A proteomics study to explore differential proteins associated with the pathogenesis of ameloblastoma.

BACKGROUND: Reports on the proteomic studies of ameloblastoma and other common odontogenic lesions are limited. We thus explored the differential proteins among ameloblastoma, odontogenic keratocyst, dentigerous cyst, and normal gingival tissue using proteomics and identified hub proteins involved in the local aggressiveness and recurrence of ameloblastoma. METHODS: Samples were obtained from 14 patients with ameloblastoma, 6 with odontogenic keratocyst, 9 with a dentigerous cyst, and 5 with normal gingival tissue. Proteins were then extracted, purified, quantified, and analysed using Easy-nLC chromatography and mass spectrometry. Further functional annotation and enrichment analyses were performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes on the target protein collection. Protein clustering and protein-protein interaction network analyses were used to screen the hub proteins. Proteins with significant interactions were screened according to their degree index. These results were verified by immunohistochemical staining. Proteins meeting the screening criteria of expression difference ploidy >1.2-fold (upregulation and downregulation) and p < 0.05 were considered differential proteins. RESULTS: In ameloblastoma, 808 differential proteins were upregulated and 505 were downregulated compared with those in odontogenic keratocyst; 309 were upregulated and 453 were downregulated compared with those in dentigerous cyst; and 2210 were upregulated and 829 were downregulated compared with those in normal gingival tissue. The three groups of differential proteins were associated with cellular exosomes, antigen binding, complement activation, human papillomavirus infection, focal adhesion, cell adhesion molecules, and metabolic pathways. CONCLUSION: CDH3 is associated with the local aggressiveness and recurrence of ameloblastoma and is a potential therapeutic target.

Altered expression of cell-cell adhesion molecules ß-catenin/E-cadherin and related Wnt-signaling pathway in sporadic and syndromal keratocystic odontogenic tumors.

Differential diagnosis of the keratocystic odontogenic tumor (KCOT) still represents a challenging problem especially if compared with the dentigerous cyst, which is similar in clinical and radiological course. Histological assessment of this entity may therefore draw crucial attention since various radical procedures are recommended for such lesions in contrast to dentigerous cysts. Since recent reports could prove the involvement of wingless(Wnt)-signaling pathway and ß-catenin in the pathogenesis of many odontogenic and neoplastic lesions indicating impairment of cell-cell adhesion, we investigated the expression of two Wnt-signaling pathways, Wnt-1 and Wnt-10A as well as ß-catenin and E-cadherin along with other related proteins in both lesions. We found a significant down-regulation in the expression of cell adhesion proteins ß-catenin and E-cadherin along with alteration of Wnt-1 and Wnt-10A expression in the epithelium of KCOT. We assessed a specific focal distribution pattern of p63 in the suprabasal cell layer and a significant up-regulation of cyclin D1. Furthermore, laminin α-2 was a characteristic marker labelling only the basement membrane of dentigerous cysts. These results provide a new hypothesis explaining a molecular mechanism to understand initiating and development of KCOTs and an alternative therapeutic approach, especially for syndromal patients, where these multilocal lesions may involve and destroy wide orofacial bony structures.

Genetic factors in the pathogenesis of ameloblastoma, dentigerous cyst and odontogenic keratocyst.

Ameloblastoma (AB), dentigerous cyst (DC) and Odontogenic keratocyst (OKC) are odontogenic lesions with propensity to malignant transformation or local invasion. The molecular mechanisms of development of these lesions are not fully understood. However, some researches have reported dysregulation of tumor suppressor genes or oncogenes in these lesions. Down-regulation of P53 gene has been reported in AB, DC and OKC. Moreover, several long non-coding RNAs such as ENST00000512916 and KIAA0125 have been dysregulated in AB tissues. Single nucleotide polymorphisms within a variety of genes have been associated with certain types of odontogenic lesions. In the current review, we summarize the current data about the expression pattern of genes in these lesions and the observed association between genetic polymorphisms and development of these lesions.

Immunoexpression of DNA base excision repair and nucleotide excision repair proteins in ameloblastomas, syndromic and non-syndromic odontogenic keratocysts and dentigerous cysts.

OBJECTIVE: To evaluate the immunoexpression of DNA base excision repair (BER) [apurinic/apyrimidinic endonuclease 1 (APE-1), X-ray repair cross complementing 1 (XRCC-1)] and nucleotide excision repair (NER) [xeroderma pigmentosum complementation group (XPF)] proteins in benign epithelial odontogenic lesions with different biological behaviors. DESIGN: Thirty solid ameloblastomas, 30 non-syndromic odontogenic keratocysts (NSOKCs), 29 syndromic odontogenic keratocysts (SKOCs), 30 dentigerous cysts (DCs) and 20 dental follicles (DFs) were evaluated quantitatively for APE-1, XRCC-1 and XPF through immunohistochemistry. RESULTS: Nuclear expression of APE-1 was significantly higher in NSOKCs, SOKCs, and ameloblastomas in comparison to DCs (p < 0.001). Nuclear expression of XRCC-1 was higher in NSOKCs and SOKCs than in DCs (p < 0.05). At the nuclear level, XPF expression was higher in NSOKCs and SOKCs than in DCs and ameloblastomas (p < 0.05). A statistically significant higher expression of APE-1 (nuclear), XRCC-1 (nuclear), and XPF (nuclear and cytoplasmic) was found in all odontogenic lesion samples as compared to DFs (p < 0.05). For all lesions, there was a positive correlation between nuclear expression of APE-1 and XRCC-1 or XPF (p < 0.05). CONCLUSIONS: Our results suggest a potential involvement of APE-1, XRCC-1 and XPF proteins in the pathogenesis of benign epithelial odontogenic lesions, especially in those with more aggressive biological behavior, such as ameloblastomas, NSOKCs, and SOKCs. We also showed that the expression of APE-1 was positively correlated with the nuclear expression of XRCC-1 and XPF, which may suggest an interaction between the BER and NER pathways in all odontogenic lesions studied herein.

Relationship between the prevalence of the dentigerous cyst and the odontogenic keratocyst tumor and the current etiologic hypothesis.

Cysts are considered as nonneoplastic benign lesions that, when present for a long period of time, can cause some discomfort, especially related to the treatment form. Among the types of cysts of the maxilla, the dentigerous cyst (DC) presents substances between the dental follicle and the crown of the tooth with high potential for resorption, and the odontogenic keratocyst tumor (OKT) characterizes for its noticed rapid growth pattern and the possibility to develop carcinomas in the lesion wall. The DC is the most common type among the developing odontogenic cystic lesions, while the OKT represents 10% of these lesions. The prevalence of the OKT found in the current study was superior to the DC, opposing data of the evaluated literature, as well as the predominance in relation to the age group. Dentigerous cyst cases were found mostly in younger individuals, whereas the OKT was observed mainly in individuals between the third and fourth decades of life. This fact reflects the fragility of these features while establishing the presumptive diagnosis and insinuates the strong relation with a probable genetic predisposition. In relation to sex and race, the findings in this article were similar to those found in the literature, highlighting the possibility of a hormonal involvement. However, the anatomopathologic examination remains essential to define the main diagnosis of the lesions observed by means of imaging examinations, providing for safer diagnoses to plan the treatment.

Assessment of fragile histidine triad expression in ameloblastoma, odontogenic keratocyst and dentigerous cyst.

BACKGROUND: FHIT (Fragile histidine triad) a member of tumor suppressor family, has been extensively studied in many solid tumors including head and neck squamous cell carcinoma. Among all head and neck cyst and tumors odontogenic lesions account approximately 3%-9%. The molecular pathogenesis of these lesions is less explored. Defects in cell cycle regulators and tumor suppressor genes could result in the development of odontogenic cyst and tumors. Hence, we aimed to determine the significant role of a tumor suppressor gene FHIT in most commonly occurring odontogenic lesions mainly ameloblastoma, odontogenic keratocyst and dentigerous cyst. SUBJECTS AND METHODS: Immunohistochemical analysis of FHIT was done in ameloblastoma, odontogenic keratocyst, dentigerous cyst and dental follicle. Interpretation of the stained slides were done using standard scoring criteria by two pathologist. The results were subjected for statistical analysis. RESULTS: Expression of FHIT varied among the groups, with highest negative expression in ameloblastoma 44.4% followed by odontogenic keratocyst 14% and 100%positive expression was seen in dentigerous cyst. The expression levels between the groups were statistically insignificant. CONCLUSION: The varied expression or negative expression of FHIT could be considered as an indicator for aggressive behavior and transformation of preneoplastic/cystic epithelium.

Alterations of FHIT and P53 genes in keratocystic odontogenic tumor, dentigerous and radicular cyst.

BACKGROUND: The purpose of this study was to determine fragile histidine triad (FHIT) and p53 protein expression, and to analyze FHIT and p53 gene status in keratocystic odontogenic tumor (KOT), dentigerous cysts (DC) and radicular cysts (RC). METHODS: The methods used were immunohistochemistry and molecular genetic methods including loss of heterozygosity (LOH) and gene sequencing. RESULTS: FHIT protein expression was different among groups. Aberrant expression was the highest in KOT, then in RC and DC. p53 protein expression was different among groups. LOH in paraffin-embedded specimens was detected in 22.6% and 12.9% for FHIT and p53 respectively. Mutation of p53 gene at codon 237 was observed in only two specimens (one KOT and one DC). Of the six frozen specimens, three exhibited FHIT gene LOH (two RC and one KOT). KOT showed loss of exons 6-7 at FHIT locus and mutation at codon 237 at p53 locus, but this could be a chance result. CONCLUSION: Aberrations of FHIT and p53 genes/proteins could be considered markers responsible for the development of odontogenic lesions.

Genetic basis of dentigerous cysts associated with supernumerary teeth: A narrative review.

In the present study, we sought to: (a) analyze the p53 gene status in dentigerous cysts (DC) associated with supernumerary teeth (ST) in a pair of siblings with ST, of whom one developed a DC; and (b) conduct a narrative review of the literature on ST associated with DC. Blood samples were obtained, and the isolated DNA was used to amplify exons 4-8 of the p53 gene using specific primers, and subsequently sequenced. No mutations were identified in the coding regions of the p53 gene. A review of the literature revealed a prevalence of DC associated with ST to be as high as 13.6%, and that 83% of the case reports performed enucleation of DC and removal of ST. Dentigerous cysts associated with ST in one sibling demonstrated that variations in phenotypes exist, and the absence of mutations cannot eliminate the potential influence of genetic risk factors.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell­derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor­κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and tartaric­resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T­cells 1 (NFATc1) and TRAP. Moreover, TRAP­positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony­stimulating factor (M­CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.

Involvement of PTCH gene in various noninflammatory cysts.

Constitutional hemizygous inactivation of PTCH, the Shh signaling pathway gene that moderates the signal, manifests itself as nevoid basal cell carcinoma syndrome or Gorlin syndrome, a condition variably characterized by a number of developmental disorders and malformations, and by predisposition to some malignancies, basal cell carcinoma in particular. Loss of heterozygosity for the PTCH region was found several years ago in the epithelial lining of odontogenic keratocysts, the cyst type with highly increased incidence in nevoid basal cell carcinoma syndrome. This finding confirmed the expectations that the gene responsible for the syndrome would have a decisive role in the genesis of these cysts even when they are not syndrome related. Suggestive temporal distribution of Shh signaling, recently observed during tooth development, lead us to investigate PTCH association with dentigerous cysts, the other major noninflammatory cyst of odontogenic origin. We report here that PTCH appears to be inactivated in dentigerous cysts, suggesting that it is responsible for their genesis as well. More generally, if our similar observations of incomplete heterozygosity in this region for dermoid cysts can be interpreted as loss of heterozygosity, PTCH alterations may prove to be a necessary, and perhaps the initiating event, in formation and growth of various noninflammatory cysts. This would be consistent with our view that local PTCH inactivation can, under favorable circumstances, lead to persistent though not by itself truly aggressive cell proliferation.

P53 protein expression in odontogenic cysts.

p53 protein seems to be related to the suppression of cell proliferation. p53-positive tissues seem to have a higher proliferative activity than p53-negative ones. Odontogenic keratocyst (OKC) has a different behavior from other types of cysts because it is more aggressive, with a tendency to recurrence. Twenty-two dentigerous cysts, 24 radicular cysts, and 20 OKCs were used in the present study. Two dentigerous cysts (9.1%), 2 radicular cysts (8.3%), and 9 OKCs (45%) expressed the p53 protein. The differences between the three groups were statistically significant (p = 0.003). In 10 cases of OKCs epithelial dysplasia was found. One of the 10 OKCs without dysplasia and 8 of the 10 OKCs with dysplasia were p53-positive: the difference between the two groups was statistically significant (p = 0.007). The overexpression of p53 protein was not on the other hand correlated with the occurrence of multiple, bilateral, and recurrent OKCs. Moreover the distribution of p53-positive cells was parabasal in contrast with other types of cysts. These qualitative and quantitative differences in proliferative activity in OKCs seem to point to an alteration in cell cycle control.

Bilateral dentigerous cyst associated with polymorphism in chromosome 1qh+.

Non-syndromal bilateral dentigerous cysts are rare. It is conceivable that they are either under-recognized or under-reported. In this article we report the unusual occurrence of non-syndromic bilateral dentigerous cysts associated with mandibular third molars with polymorphism in chromosome 1qh+. It is suggested that in non syndromal cases of bilateral dentigerous cysts karyotyping should be done to confirm the association with chromosomal 1 anomaly.

One germline mutation of PTCH gene in a Chinese family with non-syndromic keratocystic odontogenic tumours.

Keratocystic odontogenic tumours (KOCTs) are common benign cystic tumours that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). PTCH mutation can be found in sporadically or NBCCS associated KOCTs. Few PTCH mutations in families with non-syndromic KOCTs have been reported. Through PCR and gene sequence analysis, the authors discovered one missense mutation c.3277G>C in exon 19 of PTCH gene in a Chinese family with non-syndromic KOCTs. This mutation causes one highly conserved glycine residue transit to arginine on the 10th transmembrane region of PTCH protein. This work revealed that the missense mutation of PTCH is the causative and dominant gene of KOCTs in this family.

Primary cilia in the pathogenesis of dentigerous cyst: a new hypothesis based on role of primary cilia in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), an inherited disease, leads to cyst formation in the kidneys. In this condition, the kidneys are grossly enlarged with multiple cysts that result in kidney failure in a majority of individuals. This condition is also associated with cysts in other organs. Recent research has focused on defects in signaling mediated by the primary cilia as the causative factor in ADPKD. Primary cilia are also present in odontogenic epithelium. Dentigerous cyst also is a developmental cyst whose pathogenesis is controversial. Recent studies have shown that loss of Ptch and Shh signaling pathways are involved in the cystogenesis of dentigerous cyst. The Shh signaling pathway is active in the primary cilia. A scanning electron microscopic study of a dentigerous cyst wall in an ADPKD patient showed structures similar to primary cilia. Based on the presentation of a dentigerous cyst in an autosomal dominant polycystic kidney patient and the demonstration of primary cilia like structures on the cyst wall by using a scanning electron microscope, a new hypothesis for the pathogenesis of dentigerous cyst is proposed.

Comparative immunohistochemical expression of RANK, RANKL and OPG in radicular and dentigerous cysts.

OBJECTIVE: Receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) are members of the superfamily of ligands and receptors of tumour necrosis factor family involved in bone metabolism. The formation, differentiation and activity of osteoclasts are regulated by these proteins. To clarify the roles of osteoclast regulatory factors in cystic expansion of odontogenic cysts, expression of these proteins were analysed in radicular and dentigerous cysts. DESIGN: The immunohistochemistry expression of these biomarkers were evaluated and measured in lining epithelium and fibrous capsule of the radicular (n=20) and dentigerous cysts (n=20). RESULTS: A similar expression in lining epithelium was observed in the lesions. The fibrous capsule of dentigerous cyst showed a higher content of RANK-positive and RANKL-positive cells than fibrous capsule of radicular cyst. In the lining epithelium the RANKL/OPG ratio showed higher numbers of OPG-positive than RANKL-positive cells, whereas fibrous capsule of the cysts had a tendency to present a similar expression (OPG=RANKL). CONCLUSION: Ours findings indicate the presence of RANK, RANKL and OPG in cysts. Moreover, increased expression of OPG compared to RANKL in the lining epithelium could contribute to the differential bone resorption activity in theses lesions.

Oligonucleotide microarray analysis of ameloblastoma compared with dentigerous cyst.

BACKGROUND: Ameloblastoma is a benign, but locally invasive tumor known for its high rate of recurrence. However, few comprehensive genetic studies have been conducted about its tumorigenesis. Our aim was to identify possible genes involved in the development and progression of ameloblastoma, using microarray analysis with dentigerous cyst as a control. METHODS: Total RNA was extracted from two fresh dentigerous cysts and ameloblastoma specimens. Following microarray analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were performed on selected genes. RESULTS: Seventy-three genes were overexpressed and 49 were underexpressed. These genes were divided into categories according to function. The microarray results for 13 selected genes were verified with semiquantitative RT-PCR. CONCLUSIONS: We identified important genes related to the development and progression of ameloblastoma through a large-scale gene expression analysis. This study will stimulate further investigations on genes significant for early diagnosis and prognosis of ameloblastoma.

Basal cell nevus syndrome. A case report.

An 11-year-old boy with multiple dentigerous cysts in the maxilla and mandible is described. Other findings seen in the face, plantar skin, skeletal system and oral cavity indicated the lesions to be due to the basal cell nevus syndrome. This was further confirmed by the presence of similar abnormalities in his father and brother.

Immunohistochemical study of bcl-2 protein, Ki-67 antigen and p53 protein in epithelium of glandular odontogenic cysts and dentigerous cysts.

The aim of the present study was the evaluation of the immunohistochemical expression of the apoptosis-inhibiting protein bcl-2, the cell-cycle-related antigen Ki-67 and the p53 protein, which is involved both in cell cycle and apoptosis regulation, in the lining epithelium of glandular odontogenic cysts of the jaws. Formalin-fixed and paraffin-embedded tissue sections of three glandular odontogenic cysts and six dentigerous cysts were immunostained with a standard avidin-biotin peroxidase procedure, after microwave antigen retrieval. The glandular odontogenic cysts showed immunoreactivity for bcl-2 protein in the basal and suprabasal layers, while staining in dentigerous cysts was basal or focal. Most mucous cells and superficial cuboidal cells were negative. The percentage of Ki-67- or p53-positive cells was lower in glandular odontogenic cysts compared with dentigerous cysts. The findings suggest that the biological behavior of glandular odontogenic cysts may be associated with deregulation of cell death in the lining epithelium, while cell proliferation and p53 status do not seem to play a significant role.

p53 expression in odontogenic keratocyst epithelium.

The expression of p53 protein was studied in odontogenic keratocysts (OKC, 11 solitary, 5 recurrent and 6 NBCCS cysts), radicular (RC, n = 5) and dentigerous (DC, n = 5) cysts, using a panel of antibodies to p53 (clone BP53-12, clone 1801 and polyclonal CM1) and a sensitive biotin-streptavidin method on paraffin embedded sections. Of the three antibodies tested, clone BP53-12 gave the most intense and consistent nuclear staining pattern. Clone 1801 and polyclonal CM1 stained only 38% and 71% OKC linings, respectively, but not RC and DC linings. However, BP53-12+ cells were detected in the epithelial linings of all cyst types. Quantification of BP53-12+ cells was performed by manual counting and by relating cell number to unit length of basement membrane as determined by TV image analysis. BP53-12+ cell counts in solitary OKC linings (25.5 +/- 11.0 cells/mmBM) were significantly greater than those in DC (9.3 +/- 4.9 cells/mmBM, P < 0.01) and RC (6.7 +/- 2.6 cells/mmBM, P < 0.01) linings. The epithelial distribution of positive cells in OKC was predominantly suprabasal, which also varied from that of DC and RC linings (P < 0.005). There were no detectable differences in BP53-12 reactivity between the different subtypes of OKC (i.e., solitary, recurrent and NBCCS-associated OKC; P > 0.1). When data for the NBCCS-related OKC group were excluded, there was a significant correlation (r = 0.55, P < 0.01) between p53 and Ki67 labelling. To detect the presence of p53 gene mutations, genomic DNA, extracted from paraffin sections of OKC (4 solitary, 2 recurrent and 4 NBCCS cysts), RC (n = 3) and normal oral mucosa (n = 1), was subjected to a combination of polymerase chain reaction and single-stranded conformation polymorphism (PCR-SSCP) analysis for exons 5-10 of the p53 gene. Exon 4 was not analysed because of compromised DNA quality. No abnormality in banding patterns was found and all samples gave results similar to DNA from known, sequenced, normal p53 gene controls. Absence of p53 mutations within exons 5-9 was confirmed by the direct sequencing of 2 fresh frozen OKC samples (1 solitary and 1 NBCCS cyst). These results suggest that overexpression of p53 protein in OKC epithelium, detected by immunocytochemistry, is not reflected by alteration of the p53 gene and presumably reflects overproduction and/or stabilisation of normal p53 protein.