Three Different Types of Tumors in Salientia

E. ELKAN

(Group IX Laboratories, Shrod4ts Hospital, Watford, Herts. England)

SUMMARY Three tumors of widely differing structure occurring in, Salientia (Amphibia) are described. They are: (a) a nephroblastoma, (b) an atypical facial papilloma or melano carcinoma, and (c) an epithelioma resembling ulcus rodens. The first two tumors oc curred in specimens of the South African claw-footed toad (Xenopus laevis D.), the third in a specimen of the common grass frog (Rana temporaria L.).

A NEPHROBLASTOMAIN Xenopus laevis DAUDIN any of these animals falls ill, it hides in the most Neoplasms of various kinds are common in fish, inaccessible corner of the cage; many are, in any much rarer in reptiles, very rare in amphibians. case, nocturnal in their habits, and when their The first comprehensive review of the tumors de death is discovered, decomposition has usually ad scribed from cold-blooded animals was made by vanced to such a degree as to make further inves LuckÃ©and Schlumberger (Balls [1] lists 68 relevant tigation impossible. Much interesting material is titles) in 1948â€”1949.Cohrs et a!. in their textbook lost in this way. Circumstances are a little more Pathology ofLaboratory Animak reviewed the liter promising in laboratories where less attention is ature up to 196g. Finally Balls (1), reporting on paid to the imitation of natural surroundings and six new cases in Xenopus, reviewed the literature more to the health of the animals. However, even up to 196g. The present paper gives a short decrip those departments which keep large numbers of tion of three types of tumors not previously seen frogs under constant close supervision have re in Salientia. ported few cases of true neoplasms in amphib The most common neoplastic tumor in frogs and ians. toads is still the LuckÃ© (7) renal adenocarcinoma The tumor here described occurred in a fully which frequently occurs in the American leopard grown female specimen of Xenopus laevis D., the frog Rana pipiens and which has been shown to be South African claw-footed toad (Aglossa), of 1@0- transmissible (9) . All the other neoplasms seen in gm. weight, which had been kept in stock for the amphibians, and particularly in Salientia, are very performance of pregnancy tests for over a year. rare. The toad, originally imported from Cape Town, Embryonic blastomasâ€”in particular nephro South Africa, was kept in a galvanized steel tank blastomasâ€”are, according to Willis (f), commonly together with 59 other females of the same species, found in the pig, the rabbit, and the domestic fowl. and it yielded, during the period of its captivity, They are less commonly found in hares, rats, and correct results when used for these tests. None of mice. Thus far they have not been listed as occur the other toads sharing the tank showed any signs ring in poikilothermic animals. Their classification of disease, but the specimen in question attracted into renal teratomas and nephroblastomas depends the attention of the staff because, when it was on the type and the degree of differentiation the handled, a large firm mass could be palpated tissues present. Since they have thus far escaped through the abdominal wall. This toad was neither description in cold-blooded animals, we might be discolored in any way nor disfigured by hydrops, inclined to regard such a tumor as a rarity. In this symptoms which usually herald the start of any type of animal, however, such rarity may be more serious disorder in Xenopus. It was therefore killed @ apparent than real. Tropical and subtropical spe with per cent urethan. cies must be kept in heated cages either, like Appearances at post mortem examination.â€”A Xenopus, permanently submerged or, like most view of the abdominal viscera in situ after removal terrestrial species, in a warm, moist atmosphere. If of the anterior abdominal wall, the right ovary, ReceivedforpublicationMarch27,1968. and the right lobe of the liver, is presented in Fig 1641

ure 1. It varies little from that of true renal carci endothelium lining Bowman's capsule; ciliated noma seen before (6), and it seemed at first cuboidal epithelium lining the neck of the nephron; sight to be almost identical with the material high-columnar epithelium lining the convoluted published by LuckÃ©and Schlumberger (14), with tubules; low-columnar epithelium and flask cells the only exception that, in the present case, the lining the collecting ducts (Fig. 6). Of all these tumor was much bulkier than those previously cell types only the cuboidal, nonciiated epithelium seen. Its retroperitoneal origin was obvious from appears in the sections (Fig. 7, Bâ€”D,F, G). In the fact that the inferior vena cava, the large in stead of the other cell types two, or perhaps three, testine, and the right oviduct were displaced yen new kinds of cells appear which are not found in trally. No trace of the right kidney could be found the normal aglossan mesonephros: behind the tumor even with the aid of a hand lens. 1. Undifferentiated renal blastema, occurring The left kidney was found compressed between the either in loose concentric rings (Fig. 7, A) or in tumor and the left lumbo-dorsal muscles, but it dense conglomerations in the place of glomerular showed no histological abnormality and was, ap capillary loops (Fig. 7, H). parently, fully functional. Both the liver and the 2. Another kind of undifferentiated tissue made spleen were found considerably congested and en up of large, clear cells with small nuclei and well larged to twice their normal size. Both ovaries defined cell walls. These cells are always seen to were atrophic and reduced to less than half their fill some kind of cavity partly or wholly. These normal bulk; but both fat bodies were found very cavities may be abortive Bowman's capsules (Fig. well preserved. These observations are at variance 4, Fig. 7, H). with those usually made in diseased toads where, if 3. High-columnar epithelium of the â€œpalisadeâ€• the ovaries atrophy, the fat bodies do so as well. type. Here the nuclei are long and thin and lie so No metastases of the tumor were found any closely together that no cytoplasm can be seen where, nor could an invasion of abdominal veins between them (Fig. 7, C and G). The palisade cells be demonstrated. The tumor seemed to have are seen either to form the walls of an abortive grown, entirely by expansion, to its final size of tubule or to protrude, papillary fashion, into 4 X 5 X 3 cm., not invading any other organ but smaller and larger cystic spaces (Figs. 4, 5, and interfering severely with the abdominal circulation Fig. 7, G and H). and causing severe congestion in the portal circuit. Among these palisade cells there appear occa The neoplastic mass (Fig. 1) was firm and sionally larger, oval cells whose nuclei do not fill white, with an irregularly bulging surface of the the whole space available, leaving a clear gap of cauliflower type. Cross-section showed a few small cytoplasm between the nudeus and the cell wall darkly stained patches but presented no signs of (Fig. 7, D). The numerous cystic cavities, perhaps softening or degeneration. The whole tumor was representing attempts at the formation of Bow contained in a thin capsule which, in the right man's capsules, can be seen in every field (Figs. dorso-lumbar region, contained the compressed and 2, 5), and they are lined with all kinds of cu thinned-out right kidney. boidal epithelium. Their nuclei, too, vary greatly Histology.â€”Cellsin all stages of differentiation, in shape and size, and the palisade arrangement, obviously derived from the renal blastema, made though prevalent, is not always present (Fig. 7, E) up the bulk of the tumor. The stroma consisted of The two types of epithelial cell most typical of the a loose network of fibroblasts embedded in abun aglossan kidney, the ciliated columnar and the dant intercellular matrix which accepted periodic flask cell (Fig. 6), are not represented in the acid-Schiff stain as well as Alcian blue and muci tumor at all. carmine (Fig. 2). The nuclei, on the whole, stained more heavily Clear spaces within the stroma were filled with than normal with basic dyes. Mitotic figures were phagocytes containing fatty droplets (Fig. 3). In not seen. The tumor apparently grew slowly from spite of the extremely scanty vascularization of a small center of abnormal cells to its final size. the tumor no areas of overt necrobiosis were found. It was not malignant and pathogenic only insofar All parts of the tumor stained equally well and as it displaced neighboring organs, causing pres were obviously of good viability even when they sure atrophy of the affected kidney and extreme seemed to lie far removed from the nearest capil congestion of liver and spleen. The general histo lary vessel. logical picture of the neoplastic tissue is that of In the parenchyma every degree of epithelium ad infinitum repeated, but always abortive, at from the most primitive to the almost completely tempts at kidney formation. organized could. be seen. The normal epithelial â€˜rhedevelopment of the normal mesonephros in' components â€˜ofan aglossan nephron would be: Xenopus laevi@takes place between the 5th and

58th day of development (Nieuwkoop and Faber a homogeneous layer of squamous cells (Fig. 9) [16]). If one assumes that the development of this but has, in the area covering the polyp, been nephroblastoma paralleled the growth of the eroded so as to form numerous pockets of varying normal organ, its beginnings must lie far back in depth (Figs. 11, 12). These pockets are sharply de the very early days of the development of this fined and in many instances are seen to approach particular toad. the surface so closely as to leave only one layer of epithelial cells covering them. The normal aglossan AN ATYPICAL FACIAL PAPILLOMA IN epithelium, shown in Figure 9, is seven to ten cells Xenopus laevi. D. thick. At the surface of the polypous mass the An adult female of 110-gm. weight, which had epithelial covering has become completely eroded been kept with others of the same species for over (Fig. 10, arrow), but this area of ulceration was 12 months and which during that time had been small. used several times for pregnancy tests, developed The glandular and the connective tissue layers a tumor in the rostral region. The animal had of the dermis, which together form a very tough originally been imported from Cape Town; none coat in amphibians, are seen to be completely de of the other toads from this batch has shown any stroyed in the affected area by the abnormal tissue similar symptoms. The animal was isolated be produced by the tumor. Instead of the regular cause it lost color, refused to feed, and seemed to strands of connective tissue the following features have difficulties in breathing. Closer inspection were seen in the place of the dermis: (a) a network revealed some swelling and a pink discoloration in of fibroblasts with narrow, elongated nuclei ; (b) the region of the right nostril, the lining of which capillary loops, either single or in convolutions; seemed to be thrown into folds obstructing the (c) cells with large, oval, badly staining nuclei entrance (Figs. 8, 10). The condition was not obvi (Figs. 11, 12). ous at first, and details could not be ascertained The undifferentiated fibroblasts which, in other without a hand lens. The toad continued to refuse circumstances, one might have defined as sarco all food, and after 4 weeks of isolation, during matous, made up the mass of the tumor, particu which time the toad became paler and paler, it died. larly in the deeper part where it adjoined the General pallor, can, in Xenopus, always be taken underlying structures. There was, however, no evi as a sign of serious illness unless the toad happens dence of any invasion or destruction of neighboring to be kept in a vessel with a white bottom. General tissues, nor were any metastases found in any part post mortem dissection revealed no abnormality of the body. of the viscera apart from an almost complete The capillary loops were most numerous near atrophy of the ovaries and the fat bodies, signs the surface. In Figure 11 they are recognized by which may again be taken as typical of a severe the nucleated erythrocytes they contain. They systemic illness. After decalcification serial see formed either single loops or dense convolutions, tions were made through the rostral part of the filling some of the epidermal pockets and being skull. separated from the sui@face by a one -or two-cell At low-power magnification these sections re layer of epithelium only. Their presence here vealed the presence of a papillomatous growth at probably explains the pink discoloration of the the edge of the nostril (Fig. 10). In the sections the area observed in vivo. tumor did not seem to obstruct the nostril, but the The cells with large, badly staining nuclei were material must have shrunk considerably during found mainly but not exclusively in the epithelial preparation. Of all the skin tumors one may see, pockets, and the question arose whether these papillomas are perhaps the most common, and cells were derived from the disintegrating epithe little attention might have been paid to this one hum or from the eroding tumor. The epithelium had it not been for the fact that it had, to all overlying the pockets looked as if it were yielding appearances, caused the death of the toad and, more to pressure than to disintegration. On the further, that an unusual relation existed between other hand, round melanophores, typical of the the dermal tumor and the overlying epidermis. In epidermis of this region could in many instances a typical papilloma which arises from the dermis be seen lying free within the pockets as if they had or the subcutis the epidermis, otherwise unaffected, been dissolved out of the epithelium. Their pres is thrown into folds which cover the papillae and ence here would, however, be no proof of epidermal may show a greater or lesser degree of keratiniza disintegration, since the melanophores may have tion on the surface. In this case the epidermis pre wandered actively into the pockets (Fig. 12). sents a totally different picture. It does indeed Remains of dermal glands could be found em cover the polypous mass, yet it no longer presents bedded in the tumor, the glandular tissue appar

ently resisting the dissolving activity longer than phenomenology @fboth tumors is not to be ex the rest of the epithelium. Why this action did not pected. On the whole, enough similarities remain take place on a broad front but confined itself to to bring this tumor into the group of dermal mela the formation of isolated, well defined pockets noma or melano-carcinoma, a condition frequently could not be explained. It is not easy to explain seen in fish, sometimes in reptiles, but rarely in the fact that' there were two kinds of such pockets, amphibians. In Xenopus, in particular, sponta one filled with capillary loops, the other with the neous dermal tumors are extremely rare, and those large, badly staining cells mentioned. that are seen are usually due to infections or to It was not apparent why this trivial-looking encysted parasites. Because of the location of the condition should cause the animal to refuse all tumor it seems possible that it may have been food and eventually to die. Breathing was pre initiated by trauma. Frogs frequently injure the sumably still unimpaired via the other nostril, and region of the nostrils in collisions with the walls of no damage to any of the major viscera could be their cages when trying to escape. These injuries demonstrated. always heal badly, and an accident of this nature There remains the question how this tumor may have started this atypical papilloma. should be classified. Its closest counterpart in human dermatological pathology may be found in AN EPITHELIOMA RESEMBLING ULCUS Dawson's (3) classical treatise on the melanomas. RODENS IN Rana Under the heading of â€œMelano-Carcinomaâ€• Daw From the point of view of comparative pathol son describes the development of a tumor similar ogy this tumor has some interesting features. It to the one observed in Xenopus laevi@. His draw occurred in a common grass frog caught as an ings show how cells in the basal layer of the epi adult frog in the south of England by a naturalist dermis lose their cell bridges, become detached and subsequently kept in a cage as a pet. Since it from one another, and form â€œcellnestsâ€•which are was fully grown at the time of capture and since at first without contact with the dermis. Later this the owner kept the frog 4 years, it must have barrier is broken down, and the cells invade the been about 6 years old when the first signs of dis dermis through an opposing layer of lymphocytic ease became apparent. This, for a frog, must be infiltration. Pockets of cell nests are formed in the regarded as equivalent with old age. epidermis, and a comparison of the pictures pre The growth started in the right maxillary region sented by the human and the amphibian material between the bone and the orbit, where it gradually seems possible. However, the analogy breaks down reached a diameter of 8 mm. Although the right in the examination of detail. There is no evidence eye does not look damaged in sections, the frog in our sections to show that in Xenopus the degen became gradually blind in this eye. This may have erative process starts in the epithelium. On the been owing to the gradual encroachment of the contrary, it seems to start in the dermis. Also, in tumor on the cornea (Figs. 13, 15). As long as the the human material, the epithelial cells starting the frog could see with the left eye, it continued to process arose from a nevus, were heavily pigment feed. When eventually it stopped feeding after an ed, and had hyperchromatic nuclei. All these fea illness of 6 weeks it was, to all appearances, com tures are absent in the amphibian material. The pletely blind. Sections through the left half of the excessive vascularity of the invading dermal tissue skull show not only a massive retroretinal hemor is not mentioned by Dawson, while the cellular rhage filling the whole intraocular space but also polymorphism of the malignant tissue was promi an extreme congestion and distension of all the nent in both Homo and Xenopus. veins in that part of the skull. This condition was If one considers the evolutionary distance be probably due to venous thrombosis caused by the tween the two species, complete analogy in the advancing tumor.

Fia. 1.â€”Xeno pus taevis. Adult female with nephroblasto ma. Ventral view of intestines after removal of the right ovary and the rightlobeof the liver.Notethe displacementofthe inferiorvenacava,the rightoviduct,and the largeintestine. Note alsothe preservationofboth fat bodies.d, oviduct; ft right,fl left, fat body;i, largeintestine;o,ovary;a,spleen; 1, tumor; v, ventricle; ye, vena cava inferior.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1963 American Association for Cancer Research. FiG. 2.â€”Nephroblastoma in Xenopus laevis. Survey picture of central part of the tumor. X40. FIG. 3.â€”as Figure 2. Phagocytes filled with Sudan-stained droplets. Frozen section. FIG. 4.â€”as Figure @.Attempts at the formation of urinifer ous tubules. FIG. 5.â€”as Figure 2. Attempts at glomerulus formation. FIG.6.â€”Xenopuslaevis.Sectionthroughthenormalkidney showing flask cells surrounding the collecting tubules. Periodic acid-Schiffreaction.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1963 American Association for Cancer Research. FIG. 8.â€”Xenopus iaevis, adult female with tumor of the right nostril. Slightly enlarged. FIG. 9.â€”Xenopu@ laevis. Normal epidermis. Compare with Figs. 11 and 1'2. FIG. 10.â€”Xenopus laevis. Transverse section through the region of the right nostril showing facial papilloma. Eroded surface arrowed. XiS. FIG. 11.â€”As Figure 10. Epidermal pocket filled with capil laries, recognizable by the presence of the nucleated erythro cytes they contain. FIG. 1'2.â€”As Figure 10. Epidermal pocket formed by the advancing tumor. Note presence of spherical melanophores in side and outside the pocket. FIG. 13.â€”Rana ternporaria. Facial epithelioma invading the cornea. FIG. 14.â€”As Figure 13. Well differentiated epithelial cells from one of the advancing columns of the tumor. Notice pres ence of cell bridges.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1963 American Association for Cancer Research. FIG. 15.â€”Rana temporaria, adult female. Transverse sec tion through the right maxillary region at the level of the eye, showing an eroding epithelioma below the eye. c, cornea; e, ulcerating epithelioma; h, Harderian gland, invaded by the infiltrating tumor; i, iris; 1, lens; n, nictitating membrane; o, orbital cartilage; r, retina. The region marked X is shown at higher magnification in Figure 13.

Sections through the tumor itself show all the REFERENCES features of an ulcerating epithelioma. The base 1. BALL, H. Melanosarcoma and Rhabdomyoma in Two Pine of the ulcerated area, which was small, consists Snakes, Pituophis melanoteucus. Cancer Res., 6:134-38, of a mass of typical epithelial cells spreading out, 1946. 2. BAua, M. SpontaneousNeoplasmsinAmphibia.A Review in the form of thick strands toward the maxillary and Description of Six New Cases. Cancer Res., 22:442â€” process, the Harderian gland, the ocular muscles, 54, 1962. and the cornea. In some of the most advanced 3. DAWSON, J. W. The Melanomata. Edinburgh: Oliver & strands, found between the ocular muscles, the Boyd, 1925. 4. Doui'uss, R. P.; TIMON-DAVID,J.;and MosINoan, M. features of the epithelial cells were so well pre A Proposdestumeurs mÃ©laniquesdespoissons.Bull. Assoc. served that even the intercellular bridges could be Franc. Ã©tudeCancer, 27:37â€”50, 1958. seen (Fig. 14). Mitotic figures were seen, but they 5. DowNs,A. W. An EpithelialTumour of the Intestinesof were not very frequent. the Frog. Nature, 130:778, 1982. The laminar structure of the cornea did not 6. ELKAN, E. Some Interesting PathologicalCases in A.mphibi am. Proc. Zool. Soc. Load., 134:275â€”96, 1960. allow the tumor to advance in the shape of thick 7. LucicÃ©,B. A Neoplastic Disease of the Kidney of the Frog strands as elsewhere. It had to split up into sheets Ilana pipiens. Am,. J. Cancer, 22:826-84, 1934. which, at the advancing point, are only one cell 8. . Carcinoma of the Kidney of the Leopard Frog. thick (Fig. 13). The Occurrenceand Significanceof Metastases. Am. J. Cancer, 34: 15â€”20,1938. No metastases were discovered in any other 9. . Carcinoma of the Leopard Frog. Its Probable part of the frog. The growth of the tumor was Causation by a Virus. J. Exp. Med., 68:475â€”68, 1938. f@l1owed for 6 weeks, at the end of which time the 10. . Characteristics of Frog Carcinoma in Tissue Cul frog was killed because it had become completely ture. J. Exp. Med., 70:269â€”76, 1939. blind. Although malignant, the tumor was growing 11. Luc@cÃ©,B., and SCHLUMBERGER, H. The Manner of Growth of Frog Carcinoma Studied by Direct Microscopic Ex slowly and did not, apparently, interfere with the amination of Living Intraocular Transplants. J. Exp. general health of the frog. Med., 70:257â€”68,1939. In their natural surroundings frogs have so 12. . Heterotransplantation of Frog Carcinoma. Char many enemies that few @fthem survive to reach acter of Growthin the EyesofAlienSpecies.Ibid., 72:311â€” 20,1940. old age, and no descriptions of senile epithelioma 13. . The Effect of Temperature on the Growth of Frog or rodent ulcers @nongeither free-living or cap Carcinoma. I. Direct Microscopic Observations on Living tive frogs have so far been published. Clinically Intraocular Transplants. Ibid., pp. 321â€”80. and histologically the tumor here described can 14. . Common Neoplasms in Fish, Amphibians and hardly be distinguished from a rodent ulcer, and Reptiles. J. Tech. Math. BU1.mt. Assoc.Med. Mus., 22: 4â€”17,1942. it would be of great interest to know whether 15. . Induction of Metastasis of Frog Carcinoma by this tumor would appear more frequently if a pop Increase of Environmental Temperature. J. Exp. Med., ulation of frogs were allowed to live to the end of 89:269â€”78,1949. their natural life span. 16. NIEUWKOOP, P. D., and FABER, J. Normal Table of Xeno pus taevia (Daudin). Amsterdam: North-Holland Publish ing Co., 1956. ACKNOWLEDGMENTS 17. STOLK, A. Tumours of Fishes. V. Melanoma of the Skin I wish to thank Dr. E. Cotchin of the Royal Veterinary of the Cyprinid Brachydanio rena Hamilton-Buchanan. College,London,forhiskind assistanceintheevaluationof Proc. Kon. Med. Akad. Wetensch., Amsterdam. C, 56: thesetumors. 152â€”56,1953.

E. Elkan

Cancer Res 1963;23:1641-1645.

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