Genome-wide association studies, focusing primarily on unilateral breast cancer, have identified single nucleotide polymorphisms (SNPs) in a number of genomic regions that have alleles associated with a significantly increased risk of breast cancer. In the current study we evaluate the contributions of these previously identified regions to the risk of developing contralateral breast cancer. The most strongly disease-associated SNPs from prior studies were tested for association with contralateral breast cancer. A subset of these SNPs, selected upon their main effects on contralateral breast cancer risk was further evaluated for interaction with treatment modalities and estrogen receptor (ER) status.We genotyped 21 SNPs in 708 women with contralateral breast cancer and 1394 women with unilateral breast cancer who serve as the cases and controls in the Women's Environment, Cancer and Radiation Epidemiology (WECARE) Study. Records of treatment and ER status were available for most of WECARE Study participants. Associations of SNP genotypes and risk for contralateral breast cancer were calculated with multivariable adjusted conditional logistic regression methods.FGFR2 locus were significantly associated with contralateral breast cancer, including rs1219648  = 1.25, 95%CI = 1.08-1.45). Statistically significant associations with contralateral breast cancer were also observed at rs7313833, near the PTHLH gene , rs13387042 (2q35) , rs13281615 (8q24) , and rs11235127 near TMEM135 . The A allele of rs13387042 (2q35) was significantly associated with contralateral breast cancer in ER negative first tumors while the A allele of rs11235127 (near TMEM135) was significantly associated with contralateral breast cancer in ER positive first tumors. Although some SNP genotypes appeared to modify contralateral breast cancer risk with respect to tamoxifen treatment or particular radiation doses, trend tests for such effects were not significant.Multiple SNPs in the Our results indicate that some common risk variants associated with primary breast cancer also increase risk for contralateral breast cancer, and that these risks vary with the ER status of the first tumor. Patients with breast cancer are two to five times more likely to develop a second primary cancer in the contralateral breast than are unaffected women to develop an initial breast cancer -4. Estabn = 708) serve as cases and women with unilateral breast cancer (UBC)  serve as controls [ATM gene were associated with an increased risk of CBC in women who were treated with radiation for their first primary breast tumors [Our study builds on an existing, population-based, epidemiologic study of CBC: the Women's Environment, Cancer, and Radiation Epidemiology (WECARE) Study. The WECARE study is a nested case-control study in which women with CBC  and 1,394 controls (women with UBC) recruited through five population-based cancer registries: one registry covering all of Denmark and, in the US, one registry covering Iowa, two registries covering three counties in Southern California, and one registry covering three counties in Washington State. The controls were individually matched 2:1 to cases on date and age at diagnosis of the first primary breast tumors, race, and registry region and were counter-matched on registry-reported RT. Each case-control triplet consists of two women who received RT and one who did not, maximizing informativeness for radiation exposure . For 12 Medical records were retrieved to obtain detailed information on the treatment of the first breast cancer  during the at-risk period (time between first and second primary diagnoses) and other tumor characteristics, including ER status ,6.Estimated absorbed radiation doses to various specific contralateral breast locations were reconstructed for each treatment regimen by using tissue-equivalent phantoms and modeling procedures as described previously ,12. DosaP < 10-7). These were rs2981582, rs12443621, rs8051542, rs889312, rs3817198, and rs13281615 from Easton and colleagues [P = 0.001 in three populations) in Easton and colleagues [Twenty-one SNPs were selected from among those most associated with breast cancer in three prior GWASs -9. All oP value of less than 0.001 were also excluded.Multiplex SNP genotyping was carried out with the Illumina Golden Gate™ assay on the Sentrix Array Matrix scanned with the Bead Array Reader . To avoid any bias in the calling of genotypes, all DNA samples were coded and laboratory personnel were blinded to case-control status. A blinded 24% re-sampling was carried out for quality control purposes and these samples were interspersed throughout the plates. Negative controls lacking DNA were included on each plate. Results from a subset of SNPs previously genotyped by an individual SNP method, the MGB Eclipse™ Probe System , were concordant with the multiplex genotyping results. Samples with more than 10% missing genotypes and SNPs with more than 5% missing genotypes were excluded from analysis. Although Hardy-Weinberg equilibrium may not strictly apply given that all participants in the study were affected with breast cancer, SNPs deviating from Hardy-Weinberg equilibrium with a FGFR2 intron 2 region near SNP rs2981582 were selected from the HapMap Project [2 rof greater than 0.8. The program PLINK [Common SNPs  in the  Project . The Tagam PLINK  was usedP value for trend was from the log-additive model. The test for trend in Table The WECARE Study is based on a standard nested case-control design with the additional feature of counter-matching on radiotherapy status. All analyses were conducted with multivariable adjusted conditional logistic regression methods, incorporating an 'offset term' or weight to adjust appropriately for the counter-matched sampling . For theP < 10-7): rs2981582  = 1.20, 95% confidence interval (CI) = 1.04 to 1.40) and rs1219648  at 10q26 in the FGFR2 gene, rs13281615 in the 8q24 region , and rs13387042 in the 2q35 region . We observed statistically significant associations with CBC at two of the remaining 12 SNPs: rs7313833, located on chromosome 12p in the vicinity of the PTHLH gene , and rs11235127 on chromosome 11q14.2 near TMEM135 . Excluding BRCA1/2 carriers  fromFGFR2, we genotyped five haplotype-tagging SNPs, in addition to rs2981582, in all 2,102 available WECARE Study participants and found alleles at four of the six SNPs that were individually associated with risk of CBC . The risk variant at rs11235127 (near TMEM135) was associated with CBC only in individuals with ER-positive first tumors .For SNPs with a TOX3 (TNRC9), rs7313833 near PTHLH, and rs2107425 (H19) appeared to be associated with increased risk for CBC in particular dose-allele combinations , there were no obvious trends by either gene or radiation dose. Finally, none of the SNPs studied significantly modified the effects of tamoxifen use or chemotherapy (data not shown) on the incidence of CBC.There were no significant modifying effects of SNP genotype on CBC risk associated with radiation dose to the contralateral breast for those patients receiving RT for their first cancer Table . AlthougBreast cancer GWASs have tended to focus on high-risk individuals such as women from multiple-case breast cancer families or with early age at onset. The present study extends findings from these studies by examining another group of genetically predisposed women, those with CBC, and considers important treatment co-factors such as RT, chemotherapy, and hormonal exposures as well as ER status of the first tumor.FGFR2), 8q24, and 2q35, all of which had been previously replicated in studies of UBC [We observed nominally significant associations between CBC and SNPs in three regions previously associated with breast cancer, 10q26 (s of UBC -28. Rs13FGFR2 gene (rs2981582) and CBC with an RR of a magnitude similar to that observed in the original GWAS report [FGFR2 intron 2 region with haplotype-tagging SNPs in the WECARE Study population suggested that increased risk for CBC was not restricted to rs2981582 but was associated with a single haplotype, carrying risk alleles at multiple SNPs, consistent with other reports [Our initial genotyping data revealed an association between an SNP in the S report . Further2 gene rs81582 and reports .PTHLH, parathyroid hormone-like hormone, which is located 28 kb centromeric to the reference SNP, and KLHDC5, Kelch domain-containing protein 5, which is located 127 kb telomeric to the reference SNP. In HapMap CEU (Utah residents with Northern and Western European ancestry from the Centre d'Etude du Polymorphism Humain collection) { data, rs7313833 is contained within a region of linkage disequilibrium extending in the centromeric direction to include the relatively small PTHLH gene. PTHLH has a broad range of functions, including roles in mammary gland development and tumor progression [KLHDC5 suggests no obvious connection to breast development or breast cancer.Among SNPs genotyped in the WECARE Study which were as yet unconfirmed in other studies, the strongest associations with CBC risk were at rs7313833 on chromosome 12p11.22 and at rs11235127 on chromosome 11q14.2. For both of these SNPs, risk was particularly elevated in homozygotes for the minor allele. Although these two SNPs were among the 30 most significantly associated with breast cancer risk in a prior GWAS [gression ,33, makiTMEM135, which is located 54 kb centromeric to the reference SNP and which encodes a transmembrane protein that is not well characterized. The nearest gene in the telomeric direction is RAB38, a Ras family member that is expressed exclusively in melanocytes and that is located more than 700 kb away. We considered TMEM135 to be a candidate gene since studies in the mouse indicate that it is transcriptionally regulated upon treatment with tamoxifen, an estrogen analog and therapeutic agent that is associated with a decreased risk of CBC in the WECARE Study population [TMEM135, or any other SNP studied on the effect of tamoxifen on CBC risk.On chromosome 11, rs11235127 is not located in an evolutionarily conserved region, but there are several uncharacterized, apparently spliced ESTs (expressed sequence tags) in the region. The nearest annotated gene is pulation . HoweverATM gene further increased the risk of CBC specifically in those women who received RT for their first cancer. Accordingly, in the present study, we assessed whether radiation dose-associated CBC risk was modified by genotype; owing to small sample numbers in individual categories, we could not further stratify by age. Overall, the tests for interaction were not statistically significant, although some specific genotype-dose combinations were. For rs3803662 near the TOX3 gene, homozygotes for the reference allele had a statistically significantly increased risk for CBC with radiation dose, whereas carriers of the minor allele did not show statistically significantly increased risk of CBC or dose response. Currently, the function of TOX3 is not well understood and its relevance to radiation response is not clear. The only evident connection between TOX3 and breast cancer is that the gene has been implicated in breast tumor metastasis to bone [PTHLH, who received RT had a significantly increased risk of CBC. Interestingly, PTHLH has also been implicated in breast tumor metastasis to bone in a mouse model [H19 [In WECARE Study participants who had received RT, the mean radiation dose to the contralateral breast during treatment was 1.1 Gy. In our previous studies of this population, we observed that women who were under 40 years of age and who received more than 1.0 Gy of absorbed dose to a specific quadrant of the contralateral breast had a significant, 2.5-fold greater risk for CBC developing in that quadrant than unexposed women . The pre to bone . Homozygse model . Owing tFGFR2), rs3803662 in (TOX3), rs13387042 (in 2q35), and rs13281615 (in 8q24) were more strongly associated with ER-positive disease than ER-negative disease [TMEM135) associations with CBC risk displayed statistically significant heterogeneity with regard to ER status. In our study, 27.4% of the CBC cases had an ER-negative first primary tumor and 23.6% of the UBCs were ER-negative overall [TMEM135) with regard to ER status.Previous studies have assessed whether SNP associations with breast cancer vary with ER status: rs2981582  control population; controls in the study are women with diagnosed UBC. Although women with CBC represent a high-risk breast cancer group, the power to detect main effects of common risk alleles on CBC is limited by the likely enrichment for these same alleles among the UBC patients who serve as controls in the study.Our study demonstrates that a subset of common variants with moderate effects on breast cancer risk is also associated with an increased risk for CBC. In particular, we identified nominally significant associations with CBC for six of 21 SNPs implicated in prior GWASs of UBC. Two of these SNPs were significantly associated with CBC risk, depending on ER status of the first tumor. Particular combinations of SNP genotypes with radiation dose were associated with increased risk for CBC, although there were no statistically significant interactions with increasing dose and genotype. These findings underscore the need to consider breast cancer tumor characteristics and treatment modalities when examining the risk associated with individual SNPs in order to help characterize their role in breast carcinogenesis.CBC: contralateral breast cancer; CI: confidence interval; ER: estrogen receptor; GWAS: genome-wide association study; RR: rate ratio; RT: radiation therapy; SNP: single-nucleotide polymorphism; UBC: unilateral breast cancer; WECARE: Women's Environment: Cancer: and Radiation Epidemiology.Breast Cancer Research. All other authors declare that they have no competing interests.LB is an editor of SNT participated in study design, data acquisition (molecular genetics) and interpretation, and manuscript drafting and revision. JLB participated in study design, manuscript revision, and study supervision. ASR and MC participated in statistical analysis and interpretation of the data and in manuscript revision. RWH, LB, CFL, MS, SAS, and LM participated in data acquisition and manuscript revision. KEM participated in data acquisition and manuscript revision and provided critical manuscript revision. XL, JM, and XH participated in data analysis and manuscript revision. AS and DD participated in data acquisition (molecular genetics) and manuscript revision. JDB participated in data interpretation and critical manuscript revision. DCT participated in statistical analysis and interpretation and in critical manuscript revision. PC participated in study design, data interpretation, critical manuscript revision, and study supervision. The WECARE Study Collaborative Group participated in recruitment and data acquisition. All authors read and approved the final manuscript.Memorial Sloan-Kettering Cancer Center : Jonine L Bernstein , Colin Begg, Marinela Capanu, Xiaolin Liang, Jennifer Brooks, and Anne R ReinerCity of Hope  : Leslie Bernstein and Laura Donnelly-AllenDanish Cancer Society : Jørgen H Olsen, Michael Andersson, Lisbeth Bertelsen, Per Guldberg, and Lene MellemkjærFred Hutchinson Cancer Research Center : Kathleen E MaloneInternational Epidemiology Institute  and Vanderbilt University : John D Boice Jr.Lund University : Åke BorgMount Sinai School of Medicine : Barry S Rosenstein and David P AtencioNational Cancer Institute : Daniela SeminaraNew York University : Roy E ShoreNorwegian Radium Hospital and University of Oslo : Anne-Lise Børresen-DaleTranslational Genomics Research Institute (TGen) : David Duggan and Ashley SiniardUniversity of California at Irvine : Hoda Anton-Culver  and Joan LargentUniversity of California at Los Angeles : Richard A GattiUniversity of Iowa : Charles F LynchUniversity of Southern California : Robert W. Haile, Graham Casey, Daniel Stram, Duncan C Thomas, Jane Figueiredo, Shanyan Xue, Nianmin Zhou, and Anh T DiepUniversity of Southern Maine : W Douglas ThompsonThe University of Texas, M.D. Anderson Cancer Center : Marilyn Stovall and Susan SmithUniversity of Virginia  : Patrick Concannon, Sharon N Teraoka, Eric R Olson, and Nirasha RamchurrenFGFR2 SNPsTable S1. Risk of Contralateral breast cancer associated with . Table S2: Risk of Contralateral breast cancer associated with FGFR2 haplotypes.Click here for file


Three intron located SNPs with heterozygous genotypes also had decreased risk of breast cancer. Seven of the 51 IGF1R SNPs were in LD and in one haplotype block, and were likely to be associated with breast cancer risk. Overall, this case-control study demonstrates statistically significant associations between breast cancer risk and polymorphisms in IGF1R gene.The insulin-like growth factor (IGF) signaling pathway plays an important role in cancer biology. The IGF 1 receptor (IGF1R) overexpression has been associated with a number of hematological neoplasias and solid tumors including breast cancer. However, molecular mechanism involving IGF1R in carcinogenic developments is clearly not known. We investigated the genetic variations across the IGF1R polymorphism and the risk of breast cancer risk in Korean women. A total of 1418 individuals comprising 1026 breast cancer cases and 392 age-matched controls of Korean were included for the analysis. Genomic DNA was extracted from whole blood and single nucleotide polymorphisms (SNPs) were analyzed on the GoldenGate Assay system by Illumina’s Custom Genetic Analysis service. SNPs were selected for linkage disequilibrium (LD) analysis by Haploview. We genotyped total 51 SNPs in the IGF1R gene and examined for association with breast cancer. All the SNPs investigated were in Hardy-Weinberg equilibrium. These SNPs tested were significantly associated with breast cancer risk, after correction for multiple comparisons by adjusting for age at diagnosis, BMI, age at menarche, and age at first parturition. Among 51 IGF1R SNPs, five intron located SNPs  with homozygous genotype (variant genotype) were associated with decreased risk of breast cancer. Fisher’s combined  Breast cancer is commonest among malignancies affecting women worldwide and has become second most common cancer among Korean women IGF1 and IGF1R have been reported to play roles in the early transformation of mammary cells, induction of mammary epithelial hyperplasia in a transgenic mouse model, and breast cancer cell growth cells Population-based studies show that circulating IGF-I and IGFBP-3 concentrations as well as genes for IGF and IGF1R have been associated with longevity, cancer and common chronic diseases The ethical approval for this study was granted from the institutional review boards at both Asan Medical Center and at National Cancer Center, Republic of Korea. All participants completed in-person interviews with structured questionnaires after they had given informed consent. Participants provide their written informed consent to participate in this study.A total of 1418 individuals comprising 1026 breast cancer cases and 392 healthy controls of Korean were included for the analysis. The breast cancer cases were recruited from the Breast Cancer Clinic at Asan Medical Center and National Cancer Center, Korea between March 2001 and June 2008. This case population had been identified as having a histopathologically confirmed diagnosis with a first primary breast cancer. Ineligibility criteria included previous malignancy (at either the same site or a different site) and old age greater than 80 years. Control subjects were randomly selected from a cohort of 6,000 health examinees. Controls were women free of any malignancy neoplasms. Data were collected on many potential risk factors of breast cancer, such as years of education, religion, age at menarche, menopausal status, age of first pregnancy, parity, history of benign breast disease, family history of breast cancer, oral contraceptive use, estrogen replacement therapy, smoking, and alcohol consumption.Genomic DNA was extracted from 4 ml whole blood using the QIAamp DNA Blood Maxi Kit  according to the manufacturer’s instructions.http://www.hapmap.org] with HapMap option for selection of tagging SNP; location within 10 kilobases (kb) of one of the candidate genes  as well as all of the exons and introns; and minor allele frequency of at least 5% in a Han Chinese or Japanese population (to ensure sufficient power). Also included were several non-synonymous SNPs listed on Cancer panel [http://www.illumina.com] on the GoldenGate Assay system by Illumina’s Custom Genetic Analysis service  that were not in HapMap but had been validated and had a minor allele frequency of above 5% in a Han Chinese or Japanese population. Subsequently, we selected genes that encode proteins in cellular pathways that are likely to be involved in breast carcinogenesis. The major pathways we studied were steroid hormone metabolism and signaling, cytokine and growth factors, transcription factor, double strand break DNA repair, oxidative damage repair, drug response, xenobiotics metabolism, and cell-cycle control. Based on a subsequent screen using PubMed [http://www.ncbi.nlm.nih.gov/pubmed], 285 SNPs were selected from are functional or probably functional, have demonstrated a previous association with breast cancer.The selection of specific single nucleotide polymorphisms (SNPs) for this study involved several steps. The list of candidate SNPs was created based on the following criteria: inclusion in the International HapMap Project database . Linkage disequilibrium (LD) was assessed by Haploview [http://www.broad.mit.edu/mpg/haploview/]. Hardy–Weinberg equilibrium (HWE) for each SNP was tested by comparing the observed and expected genotype frequencies of the controls (χ2-test). Differences in demographic variables, selected variables, and frequencies of the genotypes between the cases and controls were evaluated by using the t-test for continuous variables and the chi-square test for categorical variables. Associations with breast cancer risk were evaluated by computing odd’s ratios (OR) and corresponding 95% confidence intervals (95% CI) by logistic regression . Covariates considered included age at diagnosis, BMI, age at menarche, age at first parturition. Additive models of effect were applied to all SNPs; tests for trend were conducted by coding for the number of variant alleles and reporting the p-value from models based on logistic regression analyses. Dominant or recessive effect models and p-values were also calculated when suggested to be appropriate for particular SNPs. Associations between haplotypes and breast cancer risk were analyzed with Haploview; additive, dominant, and recessive models of effect were also evaluated. All statistical tests were two tailed, and p-values ≤0.05 were interpreted as statistically significant. The p-value for each gene was based on Fisher’s combination.Haplotypes were constructed from genotypes of 51 SNPs of IGF1R gene by using PHASE [A total of 1418 women were included in the current study; enrolled from two hospitals of the study, National Cancer Center, Ilsan and Asan Medical Center, Seoul, Korea . The numTotal fifty one SNPs in the IGF1R gene were examined for association with breast cancer in the study. Information and estimates of frequency and distribution of the 51 IGF1R polymorphisms are shown in Polymorphisms of interest were then selected for further analysis to address whether associations with breast cancer risk were consistent when stratified by study confounders. r2 greater than 0.72. Minor allele homozygotes for these SNPs tended to be associated with breast cancer risk and were found to be in one haplotype block  and haplotype distribution among these SNPs. The LD structure of the 7 polymorphic IGF1R SNPs evaluated in the current study is shown in pe block . A signipe block . The disp = 0.0003). Eight SNPs with heterozygous genotypes also had decreased risk of breast cancer. Considering the status of IGF1R polymorphisms our results may help in improving the ability to develop effective treatment modalities with reference to associated risk breast cancer. To the best of our knowledge, these IGF1R gene polymorphisms have not been previously evaluated for associations with breast cancer susceptibility.The IGF pathway and its receptor component IGF1R appears to play important roles in breast cancer progression and tumor growth. A case-control study was conducted to first comprehensively evaluate IGF1R genetic variants in relation to breast cancer risk and then to assess any promising associations among study population. Four IGF1R SNPs  were found to be associated with breast cancer risk in the study populations as well as significant in combined analyses , and one in coding region (rs12916884) were significantly associated with the breast cancer risk. Also four IGF1R SNPs with homozygous genotype (variant genotype) had decreased risk of breast cancer. The multivariate analysis adjusted for age at diagnosis, BMI, age at menarche, and age at first parturition manifested the association of risk with rs11635251 (0.57(0.38–0.89)) and rs12916884 (0.54(0.35–0.81)). The pairwise LD and haplotype distribution analyses showed that seven polymorphic IGF1R were in LD and in one haplotype block. These seven IGF1R SNPs  were likely to be associated with breast cancer risk. The most common haplotypes in the IGF1R gene were ‘‘AAG’’ and ‘‘GCT’’ among over all subjects in the study. Overall, these results confirmed a significant association between IGF1R SNPs and the risk of breast cancer.Cytoplasmic IGF1R expression in epithelial cells of normal breast tissue has been positively associated with subsequent risk of breast cancer A limitation of this study is smaller sample size and thus statistical power of our study was limited in the stratified analyses in the small sample size of the subgroups. Although this study reports genetic polymorphisms on the IGF1R on breast cancer in Korea for the first time, the findings need to be validated in further studies with larger sample size or in meta-analyses, which is also aimed in our laboratory for years to come.This case-control study identifies tagSNPs in the IGF1R gene and demonstrates their statistically significant associations with the risk of breast cancer risk in a population of Korean women. The identification of single biomarker correlating with the response to IGF inhibitors is unlikely. However, the status of IGF1R gene polymorphisms and their plausible roles in the IGF signaling promoting cancer growth augments the importance of our findings. This may help in improving the ability to develop effective treatment modalities for those conditions in which IGF1R gene is involved. To the best of our knowledge, this study is the first to demonstrate the association between IGF1R polymorphisms with the risk of breast cancer.


BRCA1 or BRCA2 (BRCA1/2) mutation were four times more likely to develop breast cancer compared to women in the general population. Preventive measures and risk factors for breast cancer development in these high-risk women have not been evaluated to the same extent as BRCA1/2 positive women. Further, there is virtually no scientific evidence about best practices in their management and care. The proposed study will examine a role of genetic and non-genetic factors and develop the systems and parameters for the monitoring and surveillance necessary to help establish guidelines for the care of this high-risk population.We previously reported that women from high-risk families who tested negative for a BRCA1/2 mutation in the family). All eligible participants will be mailed a study package including invitation to participate, consent form, a research questionnaire to collect data regarding family history, reproductive and lifestyle factors, as well as screening and surgery. Usual dietary intake will be assessed by a diet history questionnaire. Biological samples including toenail clippings, urine and blood samples will be collected. These women will be followed every two years by questionnaire to update exposure information, screening practices, surgical and chemoprevention, and disease development.To achieve our goals, we will assemble and follow a Canadian cohort of 1,000 cancer-free women with a strong family history breast cancer (defined as two or more relatives affected by breast cancer under the age of 50, or three or more relatives diagnosed with breast cancer at any age from one side of the family and with no BRCA1/2 mutation.Findings from this study will serve to help establish clinical guidelines for the implementation of prevention, counseling, and treatment practices for women who face an elevated risk of breast cancer due to family history, but who do not carry a  BRCA1 and BRCA2 (BRCA1/2). Genetic screening provides the opportunity for women identified as carriers of a deleterious mutation to consider various risk-reducing options  [BRCA1/2 mutation [BRCA mutation-negative should follow the same preventive measures as women with an identified BRCA1/2 mutation.Approximately 10-15% of all breast cancers are estimated to have a hereditary component  and a famoxifen) . Despitemutation . This fiBRCA1/2 mutation [BRCA1/2 mutation carriers, no elevated risk of ovarian cancer or any other cancer was found in these females.We previously evaluated the risk of breast cancer among 1,492 women with a strong family history of breast cancer , but who tested negative for a mutation . We repoBRCA1/2 mutations. Preventive measures and risk factors for breast cancer development have not been evaluated to the same extent and there is currently virtually no scientific evidence about best practices in their management and care. Although risks in these women are not as high as those for BRCA mutation carriers (40% lifetime risk compared to 80%), they remain significantly higher than risks in the general population (11% lifetime risk) and thus highlight the need to improve surveillance and preventive measures specifically for these women [BRCA1 or BRCA2 mutation, or at moderate risk of cancer due to a strong family history [There are currently no protocols in place for the counseling of women from high-risk families without se women -11. The se women . Until nse women -15. Year history -19. Desp(‘Negative Study’) (funded by the Canadian Breast Cancer Foundation). This study was designed to address various research questions including: 1) what factors  influence risk for breast cancer development; 2) to what extent are screening or preventive measures being utilized in this high-risk group; 3) what are the benefits of preventive programs in reducing the incidence of breast cancer development in this high-risk group?To this end, we initiated a multi-centered, hospital-based, cohort study of high-risk/mutation negative women identified through genetic counseling clinics in Ontario BRCA1 or BRCA2[BRCA1 and BRCA2, which represents the current testing protocol in cancer genetic clinics.Approximately 20-25% of familial breast cancers can be attributed to a germline mutation in  or BRCA2,20. Thus or BRCA2 resultin or BRCA2-36. A coBRCA1 c.5279_5333del55) which is difficult to detect with short read sequences (unpublished data). The prevalence and importance of genetic variation in these additional genes has yet to be evaluated in a large sample of women from families with a strong family history of breast cancer. Using this panel, we plan to evaluate the prevalence of these mutations within our cohort.Our group has developed and previously validated the BrOCaP gene panel sequencing in our molecular genetics research laboratory at the Women’s College Research Institute for the screening of all the known BrOCaP genes. This panel targets 411 coding exons (plus 10 bp from the introns in each side of an exon) of 25 genes . Theactivity ,41 as weactivity  and folaactivity .Biological markers  of risk including mammographic density and levels of circulating sex hormone levels will also be assessed as these have been shown to be important predictors of breast cancer risk in the general population as well as other high-risk populations -46. It i‘Negative Study’ is a large prospective cohort study on high risk women from BRCA1/2 mutation negative families. A detailed study design is presented in Figure The 1. To determine whether the probands of the participants in the ‘Negative Study’ carry a mutation in any of the genes in the BrOCaP gene panel.2. To evaluate the association between hormonal and reproductive factors and the risk of breast cancer.3. To evaluate the association between dietary and lifestyle factors and the risk of breast cancer.4. To evaluate the level of breast cancer risk reduction with prophylactic surgery and use of selective estrogen receptor modulators or other chemopreventive agents .5. To evaluate the extent to which screening measures  are being utilized and whether they influence breast cancer risk.6. To evaluate the association between various biomarkers of risk  and the risk of breast cancer.BRCA1/2 mutation. An example of an eligible family and relatives is displayed in Figure BRCA1/2 mutation if one female relative with breast cancer has been confirmed by direct DNA sequencing to not be a carrier of a mutation. Families are ineligible for the study if any relative has tested positive for BRCA1/2 mutations. Women in families that meet the above criteria are evaluated for their eligibility based on the eligibility and exclusion criteria outlined.Participants are currently being recruited from the following cancer genetic centres in Southern Ontario: Hamilton Regional Cancer Centre, Mt. Sinai Hospital, Sunnybrook Health Sciences Centre, Princess Margaret Hospital, Lakeridge Health and Women’s College Hospital (WCH), the latter of which is the main research site for this study. Recruitment at the cancer genetic centres occurs by one of two methods: 1) prospective enrolment in clinic or by 2) retrospective chart review 5. Have at least one first degree relative with breast cancer 6. Have at least one relative with breast cancer receive a negative result for a BRCA1/2 mutation7. No prophylactic mastectomy at recruitmentExclusion criteria:1. Women from families where a BRCA1/2 mutation has been identified after enrolment2. Women who cannot provide a blood and urine sample at a local laboratoryThe recruitment methods are:1. Prospective Recruitment: Eligibility of study participation is determined at the time of genetic test results disclosure. If the proband or one of her first degree female relatives is eligible, a brief explanation of the study goals, rationale and what study participation entails is provided by the genetic counsellor. Probands that are interested in the study are given a study package with contact information for the research coordinator at WCH for additional information about the study, and are asked to contact WCH directly.2. Retrospective Recruitment: The study coordinator from WCH reviews pedigrees of probands that have had genetic testing at one of the participating centres in the past and have tested negative for a BRCA1/2 mutation. Once eligibility of the proband or her family has been determined, the proband is sent a study invitation letter describing the goals of the study and whether she is or any of her relatives are eligible. If the proband or a relative is interested in participating, she is asked to contact WCH directly using the information provided in the letter. All contacted probands who have not responded to the letter after 6 months are contacted by a research coordinator by telephone as a follow-up.Once an eligible participant has been enrolled in the study, they are required to provide informed consent, complete a baseline research and diet history questionnaires (DHQ), provide a blood, urine and toenail clippings samples, and consent to a follow-up questionnaire every two years to update exposure information and ascertain incident diseases Figure .i) Exposure Data Collected By Questionnaire: Data collection by questionnaire occurs at enrolment and every 2 years thereafter until death or completion of a 10 year period, whichever comes first. All study participants are mailed a consent form, a baseline questionnaire and a DHQ at the time of enrolment. The baseline questionnaire collects extensive information on many potentially important exposures and covariates, as well as family history of cancer screening and prophylactic surgeries, and has been used by the “Risk factor analysis for Hereditary Breast Cancer” study which currently has over 15,000 women enrolled. Participants are also sent a DHQ which is a food frequency questionnaire that was developed by staff at the Risk Factor Monitoring and Methods Branch at the National Cancer Institute and reflects Canadian food availability and food fortification practices . The cohii) Biological Specimen Collection: Participants have the option to have their samples collected at one of several LifeLabs locations across Ontario. All women are encouraged to come to the centre as close to the time of completion of the baseline questionnaire as possible (within the first 2 years that they are actively enrolled in the study). Briefly, two blood samples are collected in tubes (one tube without additives and one EDTA-containing tube) labelled with the subject’s study number and delivered immediately to the laboratory for processing. Serum is collected, aliquotted and stored at -80°C. The EDTA-containing tube is inverted several times and stored at -80°C for future DNA extraction. An 80 mL urine sample is also collected, aliquotted and stored at -80°C. Samples are similarly collected at LifeLabs and are delivered to WCH every three months on dry ice. All samples are stored in -80°C freezers located in WCH which are alarmed and continuously monitored. Toenail clippings are collected at the time the baseline questionnaire is completed and are mailed to WCH in a sealed envelope along with the completed consent and questionnaire at the time of enrolment.iii) Diagnostic Information: All study participants are asked for written permission at the time of enrolment to review their medical records and pathology reports to collect detailed information on screening, treatment, and diagnoses. Once a participant reports a cancer diagnosis during any follow-up cycle, the hospital/clinics are sent a requisition for the patient’s medical records. Necessary data will be extracted from the records by the investigator or trained study personnel on as needed basis. Cancers can be diagnosed through clinical means, including physical examination, through screening  or through prophylactic surgery. To identify cases in non-respondents who died, we will request permission from the next of kin to seek medical records for incident cancers.Cumulus 4 software as previously described [iv) Mammographic Density: Digital mammograms from the providing centres will be requested and computer-assisted measurement of mammographic density will be carried out using escribed . Measurei) DNA Extraction: In the WCH lab, the extracted PBL is thawed and then processed to extract archive quality germline DNA. Gentra Puregene DNA extraction kits from Qiagen are used for DNA extraction. The concentration of the extracted DNA is measured by a Nanodrop instrument. Extracted DNA will be stored in a lab standard 4°C refrigerator which allows for storage of whole DNA samples in a single tube without the need to frequently freeze and thaw the sample which will reduce its quality dramatically. This germline DNA sample will be used for all genetic tests in this cohort study.‘Negative Study’ cohort will also be tested for the same mutation. The BrOCaP gene panel sequencing has been developed and validated by Dr. Akbari in our molecular genetics research laboratory at WCRI for screening all the known BrOCaP genes (Table ii) BrOCaP Gene Panel Sequencing: The BrOCaP gene panel will be tested on identified probands with breast cancer from eligible families which will allow identification of a larger proportion of mutation carriers of BrOCaP genes. DNA has been collected from these women previously at the time of their genetic testing, and is available for research purposes. All probands have provided written consent for research testing of their sample. If a proband is found to have one of the BrOCaP gene mutations, the unaffected relative who is a part of the es Table .iii) Sex Hormone Levels: Plasma estrogen, androgen and progesterone levels will be assayed in the laboratory of Dr. R. Casper  using radioimmunoassay kits (Vitros immunodiagnostics).The collection, storage, updating, and retrieval of all clinical data for the study subjects occur at the WCRI in Toronto. Upon consent to participate, subjects are assigned a study number which will identify their data. Names are removed from the data, and are verified and edited before entered into a computer file. The data are entered into an Access Database and are secured on a server that is only accessible to members of the centre. Appropriate backup is regularly maintained. Cyrillic 3 software  is used to update pedigrees based on the information provided at follow-up. To protect the privacy of our participants, any files, biological specimens, results or questionnaires with identifying data are stored in locked cabinets for up to 15 years, after which, they will be destroyed and disposed of properly. Names are removed from biological specimens and are identified by subject number and appropriately disposed of after analysis.BRCA mutation carriers. For continuous variables, we calculated the RR for extreme quantiles using the formula by Chapman and Nam [Our target enrollment is 1,000 women. Based on preliminary data, we expect the incidence of breast cancer to be 1% per year, or 10% over a 10-year period with an expected number of 250 cancers. For all power calculations, we performed 2-sided tests with α = 0.05 and estimated the minimum detectable risk estimate at a power of 80%. Given that exposure data for the cohort to be evaluated in the current study is not yet available, we utilized exposure information from our prior publications of  and Nam . For theP-values for interaction.Data analyses will be performed to complement specific objectives 1 through 6 outlined above. Various exposures which are planned to be assessed have been summarized in Table Multivariate modeling will take into account both plausibility of biological effects and statistical evidence of confounding. We will include well-established predictors of breast cancer in multivariate modeling . Other covariates whose biological effects or potential to confound are less certain will be examined individually in stratified analyses as applicable. We will use a 10% change in the parameter of interest as the criterion to select covariates into the final model .BRCA negative families, we initiated a prospective cohort study to evaluate genetic and non-genetic determinants of risk and to determine the appropriate screening and prevention practices for these women [BRCA1/2 mutation, there is little evidence to guide practitioners on how women from families with breast cancer history and without BRCA1/2 mutations should be managed [Based on our earlier findings of a significantly increased risk of breast cancer among women from high-risk/se women . While t managed . Such op managed . Of critIn the current study, priority will be given to the integration of the comprehensive BrOCaP gene panel into our protocol. Since recruitment is currently underway, we are proposing to sequence the probands of the participants for possible mutations in the additional 23 genes known to contribute to risk of breast and ovarian cancer as a first step. Should mutations be identified, this will allow us to offer relatives from these high-risk families additional genetic testing, and help identify those women who may also be at risk of developing breast cancer due to a mutation. This will be a significant finding for these families. Meanwhile, by collecting information on reproductive, hormonal and lifestyle factors, as well as chemoprevention with aromatase inhibitors or selective estrogen receptor modulators, we will be in a position to evaluate a role of modifiable factors that may lead to accurate and effective preventive interventions specifically for this high-risk group of women. We also propose to address the relationship between various purported biomarkers of breast cancer  and whether endogenous or exogenous exposures modify any possible relationships.BRCA-negative women thus having high potential to influencing public health recommendations surrounding genetic testing, screening and prevention. The multidisciplinary approach of this study will allow for communication with other researchers and departments to share ideas and foster collaborations. In addition, the interaction with many health centers will support the translation of our research findings into clinical practice.The proposed project will improve the understanding of the biology of this disease and the mechanisms through which risk factors may alter risk. More importantly, our findings stand to make significant contributions to the field of cancer prevention in high-risk/BRCA1/2 mutation status) or the lack of eligible females in the family . Additional barriers to recruitment have included the inability to contact eligible subjects directly due to the ethical need to recruit only through the proband directly, along with incorrect or missing contact information for the proband. The need for research ethics board approval at each centre has also been a constraint. Nonetheless, by including additional centres and introducing prospective recruitment, we have been able to enhance our participation dramatically. Strengths of the current study include a strict definition of ‘high-risk’ and the exclusion of families with a known BRCA mutation.To date, we have six centres that are actively involved in subject recruitment and 119 women already enrolled in the study. A major limitation to recruitment has been the large number of ineligible pedigrees (mostly due to lack of BRCA-mutation negative families. Ultimately, our goal is to develop viable strategies for risk reduction. Even though genetic factors are clearly important given these women’s familial connections to known breast cancer patients, the potential to modify cancer risk in these high-risk women will also come from enhanced knowledge of non-genetic, modifiable factors, including a role of dietary and lifestyle factors, as well as chemoprevention and screening. This information will aid in identifying preventive measures that are successful in reducing the risk of breast cancer development and will lead to future clinical guidelines for the management of these high-risk women.In summary, the establishment of this cohort study, along with the wealth of epidemiological and biological evidence to be collected, will lead to a much-needed understanding of familial breast cancer and provide evidence to guide health professionals in the management and care of women from high-risk/BRCA1: Breast cancer susceptibility gene 1; BRCA2: Breast cancer susceptibility gene 2; DHQ: Diet history questionnaire; BMI: Body mass index; WCH: Women’s College Hospital; WCRI: Women’s College Research Institute; OC: Oral contraceptive; HRT: Hormone replacement therapy; BrOCaP: Breast and ovarian cancer predisposing.The authors declare that they have no competing interests.JK is the principal investigator of the study. SAN, KM and JK contributed to the design of the protocol. JA and DN are responsible for data collection, data entry and management of the study. JK, JA and DN drafted this manuscript. All authors approved the final version of the study. OG, AE, RD and KZ participated in subject recruitment.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/221/prepub


The female sex steroids estrogen and progesterone are important in breast cancer etiology. It therefore seems plausible that variation in genes involved in metabolism of these hormones may affect breast cancer risk, and that these associations may vary depending on menopausal status and use of hormone therapy.P values were adjusted for multiple correlated tests (PACT).We conducted a nested case-control study of breast cancer in the California Teachers Study cohort. We analyzed 317 tagging single nucleotide polymorphisms (SNPs) in 24 hormone pathway genes in 2746 non-Hispanic white women: 1351 cases and 1395 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by fitting conditional logistic regression models using all women or subgroups of women defined by menopausal status and hormone therapy use. SLCO1B1, a solute carrier organic anion transporter gene, which transports estradiol-17β-glucuronide and estrone-3-sulfate from the blood into hepatocytes. Ten of 38 tagging SNPs of SLCO1B1 showed significant associations with postmenopausal breast cancer risk; 5 SNPs  remained statistically significant after adjusting for multiple testing within this gene (PACT = 0.019-0.046). In postmenopausal women who were using combined estrogen-progestin therapy (EPT) at cohort enrollment, the OR of breast cancer was 2.31 (95% CI = 1.47-3.62) per minor allele of rs4149013 in SLCO1B1 . SNPs in other hormone pathway genes evaluated in this study were not associated with breast cancer risk in premenopausal or postmenopausal women.The strongest associations were observed for SNPs in SLCO1B1 is associated with breast cancer risk in postmenopausal women, particularly among those using EPT.We found evidence that genetic variation in  Reproductive and hormonal factors, including age at menarche, parity, number of full-term pregnancies, age at first full-term pregnancy, breastfeeding, age at menopause, body mass index (BMI), and physical activity, are associated with breast cancer risk ,2. ConsiCYP19A1 and COMT, have been associated with endogenous hormone levels [Sex steroid hormones, whether endogenous or exogenous, are synthesized and metabolized by many different enzymes (reviewed in ). Therefe levels -16; howee levels ,17-29. Re levels , suggestEPT use increases breast cancer risk to a much greater extent than estrogen-only therapy (ET) -11. TherThe California Teachers Study has been previously described in detail . Brieflyin situ breast cancer. Cases were women who had a histologically confirmed invasive primary carcinoma of the breast  and who were 80 years old or younger between 1 January 1998 and 31 May 2007. One control participant per case was randomly selected from the cohort and frequency-matched to the case on age at baseline (within 5-year age groups), self-reported race/ethnicity , and three broad geographic regions . Cancer cases were identified through quarterly linkages of the cohort to the California Cancer Registry database. Control selection was conducted on a quarterly basis without replacement. For each wave of control selection, a reference date was determined . Nearly equal numbers of controls were selected in each wave. One control participant had her breast cancer diagnosed after her control selection and was included in the analysis as both a control and a case.The nested breast cancer case-control study was designed to obtain biospecimens from breast cancer cases and unaffected controls within the 113,590 members of the cohort who were less than 80 years old at baseline, had continued residence in California during the study period (1995 to time of blood draw), and, before 1998, had no prior history of invasive or Collection of biological specimens was conducted at three study centers . Women who declined blood draw were asked whether they were willing to provide a saliva sample, and, if so, an Oragene DNA self-collection kit  was mailed to the participant with informed consent and return postage paid mailing materials. From the 8,118 eligible participants , we collected biological specimens for 74% of the cases  and 61% of the controls . All biologic samples were sent via overnight courier to the UCI laboratory for DNA extraction. DNA was extracted from blood clots by using Qiagen Clotspin Baskets and DNA QIAmp DNA Blood maxi kits  in accordance with Qiagen protocols. DNA was extracted from saliva samples by using the Oragene protocol (DNA Genotek). The nested breast cancer case-control study of the California Teachers Study has been approved by the institutional review boards at all participating institutions, and all participants provided written informed consent.2 of at least 0.80 by using the Snagger software [2 of at least 0.8. For the remaining 3 of the 24 genes, we genotyped a few selected SNPs due to space limitations of our genotyping platform. For CYP19A1, the selected SNPs were shown to be associated with circulating estrogen concentrations in a comprehensive analysis [We investigated 24 genes that are involved in female sex steroid hormone biosynthesis, metabolism, or excretion. Reviews on the function of these genes are available elsewhere ,35,36. Fsoftware  and the software ). Tagginsoftware ,39. To fanalysis   to monitor reproducibility and track plate flips or switches. The DNA samples were genotyped for the selected tagging SNPs by using the Illumina Golden Gate Assay  in the USC Core Facility. About 10% of the genotyped samples, including 189 cases and 150 controls, had a genotyping success rate  of less than 90% and were excluded from the analyses. The genotyping concordance rate based on the 160 duplicate samples with a call rate of at least 90% was 99.9%. Call rates were lower when the DNA was obtained from saliva samples: 23% of saliva samples and 8% of blood samples had a call rate of less than 90% and these samples therefore were excluded from the analyses. However, the genotyping concordance after excluding the low call rate samples was excellent for saliva samples (greater than 99.9%). In addition, results from sensitivity analyses excluding saliva samples were similar to those using all samples. For the present study, we also excluded 88 women (52 cases and 36 controls) who self-reported to have had a previous history of cancer, leaving 1,510 cases and 1,511 controls. Because the majority (approximately 91%) of participants were non-Hispanic whites, we restricted the analyses to 2,746 non-Hispanic white women .COMT, CYP11A, SULT1A1; SULT1A2, UGT1A8 not in Hardy-Weinberg equilibrium (P < 0.001); thus, we analyzed 317 SNPs in the present study.Of the 355 SNPs genotyped, 332 had an SNP call rate of at least 90%. We excluded an additional three SNPs that had discordant readings in more than two duplicate pairs, eight SNPs with an MAF of less than 1% among non-Hispanic white controls, and four SNPs in 2 of at least 0.80  in the HapMap dataset of European ancestry  with pairwise RP < 0.01 before multiple testing correction) with breast cancer risk among all women or among subgroups defined by menopausal status. To do this, we used publicly available HapMap genotype data in the CEPH population as the reference sample  and MACbuild 36 . We exclhan 0.30 .P values associated with each SNP by using log-additive models. The results did not change after further adjustment for potential confounders including menopausal status , HT use status at baseline , BMI , parity , and oral contraceptive use . Therefore, we presented the results from the conditional logistic regression models not adjusting for these potential confounders.We used conditional logistic regression models with strata defined by 5-year age group and the three specimen collection centers to estimate the odds ratios (ORs), 95% confidence intervals (CIs), and P for interaction by likelihood ratio test comparing the model with and without the product term of genotype  and menopausal status or HT use. The interaction tests for HT use were done separately for current EPT use (as compared with never HT use) and for current ET use (as compared with never HT use).We performed subgroup-specific analyses by menopausal status and, among postmenopausal women, by HT use at baseline, defining the groups of interest as never used HT, currently using ET, and currently using EPT. We calculated A greater proportion of breast cancer cases than controls were currently using EPT at baseline; a lower proportion of cases than controls had high parity. Cases also had slightly earlier age at menarche than control women Table .P values for the association between the 317 SNPs in the 24 hormone metabolism genes and breast cancer risk showed no evidence of systematic bias . However, after multiple testing across all genes was corrected for, none of these associations was statistically significant. The ORs and 95% CIs associated for all tested SNPs are provided in Supplementary Table S2 in Additional file SLCO1B1 was associated with breast cancer risk in postmenopausal women .We observed statistically significant associations at a is Table . HoweverSLCO1B1 and postmenopausal breast cancer risk in current EPT users . This association was statistically significant even after PACT adjustment across all SNPs studied (PACT = 0.023). The P value for interaction (EPT versus never HT use) was 0.019 (not corrected for multiple testing). When we combined the homozygous and heterozygous minor allele carriers , we observed similar OR estimates and P values   of 24 genes in the hormone metabolism pathway genes was associated with breast cancer risk. SLCO1B1, a gene involved in the hepatic uptake of female sex steroids, seemed to be associated with breast cancer risk among postmenopausal women. This association was statistically significant after correcting for multiple testing within the gene but was not statistically significant after we corrected for multiple testing across genes. However, there was also an indication that EPT may interact with SNPs in SLCO1B1; one variant in SLCO1B1 (rs4149013) was statistically significantly associated with breast cancer risk in EPT users.In this case-control study nested within the California Teachers Study cohort, genetic variation in only 1 . Endogeiewed in ,62. Seruiewed in . E1S is iewed in . Genetic several ,65 but n several  studies.ucasians , suggestSLCO1B1. In CGEMS, five genotyped SNPs in SLCO1B1  were associated with breast cancer risk with a P value of less than 0.05. These 5 SNPs, as imputed genotypes, were null in our dataset (data not shown), but this could be due to misclassification from imputation or false-positive associations across both CGEMS and our data. However, our findings and those of CGEMS, combined with the previous literature on the role of this gene in affecting estrone absorption, suggest that further investigation of the role of SLCO1B1 genetic variation and its interaction with EPT on breast cancer risk is warranted.In the publicly available Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer data , we founAKR1C4, ARSC, and CYP19A1. Thus, we cannot exclude the possibility that the lack of associations for these loci was due to incomplete tagging. Overall, we had 80% statistical power to detect ORs ranging from 1.17 to 1.40 for SNPs with an MAF of 0.05 to 0.49 by using log-additive models and an alpha of 0.05. For the subset analyses among postmenopausal women, the minimum detectable OR ranged from 1.20 to 1.46 for SNPs with an MAF of at least 0.05. The statistical power to detect associations in premenopausal women or to detect interactions with menopausal status or HT use was limited. Another limitation of this study is that HT use status assessed at baseline may have changed during follow-up. The participation rates  among the potentially eligible cohort members were moderate (74% for cases and 61% for controls). However, it is unlikely that the participation was differential according to genotype and case status, and thus selection bias is unlikely to have influenced our findings.The strengths of this study include the systematic investigation of a large number of hormone metabolism genes and the detailed information on HT use collected at baseline. A limitation of our study was the inability to genotype all tagging SNPs for several of the genes of interest, including SLCO1B1 may be associated with breast cancer risk in postmenopausal women, particularly in EPT users. The known effects of variants in SLCO1B1 on estrogen metabolism suggest that further study of the role of SLCO1B1 is warranted.Common genetic variations in ACT: P value adjusted for multiple correlated tests; SNP: single-nucleotide polymorphism; UCI: University of California at Irvine; USC: University of Southern California; UTR: untranslated region.BMI: body mass index; BPC3: Breast and Prostate Cancer Cohort Consortium; CEPH: Utah residents with ancestry from northern and western Europe; CGEMS: Cancer Genetic Markers of Susceptibility; CI: confidence interval; CPIC: Cancer Prevention Institute of California; E1S: estrone sulfate; EPT: estrogen progestin combined therapy; ET: estrogen therapy; HT: hormone therapy; MAF: minor allele frequency; OR: odds ratio; PThe authors declare that they have no competing interests.EL performed the statistical analysis and drafted the manuscript. FS, JPL, and SLN participated in the statistical analysis and drafted the manuscript. PLH-R, HAC, and LB participated in the design of the nested case-control study, were responsible for specimen collection, and obtained grant funding for the study. KDH prepared analytic variables, including menopausal status and hormone therapy use. AZ participated in coordinating the specimen process and SNP selection. DVDB coordinated tagging SNP selection and performed the genotyping. GU participated in the design of the study and in SNP selection and supervised the analysis and manuscript preparation. All authors edited the manuscript and provided comments on the intellectual content and read and approved the final manuscript.Supplementary Table S1. A word document of Supplementary Table S1.Click here for fileSupplementary Figure S1. A word document of Supplementary Figure S1.Click here for fileSupplementary Table S2. An excel document of Supplementary Table S2.Click here for fileSupplementary Table S3. A word document of Supplementary Table S3.Click here for file


There is extensive epidemiological evidence that menopausal hormone therapy (MHT) increases breast cancer risk, particularly combinations of estrogen and progestagen (EP). We investigated the effects of the specific formulations and types of therapies used by French women. Progestagen constituents, regimen , and time interval between onset of menopause and start of MHT were examined.We conducted a population-based case-control study in France in 1555 menopausal women (739 cases and 816 controls). Detailed information on MHT use was obtained during in-person interviews. Odds ratios and 95% confidence interval adjusted for breast cancer risk factors were calculated.We found that breast cancer risk differed by type of progestagen among current users of EP therapies. No increased risk was apparent among EP therapy users treated with natural micronized progesterone. Among users of EP therapy containing a synthetic progestin, the odds ratio was 1.57 (0.99-2.49) for progesterone-derived and 3.35 (1.07-10.4) for testosterone-derived progestagen. Women with continuous regimen were at greater risk than women treated sequentially, but regimen and type of progestagen could not be investigated independently, as almost all EP combinations containing a testosterone-derivative were administered continuously and vice-versa. Tibolone was also associated with an increased risk of breast cancer. Early users of MHT after onset of menopause were at greater risk than users who delayed treatment.This study confirms differential effects on breast cancer risk of progestagens and regimens specifically used in France. Formulation of EP therapies containing natural progesterone, frequently prescribed in France, was not associated with increased risk of breast cancer but may poorly protect against endometrial cancer. Menopausal hormone therapy (MHT) has been prescribed for more than fifty years to prevent discomfort caused by the menopause. When MHT started to be used, most preparations contained estrogen alone. In the 1970s, it was shown that the use of estrogen-alone therapy was associated with risk of endometrial cancer . ProgestThere is a large variety of MHTs available around the world, with country-specific types of molecules and regimens . It has been shown that users of combined estrogen-progestin MHT have a higher risk of breast cancer than estrogen-only users, that the risk is particularly elevated in current users and that it increases with duration of use ,12. FixeIn combined EP therapies used in France, the estrogen component is usually estradiol, but preparations may contain a large variety of progestagens ,19. WhilData on breast cancer risk associated with MHT use in France are based primarily on the E3N prospective cohort study of French female teachers . It was In the present study, we aimed at examining breast cancer risk by type of MHT used in France from the data of a large population-based case-control study. We were interested in particular in the type of progestagen, regimen and delay between onset of menopause and start of therapy. Ethics statement: The study was approved by the Ethic Committee of Kremlin-Bicêtre, France (Jan 2005) and by the National Data Protection Agency (Dec 2004). All participants signed informed consent.Côte d'Or and in Ille-et-Vilaine, two French administrative areas (départements) located in Eastern and Western part of France, respectively. The CECILE study is a population-based case-control study in The case group included incident cases of in situ or invasive breast cancer diagnosed between April 2005 and March 2007 in women aged 25-75 who resided in one of the two study areas. Patients were recruited in the main cancer hospital in each area (Centre Eugène Marquis in Rennes and Centre Georges-François Leclerc in Dijon), as well as from smaller public and private hospitals that also recruited breast cancer patients. Among the 1553 eligible cases identified during the study period, 163 refused to participate, 151 could not be contacted, and 7 died before the interview, leaving 1232 cases included in the study (participation 79.3%).Controls were women without a previous history of breast cancer recruited in the general population, and frequency-matched to the cases by 10-year age group and study area. To select the controls, we contacted a random sample of private homes by telephone. Phone numbers were selected from the telephone directory where unlisted numbers had previously been re-created. If a woman was living in the residence reached by telephone, she was invited to participate in the study within predefined quotas by age and socio-economic status (SES). Quotas by age were applied to obtain similar distributions by age among controls and among cases (frequency-matching). Quotas by SES in control women were applied to reflect the distribution by SES of women in the general population in each study area, conditionally to age. Using predefined proportion of controls by SES was used to prevent selection biases that could arise from differential participation rates across SES categories. Among 1731 controls identified by telephone fulfilling eligibility criteria, 260 declined participation for an in-person interview and 154 could not be re-contacted, leaving 1317 women available for the study (participation 76.1%). Only menopausal women were included in the analysis. Women were considered menopausal if they had had no menstruation for twelve months or more , if they had bilateral oophorectomy , or if they used MHT before natural cessation of menstruation (n=352). Women with unknown menopausal status (n=199), because of hysterectomy before cessation of menstruations or unknown date of last menstruation, were considered menopausal if they were 50 years old or more . Women with unknown menopausal status below 50 years old were excluded from the analysis (n=25). In total, the study included 1555 women, 739 cases and 816 controls .Data pertaining to each study subject were obtained from a structured questionnaire during in-person interviews conducted by trained interviewers. We elicited information on sociodemographic characteristics, history of previous diseases, family history of cancer, menstruations, oral contraceptives, infertility, reproductive history, residential and occupational history, lifetime consumption of alcohol and tobacco, recreational activities, and dietary habits. A blood sample was collected for each case and control to collect DNA and serum samples. For each MHT, we obtained information on name, dates of start and end of use. To help women remember drug names during the interview, they were given a list of fifty MHTs commonly prescribed in France. Information on in situ or invasive tumor type, histology , estrogen (ER) and progesterone receptor (PR) status was obtained from the pathology report. We classified women according to the time since last use of MHT. Current users were women treated by MHT at reference date (date of diagnosis for the cases and date of interview for the controls) or who stopped treatment less than one year before reference date.MHTs were classified in estrogen-only therapy, EP therapy, and tibolone. EP therapy was subdivided in 3 subtypes according to the progestagen constituent: natural micronized progesterone, progesterone-derivatives, and testosterone-derivatives. We also determined the time interval between age at onset of menopause and age at first use of MHT. For women who used MHT before cessation of menstruations, the time interval was set to zero. Menopausal women with unknown age at menopause and/or unknown age at first use of MHT (17 cases and 10 controls) were excluded from these analyses. The odds ratios (OR) and their 95% confidence intervals were estimated using unconditional logistic regression models. Polytomous logistic regression was also used in the analyses where the case group was subdivided according to histological type  and according to the estrogen and progesterone receptor status.2).All analyses were adjusted for age (5-year groups) and study area. We also adjusted for the following breast cancer risk factors: age at menarche , parity , age at first full-term pregnancy , duration of breast-feeding , oral contraceptive use (ever/never), personal history of benign breast disease (yes/no), family history of breast cancer (yes/no) and body mass index (BMI) .The distribution of the 739 cases and 816 controls by socio-demographic characteristics and selected breast cancer risk factors are shown in Odds ratios for breast cancer by type and duration of MHT use are shown in Elevated odds ratios for EP therapy with synthetic progestagen and for tibolone were observed for ER-positive and for PR-positive tumors. Odds ratios were slightly smaller for ER and PR-negative cancers or did not reach statistical significance, but they are based on small numbers. Elevated odds ratios were observed for the two main histological types of breast cancer, with odds ratio of 5.87 for lobular carcinoma among tibolone users .Our findings suggest that the type of progestagen in combined EP therapies used in France may modify the risk of breast cancer, and that tibolone, a molecule used as an alternative to EP therapy, increases risk. In addition, we found that women who start MHT early after onset of menopause were at increased risk as compared to women who delay treatment beyond one or more year. We also found that EP combinations were more strongly associated with ER positive than with ER negative tumors. We found no significantly increased risk of breast cancer in estrogen-only therapy users, a result in line with large observational cohort studies that reported small increased risks with hazard ratios in the range of 1.3-1.4 ,22. ContWhile estradiol is virtually the sole estrogenic component of combined EP therapy in France , a largeCombined EP therapies that do not contain micronized progesterone were used by 42% of current MHT users in our study. Among these, the progestogenic constituent was a progesterone-derivative (90%) or a testosterone-derivative (10%). By contrast, testosterone-derivatives are more commonly used in Northern European countries ,18,20. ITibolone is a synthetic steroid hormone prescribed to menopausal women, as an alternative to classical MHT. Large epidemiologic studies reported that tibolone use was associated with an increased breast cancer risk ,22,23,31Stratification of breast cancer patients by receptor status (ER and PR) indicated that EP combinations was slightly stronger with ER and PR-positive than with ER and PR-negative tumors. EP combination containing synthetic progestagens was also more strongly associated with lobular than with ductal carcinoma. These results are consistent with the literature -34. InteSeveral cohort studies have reported that the time interval between onset of the menopause and start of MHT treatment may influence breast cancer risk in menopausal women, with shorter treatment-free time interval being associated with higher risk -17. In tIn this population-based study, we sought to include all incident cases diagnosed in the study populations during the study period. Cases were identified from active real-time search in the main cancer hospital of each area, and from smaller public and private hospitals, with a high participation rate. The number of eligible cases identified was close to the expected number based on age-specific incidence rates for France . ControlOne important finding is the absence of increased breast cancer risk in women using EP treatment containing natural progesterone, a formulation which is used by a large number of MHT treated women in France. However this type of treatment may not protect against endometrial cancer. We also suggested differences in breast cancer risk according to the type of synthetic progestagen in the EP treatment, with indications of higher risks associated with testosterone derivatives that may be related to the dose rather than to the type of progestagen itself. Our results also indicate that early users of MHT after start of menopause may further increase their risk of breast cancer. Pooled analyses of several studies conducted in France may help to further scrutinize the associations between type of MHT and breast cancer.


GPX1, GPX2, GPX3, GPX4, SELS, SEP15, SEPN1, SEPP1, SEPW1, TXNRD1, and TXNRD2 among Hispanic/Native American  and non-Hispanic white (NHW)  women in the Breast Cancer Health Disparities Study. Adaptive Rank Truncated Product (ARTP) analysis was used to determine both gene and pathway significance with these genes. The overall selenoprotein pathway PARTP was not significantly associated with breast cancer risk (PARTP = 0.69), and only one gene, GPX3, was of borderline significance for the overall population (PARTP =0.09) and marginally significant among women with 0-28% Native American (NA) ancestry (PARTP=0.06). The SEPP1 gene was statistically significantly associated with breast cancer risk among women with higher NA ancestry (PARTP=0.002) and contributed to a significant pathway among those women (PARTP=0.04). GPX1, GPX3, and SELS were associated with Estrogen Receptor-/Progesterone Receptor+ status . Four SNPs  significantly interacted with dietary oxidative balance score after adjustment for multiple comparisons to alter breast cancer risk. GPX4 was significantly associated with breast cancer survival among those with the highest NA ancestry (PARTP = 0.05) only. Our data suggest that SEPP1 alters breast cancer risk among women with higher levels of NA ancestry. Selenoproteins are a class of proteins containing a selenocysteine residue, many of which have been shown to have redox functions, acting as antioxidants to decrease oxidative stress. Selenoproteins have previously been associated with risk of various cancers and redox-related diseases. In this study we evaluated possible associations between breast cancer risk and survival and single nucleotide polymorphisms (SNPs) in the selenoprotein genes  Selenium is a trace element essential for essential health that has been suggested to play a preventive role for a variety of chronic diseases, including various cancers -6. This The known human selenoproteins include the glutathione peroxidases (GPX), thioredoxin reductases (TXNRD), iodothyronine deiodinases (DIO), and selenophosphate synthetases 2 (SPS2) . The othGPX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 3 (GPX3), glutathione peroxidase 4 (GPX4), SELS, SEP15, SEPN1, SEPP1, SEPW1, thioredoxin reductase 1 (TXNRD1), and thioredoxin reductase 2 (TXNRD2). These selenoproteins were selected for analysis because their functions have been characterized and many of them have been associated with risk of various types of cancer and/or oxidative stress [In this study we evaluated single nucleotide polymorphisms (SNPs) in several selenoprotein coding genes for an association with breast cancer: glutathione peroxidase 1  genetic ancestry. Higher levels of NA ancestry have been associated with reduced breast cancer risk ,17 and sAll participants signed informed written consent prior to participation and each study was approved by the Institutional Review Board for Human Subjects at the participating institutions: University of Utah, University of Arizona, University of Colorado, University of New Mexico, Comisión de ética, and Institutional Review Board of the Cancer Prevention Institute of California. in situ (n=341) or invasive (n=1492) cancer between October 1999 and May 2004; controls were selected from the target populations of cases living in Arizona, Colorado, New Mexico, and Utah and were frequency matched to cases on ethnicity and 5-year age distribution [in situ or invasive breast cancer between January 2004 and December 2007 at 12 participating hospitals from three main health care systems; controls were randomly selected from the catchment area as the cases and frequency matched to cases based on 5-year age distribution, membership in health care institution, and place of residence. The San Francisco Bay Area Breast Cancer Study included women aged 35 to 79 years from the San Francisco Bay Area diagnosed with a first primary histologically confirmed invasive breast cancer between April 1995 and April 2002; controls were identified by random-digit dialing and frequency-matched to cases based on the expected race/ethnicity and 5-year age distribution [The Breast Cancer Health Disparities Study includes participants from three population-based case-control studies, the 4-Corners Breast Cancer Study, the Mexico Breast Cancer Study, and the San Francisco Bay Area Breast Cancer Study  who compribution . Data were harmonized across all study centers and questionnaires as previously described . Women wth percentile of consumption, all other drinkers, and non-drinkers. Referent year alcohol consumption was used for those women who did not have long-term alcohol measurements. In creating the DOBS, participants were assigned values of zero for low levels (first quartile) of exposure to anti-oxidants or high exposure to pro-oxidants (fourth quartile), one for intermediate levels (second and third quartiles) of exposure, and two for high levels (fourth quartile) of exposure to anti-oxidants and low exposure (first quartile) to pro-oxidants. A dietary oxidative balance score (DOBS) that included nutrients with anti- or pro-oxidative balance properties was developed as previously reported . Anti-ox2=0.8; minor allele frequency (MAF) >0.1; range= -1500 bps from the initiation codon to +1500 bps from the termination codon; and 1 SNP/LD bin. We distinguished European and NA ancestry in the study population by using 104 Ancestry Informative Markers (AIMs) [GPX1 (2 SNPs), GPX2 (4 SNPs), GPX3 (3 SNPs), GPX4 (1 SNP), SEPP1 (2 candidate SNPs and 1 tagSNP), SELS (2 SNPs), SEP15 (4 SNPs), SEPN1 (5 SNPs), SEPW1 (3 SNPs), TXNRD1 (7 SNPs), and TXNRD2 (20 SNPs). A description of these genes and SNPs is shown in online SEP15 rs9433110 was not analyzed since it was not in Hardy-Weinberg Equilibrium (HWE) among NHW participants. Online SEPP1 rs6865453, we observed a reversal in the major and minor allele from the most European to the most Native ancestry groups.DNA was extracted from either whole blood (n=7287) or mouthwash (n=634) samples. Whole Genome Amplification (WGA) was applied to the mouthwash-derived DNA samples prior to genotyping. A tagSNP approach was used to characterize variation across candidate genes. TagSNPs were selected using the following parameters: linkage disequilibrium (LD) blocks were defined using a Caucasian LD map and an rs (AIMs) . A multiData for survival and ER/PR tumor status were available for cases from the 4-Corners Breast Cancer Study and the San Francisco Bay Area Breast Cancer Study. Information on stage at diagnosis, months of survival after diagnosis, cause of death, and ER and PR status were available from cancer registries in Utah, Colorado, Arizona, New Mexico, and California. Information on ER and PR status of tumors was available for 1019 (69%) NHW and 977 (75%) Hispanic/NA cases. Surveillance Epidemiology and End Results (SEER) summary disease stage, based on three codes of local, regional, and distant, was used. Data on survival and ER and PR tumor status were not available from the Mexico Breast Cancer Study. The program STRUCTURE was used to compute individual ancestry for each study participant assuming two founding populations ,24. A th2) during referent year, and parity. The generalized logit link function was used when estimating breast cancer risk by ER/PR status. Associations with SNPs were assessed assuming co-dominant models. Based on the initial assessment, SNPs which appeared to have a dominant or recessive mode of inheritance were evaluated with those inheritance models in subsequent analyses. Stratified analyses tests for interactions were calculated using a 1 degree of freedom (df) Wald chi-square tests; p values based on 4-df Wald tests measure the overall SNP (treated as continuous) association with breast cancer risk by ER/PR status. Adjustments for multiple comparisons within the gene used the step-down Bonferroni correction  taking into account the correlated nature of the data using the SNP spectral decomposition method proposed by Nyholt [Genes and SNPs were assessed for their association with breast cancer risk by strata of menopausal status and genetic ancestry in the whole population and by ER/PR status for the 4-Corners Breast Cancer Study and the San Francisco Bay Area Study. Statistical analyses were performed using SAS version 9.3  unless otherwise noted. Logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for breast cancer risk associated with SNPs, adjusting for age, study center, genetic ancestry, body mass index  and 95% CI among all cases and by genetic ancestry strata. The upper two ancestry strata were combined to evaluate survival by ancestry groups since survival data were not available for the Mexico study site. In the analysis of breast cancer survival, individuals were censored when they died of causes other than breast cancer or were lost to follow-up. In addition to the minimal adjustments for age, study center, genetic ancestry, BMI during referent year, and parity, models were also adjusted for SEER summary stage. ARTP). We used the adaptive rank truncated product (ARTP) method that is based on a highly efficient permutation algorithm to determine the significance of association of each gene and of the pathway with breast cancer overall, by genetic ancestry, and by ER/PR strata. The gene p values were generated using the ARTP package in R, permuting outcome status 10,000 times while adjusting for age, BMI during referent year, and genetic ancestry ,28. The ARTP was not significantly associated with breast cancer risk (PARTP = 0.69), and the association with only one gene, GPX3, was of borderline significance for the entire population (PARTP =0.09). However, we observed several significant associations prior to adjustment for multiple comparisons between selenoprotein SNPs and breast cancer risk  was associated with breast cancer risk for the entire population. GPX3 was marginally significant among individuals with ≤28% NA ancestry (PARTP =0.06) and SEPP1 was significant among those with >28% NA ancestry (PARTP =0.002). The overall pathway was statistically significant (PARTP =0.04) among women with higher NA ancestry mainly because of the strong association with SEPP1 and breast cancer risk. GPX3 rs8177447  and SELS rs4965814  were significant among individuals of <28% NA ancestry; SEPP1 rs230812 , and SEPP1 rs6865453  were associated with breast cancer risk in individuals of 28-70% NA ancestry; and SEPN1 rs718391  was marginally associated with breast cancer risk among women of >70% NA ancestry. Associations were similar for women with >28% NA ancestry for SEPP1 rs230812 and rs6865453; when combining the upper ancestry groups these two SNPs were statistically significant . Despite apparent differences between SNP associations and breast cancer risk among different NA ancestry groups, only SEPN1 rs718391 (p=0.03 padj =0.10), SEPP1 rs230812 , and SEPP1 rs6865453  showed significant interaction by ancestry. The overall selenoprotein pathway Pcer risk . GPX3 rsARTP values . GPX1 rs1800668 was strongly associated with increased likelihood of ER-/PR+ phenotype p<0.001; padj=0.002), while rs3448 was associated with a decreased likelihood of this phenotype (p=0.04). GPX2 rs11623705 was associated with increased risk of ER-/PR+ . GPX3 rs8177447 showed a significant association with an increased likelihood of an ER-/PR+ phenotype  as did SELS rs9874  and SELS rs4965814 . SELS was also significantly associated with ER+/PR- tumors (PARTP = 0.009); SELS rs4965814 was positively associated with ER+/PR- . Multiple SNPs in SEP15 showed an association with increased likelihood of ER+/PR- tumors: rs5859 , rs486133 , and rs1407131 . TXNRD2 rs2073750 was marginally inversely associated with ER-/PR+ tumors  with an overall gene PARTP of 0.08. None of the genes evaluated showed a significant association with ER-/PR- tumor phenotype according to ARTP. Assessment of SNP associations by ER/PR status indicated that some SNPs were associated with breast cancer risk by ER/PR status; several genes had statistically significant PP values . GPX1, GGPX3 rs2070593 (p=0.002), GPX4 rs2074451 (p=0.046), SELS rs9874 (p=0.029), TXNRD1 rs17202060 (p=0.007) and TXNRD2 rs7322262 (p=0.025), significantly interacted with DOBS to alter breast cancer risk (TXNRD2 rs732262 (padj =0.38) did not remain statistically significantly associated with breast cancer risk after multiple comparison adjustment. Five SNPs, cer risk . Of thesGPX4 was significantly associated with better breast cancer survival among those with the highest NA ancestry (PARTP = 0.05). GPX4GT/TT rs2074451 showed a marginal inverse association with survival for individuals among women with >28% NA ancestry . Several SNPs in TXNRD2 were associated with survival prior to adjustment for multiple comparisons. Additionally, TXNRD2AA rs3788314 and TXNRD2CT/TT rs4333017 showed significant interaction across genetic ancestry .Multiple genes and SNPs in our analysis showed an association with breast cancer survival . GPX4 waGPX3 was borderline statistically significantly associated with breast cancer risk for all women and for women with lower levels of NA ancestry specifically. SEPP1 showed a statistically significant interaction by NA ancestry, with a strong association with breast cancer risk among women with higher NA ancestry. Some differences in association were observed by ER/PR tumor status. GPX1, GPX3, and SELS were significantly associated with ER-/PR+ tumors and SELS was significantly associated with ER+/PR- tumors. GPX4 was significantly associated with survival among those with higher NA ancestry and SEPW1 was marginally associated with survival among women in the low NA ancestry group. Although we hypothesized that DOBS would modify associations with selenoprotein genes, only one SNP in GPX4, SELS, and TXNRD1 that interacted with DOBS remained statistically significantly associated with breast cancer after adjustment for multiple comparisons.Based on ARTP results, GPX3 showed a borderline significant association overall as determined by the PARTP, and GPX3 rs8177447 was statistically significant after adjustment for multiple comparisons. This SNP is in high LD with rs3792797 and has been associated with Barrett’s Esophagus [GPX3 is one of multiple glutathione peroxidases, all of which are selenoproteins that play a role in catalyzing the reduction of hydrogen peroxides to minimize oxidative stress which can damage cells [GPX3 may also play a role in the development and progression of breast cancer. Among the genes evaluated in this study, only sophagus   showed significant differences in survival by ancestry. TXNRD2 has been associated with oxidative stress and our previous analysis of dietary factors of oxidative stress found the strongest associations among women with higher NA ancestry [TXNRD1 or TXNRD2 SNPs and breast cancer survival [We also observed associations between selenoprotein SNPs and survival. Notably, lleagues , where tSNPs see . Additiosurvival .GPX3 rs2070593, GPX4 rs2074451, SELS rs9874, and TXNRD1 rs17202060 showed significant interactions with DOBS after adjusting for multiple comparisons. Oxidative stress and high levels of reactive oxidative species have been suggested to play an important role in breast cancer development because free radicals damage DNA, thereby decreasing genomic integrity [Given their role as antioxidants and mediators of oxidative stress, we evaluated selenoprotein SNPs for interactions with DOBS and found that GPX1 [GPX4 [GPX3 was associated marginally with breast cancer risk and SEPP1 was associated with risk among women with higher NA ancestry. GPX4 was associated with breast cancer survival. Glutathione peroxidases carry out similar functions and have similar mechanisms; they contain a conserved catalytic triad of Sec, Gln, and Trp that acts by sequential oxidation and reduction of the Sec residue during catalysis [The selenoprotein genes analyzed in this study were selected due to previous studies reporting on their roles in regulating oxidative stress and/or carcinogenesis; however, the majority of the genes and SNPs had not been studied in relation to breast cancer. GPX1  and GPX4X1 [GPX4 . We did atalysis . The priARTP. These strengths allowed us to show that both GPX3 and SEPP1 were associated with breast cancer; these associations warrant further study in other populations. An additional strength is our genetically admixed population that allowed us to evaluate associations across the spectrum of European to Native ancestry. Our study has two primary limitations. First, we only evaluated a subset of the known selenoproteins and did not evaluate any DIOs or SPS2s. Since these selenoproteins, along with others, play a role in limiting oxidative stress it is possible that they may be associated with breast cancer risk. Our second limitation was in our analysis of ER/PR status and survival where we were unable to include data from Mexico. While our study population was large, the sample sizes for the different ER/PR subtypes were small, thereby decreasing the statistical power of our analysis. Nevertheless, our study has multiple strengths: our analytic approach that evaluated the pathway as a whole, and our analysis of genes beyond individual SNPs via PGPX3 was marginally significant among women with lower NA ancestry and SEPP1 was statistically significant among women with higher NA ancestry. Additionally, several genes were associated with ER/PR status. While we hypothesized that selenoprotein genes would interact with DOBS, only four SNPs significantly interacted with DOBS after adjustment for multiple comparisons. In conclusion, this study provides limited support for an association between selenoprotein genes and risk of breast cancer.While we observed few significant associations between selenoprotein genes and breast cancer risk, Table S1List of SNPs assessed.(DOCX)Click here for additional data file.Table S2MAF and HWE by level of Native American ancestry.(DOCX)Click here for additional data file.