Assessment of PI3K/AKT and MAPK/ERK pathways activation in oral lymphatic malformations.

OBJECTIVE: Lymphatic malformations are characterized by the overgrowth of lymphatic vessels during development. Activation of PI3K/AKT and MAPK/ERK signaling pathways occur in isolated lymphatic malformation and in those associated with syndromes such as CLOVES and Klippel-Trenaunay. We aimed to assess the activation of these pathways in sporadic oral lymphatic malformations. STUDY DESIGN: A convenience sample of 14 formalin-fixed paraffin-embedded samples of oral lymphatic malformations underwent immunohistochemical reactions for the phosphorylated forms of AKT1 (pAKT-Ser473) and ERK1/2 (pERK1/2-Thr202/Tyr204), which are markers of PI3K/AKT and MAPK/ERK pathways activation, respectively. RESULTS: Positive staining for pAKT1 and pERK1/2 was observed in the endothelial cells in all samples of oral lymphatic malformations evaluated. CONCLUSIONS: Our results suggest that activation of PI3K/AKT and MAPK/ERK signaling pathways participates in the pathogenesis of oral lymphatic malformations.

BRAF V600E and loss of heterozygosity assessment in benign oralneural tumours.

BACKGROUND: The purpose of this study was to evaluate loss of heterozygosity (LOH) and to assess BRAF V600E mutation in oral neurofibromas, palisaded encapsulated neuromas (PEN) and schwannomas. METHODS: Six oral neurofibroma, 5 PEN and 3 schwannoma samples were included in the study. LOH was assessed using polymorphic microsatellite markers at chromosome regions 3p (marker D3S1029), 9p (markers D9S171, D9S162, D9S157), 11q (marker D11S1369), and 17p (markers AFM238WF2 and P53), and results were evaluated after capillary electrophoresis. BRAF mutation encoding V600E was assessed by real-time PCR with a specific TaqMan probe to detect the T>A transversion at position c.1799. RESULTS: LOH occurred at chromosomes 3p (marker D3S1029), 11q (D11S1369) and 17p (AFM238WF2 and P53). LOH occurred in 2/6 neurofibromas, 2/5 PEN and in none of the 3 schwannoma samples. The 6 neurofibromas, 2/2 PEN evaluated and the 3 schwannomas were BRAF wild type. CONCLUSION: According to our results, oral benign peripheral nerve sheath tumours have a low LOH rate, but P53 locus alteration is occasionally found. Additionally, BRAF V600E mutation is either not relevant to the molecular pathogenesis of this group of lesions of the oral cavity, or may occur at very low rates.

Assessment of BRAFV600E and SMOF412E mutations in epithelial odontogenic tumours.

The classification of ameloblastoma in multicystic or unicystic variants is associated with its clinical behaviour. Recently, BRAF and SMO mutations have been reported in ameloblastomas. However, it is not clear if such mutations are shared by the multi- and unicystic variants of ameloblastoma or by odontogenic carcinomas. We assessed BRAFV600E and SMOF412E in multicystic, unicystic and desmoplastic ameloblastomas. In addition, we investigated whether the BRAFV600E mutation occurs in odontogenic carcinomas. A total of 28 formalin-fixed paraffin-embedded samples, comprising 17 ameloblastomas and 11 odontogenic carcinomas, were included. The BRAFV600E mutation was assessed by real-time PCR with a specific TaqMan probe and confirmed by Sanger sequencing. The SMOF412E mutation was assessed by Sanger sequencing. Fourteen out of 17 (82 %) ameloblastomas showed the BRAFV600E mutation, specifically, 5/6 (83 %) unicystic, 7/9 (78 %) multicystic and 2/2 desmoplastic ameloblastomas. BRAFV600E mutation was detected in 4/11 (36 %) malignant tumours, specifically, 3/8 (38 %) ameloblastic carcinomas and 1/1 clear cell odontogenic carcinoma, while the two ghost cell odontogenic carcinomas did not harbour this mutation. The SMOF412E mutation was not detected in ameloblastoma. The BRAFV600E-activating mutation is a common event in ameloblastomas, occurring regardless of site or histological type. This mutation is also detected in odontogenic carcinomas. SMO somatic mutation is a secondary genetic event in the ameloblastoma pathogenesis. Our findings support the possibility for personalised, molecular-targeted therapy for ameloblastomas and odontogenic carcinomas harbouring the BRAFV600E mutation.

Assessment of TP53 mutations in benign and malignant salivary gland neoplasms.

Despite advances in the understanding of the pathogenesis of salivary gland neoplasms (SGN), the molecular pathways associated with enhanced tumor growth and cell survival remain to be established. The aim of the present study was to investigate whether TP53 mutations are relevant to SGN pathogenesis and if they impact on p53 protein expression. The study included 18 benign and 18 malignant SGN samples. Two polymorphic microsatellite markers at the TP53 genetic locus were chosen to assess loss of heterozygosity (LOH) in the samples that had matched normal DNA. The TP53 exons 2-11 were amplified by PCR, and all of the products were sequenced. Reverse transcription-PCR of the TP53 open reading frame (ORF) was carried out in the samples that had fresh tissue available, and immunohistochemistry for the p53 protein was performed in all samples. TP53 LOH was only found in two pleomorphic adenomas. We found two missense mutations in exon 7 (one in a pleomorphic adenoma and the other in a polymorphous low grade adenocarcinoma), another in exon 8 (in a carcinoma ex pleomorphic adenoma) and a fourth missense mutation in exon 10 (in a mucoepidermoid carcinoma). In addition, a nonsense mutation was found in exon 8 of an adenoid cystic carcinoma. Several intronic and exonic SNPs were detected. Although almost all of the malignant samples were immunopositive for p53, approximately 37% of the benign samples were positive, including the sample harboring the missense mutation and one of the samples that showed LOH. The complete TP53 ORF could be amplified in all samples analyzed, including the IHC negative samples, the samples showing LOH and one sample displaying a missense mutation. In summary, our results show that TP53 mutations are not a frequent event in SGN and that p53 immunopositivity might not be associated with sequence mutations in SGN.