Association of Fusobacterium nucleatum Levels by ddPCR in Oral Rinse Samples With Periodontal Disease in Oral Squamous Cell Carcinoma Patients and in Controls.

BACKGROUND: The role of microbiome, particularly Fusobacterium nucleatum (Fn), in periodontal disease and oral squamous cell carcinoma (OSCC) has been recently explored. This study aimed to evaluate the Fn presence and its levels in oral rinse samples from Brazilian OSCC patients and healthy individuals and its association with sociodemographic, clinical, and oral health features. METHODS: In this case-control study, 80 participants were included, 31 OSCC patients and 49 individuals without a cancer history. Clinical data were collected, and an oral exam was done on a subset of the cohort. Fn levels were evaluated by droplet digital PCR (ddPCR) in oral rinse samples and were categorized as Fn-high or Fn-low based on the median number of copies per reaction. RESULTS: OSCC patients showed higher levels of Fn (68%, p = 0.03) than controls, and all OSCC cases were diagnosed with periodontal disease (100%, p = 1.0). In the univariate analysis, Fn-high level was more frequently present in OSCC cases compared to controls (p = 0.01). It was also observed that Fn-high level OSCC cases were significantly associated with self-reported non-white ethnicity (71.4%, p = 0.01) and had more infiltrative lesions (57.1%, p = 0.02) than Fn-low OSCC cases. Fn-high levels in oral rinse samples, were significantly more prevalent among OSCC than in controls. CONCLUSIONS: In OSCC patients, Fn-high levels were associated with non-white ethnicity and lesions with infiltrative clinical aspects. Among OSCC cases, all had periodontal disease.

Immune-checkpoint gene expression and BCG response in non-muscle invasive bladder cancer.

METHODS: One-hundred-six patients diagnosed with non-muscle invasive bladder cancer and treated with intravesical BCG were included and divided into two groups, BCG-responsive (n = 47) and -unresponsive (n = 59). Immunohistochemistry was used to evaluate PD-L1 expression and MSI was assessed by a commercial multiplex PCR kit. The mRNA expression profile of 15 immune checkpoints was performed using the nCounter technology. For in silico validation, two distinct cohorts sourced from the Gene Expression Omnibus (GEO) database were used. RESULTS: Among the 106 patients, only one (<1 %) exhibited MSI instability. PD-L1 expression was present in 9.4 % of cases, and no association was found with BCG-responsive status. We found low gene expression of canonic actionable immune checkpoints PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, while high expression was observed for CD276 (B7-H3), CD47, TNFRSF14, IDO1 and PVR (CD155) genes. High IDO1 expression levels was associated with worst overall survival. The PDCD1, CTLA4 and TNFRSF14 expression levels were associated with BCG responsiveness, whereas TIGIT and CD276 were associated with unresponsiveness. Finally, CD276 was validated in silico cohorts. CONCLUSION: In NMIBC, MSI is rare and PD-L1 expression is present in a small subset of cases. Expression levels of PDCD1, CTLA4, TNFRSF14, TIGIT and CD276 could constitute predictive biomarkers of BCG responsiveness.

Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research.

In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.

An industrialized diet as a determinant of methylation in the 1F region of the NR3C1 gene promoter.

Background: Dietary composition can modify gene expression, favoring the development of chronic diseases via epigenetic mechanisms. Objective: Our study aimed to investigate the relationship between dietary patterns and NR3C1 gene methylation in users of the Brazilian Public Unified Health System (SUS). Methods: We recruited 250 adult volunteers and evaluated their socioeconomic status, psychosocial characteristics, lifestyle, and anthropometrics. Peripheral blood was collected and evaluated for cortisol levels, glycemia, lipid profile, and insulin resistance; methylation of CpGs 40-47 of the 1F region of the NR3C1 gene was also measured. Factors associated with degree of methylation were evaluated using generalized linear models (p < 0.05). Lifestyle variables and health variables were included as confounding factors. Results: The findings of our cross-sectional study indicated an association between NR3C1 DNA methylation and intake of processed foods. We also observed relevant associations of average NR3C1 DNA across the segment analyzed, methylation in component 1 (40-43), and methylation in component 2 (44-47) with a pattern of consumption of industrialized products in relation to BMI, serum cortisol levels, and lipid profile. These results may indicate a relationship between methylation and metabolic changes related to the stress response. Conclusion: These findings suggest an association of methylation and metabolic alterations with stress response. In addition, the present study highlights the significant role of diet quality as a stress-inducing factor that influences NR3C1 methylation. This relationship is further linked to changes in psychosocial factors, lifestyle choices, and cardiometabolic variables, including glucose levels, insulin resistance, and hyperlipidemia.

HOXA1 3'UTR Methylation Is a Potential Prognostic Biomarker in Oral Squamous cell Carcinoma.

BACKGROUND: HOXA1 is a prognostic marker and a potential predictive biomarker for radioresistance in head and neck tumors. Its overexpression has been associated with promoter methylation and a worse prognosis in oral squamous cell carcinoma (OSCC) patients. However, opposite outcomes are also described. The effect of the methylation of this gene on different gene regions, other than the promoter, remains uncertain. We investigated the methylation profile at different genomic regions of HOXA1 in OSCC and correlated differentially methylated CpG sites with clinicopathological data. METHODS: The HOXA1 DNA methylation status was evaluated by analyzing data from The Cancer Genome Atlas and three Gene Expression Omnibus datasets. Significant differentially methylated CpG sites were considered with a |∆ß| ≥ 0.10 and a Bonferroni-corrected p-value < 0.01. Differentially methylated CpGs were validated by pyrosequencing using two independent cohorts of 15 and 47 OSCC patients, respectively. RESULTS: Compared to normal tissues, we found significantly higher DNA methylation levels in the 3'UTR region of HOXA1 in OSCC. Higher methylation levels in tumor samples were positively correlated with smoking habits and patients' overall survival. CONCLUSIONS: Our findings suggest that HOXA1 gene body methylation is a promising prognostic biomarker for OSCC with potential clinical applications in patient monitoring.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. METHODS: We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. RESULTS: MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. CONCLUSION: These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

Biomarkers for Immune Checkpoint Inhibitor Response in NSCLC: Current Developments and Applicability.

Lung cancer has the highest mortality rate among all cancer types, resulting in over 1.8 million deaths annually. Immunotherapy utilizing immune checkpoint inhibitors (ICIs) has revolutionized the treatment of non-small cell lung cancer (NSCLC). ICIs, predominantly monoclonal antibodies, modulate co-stimulatory and co-inhibitory signals crucial for maintaining immune tolerance. Despite significant therapeutic advancements in NSCLC, patients still face challenges such as disease progression, recurrence, and high mortality rates. Therefore, there is a need for predictive biomarkers that can guide lung cancer treatment strategies. Currently, programmed death-ligand 1 (PD-L1) expression is the only established biomarker for predicting ICI response. However, its accuracy and robustness are not consistently reliable. This review provides an overview of potential biomarkers currently under development or in the validation stage that hold promise in improving the classification of responders and non-responders to ICI therapy in the near future.

miR-99a-5p as a biomarker for lymph node metastasis prediction in oral squamous cell carcinoma patients.

BACKGROUND: Metastatic lymph node involvement influences therapy decisions and serves as a prognostic indicator in oral squamous cell carcinoma (OSCC). However, many early-stage patients with clinically negative lymph nodes exhibit no metastasis upon surgical staging. This study aimed to identify differentially expressed miRNAs capable of distinguishing pathologically positive (pN+) from negative (pN0) nodes in OSCC patients without clinical evidence of lymph node metastases (cN0). METHODS: Expression levels of 798 miRNAs were assessed in tumor samples from 10 pN+ and 10 pN0 patients using the Nanostring nCounter platform. Validation was performed in an independent cohort of 15 pN+ and 24 pN0 patients through RT-qPCR. RESULTS: Eight miRNAs exhibited differential expression between pN0 and pN+ patients. Notably, hsa-miR-99a-5p demonstrated high sensitivity and specificity in predicting patients at higher risk of positive lymph nodes. CONCLUSIONS: These findings highlight hsa-miR-99a-5p as a potential biomarker for detecting lymph node metastasis in primary OSCC tumors.

Mature tertiary lymphoid structures are key niches of tumour-specific immune responses in pancreatic ductal adenocarcinomas.

OBJECTIVE: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity. DESIGN: We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. RESULTS: We found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-ß may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. CONCLUSION: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

Methylation of BDNF gene in association with episodic memory in women.

Brain-derived neurotrophic factor (BDNF) gene regulation plays an important role in long-term memory formation, and the DNA methylation (DNAm) level of BDNF promoters has been associated with episodic memory deficits. Our aim was to explore the association between DNAm levels in BDNF promoter IV with verbal learning and memory performance in healthy women. We conducted a cross-sectional study by recruiting 53 individuals. Episodic memory was assessed by using the Rey Auditory Verbal Learning Test (RAVLT). Clinical interviews, RAVLT, and blood sample collection were assessed in all participants. DNAm was measured on DNA from whole peripheral blood using pyrosequencing. According to generalized linear model (GzLM) analyses, cytosine guanine dinucleotide (CpG) site 5 showed significant associations between learning capacity (LC, p < 0.035), that is, every 1% of DNA methylation at CpG site 5 results in a 0.068 reduction in verbal learning performance. To the best of our knowledge, the current study is the first to show that BDNF DNAm plays an important role in episodic memory.

PD-L1 Tumor Expression as a Predictive Biomarker of Immune Checkpoint Inhibitors' Response and Survival in Advanced Melanoma Patients in Brazil.

Immune checkpoint blockade (ICB) agents are prominent immunotherapies for the treatment of advanced melanoma. However, they fail to promote any durable clinical benefit in a large cohort of patients. This study assessed clinical and molecular predictors of ICB response and survival in advanced melanoma. A retrospective analysis was performed on 210 patients treated with PD-1 or CTLA-4 inhibitors at Barretos Cancer Hospital, Brazil. PD-L1 expression was assessed by immunohistochemistry using formalin-fixed paraffin-embedded tumor tissues collected prior to ICB therapy. Patients were divided into responders (complete and partial response and stable disease for more than 6 months) and non-responders (stable disease for less than 6 months and progressive disease). Among them, about 82% underwent anti-PD-1 immunotherapy, and 60.5% progressed after the ICB treatment. Patients that received ICB as first-line therapy showed higher response rates than previously treated patients. Higher response rates were further associated with superficial spreading melanomas and positive PD-L1 expression (>1%). Likewise, PD-L1 positive expression and BRAF V600 mutations were associated with a higher overall survival after ICB therapy. Since ICBs are expensive therapies, evaluation of PD-L1 tumor expression in melanoma patients should be routinely assessed to select patients that are most likely to respond.

Psychotropic drug use and suggestive depression symptoms associated with NR3C1 DNA methylation / Uso de drogas psicotrópicas e sintomas sugestivos de depressão associados à metilação do DNA do NR3C1

Introduction: Psychiatric disorders have become a global problem that leads millions of people to use psychotropic medications, especially benzodiazepines. The effects of these substances are widely known regarding tolerance and chemical dependence, however, from epigenetics perspective, there are still little known.Objective: To evaluate the association between psychotropic drug use, NR3C1 gene methylation and its relation with symptoms suggestive of depression in adult individuals assisted in the public health system.Methods: 385 adult volunteers (20-59 years) users of the Brazilian Unified Health System were recruited to evaluate socioeconomic, health, lifestyle conditions in a cross sectional study. BDI-II evaluated symptoms suggestive of depression and pyrosequencing evaluated NR3C1 DNA methylation. Bivariate and multivariate Poisson regression model with robust variance (p < 0.05) evaluated the association between psychotropic drug use and NR3C1 gene methylation.Results: Specific depressive symptoms such as irritability, insomnia and fatigability were associated with psychotropic drug use. Symptoms of past failure, indecision and loss of appetite were associated with hypermethylation patterns in CpGs 40 to 47 of NR3C1 gene. Moreover, psychotropic drug use is associated with 50% reduction in NR3C1 gene methylation, through model adjusted with socioeconomic, health and lifestyle confounding variables.Conclusions: Psychotropic drug use and depressive symptoms was associated with changes in NR3C1 DNA methylation. In this context, epigenetic modification resulting from psychotropic drug use and depressive symptoms could be considered, mainly in population studies with epigenetic evaluation, where these factors may be influencing the findings of future studies.

Introdução: os distúrbios psiquiátricos tornaram-se um problema global que leva milhões de pessoas ao uso de medicamentos psicotrópicos. Os efeitos dessas substâncias são amplamente conhecidos quanto à tolerância e dependência química, porém, do ponto de vista epigenético, ainda são pouco conhecidos.Objetivos: avaliar a associação entre o uso de drogas psicotrópicas, metilação do gene NR3C1 e sua relação com sintomas sugestivos de depressão em indivíduos entre 20 a 59 anos usuários da rede pública de saúde.Método: 385 voluntários de 20-59 anos, usuários do Sistema Único de Saúde brasileiro foram recrutados para avaliação das condições socioeconômicas, de saúde e de estilo de vida em estudo transversal. O BDI-II avaliou sintomas sugestivos de depressão e o pirosequenciamento avaliou a metilação do DNA de NR3C1. Modelo de regressão de Poisson bivariado e multivariado com variância robusta (p < 0,05) avaliou a associação entre o uso de drogas psicotrópicas e metilação do gene NR3C1.Resultados: sintomas depressivos específicos como irritabilidade, insônia e fadiga foram associados ao uso de medicamentos psicotrópicos. Sintomas de fracasso passado, indecisão e perda de apetite foram associados a padrões de hipermetilação nos CpGs 40 a 47 do gene NR3C1. Além disso, o uso de psicofármacos está associado à redução de 50% na metilação do gene NR3C1, por meio de modelo ajustado com variáveis de confusão socioeconômicas, de saúde e estilo de vida.Conclusão: o uso de drogas psicotrópicas e sintomas específicos depressivos foram associados a alterações na metilação do DNA de NR3C1.

Tobacco use modify exon IV BDNF gene methylation levels in depression.

This study aimed to investigate BDNF gene methylation in individuals with depression based on tobacco use. Therefore, 384 adults from southeastern Brazil were recruited to assess depression, socioeconomic status, lifestyle, and methylation by pyrosequencing exon IV promoter region of the BDNF gene. The Generalized Linear Model (GzLM) was used to check the effect of depression, tobacco, and the interaction between depression and tobacco use in methylation levels. In addition, the Kruskal-Wallis test, followed by Dunn's post hoc test, was used to compare methylation levels. Interaction between depression and tobacco use was significant at levels of BDNF methylation in the CpG 5 (p = 0.045), 8 (p = 0.016), 9 (p = 0.042), 10 (p = 0.026) and mean 5-11 (p < 0.001). Dunn's post hoc test showed that individuals with depression and tobacco use compared to those with or without depression who did not use tobacco had lower levels of BDNF methylation in CpG 5, 6, 7, 8, 9, 11, and mean 5-11. Therefore, we suggest that tobacco use appears to interfere with BDNF gene methylation in depressed individuals.

Lifestyle and NR3C1 exon 1F gene methylation is associated with changes in glucose levels and insulin resistance.

AIMS: the present study aimed to investigate how glucose and insulin levels may be associated with changes in NR3C1 gene methylation levels in adults. MAIN METHODS: 375 volunteers users of the Brazilian Public Unified Health System (SUS) were recruited to assess socioeconomic status, lifestyle, anthropometric data, blood glucose and serum cortisol levels, insulin resistance, and NR3C1 gene methylation assessment. Factors associated with glucose levels and insulin resistance were investigated using multivariate analysis GLzM at 5% significance (p<0.05). KEY FINDINGS: our results verified that glucose levels and insulin resistance were directly related to NR3C1 gene methylation and age, while not being overweight and obese and no tobacco consumption were indirectly related to glucose levels and insulin resistance. SIGNIFICANCE: habits and lifestyle may influence NR3C1 gene regulation, revealing the complexity of environmental impacts on NR3C1 methylation. Furthermore, associated risk factors must be taken into account in epigenetic studies as they directly interfere with blood glucose levels and insulin resistance.

Distinct global DNA methylation and NF-κB expression profile of preimplantation biopsies from ideal and non-ideal kidneys.

BACKGROUND: Epigenetic mechanisms may affect the ideal and non-ideal kidneys selected for transplantation and their inflammatory gene expression profile differently and may contribute to poor clinical outcomes. OBJECTIVE: Study the Global DNA methylation and the expression profiles of the DNA methyltransferases (DNMTs) and nuclear factor kappa B (NF-κB) in preimplantation kidney biopsies from ideal and non-ideal kidneys (expanded criteria donor (ECD) and with KDPI > 85%). METHODS: In a sample consisting of 45 consecutive pre-implantation biopsies, global DNA methylation levels were detected by LINE-1 repeated elements using bisulfite pyrosequencing. DNMT gene expression was assessed by real-time quantitative polymerase chain reaction, and NF-κB protein expression by immunofluorescence. RESULTS: ECD kidneys displayed increased methylation levels in LINE-1, and DNMT1 and DNMT3B expression was upregulated when comparing ECD to standard criteria donor kidneys. Similarly, kidneys with KDPI > 85% exhibited increased LINE-1 methylation and DNMT1 upregulation when compared to a KDPI ≤ 85%. NF-κB protein expression levels were greatly increased in both types of non-ideal kidneys compared to ideal kidneys. Moreover, hypermethylation of LINE-1 was associated with cold ischemia time > 20 h and ECD kidney classification. CONCLUSIONS: This study shows that global DNA hypermethylation and high expression of NF-κB occurred in both types of non-ideal kidneys and were associated with prolonged cold ischemia time. Global DNA methylation can be a useful tool to assess non-ideal kidneys and hence, could be used to expand the pool of kidneys donors.

Evaluation of acetylation and methylation in oral rinse of patients with head and neck cancer history exposed to valproic acid.

Evaluate the biological action of valproic acid in the acetylation of histones and in the methylation of tumor suppressor genes via oral rinse in patients with a previous history of head and neck squamous cell carcinoma (HNSCC). Forty-two active or former smokers were included in this randomized, double-blind, placebo-controlled trial. Oral rinse samples were collected prior to treatment with valproic acid or placebo and after 90 days of treatment. The methylation status of five tumor suppressor genes and histone acetylation were evaluated by pyrosequencing and ELISA techniques, respectively. Differences between the 90-day and baseline oral rinse acetylation and methylation results were analyzed by comparing groups. Thirty-four patients were considered for analysis. The mean percentage adherence in the valproic and placebo groups was 93.4 and 93.0, respectively (p = 0.718). There was no statistically significant difference between groups when comparing the medians of the histone acetylation ratio and the methylation ratio for most of the studied genes. A significant reduction in the DCC methylation pattern was observed in the valproic group (p = 0.023). The use of valproic acid was safe and accompanied by good therapeutic adherence. DCC methylation was lower in the valproic acid group than in the placebo group.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

DNA Methylation Markers from Negative Surgical Margins Can Predict Recurrence of Oral Squamous Cell Carcinoma.

The identification of molecular markers in negative surgical margins of oral squamous cell carcinoma (OSCC) might help in identifying residual molecular aberrations, and potentially improve the prediction of prognosis. We performed an Infinium MethylationEPIC BeadChip array on 32 negative surgical margins stratified based on the status of tumor recurrence in order to identify recurrence-specific aberrant DNA methylation (DNAme) markers. We identified 2512 recurrence-associated Differentially Methylated Positions (DMPs) and 392 Differentially Methylated Regions (DMRs) which were enriched in cell signaling and cancer-related pathways. A set of 14-CpG markers was able to discriminate recurrent and non-recurrent cases with high specificity and sensitivity rates (AUC 0.98, p = 3 × 10-6; CI: 0.95-1). A risk score based on the 14-CpG marker panel was applied, with cases classified within higher risk scores exhibiting poorer survival. The results were replicated using tumor-adjacent normal HNSCC samples from The Cancer Genome Atlas (TCGA). We identified residual DNAme aberrations in the negative surgical margins of OSCC patients, which could be informative for patient management by improving therapeutic intervention. This study proposes a novel DNAme-based 14-CpG marker panel as a promising predictor for tumor recurrence, which might contribute to improved decision-making for the personalized treatment of OSCC cases.

Methylation profile of colon cancer genes in colorectal precursor lesions and tumor tissue: perspectives for screening.

AIMS: Epigenetic alterations of genes involved in colorectal carcinogenesis are likely to be informative biomarkers for early detection. We assessed the methylation profile of a panel of seven colon cancer-related genes comparing normal colon, colorectal cancer (CRC) precursor lesions and cancer tissues from a Brazilian cohort. METHODS: The cohort comprised 114 CRC patients, including 40 matched normal tissue, 47 patients with adenomas, 33 with serrated polyps and 8 with normal colonic biopsy. DNA methylation status of SEPT9, ALX4, NDRG4, BMP3, APC, p16 and MLH1 was determined by pyrosequencing and correlated with clinicopathological features. Sensitivity, specificity, positive predictive value and negative predictive value were calculated for all genes using cancer endpoint. RESULTS: The most frequently methylated genes in cancer and in precancer lesions were SEPT9, ALX4, NDRG4, and BMP3, ranging from 55.3 to 95% of the samples. Overall, the frequency of methylation of these four genes in normal colonic tissue was significantly lower as compared to cancer or precursor lesions both in adenoma-carcinoma (p < .001 and p < .050) and serrated (sessile-serrated lesion) (p < .001 and p < .050) pathways. Additionally, sensitivity for the cancer endpoint ranged from 65.6 to 91.8%, and specificity from 17.9 to 62.9% for SEPT9, ALX4, NDRG4, and BMP3 genes. Moreover, the comethylation of ≥4 genes was higher in sessile-serrated lesion (87.5%) and conventional adenomas (78.7%) than in hyperplastic polyps (43.7%) (p = .025) and was significantly associated with proximal cancers (p = .042). CONCLUSIONS: Our study suggests the DNA methylation can constitute potential biomarkers in CRC screening of Brazilian population.

Food and nutritional insecurity is associated with depressive symptoms mediated by NR3C1 gene promoter 1F methylation.

The aim of this study was to investigate socioeconomic stressors predictive of depressive symptoms and possible epigenetic changes in the glucocorticoid receptor - NR3C1-1F - an encoding gene involved in depressive symptoms. A total of 321 adult volunteers from southeastern Brazil were recruited to evaluate depressive symptoms, socio-demographic and economic factors, including food and nutritional security (FNS) or insecurity (FNiS) status, and NR3C1-1F region methylation by pyrosequencing. Depressive symptom determinants were investigated using a Poisson regression model with robust variance. Mann-Whitney tests and structural mediation equation models were used to evaluate the relationship between NR3C1 DNA methylation, FNiS, and depressive symptoms. Multivariate Poisson regression with robust variance adjusted for sex and FNiS and NR3C1-1F region methylation predicted risk factors for depressive symptoms. Mediation analysis revealed that NR3C1-1F region methylation mediated the relationship between FNiS exposure and depressive symptoms as an outcome, and depressive volunteers and FNiS individuals exhibited a significant increase in NR3C1 methylation when compared to healthy individuals and FNS volunteers, respectively. Therefore, we suggest that stress caused by FNiS may lead to depressive symptoms and that NR3C1-1F DNA methylation can act as a mediator of both FNiS and depressive symptoms.