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Notes: Method and Results: Calcifying odontogenic cysts (COCs) were examined histopathologically and immunohistochemically to characterize the histological and cytological properties of these lesions. Histopathologically, COCs showed thin or thick lining epithelium with ghost cells. COCs were classified according to proliferative type or nonproliferative type lining epithelium, the presence or absence of ameloblastomatous appearance, and the presence or absence of odontoma in the cyst walls. Immunohistochemically, amelogenin protein was expressed chiefly in ghost cells, whereas cytokeratin 19 (CK19) and bcl-2 proteins were expressed chiefly in lining epithelial cells. The proportion of cases positive for bcl-2 protein was slightly higher in COCs with odontoma than in those without odontoma. Lining epithelial cells sporadically showed positive reactions for Ki-67 antigen. Mean Ki-67 labeling index was slightly greater in COCs with proliferative type lining epithelium, COCs with ameloblastomatous appearance of the cyst walls, and COCs with odontoma of the cyst walls than in COCs without these histological features. Our results suggest that ghost cells or lining epithelial cells show ameloblastic cytodifferentiation or odontogenic epithelial characteristics, that bcl-2 protein is associated with survival of lining epithelial cells in COCs, and that high proliferation potential is associated with ameloblastomatous proliferation or combined odontoma. COCs exhibited various histological features with several transitional forms, and immunohistochemical examinations revealed little or no difference in cytodifferentiation and cellular activity among COCs.Conclusion: We conclude that COCs with various histological features have neoplastic potential and may not be separate entities within the same histological spectrum.

Notes: Background: To clarify the possible role of apoptotic cell death in oncogenesis and cytodifferentiation of odontogenic epithelium, apoptosis-related factors – Fas, Fas ligand (FasL), caspase-3 and single-stranded DNA (ssDNA) – were analyzed in ameloblastomas as well as in tooth germs.Methods: Specimens of 5 tooth germs, 29 benign ameloblastomas and 5 malignant ameloblastomas were examined by immunohistochemistry using anti-Fas, FasL, caspase-3 and ssDNA polyclonal antibodies.Results: Immunoreactivity for Fas and FasL was detected in normal and neoplastic odontogenic epithelial cells. Fas expression in ameloblastomas was slightly lower than that in tooth germs, whereas FasL expression was similar in tooth germs and ameloblastomas. Malignant ameloblastomas showed downregulation of Fas expression and upregulation of FasL expression, as compared with benign ameloblastomas, indicating escape from cell death attack by immune cells. Immunoreactivity for caspase-3 was detected chiefly in cells neighboring the basement membrane in tooth germs and ameloblastomas. Expression of caspase-3 and Fas tended to be low in basal cell ameloblastomas and high in desmoplastic ameloblastomas, as compared with other variants of ameloblastomas. Caspase-3 expression was more intense in malignant ameloblastomas than in tooth germs and benign ameloblastomas. Apoptotic bodies reactive with anti-ssDNA antibody were detected in normal and neoplastic odontogenic epithelial cells detached from the basement membrane. Keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas showed increased numbers of apoptotic bodies and increased expression of Fas and caspase-3, as compared with other neoplastic cells. Apoptotic reactions in malignant ameloblastomas were less frequent than in benign ameloblastomas, indicating abnormal regulation of cell turnover in odontogenic epithelial cells.Conclusion: These apoptosis-related factors were detected in various patterns in normal and neoplastic odontogenic epithelium, suggesting that these factors might be associated with oncogenesis and cytodifferentiation of epithelial odontogenic tumors.

Notes: Abstract: A case of clear cell odontogenic tumor with unusual histological features is presented. A 61-year-old Japanese man was admitted because of swelling of the left premolar-molar region of the mandible. Radiological examination revealed a multilocular radiolucency with irregular margins. Histological examination of the resected specimen showed infiltrative proliferation of both clear and eosinophilic cells into the adjacent soft tissue without encapsulation, suggesting the malignant potential of the tumor. The tumor cells sporadically formed cystic lesions. In addition, several tumor cell nests showed duct-like characteristics, and many eosinophilic dentin-like structures were attached to the tumor cell nests, suggesting the potential for epithelial-mesenchymal induction. Histochemically, the clear tumor cells possessed cytoplasmic glycogen granules. Both clear and eosinophilic tumor cells showed positive immunoreactivities for cytokeratin 19, epithelial membrane antigen and filaggrin, indicating an odontogenic epithelial origin.

Notes: Background:  To investigate the roles of the apoptosis signaling pathway mediated by mitochondria in oncogenesis and cytodifferentiation of odontogenic tumors, expression of pathway signaling molecules was analyzed in ameloblastomas as well as in tooth germs.Methods:  Tissue specimens of 12 tooth germs, 41 benign ameloblastomas, and five malignant ameloblastomas were examined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry to determine the expression of cytochrome c, apoptotic protease-activating factor-1 (APAF-1), caspase-9, and apoptosis-inducing factor (AIF).Results:  The mRNA expression of APAF-1, caspase-9, and AIF was detected in all samples of normal and neoplastic odontogenic tissues. Immunohistochemical reactivity for cytochrome c, APAF-1, caspase-9, and AIF was detected in both normal and neoplastic odontogenic tissues. Expression of cytochrome c and AIF was evident in odontogenic epithelial cells neighboring the basement membrane, and APAF-1 and caspase-9 were detected in most odontogenic epithelial cells. Immunoreactivity for cytochrome c in tooth germs was slightly weaker than that in benign and malignant ameloblastomas. Keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas showed a decrease or loss of immunoreactivity for these mitochondria-mediated apoptosis signaling molecules. Expression of AIF was obviously low in ameloblastic carcinomas.Conclusion:  Expression of cytochrome c, APAF-1, caspase-9, and AIF in tooth germs and ameloblastomas suggests that the mitochondria-mediated apoptotic pathway has a role in apoptotic cell death of normal and neoplastic odontogenic epithelium. Expression of these mitochondrial apoptosis signaling molecules might be involved in oncogenesis, cytodifferentiation, and malignant transformation of odontogenic epithelium.

Notes: Background:  To clarify the role of p53 homologs in oncogenesis and cytodifferentiation of odontogenic tumors, expression of p63 and p73 was analyzed in ameloblastomas as well as tooth germs.Methods:  Tissue specimens of nine tooth germs and 48 benign and five malignant ameloblastomas were examined by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) for the expression of p63 and p73.Results:  Immunoreactivity for p63 and p73 was evident in epithelial cells neighboring the basement membrane in developing and neoplastic odontogenic tissues. p63 expression in desmoplastic ameloblastomas was significantly higher than in acanthomatous and granular cell ameloblastomas, and ameloblastic carcinomas showed higher p63 expression than metastasizing ameloblastomas. p73 expression was significantly higher in plexiform ameloblastomas than in follicular ameloblastomas, and basal cell ameloblastomas showed higher p73 expression than granular cell ameloblastomas. mRNA transcripts for ΔNp63 and TAp73 were detected in all developing and neoplastic odontogenic tissues. TAp63 mRNA was expressed in five of eight tooth germs, 16 of 34 ameloblastomas, and one of one malignant ameloblastoma, whereas ΔNp73 mRNA was recognized in one of eight tooth germs, nine of 34 ameloblastomas, and one of one malignant ameloblastoma.Conclusion:  The expression of p63 and p73 suggests that these p53 homologs play a role in differentiation and proliferation of odontogenic epithelial cells. Variations of predominantly expressed isoforms suggest that p63 and p73 might differentially function in odontogenic tissues.

Notes: Background:  To clarify the roles of the p53–MDM2–p14ARF cell cycle regulation system in oncogenesis and cytodifferentiation of odontogenic tumors, p53 gene status and expression of p53, MDM2, and p14ARF proteins was analyzed in ameloblastomas as well as tooth germs.Methods:  Paraffin sections of 16 tooth germs and 46 benign and 5 malignant ameloblastomas were examined immunohistochemically for the expression of p53, MDM2, and p14ARF proteins. Frozen tissue samples of 10 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect p53 gene alteration.Results:  Immunohistochemical reactivity for p53 was detected in 2 of 13 tooth germs, 13 of 29 ameloblastomas, and 5 of 5 malignant ameloblastomas, and the expression ratio of p53 in tooth germs was significantly lower than those in benign and malignant ameloblastomas. Direct DNA sequencing showed no alteration of p53 gene exons 5–8 in any sample of 10 benign ameloblastomas and 1 metastasizing ameloblastoma. Expression of MDM2 and p14ARF was detected in all samples of normal and neoplastic odontogenic epithelium, and the expression ratios in tooth germs tended to be lower than those in benign and malignant ameloblastomas. In ameloblastomas, expression of p53, MDM2, and p14ARF was significantly higher in plexiform cases than in follicular cases. Markedly decreased reactivity for p53, MDM2, and p14ARF was detected in keratinizing and granular cells in ameloblastoma subtypes. Basal cell ameloblastoma showed slightly higher reactivity for p53, MDM2, and p14ARF as compared with other subtypes.Conclusion:  Elevated expression of p53, MDM2, and p14ARF in benign and malignant ameloblastomas suggests that alteration of the p53–MDM2–p14ARF cascade is involved in oncogenesis and/of malignant transformation of odontogenic epithelium. p53 gene status implied that p53 mutation might play a minor role in neoplastic changes of odontogenic epithelium. Immunoreactivity for p53, MDM2, and p14ARF in ameloblastoma variants suggests that these factors might be associated with tissue structuring and cytodifferentiation of ameloblastomas.

Notes: Background:  To clarify the roles of Sonic hedgehog (SHH) signal transduction in oncogenesis and cytodifferentiation of odontogenic tumors, expression of SHH, Patched (PTC), Smoothened (SMO), and GLI1 was analyzed in ameloblastomas as well as in tooth germs.Methods:  Tissue specimens of 9 tooth germs, 36 benign ameloblastomas, and 1 malignant ameloblastoma were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of SHH, PTC, SMO, and GLI1.Results:  Expression of SHH, PTC, SMO, and GLI1 mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for SHH, PTC, SMO, and GLI1 was detected in both normal and neoplastic odontogenic tissues. Expression of SHH, PTC, and GLI1 was more evident in epithelial cells than in mesenchymal cells, whereas SMO reactivity was marked in both epithelial and mesenchymal components in tooth germs and ameloblastomas. In ameloblastomas, these SHH signaling molecules were expressed more intensely in peripheral columnar or cuboidal cells than in central polyhedral cells; keratinizing cells and granular cells showed no or little reactivity.Conclusion:  Expression of SHH, PTC, SMO, and GLI1 in tooth germs and ameloblastomas suggests that these SHH signaling molecules might play a role in epithelial–mesenchymal interactions and cell proliferation in tooth development as well as in growth of these epithelial odontogenic tumors.

Notes: Background:  To clarify the roles of osteoclast regulatory factors in progression of odontogenic tumors, expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-κB ligand (RANKL), and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) were analyzed in ameloblastomas as well as tooth germs.Methods:  Tissue specimens of nine tooth germs and 36 benign and one malignant ameloblastomas were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of PTHrP, ODF/RANKL, and OCIF/OPG.Results:  Expression of PTHrP, ODF/RANKL, and OCIF/OPG mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for PTHrP was recognized in both normal and neoplastic odontogenic epithelial cells. In ameloblastomas, PTHrP reactivity in peripheral columnar or cuboidal cells was stronger than that in central polyhedral cells, and keratinizing cells showed increased PTHrP reactivity. ODF/RANKL and OCIF/OPG were expressed predominantly in mesenchymal cells rather than in odontogenic epithelial cells in both tooth germs and ameloblastomas. Epithelial ODF/RANKL and OCIF/OPG expression was slightly lower in ameloblastomas than in tooth germs. Tumor cells in plexiform ameloblastomas showed slightly higher reactivity for PTHrP and ODF/RANKL than tumor cells in follicular ameloblastomas.Conclusion:  Expression of PTHrP, ODF/RANKL and OCIF/OPG in tooth germs and ameloblastomas suggests that these factors might locally regulate bone metabolism and dynamics in tooth development as well as in progression of ameloblastomas. These factors might also be involved in tumor cell differentiation and/or tumor tissue structuring in ameloblastomas.

Notes: Background:  Alterations of human patched (ptc) homolog have been proven to be responsible for basal cell nevus syndrome (BCNS). Mandibular cysts in heterozygous ptc knockout mouse (ptc+/− mouse) were microradiologically, histologically, immunohistochemically, and genetically examined to investigate the possible role of the ptc gene and its associates in the jaw cysts.Methods:  The mandibular bones were prepared from 63 ptc+/− mice and 6 ptc+/+ mice. Soft X-ray radiographs and histological sections were examined for detection of the presence of mandibular cysts. The mandibular cysts were immunohistochemically investigated using anti-ptc, shh, and smo antibodies. PCR analysis of loss of heterozygosity (LOH) of ptc was performed in genomic DNA from the mandibular cysts.Results:  Six ptc+/+ mice showed no pathologic change in any examinations. Microradiologically, ptc+/− mice did not show any apparent lesion. Mandibular cysts were often multiple, and were histologically detected in the alveolar bones or periodontal ligaments of the molars in 16 (25.4%) ptc+/− mice. The mandibular cysts were lined by thin parakeratotic stratified squamous epithelium and contained keratinized materials. Immunohistochemical examination showed sonic hedgehog (shh) protein mainly in cyst lining epithelium, and ptc and smoothened (smo) proteins in cyst lining epithelium, and surrounding fibrous connective tissue. Expression of ptc protein in the cyst lining epithelium tended to be weak as compared with incisor enamel organs and gingival stratified squamous epithelium. LOH of the ptc gene couldn't be found in lining epithelium of mandibular cysts in any ptc+/− mice.Conclusions:  Ptc +/− mouse is a useful model of BCNS from the standpoint of occurrence of jaw cysts, and downregulation of ptc protein in cyst lining epithelium caused by gene targeting would be associated with formation of jaw cysts in ptc+/− mice.

Notes: Background:   Tumors derived from odontogenic epithelium exhibit considerable variation and are classified into several benign and malignant entities. To clarify the role of growth factors in oncogenesis, cytodifferentiation and progression of epithelial odontogenic tumors, expression of hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β) and their receptors were analyzed in these tumors as well as in tooth germs.Methods:   Specimens of five tooth germs, 34 ameloblastomas, three calcifying epithelial odontogenic tumors (CEOTs), two clear cell odontogenic tumors (CCOTs), five adenomatoid odontogenic tumors (AOTs), six calcifying odontogenic cysts (COCs) and six malignant ameloblastomas were examined immunohistochemically with the use of antibodies against HGF, TGF-β and their receptors.Results:   In tooth germs and epithelial odontogenic tumors, immunoreactivity for HGF and TGF-β was detected in both epithelial and mesenchymal cells, while expression of their receptors was found only in epithelial cells. In tooth germs and main types of ameloblastomas, HGF and TGF-β reactivity was marked in epithelial cells near the basement membrane, and their receptors were diffusely positive in most epithelial cells. In subtypes of ameloblastomas, reduced expression of HGF, c-Met and TGF-β and increased reactivity for TGF-β receptors were detected in keratinizing cells in acanthomatous ameloblastomas, and granular cells in granular cell ameloblastomas demonstrated little or no expression of HGF, TGF-β or their receptors. As compared with main types of ameloblastomas, basal cell ameloblastomas showed high HGF reactivity, and desmoplastic ameloblastomas exhibited elevated reactivity for TGF-β and its receptors. Neoplastic cells in CEOTs, AOTs and COCs showed reactivity for HGF, TGF-β and their receptors. Elevated HGF and TGF-β reactivity was found in pseudoglandular cells in AOTs, and high expression of their receptors was noted in ghost cells in COCs. Metastasizing ameloblastomas showed similar expression patterns of HGF, TGF-β and their receptors to those of benign ameloblastomas, while CCOTs and ameloblastic carcinomas had increased HGF expression and low reactivity for TGF-β and its receptors as compared with benign ameloblastomas.Conclusions:  Immunohistochemical localization of HGF, TGF-β and their receptors in tooth germs and epithelial odontogenic tumors supports the hypothesis that HGF and TGF-β act on epithelial cells via paracrine and autocrine mechanisms. Altered expression of the agents in these epithelial odontogenic tumors, especially subtypes of ameloblastomas, AOTs and COCs, suggests that HGF and TGF-β signaling might affect differentiation of neoplastic odontogenic epithelial cells. Activated HGF/c-Met pathway and reduced TGF-β signaling in CCOTs and ameloblastic carcinomas may be associated with the malignant potential of these epithelial odontogenic tumors.