Case reports
A variant of calcifying epitheiial odontogenic tumor with Langerhans celis
Masatake Asano\ Tomihisa TakahashP, Kaoru Kusama\ Takashi lwase\ Minoru Hori^ Hiromitsu Yamanoi^ Hiroshi Tanaka^ and Itaru Moro^ Departments of 'Pathology and 'Oral Surgery, Nihon University School ol Dentistry, Tokyo, Japan
Asano M, Takahashi T, Kusama K, Iwase T, Hori M, Yamanoi H, Tanaka H, Moro I; A variant of ealcifying epithelial odontogenie tumor with Langerhans cells. J Oral Pathol Med 1990; 19; 430-4. A variant of calcifying epithelial odontogenic tumor (CEOT) with Langerhans eells is reported. Compared to a typieal CEOT, the tumor islands of this case were thin and composed of a small number of polyhedral epithelial eells. Almost no ealcifieation of homogenous eosinophilie materials was observed. In addition, clear cells which structurally corresponded to Langerhans cell were intermingled in the epithelial islands. These eells stain positively for S-IOO protein, lysozome, MT 1, LN-3 and OKT 6 antibodies, but not for keratin antibody. Electrontnicroseopie examination revealed the rod-shaped and racket-shaped structures called Birbeek's granules in the cytoplasm of these elear cells. Our observations indieate a variant case of CEOT with Langerhans cells in tumor nests.
The calcifying epithelial odontogenic tumor (CEOT) is a relatively rare tumor described by PINDHORG as a distinct entity (1, 2). CEOT, often referred to as Pindborg tumor, is composed of polyhedral epithelial cells with round, hyperchromatic nuclei and fibrous connective tissue. The presenee of homogenous eosinophilie material is a characteristic feature of this tumor (1-5). Caleification in the eosinophilie material is often observed, but there are also reported eases with a paucity of calcification (6-8). The nature of the eosinophiiic material is generally considered to be amyloid-like, but others find the similarities to enamel matrix or pre-enamel, basal lamina, keratin protein and tuft enamel protein (9-11). The origin of this tumor is eontroversial, but it is considered to be derived from reduced enamel epithelium, oral epithelium or stratum intermedium (2, 5). Several reports have indieated the presenee of elear eells with glycogen granules in the CEOT (7, 12-15). These eells are eonsidered to be either degenerating, or eells with some features of cytodifferentiated epithelial eells (12, 15). It is well known that Langerhans cells are found in the oral epithelium and
skin (16-19). However, there have been no reports of Langerhans cells in a CEOT. We report here a variant of CEOT with many Langerhans cells in epithelial tumor nests.
Key words: calcifying epithelial odontogenic tumor; jaws, neoplasms; Langerhans cell; S100 protein. Itaru Moro, Department of Pathology, Nihon University School of Dentistry, 1-8-13, KandaSurugadai, Chiyoda-ku, Tokyo 101, Japan Accepted for publication July 22, 1990.
Material and methods Clinical history
A 44 yr-old woman visited the Division of Oral Surgery, Kasukabe Hospital with a chief complaint of gingival swell-
Fig. I. Preoperative panoramic radiograph showing unilocular, well-defined, radiolucent area extending from maxillary right central incisor to right first molar.
CEOT with Langerhans cell 431 ing in the region extending from the maxillary left central incisor to the right molar. She had noted this slow-growing lesion for several years, but had neglected to report it to her dentist beeause of no pain. Her past medical history was completely unremarkable and indicated no evidence of systemic diseases. Oral examination revealed one tnissing tooth in the maxillary right first premolar area that was extracted because of dental caries about 10 yr previously. The other teeth were all vital except for the maxil-
lary right central incisor. Radiographic examinations revealed unilocular, well defined, radiolucent area from the maxillary right central incisor to the right first molar (Fig. I). There were no radiopaque foci in the lesion, Resorption of the roots of the tnaxillary central and lateral incisors and canine was detected. A clinical diagnosis of ameloblastoma was made while histologic examination of biopsied tissue by Congo red staining revealed CEOT. Under general anesthesia, partial re-
Antibodies
Company
Dilution
Reactivity
Keratin S-100 Lysozyme MT 1 LN 3 OKT 6
DAKO Co. DAKO Co. DAKO Co. Bioscience Co. Seikagaku Kogyo Ortho Co.
X50 X50 X250 X50
Keratin filament S-100 protein Lysozyme 190 KD, 110 KD antigen HLA-DR antigen CD 1 antigen
X50 X 50
Table 2. Histochemical and Immunohistologic Stainings of CEOT Epithelial islands
Congo red Congo red (birefringence) Thioflavin T Methyl violet Keratin S-100 Lysozyme MT I LN3 0KT6
Histologic procedure
The divided samples were fixed in 10% buffered formalin, embedded in paraffin and processed for routine histologic examination. Immunologic procedure
Table I, First antibodies
Staining
section of the maxilla was performed. The whole neoplasm obtained after operation was divided into several portions and processed for histologic, immunologie and electron microscopie examinatiotis.
Clear cell — — — — — + -H -1-h +
Tumor eell
Homogenous eosinophyilic Materials
Light pink Blue birefringence Yellow fluorescence Metaehrotnasia
Light pink Blue birefringence Yellow fluorescence Metaehromasia
-ij-
Sorne samples were embedded in OCTeompound and quickly frozen with dry ice and acetone. Six iitn frozen sections were cut by cryostat,fixedin acetone for 10 min and processed for immunologic staining. In addition, some of the fortnalin-fixed, paraffin embedded sections were also used for irnmunologic staining. In this case, deparaffinized seetions were pretreated with 0.1% pronase to increase antigenicity. An avidin-biotin-peroxidase complex (ABC) method was used to detect antigens in tumor tissues. Briefly, sections were immersed in methanol containing 0.3% HjOj for 20 min to block endogenous peroxidase aetivity and washed with phosphate buffered saline (PBS), pH 7.2, three times. Washed sections were incubated with normal horse serum (for monoclonal antibodies) or normal goat serum (for polyelonal antibodies) for 20 min and subsequently with first antibodies for 45 min at room temperature. After washing with PBS, biotinylated horse anti-mouse IgG or biotinyiated goat anti-rabbit IgG were applied to tissue sections, then avidinbiotin-peroxidase cotnplex using Vectastain Kit (Vector) and 0.05% 3,3'-diaminobenzidine tetrahydrochloride containing 0.05% H,0, substrate was added for 10 min. Sections were washed, counterstained with hematoxylin and mounted. First antibodies used in this study are given in Table 1. As positive controls, hutnan liver with amyloidosis and human normal gingiva were used. Negative controls were prepared by substituting PBS instead of first antibodies. Electron microscopic procedure
Fig. 2. Overall view of this tumor showing small epithelial islands and eosinophiiic materials with almost no ealeifieation. H-E stain, x 4
Some portions of the tutnor tissue were cut into small pieces with a razor blade and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH.7.4) for 2 h.
432
ASANO et al.
F/g. 5. Immunohistochemical staining of various antigens in CEOT. A, distribution of keratin in the epithelial island. Keratin was detected only in the tumor cells, x 10. B, higher magnification of A. Clear eell (arrow) in epithelial island stained negatively for anti-keratin antibody. x20. C, distribution of S-100 protein in epithelial island. Arrows pointed out S-100 protein positive cells in epithelial island, x 20. D, lysozyme positive cells (arrows) were intermingled in the epithelial island. x40. E, MT-1 positive eells were localized in epithelial island (arrow) as well as in stroma. x 40. F, LN3 in epithelial island (arrow) and in infiltrated cells of the stroma. x40.
then fixed again in 1 % osmium tetraoxide for 1 h. Specimens were dehydrated with graded ethanol and propylene oxide, and embedded in Spurr's resin. Thin sections were cut with a diamond knife, stained with uranyl aeetate and lead citrate and observed under a JEOL T-lOO eleetron microscope. Results The tumor masses consisted of thin strands of polyhedral epithelial eells and loose fibrous connective tissue (Fig. 2). The epithelial islands were composed of five to twenty cells and clear cells were frequently found in the islands. The cy-
toplasm of the polyhedral cells was eosinophiiic and finely-granular. The nuclei were relatively dense and contained a single nucleolus. There was moderate inflammatory cell infiltration with lymphocytes and plasma eells in the fibrous connective tissue. In addition, a large number of globular masses of pale, homogenous eosinophiiic material were localized within the epithelial cells as well as within the fibrous connective tissue. It was very rare to find ealeifieation in the homogenous eosinophiiic material. Histoehemieal examination of this material revealed metaehromasia with crystal violet and methyl violet stains and positive reac-
tion with Congo red stains. Weak birefringence was observed under polarized microscopy after staining with Congo red. Thioflavin T stained seetions exhibited yellow fluorescence under fluorescent microscopy. Immunohistochemically, keratin was deteeted only in tumor eells of the epithelial islands (Fig. 3A), while there was no positive reaction in the clear eells, homogenous eosinophiiic material or inflammatory cells infiltrating the stroma (Fig. 3B). Occasionally, one or two cells positive for S-100 protein (Fig. 3C), lysozyme (Fig. 3D), MTl (Fig. 3E), LN3 (Fig. 3F) or OKT 6 antibodies were identified in an epithelial island corresponding to the area of clear eells. Variable numbers of cells positive for these antibodies were also present in the inflammatory cells in the stroma. However, there were no positive reaetions for these antibodies in epithelial tumor cells nor the homogenous eosinophiiic material. The histoehemieal and immunohistochemical Staining results of this case are given in Table 2. Eleetronmieroscopically, the epithelial islands were composed of polyhedral eells with round nuelei and prominent nueleolus. The cytoplasm contained several mitochondria, bundles of tonofilaments, dense bodies, polyribosomes and small ill-developed endoplasmic reticulum. A large number of irregularly interdigitating microvilli were present between tumor eells and desmosomes were occasionally found in adjaeent cells. In some epithelial islands, clear eells with indented nuelei which corresponded to Langerhans eells, were intermingled among polyhedral tumor cells (Fig. 4). The clear cytoplasm contained a few mitochondria, dense bodies, small vesicles, multivesicular bodies, endoplasmie retieulums, small Golgi apparatuses, but no tonofilaments nor melanosome. No desmosomes were observed between elear cells and neighbouring tumor cells. The rod-shaped and racketshaped structures called Birbeek's granules were often seattered throughout the cytoplasm (Fig. 5, arrows). The homogenous material was located both intracellularly and extracellularly. It consisted of densely packed accumulations of fine-fibrillar material. Discussion It has been well-documented that the histologic characteristics of CEOT are the presence of homogenous eosinophil-
CEOT with Langerhans cell 433
l>Ai%^s:
cell in CEOT. Our histologic and electron microscopic results clearly revealed the presence of Langerhans eell in the CEOT of our ease. The Langerhans eell, whieh belongs to the mononuclear phagocyte system, is of bone marrow origin and is often found in the skin and oral mucosa (16-18). It has been clearly ascertained that Langerhans cells function as antigen presenting cells and as allogenic stimulatory cells to primed T lymphocytes in the epithelium (16, 20). Immunologically, Langerhans cells express la surface antigen and Fey and C3 receptors (20-22). It has been reported that Langerhans cells stain positively for anti-CD 1, CD3, CD4and S-100 protein antibodies (23, 24). Our immunologic examination confirmed the presence of Langerhans cells in CEOT. In addition, T6 as well as lysozyme positive cells were observed in the epithelial nest. These results probably indicated the presenee of T eell and maerophage in the epithelial nest. Neoplastic proliferation of Langerhans cells has been observed in histiocytosis X which includes eosinophiiic granuloma of bone, Hand-SchlillerChristian disease and Letterer-Siwe disease (25). In the field of dermatology, Langerhans cells have often been found in keratoacanthoma and squamous cell carcinoma (26). It has been reported that increased numbers of Langerhans cells in some cases of keratoacanthoma resulted in the regression of lesions (26). BROCKER et al. (27) reported a signifi-
cant correlation between the number of Langerhans eell and aetivation of T lymphocyte in malignant tnelanoma. MEISSNER et al. (28) reported that the
number of T6 positive Langerhans eell was lower in epithelial skins tumors such as basal and squamous cell earcinoma compared with a sex and age Schiff (PAS) reaction and were consid- matched control group and suggested ered to be either degenerating cells or that the development of skin tumor may to represent a feature of cytodifferentia- be favored by impaired immunologie tion (7, 12, 15). The clear cells found in surveillance of neoantigen-expressing our case were different from those of tumor cells resulting from reduction of previously reported eases. The clear Langerhans eell number and function. eells in our case exhibited no positive TsujiTANi et al. (29) reported that the stain for PAS reaction and had clear number of Langerhans cells influences cytoplasm and indented nuclei. No to- the prognosis of gastric carcinoma. nofilaments and melanosomes were ob- These reports suggest some correlation served and no formation of desmo- between tumor regression arid the somes between neighboring cells was number of Langerhans cell. In our case, found. The Birbeek's granules, whieh a fairly large number of Langerhans are a morphologic characteristic of cells were observed. These Langerhans Langerhans cell, were deteeted (19). To cells may play an important role in our knowledge, there has been no report antigen presentation or regression of concerning the presence of Langerhans CEOT.
F/g. 4. Electron micrograph of Langerhans cell in epithelial island. Langerhans eell had clear cytoplasm and no desmosome between neighbouring eells. Arrows indieating Birbeek's granules, x 19600.
ic material that show amyloid-like reaction and often exhibit calcification (1-8). In addition,tumor islands are generally composed of densely-arranged polyhedral epithelial cells. The case reported here is unusual eompared with that of a typieal CEOT. Although the homogenous eosinophiiic tnaterial showed amyloid-like reactions, there was no calcification. The tumor islands were composed of small numbers of epithelial cells with clear cells corresponding to Langerhans eells. The presence of clear cells in CEOT has been described (7, 12-15). These clear cells contained glycogen granules, which stained positively with periodic aeid
434
ASANO et al. 15. YAMAGUCHI A , KOKUBU JM, TAKAGI M ,
ISHIKAWA G. Calcifying epithelial odontogenie tumor: histochemical and eleetron microscopic observations on a case. Butt Tokyo Med Dent Univ 1980; 27: 129-35. 16. LASSER A . The mononuclear phagocyte system. A review. Hutn Pathot 1983; 14: 108-26. 17. SLOBERG K , JONSSON R , JONTELL M . As-
sessment of Langerhans cells in oral lichen pianus using monoclonal antibodies. J Orat Pattwt 1984; 13: 516-24. 18. PITIGALA-ARACHCHI A, CRANE IJ, SCUL-
LY C, PRIME SS. Epithelial dendritie eells in pathological human oral tissues. J Otat Pattwt 1989; 18: 11-6. 19. SAEBIEL RW, REED T H . Serial reconstruc-
tion of the characteristic granule of the Langerhans eell. J Cell Biol 1968; 36: 595-602. 20. SHELLEY W, JUHLIN L . Langerhans cells
form a reticuloendothelial trap for external contact allergens. Nature 1976; 261: 46-7. 21. ROWDEN G , LEWIS MG, SULLIVAN AK.
la antigen expression on human epidermal Langerhans cell. Nattire 1977; 268: 247-8. 22. STINGL G , KATZ SJ, CLEMENT L , GREEN
J, SHEVACH EM. Immunological functions of la - bearing epidermal Langerhans cells. J Itnmunot 1978; 121: 200513. /^/g. 5. Higher magnification of Fig. 3. Rod-shaped and racket-shaped Birbeek's granules were seen, x 65500.
References 1. PINDBORG JJ. Calcifying epithelial odontogenic tumor. Acta Pathol Microbioi Seand 1955; 3 (suppl): 71 (abstr.). 2. PINDBORG JJ. A calcifying epithelial odontogenic tumor. Catteer 1958; 2: 838-43. 3. VicKERS RA, DAHLIN DC, GORLIN RJ.
Amyloid containing odontogenic tumor. Oral Surg Oral Med Orat Pathot 1965; 20: 476-80. 4. RANLOV P, PINDBORG JJ. The amyloid
nature of the homogeneous substance in the CEOT. Aeta Patttot Mierobiot Scand 1966; 68: 169-74. 5. MAINWARING AR, AHMED A, HOPKIN-
SON TM, ANDRESON P. A clinical and
elctron microscopie study of a calcifying epithelial odontogenic tumor. J Clin Pathol 1971; 24: 152-8. 6. MopsiK ER, GABRIEL SA. Calcifying odontogenie tumor (Pindborg tumor). Oral Surg Oral Med Orat Pathot 1971; 32: 15-21. 7. WALLACE J. Calcifying epithelial odontogenie tumor ("Pindborg tumour"): a ease report. Br J Pta.it Surg 1974; 27: 2 8 30. 8. SOLOMON MP, VULETIN JC, PERTSCHUK
LP, GoRMLEY MB, ROSEN Y. Calcifying
epithelial odontogenie tumor, a histologic, histochemical, fluorescent, and ultrastruetural study. Oral Surg Oral Med Otal Pathol 1975; 40: 522-30. 9. CHAUDHRY AP, HANKS CT, LEIFER C ,
GARGIULO EA. Calcifying epithelial odontogenic tumor, a histochemical and ultrastructural study. Cancer 1972; 30: 519-29. 10. MORI M , MAKINO M , IMAI K . The histo-
chemical nature of homogeneous amorphous materials in odontogenic epithelial ttimor. J Orat Surg 1980; 38: 96-102. 11. FRANKLIN CD, MARTIN MV, CLARK A, SMITH CJ, HINDLE MO: An investigation
into the origin and nature of "amyloid" in a calcifying epithelial odontogenie tumor. / Orat Pathot 1981; 10: 417-29. 12. ANDERSON HC, KIM B , MINKOWITZ S.
Calcifying epithelial odontogenie tumor of Pindborg. An electron microscopic study. Cancer 1969; 24: 585-96. 13. KROLLS S O , PINDBORG JJ. Calcifying epi-
23. MURPHY GF, BHAN HK, SATO S, HARRIS
TJ, MiHM MC. Characterization of Langerhans cells by the use of monoclonal antibodies. Lab Invest 1981; 45: 465-8. 24. NAKAJIMA T, WATANABE S, SATO Y, SHI-
MOSATO Y, MoTot M, LENNERT K . S-IOO
protein in Langerhans eells, interdigitating reticulum cells and histiocytosis X cells. Gann 1982; 73: 429-32. 25. IDE F , IWASE T, SAITO I, UMEMURA S,
NAKAJIMA T. Immunohistoehemieal and
ultrastructural analysis of the proliferating cells in Histiocytosis X. Catteer 1984; 53: 917-21. 26. KORENBERG R, PENNEYS NS, KOWALCZYK A, NADJI M . Quantitative of SlOO protein - po.sitive cells in inflamed and non-inflamed keratoacanthoma and squamous eell carcinoma. / Cutan Pathol 1988; 15: 104-8. 27. BROCKER EB, ZWADLO G , HOLZMAN B ,
MACHER E , SORG C . Inflammatory eell
infiltrates in human melanoma at different stages of tumor progression. /;;/ J Cancer 1988; 41: 562-7. 28. MEISSNER K , HAETEK M , ARLOT M , MAUDUIT G , THIVOLET J. Quantitative
thelial odontogenic tumor: a survey of 25 cases and discussion of histomorphologic variations. Arch Pathol 1974; 98: 206-10.
analysij of T6-positive Langerhans cells in human skin eaneers. Virchow Arch A (Pathol Attat) 1986; 410: 57-63. 29. TSUJITANI S, FURUKAWA T, TAMADA R ,
14. GREER R O , RICHARDSON JF. Clear cell
OKAMURA T, YASUMOTO K , SUGIMACHI
calcifying odontogenic tumor viewed relative to the Pindborg tumor. Oral Surg Oral Med Orat Pathot 1976; 42: 775-9.
K. Langerhans cells and prognosis in patients with gastrie eareinoma. Catteer 1987; 59: 501-5.
A variant of calcifying epitheiial odontogenic tumor with Langerhans celis
Masatake Asano\ Tomihisa TakahashP, Kaoru Kusama\ Takashi lwase\ Minoru Hori^ Hiromitsu Yamanoi^ Hiroshi Tanaka^ and Itaru Moro^ Departments of 'Pathology and 'Oral Surgery, Nihon University School ol Dentistry, Tokyo, Japan
Asano M, Takahashi T, Kusama K, Iwase T, Hori M, Yamanoi H, Tanaka H, Moro I; A variant of ealcifying epithelial odontogenie tumor with Langerhans cells. J Oral Pathol Med 1990; 19; 430-4. A variant of calcifying epithelial odontogenic tumor (CEOT) with Langerhans eells is reported. Compared to a typieal CEOT, the tumor islands of this case were thin and composed of a small number of polyhedral epithelial eells. Almost no ealcifieation of homogenous eosinophilie materials was observed. In addition, clear cells which structurally corresponded to Langerhans cell were intermingled in the epithelial islands. These eells stain positively for S-IOO protein, lysozome, MT 1, LN-3 and OKT 6 antibodies, but not for keratin antibody. Electrontnicroseopie examination revealed the rod-shaped and racket-shaped structures called Birbeek's granules in the cytoplasm of these elear cells. Our observations indieate a variant case of CEOT with Langerhans cells in tumor nests.
The calcifying epithelial odontogenic tumor (CEOT) is a relatively rare tumor described by PINDHORG as a distinct entity (1, 2). CEOT, often referred to as Pindborg tumor, is composed of polyhedral epithelial cells with round, hyperchromatic nuclei and fibrous connective tissue. The presenee of homogenous eosinophilie material is a characteristic feature of this tumor (1-5). Caleification in the eosinophilie material is often observed, but there are also reported eases with a paucity of calcification (6-8). The nature of the eosinophiiic material is generally considered to be amyloid-like, but others find the similarities to enamel matrix or pre-enamel, basal lamina, keratin protein and tuft enamel protein (9-11). The origin of this tumor is eontroversial, but it is considered to be derived from reduced enamel epithelium, oral epithelium or stratum intermedium (2, 5). Several reports have indieated the presenee of elear eells with glycogen granules in the CEOT (7, 12-15). These eells are eonsidered to be either degenerating, or eells with some features of cytodifferentiated epithelial eells (12, 15). It is well known that Langerhans cells are found in the oral epithelium and
skin (16-19). However, there have been no reports of Langerhans cells in a CEOT. We report here a variant of CEOT with many Langerhans cells in epithelial tumor nests.
Key words: calcifying epithelial odontogenic tumor; jaws, neoplasms; Langerhans cell; S100 protein. Itaru Moro, Department of Pathology, Nihon University School of Dentistry, 1-8-13, KandaSurugadai, Chiyoda-ku, Tokyo 101, Japan Accepted for publication July 22, 1990.
Material and methods Clinical history
A 44 yr-old woman visited the Division of Oral Surgery, Kasukabe Hospital with a chief complaint of gingival swell-
Fig. I. Preoperative panoramic radiograph showing unilocular, well-defined, radiolucent area extending from maxillary right central incisor to right first molar.
CEOT with Langerhans cell 431 ing in the region extending from the maxillary left central incisor to the right molar. She had noted this slow-growing lesion for several years, but had neglected to report it to her dentist beeause of no pain. Her past medical history was completely unremarkable and indicated no evidence of systemic diseases. Oral examination revealed one tnissing tooth in the maxillary right first premolar area that was extracted because of dental caries about 10 yr previously. The other teeth were all vital except for the maxil-
lary right central incisor. Radiographic examinations revealed unilocular, well defined, radiolucent area from the maxillary right central incisor to the right first molar (Fig. I). There were no radiopaque foci in the lesion, Resorption of the roots of the tnaxillary central and lateral incisors and canine was detected. A clinical diagnosis of ameloblastoma was made while histologic examination of biopsied tissue by Congo red staining revealed CEOT. Under general anesthesia, partial re-
Antibodies
Company
Dilution
Reactivity
Keratin S-100 Lysozyme MT 1 LN 3 OKT 6
DAKO Co. DAKO Co. DAKO Co. Bioscience Co. Seikagaku Kogyo Ortho Co.
X50 X50 X250 X50
Keratin filament S-100 protein Lysozyme 190 KD, 110 KD antigen HLA-DR antigen CD 1 antigen
X50 X 50
Table 2. Histochemical and Immunohistologic Stainings of CEOT Epithelial islands
Congo red Congo red (birefringence) Thioflavin T Methyl violet Keratin S-100 Lysozyme MT I LN3 0KT6
Histologic procedure
The divided samples were fixed in 10% buffered formalin, embedded in paraffin and processed for routine histologic examination. Immunologic procedure
Table I, First antibodies
Staining
section of the maxilla was performed. The whole neoplasm obtained after operation was divided into several portions and processed for histologic, immunologie and electron microscopie examinatiotis.
Clear cell — — — — — + -H -1-h +
Tumor eell
Homogenous eosinophyilic Materials
Light pink Blue birefringence Yellow fluorescence Metaehrotnasia
Light pink Blue birefringence Yellow fluorescence Metaehromasia
-ij-
Sorne samples were embedded in OCTeompound and quickly frozen with dry ice and acetone. Six iitn frozen sections were cut by cryostat,fixedin acetone for 10 min and processed for immunologic staining. In addition, some of the fortnalin-fixed, paraffin embedded sections were also used for irnmunologic staining. In this case, deparaffinized seetions were pretreated with 0.1% pronase to increase antigenicity. An avidin-biotin-peroxidase complex (ABC) method was used to detect antigens in tumor tissues. Briefly, sections were immersed in methanol containing 0.3% HjOj for 20 min to block endogenous peroxidase aetivity and washed with phosphate buffered saline (PBS), pH 7.2, three times. Washed sections were incubated with normal horse serum (for monoclonal antibodies) or normal goat serum (for polyelonal antibodies) for 20 min and subsequently with first antibodies for 45 min at room temperature. After washing with PBS, biotinylated horse anti-mouse IgG or biotinyiated goat anti-rabbit IgG were applied to tissue sections, then avidinbiotin-peroxidase cotnplex using Vectastain Kit (Vector) and 0.05% 3,3'-diaminobenzidine tetrahydrochloride containing 0.05% H,0, substrate was added for 10 min. Sections were washed, counterstained with hematoxylin and mounted. First antibodies used in this study are given in Table 1. As positive controls, hutnan liver with amyloidosis and human normal gingiva were used. Negative controls were prepared by substituting PBS instead of first antibodies. Electron microscopic procedure
Fig. 2. Overall view of this tumor showing small epithelial islands and eosinophiiic materials with almost no ealeifieation. H-E stain, x 4
Some portions of the tutnor tissue were cut into small pieces with a razor blade and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH.7.4) for 2 h.
432
ASANO et al.
F/g. 5. Immunohistochemical staining of various antigens in CEOT. A, distribution of keratin in the epithelial island. Keratin was detected only in the tumor cells, x 10. B, higher magnification of A. Clear eell (arrow) in epithelial island stained negatively for anti-keratin antibody. x20. C, distribution of S-100 protein in epithelial island. Arrows pointed out S-100 protein positive cells in epithelial island, x 20. D, lysozyme positive cells (arrows) were intermingled in the epithelial island. x40. E, MT-1 positive eells were localized in epithelial island (arrow) as well as in stroma. x 40. F, LN3 in epithelial island (arrow) and in infiltrated cells of the stroma. x40.
then fixed again in 1 % osmium tetraoxide for 1 h. Specimens were dehydrated with graded ethanol and propylene oxide, and embedded in Spurr's resin. Thin sections were cut with a diamond knife, stained with uranyl aeetate and lead citrate and observed under a JEOL T-lOO eleetron microscope. Results The tumor masses consisted of thin strands of polyhedral epithelial eells and loose fibrous connective tissue (Fig. 2). The epithelial islands were composed of five to twenty cells and clear cells were frequently found in the islands. The cy-
toplasm of the polyhedral cells was eosinophiiic and finely-granular. The nuclei were relatively dense and contained a single nucleolus. There was moderate inflammatory cell infiltration with lymphocytes and plasma eells in the fibrous connective tissue. In addition, a large number of globular masses of pale, homogenous eosinophiiic material were localized within the epithelial cells as well as within the fibrous connective tissue. It was very rare to find ealeifieation in the homogenous eosinophiiic material. Histoehemieal examination of this material revealed metaehromasia with crystal violet and methyl violet stains and positive reac-
tion with Congo red stains. Weak birefringence was observed under polarized microscopy after staining with Congo red. Thioflavin T stained seetions exhibited yellow fluorescence under fluorescent microscopy. Immunohistochemically, keratin was deteeted only in tumor eells of the epithelial islands (Fig. 3A), while there was no positive reaction in the clear eells, homogenous eosinophiiic material or inflammatory cells infiltrating the stroma (Fig. 3B). Occasionally, one or two cells positive for S-100 protein (Fig. 3C), lysozyme (Fig. 3D), MTl (Fig. 3E), LN3 (Fig. 3F) or OKT 6 antibodies were identified in an epithelial island corresponding to the area of clear eells. Variable numbers of cells positive for these antibodies were also present in the inflammatory cells in the stroma. However, there were no positive reaetions for these antibodies in epithelial tumor cells nor the homogenous eosinophiiic material. The histoehemieal and immunohistochemical Staining results of this case are given in Table 2. Eleetronmieroscopically, the epithelial islands were composed of polyhedral eells with round nuelei and prominent nueleolus. The cytoplasm contained several mitochondria, bundles of tonofilaments, dense bodies, polyribosomes and small ill-developed endoplasmic reticulum. A large number of irregularly interdigitating microvilli were present between tumor eells and desmosomes were occasionally found in adjaeent cells. In some epithelial islands, clear eells with indented nuelei which corresponded to Langerhans eells, were intermingled among polyhedral tumor cells (Fig. 4). The clear cytoplasm contained a few mitochondria, dense bodies, small vesicles, multivesicular bodies, endoplasmie retieulums, small Golgi apparatuses, but no tonofilaments nor melanosome. No desmosomes were observed between elear cells and neighbouring tumor cells. The rod-shaped and racketshaped structures called Birbeek's granules were often seattered throughout the cytoplasm (Fig. 5, arrows). The homogenous material was located both intracellularly and extracellularly. It consisted of densely packed accumulations of fine-fibrillar material. Discussion It has been well-documented that the histologic characteristics of CEOT are the presence of homogenous eosinophil-
CEOT with Langerhans cell 433
l>Ai%^s:
cell in CEOT. Our histologic and electron microscopic results clearly revealed the presence of Langerhans eell in the CEOT of our ease. The Langerhans eell, whieh belongs to the mononuclear phagocyte system, is of bone marrow origin and is often found in the skin and oral mucosa (16-18). It has been clearly ascertained that Langerhans cells function as antigen presenting cells and as allogenic stimulatory cells to primed T lymphocytes in the epithelium (16, 20). Immunologically, Langerhans cells express la surface antigen and Fey and C3 receptors (20-22). It has been reported that Langerhans cells stain positively for anti-CD 1, CD3, CD4and S-100 protein antibodies (23, 24). Our immunologic examination confirmed the presence of Langerhans cells in CEOT. In addition, T6 as well as lysozyme positive cells were observed in the epithelial nest. These results probably indicated the presenee of T eell and maerophage in the epithelial nest. Neoplastic proliferation of Langerhans cells has been observed in histiocytosis X which includes eosinophiiic granuloma of bone, Hand-SchlillerChristian disease and Letterer-Siwe disease (25). In the field of dermatology, Langerhans cells have often been found in keratoacanthoma and squamous cell carcinoma (26). It has been reported that increased numbers of Langerhans cells in some cases of keratoacanthoma resulted in the regression of lesions (26). BROCKER et al. (27) reported a signifi-
cant correlation between the number of Langerhans eell and aetivation of T lymphocyte in malignant tnelanoma. MEISSNER et al. (28) reported that the
number of T6 positive Langerhans eell was lower in epithelial skins tumors such as basal and squamous cell earcinoma compared with a sex and age Schiff (PAS) reaction and were consid- matched control group and suggested ered to be either degenerating cells or that the development of skin tumor may to represent a feature of cytodifferentia- be favored by impaired immunologie tion (7, 12, 15). The clear cells found in surveillance of neoantigen-expressing our case were different from those of tumor cells resulting from reduction of previously reported eases. The clear Langerhans eell number and function. eells in our case exhibited no positive TsujiTANi et al. (29) reported that the stain for PAS reaction and had clear number of Langerhans cells influences cytoplasm and indented nuclei. No to- the prognosis of gastric carcinoma. nofilaments and melanosomes were ob- These reports suggest some correlation served and no formation of desmo- between tumor regression arid the somes between neighboring cells was number of Langerhans cell. In our case, found. The Birbeek's granules, whieh a fairly large number of Langerhans are a morphologic characteristic of cells were observed. These Langerhans Langerhans cell, were deteeted (19). To cells may play an important role in our knowledge, there has been no report antigen presentation or regression of concerning the presence of Langerhans CEOT.
F/g. 4. Electron micrograph of Langerhans cell in epithelial island. Langerhans eell had clear cytoplasm and no desmosome between neighbouring eells. Arrows indieating Birbeek's granules, x 19600.
ic material that show amyloid-like reaction and often exhibit calcification (1-8). In addition,tumor islands are generally composed of densely-arranged polyhedral epithelial cells. The case reported here is unusual eompared with that of a typieal CEOT. Although the homogenous eosinophiiic tnaterial showed amyloid-like reactions, there was no calcification. The tumor islands were composed of small numbers of epithelial cells with clear cells corresponding to Langerhans eells. The presence of clear cells in CEOT has been described (7, 12-15). These clear cells contained glycogen granules, which stained positively with periodic aeid
434
ASANO et al. 15. YAMAGUCHI A , KOKUBU JM, TAKAGI M ,
ISHIKAWA G. Calcifying epithelial odontogenie tumor: histochemical and eleetron microscopic observations on a case. Butt Tokyo Med Dent Univ 1980; 27: 129-35. 16. LASSER A . The mononuclear phagocyte system. A review. Hutn Pathot 1983; 14: 108-26. 17. SLOBERG K , JONSSON R , JONTELL M . As-
sessment of Langerhans cells in oral lichen pianus using monoclonal antibodies. J Orat Pattwt 1984; 13: 516-24. 18. PITIGALA-ARACHCHI A, CRANE IJ, SCUL-
LY C, PRIME SS. Epithelial dendritie eells in pathological human oral tissues. J Otat Pattwt 1989; 18: 11-6. 19. SAEBIEL RW, REED T H . Serial reconstruc-
tion of the characteristic granule of the Langerhans eell. J Cell Biol 1968; 36: 595-602. 20. SHELLEY W, JUHLIN L . Langerhans cells
form a reticuloendothelial trap for external contact allergens. Nature 1976; 261: 46-7. 21. ROWDEN G , LEWIS MG, SULLIVAN AK.
la antigen expression on human epidermal Langerhans cell. Nattire 1977; 268: 247-8. 22. STINGL G , KATZ SJ, CLEMENT L , GREEN
J, SHEVACH EM. Immunological functions of la - bearing epidermal Langerhans cells. J Itnmunot 1978; 121: 200513. /^/g. 5. Higher magnification of Fig. 3. Rod-shaped and racket-shaped Birbeek's granules were seen, x 65500.
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