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  • 1. Oral Med Pathol 6(2001) 51 Calcifying Odontogenic Cyst Associated with Complex and Compound Odontoma: Report of a case and immunohistochemical study. Yoshinori Jinbu, Keiichi Tsukinoki*, Ken Tanobe, Kazuro Yamaguchi, Hiroto Itoh, Yoshihisa Watanabe* and Yoko Akasaka Department of Dentistry and Oral Surgery, Jichi Medical School, Tochigi, Japan *Department of Oral Pathology, Kanagawa Dental College, Kanagawa, Japan Jinbu Y, Tsukinoki K, Tanobe K, Yamaguchi K, Itoh H, Watanabe Y and Akasaka Y. Calcifying Odontogenic cyst associated with complex and compound odontoma: Report of a case and immunohistochemical study. Oral Med Pathol 2001; 6: 51-55 , ISSN 1342-0984. A case of calcifying odontogenic cyst associated with complex and compound odontoma which appeared in the maxilla of a 15-year-o1d Japanese girl was reported with immunohistological examination. Radiologically, the lesion showed a cystic radiolucent area with odontoma-like radiopaque masses. Histopathological examination revealed the presence of ghost cells, calcified tissues and structures of complex and compound odontoma. Immunohistochemically, MIB-1 positive ratio was very low, but significant expression of Bcl-2 was observed in the neoplastic odontogenic epithelium. These results suggest that inhibition of apoptotic cell loss is more important than cell proliferation activity to the growth of the lesion in our case. Key words: Calcifying odontogenic cyst associated with odontoma, immunohistochemistry, MIB- 1 (Ki67), Bcl-2 Correspondence: Yoshinori Jinbu, Department of Dentistry and Oral Surgery. Jichi Medical School, Yakushiji 3311-1, Minamikawachi-machi, Kawachi-gun, Tochigi, 329-0498, Japan Phone: +82-285-58-7390, Fax: +82-285-44-8669 Introduction Calcifying odontogenic cyst (COC) was first catego- rized as a distinct entity by Gorlin et al. in 1962 (1). Ac- cording to the WHO classification on histological typing of odontogenic tumours (2), COC is described as a neo- plastic cyst-forming lesion in which the epithelial lining shows a well-defined basal layer of columnar cells, an overlying layer that is often many cells thick and that may resemble stellate reticulum, and masses of ghost epithelial cells that may be in the epithelial cyst lining or in the fibrous capsule. COC is considered to occupy a position between a cyst and an odontogenic tumor, hav- ing some characteristics of both. COC, accounting for only 2% of all the odontogenic tumors (3), is a relatively rare lesion which has not been fully analyzed. We report a case of COC associated with complex and compound odontoma and examine the lesion immunohistochemically to understand the cellular mechanism of its growth. Case Report The patient was a 15-year-old Japanese girl who complained of swelling in the left maxillary gingiva and delayed eruption of the maxillary left canine. She first noticed gingival swelling with discomfort in the left max- illary anterior region 11 years earlier. The swelling gradually increased in size. She visited her family den- tist and was referred to the Department of Dentistry and Oral Surgery at Jichi Medical School Hospital. Oral examination revealed a firm swelling in the labial gingiva and alveolar mucosa in the left upper an- terior region. The gingiva and alveolar mucosa covering the lesion was normal and the lesion was elastic hard to palpation. Panoramic radiograph showed an oval-shaped cyst-like radiolucency in the left upper anterior region, as well as canine impaction. The cyst-like radiolucent lesion had a well-defined margins with sclerotic rim, and multiple small radiopaque calcific masses were observed within the lesion. The axial and coronal CT images showed facial bony expansion with continuous thinned- out cortex and multiple radiopaque calcific specks (Fig. 1). There were no abnormal values in the blood examina- tion. Under a clinical diagnosis of left maxillary calcify- ing odontogenic cyst, the lesion was surgically removed and the uneruped left maxillary canine was extracted. The cystic lesion was easily removed from bone without
  • 2. 52 Jinbu et al. Calcifying odontogenic cyst adhesion, and the canine was not associated with the cys- tic lesion (Fig. 2). The removed lesion was 30¥20¥20mm in size, had a smooth surface and was elastic soft to pal- pation. Sectional view showed an unicystic lesion con- taining serous fluid and tooth-like calcifications in the cyst wall (Fig. 3). Microscopically, the surgical material taken from the cystic lesion showed proliferation of neoplastic odon- togenic epithelium composed of stellate reticulum, coboidal cells and ameloblast-like cells (Fig. 4). Typical ghost cells were observed in the lumen of the tumor mass (Fig. 5). No apparent invasion was noted in the stroma. Dental hard tissues of the abortive enamel, dentin and cementum tissue were seen in the tumor mass. These hard tissues showed a tooth-like (Fig. 6) or irregular tooth- like (Fig. 7) arrangement. These features were diagnosed as calcifying odontogenic cyst with complex and compound odontoma. Fig. 1: Axial and coronal CT image showing facial bony expansion and multiple small radiopeque masses Fig. 2: The lesion was surgically removed Fig. 3: Sectional view showing an unicystic lesion containing tooth like calcifications Fig. 4: Proliferation of neoplastic odontogenic epithelium was observed
  • 3. Oral Med Pathol 6(2001) 53 Immunohistochemistry Immunohistochemical staining for Bcl-2 and MIB- 1 (Ki-67) were performed using the antigen retrieval sys- tem of Piattelli et al. (4). After the slides were autoclaved for 5 minutes at 121˚C in 10 mM sodium citrate buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0), they were at room temperature kept for 1 hour . The slides were incubated in 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity and were pretreated for 10 minutes with 10% normal goat serum. As primary antibodies, anti-Bcl-2 antibody (dilu- tion of 1:20, DAKO, Japan) and anti-Ki67 antibody MIB- 1 (dilution of 1: 50, Immunotech S. A., Marseille, France) were used. The slides were incubated with primary anti- bodies for 1 hour. Secondary biotinylated antibody and avidin-biotin complex reagents were reacted for 10 min- utes. Color was developed with 0.02% 3, 3- diaminobenzidine-tetrahydrochloride containing 0.003% hydrogen peroxide in PBS for 10 minutes. After wash- ing, the immunostained sections were counterstained in hematoxylin, dehydrated, cleared, and mounted. Sections from a follicular hyperplasia of a tonsil served as positive control. For negative controls, PBS was used instead of primary antibodies. Assessment of Bcl-2 immunostaining was classified into 3+ for strong positive staining, 2+ for weak positive staining and 0 for negative staining, re- spectively. MIB-1 immunostaining was examined in at least 800 neoplastic cells in 8 random microscopic fields and the positive stained nuclei were using a high power (¥40) objective lens. Only strong nuclear staining was regarded as positive, and weak nuclear or cytoplasmic staining was regarded as negative. The MIB-1 labeling index (MIB-1-LI) was the percentage of the immunopositive nuclei of all neoplastic cells. Immunoreactivity for Bcl-2 was observed in the neoplastic odontogenic epithelium composed of stellate reticulum, coboidal cells and ameloblast-like cells (Fig.8). Ghost cells were completely immunonegative for Bcl-2. Bcl-2 protein was also expressed in the odontogenic epi- thelium of the area associated with odontoma. Expres- Fig. 5: The typical ghost cells in the lumen of tumor mass Fig. 6: Complex odontoma in the cyst wall Fig. 7: Compound odontoma in the cyst wall Fig. 8: Immunoreactivity for Bcl-2 was observed in the neoplastic odontogenic epithelium Fig. 9: Immunolocalization of MIB-1 was focally identified in the solid nest areas of neoplastic odontogenic epithelium
  • 4. 54 Jinbu et al. Calcifying odontogenic cyst sion level of Bcl-2 indicated strong positive staining (3+) in the ameloblastic-like cells of the solid area. The neo- plastic odontogenic epithelium composed of stellate reticu- lum, coboidal cells and basal cells of cyst wall were Bcl-2 weak positive staining (2+). Immunolabeled nuclei with MIB-1 were focally identified in the solid nest areas com- posed of neoplastic odontogenic epithelium without den- tal hard tissues and basal cells of cyst wall (Fig.9). No expression of MIB-1 was noted in the ghost cells and neo- plastic odontogenic epithelium of odontoma regions. MIB- 1-LI of the neoplastic cells in solid area was 2.85%. MIB- 1-LI of cystic area was 1.26%. Odontoma area was 0% for MIB-1-LI. Discussion Calcifying odontogenic cyst (COC) is a relatively rare lesion which was first reported by Gorlin et al (1). COC occurrs with equal frequency in the maxilla and mandible (3). Before the age of 41, the cyst is much more common in females while after 41 it is much more com- mon in males. About 75% of the lesions are situated an- terior to the first molar. It may vary in size and enlarge sufficiently to expand the bone. The lesion usually oc- curs as slowly enlarging, frequently painless and non- tender swelling of the jaw. There may be an associated unerupted tooth. The lesion is frequently cystic but occa- sionally it presents as a solid mass. The cyst wall is lined by odontogenic epithelium composed of the basal layer of columnar cells, the adjacent stellate cells and ghost cells. Occasionally dysplastic dentin may be seen, and approxi- mately 10% of COCs are associated with odontomas. Ng and Siar (5) reported that odontoma-producing COC oc- curred in younger patients and showed an even sex dis- tribution, while the non-odontoma-producing variant oc- curred in older patients and showed a female preponder- ance. COC was defined as a neoplasm by WHO classifi- cation in 1991 (2). COC is considered to behave like be- nign tumors because COC has the potential for contin- ued growth and incomplete removal can be followed by recurrence. Several classifications of COC have been pro- posed (6, 7), demonstrating the diversity in the appear- ance of lesions and the ambiguity the correlation between histopathology and behavior. In 1981, Praetorius et al. (6) divided COC into two entities: a cyst and a neoplasm. The former was further subclassified into three subtypes: simple unicystic subtype, unicystic odontoma-producing subtype and unicystic ameloblastomatous proliferating subtype. Hong et al. (7) proposed a new classification in which the cystic entity was subclassified into four sub- types; non-proliferative subtype, proliferative subtype, ameloblastic subtype and COC associated with odontoma. Recently, Takata et al. (8) subclassified COC into benign and malignant COC. The benign COC was divided into a cystic subtype and a neoplastic one. The former was fur- ther subclassified histologically into non-proliferative subtype, proliferative subtype, ameloblastomatous type, and COC associated with odontoma. They analyzed the proliferating cell nuclear antigen (PCNA) labeling index immunohistochemically in 25 cases of COC. They reported that PCNA labeling index is a possible parameter for dif- ferentiating malignant COC from benign COC and, what- ever the subtypes, the proliferative features in the lining are the main factor influencing the proliferating activity of COC. Toida (9) also proposed a new subclassification of COC, i.e. calcifying ghost cell odontogenic cyst, cystic calcifying ghost cell odontogenic tumor (CGCOT), solid CGCOT, malignant CGCOT and combined lesions, and he cited that his new subclassification can be validated further than that of Takata et al. (10). Piattelli et al. (4) showed strong positivities of MIB-1 and Bcl-2 in the od- ontogenic epithelial cells of dentinogenic ghost cell tu- mor of in an 80-year-old male. However, the nature of COC still is not fully understood. Recently, it has been reported that over expression of Bcl-2 protein exhibits the inhibition of apoptosis found on odontogenic epithelium, and over expression of Bcl-2 protein is presumed to participate in the development and evaluation of odontogenic lesions (11). On the other hand, the review of growth activity has extreme signifi- cance in the assessment of the development of tumors (12). So, to find out biological behaviors of the calcifying odontogenic cyst in our case, we compared the expression of Bcl-2 protein and growth activity. In consequence, MIB-1 was localized in the portion showing solid growth of tumors and was not found in the areas of odontoma. Takata et al. (8) has reported that the assessment of growth activity is an important parameter involved in the developmental progression of this tumor. Because the MIB-1-LI was lower in our case than in their report (8), we believe it might be better to characterize the growth as a cyst rather than as a neoplasm. Furthermore, the reason for no apparent MIB-1 positive cells in the areas of odontoma seemed to be that odontogenic differentia- tion was higher. On the other hand, Bcl-2 protein was expressed on most of the neoplastic odontogenic epithe- lium. Though a role of Bcl-2 protein has not adequately been proved, it is conceivable that the expression of Bcl-2 protein could inhibit cell death on odontogenic epithe- lium, and could indirectly support the developmental pro- gression in the case of weak proliferative activity. References 1. Gorlin RJ, Pindborg JJ, Clausen F, et al. The calcifying od- ontogenic cyst: A possible analogue of the cutaneous calcify- ing epithelioma of Malherbe. Oral Surg Oral Med Oral Pathol 1962; 15 : 1235-43. 2. Krame IRH, Pindborg JJ and Shear M. World Health Orga- nization classification of tumours. Histological typing of od-
  • 5. Oral Med Pathol 6(2001) 55 ontogenic tumour. 2nd ed. Springer-Verlag, London,1992; 1- 68. 3. Wood NK and Goaz PW. Differential diagnosis of oral le- sions. 2nd ed. The C.V.Mosby Company, St. Louis, 1980; 505- 7. 4. Piattelli A, Fioroni M, Alberti LD, et al. Immunohistochemi- cal analysis of a dentinogenic ghost cell tumour. Oral Oncol 1998; 34 : 502-7. 5. Ng KH and Siar CH. Morphometric analysis of epithelial components and dentinoid formation in non-neoplastic cal- cifying odontogenic cyst. J Nihon Univ Sch Dent 1995; 37: 156-62. 6. Praetorius F, Hjorting-Hansen E, Gorlin RJ, et al. Calcifying odontogenic cyst: Range, variations and neoplastic potential. Acta Odontol Scand 1981; 39: 227-40. 7. Hong SP, Ellis GL and Hartman KS. Calcifying odontogenic cyst: A review of ninety-two cases with reevaluation of their nature as cysts or neoplasms, nature of ghost cells, and sub- classification. Oral Surg Oral Med Oral Pathol 1991; 72 :56- 64. 8. Takata T, Lu Y, Ogawa I, et al. Proliferative activity of calci- fying odontogenic cysts as evaluated by proliferating cell nuclear antigen labeling index. Pathol Int 1998; 48 :877-81. 9. Toida M. So-called calcifying odontogenic cyst: Review and discussion on the terminology and classification. J Oral Pathol Med 1998; 27: 49-52. 10. Toida M. Proliferative activity and subtyping of calcifying odontogenic cyst. Pathol Int 2000; 50:81-3. 11. Komatsu S and Tsukinoki K. Bcl-2 expression and its role in odontogenic tumor genesis. Kanagawa Shigaku 1999; 34: 193-208. 12. Tsukinoki K, Yasuda M, Shimamura K, et al. Odontogenic myxofibroma arising in an elderly male: cell kinetics of epi- thelium and stroma. Histopathology 1999; 35: 280-1. (Accepted for publication April 20, 2001)