Thaís dos Santos Fontes Pereira

Diffuse large B-cell lymphoma, not otherwise specified of the palate: a case report

Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin´s lymphoma found in oral and maxillofacial regions. A large number of cases may be biologically heterogeneous, which are commonly defined as DLBCL, not otherwise specified (NOS) by the World Health Organization (WHO-2008). The present case reports on an ulcer of raised and irregular edges, found on the border between the hard and soft palate, as the first and only manifestation of an extranodal non-Hodgkin lymphoma in an 85-year-old patient. Incisional biopsy was carried out, and the specimen revealed a proliferation of large lymphoid cells suggestive of diffuse large cell lymphoma. An immunohistochemical analysis was performed. EBV-RNA was assessed by in situ hybridization that also proved to be negative. Immunohistochemical and EBV analyses are important to avoid delays and inappropriate treatment strategies. Although advanced age is considered an adverse prognostic factor, early diagnosis did prove to be a key contributory factor in the cure of non-Hodgkin lymphoma. Key words:Diffuse large B-cell lymphoma, elderly, EBV.

The Wnt/β-catenin pathway is deregulated in cemento-ossifying fibromas

OBJECTIVE The molecular pathogenesis of cemento ossifying fibroma (COF) is unclear. The purpose of this study was to investigate mutations in 50 oncogenes and tumor suppressor genes, including APC and CTNNB1, in which mutations in COF have been previously reported. In addition, we assessed the transcriptional levels of the Wnt/β-catenin pathway genes in COF. STUDY DESIGN We used a quantitative polymerase chain reaction array to evaluate the transcriptional levels of 44 Wnt/β-catenin pathway genes in 6 COF samples, in comparison with 6 samples of healthy jaws. By using next-generation sequencing (NGS) in 7 COF samples, we investigated approximately 2800 mutations in 50 genes. RESULTS The expression assay revealed 12 differentially expressed Wnt/β-catenin pathway genes in COF, including the upregulation of CTNNB1, TCF7, NKD1, and WNT5 A, and downregulation of CTNNBIP1, FRZB, FZD6, RHOU, SFRP4, WNT10 A, WNT3 A, and WNT4, suggesting activation of the Wnt/β-catenin signaling pathway. NGS revealed 5 single nucleotide variants: TP53 (rs1042522), PIK3 CA (rs2230461), MET (rs33917957), KIT (rs3822214), and APC (rs33974176), but none of them was pathogenic. CONCLUSIONS Although NGS detected no oncogenic mutation, deregulation of key Wnt/β-catenin signaling pathway genes appears to be relevant to the molecular pathogenesis of COF.

Absence of BRAFV600E mutation in odontogenic keratocysts

BACKGROUND Mutations in the patched 1 (PTCH1) gene are the main genetic alteration reported in sporadic and nevoid basal cell carcinoma-associated odontogenic keratocyst (OKC). Oncogenic mutations, including BRAFV600E, previously considered exclusive of malignant neoplasms have been reported in odontogenic tumors. Recently, a high frequency of BRAFV600E mutation has been reported in OKC. Because of the considerable recurrence rate of OKC, the identification of druggable genetic mutations can be relevant in the management of extensive lesions. METHODS A set of 28 OKCs was included in this work. Initially, 10 sporadic and eight OKC samples from four NBCCS patients (a pair of lesions from each syndromic patient) were submitted to targeted next-generation sequencing (NGS) of 2800 different mutations in 50 oncogenes and tumor suppressor genes, including BRAF. Ten extra sporadic OKC samples were included to assess BRAFV600E mutation using TaqMan allele-specific qPCR. RESULTS The following missense mutations occurred in one case each: ATM p.Ser333Phe, SMO p.Gly416Glu, PIK3CA p.Ser326Phe, FBXW7 p.Ser438Phe, JAK2 p.Ser605Phe, PTEN p.Arg173His, ATM p.Cys353Arg, PTEN p.Ser294Arg, MET p.His1112Tyr. None of the 18 samples showed the BRAFV600E (or any other V600) mutation in the NGS. BRAFV600E mutation was detected by qPCR in one of the 10 OKC. Collectively, our results show BRAFV600E mutation in 1 of 28 OKC cases. CONCLUSION On the basis of our results, OKCs do not present recurrent hotspot mutations in these 50 genes commonly mutated in cancer. In addition, BRAFV600E does not play a central role in OKC pathogenesis.

MicroRNA profiling reveals dysregulated microRNAs and their target gene regulatory networks in cemento-ossifying fibroma

BACKGROUND Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis, and its treatment results in morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. METHODS Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human microRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. RESULTS Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p and hsa-miR-200c-3p), and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p and hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant KEGG pathways revealed by enrichment analysis were proteoglycans in cancer, miRNAs in cancer, pathways in cancer, p53-, PI3K-Akt-, FoxO- and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. CONCLUSION miRNA dysregulation occurs in COF, and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation.

ST2 regulates bone loss in a site-dependent and estrogen-dependent manner: MACARI et al.

Interleukin‐33 (IL‐33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2‐deficient mice (ST2−/−) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary‐intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real‐time polymerase chain reaction (qPCR). Deletion of IL‐33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL‐33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2−/− OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin‐10 expression was decreased in both WT OVX and ST2−/− mice, and this reduction was completely restored in ST2−/− OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy‐induced bone loss is IL‐33/ST2‐dependent in the maxilla but not in the femur, indicating a bimodal and site‐specific role of ST2 in bone remodeling.

Cancer genes mutation profiling in calcifying epithelial odontogenic tumour

Aims To identify calcifying epithelial odontogenic tumour (CEOT) mutations in oncogenes and tumour suppressor genes. Methods A panel of 50 genes commonly mutated in cancer was sequenced in CEOT by next-generation sequencing. Sanger sequencing was used to cover the region of the frameshift deletion identified in one sample. Results Missense single nucleotide variants (SNVs) with minor allele frequency (MAF) <1% were detected in PTEN, MET and JAK3. A frameshift deletion in CDKN2A occurred in association with a missense mutation in the same gene region, suggesting a second hit in the inactivation of this gene. APC, KDR, KIT, PIK3CA and TP53 missense SNVs were identified; however, these are common SNVs, showing MAF >1%. Conclusion CEOT harbours mutations in the tumour suppressor PTEN and CDKN2A and in the oncogenes JAK3 and MET. As these mutations occurred in only one case each, they are probably not driver mutations for these tumours.

Desmoplastic fibroblastoma (collagenous fibroma) of the oral cavity

Desmoplastic fibroblastoma is benign soft tissue tumor, with fibroblastic or myofibroblastic origin, that rarely occurs in oral cavity. We reported the case of a 56-year-old man who presented a tumor in the left mandibular alveolar ridge, with slow and asymptomatic growth, with no osseous involvement. The tumor was sessile with lobulated surface, covered by healthy mucosa with erythematous areas. The lesion was excised and specimens sent to histopathology and immunohistochemistry. Histopathological exam showed a non-encapsulated fibroblastic proliferation, characterized by myofibroblasts, spindle and stellate fibroblasts with large or oval nuclei and bi or tri nucleation, immersed in an abundant hypocellular dense collagen stroma. Tumor cells were positive for vimentin, HHF35, α-smooth muscle actin and factor XIIIa. The diagnosis of desmoplastic fibroblastoma was based in the clinical history of absence of trauma related to the growth in the alveolar ridge, associated with macroscopic, microscopic and immunohistochemical features. The patient is free-diseases by eight months. Key words:Collagenous fibroma, desmoplastic fibroblastoma, neoplasm of connective and soft tissue.

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.