Genetic variants for breast cancer risk identified in genome-wide association studies (GWAS) in Western populations require further testing in Asian populations. A risk assessment model incorporating both validated genetic variants and established risk factors may improve its performance in risk prediction of Asian women.A nested case-control study of female breast cancer  within the Singapore Chinese Health Study was conducted to investigate the effects of 51 genetic variants identified in previous GWAS on breast cancer risk. The independent effect of these genetic variants was assessed by creating a summed genetic risk score (GRS) after adjustment for body mass index and the Gail model risk factors for breast cancer.P for trend <0.001). In addition to established risk factors, the GRS improved the classification of 6.2% of women for their absolute risk of breast cancer in the next five years.The GRS was an independent predictor of breast cancer risk in Chinese women. The multivariate-adjusted odds ratios  of breast cancer for the second, third, and fourth quartiles of the GRS were 1.26 (0.90 to 1.76), 1.47 (1.06 to 2.04) and 1.75 (1.27 to 2.41) respectively (Genetic variants on top of conventional risk factors can improve the risk prediction of breast cancer in Chinese women. Breast cancer is a heterogeneous disease that is associated with genetic and environmental factors. Prior to genetic studies, investigations have mainly revolved around the presence of a family history, hormonal and reproductive-related risk factors -3 with tIn an attempt to increase the clinical utility of these findings , statistDespite having a much lower breast cancer incidence in Asia than in Europe , a rapidThe subjects included in this study are women enrolled in the Singapore Chinese Health Study (SCHS), a population-based cohort study which has been described in detail previously . BrieflyBetween April 1994 and December 1999, blood and single-void urine specimens were collected from a random 3% sample of study enrollees. Details of the biospecimen collection, processing and storage procedures have been described previously . BetweenInformed consent was obtained from all participants at baseline interview, as well as at time of biospecimen collection. The Institutional Review Board at the National University of Singapore has approved this study.Incident breast cancer cases were identified through the population-based cancer registry in Singapore. As of 28 June 2010, 941 had developed breast cancer in this cohort and among them, 414 donated blood previously and were included in this study. Compared with breast cancer patients who did not donate a blood sample, those who donated were younger at diagnosis (54.9 versus 56.0 years). Patients who did not donate blood samples were less educated  than those who did . There was also a higher proportion of family history of breast cancer among those who donated blood  compared to those who did not donate .For each of the 414 breast cancer cases, up to three control subjects were randomly selected among all female cohort participants who had donated blood samples, and who were alive and free of breast cancer history at the time of cancer diagnosis of their index case. The chosen controls were matched to the index case on age at study enrollment (±3 years), dialect group , menopausal status at sample collection, dates of study enrollment (±2 years) and of blood collection (±6 months). For the 414 cases, there were six cases where only two eligible controls were found for each of them, and 408 cases where three controls were found for each of them.2) of more than 0.8 in HapMap Han Chinese in Beijing (CHB) population , where βj is the log odds ratio (OR) of the risk conferred by a variable j, and xij refers to the value of variable j for individual. The average for the variable in the population, μj, was approximated using the average among controls. The benefit of adding genetic markers into the predictive model was assessed using a net reclassification improvement (NRI) index [Variables in the Gail model and BMI were used to construct the conditional logistic regression model. The models with and without a GRS were compared in terms of their ability to accurately assess a woman’s five-year absolute risk. The probability that an individual I) index  that comI) index .As there is general expectation of a more reliable risk prediction model as additional risk variants become identified ,54-56, wP <0.05 was considered statistically significant. In the test for heterogeneity, P <0.007 was used after accounting for multiple testing through Bonferroni correction.Conditional logistic regression for the association between SNPs and breast cancer risk, NRI calculation and histogram plots were performed using R version 2.13.0. All other statistical analyses were performed using IBM SPSS version 21.0 . Statistical tests were two-sided and P = 0.003), older at first live birth (P = 0.022), report a younger age at menarche (P = 0.033), tend to be current estrogen users (P = 0.042) and fall under a higher GRS quartile (P < 0.001) compared to controls. They also have higher BMI (P = 0.066) and a positive family history of breast cancer (P = 0.063). The remaining factors comprising sleep and dietary patterns did not differ significantly between the two groups. Figure In total, 411 cases and 1,212 controls were used in the analyses. The distribution of subjects by background characteristics is shown in Table ESR1 loci - rs2046210 and rs3757318, presented ORs that were statistically significant at the 5% level. Associations with FOXQ1 (P = 0.088) and TOX3 (P = 0.098) displayed marginal significance. Apart from two tag SNPs for ESR1, another six SNPs  were also significantly associated with breast cancer in our study. Table S1 in Additional file The ORs of the 51 SNPs from the current study and their pooled estimates with previous GWAS studies are displayed in Table The associations between breast cancer risk, established risk factors and GRS were evaluated. Compared to the lowest quartile, women in the highest GRS quartile were close to 80%  = 1.27 to 2.41) more likely to have breast cancer Table . The magP <0.001), while that for controls was 2.2% (P = 0.04), though the latter was not statistically significant. This led to an NRI of 13.4% (P = 0.006), which decreased to 6.2% after correcting for optimism using a bootstrap method. This meant, that overall, 6% of women were reclassified into more appropriate risk groups when a genetic component was considered.Figure A NRI index was used to assess the improvement in risk classification that would result from adding GRS to a model comprising established risk factors only. Table We have evaluated a total of 51 SNPs and constructed a GRS to reflect their cumulative effect on breast cancer risk. The genetic score was independently associated with breast cancer risk after adjusting for education and other established risk factors. These common genetic markers, when considered in aggregate, together with reproductive factors and BMI, can improve the risk stratification for close to 10% of Singapore Chinese women. Similar to past SCHS studies ,62, caseet al., Sueta et al., and Zheng et al. have reported the discriminatory power of using five, seven and eight SNPs respectively [To our knowledge, we have investigated the largest number of SNPs for use in risk assessment in an Asian population. Studies by Dai ectively ,54,55. Aectively . LikewisTo gauge how much value a genetic component can add to an individual’s risk assessment, we included the GRS into a model consisting of Gail variables and compared its performance with the model without GRS. Many of the studies published thus far have reported the accuracy of their models in terms of area-under-the-curve (AUC) values . HoweverESR1 gene, which codes for estrogen receptor alpha (ESRα), presented statistically significant associations and consistent ORs in our study. This is in concordance with findings from other groups, which showed substantial effect sizes for SNPs in this region, highlighting a likely association with breast cancer among Chinese and Japanese women [et al., which found significant association of the 6q25.1 locus in both Asians and Europeans but greater effects in the former [MAP3K SNP, rs889312, does not increase the risk of breast cancer among the Chinese [SNPs that tagged the se women . The stuse women . This ise former . Our stu Chinese ,56, cont Chinese ,33. Howe Chinese ,38, statFOXQ1) reported here was 1.15. This is contradictory to the findings of many published works, which have reported statistically significant protective effects, but the risk effect we found is supported by the most recent BCAC study [The OR of rs11242675  was rs4784227. Rs4784227 is situated at 16q12.1 [TOX3 promoter enables it to disrupt the expression of this gene, which in turn alters chromatin structure and DNA-protein binding patterns essential for cell survival [Another SNP that was also considered in the GRS due to its marginally significant  16q12.1  and has  16q12.1 , to its  16q12.1 . Its possurvival . An OR oThis study has several strengths. The study was nested within a population-based prospective cohort that provides the use of questionnaire data collected before the occurrence of breast cancer to reduce recall and reverse causality bias. The inclusion of genetic variants in risk assessment is advantageous as it is not subjected to time-dependent errors in measurement, unlike environmental exposures such as BMI or smoking. We have also shown the strength of the association between GRS and breast cancer risk; it remains virtually unchanged even after all other established risk factors have been considered, highlighting the importance of genetics in this aspect.There are also several limitations in our study. The small sample size of approximately 1,600 women has made it difficult to attain statistical significance for most of the SNPs that were identified in GWAS studies. Nevertheless, the direction of the effects of most SNPs was consistent with the published literature. As all the subjects recruited were Chinese women, this could restrict the generalizability of our results. Studies will need to be conducted in larger populations and among women of other ethnicities to validate the effect of these polymorphisms. We were not able to consider two factors in this study: 1) the presence of copy number variations (CNVs) and their potential effects on breast cancer risk and 2) the various subtypes of the disease. Given the proximity of some SNPs to CNV regions , and theIn summary, we have shown the extent to which 51 SNPs may improve the current assessment of breast cancer risk. Most of the SNPs identified in other Western and Asian studies have presented similar effect sizes in our Singapore Chinese population. Despite conferring minimal increase in risk, individual genetic variants when considered cumulatively can result in considerable effects, leading to improved risk stratification. By including a genetic component for risk assessment, more targeted measures of prevention and screening can be implemented. For countries such as Singapore where breast cancer incidence is relatively low and mammography screening is not as well-received, cost-efficiency and ethical issues can be more aptly addressed.AUC: area under the receiver operating characteristic curve; BCAC: Breast Cancer Association Consortium; BMI: body mass index; CHB: Han Chinese in Beijing; CI: confidence interval; CNV: copy number variation; ESRα: estrogen receptor alpha; FDR: false discovery rate; GRS: genetic risk score; GWAS: genome-wide association studies; MAF: minor allele frequency; NRI: net reclassification improvement; OR: odds ratio; pOR: pooled odds ratio; RAF: risk allele frequency; SCHS: Singapore Chinese Health Study; SGVP: Singapore Genome Variation Project; SNP: single nucleotide polymorphism.The authors declare that they have no competing interests.MH directed the study and was responsible for the study design. CPLL performed data management, statistical analyses, interpretation of results and drafted the initial manuscript. AI managed the genotype data and performed statistical analyses. AS was involved in the statistical analyses and interpretation of results. JL administrated the genotyping analysis. JY and WPK were responsible for biospecimen and data collection for the Singapore Chinese Health Study cohort, on which this study is based. All authors read, critically revised and approved the final manuscript.P-het of each SNP from published studies. The pooled ORs for each SNP were obtained from published studies only.The corresponding individual and pooled ORs from published GWAS studies for 51 SNPs. This table presents the individual and pooled ORs, as well as FDR-corrected Click here for file


The purpose of this exploratory candidate gene association study was to examine relationships between polymorphisms in oxidative stress and DNA repair genes and pre-adjuvant therapy cognitive function (CF) in postmenopausal women diagnosed with early stage-breast cancer.CAT, GPX1, SEPP1, SOD1, and SOD2) and DNA repair  genes. Multiple linear regression was used to determine if the presence or absence of one or more minor alleles account for variability in CF composite scores. Based on regression findings from the analysis of individual SNPs, weighted multi-gene, multi-polymorphism genetic risk scores (GRSs) were calculated to evaluate the collective effect of possession of multiple protective and/or risk alleles.Using a neuropsychological test battery, CF was assessed in 138 women diagnosed with breast cancer prior to initiation of adjuvant therapy and 81 age- and education-matched controls and summarized across eight composites. Participants were genotyped for 39 functional or tagging single nucleotide polymorphisms (SNPs) of select oxidative stress (Each CF composite was significantly (p < 0.05) associated with one or more oxidative stress and DNA repair gene polymorphisms evaluated either by SNP main effects and/or SNP-by-prescribed breast cancer treatment group interactions. Each computed GRS was found to be significantly (p < 0.001) related to its corresponding CF composite. All associations were positive suggesting that as overall genetic protection increases, CF composite score increases (indicating better performance).These findings suggest that genetic variation in the oxidative stress and DNA repair pathways may play an important role in pre-adjuvant therapy CF in breast cancer survivors.The online version of this article (doi:10.1186/s40064-016-2061-4) contains supplementary material, which is available to authorized users. Pretreatment cognitive dysfunction has been well documented in women diagnosed with breast cancer  and DNA repair  and pre-adjuvant therapy CF in postmenopausal women with early stage breast cancer. Furthermore, cumulative multi-gene, multi-polymorphism genetic risk scores (GRSs) were calculated to evaluate the collective effect of possessing multiple significant polymorphisms.The reported variability in oxidative stress and DNA damage profiles warrants investigation of genetic variation to account for differences in cognitive phenotypes of women diagnosed with breast cancer. Considering how increased oxidative stress and decreased DNA repair capacity impacts more extreme cognitive phenotypes as well as the vulnerability of the brain within the context of increased oxidative stress due to breast cancer, we hypothesize that variability in protection from oxidative damage and capacity to repair DNA may inform variability in the extent of cognitive dysfunction among breast cancer survivors. Thus, the purpose of this exploratory candidate gene association study was to examine relationships between variation in genes involved in oxidative stress  Rapid Visual Information Processing . Women with cancer completed cognitive assessment after primary surgery but prior to initiation of adjuvant therapy. Control women completed the same cognitive assessment. The neuropsychological test battery was individually administered to study participants by trained research nurses. The selection of neuropsychological tests for the battery and reduction of individual neuropsychological test data into the eight following CF composites based on exploratory factor analysis have been described in detail previously  Nelson , and levCAT; Glutathione Peroxidase 1, GPX1; Selenoprotein P, Plasma 1, SEPP1; Superoxide Dismutase 1, Soluble, SOD1; and Superoxide Dismutase 2, Mitochondrial, SOD2) and four candidate DNA repair genes  Polymerase 1, PARP1) were identified from the literature . A detailed descriptive analysis of all data was first performed to identify any anomalies prior to modeling. Each SNP was tested for Hardy–Weinberg equilibrium using a Chi square goodness-of-fit test. To account for the heterogeneity of breast cancer tumors, women diagnosed with breast cancer were further classified prior to analysis using prescribed treatment regimen as a surrogate for disease characteristics, such as disease stage and aggressiveness. Subsequently, the analysis featured two groups of women diagnosed with breast cancer, Group A  and Group B , as well as the reference, healthy age- and education-matched control group (n = 81). Hierarchical multiple linear regression modeling was employed to estimate relationships between individual SNPs and each CF composite score. Both main SNP effect only and SNP-by-group interaction models were fitted. In all models, the prescribed treatment groups, Group A and Group B, were compared to the reference, control group. Likewise, possession of one or more minor alleles  was compared to the reference, wildtype genotype. All models were adjusted for age, estimated verbal intelligence, and levels of depressive symptoms, anxiety, fatigue, and pain at study entry. Regression diagnostics were examined for each model. Potentially influential cases were identified and sensitivity analyses were conducted to evaluate the robustness of findings. In order to retain cases found to be influential due to extreme CF scores, scores were modified to be less extreme but still the highest/lowest CF score(s) for the affected composite. Unstandardized regression b-coefficients were obtained and tested at a two-tailed significance level of 0.05.Analyses were performed using IBMGRSs were then calculated for each participant to evaluate the collective effect of multiple DNA repair and oxidative stress polymorphisms on CF composite scores. Separate GRSs were calculated for each CF composite. SNP minor alleles found to be significantly (p < 0.05) negatively or positively associated with CF composites in the individual main effect only and/or interaction effect models were utilized in GRS calculations. In order to assign greater risk/protection to alleles with stronger associations, a weighted method was employed. Unstandardized regression b-coefficients from the individual SNP models were multiplied by 0 (absence) or 1 (presence) based on a participant’s genotype and prescribed treatment group membership and then summed. For example, the equation to calculate the verbal memory GRS would be as follows:CATrs566979 and ERCC5rs4150360 would have a verbal memory GRS of 0.43 calculated as follows:Thus, a participant prescribed chemotherapy plus anastrozole (Group A) who possessed the minor alleles for A lower GRS indicates greater genetic risk for poorer CF and a higher GRS indicates greater genetic protection. Please note that if influential observations were identified by the sensitivity analysis, b-coefficients from the models with modified extreme CF scores were used. The unique contributions of GRSs in explaining the variance in CF composite scores were evaluated in the final block in a hierarchical multiple linear regression model, adjusted for age, estimated verbal intelligence, and levels of depressive symptoms, anxiety, fatigue, and pain. Participants missing genetic data necessary for completion of a GRS calculation were not included in the GRS analysis.ERCC2rs1799786 , ERCC2rs238416 , ERCC2rs50871 , PARP1rs1136410 , PARP1rs3219090 , and SEPP1rs28919892 . Of these six SNPs, only one, ERCC2rs238416  was not in Hardy–Weinberg equilibrium when considering the control group alone. This deviation is most likely due to lack of random sampling from the population for both the cases and controls. Group-wise comparisons of participant characteristics revealed that study groups differed statistically, but not clinically significantly by age and estimated verbal intelligence (Table Genotyping rates of the 39 SNPs ranged from 85.5 to 100 %. When considering the entire cohort (cases and controls), six SNPs were not in Hardy–Weinberg equilibrium: ERCC3rs2134794  or ERCC5rs873601  minor alleles was associated with poorer performance regardless of group membership. SNP main effects also influenced mental flexibility, psychomotor speed, and concentration performance. For mental flexibility, a number of significant SNP main effects were observed over multiple oxidative stress and DNA repair genes: ERCC2rs13181 , ERCC3rs4150407 , ERCC3rs4150477 , PARP1rs2271347 , SEPP1rs230819 , and SOD1rs1041740 . Significant SNP main effects were also observed for psychomotor speed: CATrs511895 , CATrs769214 , ERCC5rs11069498 , ERCC5rs751402 , ERCC5rs873601 , and SEPP1rs3877899 . For concentration, possession of one or more minor alleles for every SOD2 polymorphism evaluated, SOD2rs4880 , SOD2rs5746136 , SOD2rs8031 , contributed to poorer concentration performance regardless of group membership. In addition, the combination of Group B membership and possession of one or more ERCC2rs3916874 , ERCC2rs50872 , ERCC3rs4150407 , or ERCC5rs2296147  minor alleles contributed positively or negatively to concentration scores. The combination of DNA repair gene variation and Group A membership was found to be associated with executive function performance with multiple significant SNP-by-Group A interaction effects observed: ERCC3rs2134794 , ERCC3rs4150407 , ERCC3rs4150477 , ERCC5rs2296147 , and PARP1rs2271347 . In contrast, the combination of Group B membership and possession of one or more ERCC5rs2296148  or SOD1rs1041740  minor alleles was associated with psychomotor speed scores. The combination of group membership and genetic variation was found to be important for all three memory-related cognitive composites as well. Specifically, the combination of Group A membership and possession of one or more minor alleles for: ERCC5rs11069498 contributed positively to verbal memory  and visual working memory  scores; ERCC5rs4150360 contributed positively to verbal memory  and visual working memory ; and ERCC5rs751402 contributed positively to verbal memory  and visual memory . Additionally, the combination of Group A membership and CAT variation was found to be associated with visual memory: CATrs1001179 , and CATrs769214 . Two CAT SNP main effects, CATrs525938  and CATrs566979  were also observed with visual memory.Results from the individual SNP variant regression analyses are reported in the table found in Additional file Each computed GRS was found to be significantly p < 0.001) related to its respective CF composite Table . All ass related To our knowledge, this study represents the first investigation of relationships between oxidative stress and DNA repair gene variation and pre-adjuvant therapy CF in postmenopausal women diagnosed with early-stage breast cancer. Overall, our results revealed that performance for every CF composite was significantly (p < 0.05) associated with one or more oxidative stress and DNA repair gene polymorphisms by either SNP main effects  and/or SNP-by-group interaction effects .ERCC5 appeared to influence cognitive performance most globally, with significant relationships noted between one or more ERCC5 SNPs and every CF composite with the exception of mental flexibility. The function of ERCC5 has been most widely investigated in xeroderma pigmentosum and DNA excision repair following UV-induced damage. More generally, ERCC5 participates in nucleotide excision repair, encoding an endonuclease that makes 3’ incisions  on concentration performance within our study sample regardless of cancer diagnosis or prescribed treatment group. The SOD2 gene encodes an enzyme, manganese-dependent superoxide dismutase, that confers cell protection by eliminating mitochondrial reactive oxygen species , the SNP-by-group interaction regression coefficients for women in Group A (scheduled to received chemotherapy plus anastrozole)  and women in Group B   contributed negatively to the model, nullifying the main effect and contributing an overall negative input to executive function performance. Interactions, like the one presented, illustrate how not only genetic variation, but the combination of genetic variation and a breast cancer diagnosis can impact CF at diagnosis; alternatively, these findings highlight how factors that increase one’s risk for development of cancer may also contribute to changes in CF.Our analyses also revealed a number of significant allele effects specific to the groups of women with breast cancer compared to control women without cancer. For example, significant SNP main effects and SNP-by-group interaction effects were observed for 2 = 0.048 to 0.150. These findings not only point to the potential importance of oxidative stress response and DNA repair capacity to pretreatment changes in CF in breast cancer survivors, but, more broadly, to the value of evaluating the effect of multiple SNPs at the same time in association studies in general.In order to get a sense of the effect of the oxidative stress and DNA repair candidate pathway as a whole on pretreatment CF in women with breast cancer, we calculated weighted GRSs for each CF composite based on our individual SNP analysis. Similar to how a total score from an instrument  summarizes a given concept, GRSs “summarize the potential multiple risk genetic influences into a single quantitative parameter and do not depend on single genetic variants” , of the nine candidate oxidative stress and DNA repair genes analyzed within this study was conducted. This analysis reiterated the interconnectedness of the genes and, consequently, endorsed evaluation of the collective effect of multiple SNPs from a single pathway simultaneously. Two unique networks were identified through our analysis  often characteristic of genetic association studies. In addition, the small sample size did not allow for the evaluation of allelic dose–response relationships. Because the sample was comprised of postmenopausal women with hormone receptor positive, early-stage breast cancer who were primarily white and married, the generalizability to other more diverse populations and breast cancers is unknown. Findings from this study should be replicated in a larger, more diverse sample. In addition, differences in anesthesia exposure (or lack thereof for control women who did not undergo surgery) and its potential confounding cognitive effects were not controlled for in our analysis. Future studies and analyses should also focus on the collective effect of multiple oxidative stress and DNA repair gene variants on CF throughout and following completion of adjuvant therapy in women with breast cancer.While this study had a number of strengths, including mechanistic pathway-driven candidate gene selection, inclusion of a matched control group of women without a breast cancer diagnosis, assessment of SNP-by-prescribed treatment group interaction effects, and evaluation of the collective effect of multiple SNPs using weighted GRSs, limitations should also be acknowledged. To begin, the small study sample size, while appreciable, did not allow for detection of small effect sizes (i.e., RIn conclusion, our goal in this study was to provide data on a possible biologic mechanism to account for variability in cognitive changes in breast cancer survivors. Results from this preliminary study reveal that genetic variation in the oxidative stress and DNA repair pathways appears to play an important role in CF in women with breast cancer prior to initiation of adjuvant therapy and give reason to investigate whether polymorphisms influence cognitive decline with therapy as well. In the future, evaluation of a panel of oxidative stress and DNA repair gene polymorphisms could offer healthcare providers a means of predicting which women diagnosed breast cancer are most at risk for poorer CF and candidates for additional interventions, such as antioxidant therapy.


A population-based case-control was performed to explore familial aggregation of esophageal squamous cell carcinoma (ESCC). Family history of cancer was assessed by a structured questionnaire, and from which 2 cohorts of relatives of cases and controls were reconstructed. Unconditional logistic regression and Cox proportional hazards regression were applied for case-control design and reconstructed cohort design, respectively. We observed a close to doubled risk of ESCC associated with a positive family history of esophageal cancer among first degree relatives , after adjusting age, sex, family size and other confounders. The excess risks of ESCC increased with the increasing of first-degree relatives affected by esophageal cancer (p < 0.001). In particular, those individuals whose both parents with esophageal cancer had an 8-fold excess risk of ESCC (95% CI: 1.74–36.32). The reconstructed cohort analysis showed that the cumulative risk of esophageal cancer to age 75 was 12.2% in the first-degree relatives of cases and 7.0% in those of controls . Our results suggest family history of esophageal cancer significantly increases the risk for ESCC. Future studies are needed to understand how the shared genetic susceptibility and/or environmental exposures contribute to the observed excess risk. The data of GLOBOCAN 2012 have shown a great international variation of esophageal cancer incidence, and China is one of areas with the highest esophageal cancer incidence rates in the world24567810We thus conducted a population-based case-control study in Taixing of China, a high-incidence area of ESCC, aiming to explore the role of family history of cancer in ESCC occurrence, using two different approaches, i.e. case-control design and reconstructed cohort design.We conducted a population-based case-control study on the etiology of esophageal cancer in Taixing of Jiangsu Province from October 2010 to March of 2012. We recruited cases mainly from endoscopy units at the four largest hospitals of Taixing . More than 90% of the patients in this area are referred to these hospitals and were invited to participate. We also supplemented case recruitment by additional linkage to the local Cancer Registry. During the same time period, we enrolled population controls, which were frequency matched to the cases of ESCC on sex and age (in 5-year groups) and were randomly selected from the Taixing Population Registry. All subjects enrolled in the study were local inhabitants ages 40–85 who have lived in Taixing for at least 5 years prior to diagnosis date for cases or interview date for controls.From October of 2010 to March of 2012, we found 777 suspected cases from the hospitals, among which 752 were recruited. In addition, by linkage with the local Cancer Registry, 485 cases were further identified among whom 226 died before contact, and 101 refused or were too ill to participate, leaving 158 being recruited into the study. For each case, we also tried to collect sections from formalin-fixed and paraffin-embedded tissue blocks. All sections were stained by H.E. method and reviewed by one study pathologist. Finally we enrolled a total of 775 esophageal cancer cases into the study, and we estimated that about 81% incident cases in the study base were included according to estimated number from local Cancer Registry. Among enrolled cases, 718 (93%) were confirmed histopathologically by the study pathologist, including 648 cases of ESCC, 63 cases of esophageal adenocarcinoma, and 7 cases of other types of esophageal cancer . In total 1311 population-based controls were randomly selected, among whom 221 were excluded due to death before contact, outmigration or unfound, leaving 1090 eligible subjects. Finally, 779 controls were recruited to current study (participation rate: 71.0%).Trained staff interviewed study subjects face-to-face using a structured questionnaire, which covers information on demographic characteristics, lifestyles and family history of cancer. The staff inquired thoroughly about how many brothers, sisters and children they had, and whether their parents, siblings or children had ever been afflicted with any cancer. For those relatives without a history of cancer, we collected their vital status at the time of interview, including current age or age at death. For those relatives with a history of cancer, we further gathered the information about site of the tumor and age at cancer diagnosis.P value for trend was derived from Wald test by entering regression models the number of first-degree relatives with cancer as a continuous variable. In analysis of any cancers, unexposed subjects were those without a positive family history of any cancer. In analysis of digestive cancers, unexposed subjects were those without a positive family history of these cancers. In multivariate analysis, we adjusted for age (continuous), sex, family size (continuous), education (illiteracy/primary school/primary high school/secondary high school and above), tobacco smoking (never/ever smoker of any tobacco), alcohol drinking (never/ever), missing & filled teeth , times of tooth brushing per day (<2 times/ ≥ 2 times), daily consumption of pickled vegetables (<10 g/ ≥ 10 g) and daily consumption of fresh fruits (<27.5 g/ ≥ 27.5 g).We used unconditional logistic regression models to estimate the odds ratios (ORs) with 95% confidence intervals (CIs) of ESCC associated with a family history of cancers of the esophagus, stomach, liver, colorectum, pancreas, and all sites combined. We defined a positive family history of cancer as having at least one first-degree relative affected with cancer. We separately assessed the associations of cancers in parents and siblings with ESCC risk. We also estimated whether cases and controls differed with the number of first-degree relatives with cancer. Based on the kin-cohort data, two cohorts were reconstructed, containing the first-degree relatives of cases and those of controls (171 first-degree relatives were excluded due to missing information of age). Study subjects were followed from birth until the occurrence of esophageal cancer, death, age of 85, or the date of the study interview, whichever occurred first. The exposed cohort included first-degree relatives of cases, and the unexposed cohort those relatives of controls. Cumulative risks of esophageal cancer over time among 2 kin-cohorts were calculated and plotted using Kaplan-Meier method. The relative risk (in term of hazard ratio) of esophageal cancer for the relatives of cases compared to those of controls was derived from Cox proportional hazards regression, which included exposure status (case/control) and sex as covariates, and relative-type (parent/sibling/offspring) as stratification variable. The proportional hazards assumption was checked for each covariate in the Cox model by the method of cumulative sums of Martingale-based residualsAll statistical analyses were carried out with SAS version 9.3 .This study was approved by the Institutional Review Board of School of Life Sciences, Fudan University and the Institutional Review Board of Qilu Hospital, Shandong University. This study was carried out in accordance with the approved guidelines, and all participants provided written informed consent.Current study was based on 648 ESCC cases which were independently reviewed and confirmed by the study pathologist and 779 frequency-matched controls. After deleting 36 records (29 cases and 7 controls) with incomplete questionnaire information on family history of cancer, 619 cases and 772 controls were finally include in the analysis.The first-degree relatives of the cases were more often reported to have been affected by esophageal cancer than those relatives of the controls (34.7% vs. 21.9%), which renders an OR of 1.85(95% CI: 1.42–2.41) . The resAmong 4803 first-degree relatives of cases, 244 (5.1%) were reportedly diagnosed with esophageal cancer. The corresponding figure was 2.9% for those relatives of the controls (171 out of 6010). The cumulative risk of esophageal cancer to age 75 was 12.2% in the first-degree relatives of cases and 7.0% in those of controls  .We also performed a sensitivity analysis by excluding 70 cases whose information of family history was gathered after pathological diagnosis had been made and/or treatment had started . In this sub-analysis, 33.91% of cases reported a positive family history of esophageal cancer. The adjusted OR by unconditional logistic regression was 1.79 (95% CI: 1.35–2.37), and the hazard ratio by the Cox regression was 1.90 (95% CI: 1.53–2.37). In another sensitivity analysis, we treated missing family history of cancer as ‘no reported history of cancer’. The results remained virtually unchanged compared with those in the main analysis (data not shown).In this large population-based case-control study, we confirmed a strong association between a family history of esophageal cancer and the risk of ESCC. The excess risks increased monotonically with increasing number of affected relatives. In particular, if both parents were affected, it rendered an 8-fold excess risk of ESCC for their offspring. By the age of 75, it was estimated that about 12% of the first-degree relatives of ESCC patients might develop the malignancy, while the corresponding figure was 7% for those relatives of the normal control subjects.Previous epidemiological studies conducted in endemic areas (including mainly Linxian and Shanxi) in China have demonstrated that individuals with a family history of esophageal cancer have a higher risk of such cancer682021In the present study, we found elevated risks of ESCC among individuals with affected parents (OR = 1.63) or siblings (OR = 2.04) and an obvious dose-response relationship with the increase of affected relatives. The OR was slightly higher for siblings, compared with parents, indicating that recessive or X-linked susceptibility genes might be involved in the occurrence of ESCC, or that siblings share more lifestyle risk factors. In addition, we found the excess risk was remarkably higher if both parents were affected than that of individuals with more than one sibling was affected. This suggested that heredity does seem to take a more important role in the etiology of esophageal cancer in endemic areas.et al. have estimated the cumulative risk of esophageal cancer to age 75 was 34% in the first-degree relatives of cases and 14% in those of controls in northern IranIn many case-control studies of familial aggregation of esophageal cancer, positive family history among first-degree relatives is often used as a risk factor, and odd ratio is calculated. Instead of treating family history information as an “exposure” in case-control design, an alternative analytic strategy is to transform the case-control design into a cohort design. Based on reconstructed cohort design, Akbari There is controversy about the validity of different designs for assessing familial aggregation. Khoury and Flanders demonstrated that the case-control design would yield a biased risk estimate, with age of relatives and family size being viewed as confounding variables in assessing the disease risk estimateThere are several strengths to this study. First, the detailed information about the family history allowed us to reconstruct cohorts of the relatives of cases and controls, from which we obtained the cumulative risk estimates of esophageal cancer among exposed relatives compared with that among the unexposed relatives. Second, all of the ESCC cases were carefully reviewed and verified by the study pathologist. All subjects underwent detailed in-person interviews which provided necessary information of potential confounders. Finally, the sample size is relatively large and response rates were reasonably high among both cases and controls.Relying on self-reported information about family history of cancer is a major shortcoming in this study, especially when no validation study has been performed in the study area. This might result in potential misclassification of cancer types in first-degree relatives. However, validation studies in other settings have proved that self-reported family history of cancer could reflect the actual information when compared with retrieving information from medical records25In conclusion, our results indicate that familial aggregation of ESCC in endemic area is notable. The shared genetic susceptibility and environmental exposures, or possibility their interaction, might contribute to this phenomenon which urges future studies to explore the underlying mechanisms.How to cite this article: Chen, T. et al. Family history of esophageal cancer increases the risk of esophageal squamous cell carcinoma. Sci. Rep.5, 16038; doi: 10.1038/srep16038 (2015).


The prevalence of metabolic disorders is increasing and has been suggested to increase cancer risk, but the relation between metabolic disorders and risk of cancer is unclear, especially in young adults. We investigated the associations between diabetes, hypertension, and hypercholesterolemia on risk of all-site as well as site-specific cancers.We consecutively included men and women from nationwide Danish registries 1996–2011, if age 20–89 and without cancer prior to date of entry. We followed them throughout 2012. Metabolic disorders were defined using discharge diagnosis codes and claimed prescriptions. We used time-dependent sex-stratified Poisson regression models adjusted for age and calendar year to assess associations between metabolic disorders, and risk of all-site and site-specific cancer (no metabolic disorders as reference).Over a mean follow-up of 12.6 (±5.7 standard deviations [SD]) years, 4,826,142 individuals (50.2 % women) with a mean age of 41.4 (±18.9 SD) years had 423,942 incident cancers. Incidence rate ratios (IRRs) of all-site cancer in patients with diabetes or hypertension were highest immediately following diagnosis of metabolic disorder. In women, cancer risk associated with diabetes continued to decline albeit remained significant (IRRs of 1.18–1.22 in years 1–8 following diagnosis). For diabetes in men, and hypertension, IRRs stabilized and remained significantly increased after about one year with IRRs of 1.10-1.13 in men for diabetes, and 1.07–1.14 for hypertension in both sexes. Conversely, no association was observed between hypercholesterolemia (treatment with statins) and cancer risk. The association between hypertension and cancer risk was strongest in young adults aged 20–34 and decreased with advancing age.Diabetes and hypertension were associated with increased risk of all-site cancer.The online version of this article (doi:10.1186/s12885-016-2122-7) contains supplementary material, which is available to authorized users. Cancer is among the leading causes of death across all age groups in the western world , and as This nationwide cohort study aimed to investigate the associations between metabolic disorders, namely diabetes, hypertension and hypercholesterolemia, and risk of cancer  as well as selected cancer subtypes in men and women aged 20–89 years over 17 years of follow-up.The National Population Register includes information on vital status, date of birth, and immigration/emigration. The Danish National Patient Register (DNPR) holds information on all hospitalizations since 1977, including dates and discharge diagnoses according to the International Classification of Diseases (ICD); ICD-8 was used until 1993 and ICD-10 from 1994 onwards. The Danish Register of Causes of Death holds information on diagnoses related to the cause of death. Finally, the Danish Register of Medicinal Product Statistics  keeps records of all prescriptions dispensed from Danish pharmacies since 1995; all drugs are coded according to the Anatomical Therapeutic Chemical (ATC) classification system, and the register has been found to be accurate [In Denmark, the health care system is governmentally financed, and for administrative purposes the Danish government keeps comprehensive and nationwide registers on several health care and population related variables. Each resident is given a unique and permanent identification number; therefore, cross-linkage of the different national registries at an individual level is possible. For this study, we cross-linked data from four different registers. accurate .The study population of interest consisted of all Danish residents, included consecutively during January 1, 1996 through December 31, 2011. Each individual was included in the study population at the last of the following events: on January 1 1996, the date the individual turned 20 years old or the date of immigration to Denmark. The population was followed from date of entry until first cancer event, emigration, death, date of 90th birthday or December 31, 2012, whichever occurred first. We excluded individuals with a history of cancer  prior to date of entry.Non-melanoma skin cancer was not included in the definition of cancer , as these common and generally non-fatal cancers are most often diagnosed and treated by general practitioners, from whom data are not included in the Danish registers.Our primary outcome was first occurrence of any incident cancer (ICD-10 codes C). Our secondary outcomes were specific cancer types: breast (ICD-10 code C50), ovarian (ICD-10 code C56), endometrial , cervical (ICD-10 code C53), kidney (ICD-10 code C64), lower urinary tract , pancreatic, (ICD-10 code C25), hepatic (ICD-10 code C22), gall bladder (ICD-10 code C23), colorectal (ICD-10 code C18–C20), prostate (ICD-10 code C61), esophageal (ICD-10 code C15), and lung cancer . In this study, we identified cancer events from the DNPR and the Danish Register of Causes of Death. A recent validation study reported that ICD-10 codes of cancer registered in the DNPR have a positive predictive value of 98.0 − 100 % .diabetes, hypertension and hypercholesterolemia, were defined using ICD codes and claimed prescriptions from nationwide registers. Diabetes was defined as diabetes requiring glucose-lowering medication. Since statin is the drug of choice when initiating treatment of uncomplicated hypercholesterolemia or prophylactic in e.g. cardiovascular disorders or diabetes, we defined hypercholesterolemia as two claimed prescriptions for statins. In contrast, other lipid-lowering drugs, e.g. fibrates, as the first drug are more likely prescribed to patients with severe hypertriglyceridemia. Diabetes and hypercholesterolemia were thus defined as claiming two prescriptions of glucose-lowering medication (ATC code A10), and statins (ATC code C10AA) respectively (date of diagnosis as date of the second claimed prescription). Hypertension was defined as either 1) a diagnosis of hypertension (ICD-10 codes I10-I15) followed by a subsequent prescription claim for an antihypertensive drug within 90 days, or 2) as claimed prescriptions for two different classes of antihypertensive drugs, as described in details previously [The metabolic disorders of interest, eviously  , and duration of each metabolic disorder . Dichotomous variables were then created for each metabolic disorder (e.g. diabetes first 3 months yes/no using the left end point as reference). Age was calculated at the beginning of each interval and rounded in 2-year age intervals. In all analyses, being without the metabolic disorder of interest was used as reference (e.g. diabetes in month 9–12 was compared with no diabetes).Associations between metabolic disorders and cancer were assessed using multivariable Poisson regression models with the different metabolic disorders  included in the same model. We conducted predefined interaction analyses, specifically assessed interaction between each metabolic disorder, and age, calendar year, sex and duration of the metabolic disorder with all interaction analyses included in the same model. We assessed the cancer risk stratified by four pre-defined age-categories, i.e. 20– < 35, 35– < 50, 50– < 65 and ≥65 years.All statistical calculations were performed using SAS, version 9.4® .Metformin is sometimes prescribed to women of fertile age due to polycystic ovary syndrome. We therefore conducted a sensitivity analysis in which we excluded all prescriptions of metformin  to women between 20 and 39 years of age .Data on life style habits are not available in the administrative registries, but are correlated with socioeconomic status . We therIn Denmark, no ethics approval is needed for retrospective register-based studies. This study was approved by the Danish Data Protection Agency (j.nr.: 2007-58-0015/GEH-2014016 I-Suite nr: 02734).n = 369,400), individuals with a history of cancer prior to date of entry , and misregistered cancers (n = 2). The final study population consisted of 4,826,142 (49.8 % men and 50.2 % women). Over a mean follow-up of 12.6 years, there were a total of 423,942 incident cancers, corresponding to 30,708,457 person-years  for women and 30,282,029 person-years  for men . The age-stratified analyses showed that the highest relative risks of cancer for both men and women were observed in the younger age groups Fig. -f.In women, diabetes was associated with increased risk of kidney and hepatic cancer, and strongly associated with endometrial and pancreatic cancer risk .n = 4,223,006 with n = 408,353 cancer events), and the variable was not retained in the final models.Adjustment for educational level did not attenuate the associations between metabolic disorders and cancer  over the course of follow-up was associated with decreased or neutral cancer risk. The relative risk of cancer was particularly high among young adults with metabolic dysfunction and this risk declined steadily with higher age. Given the observational nature of the data, it is not possible to study causality.However, the associations could be caused by 1) reverse causality due to undiagnosed cancer prior to diagnosis of a metabolic disorder; 2) the metabolic disorder itself increasing the risk of cancer; 3) the risk factors increasing the risk of developing a metabolic disorder also increase the risk of cancers, thus confounding by unmeasured risk factors most importantly life style factors; or 4) simply being in contact with the health care system increases the risk of a cancer being identified.Our results showing elevated cancer risk associated with diabetes are consistent with earlier findings –23, and We observed hypertension to be associated with significantly increased cancer risks, and although previous studies on the association between hypertension and cancer risk are sparse, our finding of a stronger association in men was in accordance with an observational cohort study based on health examinations comprising nearly 580,000 adults. The study found elevated systolic blood pressure to be associated with an increase in risk of total incident cancer among men  .We observed that the risk of being diagnosed with a cancer diagnosis was significantly higher following initiation of medical treatment for a metabolic disorder, which we interpret as a situation of surveillance bias, as individuals with a known metabolic disorder may have more frequent contact with health professionals and thereby a higher likelihood of having other metabolic disorders or preclinical cancers detected. However, even in the long-term we found diabetes and hypertension to be associated with increased cancer risk. These findings are in line with previous studies –37, inclIn this study, hypercholesterolemia defined as initiation of treatment with statins, was associated with decreased or neutral cancer risk in both men and women. Statins are by far the most commonly used group of lipid medications, prescribed for elevated total-cholesterol and LDL-cholesterol, and their use has been associated with reduced risk of incident cancer in several, but not all, studies –41.n = 16,755) of the cancer events in this study were detected in individuals <40 years of age (3.7 % when excluding cervix cancers). Previous studies on associations between diabetes, hypertension and hypercholesterolemia, and overall as well as site-specific cancer, are limited, and given the low absolute cancer incidence rate, a large-scale study is needed to conduct reliable analyses. The most prevalent cancer types also differ between younger and older age-groups [The absolute cancer incidence in young individuals were low, albeit far from nonexisting, and 4.1 % (e-groups . Young aThe major strengths of this nationwide register-based study are the completeness of the records of hospital diagnoses and prescription claims; the minimal risk of selection bias and loss to follow-up; and the large population of both men and women, which provide sufficient power to conduct detailed analyses even in low-risk subgroups. Nonetheless, our study has some important limitations, primarily that the definition of metabolic disorders primarily relied on claimed prescriptions, and the true number of people diagnosed with these metabolic disorders may therefore be underestimated . This stFor hypertension, 23.3 % of our study population was defined as having hypertension either at study entry or developed during follow-up, which is comparable to the age-adjusted prevalence of 22.3 % found by a comprehensive clinical study of Danish adults aged 20-89 . AngioteCancer events were primarily identified from DNPR with supplement from the Danish Register of Causes of Death. We did not have access to The Danish Cancer Registry, which primarily gathers information from DNPR, in addition to the Danish Register of Causes of Death, and finally, from the Danish Pathology Registry. Since we do not have access to the pathology registry, our study may underestimate the number of cancer incidences, although the number is considered low, since the Danish Pathology Registry forward information to the Danish Register of Causes of Death.Data on anthropometric measures, life style habits and smoking are unfortunately not available from the Danish nationwide registers. Presence of metabolic disorders may be a marker of obesity, poor diet, physical inactivity and smoking, all of which are known to increase the risk of cancer, i.e. smoking strongly increases the risk of several cancers, particularly lung cancer and cancer of the urinary bladder. Socioeconomic status is correlated to life style factors such as obesity and smoking, and although only an approximation, our results were not altered by adjustment for socioeconomic status.In this large cohort of more than 4.6 million men and women of ages 20–89, we found that diabetes and hypertension were associated with an increased risk of incident all-site cancer across sex, age and calendar year with particularly high relative risks in the youngest age groups. Diabetes was associated with the highest cancer risk in both sexes, followed by hypertension, whereas hypercholesterolemia treated with statins was not associated with cancer risk. Our results stress the importance of prophylactic awareness among patients with diabetes and/or hypertension, not only because of increased cardiovascular risk, but also because of increased associated cancer risk.


More common is atypical hyperplasia (AH), which carries a 4–5-fold risk of breast cancer as compared to general population. Even more common is hyperplasia of the usual type and carries a roughly two-fold increased risk. Women with aspirated cysts are also at increased risk of subsequent breast cancer.There are three main ways in which women can be identified as being at high risk of breast cancer i) family history of breast and/or ovarian cancer, which includes genetic factors ii) mammographically identified high breast density, and iii) certain types of benign breast disease. The last category is the least common, but in some ways the easiest one for which treatment can be offered, because these women have already entered into the treatment system. The highest risk is seen in women with lobular carcinoma Tamoxifen has been shown to be particularly effective in preventing subsequent breast cancer in women with AH, with a more than 70% reduction in the P1 trial and a 60% reduction in IBIS-I. The aromatase inhibitors (AIs) also are highly effective for AH and LCIS. There are no published data on the effectiveness of tamoxifen or the AIs for breast cancer prevention in women with hyperplasia of the usual type, or for women with aspirated cysts.Improving diagnostic consistency, breast cancer risk prediction and education of physicians and patients regarding therapeutic prevention in women with benign breast disease may strengthen breast cancer prevention efforts. Ductal carcinoma in situ (DCIS) is now considered a precursor lesion as invasive cancer is known to arise directly from it as it is often seen adjacent to invasive cancers and when DCIS is not fully excised invasive cancer often occurs in the same region of the breast. Lobular carcinoma in situ (LCIS) however exhibits different properties and is indicative of a generalised abnormality affecting the whole breast. Varying estimates from 2-fold to 13-fold It has been known for some time that women with certain types of benign breast disease are at an increased risk of developing breast cancer. A seminal paper was published in 1985 by Dupont and Page More recently Hartmann and colleagues Several reports of women with fibroadenoma have also been published Most studies show little if any excess risk in untreated benign conditions but an increased risk of cancer in women with aspirated cysts has been consistently reported Two major prevention trials (NSABP-P1 and IBIS-I) have included women with atypical hyperplasia as a specific entry criterion making them eligible to join these placebo controlled trials of tamoxifen for breast cancer prevention Both trials have reported on the effect of tamoxifen in women with AH or LCIS. In the P1 trial, a larger effect of tamoxifen was seen for women with AH (86% reduction) compared to those in the trial overall (49% reduction) No details are available specifically for women with benign biopsies without histologic evidence of proliferation. Two trials have evaluated aromatase inhibitors for breast cancer prevention In the IBIS-II trial, women with proliferative disease were also entered on the same basis as for the IBIS-I trial. Here again the risk was higher than for women without benign disease (10 year risk in placebo arms 14.8% vs. 5.6%) and the effect of anastrozole was clearly larger for those with benign breast disease than in those without  A major challenge is full utilisation of preventive therapy in benign breast disease From these results it is clear that women with AH or hyperplasia without atypia are at an increased risk of breast cancer and preventive therapy is particularly effective in this group. Improving diagnostic consistency and breast cancer risk prediction in women with benign breast disease are important research areas. A larger effect on breast cancer prevention for women with benign breast disease was observed with aromatase inhibitors compared to tamoxifen, making this an attractive agent for postmenopausal women with proliferative disease. Furthermore, these women are also an ideal group in which to offer preventive therapy as they have already entered the medical system and have a right to know that they are at an increased risk of developing breast cancer and effective agents exist for reducing that risk. Most women are not aware of these facts and many clinicians are also insufficiently informed about breast cancer preventive therapy. Now that effective strategies for risk reduction have been demonstrated, more effort needs to be expended to make women and their clinicians aware of these possibilities and to provide help in making decisions about preventive therapy.None.All authors contributed to writing of the manuscript and approved the final version of the manuscript.The findings and conclusions in this report are those of the authors and do not represent the official position of the authors' respective institutions.JC: Received funding for trials from AstraZeneca. Speakers bureau for AstraZeneca.All remaining authors have declared no conflicts of interest.


Background: MDM2 (Murine Double Minute2) is an oncoprotein that inhibits the P53 activity. Overexpression of MDM2 gene has been reported in several human tumors. In the present study, we aimed to evaluate the impact of 40-bp insertion/deletion (ins/del) polymorphism on the promoter of MDM2 and susceptibility to breast cancer in a sample of Iranian population. Methods: This case-control study was carried out on 236 patients with breast cancer and 203 healthy individuals. Genomic DNA was extracted from the whole blood by the salting-out method. The 40-bp ins/del polymorphism was determined by using polymerase chain reaction. Results: The findings indicated that MDM2 ins/del variant increased the risk of breast cancer in co-dominant- , dominant- , and recessive-  tested inheritance models. The del allele increased the risk of breast cancer  compared with ins allele. Conclusions: Our result revealed that 40-bp ins/del polymorphism in the promoter of MDM2 increased the risk of breast cancer in an Iranian population. Further investigations with larger sample sizes and diverse ethnicities are needed to verify our findings.  Therefore, the present study was aimed to find out the possible association between 40-bp ins/del polymorphism in the promoter region of MDM2 and breast cancer in a sample of Iranian population.There is little and controversial data regarding the impact of 40-bp insertion/deletion (ins/del) poly-morphism on the constitutive promoter of Patients. This case-control study was performed on 236 histopathologically confirmed breast cancer patients and 203 age-matched women with no history of cancer of any type (as the control group) in a Southeast Iranian population. The clinicopathologic characteristics of the patients have been described in detail previously . Since MDM2 is a key component of the p53-mediated DNA-damage response, promoter polymorphism in this gene might influence this highly regulated pathway by modifying cellular MDM2 protein levels .It has been proposed that functional variants in promoter regions can lead to variable gene expression levels . PolymorIn summary, we have provided the evidence that the 40-bp ins/del polymorphism in the promoter of MDM2 gene increases the risk of breast cancer in a sample of Iranian population. However, larger sample sizes with different ethnicities are desired to validate our findings.


Introduction: Experimental studies suggest vitamin D inhibits ovarian carcinogenesis. Yet, epidemiologic studies of ovarian cancer risk and lifestyle correlates of vitamin D status, plasma 25-hydroxyvitamin D [25(OH)D], or vitamin D receptor (VDR) variants have been inconsistent.Objective: To evaluate VDR genetic associations by high vs. low predicted 25(OH)D, scores derived from known determinants of plasma 25(OH)D. To assess ovarian cancer associations with variants identified in genome-wide association studies (GWAS) of plasma 25(OH)D.Methods: We genotyped up to seven VDR and eight 25(OH)D GWAS variants in the Nurses’ Health Studies  and New England Case–Control study . We estimated haplotype scores using expectation-maximization-based algorithms. We used unconditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CI). We combined study results using DerSimonian and Laird meta-analysis.Results: Ovarian cancer risk increased per A allele of rs7975232  among all women. When stratified by predicted 25(OH)D, ovarian cancer was associated with rs731236  and rs7975232 (OR = 1.38) among women with high predicted 25(OH)D, but not among women with low levels (P ≤ 0.009). We also observed heterogeneity by predicted 25(OH)D for the ovarian cancer association with VDR 3′ end haplotypes (P = 0.009). Of 25(OH)D-associated GWAS loci, rs7041 was associated with reduced ovarian cancer risk , which did not differ by predicted 25(OH)D status.Conclusion: Our study suggests an influence of VDR 3′ end variants on ovarian cancer risk may be observed in women with high predicted 25(OH)D, which remained even after taking multiple comparisons into consideration. Future studies are needed to confirm our results and explore further the relation between vitamin D exposure, genetic variants, and ovarian cancer risk. Experimental studies suggest that vitamin D and its synthetic derivatives protect against ovarian carcinogenesis, exhibiting anti-proliferative, and pro-apoptotic effects in ovarian cancer cell lines –4 and anVDR are suspected to either directly affect or be in linkage disequilibrium (LD) with variants that influence vitamin D signaling  “f” allele] creates a VDR protein that is three amino acids longer and less transcriptionally active than the protein product of the C allele  –34. Whilignaling , which wC allele . A recenC allele . While sC allele . AdditioC allele –41, whicC allele –44, a reC allele .To explore these gaps, we assessed whether associations between variants presumed to lower VDR bioactivity or contribute to vitamin D insufficiency, and ovarian cancer risk differed by predicted 25(OH)D status in a retrospective case–control study  and two case–control studies nested within the prospective Nurses’ Health Study (NHS) and NHSII cohorts.The NHS is a prospective cohort study of 121,700 female registered nurses from 11 US states who were 30–55 years of age in 1976. NHSII began in 1989 and enrolled 116,430 female US registered nurses aged 25–42 from 14 US states. In both cohorts, biennial self-administered questionnaires gathered detailed information on lifestyle, menstrual and reproductive factors, and medical history since baseline. Blood samples were collected from a subset of 32,826 NHS participants in 1989–1990 and from 29,611 NHSII participants from 1996 to 1999. Among women who did not provide a blood sample, buccal cell samples were collected from 29,864 NHS women in 2000–2002 and 29,859 NHSII women in 2004–2006. Eligible cases consisted of women with biospecimen samples diagnosed with pathologically confirmed epithelial ovarian cancer within 4 years before sample collection up to June 1st of either 2009  or 2011 (blood) in NHSII and 2010  or 2012 (blood) in NHS with no prior cancer diagnosis except non-melanoma skin cancer. Controls were randomly selected from among women who had at least one ovary and were free of cancer (except non-melanoma skin cancer) up to and including the questionnaire cycle in which the case was diagnosed. Controls were matched to cases by cohort, biospecimen type, age (±1 month), and menopausal status at diagnosis. For participants with blood samples, controls were additionally matched to cases on postmenopausal hormone use (current vs. not current), menopausal status, month/year, time of day, and fasting status at blood collection. Completion of the self-administered questionnaire and submission of the biospecimen was considered to imply informed consent. The NHS and NHSII protocols were approved by the Institutional Review Board of Brigham and Women’s Hospital, Boston, MA, USA.The NECC Study is a population-based study of ovarian cancer. Women residing in eastern Massachusetts or New Hampshire were recruited during three enrollment phases , corresponding to three funding periods, the details of which were reported previously , 47. Bri2), total leisure-time physical activity , energy-adjusted D scores as previously described , 48. Briadjusted  vitamin adjusted . SimilarVDR gene that either have known or hypothesized functional effects on VDR activity, expression, and/or cancer risk  reside in an area of strong LD, and form several haplotypes of which G-C-A  and A-A-C  are most common. These haplotypes are in LD with long and short polyA variable number of tandem repeat (VNTR) alleles in the 3′ untranslated region (UTR) of the gene, respectively, which may alter VDR mRNA stability or translational activity (VDR 3′ end SNP rs739837 (RFLP BglI) that had been associated with advanced breast cancer risk ; NAD synthase 1 (NADSYN1)/7-dehydrocholesterol reductase ; group-specific component , which is also commonly known as vitamin D binding protein (VDBP); cytochrome P450, family 24, subfamily A, polypeptide 1 ] (We selected commonly studied Single nucleotide polymorphism (SNPs) within the cer risk . Variantactivity . We selecer risk , and rs1cer risk . Additio013897)] –44. Thre013897)] . VariantVDR and vitamin D insufficiency GWAS SNPs were genotyped in NHS and NHSII samples. To increase sample size for haplotype analyses, we additionally genotyped rs1544410, rs4588, rs7041, rs731236, and rs7975232 in NECC Phase 3 samples. Sample sizes for each SNP analyzed are reported in Table S1 in Supplementary Material. Genomic DNA was extracted from buffy coat or buccal cell samples using the QIAamp  96-spin blood protocol. DNA from NHS and NHSII participants was whole genome amplified with GE Healthcare Genomiphi . Genotyping was performed at the Dana Farber/Harvard Cancer Center High-Throughput Genotyping Core using the 5′ nuclease assay  on the OpenArray® Real-Time PCR System  on amplified DNA or the ABI PRISM® 7900HT Sequence Detection System on genomic DNA (NECC Phase 3 samples). Laboratory personnel were blinded to case–control status, and each plate included replicate samples for quality control, which had 100% concordance.Existing genotype data on rs11568820, rs1544410, rs2282679, rs228570, and rs7041 were available for NECC Phase 1 and 2 participants from previous analyses , 52, wheN = 570), as described previously (Existing plasma 25(OH)D levels were available for a subset of NHS and NHSII participants  and excluded from analyses. We excluded NHS/NHSII samples that failed genotyping of five or more SNPs . Among NECC participants, rs1544410 was out of HWE (P = 0.004). Review of screen shots for these plates revealed appropriate clustering and so rs1544410 was retained to generate VDR 3′ end haplotypes. The rs1544410 risk allele frequency (RAF) among NECC controls was similar to the RAF among NHS/NHSII controls and the HapMap Phase II + III, Release 28 Caucasian European (CEU) population .We used chi-square tests to assess Hardy–Weinberg equilibrium (HWE) among white controls within NHS/NHSII and NECC. Three of 15 SNPs  were out of HWE among NHS/NHSII participants D binding affinity constants based on published estimates [Table P values for trend.We used unconditional logistic regression models adjusted for matching factors and race to calculate odds ratios (ORs) and 95% confidence intervals (CIs). For each SNP, gene dosage effects were modeled by assigning a value of 0, 1, or 2 to a genotype trend variable according to a participant’s number of risk alleles. We estimated frequencies of plotypes , 55 to pP value for statistical interaction of VDR 3′ end haplotypes by predicted 25(OH)D status, we pooled individual-level NHS/NHSII and NECC data, additionally adjusted for study and predicted 25(OH)D scores, and compared models with interaction terms to a model without interaction terms using the likelihood ratio test.Predicted 25(OH)D scores were dichotomized at the median values of menopause-specific control distributions in NHS/NHSII and NECC. Women missing predicted 25(OH)D values were excluded from stratified analyses and tests for interaction. Within each study, statistical tests for interaction by predicted 25(OH)D were performed by the Wald test using cross-product terms. DerSimonian and Laird random effects meta-analysis  was usedP values were based on two-sided tests and considered statistically significant at P < 0.05. All analyses were conducted using SAS version 9.3 . Power calculations were performed using QUANTO  cases and 3,423  controls were available for this analysis. Women ranged in age from 34 to 72 years in NHS/NHSII and from 18 to 79 years in NECC at the time of blood collection. As expected based on the matched designs, cases, and controls were of similar age within each study. On average, NHS/NHSII participants were slightly older than NECC participants at the time of blood collection. Within each study, cases had shorter mean duration of oral contraceptive use and were less likely to have had a tubal ligation than controls. BMI, total leisure-time physical activity, total vitamin D intake , and alcohol consumption were similar between case and control groups. Total vitamin D intake was higher in NECC than in NHS/NHSII (Table r2 = 0.79) at the NADSYN1/DHCR7 locus and between rs2282679 and rs4588 (r2 = 0.95) at the GC locus among NHS/NHSII participants, we selected the SNP with the higher genotyping success rate for analysis (rs3829251 and rs4588). We did not observe significant heterogeneity in estimates between NHS/NHSII and NECC in the main effect meta-analysis of SNPs. Of the VDR SNPs assessed, we observed a significant 12% increased risk of ovarian cancer associated with each rs7975232 A allele (Table Ptrend = 0.06). The GWAS-identified rs7041 T allele was significantly associated with reduced ovarian cancer risk . We confirmed that the rs7041 T allele and the vitamin D insufficiency GRS were associated with reduced 25(OH)D levels  among the subset of NHS/NHSII participants with existing 25(OH)D measurements (N = 570). The vitamin D insufficiency GRS was not associated with ovarian cancer risk (data not shown).Risk allele frequencies observed in our control groups were comparable to those in the CEU HapMap population . Genotyping success rates were ≥95% for all SNPs except rs7975232 in NHS/NHSII (93%) and rs2228570 in NECC (94%). Given high LD between rs1790349 and rs3829251 (VDR gene (VDR 3′ end (rs1544410–rs7975232–rs731236) formed three common haplotypes and five haplotypes with frequencies <5% that were grouped into a “rare” haplotype variable. Ovarian cancer risk was not associated with the VDR 3′ end haplotypes compared to the most common haplotype (G-C-T) in the meta-analysis of NHS/NHSII and NECC  that vary in binding affinity for vitamin D analytes (Ptrend = 0.003). Estimated GC phenotypes were not associated with ovarian cancer risk in NHS/NHSII, NECC, or the meta-analysis D strata. Among women with high (above the median) predicted 25(OH)D levels, we observed significant increased risk of ovarian cancer associated with VDR variants rs731236  and rs7975232 . Similarly, heterogeneity in ovarian cancer risk by predicted 25(OH)D was observed for the VDR 3′ end haplotypes , total vitamin D intake (below vs. above median), total leisure-time physical activity (below vs. above median), menopausal status and HT use , and alcohol intake (below vs. above median). Except for heterogeneity in the rs731236 association with ovarian cancer risk by alcohol intake (P = 0.03), in which the increased risk was restricted to women with higher than the median alcohol intake, we did not observe significant effect modification by predicted 25(OH)D score components (data not shown). The GC rs7041 T allele was associated with reduced ovarian cancer risk among women with low predicted 25(OH)D (Ptrend = 0.03), but the association was not significantly different from that of women with high predicted 25(OH)D  may influence ovarian cancer risk among women with higher predicted 25(OH)D levels, but not among women with lower levels. This also is the first report to assess whether vitamin D insufficiency GWAS loci are associated with risk. Based on the anti-proliferative and pro-apoptotic effects of vitamin D on ovarian cancer cell lines D status for the other examined variants.In this study, we examined whether the vitamin D status of individuals modifies genetic associations between ll lines , the rs7VDR gene is associated with a modestly increased risk of ovarian cancer. Known vitamin D pathway genes do not appear to reside at the nine susceptibility loci newly identified by ovarian cancer GWAS studies conducted among women from North America and the UK , there was no association between the 3′ end haplotypes and ovarian cancer risk (unpublished data). However, in our study, we only observed a positive association for individuals with higher predicted 25(OH)D status. Since a substantial portion of the US and UK populations have insufficient vitamin D levels , which begins 21.6 kb downstream of the VDR gene. HDAC7 has been shown to attenuate 1,25(OH)D-mediated gene transcription in malignant breast cells D-stimulated VDR activity differed by naturally occurring rs2228570 genotype in peripheral blood mononuclear cells in vitro . The T aplotypes . The autple size , which mple size , we cannP = 0.03). The association could be due to chance considering the number of statistical tests performed in this study. Another possibility is that because rs7041, in combination with rs4588, forms three major VDBP isoforms , and the rs7041 T allele codes for isoforms with both the lowest (GC2) and highest (GC1f) binding affinity D levels would result in a lower lifetime average plasma 25(OH)D levels, increasing risk of ovarian cancer. Individual vitamin D insufficiency variants and the GRS were associated with plasma 25(OH)D in the expected direction among the subgroup of NHS/NHSII women with measured levels, but were not associated with ovarian cancer risk. Contrary to expectations, the rs7041 T allele was associated with reduced ovarian cancer risk D scores remained significant after adjusting the significance level using a Bonferroni correction (0.05/54 = 0.0009). The risk associated with VDR variant rs731236 among this same subgroup of women also remained significant using the less conservative False Discovery Rate procedure D status was generally limited. Moreover, the numerous hypotheses tested relating to SNP main effects, haplotypes, predicted 25(OH)D strata, and interactions increased the likelihood of observing false positive results. Even so, increased ovarian cancer risk associated with rs7975232 and the A-A-C 3′ end haplotype at the rocedure . Our anarocedure . Use of VDR gene. Our results indicate that genetic associations may be missed if 25(OH)D status is not considered, potentially contributing to inconsistency in the literature. Larger studies assessing heterogeneity in ovarian cancer risk by vitamin D status associated with VDR variants are required to validate our results. If resources are available, use of plasma 25(OH)D levels instead of predicted 25(OH)D scores may provide additional information on whether genetic variants are likely to influence risk over a particular threshold or range of 25(OH)D. Moreover, functional studies assessing the influence of the VDR 3′ end variants on expression and activity of VDR and neighboring genes in ovarian tissue may shed light on the degree of involvement of the vitamin D pathway compared to alternate pathways that may involve other genes in the region.In summary, we observed heterogeneity in genetic associations with ovarian cancer risk by predicted 25(OH)D status that was limited to variation at the 3′ end of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fonc.2014.00286/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit several pathways in experimental models of breast carcinogenesis, but epidemiological evidence remains insufficient to support their use for breast cancer prevention. We examined the association between use of NSAIDs and breast cancer risk in a prospective cohort.The Sister Study is a prospective cohort study of women who had a sister(s) with breast cancer. As of December 2013, 2118 incident breast cancers were ascertained from 50,884 women enrolled between 2003 and 2009. Lifetime history of NSAID use was estimated from self-reported data in pill-years, with 1 pill per week for a year equivalent to 1 pill-year. Cox regression models were used to estimate hazard ratios (HRs) of breast cancer in relation to pill-years of use for different NSAIDs, with adjustment for potential confounders.4vs1 = 0.66, 95 % CI: 0.50–0.87) and specifically for aspirin , with small, but non-significant reductions in risk for other drug classes.In the full cohort, although there was some evidence that use of non-aspirin, non-COXib NSAIDs was associated with lower breast cancer risk, there was little evidence of overall association for most categories of NSAID use. Among postmenopausal women NSAID use was not associated with reduced risk of breast cancer. However, among premenopausal women there was significantly reduced risk for any NSAID  contains supplementary material, which is available to authorized users. Based oaglandin , can alse tissue .Numerous epidemiologic studies have investigated the relation between use of NSAIDs and breast cancer. Although previous meta-analyses have suggested a modest reduction in breast cancer risk in relation to use of aspirin and other NSAIDs , 8, a raTo further clarify the role of NSAID use in breast cancer prevention, we examined the association between lifetime use of NSAIDs and breast cancer risk in a large prospective cohort of women with a sister with breast cancer.The Sister Study (NCT00047970) is a prospective cohort of 50,884 women aged 35–74 years who had a sister with breast cancer but who did not have breast cancer themselves , 19. TheInformation on NSAID use was obtained during the baseline interview in multiple ways. Prior to telephone interview, study participants were given a medications booklet listing common medications and were told to use them as a reference to identify specific medications that they used. During the computer assisted telephone interview, participants were first asked about medications used for 25 major health conditions including some for which NSAIDs may be indicated . Participants were asked the total number of years they took any medication for a specific condition and the names of each medication currently taken at least once a week for that condition. For each medication reported they were asked to report the age at first use, the number of days per week, times per day on days they took it, and total years or months of use. If the medications reported did not span at least half of the interval between first use of medicine for a condition and the date of interview, participants were asked for similar information for the medication they took for that condition for the longest time. Participants were also asked about any medications they were currently taking at least once a week that were not covered in the medical history section of the questionnaire. Finally, participants were asked if they had ever taken any pain or inflammation medications at least three times a week for 3 months in a row or longer that might not have been reported elsewhere in the interview. Again, participants were asked to report age at first use, whether they had used it regularly in the past 12 months, how many days per week they usually took the medication, and how many times a day they took the medication on days that they used it.Each reported medication was coded by product and class using the Slone Drug Dictionary . ProductUsing the detailed medication use histories provided, we constructed age-specific calendar grids for use of each specified NSAID. Total years of use for each subgroup of NSAIDs were computed by summing across the occupied ages in the calendar grid. To quantify lifetime use of NSAIDs, the frequency of use during the period was entered into the corresponding age grids and pill-years of use were computed as a product of frequency and years of use, where one pill-year represents use of one pill per week for a year.N = 1638). Because of high agreement between self-reports and medical records , we retained self-reported breast cancers in this analysis.Participants have been followed through 2013 with an average of 5.3 years of follow-up. As of December 26, 2013, a breast cancer diagnosis was documented in 2118 women in the cohort, with an average time to diagnosis of 3.2 years. Women who report a new breast cancer diagnosis were contacted 6 months after diagnosis for additional information about their diagnosis and treatment and asked to authorize release of pertinent medical records. To date, pathology reports or complete medical records have been obtained for 77 % of self-reported breast cancers  and 95 % confidence intervals (CI) for the associations between lifetime use of NSAIDs and breast cancer risk. Pill-years of NSAID use was categorized into 4 groups: <0.75 pill-years ; 0.75- < 14 pill-years; 14- < 49 pill-years; and ≥ 49 pill-years to reflect similar levels of frequency and duration of use that were used to define use of NSAIDs in previous studies , 22. Thest degree family members with breast cancer , BMI , time since the last mammogram  and age at 1st term birth . Additional adjustment for years of oral contraceptive use, alcohol intake, current physical activity and years of hormone replacement therapy (HRT) use did not change the results from the specified multivariable models  analysis. Briefly, we postulated that “health consciousness” and “pain” were two immediate, but unmeasured, factors leading to use of NSAIDs, and considered variables that were related with breast cancer risk and at least one of the unmeasured variables, as potential confounders. The final adjusted models included: race/ethnicity , level of education , history of benign proliferative breast disease , number of 1a priori subgroup analyses by tumor characteristics by treating tumor occurrences that were assigned to types not under consideration as censoring events. Specifically, we evaluated whether an association with NSAID use might vary between invasive versus in situ tumors, or according to estrogen receptor (ER), progesterone receptor (PR) and HER2 status. Prespecified  and data-driven post hoc (parity and age at first birth) subgroup analyses were also performed. Differences in the HRs between levels of these factors were evaluated by testing for multiplicative interactions or for homogeneity of trends on exposure categories as an ordinal variable. Significance tests were two-sided with the level of significance at 0.05. Stata 12.1  was used for all analyses.In addition to an analysis including all incident breast cancers, we conducted Approximately 11 % of premenopausal women and 21 % of postmenopausal women had at least 49 pill-years of NSAID use in their lifetime . Associations with NSAID use did not differ among in situ tumors, localized and advanced breast cancers  of invasive cancers were ER+/PR+/HER2-, followed by ER-/PR-/HER2- (11 %) and ER+/PR+/HER2+ (10 %). There were too few ER-/PR-/HER2+ tumors to evaluate (N = 53). There was an indication of inverse associations between long-term regular use of any NSAIDs and ER+/PR+ breast cancer regardless of HER2 status. While most associations were not statistically significant, there was a significant inverse trend for increasing pill years of non-aspirin, non-COXib NSAIDs and ER+/PR+/HER2- breast cancer (p = 0.039). The relationship between use of NSAIDs and breast cancer risk did not vary by degree of family history of breast cancer or BMI (data not shown).Approximately 26 % of all incident breast cancers were p for homogeneity of trends = 0.038): The HRs associated with the highest category of pill-years of aspirin use were 0.57 (95 % CI: 0.33–0.98) in premenopausal women and 1.03 (95 % CI: 0.87–1.23) among postmenopausal women. Although results were not statistically significant, the inverse associations with the highest category of pill-years of use of COXibs and other non-aspirin, non-COXib NSAIDs also tended to be more evident among premenopausal women.In contrast to results for postmenopausal women, lifetime use of NSAIDs was associated with reduced risk of breast cancer among premenopausal women Table . The mulSeveral exploratory analyses were conducted to better understand the unexpected subgroup finding. First, our analysis used age as the time scale for calculating HRs rather than time since enrollment, based on the results from a simulation study of different time scales in Cox regression analysis . Since wp = 0.89 in postmenopausal women). We also examined breast cancer risk in relation to pill-years of NSAID use within the past 5 years . Third, separate analysis, stratified by menopause status at study follow-up, was conducted because 46 % of premenopausal women at baseline  became postmenopausal by the end of study follow-up. This analysis did not affect the lack of the association among postmenopausal women: the HRs for the highest pill-years of aspirin were 0.55 (95 % CI: 0.24–1.23) vs 0.99 (95 % CI:0.83–1.17) for those who were premenopausal versus postmenopausal at follow-up (p for homogeneity of trends =0.067).Second, median pill-years of NSAID use were lower for premenopausal than for postmenopausal women (18.7 vs 28 years). To make the analyses in the two groups more comparable, we further divided the highest category of NSAID use (≥21 pill-years for COXibs and ≥ 49 pill-years for other types of NSAIDs) into two groups using the median among postmenopausal women (not shown), but the association with breast cancer risk remained unchanged . However, the HRs did not vary in the two strata.Another possible explanation for the observed difference may lie in the interaction between NSAIDs and timing of exposure. The development of human breast tissue progresses over the lifespan, and menopause represents one lifecycle event associated with lobular involution and decrease in glandular or dense parenchymal features. Because hormone responsiveness and cell proliferation are the highest in the undifferentiated lobules –46, chemStrengths of the present study include its prospective design, the large sample size and detailed information on potential risk factors for breast cancer. We also were able to characterize lifetime use of NSAIDs based on comprehensive questionnaire data about both medical history and medication use. In addition to questions about the general use of pain and inflammation medications, participants could report NSAID use in connection with medical histories, which included questions about all medications used in relation to 25 specific health conditions, and as part of a listing of any other medications that were currently being taken or were used within 1 year of enrollment. Despite various approaches  used to improve the accuracy of recall, however, some nondifferential measurement error is unavoidable and is hard to quantify.A few other issues concerning the interpretation of our study need to be discussed. First, we defined lifetime NSAID use based on the information collected at baseline and did not include information on changes in NSAID use during the study follow-up. However, given the relatively short time to breast cancer diagnosis in our study, it seems unlikely that change in NSAID use pattern is sufficiently large to influence the association between use of NSAIDs and breast cancer risk. Second, the present study involves multiple exposure and outcome measures, and the resulting larger number of statistical tests may have yielded spurious associations. Although the subgroup analyses were guided by biological plausibility and conducted in order to obtain a more comprehensive picture of the relationship between use of NSAIDs and breast cancer risk, findings from the subgroup analyses should be interpreted with caution. Lastly, our study population is unique in that all women had a sister with breast cancer at the time of enrollment. Because women with a family history of breast cancer on average have a 2–4 fold increased risk of developing breast cancer , women wIn this analysis of more than 50,000 women at higher risk of breast cancer on the basis of family history, there was little evidence that use of NSAIDs can protect against breast cancer. However, among premenopausal women, use of NSAIDs, particularly aspirin, was associated with reduced risk of breast cancer, raising the intriguing hypothesis of chemopreventive benefit for this subgroup of at-risk women.