Rare copy number alterations and copy-neutral loss of heterozygosity revealed in ameloblastomas by high-density whole-genome microarray analysis.

BACKGROUND: Ameloblastoma (unicystic, UA, or multicystic, MA) is a rare tumor associated with bone destruction and facial deformity. Its malignant counterpart is the ameloblastic carcinoma (AC). The BRAFV600E mutation is highly prevalent in all these tumors subtypes and cannot account for their different clinical behaviors. METHODS: We assessed copy number alterations (CNAs) and copy-neutral loss of heterozygosity (cnLOH) in UA (n = 2), MA (n = 3), and AC (n = 1) using the CytoScan HD Array (Affymetrix) and the BRAFV600E status. RT-qPCR was applied in four selected genes (B4GALT1, BAG1, PKD1L2, and PPP2R5A) covered by rare alterations, also including three MA and four normal oral tissues. RESULTS: Fifty-seven CNAs and cnLOH were observed in the ameloblastomas and six CNAs in the AC. Seven of the CNAs were rare (six in UA and one in MA), four of them encompassing genes (gains of 7q11.21, 1q32.3, and 9p21.1 and loss of 16q23.2). We found positive correlation between rare CNA gene dosage and the expression of B4GALT1, BAG1, PKD1L2, and PPP2R5A. The AC and 1 UA were BRAF wild-type; however, this UA showed rare genomic alterations encompassing genes associated with RAF/MAPK activation. CONCLUSION: Ameloblastomas show rare CNAs and cnLOH, presenting a specific genomic profile with no overlapping of the rare alterations among UA, MA, and AC. These genomic changes might play a role in tumor evolution and in BRAFV600E-negative tumors.

Quantitative expression analysis of apoptotic/antiapoptotic genes and association with immunolocalization of BAX and BCL-2 in peripheral and central giant cell lesions of the jaws.

Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) of the jaws are characterized by multinucleated osteoclast-like giant cells in a background of mononuclear cells. While mononuclear cells retain proliferative activity in both lesions, giant cells are Ki-67 negative. This observation raised the theory that giant cells are formed by cytoplasmic fusion of mononuclear cells, and also that these lesions are of reactive nature. As the giant cells are not proliferating in CGCL and PGCL, apoptosis of such cells should be investigated. We investigated the transcription of BAX and BCL-2 mRNAs in six fresh samples of CGCL and six fresh samples of PGCL by qRT-PCR (quantitative reverse transcription PCR) and used immunohistochemistry to demonstrate the localization of these proteins, as well as caspase 3 active in six paraffin-embedded samples of CGCL and nine paraffin-embedded samples of PGCL. While both groups showed increased expression of BAX and BCL-2 mRNA, PGCL showed a higher apoptotic index (ratio BAX/BCL-2) than CGCL. The three proteins investigated were expressed almost exclusively in the cytoplasm of giant cells. To further confirm apoptotic activity, we performed TUNEL analysis in the same samples of the immunohistochemistry and found a higher positivity in the giant cells of PGCL compared to the giant cells of CGCL. Our results show increased expression of apoptotic-related genes in both PGCL and CGCL and that the giant cells are probably the main source of these events. Also, it raises a hypothesis that differences in the apoptotic activity might be associated with the different clinical behavior of CGCL and PGCL.

Review article: Current concepts of ameloblastoma pathogenesis.

Ameloblastoma is a locally destructive and invasive tumour that can recur despite adequate surgical removal. Molecular studies have offered interesting findings regarding ameloblastoma pathogenesis. In the present review, the following topics are discussed regarding its molecular nature: clonality, cell cycle proliferation, apoptosis, tumour suppressor genes, ameloblastin and other enamel matrix proteins, osteoclastic mechanism and matrix metalloproteinases and other signalling molecules. It is clear from the literature reviewed that translational studies are necessary to identify prognostic markers of ameloblastoma behaviour and to establish new diagnostic tools to the differential diagnosis of unicystic from multicystic ameloblastoma. Finally, molecular biology studies are also important to develop more effective alternative approaches to the treatment of this aggressive odontogenic tumour.

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.

Increased expression of NFATc1 in giant cell lesions of the jaws, cherubism and brown tumor of hyperparathyroidism.

A variety of diseases of the jaws may present multinucleated giant cells. These diseases include central giant cell lesions (CGCL), peripheral giant cell lesions (PGCL), brown tumor of hyperparathyroidism (BTH), and cherubism. The multinucleated giant cells in these lesions are osteoclast-like. Since NFATc1 plays a significant role in osteoclast differentiation, the present study aimed to compare the expression of NFATc1 in CGCL, PGCL, BTH and cherubism. A total of 14 formalin-fixed and paraffin-embedded tissue samples of CGCL (n=4), PGCL (n=5), BTH (n=3) and cherubism (n=2) were included in the study. An immunohistochemical analysis was performed to investigate the NFATc1 protein. The majority of giant cells in all of the cases were positive for nuclear NFATc1 and the immunostaining pattern was similar in all of the groups. Although our study supports the hypothesis that giant cell accumulation in PGCL, CGCL, BTH and cherubism is mediated by NFATc1, functional studies are required to investigate this hypothesis.

Molecular review of odontogenic myxoma.

Odontogenic myxoma (OM) is a benign odontogenic neoplasm that tends to recur due to bone infiltration. This review focuses on the molecular aspects of the OM. The following topics are discussed: clonal nature, matrix metalloproteinases, apoptosis and cell proliferation, genetic alterations, and other markers. Translational studies are necessary to identify the prognostic markers of this lesion, and also, molecular biology studies may help to identify the etiologic factors and to develop more effective and less aggressive approaches, other than surgery, to the treatment of this infiltrating odontogenic tumor.