We previously developed the preferential LD approach, a novel method that successfully identified causal variants driving the GWAS signals within European-descent populations even when the causal variants were only weakly linked with the GWAS-discovered variants. To evaluate the performance of our approach in a trans-ethnic setting, we applied it to follow up breast cancer GWAS hits identified mostly from populations of European ancestry in African Americans (AA). We evaluated 74 breast cancer GWAS variants in 8,315 AA women from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Only 27% of them were associated with breast cancer risk at significance level α=0.05, suggesting race-specificity of the identified breast cancer risk loci. We followed up on those replicated GWAS hits in the AMBER consortium utilizing the preferential LD approach, to search for causal variants or better breast cancer markers from the 1000 Genomes variant catalog. Our approach identified stronger breast cancer markers for 80% of the GWAS hits with at least nominal breast cancer association, and in 81% of these cases, the marker identified was among the top 10 of all 1000 Genomes variants in the corresponding locus. The results support trans-ethnic application of the preferential LD approach in search for candidate causal variants, and may have implications for future genetic research of breast cancer in AA women.Leveraging population-distinct linkage equilibrium (LD) patterns, trans-ethnic follow-up of variants discovered from genome-wide association studies (GWAS) has proved to be useful in facilitating the identification of  Genome-wide association studies (GWAS) premised on the “common disease, common variants” hypothesis have made great strides in identifying common genetic variants associated with a variety of phenotypes . TypicalWe previously developed the novel preferential LD approach to identify causal variants that drive the GWAS signal of interest from a comprehensive genome-wide variant catalog . This apIn this study, we evaluated the performance of the preferential LD approach in an African American (AA) population, following up breast cancer GWAS hits. We carried out the study in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium, which provides rich genetic and epidemiological resources for investigating breast cancer risk in AA women –36. To dWe first sought to evaluate the 74 GWAS-identified breast cancer risk variants  in 8,315Out of the 74 GWAS-discovered variants, only 18 were associated with overall breast cancer risk in AMBER with nominal p-value < 0.05  candidate causal variants or better markers with lower association p-values for the 20 GWAS-discovered variants replicated in the AMBER consortium. A total of 5,451 candidates were identified by our approach from 127,697 variants in the 1000 Genomes variant catalog that lie within 500 kb of the GWAS-discovered variants (0.29−0.85 candidates per 1 kb for each GWAS-discovered variant). Among these, 77,608 of the 1000 Genomes variants including 4,932 of the preferential LD selected candidates were successfully genotyped or imputed in the AMBER consortium and were tested for association with breast cancer risk. After accounting for the pairwise correlations among these variants, the effective number of independent tests was 19,617 . Using a−6) in TERT locus and three variants in ANKLE1-BABAM1 locus, rs11668840, rs8100241, and rs12982058  (Table 2>0.6). For an additional six loci, the best candidate identified by the preferential LD approach was among the top 10 best markers in the corresponding locus. It is worth noting that for 11 of the above 13 loci, the best candidates identified by this approach were only in weak LD (r2<0.6) with the GWAS-discovered variants, which demonstrates the ability of this approach to pinpoint candidates even when they were not strongly linked with the GWAS-discovered variants. For example, rs73731716, the best marker identified by preferential LD approach by following up the GWAS signal rs10069690, is also the best marker among the 6,912 tested 1000 Genomes variants at the surrounding TERT locus. It is significantly associated with overall breast cancer risk with p-value 1.33×10−6 but only has weak LD with the GWAS signal (r2=0.015).We first focused on the 18 loci where the GWAS-discovered variants were associated with overall breast cancer risk in the AMBER consortium Table . In gene−5, ranked no.2 among 6438 tested 1000 Genomes variants) for the locus surrounding GWAS signal rs2981579 (p=0.0224), and rs56269701  for the locus surrounding GWAS signal rs13387042 (p=0.0287). The association between ER- breast cancer and seven 1000 Genomes variants passed study-wide significance, including the GWAS-discovered variant rs10069690 and six variants in ANKLE1-BABAM1 locus , but the correlation is much lower in AAs (r2=0.05). Consistent with the LD, the association between rs2981582 and breast cancer risk is very strong in European individuals, more modest in Asian individuals, and mostly attenuated in AAs [−6). After conditioning on the causal variant rs2981578, the major association signal in this locus was completely eliminated , as a candidate causal variant. Variant rs2912778 is also a non-coding variant in intron 2 of FGFR2 and it is highly correlated with the causal variant rs2981578 . This finding further confirms the ability of the preferential LD approach to identify rarer and weakly tagged causal variants in a trans-ethnic setting.FGFR2 locus was one of the first breast cancer loci identified by GWAS. The most strongly associated GWAS-variant is rs2981582, a non-coding variant in intron 2 of ceptor 2 . Furtherceptor 2 , 61. Theceptor 2 , 61. Theet al. investigated 67 GWAS-discovered variants in 1,231 AA cases and 2,069 controls, and found that only 10 of them (14.9%) were significantly associated (p<0.05) with overall breast cancer risk and by subtype. Chen et al. examined 19 risk variants identified by GWAS and Feng et al. further tested an additional 54 GWAS-discovered variants in AA women [In this study, we investigated the associations of 74 breast cancer risk variants previously discovered by GWAS in 8,315 AA women from the AMBER consortium. We found that the majority of the GWAS-discovered variants identified from non-African populations could not be replicated in AA women in the AMBER consortium, which is consistent with the literature. Previously, Long AA women , 62. TogAA women , 64 reinAPOL1 gene are common in AAs but absent in European Americans [MYBPC3 is common in individuals from South Asia but not observed elsewhere [ABCA1 variant that reduces cholesterol efflux is Native-American ancestry specific [FGFR2 locus, the GWAS-discovered variant in European population, rs2981582, is no longer a good proxy of the causal variant in AAs. With all of these considerations, well-powered breast cancer GWAS in African populations will not only improve the replication rate of the GWAS findings from other populations, but will also lead to identification of novel breast cancer loci that are specific to African ancestry. To date, there are only two moderately sized  GWAS of breast cancer focused on women of African ancestry [Besides sample size and statistical power, a number of additional factors could contribute to the low replication rate and therefore have direct influence on the trans-ethnic application of the preferential LD approach. First, the causal variants can be population specific , 14. Formericans ; the carlsewhere ; and thespecific . As the specific , ovarianspecific , glioma specific , prostatspecific , 29, andspecific –70. As rspecific . As has ancestry , 41, indFor the 20 loci where the GWAS-discovered variants were replicated in AA women in the AMBER consortium, we applied the preferential LD approach to search for causal variant candidates or better markers for AA breast cancer risk. The preferential LD approach could identify better markers in 16 of the 20 loci, and in 13 of them, the approach could identify markers that were top 10 among all genotyped and imputed 1000 genomes variants in the corresponding locus, which indicates the ability of this approach to follow up GWAS hits trans-ethnically. The preferential LD approach was developed to identify causal variants by following the GWAS-discovered variants even if the causal variants are much rarer. It is important to note that the goal of the preferential LD is to follow up a particular GWAS signal to search causal variants driving the corresponding association instead of fine mapping a GWAS locus (eg. selecting variants based on LD pruning) where additional risk variants independent of the GWAS signal can exist. The success of this approach relies on two factors: the presence of the causal variant in the variant catalog used by the approach, and the GWAS-discovered variant being the best tag for the causal variant. However, there were limitations for both of these two assumptions in the current trans-ethnic study. First, the currently best publicly available variant resource for African Population is from the 1000 Genomes Project, which includes 246 individuals of African-descent. As the causal variants of breast cancer are likely rare in the populations without breast cancer, they are expected to be depleted in this variant catalog, which was compiled from a relatively small number of healthy individuals. In our previous intra-ethnic application of the preferential LD approach, we utilized a comprehensive genome-wide variant catalog from 479 deeply sequenced individuals of European ancestry . Being aThe AMBER Consortium , 33 was The CBCS is a North Carolina population-based case control study of breast cancer. Breast cancer cases were identified using Rapid Case Ascertainment in cooperation with the NC Central Cancer Registry. Controls were identified using Division of Motor Vehicles lists for women under age 65 and Health Care Financing Administration lists for women 65 and older. The WCHS is a multi-site case control study in New York City (NYC) and New Jersey (NJ) aimed at evaluating risk factors for early and aggressive breast cancer in AA and European American (EA) women. Recruitment in NYC involved hospital-based ascertainment of cases, while controls were identified through random digit dialing (RDD). Cases in NJ were identified by the NJ State Cancer Registry using rapid case ascertainment. Controls were recruited though RDD and community-based efforts . The BWHAll study participants provided consent for using their data and specimens for research purposes and the study was approved by Institutional Review Boards at participating institutions. ER status for cases was determined using pathology data from hospital records or cancer registry records.Our approach identifies candidate causal variants from a comprehensive variant catalog with four major steps . First, We used the preferential LD approach to follow up the GWAS-discovered breast cancer risk variants that were replicated in the AMBER consortium by utilizing the variant catalog from the 1000 Genomes African population (phase I release 3). As the preferential LD approach excludes variants that were already interrogated in the corresponding GWAS from the search of candidate causal variants, we used the variants in the HapMap phase II YRI  samples as the variants analyzed for meta-GWAS, where multiple genotyping platforms and imputation were used, or when the GWAS platform content was unavailable . When asThe GWAS-discovered variants and their corresponding followed-up variants selected by the preferential LD approach were added as part of the custom content to the Illumina Human Exome Beadchip v1.1 –51, and As principal component analysis (PCA) using variants genotyped in all four studies of the AMBER consortium revealed little heterogeneity across studies, we analyzed all studies jointly. PCA analysis identified 35 population outliers. These outliers and six additional samples with missing phenotype information were omitted from association analyses. We used PLINK  to test


We identified a comprehensive range of factors related to breast cancer, among which several manageable factors may contribute to breast cancer prevention. Further prospective studies concerning psychological interventions, sleep regulation, health guidance, and physical exercise are required. A screening model for high-risk populations should be put on the agenda.This study aimed to investigate risk factors associated with breast cancer among Han Chinese women in northern and eastern China. A matched case-control study involving 1489 patients with breast cancer and 1489 controls was conducted across 21 hospitals in 11 provinces in China, from April 2012 to April 2013. We developed a structured questionnaire to record information from face-to-face interviews with participants. Student’s t-tests, Pearson’s chi-square tests, and univariate and multivariate conditional logistic regression analyses were used to identify variables with significant differences between the case and control groups. Ten variables were identified (P Breast cancer is the most common type of cancer worldwide; the incidence is continuing to rise, and it is the leading cause of cancer-related death among women , 2. WorlCharacteristics of established risk factors for breast cancer may vary among countries. Better understanding the characteristics of local risk factors may inform more effective breast cancer prevention strategies . When riWorldwide, numerous studies have sought understand the risk factors for breast cancer. However, there has been no consensus because of differences in sample sizes, races that comprised study populations, and local customs. Most epidemiological studies have evaluated risk factors for breast cancer based on large sample sizes in Western populations. However, these risk factors are not based on Chinese women and cannot be directly applied in China, because risk factors may differ across different populations –14. In CFigure Among the 1489 patients with breast cancer, there were 1128 cases with invasive ductal carcinoma (accounting for 75.8% of the study population), 127 (8.5%) with intraductal carcinoma, 24 (1.6%) with invasive lobular carcinoma, and 194 (10.7%) with other types of cancer . Luminal A type, luminal B type, HER-2 type, and triple negative types accounted for 10.7% (n=159), 49.9% (n=743), 8% (n=119), and 8.5% (n=126) of cases, respectively. In addition, 322 cases were estrogen receptor negative (21.63%), 1018 were estrogen receptor positive (68.37%), 149 had lost estrogen receptor status (10.01%), 417 were progesterone receptor negative (28.01%), 951 were progesterone receptor positive (63.09%), and 121 had lost progesterone receptor status (8.13%).2=5.172, P=0.222). However, there were differences in education levels , location , family average revenue , economic status , social status , and awareness of breast cancer  between the two groups.Demographic characteristics for the case and control groups are shown in Table 2=8.244, P=0.004) and number of births . No significant differences were found for breastfeeding, number of miscarriages, and use of oral contraceptives , menstrual pattern , and marital status . However, there were significant differences between the two groups in postmenopausal status , benign tumor of the breast , galactophore hyperplasia , nipple discharge , and family history of breast cancer . Variables not associated with significant differences were diabetes mellitus (4.0% vs. 3.4%), inverted nipple (1.3% vs. 0.8%), and multiple breasts (1.3% vs. 2.0%).Table 2=5.862, P=0.015), tea drinking , and sleep satisfaction , but no differences in alcohol drinking (1.0% vs. 0.8%), coffee drinking (4.8% vs. 5.6%), and physical activity (71.6% vs. 74.1%)  were observed for: family history and present life satisfaction; WHR and present life satisfaction; and WHR and sleep satisfaction Table . It is iDevelopment of breast cancer is a complicated and continuous progress, characterized by multi-step, multi-factor, and environment-gene interactions in origin. Although many studies on breast cancer development have been conducted, reported results varied widely. This may be related to disparities in study designs, geographical features, and lifestyle and healthcare factors. It is important to investigate and clarify risk factors for breast cancer, especially manageable factors, with which better prevention strategies could be formulated.We described a case-control study involving 2978 Chinese Han women. In total, 75.8% of breast cancer cases were diagnosed as invasive ductal carcinoma, which is consistent with national and international reports. In China, invasive ductal carcinoma accounts for about 70% of all female breast cancers, whereas other tumor types  account for no more than 5% –18. In oOur study Figure  showed tPrevious studies demonstrated a genetic susceptibility to breast cancer. Females with a family history of breast cancer, especially among first-degree relatives, were more likely to develop breast cancer. Moreover, the risk was further increased in cases where more than one breast cancer case had been diagnosed among first-degree relatives , 23. In Obesity is another factor that contributes to the increasing incidence of breast cancer –27. The A dietary pattern that includes a high-fat component, soy, dairy products, meat, fruits, and vegetables is supposed to affect breast cancer development and progress, although no consistent conclusions have been reached –36. In oPsychological status should not be overlooked as a potential factor related to breast cancer development , 40. ManPrevious studies showed that poor sleep quality , was related to elevated risk of a variety of tumors –45. In oWe also investigated awareness of and knowledge about breast cancer-related symptoms and risk factors. Only 72.8% of participants knew breast cancer was a common cancer among females; 83.3% reported low awareness, and only 16.7% had high awareness. About 52.7% of women recognized a lump as a clinical manifestation of breast cancer, although only about 30.0% recognized other breast cancer-related symptoms such as breast discomfort, enlarged lymph nodes, nipple inversion, and nipple discharge. In addition, 63.3% knew that family history of breast cancer and long-term use of estrogen-like medicines were risk factors for breast cancer. The rates of awareness of other risk factors were below 30%. Correlation analysis suggested that high awareness was a protective factor for breast cancer, highlighting the importance and necessity of targeted publicity and education programs.Based on previous findings that obesity may be related to increased breast cancer risk and poorer outcomes, we explored the association between adipokines and breast cancer. Adiponectin is considered the key link between obesity and breast cancer , especiaOur study was a retrospective case-control study. As women self-reported their parity, breastfeeding, disease, and alcohol use histories, our findings may be subject to recall bias. To minimize recall bias, several similar questions were asked in different sections of the questionnaire. A 1:1 matched case-control design  was used to control for possible confounders, and all interviewers were required to complete standardized training. In future, we aim to validate the risk and protective factors identified in this study using a case-cohort study.We identified a comprehensive range of factors related to breast cancer. Among these there were several manageable factors that may contribute to breast cancer prevention. Future prospective studies are needed that consider psychological interventions, sleep regulation, health guidance, and physical exercise. In addition, a screening model for high-risk populations should be put on the agenda.We conducted a multi-center, hospital-based, case-control study of breast cancer among women in northern and eastern China. This study was funded by the Ministry of Health of the People’s Republic of China, and took place in 21 hospitals located in 11 provinces, from April 2012 to April 2013.The target population was female outpatients with breast cancer aged 25–70 years in 21 hospitals. Cases and controls were matched (1:1) on age (± 3 years), diagnosis hospital , and timing of examination (within 2 months). Inclusion criteria for breast cancer cases were: (1) newly diagnosed and histologically confirmed breast cancer; (2) Han ethnic group; and (3) females aged 25–70 years. Exclusion criteria for patients with breast cancer were: recurrent or metastatic breast cancer, complication of other malignant tumors by clinical or pathological diagnosis, and <25 or >70 years of age. Inclusion criteria for the control group were: (1) negative physical examination results; (2) negative ultrasound scans of breast and/or mammographic screening results; (3) no evidence of cancer or history of cancer; and (4) Han ethnic group. Patients who had a neoplastic disease at any other site, or history of cancer or other major chronic disease were excluded from the study. Data collection strictly adhered to the inclusion and exclusion criteria. After excluding those with inadequate information or missing data, 1489 case-control pairs were involved in this study.We developed a self-designed structured questionnaire to record information obtained from participants during face-to-face interviews. The interview questionnaire was based on: published articles; the Gail, Claus, and international models; and discussions with experts in breast surgery, epidemiology, statistics, nutrition, and molecular biology. To minimize recall bias, several similar questions were asked in different sections of the questionnaire. A preliminary investigation was performed to assess the practicality and effectiveness of the survey. After repeated revisions, the final interviewer-administered questionnaire comprised seven parts. (1) Demographic characteristics and female physiological and reproductive factors . (2) Chronic diseases and family history . (3) Lifestyle habits, including smoking (including passive smoking), alcohol intake, and dietary habits. (4) Medication and chemical exposure history . (5) Breast cancer-related knowledge . (6) Medical records, specifically, information gathered from the clinical breast examination . (7) Physical measurements .For each participant, a 4-ml non-fasting blood sample was collected using an EDTA vacutainer. After sedimentation, each blood sample was stored vertically in a freezer at −80°C. Total and HMW adiponectin levels were assayed from plasma using human total adiponectin and HMW adiponectin quantitative ELISA kits, respectively . All analyses were performed at the Central Research Laboratory, the Second Hospital of Shandong University. Testing of fasting plasma glucose, triglyceride, and total cholesterol were performed by the collaborating hospitals’ clinical laboratories.Interviewers were medical professionals and medical post-graduates. All interviewer candidates were required to complete standardized training and were certified to conduct independent surveys. To minimize recall bias, several similar questions were asked in different sections of the questionnaire; for example, we used date of birth and age (years) to express actual age, years of schooling and highest degree to express education level, number of pregnancies = number of births + number of abortions, number of children = number of boys + number of girls. Solutions to contradictions are shown in All procedures performed involving human participants were in accordance with the ethical standards of the Second Hospital of Shandong University Research Committee. Written informed consent was obtained from all participants by investigators as part of the interview.The database was established using Epidata 3.1 software . Frequencies and percentages were calculated for variables such as demographic characteristics, physiological and reproductive factors, chronic diseases and family history, lifestyle habits, medication and chemical exposure history, breast cancer-related knowledge, medical records, and physical measurements. We used Student’s t-tests and Pearson’s chi-square tests for the univariate analysis, and found 17 variables had significant differences . Multivariate conditional logistic regression analyses were used to stratify independent variables with ORs and 95% CIs. All data were analyzed using SPSS version 16.0 . A two-sided P-value <0.05 was considered to be statistically significant.


The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies.n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions.Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank  with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive  and invasive breast tumors .We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations  contains supplementary material, which is available to authorized users. An effective way to decrease breast cancer-related morbidity and mortality is to identify individuals who may be at increased risk of developing breast cancer and apply early intervention strategies. In addition to inherited gene mutations, there are several demographic factors that are associated with an increased risk of breast cancer including increasing age, being overweight after menopause, alcohol intake, having never been pregnant , earlier age at menarche, and a family history of breast cancer –4. HowevAPC and RASSF1 compared with women at low risk ), ranging from 0 (non-methylated) to 1 (completely methylated), using average probe intensity for the methylated (M) and unmethylated (U) alleles. Normalization and background correction of raw signals was performed using the  1.10.2) . Illumin 1.10.2) . Prior t 1.10.2) .n = 18) and The Cancer Genome Atlas Database  [n = 55, GSE66313) and invasive ductal carcinoma  to compare DNA methylation differences between normal-adjacent tissue and pre-invasive or invasive lesions [Independent breast tissue samples were available from the National Disease Research Interchange  . Raw intAll data analysis was conducted in R version 3.3.1.Differences in cellular composition across samples represent a potential confounder when testing associations between DNA methylation and quantitative traits in EWAS . Cellulalimma [M values) and breast cancer risk factors . Genome-wide significance was determined by taking into account the false discovery rate with a threshold of statistical significance set at Q = 0.01. We ran separate multivariate limma models both unadjusted and adjusted for putative cell proportions to assess the impact of cell proportion differences on significant associations and effect-size estimates. To identify loci that may be most confounded by differences in cell type we calculated the difference in the effect-size estimates  between the cell-type unadjusted and adjusted models.We used a multivariable linear models for microarray data (limma) procedure as described in the R/bioconductor library limma  to modelP value. We then computed the permutation distribution of these minimum P values .To test the associations between putative cellular proportions and subject metadata  we applied the methods described in Houseman et al. to fit a quasi-binomial model for each putative cell-type across the data set . More spQ < 0.01) and the background set was the genomic locations of the 390,262 CpGs used in the entire analysis. For the LOLA, the ENCODE transcription factor binding sites included 42 different chromatin immunoprecipitation sequencing experiments.To assess the enrichment of risk-factor-related CpG sites at cell-type-specific histone modifications we used the eFORGEv1.2 tool with the selected option of all H3 marks measured for the consolidated Roadmap to Epigenomics data set . To examDNA methylation age  of the Komen breast tissues was calculated using the Horvath and epigenetic timer of cancer (EpiTOC) methods , 30.P value = 2.0E-03), but not subject BMI or parity  in the unadjusted limma model for subject age was 4099 CpG sites compared with 787 from the adjusted model, suggesting that a large number of false-positives are likely to be reported when differences in cell proportions are not considered  the DNA methylation patterns across purified cell populations of myoepthial cells, luminal cells, and adipocytes were consistent, suggesting that age-related changes may occur largely independent of tissue type in the normal human breast  to examine the influence of subject age, BMI, and parity on the DNA methylome. Since the estimated cellular proportions for each sample sum to nearly one, we included all but the estimated cell-type with the smallest proportion to avoid multi-collinearity in our models. In a multivariable limma model adjusted for differences in cellular mixtures, 787 CpG sites were significantly associated with age, 0 CpG sites were associated with BMI, and 0 CpG sites were associated with parity, after correcting for multiple hypothesis testing , and found no significant associations (Q > 0.01) between family history and DNA methylation differences after correcting for multiple comparisons . In both validation cohorts, putative cell-mixture proportions were significantly associated with subject age (permutation P < 0.05)  and 18 normal breast tissues from disease-free women . Subject demographics and characteristics for these two data sets are presented in Table cis-regulation of gene transcription [n = 88). Many of the age-related CpG sites that localize to gene regions (n = 630 CpG sites) demonstrated strong associations with gene expression , and modest associations with other histone modifications  . We next used the genomic coordinates of age-related CpGs as a query set against the background of the 450 K array in LOLA scanning for enrichments of TFBSs. Since hypermethylation events are likely to be biologically distinct from hypomethylation events at TFBS we stratified our LOLA into a hypermethylation and a hypomethylation enrichment analysis  and hypomethylated with age  . First, we employed the eFORGE tool to identify cell-type-specific signals in diverse tissues profiled by the Roadmap to Epigenomics Consortium. We observed robust enrichment of H3K4me1, histone modifications that mark enhancers, in both fetal tissues and mammary epithelial cells . Age acceleration was not associated with any other of the evaluated risk factors (P > 0.05). In a multivariate model considering all measured cancer risk factors, we found that race was significantly associated with increased epigenetic aging (African American P = 4.9E-02). In contrast to the Horvath clock, there was no significant correlation between chronological age and epiTOC-predicted age  and in multivariate models including all risk factors shown in Table P = 2.7E-02, Hispanic P = 2.7E-02). The remaining breast cancer risk factors were not associated with epiTOC-defined biological aging in either univariate or multivariate models (P > 0.05).To examine whether the subject-specific differences between biological and chronological age  are associated with breast cancer risk factors we first calculated DNA methylation age from the 100 Komen normal breast tissue samples using two distinct epigenetic clocks , 30. BriP < 0.05). Importantly, changes at the age-related CpGs were greater . If the epigenetic defects in age-related DNA methylation are further deregulated in pre-invasive breast cancer it would be expected that progressive changes would occur in invasive breast cancer. To test this, we assessed differential methylation using limma models adjusted for subject age in TCGA breast cancer data set. A large proportion of the age-related CpGs exhibited significant differential DNA methylation changes in breast cancer )  , there were 268 CpG sites among 775 CpGs available for measure (34.5%) that demonstrated differential methylation between DCIS and normal tissue using limma models adjusted for subject age Fig. 5)) Fig. . Again, 5)) Fig. C and D a13) Fig. .Fig. 4DNIn this study, we identified perturbations in the normal breast epigenome that may contribute to age-related increases in breast cancer risk. Age is the strongest demographic risk factor for breast cancer and is robustly associated with DNA methylation changes. Emerging literature has demonstrated that aging exerts its profound effects on the epigenome through a lifetime accumulation of environmental exposures that interfere with the placement or removal of methyl groups , 38, 39.The ability to produce reliable biological age predictions in an individual and in specific tissues holds promise for monitoring health, predicting disease risk, and providing insights into modifiable lifestyle factors that promote healthy aging. Indeed, discrepancies between chronological and biological age may suggest deregulation in DNA methylation marks and indicate increased disease risk. Horvath et al. demonstrated this phenomenon of age acceleration in a recent publication, whereby researchers found that the epigenetic age of the liver was increased by 2.7 years for every 10 units of BMI . Using 4While our findings provide strong evidence for a link between epigenetic deregulation and the two processes of aging and cancer, our study had a few limitations. For example, the questionnaire administered by the Susan G. Komen Tissue Bank is well-equipped to accurately classify a subject’s age, while other cancer risk factors such as average alcohol consumption may be impacted by recall bias. This same limitation applies to the missing data on family history in 10 subjects. Further, the small number of non-white subjects decreased the power to identify relationships between age acceleration and race. Separately, although the RefFreeEWAS method effectively accounts for the largest sources of variation in the DNA methylation data set, the method is unable to discern the particular cell types in which epigenetic changes occur. That said, the robustness of cell-type-independent observation across multiple populations and progressive alterations in cancer gives us confidence that a subset of the epigenetic defects may be important in carcinogenesis. To this end, future prospective studies are needed to investigate the relationship between DNA methylation in normal tissue and the risk of developing breast cancer. Finally, mechanistic studies will also be needed to elucidate the epigenetic contribution to increased breast cancer risk. Research aimed at early detection and disease prevention would serve to relieve patient morbidity and reduce the cost burden to the healthcare system.In summary, we have shown that epigenetic differences are strongly associated with aging and these differences may reflect epigenetic defects that predispose women at an older age to an increased risk of breast cancer.Epidemiological studies have firmly established factors of personal choice and factors beyond personal choice that alter the risk of breast cancer. Established risk factors for breast cancer include age, reproductive and family history, and BMI , 40. IndAdditional file 1:Analytic framework for reference-free epigenome-wide association study between DNA methylation and breast cancer risk factors. (PPTX 77 kb)Additional file 2:A Hierarchal clustering and heat map representation of cellular proportions of putative cell-types (K = 6) in Komen normal breast tissue (n = 100). B Metadata associations with cellular proportions when K is estimated over a range of cell types. Permutation P values presented adjacent to the colored line representing each covariate . (PPTX 424 kb)Estimation of cellular proportions and its association with subject covariates. Additional file 3:Age-related CpGs and genomic annotation. (XLSX 169 kb)Additional file 4:right panels) the intensity of blue and red points indicate shift in the effect size of the limma coefficient estimate between adjusted and unadjusted models. That is, gray points in the right panels indicate CpG sites that are not impacted by differences in cellular proportions across subject age (n =100) (A), subject BMI (n = 100) (B), parity status (n =100) (C), and family history of disease (n = 90) (D). (PPTX 1367 kb)Volcano plots representing both cell-type proportions adjusted and unadjusted limma models for each covariate evaluated in the present study. In each cell-type-adjusted volcano plot  population (n = 97) (A), and normal breast tissue from the National Disease Research Interchange (NDRI) population (n = 18) (B). Volcano plots indicate CpG-specific associations between DNA methylation and subject age. Permutation testing of subject covariate data across estimated cell-types (K) in the TCGA population (C) and NDRI population (D). (PPTX 923 kb)Age-related DNA methylation in the human normal breast validated in adjacent-to-tumor normal breast from The Cancer Genome Atlas Age-related CpG sites are associated with gene transcription. Additional file 7:n = 787). (XLSX 136 kb)Complete results from eFORGE analysis of age-related CpGs . (XLSX 71 kb)Complete results from LOLA analysis of age-related CpGs  for age-related (n =787) and randomly selected loci (n = 787). C, D DNA methylation differences between invasive breast cancer and normal adjacent tissue in limma coefficients  for age-related (n = 787) and randomly selected loci (n = 787). (PPTX 970 kb)


It has been suggested that vitamin D might protect from breast cancer, although studies on levels of vitamin D in association with breast cancer have been inconsistent. Genome-wide association studies (GWASs) have identified several single-nucleotide polymorphisms (SNPs) to be associated with vitamin D. The aim of this study was to investigate such vitamin D-SNP associations in relation to subsequent breast cancer risk. A first step included verification of these SNPs as determinants of vitamin D levels.The Malmö Diet and Cancer Study included 17,035 women in a prospective cohort. Genotyping was performed and was successful in 4058 nonrelated women from this cohort in which 865 were diagnosed with breast cancer. Levels of vitamin D (25-hydroxyvitamin D) were available for 700 of the breast cancer cases and 643 of unaffected control subjects. SNPs previously associated with vitamin D in GWASs were identified. Logistic regression analyses yielding ORs with 95% CIs were performed to investigate selected SNPs in relation to low levels of vitamin D (below median) as well as to the risk of breast cancer.rs12239582) were found to have a statistically significant association with a low risk of breast cancer , and minor homozygotes of the same SNP were found to have a tendency towards a low risk of being in the group with low vitamin D levels . Results from stratified analyses showed diverse associations with breast cancer risk for a few of the tested SNPs, depending on whether vitamin D level was high or low.The majority of SNPs previously associated with levels of vitamin D showed a statistically significant association with circulating vitamin D levels. Heterozygotes of one SNP  contains supplementary material, which is available to authorized users. About 5–10% of breast cancers are considered hereditary, from which the known breast cancer genes account for 3–4% . It has Ecological and epidemiological studies have suggested a beneficial effect of relatively high vitamin D levels, owing to solar exposure, on breast cancer risk and survival –6. ThereResearchers investigating vitamin D-related SNPs and breast cancer risk have focused mainly on SNPs located in the vitamin D receptor (VDR) gene. Results derived from these studies have also been conflicting –16. SNPsBetween 1991 and 1996, all residents of Malmö, Sweden, born between 1923 and 1950 were invited to participate in a prospective cohort study. Among invited women, 43% participated, resulting in a female cohort of 17,035 women . The Maln = 764), were matched on age and date of inclusion with control subjects (n = 764), based on 1482 individuals (some were included twice because incidence density matching was applied)  in chromosome 11); compared with major homozygotes, heterozygotes in the group with lower levels of vitamin D had a slightly decreased risk of breast cancer , but in the high vitamin D group, a reverse effect with increased breast cancer risk was seen  . Heterozygotes with low levels of vitamin D were associated with an increased risk of breast cancer , whereas heterozygotes with higher vitamin D levels had a decreased risk of breast cancer , with a p value for interaction of 0.08 . Another locus  showed a tendency towards an association with breast cancer risk. The analyses stratified for level of vitamin D showed diverse associations with breast cancer risk for three of the tested loci: rs156299 , rs10741657  and rs10485165, depending on whether vitamin D level was high or low.In the present study, a majority of the selected SNPs were associated with vitamin D levels that were in agreement with those reported in previous studies. As for breast cancer risk, a statistically significant association was observed with one of the tested loci  were statistically significantly associated with a low level of vitamin D. Equally confirming results were seen for several other SNPs of other genome sites, such as PDE3B intron, CYP2R1, and NADSYN1 in chromosome 11. Borderline statistically significant associations were seen for further SNPs on ST6GALNAC3 in chromosome 1 and on MTMR4 in chromosome 17. SNPs in chromosome 6, chromosome 7 and chromosome 10 did not show any association with low vitamin D, in contrast to previous studies of children in Western Australia [There is no current consensus regarding which level of vitamin D should be considered insufficient; in addition, the level varies over the year owing to sun exposure. Grouping individuals by high or low vitamin D level by calendar month was considered pragmatic to calculate differences in vitamin D level owing to allele variants. SNPs located in the intron of the ficiency , 25–27. ustralia , 24. Thirs12239582 and rs2209458 in ST6GALNAC3, in chromosome 1, where minor homozygotes had a borderline statistically significant association with a decreased risk of low vitamin D level and heterozygotes with a statistically significant decreased risk of breast cancer  in chromosome 1 and breast cancer risk has not been reported previously. This SNP is positioned in ST6GALNAC3, a gene encoding a sialyltransferase which might affect DBP synthesis, concentration and function and that has previously been associated with levels of DBP [rs12144344 and vitamin D levels have not been reported.The association found in our study between s of DBP . No statGC, which is the gene encoding DBP [rs7041 and rs4588, both located in the GC gene, were previously studied in association with breast cancer risk [rs7041 and no association of rs4588 are consistent with those of one previous study in which researchers showed a similar statistically significant association [rs7041 and rs4588 and found a protective effect of some combinations for post-menopausal breast cancer [Previous studies on vitamin D-associated SNPs and breast cancer risk have been focused mainly on genetic variants of the vitamin D receptor, and a few studies have been done on ding DBP . The precer risk –35. Our ociation , as wellt cancer . Howevert cancer , 33, 35.In the present study, the common homozygote was used as a reference, and relative risks were calculated individually for heterozygotes and less common homozygotes. Others have used other approaches, calculating on the basis of risk alleles and/or grouping several genotypes together. Sometimes it is not clear which allele was used as a reference, which means that comparing results between studies is difficult.rs198300 and rs13245518, both proxies for rs156299 located on chromosome 7 upstream of gene encoding neuropeptide Y, a neurotransmitter involved in mediating physiological processes, including food intake and bone homeostasis, previously associated with levels of vitamin D [rs2060793 and rs1993116, proxies for GWAS rs10741657 located in CYP2R1, which encodes cytochrome P450, an enzyme that converts vitamin D to the circulating form 25(OH)D. rs10741657 was previously associated with vitamin D levels and insufficiency [rs10485165, a proxy for rs7763511 (in chromosome 6) previously associated with vitamin D level but with unknown function [When the association of analysed SNPs with breast cancer risk was stratified by level of vitamin D, some results differed depending on vitamin D level. This association was found for itamin D . In non-ficiency , 25, 26.We found no previous reports of associations of this kind. Because this was an exploratory part of the present study, and because vitamin D level was available for only a smaller fraction of cases and control subjects, we suggest that these findings ought to be regarded with caution. The findings must be replicated, but at the same time they indicate that vitamin D level may interact with genetic traits in breast cancer risk.All patients diagnosed with cancer within the boundaries of Sweden are reported to the Swedish Cancer Registry, which is regarded as a highly validated source of information . Thanks An individual’s vitamin D level is influenced by several factors, not only sun exposure. It has also been shown that levels decrease with increasing age and increasing BMI –40, whicDifferences noted when study populations were compared with regard to risk  previously associated with levels of DBP was associated with a slightly decreased risk of breast cancer, as was a tendency towards a decreased risk of low vitamin D level, in the present study. The allele frequency of the SNP variants is high, and therefore even a small increase in risk per individual may be a substantial population attributable risk. Further results of the present study show vitamin D level to be an effect modifier of the risk of breast cancer associated with certain SNPs. This suggests that an individual’s composition of SNPs may affect the extent to which levels of vitamin D are associated with breast cancer risk.Many previous findings of SNPs associated with vitamin D levels were reassuringly replicated in this study. One SNP Additional file 2: Table S1.Baseline characteristics of control subjects and subjects with and without data on SNPs/vitamin D. (DOCX 24 kb)


Breast cancer survivors are at high risk for fracture due to cancer treatment-induced bone loss, however, data is scarce regarding the scope of this problem from an epidemiologic and health services perspective among Chinese women with breast cancer.Breast Cancer Survivors cohort were enrolled from a large cancer hospital in Beijing. Eligibility criteria included age 50–70 years, initiation of treatment for breast cancer at least 5 years prior to enrollment, and no history of metabolic bone disease or bone metastases. Data collected included sociodemographic characteristics; fracture-related risk factors, screening and preventive measures; breast cancer history; and thoracolumbar x-ray. The matched comparator group was selected from participants enrolled in the Peking Vertebral Fracture Study, an independent cohort of healthy community-dwelling postmenopausal women from Beijing.We designed a cross-sectional study comparing prevalence of vertebral fractures among age- and BMI-matched women from two cohorts. Women in the p < 0.01). The majority had early stage (85.3%) and estrogen and/or progesterone receptor positive (84.6%) breast cancer. Approximately half of breast cancer survivors reported taking calcium supplements, 6.1% reported taking vitamin D supplements, and only 27% reported having a bone density scan since being diagnosed with breast cancer.Two hundred breast cancer survivors were enrolled (mean age 57.5 ± 4.9 years), and compared with 200 matched healthy women. Twenty-two (11%) vertebral fractures were identified among breast cancer survivors compared with 7 (3.5%) vertebral fractures in the comparison group, yielding an adjusted odds ratio for vertebral fracture of 4.16 (95%CI 1.69–10.21, Despite a four-fold increased odds of prevalent vertebral fracture among Chinese breast cancer survivors in our study, rates of screening for osteoporosis and fracture risk were low reflecting a lack of standardization of care regarding cancer-treatment induced bone loss. Breast cancer incidence worldwide has risen by 20% and mortality rates by 14% since 2008, with the bulk of this increase sustained by women in low- and middle-income countries (LMICs) due to increasing life expectancy, urbanization, and adoption of Western lifestyles . Data regarding history of recurrence were also obtained.Genant semi-quantitative technique [Thoracic and lumbar lateral spine x-rays were performed to identify prevalent vertebral fractures at the diagnostic imaging department of CHCAMS. For each participant, presence of vertebral fracture was ascertained by two radiologists using the validated echnique . In thisFasting serum samples were collected from each participant and stored at -80 °C until batch testing at the end of the study period. We tested 25-hydroxy vitamin D (25OHD) levels, and the bone formation marker pro-collagen type 1 N propeptide (P1NP), and the bone resorption marker serum β-c-terminal telopeptide of type 1 collagen (CTx) to assess bone metabolism. All biomarkers were assayed at the Di’an laboratory in Beijing, China using an automated Roche electrochemiluminescence immunoassay .For each participant, data were extracted from the PK-VF Study database regarding participant age, BMI, education level, parity, menstrual history, smoking, alcohol use, history of bilateral ovariectomy, parental history of fracture, personal history of fracture, and calcium supplement use. As part of the PK-VF Study, prevalent vertebral fractures were also ascertained using lateral thoracolumbar x-ray, and evaluated by two experienced radiologists using the Genant semi-quantitative method described above. Serum biomarkers, CTx, P1NP, and 25OHD, were batch tested at the central laboratory of the Department of Endocrinology, Peking Union Medical College Hospital, by an automated Roche electrochemiluminescence immunoassay  .2 or Fisher’s exact test, and continuous variables were compared using the t-test. Univariate logistic regression was further used to calculate the odds of vertebral fracture based upon breast cancer status, age, BMI, level of education, parity, personal history of fracture, calcium supplement use, and 25OHD level. We fit the multivariable model using backward elimination beginning with all variables that were significant  in the univariate analyses  [p > 0.05) one at a time beginning with the least significant; in each step, remaining parameter estimates remained largely unchanged (< 20%). Among the cohort of breast cancer survivors, we further performed subgroup analyses based upon fracture status using χ2, Fisher’s exact test, and the t-test as appropriate.All statistical analyses were performed using Stata Intercooled 13 . Descriptive statistics were used to report the sociodemographic characteristics, frequency of fracture, and fracture-related risk factors in the two cohorts. Categorical variables were compared using χ2 (Table p = 0.004). Among breast cancer survivors the most commonly reported site of fracture was the wrist . One patient reported a history of hip fracture and only one self-reported a history of vertebral fracture. Other reported fracture sites included the ankle, lower leg, foot, finger, coccyx, knee, toes, and ribs. Thirty-three percent (6/18) of fractures were reported as low-trauma fractures. More breast cancer survivors reported using calcium supplements . Only 12/200 (6%) of women with breast cancer reported using vitamin D supplements.In total, 200 survivors of breast cancer were enrolled and 200 matched healthy women were selected from the PK-VF cohort. The mean age of both cohorts was 57.5 ± 4.9 years and over half of women had a BMI above 24 kg/m2 Table . Women ip < 0.001) (Table p < 0.001) among breast cancer survivors, and 0.78 (p < 0.001) among the PK-VF cohort demonstrating appropriate coupling of bone formation and resorption in both groups.Mean levels of 25OHD were significantly higher among the breast cancer survivors compared with women in the PK-VF Study  (Table p = 0.002) compared with women in the PK-VF cohort.Table 6) Table . In the Table p = 0.036), and had a lower level of education . Although our sample was not powered to formally evaluate differences in fracture status based upon treatment class, we observed that vertebral fractures were detected among 7/74 (9.4%) of women receiving AIs only, 5/54 (9.3%) receiving SERMs only, 1/16 (6.3%) who had received both an AI and SERM, and in 3/16 (18.8%) women who had been treated with ovariectomy. Of note, all three of the ovariectomized women with fractures were premenopausal at the time of diagnosis and had concurrently been treated with a SERM. 8/54 (14.8%) of women who received radiation treatment had a vertebral fracture, however 7 of those women were also concurrently treated with some form of endocrine therapy.Stratified analyses based upon fracture status, showed that women with fracture were older and therefore more likely to be postmenopausal at the time of diagnosis . By contrast, incidence of fractures among those treated with tamoxifen did not differ  compared with those not treated with endocrine therapy. Unfortunately, this study is significantly limited by the lack of description regarding how osteoporotic fractures were defined and measured, and sparse risk factor data.To our knowledge only one prior study has been published from mainland China examining this issue . This stThree studies from Taiwan using retrospective data from their National Insurance Research Database have also examined this issue among ethnically Chinese women with breast cancer. Tzeng et al. reported tamoxifen use was associated with reduced risk of osteoporotic fractures, however did not specify whether women in their cohort were pre- or postmenopausal . Chang ePrevious epidemiologic studies from Asian countries have demonstrated that the peak age of breast cancer diagnosis occurs approximately a decade earlier among Asian women compared with their Western counterparts , 21. TheAs a population, Chinese women undergoing treatment for breast cancer will become vulnerable to fracture at a younger age relative to their Western counterparts, during a period when comparatively little attention is typically given to fracture risk reduction. Furthermore, even when increased fracture risk is identified, management algorithms are less straightforward for premenopausal women compared with postmenopausal women. In 2012, Hadji et al. published a review of the complexities of cancer treatment-induced bone loss (CTIBL) among premenopausal women, and proposed a framework with which to approach this problem including indications for DXA screening and calcium and vitamin D supplementation . HoweverFrom a practice perspective, our study underscores the importance of awareness of fracture risk associated with breast cancer therapies both on the part of the provider and patient. Although fracture rates were relatively high, due to the asymptomatic nature of most vertebral fractures, the vast majority of women and their providers were not aware of these fractures. While guidelines exist in China for screening and management of CTIBL, it is apparent that gaps remain in terms of uptake of these recommendations in practice. Indeed, among breast cancer survivors with a prevalent vertebral fracture, less than 50% had obtained a DXA scan. While 49% of breast cancer survivors were on calcium supplements, only 9% were taking vitamin D supplements. Given age and postmenopausal status are known risk factors for fractures, it is not surprising that overall, more fractures occurred among women who were postmenopausal at the time of diagnosis. However, it is important to note that 8/108 (7.4%) of breast cancer survivors who were premenopausal at the time of diagnosis were also found to have vertebral fractures, which underscores the excess risk for fractures in this population. While studies have demonstrated bisphosphonates may be effective for mitigating CTIBL, such studies have not been powered to measure impact on fracture rates –25.Our study has several limitations that warrant mention. First, the PK-VF cohort has a few key differences compared with the breast cancer survivors cohort. It was enrolled 5 years earlier than the breast cancer survivors cohort, and carried out by a separate group of investigators and radiologists. However, the principal investigator of the PK-VF was a collaborator on this study and extensive care was taken to parallel the methodologies of the two studies to ensure comparability of findings. Second, although all breast cancer survivors were > 50 years of age, at baseline 6.5% of women in the breast cancer survivors cohort were not yet in menopause. This group also had higher education levels, earlier menarche, lower parity rates, lower personal fracture history, higher rates of calcium supplement use, and higher baseline 25OHD levels. During the design of our study, to avoid potential bias due to overmatching, we did not choose to match our controls based upon each of these factors and instead, took them into account in our regression analyses. As these characteristics would have been expected to be associated with lower risk of fracture, if anything, our findings would underestimate the risk of fracture among Chinese breast cancer survivors. Third, given the cross-sectional design of our study, our data do not allow for calculation of incidence rates of vertebral fracture over time, nor change in laboratory parameters. Our study was also not powered to look for subgroup analyses based upon treatment regimen, therefore we are unable to provide formal comparisons of fracture rates by type of treatment or duration of those treatments. Finally, our study population was limited to breast cancer survivors presenting for routine follow up at a major cancer hospital in Beijing, and therefore may not generalizable to all Chinese women with breast cancer, nor to other regions of China.In summary, our study found breast cancer survivors in China have a four-fold increased odds of prevalent vertebral fracture compared with age- and BMI-matched healthy women. However, rates of screening for osteoporosis and fracture risk by DXA were low reflecting a lack of standardization of care regarding screening, prevention and treatment of this problem. Future longitudinal studies are needed to elucidate the fracture risk specific to women who are premenopausal at the time of diagnosis and how risk relates to treatment regimens. Finally, infrastructure limitations such as lack of access to DXA imaging at cancer hospitals remain important barriers to timely intervention.


More than 15 years after the publication of the sequence of the human genome, the resulting changes in health care have been modest. At the same time, some promising examples in genetic services become visible, which contribute to the prevention of chronic disease such as cancer. These are discussed to identify barriers and facilitating factors for the implementation of genetic services. Examples from oncogenetics illustrate a high risk of serious disease where prevention is possible, especially in relatives. Some 5% of breast cancers and colorectal cancers are attributable to an inherited predisposition. These cancers occur at a relatively young age. DNA testing of relatives of affected patients may facilitate primary and secondary prevention. Training of non-genetic health care workers and health technology assessment are needed, as is translational research in terms of bringing genomics to health care practice while monitoring and evaluating. Stratified screening programs could include cascade screening and risk assessment based on family history. New roles and responsibilities will emerge. A clear assessment of the values implied is needed allowing to balance the pros and cons of interventions to further the responsible innovation of genetic services. In 2016, the cost of a whole genome sequence was close to 1,000$. However, this does not include the analysis of the enormous amount of data. Those who were disappointed about the modest consequences for clinical medicine should remember the First Law of Technology: we invariably overestimate the short-term impacts of new technologies and underestimate their longer term effects . The aim of this study is to discuss how public health can benefit from promising examples of genetic testing, such as in cases of hereditary forms of breast and colorectal cancer, and what barriers and facilitating factors should be addressed for a successful implementation.BRCA-related breast cancer and colorectal cancer in Lynch syndrome families as monogenic subtypes of common cancers (If the limited resources (both staff and funding) available in clinical genetics require a focus on priorities, considerations of medical benefit, health need, and costs apply . This is cancers . A risk Ethical and legal issues have been discussed in relation to cascade screening strategies, especially the active approach of family members. To what extent is there a right “not to know” vs. a “duty to warn” , 6? NewsBRCA1 and BRCA2 predispose for breast cancer. Many countries have breast cancer screening programs where mammography is offered to women above a certain age limit, for instance, starting at 50 years in the Netherlands. Women with a mutation in the BRCA1-gene or BRCA2-gene may develop cancer at a younger age. Their risk to develop breast cancer before 80 years of age is 66–67%  or primary prevention (chemoprevention by aspirin). Since the cancers prevented in these inherited cancer syndromes tend to occur at a relatively young age, the number of  life years saved is relatively high.Public awareness is another facilitating factor. It can be increased by a famous person such as Angelina Jolie who found herself in the position of being at risk of hereditary breast and ovarian cancers.Furthermore, initiatives to train relevant health care professionals including public health genetics can open doors. If already some ground work has been done, the next steps may be easier.A first field where public health and genetics meet is cancer screening. Cascade screening as discussed above could be developed in a program, but risk assessment in a broader sense is conceivable. Would it be possible to develop a breast cancer screening program stratified according to risk profile ? As soonIn colon cancer screening, many countries are now integrating universal colorectal cancer tumor screening in health care to identify families at higher risk of colorectal cancer due to Lynch syndrome . An impoA second field where public health and genetics meet is genetic education. If the public at large is to profit from recent developments in genetics, any physician or health worker should be able to answer questions about genetics relevant for daily practice. The relatively small number of registered clinical geneticists will not be able to teach all physicians in face-to-face training sessions. Smart solutions such as online modules, webinars, and websites are needed , 18. TheA third field where public health and genetics meet is the study of the cost-effectiveness or health technology assessment in a broader sense. This would include the assessment of values such as the extent to which patient perspectives have been taken into account, life years gained, quality adjusted life years, and percentage of cases identified. We need to discern which applications are hype vs. hope. If limited resources are available, which need prioritization? Both naïve believers and individuals who are resistant to change may be encountered as barriers for implementation of genetic services. To further the responsible innovation of genetic services a clear assessment of the relevant indicators and the implied values is needed allowing to balance the pros and cons of interventions . A clearTo conclude, in cancer screening, genetic education, and (economic) evaluations, much work needs to be done for both the public health and genetics communities to address barriers and make use of promising developments to further a successful and responsible implementation of genetic services in public health.Both MC and CE were involved in conception and design of the work for the PRECeDI project on integrating genetics in the prevention of chronic diseases. MC drafted the presentation and manuscript. CE revised it critically. Both authors approve the final version and accountable for all aspects of the work.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.


Early age at menarche, nulliparity, late age at first completed pregnancy, and never having breastfed, are established breast cancer risk factors. However, among breast cancer subtypes, it remains unclear whether all of these are risk factors for triple-negative breast cancer (TNBC).in situ Study, or the Women’s Learning the Influence of Family and Environment Study. We used multivariable polychotomous unconditional logistic regression methods to conduct case-control comparisons among breast cancer subtypes defined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression status.We evaluated the associations of these reproductive factors with TNBC, in 2658 patients with breast cancer (including 554 with TNBC) and 2448 controls aged 20–64 years, who participated in one of the three population-based case-control studies: the Women’s Contraceptive and Reproductive Experiences Study, the Women’s Breast Carcinoma Ptrend = 0.006), but age at menarche, age at first completed pregnancy, and nulliparity were not associated with risk of TNBC. Parous women who breastfed for at least one year had a 31% lower risk of TNBC than parous women who had never breastfed . The association between breastfeeding and risk of TNBC was modified by age and race. Parous African-American women aged 20–44 years who breastfed for 6 months or longer had an 82% lower risk of TNBC than their counterparts who had never breastfed .TNBC risk decreased with increasing duration of breastfeeding (Our data indicate that breastfeeding decreases the risk of TNBC, especially for younger African-American women. Breast cancer occurs more frequently than any other type of cancer in women worldwide, with an estimated 1.4 million new cases annually –10.The luminal A-like subtype is the most frequent subtype and accounts for 62–67% of invasive cases; TNBC is the second most common subtype and accounts for 10–25% of invasive cases , 12. ComBRCA1 mutation [Here, we evaluated the associations between TNBC and age at menarche, number of completed (longer than 26-week gestation) pregnancies, age at first completed pregnancy, and breastfeeding in an analysis pooling data from three case-control studies. We compared the risk estimates for TNBC with other ER/PR/HER2-defined specific subtypes, especially luminal A-like breast cancer. We also explored whether the associations between these factors and risk of TNBC differ by race  and age . Among our three source studies, one has published data on reproductive factors  and the risk of breast cancer by ER/PR/HER2 status  and has mutation . No datamutation .in situ (BCIS) Study [Eligible participants for this analysis were women who had previously participated in one of the three population-based case-control studies - the Women’s Contraceptive and Reproductive Experiences (CARE) Study , the WomS) Study , or the S) Study .The Women’s CARE Study, which was supported by National Institute of Child Health and Human Development (NICHD), was a population-based, case-control study designed to examine risk factors for invasive breast cancer among USA-born white women and African-American women . The agein situ breast cancer who were identified by random digit dialing from August 1994 through December 1998 and were frequency-matched to the expected distribution of patients with breast cancer in strata defined by 5-year age groups, race (white or African-American) and geographic region of residence [Control participants were women with no history of invasive or esidence . The paresidence . The Womn = 567). The questionnaire developed for the Women’s CARE Study was used to interview women with BCIS, and both studies were conducted during the same time period by the same interviewers and with ER, PR and HER2 status determined in the same central laboratory using the same classifications (see subsequent text). No additional controls were recruited for the BCIS Study. LA control participants from the Women’s CARE Study were deemed eligible to be controls for the BCIS Study. For the analysis presented here, we excluded 37 case participants with lobular carcinoma in situ  because LCIS is not included in the clinical definitions of in situ breast cancer [The Women’s BCIS Study investigated risk factors for BCIS among USA-born white women and African-American women who resided in LA County . Case pan = 1794). Of 1794 case participants, 1167 had ER/PR/HER2 status abstracted from pathology reports. Control participants were women ages 20–49 years who had no history of invasive or in situ breast cancer. Recruitment of control participants did not begin until 1 July 2000. Control participants were individually matched by race (white and African-American), age (within 5 years and ages 20–49 years), and neighborhood to the subset of case participants who were diagnosed between 1 July 2000 and 31 May 2003 (n = 444). The Women’s LIFE Study used an expanded version of the Women’s CARE Study questionnaire, which was modified to include additional risk factors .The Women’s LIFE Study investigated genetic and epidemiologic risk factors for invasive breast cancer in USA-born white women and African-American women who resided in LA County , 38. CasFor all three studies, detailed information prior to the reference date on reproductive factors and covariates involved in this analysis was collected by trained staff who administered standardized, in-person interviews using structured questionnaires. The reference date for a case participant was the date of breast cancer diagnosis; the reference date for a control participant was the date on which she was identified by random digit dialing in the Women’s CARE Study, or the date of initial contact in the Women’s LIFE Study.After pooling the data from three source studies, 2716 case participants with data on receptors and 2478 control participants were potentially eligible. We excluded 58 case participants and 30 control participants for whom information was missing on age at menarche , parity , duration of oral contraceptive use , education , body mass index , recreational physical activity , smoking status (2 cases), and alcohol intake . This resulted in 2658 case participants  and 2448 control participants  available for the current pooled analysis.The ER/PR/HER2 status in breast tumors in the Women’s CARE Study and the Women’s BCIS Study was determined in a centralized pathology laboratory at USC using immunohistochemistry (IHC) methods , 40. ForThe ER/PR/HER2 status abstracted from pathology reports for case participants in the Women's LIFE Study was assessed by many pathology laboratories. These laboratories might have used different methods or different cut-off points for positive receptor status, which could cause concerns about consistency with the biomarker data from the centralized pathology laboratory at USC. We previously validated the SEER registry ER/PR status data for 1048 Women’s CARE case participants in the centralized pathology laboratory at USC, which showed that the agreement between the centralized laboratory and SEER registry classification was substantial for both ER/PR , and that the associations between risk of ER/PR breast cancer subtypes and parity, age at first completed pregnancy, and breastfeeding were similar regardless of the source of information on ER/PR .We assessed the associations between TNBC (ER-/PR-/HER2-) or the other three subtypes of breast cancer defined by ER/PR/HER2 status , and the following factors: age at menarche, number of completed (longer than 26-week gestation) pregnancies, age at first completed pregnancy (defined for each woman as the age at which that pregnancy ended), and duration of breastfeeding. We estimated odd ratios (ORs) and corresponding 95% confidence intervals (95% CIs) using multivariable polychotomous unconditional logistic regression for case-control comparisons .Tests for trend were conducted by fitting ordinal values corresponding to exposure categories and testing whether the slope coefficient differed from zero. We also conducted Wald chi-square tests for homogeneity of the associations with reproductive factors across breast cancer subtypes by fitting a multivariable polychotomous unconditional logistic regression model for dichotomous or ordinal variables.2), a variable combining menopausal status and hormone therapy use , lifetime recreational physical activity  hours/week), alcohol intake , cigarette smoking status , and oral contraceptive use .We included the following factors, selected a priori, as potential confounders in all multivariable models: source study , study site , race , education as a proxy for socioeconomic status , age , family history of breast cancer , no first-degree family history), BMI  and number of completed pregnancies  as potential confounders when they were not the exposure of interest. When parity was the exposure of interest, we chose women who had never been pregnant as our reference group and treated women who had been pregnant but had never carried to completed pregnancy as a separate group that was excluded when testing for trend across categories of parity. Only parous women were included in analyses of age at first completed pregnancy and breastfeeding. Models for parous women were also mutually adjusted for number of completed pregnancies , age at first completed pregnancy , and duration of breastfeeding .Using two major subtypes - TNBC and luminal A-like breast cancer, we explored whether any associations of reproductive factors differed by race (white women or African-American women) or age group (younger (<45 years) or older (≥45 years) women). In stratified analyses by age, the variable for combining menopausal status and hormone therapy use was only included in the models for older women. When testing the effect of breastfeeding by both race and age, we combined two categories  of longer duration of breastfeeding into one (≥6 months) to avoid having groups with small numbers of study participants after stratification.th St. Gallen International Breast Cancer Conference (2013) Expert Panel recommendation to define ER+/PR+/HER2– as the luminal A-like subtype [in situ breast cancer are different from those associated with the results presented here, we repeated our analyses after excluding all in situ breast cancer cases.As the commonly used definition (ER+ and/or PR+ plus HER2–) of the luminal A-like subtype probably includes both luminal B-like and luminal A-like tumors, we also used the 13 subtype , and repP value <0.05 as statistically significant. All analyses were performed using the SAS statistical package .In reporting the results of trend tests or homogeneity tests, we considered a two-sided n = 554, 20.8%), luminal A-like , luminal B-like , and HER2-enriched  , or HER2-enriched (Ptrend = 0.56) breast cancer, but was associated with luminal A-like subtype (Ptrend = 0.009). Women whose menarche occurred at age 14 years or later had a 23% lower risk of luminal A-like breast cancer  than women whose menarche occurred at age 12 years or earlier. Number of completed pregnancies was not associated with the risk of TNBC (Ptrend = 0.26), but was inversely associated with the risk of the other three subtypes .Age at menarche was not associated with TNBC (Ptrend = 0.05). There was a statistically significant inverse association between longer duration of breastfeeding and TNBC (Ptrend = 0.006) and luminal A-like cancer (Ptrend = 0.004), but no association with the other two subtypes of breast cancer (both Ptrend ≥0.28).Among parous women, older age at first completed pregnancy was not associated with any ER/PR/HER2-defined specific subtypes, except for the luminal A-like subtype (P for homogeneity of trends = 0.19), parity (P for homogeneity of trends = 0.09), age at first completed pregnancy (P for homogeneity of trends = 0.24), or duration of breastfeeding (P for homogeneity of trends = 0.08).None of the differences in trends across the four subtypes of breast cancer was statistically significant for age at menarche (Ptrend = 0.02) and number of completed pregnancies (Ptrend = 0.0005).Among either white women or African-American women, risk of TNBC was not associated with age at menarche number of completed pregnancies, or age at first completed pregnancy Table . HoweverPtrend = 0.01) and the luminal A-like subtype (Ptrend = 0.04) among the African-American women. Among parous African-American women, breastfeeding for 12 months or longer compared to never breastfeeding was associated with a 45% lower risk of TNBC , and 43% lower risk of luminal A-like breast cancer .Longer duration of breastfeeding was modestly associated with a lower risk of TNBC in parous African-American women and a lower risk of the luminal A-like subtype in both parous white women and parous African-American women; however, statistically significant dose-response relationships were only observed in TNBC (P for homogeneity of trends = 0.02); none of the other factors differed significantly between these two subtypes (P for homogeneity of trends ≥0.10).The difference in trends across TNBC and luminal A-like breast cancer was only statistically significant for age at first completed pregnancy among parous white women . Number of completed pregnancies was inversely associated with the risk of luminal A-like subtype cancer among both younger (Ptrend = 0.02) and older women (Ptrend = 0.006). Among older parous women, age at first completed pregnancy was positively associated with the risk of the luminal A-like subtype (Ptrend = 0.01); those who had their first completed pregnancies at or after age 30 years had a higher risk of the luminal A-like subtype than those who had their first completed pregnancies at or prior to age 20 years . Duration of breastfeeding was inversely associated with the risk of both TNBC and luminal A-like breast cancer subtypes in both younger and older parous women.Similar to the results for all age groups combined, early age at menarche, nulliparity, and older age at first completed pregnancy were not associated with risk of TNBC in either younger or older women (P for homogeneity of trends = 0.03).The differences in trends across TNBC and luminal A-like breast cancer were statistically significant only for the number of completed pregnancies among younger women  Expert Panel, were similar to data presented in the tables for the more commonly used definition (ER+ and/or PR+ plus HER2–) of the luminal A-like breast cancer subtype. Moreover, when we excluded women diagnosed with in situ breast cancer, we found that the results for TNBC were unchanged.The results for ER+/PR+/HER2– breast cancer, the modified definition of the luminal A-like subtype recommended by the 13In our pooled analysis of data from three population-based case-control studies of women aged 20–64 years, we found that longer duration of breastfeeding was associated with decreased risk of both TNBC and the luminal A-like subtype, especially for TNBC among younger (20–44 years) parous African-American women. Younger parous African-American women who breastfed for 6 months or longer had an 82% lower risk of TNBC than their counterparts who had never breastfed. If this is verified in future research, promotion of breastfeeding, especially in younger African-American women may have a particularly strong impact given the higher risk of TNBC among African-American women –16.Breastfeeding has been proposed to protect against breast cancer through hormonal mechanisms that include postponing the resumption of ovulatory menstrual cycles after a pregnancy , reducinOur finding of a protective effect of a longer duration of breastfeeding against the risk of TNBC agrees with results from six previous case-control studies , 27, 32,Longer duration of breastfeeding was also associated with decreased risk of the luminal A-like subtype in our pooled analysis. Among previous studies that reported results on the association between breastfeeding and the luminal A-like subtype, breastfeeding was associated with decreased risk of the luminal A-like subtype in three case-control studies , 15, 22,It has been well-documented that estrogen and progesterone play important roles in breast tumorigenesis –60, and Previous epidemiologic data on the impact of these three factors on the risk of luminal A-like cancer and TNBC are inconsistent, particularly for TNBC. Early age at menarche was not associated with risk of TNBC in three case-control studies , 23, 27,African-American women are more likely than white women to be diagnosed with TNBC, especially at a young age (<45 years) –16. In tOur data also provide some evidence that breastfeeding may be important for limiting the increased risk of TNBC among parous African-American women, but not among parous white women (data not shown). The observed racial difference in our results for the inverse association between breastfeeding and risk of TNBC could not be explained by breastfeeding duration; African-American controls and case participants with TNBC had a shorter duration of breastfeeding on average than did white controls and case participants with TNBC . The observed difference in breastfeeding results between African-American women and white women could be related to the differences in other characteristics, such as genetic susceptibility, and requires further study.Strengths of this pooled analysis include its size, especially the large number of case participants with incident TNBC. Furthermore, the data used in this analysis were collected by trained interviewers, who administered standardized, in-person interviews using structured questionnaires, which were nearly identical across three source studies.Several limitations of the current study must be considered. First, approximately 36% of our case participants had missing data on at least one of the receptors . We compared our measures of reproductive factors in case patients with and without known ER, PR, or HER2. No statistically significant differences were detected for age at menarche, number of completed pregnancies, or duration of breastfeeding, whereas case patients with information on ER/PR/HER2 were 0.9 years older on average at first completed pregnancy than those with missing information on ER, PR, or HER2 (data not shown). The small difference in average age at first completed pregnancy is unlikely to have altered the observed associations differentially by tumor subtypes.Second, ER/PR/HER2 status in two of our source studies , 35 was in situ hybridization (FISH) analysis in the Women’s CARE Study and the Women’s BCIS Study. Based on previous validation results from the same centralized USC pathology laboratory, 7.4% of breast tumors with HER2 gene amplification in FISH analysis were false negative by 10H8-IHC (scored as 0 or 1+) and 9.7% of breast cancers without HER2 gene amplification in FISH analysis were false positive [A final limitation is that IHC was used to assess HER2 protein overexpression without validation by fluorescent positive . These mIn this pooled analysis, longer duration of breastfeeding was associated with decreased risk of TNBC especially in younger parous African-American women, suggesting a potential role of breastfeeding in prevention of TNBC among these women.


Breast education programs that lead to increased awareness have an impact on reducing breast cancer–related mortality and is an inexpensive and important strategy,2 regardless of the availability of mammography and adjuvant therapy. We believe that in addition to education about self- and clinical breast examination, emphasis should be focused on the importance of reducing modifiable risk factors and, more importantly, the education of health care providers. This is particularly important as low- and middle-income countries increasingly adopt a Western lifestyle, which increases the risk of breast cancer.3 In addition, community-level health care providers should be a reliable source of breast health education in any setting.The recent article by Kennedy et al4 To better understand breast cancer awareness and knowledge of risk factors in the general population, we surveyed 98 individuals in Arusha, Tanzania. The participants were patients, visitors, and nurses present in a general outpatient waiting room at a private general hospital. The survey was written in both English and Swahili  and asked dichotomous questions about whether patients were aware of breast cancer and whether they believed that it could be treated if diagnosed early. Breast cancer risk factor knowledge was assessed by a multiple-choice question that listed both true risk factors and known common regional misconceptions. Most of the participants were female (62.2%), the median age was 35 years , 69% had a secondary education or higher, and 85.7% reported being employed or a student , and most were aware it could be treated if detected early (93%). However, in the multiple-choice question, a notable 65% chose keeping money under the brassiere as a risk factor for breast cancer, including 77% of nine nurses surveyed . Fifty pOn the basis of the survey findings, we extended the risk factor questions to 44 medical staff members in the community, including physicians, clinical officers, assistant medical officers, and nurses. We found that 76% identified keeping money under the brassiere as a risk factor for breast cancer, which was consistent with the belief of the general population. We then provided an educational session to health care providers with a focus on risk factors and early signs of cancer.1 our study highlights the emergent need for breast cancer education of the general population in countries with limited health care resources. The study also emphasizes the need for cancer-related risk factor education for community-level health care providers because providers are key in educating patients about prevention and early detection of breast cancer, especially in areas with limited screening and adjuvant treatment capabilities.Similarly to Kennedy et al,