Odontogenic tumors and epidermoid carcinomas of the oral cavity

Odontogenic tumors and epidermoid carcinomas of the oral cavity

Research Odontogenic tumors and epidermoid carcinomas of the oral cavity An experimental Katherine LABORATORY study McD. Herrold, OF PATHOLOGY, ...

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Odontogenic tumors and epidermoid carcinomas of the oral cavity An









Bethesda, Md.




he subject of odontogenic tumors in man and domesticated animals was extensively reviewed by Gorlin, Meskin, and Brodeyl in 1963. According to those authors, the field of odontogenic tumors in laboratory animals has been largely unexplored. Schour and Massleg have stated that valuable clues to the solution of physiologic and metabolic problems are waiting to be discovered in the teeth of experimental animals, The teeth of rodents are much more than masticatory organs. They are valuable biologic indicators which, during their development, mirror and record the metabolic status of the animal. The carcinogenic property of N-methyl-N-nitrosourea (NMIJ) is well documented by many publications, but its mechanism of action is unknown.3-5 It is possible that clues to the metabolic effects of carcinogenic substances may be found in the kymographic records engraved on the teeth of rodents. The present report describes odontogenic tumors and epidermoid carcinomas of the oral cavity induced in Syrian hamsters with NMU. The similarity of these induced tumors to human odontogenic tumors may shed light on their histogenesis. MATERIALS Experimental


Syrian hamsters, 1 month old, of either sex were used. The animals were separated by sex and housed in plastic cages in groups of five. N-methyl-N-nitrosourea

The N-methyl-N-nitrosourea solutions (K & K Laboratories, Inc., Plainview, N. Y.) were always freshly prepared prior to use. The chemical (NMU) was dis-

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solved in distilled water for intragastric sodium chloride solution for intravenous Experimental

a.nd epide~rmoid

administration administration,



and in 0.85 per cent


There were two groups, designated A and B. Group A (sixteen animals) received 0.1 to 0.2 ml. (2.5 to 5.0 mg.) of NMU intravenously by means of injections into the lateral vein of the foot at monthly intervals for 3 to 4 months. The NMU was administered intragastrically to Group B (ten animals). Prior to feeding by intragastric tube, the hamsters were fasted from 4 P.M. of the preceding day. An Argyle infant feeding tube (size 8, French, 15 inches long) with attached tuberculin syringe was filled with the solution. Before insertion, the outside of the tube was wiped clean to reduce the possibility of a local action of NMU on the mueosa of the oral cavity. The tip of the tube was guided into the forestomach and 0.5 ml. (1 mg.) NMU was administered. The schedule for intragastric administration was twice a week for 4 months. Complete autopsies were performed on all animals killed or found dead. The tissues were fixed in 10 per cent buffered formalin solution. Prior to fixation of the head, the skin and vertex of the skull were removed. Decalcification of the intact skull was carried out in 5 per cent formic acid. Multiple frontal sections of the head, 3 to 4 mm. thick, were made. In most instances step sections at three levels were made of each block. Paraffin sections cut at 6 microns were stained with hematoxylin and eosin. RESULTS

The present report describes only the odontogenic tumors and epidermoid carcinomas of the oral cavity that were induced in Syrian hamsters by NMU, Specific lesions and benign and malignant tumors produced by this carcinogen at other sites will be the subject of a separate report. A summary of the results-including the effective number of hamsters in each experimental group, the average life span, total dose (in milligrams) oi’ NMU received, the lesions, and the frequency of tumors of the odont,ogenic apparatus and epidermoid carcinomas of the oral ca.vity---is shown in Table 1. he animal in Group A died at 51/z weeks, and sections were not available from two animals (one in Group A and one in Group B) because of cannibalism. The r;lnpv Table I. Effect of N-methyl-N-nitrosourea in Syrian hamster

Effective number








on the incisor teeth and oral carit!

Route of





spas (months)

tration NYU

NMU (w.)


Alteration of dentine

Reaotion Of periodo&al

1 E’$-. carci-


Odontogenie tzlmors

of oral t?avity





7.5 to 12.5








w 9

9/ 9

6/ 9

6/ 9


O.S., O.M. 6%O.P. February, 1968


Table II. Odontogenic tumors induced in Syrian hamsters by N-nitroso-Nmethylurea Number of twmors Cla.ssifioation

Type oftwtncw

Epithelial ductive tissue

tumors without inchanges in connective


Epithelial ductive tissue

tumors with inchanges in connective

Ameloblastic fibroma Ameloblastic odontoma Complex odontoma Compound odontoma Totals




Grozlp B




3 4 0 1 -iiT

: 0 -ii-

in life span for Group A animals was 4 to 14 months, with an average survival time of Sl/, months; for Group B, the range was 6 to 10 months, with an average of 8 months. The total dose of NMU received was 7.5 to 12.5 mg. for Group A and 32.0 mg. for Group B. ODONTOGENIC


The odontogenic tumors induced in Syrian hamsters by NMU were classified according to the scheme proposed by Pindborg and Clausen in 1958, which is based mainly on embryologic principles. Table II shows the method of classification, the types of odontogenic tumors, and the number of tumors observed in each experimental group. Most of the tumors originated in the mandibular incisors, and only occasionally did lesions originate in the upper incisors. With the exception of the ameloblastomas, which were found only in Group B animals, the same tumor types were observed in both experimental groups. Four animals had more than one type of tumor. Ameloblastoma

Grossly, the animals showed unilateral enlargement of the mandibular region, with marked swelling and induration of the adjacent tissues. These tumors had the characteristic microscopic appearance of the acanthomatous type of ameloblastoma. Interlacing strands and islands of cells were embedded in a connective tissue stroma. The cells outlining the epithelial aggregates were clearly odontogenie in character and were characterized by peripheral palisading of the cells. The central portion of the epithelial islands revealed keratinized and parakeratinized epithelial pearls (Fig. 1). Cystic degeneration was frequent in the epithelial masses.The tumors were locally invasive, destructive, and extended into the opposite side of the jaw. No distant metastases were observed. Microscopic examination revealed nests of odontogenic epithelium located in the periodontal membrane of the mandibular incisors of three animals. Histologically, these lesions bear a striking similarity to the ameloblastomas observed, and it is possible that this lesion is an early stage in the development of this type of tumor (Fig. 2).

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tumors and epidermoid carcinomas 265

Fig. 1. Ameloblastoma with acanthomatous pattern. (Magnification, x125.) Fig. 8. Island of odontogenic epithelium in periodontal membrane of mandibular incisor. Note peripheral palisadin of cells. (Magnification, x225.) Fig. PI. Ameloblastic fi f roma with ramifying islands of odontogenic epithelium. (Magnifb:ation, x380.) Fig. 4. Ameloblastic odontoma showing ameloblastoma-like islands of proliferating odontogcnic epithelium with irregular zones of dentine and pre-enamel. (Magnification, x125.) (All sections stained with hematoxylin and eosin.)



O.S.,O.M. &O.P. February, 1968




Microscopically, these tumors were composed of ramifying islands and nests of epithelial cells in a loose, cellular, connective tissue stroma. For the most part, the cells composing the strands were one or two cell layers in thickness, cuboidal in shape, and resembled the dental lamina (Fig. 3). Ameloblastic


These tumors showed ameloblastoma-like islands of proliferating odontogenic epithelium, together with irregular zones of matrix histologically resembling dentine and pre-enamel (Fig. 4). Complex


The complex odontoma differs from the ameloblastic odontoma in the absence of ameloblastic tissue. Microscopically, these tumors consisted of conglomerate massesof a matrix resembling dentine and cementum (Fig. 5). Compound


The calcified structure in these tumors showed an anatomic similarity to normal teeth, although they were small and of abnormal shape (Fig. 6). Ectodermal



On gross examination, three animals were thought to have tumors of the lower jaw because of the swelling and induration of the tissues. Microscopic examination revealed cysts lined with noncornified squamous epithelium and an osteoblastic reaction of the periodontal membrane (Fig. 7). In two animals early cystic lesions were developing in the stratum intermedium of the enamel organ. The cysts were lined with squamous epithelium, and there was an associat,edinflammatory component. The ameloblastic epithelium was atrophied. PERIODONTAL


All animals in both experimental groups revealed nests of cells, which varied in number and type in the periodontal membrane predominantly of the lower incisors. The most frequent type observed resembled the epithelial rests of Malassez. The peripheral cells in these oval nests showed a tendency to palisade (Fig. 8). In my experience, these so-called epithelial rests have never been seen in the periodontal membrane in untreated hamsters, but they have been noted in hamsters treated with diethylnitrosamine (DENA). Other clusters of cells had a squamous appearance (Fig. 9). Sometimes there was partial condensation or hyalinization of fibrous connective tissue about these epithelial islands. The possibility that in some instances these cells represented direct local invasion of the periodontal membrane by epidermoid carcinomas of the oral cavity was considered. Definite invasion by local extension of these malignant tumors was noted (Fig. 10). However, the islands of squamous epithelium were observed in animals that did not have tumors and showed only epithelial atypism of the oral mucous membrane. The central portion of the epithelium sometimes showed cystic degeneration and keratinization (Fig. 11). Small denti-

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Fig. 5. Complex odontoma. Conglomerate masses of matrix resembling dentine, enamel, and x108.) cer nentum. (Magnification, Fig. 6. Compound odontoma. Small and abnormally shaped calcified stru&res which to normal teeth. (Magnification, x60.) sherw anatomic similarity Fig. 7. EctodermaI odontogenic cyst. Lumen of cyst contains inflammatory cells and lining epithelium is noncornified squamous. Note prominent, ostcoblastic reaction of periodontal numrx40.) brr tne. (Magnification, Fig. 8. Odontogenie epithelial rests of Malassez in periodontal membrane of lower in&or. [agnifieation, x380.) (All sections stained with hematoxylin and nosin.)



Fig. 11


OS, O.M.&O.P. February, 1968





Fig. 9. Nests of cells with squamous appearance in periodontal membrane. Clusters cIf cells in upper right resemble epithelial rests of Malassez. (Magnification, x150. ) Fig. 10. Epidermoid carcinoma of gingiva which has invaded periodontal membrane. Wagni fication, x55.) Fig. 11. Nests of squamous epithelium in periodontal membrane showing cystic deg:eneraNote irregular mass of matrix to left which resembles dentine. Wag ti on and keratinization. ni .fication, x135.) Fig. 18. Denticles in periodontal membrane of lower incisor. (Magnification, x225.) (All sections stained with hematoxylin and eosin.)

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tumors and epidermoid




Fig. in Fig. 100.) Fig. n.ifieation, Fig. n.ification, 0 rigin

13. Lingual side of upper incisor with clusters of cells which suggest enamel organ periodontal membrane. (Magnification, x125.) 14. Upper incisor with primitive type of dentine with cell inclusions. (Magnification. 25. Lower incisor showing infolding of dentine and inductive effect in pulp. x40.) 16. Epidermoid ‘carcinoma of palate which has invaded nasopharyngeal tube. x50.) (All sections stained with hematoxylin and eosin.)

(Msg. (&lag



O.S., O.&f. & O.P. February, 1968

cles or toothlike structures were noted occasionally (Fig. 12,). Incisors that showed either extreme thinning or complete absence of dentine on the lingual side revealed clusters of cells suggestive of enamel organ origin (Fig. 13). DISTURBANCES IN ODONTOGENESIS

Faulty odontogenesis is considered to be the basis for the development of odontogenic tumors. The alterations in the incisor teeth, which indicated disturbed odontogenesis, were observed primarily in the dentine and, to a lesser extent, in the enamel organ and pulp. The dentine was often irregular in width, with areas of extreme thinning or absence on the lingual side. The thickness of the dentine on the labial side was usually normal as contrasted with the remainder of the tooth. There were areas of interglobular dentine and formation of an immature, primitive type of dentine with cell inclusions, the so-called osteodentine (Fig. 14). Often the dentine sharply folded, and the disorganized dentine lacked proper morphodifferentiation (Fig. 15). The odontoblasts were normal, for the most part, but sometimes appeared elongated and hyperplastic. An occasional incisor and mandibular molar revealed necrosis of the pulp tissue (including the odontoblasts), distortion of the pulpal outline, and small areas of osteoid tissue in the pulp. The ameloblasts generally were unaffected except for an occasional focal area of atrophy, flattening of the epithelial cells, or squamous metaplasia. EPIDERMOID CARCINOMAS


The site of origin for epidermoid carcinomas of the oral cavity included the buccal mucosa, gingiva, tongue, and hard palate. Frequently the tumors were multicentric. Histologically, all the tumors were epidermoid carcinomas, but the degree of differentiation varied. Keratinization was prominent in some, whereas others were less well differentiated. The tumors were locally invasive and extended into the soft tissues of the face, alveolar bone, bone marrow, and periodontal membrane, and polypoid massesof tumor projected into the lumen of the nasopharyngeal tube (Fig. 16). Nests of tumor cells were noted in blood vessels, and one animal in Group B had metastatic nodules of epidermoid carcinoma in the lung, It was impossible to determine the primary site for the metastases, for this animal also had a primary epidermoid carcinoma of the esophagus and forestomach. In addition to the tumors, the epithelium of the oral cavity revealed focal areas of hyperkeratosis, basal-cell hyperplasia, epithelial atypism, and squamouscell papillomas. DISCUSSION

The tumors described in this report that were induced in Syrian hamsters by NMU are histologically identical to types of odontogenic tumor which occur in man. These tumors were classified according to their human counterparts. Spontaneous tumors of this type have never been observed or reported to occur in Syrian hamsters. Bullock and Curtis’ described the types of spontaneous tumor which they found in 489 rats of the August strain. Two animals had lower

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jaw lesions which were classified as odontomas. These were epithelial tumors showing inductive change in the connective tissue, and one of the tumors contained rudimentary teeth. All of the nitrosamine compounds so far investigated, with the exception of N-nitroso-N-methylurea, have only an indirect carcinogenic effect, whereas NMU has both an indirect and a direct carcinogenic action. NMU procuces malignant tumors at the site of local application. Epidermoid carcinomas of the skin, trachea, bronchi, larynx, nasopharyngeal tube, pharynx, and esophagus arc induced by topical application, and undifferentiated sarcomas develop at the site following subcutaneous injection. If NMU is injected parenterally, the indirect carcinogenic effect shows a high degree of specificity for the squamous epithelium of the oral cavity, esophagus, forestomach, lower third of the vagina (which, in hamsters, is of the squamous type), the mucosa of the small intest,ine and colon, and the central and peripheral nervous systems. The acute toxic effects involve the glandular stomach with ulceration and hemorrhage and the hemat,opoietic system with aplasia of the bone marrow and cystic hemorrhagic lesions of the spleen complicated by rupture with hemoperitoneum and pigmentary degeneration of the retina.5, s, Q The effects induced by NMU, as described in this report, include disturbances in odontogenesis, odontogenic tumors, and epidermoid carcinomas of the oral cavity. The mechanism by which NMU exerts its carcinogenic action and biologic effects is unknown. It is possible and likely that several distinct and different, mechanisms may be operative at either t,he biochemical or intracellular level. Vitamin A is concerned primarily with t.he process of differentiation of epithelial cells, rod function in the retina, and the normal development of teeth. The mechanism by which vitamin A exerts its biologic functions is obscure, but rarcinogenic substances have been shown to alter its metabolism.10 Vitamin A deficiency is the most important of the known vit.amin deficiencies in its effect upon the normal development of the incisor teeth in rats, guinea pigs, and hamsters.lll I2 Burn, Orten, and SmithI” described odontomas in 62 per cent of rats in which a. chronic vitamin A deficiency was maintained for periods up to 365 days. In a few animals supernumerary incisor teeth were also observed.13 NMMU or one of its metabolites could function as a vitamin A antagonist, and this action might be a possible explanation for the induction of odontogenic tumors and the disturbances in odontogenesis caused by this carcinogen. There are, however, striking differences between the effects produced in the incisors of rodents by a chronic vitamin A deficiency and by NMU. The tumors or odontoman described by Burn, Smith, and Orten which developed in rats with a chronic vitamin A deficiency resulted from an overgrowt,h of pulp tissue, Although these tumors sometimes contained dentinal or osteodentinal structures and inclusions of odontoblasts, histologically they resembled cellular fibromas. The only reaction observed in the periodontal membrane was an occasional slight inflamm;ltory response. With NMU, the target tissue is primarily the periodontal membrane, and the odontogenic tumors are definitely of epithelial rather than connective tissue origin. This experimental model of induced odontogenic tumors a.nd assoeiatcd

272 Herrold

OS., O.M. & O.P. February, 1968

disturbances in odontogenesis produced by NMU in Syrian hamsters may provide not only a means for studying the histopathogenesis of these unusual tumors but also a method for determining whether certain types, such as an ameloblastic fibroma, mature into a compound odontoma. SUMMARY

Odontogenic tumors and epidermoid carcinomas of the oral cavity induced in Syrian hamsters by N-nitroso-N-methylurea (NMU) have been described. The induced odontogenic tumors show a striking histologic similarity to the human counterparts of this type of tumor. The epidermoid carcinomas were often multicentric and locally invasive. The sites of origin included the gingiva, buccal mucosa, palate, and tongue. The incisor teeth revealed disturbances in odontogenesis. The changes were characterized principally by alterations in morphodifferentiation and histodifferentiation of the dentine. The periodontal membrane reacted by proliferation of nests of epithelial cells which histologically resembled odontogenic epithelium. Occasionally, the clusters of cells had a squamous appearance. REFERENCES

1. Gorlin, R. J., Meskin, L. H., and Brodey, R.: OdontogenicTumors in Man and Animals: Patholoaic Classification and Clinical Behavior-a Review, Ann. New York Acad. SC. 108: 722-771; 1963.

2. &hour, I., and Massler, M.: The Teeth. Irt Farris, E. J., and Griffith, J. Q. (editors) : The Rat in Laboratory Investigation, ed. 2, Phildelphia, 1949, J. B. Lrppincott Company, pp. 104-165. 3. Druckrey, H., Steinhoff, D., Preussmann, R., and Ivankovib, S.: Erzeugung von Hrebs durch eine einmalige Dosis von Methylnitrosoharnstoff und Verschiedenen Dialkylnitrosaminen an Ratten, Ztschr. Krebsforsch. 66: l-10, 1964. 4. Druckrey, H,, Ivankovib, S., and Preussmann, R.: Selektive Erzeugung maligner Tumoren im Gehirn und Riickenmark von Ratten durch N-Methyl-N-Nitrosoharnstoff, Ztschr. Krebsforsch. 66: 389-408, 1965. Administered Subcutane5. Herrold, K. M.: Carcinogenic Effect of N-Methyl-N-Nitrosourea ously to Syrian Hamsters, J. Path, & Bact. 92: 35-41, 1966. 6. Pindborg, J. J., and Clausen, F.: Classification of Odontogenic Tumors; a Suggestion, Aeta odont. scandinav. 16: 293-301, 1958. 7. Bullock, F. D., and Curtis, M. R.: Spontaneous Tumors of the Rat, J. Cancer Res. 14: l-115, 1930. 8. Herrold, K. M.: Pigmentary Degeneration of the Retina Induced by N-Methyl-N-Nitrosourea; an Experimental Study in Syrian Hamsters, Arch. Ophth. 78: 650-653, 1967. 9. Herrold, K. M.: Unpublished data. Green, J.: Antagonists of Vitamin A, Bibl. nutr. et dieta 8: 33-43, 1966. :1”: Wolbach, 5. B., and Howe, P. R.: The Incisor Teeth of Albino Rats and Guinea Pigs in Vitamin A Deficiency and Repair, Am. J. Path. 9: 275293, 1933. 12. Salley, J. J., and Bryson, W. F.: Vitamin A Deficiency in the Hamster, J. D. Res. 36: 935.944, 1957. 13. Burn, C. G., Orten, A. U., and Smith, A. II.: Changes in the Structure of the Developing Tooth in Rats Maintained on a Diet Deficient in Vitamin A, Yale J. Biol. & Med. 13: 817830, 1941.