The mechanisms underlying the strong association between percentage dense area on a mammogram and the risk of breast cancer are unknown. We investigated separately the absolute dense area and the absolute nondense area on mammograms in relation to breast cancer risk.We conducted a nested case-control study on prediagnostic mammographic density measurements and risk of breast cancer in the Nurses' Health Study and the Nurses' Health Study II. Premenopausal mammograms were available from 464 cases and 998 controls, and postmenopausal mammograms were available from 960 cases and 1,662 controls. We used a computer-assisted thresholding technique to measure mammographic density, and we used unconditional logistic regression to calculate OR and 95% CI data.tertile 3 vs 1 = 2.01, 95% CI = 1.45 to 2.77) and among postmenopausal women . However, increasing absolute nondense area was associated with a decreased risk of breast cancer among premenopausal women  and among postmenopausal women . These associations changed minimally when we included both absolute dense area and absolute nondense area in the same statistical model. As expected, the percentage dense area was the strongest risk factor for breast cancer in both groups.Higher absolute dense area was associated with a greater risk of breast cancer among premenopausal women (OROur results indicate that absolute dense area is independently and positively associated with breast cancer risk, whereas absolute nondense area is independently and inversely associated with breast cancer risk. Since adipose tissue is radiographically nondense, these results suggest that adipose breast tissue may have a protective role in breast carcinogenesis. Mammographic density is one of the strongest predictors of breast cancer risk. Breast epithelium and stroma are radiographically dense and appear light on a mammogram, whereas adipose tissue is nondense and appears dark. Women with more than 75% of the total area on a mammogram occupied by dense area have a four- to sixfold greater risk of breast cancer compared to women with little or no dense area on a mammogram ,2. AlthoThe biological mechanisms underlying the association between mammographic density and breast cancer risk are unclear. It is plausible that mammographic density is related to breast cancer risk because it reflects the total amount of epithelium and stroma in the breasts . The assA priori we expected there to be a positive association between absolute dense area and percentage dense area and risk of breast cancer. We had no a priori hypothesis regarding the association between nondense mammographic area and risk of breast cancer.In this study, we separately investigated absolute dense area, absolute nondense area and percentage dense area in relation to breast cancer risk. The aim of the study was to better understand the aspects of the association between mammographic density and risk of breast cancer. We conducted a nested case-control study on mammographic density and risk of breast cancer in the Nurses' Health Study (NHS) and Nurses' Health Study II (NHS II).The NHS started in 1976, when 121,700 US registered nurses ages 30 to 55 years completed a questionnaire about their medical history, reproductive history and lifestyle habits . Follow-In the current study, we made use of an existing breast cancer case-control study nested within the subcohort of women who provided a blood sample and had no history of breast cancer at the time they provided the sample. During follow-up to 1 June 2004, there were 1,897 incident cases of breast cancer reported on the biennial questionnaire and confirmed by medical record review. These cases were matched to 3,165 controls on age, menopausal status, current use of postmenopausal hormones, month, time of day and fasting status at the time of blood collection. At the time of mammographic data collection, 1,612 cases and 2,857 controls were alive and were sent letters requesting their participation in the study. We attempted to obtain mammograms taken as close to the date of blood collection as possible from 1,504 cases and 2,512 controls who gave their permission to participate. We obtained mammograms from 1,446 cases and 2,406 controls. We excluded 37 cases and 35 controls because of poor-quality mammograms, 104 cases whose date of mammogram was after the date of the breast cancer diagnosis and 9 controls with discrepant information regarding menopausal status at the date of the mammogram and at the date of diagnosis/index date. In total, we had usable mammograms for 1,305 cases and 2,362 controls. The median time between mammography and blood draw was 11 months before blood collection (IQR = 46 months). The median time between mammography and breast cancer diagnosis was 4.6 years (IQR = 5.3 years).The NHS II started in 1989, when 116,686 US registered nurses ages 25 to 42 years completed a questionnaire about their medical history, reproductive history and lifestyle habits [As in the NHS, our current study was nested within the subcohort of women who had no history of breast cancer at the time they provided a blood sample. During follow-up to 1 June 2003, there were 318 incident cases of breast cancer reported on the biennial questionnaire and confirmed by medical record review. These cases were matched to 635 controls on age, menopausal status, current use of postmenopausal hormones, month, time of day and fasting status at the time of blood collection. At the time of mammographic data collection, 304 cases and 624 controls were alive and were sent letters requesting their participation. We attempted to obtain mammograms taken as close to the date of blood collection as possible from 274 cases and 519 controls who gave their permission to participate. We obtained mammograms from 253 cases and 502 controls. We excluded 13 cases and 15 controls because of poor-quality mammograms, 17 cases whose date of mammogram was after the date of the breast cancer diagnosis, and 1 case and 5 controls with discrepant information regarding menopausal status at the date of the mammogram and at the date of diagnosis/index date. In total, we had usable mammograms for 222 cases and 482 controls. The median time between mammography and blood draw was 3 months before blood collection (IQR = 18 months). The median time between mammography and breast cancer diagnosis was 2.1 years (IQR = 2.6 years).The detailed methods used for mammographic density measurement in this study have been published previously . Briefly2), current postmenopausal hormone use (PMH) and alcohol intake (g/day). Thus information on BMI, PMH use and alcohol intake was extracted from questionnaires returned by the participants 0 to 4 years before mammography. For the covariates age at menarche, menopausal status, parity, age at first childbirth, age at menopause and family history of breast cancer, we used the most recent information from any biennial questionnaire returned by the participant before the mammography.We used the most recent information from the two biennial questionnaires preceding the date of the mammogram to obtain data on the following covariates: body mass index (BMI in kg/mn = 287). After these exclusions, 464 cases and 998 controls with premenopausal mammograms and 960 cases and 1,662 controls with postmenopausal mammograms remained for analysis.We combined the cases and controls from NHS and NHS II to increase the power of our study. We excluded women with unknown menopausal status at the time of mammogram (n = 232) or PMH use (n = 210) were assigned a missing indicator variable. As a sensitivity analysis, we restricted the analysis to women with no missing information on any covariates .Among women with premenopausal mammograms, we divided the mammographic density measurements into tertiles based on the distribution among the controls. Given the larger sample size of postmenopausal women compared with premenopausal women, we were able to examine finer categories of the distribution among postmenopausal women. Among women with postmenopausal mammograms, the mammographic density measurements were divided into quintiles based on the distribution among the controls. All covariates except for alcohol consumption and PMH use had missing data for less than 2% of both cases and controls and were assigned the reference category or median value. Women with missing information on alcohol consumption  and study (NHS or NHS II). The multivariate model was additionally adjusted for the following categorical and continuous variables using data reported closest in time to the mammography: BMI , age at menarche , family history of breast cancer (yes or no), parity and age at first childbirth , alcohol use , and, for women who were postmenopausal at the time of mammography, PMH use  and age at menopause (continuous). We also adjusted for absolute dense area and absolute nondense area simultaneously in the multivariate model. We assessed the interaction between absolute dense area and nondense area with respect to breast cancer risk on the continuous scale. Absolute dense area, absolute nondense area and the cross-product of these factors were entered to the multivariate models as continuous variables, and we used the Wald test to assess the statistical significance of the interaction terms. We also investigated, on the continuous scale, whether there was an interaction between the different measurements of mammographic density and BMI.Since premenopausal and postmenopausal breast cancer may have, in part, different etiologies, and because some women who were premenopausal at the time of mammography were postmenopausal at the time of breast cancer diagnosis, we conducted a sensitivity analysis in which we restricted that analysis to women who were premenopausal at the time of mammography and at the time of breast cancer diagnosis (263 cases and 586 controls). This study was approved by the Committee on the Use of Human Subjects in Research at Brigham and Women's Hospital. All participants in this study provided informed consent.In this study, there were 464 breast cancer cases and 998 controls with premenopausal mammograms, as well as 960 breast cancer cases and 1,662 controls with postmenopausal mammograms. Among controls, premenopausal women with higher absolute dense area were younger at the time of mammography, were somewhat older at the time of menarche, were more often nulliparous, were older when they had their first child, had fewer children and were leaner  and postmenopausal women   and postmenopausal women . When we additionally mutually adjusted for absolute dense area and absolute nondense area, the results were generally similar. Among both pre- and postmenopausal women, the percentage dense area was most strongly associated with breast cancer. We found no significant interactions between absolute dense area, absolute nondense area, or percentage dense area and BMI among premenopausal or postmenopausal women .Increasing absolute dense area was associated with a greater risk of breast cancer after we adjusted for potential confounding factors among premenopausal women .Table In a sensitivity analysis restricted to women who were premenopausal both at the time of mammography and at the time of breast cancer diagnosis, the results were generally similar  for breast cancer, but reported no risk estimates for absolute nondense area adjusted for absolute dense area [P for trend = 0.02) for breast cancer. After adjustment for absolute dense area, the OR changed to 1.08 . Although both of these studies suggest an inverse association between nondense area and breast cancer risk, it is unclear why adjustment for dense area in the Stone et al. study had a different effect than in the current study. Both studies used the same computer-assisted thresholding techniques to measure mammographic density; however, Stone and colleagues used the mediolateral oblique mammogram view, whereas we used the craniocaudal view. Although the mammogram view may affect the mammographic density measurements and thus their ability to predict breast cancer risk, it seems unlikely to explain our different study findings [Two previous studies have reported the association between absolute nondense area and the risk of breast cancer. The UK Guernsey prospective studies (111 cases of breast cancer) found that women in the highest versus lowest quartile of absolute nondense area had a hazard ratio of 0.56 (95% CI = 0.29 to 1.11; nse area . Stone afindings . Taken tPrior findings published by Stuedal and colleagues  lend indPrevious research has led to the hypotheses that mammographic density reflects the proliferation of epithelial and stromal cells ,16, the Several different factors or mechanisms can explain the inverse association between absolute nondense area and breast cancer risk that we found. Absolute nondense area may be a marker of some other factors that in turn are true protective factors for both premenopausal and postmenopausal breast cancer. Although BMI is the strongest confounder of this association, adjusting for BMI did not explain but a small fraction of the association. In addition, we controlled for several other potential confounding factors, and adjusting for these factors did not materially alter the results, although some residual confounding cannot be excluded.per se, is that the dense area and the nondense area on a mammogram reflect the extent of conversion of the breast tissue into either dense tissue that is associated with breast cancer risk or nondense tissue that is not. Lobular involution is inversely associated with breast cancer risk. Ghosh and colleagues [Our results support the view that breast fat could have a protective effect on breast cancer risk . Little lleagues  reportedA limitation of this study is that we were unable to collect mammograms from all eligible women. However, we have no reason to suspect that women with missing mammograms are systematically different from women for whom we were able to collect mammograms. In addition, our findings regarding absolute dense area and percentage dense area in relation to breast cancer risk are in line with those of numerous previous studies, indicating that our measurements of mammographic density, including absolute nondense area, are valid. Another limitation of this study is that information on some covariates that can change substantially over a short period of time, notably BMI, PMH use and alcohol consumption, was retrieved from questionnaires returned by the participants up to four years before mammography. This could have led to misclassification of the covariates and thus to residual confounding.Our results indicate that absolute dense area is independently and positively associated with breast cancer risk, whereas absolute nondense area is independently and inversely associated with breast cancer risk. We propose that the nondense area on a mammogram should be investigated as a distinct factor when studying the association between mammographic patterns and risk of breast cancer. Furthermore, our results suggest that adipose breast tissue may have an important protective role in breast carcinogenesis.BMI: body mass index; CI: confidence interval; IQR: inter-quartile range; NHS II: Nurses' Health Study II; NHS: Nurses' Health Study; OR: odds ratio; PMH: postmenopausal hormone.The authors declare that they have no competing interests.RMT designed the case-control study used for this analysis, came up with the analytical approach used for this analysis and drafted the manuscript. AP came up with the analytical approach used for this analysis, conducted the data analysis and drafted the manuscript. SEH and WCW designed the case-control study used for this analysis. PL and DT came up with the analytical approach used for this analysis. All authors interpreted the results. All authors edited the manuscript and approved the final manuscript submitted for publication.Table S1: Odds ratios for breast cancer among women who were premenopausal at the time of mammography and at the time of breast cancer diagnosis according to tertiles of different mammographic characteristics among 263 cases and 586 controls.Click here for file


American women of African ancestry (AA) are more likely than European Americans (EA) to have estrogen receptor (ER)-negative breast cancer. 25-hydroxyvitamin D (25OHD) is low in AAs, and was associated with ER-negative tumors in EAs. We hypothesized that racial differences in 25OHD levels, as well as in inherited genetic variations, may contribute, in part, to the differences in tumor characteristics.n = 928)-control (n = 843) study of breast cancer in AA and EA women, we measured serum 25OHD levels in controls and tested associations between risk and tag single nucleotide polymorphisms (SNPs) in VDR, CYP24A1 and CYP27B1, particularly by ER status.In a case , but had no effect in EAs. The majority of associations were for ER-negative breast cancer, with seven differential associations between AA and EA women for CYP24A1 (p for interaction < 0.10). SNP rs27622941 was associated with a > twofold increased risk of ER-negative breast cancer among AAs , but had no effect in EAs. rs2209314 decreased risk among EAs , with no associations in AAs. The increased risk of ER-negative breast cancer in AAs compared to EAs was reduced and became non-significant  after adjusting for these two CYP24A1 SNPs.More AAs had severe vitamin D deficiency (< 10 ng/ml) than EAs (34.3% vs 5.9%), with lowest levels among those with the highest African ancestry. Associations for SNPs differed by race. Among AAs, These data suggest that genetic variants in the vitamin D pathway may be related to the higher prevalence of ER-negative breast cancer in AA women. Skin piCYP27B1 and CYP24A1, respectively. Binding of vitamin D to the vitamin D receptor (VDR) activates or suppresses gene transcription, depending on the type of response elements [Endogenous 25OHD may also be affected by variability in metabolic pathways, and synthesis and metabolism catalyzed by two major enzymes, 1α-hydroxylase and 24-hydroxylase, which are encoded by elements , and genelements , is likeVdr knockout mice were more likely than their wild-type littermates to develop Er/Pr- tumors [- compared with ER+ tumors [Laboratory, preclinical, and clinical findings support the hypothesis that low levels of vitamin D are related to breast cancer risk. In the human mammary gland, VDR is expressed in all cell types , and vit- tumors . Consist+ tumors -21. Rece+ tumors .VDR, CYP27B1, and CYP24A1) in relation to breast cancer risk, particularly in relation to self-reported race and estrogen receptor status. We also tested whether vitamin D-related genetic variants could explain, in part, the higher prevalence of ER- breast cancer among AA women.Here, we examined levels of 25OHD in AA and EA women without breast cancer in relation to self-reported race as well as ancestry, which was estimated by using ancestry informative markers (AIMs). 25OHD levels in women with breast cancer could be a result of disease processes, and some samples were obtained after chemotherapy was initiated; thus, we did not compare serum levels of 25OHD between cases and controls. Instead, we evaluated variants in vitamin D activity and major metabolism  is an ongoing study designed specifically to examine the role of genetic and non-genetic factors in early/aggressive breast cancer in AA and EA women. Study design, enrollment, and collection of data and biospecimens have been described in detail previously . BrieflyVDR in both AA and EA populations. First, 122 SNPs in the VDR region plus 15-kb regions from both 3' and 5' ends were selected from HapMap [VDR variants finally selected were commonly studied SNPs, including Cdx2 (rs11568820), Fok1 (rs2228570), Bsm1 (rs1544410), Apa1 (rs7975232), and Taq1 (rs731236). For CYP24A1, 15 multi-population tag SNPs were selected by using publicly available CEU (Utah residents with ancestry from Northern and Western Europe) and YRI (Yoruba in Ibadan) genotype data from HapMap. For CYP27B1, only one SNP had a minor allele frequency of at least 0.05 in the dbSNP database and was thus included. To control for potential bias due to population admixture and to examine serum 25OHD levels in relation to ancestry, a panel of 108 AIMs that have been shown to be effective in correcting for admixture in case-control studies [We used a two-step approach to select a set of multi-population tag single-nucleotide polymorphisms (SNPs) that represent common genetic variations - minor allele frequency of at least 0.05 - in m HapMap  and othem HapMap . These Sm HapMap , 49 multpost hoc to ensure that the calls were robust.DNA was extracted from blood samples by using FlexiGene™ DNA kits  in accordance with the instructions of the manufacturer and from saliva collected in Oragene™ kits . Genomic DNA was evaluated and quantified by a Nanodrop UV spectrometer  and a PicoGreen-based fluorometric assay  and stored at -80°C until analysis. Selected tag SNPs and AIMs were genotyped by an Illumina GoldenGate assay  at the Genomics Core Facility at the Roswell Park Cancer Institute. Five percent duplicates and two sets of in-house trio samples were included for quality control purposes. The average successful genotyping rate for each sample and each SNP was at least 99%, and no SNPs violated Hardy-Weinberg equilibrium in controls or mendelian inheritance. Clustering plots of SNPs that were significant in the statistical analysis were manually re-inspected Serum samples were available from 242 AA and 187 EA women in the control group and were used to measure levels of 25OHD by immunochemiluminometric assay. The assay coefficient of variation was 10.3%.n = 13); therefore, 547 AA cases and 461 AA controls and 381 EA cases and 382 EA controls were included in the final analyses for SNPs and breast cancer risk. There were no exclusions due to conflicting self-report and marker ancestry in the serum analyses performed in controls only. To compare serum levels of 25OHD between AA and EA women, least squares means and standard errors were calculated with adjustment for age, body mass index (BMI), and season of blood collection (four seasons). To test genotype-phenotype correlations between SNPs and 25OHD levels in controls, the Pearson correlation test was used and genotypes were coded as 0, 1, and 2 to reflect the number of copies of the minor allele.Individual ancestral proportions for EA and AA were estimated with the Bayesian Markov chain Monte Carlo clustering algorithm implemented in STRUCTURE 2.3 . We inclt test or chi-squared test. All genotype analyses were performed for AA and EA populations separately. A genotypic (co-dominant) model was assumed for SNP effects. When genotype frequencies of the rare homozygote were not more than 5% in both populations, categories were collapsed (homozygote rare and heterozygote) for power considerations. To test whether there was a linear dose effect of the variant alleles (log-additive genetic model for trend test), SNPs were coded as 0, 1, and 2 as described above. Univariate single SNP analysis was performed, and resulting P values were plotted after logarithm transformation with accompanying linkage disequilibrium (LD) map by using the snp.plotter R package [Descriptive variables were analyzed by Student  package . CovariaVDR and CYP24A1, haplotype structure was determined by using the method of Gabriel and colleagues [n = 10,000) as implemented in PLINK [For lleagues , and eacin PLINK .To examine whether the associations of SNPs with breast cancer risk differed between AA and EA women, interaction with race was tested by including a race*SNP term in the logistic regression model without an estimate of ancestry. A similar approach was used to test modification effects by menopausal status.- breast cancer between AA and EA women, OR of ER- breast cancer by race was first estimated from a base model containing race and other covariates. SNPs that were differentially associated with ER- breast cancer risk by race (P for interaction was less than 0.10) were then entered in the base model and subjected to backward selection. A substantial reduction of OR (≥ 10%) for AA versus EA race by adding selected SNPs would indicate that those SNPs explained, in part, the higher risk of ER- breast cancer in AA than in EA women.To test whether selected SNPs contributed to differential risk of ER2) and were less likely to have a college education or beyond (57.5% versus 82.0%), to take hormone replacement therapy after menopause (14.0% versus 24.1%), or to have a family history of breast cancer in first-degree relatives (13.5% versus 22.4%) . There were no significant case-control differences in AAs or EAs, except that in EA women, cases were less likely than controls to have a college education and more likely to have a positive family history of breast cancer (P ≤ 0.001).Table P < 0.001). As shown in Figure 2-transformed level mean ± standard deviation: 6.54 ± 0.47 versus 6.96 ± 0.53; q value after controlling for multiple comparison: 1.30 × 10-5) . When correlations between SNPs and serum 25OHD levels in AA and EA women were tested, the minor allele of VDR SNP rs2239186 was significantly associated with increased levels of 25OHD in AAs. For the AA, AG, and GG genotypes, the means and standard deviations of serum 25OHD were 13.5 ± 6.5, 16.3 ± 8.7, and 21.2 ± 12.2 ng/mL, respectively (P = 0.006). However, the differences were not significant in EA women .Among controls, serum levels of 25OHD were lower in AA than EA women  Figure . We cateP < 0.05); 12 of these SNPs were the rare variant in one group (AA or EA) but the common allele in the other group  - but none in CYP24A1 were associated with breast cancer risk at a nominal significance level of 0.05 . In EA women, two SNPs in VDR - rs11608702 and rs7975332 (Apa1) - and three SNPs in CYP24A1 - rs912505, rs3787555, and rs2244719 - were associated with breast cancer risk (P < 0.05)  displayed significantly different allele frequencies by self-reported race  in CYP24A1 which had differential associations between AA and EA women (P for interaction by race was not more than 0.10) after adjustment for age, proportion of European ancestry, BMI, family history of breast cancer, and education. In AA women, the combined GG and AG genotypes of rs2239186, which remained significant after correction for multiple testing and were also related to increased levels of 25OHD, were associated with an almost 50% reduction of risk of breast cancer in comparison with homozygotes for A alleles . Among AA women, a reduced risk associated with VDR rs12721364  and a marginally increased risk with SNP rs11568820 (Cdx2)  were observed.Table VDR 'at-risk' G allele for rs2239186 was more common in EA women but was not associated with breast cancer risk , nor were VDR rs12721364 SNPs. There were increases in risk by the VDR SNP rs11608702 and significant decreases in risk by two CYP24A1 variants: rs912505 and rs3787555; however, these did not remain significant after correction for multiple testing.Among EA women, the VDR rs2239186. Among AA women, a G-G-G haplotype consisting of this SNP and two neighboring variants was associated with a decreased risk of breast cancer after adjustment for multiple testing  .Results from haplotype analysis were consistent with those from single SNP analysis for - breast cancer   and there were no associations in EAs . Conversely, CYP24A1 rs2209314 was associated with an almost threefold decreased risk of ER- breast cancer in EA women  and there were no associations in AA women .Stratification by ER status revealed associations that were not observed in the overall analysis, and the majority of findings were observed only for ER- breast cancer in AA women in comparison with EA women, a base model containing self-reported race and other covariates was developed . The eight SNPs that showed significant interactions with race were tested in the base model. After backward selection, the two CYP24A1 SNPs shown above, rs2209314 and rs2762941, remained significant in the final model, reducing the risk associated with AA race by 22% and rendering it non-significant .To determine whether these SNPs contributed to the observed higher risk of ERed Table . The basVDR with menopausal status , or the 3' untranslated region poly(A) microsatellite in either AA or EA women [To date, three studies have examined selected SNPs in EA women ,41, and EA women . HoweverVDR and two SNPs in CYP24A1 had differential associations with breast cancer by race (P for interaction was not more than 0.10). The fact that the associations were not consistent in AA and EA populations corroborates the differences in blood levels of vitamin D and frequency and LD pattern of vitamin D-related genetic variants, implying that the race-specific associations might be the result of gene-environment interactions. In further analyses stratified by ER status, one SNP in VDR and seven SNPs in CYP24A1 were specifically associated with ER- but not ER+ cancer risk, and the associations differed between AA and EA women. Controlling for the two SNPs in CYP24A1 in a multivariate model substantially reduced the increased ER- breast cancer risk associated with AA race and made the association no longer significant. This provides the first evidence supporting the contribution of vitamin D-related genetic variants to higher risk of more aggressive breast cancer in AA women.We found that four SNPs in VDR without previously known functionality. However, in our analyses, SNP rs2239186 was associated with increased serum levels of 25OHD in AA women without breast cancer. This SNP and a haplotype containing it were also significantly associated with reduced breast cancer risk in AA women, irrespectively of ER status. This SNP resides in an intronic region and thus is unlikely to be the causal variant. However, it may be a marker for a causal SNP outside of the VDR gene. This SNP has not been implicated in other breast cancer studies but has been shown to be associated with reduced risk of colorectal cancer in individuals with low vitamin D intake [We found significant associations between breast cancer risk and a number of tag SNPs in D intake  and was D intake . This SNCYP24A1 shown to be associated with racial differences in ER- breast cancer risk are intronic. Although these SNPs have not been implicated previously in breast cancer, elevated expression of CYP24A1 was found in breast cancer tissues [CYP24A1 with serum 25OHD levels in either AA or EA populations, indicating that these two SNPs themselves or linked causal variants may affect ER- breast cancer not through altering circulating 25OHD levels but availability of vitamin D in local mammary tissues.The two SNPs, rs2209314 and rs2762941, in  tissues , indicatVDR SNP. Another limitation is that only three genes in vitamin D-related pathways were included in this study. Although VDR, CYP27B1, and CYP24A1 are the three key genes in this pathway, genes encoding for some other vitamin D metabolizing enzymes, particularly GC encoding for vitamin D binding protein (which has been related to circulating vitamin D levels), may also be related to breast cancer risk and warrant further studies. AA women are more likely to develop breast cancer at a younger age than EAs; we enrolled all eligible AA women but randomly selected eligible EA women, frequency-matching by 5-year age categories. We also initially limited eligibility to women 65 years or younger because of low participation of older women without breast cancer to case-control studies. Thus, the overall study population is younger than that of some other studies. Although we found no evidence of modification effects by menopausal status for any but two SNPs (Table S5 of Additional file One limitation of our study is the lack of validation for the significant findings. The number of patients with breast cancer and controls was relatively limited, especially after stratification by race and ER status. None of the associations with SNPs, except for rs2239186, remained significant after correction for multiple comparisons. We thus could not exclude the possibility of false-positive findings in our data. However, the fact that rs2239186 was associated with higher serum 25OHD levels in AA women as well as reduced breast cancer risk in this population is biologically coherent and reduces the likelihood of spurious findings for this VDR and CYP24A1 between AA and EA women. Specifically, we found race-specific associations with breast cancer risk and these associations may be due to distinct genetic background and differences in 25OHD levels between the two populations. Our study provides evidence that variants in vitamin D-related genes may contribute to higher risk of ER- breast cancer in AA than EA women. Future studies are warranted to validate our findings and to investigate whether pre-diagnostic blood levels of 25OHD may also be related to racial differences in risk of ER- breast cancer.We found notable differences in blood levels of 25OHD and genetic variants in 25OHD: 25-hydroxyvitamin D; AA: African-American; AIM: ancestry informative marker; BMI: body mass index; CEU: Utah residents with ancestry from Northern and Western Europe; CI: confidence interval; EA: European-American; ER: estrogen receptor; LCL: lymphoblastoid cell line; LD: linkage disequilibrium; OR: odds ratio; PR: progesterone receptor; SNP: single-nucleotide polymorphism; UV: ultraviolet; VDR: vitamin D receptor; WCHS: Women's Circle of Health Study; YRI: Yoruba in Ibadan.The authors declare that they have no competing interests.SY helped to develop the hypothesis, design the study, and perform the data analysis and interpretation. CBA helped to develop the hypothesis, design the study, provide the data and biospecimen from the WCHS, perform the data analysis and interpretation, and provide financial support for the study. GZ helped to provide the data and biospecimen from the WCHS and to perform the data analysis and interpretation. DHB, LJ, MR, GC, WD, KSP, and EVB helped to provide the data and biospecimen from the WCHS. HH provided pathological data of tumor samples. C-CH, LES, LT, CSJ, DLT, SEM and FA helped to perform the data analysis and interpretation. HZ helped to provide financial support for the study. All authors read and approved the final draft of the manuscript.Supplementary Tables S1-S5. The file contains the following five supplementary tables. Table S1. Breast cancer risk associated with SNPs in VDR, CYP27B1, and CYP24A1 in African American and European American women. Table S2. Haplotypes of VDR and CYP24A1 in significant association with breast cancer risk in African American and European American women. Table S3. Risk of estrogen receptor positive breast cancer associated with SNPs in VDR, CYP27B1, and CYP24A1 in African Americana and European American women. Table S4. Risk of estrogen receptor negative breast cancer associated with SNPs in VDR, CYP27B1, and CYP24A1 in African Americana and European American women. Table S5. SNPs in VDR that show differential associations with breast cancer stratified by menopausal status in African American and European American women.Click here for file


Breast cancer is among the most common cancers worldwide. Diabetes is an important chronic health problem associated with insulin resistance, increased insulin level, changes in growth hormones and factors, and activation of mitogen-activating protein kinase (MAPK) pathways, leading to an increased breast cancer risk. This paper looked at the epidemiologic studies of the association between type 2 diabetes and risk of breast cancer and its effect on overall cancer-specific survival. The combined evidence overall supported a modest association between type 2 diabetes and the risk of breast cancer, which was found to be more prevalent among postmenopausal women. Effect of oral diabetics and insulin therapy on breast cancer risk was also evaluated. It was found that metformin and thiazolidinones tended to have a protective role. Metformin therapy trials for its use as an adjuvant for breast cancer treatment are still ongoing. Sulfonylurea and insulin therapy were found to be mildly associated with increased overall cancers. No evidence or studies evaluated the association of DPPIV inhibitors and GLP 1 agonists with breast cancer risk because of their recent introduction into the management of diabetes. Breast cancer is among the most common cancers worldwide and is the second leading cause of cancer death for women in the United States, after lung cancer, with an estimated incidence of 226,870 cases and estimated deaths of 39,510 cases in the year of 2012. The National Cancer Institute also estimated that 1 in 8 women in the United States has the chance of developing invasive breast cancer throughout her lifetime . AssociaDifferent mechanisms contribute to the association between diabetes and breast cancer. Diabetes induces several changes in different hormonal systems including insulin, insulin-like growth factors, estrogen, and other growth factors, all of which may affect the risk for breast cancer development. As shown in With regards to the complex association between type 2 diabetes and incidence of breast cancer, several prospective and case-control studies reported increased risk ratios for breast cancer among women with type 2 diabetes –14. HoweP < 0.001) [ A cohort study within the UK General Practice Research Database found that diabetes was associated with 29% increased breast cancer risk (95% CI: 1.16–1.44), but the association was markedly attenuated when adjusted for age, region, and body mass index (BMI) . This cohort also showed that women with breast cancer who had preexisting diabetes had an overall increased mortality of 49%, (95% CI: 1.17–1.88), as compared to breast cancer without diabetes and this persisted even after controlling for age, period, region, BMI, smoking, alcohol, and deprivation . In anot< 0.001) . On the < 0.001) . Moreove< 0.001) . A recent meta-analysis showed that the relative risk for breast cancer in women with diabetes was 1.27 , 1.16–1.39). Prospective studies showed a lower risk ) as compared to retrospective studies ). Type 1 diabetes, or diabetes in premenopausal women, was not associated with the risk of breast cancer  SRR 0.86 , resp.). Studies adjusting for body mass index (BMI) showed lower estimates ) as compared with those studies that were not adjusted for BMI ). It was concluded that the risk of breast cancer in women with type 2 diabetes is increased by 27%, but decreased to 16% after the adjustment for BMI. No increased risk was seen for women at premenopausal ages or with type 1 diabetes [In a recent review of the association of diabetes, metabolic syndrome, and breast cancer risk, included were 26 studies, of which 10 were case-control studies, 3 of which directly looked at the association of diabetes and breast cancer , 37, 38,Several observational studies suggested that metformin use decreases the incidence of several cancers overall –50. For As for the proposed protective effect of metformin, it is well known that metformin acts by increasing glucose uptake by skeletal muscle and thus reducing hyperglycemia by improving insulin sensitivity through the stimulation of the adenosine monophosphate-activated protein kinase (AMPK), leading to the suppression of gluconeogenesis, protein, and fatty acid synthesis resulting in partial metabolic normalization of hyperglycemia and insulin resistance , 55. StiRecently, an epidemiological study of 2,529 women with breast cancer reported higher pathologic complete response in diabetic patients on neoadjuvant systemic therapy and receiving metformin (pCR 24%) as compared to diabetic patients not receiving metformin (pCR 8%) and nondiabetic patients not receiving metformin (pCR 16%) . HoweverA currently ongoing phase III trial of metformin versus placebo in early-stage breast cancer, where nondiabetic women or men, younger than age 75 with newly diagnosed early-stage breast cancer, within the previous 12 months, and following their surgery to remove their tumor, will be randomly assigned to take metformin or placebo pills twice a day for 5 years. Participants in the trial may also receive adjuvant hormone and/or radiation therapy, but if chemotherapy (adjuvant or neoadjuvant) was given, it must have been completed prior to joining the study. This study will mainly monitor to see if metformin would improve disease-free survival, overall survival, and a number of other medical, biological, and quality-of-life endpoints .Other 6 ongoing studies are currently done evaluating the efficacy and safety of treating cancer patients with the metformin. The European Institute of Oncology in Italy is currently planning a presurgical randomized, double-blind, placebo-controlled phase II trial in which 100 histologically confirmed that breast cancer patients not suitable for neoadjuvant therapy will be assigned randomly to either metformin  as compared with other antidiabetic agents and they showed a neutral effect of TZDs on the likelihood of the development of cancers including colon, prostate, and breast cancers .P = 0.041, HR = 0.52, 95% CI 0.28–0.97) and thiazolidinediones  significantly lengthened survival and decreased breast cancer-specific mortality  [The therapeutic effects of troglitazone, in patients with refractory metastatic breast cancer to at least one chemotherapy regimen (ER negative tumors) or two hormonal regimens (ER positive tumors), were evaluated before it was withdrawn from the market following FDA warnings on hepatic toxicity. No objective responses were observed and it was found to have little apparent clinical value among patients with treatment-refractory metastatic breast cancer . In a st, resp.) . It was , resp.) .In a recent review, it was found that the first and second generation sulfonylureas, but not the third generation glimepiride, and glinides increased the risk of overall cancer, specifically hepatocellular cancer but it was less frequently associated with breast cancer, pancreatic cancer, or bladder cancer. It was also found that this increased but slightly less risk as compared to insulin supported the hypothesis that an increasing insulin level plays an important role in carcinogenesis . No otheβ-cell mass by improving islet cell function through inhibiting apoptosis. Dipeptidyl peptidase-IV (DPP-IV) inhibitors, as well as glucagon-like peptide-1 (GLP-1) agonists, are relatively new medications used in the treatment of DM2 where GLP-1 agonists, exenatide, and liraglutide were first introduced in the USA in 2005 and sitaglipitin, first DPP IV inhibitor introduced in 2006. These therapies are effective in preserving Their relatively short-term use clinically does not permit any meaningful data on their malignancy risks. Sitagliptin did show increased pancreatic ductal hyperplasia in a small rodent model study which may predispose to pancreatic cancer risk although one short-term study involving human pancreatic cancer cell lines did not show this –77. No dP = 0.97). It is important to note that this study included 12,537 participants whose mean age was around 65 who had cardiovascular risk factors plus impaired fasting glucose, impaired glucose tolerance, or early diabetes. Insulin glargine was shown to have a neutral effect on cardiovascular outcomes and cancers [A recent meta-analysis, including 562,043 participants and 14,085 cases of cancer, was published assessing the risk of cancer during treatment with insulin. It showed that insulin treatment was associated with an increased risk of overall cancer (RR (95% CI) = 1.39 ) especially with pancreatic cancer (RR (95% CI) = 4.78 ) . Signifi cancers .


Risk reduction was even greater (87%) in age group <60 .Pattern of finger lengths may be a simple marker of prostate cancer risk, with length of 2D greater than 4D suggestive of lower risk. A postal questionnaire was sent to all eligible participants to collect information on selected exposures including right-hand pattern. Subjects were asked to identify the finger length pattern on their right hand as nearest to series of pictures, with clear instruction of how best to compare their hand with the pictures provided. There were three illustrations indicating: the index finger longer than the ring finger, the index equally as long as the ring finger and the index shorter than the ring finger. The latter was used as the reference category. The study received ethical committee approval .Unconditional logistic regression (SPSS version 16) was used to generate odds ratios (ORs) and 95% confidence intervals (CIs). To control for confounding, age and social class were added to the model; age was included as a continuous variable, whereas social class was fitted as a categorical variable.Response rates to the questionnaire for cases and controls were 83 and 70%, respectively. The general characteristics of the study population are shown in The study was a large case–control study with data collected over a period of 15 years, with similar rates of cases and controls recruitment. The subjects were asked to self-identify their pattern of index (2D) as compared to ring finger (4D). The results showed a negative association between length of 2D greater than 4D and prostate cancer risk  in all ages and at ages <60  (results not shown). These negative associations suspect that lower prenatal activity of testosterone is protective against prostate cancer later in life.r=−0140, P=0.007) (The only study to investigate the relationship between digit length pattern and prostate cancer is the Korean Cohort study (366 subjects), which found a significant negative association between digit ratio and PSA  are at greater risk of breast cancer. Women with the more ‘feminine’ pattern of digit length (2D : 4D high – ring finger closer in length or shorter than the index finger) were also more likely to present at a younger age , which was not seen in the left hand (Although finger length in humans has been studied for decades, its relationship with hormones has been determined only by one relatively small-scale study (utero on prostate cancer risk. Hand pattern might represent a simple marker for prostate cancer risk, particularly in men age under 60 years.A protective effect of a high 2D : 4D hand pattern on prostate cancer risk was observed. High 2D : 4D hand pattern may be the marker of low prenatal androgenic activity, suggesting the importance of hormone modulation in


The intestinal microbiome plays an important role in human physiology. Next-generation sequencing technologies, knockout and gnotobiotic mouse models, fecal transplant data and epidemiologic studies have accelerated our understanding of microbiome abnormalities seen in immune diseases and malignancies. Dysbiosis is the disturbed microbiome ecology secondary to external pressures such as host diseases, medications, diet and genetic conditions often leading to abnormalities of the host immune system. Specifically dysbiosis has been shown to lower circulating lymphocytes, and increase neutrophil to lymphocyte ratio, a finding which has been associated with a decreased survival in women with breast cancers. Dysbiosis also plays a role in the recycling of estrogens via the entero-hepatic circulation, increasing estrogenic potency in the host, which is another leading cause of breast malignancy. Non-modifiable factors such as age and genetic mutations disrupt the microbiome, but modifiable factors such as diet may also lead to profound disruptions as well. A better understanding of dietary factors and how they disrupt the microbiome may lead to beneficial nutritional interventions for breast cancer patients. The human digestive tract is known to host trillions of microbes collectively called the intestinal microbiota –4. A comThe intestinal microbiota is maternally inherited at birth as the newborn is delivered through the vaginal canal –9. LaterInsights into the dynamic structure of intestinal microbiota have become possible with the advent of next-generation sequencing technologies; such technologies are able to fully characterize the polymorphism of bacterial communities inhabiting the human intestines using bacterial 16S RNA ribosomal sequences –13. ThisBacteroides and increased proportion of the minor phylum Proteobacteria are observed [The ratio of Firmicutes to Bacteroidetes changes with age. Newborns and infants have more Bacteroidetes; in adults Firmicutes are the predominant intestinal microbiome phylum, and in the elderly more observed . Major sEnvironmental and external factors may act on the composition of the microbiome. The term dysbiosis defines the disturbed microbiome ecology which may be secondary to external factors such as disease, medications, and diet as well as to nonmodifiable genetic conditions . KnockinIn general, several studies have shown that the microbiome structure suffers if genetic defects interfere with the normal development of the immune system of the animal –31.  TabFaecalibacterium prausnitzii and Roseburia intestinalis in their ileum [Escherichia coli and Salmonella typhimurium [Faecalibacterium and Roseburia to survive in the intestinal microbiome of patients with Crohn's disease deprives these patients of bacteria which produce short chain fatty acids (SCFA) used as nutrition and energy for colonocytes [Human research has shown that patients with mutations in Crohn's disease genes such as nucleotide binding oligomerization domain 2 (NOD2)  and autoir ileum . This dehimurium , 37. Theonocytes . Decreasonocytes , 40. Fiber represents the indigestible portions of plant cell walls and may be either soluble or insoluble. The insoluble fibers, such as lignins, cellulose, dextrins, waxes, and chitins are not fermented by the human commensal microbiota. Soluble fiber such as inulin, arabinoxylans, pectins, beta-glucans, amylase resistant starches, fructans, and lignans are fermented by the intestinal microbiota into short chain fatty acids . LignansBifidobacterium (from Actinobacteria phylum) the anti-inflammatory Faecalibacterium prausnitzii (a Firmicute) as well as commensal species from Bacteroidetes phylum [The presence of high concentrations of soluble fiber in the distal ileum and colon favors the growth and maintenance of beneficial s phylum , 55. By-s phylum . Faecalibacterium prausnitzii, Roseburia intestinalis (from Firmicute phylum) were quantified by 16S ribosomal tagged probes by fluorescent in situ hybridization concomitantly with the quantity of short chain fatty acids (SCFA) per gram of dry fecal weight [Faecalibacterium prausnitzii, Roseburia intestinalis, and SCFA (P < 0.001). At 2 weeks, the fiber rich diet increased ten-folds the quantity of SCFA and of both F. prausnitzii and Roseburia (P < 0.01) [In one study, fourteen healthy volunteers consumed for 2 weeks a high fiber diet (19 grams/day) followed by 2 weeks of a low fiber diet (under 5 grams/day). The microbiome was analyzed and l weight . The low < 0.01) . The promotion of growth of Firmicutes and Bacteroidetes within the intestinal microbiome by dietary fibers has another important effect: these bacteria are able to metabolize dietary lignans into the potent phytoestrogens enterodiol (END) and its oxidation product enterolactone (ENL), which are then readily absorbed into bloodstream , 59. Antibiotic use influences the bioavailability of enterolactone due to disruptions in the intestinal microbiome . Unlike estrogen, high blood levels of phytoestrogens have been shown to be inversely associated with risk of breast cancer in epidemiologic studies , 62. In Research on more than 35,000 women participants in the UK Women's Cohort Study showed that premenopausal women who ate most of their fiber from whole grains (at least 13 grams per day of whole grain fiber) and 30 grams per day of total fiber had 50% less breast cancer. If the majority of soluble fiber came from fruits rather than whole grains the protection from breast cancer was slightly lower 35% for those women who ate 30 grams of fiber compared to those who ate only 2 grams of fiber daily . The benefit of high fiber diets extends to postmenopausal women, despite the fact that with age the intestinal microbiota ratio of Firmicutes to Bacteroidetes phylums changes in an unfavourable direction . A study of over 51,000 postmenopausal women followed over more than 8 years showed that women who consumed 30 grams or more of fiber from fruit and whole grains had 34% less breast cancer than those who consumed less quantities of those items. Notably, lignan fiber seemed to be more protective than fruit and vegetable fibers . In another study, premenopausal women at risk for breast cancer because of benign breast disease  were asked to undergo a baseline periareolar fine needle aspiration (RPFNA) and were then followed on a diet which included daily plant lignans (intervention) for one year. Serum enterolactone was measured before and after the intervention. After intervention serum enterolactone increased ninefold from the baseline. After one year, a repeated RPFNA showed atypia in only half of the women treated with diets rich in lignans in whom the serum enterolactones increased ninefold from baseline levels . A Finnish case-control study also looked at serum levels of enterolactone and their association with breast cancer. Women with the highest dietary intake on lignans (at least two slices of rye bread per day) were in the highest quintile of serum enterolactone (54 nmol/L) while with the lowest consumption were in the lowest quintile (3 nmol/L). After eight years of followup, women in the highest quintile of enterolactone had 62% less breast cancer diagnosed compared to the lowest quintile . In a study of more than 300 women diagnosed with breast cancer, serum enterolactone was used to quantify the amount of dietary lignans consumed. At a median followup of 276 months there was 30% decrease in breast cancer specific mortality in patients with enterolactone levels more than 10 nmol/L . A recent meta-analysis of serum enterolactones and breast cancer showed a protective effect for breast cancer for women with serum levels in the highest quartile compared to the lowest  . The effSerum enterolactone, a product of microbiome fermentation of dietary lignans, appear to have protective effects in breast cancer patients and in breast cancer prevention. Saturated fats are the source of cholesterol used by the ovaries for estrogen synthesis. In postmenopausal women the adipose tissue, adrenal glands, and other organs transform circulating androgens into estrogens using the aromatase enzymes . In the E. coli, Klebsiella, Enterobacter, and Citrobacter and are detrimental to certain species of Firmicutes and Bacteroidetes in the intestinal microbiota. This process in turn produces a dysbiotic state [E. coli from Proteobacteria phylum, an organism that is able to produce potent beta-glucuronidases [Diets rich in fats and red meat promote the production and excretion of bile acids necessary for fat digestion and absorption. Commensal bacteria then break down these bile acids into deoxycholic and lithocholic acids, metabolites that favor the growth of Proteobacteria species such as ic state  that favonidases , deconjuonidases . A direconidases . 2, as compared to normal weight controls [Diet-induced obesity changes the balance of Firmicutes to Bacteroidetes phylums. Studies in twins, of which one is obese, the other nonobese, have shown smaller Bacteroidetes phylums and more Firmicutes in the obese twin as compared to the lean twin , 78. Notcontrols . Much of what we know about the symbiotic interaction between the intestinal microbiome and systemic immune system development has come from the study of germ-free (knows as gnotobiotic) rodents. Lack of intestinal microbiota in gnotobiotic rodents decreases the size of the small and large intestine Peyer's patches of the spleen and also affects distant immune organs. The size of the pancreas and the number of beta-cells are decreased , and theThe colonization of gnotobiotic rodents with known strains of human microbiome allows measuring the effects of known bacterial species on the immune system development. Sphingomonas species (of Proteobacteria phylum) [Sphingomonas in the Proteobacteria phylum and prevents proper development of CD8+ antitumor cytotoxic T cells [Normal microbiome contributes to maturation of effector CD8+ T cells (known as killer T cells) via contact with  phylum) . Inflamm T cells , 87. CD8 T cells . T cell differentiation takes place in the thymus from 12 weeks of gestation until the thymus regresses through involution by 9 months of age . The rolE. coli, Klebsiella, Enterobacter, and Citrobacter) and Fusobacterium nucleatum [Fusobacterium nucleatum species are able to kill maturing lymphocytes via M cells in the Peyer's patches via direct contact, lowering the number of circulating systemic lymphocytes [It was previously noted that diets rich in fats and red meats result in dysbiosis, favoring the growth of Proteobacteria phylum  at diagnosis predicts long-term cancer outcome, with higher ratios predicting worse outcomes independently of patients' age or stage at diagnosis –109. P < 0.001) [P < 0.0001) [In a study of early stage breast cancer  a neutrophil-to-lymphocyte ratio of more than 2.5 was associated with 4-fold risk of disease relapse at 10 years compared to patients with a ratio lower than 2.5 regardless of stage or age at diagnosis (< 0.001) . A retro 0.0001) . P < 0.0001) [In BIG-02-98 study of more than 2000 patients with node positive breast cancer more than 50% infiltration of tumor stroma with lymphocytes was associated with reduced risk of breast cancer relapse and death. For each additional 10% increase in percentage of lymphocytes the risk of relapse was 17% lower and risk of death was 27% lower independent of stage at diagnosis and patients' age ( 0.0001) . Sphingomonas species (of Proteobacteria phylum) [Sphingomonas in the Proteobacteria phylum and prevents proper development of CD8+ antitumor cytotoxic T cells detected in the peripheral blood [Most chemotherapy decreases tumor burden by 1-2 logs. The decrease is followed by immune recognition of tumor antigens, with the desirable response being that the rest of the tumor burden is dealt with by the patient's own immune system . CD8+ T  phylum) . Inflammal blood , 87. P < 0.001), than those having fewer than five [2 were associated with a 60% recurrence-free survival at 8 years versus 20% in patients with fewer than 20/mm2 (P < 0.0019) [Research correlating the number of CD8+ effector T cells infiltrating breast cancer tumors with patients survival shows that patients with higher numbers of effector T cells in their breast tumors have better chance of being successfully treated for their disease or being long-term survivors than those without these immune cells. In a study of over 1,300 patients followed for over 10 years, patients whose breast tumors had more than 24 of CD8+ cells per high power field of the tumor had better breast cancer specific survival, 75% versus 45% (han five . In over 0.0019) . The incidence of breast cancer around the world is vastly different in the USA and Western Europe compared to Asia and Africa. In the industrialized world, the incidence is around 120 women per 100,000 individuals per year; in the less developed parts of the world it is 17 per 100,000 . The wes


Research on the association between dietary patterns and breast cancer survival is very limited.A prospective follow-up study was conducted in Germany, including 2522 postmenopausal breast cancer patients diagnosed in 2001–2005 with available food frequency questionnaire data. Vital status, causes of death, and recurrences were verified through the end of 2009. Principle component factor analysis was used to identify pre-diagnostic dietary patterns. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated with Cox proportional hazards models.vs lowest quartile: HR, 3.69; 95% CI, 1.66–8.17; P-trend <0.001). No associations with breast cancer-specific mortality and breast cancer recurrence were found. The ‘healthy’ dietary pattern was inversely associated with overall mortality  and breast cancer recurrence  in stage I–IIIa patients only.Two major dietary patterns were identified: ‘healthy’  and ‘unhealthy’ . Increasing consumption of an ‘unhealthy’ dietary pattern was associated with an increased risk of non-breast cancer mortality (highest Increasing intake of an ‘unhealthy’ pre-diagnostic dietary pattern may increase the risk of non-breast cancer mortality, whereas increasing intake of a ‘healthy’ pattern may reduce the risk of overall mortality and breast cancer recurrence. Two prospective cohort studies examined the association of dietary patterns with prognosis in breast cancer patients in the United States. In both studies, a lower pre- and post-diagnostic intake of the Western dietary pattern and a higher post-diagnostic intake of the prudent dietary pattern were associated with decreased mortality from causes unrelated to breast cancer , previous cancer (n=207), in situ breast cancer (n=165), or energy intake in the bottom or top 1.0 percentile (n=50). This study was approved by the ethics committee of the University of Heidelberg, the ethical review board of Hamburg Medical Council, and the Medical Board of the State of Rheinland-Pfalz, and conducted in accordance with the Declaration of Helsinki.Patients were recruited in 2002–2005 within a large population-based case–control study on breast cancer in Hamburg and the Rhein-Neckar-Karlsruhe region in Germany . Participants without event of interest were censored at the date of last contact or 31 December 2009, whichever came first.Vital status of participants was determined through population registries up to the end of 2009. Causes of death were extracted from death certificates. Medical records were checked or treating physicians were contacted to identify recurrences and to verify self-reported events collected during a follow-up telephone interview conducted from May to September 2009 (>90% self-reported events verified). Recurrence included ipsilateral/contralateral/local/regional invasive recurrence and distant recurrence, and analyses for this endpoint were restricted to participants with stage I–IIIa disease, as well as information on recurrences occurring after recruitment into the study . The number of factors to be retained in the model was determined by eigenvalues of the correlation matrix (>1), Scree plots, and the natural interpretability of each factor. An orthogonal transformation (varimax) was used to rotate the correlation matrix to get a simpler data structure with greater interpretability . Each inP-value <0.05. All statistical analyses were performed using the SAS software 9.2 (SAS Institute).Delayed-entry Cox proportional hazards models, based on time since recruitment until event/censoring, were used to examine the association of the factor score with survival and recurrence . Hazard Women were enroled in the study a median time of 97 days after diagnosis, and median follow-up time was 5.5 years. Overall, 316 deaths occurred, 235 due to breast cancer and 81 due to non-breast cancer causes . Of the 2184 patients with stage I–IIIa disease and available data on recurrence status, 247 had a breast cancer recurrence.Two major pre-diagnostic dietary patterns were defined: (1) high vegetables, fruits, vegetable oil, sauces/condiments, and soups/bouillons intake , explaining 45.8% of the variance; and (2) high red meat, processed meat, and deep-frying fat intake , explaining 29.8% of the variance . Women iP-trend<0.001; P-trend=0.02) and breast cancer recurrence  was found (Women in the highest compared with the lowest quartile of the ‘unhealthy’ pattern were at an increased risk of overall mortality, driven by an increased risk of non-breast cancer mortality (HR, 3.69; 95% CI, 1.66–8.17; as found . We obseIn our study of German postmenopausal breast cancer patients, a higher pre-diagnostic intake of an ‘unhealthy’ dietary pattern was associated with an increased risk of non-breast cancer mortality, whereas the ‘healthy’ pattern was inversely associated with risk of overall mortality and breast cancer recurrence among stage I–IIIa patients only.Similar results were found by two previous US studies for the mortality outcomes with either pre-diagnostic (The dietary patterns we identified are somewhat different from those in the US studies. One explanation is that factor analysis is subject to several subjective decisions (Strengths of our study are the relatively large sample size, population-based design, and nearly complete follow-up. Limitations include the use of the FFQ. Although this is a validated dietary assessment methodology, non-captured dietary factors may have gone undetected, and recall bias and measurement errors cannot be ruled out. Also, potential changes in dietary patterns after diagnosis cannot be accounted for. We cannot exclude residual or uncontrolled confounding, and had a limited sample size for subgroup analyses.In conclusion, higher intake of a pre-diagnostic ‘unhealthy’ dietary pattern may increase the risk of non-breast cancer mortality, whereas a higher intake of a ‘healthy’ dietary pattern may decrease the risk of overall mortality and breast cancer recurrence.


Objective. This study assessed the initial experiences, symptoms, and actions of patients in Ethiopia ultimately determined to have breast cancer. Methods. 69 participants in a comprehensive breast cancer treatment program at the main national cancer hospital in Ethiopia were interviewed using mixed qualitative and quantitative approaches. Participants' narratives of their initial cancer experience were coded and analyzed for themes around their symptoms, time to seeking advice, triggers for action, and contextual factors. The assessment was approved by the Addis Ababa University Faculty of Medicine Institutional Review Board. Results. Nearly all women first noticed lumps, though few sought medical advice within the first year (average time to action: 1.5 years). Eventually, changes in their symptoms motivated most participants to seek advice. Most participants did not think the initial lump would be cancer, nor was a lump of any particular concern until symptoms changed. Conclusion. Given the frequency with which lumps are the first symptom noticed, raising awareness among participants that lumps should trigger medical consultation could contribute significantly to more rapid medical advice-seeking among women in Ethiopia. Primary care sites should be trained and equipped to offer evaluation of lumps so that women can be referred appropriately for assessment if needed. Breast cancer is an increasingly visible disease, and a rapidly growing cause of mortality, in developing countries . In AfriIn Ethiopia, breast cancer is typically a fatal disease with high mortality , 5, unliThis evaluation includes a qualitative and quantitative assessment of the experience of participants with breast cancer who, ultimately, successfully accessed the services of the Ethiopia Breast Cancer Project (EBCP) and reports on their recognition of symptoms, their attitudes upon noticing those symptoms, and their motivations for and experiences with taking action.This project focuses upon women who accessed care through the Ethiopia Breast Cancer Project (EBCP). EBCP aims to strengthen human resource capacity, technical competency, and advocacy and improve access to treatment for breast cancer in Ethiopia, working closely with all related departments and services of Tikur Anbessa Hospital (TAH), the Ministry of Health, and the Ethiopian Cancer Association . PatientInterviewers were fluent in written and spoken Amharic and English and sequentially identified EBCP program participants to interview over a one-month span. Informed, verbal consent was obtained prior to interviewing. Interviewers participated in a one-day training session that included: study overview, ethical conduct of research, role play, and pilot test interviews and review. The teams debriefed each day with investigators for major points and discussion items for the group. Qualitative data was analyzed through theme analysis predominantly using ATLAS.ti, Version 5.5 , and quantitative data was analyzed using JMP, Version 8 . Where necessary, translated English grammar in direct quotes is corrected from transcriptions to ease readability and where necessary, placed in the first person context, though content remains unchanged.In total, 55 patients directly participated in the study, in addition to 14 proxies  representing other patients, for a total of 69 patients represented. Most participants were married and female, and more than half were age 50 years or younger. About two-thirds of participants lived in Addis Ababa, and almost three-quarters of the population was Ethiopian Orthodox. Two-thirds of the participants were diagnosed with breast cancer in the immediate two years prior to the assessment. The average interview lasted 41 minutes. When compared to the clinic treatment population for the first half of 2008 from EBCP programmatic data (unpublished), the study sample did not differ significantly from the clinic treatment population on any of the following parameters: age, gender, or residence of patient.Participants were asked in an open-ended way to tell their story of how they came to learn they had breast cancer. If necessary, participants were prompted for relative dates when they first noticed symptoms, when they first accessed care, and their navigation of the care system . These https://www.citiprogram.org/). Participant names were not collected as part of this project, and indirect identifying information was grouped and presented in general categories or in aggregate.This project was reviewed and approved by the Addis Ababa University Faculty of Medicine IRB. Additionally, project team members were trained in research ethics using materials from the CitiProgram ”“I came with my sister in law. She had a lump starting a year back. At first it was painless. Around 6 months later the lump become harder and painful. (Case  56)”“Three years back, I started feeling sharp, tingling kind of pain in my right breast. I went to a nearby clinic and was diagnosed to have a cold, and I started oral medication. Then after some time, I developed a pea-sized hard nodule over the same breast, which was getting bigger at time passed on by. (Case  23)”“I came with my sister. She told me she had a lump on her breast about two years back. We were not worried that much because it was painless. But, starting last year, the lump become painful and we took her to [a clinic]. (Case  28)”Similarly, as shown in “About two years ago, I noticed a small lump on my right breast. I just ignored it, considering it a simple swelling. After a year, I went to [Eastern Ethiopia] where my biggest child lives. I told him that I had a lump on my breast. I think he heard about breast cancer. He immediately took me to nearby clinic… (Case  37)”“I saw a lump on my left breast. It was painless at that time and I thought it will resolve by its own. Finally the lump became harder… it be come [sic] very painful within two years. I went to a health center the physician examined me and told me that it is breast cancer, and I will not live more than 2 months… (Case  41)”Finally, participants indicated that the most common reason for initiating some form of action to address their symptoms —includin“Before 2 years, I started feeling sharp pain over my left breast with a little swelling. But I did not do anything about it or told anybody because I was under the consideration that it will go away by itself. But after about 4 months, I had to go to a nearby health center because the mass was getting bigger and the pain was getting worse. (Case  49)”“Three years back, I started nursing my last child. After 1 month, I started to experience severe pain on my left breast. I went to a physician [who] told me that it is cold and gave me an antibiotic. Three month later I experienced a similar problem and got similar medication. After a month of this medication I noticed a small lump on the same breast. I showed it to my husband, and my sisters they told me that it simple lump and not to worry about it. After 2 and 1/2 years, I met my old friend who works in Addis Ababa as a nurse and I showed her too. She told me to go to the health center and get treated. After doing some investigation it is found to be cancer. I was shocked. (Case  27)”“Two years back, I noticed a small nodule over my left breast but I was not that much concerned about it. Then when I got pregnant in that year, the mass started becoming very painful which forced to seek medical attention. Then I was told that it could be cancer and that I would get the treatment after giving birth. (Case  65)”Clearly, in Ethiopia lumps are the first noticeable signs of breast cancer typically recognized by participants. Almost all of the participants in this study noticed a lump at some point and most participants also dismissed the lump, at first, as nothing to be concerned with. In fact, some participants ignored their lump for several years. In time, participants noticed more lumps or changes in symptoms , and that triggered them to seek advice, from a traditional healer, clinic, or other health care source. A few women were motivated to seek advice from their friends or family, and for some their cancer was discovered in the course of obtaining medical care for something else.Several other studies have similarly found that lumps are the dominant symptom noticed by women with breast cancer and that most women find lumps as their primary symptom –15. FurtStudies also indicate that women in low-resource areas delay seeking care longer than women in other parts of the world, with delays of a year or more from detection of symptom to seeking advice , 15, 17.Notably, this study included participants who, at least eventually, obtained care at the national cancer hospital in Ethiopia. Even among these women who obtained care, delay from noticing to acting on symptoms was considerable. This study is limited in that delay, symptom recognition, and the dynamics of triggers for action are unknown among women not entering the care system in Ethiopia.Given the importance of lumps in initiating the cascade of events that could lead to breast cancer diagnosis, increasing women's knowledge of lumps as a trigger for advice may help reduce the lag time between initial notice of the symptom and eventual action in Ethiopia. Focused public campaigns and other strategies to increase awareness may be effective in promoting action when confronted with a lump . Streng


Mechanistic data suggest that different types of fatty acids play a role in carcinogenesis and that antioxidants may modulate this relationship but epidemiologic evidence is lacking. Our aim was to investigate the association between plasma saturated, monounsaturated and polyunsaturated fatty acids  and overall and breast cancer risk and to evaluate the potential modulatory effect of an antioxidant supplementation on these relationships.A nested case-control study included all first incident cancer cases diagnosed in the SU.VI.MAX study between 1994 and 2002 . Participants to the SU.VI.MAX randomized controlled trial received either vitamin/mineral antioxidants or placebo during this intervention period. Baseline fatty acid composition of plasma total lipids was measured by gas chromatography. Conditional logistic regression was performed overall and stratified by intervention group.trend = 0.002), the dihomo-γ-linolenic/linoleic acids ratio (Ptrend = 0.001), mead acid (Ptrend = 0.0004), and palmitoleic acid (Ptrend = 0.02) were inversely associated with overall cancer risk. The arachidonic/dihomo-γ-linolenic acids ratio (Ptrend = 0.02) and linoleic acid (Ptrend = 0.02) were directly associated with overall cancer risk. Similar results were observed for breast cancer specifically. In stratified analyses, associations were only observed in the placebo group. Notably, total PUFAs were directly associated with overall (Ptrend = 0.02) and breast cancer risk in the placebo group only.Dihomo-γ-linolenic acid (PSpecific SFAs, MUFAs and PUFAs were prospectively differentially associated with cancer risk. In addition, this study suggests that antioxidants may modulate these associations by counteracting the potential effects of these fatty acids on carcinogenesis. Mechanistic data suggest that different types of dietary fatty acids may influence carcinogenesis in different ways. For instance, n-3 polyunsaturated fatty acids (PUFAs) may be involved in several mechanisms that counteract carcinogenic processes However, epidemiological data remain inconsistent. As estimation of usual dietary fatty acid intake may be prone to measurement errors Moreover, this high level of heterogeneity within epidemiological data may support the existence of other factors that could modulate the relationship between circulating fatty acids and cancer risk, explaining contrasted results across different populations. Mechanistic data from animal models suggest that dietary antioxidants may be good candidates for this modulatory role Thus, the objectives of the present study were 1) to prospectively investigate the relationships between plasma SFAs, MUFAs, PUFAs and the risk of overall and breast cancer; and 2) to assess the potential modulatory effect of an antioxidant supplementation on these relationships.The “Supplementation en Vitamines et Minéraux Antioxydants” study (SU.VI.MAX) is a population-based, double-blind, placebo-controlled, randomized trial  initially designed to assess the effect of a daily antioxidant supplementation on the incidence of cardiovascular disease and cancer At enrollment, all participants underwent a clinical examination and anthropometric measurements by the study nurses and physicians. They completed questionnaires regarding sociodemographic data, smoking, physical activity, and medication use. Fasting blood samples were taken up at inclusion from all subjects (before randomization and start of the intervention). Samples were centrifuged immediately after blood draw, and plasma aliquots were then preserved in sodium heparin. Less than one hour after blood draw, plasma aliquots were stored at −20°C in dry ice for shipment to the central biobank (maximum 24 hours), where they were stored frozen in liquid nitrogen (−70°C).Major health events were self-reported by subjects during follow-up. Investigations were conducted in all declared cancer cases to obtain medical data from participants, physicians, and/or hospitals. All information was reviewed by an independent expert committee and cases were validated by pathological reports and classified using the International Chronic Diseases Classification, 10th Revision, Clinical Modification (ICD-10) A case-control study nested in the SU.VI.MAX cohort was designed to include all first primary incident cancer cases diagnosed between baseline in 1994 and December 2002. Controls (one per case) were randomly selected among participants with complete follow-up data and without cancer diagnosis by the end of follow-up and were matched by gender, age (±6 months), number of dietary records and intervention group of the trial (antioxidants or placebo).Baseline plasma samples of selected subjects were used to determine the fatty acid composition of total lipids. Lipids were extracted from 150 µl aliquots of plasma with hexane/isopropanol , saponified with NaOH in dry methanol at 100°C, and the fatty acids were methylated with boron trifluoride (14%) in methanol. The fatty acid methyl esters were quantified by gas chromatography using a capillary column , and hydrogen as carrier gas. Peak identification was made by comparison of their elution times with that of a mixture of commercial standards. Fatty acid composition was expressed as percentages of the total area of all fatty acid peaks. The coefficients of variation (CVs) were <23.8% for the saturated fatty acids (SFAs), <8.0% for cis MUFAs, <12.2% for n-6 PUFAs, <7.7% for n-3 PUFAs and 10.3% for Mead acid.We calculated the following ratios: total n-3 PUFAs to total n-6 PUFAs, arachidonic acid to dihomo-γ-linolenic acid (indicator of the activity of Δ5-desaturase), dihomo-γ-linolenic acid to linoleic acid (indicator of the activity of Δ6-desaturase and elongase), and oleic acid to stearic acid (indicator of the rate-limiting enzyme Δ9-desaturase).Baseline characteristics of participants were compared between cancer cases and controls by using conditional logistic regression analyses. ORs and 95% confidence intervals for overall and breast cancer risk associated with quartiles of each plasma fatty acid, fatty acid categories and ratios were examined by using conditional logistic regression models. Multivariate models were adjusted for gender, age, body mass index (BMI), height, intervention group, alcohol intake, physical activity, smoking status, family history of cancer, and educational level. In breast cancer analyses, multivariate models were further adjusted for family history of breast cancer, number of children, menopausal status and use of menopausal hormone therapy at baseline. There was no missing data for covariates except for smoking status, physical activity and educational level for which missing values (less than 5% for each variable) were replaced by the modal value. Adjustment variables were coded as indicated in Since cases and controls were matched for the antioxidant supplement group, statistical interaction between antioxidant supplementation and plasma fatty acids could not be formally tested. However, stratified analyses were performed by running the models separately in supplemented and non-supplemented subjects. In sensitivity analyses, models were also performed on the absolute values of fatty acids. All statistical tests were two-sided, and P<0.05 was considered statistically significant, however, we also pointed out results that remained statistically significant with a more conservative threshold (P<0.01). Analyses were performed with SAS software .A total of 250 incident cancer cases were diagnosed during follow-up: 154 breast and 96 other cancer cases . In breast cancer cases, 63 were premenopausal and 91 were postmenopausal. 81% were estrogen receptor positive (ER+) and 71% were progesterone receptor positive (PR+). 69% of breast cancers were ductal, 13% were lobular and 18% derived from other histological types. Mean tumor size was 16.2 (±11.75) mm for breast tumor. 250 controls were randomly selected and matched to cases. Median follow-up time was 3.7 y for cancer cases and 7.9 y for controls. The characteristics of overall and breast cancer cases and controls are described in Q4vs.Q1 = 0.49 95%CI = 0.28–0.85, Ptrend = 0.002), the dihomo-γ-linolenic/linoleic acids ratio , mead acid , and palmitoleic acid  were inversely associated with overall cancer risk. The arachidonic/dihomo-γ-linolenic acids ratio  and linoleic acid  were directly associated with overall cancer risk. The associations between mead acid, linoleic and dihomo-γ-linolenic acids and cancer risk persisted after mutual adjustment for each other. Results for dihomo-γ-linolenic acid, mead acid and the dihomo-γ-linolenic/linoleic acids ratio remained statistically significant when a p-value of 0.01 for significance was considered.Associations between plasma fatty acids and overall cancer risk are presented in Q4vs.Q1 = 2.88, 95%CI = 1.20–6.92, Ptrend = 0.02) whereas γ-linolenic acid , total SFA , and palmitic acid  were associated with decreased overall cancer risk. Among these results, those regarding γ-linolenic and palmitic acids remained statistically significant when a p-value of 0.01 for significance was considered.When analyses were stratified by intervention group Table3,Q4vsQ1 = 4.10, 95%CI = 0.92–18.39, Ptrend = 0.03). However the later result was no longer statistically significant if a p-value threshold of 0.01 was considered.Similar trends were observed for breast cancer specifically Table4 Further adjustment for energy, total lipid, and fruit and vegetable intakes, and number of dietary records did not modify the findings, neither did the sensitivity analyses excluding cases (n = 30) diagnosed during the first year of follow–up nor excluding the in-situ breast cancer cases (n = 20) (data not shown). Results were also similar when analyses were performed on the absolute values of fatty acids, for subjects with such available data (n = 174 cases and 174 controls) (data not shown).In this prospective study, we observed inverse associations between dihomo-γ-linolenic acid, the dihomo-γ-linolenic/linoleic acids ratio, γ-linolenic acid (placebo group), mead acid, palmitoleic acid and overall cancer risk, and direct associations between the arachidonic/dihomo-γ-linolenic acids ratio, linoleic acid and overall cancer risk. Similar results were observed for breast cancer specifically. In addition, to our knowledge, this study was the first to prospectively examine the potential modulatory role of an antioxidant supplementation on the relationships between circulating SFAs, MUFAs and PUFAs and overall and breast cancer risk. Interestingly, no association was observed in the antioxidant-supplemented group, whereas all previously described associations were found in the placebo group. Some associations were even observed only in the placebo group, such as a direct association between total PUFAs and overall and breast cancer risk.We observed inverse associations between dihomo-γ-linolenic acid, the ratio of dihomo-γ-linolenic/linoleic acids (indicator of the Δ6 desaturase and elongase which converts linoleic acid into dihomo-γ-linolenic acid), γ-linolenic acid (placebo group) and overall cancer risk. Consistently, a prospective case-control study nested in the Carotene and Retinol Efficacy Trial (CARET), including 641 cases, reported an inverse association between dihomo-γ-linolenic acid and non-aggressive prostate cancer risk To our knowledge, the inverse association observed in the present study between mead acid and the risk of overall and breast cancer has not been previously documented in epidemiologic studies. Mechanistic data support this result. Mead acid is converted to C3 and D3 leukotrienes, which have an anti-inflammatory effect Linoleic acid was directly associated with overall and breast cancer risk. Prospective epidemiological studies have generally failed to establish clear evidence of an association between linoleic acid and cancer risk This result is also consistent with the previously discussed inverse association observed between mead acid and cancer risk. Indeed, under normal physiological conditions, n-9 derivatives are formed in small amounts, and a significant increase in mead acid status (a metabolite of oleic acid) suggests a deficiency of n-6 (and n-3) essential fatty acids We found that an increasing concentration of palmitoleic acid was associated with a decreased risk of overall and breast cancer. Consistent with our findings, a case-control study The ratio of arachidonic/dihomo-γ-linolenic acids (indicator of the Δ5 desaturase activity) was associated with an increased risk of overall cancer. In line with this finding, a Swedish nested case-control study observed a borderline non-significant increase in breast cancer risk associated with this ratio One of the most salient and original findings of our study is the fact that antioxidant supplementation strongly modulated the associations between circulating SFAs, MUFAs, PUFAs and cancer risk. In the ATBC Study, serum linoleic acid was inversely associated with prostate cancer risk only among men who received high-dose α-tocopherol supplements (50 mg/day) Similarly, the previously discussed inverse association between mead acid and overall and breast cancer risk was observed in the placebo but not in the antioxidant-supplemented group. Indeed, antioxidant supplementation, by preserving essential PUFAs from peroxidation, may limit the synthesis of mead acid.This modulation by antioxidant intake may explain discrepancies between previous studies investigating the associations between circulating PUFAs and cancer risk Our results showed an inverse association between SFAs  and the risk of overall and breast cancers in the placebo group only. In contrast, several prospective epidemiological studies have reported direct associations between palmitic acid and prostate Strengths of our study include its prospective design, the wide range of circulating SFAs, MUFAs and PUFAs studied and, for the first time in an epidemiological study, the investigation of a potential modulatory role of an antioxidant supplementation in the association between plasma fatty acids and overall and breast cancer risk.trans-MUFAs trans fatty acids in the present study. Fourth, as participants received a combination of antioxidants, it was not possible to identify if one of them was more particularly involved in the modulation of the association between plasma fatty acids and cancer risk. However, it can be postulated that the fat-soluble vitamin E may have played a central role, as previously suggested Some limitations should be acknowledged. First, plasma composition of fatty acids was evaluated only once, at baseline. It would have been interesting to evaluate how this composition varied in time after inclusion, overall and by antioxidant supplementation group, but this information was not available. Indeed, several factors may have modified plasma fatty acids profiles during follow-up, such as variation in endogenous lipogenesis or dietary factors. In addition, it cannot be ruled out that other factors than antioxidant supplementation may also have modified the associations between fatty acid levels and cancer risk during follow-up, such as use of specific drugs or weight change over time. However, these could not be investigated in the present study. Second, the fatty acid composition of plasma was determined based on total lipids. Other biomarkers such as fatty acid composition of plasmatic phospholipids or fatty acids from adipose tissue are more appropriate to reflect long-term fatty acid intake In conclusion, this prospective study highlighted several inverse or direct associations between specific plasma SFAs, MUFAs, PUFAs and cancer risk that were supported by mechanistic plausibility. Notably, for the first time, we have found a negative association between mead acid and overall and breast cancer risk. Our initial hypothesis of a modulatory effect of an antioxidant supplementation on these relationships was verified. To our knowledge, this had never been investigated before in any epidemiological study on circulating fatty acids and overall or breast cancer risk. Additional prospective studies and mechanistic data are needed to better apprehend the influence of antioxidants on the potential pro- and anti-carcinogenic effects of fatty acids.