Hypermethylation status of DAPK, MGMT and RUNX3 in HPV negative oral and oropharyngeal squamous cell carcinoma.

Squamous cell carcinoma of the oral cavity and oropharynx is the sixth most common type of cancer in the world. During tumorigenesis, gene promoter hypermethylation is considered an important mechanism of transcription silencing of tumor suppressor genes, such as DAPK, MGMT and RUNX3. These genes participate in signaling pathways related to apoptosis, DNA repair and proliferation whose loss of expression is possibly associated with cancer development and progression. In order to investigate associations between hypermethylation and clinicopathological and prognostic parameters, promoter methylation was evaluated in 72 HPV negative oral and oropharyngeal tumors using methylation-specific PCR. Hypermethylation frequencies found for DAPK, MGMT and RUNX3 were 38.88%, 19.44% and 1.38% respectively. Patients with MGMT hypermethylation had a better 2-year overall survival compared to patients without methylation. Being MGMT a repair gene for alkylating agents, it could be a biomarker of treatment response for patients who are candidates for cisplatin chemotherapy, predicting drug resistance. In view of the considerable levels of hypermethylation in cancer cells and, for MGMT, its prognostic relevance, DAPK and MGMT show potential as epigenetic markers, in a way that additional studies may test its viability and efficacy in clinical management.

Potential biomarkers of ductal carcinoma in situ progression.

BACKGROUND: Ductal carcinoma in situ is a non-obligate precursor of invasive breast carcinoma and presents a potential risk of over or undertreatment. Finding molecular biomarkers of disease progression could allow for more adequate patient treatment. We aimed to identify potential biomarkers that can predict invasiveness risk. METHODS: In this epithelial cell-based study archival formalin-fixed paraffin-embedded blocks from six patients diagnosed with invasive lesions (pure invasive ductal carcinoma), six with in-situ lesions (pure ductal carcinoma in situ), six with synchronous lesions (invasive ductal carcinoma with an in-situ component) and three non-neoplastic breast epithelium tissues were analyzed by gene expression profiling of 770 genes, using the nCounter® PanCancer Pathways panel of NanoString Technologies. RESULTS: The results showed that in comparison with non-neoplastic tissue the pure ductal carcinoma in situ was one with the most altered gene expression profile. Comparing pure ductal carcinoma in situ and in-situ component six differentially expressed genes were found, three of them (FGF2, GAS1, and SFRP1), play a role in cell invasiveness. Importantly, these genes were also differentially expressed between invasive and noninvasive groups and were negatively regulated in later stages of carcinogenesis. CONCLUSIONS: We propose these three genes (FGF2, GAS1, and SFRP1) as potential biomarkers of ductal carcinoma in situ progression, suggesting that their downregulation may be involved in the transition of stationary to migrating invasive epithelial cells.

Effects of glyphosate exposure on human health: Insights from epidemiological and in vitro studies.

Glyphosate (GLY) is a broad-spectrum, post-emergent, non-selective and synthetic universal herbicide, whose commercial formulations are referred to as glyphosate-based-herbicides (GBHs). These chemicals and their metabolites can be found in soil, air, water, as well as groundwater and food products. This review summarizes to summarize current in vitro and epidemiological studies investigating the effects of GLY exposure on human health. Recent human cell studies have reported several GLY and GBH toxicological effects and have contributed to a better understanding of the deleterious consequences associated with their exposure. However, these detrimental effects are dependent on the cell type, chemical composition, as well as magnitude and time of exposure, among other factors. Moreover, the deleterious effects of GLY exposure on human health were observed in epidemiological studies; however, most of these studies have not determined the GLY dosage to confirm a direct effect. While GLY toxicity is clear in human cells, epidemiological studies investigating individuals exposed to different levels of GLY have reported contradictory data. Therefore, based on currently available in vitro and epidemiological data, it is not possible to confirm the complete safety of GLY use, which will require additional comprehensive studies in animal models and humans.

Glyphosate-based herbicides at low doses affect canonical pathways in estrogen positive and negative breast cancer cell lines.

Glyphosate is a broad-spectrum herbicide that is used worldwide. It represents a potential harm to surface water, and when commercially mixed with surfactants, its uptake is greatly magnified. The most well-known glyphosate-based product is Roundup. This herbicide is potentially an endocrine disruptor and many studies have shown the cytotoxicity potential of glyphosate-based herbicides. In breast cancer (BC) cell lines it has been demonstrated that glyphosate can induce cellular proliferation via estrogen receptors. Therefore, we aimed to identify gene expression changes in ER+ and ER- BC cell lines treated with Roundup and AMPA, to address changes in canonical pathways that would be related or not with the ER pathway, which we believe could interfere with cell proliferation. Using the Human Transcriptome Arrays 2.0, we identified gene expression changes in MCF-7 and MDA-MB-468 exposed to low concentrations and short exposure time to Roundup Original and AMPA. The results showed that at low concentration (0.05% Roundup) and short exposure (48h), both cell lines suffered deregulation of 11 canonical pathways, the most important being cell cycle and DNA damage repair pathways. Enrichment analysis showed similar results, except that MDA-MB-468 altered mainly metabolic processes. In contrast, 48h 10mM AMPA showed fewer differentially expressed genes, but also mainly related with metabolic processes. Our findings suggest that Roundup affects survival due to cell cycle deregulation and metabolism changes that may alter mitochondrial oxygen consumption, increase ROS levels, induce hypoxia, damage DNA repair, cause mutation accumulation and ultimately cell death. To our knowledge, this is the first study to analyze the effects of Roundup and AMPA on gene expression in triple negative BC cells. Therefore, we conclude that both compounds can cause cellular damage at low doses in a relatively short period of time in these two models, mainly affecting cell cycle and DNA repair.

A view of the maternal inheritance of Espírito Santo populations: The contrast between the admixed and Pomeranian descent groups.

In addition to its valuable utility in forensic investigations, mitochondrial DNA (mtDNA) analysis is a reliable tool to uncover the origins of admixed populations, such as Brazilians. The state of Espírito Santo (ES), similar to other coastal Brazilian states, has a population shaped by 3 main ancestral roots: Amerindian, African and European. Among the latter, the Pomeranian descendants stand out due to the preservation of the traditional aspects of their culture, especially the Pomeranian language. Despite the genetic data already available, there is no mtDNA database that adequately reflects the diversity, the geographic distribution, and the origins of the maternal lineages from ES. To increase the knowledge of maternal ancestry and to investigate the population's genetic stratification, a total of 291 samples were collected in the 4 macroregions (Metropolitan, South, Central and North) of ES and in the Pomeranian communities. Complete control region data were produced for the general (N=214) and Pomeranian (N=77) groups. Regarding the general population, the high values of haplotype diversity (H=99.9%) and pairwise differences (MNPD=16.9) found are in agreement with those reported for other populations in the southeast region of the country. Regarding maternal inheritance, the ES populations stood out due to the predominance of European haplogroups (49.5%), although the North macroregion had a higher African ancestry (47.1%). Among the Pomeranians, the lowest MNPD value (11.2) and the high percentage of shared haplotypes (15%) were indicative of founder events. The FST analysis showed that the Pomeranians (98.7% of European lineages) are genetically isolated from the other admixed populations in Brazil. This study demonstrated that the ES state contains singularities regarding the intrapopulational and interpopulational diversity of mtDNA. Even after 5 centuries of interethnic admixture, the present-day population of Espírito Santo harbors genetic marks that trace back to the historical aspects of its formation.

PAI-1, CAIX, and VEGFA expressions as prognosis markers in oral squamous cell carcinoma.

BACKGROUND: In oral squamous cell carcinoma (OSCC), the HIF-1 complex promotes the expression of genes involved in specific mechanisms of cell survival under hypoxic conditions, such as plasminogen activator inhibitor-1 (PAI-1), carbonic anhydrase 9 (CAIX), and vascular endothelial growth factor A (VEGFA). The study aimed to investigate the presence and prognostic value of PAI-1, CAIX, and VEGFA in OSCC. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expressions of these proteins in 52 tumoral tissue samples of patients with OSCC, surgically treated and followed by a minimum of 24 months after surgery. The correlations between protein expressions and clinicopathological parameters and prognosis were analyzed. RESULTS: Positive PAI-1 membrane expression was significantly associated with local disease relapse (P = .027). Multivariate analysis revealed that the positive PAI-1 membrane expression is an independent marker for local disease relapse, with approximately 14-fold increased risk when compared to negative expression (OR = 14.49; CI = 1.40-150.01, P = .025). Strong PAI-1 cytoplasmic expression was significantly associated with the less differentiation grade (P = .027). Strong CAIX membrane expression was significantly associated with local disease-free survival (P = .038). Positive CAIX cytoplasmic expression was significantly associated with lymph node affected (P = .025) and with disease-specific survival (P = .022). Multivariate analysis revealed that the positive CAIX cytoplasmic expression is an independent risk factor for disease-related death, increasing their risk approximately 3-fold when compared to negative expression (HR = 2.84; CI = 1.02-7.87, P = .045). Positive VEGFA cytoplasmic expression was significantly associated with less differentiation grade (P = .035). CONCLUSION: Our results suggest a potential role for these expressions profiles as tumor prognostic markers in OSCC patients.

JMJD1A, H3K9me1, H3K9me2 and ADM expression as prognostic markers in oral and oropharyngeal squamous cell carcinoma.

AIMS: Jumonji Domain-Containing 1A (JMJD1A) protein promotes demethylation of histones, especially at lysin-9 of di-methylated histone H3 (H3K9me2) or mono-methylated (H3K9me1). Increased levels of H3 histone methylation at lysin-9 (H3K9) is related to tumor suppressor gene silencing. JMJD1A gene target Adrenomeduline (ADM) has shown to promote cell growth and tumorigenesis. JMJD1A and ADM expression, as well as H3K9 methylation level have been related with development risk and prognosis of several tumor types. METHODS AND RESULTS: We aimed to evaluate JMJD1A, ADM, H3K9me1 and H3K9me2expression in paraffin-embedded tissue microarrays from 84 oral and oropharyngeal squamous cell carcinoma samples through immunohistochemistry analysis. Our results showed that nuclear JMJD1A expression was related to lymph node metastasis risk. In addition, JMJD1A cytoplasmic expression was an independent risk marker for advanced tumor stages. H3K9me1 cytoplasmic expression was associated with reduced disease-specific death risk. Furthermore, high H3K9me2 nuclear expression was associated with worse specific-disease and disease-free survival. Finally, high ADM cytoplasmic expression was an independent marker of lymph node metastasis risk. CONCLUSION: JMJD1A, H3K9me1/2 and ADM expression may be predictor markers of progression and prognosis in oral and oropharynx cancer patients, as well as putative therapeutic targets.

ATM, BCL2, and TGFß Gene Polymorphisms as Radiotherapy Outcome Biomarkers in Head and Neck Squamous Cell Carcinoma Patients.

AIMS: Polymorphisms in cell cycle genes are considered prognostic as radiosensitivity markers in patients with head and neck squamous cell carcinoma. Therefore, we aimed to investigate the relationship of ATM 5557G>A, ATM IVS62 + 60G>A, TP53 215G>C, BCL2-938C>A, TGFß-509C>T, and TGFß 29C>T with radiotherapy response. MATERIALS AND METHODS: Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism in 210 patients with oral cavity/oropharyngeal carcinoma and 101 patients with laryngeal tumors. RESULTS: In irradiated oral cavity/oropharyngeal tumors, the ATM IVS62 + 60G>A AA genotype significantly increased local recurrence risk (odds ratio [OR] = 4.43; confidence interval [CI] = 1.22-16.13) and the BCL2-938C>A C allele and the TGFß-509C>T T allele were associated with worse disease-specific survival (hazard ratio [HR] = 0.46; CI = 0.24-0.90 and HR = 2.20; CI = 1.12-4.29, respectively). In irradiated laryngeal carcinoma, the TGFß 29C>T C allele was associated with increased local recurrence risk (OR = 0.09; CI = 0.02-0.53), death rate (OR = 0.18; CI = 0.04-0.86), and worse local disease-free and disease-specific survival rates (HR = 0.13; CI = 0.03-0.59 and HR = 0.21; CI = 0.07-0.60, respectively), while the BCL2-938C>A C allele was related to a worse disease-specific survival (HR = 0.32; CI = 0.12-0.83). DISCUSSION: These results can help individualize treatment according to a patient's genetic markers. We demonstrated that ATM IVS62 + 60G>A, TGFß 29C>T, TGFß-509C>T, and BCL2-938C>A can function as biomarkers of tumor radiosensitivity, being candidates for a predictive genetic profile of radiotherapy response.

HIF-1alpha expression profile in intratumoral and peritumoral inflammatory cells as a prognostic marker for squamous cell carcinoma of the oral cavity.

The HIF-1 transcriptional complex is responsible for controlling transcription of over 100 genes involved in cell hypoxia response. HIF-1alpha subunit is stabilized in hypoxia conditions, creating the HIF-1 nuclear transcription factor. In inflammatory cells, high HIF-1alpha expression induces lymphocytic immunosuppression, decreasing tumoral antigen recognition, which promotes tumor growth. The present work investigated the relationship between HIF-1alpha expression in lymphocytes populating the intratumoral and peritumoral region of 56 patients with oral cancer. Our data indicates a prognostic value for this expression. High HIF-1alpha expression in peritumoral inflammatory cells is significantly related to worse patient outcome, whereas high expression in the intratumoral lymphoid cells correlates with a better prognosis. A risk profile indicating the chance of disease relapse and death was designed based on HIF-1alpha expression in tumoral inflammatory cells, defining low, intermediate and high risks. This risk profile was able to determine that high HIF-1alpha expression in peritumoral cells correlates with worse prognosis, independently of intratumoral expression. Low HIF-1alpha in tumor margins and high expression in the tumor was considered a low risk profile, showing no cases of disease relapse and disease related death. Intermediate risk was associated with low expression in tumor and tumor margins. Our results suggest that HIF-1alpha expression in tumor and peritumoral inflammatory cells may play an important role as prognostic tumor marker.

Genetic variability of inflammatory genes in the Brazilian population.

Inflammatory gene variants have been associated with several diseases, including cancer, diabetes, vascular diseases, neurodegenerative diseases, arthritis, and others. Therefore, determining the population genetic composition of inflammation-related genes can be useful for the determination of general risk, prognostic and therapeutic strategies to prevent or cure specific diseases. We have aimed to identify polymorphism genotype frequencies in genes related to the inflammatory response in the Brazilian population, namely, IκBL -62AT, IκBL -262CT, tumor necrosis factors alpha (TNFa) -238GA, TNFa -308GA, lymphotoxin-alpha (LTa) +80AC, LTa +252AG, FAS -670AG, and FASL -844TC, considering the white, black, and Pardo ethnicities of the São Paulo State. Our results suggest that the Brazilian population is under a miscegenation process at the current time, since some genotypes are not in the Hardy-Weinberg equilibrium. In addition, we conclude that the Pardo ethnicity is derived from a complex mixture of ethnicities, including the native Indian population.

Analysis of genome instability in breast cancer.

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order to determine patterns and levels of instability, as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-ß were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-ß were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.

Histological profile and age at diagnosis of breast and ovarian tumors: A register-based study in Espirito Santo, Brazil.

Breast and ovarian cancers are important public health problems in Brazil. However, in various locations in the Brazilian territory these types of cancer remain poorly characterized. Therefore, this study aimed to compare data collected from the Pathology Services of two Oncology Reference Hospitals in Espirito Santo state (Brazil) with the data in previous Brazilian studies. Histological type frequency and age at diagnosis of breast and ovarian tumors between 2001-2004 and 2009-2010 were analyzed. Tumor size, histological grade, lymph node status, hormone receptor status [estrogen (ER) and progesterone receptors (PR)] as well as HER2 and Ki-67 marker status were obtained for the cases of infiltrating ductal carcinomas of the breast during the period 2009-2010. Categorical variables were described by their absolute and/or relative frequencies, while quantitative variables were expressed as the mean ± standard deviation and median, using 95% confidence intervals. Chi-square tests were performed to examine whether or not the studied variables differed by age at the time of diagnosis. Malignant breast tumors (1,758) and 119 malignant ovarian tumors were examined. Mean ages for malignant breast and ovarian tumors were 53.59 and 52.98 years, respectively. An increased tumor frequency in the age group of ≤35 years was observed for other malignant tumors of the breast during the period of 2009-2010, compared to 2001-2004. When considering only infiltrating ductal carcinomas with immunohistochemistry records available, there were 82.1% (133 cases) of ER+/PR+ tumors, 1.9% (3 cases) of ER-/PR-/HER2 3+ and 16.04% (26 cases) of ER-/PR-/HER2- tumors. The findings of this study demonstrated detailed information concerning breast and ovarian tumor behavior in the Espirito Santo state (Brazil), allowing for a precise comparison with other populations (Brazilian or international), which may be helpful in the planning of prevention and treatment strategies.

Promoter hypermethylation in primary squamous cell carcinoma of the oral cavity and oropharynx: a study of a Brazilian cohort.

Epigenetic silencing of cancer-related genes plays an important role in oral/oropharyngeal squamous cell carcinoma (OSCC). We evaluated promoter hypermethylation of 4 cancer-related genes in OSCCs of a Brazilian cohort and determined its relationship with exposure to alcohol, tobacco, HPV infection and clinicopathological parameters. CDKN2A (cyclin-dependent kinase inhibitor 2A or p16), SFN (stratifin or 14-3-3 σ), EDNRB (endothelin receptor B) and RUNX3 (runt-related transcript factor-3) had their methylation patterns evaluated by MSP analysis in OSCC tumors (n = 45). HPV detection was carried out by PCR/RFLP. Aberrant methylation was detected in 44/45 (97.8 %) OSCC; 24.4 % at CDKN2A, 77.8 % at EDNRB, 17.8 % at RUNX3 and 97.8 % at SFN gene. There was no significant association between methylation patterns and clinical parameters. HPV (subtype 16) was detected in 3 out of 45 patients (6 %). Our findings indicate that HPV infection is uncommon and methylation is frequent in Brazilian OSCCs, however, EDNRB and SFN gene methylation are not suitable OSCC biomarkers due to indistinct methylation in tumoral and normal samples. In contrast, CDKN2A and RUNX3 genes could be considered differentially methylated genes and potential tumor markers in OSCCs.

Polymorphism analysis of MTHFR, factor II, and factor V genes in the Pomeranian population of Espirito Santo, Brazil.

Pomeranian populations worldwide immigrated originally from the north of Europe, and because of their preferential marriage, religion, and cultural habits, they show little or no reproductive mixing with local populations. Methylenetetrahydrofolate reductase gene (MTHFR) C677T, Factor V Leiden, and Factor II G20210A polymorphisms are linked to augmented clotting and their frequencies may vary according to population ethnicity. We aimed to assess the frequencies of these thrombophilic alleles in the Pomeranian population residing in Espirito Santo and compare with the general population of the Espirito Santo state, Brazil. A total of 200 individuals were analyzed. The intrapopulation fixation index of the MTHFR C677T polymorphism was 0.03736. The observed heterozygosity was 0.44 and 0.4 for the general and Pomeranian populations, respectively. According to the chi-square test, both populations are in Hardy-Weinberg equilibrium. Four polymorphic alleles were detected for Factor II (2.02%) and 8 for Factor V (4.81%). Our results show that there is gene flow between the general and the Pomeranian population of Espirito Santo, which should no longer be considered an isolated population.