miRNA expression profiling of hereditary breast tumors from BRCA1- and BRCA2-germline mutation carriers in Brazil.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene expression regulation and have been described as key regulators of carcinogenesis. Aberrant miRNA expression has been frequently reported in sporadic breast cancers, but few studies have focused on profiling hereditary breast cancers. In this study, we aimed to identify specific miRNA signatures in hereditary breast tumors and to compare with sporadic breast cancer and normal breast tissues. METHODS: Global miRNA expression profiling using NanoString technology was performed on 43 hereditary breast tumors (15 BRCA1, 14 BRCA2, and 14 BRCAX), 23 sporadic breast tumors and 8 normal breast tissues. These normal breast tissues derived from BRCA1- and BRCA2- mutation carriers (n = 5) and non-mutation carriers (n = 3). Subsequently, we performed receiver operating characteristic (ROC) curve analyses to evaluate the diagnostic performance of differentially expressed miRNAs. Putative target genes of each miRNAs considered as potential biomarkers were identified using miRDIP platform and used for pathway enrichment analysis. RESULTS: miRNA expression analyses identified several profiles that were specific to hereditary breast cancers. A total of 25 miRNAs were found to be differentially expressed (fold change: > 2.0 and p < 0.05) and considered as potential biomarkers (area under the curve > 0.75) in hereditary breast tumors compared to normal breast tissues, with an expressive upregulation among BRCAX cases. Furthermore, bioinformatic analysis revealed that these miRNAs shared target genes involved in ErbB, FoxO, and PI3K-Akt signaling pathways. CONCLUSIONS: Our results showed that miRNA expression profiling can differentiate hereditary from sporadic breast tumors and normal breast tissues. These miRNAs were remarkably deregulated in BRCAX hereditary breast cancers. Therefore, miRNA signatures can be used as potential novel diagnostic biomarkers for the prediction of BRCA1/2- germline mutations and may be useful for future clinical management.

Anti-erosion effect of an experimental varnish on eroded dentin.

This in vitro study evaluated the effect of an experimental varnish containing 20% nano-hydroxyapatite (nHAP) associated with 5% stannous chloride (SnCl2) against erosive-abrasive wear on bovine dentin. Samples of bovine cervical dentin were pre-eroded (0.3% citric acid, pH 2.6 for 10 minutes) and randomized into 4 groups (n=10): Control group - experimental varnish without active ingredient (CG); experimental varnish containing 20% nHAP (nHG); experimental varnish containing 5% SnCl2 (24.800 ppm Sn2+) (SnG); experimental varnish containing 20% nHAP associated with 5% SnCl2 (18.300 ppm Sn2+) (nHSnG). After applying the materials, the erosive-abrasive challenges were performed for five days. Erosive dentin loss and analysis of the pattern of dentinal obliteration were performed by 3D confocal laser microscopy. A one-way ANOVA/Bonferroni test was performed to analyze the data (α=0.05). The SnG and nHSnG experimental groups presented more effectiveness in preventing erosive wear when compared to the other groups (p<0.05). There was no statistically significant difference between the SnG and nHSnG groups (p = 0.731) in tooth structure dentin loss. Regarding the amount of open dentinal tubules, the highest amount of obstructed dentinal tubules was demonstrated in SnG and nHSnG (p < 0.05) when compared to the others. Between SnG and nHSnG there was no significant difference (p = 0.952) in the amount of closed dentinal tubules in the dentin. Experimental varnishes containing 5% SnCl2 associated or not with 20% nHAP showed to be a promising strategy in preventing erosive-abrasive wear of dentin. In addition, nHSnG was able to obliterate dentinal tubules.

Clinicopathological and molecular characterization of Brazilian families at risk for Lynch syndrome.

Lynch syndrome (LS), is the most common hereditary colorectal cancer syndrome. However, it is poorly characterized in Brazil. Therefore, we aimed to determine the spectrum of pathogenic variants in Mismatch Repair (MMR) genes and investigate the MLH1 promotor methylation role as a second hit in LS tumors. Tumor screening through microsatellite instability and immunohistochemistry for MMR proteins was performed in 323 cases who met clinical criteria. BRAF-V600E and MLH1 promoter methylation were analyzed for all MLH1-deficient tumors. Patients with MMR deficient tumor proceeded to germline genetic testing. MMR deficient tumors were detected in 41% of patients recruited. About half of patients carried a pathogenic germline variant. Two recurrent variants in MLH1 and three novel pathogenic variants were identified. Furthermore, pathogenic germline variants with concomitant somatic MLH1 hypermethylation were found in 6% of cases. Predictive genetic testing was offered to 387 relatives. Overall, 127 tumors were diagnosed in 100 LS patients, from 62 unrelated families. Our molecular data provide new information about the spectrum of MMR mutations, which contributes to a better characterization of LS in Brazil. Furthermore, we call attention to the possibility of failure in the diagnosis of germline MLH1 mutation carriers when somatic MLH1 hypermethylation is used to rule out LS.

Using Co-segregation and Loss of Heterozygosity Analysis to Define the Pathogenicity of Unclassified Variants in Hereditary Breast Cancer Patients.

The use of gene panels introduces a new dilemma in the genetics field due to the high frequency of variants of uncertain significance (VUS). The objective of this study was to provide evidence that may help in the classification of these germline variants in terms of their clinical impact and association with the disease in question. A total of 52 unrelated women at-risk for HBOC and negative for BRCA1/BRCA2 pathogenic variants were evaluated through a gene panel comprising 14 breast and/or ovarian cancer susceptibility genes. Of the 453 germline variants identified, 15 variants (classes 3, 4, and 5) in the ATM, BRIP1, CHEK2, MRE11A, MUTHY, PALB2, RAD50, and RAD51C genes were evaluated via databases, co-segregation studies and loss of heterozygosity in the tumor. The co-segregation analysis allowed the establishment of an association with the presence of variants and the risk of cancer for variant c.316C>T in the BRIP1 gene. Four variants of uncertain significance showed loss of heterozygosity in the tumor (ATM c.4709T>C; CHEK2 c.1036C>T; PALB2 c.1001A>G, and RAD50 c.281T>C), which is an indication of pathogenicity. Thus, the present study provides novel evidence that favors the association of variants in moderate-risk genes with the development of hereditary breast cancer.

Impact of genetic variants in clinical outcome of a cohort of patients with oropharyngeal squamous cell carcinoma.

Tobacco- or human papillomavirus- driven oropharyngeal squamous cell carcinomas (OpSCC) represent distinct clinical, biological and epidemiological entities. The aim of this study was to identify genetic variants based on somatic alterations in OpSCC samples from an admixed population, and to test for association with clinical features. The entire coding region of 15 OpSCC driver genes was sequenced by next-generation sequencing in 51 OpSCC FFPE samples. Thirty-five percent of the patients (18/51) were HPV-positive and current or past tobacco consumption was reported in 86.3% (44/51). The mutation profile identified an average of 2.67 variants per sample. Sixty-three percent of patients (32/51; 62.7%) were mutated for at least one of the genes tested and TP53 was the most frequently mutated gene. The presence of mutation in NOTCH1 and PTEN, significantly decreased patient's recurrence-free survival, but only NOTCH1 mutation remained significant after stepwise selection, with a risk of recurrence of 4.5 (HR 95% CI = 1.11-14.57; Cox Regression p = 0.034). These results show that Brazilian OpSCC patients exhibit a similar clinical and genetic profile in comparison to other populations. Molecular characterization is a promising tool for the definition of clinical subgroups, aiding in a more precise tailoring of treatment and prognostication.

Differential Profile of BRCA1 vs. BRCA2 Mutated Families: A Characterization of the Main Differences and Similarities in Patients.

The identification of families at-risk for hereditary breast cancer (BC) is important because affected individuals present a much higher cancer risk than the general population. The aim of this study was to identify the most important factors associated with the presence of a pathogenic BRCA1/BRCA2 mutation. Family history (FH), histopathological and immunohistochemical characteristics were compared among BC women with pathogenic BRCA1/BRCA2 variants; VUSs in BRCA1/BRCA2; BRCA1/BRCA2 WT and sporadic BC. The most significative differences observed concerned the molecular subtype of the tumors, age at cancer diagnosis and FH of cancer. The presence of bilateral breast cancer (BBC), number of BC cases and the presence of ovarian cancer (OC) increased (respectively) 5.797, 5.033 and 4.412 times the risk of being a BRCA1/BRCA2 mutation carrier. Besides, women with BRCA1 or BRCA2 mutations presented different tumor and FH profiles. The main characteristics associated with a BRCA1 mutation were triple negativity (OR: 17.31), BBC history (OR: 4.96) and occurrence of OC (OR: 4.32). There were no major discerning components associated with BRCA2 mutations. Thus, we conclude that tumor pathology and FH of cancer might be considered together at the time of genetic testing mainly in countries where access to genetic testing is still restricted.

Anti-erosion effect of an experimental varnish on eroded dentin

Abstract This in vitro study evaluated the effect of an experimental varnish containing 20% nano-hydroxyapatite (nHAP) associated with 5% stannous chloride (SnCl2) against erosive-abrasive wear on bovine dentin. Samples of bovine cervical dentin were pre-eroded (0.3% citric acid, pH 2.6 for 10 minutes) and randomized into 4 groups (n=10): Control group - experimental varnish without active ingredient (CG); experimental varnish containing 20% nHAP (nHG); experimental varnish containing 5% SnCl2 (24.800 ppm Sn2+) (SnG); experimental varnish containing 20% nHAP associated with 5% SnCl2 (18.300 ppm Sn2+) (nHSnG). After applying the materials, the erosive-abrasive challenges were performed for five days. Erosive dentin loss and analysis of the pattern of dentinal obliteration were performed by 3D confocal laser microscopy. A one-way ANOVA/Bonferroni test was performed to analyze the data (α=0.05). The SnG and nHSnG experimental groups presented more effectiveness in preventing erosive wear when compared to the other groups (p<0.05). There was no statistically significant difference between the SnG and nHSnG groups (p = 0.731) in tooth structure dentin loss. Regarding the amount of open dentinal tubules, the highest amount of obstructed dentinal tubules was demonstrated in SnG and nHSnG (p < 0.05) when compared to the others. Between SnG and nHSnG there was no significant difference (p = 0.952) in the amount of closed dentinal tubules in the dentin. Experimental varnishes containing 5% SnCl2 associated or not with 20% nHAP showed to be a promising strategy in preventing erosive-abrasive wear of dentin. In addition, nHSnG was able to obliterate dentinal tubules.

Resumo Este estudo in vitro avaliou o efeito de um verniz experimental contendo 20% de nano-hidroxiapatita (nHAP) associado a 5% de cloreto estanoso (SnCl2) contra o desgaste erosivo-abrasivo da dentina bovina. As amostras de dentina cervical bovina foram pré-erodificadas (0,3% de ácido cítrico, pH 2,6 durante 10 minutos) e aleatorizadas em 4 grupos (n=10): Grupo controle - verniz experimental sem ingrediente ativo (GC); verniz experimental contendo 20% nHAP (GnH); verniz experimental contendo 5% SnCl2 (24.800 ppm Sn2+) (GSn); verniz experimental contendo 20% nHAP associado a 5% SnCl2 (18.300 ppm Sn2+) (GnHSn). Após a aplicação dos materiais, os desafios erosivo-abrasivos foram realizados durante cinco dias. Perda de dentina erosiva e análise do padrão de obliteração dentinária foram realizadas por microscopia laser confocal 3D. Foi realizado o teste ANOVA/Bonferroni unidireccional para analisar os dados (α=0,05). Os grupos experimentais GSn e GnHSn apresentaram maior eficácia na prevenção do desgaste erosivo quando comparados com os outros grupos (p<0,05). Não houve diferença estatisticamente significativa entre os grupos GSn e GnHSn (p = 0,731) na perda de dentina da estrutura dentária. Relativamente à quantidade de túbulos dentinários abertos, a maior quantidade de túbulos dentinários obstruídos foi demonstrada em GSn e GnHSn (p < 0,05) quando comparada com os outros grupos. Entre GSn e GnHSn, não houve diferença significativa (p = 0,952) na quantidade de túbulos dentinários fechados na dentina. Os vernizes experimentais contendo 5% de SnCl2 associados ou não a 20% de nHAP mostraram ser uma estratégia promissora na prevenção do desgaste erosivo-abrasivo da dentina. Além disso, o GnHSn conseguiu obliterar os túbulos dentinários.

Screening and characterization of BRCA2 c.156_157insAlu in Brazil: Results from 1380 individuals from the South and Southeast.

Portuguese immigration to Brazil occurred in several waves and greatly contributed to the genetic composition of current Brazilian population. In this study, we evaluated the frequency of a Portuguese founder Alu insertion in BRCA2 exon 3 (c.156_157insAlu) among individuals fulfilling Hereditary Breast and Ovarian Cancer (HBOC) syndrome criteria in 1,380 unrelated families originated from three distinct Brazilian States. We identified the c.156_157insAlu BRCA2 mutation in nine (9/1,380; 0.65%) probands analised. In carrier probands, European ancestry had the highest proportion (80%), followed by the African (10%) and Amerindian and in most families with the rearrangement, haplotype analyses were compatible with the Portuguese ancestral haplotype. In conclusion, the present study reports a low albeit relevant frequency of the Portuguese BRCA2 founder mutation c.156_157insAlu in Brazilian patients at-risk for HBOC Brazilian population.

Genetic and epigenetic characterization of the BRCA1 gene in Brazilian women at-risk for hereditary breast cancer.

This study aimed to characterize women at-risk for hereditary BC regarding their clinical and molecular characteristics (mutation and methylation in the BRCA1 gene) and correlate the gene expression levels with histopathological, clinical and family history information. BRCA1 real time qPCR was performed to evaluate methylation status and gene expression. The study included 88 women grouped according to the BRCA1 mutational status: 23 BRCA1 mutated, 22 with a Variant of Unknown Significance (VUS) in BRCA1 and 43 BRCA1 WT. Most BRCA1 mutated tumors were triple negative (69.6%) and had histologic grade III (61.0%). Patients with VUS/WT BRCA1 were predominantly of luminal B subtype with histological grades I and II. Regarding the methylation profile, BRCA1 hypermethylation was observed in only two patients (both WT) and none had association with pathogenic BRCA1 mutation. In one patient methylation was present in both, tumor and normal tissues. Hypermethylated tumors had ductal histology, negativity for ER and occurred in < 50 years patients. Gene expression profile showed in all groups lower BRCA1 mRNA levels in tumor tissue compared to the adjacent breast tissue, thereby indicating the loss/decrease of gene function. No association was found between the levels of BRCA1 gene expression and family history of cancer. In summary, our findings suggested that methylation at the BRCA1 gene is not the "second" event in the development of BC in patients with germline mutations in BRCA1 and, although rare, BRCA1 epimutations can constitute an explanation for a fraction of HBOC families.