Association of breast and gut microbiota dysbiosis and the risk of breast cancer: a case-control clinical study.

BACKGROUND: Breast cancer ranks first in women, and is the second cause of death in this gender. In addition to genetics, the environment contributes to the development of the disease, although the factors involved are not well known. Among the latter is the influence of microorganisms and, therefore, attention is recently being paid to the mammary microbiota. We hypothesize that the risk of breast cancer could be associated with the composition and functionality of the mammary/gut microbiota, and that exposure to environmental contaminants (endocrine disruptors, EDCs) might contribute to alter these microbiota. METHODS: We describe a case-control clinical study that will be performed in women between 25 and 70 years of age. Cases will be women diagnosed and surgically intervened of breast cancer (stages I and II). Women with antecedents of cancer or advanced tumor stage (metastasis), or who have received antibiotic treatment within a period of 3 months prior to recruitment, or any neoadjuvant therapy, will be excluded. Controls will be women surgically intervened of breast augmentation or reduction. Women with oncological, gynecological or endocrine history, and those who have received antibiotic treatment within a period of 3 months prior to recruitment will also be excluded. Blood, urine, breast tissue and stool samples will be collected. Data regarding anthropometric, sociodemographic, reproductive history, tumor features and dietary habits will be gathered. Metabolomic studies will be carried out in stool and breast tissue samples. Metagenomic studies will also be performed in stool and breast tissue samples to ascertain the viral, fungal, bacterial and archaea populations of the microbiota. Quantitation of estrogens, estrogen metabolites and EDCs in samples of serum, urine and breast tissue will also be performed. DISCUSSION: This is the first time that the contribution of bacteria, archaea, viruses and fungi together with their alteration by environmental contaminants to the risk of breast cancer will be evaluated in the same study. Results obtained could contribute to elucidate risk factors, improve the prognosis, as well as to propose novel intervention studies in this disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT03885648 , 03/25/2019. Retrospectively registered.

Analysis of population characteristics related to the total effective xenoestrogen burden: a biomarker of xenoestrogen exposure in breast cancer.

To analyse the link between breast cancer and the combined effect of environmental xenoestrogens, we developed, standardised and applied a biomarker of exposure to assess the total effective xenoestrogen burden (TEXB) in human adipose tissue in a case-control study. Environmental oestrogens (TEXB-alpha) are separated from endogenous oestrogens (TEXB-beta), and the combined oestrogenic effect is determined from its proliferative effect (E-Screen assay). The aim of the study was to identify potential confounders, effect modifiers or other covariates associated with higher TEXB levels. In cases, age, family history of breast cancer, lactation experience and smoking were associated with TEXB-alpha. In controls, only age was associated with TEXB-alpha levels. In cases, age, educational level, age at menarche, menopausal status, marital status, lactation experience and smoking were associated with TEXB-beta. In controls, only menopausal status was significantly associated with TEXB-beta levels. In conclusion, TEXB, as a biomarker of exposure, takes account of environmental, dietary, lifestyle, genetic and reproductive factors, which are not usually systematically measured across studies.

Biomonitoring of environmental estrogens in human tissues.

Two examples are presented for the application of the total effective xenoestrogen burden as biomarker of chemical exposure measured in tissue samples from patients recruited for two case-control studies. The first study focused on environmental chemicals with hormone mimicking activity, the so-called environmental estrogens, and their participation in the etiology of breast cancer. The second study investigated mother-child exposure to persistent organochlorine chemicals and assessed their combined effect on the risk of male urogenital malformations in the newborn.

Valproic acid modulates radiation-enhanced matrix metalloproteinase activity and invasion of breast cancer cells.

PURPOSE: To evaluate matrix metalloproteinase (MMP) activity and invasion after ionizing radiation (IR) exposure and to determine whether MMP could be epigenetically modulated by histone deacetylase (HDAC) inhibition. MATERIAL AND METHODS: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were cultured in monolayer (2D) and in laminin-rich extracellular matrix (3D). Invasion capability, collagenolytic and gelatinolytic activity, MMP and TIMP protein and mRNA expression and clonogenic survival were analyzed after IR exposure, with and without a HDAC inhibition treatment [1.5 mM valproic acid (VA) or 1 µM trichostatin-A (TSA)]. RESULTS: IR exposure resulted in cell line-dependent stimulation of invasion capacity. In contrast to MCF-7 cells, irradiated MDA-MB-231 showed significantly enhanced mRNA expression of mmp-1, mmp-3 and mmp-13 and of their regulators timp-1 and timp-2 relative to unirradiated controls. This translated into increased collagenolytic and gelatinolytic activity and could be reduced after valproic acid (VA) treatment. Additionally, VA also mitigated IR-enhanced mmp and timp mRNA expression as well as IR-increased invasion capability. Finally, our data confirm the radiosensitizing effect of VA. CONCLUSION: These results suggest that IR cell line-dependently induces upregulation of MMP mRNA expression, which appears to be mechanistically linked to a higher invasion capability that is modifiable by HDAC inhibition.

[Disease-free survival related factors in breast cancer]. / Factores relacionados con la supervivencia libre de enfermedad en el cáncer de mama.

OBJECTIVE: To evaluate the relationship between the clinical and pathological parameters of the primary tumor and disease-free survival (DFS) in a sample of hospital cases of invasive breast cancer. MATERIAL AND METHOD: We performed a retrospective cohort study in 635 patients recruited at San Cecilio University Hospital in Granada (Spain) between 1994 and 2006. Information on the primary tumor and the outcomes of patients was collected by reviewing the medical records. Predictors of recurrence and/or metastasis and DFS (follow up of 3, 5 and 10 years) were analyzed by using Cox regression analysis. RESULTS: Multivariate models adjusted for age, tumor size, lymph nodal status, histological grade and estrogen and progesterone receptor expression showed a higher risk of recurrence and/or metastasis and lower DFS (adjusted relative risk, 95% confidence intervals) with tumor size (3 yrs: 3.00, 1.79-5.03; 5 yrs: 2.56, 1.65-3.98; 10 yrs: 2.16, 1.44-3.24), lymph nodal status (3 yrs: 4.58, 2.42-8.65; 5 yrs: 3.84, 2.35-6.30; 10 yrs: 3.08, 2.05-4.61), lymphovascular invasion (5 yrs: 1.88, 1.16-3.04; 10 yrs: 2.19, 1.43-3.35), multifocal and/or multicenter tumors (3 yrs: 2.69, 1.46-4.96; 5 yrs: 1.90, 1.08-3.35) and p53 protein expression (3 yrs: 2.03, 1.00-4.09). DFS was positively associated with an increased expression of progesterone receptor (3 yr: 0.48, 0.26-0.89; 5 yrs: 0.58, 0.35-0.97; 10 yrs: 0.59, 0.38-0.90). CONCLUSIONS: The biological characteristics of the primary tumor can be used to identify patients with distinctive prognoses and DFS, and could be helpful in making individual follow up strategies.

Assessment of estrogenic and anti-androgenic activities of the mycotoxin zearalenone and its metabolites using in vitro receptor-specific bioassays.

Zearalenone (ZEN) is a well-known mycotoxin present in numerous agricultural products. Humans and animals are therefore at a risk of exposure to zearalenone through consumption of contaminated food. After intake, ZEN is reduced to α- and ß-zearalenol (α-ZEL and ß-ZEL), zearalanone (ZAN), and α- and ß-zearalanol (α-ZAL and ß-ZAL). Although their estrogenicity has been well characterized, much less is known about their interaction with other nuclear receptors. This study was undertaken to investigate interactions of ZEN and its five metabolites, with the human androgen receptor (hAR) and estrogen receptor alpha (hERα). Their ability to induce hAR-mediated reporter gene expression was examined in androgen-sensitive PALM cells, whereas the effects on hERα function were assessed in MCF-7 cells using the E-Screen bioassay. We confirm that ZEN and its metabolites are full agonists for hERα and demonstrate that all six compounds tested possess hAR-mediated antagonistic activity in PALM cells, in which ZAN, α-ZAL, and ß-ZAL were the most effective hAR antagonists. Overall, the observed estrogenic and anti-androgenic potencies of ZEN and its metabolites suggest that these compounds may interfere with the endocrine system by various modes of action and that further investigation is warranted into their role as endocrine disrupters in animals and humans.

Fiabilidad de un examen clínico objetivo estructurado para predecir la futura competencia profesional de estudiantes de medicina / Reliability of an objective structured clinical examination to assess the future professional competence of medical students

Introducción: Cuando con los resultados de un examen clínico objetivo estructurado (ECOE) se decide sobre la futura competencia profesional de estudiantes de medicina, la fiabilidad de dicha prueba debe adecuarse a esta finalidad. Objetivo: Calcular la fiabilidad (alfa de Cronbach) de una serie de ECOE y su relación con la duración, número de participantes, estaciones, ítems y evaluadores. Sujetos y métodos: Se analizan 14 ECOE realizados a 2.995 estudiantes de cuarto y quinto curso de la Facultad de Medicina de Granada desde 2004 a 2013. Resultados: La fiabilidad fue ≥ 0,70 en el 92,84% de los ECOE. También fue significativamente ≥ 0,70 cuando la duración total fue ≥ 60 minutos (p = 0,042), el número de estaciones ≥ 10 (p = 0019), el número de ítems ≥ 50 (p = 0,018) y el número de evaluadores ≥ 6 (p = 0,018). No se observaron diferencias con el número de estudiantes ni con las opciones al ítem utilizadas. Conclusiones: Los ECOE cuyos resultados se utilicen para aprobar asignaturas de la carrera de medicina deben tener una fiabilidad ≥ 0,70. Para alcanzar dicha fiabilidad o mayor, el formato debe constar de al menos 10 estaciones, durar ≥ 60 minutos, tener ≥ 50 ítems y ≥ 6 evaluadores

Introduction: When the future professional competence of medical students is decided based on results of an objective structured clinical examination (OSCE), the reliability of this test should be adequate to this purpose. Aim: To calculate the reliability (Cronbach's alpha) of each one of OSCEs we performed and its relationship with the duration, number of participants, stations, items and evaluators. Subjects and methods: Fourteen OSCE tests performed to 2995 medical students of 4th and 5th year of the Faculty of Medicine of Granada between 2004 to 2013 were analyzed. Results: The reliability was ≥ 0.70 in 92.84% of the OSCEs. It was also significant ≥ 0.70 with a total duration ≥ 60 minutes (p = 0.042), and a number of stations ≥ 10 (p = 0.019), a number of items ≥ 50 (p = 0.018) and a number of evaluators ≥ 6 (p = 0.018). No differences with the number of students, neither with the options to the item were observed. Conclusions: The OSCEs carried out in centers which results are used to approve subjects of the medical career, must have a reliability ≥ 0.70. To achieve this reliability or greater, the format should consist of at least: 10 stations, a duration ≥ 60 minutes, and having ≥ 50 items and ≥ 6 evaluators

Gene expression signatures in breast cancer distinguish phenotype characteristics, histologic subtypes, and tumor invasiveness.

BACKGROUND: The development of reliable gene expression profiling technology is having an increasing impact on the understanding of breast cancer biology. METHODS: In this study, microarray analysis was performed to establish gene signatures for different breast cancer phenotypes, to determine differentially expressed gene sequences at different stages of the disease, and to identify sequences with biologic significance for tumor progression. Samples were taken from patients before their treatment. After microarray analysis, the expression level of 153 selected genes was studied by real-time quantitative polymerase chain reaction analysis. RESULTS: Several gene sequences were expressed differentially in tumor samples versus control samples and also were associated with different breast cancer phenotypes, estrogen receptor status, tumor histology, and grade of tumor differentiation. In lymph node-negative tumors were identified a set of genes related to tumor differentiation grade. CONCLUSIONS: Several differentially expressed gene sequences were identified at different stages of breast cancer.

Factores relacionados con la supervivencia libre de enfermedad en el cáncer de mama / Disease-free survival related factors in breast cancer

Objetivo: Evaluar la relación entre los parámetros clínicos y anatomopatológicos del tumor primario y la supervivencia libre de enfermedad (SLE) en una serie hospitalaria de casos de cáncer de mama invasivo. Material y método: Estudio de cohortes retrospectivo con 635 pacientes diagnosticadas en el Hospital Universitario San Cecilio de Granada entre 1994 y 2006. La información relativa al tumor primario y a la evolución de la enfermedad se recogió mediante revisión de historias clínicas. La identificación de factores relacionados con el riesgo de recidiva y/o metástasis, y la SLE se realizó, a corto (3 y 5 años) y medio plazo (10 años), mediante análisis de regresión de Cox. Resultados: Tras ajustar por edad, tamaño tumoral, afectación ganglionar, grado histológico y expresión de receptores de estrógenos y de progesterona, se relacionan con mayor riesgo de recaída y menor SLE: el tamaño tumoral (3 años: riesgo relativo ajustado 3,00, intervalo de confianza del 95% 1,79-5,03; 5 años: 2,56, 1,65-3,98; 10 años: 2,16, 1,44-3,24), la infiltración ganglionar (3 años: 4,58, 2,42-8,65; 5 años: 3,84, 2,35-6,30; 10 años: 3,08, 2,05-4,61), la invasión linfovascular (5 años: 1,88, 1,16-3,04; 10 años: 2,19, 1,43-3,35), la multifocalidad/multicentricidad (3 años: 2,69, 1,46-4,96; 5 años: 1,90, 1,08-3,35) y p53 (3 años: 2,03, 1,00-4,09). Se relacionan con mayor SLE, la expresión de receptores de progesterona (3 años: 0,48, 0,26-0,89; 5 años: 0,58, 0,35-0,97; 10 años: 0,59, 0,38-0,90). Conclusiones: Las características biológicas del tumor primario permiten identificar pacientes con diferente pronóstico y SLE, pudiendo contribuir a la planificación de estrategias de seguimiento más personalizadas (AU)

Objective: To evaluate the relationship between the clinical and pathological parameters of the primary tumor and disease-free survival (DFS) in a sample of hospital cases of invasive breast cancer. Material and method: We performed a retrospective cohort study in 635 patients recruited at San Cecilio University Hospital in Granada (Spain) between 1994 and 2006. Information on the primary tumor and the outcomes of patients was collected by reviewing the medical records. Predictors of recurrence and/or metastasis and DFS (follow up of 3, 5 and 10 years) were analyzed by using Cox regression analysis. Results: Multivariate models adjusted for age, tumor size, lymph nodal status, histological grade and estrogen and progesterone receptor expression showed a higher risk of recurrence and/or metastasis and lower DFS (adjusted relative risk, 95% confidence intervals) with tumor size (3 yrs: 3.00, 1.79-5.03; 5 yrs: 2.56, 1.65-3.98; 10 yrs: 2.16, 1.44-3.24), lymph nodal status (3 yrs: 4.58, 2.42-8.65; 5 yrs: 3.84, 2.35-6.30; 10 yrs: 3.08, 2.05-4.61), lymphovascular invasion (5 yrs: 1.88, 1.16-3.04; 10 yrs: 2.19, 1.43-3.35), multifocal and/or multicenter tumors (3 yrs: 2.69, 1.46-4.96; 5 yrs: 1.90, 1.08-3.35) and p53 protein expression (3 yrs: 2.03, 1.00-4.09). DFS was positively associated with an increased expression of progesterone receptor (3 yr: 0.48, 0.26-0.89; 5 yrs: 0.58, 0.35-0.97; 10 yrs: 0.59, 0.38-0.90). Conclusions: The biological characteristics of the primary tumor can be used to identify patients with distinctive prognoses and DFS, and could be helpful in making individual follow up strategies (AU)

Breast cancer risk and the combined effect of environmental estrogens.

OBJECTIVE: The present study aimed to determine whether the combined effects of environmental estrogens measured as the total effective xenoestrogen burden (TEXB-alpha) are a risk factor for breast cancer over and above the risk potentially linked to specific pesticides. METHODS: We measured the levels of 16 organochlorine pesticides as well as TEXB in adipose tissue of 198 women at the time of breast cancer diagnosis. These were compared with findings in 260 age and hospital matched control women without breast cancer. RESULTS: The median levels of p,p'-DDE (1,1-dichloro-2,2-bis( p -chlorophenyl)ethylene), aldrin, endosulfan ether and lindane (the pesticides detected in > 40% of the study population) were higher in cases than controls, although the differences did not reach statistical significance. After adjusting for potential confounders, the odds ratio (OR) for breast cancer in women with detectable levels of aldrin was 1.55 (95% confidence interval (CI) 1.00-2.40). Among the postmenopausal women, the OR for aldrin and lindane was 1.84 (95% CI 1.06-3.18) and 1.76 (95% CI 1.04-2.98), respectively. Among cases with body mass index (BMI) below the median (28.6 kg/m2), the OR was 3.42 (95% CI 1.22-9.58) for women in the highest quartile of TEXB-alpha versus those in the lowest. The subgroup of leaner postmenopausal women showed an increased risk (OR: 5.67; 95% CI 1.59-20.21) for those in the highest tertile versus those in the lowest. CONCLUSIONS: We found an increased risk for breast cancer in the leaner women, especially in the leaner postmenopausal subgroup, related to the TEXB-alpha. The pesticides aldrin and lindane are also individually associated with risk.