Breast cancer among women is an increasing public health issue. Early detection is the backbone of treatment thus reducing late presentation, morbidity and mortality. In this study, we explored knowledge and use of breast cancer screening among women of reproductive age in Oyo State Secretariat, Ibadan.Descriptive cross-sectional research design was employed to gather data from 204 female workers who consented through purposive sampling technique. A validated semi-structured questionnaire with reliability coefficient of 0.75 was used. Ethical protocols were duly followed. Data were analysed using frequencies, percentages and chi-squared tests at significant level of 0.05 using the Statistical Package for Social Science version 20.Most of the respondents (82.2%) had adequate knowledge of breast self-examination (BSE) and mammogram, but there was a relatively lower level of utilization of these screening measures. Also, there were significant associations between knowledge, level of education and use of BSE and mammogram (p<0.05).In addition to educating women on BSE and mammogram, continuous efforts should be made through workshops and various media to emphasize the significance of practice. This could increase the use of the screening measures leading to a reduction in morbidity and mortality. About 1.67 million new cases were diagnosed and 0.522 million deaths from breast cancer occurred in 20121. Although breast cancer is rare in men, it has been estimated that 2470 men were diagnosed with breast cancer worldwide and approximately 460 die each year2. The prevalence is rising in most countries and is estimated to rise further over the next 20 years despite current efforts to prevent the disease3.The increased incidence is not surprising since there has been, in most countries, an increase in the number of women with major breast cancer risk factors. The risk factors for breast cancer include advancing age4, family history of breast cancer, early menarche, late menopause and the use of hormonal replacement therapy (HRT) with combined estrogen and progesterone5, late age of first pregnancy, few pregnancies, and short or no periods of breastfeeding. Other risk factors that could be implicated in the burden of breast cancer are obesity, alcohol consumption, and inactivity6.Breast cancer is the most frequent cancer that occurs among women worldwide, and the second leading cause of death, claiming the lives of thousands of women each year and affecting countries at all levels of development6. The earlier breast cancer is detected, the better the effectiveness of the treatment and the likelihood of survival. Breast cancer screening methods include BSE, Clinical Breast examination (CBE) and mammography, and these are usually done in combination7. Among these methods, mammography is the only one that has been proven to be effective, but the method is only cost-effective and feasible in countries with good health infrastructure8. BSE is the recommended method in developing countries because it is easy, convenient, private, safe and requires no specific equipment9. Its purpose is to make women familiar with both the appearance and feel of their breasts as early as possible, so that they will be able to easily detect changes in their breast. Several studies have revealed that a positive association exists between the performance of BSE and detection of breast cancer, as most of the early breast tumor detections have been self-discovered11.The control of breast cancer in most developing countries including Nigeria and Cameroon is under the auspices of national control programs promoted by the World Health Organization and this involves educating and screening young women for clinical manifestations of breast cancer13 affirm only 17% and 12% of women, respectively, were observed to perform BSE monthly. Such an observation has equally been noted among health personnel. For instance, a study conducted among nurses and midwives reported only 14% compliance14. As a result, most cases of breast cancer in women are diagnosed at an advanced stage due to late presentation. Although BSE is a simple, quick and cost-free procedure, it appears that many women either perform it incorrectly or not at all.BSE for the early detection of breast cancer is not often done by many women. Studies8. Combined results from randomized screening trials suggest that mammography reduces the risk of dying from breast cancer by 15–20%, and studies of modern mammography screening programs in Europe found that the risk of death from breast cancer was reduced by more than one-third15. Thus, knowledge and practice of breast cancer preventive measures and screening is therefore critical in the reduction of breast cancer morbidity and mortality.Early diagnosis of breast cancer has been clearly shown to reduce mortality and improve survival16. To plan critically needed breast cancer awareness and education programs that optimally address Nigerian women civil servants, healthcare professionals and planners must know the women’s current level of understanding. Therefore, this cross-sectional study in State Secretariat, Ibadan, was performed to explore public knowledge of breast self-examination (BSE) and mammogram amongst women workers in Oyo State.However, poor awareness and knowledge about breast cancer symptoms and screening methods have been previously reported by several different studiesThe main aim of this study was to assess the level of knowledge and use of breast self-examination and mammogram among women of reproductive age in Oyo State Secretariat, Ibadan, Oyo State. Other objectives included: 1) Assess the level of knowledge of breast cancer, its risk factors and prevention measures; 2) Assess level of utilization of mammogram and breast self-examination; and 3) Identify the perceived barriers to the use of BSE and mammogram.The study was a descriptive cross-sectional design that used a purposive sampling technique to select 204 consenting women from all specialities working in the State Secretariat, Agodi, Ibadan, to assess their views on breast cancer prevention actions (BSE and mammogram). Ethical approval was obtained from the Ethical Review Committee of the University of Ibadan, University College Hospital and from the Head of Service, Oyo State. With approval from the Head of Service, respondents’ consent was obtained, confidentiality of information was ensured and participants were not coerced to participate in the study and free to withdraw their participation at any stage of the study if they so desired without any negative consequences.17. The calculated sample size was 184. Adjusting the sample size for 10% attrition, brought it to 204. Purposive sampling technique was used to select all participants who met the eligibility criteria.The subjects for the study were women workers of reproductive age (20–62 years) in Oyo State Secretariat, Agodi, Ibadan. The total number of female workers in Oyo State Secretariat were 340 and the sample size for this study was determined using the Yamane formulaData were collected using a structured validated close-ended questionnaire. The questionnaire consisted of five sections. Section A consisted of sociodemographic data. Section B asked questions on knowledge of breast cancer, its risk factors, signs and symptoms and preventive measures. Section C was on questions related to knowledge of BSE and mammogram. Section D was on level of utilization of mammogram and self-breast examination. Section E was concerned with barriers to the use of breast self-examination and mammogram.The instrument was presented to research experts to ensure validity, while reliability was determined using a test-retest method among 10 female workers that were picked randomly from outside the study centre and yielded a coefficient of 0.75. The researchers administered the questionnaire to the respondents face-to-face and collected them after they were completed.Data obtained were coded and entered into a spread-sheet. Analysis was performed using the Statistical Package for Social Science, version 20. Descriptive statistics such as frequency counts, percentages, mean and standard deviation were used to summarize and present the results. Student’s t-test was used to compare the categorical variables at p<0.05. Logistics regression was used to identify awareness and level of utilization of breast self-examination and mammogram among female workers in State Secretariat, Oyo State, at 0.05 level significance.The 204 women who participated in the study had their questionnaires checked to ensure completion before retrieval. The return rate was 100%.Of the respondents, 40.7% were in the age group 20–32 years . Ethnic Data on knowledge and risk of breast cancer revealed that 192 (94.1%) respondents heard about breast cancer, while 12 (5.9%), claimed they never heard of it. The sources of the 192 respondents’ information were: health workers , newspapers , parents , and social media . On the risk factors for breast cancer, 72 (37.5%) respondents reported genetics 23 (12.1%), diet 36 (18.6%), while others gave metaphysical reasons. On the symptoms, the majority 170 (88.6%) claimed a lump in the breast, change in breast shape 11 (5.7%) and shortness of breath. Genetic mutations were the most identified risk factor for breast cancer; 59 (30.7%) respondents claimed age, while 13 (6.8%) felt it resulted from post-menopausal hormone therapy.The response of knowledge about the risk factors, causes, and symptoms of breast cancer showed that 17.2% (n=35) of the study population had poor knowledge of breast cancer and its risk factors. The cause, symptoms and risk factors of breast cancer were known by 82.8% (n=169) of the respondents. This means that the vast majority of women in the study had good knowledge of breast cancer. They were women who scored above the average of the total score, while those who scored below the average were women with poor knowledge (17.2%).On the barriers to using the mammogram and self-breast examination, 193 (94.6%) respondents maintained that breast cancer screening is time consuming. Fear of exposure to radiation discouraged 180 (88.2%) from doing mammography, while 122 (59.8%) claimed fear of the outcome of the mammogram and breast self-screening. Another 133 (65.2%) claimed they did not understand the process of breast self-examination and 168 (82.4%) suggested that they needed to know more about breast cancer screening.19, as well as in Turkey20. However, this level of knowledge is in contrast to other studies conducted in Turkey, where the majority of the women had little knowledge21, and among minorities in the United State23. Poor knowledge of the risk factors was also noted by studies in Nigeria24 and in the Middle East27.The high level of knowledge demonstrated by the respondents is in agreement with studies in similar settings30 while social media have also been identified as sources31. This view is also held by studies in developed countries33. However, information from family members and friends could be unreliable, thereby leading to fears and poor compliance. It is therefore important that oncology related health workers be trained in giving information to clients in various settings. This will be more correct, increase knowledge, improve use and reduce morbidity.Health workers and family members were the major sources of information on screening and knowledge of breast cancer according to researchers in similar settings34, but in contrast with a study in a different setting33. Similarly, the participants’ knowledge of the value of mammography was supported by other studies. A Nigerian study noted that poor knowledge among their participants about mammography being an early detection measure24 was due to the fact that it was not readily available in a rural setting34. This could be attributed to the socioeconomic condition experienced in the countryside, especially among rural dwellers, where there is poor access, geographically and financially. Most of the rural dwellers are farmers with poor educational background that reduces their ability to access relevant information31. Also poor access roads prevent the sale of farm produce resulting in limited income, which further compounds the issue of knowledge about and travelling for a mammography. Improving accessibility to the rural areas could improve flow of information and increase knowledge of the value of a mammogram.The fact that majority of the respondents were aware of the value of BSE in early detection of breast cancer and as a non-invasive procedure is in the right direction, in line with results from another study30. However, an earlier study in the region found higher proportions of the practice of BSE but poor adherence to the process32. This could be attributed to increased awareness over the years, which led to more exposure but with no corresponding level of utilization. This suggests that the issue of wider utilization requires more than awareness and should be addressed by examining other sociocultural factors.Findings from this study revealed low utilization of BSE and mammogram despite the high level of knowledge, in line with other studies35. Also, the reported issue of not having enough time could be attributed to the long waiting experienced in most federal and state centers that offer screening services. The implication of this is that follow-up appointments should be streamlined in order to reduce waiting time and enhance compliance.Among factors that constituted barriers were views of low risk and fear of having abnormal results; this has been corroborated by findings that views of not being at risk, fear, and anxiety of abnormal results are major barriers37. A woman with a higher level of education is more likely to report promptly to the hospital for proper examination if any symptom suggestive of cancer is seen or felt; although earlier studies reported that educated women often forget to practise BSE.The issue of clinical and risk factors being significantly linked to respondents’ knowledge and educational level is in line with other studiesThere is a need to educate women on BSE and mammography on the significance of the practice and use of these methods for the early detection of breast cancer. Nurses and midwives should organize training sessions, especially on BSE, so that women can practise it on their own.The research project aimed at assessing the knowledge and use of BSE and mammogram among women of reproductive age in Oyo State Secretariat, Ibadan, however the sample size constitutes only a small percentage of the civil servants in Nigeria, which limits the generalization of the study.Findings from this study demonstrate that although the majority of the respondents had adequate knowledge of BSE and mammogram, the level of utilization of these methods was still low. More so, a significant association was found between knowledge, level of education and use of BSE and mammogram. Identified barriers to wider utilization of the screening methods, were time, fears of the result of the screening, and other sociocultural factors, and these should be taken into account when designing interventions. This may reduce the barriers, facilitate wider utilization and ultimately lead to a decrease in the morbidity and mortality rate of breast cancer.In as much as there are strategies in place for increasing awareness and sensitization about breast cancer screening, measures should also be in place to educate, motivate and encourage women to practise BSE and mammogram as preventive measures for the early detection of breast cancer.Midwives should reinforce the need to practise these screening measures among women. Governments and other stake holders should provide support to facilitate the education of women and provide adequate information about preventive screening for breast cancer. Efforts should be directed to making mammogram more accessible, available and affordable for all, especially in rural areas.


They play a role in various diseases, including development and progression of breast cancer; the objective of this study was to evaluate the association of common variants in the DEAR1 with breast cancer in women from Colombia. Single nucleotide polymorphisms (SNPs) rs584298, rs2927970, rs59983645, and rs599167 were genotyped in 1022 breast cancer cases and 1023 healthy controls using the iPLEX® and Kompetitive Allele Specific PCR (polymerase chain reaction) (KASP) method. The associations between SNPs and breast cancer were examined by conditional logistic regression. The associations between SNPs and epidemiological/histopathological variables were examined by multinomial logistic regression.We carried out a case-control study to investigate associations of variants in P = .048 and P = .004, respectively). The analysis of the disease characteristics showed that SNP rs584298 (genotype AG) (P = .015) shows association with progesterone receptor (PR) status and (genotype AA) (P = .048) shows association with human epidermal growth factor receptor 2 (HER2) status.Associations were found between tag SNPs and breast cancer adjusted for the epidemiological risk factors rs584298 genotypes AG and GG (DEAR1 showed associations with breast cancer and the expression of HER2 receptor; when this receptor is amplified, the result is aggressive tumoral subtype and expression of PR receptor that is associated with high-proliferative tumor grade. Validation of this SNP is important to establish whether this variant or the tagged variant is the cause for the risk association showed.The SNP rs584298 in  The members of this family participate in several cellular processes, such as proliferation, differentiation, oncogenesis, apoptosis, intracellular trafficking, innate cellular responses to retroviral infection, and inflammation. The DEAR1 gene in breast cancer has been considered a predictor of local recurrence-free survival in early-onset breast cancer; it regulates polarity and tissue architecture1 and is a master regulator of transforming growth factor β (TGF-β)-driven epithelial-mesenchymal transition (EMT).3 In breast cancer, DEAR1 is downregulated in ductal carcinoma in situ lesions and infiltrating ductal carcinoma.1Breast cancer is the most common cancer in women worldwide and is the leading cause of death among cancer cases. Different genetic and nongenetic factors are associated with risk of breast cancer. Ubiquitin ligase proteins are engaged in the regulation of the turnover and the activity of many target proteins. The 4 different to the genotype data from 116 CEU individuals (CEU: Utah residents with ancestry from northern and western Europe) that contain 94 SNPs in HapMap (haplotype map).5 This difference confirms that SNP frequencies change across different populations, and it is important to define which SNPs are associated with risk in each population. Most of the breast cancer cases are sporadic, and in the 10% of the hereditary cancer, BRCA1 and BRCA2 (breast cancer genes 1 and 2) are the best-known susceptibility genes; they are associated with the risk of up to 85% of developing breast cancer in mutation carriers.6,7In total, 230 single nucleotide polymorphisms (SNPs) have been identified based on genotype data from 60 CLM individuals (CLM: Medellin-Colombia) in the 1000 Genomes Project,811 Analyses performed on different populations allow discovering causal factors.12 The association of Tag SNPs rs584298, rs2927970, rs59983645, and rs599167 in DEAR1 with risk of breast cancer was analyzed in the total of cases and controls in women from the Colombian population by genotyping. This is the first study conducted on the association between SNPs in DEAR1 and breast cancer.Although the impact of high-risk mutations is important, they account for less than 25% of the cases of breast cancer predisposition; other risk factors can be explained by the addition of multiple common variants, known as low-penetrance variants in the polygenic model.Case-control study was carried out; cases were patients with breast cancer with different histopathological reports, and controls were patients without breast cancer or any breast injury.13 Cases were women with a diagnosis of breast cancer after January 1, 2004. Controls were recruited in the period of 06/2007 to 06/2011. Clinical, epidemiological, and histopathological data were collected as well as informed consent. The data included age at breast cancer diagnosis, histology, histological grade, tumor size, lymph node status, estrogen receptor (ER) status, progesterone receptor (PR) status, and human epidermal growth factor receptor 2 (HER2) status; epidemiological data included menopausal status, family history of breast and ovarian cancer, postmenopausal hormone therapy, oral contraceptive (OC) use, body mass index (BMI), and smoking. Data on follow-up of the breast cancer patients were collected ±5 years after the date of breast cancer diagnosis during the course of this study, including the date of last follow-up, date of locoregional relapse, date of distant metastasis relapse, date of contralateral breast cancer, date of death, and cause of death. This study was approved with the code MED-119-2009 by the ethics committee of Pontificia Universidad Javeriana.In total, 1022 cases were included. Patients with breast cancer were enrolled at different hospitals throughout the country, mainly at hospitals in Bogotá, Neiva, and Villavicencio. Unselected patients  were recruited during the period 03/2007 to 02/2011 within the Col-BCCC (Colombian breast cancer case-control study), at PUJ (Pontificia Universidad Javeriana Bogotá-Colombia)14 All study participants gave written informed consent. Controls were matched to cases with a range of up to 2 years.In total, 1022 controls were included. The participants were healthy and unrelated women, who participated in the National Pap Smear Program.Cases and controls were eligible if they were of Hispanic origin. Cases were included if breast cancer diagnosis was in or after January 1, 2004. Controls without any relationship with the cases, without a family history of breast cancer, and without any breast disease were included.15 DNA samples were available from all study participants.Eight milliliters of blood were drawn from each participant of the Col-BCCC; tubes with the anticoagulant EDTA (ethylenediaminetetraacetic acid) were used. Genomic DNA was extracted using a salting-out method.r2 ⩾0.8 with at least 1 tag SNP were selected. The comparison revealed that the DEAR1 linkage patterns differ between HapMap’s CEU population  and the CLM population from the1000 Genomes Project . As a consequence, we decided to proceed with data from the 1000 Genomes Project, because the CLM population is expected to be more genetically related to study population under investigation. The tagger program (http://www.broad.mit.edu/mpg/tagger/) was used to select SNPs using pairwise tagging methods.The data set in HapMap (CEU population) and 1000 Genomes Project (CLM population) were compared; SNPs with minor allele frequency (MAF) ⩾5% and DEAR1 gene spans the region from position 33611003 to 33647660 on chromosome 1 (GRCh37). Based on genotype data from the CLM population , the DEAR1 gene region and a 5-kbp putative promoter region (position 33611003 to 33652660) comprise 230 SNPs . Of these 230 SNPs, there were 65 SNPs with an MAF ⩾5%. Eight tag SNPs captured 100% of the alleles of these 65 SNPs at a correlation of r2 ⩾0.8. The selected tag SNPs are shown in Supplemental Table S4. From a previous tagging SNP approach, genotypes were obtained for 1 SNP in tagging group 1 (rs59983645), for 2 SNPs in tagging group 2 (rs584298 and rs628466), and for rs2927970. Genotypes for rs599167 in tagging group 3 were obtained using the KASP assay. Four SNPs  were not genotyped. In total, 4 of 65 SNPs capture 93.8% (61/65) of the genetic variation in the investigated region considering an MAF ⩾5% and r2 ⩾0.8.According to the 1000 Genomes Project (phase 3), the http://agenabio.com/products/massarray-system/).Genotyping of rs584298, rs2927970, and rs59983645 was performed by MALDI-TOF (Matrix-Associated Laser Desorption Ionization Time of Flight) mass spectrometry using Sequenom’s MassArray iPLEX system  and the iPLEX Gold Chemistry. Post-run data were analyzed using Sequenom’s Typer Analyzer software version 4.0.20 (http://www.lgcgroup.com/products/kasp-genotyping-chemistry/overview/#.VhY0UEa6KSo). All assay plating was performed on 384-well plates. Primers for each tag SNP were designed using Assay Design Suite software from Agena Bioscience.Genotyping of rs599167 was performed using a KASP genotyping (Kompetitive Allele Specific PCR (polymerase chain reaction)) assay, and the post-run data were analyzed using the SNPviewer software version 4.0.0.0  criteria were in accordance with the BCAC (Breast Cancer Association Consortium) and CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1/2). The QC for samples analyzed by MALDI-TOF includes 5.8% of duplicate samples and at least 95% of the concordance rate. The QCs for samples analyzed by KASP Genotyping Assay included 6.0% of duplicate samples (61 cases and 61 controls). At least 5% duplication with ⩾98% concordance was required.Supplemental Tables S5 to S8.Four non-DNA controls (NTC—no template control) were included per 384-well plate. A minimum of ⩾2 NTCs was used per plate. DNA samples that failed for >2 of the 4 analyzed SNPs were excluded (8 samples). After exclusion of these samples, the call rate for SNPs was rs584298: 98.5%; rs2927970: 98.7%; rs59983645: 98.3%; rs599167: 95.6%. A minimum call rate of ⩾95% was required. The sample had no significant deviation from HWE (Hardy-Weinberg equilibrium); the HWE analysis data are shown in 2 test.Differences between breast cancer cases and controls in the frequencies of general characteristics, such as family history, number of childbirths, breastfeeding experience, OC use, BMI, smoking, and age at first full-term pregnancy, were tested using the χ2 with 1 degree of freedom (1 df). Associations between genetic variables and breast cancer risk model adjusted for 6 potential epidemiological breast cancer risk factors  were analyzed by conditional logistic regression with odds ratios (ORs) and 95% confidence intervals (CIs). All variables were included into the complete model, and to determine which terms were significant, a hierarchical backward elimination approach was carried out. The variables not statistically significant during this procedure were evaluated as possible confounding factors. To select the best model, the Bayesian information criterion and the Akaike information criterion were used. The associations between DEAR1 genotypes and 7 clinical and histopathological tumor characteristics  of breast cancer cases were analyzed by multinomial logistic regression with relative risk ratio and 95% CIs. All tests were 2-sided. Multicollinearity between independent variables was evaluated. All the statistical analyses were performed using Stata software version 15.16 Significance was settled at a P = .05.For each SNP, deviation from HWE was assessed by a χA multivariate analysis of survival, using Kaplan-Meier approach, was conducted, with right censored observations. The survival rates are presented with 95% CIs.P > .005) were found. Significant differences between case and control groups are evident for the following variables, smoking habit (P = .000388), family history of breast cancer (P = .00001), use of OCs (P = .0291) and use of hormone replacement therapy (HRT) (P = .00001), parity (P = .0128), and breastfeeding (P = .0265); BRCA1 or BRCA2 mutations also show differences between case and control groups (P < .0001). The frequency of the variables for case and control groups is shown in The ages of the case and control groups were similar with a mean age of 52 years for the interviewees; the case group has a mean age of diagnosis of 49.75 years. Case and control groups show principally a tendency to overweight; no significant statistical differences . The genotype distributions of all SNPs in controls did not deviate from HWE. The results showed association of tag SNP rs584298 with breast cancer risk genotype AG and GG (P = .048 and P = .004 respectively) after adjusting for age, family history, BMI, menopausal status, replacement hormonal therapy, and breastfeeding. The 3 other tag SNPs individually and adjusted for the epidemiological risk variables show association, but in the complete model including all 4 tag SNPs did not show the same result because of the correlation between each other. Furthermore, no statistically significant difference was found. The conditional logistic regression analysis for the full model and the reduced model with epidemiological risk variables is shown in Four tag SNPs in the An evaluation of multicollinearity between risk factors and genotypes was performed. Risk variables age and menopausal status show high correlation but not collinearity. Tag SNPs rs599167 and 59983645 show collinearity; for this reason, these 2 SNPs were not included in the final conditional logistic regression model; no other SNPs showed significance level for entry into the model.DEAR1 gene were associated with some specific tumoral characteristics, a multinomial logistic regression was carried out between DEAR1 genotypes and 5 clinical and histopathological tumor characteristics  (data are shown in P = .048), and genotype AG with PR expression (P = .015); therefore, if 1 patient has HER2 expression, the relative risk of having the genotype AA over GG (referent group) would increase the risk by a factor of 3.48 times when the other variables in the model are held constant. The association with PR status will also increase: if a patient has a PR expression, the relative risk to have the genotype AG over GG (referent group) would be expected to increase by a factor of 2.78 times when the other variables in the model are held constant  time for recurrence: defined as the time from diagnosis to a random documentation of a breast event; any local, regional, or distant recurrence of breast cancer; or a contralateral breast cancer; (2) breast cancer–specific survival (BCSS): defined as the time from diagnosis to death from breast cancer; (3) overall-free survival (OSS): estimated as the time from registration to death of any cause.DEAR1 region and BCSS, OSS, and time to recurrence. Survival Kaplan-Meier data are shown in Supplemental Tables S1 to S3.The first end-point, time to recurrence, evidenced that 42 patients had contralateral breast cancer or locoregional disease. From these 42 patients, 31 were censored and 11 died. Data available for BCSS and OSS analyses were as follows: 77 (7.5%) patients died from the total of 1022. After 5-year follow-up, from these 75 patients, 26 (33.7%) patients died due to breast cancer, 18 (23.37%) died for a cause different to breast cancer, and 33 (42.85%) died for unknown cause. A total of 318 (31.11%) patients had a follow-up missed out from the total. The number of patients alive until the date of the last follow-up was 627 (61.35%). The multiple analyses of survival were conducted using the Kaplan-Meier survival method. There was no evidence of association between SNPs in the 17,18 Cases were mainly diagnosed in their fourth and fifth decades, and the majority shared characteristics considered as protective factors for the disease such as parity, age of women at birth of first child (less than 30 years old), breastfeeding, and no use of HRT or OCs. Most of the established breast cancer risk factors evaluated among Colombian women in the current study had strong associations with breast cancer.Most cases and controls were in postmenopausal status and were overweight with a BMI > 25. Those both elements are considered as a risk factor for breast cancer.DEAR1 gene located in the region 1p35.1 encodes for a member of the tripartite motif (TRIM) protein, one of the subfamilies of the RING type E3 ubiquitin ligases, which is associated with the development of breast cancer and invasion.3 Functional cell line mutational analysis provides evidence that DEAR1 is a key regulator of acinar morphogenesis in the mammary gland and an independent predictor of local recurrence-free survival. The association between DEAR1 and breast cancer suggests that DEAR1 polymorphisms could be a susceptibility factor for this disease.3 Besides the obvious role as a key regulator of acinar morphogenesis, DEAR1 gene has recently been considered a regulator of p27 stability, and an inhibitor of cyclin-dependent kinases. Decreased TRIM62 expression by RNA interference in cancer HER2 + cell lines not only induces cell cycle arrest but also causes the complete relocation of p27 to the nucleus.19 The loss of function of DEAR1 gene in the presence of TGF-β results in a failure in the acinar morphogenesis, upregulation of EMT markers, resistance to anoikis, migration, and invasion; therefore, DEAR1 is a master regulator of EMT.3 The loss of DEAR1 expression has also been considered an adverse prognostic factor in acute myeloid leukemia.20 In nonsmall cell lung cancer lesions, the loss of TRIM62 levels is lower during disease progression, which has been associated with poor clinical outcomes.21 The effect of this gene on breast cancer, as determined by functional analysis, increases the probability of one or many variants being associated with breast cancer risk. Until now, no reports about variants in DEAR1 associated with breast cancer risk adjusted for different epidemiological risk factors had been conducted and it is important to highlight the role of interactions between genotype, risk factors, and breast cancer risk, but the current approaches to identify such interactions are scarce, and new methodologies should be developed. This is the first report of DEAR1 variants associated with breast cancer risk; further studies in other populations are needed to confirm these slight associations between breast cancer risk and rs584298 as well as PR status and HER2 status.The DEAR1 gene were genotyped. The association with breast cancer risk was adjusted with epidemiological risk factors in a sample of the Colombian population. After adjusting for the covariates of epidemiological risk, the association with the tag SNPs rs584298 and breast cancer risk was found. This SNP despite of the similar frequency in different populations has no report of clinical significance.4 The rs584298 tagged an SNP (rs689187) located in 3′-UTR region of TRIM62, an miRNA binding site therefore probably causal variant associated with breast cancer risk. These 2 variants have a high r2 and D′ values, showing that they are coinherited. Further analyses are needed to confirm whether rs689187 is affecting the miRNA binding site and whether this could be a causal variant associated with breast cancer progression. Other replication studies are required to confirm that these variants could be a prognostic factor for patients with in situ breast cancer.22 Some genome-wide association studies have evaluated interactions between identified genetic variants and traditional breast cancer risk factors; none found significant interactions after correction for the number of comparisons made, and all were conducted among predominantly European-ancestry populations.23 In this case, probably the lack of strong associations should be due principally to the size and diversity of the populations evaluated, the number of SNPs tagged, and the missing data for each variable. Additional studies in other populations should be conducted.Four SNPs  in the 24 Several miRNA have been associated with overexpression of HER2.25 Also, the genotype AG of rs584298 in TRIM62 shows association with expression of PR status, possibly the causal variant could be the SNP in the miRNA binding site rs689187 affecting the expression of PR. Further studies are needed to identify the possible miRNA associated with the 3-UTR region in TRIM62 and the pathway involved in the HER2 dysregulation to establish it as a possible prognostic marker in breast cancer.The allele A of rs584298 was associated with the expression of HER2 receptor; therefore, it would be expected that rs689187 would be the causal variant associated; several studies had shown the importance of SNPs or miRNA binding sites polymorphism, mainly SNPs within the 3′-UTR gene region which may influence and regulate the posttranscription modulation on gene expression and in this manner confer susceptibility to the disease. HER2 overexpression has an important prevalence, nearly to ~22% in breast cancer; the upregulation of HER2 is associated with different histological characteristics of the tumor, metastasis and invasiveness, angiogenesis, and a poor overall prognosis.26Survival analysis show more than 90% of censored data with more than 30% unknown data, making it difficult to perform Kaplan-Meier analysis and probably this is one of the reasons for nonassociation.DEAR1 showed association with positive HER2 and PR receptors breast cancer risk in a sample of Colombian population; therefore, this information could be relevant for therapeutic decisions and prognosis. This SNP should be tested in other populations to confirm the association in a larger sample. In the future, those SNP could be considered as part of cancer risk and prognosis panels. It is crucial to perform SNP validation (rs689187) of the SNP in the miRNA binding site with functional analysis to determine the SNP targets.SNP rs584298 in Some limitations in the study were related principally with the tumor pathological information and follow-up. Shortly after the patients were diagnosed, they were referred to the study; therefore, they did not have all the pathological information, making it difficult to fill all the required information.Click here for additional data file.DEAR1 Tagging Single Nucleotide Polymorphisms With Breast Cancer in a Sample of Colombian Population: A Case Control Study by Angela P Beltrán, Edgar Benitez, Martin Rondon, Yeimy V Ariza, Fabio A Aristizabal and Ignacio Briceño in Breast Cancer: Basic and Clinical ResearchSupplemental material, SUPPLEMENTAL_INFORMATION_DEAR1_xyz3051994c94f72 for Association of


Ptrend < 0.001) for total polyamines, 0.35  for putrescine and 0.79  for spermidine, respectively. However, higher intake of spermine was associated with increased risk of colorectal cancer, with an adjusted OR of 1.58 . This data indicate that higher intake of total polyamines, putrescine and spermidine, as well as lower intake of spermine, is associated with a decreased risk of colorectal cancer.Polyamines  are small, cationic molecules that are necessary for cell proliferation and differentiation. Few studies have examined the association of dietary polyamines intake with colorectal cancer risk. The aim of this study was to evaluate total polyamines, putrescine, spermidine, and spermine intake in relation to colorectal cancer risk in China. In total, 2502 colorectal cancer cases and 2538 age- and sex-matched controls were recruited from July 2010 to April 2019. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated by multivariable unconditional logistic regression after adjustment for various potential confounding factors. Higher intake of total polyamine, putrescine and spermidine was significantly associated with reduced risk of colorectal cancer. The adjusted ORs for the highest compared with the lowest quartile of intake were 0.60 (95% CI 0.50, 0.72;  Globally, colorectal cancer is the third most commonly diagnosed cancer and is the second leading cause of death from cancer . It has Polyamines are small, cationic molecules that are necessary for cell proliferation and differentiation. Dietary polyamines and their metabolites by intestinal microorganisms, as well as endogenous polyamines, have been shown to be major determinants of the total body polyamines pool . AlthougSo far, few epidemiological studies have reported the association between dietary polyamines intake and colorectal cancer risk. Only one prospective study examined the association between dietary polyamines intake and colorectal cancer risk in postmenopausal women . An inveThis ongoing case-control study started in July 2010 and the design of the study has been previously reported . In brieCancer-free controls were frequency matched to cases by 5-year age group and sex. These came from two control groups. The first group of hospital-derived controls were recruited from the inpatients admitted to the Departments of Otorhinolaryngology, Vascular Surgery and Plastic and Reconstructive Surgery in the affiliated Hospital of Sun Yat-sen University during the same period as the cases. They were mainly admitted for medical conditions as follows: sudden deafness, sinusitis, otitis media, trigeminal neuralgia, facial paralysis, and varicose veins. Out of 1533 eligible hospital-derived controls, 1357 were successfully interviewed, a participation rate of 88.52%. The second control group was recruited through flyers, advertisements, invitations or referrals. A random selection occurred of 1181 community-derived controls by sampling from 3089 successfully interviewed community residents from a community-based cohort , matchedThis study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Ethical Committee of the School of Public Health, Sun Yat-sen University (No. 2019-018). All study subjects signed informed consent forms before the interview.Information on demographic situation, lifestyle, body weight and height and family history of cancer was collected through face-to-face interviews by trained interviewers using a structured questionnaire. Detailed menstrual and reproductive histories were obtained from women. Clinical diagnoses and histological findings were abstracted from the hospital medical records. Body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters. Regular smokers were defined as those who had smoked at least 1 cigarette per day for more than 6 months consecutively. Passive smokers were non-smokers who reported being exposed to others’ tobacco smoke for at least 15 min/day during the previous 5 years. Regular alcohol consumption was defined as alcohol drinking at least once per week during the past year. The intensity of physical activity was estimated based on occupational activity and household and leisure-time activities. The mean metabolic equivalent (MET)-hour value of each activity was calculated according to the Compendium of Physical Activities ,22. MET-A validated 81-item food frequency questionnaire (FFQ)  was usedt-test or Wilcoxon rank-sum test for continuous variables, and the χ2-test for categorical variables. The residual method was used to adjust for total energy intake [Comparison between cases and controls were performed using the y intake . Dietaryy intake ,32, and y intake ,34. Howep values were two-sided and a p < 0.05 was considered to be statistically significant.Analysis stratified by sex was conducted. The interaction was evaluated by including a product term in multivariable models. Subgroup analyses by cancer site  and different sources of controls  were also performed. All statistical analyses were done with SPSS 22.0 . All Among the 2502 cases, 1425 were men and 1077 were women. In total, there were 1565 colon cancers and 937 rectal cancers. More cases than controls were married, lived in rural areas, and had active smoking, regular alcohol drinking and family history of cancer. Case subjects were also likely to have a larger proportion of farmers, less education, lower income, lower BMI, heavier occupational activity and fewer household and leisure activities. No significant differences were found in passive smoking between cases and control subjects. Age at menarche and menopausal status were also not significant differences between cases and controls among women .Compared with the controls, intake of energy-adjusted total polyamines, putrescine and spermidine was significantly lower among cases, and spermine intake was significantly higher among cases .Ptrend < 0.001) for total polyamine, 0.35  for putrescine and 0.79  for spermidine. However, higher intake of spermine was associated with increased risk of colorectal cancer, with an adjusted OR of 1.58  comparing the highest quartile with the lowest quartile.As presented in Ptrend = 0.002) for spermidine and 2.13  for spermine, comparing the highest quartile with the lowest quartile, respectively.Analysis stratified by sex showed that intakes of total polyamines and putrescine were inversely associated with colorectal cancer both in men and women . A signiSubgroup analysis by cancer site showed that intakes of total polyamines, putrescine and spermidine were inversely associated, while spermine intake was positively associated with colon and rectal cancer risk . SubgrouThis is the first study to evaluate the association between intake of total polyamines, putrescine, spermidine, and spermine and risk of colorectal cancer both in men and women. The results showed that higher intake of total polyamines, putrescine and spermidine was significantly associated with reduced risk of colorectal cancer. However, higher intake of spermine was associated with increased risk of colorectal cancer.Dietary intake, cellular synthesis, and microbial synthesis in the gut are three main sources for polyamines in organisms. Cellular polyamines were polycationic compounds and could bind to negatively charged biomolecules, change their structures and modulate their functions ,36. ThusOur observation of an inverse association between total polyamines intake and colorectal cancer risk was consistent with one prospective study which reported an inverse association of high polyamine intake with risk of colorectal cancer in postmenopausal women with BMI ≤ 25 . Experim2+ channel remodeling in colorectal cancer and induce colon cancer cell proliferation and apoptosis resistance [So far, no epidemiological study has reported the association between putrescine and spermidine intake and colorectal cancer risk. Our observation of inverse associations between putrescine and spermidine intake and colorectal cancer risk were supported by some animal studies. Autophagy can suppress oncogenesis through improving genomic stabilization, limiting inflammation, and facilitating adequate immune responses against cancer cells , and onesistance . Some epsistance  and all-sistance . Due to N1-acetyltransferase and were either excreted from the cell or oxidized by peroxisomal N1-acetylpolyamine oxidase (PAOX), resulting in H2O2 [. Spermine can also be oxidized directly by spermine oxidase (SMOX) to produce H2O2, which was localized in the cytoplasm and nucleus of mammalian cells [2O2 produced by SMOX had greater potential for generating genetic damage than that produced by PAOX, which was produced in the presence of catalase in a normally functioning peroxisome [2O2 produced by up-regulated SMOX is sufficient to evoke oxidative stress and DNA damage, which had been linked to subsequent carcinogenesis [The observation of a positive association between spermine intake and colorectal cancer risk in the present study is contrary to our initial hypothesis. Generally, ingested spermine could be quickly absorbed and distributed from the intestinal gut without degradation , and int in H2O2 ,59. Speran cells . Thus, troxisome . Since sogenesis ,62. ThisAnalysis stratified by sex showed that high intake of total polyamines and putrescine were associated with reduced risk of colorectal cancer in both men and women. A sex-modified interaction was found in the association of colorectal cancer risk with putrescine intake, but not with total polyamines intake. Besides, the inverse association of spermidine and positive association of spermine intake with the risk of colorectal cancer were observed only among men. One possible explanation for this sex difference may be the complexity of diet. A previous study suggested that differences existed in the dietary patterns of men and women , indicatThe present study has several strengths. This is the first to investigate the association between dietary polyamines intake and the risk of colorectal cancer both in men and women, and evaluate the association between putrescine, spermidine and spermine and colorectal cancer risk separately. A relatively large sample was included in our study, which provided adequate statistical power to detect associations with the risk of colorectal cancer. Moreover, a wider range of potential confounders were collected and adjusted in the multivariable logistic regression analysis.Some limitations should also be considered. Firstly, selection bias is a common concern in case-control studies. In the present study, although colorectal cancer patients were recruited from only one hospital, patients at this hospital shared similar clinical characteristics with those from other large hospitals in mainland China . BesidesThe present study indicated that higher intake of total polyamines, putrescine and spermidine was associated with reduced risk of colorectal cancer, while higher intake of spermine was associated with increased risk of colorectal cancer in the Chinese population. More studies, especially prospective studies, are needed to further confirm the effects of polyamines intake on colorectal cancer risk.


Breast cancer patients with metastatic disease have a higher incidence of deaths from breast cancer than patients with early-stage cancers. Recent findings suggest that there are differences in immune cell function between metastatic and non-metastatic cases, even years before diagnosis. We have analyzed whole blood gene expression by Illumina bead chips in blood samples taken using the PAXgene blood collection system up to two years before diagnosis. The final study sample included 197 breast cancer cases and 197 age-matched controls. We defined a causal directed acyclic graph to guide a Bayesian data analysis to estimate the risk of metastasis associated with the expression of all genes and with relevant sets of genes. We ranked genes and gene sets according to the sign probability for excess risk. Among the screening detected cancers, 82% were without metastasis, compared to 53% of between-screening detected cancers. Among the highest ranking genes and gene sets associated with metastasis risk, we identified plasmacytiod dentritic cell function, the SLC22 family of transporters, and glutamine metabolism as potential links between the immune system and metastasis. We conclude that there may be potentially wide-reaching differences in blood gene expression profiles between metastatic and non-metastatic breast cancer cases up to two years before diagnosis, which warrants future study. In recent decades, survival of breast cancer has increased substantially . HoweverThe host immune response plays an important role in modulating the progression of cancer, including the progression of metastasis . The fatThe diagnostic potential of blood gene expression profiles for breast cancer has been investigated in blood samples taken at the time of diagnosis . But so In this study we used whole-genome gene expression data from 197 breast cancer cases and age-matched controls from the Norwegian Women and Cancer (NOWAC) Post-genome cohort. Our aim was to investigate the potential differences in blood gene expression profiles between patients with metastasized cancer and patients with non-metastasized cancer. This is an exploratory analysis to uncover promising avenues for future research. Hence, we do not focus on hypothesis testing and control of the error rates associated with these procedures. Instead, we apply Bayesian modeling to shrink estimates toward reasonable ranges.The NOWAC study is a nationally representative, prospective questionnaire-based cohort of approximately 170 000 middle-aged women . Among NThe Cancer Registry of Norway provided information on mammography screening attendance and clinical information on cancer diagnoses. The most recent cancer registry update for the present study is from 2017. We defined breast cancer cases with a positive lymph node status as metastatic cases. Breast cancer subtypes were defined in accordance with the consensus  on cliniFor our study, we started out with 231 women who were diagnosed with breast cancer at most two years after providing a blood sample. We drew age-matched, healthy controls from the NOWAC Post-genome cohort. Due to missing data on height, weight, or HRT use, we excluded seven cases and their corresponding controls. We also excluded nine case/control pairs due to missing information on screening attendance, and three due to missing metastasis status. Finally, we excluded 15 case/control pairs due to missing gene expression data for the control. This left a final study sample for data analysis of 197 breast cancer cases and 197 age-matched controls. Since we compared cases with metastasis to cases without metastasis, the controls served merely as a normalization of expression levels. This is primarily useful to mitigate batch effects.https://www.r-project.org/). The code we produced for this project is available online at https://github.com/uit-hdl/holsbo_olsen_2020.We performed all data processing and analysis in R, using the Bioconductor and rstan packages . RNA purity was assessed by NanoDrop ND 8000 spectrophotometer , and RNA integrity by Bioanalyzer capillary electrophoresis . mRNA was amplified and labeled using the Illumina TotalPrepT-96 RNA Amplification Kit , and hybridized to Illumina HumanWG-6 v.3 Expression BeadChip microarrays . The raw microarray images were processed in Illumina GenomeStudio.We performed preprocessing of the raw microarray data according to the NOWAC standard procedure . BroadlyBackground correction of expression values using negative control probes.x2 = log2 (rawx)Transform the expression values by p < 0.01).Filter out genes expressed below detection threshold . We did this by a Bayesian hierarchical model, stratifying by mode of detection, s ∈{screening, interval} with partial pooling between strata. For the observed metastatic status of person i, yi ∈{0, 1}, we specified the following model:We modeled metastasis probability for a single gene p) = log(p) − log(1 − p) is the logarithmic odds of metastasis. Equations 1–4 describe a logistic regression with varying slopes between detection methods. I.e. we considered the association between risk and differential expression to be similar but not identical for screening and interval cancers. Equations 5–7 define the hyperpriors for the slopes. Equations 6, 7 imply a lognormal distribution for gσ. We used this non-centered parameterization because it makes the estimation faster and more reliable. We estimated these models using the NUTS sampler implemented in Stan (The function logit( in Stan . Having gχ to have mean zero and standard deviation of unity. This makes logit−1 (gα) the metastasis probability, or risk, for an individual with average differential expression of gene g. Likewise the quantity logit−1 (gα + gzβ) is the risk for someone with differential expression z standard deviations higher than the average. We call the quantityWe standardized g. We chose Z = 0.1 because in our data most differences in means between metastases and non-metastases fall between ± 0.1 standard deviations. Hence we considered this a reasonable increase in differential expression for our investigation.the excess risk of metastasis for gene Excess risk is a signed quantity on the absolute scale. An excess risk of 0.01, or 1%, means that the risk of someone with elevated expression in a certain gene has a risk of 1% more than someone with average expression. I.e. if the risk associated with average expression is 25%, which it roughly tends to be, the risk of someone with 2% excess risk is 27%. A negative excess risk suggests that decreased expression has a higher metastatic risk, which implies under-expression among metastatic cases. We use excess risk throughout to assess how important the variation of a certain gene’s expression is for metastatic spread.ad hoc to provide a relaxed coverage of the parameter sizes we see fitting gene-wise maximum likelihood regressions for other outcomes (smoking and similar). This discourages outrageous estimates while still lending credence to realistic sizes. gsα and gsβ along with the implied prior predictive distribution for excess risk. We have centered the prior distribution for gsα , the log odds for someone with average expression, on what roughly corresponds to a risk of 25%, which is what is seen in the population. The prior implied excess risk is sharply peaked around 0 and has the middle 60% of its mass in the range ± 0.014.We chose our priors to provide a slight shrinkage toward the null effect. The prior parameters are chosen sign probability of an excess risk is p (gρ > 0) when the median of gρ is positive and p(gρ < 0) when the median is negative. This probability lies between.5 and 1 and expresses how much of the density for gρ lies away from zero. A high sign probability means that we are quite sure of the direction of an excess risk but does not say anything about its magnitude. We used sign probability both to rank genes and to rank gene sets.The We ranked genes in decreasing order by sign probability and examined the first 100.We ranked gene sets by the average sign probability in a given set. We extracted gene sets from the Molecular Signatures Database v.7.0 , using The NOWAC study was approved by the Norwegian Data Inspectorate and the Regional Ethical Committee of North Norway (reference: REK NORD 2010/2075). All women gave written, informed consent. Collection and storage of biological material was approved by the REK in accordance with the Norwegian Biobank Act (reference: P REK NORD 141/2008 Biobanken Kvinner og Kreft ref. 200804332-3).In our study population of 394 middle-aged women , age, BMI, smoking, and parity were similar between breast cancer cases and controls  after vaccination against influenza . In geneFour members of the SLC22 family were among the genes most frequently found in our gene set data, all of which were associated with decreased risk of metastasis (i.e. down-regulated). Two were among the top 100 single genes associated with metastasis risk. This family of transporters are involved in diverse and ubiquitous processes like metabolism, and inter-organ and inter-organism signaling. A recent review suggests they have equal importance as the neuroendocrine system and growth factor-cytokine system . Their rWe identified three single genes of glutamine metabolism associated with increased risk of metastasis, and related gene sets were among our top identified gene sets. Glutamine is the body’s most abundant amino acid and is considered a “fuel for the immune system”. It is an essential amino acid for lymphocyte proliferation, cytokine production, and activities of macrophages and neutrophils . Low gluDAGs are helpful for translating causal relationships into associations and create an overview of the subject matter as a basis for interdisciplinary discussions with the aim of designing an analysis strategy. But as with any method, there are strengths and weaknesses. Drawing the diagram to include all relevant assumptions is a challenge, also because absence of an arrow is a strong assumption. Nevertheless, drawing the DAG forces clarity about the underlying assumptions. To the extent that the diagram represents the true causal relationships, it helps identifying key sources of bias.Our DAG is based on well-established risk factors for breast cancer. In the broad context, these risk factors act through two main mechanisms: either through DNA damage, or through hormone-related processes. Age, alcohol use, and smoking ; and excOur data is from a case-control study nested within the prospective NOWAC Post-genome cohort. The advantage of this study design is that recall bias is reduced because exposure information is collected prospectively, before the onset of disease. Also, selection bias is reduced due to the prospective cohort being population-based. However, the nested case-control study cannot be used to infer causality between the exposure and the outcome. Specifically, it is not possible to measure and statistically control for all variables that may affect breast cancer metastasis. Hence the observed associations may be confounded.via direct cell-cell contact and via excreted factors. Extracellular and intracellular signaling pathways are often redundant, two-way, and containing feedback loops. None of these mechanisms can be easily expressed via a DAG. One solution might be to map out the molecular two-way interactions and feedback loops as linear sequences of events in time  and/or the host institution (info.helsefak@uit.no). Requests to access these datasets should be directed to Helsefak UiT (info.helsefak@uit.no); EH (einar.j.holsbo@uit.no).The data analyzed in this study is subject to the following licenses/restrictions: Due to ethical restrictions on this dataset, which contains potentially sensitive patient information, the data will be made available upon request. Please contact the corresponding author . All women gave written, informed consent. Collection and storage of biological material was approved by the REK in accordance with the Norwegian Biobank Act (reference: P REK NORD 141/2008 Biobanken Kvinner og Kreft ref. 200804332-3). The patients/participants provided their written informed consent to participate in this study.Both authors designed the study. EH analyzed the data, and KO interpreted the results and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by grants from UiT The Arctic University of Norway, and the European Research Council (ERC-AdG 232997-Tice).Some of the data in this article are from the Cancer Registry of Norway. The Cancer Registry of Norway is not responsible for the analysis or interpretation of the data presented. The laboratory work and Illumina bead chip service was provided by the Genomics Core Facility (GCF), Norwegian University of Science and Technology (NTNU). GCF is funded by the Faculty of Medicine and Health Sciences at NTNU and Central Norway Regional Health Authority.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility.We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands.We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not.As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood. In utero exposure to estrogenic endocrine disruptors (EDCs) is associated with increased risk of breast cancer. Women exposed in the womb to diethylstilbestrol (DES) have twice the risk of developing the disease after the age of 40 , 2. WhilRodent models demonstrate that early life exposures to estrogenic EDCs have long-term consequences on mammary gland development and promote tumorigenesis. In utero BPA exposures induce preneoplastic lesions and hyperplasia in rat mammary glands , 9. MiceAs many of the EDCs that alter mammary gland development are estrogenic, the ability of these chemicals to stimulate the estrogen receptor is used as the fundamental characteristic to identify them as potentially harmful compounds. Attempts to link the estrogenic action of BPA to the phenotypic changes in the mammary gland implicate the developing mesenchyme as a potential cellular target of EDC action . The devAnimal experiments were performed in compliance with protocols approved by The Ohio State University Institutional Animal Care and Use Committee  and in accordance with the accepted standard of humane animal care. CD1 mice  were maintained in polysulfone cages and fed a diet containing minimal levels of phytoestrogen (Harlan2019X). BPA dissolved in sesame oil was administered to pregnant mice via intraperitoneal (IP) injection as previously described , unless Luciferase reporter assay was performed as previously described , with mig for 3 min) to collect the epithelial organoids, stromal cells, fibroblasts, and red blood cells in the pellet. This pellet was rinsed in red blood cell lysis buffer (Sigma-Millipore) two times and resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were plated onto a cell culture dish for 1 h to separate the epithelial organoids from fibroblasts. Adherent fibroblasts were maintained at 37 °C/5% CO2/5% O2 until 80% confluent. Plates were washed with 1× phosphate-buffered saline (PBS) and DNA and RNA harvested using the ZR-Duet DNA/RNA mini prep kit (Zymo Research).Mammary fibroblast cells were isolated from adult mice, as previously described . BrieflyFibroblasts were collected by trypsinization, washed with 3% bovine serum albumin (BSA) in PBS, and then incubated with Ghost Dye Red 780 Viability Dye stain  for 10 min at room temperature. Cells were washed with 3% BSA in PBS, fixed with 0.1% paraformaldehyde for 15 min at room temperature, washed again with 3% BSA in PBS, and permeabilized with 2% saponin for 15 min at room temperature. Cells were then incubated with Fc blocking anti-mouse CD16/CD32 antibody  for 10 min. After an additional 3% BSA in PBS wash, cells were labeled with primary antibodies to fibroblast-specific protein 1 , fibroblast activation protein α , α smooth muscle actin , platelet-derived growth factor receptor α , platelet-derived growth factor receptor β , Vimentin , or secreted protein acidic and rich in cysteine  for 30 min at 4 °C. Again, cells were washed with 3% BSA in PBS and then followed by incubation with appropriate fluorescent secondary antibodies for 30 min at 4 °C. Cells were washed with 3% BSA in PBS three times prior to analysis on a flow cytometer (BD LSRFortessa).p ≤ 0.05, FDR 5%) . M. M43]. MAs BPA exposure increases ECM collagen content and gland stiffness, which are associated with increased breast cancer risk , 44, we In this study, we focused our efforts to analyze the changes within the stroma induced by in utero BPA exposures that persist into adulthood when carcinogenic transformation occurs. We identified fibroblast-specific reprogramming of the transcriptome associated with ECM organization. These changes are highlighted by increased collagen deposition and collagen fiber width, decreased hydraulic permeability, and a stiffer mammary gland. Numerous models have demonstrated that altering the ECM alone can contribute to oncogenesis . In factEarly life EDC exposure has life-long consequences towards human health. Interestingly, we found that these phenotypes of increased ECM density and gland stiffness, known to be risk factor for breast cancer, were not evident in young mice. These data support the idea that carcinogenic risk progresses throughout life and the impact of these exposures is not necessarily apparent at the time of exposure. In line with this hypothesis is the fact that DES daughters do not display increased breast cancer risk until the women have reached 40 years of age , 2.Identification of new EDCs is mostly driven by the ability to drive a hormone response in various in vitro and in vivo assays. Furthermore, much of the analysis of BPA and its alternatives have used surrogate phenotypes other than tumor formation to implicate cancer risk, such as developmental abnormalities. There is a clear need to understand which EDC-driven phenotypes are actually associated with cancer risk and if those phenotypes are being driven by estrogenic properties of the compounds. Herein, we demonstrate that BPA increases collagen production and mammary gland stiffness, which are known risk factors for breast cancer , 42, 44.Herein, we identified long-term reprogramming events within the mammary fibroblast population associated with in utero EDC exposures. These events lead to increased collagen production resulting in a stiffer mammary gland. As mammary gland stiffness is associated with human breast cancer risk , these dAdditional file 1.Additional file 2.Additional file 3.


TM) scores were calculated based on food and supplement intake. Cox regression and competing risk models were used to estimate multivariable-adjusted hazards ratios (HRs) and 95% confidence intervals (95% CIs) by E-DII tertile (T) for all-cause and breast cancer-specific mortality. With median follow-up of 14.6 years, there were 296 (27.8%) deaths from all causes and 100 (9.4%) breast cancer-specific death. The E-DII was associated with all-cause mortality . Non-linear positive dose–response associations with mortality from all causes were identified for E-DII scores (Pnon-linearity < 0.05). The post-diagnosis E-DII was statistically significantly associated with mortality risk among breast cancer survivors. Long-term anti-inflammatory diet might be a means of improving survival of breast cancer survivors.Inflammation-modulating nutrients and inflammatory markers are established cancer risk factors, however, evidence regarding the association between post-diagnosis diet-associated inflammation and breast cancer survival is relatively sparse. We aimed to examine the association between post-diagnosis dietary inflammatory index (DII®) and risks of all-cause and breast cancer-specific mortality. A total of 1064 female breast cancer survivors in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening (PLCO) Trial prospective cohort, were included in this analysis if they had completed the diet history questionnaire (DHQ). Energy-adjusted DII (E-DII Breast cancer is rapidly becoming more common in other parts of the world2. In the past 40 years, breast cancer screening and treatments have improved substantially. Previous studies suggest that screening for early detection and improved treatment, including adjuvant therapy, have contributed to about one-half of the decline in breast cancer-related mortality3. Mortality can be lowered yet more by applying comprehensive disease management4. The importance of healthful diets for survivorship had been indicated in several studies6. However, debate continues about the best strategies for the dietary management of breast cancer patients to improve long-term survival7.Approximately 268,600 women were diagnosed with invasive breast cancer in the United States in 201911. More recently, literature has emerged that offers contradictory findings regarding the relationship between survival after a breast cancer diagnosis and specific nutrients known to modulate inflammation, such as dietary fat, fruits, vegetables, fiber, and alcohol15. The overall inflammatory potential of the diet may provide better insights into the effect of diet on breast cancer survival than assessing only a single nutrient; after all, a typical human diet consists of a variety of both proinflammatory and anti-inflammatory foods and nutrients16. The dietary inflammatory index (DII®) reflects both a robust literature base and standardization of individual intakes to global referent values17. To date, a prospective cohort study from the Women’s Health Initiative Study has used the DII to assess the association between post-diagnosis dietary inflammatory potential and survival of invasive breast cancer patients, indicating that consuming a more anti-inflammatory diet after breast cancer diagnosis reduced risk of death from cardiovascular disease18. Two retrospective cohort studies found inconsistent results: Jang et al. showed that anti-inflammatory diets may decrease the risk of cancer recurrence and overall mortality in patients with breast cancer19, while findings from Italy did not suggest an association between the inflammatory potential of diet and the survival of female breast cancer patients20. Retrospective studies are susceptible to both selection and information biases. For example, it has been noted that positive dietary changes were more common among younger women or those who underwent chemotherapy21, and it is commonly assumed that dietary data are susceptible to biases associated with known outcomes, as in case-control studies23. Furthermore, some clinicopathological or lifestyle factors may mediate the association of diet and disease risk or survival, such as age24, hormone receptor status25, body mass index (BMI)26, gene polymorphisms27, and smoking status28.Chronic inflammation is implicated in breast cancer, and studies suggest a link between inflammation and breast cancer outcomesTM) is a composite measure of diet-associated inflammation which takes into account inflammatory effect of overall diet of an individual as opposed to looking at specific nutrients or food items. In this study, we aimed to comprehensively assess the association between inflammatory potential of post-diagnosis diet, as estimated by the E-DII, and all-cause and breast cancer-specific mortality among post-menopausal breast cancer patients from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) prospective cohort.The energy-adjusted DII , HR, 1.53; 95% CI, 1.15–2.03 (T3 vs. T1), Table P < 0.001; HR, 1.76; 95% CI, 1.10–2.82 (T2 vs. T1), HR, 1.70; 95% CI, 1.17–2.47 (T3 vs. T1), Table After a median follow-up time of 14.6 years from breast cancer diagnosis , a total of 1064 eligible female breast cancer cases were available for analyses , both the HRs for all-cause mortality and breast cancer-specific mortality in model2 became much stronger . Women in the most pro-inflammatory diet (T3) subgroup with more than 15 person-years follow-up had a 2.18-fold higher risk of all-cause death than those with the most anti-inflammatory diet  than theirs (−6.8 to 3.8). Another retrospective study found anti-inflammatory diets might improve the survival of breast cancer patients, particularly among younger women, those who were premenopausal, obese, had HR + breast cancer, had tumor more than 2 cm, and had lymph node metastasis7. However, they used the DII (not the E-DII) and did not consider dietary supplements19. Three studies31 considered other cancer types in relation to post-diagnosis E-DII score on the risk of mortality, however, a post-diagnosis proinflammatory diet was not statistically significantly associated with mortality risk in cancer patients. Relatively short-term follow-up time and retrospective study designs maybe contribute to above-mentioned null results, which are similar to what we found when we examined the short duration (≤15 person-years) subgroup. Further studies with extended follow-up time will be warranted in the future.At present, only one prospective study has evaluated the influence of post-diagnosed dietary inflammatory potential on breast cancer outcomes33. A previous study reported that dietary modifications were observed in women with breast cancer in Malaysia, and 66.7% were found to have decreased intake of energy, protein, total fat and vitamin E, and increased intake of carotene and vitamin C intake34. A prospective analysis from the Women’s Health Initiative35 reported that dietary inflammatory potential before diagnosis is related to breast cancer death; however, future studies are needed to examine the inflammatory potential of post-diagnosis diet that is an important approach to conduct dietary intervention in process of the secondary breast cancer prevention. Improved recurrence-free survival and disease-free survival were observed in a large dietary intervention trial among women diagnosed with breast cancer who received dietary fat reduction treatment for breast cancer in the USA12. Furthermore, the Nurse’s Health Study (NHS)36 with a median of 9.3 years follow-up, including breast cancer patients more than 60 years old, investigated Dietary Approaches to Stop Hypertension (DASH) score and the Alternative Healthy Eating Index (AHEI)-2010 and risks of deaths from breast cancer and all causes. This NHS study reported better adherence to a priori dietary indices after breast cancer diagnosis was associated with a 28% (DASH) and 43% (AHEI) reduced risk of non-breast cancer mortality. The WHI reported that in breast cancer survivors, postdiagnosis higher Healthy Eating Index (HEI)-2005 scores that reflected better quality diets were associated with better overall and cause-specific survival37. Because a lower E-DII score, indicating a more anti-inflammatory diet pattern, is associated with better diet quality score , our results are consistent with what was observed in those studies.Prior studies have suggested that patients tend to make changes in their diet after a cancer diagnosis38. In another WHI study, better dietary quality also was associated with a reduced risk of all-cause mortality among women with ER-positive tumors rather than ER-negative tumors37. Due to unbalanced distribution of the incidence rate and significantly different prognosis of ER-positive and ER-negative tumors39, we suggest that further experimental and epidemiological studies are warranted to validate associations between diet and breast cancer subtypes.We did not find any interaction effects of ER/PR status on the association between E-DII score and risk of all-cause mortality, perhaps due to limited sample size and resulting loss in statistical power. A prior WHI study indicated that a positive relationship between E-DII scores and all-cause mortality risk was seen among ER-positive breast cancer cases and among the combined ER-positive and/or PR-positive cases but not ER-negative /PR-negative cases31. Biologically, cigarette smoke contains many oxidants and free radicals and pro-inflammatory compounds that may activate endogenous mechanisms such as recruitment of neutrophils and macrophages to further increase the oxidant injury40. The shift in balance between oxidant/antioxidant in favor of oxidants, termed “oxidative stress”, results in many pathological conditions including cancers41. Thus, anti-inflammatory diets, including supplements such as vitamins C and E), and β-carotene could protect smokers from experiencing oxidative stress41.We failed to show that the effect of the E-DII on all-cause mortality in breast cancer survivors differed between smokers and non-smokers; a study focusing on high-grade serous ovarian carcinoma found a protective effect of anti-inflammatory diet on all-cause mortality risk among smokers18. Third, the FFQ in the PLCO provided only 35 out of 45 DII components, which might lead to under- or over-estimation of the relationship of E-DII with mortality17. In reality, we had demonstrated that missing even 15–20 parameters had little effect on DII scores in our prior work42. Fourth, the FFQ is known to suffer from a variety of biases associated with structured questionnaires. Most prominent among these are response sets such as social approval and social desirability44. This leads to the fifth weakness, which is that we had no information on any of these potentials in biasers46. Missing information on HER2 status and adherence to treatments resulted in missing data in the multivariable models, with the potential to bias HR estimates. Participants were excluded who did not have valid DHQ responses. This could potentially bias the results. When analyzing subjects who were excluded from the current analysis due to an invalid or missing DHQ, we found that they were likely to be older, black, obese, divorced or widowed, have physical activity less than one time per month, and experience hormone therapy than those who were included in the risk estimates  of otherwise eligible women were excluded because they had a cancer diagnosis before completing the DHQ. In this process of exclusions, some selection bias might exist. Second, related to this first issue, the number of deaths was relatively small, thus precluding stratified analyses with sufficient statistical power to observe significant associations, especially in subgroups for breast cancer-specific mortality. We failed to detect a meaningful association between E-DII and the few observed cardiovascular disease mortality events, even though there is considerable evidence showing that consuming a more anti-inflammatory diet after breast cancer diagnosis can reduce the risk of death from cardiovascular diseaseIn conclusion, this study evaluated the association of the inflammatory potential of diet with all-causes and breast cancer-specific mortality risks in a prospective cohort study of breast cancer survivors. Our findings support that anti-inflammatory potential of a post-diagnosis diet may be a means for reducing risk of breast cancer and all-causes death among breast cancer survivors. To precisely tailor the dietary interventions for breast cancer survivors in the future, additional well-designed cohort studies with large number of cases are warranted to validate our results and identify specific subgroups who would have a survival benefit of post-diagnosis anti-inflammatory diets.47. In total, 154,897 eligible participants , aged 55–74 years were enrolled from November 1993 to July 200147. Participants were individually randomized to the intervention group or the control group in equal proportions. Data were collected on cancer diagnoses and deaths from all causes occurred through July 31, 2011.PLCO was sponsored by the National Cancer Institute (NCI), conducted in the United States, and its purpose was to determine the effects of screening on cancer-related mortality and secondary endpointshttps://biometry.nci.nih.gov/cdas/plco/) was approved by the Institutional Review Board of the United States National Cancer Institute (NCI).All the participants provided written informed consent to participate in the study, and the study protocol . According to the exclusion criteria, we removed from consideration 2319 women whose dietary information were collected prior to their breast cancer diagnosis; 1017 cases with invalid DHQ responses ; 14 cases who did not return baseline questionnaires and 147 cases with IV stage or unknown stage tumor. After these exclusions, the analytical cohort included 1064 breast cancer patients with follow-up data.48. Diet was assessed by a self-reported food frequency questionnaire (FFQ), the DHQ version 1.0  introduced in 1998 to the control and intervention arms within a median of three years after randomization in this trial50. On the DHQ, participants reported their frequency of intake and portion size of 124 food items and supplement use over the previous year51. Daily nutrient intake was calculated by the DietCalc software52, which integrated responses of food frequency, portion size, and other responses with a nutrient database based on national dietary data 49. The DHQ has been validated against four 24-h dietary recalls (one in each season) among 1640 nationally representative participants in the Eating at America’s Table Study where the energy-adjusted correlation coefficients for dietary factors ranged from 0.51 for vitamin E to 0.78 for magnesium among women and from 0.41 for sodium to 0.83 for thiamin among men51.Dietary assessment was similar to our previous study17. The energy-adjusted DII (E-DII) score was calculated based on reported nutrient and food intake from the DHQ which were linked to the corresponding inflammatory effect scores designated in the DII53  E-DII scores indicate more anti-inflammatory diets. The DII score has been construct-validated with different dietary assessment instruments and found to be associated with higher levels of IL-655, high-sensitivity CRP42, and homocysteine56. Because most of the participants (79%) in the PLCO consumed supplements, and many dietary factors used in supplements have anti-inflammatory properties57, we report E-DII from food plus supplements.Eight DII components including ginger, turmeric, garlic, oregano, rosemary, eugenol, saffron, and flavonols were not available from the DHQ. The remaining 37 components were available for E-DII score calculation in our analysis. The food and nutrient consumption estimated at the DHQ was first adjusted for total energy per 1000 calories. To avoid the arbitrariness as a result of simply using raw intake amounts, the energy-adjusted dietary intake was subsequently standardized to a composite dietary database representing energy-adjusted dietary intake from 11 populations living in different countries across the world2 and categorized based on the World Health Organization criteria. Years from breast cancer diagnosis to DHQ completion was defined as interval time between breast cancer diagnosis and DHQ completion.Information on age at breast cancer diagnosis, race/ethnicity, marital status, education level, smoking status, income level, number of living birth, physical activity, breast feeding, oophorectomy status, family history of breast cancer and history of diabetes were assessed at baseline using self-administered questionnaires. Hormone therapy, birth control pills, aspirin use and total energy intake were acquired from DHQ. BMI was calculated as weight (kg)/height(m)Detailed cancer characteristics such as diagnosis date, stage, tumor morphology  and hormone receptor results were acquired. The cutoff for estrogen receptor (ER)-negative and progesterone receptor (PR)-negative IHC status was less than 1% staining in the nuclei.58, death was primarily ascertained through a mailed annual study update questionnaire, with next of kin notifying the trial of death, which were verified by obtaining death certificates; searches of the National Death Index also were conducted to ascertain death. Autopsy and hospitalization records were used to determine the underlying cause of death. If these were unavailable, death certificates, medical records or other records were utilized.Outcomes included death from breast cancer and any cause. As previously describedAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the PLCO study.P < 0.05) and frequencies (percentages) for categorical variables was calculated. Accordingly, Kruskal–Wallis test and Chi-Square test (Fisher’s exact if needed) were employed to test differences of continuous and categorical-co-variates, respectively, between the three groups.We divided the eligible patients into tertiles according to E-DII from food plus supplements with cutoff points determined from the distribution of the entire cohort. To present the baseline characteristics of the study cases, median ((interquartile range (IQR)) for continuous variables that are not normally distributed as indicated by Shapiro–Wilk normality test  or they changed the crude risk estimate by >10% in bivariate analyses60, in addition, prognostic factors for breast cancer patients from literature review also were treated as confounding factors. In model 1, we adjusted for age at time of breast cancer diagnosis and total energy intake. Model 2 additionally adjusted for years from breast cancer diagnosis to DHQ completion, body mass index, trial arm, race, marital status, income, educational level, hormone replacement therapy, history of diabetes, physical activity, stage, and ER/PR status. Cancer stage and ER/PR status were used as substitutes for the currently unavailable cancer treatment data, as breast cancer stage and hormone receptor status may influence types of treatment received62. The proportional hazards (PH) assumption was examined using the Schoenfeld residual test63, and we only find that co-variables like age, years from breast cancer diagnosis to DHQ completion and stage violated the PH assumption in multivariable analyses. Thus, we fitted an extended Cox proportional hazards models stratified by age , years from breast cancer diagnosis to DHQ completion  and stage .For each mortality outcome, women were followed from diagnosis of primary invasive breast cancer until death, loss to follow-up or the end of follow-up. Cox proportional modeling was fitted to estimate crude, age and total energy intake-adjusted and multivariable-adjusted HRs and 95% confidence intervals (CI) for breast cancer patients in the lowest E-DII tertile (most anti-inflammatory diet) as the referent. To minimize the potential impacts of competing risk bias on the association of E-DII with breast cancer-specific mortality risk, competing risk regression models were employed to estimate sub-distribution HR and 95% CIs, with non-breast cancer causes of death as competing risk eventsp values < 0.05 were considered as an indicator of significant effect modification. We planned a priori stratified analysis by important co-variables that were considered clinically relevant, including age , tumor stage (carcinoma in situ and invasive), smoking status , ER/PR status and follow-up time  whose median value is 15 person-years on the association between E-DII and all-cause mortality. Given the small number of breast cancer-specific death in this study, we did not further stratify analyses of breast cancer-specific mortality in interaction analyses.Effect modification by baseline characteristics and clinicopathological variables was examined by adding the cross-product of each effect modifier with E-DII tertile in the multivariable-adjusted model. Likelihood ratio tests were conducted, and 64, and the reference level was set at −7.9 . Specifically, a Pnon-linearity was obtained by testing the null hypothesis that the regression coefficient of the second spline was equal to zero.We further assessed the potential non-linear dose–response relationship of E-DII to all-causes mortality through restricted cubic spline models with 3 knots at the 10th, 50th, and 90th percentiles65, we excluded cases with less than 1 year from breast cancer diagnosis to DHQ completion.In sensitivity analyses, considering dietary changes due to adjuvant therapies involving chemotherapy and radiotherapy for the disease might affect appetiteAll P values reported are two-sided. Those less than 0.05 were considered statistically significant. All analyses were conducted using R software (version 3.4.1).Further information on research design is available in the Supplementary InformationReporting Summary