Investigação de mutações hotspot nos genes KRAS, BRAF e CTNNB1 no ameloblastoma adenoide e dos padrões de expressão de KRAS E BRAF na odontogênese murina

Abstract

Odontogenic cysts and tumours comprise a heterogeneous group of lesions arising from the odontogenic apparatus. Oncogenic mutations have been reported in these lesions, with molecular alterations predominantly affecting three major signalling pathways: MAPK/ERK, Wnt/beta-catenin, and Sonic Hedgehog. Adenoid ameloblastoma is a recently recognised odontogenic neoplasm, included in the fifth edition of the WHO Classification of Head and Neck Tumours. Histologically, it resembles ameloblastoma, adenomatoid odontogenic tumour, and lesions containing ghost cells. However, its molecular pathogenesis remains poorly understood. In this study, we investigated the presence of mutations in BRAF, KRAS, and CTNNB1 — genes commonly mutated in odontogenic tumours — in a cohort of 11 adenoid ameloblastoma samples derived from 9 cases, using Sanger sequencing. Immunohistochemistry for beta-catenin was also performed to assess its staining pattern in adenoid ameloblastoma. Given the high prevalence of hotspot mutations in KRAS and BRAF in certain odontogenic tumours, and the limited knowledge regarding the expression patterns of these genes during odontogenesis, we further explored the expression of the murine homologues Braf and Kras in developing CD1 mouse embryos (E12.5–E19.5) via digoxigenin-labelled RNA in situ hybridisation. Molecular analysis of the adenoid ameloblastoma cases revealed no hotspot mutations in KRAS or BRAF in the cohort examined. In contrast, exon 3 mutations in CTNNB1 (p.Ser33Cys, p.Gly34Arg, and p.Ser37Phe) were identified in 4 of 9 cases. Nuclear immunopositivity for beta-catenin was observed in all cases assessed by immunohistochemistry. The analysis of Kras and Braf expression in developing murine molars demonstrated that Kras was predominantly expressed in the dental epithelium during the late bud, cap, and bell stages, initially localised in the mesial region of the tooth germ and subsequently concentrated in the enamel knot. Braf expression peaked at E14.5 in the enamel knot and expanded towards the inner enamel epithelium and stellate reticulum by E15.5. Scattered ectomesenchymal cells also exhibited expression of both genes. This study is the first to report CTNNB1 mutations in adenoid ameloblastoma, providing molecular evidence in support of its classification as a distinct pathological entity rather than a subtype of ameloblastoma. Furthermore, we characterised the spatial and temporal expression patterns of Kras and Braf during murine molar odontogenesis, offering insights into the potential developmental roles of these genes and contributing to a deeper understanding of the MAPK/ERK pathway in odontogenic lesion pathogenesis, as well as their prospective implications for regenerative dentistry and medicine.