells. (This could also be true of the upper limb in humans where there are more target cells due to hair follicle melanocytes compared with the skin of the dorsum of the hand.) Moreover follicular melanocytes sited in the der- mis would absorb more UVA than target cells at the epidermal level, and given the possible tumorigenic potential of long wavelength UVA ( 1 1) this may also partly explain the concentration on the dorsal ear. It is noteworthy that there was relatively little change in number and distribution of lentigines after 2-3 years of age, the age after which SCC and melanoma first occur. There is however an abundance of melanocytes in goat epidermis, as demonstrated by S-100 staining.

Lentigines were more prevalent on the exposed dorsum of the ear than on the unexposed ventral surface of the ear, akin to the greater prevalence of nevi on the dorsal compared with the ventral surface of the arms in humans. Although average numbers of lentigines were not significantly differ- ent on the exposed surface of the ear and the perineum (ad- justing for surface area) after 2 years of age, on gross appearance the lentigines on the dorsal ear were more deeply pigmented than those on either the ventral ear or the perineum. Microscopically, pigmentation was most intense in the skin of the dorsal surface of the ear as well. Although the sun exposure experiment was based on a small number of animals, it showed increased numbers of lentigines on the shorn sides of the animals over 9 months. This difference was presumably due almost entirely to differences in sun exposure of the two sides in each animal and indicates that the UV dose over 9 months was sufficient to stimulate development of proliferative melanocytic lesions in previously unexposed skin. These lentigines are considered possible but not obligate precursors of melanoma in the goat, somewhat analogous to melanocytic nevi in humans (nevi per se were not observed in goats).

There is a contrasting site predilection of SCC and melanomas in the goat model as in humans. Intriguingly in the goat model these two malignant tumors have reciprocal site distributions, which is not so clearly the case for humans (10.12). The reasons are unknown; however it does not seem on the basis of current evidence, that caprine SCC is asso- ciated with papillomavirus because vulva1 viral warts are not seen in Angoras; on the other hand the induction of bovine SCC may involve papillomavirus (1 3). It is clear however that sunlight induces melanomas in Angoras as in humans

(14) in a highly site-specific manner, and in neither can the variation be explained entirely by differences in sun exposure. It is hoped that the Angora goat model for melanoma may not only suggest paradigms for explaining the site differences observed for human melanoma, but also be useful in the clarification of possible molecular changes that are involved following carcinogenic levels of sun exposure.

Acknowledgements-We are grateful to Helen Leonard and all other goat owners who gave us their assistance. This study was supported by a grant from the National Health and Medical Research Council of Australia (NHMRC). R. N. is supported by a grant from the Public Health Research and Development Committee of the NHMRC.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

1 I .

12.

13.

14.

International Agency for Research on Cancer (1992) Solar and ultraviolet radiation. IARC Monogr. Eval. Carcinog. Risks Hu- mans, Vol. 55. IARC, Lyon, France. Levene, A. (1971) Equine melanotic disease. Tumori 57, 133- 168. Amoss, M. S., S. G. Ronan and C. W. Beattie (1988) Growth of Sinclair swine melanoma as a function of age, histopathological status and gonadal status. Cancer Res. 48, 1708-171 I . Ley, R. D., L. A. Applegate, R. S. Padilla and T. D. Stuart (1989) Ultraviolet radiation-induced malignant melanoma in Monodelphis domestica. Photochem. Photobiol. 50, 1-5. Robinson, E. S., J. L. VandeBerg, G. B. Hubbard and T. P. Dooley (1994) Malignant melanoma in ultraviolet irradiated lab- oratory opossums: Initiation in suckling young, metastasis in adults, and xenograft behaviour in nude mice. Cancer Res. 54, 5986-5991. Setlow, R. B., A. D. Woodhead and E. Grist (1994) Animal model for ultraviolet radiation-induced melanoma: platyfish- swordtail hybrid. Proc. Natl. Acad. Sci. USA 86, 8922-8926. Sutherland, B. M. and P. V. Bennett (1995) Human white blood cells contain cyclobutyl pyrimidine dimer photolyase. Proc. Natl. Acad. Sci. USA 92, 9732-9736. Parsons, P. G., H. Takahashi, J. Candy, B. Meyers, J. Vickers, W. R. Kelly, I. Smith and P. Spradbrow (1990) Histopathology of melanocytic lesions in goats and establishment of a melanoma cell line: a potential model for human melanoma. Pigment Cell Res. 3, 297-305. Ramadan, R. 0.. A. M. El Hassan and M. H. Taj El Deen ( I 988) Malignant melanoma in goats: a clinico-pathological study. J . Comp. Parhol. 98, 237-246. Green, A., R. MacLennan, P. You1 and N. Martin (1993) Site distribution of cutaneous melanoma in Queensland. Int. J . Can- cer 353, 232-236. Sterenborg, H. J. C. M. and J. C. van der Leun (1990) Tumor- igenesis by a long wavelength UV-A source. Photochem. Pho-

Gallagher, R. P., B. Ma, D. I. McLean, C. P. Yang, V. Ho, J. A. Carruthers and L. M. Warshawski (1987) Trends in basal cell carcinoma, squamous cell carcinoma and melanoma of the skin from 1973 through 1987. J. Am. Acad. Dermatol. 23,413- 421. Spradbrow, P. (1987) Immune response to papillomavirus in- fection. In Papillomaviruses and Human Disease (Edited by K. Syrjanan, L. Gissma and L. G. Koss), pp. 334-370. Springer Verlag, Berlin. Green, A. (1992) A theory of the site distribution of melanomas. Cancer Causes Control 3, 5 13-5 16.

tobiol. 51, 325-330.

an animal model for human melanoma

Documents