Alessandra Pires Duarte

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.

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.

Possible Effects of Drinking and Smoking Habits on Hippuric Acid Levels in Urine of Adults with No Occupational Toluene Exposure.

Drinking and Smoking Habits and Urinary Level of Hippuric Acid in Adults not Exposed to Toluene: Edna Maria Alvarez‐leite, et al. Department of Clinical Chemistry and Toxicology, College of Pharmacy, Federal University of Minas Gerais‐UFMG, Brazil—Hippuric acid (HA) is still the biomarker most used for monitoring exposure to toluene, but it is produced by the body even in the absence of this solvent, and has the disadvantage of showing significant variation in and between individuals, depending on environmental factors and individual characteristics. A number of studies have reported the influence of individual drinking and smoking habits on toluene metabolism, but the effect on urinary excretion of HA is still controversial. This study was conducted in an attempt to examine whether these individual habits also affect HA excretion in individuals not exposed to toluene. Urine sample from 195 people (99 women and 96 men), ranging in age from 17 to 46 years old, were collected. The individuals were classified in groups according their drinking and smoking habits. The data from the current study indicate that these two social habits, either separately or combined, do not influence basal urinary HA levels in this study group.

Cell phone use and parotid salivary gland alterations: no molecular evidence

Background: The association between cell phone use and the development of parotid tumors is controversial. Because there is unequivocal evidence that the microenvironment is important for tumor formation, we investigated in the parotid glands whether cell phone use alters the expression of gene products related to cellular stress. Methods: We used the saliva produced by the parotid glands of 62 individuals to assess molecular alterations compatible with cellular stress, comparing the saliva from the gland exposed to cell phone radiation (ipsilateral) to the saliva from the opposite, unexposed parotid gland (contralateral) of each individual. We compared salivary flow, total protein concentration, p53, p21, reactive oxygen species (ROS), and salivary levels of glutathione (GSH), heat shock proteins 27 and 70, and IgA between the ipsilateral and contralateral parotids. Results: No difference was found for any of these parameters, even when grouping individuals by period of cell phone use in years or by monthly average calls in minutes. Conclusion and Impact: We provide molecular evidence that the exposure of parotid glands to cell phone use does not alter parotid salivary flow, protein concentration, or levels of proteins of genes that are directly or indirectly affected by heat-induced cellular stress. Cancer Epidemiol Biomarkers Prev; 23(7); 1428–31. ©2014 AACR.

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.

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.

Abstract 88: Recurrent KRAS G12V pathogenic mutation in adenomatoid odontogenic tumors

The adenomatoid odontogenic tumor (AOT) is a benign tumor of uncertain pathogenesis. Schimmelpenning syndrome is characterized by sebaceous nevi, which occurs often on the face, associated with variable ipsilateral abnormalities of the central nervous system, including ocular and skeletal abnormalities. It results from postzygotic autosomal dominant HRAS or KRAS lethal mutations that survive by somatic mosaicism. RAS genes mutations were previously reported in lesional tissue (including nevus sebaceous) of a patient, but not in normal skin or blood leukocytes, consistent with a somatic mosaic state. Interestingly, a case of multiple AOT was reported in a Schimmelpenning syndrome patient, which prompted us to evaluate RAS genes mutations, as well as 207 cancer genes mutations in a sample of one AOT from one Schimmelpenning syndrome patient having multiple tumors (index patient). We used the Ion AmpliSeqTM Cancer Hotspot Panel to interrogate these mutations by targeted next generation sequencing. We further included 3 sporadic AOT samples to assess if they shared similar mutations with the sample of the index patient. Additionally, molecular karyotyping analysis was performed in the index patient sample, as well as in one sporadic AOT sample by using a high-density whole genome array platform (Cytoscan® HD Array). The pathogenic KRAS G12V mutation was detected in the index patient sample of AOT, and in 2 out of 3 samples evaluated. No other pathogenic mutation was detected in the AOT samples. TaqMan® Mutation Detection probes specific to the c.35G>T KRAS gene substitution and/or Sanger sequencing were used to validate the mutation in all samples and in a panel of 4 additional sporadic AOT samples. A total of 7 out of 8 AOT samples showed the KRAS pathogenic mutation. We found a few copy number variations (CNVs), most of them common variations or alterations not encompassing genes. Loss of 7p15.3 encompassing the IGF2BP3 gene was detected in the sporadic AOT sample. In conclusion, we report for the first time the recurrent activating KRAS G12V mutation in a high proportion of investigated AOTs, while no other pathogenic mutation interrogated was detected. The importance of the 7p15.3 loss in the aetiopathogenesis of this tumor type remains to be established. Despite the benign nature of AOT, our results shed some light in future molecular targeted-therapy for the lesion. Supported by: CNPq, CAPES and FAPEMIG (Brazil). Citation Format: Carolina C. Gomes, Silvia F. Sousa, Grazielle F. Menezes, Thais S.F. Pereira, Rennan G. Moreira, Alessandra P. Duarte, Wagner H. Castro, Rolando A.R. Villacis, Silvia R. Rogatto, Marina G. Diniz, Ricardo S. Gomez. Recurrent KRAS G12V pathogenic mutation in adenomatoid odontogenic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 88.

Abstract 702: BRAF V600E mutation in benign and malignant epithelial odontogenic tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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 mutation was less frequent in odontogenic carcinomas than in ameloblastomas. In ameloblastomas, this mutation occurred at similar proportions in cases from the mandible and maxilla, as well as among unicystic, multicystic and desmoplastic variants, suggesting that there is no difference in their molecular pathogenesis. Our findings support the possibility for personalised, molecular targeted therapy for ameloblastomas and ameloblastic carcinomas harbouring the BRAFV600E mutation. Supported by: FAPEMIG (Fundacao de Amparo a Pesquisa de Minas Gerais), CNPq (National Council for Scientific and Technological Development) and CAPES (Coordination for Improvement of Higher Education Personnel), Brazil Citation Format: Marina Goncalves Diniz, Carolina Cavalieri Gomes, Bruna Viana Antonini Guimaraes, Alessandra Pires Duarte, Ricardo Santiago Gomez. BRAF V600E mutation in benign and malignant epithelial odontogenic tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2015-702