Elevated levels of circulating estrogens are linked to breast cancer risk among postmenopausal women but little is known about the importance of estrogen metabolism. A recently developed liquid chromatography tandem mass spectrometry-based method (LC-MS/MS) measuring a panel of 15 estrogen metabolites (EM) has been evaluated in one study, linking high levels of 2-pathway metabolites relative to the parent estrogens to reduced breast cancer risk. We analyzed this panel of EM in a nested case-control study of postmenopausal breast cancer.Between 1977 and 1987, 6,915 women provided blood samples to the Columbia Missouri Serum Bank and were followed for incident breast cancer through December 2002. We studied 215 postmenopausal breast cancer cases and 215 matched controls who were postmenopausal and not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway  and by ratios of the groupings. Logistic regression models controlling for matching and breast cancer risk factors were used to calculate quartile-specific odds ratios (ORs) and 95% CIs.P trend = 0.07). The OR for total EM, the parent estrogens estrone and estradiol, and 2-pathway catechol EM (2-hydroxyestrone and 2-hydroxyestradiol) were elevated but the trends were not statistically significant. Among 2-pathway metabolites, risks for the highest levels of 2-hydroxyestrone-3-methyl ether and 2-methoxyestradiol were reduced; ORs for women in the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.53 (95% CI = 0.30 to 0.96), respectively. Overall, women with higher levels of 2-pathway EM had a reduced risk of breast cancer, which remained after accounting for levels of parent EM, 4-pathway EM and 16-pathway EM .Significant elevated risks were not observed for individual EM, except for quartiles of 16-epiestriol (Women with more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer. Further studies are needed to clarify the risks for specific EM and complex patterns of estrogen metabolism. This will require aggregation of EM results from several studies. Elevated levels of circulating estrogens are linked to postmenopausal breast cancer risk , but litWhether a panel of EM provides more discrimination of risk than conventional immunoassays of only the parent estrogens is not known. To date, the only study with prospectively collected bloods to have evaluated postmenopausal breast cancer risks associated with this panel of EM  found thTo corroborate these findings, we analyzed this panel of EM in sera drawn prospectively from participants in a nested case-control study of postmenopausal breast cancer identified from the Columbia Missouri Serum Bank Cohort. For a subset of the study participants, parent estrogens had been measured previously by an indirect radioimmunassay (RIA) , which aThe Columbia Missouri Serum Bank was established as part of the National Cancer Institute's Biological Markers Project to identify serum markers of breast cancer, as has been described previously . In brieWomen were followed in two phases for incident breast cancer. The first follow-up, ending December 1989, identified 107 postmenopausal breast cancers, 70 of whom were also postmenopausal at blood collection. Breast cancer cases were identified by self report from a mailed questionnaire and medical records from the University of Missouri Hospital and Ellis Fischel Cancer Center, Missouri Cancer Registry and Missouri Department of Vital Statistics. A number of biomarker studies were conducted on the breast cancer cases and matched controls identified in this first follow-up including a study of serum estrogens and androgens [13C-labeled estrone and estradiol .Stable isotope dilution LC-MS/MS was used to quantitate15 EM including: estrone, estradiol, 2-pathway metabolites ; 4-pathway metabolites ; and 16-pathway metabolites . Details of the method have been published previously [In serum, this method detects 15 EM which circulate primarily as sulfated and/or glucuronidated conjugates. Beginning with the parent estrogens  and 95% CIs for individual EM, pathway EM and the ratios of the pathways. ORs controlling for matching factors and known breast cancer risk factors were calculated, with confounding addressed by including covariates that changed the effect estimates by more than 10% in the final model. The final model included: family history of breast cancer; body mass index (BMI); age at menarche; parity and age at first birth; age at blood collection; type of menopause; years between blood collection and menopause; season; and time of day of blood draw. For each EM, further adjustment for unconjugated estrone was made to assess whether levels of a given EM contributed to risk independent of parent levels; however, this did not alter OR estimates by >10% and results are not presented. The magnitude and patterns of risks from additional analyses, including using conditional logistic regression models, and excluding the 17 women whose breast cancer was diagnosed less than two years after blood donation, were comparable to the reported results and are not provided. No adjustment was made for multiple comparisons.We assessed EM consistency within a woman over two to three years using samples from controls with repeat blood collections, using nested analysis of variance (ANOVA) models to calculate the intraclass correlation coefficients (ICC). Pearson's correlation coefficients were used to assess comparability of RIA and LC-MS/MS measurements of the parent estrogens. All measurements were logarithmically transformed. For all analyses, all tests of significance were two-tailed and probability values of <0.05 were considered statistically significant. Analyses were performed using SYSTAT software  and foreP <0.05) than parous controls. Most women in this study were naturally menopausal (71%), with the remaining having had an ovariectomy (13%) or hysterectomy (16%). The mean age at breast cancer diagnosis was 61.8 (range 50.8 to 76.8), with mean follow-up of 11.7 years (range 0.3 to 24.8 years). The age at blood collection for both groups was similar .Table Median and interdecile ranges (10% and 90% percentiles) of each EM are presented for cases and controls Table . Among aAmong controls, levels of the parent estrogens, estrone and estradiol, were highly correlated with each other ; with estrone catechols  and with some 16-pathway EM . Correlations between the parent estrogens and the methoxycatechols were lower (rho ranged from 0.22 to 0.43), while correlations between the methoxycatechols themselves, or between the methoxycatechols and either the 16-pathway EM or the 2- and 4-catechols were low to moderate (rho ranged from 0.09 to 0.43).For the parent estrogens, risks for total, conjugated and unconjugated estrone and conjugated estradiol were elevated in all quartiles relative to the lowest quartile, but no trends were observed Figure , and onlP trend for both <0.05). ORs for the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.46 (95% CI = 0.25 to 0.84) for 2-hydroxyestrone, 3-methyl ether and total 2-methoxyestradiol, respectively.Among the 2-pathway metabolites Figure , risks fP trend = 0.099).Of the 4-pathway metabolites  of increasing risk with increasing concentration were observed for total estriol and 16-epiestriol.In the 16-pathway Figure , borderlP trend = 0.099), but the individual risks were not significant. No associations were observed for the 4-pathway or 16-pathway EM.For this component of the analysis, EM in a given pathway was summed. For total EM  and the parent estrogens (estrone plus estradiol), ORs were elevated but not significant at all levels and no trends were observed Figure . DecliniP trend = 0.11, Figure Ratios of pathways were studied to explore the relative contribution of one pathway to breast cancer risk while accounting for either the precursor estrogens (parent estrogens) or another pathway. Relative to levels of the parent EM, women with higher levels of 2-pathway EM were at lower risk (OR for quartile 4 versus quartile 1 = 0.72 (95% CI = 0.41 to 1.27)), but the trend was not significant  and methoxycatechols  were for the most part not statistically significant, and no trends were observed (data not shown). No patterns were observed for ratios of the catechol EM to methoxycatechol EM.Overall, among the 24 non-case women with two or more consecutive annual blood draws, the mean percent changes from one year to the next for estrone and estradiol were modest . However, temporal differences were observed for the other EM. The ICCs for estrone and estradiol were relatively high (72% and 65%, respectively; Additional file Figure In this nested case-control study of postmenopausal breast cancer and patterns of estrogen metabolism, increased risks for individual EM were not observed except for 16-epiestriol. Risks for estrone, estradiol and the 2-pathway catechols, 2-hydroxyestrone and 2-hydroxyestradiol, were non-significantly elevated at all levels of exposure, while risk reductions were suggested for higher levels of some 2-pathway methoxyestrogens, including 2-hydroxestrone 3-methyl ether, and conjugated and total 2-methoxyestradiol. Analysis of the pathways provided information beyond that contributed by parent estrogens alone since women with higher levels of 2-pathway EM overall tended to have a reduction in breast cancer risk, which persisted even after accounting for the contribution of their precursor hormones, estrone and estradiol. Similarly, relative to levels of both 4-pathway and 16-pathway EM, women with higher levels of 2-pathway metabolites were at lower, albeit not statistically significant, risk than those with low 2-pathway EM. Taken together, these findings suggest that women with more extensive estrogen metabolism along the 2-hydroxylation pathway may have a reduced risk of postmenopausal breast cancer.To date, the only study of postmenopausal breast cancer to report findings from this panel of estrogens observed a similar protective effect among women with high 2-pathway metabolism . In thatOur study had a number of potential limitations. Unlike most studies of postmenopausal breast cancer, our cases were not more likely than controls to be overweight or obese, potentially limiting the generalizability of our findings. In addition, hormone receptor status could only be obtained on a fraction of the cases in this study. Serum samples were stored in glass vials at -70°C for more than 30 years and the consequence of such long-term storage on sex hormones is not known. Sample evaporation and degradation do not appear to have influenced measured EM levels in this study, since as discussed, the ranges of individual EM values were quite broad, and for those estrogens commonly measured by commercial laboratories, absolute levels were consistent with normative values ,14. IronWhile the primary intent of this study was to evaluate whether this panel of EM is informative with regard to breast cancer risk, it is the first study to explore features of this panel that are pertinent to epidemiologic research. For instance, in order to reflect adequately a woman's long-term adult hormone profile, EM measurements must be relatively stable from one year to the next. The year-to-year reproducibility of parent estrogens in postmenopausal women using RIAs has been conflicting -19, withElevated levels of circulating estrogens have been convincingly linked to breast cancer risk among postmenopausal women ,18, yet Further investigations are needed to corroborate whether women with more extensive estrogen 2-pathway estrogen metabolism are at reduced risk of postmenopausal breast cancer and to identify whether breast cancer risk is associated with specific EM or other complex patterns of estrogen metabolism. This will require the aggregation of EM results from several studies. Additionally, efforts to replicate the temporal variability of the metabolites are needed to evaluate whether measures from a single blood collection are sufficient for meaningful risk assessment.ANOVA, analysis of variance; BMI, body mass index; EM, estrogen metabolites; ICC, intraclass correlation coefficient; IRB, Institutional Review Board; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NCI, National Cancer Institute; OR, odds ratio; QC, quality control; RIA: radioimmunoassay.The authors declare that they have no competing interests.All the authors made substantial contributions to the study design, conduct of the hormone assays or the analysis and interpretation of data. RTF and GLG conceived and designed the study, coordinated the research, provided guidance throughout the study process, performed the analysis, participated in interpretation of the data and drafted the manuscript. XX and TDV carried out hormone assays. JFD participated in the study design and sample acquisition, participated in the interpretation of the data and helped draft the manuscript. LAB and BJF participated in the analysis and interpretation of the data and helped draft the manuscript. All authors were involved in revising the manuscript, and have read and approved the final manuscript.Intraclass Correlation Coefficients of EM. Description: Temporal variability in EM measurements from 24 women who donated bloods annually over a three year period.Click here for file


The aim of the current study was to investigate breast cancer screening performance among Iranian Turkmen women along with their knowledge and beliefs. A cross-sectional study was carried out in June to December 2011. Through clustered sampling method, 1080 Iranian Turkmen women completed the questionnaire including breast cancer screening adherence, knowledge, fatalism beliefs, and perceived threat using Champions Health Belief Model Scale (CHBMS).The mean age of the participants was 43.04 (SD = 11.80) years. Compliance rate in a regular basis based on national guidelines was 13.1%, 2.5%, and 0.9% for SBE, CBE, and mammography, respectively. A mere 4% have been provided adequately with information about breast cancer. Having knowledge was the best predictor of breast cancer screening adherence along with high educated husbands for SBE performing. Susceptibility and fatalism were low and were influenced by participants' educational level and age. In conclusion, Iranian Turkmen women had insufficient knowledge, low perceived susceptibility, high fatalistic belief, and very poor adherence to breast cancer screening. There is a need for providing breast cancer education programs among the Iranian Turkmen women to increase their adherence rate. Breast cancer is the most common type of cancer among women and its incidence rates are increasing throughout the world , 2. AccoEssential to the success of decreasing mortality, early detection and breast cancer diagnosis is logically a significant process and it could lead consequently to an increase in the survival rate up to 95% . There hThe Health Belief Model (HBM) has provided a useful conceptual framework to prevent health behaviors. According to HBM, threat perception and behavioral evolution are the two main components which are derived from expectancy-value theory. Threat perception included two key beliefs: perceived susceptibility and anticipated severity of contracting health conditions . As idenThe model states internal and external cues such as body states and environmental factors which may also promote or inhibit health enhancing behavior (cues to action), the confidence that one can successfully practice the behavior required to produce the outcome (self-efficacy), and the desire to maintain good health status  were added to the model . HBM hasResearchers have found that the fatalistic attitudes are the major barrier to recommended breast cancer screening uptake. Fatalism is identified as a doctrine of fate, a philosophical doctrine held by individuals who believe that all events are fated to happen and that human beings have no control over their futures and are unable to change their outcomes . FatalisTurkmen people are a racial group that reside in northern and north east of the Islamic Republic of Iran and composed about 0.8% of the Iranian population. This population group has different culture, religion, and language from the most part of the country. There is not any survey that reported this racial minority with the topic of breast cancer screening behaviors. Studies indicated that along with demographic characteristics, the health belief variables such as perceived susceptibility to breast cancer, seriousness of breast cancer can influence the performance of BSE, CBE, and mammography –25. HencA cross-sectional study was carried out in June to December 2011, simultaneously in Gonbade Qabous and Turkmen districts in Golestan province where their main residents are Turkmen people. Prior to data collection, the study was approved by the Golestan University of Medical Sciences Authorities. Women gave verbal consent to participate after being given assurances of confidentiality and anonymity, and no incentives were offered for completing the survey. The inclusion criteria for participants were 30 years of age or older, women who are Turkmen ethnic group, and capable of communicating in Turkmen language. α = 0.742). The perceived susceptibility and perceived severity related to breast cancer instruments were translated using the Banville method, to develop a cultural adaptation . In brieTo recruit eligible women to participate in this study, we had recourse to a number of 10 health centers in the cities and 60 rural health houses, providing and including all family records basis on primary health cares. Participants were a clustered randomized sample of 1080 Turkmen women. Their ages ranged between 30 and 82 years, with a mean age of 43.04 ± 11.80 years. The majority of them (85.3%) were married, 5.8% unmarried, and the remainder divorced or separated. Demographic profiles of the participants are reported in The questionnaire consisted of four parts: 1) demographic variables included questions about age, level of education, current marital status, children's number, job, and breast cancer risk factors such as history of oral contraceptive use, family history of breast cancer, and whether breastfed their child/children; (2) knowledge-related breast cancer screening: hearing of BSE, CBE, and mammography, periodic time of BSE, CBE, and mammography, and receiving of first mammogram time; (3) practice for breast cancer screening behaviors: recommended frequency of BSE, CBE, mammography, and regular/irregular basis; (4) susceptibility (3 items) and seriousness (7 items) to breast cancer were assessed using the Champions Health Belief Model Scales (CHBMS) , 15. Par demograpU tests were conducted for any differences between perceived threat and fatalism with demographic variables and BSE, CBE, and mammography performance. Logistic regression analysis was performed to identify the extent to which individual variables significantly predicted breast cancer screening behaviors. In all tests, values of P ≤ 0.05 were considered significant. All data were entered in the SPSS (version 15) Windows software. Descriptive statistics were used in demographic characteristics and other variables. Chi-Square test was applied to on the difference between breast cancer screening performance and selected demographic variables. One-way analysis of variances and Mann-Whitney According to When asked about their common barrier to participating in breast cancer educational session, they listed lack of time (47.8%), followed by transportation (20.7%), did not want to think about breast cancer (10.4%), and did not believe that breast cancer is important (2.5%), and 18.6% did not report any reasons for barriers.When women were asked if they had heard something about BSE, CBE, and mammography, 42.2% reported that they heard about BSE, 32.7% about CBE, and 24.4% about mammography. Results regarding breast cancer screening behaviors based on national guidelines revealed that the majority of women (94.4%) did not know anything about monthly BSE. Only 1.9% correctly reported that CBE every three years should be performed between the ages of 20 and 39 years. The annual yearly mammography screening test started at the age of 40 was reported by 4.5% of the participants.Seventy-six point eight percent of the participants had not ever performed BSE, followed by 93% for CBE, and 97.8% for mammography. Last month performance of BSE among those who have 30 years and older was 13.1%. Among those who have 40 years and older, mammogram in the past year was .9% and CBE in the past year was 2.5%. When women were asked if they were acquainted with any person who had breast cancer, they reported only one person having breast cancer themselves, 0.8%, 0.7%, 0.1%, 10.7%, and 4.2% reported that their mother, sister, daughter, close relatives (except family members), and friends have breast cancer, respectively. 42.5% delineated they have also lie with breast cancer.χ2 = 16.208, P = 0.000) and current marital status  with performance of BSE. Fisher's Exact test results showed current marital status and exposure to breast cancer were not influencing variables with performing CBE and mammogram. The breast cancer screening behaviors were influenced by age group except for mammography  and wereThe results described that the mean and SD of susceptibility of the women were 2.82 ± 1.22, and this rate for their seriousness was 3.72 ± 0.7. The mean and SD of fatalism beliefs toward breast cancer were 3.86 ± 0.78. The ANOVA test showed there was a significant difference between susceptibility and fatalism with educational level  but not U test showed significant differences between SBE performance and susceptibility , but no significant differences emerged between the SBE practice with subscales of severity  and fatalism . This result repeated regarding CBE performance with the subscales of susceptibility , severity , and fatalism  and mammogram practice with the subscales of susceptibility , severity , and fatalism .The Mann-Whitney U = 116126, P = 0.000) and fatalistic belief , but seriousness was not impressed by exposure to breast cancer .Significant association was founded between exposure to the concept of perceived susceptibility  at the age of 40 and older did not report CBE in the past year; this rate was 99.1% for mammogram. Our findings have been identical nearly to the results of other previous studies conducted in Iran, where breast cancer screening performance in accordance with national guidelines ranged from 4.5% to 42.7% for SBE, 3% to 29.5% for CBE, and 3.3% to 30.5% for mammography in different samples and varied ethnic groups –38. AvciThe results demonstrated that SBE performance was impressed by age, marital status, and exposure to breast cancer. Inconsistent with our finding Avci reported marital status and familial breast cancer history were significantly associated with BSE performance, but not associated by educational levels and age . JarvandIn line with mammography among women of 40 years and older, the very poor performance was observed. Iranian Ministry of Health recommended mammogram every 1-2 year at age of 40 and thereafter. The finding of the current study related to mammogram practice is lower than other parts of Iran. In contrast, mammography practice is higher in western countries, where Wu et al. reported that compliance with recommended screening practice is 64% among Filipino, Chinese, and Asian-Indian women of ethnic group in the United States . SecginlMammography adherence was not significantly associated with demographic characteristics like age, education, marital status, and breast cancer family. Contrary to our findings other studies demonstrated that annual mammogram performance varied according to age, educational level, marital status, and having breast cancer exposure , 42. HowOn the basis of the selected HBM subscales, only concept of susceptibility was positively associated with BSE, CBE, and mammography compliance. Women who performed BSE, CBE, and mammography perceived higher risk to breast cancer than those who did not practice BSE, CBE, and mammography. The concept of perceived severity is not significantly associated with screening behavior that is inconsistent with HBM theory. Our findings are in agreement and supported other studies, where they concluded that perceived severity may be that the breast cancer is regarded as a serious condition by most women , 41, 42.In the current study, the education level and age were significantly associated with susceptibility; the younger and high educated women perceived higher risk to breast cancer. Meanwhile, the education level negatively correlated with seriousness and fatalism that poorly educated women are more likely to consider the breast cancer as severe condition and that its susceptibility is the will of God and depend on luck. In consistence with our findings, Katapodi et al. meta-analytic review demonstrated that perceived risk is weakly influenced by age and education. Seven of those studies concluded that younger women were more likely to perceive higher risk for developing breast cancer than older women . The perWe found that fatalism belief was higher than perceived susceptibility and seriousness which could considered as a barrier to breast cancer screening behaviors and early detection. Fatalism belief had a positive significance with age; the older women had higher perceived fatalistic belief. This result supports previous findings suggesting that fatalistic belief is an important belief in Muslim societies , 46, 47. In conclusion, the results of the current study revealed Turkmen women had poor knowledge, low perceived susceptibility, high fatalistic belief, and low adherence to breast cancer screening. For increasing breast cancer screening adherence of Turkmen women, conducting a holistic and comprehensive intervention through viable strategy is imperative.First, the study used a cross-sectional sample. Second, the self-report nature of questionnaire in spite of the anonymous nature of the questionnaires should be mentioned. Third, women's self-report in compliance with CBE and mammography screening was not validated with their medical records. Another limitation was that the concepts of perceived barriers, perceived benefits, self-efficacy, and health motivation were not survived. Thus, further studies are needed to examine these concepts in other geographic locations of Turkmen population women. In spite of this limitation, the current study demonstrated new sights for the understanding of perceived threat, fatalism, and behaviors of breast cancer screening in Iranian Turkmen women.


Breast cancer is a major cause of death for women in Japan. The objectives of this study were to estimate and project the economic burden associated with breast cancer in Japan and identify the key factors that drive the change of the economic burden of breast cancer.We calculated the cost of illness (COI) every 3 years from 1996 to 2020 using governmental statistics. COI was calculated by summing the direct costs, morbidity costs, and mortality costs.From 1996 to 2011 COI was trending upward. COI in 2011 (697 billion yen) was 1.7-times greater than that in 1996 (407 billion yen). The mortality costs accounted for approximately 65–70 % of the total COI and were a major contributing factor to increase in COI. It was predicted that COI would continue to trend upwards until 2020 (699.4–743.8 billion yen depending on the model), but the rate of increase would decline.COI of breast cancer has been steadily increasing since 1996. While the rate of increase is expected to plateau, the average age at death from breast cancer is still less than that from other cancers, and the relative economic burden of breast cancer will continue to increase in the foreseeable future. Breast cancer (ICD 10 code: C50) is the most prevalent cancer among women and the fifth leading cause of death –4, but hTo date, only a few studies have attempted to estimate the economic burden of breast cancer in Japan , 13. MorIn this study, we calculated direct costs as well as indirect costs, which include the opportunity cost because of disease and death. The goal was to adequately capture the social burden of breast cancer by estimating past trends and projecting future trends in the costs of breast cancer. We already tried to calculate COI of several cancers in the past . But thiA previous study calculated the cost of illness (COI) of stomach cancer in Japan . The COIComparing with stomach cancer, the social burden of breast cancer is expected to increase. We compare our COI estimates for breast cancer with those for stomach cancer to show an evidence for prioritizing policies for cancer control.The COI method is well described for measuring the social burden of disease –22. In tThe COI calculation is dependent on three variables: direct costs, morbidity costs, and mortality costs. The direct costs are defined as medical expenses . In this study, we used reimbursement data from the “Survey of National Medical Care Insurance Services” to calculate annual medical expenses.The morbidity costs are associated with inpatient care and outpatient care. The morbidity costs of inpatients were calculated by multiplying total person-days of hospitalization by the 1-day labor-value per person. The morbidity costs of outpatients were calculated by multiplying total person-days of outpatient visits by half the 1-day labor-value per person. Total person-days of outpatient visits and hospitalization according to sex and 5 years age-groups were calculated based on the “Patient Survey”. The labor-value was calculated according to sex and 5 years age-groups using the “Basic Survey on Wage Structure”, “Labor Force Survey”, and “Estimates of monetary valuation of unpaid work”.The mortality costs are measured as the loss of human capital , which was calculated by multiplying the number of deaths by the lifetime labor-value per person. The number of deaths caused by breast cancer according to sex and 5 years age-groups was obtained from “Vital Statistics”. The lifetime labor-value was calculated by summing the income, which the person could have earned in the future if they had not died, from the year of death to life expectancy. The future labor-value was adjusted to a present value using a 3 % discount rate.Predictions of future COI from 2014 to 2020 were based on the “Population Projection for Japan: 2011–2060 (January 2012)” by the National Institute of Population and Social Security Research. The year 2011 was selected as the benchmark for the 1-day labor-value by sex and 5 years age-groups. Two methods were utilized for the future projection of COI. The first is the “fixed” method, which fixes health-related indicators  of each age-group at the 2011 level and changes future population and age structure. The other is the “variable” method, which estimates health-related indicators in addition to population and age structure. Future health-related indicators are estimated using linear regression (linear model), logarithmic regression (logarithmic model), or a combination of regressions of higher coefficient of determination (mixed model). The details of the methodology are outlined in the COI study of stomach cancer . The preThis study used only aggregated data, and did not use human or animals. In Japan, for this kind of study no institutional review is requested .Table Increased mortality costs were the primary factor contributing to increased COI. Because the mortality cost per person  was stable (36.9–41.2 million yen), it is likely that the increase in the number of deaths directly led to increases in the mortality costs. According to the National Cancer Center of Japan, the number of deaths increased 61.2 % from 1996 to 2011 [Table Table The fixed model assumes that health-related indicators were fixed at 2011 levels and only demographic changes had any impact on COI. According to the National Institute of Population and Social Security Research, the Japanese population began to decrease in 2008 and it was estimated to continue to decrease until 2020, while the rate of aging was estimated to rise . Under tUsing a linear model, COI in 2014, 2017, and 2020 was estimated to be 714.1 billion yen, 731.1 billion yen, and 743.6 billion yen, respectively. Using a logarithmic model the predictions were valued at 699.2 billion yen, 698.5 billion yen, and 699.4 billion yen, respectively, and lastly, using a mixed model, COI in 2014, 2017, and 2020 was estimated to be 713.5 billion yen, 730.0 billion yen, and 743.8 billion yen, respectively. Figure Since the trend of each health related indicator was different, the monotype estimation (logarithmic model or linear model) might not predict future COI precisely. The mixed model was a combination of models of higher coefficient of determination and, therefore, considered the most valid model in this study. According to this mixed model, COI showed a 6.7 % (APC: 0.7 %) increase from 2011 to 2020. The direct and morbidity costs were stable, but the mortality costs increased 10.4 % (APC: 1.1 %). As the number of deaths increased by 17.2 % (APC: 1.8 %), the mortality costs per person decreased continuously. Figure Our previous study of COI of stomach cancer estimated COI in 1996, 2002, 2008, 2014, and 2020 using the year 2008 as the benchmark. In that study, COI was estimated to be 1114.2 billion yen  in 2008 and was predicted to decrease to 484.5 billion yen in 2020 using a mixed model . The decrease in COI from 2008 to 2020 was 56.5 % (APC: 8.8 %), and the mortality cost was also predicted to decrease by 55.6 % (APC: 8.6). However, there was a large variation in COI estimation (70.1 % variation to COI in 2008) by each method .Compared with COI of stomach cancer, COI of breast cancer was smaller in the benchmark year but was predicted to increase continuously to be 1.5-times that of stomach cancer by 2020. The mortality costs of breast cancer were estimated to be smaller, but were predicted to be 1.5-times those of stomach cancer. Moreover, there was less variation in the estimation of COI of breast cancer, which was only 6.4 % of the benchmark year’s COI. Each estimation method showed that COI of breast cancer was stable or increased modestly.The results of this study demonstrated that COI of breast cancer increased significantly from 1996 to 2011. The increase in the mortality costs contributed significantly. Furthermore, it was predicted that COI would continue to trend upwards until 2020, but the rate of increase would decline. The annual average rate of increase was 3.8 % from 1996 to 2011 but was predicted to be only 0.7 % from 2011 to 2020 in the mixed model. Because the variation in the model was small, we can conclude that future COI will be stable or increase only slightly.COI and the mortality costs of breast cancer are predicted to exceed those of stomach cancer in the near future. Changes in the number of deaths and the average age at death were the main causes of the increase in the mortality costs. Because the mortality costs were calculated as the number of deaths multiplied by the mortality costs per person  according to sex and 5 age-groups, the increase in the number of deaths had a direct influence on the increase in the mortality costs. Additionally, the lifetime labor-value per person  differs according to the average age at death. Because the human capital value of the 25 to 29-age-group was the highest, an increase in the average age at death resulted in decreased mortality costs. Regarding the number of deaths from benchmark year to 2020, for stomach cancer, there was a 25.1 % decrease (APC: −2.4 %), and conversely, for breast cancer, there was a 17.2 % increase (APC: 1.8). Nonetheless, the number of deaths from stomach cancer was predicted to be 37,581, whereas that from breast cancer was predicted to be only 14,986. However, the mortality costs per person were predicted to be 9.5 million yen for stomach cancer and 35.7 million yen for breast cancer. This difference is most likely because of the difference in the average age at death, which was predicted to be 79.1 years for stomach cancer and 67.5 years (11.6 years younger) for breast cancer in 2020. Moreover, while the annual rate of increase in the average age at death from stomach cancer was stable before and after the benchmark year (0.5 %), the annual rate of increase in the average age at death from breast cancer declined after the benchmark year (from 0.6 to 0.2 %).There are two types of cancers: one affects an “older group”  and the other affects a “younger group”  . The treThe effectiveness of mammography for the detection of breast cancer has already been proven by several studies , 26. WhiThere are some limitations to this study that complicate the interpretation of the data used for approximations. Firstly, the study period was relatively short and there were dramatic changes within the healthcare system during this time. However, the variation among the different methods for determining projections was small and, therefore, the projections are likely to be accurate for the near future. Additionally, we could not predict future increases in the labor-value per person, particularly with regard to the employment and compensation rates for women, which are likely to rise. When these are taken into account, COI of breast cancer is likely to increase even more, which would only further strengthen our conclusions.The findings of the present study suggest that COI of breast cancer has continuously increased up to the present and that trend is likely to continue, although the pace may be expected to decline. The average age at death from breast cancer was less than that from other cancers and the pace of aging was slow. These factors contribute to increasing the social burden of breast cancer, making it clear that policies to mitigate these effects are critical.


Being the most common cancer among women worldwide, it is vital to be well-aware of breast cancer risk factors, symptoms and curability. However, few studies have reported breast cancer literacy in students using a validated instrument.ilaka of Parbat district. Questionnaire with modified Comprehensive Breast Cancer Knowledge Test was self-administered to 516 students. Knowledge score was categorized into two categories: ‘good knowledge’ and ‘poor knowledge’ taking median score as the cut-off. Chi-square test was used to determine difference in knowledge by socio-demographic factors, including gender.A cross-sectional study was conducted among students of grades 11 and 12 from eleven higher secondary schools, one selected randomly from each χ2 = 4.02, p = 0.045). Pain in the breast (23.3 %), change in the shape of the breast (20.0 %) and discharge of pus (14.1 %) were the three most commonly recognized symptoms. Nearly one in two (47.1 %) students indicated that the school curriculum inadequately informed them on breast cancer.Only 4.8 % of the students responded correctly to at least half of the items, and 1.4 % did not respond correctly to any of the items on risk factors and curability. Physical exercise was identified as a protective factor of breast cancer by 62.4 % of the students. Presence of noncancerous breast lumps (56.6 %) and being overweight (36.4 %) were recognized as the risk factors. Knowledge of lumpectomy and radiation therapy for treatment of breast cancer was reported by 42.8 % of students, while only 39.0 % were aware of the availability of treatment therapies other than mastectomy. Males were significantly better informed than females  contains supplementary material, which is available to authorized users. Global statistics suggest that breast cancer is the most frequently diagnosed and a leading cause of death in women . In SoutA bitter truth -- Nepal has no national registries for cancer and the reported prevalence of cancer varies with different studies . A hospiInterventions promoting healthy behaviors and practices in early adolescence have a great role in preventing breast cancer as some of these behaviors are developed at this stage of life. Targeting adolescents is crucial for the success of any prevention efforts, also because they constitute a major proportion of population. Adolescent students are very receptive to information , and theIn light of the evidences mentioned above, this study aims to explore the literacy of breast cancer risk factors, curability and its symptoms among the higher secondary students of Western Nepal. We focus on the difference in knowledge by socio-demographic factors, including gender.2, population of 146,590 in 2011 of which 29,312 are aged 14–22 years and 12,645 attend a school [ilakas (sub-districts) and Parbat district consists of 11 such ilakas. Parbat represents a typical Nepalese hilly community with shades of both the urban and the rural lifestyles.A cross-sectional study among the higher secondary students of Parbat district of Nepal was conducted. Parbat is a hilly district in Western Nepal with an area of 494 kma school . Districilaka. In the second stage, the principals of respective schools were contacted, briefed about the study objectives and asked for their permission to collect data. In order to attain the desired sample size, the number of students to be recruited from each school in proportion to the enrollment size was determined. Lottery method was used to randomly select the allocated number of students from attendance register at each school. Out of 530 students approached, none declined from participation in the study. Complete information was obtained from 516 participants and the responses of 14 participants were discarded because of missing information  was used, with some contextual modifications. The original tool consists 20 items , with reliability coefficient of 0.71 . Two staDemographic information  and status of tobacco and alcohol consumption were obtained from the participants. Information regarding the family structure, family history of cancer and monthly family income of the students were also recorded. Students were asked for their perspectives on the adequacy of the breast cancer information in their curriculum. Sources of information regarding breast cancer and breast cancer symptoms, that were originally not a part of the CBCKT, were also recorded. Students were provided with a list of symptoms as options, and were asked to select potential symptoms of breast cancer.p-value <0.05 taken as statistically significant.Data entry was carried out in EpiData 3.1 while all the analyses were performed in SPSS version 21 . Frequency and percentage were generated for responses to knowledge statements and socio-demographic variables. For a correct response to any of the 23 items on the questionnaire, a score of 1 was provided while an incorrect response or a response indicating ‘Don’t know’ was scored 0. A composite score of all the items combined was calculated which could range from 0–23. Median value of the composite score was 7.0 (slightly greater than the mean: 6.65). Knowledge score, after dichotomization taking the median as the cut-off, was grouped into two categories: ‘good knowledge’ and ‘poor knowledge’. Comparison of knowledge level with personal characteristics of students was made using chi-squared test, This study was approved by the Department of Community Medicine and Public Health (DCMPH), Maharajgunj Medical Campus (Ref. No. 105-072-073). During the ethical review, the need for parental consent for participants aged <16 years was waived by the DCMPH owing to the non-interventional and non-invasive study design. Prior to data collection, permission was obtained from the District Education Office and school principals. We obtained written informed consent from all the participants after explaining the study objectives. No financial incentive was provided to the participants. Participants were assured of anonymity and were informed of their right to voluntary participation and withdraw from the study at any stage.χ2 = 4.02, p = 0.045) (Table Participants belonged to an age group of 14 to 22 years with mean of 16.92 years (SD = 1.13). More than half of the participants (54.8 %) were female, 71.3 % were Brahmin/Chhetri and 95.0 % were Hindu. A majority of the participants (62.0 %) studied in grade 12, 1.9 % were tobacco users and 1.7 % consumed alcohol. One in every five participants (20.2 %) had a family history of cancer. Knowledge of students varied significantly with gender  perceived that the school curriculum inadequately informed them on breast cancer. One-in-four students (24.8 %) had heard of Breast Self Examination (BSE) while 29.8 % had heard of mammography. Only 4.8 % of the students responded correctly to at least half of the 23 items, and 1.4 % did not respond correctly to any of the items on risk factors and curability.Breast cancer was correctly identified as the most common cancer in women by 49.0 %  of the students. Regarding the knowledge of risk factors of breast cancer, most of them  were aware of the protective role of breastfeeding and exercise . Respondents correctly reported risk factors of breast cancer like early menarche , delayed menopause , use of oral contraceptives  and being overweight . Similarly, 25.4 %  of the students incorrectly reported childbearing before age of 30 to be a risk factor for breast cancer. Further, 22.3 %  of them were aware that risk of breast cancer gets higher in older age. Only 11.6 %  of the students correctly responded that a woman without any known risk factor may also develop breast cancer later in her life. Increased risk of breast cancer with the occurrence of some fibrocystic breast disease was correctly reported by 56.6 %  of the students.Students had false perception that hard blow to breast  and irritation caused by a brassiere  triggers breast cancer. Similarly, 42.2 %  wrongly responded that most of the lumps in breast are cancerous. Also, 30.8 %  were misinformed that breast cancer is rare in older women (aged >70 years)  respondents were informed that modalities of treatment other than mastectomy are also available and 42.8 %  knew that lumpectomy and radiation therapy are available alternatives for treatment of breast cancer. Less than half  of the students held a misperception that successful treatment of cancerous breast lump after it becomes painful is next to impossible while 21.1 %  believed that treatment of breast cancer without removal of all the lymph glands around breast and under the arm is not possible.A significant proportion  of the students believed that effective treatment of breast cancer after it can be felt by a woman is not possible, and 39.5 %  had the belief that the chances of successful treatment is better upon removal of the whole breast. Similarly, 29.3 %  of the participants believed that having a family history of breast cancer decreased the likelihood of breast cancer being cured. Also, 35.9 %  thought that despite the early detection and treatment, a woman with breast cancer cannot live a normal life  reported television/radio as the most common source of information on breast cancer. Other sources, reported in descending order of frequency were: teachers 26.0 %), textbooks (25.8 %), health workers (25.2 %), friends/relatives (24.2 %), newspapers (23.6 %) and poster/pamphlets (10.5 %) .Many respondents did not know that breast cancer can be prevented at an early stage if diagnosed properly. A substantial proportion of students had myths that early detection of breast cancer has no benefits on success of treatment, and hard blow to the breast causes breast cancer. This depicts the abysmal level of awareness and ignorance of risk factors in the community. Being less aware about the screening methods and treatment modalities can be a barrier to seek screening and detection services thus causing a delay in diagnosis and treatment. In breast cancer, the more advanced the disease, the lower the chance of survival .Age, educational level, household income and family history of breast cancer are reported to be significantly associated with the knowledge level , 23. HowBy knowing a simple measure like BSE, women can decrease the risk of breast cancer by early detection and prompt treatment. The study showed that less than one-fourth of the respondents had heard of BSE. Of the people who have heard of BSE, few know the correct practice , 25. PraThe most common source of information related to the breast cancer was Television/Radio which is similar to the findings reported by previous studies , 28. ThiKnowledge regarding the symptoms of breast cancer is of importance as a layman seeks health services depending on his/her literacy of these symptoms. In this study, only less than half of the students could mention at least one symptom of breast cancer. The three most commonly cited symptoms of breast cancer were pain in breast, change in breast shape and discharge of pus from the nipple. Our findings are consistent with a previous study conducted among Australian women, in which these three symptoms were reported by a major proportion of participants .It is important to note that in most of the items, a high percentage of the students replied with a ‘Don’t know’ response. This depicts that the students have no idea regarding the risk factors and curability of breast cancer. Further, nearly half of the students perceived existing school curriculum inadequate to be well-aware on breast cancer.As higher secondary students are in age of making reproductive choices; they should, therefore, be aware about the risks linked with oral contraceptive use. While most of the biological risk factors are non-modifiable, some behaviors like use of oral contraceptive, physical exercise, breastfeeding and initiation of regular BSE at an early age can reduce the risk of developing breast cancer. There is a scope to promote BSE among the adolescent girls in schools when health education campaigns about breast cancer are launched in partnership with schools, parents and community organizations in our study population. As breast cancer screening is likely to increase flow of patients seeking care, any further effort for screening should come along with capacity building of hospitals in detection, treatment and care of people with potential cancers.The result of this study found that students have a poor knowledge on many breast cancer risk factors, symptoms and curability. There is a need to boost knowledge of students, especially female students. National Health Education, Information and Communication Centre and concerned stakeholders should design awareness programs to sensitize women as well as men, family, and the general community about breast cancer and its early detection measures.However, the study has few limitations. The instrument used for the assessment of knowledge has not been validated in the Nepalese context, so, room for questioning the construct validity of the tool is present. Besides, all the schools included in this study were public and were drawn from one hilly district of Nepal. This might limit the generalization of our findings to students from private schools and to other districts from the plains. Still, this study will be valuable to frame relevant awareness activities and to guide policy measures considering the context where no literature on breast cancer awareness is available.This study demonstrates that students have poor knowledge on many breast cancer risk factors, symptoms and curability. Furthermore, most of the students had misleading information about age, early menarche, delayed menopause and use of OCPs as the risk factors. Many myths regarding curability of breast cancer were prevalent. Breast cancer literacy was significantly better among males in comparison to females. Future studies should particularly aim at identifying measures necessary to address the inadequate knowledge, along with the gaps in school curriculum.


Previous studies suggest that polycyclic aromatic hydrocarbons (PAHs) may adversely affect breast cancer risk. Indoor air pollution from use of indoor stoves and/or fireplaces is an important source of ambient PAH exposure. However, the association between indoor stove/fireplace use and breast cancer risk is unknown. We hypothesized that indoor stove/fireplace use in a Long Island, New York study population would be positively associated with breast cancer and differ by material burned, and the duration and timing of exposure. We also hypothesized that the association would vary by breast cancer subtype defined by p53 mutation status, and interact with glutathione S-transferases GSTM1, T1, A1 and P1 polymorphisms.Population-based, case-control resources  were used to conduct unconditional logistic regression to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI).Breast cancer risk was increased among women reporting ever burning synthetic logs  in their homes , but not for ever burning wood alone . For synthetic log use, longer duration >7 years, older age at exposure  and 2 or more variants in GSTM1, T1, A1 or P1  were associated with increased risk.Burning wood or synthetic logs are both indoor PAH exposure sources; however, positive associations were only observed for burning synthetic logs, which was stronger for longer exposures, adult exposures, and those with multiple GST variant genotypes. Therefore, our results should be interpreted with care and require replication.The online version of this article (doi:10.1186/1476-069X-13-108) contains supplementary material, which is available to authorized users. Breast cancer is the most common cancer diagnosed among women in the United States (U.S.) . ExperimPAHs are formed from the incomplete combustion of organic material . The maiPrevious studies have often relied on measuring PAH exposure with the biomarker PAH-DNA adducts –9. HowevIndoor air pollution, or household air pollution, from indoor stoves/fireplaces is an important ambient PAH source and is of significant world-wide public health concern . PreviouEnvironmental exposures that occur early in life, or during hypothesized biological windows of susceptibility may be more strongly associated with the risk of breast cancer . For exaSpecific base substitutions and other transitions and transversions in the tumor suppressor gene p53 have been associated with PAH exposure . Thus, iGlutathione S-transferase (GST) enzymes aid in the metabolism of PAHs and polymorphisms in the GST genes may alter individual’s ability to metabolize PAH compounds, and to remove reactive intermediates from the body . GST varHere we report on the association between use of indoor stoves and fireplaces and breast cancer risk. We hypothesized that this association would be modestly elevated for all types of material burned, stronger with increasing years of exposure and for early-life exposure and that it may vary by the timing of exposure and among susceptible subgroups defined by GST gene variants, hormone receptor subtype and p53 tumor mutations.Our investigation builds upon population-based case-control resources from the Long Island Breast Cancer Study Project (LIBCSP). Parent study methods have been previously described in detail . IRB appThe LIBCSP included English-speaking women residing in Nassau and Suffolk counties on Long Island, New York. Cases were women newly diagnosed with a first primary in situ or invasive breast cancer between August 1, 1996 and July 31, 1997. Cases were ascertained by daily/weekly contact with pathology departments of all 28 hospitals on Long Island and three tertiary care hospitals in New York City.Controls were randomly selected female residents from the two source counties who had no history of breast cancer and were frequency matched to cases based on the expected case age distribution by 5-year age group. Potential controls were identified by random digit dialing for those less than 65 years of age and by Health Care Finance Administration rosters for those 65 years and older. The response rate for women <65 years was 76.1% and for women >65 years was 43.3% .Written signed informed consent was obtained from all participants prior to interview. A total of 1,508 cases and 1,556 controls  completed the interview. Participants ranged in age from 20 to 98 years, with one third 65 years of age or older at the time of diagnosis (for cases) or identification (for controls), and 67% were postmenopausal. In addition, 94% self-identified as white, 5% black, and 2% other, which reflects the underlying racial distribution of these two Long Island counties at the time the study was conducted.Indoor stove/fireplace use was assessed using a structured questionnaire. The LIBCSP case-control interview occurred in the respondents’ homes and, for cases, within several months of the diagnosis of the first primary breast cancer. All participants were administered the main case-control questionnaire, a comprehensive assessment of known and suspected risk factors for breast cancer. This instrument included a question asking whether a participant used an indoor stove or fireplace ≥3 times a year for each residence lived at on Long Island; and, if yes, whether they burned wood, coal, gas and/or synthetic logs. Synthetic logs also contain wood; therefore, it is important to note that future references to wood-burning exclude synthetic logs use. The date and/or ages of participants at time of living in each residence was used to calculate exposure duration and timing. Exposure frequency or exposures to stove/fireplace use at non-residences or residences outside of Long Island were not captured.To consider breast cancer subtype, we utilized data on p53 mutation status of the tumor. Archived paraffin-embedded tumor tissue was obtained and DNA was extracted (n = 859). Mutations were detected in exons 5-8 of p53, described in detail in Rossner et al. . SamplesBreast cancer subtype defined by hormone receptor status was obtained from medical records . Cases  estimated the association between breast cancer risk and residential indoor stove/fireplace use, with consideration given to potential confounders; (2) explored associations with more detailed exposure information, including material burned and timing of exposure; (3) explored associations with breast cancer subtype; and (4) considered effect modification by GST polymorphisms, menopausal status and smoking history.First, we used unconditional logistic regression  to estimSecond, associations between breast cancer risk and detailed exposure characteristics, including stove/fireplace type, duration, and timing, were also considered using unconditional logistic regression. For example, we used indicator variables to assess the association for each material burned ; subanalyses, however, were limited to wood and synthetic logs due to the limited sample sizes of women reporting burning coal or gas. To assess possible correlation between women who reported burning wood and those who reported burning synthetic logs, a kappa statistic was calculated . We usedThird, associations with the cases categorized by breast cancer subtypes, defined by p53 mutation status and hormone receptor status, were examined using polytomous logistic regression , which sFourth, to consider possible effect modification by GST polymorphisms, menopausal status or active/passive smoking status, unconditional logistic regression was used. Effect measure modification on a multiplicative scale was assessed by comparing multivariable models with and without cross-product terms of the exposure and menopausal status  and active/passive smoking  as well as using the approach described below for each GST polymorphism separately, as well as number of GST variants . For posCase-control participant characteristics, stratified by any stove/fireplace use, are displayed in Table In the LIBCSP population, 747 cases (49.5%) and 768 controls (49.4%) reported using a fireplace or indoor stove at least 3 times per year in one of their Long Island residences Table . The preAs shown in Table As shown in Table As shown in Table We did not observe any statistically evident heterogeneity by p53 mutation status  [Use of solid fuels for indoor heating and cooking is most common in Africa and Southeast Asia, with a prevalence of use at approximately 60% and thus, most research on health impacts have been conducted in countries in these geographic areas , 35. How3; orders 0 ng/m3) . This laThere has been limited testing on the emission profiles of synthetic logs, consisting of wax and sawdust, compared to natural logs. The increase in breast cancer risk we observed with synthetic log burning may be biologically plausible. A report by Gullet et al.  measuredRetrospective stove/fireplace use was collected in the mid-1990’s which would have reflected exposures before the Environmental Protection Agency began endorsing certified indoor stoves/fireplaces with lower emissions . This, cA Washington state department of health study estimated that 40% of people used some type of wood burning device  . Among tPAHs may have both mutagenic and epigenetic possible mechanisms that are relevant to breast carcinogenesis . PAHs caWe did find a suggestive evidence that any indoor stove/fireplace use and wood burning prior to age 20 years, which encompasses age before first birth for approximately 90% of the LIBCSP study population, may be more relevant for future breast cancer risk than exposure that occurs after age 20 years. This is consistent with evidence from another PAH source, active smoking, which may be particularly important to breast cancer risk if the exposure is prior to the first pregnancy . On the Reporting the use of indoor stoves/fireplaces is a surrogate measure of indoor PAH exposure, but other factors (such as exterior air pollution from vehicular traffic in urban areas) may contribute to indoor air pollution levels . TherefoWe did not find an association between stove/fireplace use and p53-mutation-positive tumors, which is consistent with previous results from the LIBCSP investigating other ambient PAH sources , but conWe observed no evidence for an interaction between individual variant GST alleles with indoor stove/fireplace use, despite the biologically plausibility . HoweverDifferential recall bias is always a potential concern for case-control studies. However, our most prominent finding is among those that burned synthetic logs in their homes. It is unlikely at the time of data collection that cases would have suspected certain wood-based fuel materials would be more carcinogenic than others. In addition, this study population had many long-term users of synthetic logs which suggest these results are not just an artifact of differential recall. Despite this, it is still possible that this association may have been impacted by recall bias. There is also the possibility of the influence of selection bias, particularly among older control women who had a lower response rate. However, the mechanism by which stove/fireplace use would affect response rates among only elderly control women is unknown. Participations rates may vary by socioeconomic status. High socioeconomic status is a risk factor for breast cancer ; howeverOur findings suggest that residential indoor air pollution from burning synthetic logs, but not wood, may be associated with an increase in breast cancer risk. The high incidence of breast cancer in the U.S. and the relatively common prevalence of indoor stoves/fireplaces suggest that this research, if confirmed, may have considerable public health importance. Our results provides new information on an important ambient PAH exposure source, which has been understudied in populations with low exposure levels such as those found in the U.S., and may guide future research on the potential carcinogenicity of PAH exposure on the breast.Indoor Air Pollution Exposure from Use of Indoor Stoves and Fireplaces in Association with Breast Cancer: A Case-Control Study.(DOCX 43 KB)Additional file 1:


HOXB13 p.G84E mutation has been firmly established as a prostate cancer susceptibility allele. Although HOXB13 also plays a role in breast tumor progression, the association of HOXB13 p.G84E with breast cancer risk is less evident. Therefore, we comprehensively interrogated the entire HOXB13 coding sequence for mutations in 1,250 non-BRCA1/2 familial breast cancer cases and 800 controls. We identified two predicted deleterious missense mutations, p.G84E and p.R217C, that were recurrent among breast cancer cases and further evaluated their association with breast cancer risk in a larger study. Taken together, 4,520 familial non-BRCA1/2 breast cancer cases and 3,127 controls were genotyped including the cases and controls of the whole gene screen. The concordance rate for the genotyping assays compared with Sanger sequencing was 100%. The prostate cancer risk allele p.G84E was identified in 18 (0.56%) of 3,187 cases and 16 (0.70%) of 2,300 controls . Additionally, p.R217C was identified in 10 (0.31%) of 3,208 cases and 2 (0.087%) of 2,288 controls . These results imply that none of the recurrent HOXB13 mutations in the Dutch population are associated with breast cancer risk, although it may be worthwhile to evaluate p.R217C in a larger study.The  Breast cancer is the second leading cause of cancer death in Western countries and the most frequently diagnosed cancer in Western women. A family history of breast cancer is a major risk factor for developing breast cancer. Approximately 10–15% of breast cancer patients have at least one first-degree relative with breast cancer. Depending on the number of affected first-degree relatives, this implies risk ratios for breast cancer of 1.80 for one affected relative to 3.90 for three or more affected relativesBRCA1 and BRCA2 were identified in the 1990s and germline mutations in these genes confer average cumulative lifetime breast cancer risks by age 70 of 65% and 45%, respectively23ATM, CHEK2, PALB2, and RAD50 confer 2- to 4-fold increased breast cancer risksPALB2 mutations may be higher than initially thought6i.e. per allele odds ratios) of up to 1.3Familial breast cancer has been associated with mutations in several high- and moderate-risk breast cancer susceptibility genes, as well as an increasing number of low-risk breast cancer susceptibility alleles. The two high-risk genes HOXB13 gene was reported to be associated with prostate cancer111213HOXB gene cluster at 17q21–22 had identified a number of highly correlated common SNPs that were associated with prostate cancer risk and tagging the rare HOXB13 p.G84E variant. This not only further established the association between HOXB13 p.G84E and prostate cancer risk, but also provided evidence that GWAS associations could actually be driven by rare variantsHOXB13 gene encodes a transcription factor that plays an important regulatory role during embryonic development, but also in tumorigenesis. For example, HOXB13 was reported to regulate the transcription of androgen receptor (AR) target genesHOXA9, HOXB13 is the most commonly deregulated gene in solid cancersHOXB13 was shown to preferentially bind a low-risk prostate cancer susceptibility allele located in an AR and FOXA1 binding site (i.e. rs339331), thereby enhancing RFX6 expression and promoting metastasisIn this respect, the rare variant c.251G > A  in the HOXB13 gene expression is regulated by estrogen in an ER dependent mannerHOXB13:IL17BR expression ratio was found to be a prognostic and predictive biomarker for ER-positive breast cancer patients20HOXB13 might also be a likely candidate for being a breast cancer susceptibility gene. So far, three studies have investigated this hypothesis but obtained contradictory results. In the study by Alanee et al.HOXB13 p.G84E mutation was shown to confer an increased breast cancer risk, however, Akbari et al. could not replicate this association in a larger studyet al. also found no association with breast cancer risk, but did observe a suggestive association in a particular high-risk subgroupIn breast cancer, HOXB13 gene in 1,250 Dutch familial breast cancer cases and 800 geographically matched controls to establish whether the p.G84E mutation or other mutations in the HOXB13 gene are associated with an increased breast cancer risk.In this study, we therefore analyzed the entire coding region of the HOXB13 gene for germline mutations in 1,250 non-BRCA1/2 breast cancer patients and 800 controls from the Rotterdam Breast Cancer Study (RBCS) study. Using PCR and Sanger sequencing, we identified a total of eleven different rare variants (i.e. c.251G > A and c.649C > T) were detected in multiple cases and controls (i.e. 87% for p.G84E and p.R217C), Meta-SNPi.e. 0.730 for p.G84E and 0.895 for p.R217C) and PON-P2i.e. 0.967 for p.G84E and 0.974 for p.R217C). Moreover, p.G84E is localized in the MEIS binding domain, whereas p.R217C is localized to the homeodomain of HOXB13, further increasing the likelihood that these mutations are pathogenici.e. to all indexes from families counselled between 1994 and 2014 for cases and between 1996 and 2010 for controls) and by including the Amsterdam Breast Cancer Study (ABCS-F).We evaluated the entire coding sequence of the variants  and two controls . The c.2controls . For allBRCA1/2 breast cancer patients and 3,127 controls were genotyped. These also included the 1,250 non-BRCA1/2 breast cancer patients and 800 controls from the RBCS study that were used in the whole gene screen to evaluate the quality of the genotyping assay. The concordance between the results from the custom-designed Taqman genotyping assay and Sanger sequencing of these patients was 100%. Interestingly, the p.G84E mutation was identified in 18 (0.56%) of 3,187 cases and 16 (0.70%) of 2,300 controls (P = 0.54). The p.R217C mutation was identified in 10 (0.31%) of 3,208 cases and 2 (0.087%) of 2,288 controls (P = 0.14). These results imply that none of the recurrent HOXB13 mutations in the Dutch population are associated with breast cancer risk.In order to facilitate fast and accurate screening of the p.G84E and p.R217C mutations, two custom-designed Taqman genotyping assays were developed for analyzing all samples from the RBCS and ABCS-F case-control studies. In total, all 4,520 non-controls . Consistcontrols . ConsistHOXB13 c.251G > A (p.G84E) mutation has been shown to confer a 4- to 5-fold increased prostate cancer risk1112HOXB13 gene mutations are also associated with breast cancer risk. Our results show that the prostate cancer risk variant p.G84E is not associated with breast cancer risk. Furthermore, another recurrent mutation in the HOXB13 gene  was also not associated with increased breast cancer risk, although it was more prevalent in cases than controls.The et al. had previously shown that the HOXB13 p.G84E mutation conferred a moderate to high breast cancer riskP = 0.02). However, in a larger study  conducted by Akbari et al., no association of the p.G84E mutation with breast cancer risk was observed among Canadian and Polish women of European originP = 1.0). Also a third study by Laitinen et al. consisting of 986 cases  and 1,449 controls found no overall association between the p.G84E mutation and  breast cancer risk among Finnish womeni.e. 86 cases from the Pirkanmaa area of Finland; OR = 3.2, 95% CI = 0.9–11.9). Here in this study, we also did not observe an increased breast cancer risk associated with the p.G84E mutation in a relatively large study of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls . It thus appears that the HOXB13 p.G84E mutation is not associated with increased breast cancer risk, although it cannot be excluded that it is associated with a specific high–risk subgroup. Considering the low population frequency, much larger studies are needed to determine whether the latter is indeed the case.Interestingly, Alanee HOXB13 also identified c.649C > T (p.R217C) as a recurrent mutation in the Dutch population, which was predicted to be pathogenic. In both the discovery as well as the validation phase of the study, the mutation was more prevalent in familial breast cases than controls, but the association with breast cancer risk was not significant. Considering the wide CIs and the very low population frequency, there is a possibility that the study was underpowered and failed to detect the association. Evaluation of HOXB13 p.R217C in a larger study or a population with a higher carrier allele frequency might therefore still be worthwhile to pursue. Since the p.G84E variant varies widely among different geographic populations (i.e. highest in North and West-Europeans and lowest in non-Europeans)2929et al. reported that p.R217C did not co-segregate with prostate cancer in the two families they identifiedThe whole gene screen for HOXB13 mutations that we identified in the Dutch population were associated with breast cancer risk, although it may be worthwhile to evaluate p.R217C in a larger study or a population with a higher allele frequency.To conclude, none of the recurrent BRCA1 and BRCA2 was assigned to be the index case and included in RBCS. Additionally, breast cancer cases were included that were diagnosed either before 40 years with unilateral breast cancer or before 50 years with bilateral breast cancer without having a first or second degree relative diagnosed with either breast or ovarian cancer. All cases and their tested relatives were negative for both BRCA1 and BRCA2 mutations. Median age of the RBCS cases was 44 years (range 18–92 years). The RBCS control population  was geographically matched and included women from cystic fibrosis families who were either spouses of individuals at risk of being carrier of a CFTR mutation or individuals who were tested negative for a CFTR mutation and were counselled between 1996 and 2010. Median age of the RBCS controls was 41 years (range 10–97 years).The samples included in this study were from two Dutch breast cancer case-control studies: RBCS and ABCS-F. RBCS cases  were selected from the database of the Clinical Genetics Centre at Erasmus University Medical Centre in Rotterdam, representing the Southwestern part of the Netherlands. Selected families included all families counselled between 1994 and 2014 that presented with at least two cases of female breast cancer or at least one case of female breast cancer and one case of ovarian cancer in first- or second-degree relatives. At least one of these two cases needed to be diagnosed before the age of 60. For each family, the youngest breast cancer patient who had been tested for BRCA1/2 mutation or unclassified variant, who were counselled in the Family Cancer Clinic and diagnosed and/or treated with cancer in the Antoni van Leeuwenhoek hospital in the period 1995–2012. For each family, only the youngest breast cancer patient who had been tested for BRCA1 and BRCA2 was included. ABCS-F controls  are healthy women of all ages (median age was 49 years (range 18–69 years)) from the general population and were recruited through the blood bank.ABCS-F cases  were selected from the linked databases of the Division of Diagnostic Oncology and the Tumor Registry of the Antoni van Leeuwenhoek hospital in Amsterdamhttp://www.fmwv.nl). The RBCS and ABCS-F studies were approved by the Medical Ethical Committes of the Erasmus Medical Center Rotterdam and the Netherlands Cancer Institute, respectively. All individuals gave written informed consent.All experiments were performed in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands (HOXB13 (RefSeq NM_006361.5) coding region was analyzed for sequence variations in 1,250 non-BRCA1/2 familial breast cancer cases and 800 controls from RBCS (i.e. indexes from families counselled between 1995 and 2009 for cases and between 1996 and 2006 for controls) using PCR and Sanger sequencing. Twenty nanograms of DNA, extracted from peripheral blood, was PCR amplified in a final volume of 15 μl containing 1X GoTaq buffer , 1.5 mM MgCl2, 200 μM dNTPs , 1 μM of each primer and 0.75 U of GoTaq polymerase (Promega) using an ABI2720 thermal cycler . First, the PCR reaction was incubated for 5 minutes at 94 °C, followed by 35 cycles of 94 °C for 30 seconds, 58 °C for 1 minute and 72 °C for 1 minute. The PCR reaction ended with a final extension at 72 °C for 5 minutes. Removal of dNTPs and primers before sequencing was done by ExoSAP-IT PCR Product Cleanup . Briefly, 2.5 μl of PCR product was incubated with 0.5 μl of ExoSAP-IT and 1x GoTaq buffer in a final volume of 12.5 μl. Incubation took place in an ABI2720 thermal cycler for 15 minutes at 37 °C. Then enzymes were inactivated at 80 °C for 15 minutes before proceeding with Sanger sequencing. The sequencing reaction contained 2 μl of ExoSAP-it treated PCR product, 1 μl BigDye Terminator v3.1 reaction mix (Thermo Scientific), 1X BigDye Terminator sequencing buffer (Thermo Scientific) and 0.16 μM of sequencing primer in a final volume of 10 μl and was carried out in an ABI2720 thermal cycler according to the following protocol: 1 cycle of 96 °C for 2 minutes and 25 cycles of 96 °C for 30 seconds, 58 °C for 30 seconds and 72 °C for 2 minutes. Subsequently, the sequencing product was precipitated with absolute ethanol and 3M of NaAc, resuspended in 20 μl of Hi-Di formamide (Thermo Scientific), and ran on an ABI3130XL Genetic Analyzer (Thermo Scientific). Sequencing electropherograms were analyzed using Mutation Surveyor v3.20 software . Sanger sequencing was successful for 96.2% of the samples and PCR and sequencing primer sequences for the two exons of the HOXB13 gene are available in The entire HOXB13 gene was performed for all 7,647 DNA samples from RBCS and ABCS-F using custom-made Taqman genotyping assays (Thermo Scientific) on a Mx3000/3005P qPCR machine . For genotyping p.G84E, 0.5X of Taqman genotyping assay and 0.5X of Taqman Genotyping Master Mix (Thermo Scientific) was added to 20 ng of genomic DNA in a final volume of 10 μl, whereas for p.R217C, 1X Taqman genotyping assay and 1X ABsolute qPCR Mix, low ROX (Thermo Scientific) was added to 20 ng of genomic DNA in a final volume of 10 μl. Cycling conditions were: 1 cycle of 10 minutes (for Taqman Genotyping Master Mix) or 15 minutes  at 95 °C and 45 cycles of 15 seconds at 92 °C and 1 minute at 60 °C. The MxPro qPCR software v4.10 (Agilent) was used to visualize the genotyping results. The call rate of the genotyping assays was 97.9% for p.G84E and 98.1% for p.R217C, respectively, and Taqman assay design is specified in Genotyping of the c.251G > A  and c.649C > T  mutations in the HOXB13 mutations (i.e. p.G84E and p.R217C) with breast cancer risk was evaluated by comparing the carrier allele frequency between cases and controls using either a χ2 test or a Fisher’s exact test (i.e. when the expected frequency ≤5 in any of the groups). Odds ratios and their 95% confidence intervals were calculated based on 2 × 2 table analysis of the cases and controls. All statistical tests were two-sided and P-values were considered statistically significant when smaller than 0.05.The association of both How to cite this article: Liu, J. et al. Recurrent HOXB13 mutations in the Dutch population do not associate with increased breast cancer risk. Sci. Rep.6, 30026; doi: 10.1038/srep30026 (2016).