J Oral Maxillofac 50:166-169.

Surg

1992

lmmunohistochemical, Ultrastructural, and Histogenetic Considerations in a Patient With Melanotic Neuroectodermal Tumor of Infancy SUZANA ORSINI MACHADO DE SOUSA, DDS, PHD,* NEY SOARES DE ARAUJO, DDS, PHD,t ANTONIO SESSO, MD, PI-ID,* AND VERA CAVALCANTI DE ARAUJO, DDS, PHD§

Report of Case

The melanotic neuroectodermal tumor of infancy (MNTI) is an uncommon neoplasm of early childhood that has been referred to by a variety of names in the past, reflecting conflicting opinions about its histogenesis. I4 Typically, the tumor arises in the jaws, especially in the maxilla of infants under 12 months old. It usually behaves in a benign fashion, but a few cases of recurrence and metastasis have been described in the literature.7-9 Krompecher,’ in 19 18, published the first report of the tumor, and it was only in 1966 that Bore110 and Gorlin6 reported that the tumor elaborated high levels of vanilmandelic acid, suggesting a neural crest origin because this laboratory finding is shared with other tumors originating from the neural crest: pheochromocytoma, ganglioneuroblastoma, neuroblastoma, and retinoblastoma. This fact led the authors to propose the term melanotic neuroectodermal tumor of infancy for the lesion. In recent years, a few ultrastructural and immunohistochemical studies have been performed to clarify the histogenesis of the tumor. Nevertheless, the results are still inconclusive.‘0-‘5 For this reason, in the present study we examined a case of the tumor using immunohistochemical and ultrastructural observations.

An MNTI located on the right maxilla of a 3-month-old black girl was studied (Fig 1). Tissue for light-microscopic examination was fixed in 10% buffered formalin for 24 hours and embedded in paraffin. Five-micrometer sections were obtained and stained with hematoxylin and eosin. For electron microscopic observation, the specimen was fixed in 2% phosphate-buffered glutaraldehyde, postfixed in 1% osmiun tetroxide, washed in sucrose phosphate buffer, and then immersed in 0.5% aqueous uranyl acetate overnight. Fragments were dehydrated in ethanol and embedded in Araldite (Balzers Tee, Lom, Brazil). Ultrathin sections were stained with lead citrate and uranyl acetate and examined in a Zeiss (D-7082 Oberkochen, Germany) EM 952 electron microscope. Immunostaining was carried out with the peroxidase-antiperoxidase (PAP) method.16 The dilutions of antiserum and each incubation condition were as follows: vimentin (Dakopatts, Copenhagen F, Denmark), 1:200, overnight at 4’C, keratin (Dakopatts), 45 to 55 kd, 1:800, overnight at 4°C; S100 protein (Dakopatts), 1:1,500, overnight at 4°C; and neuron-specific enolase (Dakopatts), l:lOO, overnight at 4°C. Aminoethylcarbazole was used as the chromogen. Positive and negative controls were included in all the preceding procedures. RESULTS

Light Microscopy The examination of sections stained with hematoxylin and eosin showed small round cells having a scanty cytoplasm and round basophilic nuclei grouped in alveoluslike structures (Figs 2, 3). Around those alveoluslike structures larger cells were seen, containing a large, clear, oval nucleus. Sometimes a granular brown pigmentation was present in the cytoplasm of the latter cells. The stroma was fibrous and contained numerous small blood vessels and fibroblasts.

Received from the University of Sao Paula, Brazil. * Assistant Professor, Department of Oral Pathology, School of Dentistry. t Professorand Chairman, Department of Oral Pathology, School of Dentistry. $ Associate Professor, Department of Pathology, School of Medicine. 4 Professorand Chairman, Department of Oral Pathology, School of Dentistry. Address correspondence and reprint requests to Dr Machado de Sousa: Disciplina de Patologia Bucal, Faculdade de Odontologia, Universidade de Sao Paulo, C.P. 8216, Sao Paulo, S.P., Brazil. p.

Electron Microscopy Under the electron microscope, the cytoplasm of the large, pigmented cells contained melanosomes in various developmental stages (Figs 4, 5). Numerous rough endoplasmic reticulum (RER) cistemae appeared stacked. Desmosomelike structures were often observed (Fig 4). Bundles of interme-

0 1992 American Association of Oral and Maxillofacial Surgeons 0278-2391/92/5002-0017$3.00/O

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FIGURE 3. General aspect of the tumor showing small cells arranged in alveolus-like structures surrounded by larger ovoid cells in a fibrous stroma (hematoxylin-eosin, original magnification X 175).

Discussion

FIGURE I.

Clinical appearance of the tumor.

diate-sized filaments frequently crossed the cytoplasm in various directions. They appeared to be bundles of tonofilaments

(Fig 5). Groups of smaller cellular profiles also were found. Nuclei in those profiles were prominent in size, with dispersed chromatin material, and occasional nucleolar transections. The cytoplasm exhibited many flattened RER cisternae.

Immunohistochemistry

The MNTI has been the subject of many studies, mainly because of its obscure origin. Several hypotheses for the origin of the tumor have been postulated, and a variety of names have been used to designate it.‘-’ The theory of the neural crest origin, proposed by Bore110 and Gorlin6 in 1966 has been the most widely accepted. Previously reported cases of MNTI show that it is composed of two cell types, which most authors consider to be a melanocytelike and a neuroblastlike cell. According to the present study, the small cells were positive for enolase, but under the electron microscope these cells did not present any typical feature. such as dendritic processes with microtubules, neurolllaments

The small cells grouped in alveoluslike structures stained positively only for neuron-specific enolase among the tested antibodies (Fig 6). On the other hand, the larger cells stained positively for keratin, vimentin, and neuron-specific enolase. Only S-100 protein, among the antibodies studied, did not stain any of the cell types of the MNTI (Figs 6-8).

FIGURE 2. Low-power view of the mucosa exhibiting the tumor (original magnification X 63).

LlGURE 4. Electron micrograph showing cytoplasmic profiles of the pigmented ceils. Melanin granules at various stages of development are seen near rough endoplasmic reticulum cistemae. Notice desmosomelike structures (arrow) (lead citrate-uranyl acetate, original magnification X30.000).

MELANOTIC NEUROECTODERMAL

TUMOR OF INFANCY

FIGURE 7. Positivity of the large tumor cells for keratin immunoreaction (PAP, original magnification X400).

FIGURE 5. Cytoplasmic detail ofa pigmented cell showing melanin granules and bundles of tonofilaments (lead citrate-many1 acetate, original magnification X40,000).

or secretory granules, that would identify them as real neuroblasts. The large cells were positive for keratin and vimentin, and under the electron microscope these cells exhibited melanosomes in various stages of development and tonofilaments. These findings also were seen in a cell of a recent case of an MNTI occurring in the soft tissue of a child’s thigh reported by Sheck et al.” It is known that the presence of melanosomes and the positivity for vimentin are classically described in melanocytic lesions, whereas the positivity for keratin and the presence of tonofilaments are unusual features for these lesions. In 1989, Miettinen and Franssila,” studying the immunohistochemical spectrum of melanomas, showed that those tumors may contain keratins.

FIGURE 6. Positivity of the small, nonpigmented cells and pigmented cells for neuron-specific enolase immunoreaction (PAP, original magnification X250).

The immunohistochemical aspects of the MNTI have already been discussed.‘4*15 Stirling et alI4 were surprised by the fact that the large tumor cells were positive for keratin and negative for S- 100 protein. These findings were confirmed by our results and by the subcellular study that demonstrated the presence of tonofilaments. Melissari et all5 and Sheck et al,” among other authors, showed ultrastructural features of nerve cells in addition to the immunohistochemical findings of enolase and S-100 protein in some of the tumor cells. The expression of multiple intermediate filaments, and also multiple ultrastructural features, might reflect the immaturity of the tumor cells. It is generally believed that when cells are primitive, many different genes are acting simultaneously, whereas in mature cells the gene expression is suppressed for programmed directions and there are fewer acting genes.” Our results allow us to share the view of many observers that the MNTI originates in the neural crest, of which some cells have shown pluripotentiality.20 The search for cells of neuroectodermal origin in the region where the tumor most commonly occurs has pointed

FIGURE 8. Positivity of the large cells for vimentin immunoreaction (PAP, original magnification X250).

MACHADO DE SOUSA ET AL

to the retinal anlage, the pineal eye, and the vomeronasal organ of Jacobson as the main focus of origin of these cells, according to the majority of authors.5*2’,22 We do not argue these possibilities, but would like to remind that the participation of the neural crest in dental papilla cells is generally admitted in mammals,23 and therefore we cannot rule out the possibility of this origin for the MNTI because clinical findings show that this tumor occurs chiefly in the jaws of young children. If this hypothesis is admissible, the tumor cell phenotype might be interpreted as the result of a failure of specific interactions during the migration of the neural crest cells to become dental papilla cells, on the one hand, and, on the other hand, of environmental influences. Acknowledgment The authors thank Dr Gilbert0 Marcucci for providing the clinical photograph.

References 1. Krompecher E: Zur Histogenese der Adamantinome und sowstiger Kiefergeschwulste. Beitr Path01 Anat 64: 165, 1918 2. Mummery .I. Pitts AT: A melanotic epithelial odontoma in a child. Br Dent J 47: 12, 1926 3. Wass SH: Melanotic adamantinoma of the mandible in a child aged 5 months. Proc Rov Sot Med 4 1:28 1. 1948 4. Sha&r WG, Frissel CT: The melanoamelobla&oma and retinal anlage tumors. Cancer 6:360. 1953 5. Stowens D: A pigmented tumor of infancy-The melanotic progonoma. J Path01 Bact 73:43, 1957 6. Bore110ED. Gorlin RJ: Melanotic neuroectodermal tumor of infancy-A neoplasm of neural crest origin. Report of a case associated with high urinary excretion of vanilmandelic acid. Cancer 19:196, 1966 7. Dehner LP, Sibley RK, Sauk JJ, et al: Malignant melanotic neuroectodemtal tumor of infancy. A clinical, pathologic, ultrastructural and tissue culture study. Cancer 43: 1389, 1979 8. Palacios JJN: Malignant melanotic neuroectodermal tumor of

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infancy. Light and electron microscopy study. Cancer 46:529, 1980 9. Young S, Gonzalez-Cruissi F: Melanocytic neuroectodermal tumor of the foot. Report of a case with multicentric origin. Am J Clin Pathol 84:37 1, 1985 10. Hayward AF, Fickling BW, Lucas RB: An electron microscope study of a pigmented tumor of the jaw of infants. Br J Cancer 231702, 1969 I 1. Cutler LS. Chaudry AP, Topazian R: Melanotic neuroectodermal tumor of infancy: An ultrastructural study, literature review and re-evaluation. Cancer 48:257, 198 I 12. Johnson RE. Scheithauer BW. Dahlin DC: Melanotic neuroectodermal tumor of infancy: A review of seven cases. Cancer 52:661, 1983 13. Dourov N, Mayer R, de Martelaere F, et al: Melanotic neuroectodermal tumor of infancy with high serum levels of alphafeto protein. J Oral Path01 16:25 1, 1987 14. Stirling RW, Powel G, Fletcher CDM: Pigmented neuroectodermal tumor of infancy: an immunohistochemical study. Histopathology 12:425, 1988 15. Melissari M, Tragni G. Gaetti L, et al: Melanotic neuroectodermal tumor of infancy. Immunohistochemical and ultrastructural study of a case. J Craniomaxillofac Surg 16:330, I988 16. Sternberger LA, Hardy PH, Cuculis JJ, et al: The unlabeled antibody enzyme method of immunohistcchemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem I8:3 15, 1970 17. Sheck 0. Ruck P, Harms D, et al: Melanotic neuroectodermal tumor of infancy occurring in the left thigh of a 6-month-old female infant. Ultrastr Path01 13:23, 1989 18. Miettinen M, Fransilla K: Immunohistochemical spectrum of malignant melanoma. The common presence of keratins. Lab Invest 61:623, 1989 19. Kimura N, Nakasto Y, Nagima H, et al: Expression of intermediate filaments in neuroendocrine tumors. Arch Pathol Lab Med 114:506, 1990 20. Noden DM: The migration and cytodifferentiation of cranial neural crest cells, in Prat RM, Christian RL (eds): Current Research Trends in Prenatal Craniofacial Development. New York, NY, Elsevier, 1980, p 3 2 1. Halpert B, Patzer R: Maxillary tumor of retinal anlage. Surgery 221837. 1947 22. Clarke BE, Parsons H: An embryological tumor of retinal anlage involving the skull. Cancer 4:78, 195 I 23. Ruth JV: Development, form and function of odontoblasts. Odontoblast differentiation and the formation of the odontoblast layer. J Dent Res 64:489, 1985

Immunohistochemical, ultrastructural, and histogenetic considerations in a patient with melanotic neuroectodermal tumor of infancy.

J Oral Maxillofac 50:166-169. Surg 1992 lmmunohistochemical, Ultrastructural, and Histogenetic Considerations in a Patient With Melanotic Neuroecto...
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