Alterations in the redox balance are involved in the origin, promotion and progression of cancer. Inter-individual differences in the oxidative stress regulation can explain a part of the variability in cancer susceptibility.The aim of this study was to evaluate if polymorphisms in genes codifying for the different systems involved in oxidative stress levels can have a role in susceptibility to breast cancer.We have analyzed 76 single base polymorphisms located in 27 genes involved in oxidative stress regulation by SNPlex technology. First, we have tested all the selected SNPs in 493 breast cancer patients and 683 controls and we have replicated the significant results in a second independent set of samples (430 patients and 803 controls). Gene-gene interactions were performed by the multifactor dimensionality reduction approach.OGG1), rs406113 and rs974334 (GPX6), rs2284659 (SOD3), rs4135225 (TXN) and rs207454 (XDH) were significant in the global analysis. The gene-gene interactions demonstrated a significant four-variant interaction among rs406113 (GPX6), rs974334 (GPX6), rs105213 (OGG1) and rs2284659 (SOD3)  with high-risk genotype combination showing increased risk for breast cancer .Six polymorphisms rs1052133 (The results of this study indicate that different genotypes in genes of the oxidant/antioxidant pathway could affect the susceptibility to breast cancer. Furthermore, our study highlighted the importance of the analysis of the epistatic interactions to define with more accuracy the influence of genetic variants in susceptibility to breast cancer. Despite breast cancer (BC) being the most frequent cancer in women in western countries and the second cause of cancer death after lung cancer, the risNumerous evidence links carcinogenesis and oxidative stress regulation, including prooxidant and antioxidant defense systems–7. OxidaPrevious studies with knockout animals that lack antioxidant enzymes support the view that ROS contribute to the age-related development of cancer. For instance, mice deficient in the antioxidant enzyme CuZnSOD showed increased cell proliferation in the presence of persistent oxidative damage contributing to hepatocarcinogenesis later in life. AnotherIn this context, single nucleotide polymorphisms (SNPs) in components of the cellular redox systems can modify the redox balance and take part in both the BC initiation and/or progression, as well as determine possible therapeutic treatments–17.Despite the importance of oxidative stress in the development and progression of cancer, few studies have evaluated the relationship between genetic modification in genes coding for enzymes relatives to the redox system and the susceptibility to develop BC. The previous studies had focused mainly on the analysis of genes related to antioxidant defense enzymes, 19, butThe aim of this study was to evaluate the association between common variants in genes coding for proteins related to the redox system (antioxidant and oxidant systems or proteins) and the susceptibility to develop BC. We hypothesized that common SNPs related to the redox pathway are associated with an altered risk for BC. We chose 76 SNPs on which to perform a two-step study: one first exploratory set and a second, independent, validation set. We also decided to investigate the impact of complex interactions between SNPs at different genes of the stress oxidative pathway. To address this issue, we analyzed the effects of gene-gene interactions by the multifactor dimensionality reduction (MDR) approach. This analysis was carried out in four SNPs that were statistically significant in the combinatorial set.The underlying analyses were carried out in a Caucasian Spanish population. The study was carried out in two steps with two population groups. A first group of 1176 samples was composed of 493 female patients diagnosed for BC between the years 1998–2008 at La Paz Hospital and Foundation Jimenez Díaz (Madrid), and 683 healthy women controls recruited at the Hospital of Valladolid (Spain).Thereupon, we chose the polymorphisms that showed marginally significant association , and we replicated the procedure in a second independent group (n = 1233) where we included 430 female patients diagnosed for BC between the years 1988–1998 at the Clinic Hospital of Valencia (Spain) and 803 samples from cancer-free women recruited at the blood donor bank at the same Hospital. Blood was collected between 2010 and 2011 during periodical patient visits. The blood from controls was extracted between the years 2009 and 2012. In both groups, the controls were women without pathology or history of cancer. Controls were not matched to cases, but were similar in age. In group 1, cases’ mean age was 57.5 (range 23.5-89.5), and that for donors was 52.7 (21.5-96.5). In group 2, cases’ mean age was 54.1 (20.5-86.5) while in donors, it was 54 (22.5-92.5).We selected this staged approach because it allowed us to analyze only those polymorphisms with indicative results and reduced the number of genotyping reactions without significantly affecting statistical power, 20.The research protocols were approved by the ethics committee of the INCLIVA Biomedical Research Institute. All the participants in the study were informed and gave their written consent to participate in the study.http://www.ncbi.nlm.nih.gov/projects/SNP/) and HapMap (http://www.hapmap.org). The selection of polymorphisms was performed by SYSNP in a second independent set.Set 2: Subsequently, and in order to better identify those polymorphisms that could be associated with BC, we replicated the 10 SNPs with a OGG1 gene maintained the statistical significance  after the Bonferroni correction.Set 1 + Set 2: Finally, we analyzed the 10 polymorphisms in the global population set 1 + set 2 . The results are listed in Table GPX6) and rs1052133 (OGG1) was the most significant (p = 0.041). The best three-loci model included rs406113 on the GPX6 gene, rs1052133 on the OGG1 gene and rs2284659 on the SOD3 gene, and it showed statistical significance (p < 0.0007) with an OR = 1.82 and 95% CI = 1.28-2.58. A four-way interaction found that between rs406113 on the GPX6 gene, rs974334 on the GPX6 gene, rs1052133 on the OGG1 gene and rs2284659 on the SOD3 gene predicts breast cancer with a testing balance accuracy of 0.5267. This four-loci model had a chi-square value of 11.284 (p = 0.0008) and an OR of 1.75 [95% CI = 1.26-2.44]. The four polymorphism combinatory model showed a higher predisposition to BC than the polymorphisms rs406113, rs974334 and rs2284659 did individually  and had values similar to the ones of polymorphism rs1052133 (ORX5 = 1.82). The summary of the multi-factor dimensionality results are listed in Table There is growing evidence that epistasis interactions between genes may play a role in cancer risk, and different variable selection approaches have been developed to analyze the potential gene-gene and gene-environment interactions. The fout p = 0.01. The beThe combined genotype AA for rs406113, CC/CG for rs974334, CC/CG for rs1052133 and GG/ GT for rs2284659 showed a higher risk for BC, which is consistent with the models described for the polymorphisms individually. Figure SOD2) gene A16V (C/T) (rs4880) led to structural alterations in the domain responsible to target the mitochondria, giving a reduction in the antioxidant potential) and p-value = 0.0004). Our results are in concordance with the meta-analysis by Yuan and collaborators that suggests that the hOGG1 326 Cys allele provides a significant protective effect for BC in European women. The impOGG1. This enOGG1, 48, 49.carriers. This daCAT gene was not significant in our analysis, but it presented a borderline tendency in the Cebrian et al. study. Moreover, they found a significant difference in genotype distribution between cases and controls in rs4135179 (TXN). We, however, were unable to confirm this in our global analysis, although we detected a marginal significance in set 1. The reason for this discrepancy can be found in the population’s characteristics, in the superior age of the population included in the Cebrian study[The epistatic analysis of the four most significant polymorphisms in relation to the susceptibility to BC was performed with the MDR method. This is a reliable approach that has been widely used, 51–54. an study.Our study has several limitations to take into consideration. Firstly, there is no data available about the lifestyle of controls and patients that could be related to oxidative stress, such as diet, exercise and the consumption of tobacco and alcohol. Secondly, polymorphisms that were not explored in our study may affect the risk to develop BC and should be taken into account in the analysis of our data and in further studies. Nevertheless, the association between SNPs and risk for BC is reliable since that power exceeded 95% in all the cases. All samples are from the same country and ethnicity, and the adjustment for age reduces variability.Additionally, MDR has 80% statistical power to detect true interactions in two-, three-, and four-way gene-gene interactions, even with a small number of cases and controls. FurtherDespite these considerations, our work, as far as we know, is the largest study in the Spanish population that analyzes the influence of polymorphisms in oxidative genes in susceptibility to BC. Overall, our data, together with that published in the bibliography, 55–62, OGG1, GPX6, SOD3, TXN and XDH genes significantly associated with predisposition to breast cancer. These associations have not been described previously, except for rs1052133 (OGG1). Concerning this polymorphism the published results in breast cancer were contradictory, and some authors found only a significant association with the risk of developing lung cancer. We have found an increment in the risk of developing breast cancer in the carriers of at least one Ser allele (recessive model) in concordance with a meta-analysis of breast cancer susceptibility in European women. In this particular case, an incorrect expression of the protein encoded by the OGG1 gene could interfere with the suitable repair of the genetic material. Furthermore, our study highlighted the importance of the analysis of the epistatic interactions in order to define the influence of genetic variants in susceptibility to breast cancer more precisely. Further studies on the relevance of these and other polymorphisms in the development of breast cancer should be performed.Our results suggest that different genotypes in genes of the oxidant/antioxidant pathway could affect the susceptibility to breast cancer. We have found six polymorphisms in


Breast density and single-nucleotide polymorphisms (SNPs) have both been associated with breast cancer risk. To determine the extent to which these two breast cancer risk factors are associated, we investigate the association between a panel of validated SNPs related to breast cancer and quantitative measures of mammographic density in a cohort of Caucasian and African-American women.In this IRB-approved, HIPAA-compliant study, we analyzed a screening population of 639 women (250 African American and 389 Caucasian) who were tested with a validated panel assay of 12 SNPs previously associated to breast cancer risk. Each woman underwent digital mammography as part of routine screening and all were interpreted as negative. Both absolute and percent estimates of area and volumetric density were quantified on a per-woman basis using validated software. Associations between the number of risk alleles in each SNP and the density measures were assessed through a race-stratified linear regression analysis, adjusted for age, BMI, and Gail lifetime risk.rs3817198 (in LSP1) was significantly associated with both absolute area (p = 0.004) and volumetric (p = 0.019) breast density in Caucasian women. In African-American women, SNPs rs3803662 (in TNRC9/TOX3) and rs4973768 (in NEK10) were significantly associated with absolute (p = 0.042) and percent (p = 0.028) volume density respectively.The majority of SNPs were not found to be associated with any measure of breast density. SNP The majority of SNPs investigated in our study were not found to be significantly associated with breast density, even when accounting for age, BMI, and Gail risk, suggesting that these two different risk factors contain potentially independent information regarding a woman’s risk to develop breast cancer. Additionally, the few statistically significant associations between breast density and SNPs were different for Caucasian versus African American women. Larger prospective studies are warranted to validate our findings and determine potential implications for breast cancer risk assessment.The online version of this article (doi:10.1186/s12885-015-1159-3) contains supplementary material, which is available to authorized users. Breast cancer is currently the most commonly diagnosed cancer and the second leading cause of cancer death in women in the US . RecentlMammographic density, the most broadly used measure of breast density, represents the relative amount of radiographically-opaque fibroglandular tissue versus radiographically-translucent adipose tissue in the breast. Commonly measured via visual assessment either qualitatively using the American College of Radiology Breast Imaging-Reporting and Data System (BI-RADS) density categories ,17, or qTo date, the etiological pathways underlying the increase in breast cancer risk due to the presence of dense tissue are not yet clearly understood ,27. BreaIn this context, we investigate associations between a panel of validated SNPs related to breast cancer risk and quantitative measures of mammographic density in a race-stratified cohort. Given the increasing interest in identifying which measures of breast density are most related to breast cancer risk , we eval2), overweight (25 kg/m2 ≤ BMI < 30 kg/m2) and obese (BMI ≥ 30 kg/m2). Race information was categorized as Caucasian, African-American or Other; however, given the relatively small number of women who identify as “Other” (N = 31), only women who identified as either Caucasian (N = 389) or African-American (N = 250) were included in this study.In this University of Pennsylvania Institutional Review Board (IRB) approved, HIPAA compliant study, we retrospectively identified a cohort of women aged 40 years or older from our routine breast screening population who had also been prospectively recruited by a separate, IRB-approved, HIPAA compliant clinical study at our institution investigating the added value of genomic markers in breast cancer risk prediction . For thers13387042), MRPS30 (rs4415084), FGFR2 (rs1219648), TNRC9/TOX3 (rs3803662), 8q24 (rs13281615), LSP1 (rs3817198), MAP3K1 (rs889312), NEK10 (rs4973768), 1p11 (rs11249433), RAD51L1 (rs999737), COX11/STXBP4 (rs6504950), and CASP8 (rs1045485), which have been consistently associated with either overall or subtype specific cancer risk, the risk for metastatic disease or age at diagnosis [For each woman, information regarding the genotype of 12 SNPs were obtained from a commercially available assay based on Illumina Infinium II whole-genome genotyping  . The deCiagnosis -49. DetaDA, is divided by the total breast area, BA, to obtain a woman’s breast percent density (PD%) using equation Breast density was measured using fully-automated methods. Area-based absolute and percent mammographic density was assessed on a per-image basis using a previously validated, fully-automated algorithm . BrieflyThese area density estimates acquired per image were averaged across each individual woman’s left and right mediolateral-oblique (MLO) and craniocaudal (CC) screening images in order to obtain a per-woman estimate of both absolute area of dense tissue and PD% for further analysis.BV, and fibroglandular dense tissue volume, DV, are then used to calculate the relative volumetric percent density (VD%) seen mammographically via equation Absolute fibroglandular breast tissue volume and volumetric percent density were also automatically assessed on a per-image basis using fully-automated, FDA-cleared software  which is based on the widely validated Highnam and Brady method adapted for digital mammography ,51. BrieAs with the area density measures, the individual volumetric density estimates acquired per-image were averaged to obtain corresponding per-woman estimates of absolute fibroglandular tissue volume and VD%.Differences in age, BMI, Gail lifetime risk, and breast density distributions between the Caucasian and African-American cohorts were assessed using two-sided t-tests with unequal variances for continuous variables and the Chi-squared test for categorical variables at an α = 0.05 significance level. Pearson’s correlation coefficient was used to assess associations between absolute dense area, absolute dense volume, PD% and VD%. Associations between the four breast density measures and each SNP were then assessed with linear regression, in which we adjusted for age, BMI, and Gail lifetime risk by including them as additional covariates in the regression model to determine the significance of the change in density due to the differences in SNP genotype between women in the presence of these additional explanatory variables.R2, for each regression model with a significant association to a breast density measure, using a recently proposed method for datasets with multiple imputation [For all analyses, breast density measures were first log-transformed to approximate a normal distribution as has been done in prior works investigating the genetic basis of breast density  as well putation . Lastly,Caucasian women were slightly older (p = 0.03), had a lower overall BMI p < 0.001), and a higher Gail lifetime risk (p < 0.001) than African-American women. When comparing breast density between the two groups, Caucasian women were denser in terms of their percent density both by the area (p < 0.001) and volumetric (p = 0.003) metrics, while African-American women had a greater absolute volume of fibroglandular tissue (p < 0.001). No significant difference was seen between the two groups in terms of absolute area density (p = 0.90). A summary of the demographic and imaging characteristics for the women in our study cohort is shown in Table , and a hrs3817198, was found to be significantly associated to absolute area density in Caucasian women at the Bonferroni level , but not in Caucasian women  and absolute dense volume (p = 0.026) in Caucasian women, and also became significantly associated with area percent density (p = 0.044). SNP rs3803662 also retained its significance in terms of its association with absolute volume density in African-American women (p = 0.048); while rs4973768 ceased to be significantly associated with volumetric percent density (p = 0.059). Lastly, complete-data analysis  with absolute measures of breast density in Caucasian women, although SNPs rs3803662 and rs4973768 only approached significance (p ≤ 0.1) with absolute volume density and volume percent density, respectively, in African-American women, likely due to the decreased sample size in the complete-data analysis leading to a loss of statistical power.When investigating joint associations between the entire panel of SNPs and each breast density measure through multivariable analysis . Specifically, SNP rs3817198 (in LSP1) was associated with absolute measures of area and volume density in Caucasian women, while in African-American women different SNPs, namely rs3803662 (in TNRC9/TOX3) and rs4973768 (in NEK10) were associated with absolute volume of dense tissue and percent volume density, respectively.We evaluated potential associations between a panel of validated breast cancer-related SNPs and quantitative measures of volumetric and area-based breast density in a cohort of Caucasian and African-American women. We found that the majority of the SNPs evaluated are not associated with breast density, and that those SNPs that are associated with breast density explain only a small fraction of the total variability in density after accounting for age, BMI and Gail lifetime risk . In addition, we only investigated a panel of 12 low-penetrance SNPs associated with breast cancer, while many more risk loci have been recently identified . To fullIn conclusion, the majority of the SNPs evaluated in our study were not found to be significantly associated with breast density. Although this may be due to the relatively small sample size of our study, and therefore limited power to detect more subtle associations, our observations suggest that these two risk factors may be capturing potentially independent information regarding a woman’s risk for breast cancer. As such our findings may have implications in the development of future breast cancer risk models by providing evidence that both SNPs and breast density could be considered simultaneously as risk predictors to potentially improve discriminatory capacity. Additionally, our study suggests that the associations between SNPs and breast density appear to differ between Caucasian and African American women. Larger prospective studies are warranted to further validate our findings and determine potential implications for breast cancer risk assessment. Ultimately, understanding the independent pathways that these different risk factors relate to breast cancer could lead to the development of improved risk assessment tools and prevention strategies.


A cross-sectional study was performed to assess bone health history among aromatase inhibitor (AI) users before breast cancer (BC) diagnosis, which may impact fracture risk after AI therapy and choice of initial hormonal therapy. A total of 2,157 invasive BC patients initially treated with an AI were identified from a prospective cohort study at Kaiser Permanente Northern California (KPNC). Data on demographic and lifestyle factors were obtained from in-person interviews, and bone health history and clinical data from KPNC clinical databases. The prevalence of osteoporosis and fractures in postmenopausal AI users was assessed, compared with 325 postmenopausal TAM users. The associations of bone health history with demographic and lifestyle factors in AI users were also examined. Among all initial AI users, 11.2% had a prior history of osteoporosis, 16.3% had a prior history of any fracture, and 4.6% had a prior history of major fracture. Postmenopausal women who were taking TAM as their initial hormonal therapy had significantly higher prevalence of prior osteoporosis than postmenopausal AI users . Among initial AI users, the associations of history of osteoporosis and fracture in BC patients with demographic and lifestyle factors were, in general, consistent with those known in healthy older women. This study is one of the first to characterize AI users and risk factors for bone morbidity before BC diagnosis. In the future, this study will examine lifestyle, molecular, and genetic risk factors for AI-induced fractures. The current third-generation AIs inhibit 96–99% a priori bone health history, age, physical activity, smoking, and alcohol intake The aforementioned guidelines vary slightly but usually include bone mineral density (BMD) testing and clinical assessment of risk factors for fracture In a cross-sectional analysis of one of the largest contemporary cohorts of breast cancer patients, we describe history of osteoporosis and fracture and the prevalence of risk factors for fracture , physical activity, smoking, alcohol intake, and calcium and vitamin D supplement use) before breast cancer diagnosis among initial AI users. We also compare prevalence of bone health history in postmenopausal AI users with postmenopausal TAM users. Lastly, we examine associations of these fracture risk factors with prior history of osteoporosis and fracture outcomes in AI users.The Pathways Study is a prospective study of 4,505 women with newly diagnosed invasive breast cancer who are members of Kaiser Permanente Northern California (KPNC), a large, integrated health care delivery system covering the San Francisco-Oakland Bay Area, Sacramento, and surrounding counties. Recruitment was from January 2006 to April 2013 through rapid case ascertainment procedures designed to enroll women prior to initiation of chemotherapy, as described elsewhere For this bone health sub-study, women were included if they had at least one hormonal therapy prescription of an AI or TAM that was indicated for treatment of their first primary breast cancer. A total of 1,159 women who had no hormonal therapy, 27 women who initiated hormonal therapy after recurrence of their original breast cancer, and 4 women who initiated hormonal therapy after their second primary breast cancer were excluded. The final study population consisted of 3,315 eligible women. Based on complete hormonal therapy prescription data through December 2013, 2,157 (65.1%) were initial AI users, and 1,158 (34.9%) were initial TAM users. For this analysis of baseline bone health history, only the initial AI users were included. In selected analyses, 325 postmenopausal women who received TAM as their initial hormonal therapy drug were also included as a comparison group to postmenopausal AI users .Clinical and diagnostic tumor characteristics were obtained from the KPNC Cancer Registry approximately four months post-diagnosis The baseline interview was conducted at enrollment into the cohort approximately two months post-diagnosis, and included interviewer and self-administered questionnaires on sociodemographics, diet, physical activity, smoking, established breast cancer risk factors, health history, and use of vitamin/mineral supplements. Anthropometric measures were also obtained at baseline.Information was collected on hysterectomy and oophorectomy and associated dates of the surgery, and age or date of last period. Menopause was defined as the absence of menses for 12 consecutive months or more relative to the date of the baseline interview, or having a complete hysterectomy or oophorectomy of both ovaries.Physical activity was assessed using an activity frequency questionnaire based on the validated Arizona Activity Frequency Questionnaire (AAFQ) Prescription drug data for nearly 100% of KPNC enrollees is recorded in the KPNC pharmacy database, including drug name, National Drug Code, dosage and therapeutic class; prescription dates and cost; dispensing and refills; and prescribing physician, thus minimizing recall bias th edition (ICD-9) outpatient and hospitalization diagnoses of bone outcomes were obtained from the KPNC electronic medical record (EMR). These diagnosis codes include: (1) osteoporosis (733.00–733.09); (2) any prior fracture involving the neck, trunk, upper and lower extremities ; and (3) any major osteoporotic fracture of the spine, humerus, wrist, or hip  were ascertained as previously described International Classification of Diseases, 9Considering potential under-diagnosis or documentation of osteoporosis by clinicians Analyses of initial AI or TAM use in postmenopausal breast cancer patients by select characteristics, including prior history of osteoporosis and fracture, were conducted using logistic regression with adjustment for age at breast cancer diagnosis as a continuous variable.In the overall initial AI user group, we calculated odds ratios (OR) and 95% confidence intervals (CI) using logistic regression to estimate the associations of lifestyle and clinical factors at breast cancer diagnosis with prior history of 1) osteoporosis, 2) any fracture, and 3) any major osteoporotic fracture. All models were adjusted for age, race/ethnicity, menopausal status, and year of breast cancer diagnosis, and all p-values were two-tailed with a significance level of 0.05. Analyses were repeated after excluding those diagnosed with breast cancer before menopause. The results were similar to those from the overall initial AI user group and thus are not presented here.All analyses were conducted in SAS version 9.3 .The study was approved by the KPNC institutional review board.2, with 31.8% being overweight and 38.2% obese. The median (IQR) of moderate-vigorous physical activity at baseline was 19.5 (7.3–39.1) metabolic equivalent (MET)-hours/week. Over half of the patients were never smokers at baseline (52.0%), 42.5% were former smokers, and only 5.5% were current smokers. Alcohol intake was light at baseline, with 27.9% being never drinkers, and among those who drank, the median intake was 3.10 grams/day. Nearly two-thirds of the patients did not take either calcium or vitamin D supplements at baseline (63.2%), 16.5% took calcium, 12.6% took vitamin D, and 7.7% took both supplements.As shown in Among the initial AI users, 11.2% had a prior history of osteoporosis, including 3.5% at 6 years or more before breast cancer diagnosis, and 7.7% within 6 years Table 1.2 vs. 29.0 kg/m2, p = 0.0001), and more physically active . No differences in race/ethnicity, smoking history, or alcohol intake were found between the two groups. For bone health history prior to breast cancer diagnosis, although initial TAM users were younger, they had a significantly higher prevalence of prior osteoporosis than initial AI users , but a similar prevalence of any prior fracture  and major prior fracture . Consistent with a higher prevalence of prior osteoporosis compared with AI users, TAM users were more likely to take calcium and/or vitamin D than AI users .Among postmenopausal patients with HR-positive breast cancer, the majority received AIs as their initial hormonal therapy , and 325 patients (n = 13.8%) received TAM as their initial hormonal therapy. No apparent secular trend was found in the use of AIs relative to TAM during the study period (2005–2013). To explore whether bone health history or other known risk factors for fractures might affect the choice of hormonal therapy drugs, comparisons were conducted between postmenopausal initial AI users and postmenopausal initial TAM users. As shown among the postmenopausal women in In Associations of patient characteristics and lifestyle factors with prior history of any fracture are given in In a large contemporary cohort of breast cancer survivors who were initially treated with AIs, we found that 11.2% had a prior history of osteoporosis, 16.3% any fracture, and 4.6% major fracture before breast cancer diagnosis. Although the majority of postmenopausal women were initially treated with AIs, a sizable proportion (13.8%) was initially treated with TAM. Furthermore, these TAM users had nearly twice the prevalence of prior osteoporosis compared with initial AI users. Finally, the associations of selected risk factors with prior history of bone health outcomes in breast cancer patients initially treated with AIs were largely consistent with those expected from the healthy older population To our knowledge, our observational study is the largest to date to describe the use of AIs as primary hormonal therapy in conjunction with prior bone morbidity. A previous study of 343 women with early-stage breast cancer about to initiate AI therapy reported 22.2% with osteoporosis and 11.4% with any fracture For postmenopausal women diagnosed with early stage, HR-positive breast cancer, AIs have been shown to have superior efficacy in lowering risk of recurrence compared with TAM, and thus have become the preferable choice for this patient subgroup. However, TAM remains a viable choice for initial hormonal therapy for those who seek to avoid AIs’ musculoskeletal effects. This is likely true for women deemed to have low risk of recurrence but are susceptible to fractures, as suggested by our results. In postmenopausal patients in our study, initial TAM users were slightly younger than initial AI users, yet the former had significantly higher prevalence of osteoporosis history. However, initial TAM users were more likely to have stage I disease than initial AI users, suggesting their risk of recurrence was lower. A lower risk of recurrence coupled with a higher risk of fracture might have influenced physicians and patients to favor TAM over AIs as their first choice of initial hormonal therapy. This speculation was further strengthened by the findings of higher usage of calcium and/or vitamin D supplement and higher physical activity in the initial TAM users than in the initial AI users. As supplement use and physical activity were surveyed soon after breast cancer diagnosis, we could not assess whether these data represent exposure status before or after the diagnosis of osteoporosis. Higher usage of supplements and being more physically active might have been in response to being diagnosed with osteoporosis . It is also interesting to note that a small proportion of initial AI users were diagnosed with breast cancer before menopause. Most likely those patients experienced menopause due to chemotherapy or radiation therapy and were subsequently eligible for AI therapy.Among initial AI users, we identified several risk factors associated with history of osteoporosis, including older age, Asian race, and lower BMI. Older age was also associated with fracture history and being physically active was associated with lower risk of major prior fracture. These associations were in the same direction as expected in a general healthy older population, suggesting common mechanisms for osteoporosis and fracture regardless of later breast cancer diagnosis. Although smoking and alcohol consumption are risk factors for osteoporosis and fracture in non-cancer patients, we did not find such associations in initial AI users, possibly due to the small proportion (5.5%) of current smokers in the study and light alcohol intake in the cohort (median intake of 3.10 grams/day among drinkers). We also found that Asian AI users had a higher risk of osteoporosis but lower risk of prior fracture than Whites. This observation potentially reflects known racial/ethnic differences of lower bone mineral density, yet decreased fracture risk, in healthy Asians compared with Whites When comparing our proportion of prior fracture to healthy postmenopausal women at a similar age in the Women’s Health Initiative (WHI) Our study was based on a large prospective breast cancer cohort from one of the nation’s largest integrated healthcare delivery systems where complete electronic medical records and pharmacy information are available on all patients. This enabled us to accurately assess bone health history through their health insurance membership with Kaiser Permanente. Moreover, the cohort was established in 2005, a time concurrent with the widespread use of AIs. Our study was further strengthened by the availability of extensive demographic and lifestyle information collected at the time of diagnosis through in-person interviews.a priori osteoporosis diagnosis, as AIs did not become the preferential choice of hormonal therapy for postmenopausal breast cancer patients until the mid-2000’s. To overcome this limitation, we classified patients with BP treatment but without an ICD-9 diagnosis of osteoporosis as osteoporotic. The results were similar when we relied only on osteoporosis ICD-9 diagnosis. Furthermore, the prevalence of prior spine fractures could have been underestimated due to asymptomatic fractures not being coded by the physician, but for our analyses, we considered spine fractures as clinically diagnosed. Finally, given our access to electronic medical records on the cohort, we estimate that only 35% had BMD measurements before breast cancer diagnosis based on Current Procedural Terminology (CPT) codes, thus precluding us from classifying osteoporosis status based on BMD data for the entire cohort. To note, the prevalence of prior BMD screening in our cohort is greater than 16% reported in a small study of BMD screening adherence among 342 breast cancer patients on AIs, and these women were also members of an integrated health care system Nevertheless, our study is not without limitations. Due to its cross-sectional nature, we could not infer causality of the selected risk factors for prevalent osteoporosis and fracture among breast cancer patients. In addition, osteoporosis was potentially under-diagnosed in our patient population. However, this is not a unique challenge to this study. Clinicians vary in their use and coding of the term osteoporosis such that it may be identified after BMD testing, after a primary care visit, at the time of a specialty visit, at the time of bisphosphonate initiation, or at the time of fracture. Further, a diagnosis may not always mean that the T-score is in the osteoporosis range, and a diagnosis could also be made in the presence of a fragility fracture while BMD is only in the osteopenic range. There have also been temporal changes in the frequency of osteoporosis diagnosis To conclude, a small proportion of breast cancer patients initially treated with AIs had a positive history of osteoporosis or fracture before their cancer diagnosis. A history of osteoporosis might influence the choice of initial hormonal therapy drug among patients with low risk of recurrence. Risk factors for osteoporosis and fracture before breast cancer diagnosis were similar to those among healthy older women. Given that adverse effects on bone health is a common comorbidity concern for many cancer survivors


PALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing.In this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls.p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3–18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism .We identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.Our findings support truncating variants in  PALB2 were subsequently shown to be associated with familial breast cancer [. used a modified segregation analysis approach to determine that the age-specific risk of breast cancer among female mutation carriers overlaps the risk conferred by BRCA2 mutations [PALB2 is the most significant breast cancer predisposition gene after BRCA1 and BRCA2.Partner and localizer of BRCA2 (PALB2) plays a central role in homologous recombination-mediated repair of double-strand DNA breaks  and bialt cancer  and numet cancer ). Most rutations  establisPALB2 c.3113G > A (p.Trp1038*) as a recurring truncating mutation among familial breast cancer index cases, and established the enrichment of c.3113G > A in cases compared to controls [PALB2 mutations have analysed significant numbers of family cancer clinic-ascertained cases or matched controls. Because early studies focused on screening just for the presumed common pathogenic mutations, in Australia . From 2003, individuals with a ≥10 % risk of carrying a BRCA1 or BRCA2 mutation, as estimated by BRCAPro, including tumour pathology, were also eligible [Cancer-affected women in the study were referred by their physician to a specialist Familial Cancer Centre (FCC) for genetic testing of eligible .BRCA1 and BRCA2, including for large rearrangements by multiplex ligation probe-dependent amplification (MLPA), returned negative results.Our final case cohort , the Peter MacCallum Cancer Centre, Hunter New England Health and The University of Newcastle. This study was carried out in accordance with all relevant regulations and guidelines.PALB2 gene was performed as part of a custom sequencing panel. All coding PALB2 exons were amplified from 225 ng of germline DNA extracted from blood or saliva using the HaloPlex Targeted Enrichment Assay  according to the manufacturer’s protocol using an Agilent Bravo Automated Liquid Handling System. Paired-end 100 or 150 bp sequence reads were generated from the indexed, pooled libraries on a HiSeq2500 Genome Analyzer . Sequence reads were trimmed of adapter using Cutadapt [Germline mutation analysis of the Cutadapt . Genome Cutadapt , 18. ProCutadapt . VariantCutadapt . All novCutadapt . The folCutadapt , SIFT [2Cutadapt  and PolyPALB2. These coding regions were well covered by sequence reads in both cases and controls. The mean read depth across the entire gene for all samples was 217 , with an average of 98.66 % of the coding regions covered by at least 20 reads (98.12 % for cases and 99.20 % for controls).A total of 1996 breast cancer index cases and 1998 non-cancer controls were screened for germline mutations in the coding regions of p = 0.0001, chi-squared test; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3–18.9)  and four among 1998 controls (0.2 %), demonstrating a significant enrichment in cases . The four most common variants (>3 % carrier frequency) were tested for association with breast cancer. Surprisingly, the most common variant, c.1676A > G , which is predicted by CADD, Condel, PolyPhen2 and SIFT to be benign, was significantly more common in cases  than controls (16.8 % non-wild-type) with a per-allele OR of 1.24 . There were 20 cases homozygous for this variant versus ten controls . The overall trend for an effect had a p value of 0.0018 (Cochrane-Armitage trend test). The minor allele frequency was 0.105 in cases and 0.086 in controls, compared to other databases where the minor allele frequency of this variant in European populations was 0.09 (1000 Genomes), 0.096 (ExAC) and 0.09 (EVS), but showed increased frequency in African and Asian populations.A large number of missense variants (n = 54) were detected in the cohort Table . There wConsidering only those rare variants present in fewer than five carriers among 3994 cases and controls (approximately 0.1 %), a similar number of missense variants were detected in both groups (40 in cases (2 %), 28 in controls (1.4 %)), which does not suggest any association of rare missense variants with risk. There was also no significant enrichment in cases when limited to rare variants that were predicted to be deleterious by any of Condel, SIFT or Polyphen2  or with a CADD score of >10 .We detected 23 synonymous variants Table . NeitherBRCA1 and BRCA2 variant could be identified. The frequency of PALB2 truncating variants in this cohort (1.1 %) is similar to other studies analysing high-risk breast cancer individuals . These data will enable evidence-based clinical guidelines to include full PALB2 screening if previously they had advised testing limited to the specific common variant only.This study screened Australian individuals with breast cancer who had been referred to a Familial Cancer Centre for genetic testing and in whom no pathogenic  overall , 26–41)  overall , 42, 43)The prevalence of truncating variants in cancer-free controls was 0.15 % in the LifePool cohort. These individuals were ascertained from women attending population-based mammographic screening, which in Australia is targeted towards women over 50, although some younger women are included. Thus, this volunteer cohort may not be entirely representative of the general population, although all were cancer-free at the time of analysis. Nonetheless, the frequencies of missense and synonymous variants are consistent with those reported in large databases such as 1000 Genomes , Exome APALB2 function. It should be noted that such a prediction was relatively common for the variants we detected in PALB2  and any effect would need to be confirmed by an RNA-based assay.We did not observe any significant enrichment in missense mutations overall, although the frequency was slightly higher in the cases when only rare, deleterious mutations were considered. The contribution of rarer variants to breast cancer risk will need to be evaluated in larger case–control cohorts. Surprisingly, the common variant c.1676A > G  was significantly enriched in cases versus controls, although with only a modest odds ratio (1.24). There was a trend towards homozygous carriers of this variant being enriched in cases versus controls with an OR of 2.08. This variant was shown to be associated with an increased breast cancer risk in multiple-case breast cancer families in Chile compared to population controls  with an PALB2, which examined p.Leu939Trp, p.Leu1143Pro and p.Thr1030Ile [There has been only one study to date that has examined the likely functional effect of missense variants in r1030Ile . The firPALB2 as a breast cancer predisposition gene when considering truncating mutations. We did not see any excess in missense mutations in cancer cases overall, although there may be individual variants that are associated with risk at low penetrance. We advise extreme caution in attributing risk to missense PALB2 mutations when determining clinical management.Our data strongly support


Recent oral contraceptive (OC) use has been consistently associated with increased risk of breast cancer, but evidence on specific breast cancer subtypes is sparse.We investigated recency and duration of OC use in relation to molecular subtypes of breast cancer in a pooled analysis of data from the African American Breast Cancer Epidemiology and Risk Consortium. The study included 1,848 women with estrogen receptor-positive (ER+) breast cancer, 1,043 with ER-negative (ER-) breast cancer (including 494 triple negative (TN) tumors, which do not have receptors for estrogen, progesterone, and human epidermal growth factor 2), and 10,044 controls. Multivariable polytomous logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for exposure categories relative to never use, controlling for potential confounding variables.OC use within the previous 5 years was associated with increased risk of ER+ , ER- , and TN  breast cancer. The risk declined after cessation of use but was apparent for ER+ cancer for 15 to 19 years after cessation and for ER- breast cancer for an even longer interval after cessation. Long duration of use was also associated with increased risk of each subtype, particularly ER-.Our results suggest that OC use, particularly recent use of long duration, is associated with an increased risk of ER+, ER-, and TN breast cancer in African American women. Research into mechanisms that explain these findings, especially the association with ER- breast cancer, is needed. A combined analysis of 54 studies observed a 24% increase in breast cancer risk for current use of oral contraceptives (OCs) and a 16% increase in risk for <5 years since stopping OC use . SubsequGene expression studies have identified at least five intrinsic subtypes of breast cancer, and the most common clinical markers of breast cancer heterogeneity to date have been presence or absence of estrogen receptors (ERs) or progesterone receptors (PRs) and overexpression of human epidermal growth factor receptor 2 (HER2) . Tumors Some risk factors, particularly hormone-related factors, appear to differentially associate with specific breast cancer subtype ,21, but The objective of the present study was to investigate OC use in relation to molecular subtypes of breast cancer in African American women using data from a large consortium, the African American Breast Cancer Epidemiology and Risk (AMBER) project. In particular, we were able to assess long duration of OC use and recency of use, including intervals as long as 30 years or more since last use.The AMBER Consortium has been described in detail elsewhere . Its purThe BWHS is a prospective cohort study of 59,000 African American women aged 21 to 69 years at baseline in 1995 . Particiin situ breast cancer cases and matched controls [The CBCS is a population-based case–control study of women aged 20 to 74 years in North Carolina conducted from 1993 through 2001 . Breast controls .The WCHS is a population-based case–control study of women aged 20 to 75 years that began in New York in 2003 and currently enrolls participants in New Jersey only ,31. BreaEach study obtained informed consent from all participants and was approved by the relevant Institutional Review Boards, which are listed in Acknowledgements. Each study prepared a data file that included questionnaire data for a specified set of variables and pathology data from medical records or state cancer registry records. Data were harmonized at the data coordinating center with regular input from each study’s principal investigator and data collection staff.International Classification of Diseases for Oncology, Third Edition), not including lobular carcinoma in situ (8520/2), phyllodes tumors (9020), or Paget’s disease (8540/3), were confirmed through medical records and state cancer registries [–/PR–/HER2–). ER data were available for 72% of cases and PR data were available for 69% of cases. Testing for HER2 has become routine more recently; therefore, HER2 data were available for only 49% of cases. There were no statistically significant differences between women with and without known receptor status by age or OC use. The proportion of ER– cases  and duration of OC use. Women with a total duration of OC use of less than 1 year were considered to be never users. Recency of OC use was categorized as within the previous 5 years, 5 to 9 years ago, 10 to 14 years ago, 15 to 19 years ago, 20 to 24 years ago, 25 to 29 years ago, or ≥30 years ago. Duration of OC use was categorized as 1 to 4 years, 5 to 9 years, 10 to 14 years, or ≥15 years.n = 60) whose periods had stopped due to use of mediation, radiation, or chemotherapy for various health conditions other than breast cancer. Women who had a hysterectomy with retention of one or both ovaries were classified as premenopausal if their index age was less than or equal to the age at which 10% of women in their study had reached natural menopause, and as postmenopausal if their index age was greater than or equal to the age at which 90% of women in their study had reached natural menopause; otherwise, these participants were classified as having unknown menopausal status.Self-reported data were available on the following covariates: history of breast cancer in a first-degree female relative , age at menarche, parity, age at first birth, lifetime duration of breastfeeding, menopausal status, age at menopause, menopausal female hormone use, height, weight, educational attainment, alcohol consumption, and cigarette smoking. For menopausal status, women who were still menstruating were classified as premenopausal. Women who had experienced a natural menopause or a hysterectomy with bilateral oophorectomy were classified as postmenopausal. Also classified as postmenopausal were women (2), educational attainment , alcohol consumption , smoking status , and geographic region . We used the missing indicator method to handle missing data. For menopausal status, 8.8% of women had missing data; for each of the other covariates, fewer than 2% had missing data. To test for trend across categories of OC duration or OC recency, the ordinal variable was treated as a continuous variable in the regression model. Trend tests were carried out among OC users only. To examine potential differences by subtype, we carried out case–case analyses, in which ORs were computed for the relation of OC use to ER– relative to ER+ breast cancer. Effect modification was explored through stratification by age, menopausal status, body mass index, and parity. Interaction on the multiplicative scale was tested by the likelihood ratio test, comparing models with and without multiplicative interaction terms. Analyses were performed using SAS 9.2 statistical software .Polytomous logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the relation of measures of OC use to ER+ and ER– breast cancer risk. For TN breast cancer risk, polytomous models had three outcomes: TN, ER+, and ER–/not TN. We controlled for age (continuous), study , time period of case diagnosis/control index date , family history of breast cancer , age at menarche , parity , age at first birth , lactation , menopausal status and age at menopause , female hormone use , body mass index . The strongest associations were for OC use within the previous 5 years . For ER+ breast cancer, the ORs were elevated through 15 to 19 years since last use and the estimate for 15 to 19 years was statistically significant . For ER– breast cancer, the pattern was less consistent; the OR declined to 1.07  at 20 to 24 years since last use, but there was a statistically significant elevation at 25 to 29 years since last use . Risk of ER– cancer increased with increasing duration of OC use, with ORs of 1.43 (95% CI = 1.13 to 1.80) and 1.54 (95% CI = 1.15 to 2.06) for 10 to 14 and ≥15 years duration, respectively. For ER+ breast cancer, the OR for 10 to 14 years duration was elevated  but the estimate for ≥15 years was approximately 1.00. Among recent users, risk increased with increasing duration of OC use for both ER+ and ER– cancer. The OR for ≥10 years of use that continued into the 5 years before diagnosis or index date was 1.65 (95% CI = 1.27 to 2.15) for ER+ cancer and 1.73 (95% CI = 1.26 to 2.37) for ER– cancer. The corresponding estimates for ≥10 years of use that ended at least 20 years previously was 0.88 (95% CI = 0.65 to 1.19) for ER+ cancer and 1.50 (95% CI = 1.03 to 2.17) for ER– cancer.Table In a case–case analysis (data not shown), ever use of OCs  and OC use within the previous 5 years  were not associated with ER– breast cancer, relative to ER+ breast cancer. Increased risk of ER– breast cancer was associated with long duration (≥15 years) of OC use relative to ER+ cancer: the OR was 1.51 (95% CI = 1.07 to 2.15).in situ breast cancer (data not shown), results for invasive cancer  were similar to the overall findings: the ORs for invasive ER+ cancer were 1.14 (95% CI = 1.00 to 1.31) for ever use, 1.41 (95% CI = 1.10 to 1.81) for use within the previous 5 years, and 0.95 (0.69 to 1.30) for duration of 15 years or more; the corresponding estimates for invasive ER– cancer were 1.36 (95% CI = 1.14 to 1.62), 1.62 (95% CI = 1.20 to 2.20) and 1.59 (1.11 to 2.29), respectively. For in situ cancer, based on 255 ER+ cases, risk of ER+ breast cancer was significantly elevated for recent OC use , but was not associated with long duration of OC use . There were too few ER– in situ cases (n = 43) for an informative analysis.In an analysis stratified by invasive and P trend =0.05). Risk increased with increasing duration of OC use, with an OR of 1.62 (95% CI = 1.11 to 2.38) for ≥15 years of use (P trend = 0.05). Analyses of a joint variable for duration and recency of use indicated that the increased risk associated with at least 10 years of OC use was statistically significant both for use less than 5 years ago  and use 5 to 9 years ago .The relation of OC use to risk of TN breast cancer is presented in Table P interaction = 0.75 and P interaction = 0.77, respectively).In analyses stratified by age , recent and long-duration OC use were associated with increased risk of breast cancer in every age group . Breast cancer risk was elevated for recent OC use in both nulliparous and parous women (Table P interaction = 0.08). Long duration of OC use was associated with breast cancer among parous women, but not among nulliparous women (Table P = 0.42).In analyses stratified by menopausal status Table , ORs weren Table : among nen Table . The int2 (Table P interaction = 0.04), but not for duration of OC use (P interaction = 0.16).Recent and long-duration OC use were associated with larger increases in breast cancer risk among women who were overweight or obese than among women with body mass index <25 kg/m2 Table . The intP value for heterogeneity was 0.57 or greater for all comparisons, indicating no statistically significant heterogeneity.Figure Both recent OC use and long duration of OC use were associated with increased risk of breast cancer in this study of African American women. The positive associations were observed for ER+, ER–, and TN breast cancer. ORs were highest for TN breast cancer: women who had used OCs in the past 5 years were estimated to have a 78% increased risk of TN breast cancer and those who had used OCs for at least 15 years had a 62% increase.While some studies have found no association of recent OC use with overall breast cancer risk ,5,38,39,There are few previous studies of OC use with molecular subtype of breast cancer. Five studies found associations of OC use with both ER+ and ER– breast cancer ,8-10,12.Combination OCs contain estrogens and progestins and have been found to increase circulating levels of estradiol ,49. A poFew studies have examined potential effect modification of the relation of OC use to breast cancer risk by age ,23,57. ODifferences in risk of breast cancer by age or menopausal status could reflect changes in OC formulations. The estrogen and progestin composition of OCs has varied, with declining doses of hormones and new types of progestin introduced over time ,60,61. TThe associations of OC use with breast cancer risk in the present study were stronger among overweight and obese women. No previous studies have assessed whether an association of breast cancer risk with recent or long duration of OC use is modified by body mass index. This finding is particularly important given the high prevalence of obesity in African American women . ObesityThe present analysis is the largest yet conducted of OC use in relation to specific subtypes of breast cancer among African American women. It allowed for informative assessment of associations within subtype overall and within some strata of interest. However, statistical power in some subanalyses was limited. Numerous important risk factors for breast cancer were controlled in the analyses. While the studies that contributed to the present analyses differed in study design and in population characteristics, a meta-analysis found no significant heterogeneity in the results. In addition, results on other risk factors, including parity and body size, have been similar across the individual studies ,21,69-72In sum, our findings suggest that recent OC use, particularly of long duration, is associated with increased risk of ER+, ER–, and TN breast cancer in African American women, with possibly a stronger relation with TN breast cancer. Increases in risk associated with OC use were apparent for up to 15 years or more after cessation of use. The association with OC use was most pronounced among overweight and obese women. Research is needed to investigate the mechanisms by which obesity may further increase the breast cancer risk associated with OC use.


Evidence from animal models shows that tissue stiffness increases the invasion and progression of cancers, including mammary cancer. We here use measurements of the volume and the projected area of the compressed breast during mammography to derive estimates of breast tissue stiffness and examine the relationship of stiffness to risk of breast cancer.Mammograms were used to measure the volume and projected areas of total and radiologically dense breast tissue in the unaffected breasts of 362 women with newly diagnosed breast cancer (cases) and 656 women of the same age who did not have breast cancer (controls). Measures of breast tissue volume and the projected area of the compressed breast during mammography were used to calculate the deformation of the breast during compression and, with the recorded compression force, to estimate the stiffness of breast tissue. Stiffness was compared in cases and controls, and associations with breast cancer risk examined after adjustment for other risk factors.After adjustment for percent mammographic density by area measurements, and other risk factors, our estimate of breast tissue stiffness was significantly associated with breast cancer  and improved breast cancer risk prediction in models with percent mammographic density, by both area and volume measurements.An estimate of breast tissue stiffness was associated with breast cancer risk and improved risk prediction based on mammographic measures and other risk factors. Stiffness may provide an additional mechanism by which breast tissue composition is associated with risk of breast cancer and merits examination using more direct methods of measurement. Physical forces generated by interactions between cells, and between cells and the extracellular matrix, influence a variety of cell functions including cell growth, survival, motility and differentiation Radiologically dense breast tissue on mammography, referred to as mammographic density, reflects variations in breast tissue composition. Epithelial and stromal tissues attenuate x-rays more than fat and appear dense or white, while fat is more radiolucent and appears dark. Compared to women with little or no density, those with extensive density have a 4–6 fold greater risk of developing breast cancer We have used measurements of the breast made in a case-control study of mammographic density and risk of breast cancer to estimate the extent to which the breast is deformed during compression and derive an estimate of the stiffness of breast tissue. We have compared the estimate of stiffness in cases and controls after adjustment for other breast cancer risk factors.Details of the recruitment of subjects and of their characteristics, and of the methods used to measure breast tissue volume and area, have been given elsewhere Written informed consent was obtained from all subjects who authorized the release of their mammograms for the purpose of density measurement and agreed to take part in a telephone interview that asked about factors related to breast cancer risk. Ethics approval for the study was obtained from the University Health Network, Mount Sinai Hospital, Sunnybrook and Women’s College Hospital and from Cancer Care Ontario (for the Ontario Breast Screening Programme).We have recruited cases and controls that had been examined on mammography units in the clinics of Mount Sinai Hospital, Women’s College Hospital, University Health Network, Sunnybrook Health Sciences Centre, and the North York and Scarborough sites of the Ontario Breast Screening Programme (OBSP), all in Toronto, Canada. The selection of cases and controls was from all subjects having mammography in these sites during the period of 13 March, 2000 and 7 July, 2003. All mammography units in these clinics were calibrated using the methods described below. All subjects examined in OBSP sites were seen for screening mammography, while those seen in hospital sites are likely to have included some for screening and some for evaluation of symptomatic breast disease. OBSP screening sites contributed only 8.2% of the cases and control subjects. The number of cases was small compared to hospital sites and only 1 control could be matched per case.Potentially eligible cases were all incident cases diagnosed between 13 March, 2000 and 7 July, 2003 in hospitals where the machines had been calibrated and with at least one screen-film mammogram performed before diagnosis. Cases with bilateral synchronous breast cancer, in which a screen-film mammogram without radiological signs of cancer was not available, were excluded. Subjects who had breast implants, or reduction mammoplasty were also excluded.Controls were selected from the same study population as cases. We attempted to identify 2 controls for each case, one examined on the same mammography machine as the case and the other from a different machine. However, some mammography clinics had only one machine and for these we recruited only controls examined on the same machine as the cases. The two types of controls were combined for this analysis.With the agreement of their physician, potentially eligible case and control subjects were contacted by mail, the study explained and they were asked for consent to the use of their mammogram and to participate in a telephone interview to provide information about risk factors for breast cancer.After consent had been obtained, screen-film mammograms for the case and control subjects selected were obtained from the participating mammography units. To “blind” the process of measurement to case or control status, we selected the image of the breast contra-lateral to the cancer, and the corresponding mammograms in the matched controls. Two methods of measurement that have been described previously Cumulus 4 software. Measurements of the areas of dense tissue and total area were generated and percent density calculated.Computer-assisted measurement of mammographic density was carried out by one reader (NFB) using Each mammography machine from which we recruited was calibrated to determine the relationship between the image signal  in each pixel, the exposure factors , tube target and beam filter) and the amount of radiation transmitted by the breast. The latter can then be related to the combination of breast thickness and composition by imaging a “phantom” composed of steps of tissue-equivalent plastics of different thicknesses and representing a range of combinations of fat and fibroglandular tissue Compressed breast thickness is the distance between the compression paddles of a mammography machine and the breast supporting tabletop when the mammogram is obtained. Breast thickness is not constant across the breast area; and we generated a thickness map for each x-ray image to calculate the total volume and dense volume of the breast. Equations to predict a thickness map for each image were developed from the readout thickness reported by each mammography machine, coordinates in the plane parallel to the breast support table, and the compression force reported by a mammography machine.We further assume that the contralateral breast in cases is representative of the subject. It is known that breast tissue composition, assessed by either mammography We defined the difference between the radius of the mammographic area semicircle, and the radius of the volumetric hemisphere as “deformation”. With the compression force recorded with each mammogram the measure of deformation was used to calculate “stiffness” by the formula: Force/Deformation (N/cm), where N denotes deca-Newtons and cm centimetres.All subjects with available volumetric breast measurements had deformation computed. We excluded from the analysis two subjects with deformation smaller than zero. For selected characteristics of the case and control subjects, we calculated mean and standard deviation (SD) for continuous variables, and proportion for categorical variables. Differences between cases and controls were ascertained by t-test for symmetrically distributed continuous variables, Wilcoxon rank-sum test for the non-symmetrical ones, and chi-square test for categorical variables.We used linear regression models to examine the association between stiffness and breast cancer status , and parity (parous/nonparous), with and without adjustment for breast density measurements. We applied natural log transformation to stiffness, square root transformation to all measurements of breast area, and cube root transformation to all measurements of breast volume to make the distributions more symmetrical with stable variance.We used logistic regression modeling to examine the association of the area and volume measurements with risk of breast cancer before and after adjustment for deformation or stiffness, in addition to adjustment for the risk factors for breast cancer mentioned above. All p-values were calculated from two-tailed tests of statistical significance.We imputed the mean value of weight for three subjects, menopausal status (post) for two subjects, and the mean value of age at birth of first child for 280 subjects. For fourteen subjects with force recorded as zero, we used the mid-point value (20) between the machine recording threshold (30) and the minimum force required to produce pressure (10). The results obtained using this imputation were very similar to those from the analysis excluding these 14 subjects. All statistical analyses were carried out using Statistical Analysis Systems (SAS) 9.2 software.The ORs and 95% CIs for quartiles of stiffness with reference to quartile 1, were 0.94  for quartile 2, 1.17  for quartile 3, and 1.35  for quartile 4.The interquartile odds ratio (IQOR) for stiffness, adjusted for other non-mammographic risk factors was 1.24 : 1.05, 1.46), p = 0.01. After adjustment for percent mammographic dense area in addition to other risk factors, the IQOR for stiffness was 1.29 , p = 0.003. After adjustment for percent dense volume, the IQOR for stiffness was 1.27 , p = 0.004.Before inclusion of the stiffness measure, percent mammographic density, by the area (p = 0.0005) and volume (p = 0.001) measures, and the area (p = 0.01) and volume (p = 0.001) of dense tissue were all significantly and positively associated with risk of breast cancer as separate predictors. After the inclusion in the model of the stiffness measure, the association of percent mammographic density by the area measure was slightly reduced, as shown by the regression coefficient and the interquartile odds ratio, although the area under receiver operating characteristic curve (AUC) increased by 1.4%. For all other mammographic measures the regression coefficients and interquartile odds ratios increased after the inclusion of stiffness, and the associated AUCs increased by between 2.4 and 3.5%.These results show that estimates of the stiffness of breast tissue during compression are associated with risk of breast cancer after adjustment for other known risk factors including percent mammographic density, which is strongly associated with risk of breast cancer Strengths of the present study include the relatively large numbers of incident cases and matched controls, with breast images acquired prospectively from calibrated machines. However, stiffness was not measured directly but was based on idealized assumptions about the shape of the breast volume and projected area that are potentially subject to error. For example, calculation of the breast volume measures requires accurate information about the thickness of the compressed breast at each pixel in the image. Yaffe et al have shown elsewhere that the measured percent density by volume is very sensitive to small errors in the measurement of breast thickness Percent mammographic density, as assessed here in the area measurement, reflects variations in breast tissue composition There are abundant data to suggest that an association between breast tissue stiffness and breast cancer risk is biologically plausible. Epithelial and stromal cells, collagen, and fat, the tissue components that contribute to variations in mammographic density, are related to each other in several ways. Epithelial and stromal cells communicate with each other by means of paracrine growth factors Collagen and the stromal matrix are products of stromal cells that may, through their mechanical and other properties, facilitate tumor invasion To date, there has been limited application of these basic science findings to understanding the association between mammographic density and risk of breast cancer. In addition to having greater amounts of collagen, epithelial and stromal cells, and larger areas that are immunohistochemically positive for Insulin-like growth factor-I (IGF-1), radiologically dense breast tissue also has greater amounts of the stromal matrix regulatory protein tissue inhibitor metalloproteinase-3 Our results suggest that knowledge of both the quantity and stiffness of breast tissue may improve prediction of breast cancer risk in individuals, and facilitate research into the tissue factors that influence breast cancer risk. Stiffness may provide an additional mechanism by which breast tissue composition influences risk of breast cancer and merits examination using more direct methods of measurement such as elastography using ultrasound or magnetic resonance An estimate of breast tissue stiffness was associated with breast cancer risk, and improved risk prediction based on mammographic measures and other risk factors. Stiffness may provide an additional mechanism by which breast tissue composition is associated with risk of breast cancer and merits examination using more direct methods of measurement.


The evidence on the relationship between breast cancer and different types of antihypertensive drugs taken for at least 5 years is limited and inconsistent. Furthermore, the debate has recently been fueled again with new data reporting an increased risk of breast cancer among women with a long history of use of antihypertensive drugs compared with nonusers.In this case-control study, we report the antihypertensive drugs–breast cancer relationship in 1,736 breast cancer cases and 1,895 healthy controls; results are reported stratifying by the women’s characteristics  tumor characteristics and length of use of antihypertensive drugs.The relationship among breast cancer and use of calcium channel blockers (CCB) for 5 or more years had odds ratio (OR) = 1.77 . Stratifying by BMI, the OR increased significantly in the group with BMI ≥ 25 . CCBs were even more strongly associated with more aggressive tumors, . On the other hand, premenopausal women were the only group in which angiotensin II receptor blockers may be associated with breast cancer  but this could not be identified with any type or stage. Use of angiotensin-converting-enzyme inhibitors, beta blockers and diuretics were not associated with risk.In this large population-based study we found that long term use of calcium channel blockers is associated with some subtypes of breast cancer (and with breast cancer in overweight women). Hypertension is a highly prevalent disease affecting around 30–45% of the general population  and antiBreast cancer is the most common cancer among women in both developed and developing countries. One in ten of all new cancers diagnosed worldwide each year is a cancer of the female breast. It is also the leading cause of cancer death among women worldwide. More than 1.67 million cases are diagnosed and more than 522,000 patients die from it worldwide every year .The carcinogenic potential of antihypertensive drugs has been debated for nearly 50 years . Even siThe discrepant results and the high prevalence of antihypertensive medication in middle-aged population justify carrying out new research in order to provide additional evidence about the relationship with cancer development. The aim of the present study is to assess the association between breast cancer and previous use of antihypertensive medication, taking into account the class of antihypertensive drug and the duration of use, in a large population-based case-control study conducted in Spain, the MCC-Spain study.This study was approved by the corresponding ethics committee of each area  and informed written consent was obtained from parents. The MCC-Spain study also followed the Declaration of Helsinki and the Spanish Personal Data Protection Act of 1999.The Multi Case-Control (MCC-Spain) study has been described in detail . BrieflyAll cases of breast cancer included were incident and pathology confirmed, with no previous diagnosis of breast cancer; they were aged between 20 and 85 years old, and resident within the influence area of the hospital for at least 6 months prior to recruitment in 10 Spanish provinces . Controls with no prior history of breast cancer were selected from the general population according to age and regional distribution of the cases included in the study. In this paper, 1736 cases of breast cancer in women and their 1895 frequency-matched controls were considered. Response rates were 71% for breast cancer and 72% for controls, with no differences in the main socio-demographic variables among those who participated and those who refused to participate.Participants were interviewed face-to-face by trained interviewers, using a comprehensive epidemiological questionnaire that collected socio-demographic information, personal and family history of cancer, anthropometric data, smoking habits, alcohol intake, occupation, physical activity, water consumption, reproductive and medical history and medication use, family history, sun exposure, sleep habits, use of hygiene products and cosmetics, signs and symptoms. Comprehensive dietary habits were obtained with the use of a validated food-frequency questionnaire.Participant’s weight was self-reported, as estimated one year before diagnosis for cases and one year before the interview for controls. Accordingly, body mass index (BMI) was calculated considering self-reported weight, referred to that date, and height. Total fat and vegetable intakes were estimated from the questionnaire using local food composition tables. Similar estimates provided total energy consumption. Physical activity was recorded for the longest occupation and also considering recreational physical exercise.Detailed information was obtained on past medical conditions and the corresponding medications used. The age at onset, the dates of diagnosis or occurrence and the type of treatment received for each condition was also registered.Drug use was recorded by indication. For each drug, the brand name, dose and duration of exposure were recorded to identify patients with regular drug consumption  and the duration of consumption.The drugs were coded following the Anatomical Therapeutic Chemical Classification System (ATC codes) to define groups with similar mechanisms of action [WHOCC Homepage. WHO Collaborating Centre for Drug Statistics Methodology].All drugs indicated for the treatment of hypertensive diseases have been considered. The ATC codes included in the present analysis are code C02 (Antihypertensive), C03 (Diuretics), C04 , C07 (Beta blocking agents), C08  and C09 (Agents acting on the renin-angiotensin system). Results will be presented for each separate group and for specific antihypertensive drugs that had a prevalence of use over 1% in controls.2). In order to study the relationship between antihypertensive drug use and different breast cancer subtypes, we applied multinomial logistic regression models; multinomial logistic regression is useful when the outcome is categorical rather than dichotomic; for instance, according to cancer stage, participants were classified in one out of three categories: control / breast cancer stage I-II / breast cancer stage III-IV. Multinomial logistic regression allows to estimate odds ratios for every category (i.e.: breast cancer stage I-II / breast cancer stage III-IV) comparing with the reference category (i.e.: control) [Unconditional binomial logistic regression was used to assess the association between antihypertensive drug use and breast cancer overall and stratifying for menopausal status and BMI (<25/≥25 kg/mcontrol) . Statist2), clinical stage (I-II / III-IV), ductal , invasive and immunohistochemistry . Results, which are reported only in strata with at least 5 cases or controls using antihypertensive drugs are shown as odds ratios (OR) with 95% confidence intervals (CI). All reported p-values are two-tailed. Statistical analysis was carried out using the package Stata 14/SE .Stratified models were developed according to menopausal status, BMI  and twice the risk in women with BMI ≥ 25 ; there is moderate confirmation of both associations in women taking CCBs for 5 years or more. Angiotensin II receptor blockers (ARB) were the only group associated with an increased risk of breast cancer in premenopausal women , but not in postmenopausal women . The use of diuretics, beta blockers and angiotensin converting enzyme inhibitors (ACEI) was not associated with breast cancer risk in any women or in different strata.Results of the association between the use of antihypertensive drugs and incident breast cancer according to tumor characteristics are shown in Looking at specific classes of antihypertensive drugs, CCBs were even more strongly associated with more aggressive tumors, multiplying by 2.7 the risk of tumors in stage III-IV , non-ductal cancers , and Erbb2+ cancer . CCBs were also associated with invasive tumors . Similar results were found in women taking CCBs for at least five years.The use of diuretics, beta blockers, ACEIs or ARBs was not associated with increased risk for specific tumor characteristics.2, cancer in stages III-IV, non-ductal cancer and Erbb2+ cancer. Previous results on CCB-breast cancer relationship have been contradictory; Fitzpatrick et al, in a study limited to women aged 65 years or more, found an elevated risk of breast cancer associated with CCB usage [In this population-based case-control study the use of antihypertensive medications as a global group was associated with higher risk of invasive breast cancer, and this risk appears to be confined to triple negative breast cancer and concentrated in premenopausal women. Our results were similar to those found in a large prospective study, the California Teachers Study (CTS) with 133,479 women . In contCB usage ; Li et aCB usage ; while aCB usage . NegativCB usage –15,23,24CCBs may increase the risk of cancer by changing intracellular calcium levels, which could affect the process of programmed cell death, not enabling the destruction of damaged cells to prevent the development of diseases such as cancer, resulting in indiscriminate replication of an impaired cell . CalciumOn the other hand, nifedipine–a CCB- has been found to increase proliferation and migration of breast cancer cells, which could be responsible for the association between CCBs and late stage cancers. This nifedipine effect–which is not shared by other CCBs such as verapamil- seems to be produced via the Erk pathway activation and is independent of the calcium channel-blocking effect .ARBs were the only antihypertensive group associated with an increased risk of breast cancer in premenopausal women in our results; using ARBs before menopause was, however, scarce; therefore, when stratifying by length of consumption the results were non-significant, although the odds ratios scaled from 3.48 for less than 5 year users to 6.64 for 5 or more year users. We have not found other papers analyzing the relationship between ARBs and breast cancer in premenopausal women; this together with the small number of premenopausal women taking ARBs make the interpretation of this result highly speculative. Bhashkaran et al (2012) found an increased risk of breast cancer associated with short-term exposure to ARBs; they suggested that such an association could not be causal but the result of confounding by indication: according to them, some early symptoms of breast cancer could induce ARBs to be indicated instead of ACEIs in hypertensive patients, leading, therefore, to a spurious ARB–breast cancer association . We haveDifferences in study design and population characteristics may explain the conflicting results reported on the antihypertensive drugs–breast cancer association. Inferring is difficult because of the small sample size in some studies, differences in the populations evaluated and designs . In many cases, randomized trials cannot identify long-term adverse effects of medication because they are usually conducted for relatively shorter periods (i.e.: 5 years or less) . SubsequThe present study has some limitations. First, recall bias should be considered as in any case-control study. Drug consumption was obtained using a standardized questionnaire in face-to-face interviews where examiners were blinded to the case-control status. If a non-differential recall bias was produced, then the odds ratios should be downward biased and the positive associations we have found for CCB, ARBs or ACEIs would actually be even stronger than reported here. If a differential recall bias were responsible for these associations, breast cancer cases would also have over-declared (or controls under-declared) their consumption of other hypertensive drugs; however, no association has been found between breast cancer and diuretics or beta-blockers, which makes a differential recall bias less probable. Second, some strata in our study have small numbers of exposed cases or controls; this could produce unstable estimates. This limitation should be especially considered regarding ARBs, as their relationship with breast cancer is confined to premenopausal women, a relatively small subgroup whose exposure to hypertensive drugs could not be too long. Third, antihypertensive consumption was reported by indication; therefore, most people taking antihypertensive drugs have hypertension. Thus, we cannot distinguish using antihypertensive drugs from having hypertension. Lastly, many comparisons have been made, raising the probability of finding some spurious results However, the consistency of some results in different subgroups, such as those for CCBs, supports the existence of a real excess risk associated with their use.2. As people with hypertension are expected to take antihypertensive drugs for many years, their relative effect on breast cancer should be taken into account when choosing the antihypertensive to be prescribed.In summary, we report that consumption of antihypertensive medications -as a global group- was associated with an increased odds of breast cancer in premenopausal women, and the use of CCBs in particular was associated with an increased odds of breast cancer in postmenopausal women and those with BMI higher than 25 Kg/mS1 TableCategory reference no antihypertensive treatment.(DOCX)Click here for additional data file.S2 TableCategory reference no antihypertensive treatment.(DOCX)Click here for additional data file.