Somatic Mutations in KEAP1-NRF2 Complex in Breast Cancer.

Breast cancer remains the leading cause of cancer deaths for women. Long-term estrogen exposure is considered carcinogenic due to semiquinone production and to compromised detoxification. Metabolic regulator polymorphisms, such as KEAP1 (rs1048290) and NRF2 (rs35652124, rs6721961, rs6706649), can be valuable in understanding the individual cytoprotection profile. Thus, we aim to genotype these polymorphisms in blood, tumours and surrounding tissue, to identify somatic mutations and correlate it to prognoses. A total of 23 controls and 69 women with histological confirmed breast cancer were recruited, and DNA from blood/surrounding/tumour tissue was genotyped. Genotyping and clinicopathological data were correlated. We verified that rs35652124 presents different genotype distribution between the blood/surrounding tissue (p-value = 0.023) and tumour/surrounding tissues (p-value = 0.041). Apart from rs35652124 and considering the histological grade, the other four polymorphisms have different distributions among different tissues. There is a tendency towards the loss of heterozygosity in the surrounding tissue when compared to blood and tumour tissues, and higher genotype variability in histologic grade 2. These somatic mutations and different distribution patterns may indicate a heterogeneous and active microenvironment, influencing breast cancer outcome. Additionally, it would be pertinent to evaluate the predictive value of the histologic grade 2 considering somatic mutation profiles and distributions.

Follicular Fluid and Blood Monitorization of Infertility Biomarkers in Women with Endometriosis.

Infertility is recognized globally as a social disease and a growing medical condition, posing a significant challenge to modern reproductive health. Endometriosis, the third-most frequent gynecologic disorder, is one of the most common and intricate conditions that can lead to female infertility. Despite extensive research, the etiology, malignant transformation, and biological therapy of endometriosis remain unknown. Blood and follicular fluid are two matrices that have been carefully studied and can provide insights into women's health. These matrices are clinically significant because they contain metabolites closely associated with women's illness stage and reproductive outcomes. Nowadays, the application of metabolomic analysis in biological matrices may be able to predict the outcome of assisted reproductive technologies with greater precision. From a molecular viewpoint on reproductive health, we evaluate and compare the utilization of human follicular fluid and blood as matrices in analysis for diagnostic and assisted reproductive technology (ART) predictors of success for endometriosis patients. In the follicular fluid (FF), plasma, and serum of endometriosis-affected women, researchers identified dysregulations of oxidative stress, upregulation of several immune factors, and aberrations in energy metabolic pathways. The altered signatures negatively correlate with the overall oocyte and embryo quality and fertilization rate.

Association Between Follicle-Stimulating Hormone Receptor (FSHR) rs6166 and Estrogen Receptor 1 (ESR1) rs2234693 Polymorphisms and Polycystic Ovary Syndrome Risk, Phenotype, and Reproductive Outcomes in an Infertile Portuguese Population.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a common endocrine disorder often leading to anovulatory infertility. PCOS pathophysiology is still unclear and several potential genetic susceptibility factors have been proposed. The effect of polymorphisms in two genesrelated to follicular recruitment and development, the follicle-stimulating hormone receptor (FSHR) and the estrogen receptor 1 (ESR1), have been studied in different populations with contradictory results. AIMS: To evaluate the influence of FSHR rs6166 (c.2039A>G) and of ESR1 rs2234693 (Pvull c.453-397 T > C) polymorphisms on PCOS risk, phenotype, and response to controlled ovarian stimulation (COS). MATERIALS AND METHODS: Genotyping of the FSHR rs6166 and the ESR1 rs2234693 polymorphisms was performed in PCOS women and a control group undergoing in vitro fertilization (IVF). Demographic, clinical, and biochemical data, genotype frequency, and IVF outcomes were compared between groups. RESULTS: We evaluated 88 PCOS women and 80 controls. There was no significant difference in the genotype distribution of FSHR rs6166 polymorphism between PCOS women and controls (AA 31.8%/AS 48.9%/SS 19.3% in PCOS women vs AA 37.5%/AS 40.0%/SS 22.5% in controls; p = 0.522). The same was true for the ESR1 rs2234693 (CC 24.1%/CT 46.0%/TT 29.9% in PCOS women vs CC 18.8%/CT 48.8%/TT 32.5% in controls; p = 0.697). In PCOS women, we found higher follicle-stimulating hormone (FSH) levels on the third day of the menstrual cycle associated with the SS variant of the FSHR polymorphism (9.2 vs 6.2 ± 1.6 and 5.6 ± 1.6 mUI/mL; p = 0.011). We did not find other associations between the baseline hormonal parameters, antral follicle count, and response measures to COS with FSHR or ESR1 genotypes. We found, however, a need for higher cumulative doses of FSH for COS in patients with the SS variant of the FSHR rs6166 polymorphism (1860.5 ± 627.8 IU for SSvs 1498.1 ± 359.3 for AA and 1425.4 ± 474.8 for SA; p = 0.046 and p = 0.046). CONCLUSION: Our data suggest that in the population, FSHR rs6166and ESR1 rs2234693 polymorphisms do not influence the risk of developing PCOS nor do they influence the patient's phenotype and IVF success. However, the SS variant of the FSHR rs6166 polymorphism may be associated with FSH resistance requiring higher FSH doses for COS.

CYP19A1 TC/CC Polymorphism, along with Deletion of GSTM1 and GSTT1 Genes, Strongly Influences Female Infertility Risk.

Oxidative stress has a fundamental role in the pathophysiology of various conditions, like infertility. This case-control study was performed to assess the potential role of CYP19A1, GSTM1, and GSTT1 in modifying individual predisposition to female infertility. Genotyping of 201 women with established infertility and 161 fertile female controls was performed, and statistical associations were analyzed. For carriers of GSTM1 null genotype along with CYP19A1 C allele, there is a significant association with female infertility risk (OR 7.023; 95% CI (3.627-13.601; p < 0.001), and, also for carriers of GSTT1 null genotype along with the CYP19A1 TC/CC genotype (OR 24.150; 95% CI (11.148-52.317; p < 0.001). A positive association with female infertility risk for carriers of the C allele in CYP19A1 and null genotypes in GTSM1 (OR 11.979; 95% CI (4.570-31.400; p < 0.001) or GSTT1 (OR 13.169; 95% CI (4.518-38.380; p < 0.001) was found. When both GSTs are deleted, the risk of developing female infertility is significant, independently of the CYP19A1 genotype; when all the presumed high-risk genotypes are present, we found a significant association with female infertility risk (OR 47,914; 95% CI (14,051-163,393; p < 0.001).

Cell-Free DNA as a New Biomarker of IVF Success, Independent of Any Infertility Factor, Including Endometriosis.

Cell-free DNA fragments detected in blood and in other biological fluids are released from apoptotic/necrotic cells. In this study, we analyzed cfDNA levels in follicular fluid (FF) samples from patients with infertility. Samples were collected from 178 infertile women and cfDNA was extracted and quantified by qPCR, using ALU115 and ALU247 primers, and statistical correlations were performed. We found that cfDNA concentration was significantly higher in FF pools from women aged 35 and over than in women under 35 years of age (p = 0.017). We also found that q247 cfDNA levels were significantly higher in women with an associated female factor, such as endometriosis, PCOS and POF, compared with women with no specific cause of infertility (p = 0.033). The concentration of cfDNA did not vary significantly in each group of women with an associated female factor. The concentration of cfDNA was significantly higher in the FF of women that obtained embryos with a high fragmentation rate, compared to embryos with a low fragmentation rate (p = 0.007). Finally, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels (p = 0.043). The quantification of cfDNA could be an important biomarker of follicular micro-environment quality to predict embryo quality and the success of IVF, making them more specific and effective.

Volatilomics as an Emerging Strategy to Determine Potential Biomarkers of Female Infertility: A Pilot Study.

Due to its high prevalence, infertility has become a prominent public health issue, posing a significant challenge to modern reproductive medicine. Some clinical conditions that lead to female infertility include polycystic ovary syndrome (PCOS), endometriosis, and premature ovarian failure (POF). Follicular fluid (FF) is the biological matrix that has the most contact with the oocyte and can, therefore, be used as a predictor of its quality. Volatilomics has emerged as a non-invasive, straightforward, affordable, and simple method for characterizing various diseases and determining the effectiveness of their current therapies. In order to find potential biomarkers of infertility, this study set out to determine the volatomic pattern of the follicular fluid from patients with PCOS, endometriosis, and POF. The chromatographic data integration was performed through solid-phase microextraction (SPME), followed by gas chromatography-mass spectrometry (GC-MS). The findings pointed to specific metabolite patterns as potential biomarkers for the studied diseases. These open the door for further research into the relevant metabolomic pathways to enhance infertility knowledge and diagnostic tools. An extended investigation may, however, produce a new mechanistic understanding of the pathophysiology of the diseases.

Follicular Fluid: A Powerful Tool for the Understanding and Diagnosis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) represents one of the leading causes of anovulatory infertility and affects 5% to 20% of women worldwide. Until today, both the subsequent etiology and pathophysiology of PCOS remain unclear, and patients with PCOS that undergo assisted reproductive techniques (ART) might present a poor to exaggerated response, low oocyte quality, ovarian hyperstimulation syndrome, as well as changes in the follicular fluid metabolites pattern. These abnormalities originate a decrease of Metaphase II (MII) oocytes and decreased rates for fertilization, cleavage, implantation, blastocyst conversion, poor egg to follicle ratio, and increased miscarriages. Focus on obtaining high-quality embryos has been taken into more consideration over the years. Nowadays, the use of metabolomic analysis in the quantification of proteins and peptides in biological matrices might predict, with more accuracy, the success in assisted reproductive technology. In this article, we review the use of human follicular fluid as the matrix in metabolomic analysis for diagnostic and ART predictor of success for PCOS patients.

Influence of Estrogenic Metabolic Pathway Genes Polymorphisms on Postmenopausal Breast Cancer Risk.

Estrogen metabolism plays an important role in tumor initiation and development. Lifetime exposure to high estrogens levels and deregulation of enzymes involved in estrogen biosynthetic and metabolic pathway are considered risk factors for breast cancer. The present study aimed to evaluate the impact of mutations acquisition during the lifetime in low penetrance genes that codify enzymes responsible for estrogen detoxification. Genotype analysis of GSTM1 and GSTT1 null polymorphisms, CYP1B1 Val432Leu and MTHFR C677T polymorphisms was performed in 157 samples of women with hormone-dependent breast cancer and correlated with the age at diagnosis. The majority of patients with GSTT1 null genotype and with both GSTM1 and GSTT1 null genotypes were 50 years old or more at the diagnosis (p-value = 0.021 and 0.018, respectively). Older women with GSTM1 null genotype were also carriers of the CYP1B1Val allele (p-value = 0.012). As well, GSTT1 null and CYP1B1Val genotypes were correlated with diagnosis at later ages (p-value = 0.022). Similar results were found associating MTHFR C677T and GSTT1 null polymorphism (p-value = 0.034). Our results suggest that estrogen metabolic pathway polymorphisms constitute a factor to be considered simultaneously with models for breast cancer risk assessment.

Ethics of DNA Vaccine Transfer for Clinical Research.

Several experimental human DNA vaccines are currently undergoing Phase I, II, and III clinical trials in order to investigate their efficacy and safety. Human clinical trials must follow guidelines and procedures that have been approved by the regulatory authorities and ethics committees. Ethical clinical research is much more than applying an informed consent to participants. In this chapter we will review the ethical standards and provide a framework to evaluate and design ethical clinical research. Despite being universal standards supported by universal guidelines, they must be adapted to the conditions in each country where the clinical research is being conducted.

Preparation of an Academic Clinical Trial.

Clinical trials are research studies performed in humans to evaluate the efficacy and safety of an intervention. They are the primary method by which researchers discover if a new treatment (drug, diet, medical device) is safe and effective in humans. DNA vaccines are considered, by definition, advanced therapy medicinal products (ATMPs). ATMPs are medicines for human use that are based on genes, tissues, or cells. They offer groundbreaking new opportunities for the treatment of disease and injury. Clinical trials using ATMPs are subject to specific regulatory requirements. This chapter will describe the most important steps when planning a clinical trial with DNA vaccines, such as regulatory and submission requirements, designing of a successful clinical trial protocol, stakeholders' responsibilities, and feasibility assessment.

Women with polycystic ovary syndrome and other causes of infertility have a higher prevalence of GSTT1 deletion.

RESEARCH QUESTION: Is GSTM1 and GSTT1 deletion associated with the development of polycystic ovary syndrome (PCOS)? DESIGN: A case-control study was designed to investigate the association between GSTM1 and GSTT1 gene polymorphisms with PCOS. Blood samples from 201 women diagnosed with infertility were taken, of which 69 women were diagnosed with PCOS. Genomic DNA was extracted, and genotyping analyses were conducted by polymerase chain reaction-based methods. Odds ratios and 95% confidence intervals were calculated by unconditional logistic regression. RESULTS: An increased risk of PCOS was found to be associated with GSTT1 null genotype (OR 4.890, 95% CI 2.261 to 9.122; P < 0.001). A strong association between GSTT1 null genotype was found with female infertility, regardless of the associated cause (OR 5.300, 95% CI 3.238 to 8.675; P < 0.001) as well as with the GSTM1 null genotype (OR 1.620, 95% CI 1.067 to 2.459; P = 0.026). A statistically significant association with the development of infertility was also found when carriers of the combined genotype GSTT1+/GSTM1+ was compared with carriers of the combined genotype GSTT1-/ GSTM1+ (OR 3.600 95% CI 1.864 to 6.956; P < 0.001). The two-way combination of GSTT1 and GSTM1 null genotypes resulted in an increased susceptibility to infertility development (OR 11.136; 95% CI 5.035 to 24.629; P < 0.001). CONCLUSIONS: Carriers of GSTT1 null genotype seem to have higher susceptibility to developing PCOS and infertility from other causes. Also, GSTT1 null genotype, alone or in association, are related with increased susceptibility to infertility development, independently of its cause. GSTM1 null genotype is only associated with all cause of infertility when the GSTT1 is null.

The prognostic value of NRF2 in breast cancer patients: a systematic review with meta-analysis.

PURPOSE: Nuclear factor E2-related factor 2 (NRF2) is a transcription factor that plays a major role in the regulation of intracellular antioxidant response. The effect of NRF2 overexpression in many malignancies is still unclear and recent meta-analysis correlated NRF2 overexpression with poor prognosis in a variety of human cancers. However, the effect of NRF2 overexpression in breast cancer is still unclear. Thus, the main goal of this work was to clarify the role of NRF2 expression in survival and relapse of breast cancer patients by performing a systematic review according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) statement, followed by a meta-analysis. METHODS: The electronic search was conducted in PubMed, Scopus, SciELO, Web of Science and Embase between November of 2017 and September of 2018. To be included, studies should evaluate NRF2 expression in breast cancer tissue, through immunohistochemistry and/or mRNA and had to report one or more of the following outcomes: overall survival (OS), disease-free survival (DFS), mean survival and median survival. RESULTS: For the meta-analysis, seven studies were included and NRF2 expression was correlated with OS and DFS. It was observed that compared to patients with low NRF2 expression, patients with NRF2 overexpression had poorer OS with a hazard ratio of 1.82 (95% CI 1.32-2.50; p value < 0.0001), and poorer DFS, with a hazard ratio of 1.79 (95% CI 1.07-3.01; p value = 0.03). CONCLUSIONS: These results suggest that tumours that overexpress NRF2 have a worse clinical outcome. Thus, NRF2 expression could be a marker for the prognostic of breast cancer patients and, in the future, it would be pertinent to focus on improving treatment efficacy for patients with NRF2 overexpression.

Prognosis of hormone-dependent breast cancer seems to be influenced by KEAP1, NRF2 and GSTM1 genetic polymorphisms.

Influence of Glutathione S-transferase Mu1 (GSTM1) has long been studied in breast cancer and GSTM1 null genotype was correlated with breast cancer risk. Nuclear factor-erythroid 2-related factor-2 (NRF2) is a transcription factor that forms a complex with Kelch-like ECH-associated protein-1 (KEAP1). Recent studies have demonstrated that expression of these proteins is deregulated in several malignancies. Thus, in the present study we aim to distinguish GSTM1 heterozygous from wild type genotype in breast cancer patients and evaluate the presence and clinical significance of NRF2 and KEAP1 polymorphisms, alone or in association, with breast cancer prognosis, in cases confirmed to have GSTM1-present genotype. Study population consisted in 52 patients with breast cancer. Genomic DNA was extracted, GSTM1 was genotyped through multiplex PCR and gene dose was evaluated through real-time PCR. All cases were sequenced, through Sanger sequencing, for specific regions of NRF2 and KEAP1. Genotyping and clinicopathological data were correlated and statistical analysis was performed. GSTM1 wild type was identified in 1 case and 26 cases were identified as heterozygous, these data were correlated with Human Epidermal growth factor Receptor 2 (HER2) status (p value = 0.017). We also verified that most cancers diagnosed at younger ages had the presence of KEAP1 and/or NRF2 polymorphisms. The association of GSTM1 heterozygous genotype with rs1048290 and rs35652124 seems to be associated with HER2+ (p < 0.05). Our results suggest that GSTM1 * 1/0 genotype and the cumulative presence of at least one allele mutated in KEAP1 and/or NRF2 polymorphisms might be associated with worse prognosis for breast cancer patients.

Positive association of polymorphisms in estrogen biosynthesis gene, CYP19A1, and metabolism, GST, in breast cancer susceptibility.

PURPOSE: This case-control study was conducted in order to evaluate the potential role of polymorphic genes encoding enzymes involved in estrogen biosynthesis (CYP19A1) and metabolism (GSTM1, GSTT1, and GSTP1), and their action in modulating individual susceptibility to breast cancer. METHODS: Genomic DNA was extracted from blood samples of 101 patients with histological diagnosis of breast cancer and 121 healthy women. Genotyping analyses of CYP19A1 codon 39 Trp/Arg (T/C), GSTM1 and GSTT1 homozygous deletions, and GSTP1 codon 105 Ile/Val (A/G) were performed by polymerase chain reaction-based methods. RESULTS: Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression. Significant statistical association of the TC/CC genotypes combined with breast cancer risk was found, with reference to TT genotype (OR=1.770; 95% CI=1.036-3.024; p=0.036). Also, CYP19A1 arginine allele in homozygosity or heterozygosity (TC/CC) was associated with a significant increased risk for breast cancer when associated to GSTM1 null genotype (OR=6.158; 95% CI=2.676-14.171; p<0.001) and GSTT1 null genotype (OR=4.870; 95% CI=2.216-10.700; p<0.001). The three-way combination of CYP19A1 TC/CC, GSTM1 null, and GSTT1 null polymorphism was related with significant increased risk for breast cancer (OR=11.429; 95% CI=3.590-36.385; p<0.001). Valine alleles compared with isoleucine alleles in codon 105 in GSTP1, in combination with CYP19A1 genotypes, were not associated with an increase of breast cancer development. CONCLUSIONS: Our results suggest that the effect of CYP19A1 T/C polymorphism in susceptibility to breast cancer development can be modulated by the presence of GSTM1 and GSTT1, but not GSTP1.

Glutathione S-transferase M1, T1, and P1 genotypes and breast cancer risk: a study in a Portuguese population.

Glutathione S-transferases are a superfamily of multifunctional enzymes that play a key role in Phase II metabolism, detoxifying therapeutic drugs, and various carcinogens by conjugation with glutathione. We undertook a case-control study in Central-Eastern Portuguese population to evaluate the association of null genotype in GSTM1 and GSTT1 along with the polymorphism in GSTP1 (A/G) and susceptibility to breast cancer. The population sample consisted of 85 patients with histological diagnosis of breast cancer and 102 healthy women. Genomic DNA was extracted from blood samples, and genotyping analyses were performed by PCR-based methods. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression. We found a increased breast cancer risk associated with GSTM1 null genotype (OR = 3.597; 95% CI = 1.849-6.999; P = 0.0001) and GSTT1 (OR = 2.592; 95% CI = 1.432-4.690; P = 0.002), but the presence of valine alleles compared to isoleucine alleles in codon 105 in GSTP1 did not increase the risk of breast cancer development. The two-way combination of GSTM1 and GTTT1 null genotypes resulted in 8-fold increase for breast cancer risk (OR = 8.287; 95% CI = 3.124-21.980; P = 0.0001) and the three-way combination of GSTP1 105AA/AG and null genotypes for both GSTM1 and GSTT1 resulted in 5-fold increase for breast cancer risk (OR = 5.040; 95% CI = 1.392-18.248; P = 0.016). Our results suggest that GSTM1 and GSTT1 null genotype alone, both combined or combined with GSTP1 valine alleles, are associated with higher susceptibility to breast cancer development.