Peripheral clear cell variant of calcifying epithelial odontogenic tumor: Report of a case and immunohistochemical investigation

Peripheral clear cell variant of calcifying epithelial odontogenic tumor: Report of a case and immunohistochemical investigation Ricardo Alves Mesquita, DDS, PhD,a Monica Andrade Lotufo, DDS, MS,b Norberto Nobuo Sugaya, DDS, PhD,c Ney Soares de Arau´jo, DDS, PhD,d and Vera Cavalcanti de Arau´jo, DDS, PhD,d Sa˜o Paulo, Brazil ˜ O PAULO UNIVERSITY SA

A case of peripheral calcifying epithelial odontogenic tumor, clear cell variant, located on the right gingival maxilla of a 48-year-old woman, presenting as a 2.0-cm solitary, firm nodule was studied. Microscopically, it was composed of polyhedral and clear epithelial cells associated with amyloid-like deposition. The clear epithelial cells exhibited granules that were positive for periodic acid-Schiff, and the amyloid-like deposit stained with Congo red showed a green birefringence in the polarized light. Polyhedral and clear epithelial cells were immunopositive for AE1/AE3 and cytokeratin 14. Immunoexpression of fibronectin and types I and III collagen were different between the amyloid-like deposits and the connective tissue stroma. Tenascin surrounded epithelial cells located inside the amyloid-like deposits. Laminin and type IV collagen were immunodetectable around the strands, cords, and nests of epithelial cells. This report represents the seventh case of peripheral calcifying epithelial odontogenic tumor, clear cell variant. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:198-204)

The calcifying epithelial odontogenic tumor (CEOT) is a rare odontogenic benign tumor, occasionally invasive, which produces a homogeneous, eosinophilic, pale-staining amyloid-like material which may become calcified.1,2 It was first described by Pindborg3 in 1955, and also is referred to as Pindborg tumor.4 By far the most common CEOT location is intraosseous, but it also may be extraosseous. The extraosseous, or peripheral, variant was first observed by Pindborg5 in 1966, and it represents approximately 6.0% (11 cases) of the total cases of CEOT.2,5-12 Under microscopic examination, the classical pattern of CEOT is a combination of cords, nests, or sheets of polyhedral epithelial cells, amyloid-like deposits, and concentric calcified structures (Liesegang’s rings).9,13-15 The clear cell variant of CEOT was first described in 1967 by Abrams and Howell.7 It is characterized by polyhedral epithelial cells alternating with large epithelial cells with a clear, foamy cytoplasm; distinct cell borders; moderated variation in nuclear size; some vacuolated nuclei; and no a Graduate Student, Department of Oral Pathology, School of Dentistry, Sa˜o Paulo University. b Graduate Student, Department of Stomatology, School of Dentistry, Sa˜o Paulo University. c Professor, Department of Stomatology, School of Dentistry. d Professor and Chairman, Department of Oral Pathology, School of Dentistry, Sa˜o Paulo University. Received for publication Mar 5, 2002; returned for revision Apr 4, 2002; accepted for publication Jun 26, 2002. © 2003, Mosby, Inc. 1079-2104/2003/$30.00 ⫹ 0 doi:10.1067/moe.2003.63

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extreme hyperchromatism or bizarre nuclei. The clear epithelial cells contain glycogen, as demonstrated by the periodic acid-Schiff (PAS) reaction, which is removed by diastase digestion.10 Fifteen cases of the clear cell variant have been described in the literature, and 6 of them (40.0%) represent the peripheral variant. The mean age of the patients with the peripheral CEOT, clear cell variant (PCEOTCC) is 34.3 years (range, 16 to 64 years) at the time of diagnosis, and the male-to-female ratio is identical (M/F ⫽ 3:3). All cases are located in the gingiva, with the mandible more commonly affected than the maxilla (Md/ Mx⫽ 4:2).2,7,10,12,16-23 This variant presents an indolent behavior and good prognosis, and none of the 11 patients have shown signs of tumor recurrence after conservative treatment.11,12 The purpose of this article is to present clinical, microscopic and immunohistochemical characteristics of a PCEOTCC occurring in the gingiva of the right maxilla of a 48-year-old woman. We evaluated the immunohistochemical profile of the epithelial cellular component for cytokeratins (CKs); the extracellular matrix proteins for fibronectin, tenascin, and types I and III collagen; the basement membranes components for laminin and type IV collagen; and the amyloid component for amyloid A component. CASE REPORT A 48-year-old woman was referred to the Stomatology Clinic of the Dentistry Service at the University Hospital (Sa˜o Paulo, Brazil) in November 1999, for evaluation of a fibrous mass in the anterior maxillary gingiva of 10 months’ duration. Intraoral physical examination revealed on the vestibule gin-

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Fig 1. A, Intraoral view showing a solitary, sessile, well-circumscribed, dome-shaped nodule covered with a smooth, regular, and erythematous mucosal surface in the right gingival maxilla (tooth 13).

giva of tooth number 13 a 2.0-cm painless, firm, sessile, well-circumscribed, dome-shaped nodule covered by a smooth, regular, and erythematous mucosal surface (Fig 1). Traumatic injury was not reported. The patient presented with caries and periodontal diseases. Radiographic examination (periapical, occlusal, and panoramic) revealed bone loss consistent with periodontal disease but no other signs of bone involvement. Her medical history included hypertension under control and diabetes under investigation. An incisional biopsy was performed after a clinical differential diagnosis of pyogenic granuloma, peripheral ossifying fibroma, and peripheral giant cell granuloma. The specimen was sent to the Oral Pathology Service of the University of Sa˜ o Paulo (Sa˜ o Paulo, Brazil), and a diagnosis of PCEOTCC was made. The specimen from incisional biopsy was fixed in 10% neutral formalin solution and embedded in paraffin. Sections 5 ␮m thick were obtained and stained with hematoxylin and eosin, with PAS, with and without preliminary diastase digestion, and by the Congo red method. Sections 3 ␮m thick were submitted for immunohistochemical study by the streptavidin-biotin standard protocol, which was performed in a universal staining system (Dako Auto Staining; Dako Corp, Carpinteria, Calif). Immunohistochemical evaluation was carried out with primary antibodies against CKs; fibronectin; tenascin; types I, III, and IV collagen; laminin; and amyloid A component. The primary antibodies used in this study and their sources, dilutions, and protocols are listed in the Table. Appropriate positive and negative controls were included. Antigen retrieval for AE1/AE3, CK-7, CK-8, CK-13, CK-14, CK-19, and amyloid A component was achieved by steamheat antigen retrieval (30 minutes in citric acid—10 mmol/L, pH 6.0). For antibody reactions with fibronectin; tenascin; laminin; and types I, III and IV collagen, antigen retrieval was achieved by using 1% pepsin (1.0 g of pepsin in 100 mL of 10% chloridric acid solution at pH 1.8; 30 minutes, 37°C).

The patient was referred to the surgical department clinic, where a total excision of the reminiscent lesion was performed. The patient has remained disease-free after a 30month follow-up.

PATHOLOGY REPORT Microscopic description Hematoxylin and eosin–stained sections revealed a mucosal fragment represented by a dense connective tissue stroma and covered along the convex surface by a parakeratinized stratified squamous epithelium. In all lamina propria, the connective tissue stroma was replaced by irregular strands, cords, and nests of epithelial cells (Fig 2, A). The polyhedral epithelial cells disclosed an abundant, eosinophilic cytoplasm; roundto-oval relatively large nuclei with dense chromatin; and evident intercellular bridging. These cells exhibited discrete pleomorphic and hyperchromatic nuclei (Fig 2, B and C). In various instances, the epithelial cells had a clear, foamy, vacuolated cytoplasm (clear epithelial cells; Fig 2, A and D). Bundles of dense connective tissue separated the epithelial component. A central area of an amorphous, homogeneous, pale, eosinophilic, amyloid-like deposit was observed. This deposit presented with a green birefringence in the Congo red stain with polarized light (Fig 2, A, E, and F). In this area, some epithelial cell nests were observed within the amyloid-like deposit. In the PAS stain, clear epithelial cells demonstrated granules of positive material, which were removed by prior diastase digestion. Mitotic figures, tumor necrosis, and calcified material were not observed. Intense chronic inflammatory cell

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Table. Primary antibodies, sources, and protocols Antibody Epithelial cell component AE1/AE3 CK⫽7 CK⫽8 CK⫽13 CK⫽14 CK⫽19 Extracellular matrix proteins Fibronectin Tenascin Type I collagen Type III collagen Basement membrane components Laminin Type IV collagen Amyloid component Amyloid A component

Source

Protocol

Dako,* code: M3515 † NeoMarkers, code: K72.7 Dako, code: M631 NeoMarkers, code: 1C7 ⫹ 2D7 NeoMarkers, code: LL002 Dako, clone: RCK 108

SHAR, SHAR, SHAR, SHAR, SHAR, SHAR,

1:70; incubation—30 min at 37°C, StABC 1:100; incubation—30 min at 37°C, StABC 1:100; incubation—30 min at 37°C, StABC 1:50; incubation—30 min at 37°C, StABC 1:1000; incubation—30 min at 37°C, StABC 1:100; incubation—30 min at 37°C, StABC

Dako, code: A 0245 Dako, code: M 0636 ‡ Novocastra, code: COLL-lp § Biogenex, clone: HWD1.1

Pepsin, Pepsin, Pepsin, Pepsin,

1:800; 1:150; 1:150; 1:300;

㛳

incubation—30 incubation—30 incubation—30 incubation—30

min min min min

at at at at

37°C, 37°C, 37°C, 37°C,

StABC StABC StABC StABC

Sigma, clone: LAM-89 Dako, clone: CIV 22

Pepsin, 1:1200; incubation—30 min at 37°C, StABC Pepsin, 1:50; incubation—30 min at 37°C, StABC

Dako, clone: mc1

SHAR, 1:100; incubation—30 min at 37°C, StABC

SHAR, Steam ⫽ heat antigen retrieval; StABC, streptavidin-biotin standard protocol; CK, cytokeration. * Dako, Dako Corporation, Carpinteria, Calif. † NeoMarkers, Fremont, Calif. ‡ Novocastra Laboratories Ltda, Newcastle, England. § Biogenex, San Ramon, Calif. 㛳 Sigma, St Louis, Mo.

infiltrate, consisting of lymphocytes or plasma cells, was present. Immunohistochemical findings Immunohistochemically, all epithelial cells (polyhedral and clear) strongly expressed CKs AE1/AE3 and 14, but they were immunonegative for all other CKs investigated (Fig 3, A). The connective tissue stroma and the amyloid-like area were immunopositive for fibronectin and type III collagen, but the appearance and intensity of the reactions were different. The fibronectin presented as short, thick, and wavy bundles with an amorphous diffuse appearance in connective tissue stroma and the amyloid-like deposit, but the immunoexpression was more intense in the area of the amyloid-like deposit than in the connective tissue stroma (Fig 3, B). Type III collagen presented as long, thick, and wavy bundles in connective tissue stroma; in the amyloid-like deposit, the immunoexpression was less intense and diffuse (Fig 3, C and D). Type I collagen presented as short, thick, and wavy bundles with an amorphous diffuse appearance in connective tissue stroma and the amyloid-like deposit, and the immunoexpression was the same in both the amyloidlike deposit and connective tissue stroma. Tenascin was immunodetectable around rare epithelial cells located inside the amyloid-like deposit (Fig 3, E). Laminin and type IV collagen were immunodetectable around the strands, cords, and nests of epithelial cells. Epithelial

cells located near or inside of the amyloid-like area were immunonegative for both laminin and type IV collagen antibodies. The amyloid-like deposit was immunonegative for amyloid A component antibody. DISCUSSION Microscopic features observed in the present case indicate that this tumor qualifies for the diagnosis of PCEOTCC. Clinically, this case appeared as a painless, firm, sessile, well-circumscribed nodule covered by smooth, regular, and erythematous mucosal surface. These clinical features, although not unique to PCEOTCC, may be helpful to differentiate PCEOTCC from inflammatory peripheral lesions commonly seen in the gingiva (ie, pyogenic granuloma, peripheral giant cell granuloma, and peripheral ossifying fibroma).24 There was no spontaneous or easy bleeding; moreover, the homogenous erythematous surface, vestibule location away from the gingival margin, lack of dental dislocation, and absence of radiopacities in this lesion might exclude the clinical diagnosis of pyogenic granuloma and peripheral ossifying fibroma. Microscopically, the irregular strands, cords, and nests of the polyhedral epithelial cells with abundant, eosinophilic cytoplasm, round-to-oval relatively large nuclei with dense chromatin, and evident intercellular bridging in association with clear epithelial cells were observed. An amyloid-like deposit, identified on polarized light as a green birefringence of the Congo red stain, was also

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Fig 2. Microscopic features of the peripheral calcifying epithelial odontogenic tumor. A, A low-power view of the gingival mucosa fragment lined by parakeratinized stratified squamous epithelium presenting in the lamina propria as irregular strands, cords, and nests of epithelial eosinophilic and clear cells and amyloid-like material (hematoxylin-eosin; original magnification ⫻25.6). B, Nests and cords of polyhedral eosinophilic cells (hematoxylin-eosin; original magnification ⫻200). C, High-power view of polyhedral cells exhibiting discrete pleomorphic and hyperchromatic nucleus and intercellular bridging (hematoxylin-eosin, original magnification ⫻320). D, Clear cells presented with a clear, foamy, and vacuolated cytoplasm (hematoxylin-eosin; original magnification ⫻320). E and F, Amyloid-like material stained by Congo red (E) and revealed by polarized light showing the characteristic green birefringence in a darkened background (F) (Congo red; original magnification ⫻200).

present. Calcified material in the form of spherules (Liesegang’s rings), a common histologic feature of the CEOT, was not found in our case, which explained the lack of radiopaque material under radiographic investigation. Both a minimal amount and total absence of calcified material in the peripheral variant of the CEOT have been described in other cases.10,24,25 The presence of clear epithelial cell component is fundamental to the diagnosis of the clear cell variant of the CEOT. Nevertheless, no difference in CK expression was found in polyhedral or clear epithelial cells in this study. The only difference was the presence of granules of material positive to PAS staining in the clear epithelial cells.2,7,10,12 Those cells have been

interpreted as an epithelial degenerative process or as a feature of epithelial cytodifferentiation.16,20,21 Considering the clear epithelial cells component of PCEOTCC, the differential diagnosis with other lesions as metastatic renal cell carcinoma and odontogenic lesions (ie, clear cell odontogenic carcinoma and peripheral ameloblastoma with clear cell differentiation) must be regarded. It is fundamental to consider microscopic features (ie, typical microscopic features and morphologic growth patterns) of the lesions. In addition, auxiliary methods as immunohistochemistry, histochemistry, and ultrastructural findings may contribute to the correct diagnosis of PCEOTCC.21,26

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Fig 3. A, Epithelial cells located inside the amyloid-like material are immunopositive for cytokeratin 14. Amyloid-like material is immunonegative to cytokeratin 14 (streptavidin-biotin technique; original magnification ⫻320). B, Fibronectin presents as short, thick, and wavy bundles. Immunoexpression is more intense in amyloid-like material (arrowhead) than in connective tissue stroma (streptavidin-biotin technique; original magnification ⫻200). C and D, Type III collagen immunoexpression. In connective tissue stroma (C) it presents as long, thick, and wavy bundles. In amyloid-like material (D), the staining is less intense and diffuse (streptavidin-biotin technique; original magnification ⫻320). E, The basal membrane of epithelial cell nests located inside the amyloid-like material is immunopositive for tenascin (streptavidin-biotin technique; original magnification ⫻320).

The relationship between the presence of clear epithelial cells in odontogenic tumors with tumor aggressiveness has been questioned.27 In cases of clear cell odontogenic carcinoma, as reported by Waldron et al,28 Hansen et al,29 and Aguiar et al,30 the presence of clear epithelial cells was proved to be a sign of increased tumor aggressiveness, indicating a more radical surgical approach. However, in the biologic behavior of the present case, in addition to the 6 other cases reported in the literature, the presence of a clear epithelial cell component was not associated with aggressiveness of the PCEOTCC.2,7,10,12 Evaluation of the epithelial component and the amyloid-like deposit has been performed in CEOT and in its variants. Although the epithelial component has been shown to be immunopositive to CKs, previous studies have used pan-CK antibodies.23,31,32 In the present study, we used pan (AE1/AE3) and specific CK (CK-7, CK-8, CK-13, CK-14, and CK-19) antibodies. Immunostaining of epithelial component was positive only for AE1/AE3 and CK-14. Immunopositivity for CK-14 was expected, because this is one of the major CKs of the other odontogenic tumors (ameloblastoma) and occurs during human enamel organ development.33,34

Although widely investigated, the true nature of the amyloid-like deposit is still controversial. Solomon et al,13 using ultrastructural and histochemical studies, indicated that the amorphous, acellular, eosinophilic deposits present in the CEOT are not amyloid. Other interpretations are basement membrane components, CKs, and enamel matrix.31,35-39 The lack of staining for antilaminin, type IV collagen, and CK antibodies, as also demonstrated by Esguep et al,40 Mori et al,32 Takata et al,41 and Kumamoto et al,23 suggests that the amyloid-like deposit is not represented by basement membrane components or CKs. Our results also showed the lack of immunopositivity for laminin and type IV collagen in the basement membrane zone around nests of epithelial cells located in the amyloidlike area. In contrast, immunopositivity for tenascin in the same area was seen. Positive staining with Congo red and immunonegative staining with amyloid A, also demonstrated by Franklin et al15 and Takata et al,41 indicate that the amyloid-like deposit represents a non-A amyloid substance. Ultrastructural evaluation of the amyloidlike deposit of the CEOT shows that they consist of dense accumulation of fibrillar collagen material

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(fibrils).41,42,43 Biochemical evaluation demonstrates that this deposit is represented by a diverse group of related proteins, but the origin of the proteins is not clear.15 In the present investigation, it was demonstrated that the amyloid-like deposit is composed of extracellular matrix proteins (fibronectin and types I and III collagen), although it had a different appearance and intensity of staining when compared with the nonamyloid extracellular matrix. The findings of this investigation are in accord with other studies suggesting that the amyloid-like deposit of the CEOT represents non-A amyloid, where the diverse group of related proteins is composed of extracellular matrix proteins. We would like to thank the technical expertise and assistance of Mrs Edna Toddai and Mrs Elisa dos Santos (Oral Pathology Laboratory, School of Dentistry, University of Sa˜ o Paulo, Brazil). REFERENCES 1. Franklin CD, Pindborg JJ. The calcifying epithelial odontogenic tumor. A review and analysis of 113 cases. Oral Surg Oral Med Oral Pathol 1976;42:753-65. 2. Ai-Ru L, Zhen L, Jian S. Calcifying epithelial odontogenic tumors: a clinicopathologic study of nine cases. J Oral Pathol 1982;11:399-406. 3. Pindborg JJ. Calcifying epithelial odontogenic tumors. Acta Pathol Microbiol Scand 1955;3(Suppl):71-6. 4. Shafer WG, Hine MK, Levy BM, editors. A textbook of Oral Pathology, 2nd ed. Philadelphia: W.B. Saunders; 1963. p. 217. 5. Pindborg JJ. The calcifying epithelial odontogenic tumor. Review of literature and report of an extra-osseous case. Acta Odontol Scand 1966;24:419-30. 6. Decker RM, Laffitte HB. Peripheral calcifying epithelial odontogenic tumor. Oral Surg Oral Med Oral Pathol 1967;23:398402. 7. Abrams AM, Howell FV. Calcifying epithelial odontogenic tumors: report of four cases. J Am Dent Assoc 1967;74:1231-40. 8. Patterson JT, Martin TH, DeJean EK, Burzynski NJ. Extraosseous calcifying epithelial odontogenic tumor. Report of a case. Oral Surg Oral Med Oral Pathol 1969;27:363-7. 9. Krolls SO, Pindborg, JJ. Calcifying epithelial odontogenic tumor. A survey of 23 cases and discussion of histomorphologic variations. Arch Pathol 1974;6:266-9. 10. Wertheimer FW, Zeilinski RJ, Wesley RK. Extraosseous calcifying epithelial odontogenic tumor (Pindborg tumor). Int J Oral Surg 1977;6:266-269. 11. Takeda Y, Suzuki A, Sekiyama S. Peripheral calcifying epithelial odontogenic tumor. Oral Surg Oral Med Oral Pathol 1983;56: 71-5. 12. Houston GD, Fowler CB. Extraosseous calcifying epithelial odontogenic tumor. Report of two cases and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;83:577-83. 13. Solomon MP, Vulentin JC, Pertschuk LP, Gormley MB, Rosen Y. Calcifying epithelial odontogenic tumor. A histologic, histochemical, fluorescent and ultrastructural study. Oral Surg Oral Med Oral Pathol 1975;40:523-30. 14. Mori M, Makino M. Calcifying epithelial odontogenic tumor: histochemical properties of homogeneous acellular substances in the tumor. J Oral Surg 1977;35:631-8. 15. Franklin CD, Martin MV, Clark A, Smith CJ, Hindle MO. An investigation into the origin and nature of “amyloid” in a calcifying epithelial odontogenic tumour. J Oral Pathol 1981;10:41729.

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Reprint requests: Vera Cavalcanti de Arau´ jo, DDS, PhD Universidade de Sa˜ o Paulo Faculdade de Odontologia Disciplina de Patologia Bucal Av. Prof. Lineu Prestes 2227 Cidade Universita´ ria Sa˜ o Paulo-SP, Brasil CEP: 05508-900 [email protected]