ATM) gene (MIM ID 208900) encodes a protein kinase that plays a significant role in the activation of cellular responses to DNA double-strand breaks through subsequent phosphorylation of central players in the DNA damage-response pathway. Recent studies have confirmed that some specific variants in the ATM gene are associated with increased breast cancer (BC) risk. However, the magnitude of risk and the subset of variants that are pathogenic for breast cancer remain unresolved.The ataxia-telangiectasia mutated , and modified segregation analysis was used to estimate the BC penetrance associated with these rare ATM variants.To investigate the role of P = 0.00008), corresponding to a 60% cumulative risk of BC by age 80 years. Analysis of loss of heterozygosity (LOH) in 18 breast tumors from women carrying likely pathogenic rare sequence variants revealed no consistent pattern of loss of the ATM variant.In the case-control analysis, we observed an odds ratio of 2.55 and 95% confidence interval  for the most likely deleterious variants. In the family-based analyses, the maximum-likelihood estimate of the increased risk associated with these variants was hazard ratio (HR) = 6.88 (95% CI, 2.33 to 20.3; ATM c.7271T > G, or truncating mutations demonstrate a significantly increased risk of breast cancer with a penetrance that appears similar to that conferred by germline mutations in BRCA2.The risk estimates from this study suggest that women carrying the pathogenic variant,  ATM) gene (MIM ID 208900) encodes a protein kinase that plays a major role in activating cellular responses to DNA double-strand breaks through downstream phosphorylation of central players in the DNA damage-response pathways, including BRCA1, p53, and Chk2 [The ataxia-telangiectasia mutated . Howeve [et al.  had hypoATM were likely to confer an increased risk of breast cancer, and to compare this with the risk conferred by protein-truncating mutations, we previously carried out a meta-analysis of published data and also mutation screened almost 1,000 breast cancer cases and a similar number of controls [in silico missense substitution analysis that provides a ranking of missense variants from evolutionarily most likely (C0) to least likely (C65). We found marginal evidence that protein-truncating (T) and splice-site junction (SJ) mutations confer on average a moderately increased risk of breast cancer (odds ratio (OR), 2.3; 95% CI, 1.1 to 4.8), but stronger evidence that a subset of rare, evolutionarily unlikely missense (rMS) C65 substitutions conferred on average a higher risk of breast cancer .To determine which rare missense variants in controls . In addiATM variants, and to determine whether they were likely to act as dominant negatives, we genotyped a large panel of rare missense variants, as well as truncating and splice-junction mutations, in breast cancer cases and controls from four large studies. We also genotyped all available relatives of breast cancer cases found to carry putative breast-cancer associated ATM variants to estimate their penetrance. In addition, we carried out loss of heterozygosity (LOH) analyses and a review of the pathology of breast tumors from these mutation carriers.To define better the risks associated with these classes of n = 2,517 invasive and 53 DCIS) and controls  from three sources: (a) population-based case and control breast cancer families from the NCI-sponsored Breast Cancer Family Registry (BCFR) [et al. [ATM study, have been approved by the relevant ethical committees.We studied Caucasian cases of breast cancer (y (BCFR) ; and (c)y (BCFR)  (Table 1y (BCFR)  but note [et al. , althoug [et al. . The indMissense variants and in-frame deletions were assessed for the degree of conservation within the ATM multiple protein sequence alignment and for the predicted severity of the amino acid substitution, according to the Align-GVGD class, as previously described ,20. We sATM variants, 129 family members of women who had been found to carry a truncating mutation (n = 10), splice-site variant (n = 1), or evolutionarily unlikely missense substitution (C65 and C55) in the FAT, kinase, and FATC domains (n = 16) were genotyped for the respective variant by direct sequencing (Table To estimate the penetrance of the likely deleterious ATM-positive breast tumors (from 21 different carriers of Group 3 ATM variants) and H&E slides of 38 control breast tumors (age matched within 6 years) ascertained from the Royal Brisbane and Women's Hospital between 2004 and 2009. The slides were scored for pathologic features by using a modified pro forma that was initially developed for studies on the pathology of BRCA-associated cancers. Specifically, we assessed for the presence of in situ disease (LCIS and DCIS), invasive tumor type, and overall nuclear grade by using the modified Nottingham Grading System [A blinded pathology review was performed by one of us (SRL) on 35 H&E slides of g System , and for2 analysis comparing each variant group against the reference, stratified by study center. All analyses were performed by using STATA 10.0 .Conditional logistic regression was used to examine the associations between variants in a given class and the risk of breast cancer, stratified by study center, by using a case-control design. To guard against results driven by individual study centers, we also performed Mantel-Haenszel χATM variants was estimated by using modified segregation analysis of family genotypes adjusted for ascertainment. Models were fitted under maximum likelihood theory by using the statistical package Mendel///version 3.2 [Penetrance of sion 3.2 . Noncarret al. [ATM variant segregating in the pedigree. P Values for the modified segregation analyses were based on the likelihood ratio test and were two-sided. Cumulative risk estimates were calculated from the hazard-ratio estimates as 1 minus the exponential of the cumulative incidence, and the corresponding confidence intervals were calculated by using a parametric bootstrap with 5,000 replications. The model assumed a dominant mode of action of the ATM variants on breast cancer risks and a combined allele frequency of 0.001 for the variants in the population.As in Antoniou et al. ,28, a miet al.  was usedATM variants compared with those associated with BRCA2, as estimated by Antoniou et al., 2003 [et al. estimate, with cumulative penetrances re-estimated at each trial value of the multiplier. This allowed a similar pattern of age-specific effects as in BRCA2, but required estimation of only a single parameter.In separate analyses, we examined the risk associated with these l., 2003 . In thesATM variants that passed QC, 29 were observed one or more times in the analyzed set of 2,570 cases and 1,448 controls , the presence of an ATM variant increased breast cancer risk by an estimated factor of 6.88  and did not depend on age (P = 0.9). The estimated cumulative risks of developing breast cancer for female carriers, assuming US SEER incidence rates, are shown in Figure ATM c.7271T > G variant found that this variant increased breast cancer risk by a factor of 8.0  compared with 4.4  for families with other variants.We genotyped additional relatives in all 27 families in which putative pathogenic variants had been identified. The specific variants included 16 missense variants, of which 15 were p.Val2424Gly (c.7271T > G) (rs28904921), seven were frameshifts (of which four were c.3802delG), three were nonsense mutations, and one was a consensus splice-site variant , indicating that the ATM alleles segregating in these 27 families were associated with risks equivalent to 75% those of BRCA2. In the 16 families with a missense variant, the penetrance estimate was 1.1 that of BRCA2, whereas in the 11 families with a truncating or splice junction (TSJ) mutation, the best estimate was 0.3, although this difference was not significant .Under the assumption that the penetrance of the ATM showed loss of the mutant allele, and the remainder showed no LOH. Of the eight cases with the C65 variant, p.Val2424Gly (ATMc.7271T > G), one showed loss of the wild-type, one showed partial loss of the mutant, and the remainder showed no LOH. Of the remaining two cases with C55 or C65 variants, one showed loss of the mutant allele, and the other had no LOH.LOH results for the 18 different affected women are summarized in Table P = 0.049), largely because of the paucity of ATM tumors with a mitotic score of 3 (two of 18 compared with 14 of 34 of the control tumors). This was also supported by analysis of the quantitative count of mitoses per 10 high-power fields, with some suggestion of lower mitotic rates in the ATM tumors than in the control tumors .We compared the ATM-positive tumors with a set of age-matched control breast tumors. No statistically significant difference was noted in overall histologic grade. Looking at the three individual components of grade , no apparent differences were related to pleomorphism or tubule scores. However, a marginally significant association was seen between the mitotic score , appears to confer a high risk of breast cancer and to act as a dominant negative [Gatti et al.  hypothesnegative ,32. Thisnegative  and subsnegative , but ininegative .ATM variants, and to examine the molecular pathologic characteristics of ATM-positive tumors, we genotyped 76 rare ATM variants in 2,570 breast cancer cases and 1,448 controls. In addition, we genotyped specific variants in the relatives of probands carrying ATM variants judged likely to be pathogenic. Because, by definition, we expect variants conferring moderate to high risks of breast cancer to be rare in the general population, traditional case-control studies of even several thousand cases and controls are typically underpowered to detect associations with these sequence variants. We could potentially pool these variants and compare the aggregate frequency of these variants to increase power. However, the power is often reduced because of the inherent heterogeneity of such variants, in which only a minority is associated with increased risk. One strategy to address this problem is to use in silico methods to group variants into categories based on their probability of representing variants that are damaging to the normal protein function. Although a number of such methods are available, we used the Align-GVGD that has been applied to a number of genes, including BRCA1, BRCA2, CHEK2, ATM, and mismatch-repair genes [To refine the risks associated with different classes of ir genes ,34. Howeir genes .ATM and breast cancer risk [ATM c.7271T > G . Renwick et al. [ATM. Bernstein et al. [ATM c.7271T > G  mutation carriers from the Northern California BCFR site that Bernstein et al. [BRCA2 mutation (and was thus excluded from our study), and for the other, no DNA sample was available for our analyses. This missense ATM variant was first reported to be associated with a mild form of AT and might have originated in the Orkney Islands in Scotland and then spread throughout populations with large numbers of Scottish immigrant populations, such as those of Australia. Our analysis of independent samples from four case-control studies provided some support of the observation by Tavtigian et al. [As in other studies of cer risk ,16, the k et al.  did not n et al.  had prevn et al.  had idenn et al. ,32 that ATM sequence variants in breast cancer, we took advantage of the fact that the resources from which the cases were drawn had also included the relatives of those cases, providing us with the ability to genotype both affected and unaffected relatives of cases in which potentially pathogenic variants had been identified. As in Bernstein et al. [ATM variant. Our analyses of family data in 27 families of carriers of either protein-truncating (n = 11) or rare, evolutionarily unlikely, potentially damaging missense mutations (n = 16) demonstrated a significantly increased risk of breast cancer with a penetrance that appears similar to that conferred by germline mutations in BRCA2. However, even in a study of this size, the confidence intervals are wide. Suggestive evidence also was noted from the family-based analysis that a higher risk was associated with the ATM c.7271T > G  mutation than with truncating mutations, although these differences were not statistically significant because of the relatively small sample size of families. The penetrance associated with truncating mutations was only marginally significant. If our estimates of breast cancer risk are correct, then women carrying the ATM c.7271T > G variant would be at sufficiently high risk to warrant screening for at least this variant in multiple-case families without mutations in BRCA1 or BRCA2. If such a variant is identified, these women could be counseled in a manner similar to that with BRCA2 carriers, and those affected with breast cancer might also be candidates for treatment with PARP inhibitors in a manner similar to that with BRCA1 and BRCA2 carriers. This suggestion is based on the evidence that the inhibition of PARP1 is synthetically lethal with mutation or loss of ATM, and the effect is mediated through mitotic catastrophe independent of apoptosis [As a second approach to verifying and characterizing the role of n et al. , even inpoptosis ,36.Consistent with the dominant-negative hypothesis ,31,32, wATM variant and a hospital-based series of 38 age-matched control breast tumors did not reveal any distinctive pattern of histopathologic characteristics, as had been previously reported in BRCA1 tumors [ATM tumors were associated with a lower mitotic index than were control tumors, which is in contrast to the clear increase in mitotic index associated with BRCA1 tumors [ATM c.7271T > G mutation carriers showed that they were all luminal A or B tumors, and we would not have expected them to share histopathologic characteristics with BRCA1 tumors [et al. [ATM carriers, we did not observe this in our series, with half the lobular or mixed lobular/ductal in each group (P = 0.66).Blinded pathology review of 35 tumors from cases who carried a likely deleterious 1 tumors . However1 tumors . In agre1 tumors . In cont [et al. , who repATM gene for association with breast cancer risk. In addition to the standard case-control approach, we used the power of a family-based design inherent in the two resources from which the cases and controls were derived (BCFR and kConFab) to estimate more precisely the risks of breast cancer through genotyping of relatives of the probands carrying the putative pathogenic variants. Although Bernstein et al. [et al. did not include genotypes of additional relatives from these families in their analysis. Taken as a whole, our study adds to the growing body of evidence that a subset of rare ATM variants confers levels of risk that may have clinical implications for the women who carry them, as well as for their at-risk family members.This is the largest study to date investigating large numbers of rare missense variants in the n et al.  used a sATM: ataxia telangiectasia mutated; BC: breast cancer; BCFR: Breast Cancer Family Registry; CI: confidence interval; DCIS: ductal carcinoma in situ; H&E: hematoxylin and eosin; HR: hazard ratio; kConFab: Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer; LCM: laser capture microdissection; LCIS: lobular carcinoma in situ; LOH: loss of heterozygosity; OR: odds ratio; rMS: rare missense; SJ: splice-site junction; T: protein truncating.AT: ataxia telangiectasia; The authors declare that they have no competing interests.GCT participated in the writing of the manuscript, conceived and designed the study, and directed the laboratory work. DEG and JLH participated in the writing of the manuscript and performed the statistical analyses. SH participated in the writing of the manuscript and performed the iPLEX genotyping and the LOH studies. MCS and EMJ participated in the writing of the manuscript and managed the data. PJO participated in the writing of the manuscript and performed the DNA sequencing. KKK participated in the writing of the manuscript. XC performed the iPLEX genotyping and the LOH studies. JGD performed the statistical analyses. MJD, SB, ABS, IA, and MBT managed the data. SL and LdS performed the pathology review. All authors read and approved the final manuscript.ATM variants genotyped in cases and controls.Click here for file


Epidemiological studies consistently indicate that alcoholic beverages are an independent risk factor for female breast cancer. Although the mechanism underlying this effect remains unknown, the predominant hypothesis implicates mutagenesis via the ethanol metabolite acetaldehyde, whose impact on the carcinogenesis of several types of cancer has been shown in both experimental models and molecular epidemiological studies. Many of the epidemiological studies have investigated genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) His48Arg and aldehyde dehydrogenase-2 (ALDH2) Glu504Lys, because of the strong impact these polymorphisms have on exposure to and accumulation of acetaldehyde. With regard to breast cancer, however, evidence is scarce.ADH1B and ALDH2, we conducted a case–control study of 456 newly and histologically diagnosed breast cancer cases and 912 age- and menopausal status-matched noncancer controls. Gene–gene and gene–environment interactions between individual and combined ADH1B and ALDH2 gene polymorphisms and alcohol consumption were evaluated.To clarify the impact on female breast cancer risk of the interaction of the effects of alcohol consumption and polymorphisms in the alcohol-metabolizing enzymes ADH1B and ALDH2 on the risk of breast cancer. Neither was there any significant gene–environment interactions between alcohol drinking and polymorphisms in ADH1B and ALDH2.Despite sufficient statistical power, there was no significant impact of Our findings do not support the hypothesis that acetaldehyde is the main contributor to the carcinogenesis of alcohol-induced breast cancer. Indeed, the carcinogenic effect of acetaldehyde has been clearly shown in experimental models of several types of cancer6–8 and its impact on human cancer risk has been confirmed in molecular epidemiological studies for several cancers,9–15 Nevertheless, evidence with regard to breast cancer is scarce.16Breast cancer now ranks first among cancers that affect women worldwide.17 Regarding the ADH1B Arg48(*1:slow)/His(*2:rapid) polymorphism, the ADH1B*2 allele is a superactive subunit of ADH1B that confers an approximately 40-times-higher Vmax than the less active ADH1B*1/*1 form.18,19 In contrast, for the ALDH2 Glu504(*1:active)/Lys504(*2:null) polymorphism, the ALDH2*2 allele encodes a catalytically inactive subunit.18,19 Individuals with the ALDH2*1/*2 genotype have only 6.25% of the normal level of ALDH2 protein. The ADH1B*1 and ALDH2*2 alleles, which are associated with prolonged exposure to or higher accumulation of acetaldehyde, are clustered in east Asian populations,20,21 including Japanese, and lead to high acetaldehyde concentrations upon alcohol consumption in affected individuals.Polymorphisms in alcohol-metabolizing enzyme genes cause marked variation in acetaldehyde concentrations among individuals. Ethanol is oxidized to acetaldehyde by the alcohol dehydrogenase enzymes (ADH), particularly ADH1B. Acetaldehyde is then further oxidized and detoxified into acetate by aldehyde dehydrogenases (ALDHs), and this oxidation owes much to ALDH2. Genes that encode these 2 representative alcohol-metabolizing enzymes display polymorphisms that modulate individual differences in alcohol-detoxifying capability and drinking behavior.ADH1B and/or ALDH2 gene polymorphisms.9–14Given this background, molecular epidemiological studies have investigated the carcinogenic impact of acetaldehyde by examining possible gene–environment interactions of the effects of alcohol consumption and polymorphisms in alcohol-metabolizing enzyme genes on the risk of several types of cancer. The results revealed effect modifications by these polymorphisms for several types of cancer in individuals expected to be rapidly or extensively exposed to acetaldehyde due to the presence of We conducted a case–control study to determine the impact of the interaction of the effects of alcohol consumption and polymorphisms in the alcohol-metabolizing enzymes ADH1B and ALDH2 on the risk of female breast cancer in Japan.ALDH2 and alcohol consumption, after calculating the odds ratio (OR) for ALDH2*2 , the OR for alcohol drinking , and the interaction OR between them (2.0) that provided greater than 95% power. All subjects were recruited within the framework of the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), as described elsewhere.22,23 In brief, information on lifestyle factors was collected using a self-administered questionnaire and checked by a trained interviewer. Outpatients were also asked to provide blood samples. Each patient was asked about his or her lifestyle when healthy or before the current symptoms developed. Approximately 95% of eligible subjects completed the questionnaire and 60% provided blood samples. We used noncancer patients at our hospital as controls, given the high likelihood that our cases were members of this population. We previously confirmed the feasibility of using noncancer outpatients at our hospital as controls in epidemiological studies by showing that their general lifestyles accorded with those of a general population randomly selected from the electoral roll in Nagoya City, Aichi Prefecture.24 The data were loaded into a HERPACC database and routinely linked with the hospital-based cancer registry system to update the data on cancer incidence. All participants gave written informed consent and the study was approved by Ethical Committee of Aichi Cancer Center.The cases were 456 patients with no previous history of cancer who were newly and histologically diagnosed with breast cancer from January 2001 through June 2005 at Aichi Cancer Center Hospital in Japan. The controls were 912 subjects who were randomly selected and matched by age (±3 years) and menopausal status (pre- or postmenopause) to cases in a 1:2 case–control ratio. This sample size was selected to enable the detection of gene–environment interactions between 25 All of the assays were done in 96-well PCR plates. Amplification reactions (5 µL) were done in duplicate with 30 ng of template DNA, 2× TaqMan Universal Master Mix buffer (Applied Biosystems), 20× primer, and probe mix (Applied Biosystems). Thermal cycling was initiated with a first denaturation step of 20 seconds at 95 °C, and then by 40 cycles of 3 seconds at 95 °C and 30 seconds at 62 °C. After PCR was completed, plates were brought to room temperature and read using a 7500 Fast Real-Time PCR System (Applied Biosystems), and the results were analyzed using the 7500 Fast System SDS software. The quality of genotyping in our department is routinely assessed by conducting re-genotyping of a randomly selected 5% of samples, and complete concordance of the results of the genotyping was confirmed. We also confirmed that there were no allelic distributions among the controls that departed from the Hardy–Weinberg frequency.DNA from each subject was extracted from the buffy coat fraction with a BioRobot EZ1 with an EZ1 DNA Blood 350-µL kit or QIAamp DNA Blood Mini Kit . Genotyping was based on Taqman Assays from Applied Biosystems . The principle of the TaqMan Real-Time polymerase chain reaction (PCR) assay system using fluorogenic probes and 5' nuclease has been described by Livak.sake, beer, shochu, whiskey, and wine) was defined as the average number of drinks per day, which was then converted into a Japanese sake (rice wine) equivalent. One drink is equal to 1 go (180 mL) of Japanese sake, which contains 23 g of ethanol and is equivalent to 1 large bottle (633 mL) of beer, 2 shots (60 mL) of whiskey, or 2.5 glasses of wine (200 mL). One drink of shochu (distilled spirit), which contains 25% ethanol, was rated as 108 mL. Total alcohol consumption was estimated as the amount of pure alcohol consumption (g/drink) of Japanese sake, beer, shochu, whiskey, and wine among current regular drinkers. Alcohol consumption was classified into 5 categories: never, former, and—for current drinkers—light, moderate, and heavy. Heavy drinkers were defined as those currently consuming at least 15 g of pure alcohol per day, moderate drinkers as those currently consuming between 5 g and less than 15 g per day, and light drinkers as those currently consuming less than 5 g per day. Cumulative smoking dose was evaluated in pack-years, ie, the product of the number of packs consumed per day and years of smoking. Smoking status was classified into 4 categories: never, former, current smoker of <20 pack-years, and current smoker of ≥20 pack-years. Former drinkers and former smokers were defined as those who had quit drinking or smoking at least 1 year before the survey, respectively. Body-mass index (BMI) was calculated as weight divided by height squared (kg/m2). Regular exercise was defined as exercise activity more than once per month, regardless of exercise time. In our study, family history was considered positive if either a mother or sister had had breast cancer.Consumption of each type of beverage  and alcohol consumption , which were then included in the model. In interaction analysis, products of scores for genotype and alcohol consumption, as described above, were included as interaction terms. A P value less than 0.05 was considered statistically significant. All analyses were performed using STATA version 10 , except for the power calculations, which were performed using QUANTO version 1.2.26To assess the strength of the associations between polymorphic genes involved in ADH1B and ALDH2 metabolism and the risk of breast cancer, odds ratios (ORs) with 95% confidence intervals (CIs) were estimated using logistic regression models adjusted for potential confounders. Although we attempted conditional logistic regression as primary analysis, we finally applied unconditional logistic regression to avoid the dropping of controls, which led to unstable estimation in stratified analysis. Consistency between conditional and unconditional logistic regression models was confirmed. Potential confounders considered in the multivariate analyses were age (as a continuous variable), alcohol consumption , smoking status , current BMI , regular exercise , family history of breast cancer , menopausal status , age at menarche , parity , past use of hormone-replacement therapy , and mode of referral to our hospital . To account for possible differences between cases and controls, we adjusted for mode of referral to our hospital. Differences in categorized demographic variables between the cases and controls were tested using the chi-square test. Mean ages of cases and controls were compared using the Data from 456 breast cancer cases and 912 controls were available for analysis. Table ADH1B and ALDH2, and individual ORs and 95% CIs for breast cancer risk. Genotype frequencies for both polymorphisms were in accordance with the Hardy–Weinberg law in controls: ADH1B His48Arg (P = 0.90) and ALDH2 Glu504Lys (P = 0.10). No significant impact on breast cancer risk by genotype was seen with individual polymorphisms for either ADH1B or ALDH2 in the analysis controlling for matching factors only or the analysis also controlling for potential confounders.Table ADH1B and ALDH2 genotype. In overall analysis, heavy drinkers (those consuming more than 15 g ethanol/day) versus nondrinkers, the point estimate of the OR was greater than unity , although not significantly so. Former drinkers with ADH1B*1/*1 and all categories with ALDH2*2/*2 could not be analyzed because of the small numbers of case and control subjects. None of the available analyses stratified by individual ADH1B and ALDH2 genotypes showed a significant association with alcohol drinking status.Table ADH1B and ALDH2 genotypes. Among all 9 genotype combinations, only the combination of ADH1B*1/*2 and ALDH2*2/*2 was significantly associated with a reduced risk of breast cancer, although it should be mentioned that all subjects with this genotype combination were never-drinkers. None of the available analyses for the combined genotype showed a significant association with increased or reduced risk (data not shown) for any type of drinker , although genotype combinations which included ALDH2*2/*2 could not be analyzed because of the small number of drinkers with the ALDH2*2/*2 genotype , mastitis (7.5%), gastrointestinal disease (4.1%), or benign gynecologic disease (4.1%),35 which indicates that any such bias would be limited. Finally, the study had sufficient power to assess the impact of ALDH2 polymorphism on the risk of breast cancer. However, careful interpretation of results from the stratified and interaction analyses is necessary due to the limited number of subjects included in these analyses.The methodological background of this study warrants discussion. First, with regard to the control population, we used noncancer patients at the ACCH because our case subjects were from this population, and this guarantees internal validity. To account for differences in background between cases and controls, we adjusted for mode of referral to our hospital. Moreover, with regard to the external validity of our results, we previously showed that individuals selected randomly from our control population were similar to the general population from which they were drawn, in terms of the exposure of interestADH1B and ALDH2 gene polymorphisms on the risk of female breast cancer and no significant gene–environment interactions of the effects of alcohol consumption and polymorphisms in ADH1B and ALDH2 on the risk of breast cancer, despite sufficient statistical power to observe such associations. These findings do not support the hypothesis that acetaldehyde is the main contributor to the carcinogenesis of alcohol-induced breast cancer.In conclusion, in this case–control study we found no significant impact of


After the five-year follow-up survey in 1995 to 1998, 350 cases were newly diagnosed in 43,639 women during 9.5 years' follow-up. The baseline questionnaire assessed the frequency of total green tea drinking while the five-year follow-up questionnaire assessed that of two types of green tea, P for trend = 0.60) in the baseline data. Similarly, compared with women who drank less than one cup of Sencha or Bancha/Genmaicha per week, adjusted HRs for women who drank 10 or more cups per day were 1.02  for Sencha and 0.86  for Bancha/Genmaicha. No inverse association was found regardless of hormone receptor-defined subtype or menopausal status.Compared with women who drank less than one cup of green tea per week, the adjusted hazard ratio (HR) for women who drank five or more cups per day was 1.12  0.81 to 1.56; In this population-based prospective cohort study in Japan we found no association between green tea drinking and risk of breast cancer. Camellia sinensis, differences in the manufacturing process mean that green tea has a higher catechin content than black tea [in vitro and animal studies, which have demonstrated various protective effects of green tea and tea polyphenols acting via strong antioxidant activity, inhibition of cell proliferation and angiogenesis, induction of apoptosis, and antiestrogenic properties [Green tea is regularly consumed in Japan and China as a traditional habit and cultural characteristic. Although produced from the same plant, lack tea -3, whichlack tea . Specifilack tea -3,5,6. Black tea , a controperties -3,5,6,8.in vitro and animal studies, few epidemiological studies have examined the association between green tea intake and risk of breast cancer, and their findings have been inconsistent [in vitro studies have suggested the inhibition of aromatase by green tea extracts [In contrast to nsistent -16. An insistent -11, whernsistent ,13 or onnsistent . This innsistent -11, whernsistent ,13. Inclnsistent ,13. Sevensistent , for exansistent . Howeverextracts  and of eTo address these issues, we conducted a large-scale population-based prospective cohort study in Japan on the association between green tea intake and the risk of breast cancer.The Japan Public Health Center-based Prospective Study (JPHC Study), which began in 1990 for Cohort I and in 1993 for Cohort II, included 140,420 inhabitants  in the municipalities supervised by 11 public health centers (PHC). Details of the study design have been described elsewhere [n = 98), we defined a population-based cohort of 67,422 women.The study population comprised registered Japanese inhabitants living in each PHC area, aged 40 to 59 years in Cohort I and 40 to 69 years in Cohort II. In the present analysis, one PHC area was excluded because data on cancer incidence were not available. Thus, after exclusion of ineligible women  returned the baseline questionnaire. Of these, 1,510 who reported a history of cancer in the baseline survey were excluded, leaving 54,376 women as the baseline questionnaire respondents. Of the 67,422 women, we identified 62,788 as eligible for the five-year follow-up survey after exclusion of those who had died, moved out of the study area, or been lost to follow-up before the survey, of whom 52,485 (84%) returned the questionnaire. We also excluded 1,860 women who had a history of cancer at the five-year follow-up survey and 5,813 women who did not respond to the baseline questionnaire, leaving 44,812 women as the five-year follow-up questionnaire respondents.Sencha  and Bancha /Genmaicha (blend of bancha and roasted brown rice), oolong tea, black tea, coffee and canned coffee was assessed in terms of frequency and amount using nine precoded categories: less than one cup per week, one to two cups per week, three to four cups per week, five to six cups per week, one cup per day, two to three cups per day, four to six cups per day, seven to nine cups per day, and ten or more cups per day. Sencha and Bancha are the two main types of green tea consumed in Japan, and are usually prepared by steeping the tea leaves in hot water. The amounts of Sencha or Bancha/Genmaicha consumed (ml per day) were computed by multiplying the frequency by the portion size for each beverage (120 ml per cup). Total green tea intake was defined as the sum of Sencha and Bancha/Genmaicha intake. The validity of green tea intake reported by the cohort was assessed using dietary records for 28 days (seven-day dietary records in four seasons) or 14 days. Spearman's correlation coefficients for green tea intake between the dietary record data and baseline questionnaire were 0.63 for Cohort I [Information on beverages, including green tea, oolong tea, black tea, and coffee, was obtained in the baseline questionnaire in terms of frequency and amount using six precoded categories: less than one cup per week, one to two cups per week, three to four cups per week, and almost daily . In the five-year follow-up questionnaire, the consumption of beverages, including two items of green tea, namely n = 54,376), 2,962 women (5.4%) moved out of the study area and 201(0.4%) were lost to follow-up. The occurrence of cancer was identified by active patient notification from major local hospitals in the study area and data linkage with population-based cancer registries, with permission from the local governments responsible for the registries. Death certificates were used to supplement information on cancer incidence. Site of origin and histologic type were coded by members of our Study Group using the International Classification of Diseases for Oncology, Third Edition (ICD-O-3), code C500-509. Information on estrogen receptor (ER) and progesterone receptor (PR) status was collected from medical records or pathology reports. Up to the end of the study period, 586 new breast cancer cases were identified among the baseline questionnaire respondents  and 362 cases among the five-year follow-up questionnaire respondents . Diagnosis was microscopically verified in 95% of 586 cases, and based on death certificates only in 1.0%. Information on ER and PR status was available for 278 (47%) and 262 (45%) of 586 cases, respectively.All registered women were followed from the start of the study period to 31 December 2006. Data on residential relocation were obtained from residential registries. Among the baseline respondents  until the date of diagnosis of breast cancer, date of relocation from the study area, date of death, or end of the study period (31 December 2006), whichever occurred first. Similarly, person-years of follow-up were calculated from the five-year follow-up survey (1995 to 1998) until the date of diagnosis of breast cancer, date of relocation from the study area, date of death, or end of the study period (31 December 2006), whichever occurred first for the five-year follow-up data analyses.P-values reported are two-sided, and significance level was set at P < 0.05.The Cox proportional hazards model was used to estimate hazard ratio (HR) and 95% confidence intervals (CI) of breast cancer by green tea intake using the SAS program (PROC PHREG) . The following variables were used for adjustment as potential confounders: age, study area, age at menarche, menopausal status at baseline and age at menopause, number of births, age at first birth, height, body mass index, alcohol intake, smoking status, physical activity, exogenous hormone use, family history of breast cancer, oolong tea intake, black tea intake, coffee intake and canned coffee intake. Linear trends for HRs were tested in the Cox proportional hazards models using the exposure categories as ordinal variables. All During 733,667 person-years of follow-up  for 53,793 women between 1990 to 1994 and 2006, a total of 581 cases of breast cancer were newly diagnosed and included in the baseline data analyses; while during 412,801 person-years of follow-up  for 43,639 women between 1995 to 1998 and 2006, a total of 350 cases of breast cancer were newly diagnosed and included in the five-year follow-up data analyses.Sencha and Bancha/Genmaicha, while 5.2% and 2.5% drank 10 or more cups per day, respectively, in the five-year follow-up data. Women who drank five or more cups per day in the baseline data tended to be older, live in towns or villages, be taller, have an earlier onset of first menstruation and fewer births, have a lower use of exogenous female hormones, drink less oolong tea and coffee, and consume more fruits and isoflavones. Similar characteristics was observed for women who drank 10 or more cups of Sencha per day in the five-year follow-up data, while women who drank 10 or more cups of Bancha/Genmaicha per day tended to be older, have later onset of first menstruation, earlier age at first birth and a larger number of births.Baseline characteristics of the study participants according to green tea intake are shown in Table P for trend = 0.60) in the baseline data analyses. Similarly, compared with women who drank less than one cup of Sencha or Bancha/Genmaicha per week, adjusted HRs for women who drank 10 or more cups per day in the five-year data analyses were 1.02  for Sencha and 0.86  for Bancha/Genmaicha. Moreover, compared with women who drank neither Sencha nor Bancha/Genmaicha, the adjusted HR for women who drank more than 1,320 ml per day was 1.29 . No substantial change was seen after further adjustment for other potential confounders such as residential area or dietary intake of meat, fish, vegetables, fruit, energy and isoflavones (data not shown). Further, no substantial change was seen in either baseline or five-year follow-up data after the exclusion of women within the first five years of follow-up to minimize the influence of existing preclinical conditions (data not shown), or after the exclusion of women who drank more than one cup of oolong tea or black tea per week to prevent the inclusion of other tea drinkers into the green tea intake reference category (data not shown).We found no inverse association between green tea intake and the risk of breast cancer regardless of green tea intake as assessed by the baseline or five-year follow-up questionnaire Table . CompareTo assess the potential influence of changes in green tea intake during the follow-up period on our findings, we first categorized subjects into three consumption groups for each survey: non-drinkers (corresponding to 'less than one cup per week'), 1 to 719 ml per day, and more than 720 ml per day (corresponding to 'more than five cup per day' in the baseline questionnaire). We next categorized subjects into three groups based on the combination of green tea intake calculated from the both the baseline and five-year follow-up questionnaires: non-drinkers for both questionnaires, more than 720 ml per day for both questionnaires, and other combinations. Compared with women who did not drink green tea for both questionnaires, adjusted HR was 1.12 (95% CI 0.45 to 2.83) for women who drank more than 720 ml per day for both questionnaires.For analysis of cases by hormone receptor status and among women grouped by menopausal status, we re-categorized women into four groups for the baseline data and three for the five-year follow-up data. In the baseline data analyses, we found no inverse association between green tea intake and the risk of breast cancer regardless of hormone receptor-defined subtype Table . StratifIn additional analyses to investigate the associations of oolong tea, black tea, and coffee intake with breast cancer risk, we found no inverse associations with the baseline Table  or five-In this population-based prospective cohort study, we found no overall association between green tea intake and the risk of breast cancer among Japanese women regardless of menopausal status. Our findings are in general agreement with those of three prospective studies, including two Japanese cohort studies, which found no association between green tea intake and breast cancer risk -14. One The other major strength of the present study was its prospective design, in which information was collected before the subsequent diagnosis of breast cancer, thereby avoiding the exposure recall bias inherent to case-control studies. Subjects were selected from the general population, the sample was large, the response rate to the questionnaire (more than 80%) was acceptable for study settings such as this, and the loss to follow-up (0.4%) was negligible. Furthermore, the cancer registry in the study population was of sufficient quality to reduce the possibility of misclassification of the outcome.Several limitations of this study warrant mention. First, since green tea intake was assessed by self-administered questionnaire, misclassification may have been unavoidable, albeit that our validation study showed relatively high validity . Changesn = 581) in the baseline data analyses, reflecting the low incidence rate in Japan  [Second, in spite of a reasonably large cohort population  and long follow-up period (average 13.6 year), the number of breast cancer cases was relatively low (parison) . AlthougThird, although we measured and adjusted for several potential confounders in the statistical model as far as possible, the effects of confounding by unmeasured variables and residual confounding cannot be totally discarded.Fourth, information on hormone receptor status was available for 45 to 47% cases in the present study. The major reason for the unknown cases is that collection of this information began in 2002, while data for 1990 to 2002 were obtained by retrospective review of medical records or pathology reports. The relatively small number of cases weakened the statistical power to detect the association. In addition, despite the generally high agreement between the enzyme-linked immunoassay and immunohistochemical techniques, differences in classification as well as interlaboratory variation may result in misclassification, which, if present, may have also attenuated the true association. These methodological limitations might partly explain our findings, which showed no inverse association between green tea intake and the risk of breast cancer regardless of hormone receptor-defined subtype. Thus, further research to clarify the differential associations by hormone receptor-defined subtype is warranted.Sencha, for example, one of most popular green teas in Japan, contains higher levels of tannin, vitamin C and folate than Bancha/Genmaicha [Our findings contradict those of three case-control studies, which showed an inverse association -11. Giveenmaicha . The preenmaicha ,10, no senmaicha . Differemethylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS) genes [Alternatively, the inconsistencies reported in previous studies might be explained by possible effect modification by dietary factors and genetic polymorphisms ,14,26. IS) genes ,13. On tMoreover, a recent cohort study in Shanghai showed a time-dependent interaction between green tea consumption and age of breast cancer onset: HRs for women who started green tea drinking at 25 years of age or younger versus non-green tea drinkers were 0.69 for premenopausal breast cancer and 1.61 for postmenopausal breast cancer . This coAlthough oolong tea, black tea, and coffee are less popular than green tea in Japan, they are also rich in phenolic compounds and have been hypothesized to have a protective effect against the development of breast cancer: oolong tea and black tea contain higher levels of theaflavins and thearubigins than catechins, and coffee contains a high level of chlorogenic acid -3. We alIn this population-based prospective cohort study, we found no overall association between green tea intake and the risk of breast cancer among Japanese women. Our findings suggest that drinking green tea as a beverage is unlikely to reduce the risk of breast cancer regardless of green tea type and number of cups within a usual drinking habit.CI: confidence interval; EGCG: (-)-epigallocatechin-3-gallate; ER: estrogen receptor; HR: hazard ratio; JPHC Study: Japan Public Health Center-based Prospective Study; PHC: public health centers; PR: progesterone receptor.The authors declare that they have no competing interests.MoI, MaI, SS, NS, TY, TS and ST were involved with the study concept and design. MoI, MaI, SS, NS, TY, TS and ST participated in the acquisition of data. MoI, MaI, SS, NS, TY, TS, WW and ST contributed to the analyses and interpretation of data. MoI conducted the statistical analyses and wrote the manuscript. All authors participated in the interpretation of results and critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.


Cancer is the second leading cause of death in the US and in Mississippi. Breast cancer (BC) is the most common cancer among women, and the underlying pathophysiology remains unknown, especially among African American (AA) women. The study purpose was to examine the joint effect of menopause status (MS) and hormone replacement therapy (HRT) on the association with cancers, particularly BC using data from the Jackson Heart Study. The analytic sample consisted of 3202 women between 35 and 84 years of which 73.7% and 22.6% were postmenopausal and on HRT, respectively. There were a total of 190 prevalent cancer cases (5.9%) in the sample with 22.6% breast cancer cases. Menopause (p < 0.0001), but not HRT (p = 0.6402), was independently associated with cancer. Similar results were obtained for BC. BC, cancer, hypertension, type 2 diabetes, prevalent cardiovascular disease, physical activity and certain dietary practices were all significantly associated with the joint effect of menopause and HRT in the unadjusted analyses. The family history of cancer was the only covariate that was significantly associated with cancer in the age-adjusted models. In examining the association of cancer and the joint effect of menopause and HRT, AA women who were menopausal and were not on HRT had a 1.97  times odds of having cancer compared to pre-menopausal women after adjusting for age; which was attenuated after further adjusting for family history of cancer. Given that the cancer and BC cases were small and key significant associations were attenuated after adjusting for the above mentioned covariates, these findings warrant further investigation in studies with larger sample sizes of cancer (and BC) cases. Breast The Jackson Heart Study was initiated as a response to American health disparities in the US with a view to gaining a better understanding of key factors in the development of cardiovascular disease in African Americans . Few stu2.The Jackson Heart Study (JHS) is a prospective cohort study of cardiovascular disease in non-institutionalized African American adults aged 21–95 residing in the Jackson, MS metropolitan area (MSA). The JHS is the largest single-site prospective epidemiologic investigation of cardiovascular disease in African Americans. The state of Mississippi has the highest percentage of African American residents (36.9%) of any state in the U.S. Participants were recruited from urban and rural areas of three counties in the Jackson MSA, which includes Hinds, Madison, and Rankin counties. Overall, 17% of JHS participants were recruited through random sampling of the Jackson MSA commercial database (Accudata), 22% as family members, 31% from the Jackson, MS site of the National Heart, Lung, and Blood Institute’s Atherosclerosis Risk in Communities study, and 30% as volunteers. The final JHS cohort included 5,301 participants, equivalent to 6.6% of all African Americans residing in the Jackson MSA. Data utilized in the analysis were obtained from JHS examination 1 (2000–2004) and the JHS examination 1 annual follow-up interview which was completed within three months of the anniversary date of the original visit. Study design has been detailed elsewhere –14.The analytic sample size for this study is 3202 and it was derived using the exclusion criteria: a) exclusion of all male participants and b) exclusion of female participants who were 21–34 and 85–95 years old to minimize the potential of have a highly correlated data set, since these women were strictly recruited for the family study. The sample size for the analysis involving the joint effects of menopause and HRT was 3142 due to further exclusion of women who were pre-menopausal on HRT. The JHS variables utilized in this study were grouped into the following: outcome measures, reproductive measures, demographic and socioeconomic measures, clinical risk factors and behavioral risk factors, which included dietary practices.The primary and secondary outcome variables were prevalent cancer and prevalent breast cancer, respectively. Both prevalent cancer and breast cancer cases were defined by the participants responses to the following questions: (1) “Has your doctor or health care professional ever said you have cancer?” This question was taken from the Personal and Family History Questionnaire; (2) “Has a doctor ever said you had cancer?”; or (3)“Have you had another cancer?” Questions two and three were taken from the annual follow-up questionnaire. The date of diagnosis was prior to the participant’s JHS Exam 1 visit date.Women who responded “yes”, at the baseline examination, to having had menstrual periods or bleeding during the past two years were classified as pre menopausal. Women who responded “no” to the same question were classified as post menopausal. Women were classified as currently taking HRT, if they responded “yes” to either of the following questions: are you currently taking: (a) first identified hormone, (b) second identified hormone or (c) third identified hormone in the reproductive history questionnaire?, and provided proof of hormone use based on medications that were brought to clinic visit. Medications were transcribed and coded as HRT by a pharmacist using the Medispan dictionary and classified according to the Therapeutic Classification System. History of contraceptive use was ascertained based on the history of taking birth control pills included in the JHS Reproductive History Questionnaire.Demographic information, including age and gender were obtained at the examination. Socioeconomic status was measured by annual family income. Income was self reported in the following categories: less than $5,000; $5,000–7,999; $8,000–11,999; $12,000–15,999; $16,000–19,999; $20,000–24,999; $25,000–34,999; $35,000–49,999; $50,000–74,999; $75,000–99,999; $100,000 or more. Income was classified as low, lower-middle, upper-middle, and high based upon family size, number of children <18 years of age and the U.S. Census designated poverty level for the year in which the income information was obtained. Low income was defined as below the poverty level for the corresponding family size/number of children combination. High income was defined as more than four times the poverty level threshold for each family size by child grouping. The two middle income categories were divided at 2.5 times the poverty level.The clinical risk factors for cardiovascular disease examined in this paper included hypertension, diabetes, and obesity. Standardized questionnaires were used to obtain medical history information and medication usage within the past two weeks of exam 1 for diabetes and high blood pressure, and participants were asked to bring their medications to the examination. To determine hypertension status, two resting blood pressure readings were taken one minute apart at the exam using a Hawksley random-zero sphygmomanometer (Hawksley and Sons Ltd.) and averaged. Hypertension was defined according to JNC VII criteria as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg at exam, or use of blood pressure lowering medication  within 2 weeks prior to the examination, or self-reported history of hypertension.2 using measurements of weight and height at the exam while participants wore light clothing and no shoes. Obesity was defined according to the World Health Organization standard of BMI ≥ 30.Fasting blood samples were also taken at the exam and blood glucose was measured at a central laboratory. Type II diabetes was defined according to American Diabetes Association 2004 criteria as fasting glucose ≥126 mg/dL, or confirmed medication inventory or self-reported use of anti-diabetic medications, or self-reported diabetes diagnosis. Body mass index (BMI) was derived to determine overweight/obesity status. BMI was calculated in kg/mCurrent smoking status was self-reported by participants at the exam as having smoked 400 cigarettes or more in his/her lifetime and having smoked within 3 months of the JHS baseline interview. Heavy alcohol drinkers were defined as persons who consumed greater than 24 grams of alcohol per day. Physical activity was assessed using the Jackson Heart Study Physical Activity Cohort instrument derived from modification of the Baecke physical activity survey . Total pDescriptive statistics were used to describe the characteristics of the study sample. Two-sample t-tests were performed to compare cancer cases and breast cancers cases with their respective controls for all the demographic, SES, lifestyle and clinical factors which were continuous in nature. For the categorical factors chi-square tests were performed to examine the association of the factors with prevalent cancer and prevalent breast cancer. These chi-square tests were performed for the purpose of identifying potential cofounders to be adjusted for in further analyses. Also, chi-square tests were used to examine the association of prevalent cancer and breast cancer with the joint effect of menopause status and hormone replacement therapy (HRT). The general linear modeling (GLM) approach was used to assess differences among the three groups defined by the joint effect of menopause and HRT. The test of association between prevalent cancer and breast cancer and the joint effect of menopause and HRT was conducted by means of multiple (multivariable-adjusted) logistic regression; which computed the odds ratios (OR) and 95% confidence intervals (CI). Age and covariates that were significantly associated with both outcome measures were included in each of the multiple logistic regression models. A significance level of 0.05 was used in all inferential analyses.3.2. Generally, the study participants were well educated with approximately 61% reporting greater than a high school education; and 55.7% having a family income that placed them in the upper-middle to affluent income class. Their health profile concerning medical and reproductive history suggested that about 60% had a family history of cancer, 73.7% were post menopausal and 22.6% reported use of hormones. The mean (SD) number of pregnancies and live-born children were 3.5 (2.5) and 3.2 (2.2), respectively. There were a total of 190 prevalent cancer cases (5.9%) in the sample with 22.6% of them being breast cancer .Similar to To examine the joint effect of HRT and menopause, the three subgroups  were compared with respect to the factors and outcome measures considered in this study . AssessiPrevalent cancer was associated with age , and family history of cancer  in age-adjusted models. However, for breast cancer it was only associated with age . None of the demographic and socioeconomic measures, and clinical and behavior risk factors were independently associated with the outcome measures (Cancer and breast cancer). Data is not presented in this paper. In unadjusted models the odds of prevalent cancer was significantly higher in post-menopausal women who were not on HRT  and those who were on HRT  compared to women who were pre-menopausal . However4.In this study, several factors in the following domains of measurement, Demographic and Socioeconomic, Clinical and Behavioral Risk Factors, were identified as factors that were associated with prevalent cancer and breast cancer. Also, several of these factors were significantly associated with the joint effect of menopause status and HRT. Though collection of cancer-related data was limited in the JHS cohort, the estimated prevalence of cancer in African American women represented by the JHS was 5.9%, slightly more than half of the prevalence of cardiovascular disease (9.5%) in the same sample population. This finding seems consistent with the ratio of CVD and cancer mortality in the State of Mississippi, the two most common causes of mortality in the state. Of the types of cancers in this sample, the most prevalent was breast cancer, which represented 22.6% of all cancers cases. These findings were consistent with the literature that refers to breast cancer as the most common form of cancer and the second leading cause of death among women in the United States .vs. non-cancer), (breast cancer vs. non-breast cancer) and  based on the cut-point of greater than 30% of total energy consumed set forth by the National Institute of Health’s National Heart, Lung, and Blood Institute [The unadjusted comparison of dietary practices, total fat intake, total vitamin E intake, Lycopene and % calories from alcohol significantly differ between cancer and non cancer cases. However, these significant associations were attenuated in the age adjusted logistic models. This study does not support earlier findings in the literature that certain lifestyles, such as dietary practices, tobacco use, alcohol consumption, weight gain, and physical activity, as well as high blood pressure, considered traditional CVD risk factors, may be linked to the development of both CVD and cancer (and particularly breast cancer). However, given the small number of cancer cases, it is important to note that the study findings underscore that there is little known association between dietary practices and cancer in African American women –19. Thusnstitute .Physical activity measured by home and yard activities was significantly associated with cancer in the unadjusted models. However these associations were attenuated in the age adjusted models. The mean age of the onset of use of birth control pills, and two classical CVD risk factors, hypertension and Type 2 diabetes mellitus, were also significantly related to cancer. However, these significant findings were not evident for prevalent breast cancer. A plausible explanation could be the low prevalence of breast cancer (1.1%) in the study sample.Since menopause, but not HRT, was independently associated with cancer, and the joint effect of menopause and HRT suggested that post-menopausal women who were not on HRT had higher odds ratios of cancer (and breast cancer) compared to pre-menopausal and post-menopausal women who were on HRT, further investigation of these findings is warranted. The pursuit of the recommended further studies in this area of scientific investigation is very important for the following reasons. The number of cancer and breast cancer cases in this study was relatively small and the findings could be validated or challenged with studies of larger numbers of cancer or breast cancer cases. Secondly,  reportedThough data for classifying cancer and especially breast cancer was limited in the Jackson Heart Study compared to extensive data on CVD outcomes and risk factors, this study provides insight into the potential of ascertaining additional data specific to cancer etiology, progression and survival, given the extensive data on biological and psychosocial determinants of cancer morbidity and mortality as outcome measures. The reproductive history coupled with repeated data collection on medication use, provides a foundation for future studies of cancer that utilizes the Jackson Heart Study data.Given that the Jackson Heart Study is a longitudinal study, it provides scientific resources for future recommended investigations that may examine the joint effects of menopause and HRT on the risk of developing cancer (breast cancer) in African American women.


Soy and red clover isoflavones are controversial due to purported estrogenic activity and possible effects on breast cancer. We conducted a systematic review of soy and red clover for efficacy in improving menopausal symptoms in women with breast cancer, and for potential impact on risk of breast cancer incidence or recurrence.We searched MEDLINE, Embase, the Cochrane Library, and AMED from inception to March 2013 for human interventional or observational data pertaining to the safety and efficacy of soy and red clover isoflavones in patients with or at risk of breast cancer. Of 4179 records, we included a total of 131 articles: 40 RCTs, 11 uncontrolled trials, and 80 observational studies. Five RCTs reported on the efficacy of soy for hot flashes, showing no significant reductions in hot flashes compared to placebo. There is lack of evidence showing harm from use of soy with respect to risk of breast cancer or recurrence, based on long term observational data. Soy intake consistent with that of a traditional Japanese diet  may be protective against breast cancer and recurrence. Human trials show that soy does not increase circulating estradiol or affect estrogen-responsive target tissues. Prospective data of soy use in women taking tamoxifen does not indicate increased risk of recurrence. Evidence on red clover is limited, however existing studies suggest that it may not possess breast cancer-promoting effects. Soy consumption may be associated with reduced risk of breast cancer incidence, recurrence, and mortality. Soy does not have estrogenic effects in humans. Soy intake consistent with a traditional Japanese diet appears safe for breast cancer survivors. While there is no clear evidence of harm, better evidence confirming safety is required before use of high dose (≥100mg) isoflavones can be recommended for breast cancer patients.  Breast cancer accounts for almost one third of cancers diagnosed among women. In the United States, there were approximately 288 thousand new cases expected for 2011 . Among bGlycine max, contains the class of phytoestrogens known as isoflavones, specifically, genistein, daidzein, glycitein, biochanin A, and formononetin , and 16 examined intake of soy isoflavones ,77,81,84On subgroup analysis, there was no clear difference in effect based on the type of soy exposure , or according to study quality (NOS score), the measure of exposure , or menopausal status S1-S2. TSeven studies examined the effect of soy by receptor status; these findings were mixed. Three studies reported no modification of effect by ER/ PR status ,58,81. TA total of 10 nested case control studies were included pertaining to soy, shown in 2 odds ratio (associated with doubling of exposure) and as a different measure was not included in the forest plots [Of eight studies assessing risk of breast cancer, one showed significantly reduced risk associated with higher plasma genistein . Grace est plots . NonetheFive studies showed no significant effects in either direction. One study examined mammographic density as a surrogate of breast cancer risk, however with mixed findings . While cA total of 24 prospective cohort studies were included, described in Two studies reported on associations with mammographic density as a predictor of breast cancer risk ,110. OneTwo studies reported on menopausal symptoms and quality of life among breast cancer patients ,95. TherNine prospective studies reported on risk of breast cancer recurrence or mortality ,106,112.Of five studies reporting on recurrence ,106,112,Of seven studies examining survival ,106,112,We included 11 uncontrolled trials examining the effect of soy on safety outcomes relevant to breast cancer risk and progression ,119. TheIn studies conducted among healthy women, there were no significant changes compared to baseline in endometrial thickness ; rates oOf the 38 randomized controlled trials, five assessed the efficacy of soy for hot flashes among breast cancer survivors -131, whiA total of 18 RCTs assessed circulating estrogens levels  Fifteen of 18 studies found no significant impact from soy compared to the control group (p>0.05) ,160,163.A total of nine RCTs assessed the impact of soy consumption on estrogen metabolism as measured through urinary excretion of urinary metabolites. Of these, five reports of six studies found no significant change in 2-hydroxyestrone, 16α-hydroxyestrone, or 2:16α hydroxyestrone ratio compared to the control group  ,161,164.None of the studies assessing endometrial thickness ,136,155,Three studies looked at molecular markers of breast cell proliferation or genetic markers of breast cancer risk. One study found no change in BRCA 1 and 2 mRNA level due to soy consumption, however there was a decrease over time in the placebo group, yielding a significant difference between groups (p<0.001) . One stuRCTs were assessed as having a moderate risk of bias, with inadequate reporting of random sequence generation and allocation concealment (selection bias) in 18 and 32 of 43 unique RCTs, respectively. The majority of RCTs showed low risk of performance bias, detection bias, attrition bias, and reporting bias; description of blinding of participants, blinding of outcome assessment, complete outcome data and reporting was adequate in over 88% of the studies. The risk of bias across studies is shown in The most common adverse event associated with soy consumption was mild to moderate gastrointestinal discomfort, but this usually occurred with comparable frequency in both the soy and the placebo groups ,147,150;Seven studies included in our review investigated the effect of soy in combination with hormonal therapies: tamoxifen and aromatase inhibitors. Four cohort studies ,106,112 Of the cohort studies, the WHEL cohort exhibited relatively low soy isoflavone intake  and yielded null results: HR for recurrence among tamoxifen users was 0.59 (0.27-1.29) . Guha etOne cohort study examined soy consumption by post-menopausal women who were on anastrozole, and found decreased risk of recurrence among those with an intake of >42mg/d isoflavones, AHR 0.65 (0.47-0.85) .Two RCTs pertaining to red clover were included ,166. TheIn brief, Atkinson et al assessed the effect of red clover on mammographic density as well as a panel of other markers of estrogenic activity, in 205 women with an increased risk of breast cancer due to their breast density pattern (Wolfe P2 or DY mammographic breast patterns); there was also a secondary assessment of hot flashes . ParticiPowles et al investigated the effect of red clover in 401 women with a family history of breast cancer (at least one first degree relative affected), assessing circulating FSH, endometrial thickness, mammographic density, and bone density . ParticiResults of the study by Atkinson showed no significant changes in hot flash score (p=0.88) or mean number of daily hot flashes (p=0.41) when groups were compared . NonetheNeither of the RCTs reported on breast cancer incidence rates, however surrogate markers of estrogenic activity were examined. Atkinson reported no significant changes in estradiol, FSH, or LH over the one year period: estradiol increased by 14.0 pmol/L in the red clover group compared to a decrease of 0.9 in the placebo group, however this was not significant, p=0.49); FSH decreased 4.2 IU/L in the red clover group compared to a decrease of 2.9 in the placebo group, p=0.83; LH decreased 4.0 IU/L in the red clover group compared to a decrease of 4.2 IU/L in the placebo group, p=0.71; and tyrosine kinase increased 1.62 units of activity/ µg protein in the red clover group compared to an increase of 0.90 in the placebo group, p=0.16 . P = 0.009) [CC/CT vs. TT: OR 0.95, 95%CI 0.89-1.00 [Neither of the two RCTs reported any significant changes in mammographic density among both pre- and post-menopausal women ,166, and= 0.009) . ESR1 co= 0.009) . For ins.89-1.00 .. It is Atkinson did not report adverse events. Powles reported adverse effects, which most commonly included breast abnormality, “skin related symptoms” (not described), and other minor adverse events, however these were equally distributed between red clover and placebo groups .According to the Cochrane risk of bias tool, the trial reported by Powles was assessed as having low risk of bias in all of the categories, except one for which information about detection bias was not provided . The triThe results of our systematic review suggest that there is a lack of real evidence showing that soy increases risk of breast cancer or breast cancer recurrence. This is an important finding given the generally perceived controversial status of soy in relation to breast cancer . Our revAmong studies included in our review, case control studies showed a stronger association between soy and reduced risk of breast cancer. As shown in The effect of soy on hot flashes in breast cancer patients is not clear. RCTs noted some improvements over time, but not in comparison to placebo -131. ThiSeveral factors influence the biological activity of soy isoflavones in the body. First, soy isoflavones show selectivity toward ER-ß over ER-α -175. ThiSecondly, preclinical evidence has shown that under conditions of high estrogen concentration similar to premenopausal levels, soy isoflavones act as ER antagonists, while under conditions of low estrogen comparable to postmenopausal levels, they are ER agonists ,181,182.Placed in this context, the clinical data reviewed by our study, which demonstrates a lack of any clear pro-estrogenic effects from use of soy, is quite noteworthy. We found no effects on circulating estradiol, and no measurable effects on estrogen-sensitive target tissues, such as breast tissue (density) and endometrium. In addition, our findings are in agreement with those reported by Hooper et al in a 2009 meta analysis of 47 trials . Hooper Despite this, and despite the fact that our study failed to show any clear estrogenic effects observable in humans overall, the possibility of soy having estrogen-like effects under some circumstances in certain subgroups of women cannot be ruled out . Of partOur review revealed no clear modification of soy’s effect based on menopausal status or ER+/- status in breast cancer patients in large observational studies. However we did find variation according to geographical locale; studies conducted in Asian countries more often reported chemopreventive effects compared to studies in Western countries, which more often reported null results. We attribute this to a difference in soy consumption between these areas. The traditional Japanese diet contains between 6-11g of soy protein and 25-50mg isoflavones; top percentiles of soy intake in Asian studies consume as much as 20g soy protein or 100mg isoflavones per day , while tThe suggestion has been made that the effect of soy depends on genetic variations present in Asian populations. Nechuta et al conducted an analysis of the effect of soy on breast cancer recurrence, investigating the possibility of ethnic variations . They reOur review is broad in its scope, assessing soy in the context of breast cancer from several perspectives, including risk of breast cancer, risk of recurrence, estrogenic effects, and risk of interactions with tamoxifen and other hormonal therapies. Our findings are generally in agreement with those of the American Cancer Society, suggesting that moderate amounts of soy intake (up to 3 servings per day) is likely safe for consumption by women with breast cancer . Although we included a large number of studies regarding soy and breast cancer risk, we were unable to pool data with respect to risk of breast cancer and risk of recurrence due to heterogeneity. There is a lack of long term interventional data assessing cancer risk. This is a particularly important shortcoming because RCTs of under 2 years duration are unlikely to reveal any serious adverse effects in breast cancer survivors, including possible interactions between soy and tamoxifen. This question deserves high priority for future research in this area. In addition, there is a need to more carefully assess the dose-response relationship between soy intake and risk of breast cancer in order to more clearly delineate the threshold of exposure needed for potential therapeutic effects. Soy does not appear to influence levels of circulating estrogen or exert estrogen-like effects at target tissues. There is a lack of evidence showing clear effects of soy consumption or supplementation on reduction of hot flashes in breast cancer patients. Observational data suggest that higher soy intake, consistent with that of a traditional Japanese diet, may be protective against the development of breast cancer as well as breast cancer recurrence and mortality, although there is a need for clinical studies to confirm this relationship. Until there is more data supporting safety, caution is warranted with high dose isoflavone supplements in patients with breast cancer. Figure S1Risk of Breast Cancer By Study Quality (Isoflavones).(TIF)Click here for additional data file.Figure S2Risk of Breast Cancer by Menopausal Status (Isoflavones).(TIF)Click here for additional data file.Figure S3Risk of Breast Cancer by Dose of Soy Isoflavones.(TIF)Click here for additional data file.Figure S4Risk of Breast Cancer Recurrence with Intake of Soy Protein or Isoflavones.(TIF)Click here for additional data file.Figure S5Risk of Bias Across Studies.(TIFF)Click here for additional data file.Table S1Medline Search Strategy.(DOCX)Click here for additional data file.Table S2Case Control Studies of Soy and Risk of Breast Cancer.(DOC)Click here for additional data file.Table S3Prospective Cohort Studies of Soy and Risk of Primary Breast Cancer.(DOC)Click here for additional data file.Table S4Trials Assessing Effect of Soy on Hormonal Biomarkers and Estrogen Dependent Tissues.(DOC)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


CXCL5-rs425535 , while among EA women, there were associations with TNFA-rs1799724  and CRP-rs1205 . For postmenopausal women, IL1B-rs1143627  and IL1B-rs16944  were associated with risk among EA women, with significant associations for TNFA-rs1799724 limited to estrogen receptor (ER) positive cancers . However, none of the SNPs retained significance after Bonferroni adjustment for multiple testing at the level of P0.0012 (0.05/42) except for TNFA-rs1799724 in ER positive cancers. In a stage II validation , we extended evaluations for four SNPs , which yielded similar results. In summary, distributions of variants in genes involved in innate immune pathways were found to differ between AA and EA populations, and showed differential associations with breast cancer according to menopausal or ER status. These results suggest that immune adaptations suited to ancestral environments may differentially influence breast cancer risk among EA and AA women.African American (AA) women are more likely than European American (EA) women to be diagnosed with early, aggressive breast cancer. Possible differences in innate immune pathways  have received little attention as potential mechanisms underlying this disparity. We evaluated distributions of selected genetic variants in innate immune pathways in AA and EA women, and examined their associations with breast cancer risk within the Women’s Circle of Health Study (WCHS). In stage I of the study (864 AA and 650 EA women) we found that genotype frequencies for 35 of 42 tested SNPs (18 candidate genes) differed between AAs and EAs (corroborated by ancestry informative markers). Among premenopausal AA women, comparing variant allele carriers to non-carriers, reduced breast cancer risk was associated with  Breast cancer is the most common cancer among women in the United States, accounting for 29% of all newly diagnosed cancers . AlthougTNFα), interleukin-1 beta (IL1β) and interleukin-6 (IL-6) [IL6, TNFα, C-reactive protein (CRP), have been associated with a greater risk for several types of cancer and cancer prognosis, including breast [CRP and IL6 levels than EAs [Chronic inflammation has been implicated in tumor initiation, promotion, progression, invasion, and metastasis . A state6 (IL-6) . Elevateg breast ,9. Severg breast . Indeed,than EAs ,12. Becathan EAs –15, whicTNF, IL1α, IL1β, IL1 receptor antagonist (IL1RN), and IL6, with mixed results.Variants in genes involved in the innate immune response pathway may influence the production or action of inflammatory cytokines and subsequently modulate inflammatory response, influencing risk of breast cancer. Racial differences in allele frequencies of single nucleotide polymorphisms (SNPs) for certain cytokine genes have been reported in several studies, and certain genetic variants associated with increased levels of pro-inflammatory biomarkers are more frequent in AAs than that in EAs ,17. WhilIn this case-control study, we used a two stage design to examine potential associations between breast cancer and variants in genes involved with chronic inflammation within innate immunity-related pathways in AA and EA women. We hypothesized that the differential distribution of ‘at-risk’ alleles could contribute to the higher incidence of aggressive breast cancer among AA women, particularly risk of ER-negative breast cancers.Analyses were conducted using data and samples from the Women’s Circle of Health Study (WCHS), a case-control study designed to evaluate risk factors for early/aggressive breast cancer in AA and EA women. Details of the study design, enrollment criteria, and collection of biospecimens and questionnaire data have been described previously ,23. In bThis study was approved by institutional review boards at Roswell Park Cancer Institute (RPCI), the Cancer Institute of New Jersey (CINJ), Mount Sinai School of Medicine , and participating hospitals in New York. Signed informed consent was obtained from each participant prior to interview and biospecimen collection.Detailed data on demographic characteristics, medical history, family history of cancer, and lifestyle factors were collected by in-person interviews. Anthropometric measures and biospecimens were collected by trained interviewers. Pathology data including ER status, grade and stage, and were collected and abstracted by trained study staff.TM DNA isolation kits  and from OrageneTM kits following the manufacturer’s protocols, but the majority of DNAs were derived from saliva samples collected using OrageneTM kits . Genomic DNA was evaluated and quantitated by Nanodrop UV-spectrometer  and PicoGreen-based fluorometric assay , and stored at -80°C until analysis.Genomic DNA was initially extracted from blood samples using the FlexiGeneForty-four SNPs were selected in eighteen candidate genes involved in innate immune response pathways by surveying the Human Genome Epidemiology (HuGE) Navigator . SNPs we2 values between each SNP pair using the program Haploview [Descriptive variables were compared between cases and controls using chi-square tests for categorical variables and t-tests for continuous variables. Multivariable unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the risk of breast cancer associated with genotype with adjustments for age at diagnosis (continuous), family history of breast cancer , body mass index (continuous), education , history of benign breast disease , cigarette smoking , and proportion of European ancestry (continuous). All analyses were performed separately for EA and AA women. Participants with the most common homozygous genotype among EA controls were treated as the referent group. Co-dominant as well as dominant models (heterozygous and rare homozygous genotypes combined) were computed for all SNPs examined. Additive genotype coding based on the number of rare allele was used as ordinal variables in tests for linear trend. Analyses also were conducted to examine whether SNP associations with breast cancer risk differed by menopausal or ER status. Interactions by self-reported race were tested by including an interaction term SNP*self-reported race in multivariable logistic models. Linkage disequilibrium (LD) was determined by calculating raploview .P0.05. All significant p-values were further adjusted for multiple comparisons using Bonferroni correction, with P0.0012 (0.05/42) considered statistically significant.All analyses were conducted using SAS V 9.3 . Statistical tests were two-sided and considered statistically significant for uncorrected P=0.007). Characteristics of 1,307 EA and 1,365 AA cases and controls in stage II of the study, after additional participant accrual into WCHS, were generally similar to those observed among stage I participants  are shown in icipants . HoweverP0.05), and for 4 of these SNPs , the rare allele variant was reversed between the two groups. ORs, 95% CIs, and P-values for trend from codominant models and from dominant models between each SNP and breast cancer risk are shown in TNFA-rs1799724 T allele (CT+TT) showed increased breast cancer risk .For 35 of the 42 SNPs, allele frequencies differed significantly between AA and EA controls . Compared to the CC genotype, the combined CT and TT genotypes of TNFA-rs1799724 were associated with a 2.3-fold increased breast cancer risk , whereas carriers of the CRP-rs1205 T allele (CT and TT) had a 46% reduced risk . Among premenopausal AA women, one SNP (rs425535) in chemokine (C-X-C motif) ligand 5 (CXCL5) was observed to be inversely associated with breast cancer risk , with AG/AA genotypes associated with a 39% reduced risk compared to the GG genotype. Suggestive associations were also observed for two TNFA SNPs in premenopausal AA women, with the variant TC/CC genotypes of TNFA-rs179964 and CA/AA genotypes of TNFA-rs1800630 being associated with a 1.7-fold increased breast cancer risk. Moreover, three SNPs in chemokine (C-C motif) ligand 2 (CCL2)  showed a suggestive non-significant elevated risk in premenopausal AA women. CCL2-rs4586 also was observed to be associated with an increased risk in premenopausal EA women, but the elevated risk was attenuated towards null in the stage II analysis in a larger sample of WCHS participants.Among premenopausal EA women, associations were observed for IL1B (rs1143627 and rs16944) were associated with increased risk  and one SNP in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) (rs2066842) was observed to be inversely associated with risk . The TC/CC genotypes of IL1B-rs1143627 or CT/TT genotypes of IL1B-rs16944 were associated with a 1.8-fold increased risk, whereas CT/TT genotypes of NOD2-rs2066842 were associated with a 43% decrease in risk. Among postmenopausal AA women, IL1RN-rs4251961 was inversely associated with breast cancer risk, with CT/CC genotypes associated with a decreased risk .Among postmenopausal EA women, SNPs in CXCL5-rs425535 and CRP-rs1205 among premenopausal women , and IL1B-rs1143627 and NOD2-rs2066842 among postmenopausal women . Of these, the interaction by race for CXCL5-rs425535 was most significant, where AG/AA genotypes were associated with a reduced risk among AA premenopausal women  and a non-significant elevated risk among EA premenopausal women .Although genotype associations with breast cancer risk differed in strength according to self-reported race, no SNP by race interactions were statistically significant except for TNFA (rs179924), NOD2 (rs2066842), and CCL2 (rs4586), associations were distinct from those observed by menopausal status , with the association remained significant after correction for multiple testing (P=0.04). Suggestive increased risk of ER positive cancer was also observed in EAs for carriers of FGF2-rs308379 TA/TT genotypes . Among AAs, carriers of the variant allele for CCL5-rs2280789 were 48% more likely to be diagnosed with ER positive breast cancer (P=0.03). CCL2-rs4586 homozygous variants (CC) were also associated with a suggestive 1.7-fold increased risk of developing ER positive cancer in AAs .Among EAs, carriers of the variant NOD2-rs2066842 CT/TT genotypes were observed to be 58% less likely to develop ER negative breast cancer (P=0.02), and among AAs, two SNPs in CCL5 (rs2107538 and rs3817655) in LD were associated with ER negative breast cancer . Carriers of CCL5-rs2107538 CT/TT genotypes or CCL5-rs3817655 AT/AA genotypes were 40% less likely to be diagnosed with ER negative breast cancer compared to women who had CC or TT genotype, respectively. The associations for TNFA-rs1799724 and NOD2-rs2066842 were also different between AA and EA women , with a significant increased risk of ER positive or decreased risk of ER negative breast cancer in EA, but not in AA women.EA women who carry CCL2-rs4586, CRP-rs1205, CXCL5-rs425535, IL1RN-rs4251961, to a larger dataset after accrual of an additional 501 AA and 657 EA cases and controls into the WCHS. Associations by menopausal status and by ER status are presented in CCL2-rs4586 TC/CC genotypes in premenopausal EA women was attenuated to the null in the larger dataset . In addition, we observed a significant interaction between AA and EA women for the association of CCL2-rs4586 with risk of ER positive cancers in the larger data set (P for interaction=0.04). In comparison with results from the smaller population, a stronger significantly increased risk  was observed for AA women who carried the CCL2-rs4586 CC genotype compared to those with TT genotypes.We extended analysis for four SNPs, TNFA-rs1799724, being significantly associated with overall breast cancer risk among EA women. SNP associations with breast cancer risk, however, were found to vary substantially between AA and EA populations when menopausal or ER status was considered. Our findings suggest that different gene networks may be associated with breast cancer in AA versus EA women, pre- versus post-menopausal women, and in ER positive versus ER negative breast cancers, and provide insights into the etiology of breast cancer within these subgroups, indicating areas for further research into reasons for early onset/aggressive breast cancer in AA women.Recently, a number of genome-wide association (GWA) studies focused on breast cancer have been completed, and have identified novel genetic variants as potentially being associated with breast cancer risk . GWA stuThe potential impact of inflammation-related susceptibility loci that are unevenly distributed within populations as a contributor to observed heterogeneity in breast cancer phenotypes and risk between different racial groups has been understudied. As reviewed by Pennington and colleagues  and ChapTNFA- rs1799724 in EA women, with combined CT+TT genotypes associated with a 2.3-fold increased breast cancer risk in premenopausal women, and a 2-fold increased risk of ER positive cancers. This variant located within the promoter region of the gene has been associated with increased [TNFα [TNFα is implicated in chronic inflammation and can support tumor growth and breast cancer progression. Positive cross-talk between 17β-estradiol and TNFα in inflammatory and angiogenic pathways, and the ability of TNFα to modulate gene regulation by 17β-estradiol may explain, in part, why associations were strongest for premenopausal EA women and ER positive breast cancers [TNFA, but no significant associations were found in EA women, similar to findings from other studies [TNFA-rs361525 was associated with a modest increase in breast cancer risk among EAs in a study of ~5300 cases and 4900 controls [TNFA- rs1800629 variant in Caucasians, although this appeared to be largely driven by results from one study (OR=0.55); no associations were observed with TNFA-rs1799964, rs1800630, or rs361525 [TNFA-rs1799964 and rs1800630 were associated with a similar 1.7-fold increased breast cancer risk in premenopausal AA women who carry the variant C allele of TNFA-rs1799964 or the variant A allele of TNFA-rs1800630, but not among EA women.The strongest association observed in the study was for ncreased  and decred [TNFα  productied [TNFα . Associaed [TNFα ,45. TNFα cancers . Because studies ,20,47. Tcontrols , but thirs361525 . In our CRP is an inflammatory effector that has been linked to breast cancer risk and poorer prognosis, with higher circulating levels observed in AA women compared to EAs [CRP-rs1205 were associated with decreased breast cancer risk in EA, but not AA women. Located in the 3’ untranslated region of the gene, this variant is consistently associated with decreased serum CRP levels [CXCL5, which encodes for epithelial neutrophil-activating peptide (ENA-78), is up-regulated in breast tumors and plays a role in regulating neutrophil homeostasis, an essential component of innate immunity, and a major contributor to inflammation-associated tissue damage [CXCL5-rs425535 variant A allele was associated with a decreased risk of breast cancer in AA premenopausal women, but not EAs, and was much more common among AA (MAF= 39.7%) than among EA women (MAF=13.5%). This SNP is located in an exon splicing enhancer site that could be important for transcriptional control and is highly linked with the CXCL5-rs352046 variant (r2=0.94), which is associated with significantly higher ENA-78 plasma concentrations [d to EAs . Among pP levels ,51, but P levels . The tote damage . In our trations . Why thiCCL2 is an important inflammatory chemokine involved in macrophage recruitment and expression of angiogenic factors that are highly expressed within breast tumors and associated with the development and progression of breast cancer [CCL2 SNPs  in LD were suggested to be associated with increased risk of premenopausal breast cancer in AA women, although an elevated risk observed for rs4586 in EA women in the initial analysis was attenuated towards the null in our second stage analysis using a larger study population. Of these, rs1024611 located in the promoter region of the gene has been demonstrated to affect CCL2 protein levels by changing transcription factor binding sites, with the variant C allele being associated with higher CCL2 levels [CCL2 levels. Studies have shown that CCL2 gene expression is inhibited by 17β-estradiol [CCL2, is most relevant with respect to breast cancer risk against a background of low CCL2 expression. Others, like us, did not observe associations between CCL2-rs1024611 and breast cancer risk among Caucasians [t cancer . In our 2 levels . The othstradiol , our finucasians ,61.IL1B gene, rs1143627 and rs16944, were associated with an increased risk of breast cancer in EA, but not AA women. Pro-inflammatory IL-1 is implicated in cancer progression, and intratumoral levels of IL1β are higher compared with normal adjacent breast tissue [IL1-rs16944 variant CT/TT genotypes are associated with higher CRP levels than the CC genotype in healthy individuals [IL1B-rs1143627 variant has been associated with increased breast cancer risk in Asian populations [IL-1β levels in breast tissue might be controlled in vivo by estradiol and is correlated with abdominal subcutaneous fat [NOD2-rs2066842 variant was associated with decreased breast cancer risk among postmenopausal EA women, and reduced overall risk of ER negative cancers. Although this SNP has not been implicated in risk of breast cancer, it has been shown to be associated with Crohn’s disease in Caucasians [Among postmenopausal women, two SNPs in high LD in the ividuals . The IL1ulations ,65, but ulations ,67. BecaCCL2, CCL5, NOD2, FGF2 were found to be associated with either risk of ER positive or negative breast cancer in either AA or EA women, although only the NOD2 SNP association with ER positive tumors differed between AA and EA women, and none have been examined in relation to breast cancer risk. Notably, variant alleles for two SNPs  in high LD in CCL5 (RANTES) were associated with approximately 35% decreased risk of ER negative breast cancer in AA women. RANTES is a chemokine that assists in the recruitment of inflammatory cells, and evolutionary pressures have been shown to have significant impact on genetic variation in this gene across various populations [CCL5 promotes breast cancer progression [Several other SNPs in ulations . Tumor egression , and botgression . OverallOne limitation of this study is that we focused on a select panel of SNPs in each gene thought to be important in cancer risk based on previous studies without including a comprehensive set of variants. This candidate gene and SNP selection approach based on limited literature for AA populations could have affected our ability to identify novel genetic variants, especially for AA women. In addition, although this is a study with a large number of AA and EA women to examine racial differences for these genetic variants with breast cancer risk, our sample size was limited when analyses were stratified by menopausal and ER status. We were able, however, to test and strengthen several promising relationships in a larger study sample after additional participant accrual. In the larger sample, controls were slightly younger than cases (stage II dataset) because eligibility criteria for cases and controls, originally up to age 65, was expanded to include older women up to age 75, fewer older controls were recruited towards the end of the study. This difference, however, was unlikely to confound or bias our findings given that all models included age as a covariate, and we performed a sensitivity analysis excluding older women (70 years old) and found results were very similar to those reported using all participants. Finally, it must be emphasized that our findings should be interpreted with caution because few associations remained significant after correction for multiple testing. Nevertheless, we expect the probability of making a type I error is attenuated by our choice to evaluate primarily functional SNPs shown to affect gene expression by changing transcriptional binding sites.In summary, this is the most comprehensive study that was designed to specifically examine putatively functional genetic variants in the innate immunity related inflammatory pathway with breast cancer risk and risk of ER-positive and ER-negative disease in AA and EA women simultaneously. Our findings indicate that genetic variants in innate-immunity pathways are associated with breast cancer risk in both AA and EA women, although susceptible and protective loci differed by race, and played a role in the etiology of both ER-negative as well as ER-positive breast cancers. Nevertheless, as the first study to comprehensively assess these genetic variants in both AA and EA women by menopausal or ER status, our findings could provide valuable information for better understanding of the etiology of this disease in both AA and EA women. Future studies with comprehensive resequencing or functional analyses are needed to further explore these associations.Table S1Characteristics of 1,307 European American (EA) and 1,365 African American (AA) cases and controls in the Women’s Health Circle of Study (WCHS).(PDF)Click here for additional data file.Table S2Single nucleotide polymorphisms (SNPs) of innate immune response related pathways and risk of breast cancer among 650 European American and 864 African American women in the WCHS.(PDF)Click here for additional data file.Table S3Single nucleotide polymorphisms (SNPs) of innate immune response related pathways and risk of breast cancer by menopausal status among 650 European American and 864 African American women in the WCHS.(PDF)Click here for additional data file.Table S4Single nucleotide polymorphisms (SNPs) of innate immune response related pathways and risk of breast cancer by estrogen receptor status among 650 European American and 864 African American women in the WCHS.(PDF)Click here for additional data file.