Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

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.

The evolving molecular characterisation, histological criteria and nomenclature of adenoid ameloblastoma as a World Health Organisation tumour type.

Adenoid ameloblastoma (AA) was recently recognised as a separate tumour type in the most recent World Health Organisation (WHO) classification of head and neck tumours. This decision has been considered controversial by several groups, who have described AA as a subtype of ameloblastoma, a hybrid odontogenic tumour or to fall within the spectrum of other recognised odontogenic tumours, including dentinogenic ghost cell tumour and adenomatoid odontogenic tumour. Here we review the reasons for the WHO decision to classify AA as a separate tumour type. We also critique molecular and histological findings from recent reports published since the WHO classification. While acknowledging that the classification of tumours is constantly evolving, the balance of current evidence suggests that AA should remain a distinct tumour type, and not a subtype of ameloblastoma, pending further molecular characterisation.

The molecular basis of odontogenic cysts and tumours.

The advances in molecular technologies have allowed a better understanding of the molecular basis of odontogenic cysts and tumours. PTCH1 mutations have been reported in a high proportion of odontogenic keratocyst. BRAF p.V600E are recurrent in ameloblastoma and KRAS p.G12V/R in adenomatoid odontogenic tumour, dysregulating the MAPK/ERK pathway. Notably, BRAF p.V600E is also detected in ameloblastic carcinoma, but at a lower frequency than in its benign counterpart ameloblastoma. Recently, adenoid ameloblastoma has been shown to be BRAF wild-type and to harbour CTNNB1 (ß-catenin gene) mutations, further suggesting that it is not an ameloblastoma subtype. CTNNB1 mutations also occur in other ghost-cell-containing tumours, including calcifying odontogenic cysts, dentinogenic ghost cell tumours and odontogenic carcinoma with dentinoid, but the link between CTNNB1 mutations and ghost cell formation in these lesions remains unclear. Regarding mixed tumours, BRAF p.V600E has been reported in a subset of ameloblastic fibromas, ameloblastic-fibrodentinomas and fibro-odontomas, in addition to ameloblastic fibrosarcoma. Such mutation-positivity in a subset of samples can be helpful in differentiating some of these lesions from odontoma, which is BRAF-wild-type. Recently, FOS rearrangements have been reported in cementoblastoma, supporting its relationship with osteoblastoma. Collectively, the identification of recurrent mutations in these aforementioned lesions has helped to clarify their molecular basis and to better understand the interrelationships between some tumours, but none of these genetic abnormalities is diagnostic. Since the functional effect of pathogenic mutations is context and tissue-dependent, a clear role for the reported mutations in odontogenic cysts and tumours in their pathogenesis remains to be elucidated.

Fusion genes aiding the diagnosis of soft tissue tumours of the oral cavity: From bench to bedside.

Soft tissue tumours (STT) are a heterogeneous group of benign, malignant, and intermediate/borderline mesenchymal tumours. In the oral and maxillofacial region, less than 3% of all lesions correspond to benign STT and <1% are sarcomas. Overlapping microscopic features may lead to quite challenging diagnostic processes. Translocations and fusion genes are frequent, and type-specific genetic alterations are detected in these tumours. The detection of such alterations by classic cytogenetic, FISH, RT-PCR or NGS can help to define the diagnosis. This narrative review aims to review fusion genes reported for STT that affect the oral cavity and their use in diagnostic molecular pathology. Basic concepts regarding mechanisms of fusion genes formation are presented to clarify this information for surgical pathologists. The chromosomal rearrangements and fusion genes of adipocytic, fibroblastic and myofibroblastic, vascular, pericytic, smooth muscle, skeletal muscle, chondro-osseous, and uncertain origin STT are summarised. The advance in molecular pathology techniques has led not only to a better understanding of the molecular pathogenesis of STT, but also to the development of helpful diagnostic tools. Therefore, it is important for the oral and head and neck pathologists to familiarise with the signature rearrangements and fusion genes for each tumour.

Analysis of KRAS, BRAF, and EGFR mutational status in respiratory epithelial adenomatoid hamartoma (REAH).

BACKGROUND: Respiratory epithelial adenomatoid hamartoma (REAH) is a sinonasal glandular overgrowth arising from the surface respiratory epithelium and invaginating into the stroma. Clinically, it appears as a polypoid mass that may cause nasal obstruction, anosmia, and epistaxis. The presence of cartilaginous and/or osseous areas move the lesion to a chondro-osseous respiratory epithelial (CORE) hamartoma subtype. Scattered small seromucinous glands may be observed between typical REAH glands and when it is the only feature, it represents seromucinous hamartoma (SH). The molecular pathogenesis of REAH has been poorly explored and remains unclear. Given that KRAS, BRAF, and EGFR mutations have been detected in a variety of sinonasal tumors, we aimed to assess these mutations in REAH and SH. METHODS: Ten REAH (including one CORE subtype), in addition to two SH cases, were Sanger sequenced by standard techniques. The targeted regions included KRAS exons 2-4 (encompassing hotspots codons 12, 13, 61, and 146), BRAF exons 11 and 15 (spanning the V600 codon), and EGFR exons 19 and 20. RESULTS: All REAH and SH samples showed wild-type sequences for KRAS, BRAF, and EGFR genes. CONCLUSION: Our results demonstrate a lack of KRAS, BRAF, or EGFR pathogenic variants with further evaluation of REAH and SH needed to elucidate driver genetic events.

Interrogation of TERT promoter hotspot mutations in ameloblastoma and ameloblastic carcinoma.

BACKGROUND: TERT promoter mutations increase telomerase activity, conferring cell immortality. The coexistence of TERT promoter mutations with BRAFV600E is associated with aggressiveness. Ameloblastoma and ameloblastic carcinoma are infiltrative neoplasms that harbor BRAFV600E; however, it remains unknown if these odontogenic tumors also show TERT promoter mutations. METHODS: Genomic DNA of paraffin-embedded ameloblastomas (n = 6) and ameloblastic carcinomas (n = 3) were Sanger-sequenced to assess the hotspot TERT promoter mutations C228T and C250T. BRAFV600E status was screened by TaqMan allele-specific quantitative polymerase chain reaction. RESULTS: None of the samples harbored TERT promoter mutations. The BRAFV600E mutation was positive in 3 of 6 of ameloblastomas and in 1 of 3 of ameloblastic carcinomas. CONCLUSION: The absence of TERT promoter mutation in the samples indicates that this molecular event is not relevant to the tumors' pathogenesis. Further studies are necessary to explore undefined genetic or epigenetic mechanisms related to TERT-upregulation in ameloblastoma, and the telomerase activity in ameloblastic carcinoma.

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.

Revisiting the human dental follicle: From tooth development to its association with unerupted or impacted teeth and pathological changes.

Dental follicles are involved in odontogenesis, periodontogenesis, and tooth eruption. Dental follicles are unique structures, considering that their remnants can persist within the jawbones after odontogenesis throughout life if the tooth does not erupt. Pathological changes may occur in these tissues as individuals age. The changes range from benign to life threatening. Thus, the assessment of age-related changes in dental follicles associated with unerupted teeth is of paramount importance. In this review, we summarize the physiological roles and changes in dental follicles in odontogenesis, tooth eruption, and aging, in addition to the pathological changes associated with these structures. We encourage investigators to consider this peculiar tissue as a unique model and explore its potential to clarify its importance from the viewpoints of developmental biology, tissue physiology, and pathology.

Adenoid ameloblastoma harbors beta-catenin mutations.

Adenoid ameloblastoma is a very rare benign epithelial odontogenic tumor characterized microscopically by epithelium resembling conventional ameloblastoma, with additional duct-like structures, epithelial whorls, and cribriform architecture. Dentinoid deposits, clusters of clear cells, and ghost-cell keratinization may also be present. These tumors do not harbor BRAF or KRAS mutations and their molecular basis appears distinct from conventional ameloblastoma but remains unknown. We assessed CTNNB1 (beta-catenin) exon 3 mutations in a cohort of 11 samples of adenoid ameloblastomas from 9 patients. Two of the 9 patients were female and 7 male and in 7/9 patients the tumors occurred in the maxilla. Tumors of 4 of these 9 patients harbored CTNNB1 mutations, specifically p.Ser33Cys, p.Gly34Arg, and p.Ser37Phe. Notably, for one patient 3 samples were analyzed including the primary tumour and two consecutive recurrences, and results were positive for the mutation in all three tumors. Therefore, 6/11 samples tested positive for the mutation. In the 6 mutation-positive samples, ghost cells were present in only 2/6, indicating beta-catenin mutations are not always revealed by ghost cell formation. Dentinoid matrix deposition was observed in 5/6 mutation-positive samples and clear cells in all 6 cases. None of the cases harbored either BRAF or KRAS mutations. Beta-catenin immunoexpression was assessed in the samples of 8 patients. Except for one wild-type case, all cases showed focal nuclear expression irrespective of the mutational status. Together with the absence of BRAF mutation, the detection of beta-catenin mutation in adenoid ameloblastomas supports its classification as a separate entity, and not as a subtype of ameloblastoma. The presence of this mutation may help in the diagnosis of challenging cases.

Angiogenesis in patient-derived xenografts of odontogenic myxoma.

Previously, by employing 3D organotypic tissue culture and patient-derived xenograft (PDX) model, oral myxoma response to a MAPK/MEK inhibitor was observed. Gross examination of the tumour fragments obtained after 55 days of PDX grafting revealed increased capsule vascularization. Microscopic analyses showed blood capillaries intermixed with myxoma cells, but the origin of these capillaries, from mice or humans, was not established. This study aimed to investigate whether the endothelial cells observed in the myxoma PDX model are derived from the mouse or from the primary human tumour. Immunohistochemistry was performed on five tumour fragments from the PDX of myxoma after 55 days of implantation in mice. Immunopositivity for antibodies against human (HLA-ABC) and mouse (H2 Db/H2-D1) major histocompatibility complex class I (MHCI) was assessed in the endothelial cells. The endothelial cells in the PDX fragments revealed a membrane staining for the human MHCI protein in the PDX tumour and adjacent connective tissue capsule, indicating that capillaries were derived from the human tumour fragment. Considering the probable human origin of the endothelial cells from capillary blood vessels in the myxoma PDX, we conclude that this PDX model is an interesting model to study myxoma angiogenesis.

Integrated proteomics, phosphoproteomics and metabolomics analyses reveal similarities among giant cell granulomas of the jaws with different genetic mutations.

BACKGROUND: Giant cell granuloma of the jaws are benign osteolytic lesions of the jaws. These lesions are genetically characterized by mutually exclusive somatic mutations at TRPV4, KRAS, and FGFR1, and a fourth molecular subgroup which is wild-type for the three mutations. Irrespective of the molecular background, giant cell granulomas show MAPK/ERK activation. However, it remains unclear if these mutations lead to differences in their molecular signaling in giant cell granulomas. METHODS: Metabolomics, proteomics, and phosphoproteomics analyses were carried out in formalin-fixed paraffin-embedded samples of giant cell granuloma of the jaws. The study cohort consisted of five lesions harboring mutations in FGFR1, six in KRAS, five in TRPV4, and five that were wild-type for these mutations. RESULTS: Lesions harboring KRAS or FGFR1 mutations showed overall similar proteomics and metabolomics profiles. In all four groups, metabolic pathways showed similarity in apoptosis, cell signaling, gene expression, cell differentiation, and erythrocyte activity. Lesions harboring TRPV4 mutations showed a greater number of enriched pathways related to tissue architecture. On the other hand, the wild-type group presented increased number of enriched pathways related to protein metabolism compared to the other groups. CONCLUSION: Despite some minor differences, our results revealed an overall similar molecular profile among the groups with different mutational profile at the metabolic, proteic, and phosphopeptidic levels.

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.

Patient-derived xenograft models for the study of benign human neoplasms.

Preclinical models are a core feature of translational research, and patient-derived xenograft (PDX) models have increasingly been used with such purpose. PDX involves the transplantation of fresh human tumor samples into immunodeficient mice to overcome immunologic rejection. It is a valuable tool for basic as well as preclinical research, contributing to the establishment of models to characterize the neoplasms to drug screening and to allow the identification of therapeutic targets. The use of these models is justified because they retain the histological and genomic features of the primary tumor. PDX models are well described for malignant neoplasms, for which the advantages are clear and include the development of drug treatments. The establishment of malignant tumors PDX is undeniably important from a medical perspective. However, few studies have used such models for benign neoplasms. The use of PDX for benign neoplasm studies can help to clarify the pathobiology of these diseases, as well as invasion and malignant transformation mechanisms, which from a biological perspective is equally important to the study of malignant tumors. Therefore, the aim of this study is to review the current methodology for PDX model generation and to cover its main applications, focusing on benign neoplasms.

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.

Whole-exome sequencing reveals novel vacuolar ATPase genes' variants and variants in genes involved in lysosomal biology and autophagosomal formation in oral granular cell tumors.

BACKGROUND: Granular cell tumors (GCTs) are rare neuroectodermal soft tissue neoplasms that mainly affect the skin of the upper limbs and trunks and the oral cavity. GCTs are derived from Schwann cells and, ultrastructurally, their intracytoplasmic granules are considered autophagosomes or autophagolysosomes and are consistent with myelin accumulation. METHODS: In this study, a convenience set of 22 formalin-fixed, paraffin-embedded samples of oral GCTs, all but one sample located at the tongue, was screened for mutations by whole-exome (WES) or targeted sequencing. RESULTS: WES revealed two novel variants in genes of the vacuolar ATPase (V-ATPase) complex: ATP6AP1 frameshift c.746_749del, leading to p.P249Hfs*4, and ATP6V1A non-synonymous c.G868A, leading to p.D290N. Each of these mutations occurred in one case. With regard to the samples that were wild type for these V-ATPase variants, at least two samples presented variants in genes that are part of endosomal/lysosomal/autophagosomal networks including ABCA8, ABCC6, AGAP3, ATG9A, CTSB, DNAJC13, GALC, NPC1, SLC15A3, SLC31A2, and TMEM104. CONCLUSION: Although the mechanisms involved in oral GCT initiation and progression remain unclear, our results suggest that oral GCTs have V-ATPase variants similarly to GCTs from other tissues/organs, and additionally show variants in lysosomes/endosomes/autophagosomal genes.

BRAF p.V600E status in epithelial areas of ameloblastoma with different histological aspects: Implications to the clinical practice.

BACKGROUND: BRAF p.V600E is reported in up to 80% of ameloblastomas. Despite the high frequency, the presence of this mutation in different histopathological areas of the tumour has not been investigated. This information has an important role in the use of BRAF p.V600E assessment as an auxiliary tool in the differential diagnosis between unicystic ameloblastoma and other odontogenic cystic lesions, especially when only incisional biopsies are available. Therefore, the purpose of the present study was to investigate BRAF p.V600E heterogeneity in unicystic ameloblastoma. METHODS: Five cases of ameloblastoma and two dentigerous cysts were analysed. The regions exhibiting different microscopic characteristics were selected from each ameloblastoma case and manually dissected. TaqMan allele-specific qPCR or Sanger sequencing was performed to determine BRAF p.V600E status. RESULTS: We screened the mutation in a small cohort of UA and no molecular heterogeneity was found. Four cases of ameloblastoma (80%) exhibited BRAF p.V600E in all different areas evaluated. One case did not harbour the mutation in any microscopic region analysed. The BRAF mutation was absent in the dentigerous cysts. CONCLUSION: Ameloblastomas appear to exhibit a homogeneous profile regarding the BRAF p.V600E no matter what histological feature is observed under light microscopy, suggesting that this molecular test may contribute to establish the correct diagnosis in cases microscopically resembling other odontogenic lesions.