Comprehensive Genomic Analysis of Cemento-Ossifying Fibroma.

Cemento-ossifying fibroma (COF) of the jaws is currently classified as a benign mesenchymal odontogenic tumor, and only targeted approaches have been used to assess its genetic alterations. A minimal proportion of COFs harbor CDC73 somatic mutations, and copy number alterations (CNAs) involving chromosomes 7 and 12 have recently been reported in a small proportion of cases. However, the genetic background of COFs remains obscure. We used a combination of whole-exome sequencing and RNA sequencing to assess somatic mutations, fusion transcripts, and CNAs in a cohort of 12 freshly collected COFs. No recurrent fusions have been identified among the 5 cases successfully analyzed by RNA sequencing, with in-frame fusions being detected in 2 cases (MARS1::GOLT1B and PARG::BMS1 in one case and NCLN::FZR1 and NFIC::SAMD1 in the other case) and no candidate fusions identified for the remaining 3 cases. No recurrent pathogenic mutations were detected in the 11 cases that had undergone whole-exome sequencing. A KRAS p.L19F missense variant was detected in one case, and 2 CDC73 deletions were detected in another case. The other variants were of uncertain significance and included variants in PC, ACTB, DOK6, HACE1, and COL1A2 and previously unreported variants in PTPN14, ATP5F1C, APOBEC1, HDAC5, ATF7IP, PARP2, and ACTR3B. The affected genes do not clearly converge on any signaling pathway. CNAs were detected in 5/11 cases (45%), with copy gains involving chromosome 12 occurring in 3/11 cases (27%). In conclusion, no recurrent fusions or pathogenic variants have been detected in the present COF cohort, with copy gains involving chromosome 12 occurring in 27% of cases.

MMP13 Expression and Activity Suggest Its Role in Bone Resorption in Ameloblastomas.

BACKGROUND: Ameloblastoma is a locally destructive benign odontogenic tumor. While the neoplastic cells of conventional ameloblastoma can infiltrate the connective tissue and bone, in unicystic ameloblastoma the epithelium is encapsulated. The mechanisms driving ameloblastoma's bone resorption remains unclear. METHODS: RNA sequencing (RNA-seq) was performed in a discovery cohort of conventional ameloblastoma, and pathway enrichment analysis was carried out. mRNA levels of MMP13, a gene associated with bone resorption, were assessed using RT-qPCR in a larger cohort of conventional ameloblastoma and in unicystic ameloblastoma. Zymogram gels and the immunoexpression profile of collagenase 3 (encoded by MMP13 gene) were evaluated as well. RESULTS: Enriched pathways related to bone mineralization and upregulation of MMP13 were observed in ameloblastomas. Collagenolytic activity of collagenase 3 was detected in the tumor lysates. Collagenase 3 immunopositivity was observed in ameloblastomatous epithelium infiltrating the fibrous capsule of unicystic ameloblastoma. At the tumor-bone interface, collagenase 3 expression was detected in stromal cells, osteoblasts, and osteocytes. CONCLUSION: The results indicate a potential involvement of MMP13 in ameloblastoma-related bone resorption and progression.

Chronic rhinosinusitis with nasal polyps does not harbor KRAS, BRAF, and EGFR mutations.

BACKGROUND: Chronic rhinosinusitis is a chronic inflammation of the nasal mucosa and nasal polyps are present in ~25%-30% of cases (chronic rhinosinusitis with nasal polyps [CRSwNP]). CRSwNP is associated with significant morbidity and decreased quality of life, making it clinically important. Inflammation leads to DNA damage and DNA mutations occur in some inflammatory diseases. Notably, mutations in KRAS, BRAF, and EGFR have been reported in different human benign and malignant neoplastic lesions. In addition, KRAS mutations have also been reported in non-neoplastic tissues under chronic inflammatory conditions. Importantly, KRAS mutations have been reported in oncocytic sinonasal papillomas and sinonasal squamous cell carcinoma associated with oncocytic sinonasal papilloma and EGFR mutations have been reported in sinonasal adenocarcinoma, inverted sinonasal papilloma, and sinonasal squamous cell carcinoma associated with inverted sinonasal papilloma. The molecular pathogenesis of nasal polyps remains unclear. Therefore, the present study aimed to investigate the presence of KRAS, BRAF, and EGFR pathogenic mutations in CRSwNP. METHODS: Fourteen chronic rhinosinusitis-associated nasal polyp samples were direct sequenced, targeting KRAS exons 2, 3, and 4 (encompassing important hotspot mutations, including codons 12, 13, 61 and 146), BRAF exons 11 and 15, and EGFR exons 19 and 20. RESULTS: No pathogenic mutations were detected in the sequenced regions of KRAS, BRAF, and EGFR genes. CONCLUSION: This finding suggests that mutations in these genes are not a frequent event in CRSwNP, and, if they occur, they might represent marginal events at best.

Can SARS-CoV-2 screening in oral biopsies aid epidemiological surveillance?

BACKGROUND: Three years after the first confirmed COVID-19 case in Brazil, the outcomes of Federal government omissions in managing the crisis and anti-science stance heading into the pandemic have become even more evident. With over 36 million confirmed cases and nearly 700 000 deaths up to January 2023, the country is one of the hardest-hit places in the world. The lack of mass-testing programs was a critical broken pillar responsible for the quick and uncontrolled SARS-CoV-2 spread throughout the Brazilian population. Faced with this situation, we aimed to perform the routine SARS-CoV-2 screening through RT-qPCR of oral biopsies samples to aid in the asymptomatic epidemiological surveillance during the principal outbreak periods. METHODS: We analyzed 649 formalin-fixed paraffin-embedded oral tissue samples from five important oral and maxillofacial pathology laboratories from the north, northeast, and southeast geographic regions of Brazil. We also sequenced the whole viral genome of positive cases to investigate SARS-CoV-2 variants. RESULTS: The virus was detected in 9/649 analyzed samples, of which three harbored the Variant of Concern Alpha (B.1.1.7). CONCLUSION: Although our approach did not value aiding asymptomatic epidemiological surveillance, we could successfully identify a using FFPE tissue samples. Therefore, we suggest using FFPE tissue samples from patients who have confirmed diagnosis of SARS-CoV-2 infection for phylogenetic reconstruction and contraindicate the routine laboratory screening of these samples as a tool for asymptomatic epidemiological surveillance.

Quantitative proteomic study reveals differential expression of matricellular proteins between fibrous dysplasia and cemento-ossifying fibroma pathogenesis.

BACKGROUND: Fibrous dysplasia (FD) and cemento-ossifying fibroma (COF) are the most common gnathic fibro-osseous lesions. These diseases exhibit remarkable overlap of several clinicopathological aspects, and differential diagnosis depends on the combination of histopathological, radiographic, and clinical aspects. Their molecular landscape remains poorly characterized, and herein, we assessed their proteomic and phosphoproteomic profiles. METHODS: The quantitative differences in protein profile of FD and COF were assessed by proteomic and phosphoproteomic analyses of formalin-fixed paraffin-embedded tissue samples. Pathway enrichment analyses with differentially regulated proteins were performed. RESULTS: FD and COF exhibited differential regulation of pathways related to extracellular matrix organization, cell adhesion, and platelet and erythrocytes activities. Additionally, these lesions demonstrated distinct abundance of proteins involved in osteoblastic differentiation and tumorigenesis and differential abundance of phosphorylation of Ser61 of Yes-associated protein 1 (YAP1). CONCLUSIONS: In summary, despite the morphological similarity between these diseases, our results demonstrated that COF and DF present numerous quantitative differences in their proteomic profiles. These findings suggest that these fibro-osseous lesions trigger distinct molecular mechanisms during their pathogenesis. Moreover, some proteins identified in our analysis could serve as potential biomarkers for differential diagnosis of these diseases after further validation.

Central giant cell granulomas of the jaws stromal cells harbour mutations and have osteogenic differentiation capacity, in vivo and in vitro.

BACKGROUND: Central giant cell granulomas (CGCG) of the jaws are osteolytic lesions that may behave aggressively and respond poorly to surgery. Microscopically, in addition to giant cells, there is a mononuclear cell population composed of macrophage/monocytic cells and spindle-shaped cells of mesenchymal origin. Seventy two percent of these tumours harbour mutually exclusive TRPV4, KRAS and FGFR1 mutations. We aimed to assess the mutational status of mononuclear and giant cells and the osteogenic potential of stromal cells in vitro and in vivo. METHODS AND RESULTS: We screened CGCG for signature mutations and used laser-capture microdissection to demonstrate that the mutations are restricted to the mononuclear cells. Additionally, we established CGCG primary cell culture and observed that the cells retained the mutations throughout passages. By flow cytometry, we observed predominance of CD14- CD51- CD61- cells, consistent with the expected profile for stromal cells. Considering the mesenchymal origin of stromal cells, we assessed the osteogenic differentiation potential of CGCG cells in culture by cytochemistry (von Kossa and alizarin red staining), alkaline phosphatase (ALP) activity assay and gene expression of osteogenic markers. CGCG cells presented self-capacity to increase ALP levels in a time-dependent manner and under osteogenic induction presented increasing number of calcium deposits, and overall higher expression of osteocalcin, RUNX2, ALPL and osteopontin than cells without osteogenic induction. A patient-derived xenograft model for CGCG was established, and osteoid material deposition was observed. CONCLUSION: Collectively, the results confirm that the signature mutations are restricted to stromal cells in CGCG, and the in vitro and in vivo results support that these cells have the capacity to differentiate into osteoblasts, in line with the bone formation often observed in the stroma of these lesions.

Unveiling metabolic changes in marsupialized odontogenic keratocyst: A pilot study.

OBJECTIVE: We aimed to assess which metabolic pathways would be implicated in the phenotypic changes of the epithelial lining of odontogenic keratocyst after marsupialization, comparing pre- and post-marsupialized lesions with adjacent oral mucosa. MATERIALS AND METHODS: Eighteen formalin-fixed and paraffin-embedded tissues from six subjects were divided into three paired groups: odontogenic keratocyst pre- (n = 6) and post-marsupialization (n = 6), and adjacent oral mucosa (n = 6). The metabolic pathways found in these groups were obtained by high-performance liquid chromatography-mass spectrometry-based untargeted metabolomics performed. RESULTS: Through putative metabolite annotation followed by pathway enrichment and predictive analysis with automated algorithms (Mummichog and Gene Set Enrichment Analysis), we found differences in many cellular processes that may be involved in inflammation, oxidative stress response, keratinocyte-basal membrane attachment, differentiation, and proliferation functions, all relevant to odontogenic keratocyst pathobiology and the phenotype acquired after marsupialization. CONCLUSION: Our study was able to identify several metabolic pathways potentially involved in the metaplastic changes induced by marsupialization of odontogenic keratocysts. An improved comprehension of this process could pave the way for the development of targeted therapies.

The genetic basis of oral leukoplakia and its key role in understanding oral carcinogenesis.

Oral leukoplakia (OL) is the most common oral potentially malignant disorder, with a global prevalence of 2%-3%, variable malignant transformation rate and incompletely understood aetiology. Considering the subjectivity in oral dysplasia grading, other evaluation methods have been tested as predictors of malignant transformation. DNA ploidy status and loss of heterozygosity signatures have been shown to be good predictive markers of malignant transformation. However, effective markers to predict which lesions will progress to invasive carcinoma and by which mechanisms remain unclear. Recent evidence suggests that dysplasia progression to carcinoma occurs through neutral clonal evolution (i.e. randomly). We focus on the genetic basis of OL, encompassing the gross chromosomal alterations and single-gene mutations, and discuss such alterations in the context of aetiology, clinical presentation and progression. The deeper we understand the genetic basis of OL, the more we approach a better comprehension of the complex and poorly understood process of oral carcinogenesis.

Deregulation of desmosomal proteins and extracellular matrix proteases in odontogenic keratocyst.

OBJECTIVE: Odontogenic keratocyst (OKC) is a benign lesion that tends to recur after surgical treatment. In an attempt to clarify the molecular basis underlining the OKC pathobiology, we aimed to analyze its proteomic profile. MATERIALS AND METHODS: We compared the proteomic profiles of five OKC and matched normal oral mucosa by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, we performed enrichment analysis and a literature search for the immunoexpression of the proteomics targets. RESULTS: We identified 1,150 proteins and 72 differently expressed proteins (log2 fold change ≥ 1.5; p < .05). Twenty-seven peptides were exclusively detected in the OKC samples. We found 35 enriched pathways related to cell differentiation and tissue architecture, including keratinocyte differentiation, keratinization, desmosome, and extracellular matrix (ECM) organization and degradation. The immunoexpression information of 11 out of 50 proteins identified in the enriched pathways was obtained. We found the downregulation of four desmosomal proteins (JUP, PKP1, PKP3, and PPL) and upregulation of ECM proteases (MMP-2, MMP-9, and cathepsins). CONCLUSIONS: Proteomic analysis strengthened the notion that OKC cells have a similar proteomic profile to oral keratinocytes. Contextual investigation of the differentially expressed proteins revealed the deregulation of desmosome proteins and ECM degradation as important alterations in OKC pathobiology.

First insights for targeted therapies in odontogenic myxoma.

OBJECTIVE: Odontogenic myxoma (OM) occasionally responds poorly to surgical treatment. The MAPK pathway is constitutively activated in several neoplasms and we aimed to test if the MAPK pathway is activated in OM, in order to pave the way for an alternative therapy for aggressive and recurrent cases. MATERIALS AND METHODS: The immunoexpression of phosphorylated ERK1/2 (pERK1/2) was assessed in OM. We established a 3D organotypic culture model for the in vitro study and patient-derived xenografts (PDX) in mice for the in vivo study. The MEK inhibitor U0126 was used to inhibit phosphorylation of ERK1/2 in the in vitro and in vivo models. RESULTS: All OM showed strong pERK1/2 immunoexpression, consistent with MAPK pathway activation. Treatment of the 3D culture with U0126 resulted in a reduced pERK1/2/ERK1/2 ratio. Consistent with the in vitro results, all PDX of animals treated with U0126 showed a decreased volume fold change compared with controls. CONCLUSIONS: The MAPK pathway is activated in OM and its inhibition leads to tumor shrinkage in PDX and cell culture models. CLINICAL RELEVANCE: Our results offer a pre-clinical frame for OM-targeted therapy. Further work is needed to determine if this initial finding holds clinical promise.

KRAS mutations drive adenomatoid odontogenic tumor and are independent of clinicopathological features.

Adenomatoid odontogenic tumor is a benign encapsulated epithelial odontogenic tumor that shows an indolent clinical behavior. We have reported in a few adenomatoid odontogenic tumors mutations in KRAS, which is a proto-oncogene frequently mutated in cancer such as lung, pancreas, and colorectal adenocarcinomas. We aimed to assess KRAS mutations in the hotspot codons 12, 13, and 61 in a large cohort of adenomatoid odontogenic tumors and to test the association of these mutations with clinical (age, site, tumor size, follicular/extrafollicular subtypes) and histopathological parameters. Thirty eight central cases were studied. KRAS codon 12 mutations were assessed by TaqMan allele-specific qPCR (p.G12V/R) and/or Sanger sequencing, and codon 13 and 61 mutations were screened by Sanger. Histological tumor capsule thickness was evaluated by morphometric analysis. Additionally, the phosphorylated form of the MAPK downstream effector ERK1/2 was investigated. Statistical analysis was carried out to test the association of KRAS mutations with clinicopathological parameters. KRAS c.35 G >T mutation, leading to p.G12V, was detected in 15 cases. A novel mutation in adenomatoid odontogenic tumor, c.34 G >C, leading to p.G12R, was detected in 12 cases and the other 11 were wild-type. Codon 12 mutations were not associated with the clinicopathological parameters tested. RAS mutations are known to activate the MAPK pathway, and we show that adenomatoid odontogenic tumors express phosphorylated ERK1/2. In conclusion, a high proportion of adenomatoid odontogenic tumors (27/38, 71%) have KRAS codon 12 mutations, which occur independently of the clinicopathological features evaluated. Collectively, these findings indicate that KRAS mutations and MAPK pathway activation are the common features of this tumor and some cancer types. Although it is unclear why different codon 12 alleles occur in different disease contexts and the complex interactions between tumor genotype and phenotype need clarification, on the basis of our results the presence of KRAS p.G12V/R favors the adenomatoid odontogenic tumor diagnosis in challenging oral neoplasm cases.

Lack of association between denture trauma and loss of heterozygosity confronts the proposed pathologic role of chronic mucosal trauma in oral carcinogenesis.

Chronic mucosal trauma is suggested as an additional etiologic risk factor for oral squamous cell carcinoma (OSCC), but there is a lack of experimental-molecular data. If chronic trauma of the oral mucosa is carcinogenic, it should be associated with early genetic alterations seen during typical progression of OSCC, like loss of heterozygosity (LOH). We investigated LOH in the key chromosomal arms 3p, 9p and 17p in inflammatory fibrous hyperplasia associated with removable dental prosthesis and also in normal oral mucosa, by using the polymorphic microsatellite markers D3S1300 at 3p14.2, D9S1748 at 9p21, D17S1289 at 17p12 and D17S974 at 17p13 and capillary electrophoresis. LOH was detected in 2/15 (13%) fibrous hyperplasia samples similarly to other reactive and inflammatory lesions. None of the normal mucosa samples presented LOH. Our experimental-molecular results do not support the hypothesis that trauma associated with dental prosthesis has an important role in oral carcinogenesis.

Bringing benign ectomesenchymal odontogenic tumours to the lab: An in vitro study using an organotypic culture model.

BACKGROUND: Benign neoplasms exhibit most of the cellular phenomena considered hallmarks of cancer, except the capacity to metastasize. Thus, the elucidation of the mechanisms associated with the progression of benign neoplasms may complement and clarify the mechanisms involved in carcinogenesis. Benign odontogenic tumours often result in facial deformities and morbidities, and have complex pathogenesis, mainly due to the diversity of interactions between the odontogenic epithelium and the ectomesenchyme. Primary cell culture of such tumours is not only difficult to be established and maintained, but also tumour cells lose characteristic cellular morphology. Considering gene expression, growth, migration, proliferation and cellular morphology are controlled by cell-cell interactions and cell-extracellular matrix interactions, cell culture in 3D substrates has gained space as a way to overcome some of the limitations of traditional monolayer cell culture systems. METHODS: In this study, fragments obtained from mesenchymal odontogenic tumours were cultured in type I collagen scaffolds. Invasion tests were performed in these models, as well as phenotypic characterization of the cultured tumours. RESULTS: The results obtained for the odontogenic myxoma and the cemento-ossifying fibroma demonstrate a good reproduction of the growth pattern of these tumours under ex vivo conditions. Microscopic evaluation showed maintenance of cell viability in the explants for more than 30 days, without the presence of necrosis. CONCLUSION: This is the first study involving long-term 3D primary cultures of benign odontogenic tumours, which is expected to support complex approaches to cell and molecular biology, and to serve as an experimental model for testing molecular therapies.

Reticular and erosive oral lichen planus have a distinct metabolomic profile: A preliminary study using gas chromatography-mass spectrometry.

BACKGROUND: Oral Lichen Planus is a chronic inflammatory disorder that affects the oral mucosa, with the reticular and erosive forms representing the primary clinical variants of the disease. Previous studies have shown that metabolic alterations may well be involved in the pathogenesis of the disease; however, the molecular mechanisms related to the clinicopathological differences between erosive and reticular forms remain unknown. METHODS: A comparative metabolomic analysis was performed on formalin-fixed and paraffin-embedded tissue samples of erosive (n = 6) and reticular (n = 10) oral lichen planus using gas chromatography-mass spectrometry. RESULTS: The metabolomic analysis showed a distinct profile between the two clinical variants. Five metabolites (cyclohexanamine, glycine, mannitol/sorbitol, methyl palmitate and trehalose) were significantly diminished in erosive oral lichen planus as compared to the reticular form. CONCLUSIONS: Reticular and erosive forms of oral lichen planus have a distinct metabolic profile. However, further studies using a large number of fresh tissue samples are necessary to confirm this data.

The importance of BRAF-V600E mutation to ameloblastoma metabolism.

BACKGROUND: Ameloblastoma is a locally infiltrative, aggressive epithelial odontogenic neoplasm. BRAF-V600E mutation is frequently found in this tumor and has a pivotal role in its pathogenesis, but the consequences of this alteration need to be addressed. An untargeted metabolomics approach was applied to verify whether metabolic disturbances are related to tumor biology and whether BRAF-V600E mutation contributes to these alterations. METHODS: Formalin-fixed and paraffin-embedded tissue specimens from thirteen ameloblastoma and six dental follicles were included in this study. BRAF mutational status was determined by competitive allele-specific real-time PCR. Metabolite extracts were analyzed using gas chromatography coupled to mass spectrometry. Univariate and multivariate statistical methods were employed to compare the metabolic profiles of the samples. RESULTS: The abundance of eleven metabolites was significantly higher in ameloblastoma in relation to dental follicles, including amino acids, fatty acids, carbohydrates, inorganic acids, and organoheterocyclic compounds. The presence of BRAF-V600E mutations in ameloblastoma was related to decreased levels of glycerol in comparison with tumors carrying only wild-type alleles of this gene. No metabolic differences were observed between recurrent and primary manifestations of ameloblastoma. CONCLUSIONS: Ameloblastoma exhibits a distinct metabolic profile from normal odontogenic epithelium. BRAF-V600E may contribute to metabolic alterations in ameloblastoma. Collectively, our findings suggest that metabolic alterations might play a role in tumor pathogenesis.

Oral pyogenic granulomas show MAPK/ERK signaling pathway activation, which occurs independently of BRAF, KRAS, HRAS, NRAS, GNA11, and GNA14 mutations.

BACKGROUND: Pyogenic granuloma (PG) is a benign nodular lesion with a prominent vascular component which may affect different sites. Recently, BRAF, KRAS, HRAS, NRAS, GNA11, and GNA14 mutations were reported on PGs of the skin. The present study assessed the role of the MAPK/ERK pathway in oral PG pathogenesis. METHODS: Mutations in hotspot regions of genes involved in the MAPK/ERK pathway activation were investigated by Sanger sequencing. The expression of phospho-ERK1/2 was evaluated by immunohistochemistry. RESULTS: Oral PGs did not show mutations in the sequenced regions of the genes BRAF, KRAS, HRAS, NRAS, GNA11, or GNA14. Our results also showed activation of the MAPK/ERK pathway demonstrated by phospho-ERK1/2 immunohistochemical positivity. CONCLUSIONS: Although oral PG shows MAPK/ERK pathway activation, the driver molecular event remains to be elucidated.

DNA methylation patterns of genes related to immune response in the different clinical forms of oral lichen planus.

BACKGROUND: The oral lichen planus is a chronic inflammatory disease. Although its aetiology is not well understood, the role of T lymphocytes in its inflammatory events is recognised. Identifying the epigenetic mechanisms involved in the pathogenesis of this immune-mediated condition is fundamental for understanding the inflammatory reaction that occurs in the disease. The purpose of this work was to evaluate the methylation pattern of 21 immune response-related genes in the different clinical forms of oral lichen planus. METHODS: A cross-sectional study was performed to analyse the DNA methylation patterns in three distinct groups of oral lichen planus: (i) reticular/plaque lesions; (ii) erosive lesions; (iii) normal oral mucosa (control group). After DNA extraction from biopsies, the samples were submitted to digestions by methylation-sensitive and methylation-dependent enzymes and double digestion. The relative percentage of methylated DNA for each gene was provided using real-time polymerase chain reaction arrays. RESULTS: Hypermethylation of the STAT5A gene was observed only in the control group (59.0%). A higher hypermethylation of the ELANE gene was found in reticular/plaque lesions (72.1%) compared to the erosive lesions (50.0%). CONCLUSION: Our results show variations in the methylation profile of immune response-related genes, according to the clinical type of oral lichen planus after comparing with the normal oral mucosa. Further studies are necessary to validate these findings using gene expression analysis.

MicroRNA profiling reveals dysregulated microRNAs and their target gene regulatory networks in cemento-ossifying fibroma.

BACKGROUND: Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis, and its treatment results in morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. METHODS: Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human microRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. RESULTS: Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p and hsa-miR-200c-3p), and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p and hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant KEGG pathways revealed by enrichment analysis were proteoglycans in cancer, miRNAs in cancer, pathways in cancer, p53-, PI3K-Akt-, FoxO- and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. CONCLUSION: miRNA dysregulation occurs in COF, and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation.

Absence of BRAFV600E mutation in odontogenic keratocysts.

BACKGROUND: Mutations in the patched 1 (PTCH1) gene are the main genetic alteration reported in sporadic and nevoid basal cell carcinoma-associated odontogenic keratocyst (OKC). Oncogenic mutations, including BRAFV600E, previously considered exclusive of malignant neoplasms have been reported in odontogenic tumors. Recently, a high frequency of BRAFV600E mutation has been reported in OKC. Because of the considerable recurrence rate of OKC, the identification of druggable genetic mutations can be relevant in the management of extensive lesions. METHODS: A set of 28 OKCs was included in this work. Initially, 10 sporadic and eight OKC samples from four NBCCS patients (a pair of lesions from each syndromic patient) were submitted to targeted next-generation sequencing (NGS) of 2800 different mutations in 50 oncogenes and tumor suppressor genes, including BRAF. Ten extra sporadic OKC samples were included to assess BRAFV600E mutation using TaqMan allele-specific qPCR. RESULTS: The following missense mutations occurred in one case each: ATM p.Ser333Phe, SMO p.Gly416Glu, PIK3CA p.Ser326Phe, FBXW7 p.Ser438Phe, JAK2 p.Ser605Phe, PTEN p.Arg173His, ATM p.Cys353Arg, PTEN p.Ser294Arg, MET p.His1112Tyr. None of the 18 samples showed the BRAFV600E (or any other V600) mutation in the NGS. BRAFV600E mutation was detected by qPCR in one of the 10 OKC. Collectively, our results show BRAFV600E mutation in 1 of 28 OKC cases. CONCLUSION: On the basis of our results, OKCs do not present recurrent hotspot mutations in these 50 genes commonly mutated in cancer. In addition, BRAFV600E does not play a central role in OKC pathogenesis.