Objective: To detect the SMO mutations in odontogenic keratocyst (OKC) and to explore the mechanism behind. Methods: Patients with OKC who received treatment in the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology,Peking University, from September 2012 to June 2017 were enrolled. OKC samples from 10 patients diagnosed as naevoid basal cell carcinoma syndrome (NBCCS)-related OKC (4 females and 6 males) and 20 patients diagnosed as sporadic OKC (7 females and 13 males) were collected. Genomic DNAs were extracted from fibrous capsules and epithelial lining respectively. SMO mutations were detected and analyzed by Sanger sequencing. Results: Three SMO mutations were found in one NBCCS-associated OKC who carrying c.2081C>G (p.P694R) mutation) and two sporadic OKC who carrying c.907C>T (p.L303F) mutation and c.1247_1248delinsAA (p.G416E), respectively), among which the first two mutations were novel mutations that had not been reported before. Besides, two mutations in sporadic OKC were not paired with PTCH1 mutations. Conclusions: In addition to PTCH1 gene mutations, SMO gene mutations also exist in OKC which might be related to the development of OKC.
Basal Cell Nevus Syndrome/genetics* ; Female ; Humans ; Male ; Mutation ; Odontogenic Cysts/genetics* ; Odontogenic Tumors/genetics* ; Smoothened Receptor/genetics*

OBJECTIVETo study hTERT mRNA expression in ameloblastoma (AB) and odontogenic keratocyst (OKC) and to investigate genesis, development and biological characteristics of AB and OKC.

METHODShTERT mRNA expression in 54 cases of AB, 16 cases of OKC, 7 cases of oral normal mucosa was detected by in situ hybridization.

RESULTSThe positive rates of hTERT mRNA in AB, OKC, and oral normal mucosa were 94.4% (51/54), 87.5% (14/16), 1/7, respectively. There was significant statistical difference (P < 0.001). About clinicalpathology of AB, there was no difference for hTERT expression (P > 0.05) hTERT mRNA was positive in peripheral and stellate reticula cells.

CONCLUSIONS(1) hTERT activity plays an important role in genesis, development of AB. (2) hTERT positive rate is related to cell differentiation and clinical biological behavior.

Ameloblastoma ; DNA-Binding Proteins ; metabolism ; Humans ; Jaw Neoplasms ; Odontogenic Cysts ; Odontogenic Tumors ; metabolism ; RNA, Messenger ; metabolism ; Telomerase ; genetics

Odontogenic keratocysts (OKCs) are common cystic lesions of odontogenic epithelial origin that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS). OKCs are locally aggressive, cause marked destruction of the jaw bones and have a propensity to recur. PTCH1 mutations (at ∼80%) are frequently detected in the epithelia of both NBCCS-related and sporadic OKCs, suggesting that PTCH1 inactivation might constitutively activate sonic hedgehog (SHH) signalling and play a major role in disease pathogenesis. Thus, small molecule inhibitors of SHH signalling might represent a new treatment strategy for OKCs. However, studies on the molecular mechanisms associated with OKCs have been hampered by limited epithelial cell yields during OKC explant culture. Here, we constructed an isogenic PTCH1 cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1 heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency. This activation was found to be downregulated in a dose-dependent manner by the SHH pathway inhibitor GDC-0449. In addition, through inhibition of activated SHH signalling, the enhanced proliferation observed in these induced cells was suppressed, suggesting that GDC-0449 might represent an effective inhibitor of the SHH pathway for use during OKC treatment.
Anilides ; pharmacology ; Basal Cell Nevus Syndrome ; Hedgehog Proteins ; genetics ; pharmacology ; Humans ; Molecular Targeted Therapy ; Odontogenic Cysts ; genetics ; physiopathology ; therapy ; Odontogenic Tumors ; genetics ; physiopathology ; therapy ; Pyridines ; pharmacology

Odontogenic tumors are rare lesions with unknown etiopathogenesis. Most of them are benign, but local aggressiveness, infiltrative potential, and high recurrence rate characterize some entities. The MAP-kinase pathway activation can represent a primary critical event in odontogenic tumorigenesis. Especially, the BRAF V600E mutation has been involved in 80-90% of ameloblastic lesions, offering a biological rationale for developing new targeted therapies. The study aims to evaluate the BRAF V600E mutation in odontogenic lesions, comparing three different detection methods and focusing on the Sequenom MassARRAY System. 81 surgical samples of odontogenic lesions were subjected to immunohistochemical analysis, Sanger Sequencing, and Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (Sequenom). The BRAF V600E mutation was revealed only in ameloblastoma samples. Moreover, the presence of BRAF V600E was significantly associated with the mandibular site (ρ = 0.627; P value <0.001) and the unicystic histotype (ρ = 0.299, P value <0.001). However, any significant difference of 10-years disease-free survival time was not revealed. Finally, Sequenom showed to be a 100% sensitive and 98.1% specific, suggesting its high-performance diagnostic accuracy. These results suggest the MAP-kinase pathway could contribute to ameloblastic tumorigenesis. Moreover, they could indicate the anatomical specificity of the driving mutations of mandibular ameloblastomas, providing a biological rational for developing new targeted therapies. Finally, the high diagnostic accuracy of Sequenom was confirmed.
Ameloblastoma/pathology* ; Carcinogenesis ; Humans ; Mitogen-Activated Protein Kinases/genetics* ; Mutation ; Odontogenic Tumors/pathology* ; Proto-Oncogene Proteins B-raf/metabolism* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo study the expression of HOXC13 mRNA in ameloblastoma (AB), and to investigate its biological significance.

METHODSHOXC13 mRNA was examined in 47 cases of AB (primary AB 29 cases, recurrent AB 14 cases, malignant AB 4 cases). 2 cases of fibrous dysplasia of bone, 10 cases of keratocystic odontogenic tumor (KCOT) and 7 cases of normal oral mucosa were selected as control.

RESULTSThe positive rates of HOXC13 mRNA in AB, KCOT, and normal oral mucosa were 97.9% (46/47), 7/10 and 3/7, respectively. There was a significant difference among AB, OKC and normal mucosa (chi(2) = 21.665, P = 0.001). For HOXC13, the keratinizing cells and granulizing cells in AB were negative, some fibroblasts were positive, 2 cases of fibrous dysplasia of bone were positive.

CONCLUSIONSHOXC13 was highly expressed in AB. The expression of HOXC13 mRNA in AB had heterogeneity, which could improve the epithelial proliferation, and its loss may lead to the cornification and degeneration of epithelial cells.

Adolescent ; Adult ; Ameloblastoma ; genetics ; metabolism ; pathology ; Female ; Gene Expression ; Genes, Homeobox ; Homeodomain Proteins ; metabolism ; Humans ; Male ; Middle Aged ; Mouth Mucosa ; metabolism ; Odontogenic Tumors ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Young Adult

AIMTo clarify the role of PTCH in patients with NBCCS-related and non-sydromic keratocystic odontogenic tumors.

METHODOLOGYMutation analysis was undertaken in 8 sporadic and 4 NBCCS-associated KCOTs.

RESULTSFour novel and two known mutations were identified in 2 sporadic and 3 syndromic cases, two of which being germline mutations (c.2179delT, c.2824delC) and 4 somatic mutations (c.3162dupG, c.1362-1374dup, c.1012 C>T, c.403C>T).

CONCLUSIONOur findings suggest that defects of PTCH are associated with the pathogenesis of syndromic as well as a subset of non-syndromic KCOTs.

Adolescent ; Adult ; Amino Acid Sequence ; Basal Cell Nevus Syndrome ; genetics ; Chromatography, High Pressure Liquid ; Codon, Nonsense ; genetics ; Codon, Terminator ; genetics ; Conserved Sequence ; genetics ; Cytosine ; Exons ; genetics ; Female ; Frameshift Mutation ; genetics ; Gene Duplication ; Germ-Line Mutation ; genetics ; Guanine ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Mutation, Missense ; genetics ; Odontogenic Tumors ; genetics ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Deletion ; genetics ; Syndrome ; Threonine ; genetics ; Thymine

Keratocystic odontogenic tumors (KCOT) are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density (MVD) than dentigerous cysts (DC) and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing (RNA-Seq) was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 (LOXL4), the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts (P<0.05). In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells (HUVECs). There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues (r=0.882). These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.
Adult ; Amino Acid Oxidoreductases ; genetics ; Cell Movement ; genetics ; Cell Proliferation ; Dentigerous Cyst ; enzymology ; pathology ; Disease Progression ; Female ; Fibroblasts ; pathology ; Gene Expression Regulation, Enzymologic ; genetics ; Gingiva ; pathology ; Human Umbilical Vein Endothelial Cells ; pathology ; Humans ; Male ; Microvessels ; pathology ; Neovascularization, Pathologic ; genetics ; Odontogenic Tumors ; blood supply ; enzymology ; pathology ; Sequence Analysis, RNA ; Stromal Cells ; pathology ; Young Adult