Results from this study provide some support for an inverse association between serum IGFBP2 levels and risk of AH, particularly in nondiabetic women who are overweight or obese. Further studies are required to confirm these results.Atypical hyperplasia of the breast (AH) is associated with increased risk of subsequent invasive breast cancer, yet little is known about the etiology of AH. Insulin-like growth factor binding protein 2 (IGFBP-2) may contribute to the development of AH due to its proliferative effects on mammary tissue. We conducted a nested case-control study of postmenopausal women enrolled in Women's Health Initiative-Clinical Trial. Cases were 275 women who developed incident AH during follow-up, individually (1 : 1) matched to controls. Levels of IGFBP-2 were determined from fasting serum collected at baseline. Multivariable conditional logistic regression models were used to estimate odds ratios for the association of IGFBP-2 with risk of AH. Serum IGFBP-2 was associated with a nonsignificant decrease in risk for AH, when comparing the highest quartile to lowest quartile . This decrease in risk was most evident when analyses were restricted to nondiabetic, nonusers of hormone therapy (OR = 0.33, 95% CI = 0.13–0.86,  This e cancer , 6. in vitro studies have shown that it has a proliferative effect in mammary and other tumor cell lines [Little is known regarding the etiology of AH and the progression to breast cancer. However, insulin-like growth factors and their binding proteins have been shown to be critical in the development of many epithelial cancers, including breast cancer . Insulinll lines –10 and cll lines . Thus, sll lines .Potentially counterbalancing the proliferative effect of IGFBP-2 is the observation that serum IGFBP-2 levels are inversely associated with obesity . ObesityThese observations suggest a potential dual effect for IGFBP-2 in breast cancer development: IGFBP-2 may reduce the risk of AH via protection against development of insulin resistance and by virtue of its inverse association with obesity, while concurrently increasing risk of AH via its proliferative effects on breast tissue.To our knowledge, no previous studies have investigated the association between circulating IGFBP-2 levels and risk of AH. A greater understanding of the development of AH might enhance our understanding of the origins of breast cancer and help identify women who would benefit from increased surveillance and screening. To this end, we investigated the association of IGFBP-2 with risk of incident AH in a large prospective cohort of postmenopausal women.This investigation was conducted as a case-control study nested within Women's Health Initiative-Clinical Trial (WHI-CT) . BrieflyEvery 6 months, participants in the trial completed medical questionnaires on clinical events including breast procedures. In the Benign Breast Disease Ancillary Study, which was conducted in all WHI clinical centers , 23, womCases in our study were women diagnosed with incident BPBD with AH during follow-up in the WHI-CT. Of the 294 cases of AH ascertained, a total of 275 cases of BPBD with AH were included in the present study, consisting of all cases of atypical hyperplasia that had serum available. Controls were women who did not develop BPBD during the same follow-up period as that for the corresponding cases and who did not have an abnormal mammogram or abnormal clinical breast exam during the same period. Controls were selected from eligible participants using risk-set sampling (with replacement)  and werer = 0.999).Laboratory testing was performed by the Pollak Assay Laboratory at McGill University in Montreal, Quebec, Canada. Serum IGFBP-2 levels were determined by enzyme-linked immunosorbent assay (ALPCO Diagnostics), which has an assay sensitivity of 0.2 ng/mL. All samples but one which did not have sufficient volume were retested as blind duplicates and showed strong correlations  were assessed using Spearman correlation coefficients among controls only.2). Adjusting for BMI as a continuous variable did not change the results substantially when compared to the four-category adjustment (data not shown). Additional adjustment for other variables  had no discernible impact on the effect estimates and these variables were not included in the final models. A second model was then run, additionally adjusting for serum levels of insulin, C-reactive protein, and adiponectin, as well as tertiles of estradiol among nonusers of HT only, with HT users as a separate category (as standard estradiol assays cannot accurately measure equine hormones present in most HT preparations). Analyses were also conducted after stratifying separately by hormone use at baseline , BMI based on the median in controls , and age based on the median in controls . These stratified analyses were conducted by introducing interaction terms into multivariate models that also included the main effect variables. We also reran analyses after restricting the study population to those without a history of diabetes (16 cases and 17 controls reported a history of diabetes). For all analyses, tests for trend were performed by assigning median values to each quartile and modeling these categories as a continuous variable. All hypothesis tests were two-sided and all analyses were done using the statistical software Stata S/E 13.0 for Windows .Conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association of IGFBP-2 with risk of atypical hyperplasia. Analyses were first conducted with adjustment for age , and BMI  than in those classified as overweight or obese (≥25 kg/m2)  . Further5 kg/m2) . Serum I5 kg/m2) .p = 0.011) that was attenuated with adjustment for insulin, adiponectin, C-reactive protein, and estradiol , and when restricted to those without a history of diabetes .Considering all subjects together, serum IGFBP-2 was associated with a decrease in risk of AH in the age- and BMI-adjusted model only, with those in the highest quartile of serum IGFBP-2 having a 49% decrease in risk compared to those in the lowest quartile  . This asWhen stratified by use of HT, there was a decrease in risk of AH with increasing serum IGFBP-2 that was evident only among nonusers of HT , althougThe association of IGFBP-2 with BPBD also appeared to differ by BMI , althougTo our knowledge, this is the first study to investigate the association between serum levels of IGFBP-2 and the risk of atypical hyperplasia of the breast. Overall, we found a nonsignificant decrease in risk of AH with increasing serum IGFBP-2. The inverse association was of borderline statistical significance in two, prespecified subgroups: nondiabetic, nonusers of HT and women who were overweight or obese.2 showing evidence of decreased risk of breast cancer with increasing IGFBP-2, an association not seen in women under 26 kg/m2 [Although no studies have previously examined the association between IGFBP-2 and risk of BPBD, there is some epidemiological evidence for a role of IGFBP-2 in breast cancer. An early study investigated the association between IGFBP-2 and postmenopausal breast cancer and found a reduction in risk with increasing serum IGFBP-2 , althoug26 kg/m2 , whereas26 kg/m2 . In addi26 kg/m2 .Findings from our study and from these previous studies in breast cancer are in contrast with previous experimental evidence suggesting that IGFBP-2 promotes cell proliferation and tumor growth . This ma in vitro, both via inhibition of IGF-I [With respect to breast cancer and perhaps other obesity-related cancers such as colorectal cancer , the proof IGF-I  and alsoof IGF-I . In addiof IGF-I . There iof IGF-I , 41. Givof IGF-I . Estrogeof IGF-I .Our findings suggest that the effect of IGFBP-2 on risk of AH was modified by both HT use and BMI. The inverse association between serum IGFBP-2 levels and breast cancer risk observed previously seems to be restricted to women who were not using HT and to those women who are overweight or obese. Although suggestive, our study was underpowered to detect significant interactions between these subgroups with respect to risk of AH. A further limitation of this study was that IGFBP-2 was only measured once, at baseline. Repeated measurements over time might have resulted in more accurate estimates of serum levels. Also, the WHI-CT participants were a nonrandom sample of the population, which may limit the generalizability of our findings. However, because the results reported here demonstrate associations between a measured biological marker and risk of AH, it seems plausible that this marker may have similar effects in other populations. There is also the possibility of disease misclassification in this study, which may have biased our results towards the null. Major strengths of this prospective study were the collection of prediagnostic blood samples at baseline and ascertainment of subsequent (postbaseline) development of AH, thereby limiting bias.In conclusion, the results of this study are suggestive of an inverse association between serum IGFBP-2 levels and risk of atypical hyperplasia of the breast. This reduction in risk was most evident among nondiabetic, nonusers of HT at baseline and among obese and overweight women, and the reduction in risk persisted after adjustment for associated serologic factors such as insulin and estradiol. However, these results should be interpreted with caution, as the lack of statistical significance could be due to the relatively small numbers of study subjects available for analysis or could indicate that no true association exists between IGFBP-2 and AH. Nevertheless, given the association between AH and breast cancer, if our finding that serum IGFBP-2 level is associated with reduced risk of AH is confirmed in further studies, this may suggest a role for IGFBP-2 in the early stages of breast carcinogenesis.


The role of family history to the risk of breast cancer was analyzed by incorporating menopausal status in Hong Kong Chinese women, with a particular respect to the estrogen receptor-positive (ER+) type.Seven hundred and forty seven breast cancer incident cases and 781 hospital controls who had completed information on family cancer history in first-degree relatives  were recruited. Odds ratio for breast cancer were calculated by unconditional multiple logistic regression, stratified by menopausal status  and type of relative affected with the disease. Further subgroup analysis by tumor type according to ER status was investigated.Altogether 52 (6.96%) breast cancer cases and 23 (2.95%) controls was found that the patients’ one or more first-degree relatives had a history of breast cancer, showing an adjusted odds ratio (OR) of 2.41 (95%CI: 1.45–4.02). An excess risk of breast cancer was restricted to the ER+ tumor , with a relatively higher risk associated with an affected mother  than an affected sister , while the relative risk was more prominent in the subgroup of pre-menopausal women. Compared with the breast cancer overall, the familial risks to the ER+ tumor increased progressively with the number of affected first-degree relatives.This study provides new insights on a relationship between family breast cancer history, menopausal status, and the ER+ breast cancer. A separate risk prediction model for ER+ tumor in Asian population is desired. Breast cancer is one of the most commonly diagnosed cancer worldwide, and it is the third leading cause of cancer death in Hong Kong women,2. AlthoBreast cancer is a heterogeneous disease that can be divided into distinct tumor subtypes commonly classified by the status of estrogen receptor. Clinically, estrogen receptor-positive (ER+) breast cancer has better responsiveness to antiestrogens compared to ER-negative (ER-) subtype. EvidencThe study protocol was approved by both the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committees and the Kowloon West Cluster. The informed consent for both cases and controls was obtained before the interview. Chinese women, aged between 20 and 84 with newly diagnosed primary breast cancer (ICD-10 code 50), were confirmed by histology. All eligible cases were consecutively identified from the Department of Surgery or Clinical Oncology of 3 hospitals in Hong Kong during the period 11/2011 to 05/2014, and were interviewed within 3 months after diagnosis. Seventy hundred and sixty seven eligible cases were interviewed and the response rate was 91.1%, and the main reasons for the non-responses were no interest and poor medical condition. Each breast cancer incident case was frequency matched in 5-year age groups by a control patient selected from the same hospital where the cases came from. We recruited 792 eligible controls with a broad disease pattern of diagnosis, with the response rate of 93%. To be eligible, the cases and controls must have no previously physician-diagnosed cancer at any site.Personal interviews were conducted by trained interviewers using a standardized questionnaire to obtain information on age at diagnosis (or age at interview for the controls), educational attainment, smoking habits, alcohol drinking, body mass index (BMI), history of benign breast diseases, family cancer history, occupational history, and reproductive factors including age at menarche, menopausal status, parity, breastfeeding, age at first birth and hormone replacement therapy. In terms of family cancer history, each participant was required to report whether their FDR had ever been diagnosed by a doctor of having cancer at any site. If a positive answer was obtained, further information on which family member and what type of cancer was diagnosed should be collected.The data on ER status (for cases only) was extracted and the disease diagnosis for the cases and controls from the hospital medical records were verified. The quality of the data about family cancer history was assessed by re-interviewing 25% of the cases and controls about 6 weeks after the initial interviews, and the agreement between the two interviews was high (kappa = 0.62).Table 1). A case-case analysis was conducted to test the heterogeneity of family breast history in FDR by ER status. Further stratified analyses were conducted according to menopausal and ER status, while the gradient relationship of breast cancer risk with increasing number of FDR impacted was further explored by trend tests.Unconditional multivariate logistic regression models was performed to estimate the adjusted odds ratio (OR) and 95% confidence intervals (CIs) for the association with each family cancer history variable after controlling for potential confounding variables that were statistically significant at p < 0.05 from the univariate analysis (Table 1). Overall, breast cancer cases were significantly younger than the controls at the age of menarche, but they had a relatively higher proportion of hormone replacement therapy. Compared with breast cancer cases, the controls had more children, lower level of BMI, and younger age at first birth. The distribution of educational attainment, tobacco smoking, alcohol drinking, oral contraceptives usage, and menopausal status was similar between the cases and controls. ER+ tumor accounted for 76.4% (438/573) of breast cancer cases with a known status of estrogen receptor. Similar distribution of ER status appeared between cases <50 years (ER+ vs. ER-: 75.7% vs. 24.3%) and those aged 50 years or above  (ER+ vs. ER-: 76.8% vs. 23.2%) (p = 0.76).Seven hundred and forty seven breast cancer cases and 781 controls had completed information on family cancer history (Table 2), the OR was greater when the mother  was impacted. Case-case analysis by ER status showed that slightly more cases with a family history of breast cancer in the FDR presented ER+ tumor than those without a family history, whilst the heterogeneity was not statistically significant  due to a small sample size in the ER- type; nevertheless, there was no obvious difference in the age at diagnosis of breast cancer between women with (56.04±9.60 years) and without a family history .A total of 277 (37.1%) breast cancer cases and 214 (27.4%) controls were reported a family history of any type of cancer in the FDR, showing an adjusted OR of 1.47 (95%CI: 1.17–1.83). This moderate association with breast cancer risk was mainly driven by a family cancer history of the head and neck , genitourinary , and the breast . About 1.4 times more breast cancer cases than the controls having a history of breast cancer in mother or sister (6.96% vs. 2.95%). Compared with a sister impacted by breast cancer  (Table 3). There was a similar pattern of the results when subgroup analysis was conducted according to the mean age at the diagnosis of breast cancer (55 years).Subgroup analysis according to ER status showed that an excess risk of breast cancer was restricted to the ER+ cases, with an OR of 3.97 (95%CI: 1.46–10.79) for those with a mother impacted by breast cancer and 2.06 (95%CI: 1.07–3.97) for those who had an impacted sister. Stratified analysis by menopausal status demonstrates that a significantly increased risk of breast cancer was confined to the pre-menopausal women  who had mother impacted by breast cancer , and this association was enhanced for women with the ER+ tumor type . A moderate excess risk of breast cancer was also indicated in the post-menopausal women, but there was no statistical significance  were significantly modified by estrogen receptor status–23; amonResults from this hospital-based case-control study were regarded as valid and were expected to have general application. The breast cancer cases were recruited from several large hospitals, and the age and distribution of our breast cancer cases by histological subtypes were highly similar to those reported by the Hong Kong Cancer Registry. The response rate for breast cancer cases was high (92%) and the misclassification of breast cancer status was minimal as all cases included were confirmed by histology. Despite population controls were theoretically preferred to hospital controls in a case-control study, there wData from a special group of 117 hospital controls  showed a lower proportion of a family history of breast cancer (6% vs. 7.0%) or any cancer (31.6% vs. 37.1%) in the FDR than the surgically confirmed breast cancer. The differential family cancer history reported in these 2 subgroups suggested the lack of interviewer bias and, hence, provided further support for the validity of our results. Another concern is a potentially increased surveillance of breast cancer among women with a family history of the disease, which may lead this group of women to presenting less advanced staging of breast cancer than those without a family history. Little difference in early breast cancer  was noticed between women with and without a family history (11.5% vs. 10.6%) (Data not shown). The population attributable risk was estimated because the breast cancer cases in our study was a reasonable representative to the general population, and was found that about 4.1% and 11.8% of incident breast cancer cases could be attributable to having a family history of breast cancer or any cancer in FDR. NeverthIn conclusion, this study provided new insights on a relationship between family breast cancer history, menopausal status, and the ER+ breast cancer. An establishment of an individual risk factor model for ER+ and ER- breast cancer is desired for Asian population and this may advance the current Gail model in which no separation of risk prediction model was provided.


Several recent cohort studies have found an association between smoking and breast cancer, but the association between lifetime smoking exposure and breast cancer mortality is less well described. We examined whether smoking before breast cancer diagnosis is a predictor of breast cancer mortality in a large cohort with more than 4.1 million years of follow-up, with a special focus on women who initiated smoking before first childbirth. Information on smoking status was collected before breast cancer diagnosis and used to estimate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of breast cancer mortality in a cohort of 302,865 Norwegian women with 1106 breast cancer deaths. Women were enrolled between 1974 and 2003 and followed up through linkages to national registries until 31 December 2007. We found that breast cancer mortality was slightly but significantly increased for current  and ever  smokers as compared to never smokers. No statistically significantly increased mortality was found for women who initiated smoking before first childbirth, and no dose-response association was revealed for any of the different measures of smoking exposure. A large proportion of heavy smokers may have died from other causes than breast cancer during follow-up, possibly diluting our results. This study found that lifetime smoking exposure had a significantly increased risk of breast cancer mortality compared with never smokers. In the past 30 years in Norway, breast cancer incidence has almost doubled while mortality has been stable, or decreased, in the same period The International Agency for Research on Cancer recently classified cigarette smoking as possibly carcinogenic to the human breast The proportion of women who smoke daily in Norway increased from 23% in 1954 to 37% in 1970, before stabilizing at around 32% for the rest of the century Cancer mortality is a function of incidence influenced by exposure to lifetime risk factors, inherited factors and survival after diagnosis n = 7138), women without information on smoking status (n = 2808), education level (n = 6913), body mass index , and physical activity (n = 4207) were excluded from the present analysis, leaving 302,865 women in the analytical cohort.The study population comprised Norwegian women recruited into three prospective cohort studies conducted by the National Health Screening Service : the Norwegian Counties Study (1974–1988), the 40 Years Study (1985–1999) and the Cohort of Norway (CONOR) Study (1994–2003). Selection of participants in these studies was usually based on year of birth and residence . A total of 330,342 women were eligible before exclusion, and if any women participated in more than one study only the first record was used. All women with a cancer diagnosis prior to start of follow-up . Only the CONOR Study had a specific question related to age at smoking initiation. In the other two studies we calculated this variable for both current smokers (age at enrolment minus smoking duration in years) and former smokers (age at quitting smoking minus smoking duration in years). For parous women, the variable “smoking duration before first childbirth” was calculated as age at first childbirth minus age at smoking initiation. All participants who were neither current nor former smokers were classified as never smokers, and never smokers constitute the reference group throughout this paper, unless otherwise noted.Participants were categorized into three groups based on their level of physical activity at enrolment: sedentary , moderate , and heavy . The most recent information regarding duration of education obtained from Statistics Norway was used to assign participants to one of three categories according to duration of education: <10, 10–12, ≥13 years. Information on number of children and age at first childbirth was also obtained through linkages to Statistics Norway. As questions on alcohol consumption were only included from 1994 onwards, information on alcohol consumption was missing in 62% of the women in the analytical cohort.We followed all participants through linkages with the Cancer Registry of Norway, the Norwegian Cause of Death Registry, and the Central Population Register using the unique 11-digit personal identification number to identify all cancer cases, deaths and emigrations. Information about tumor stage at diagnosis, or treatment, was not considered in our analysis. These national registries are accurate and may be regarded as virtually complete We used Cox proportional hazard models  to estimate the multivariate-adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) of breast cancer mortality associated with different measures of smoking exposure . Entry time in the statistical model was defined as age at enrolment, and exit time as age at death, emigration or the end of follow-up (31 December 2007), whichever occurred first.2) and level of physical activity . We analyzed the age- and multivariate-adjusted HRs with 95% CIs for breast cancer mortality according to the selected covariates included in the multivariate analysis.The covariates included in the final models, decided a priori, were age at enrolment (continuous variable), duration of education , number of children , age at first childbirth , BMI , and SAS version 9.4 .The multivariate analysis in Tables At study enrolment, the mean age was 44 years and 59% of the 302,865 Norwegian women were ever smokers. During 14 years of median follow-up, we confirmed 1106 breast cancer deaths and 14,446 deaths by all other causes. Mean age at breast cancer death was 61 years in the Counties Study, 54 years in the 40 Years Study and 66 years in the CONOR Study (Table Ptrend < 0.01), and were positively related with age at first childbirth (Ptrend < 0.01) and with BMI (Ptrend = 0.02). Information on alcohol consumption was only available for 36.9% of the women, and showed a nonsignificant 22% increased risk of breast cancer mortality for those drinking > weekly .Table P for trends ≥0.05) and breast cancer mortality. One significant dose-response association with breast cancer mortality was observed in the Counties Study for number of cigarettes smoked per day (Ptrend < 0.01), but no other consistent pattern was revealed for the trend test when analyzed by study. For smoking duration, we found a reduction in risk increase for smoking 11–20 years from 20%  to nonsignificant 6%  for smoking ≥21 years. A subanalysis for high number of pack-years as a measure of high lifetime smoking exposure showed a nonsignificant increase in breast cancer mortality of only 3%  for ≥21 pack-years (66 cases).Table PWald = 0.80). Comparing the highest number of pack-years (≥11 pack-years) between these birth cohorts showed a 3% risk difference  and , respectively.We also did a stratified analysis by birth cohorts (≤1950>), including 149,270 and 878 breast cancer deaths for those born in and before 1950, and 153,595 women and 228 breast cancer deaths in those born after 1950. We found a 5% difference in breast cancer mortality risk for ever smokers as compared with never smokers, for the oldest birth cohort  and the corresponding figure for those born after 1950 . The test for heterogeneity revealed no significant difference . Stratification for the other exposure variables  by birth cohort (≤1950>) all showed nonsignificant results (data not shown).Stratification by birth cohort (≤1950>) showed a 15% difference in breast cancer mortality for ever smokers as compared with never smokers, for those born in 1950 or before  and the corresponding figure for those born after 1950 . The test for heterogeneity revealed no significant difference , current  and former smokers .We included alcohol consumption as a covariate in the multivariate model after excluding women without alcohol information, restricting the sample size to 114,804 women. All the results became statistically nonsignificant. For ever smokers as compared with never smokers, the estimate for breast cancer mortality increased to 20% . For former smokers the corresponding figure increased to 26% , and for current smokers it decreased to 13% . A Wald's test for heterogeneity between the subpopulation with alcohol information and the full analytical cohort yielded nonsignificant results for ever .Our finding of an increased smoking-related risk of breast cancer mortality, but without a dose-response association between the exposure and outcome variables, is probably a result of competing risks of mortality, as heavy and long-term smokers could have died from other smoking-related diseases The smoking pattern in Norway Most previous studies examining the association between smoking and death from breast cancer have assessed smoking status at breast cancer diagnosis without consideration of lifetime smoking exposure n = 114,804), increased the risk for ever and former smokers to 20% and 26%, respectively, and reduced the risk for current smokers to 13%, as compared to the main analysis. The tests for heterogeneity for smoking status , between the populations with and without alcohol information revealed no significant difference between the results for breast cancer mortality, suggesting that alcohol consumption is of limited importance in this study. However, higher alcohol consumption is a known risk factor for overall mortality before breast cancer diagnosis as a proxy for lifetime smoking exposure, finding a significantly increased mortality risk of 54% for >20 pack-years The report by Pirie et al. from the Million Women Study found a 13% significantly increased risk for breast cancer mortality associated with current smoking To our knowledge, no previous studies have examined in detail the association with smoking before first childbirth and breast cancer mortality, as presented in this paper. We consider it a major strength that smoking assessment in this study was conducted with smoking status at enrolment, and not at breast cancer diagnosis, to show the true risk between lifetime smoking exposure and breast cancer death. Other strengths include its prospective, nationwide population-based cohort design, the large size and the complete follow-up through national registries. Our analytical cohort included a large proportion of young smoking females, many of whom initiated smoking before first childbirth, which enabled us to study this period in detail. Smoking histories were obtained at enrolment and were not subject to recall bias. Other studies have found substantial differences in smoking status in studies with long follow-up Our study also has important limitations: we were not able to adjust for some covariates which may increase breast cancer incidence, such as the use of hormone replacement therapy, use of oral contraceptives, family history of breast cancer, higher mammographic density, higher age at menopause, lower age at menarche, weight gain during adult life, or alcohol consumption Although still controversial


Uruguay exhibits one of the highest rates of breast cancer in Latin America, similar to those of developed nations, the reasons for which are not completely understood. In this study we investigated the effect that ancestral background has on breast cancer susceptibility among Uruguayan women.We carried out a case–control study of 328  women enrolled in public hospitals and private clinics across the country. We estimated ancestral proportions using a panel of nuclear and mitochondrial ancestry informative markers (AIMs) and tested their association with breast cancer risk.Nuclear individual ancestry in cases was (mean ± SD) 9.8 ± 7.6% African, 13.2 ± 10.2% Native American and 77.1 ± 13.1% European, and in controls 9.1 ± 7.5% African, 14.7 ± 11.2% Native American and 76.2 ± 14.2% European. There was no evidence of a difference in nuclear or mitochondrial ancestry between cases and controls. However, European mitochondrial haplogroup H was associated with breast cancer .We have not found evidence that overall genetic ancestry differs between breast cancer patients and controls in Uruguay but we detected an association of the disease with a European mitochondrial lineage, which warrants further investigation.The online version of this article (doi:10.1186/s12905-015-0171-8) contains supplementary material, which is available to authorized users. Breast cancer is the most common malignancy among women, with an annual incidence worldwide of one million cases, of which almost 60% occur in the United States and Europe . The disAmong Uruguayan women breast cancer is the most frequent cancer type as well as being the most common cause of death related to cancer. The nation’s overall age-adjusted incidence rate is 71 cases per 100,000 women per year, and mortality rates due to breast cancer reach 23 per 100,000 women per year ,5. TheseEarlier studies have shown that European genetic ancestry estimated using a set of nuclear ancestry informative markers (AIMs) is positively associated with breast cancer risk in US Hispanic and Mexican women ,16. The In this study we investigated the association of ancestry with breast cancer risk in Uruguayan women, using nuclear and mtDNA AIMs to extensively characterize the ancestral background of patients and controls.A sample of 200 sporadic breast cancer patients and 216 controls was collected in public hospitals and private clinics across Uruguay. Women younger than 45 years of age or with more than one affected relative were excluded from participation to prevent the inclusion in the study of familial cases. The health centers where enrollment took place were Hospital de Clínicas “Manuel Quintela”, Centro Hospitalario Pereira Rossell, Hospital Central de las Fuerzas Armadas, Instituto Nacional del Cáncer and Casa de Galicia in Montevideo , and four hospitals in other regions, i.e. Asistencial de San Carlos , Hospital de Tacuarembó , Cooperativa Médica de Paysandú  and Hospital de Soriano . All patients had been diagnosed with breast cancer a year or less before inclusion in the study. Controls were selected from the same hospitals and clinics as the patients, and similarly, control women with a family history of breast cancer were excluded. Patients and controls filled in a questionnaire that gathered information on socio-demographic, lifestyle, reproductive and family history variables. Individuals recruited from centers where only cases or only controls were available were excluded from analysis. Thus, out of the 416 individuals enrolled to the study, 328 (164 cases and 164 controls) were further analyzed. Of these 141 cases and 139 controls were recruited in public hospitals, whereas 23 cases and 25 controls were recruited in private clinics. A descriptive summary of the study population, by disease status, is given in Table This study was approved by the University of Chicago and the Universidad de la República review boards and all individuals provided written informed consent to participate.www.kbioscience.co.uk), who use their own form of competitive allele specific PCR system (KASPar), and also at the University of Chicago using Sequenom MassArray technology  [Genomic DNA was extracted from peripheral blood leukocytes using a FlexiGene® DNA Kit (QIAGEN) and stored at −20°C until analysis. SNP genotyping was undertaken by KBioscience Ltd.  .We genotyped 166 AIMs located along all autosomes and the X chromosome in 400 individuals for which DNA was successfully extracted (189 cases and 211 controls). These were part of two sets of AIMs: 109 were previously defined by us , and 57 AIMs with minor allele frequencies below 1% and genotyping rate lower than 90% were excluded. After filtering there remained 160 AIMs for ancestry estimation . Complete genotyping to estimate nuclear ancestral proportions was successful in 312 participants (148 cases and 164 controls).The parental populations used to estimate admixture proportions included 42 Europeans (Coriell’s North American panel), 37 West Africans , and 30 Native Americans (15 Mayans and 15 Nahuas), which were genotyped on an Affymetrix 100 K SNP chip . Additionally we had AIM genotypes for 243 Europeans , 279 Africans , and 184 Native Americans .We calculated individual African, Native American and European ancestry in affected and unaffected women using the program Structure . Given tAll 328 samples were successfully genotyped and/or sequenced and assigned to mtDNA haplogroups. We analyzed 14 mtDNA restriction sites that define continental haplogroups and sequenced the hypervariable region I . Individuals were initially assayed for polymorphic variants characterizing major Native American haplogroups A, B, C and D   and at the Institut Pasteur Montevideo (Uruguay). Sequences were examined using the Chromas 2.01 software (Technelysium Pty Ltd.) and aligned using the Genedoc software version 2.7.000 .Haplogroup definition was established following published criteria -38, and Assessment of the association of non-genetic risk factors with susceptibility to breast cancer was carried out using t-tests, ANOVA, and chi-square tests.Differences in ancestry estimates between cases and controls were examined with the Wilcoxon rank-sum test. The association of ancestry with confounders was assessed using linear regression in the case of age, and the continuous anthropometric and reproductive variables, and Kruskal-Wallis and Wilcoxon rank-sum tests in the case of categorical variables. Chi-square tests were applied to compare mtDNA haplogroup distributions. Multivariable logistic regressions were used in the analysis of nuclear and mitochondrial ancestry and disease with adjustment for age, educational achievement and hospital where recruitment took place. Other potential confounders were not included in the models because of sample size reductions due to missing data. All analyses were carried out using the statistical package Stata .Hardy-Weinberg equilibrium was ascertained for all nuclear AIMs using the program PLINK . The assPatients were on average older, less likely to have a university education, and more likely to be unemployed, have lower income and depend more on social security and a pension than controls. Additionally, patients were more likely to have relatives with breast cancer. The mean age at diagnosis was 57 years old, the majority of affected women being postmenopausal (65% compared to 52% in controls). Other well-known breast cancer risk factors, such as early age at menarche, taking oral contraceptives, nulliparity, and breastfeeding, did not show evidence of association with the disease. Age at birth of first child and at menopause was higher in cases than in controls. Among controls, red meat consumption was slightly more frequent than in patients Table .AIMs rs7504, rs35395 and rs1341567 were out of Hardy-Weinberg equilibrium (p < 0.001), after a Bonferroni correction for multiple testing (data available on request).We confirmed the tri-hybrid nature of the Uruguayan population, which showed contributions from Africans, Native Americans and Europeans. We found that the Structure model assuming a three-way admixture process provided the best fit, compared to models considering either one or two parental populations (data not shown). Ancestral proportions in the population as a whole were (mean ± SD) 9.4 ± 7.5%, 14.0 ± 10.8% and 76.6 ± 13.7%, respectively. There was considerable variation in ancestry among participants. Nuclear DNA African ancestry ranged from 1% to 43%, Native American ancestry from 1% to 48%, and European ancestry from 33% to 96%  and hospital where receiving care, but not with place of residence or location .We ran logistic regression models to test for the effect of ancestry on breast cancer risk with adjustment for age, education and hospital. Education was strongly correlated with socioeconomic status, therefore we did not include the latter in the analyses . European ancestry was positively associated with risk, as was African ancestry, whereas Native American ancestry showed an inverse association. However, there was no strong statistical support for these effects Table .Table 3JAZF1 . No associations were detected if a correction for multiple testing was implemented.There were 16 AIMs associated with breast cancer risk as indicated by the Cochran-Armitage trend test (p ≤ 0.05). Thirteen AIMs showed an association with disease when adjustment for European and Native American ancestry was performed .Potential confounders were not associated with mtDNA ancestry with the exception of hospital and place of residence, and weakly with income, in controls. Individuals with a Native American mitochondrial haplogroup were less likely to receive medical attention at a private clinic, and to reside outside Montevideo  [JAZF1 that independently explain the associations with each disease [The strongest associated AIM, although not showing a robust enough association to overcome a Bonferroni correction for multiple testing, rs10486576, is located in the es (T2D) , height es (T2D)  and proses (T2D) . Two sep disease . In factWe did not find strong evidence of association of nuclear and mitochondrial ancestry estimates with breast cancer risk. This may reflect a lack of statistical power, consequence of a small sample size. On the other hand, it may be indicative of the role of non-genetic factors, possibly related to lifestyle and environmental conditions, on the onset of breast cancer. Epigenetic effects may also be at play . Although not robust, we observed an increased risk for breast cancer with higher European ancestry and, concurrently, a protective effect of Native American ancestry. The direction of the ancestry effects we uncovered is consistent with associations reported for US Latinas and Mexican women ,16,45 foNuclear ancestral proportions varied with educational achievement, different indicators of socioeconomic status and hospital where receiving medical attention. Individuals who attended university, reported being of middle class status and were recruited at a private clinic had on average higher European and lower African and Native American ancestry than participants of a lower socioeconomic position. Age at first child and age at menopause were strongly associated with ancestry and at the same time with socioeconomic status and education.Mitochondrial European haplogroup H showed an association with breast cancer susceptibility, with carriers of the haplogroup having approximately twice the risk of being diagnosed with the disease than non-carriers, whether considering all volunteers or only those of European mitochondrial ancestry. These results suggest a potential influence of ancestry on breast cancer risk that is more obvious with mitochondrial than with nuclear DNA polymorphisms, an actual effect of haplogroup H-linked variants on the disease, or on the other hand, they could be due to chance or inadequate adjustment for population stratification, and therefore require further investigation .BRCA2 carriers whereas haplogroup X was more frequent in BRCA1 carriers compared to patients not carrying BRCA mutations among Italian familial breast cancer cases [BRCA2 over BRCA1 mutations in breast cancer families, which may be linked to their ethnic origin [Earlier studies on the relationship of mitochondrial haplogroups and breast cancer reported an increased risk associated with haplogroups I and K and a protective role for haplogroups H and U -49. Theser cases . Althougc origin .The presence of population structure has important consequences for association studies of genetic risk factors underlying complex diseases and traits with a heterogeneous worldwide distribution as it may lead to false positive findings. Confounding by population stratification is particularly relevant in studies that examine mtDNA variants, as mtDNA is highly structured in human populations due to its lower effective population size .We have shown that in the Uruguayan population the genetic ancestral background is correlated with socioeconomic status and educational attainment, a feature shared with other Latin American populations ,55, and As the characteristics of our study sample diverge in part from those of previously published studies from Uruguay  we cannAdditionally, it is possible that, by excluding cases and controls with a family history of breast cancer, and assuming that women with such history are more likely to be of European origin ,58, the We did not find differences in biparental or maternal ancestral proportions between breast cancer patients and controls in Uruguay, but we uncovered an association of European mitochondrial haplogroup H with breast cancer risk. These findings require replication in an independent sample. In addition, we note that the presence of population structure in Uruguay, likely emerging from the admixture process that took place due to the conquest and colonization of the Americas, could affect the outcome of association studies carried out in this population.


Using data from the French E3N prospective cohort, Marina Kvaskoff and colleagues examine the association between number of cutaneous nevi and the risk for breast cancer.Please see later in the article for the Editors' Summary While melanocytic nevi have been associated with genetic factors and childhood sun exposure, several observations also suggest a potential hormonal influence on nevi. To test the hypothesis that nevi are associated with breast tumor risk, we explored the relationships between number of nevi and benign and malignant breast disease risk.ptrend = 0.04), although significance was lost after adjustment for personal history of BBD or family history of breast cancer. The 10-y absolute risk of invasive breast cancer increased from 3,749 per 100,000 women without nevi to 4,124  per 100,000 women with “very many” nevi. The association was restricted to premenopausal women , even after full adjustment , but did not differ according to breast cancer type or hormone receptor status. In addition, we observed significantly positive dose–response relationships between number of nevi and history of biopsy-confirmed BBD  and family history of breast cancer in first-degree relatives . The main limitations of our study include self-report of number of nevi using a qualitative scale, and self-reported history of biopsied BBD.We prospectively analyzed data from E3N, a cohort of French women aged 40–65 y at inclusion in 1990. Number of nevi was collected at inclusion. Hazard ratios (HRs) for breast cancer and 95% confidence intervals (CIs) were calculated using Cox proportional hazards regression models. Associations of number of nevi with personal history of benign breast disease (BBD) and family history of breast cancer were estimated using logistic regression. Over the period 15 June 1990–15 June 2008, 5,956 incident breast cancer cases  were ascertained among 89,902 women. In models adjusted for age, education, and known breast cancer risk factors, women with “very many” nevi had a significantly higher breast cancer risk . Uncontrolled cell division leads to the formation of a lump that can be detected by mammography (a breast X-ray) or by manual breast examination. Breast cancer is treated by surgical removal of the lump, or, if the cancer has started to spread, by removal of the whole breast (mastectomy). Surgery is usually followed by radiotherapy or chemotherapy to kill any remaining cancer cells. Because the female sex hormones estrogen and progesterone stimulate the growth of some tumors, drugs that block hormone receptors are also used to treat receptor-positive breast cancer. Nowadays, the prognosis (outlook) for women with breast cancer is good, and in developed countries, nearly 90% of affected women are still alive five years after diagnosis.Several hormone-related factors affect a woman's chances of developing breast cancer. For example, women who have no children or who have them late in life have a higher breast cancer risk than women who have several children when they are young because pregnancy alters sex hormone levels. Interestingly, the development of moles (nevi)—dark skin blemishes that are risk factors for the development of melanoma, a type of skin cancer—may also be affected by estrogen and progesterone. Thus, the number of nevi might be a marker of blood hormone levels and might predict breast cancer risk. In this prospective cohort study, the researchers test this hypothesis by investigating the association between how many moles a woman has and her breast cancer risk. A prospective cohort study enrolls a group (cohort) of people, determines their baseline characteristics, and follows them over time to see which characteristics are associated with the development of specific diseases.In 1990, the E3N prospective cohort study enrolled nearly 100,000 French women (mainly school teachers) aged 40–65 years to investigate cancer risk factors. The women completed a baseline questionnaire about their lifestyle and medical history, and regular follow-up questionnaires that asked about cancer occurrence. In the initial questionnaire, the women indicated whether they had no, a few, many, or very many moles. Between 1990 and 2008, nearly 6,000 women in the cohort developed breast cancer. Using statistical methods to calculate hazard ratios , the researchers report that women with “very many” nevi had a significantly higher breast cancer risk (a higher risk that was unlikely to have occurred by chance) than women with no nevi. Specifically, the age-adjusted HR for breast cancer among women with “very many” nevi compared to women with no nevi was 1.17. After adjustment for a personal history of benign (noncancerous) breast disease and a family history of breast cancer (two established risk factors for breast cancer), the association between nevi and breast cancer risk among the whole cohort became nonsignificant. Notably, however, the association among only premenopausal women remained significant after full adjustment (HR = 1.34), which corresponded to an increase in ten-year absolute risk of invasive breast cancer from 2,515 per 100,000 women with no nevi to 3,370 per 100,000 women with “very many” nevi.PLOS Medicine Research Article, they suggest that further studies into the biological mechanisms underlying the relationship between nevi and breast cancer and the association itself should be undertaken.These findings suggest that among premenopausal women there is a modest association between nevi number and breast cancer risk. This noncausal relationship may indicate that nevi and breast diseases are affected in similar ways by hormones or share common genetic factors, but the accuracy of these findings may be limited by aspects of the study design. For example, self-report of nevi numbers using a qualitative scale may have introduced some inaccuracies into the estimates of the association between nevi number and breast cancer risk. Most importantly, these findings are insufficient to support the use of nevi counts in breast cancer screening or diagnosis. Rather, together with the findings reported by Zhang et al. in an independent http://dx.doi.org/10.1371/journal.pmed.1001660.Please access these websites via the online version of this summary at PLOS MedicinePerspective by Fuhrman and CardenasThis study is further discussed in a PLOS MedicineResearch Article by Zhang et al. also investigates the relationship between nevi number and breast cancer riskAn independent US National Cancer Institute provides comprehensive information about cancer (in English and Spanish), including detailed information for patients and professionals about breast cancer; it also has a fact sheet on molesThe Cancer Research UK, a not-for profit organization, provides information about cancer, including detailed information on breast cancerbreast cancer; the not-for profit organization Healthtalkonline also provides personal stories about dealing with breast cancerThe UK National Health Service Choices website has information and personal stories about E3N prospective cohort study is available; detailed information is available in FrenchMore information about the  CDKN2A and MTAP, both located at the 9p21 locus, and PLA2G6, located at 22q13 Melanocytic nevi (hereafter referred to as nevi) are benign skin tumors resulting from epidermal melanocyte proliferation. They occur more frequently in fair- than in dark-skinned individuals and can be either congenital or acquired later in life. Nevus acquisition starts early in childhood and peaks during puberty, then declines in adulthood, with progressive loss with age CDKN2A gene CDKN2A inactivation has been shown to be common in breast cancer Number of nevi is the strongest known risk factor for cutaneous melanoma In order to investigate whether nevus count is associated with breast tumor risk, we explored the relationships between number of nevi and the risks of benign and malignant breast diseases and family history of breast cancer, in the French E3N prospective cohort.The E3N cohort received ethical approval from the French National Commission for Computed Data and Individual Freedom , and all participants in the study provided informed consent.E3N is a prospective cohort study involving 98,995 women born in 1925–1950, living in metropolitan France at inclusion and insured by the Mutuelle Générale de l'Éducation Nationale, a national health scheme primarily covering teachers. The cohort has been described in detail elsewhere n = 1,415, 27% of the tumors)—were excluded from the analyses stratified according to hormone receptor status.All cohort questionnaires inquired about the occurrence of cancer, including breast cancer, requesting contact details of the participants' physicians and permission to contact them. A small number of breast cancer cases were further identified from insurance files and death certificates. Pathology reports were obtained for 93% of incident cases. We also considered cases for which pathology reports had not been obtained, because the proportion of false-positive self-reports was low in our study population (<5%). Information on ascertained estrogen receptor (ER) and progesterone receptor (PR) status was extracted from pathology reports, and invasive breast cancer cases were classified accordingly into four categories: ER+/PR+, ER+/PR−, ER−/PR+, and ER−/PR−. Women with unknown receptor status—mostly with tumors diagnosed in the early years of follow-up, when determining hormone receptor status was not compulsory . Women also reported whether a biopsy had been performed for these diseases. This information was then collected prospectively in subsequent questionnaires . To avoid the potential selection bias of including only late-diagnosed BBD, both prevalent and incident BBDs were included in the analyses. When history of BBD was analyzed as a potential confounder, both biopsied and non-biopsied BBDs were included in order to maximize statistical power; however, we performed a sensitivity analysis excluding non-biopsied BBDs and verified the stability of the findings. When history of BBD was analyzed as an outcome, only biopsy-confirmed BBDs were considered in order to minimize misclassification.The 1990 and 1992 questionnaires asked women to report if they had ever had a personal history of benign breast disease (BBD)  before inclusion in the cohort , bilateral oophorectomy, or use of MHT, or if they self-reported being postmenopausal. Date of menopause was defined as the date of last menstrual period , date of bilateral oophorectomy, or, in decreasing order of priority, self-reported date of menopause, date MHT use began, or date menopausal symptoms began; if no information on date of menopause was available, date of menopause was defined as the date corresponding to age 47 y if menopause was known to be artificial, or age 51 y otherwise, i.e., the median ages for artificial and natural menopause in the cohort, respectively.The inclusion questionnaire asked women to self-report their number of moles, with four possible answers: none, a few, many, or very many. The questionnaire also inquired about education level, skin complexion, height and weight, and physical activity. Data on age at menarche, parity, age at first full-term pregnancy, and breastfeeding were collected in the inclusion (1990) and 1992 questionnaires. Data on use of oral contraceptives (OCs), premenopausal progestogens, and menopausal hormone therapy (MHT) were collected in 1992 and updated in each follow-up questionnaire. Body mass index (BMI) was calculated as weight in kilograms, reported at inclusion and updated at each follow-up questionnaire, divided by height in meters squared. Menopausal status, age at menopause, and history of recent mammographic exam (in the period since the previous questionnaire) were collected at inclusion and at each follow-up questionnaire. To obtain average levels of residential sun exposure during childhood and adulthood, we linked data on county of birth and county of residence at inclusion to a database from the Joint Research Centre of the European Commission containing mean daily ultraviolet (UV) radiation dose in French counties Statistical analyses were performed using SAS software . Hazard ratios (HRs) for breast cancer and 95% confidence intervals (CIs) were estimated using Cox proportional hazards regression models with age as the timescale. We also calculated absolute risks of breast cancer associated with the number of nevi. The association between number of nevi and breast cancer risk was assessed using different adjustment models. We first used age-adjusted models stratified by birth cohort in 5-y categories (Model 1) and then built a model including as covariates only those factors whose inclusion resulted in a 10% change of the HR for number of nevi. Besides age and birth cohort, this model included education , use of premenopausal progestogens (ever/never), menopausal status , age at menopause in postmenopausal women (<51 or ≥51 y), and use of MHT in postmenopausal women (ever/never) (Model 2). Because further adjustment for personal history of BBD or family history of breast cancer resulted in the largest changes in HRs for breast cancer, we evaluated their effect separately by creating two models, Model 2 further adjusted for personal history of BBD (Model 3) and Model 3 additionally adjusted for family history of breast cancer (Model 4). Finally, a fully adjusted model included all the preceding covariates along with the remaining potential confounders, including BMI, height, physical activity, age at menarche, parity, age at first full-term pregnancy, breastfeeding, use of OCs, personal history of mammographic exam, and UV dose in county of birth and of residence at cohort inclusion (Model 5). All models were generated both for in situ and invasive breast cancer cases combined, and for in situ and invasive cases separately, and homogeneity tests were used to compare estimates between in situ and invasive cases. We performed tests for linear trend across categories of number of nevi by assigning a numerical value to each category. Effect modification was evaluated using interaction tests. In addition, we stratified the analyses according to menopausal status, and we performed stratified analyses according to hormone receptor status or tumor histological type in invasive cases using competing-risk models We additionally used a case–control design to explore the relationships between number of nevi and (1) personal history of BBD and (2) family history of breast cancer in first-degree relatives. For this analysis, unconditional logistic regression models were used to estimate odds ratios (ORs) and 95% CIs. For history of BBD, potential confounders were included only if their inclusion resulted in at least a 10% change in the OR. Final models included age at cohort inclusion, age at last returned questionnaire, education, premenopausal progestogens, menopausal status, age at menopause, use of MHT in postmenopausal women, and family history of breast cancer. For family history of breast cancer, models included education and age at cohort inclusion.n = 2,211; 2.2% of the total cohort) were more likely to be older, to have a late menarche, to be younger at their first full-term pregnancy, and to be nulliparous, but were less likely to have a high education level or a high physical activity level at baseline; to be overweight or obese; to be tall; to have ever used OCs, premenopausal progestogens, or MHT; to have ever breastfed; to have ever had a history of BBD, a mammographic exam, or a family history of breast cancer; or to have a high UV dose in their county of residence at baseline compared to those with available data on this factor , with a cancer history before inclusion , lost to follow-up after they replied to the inclusion questionnaire , or with primary amenorrhea (n = 27). Our final sample for analysis consisted of 89,802 women. Woman-years were computed from the date the first questionnaire was returned to the date of diagnosis of breast cancer or any other cancer, date of last questionnaire returned, or date of end of follow-up , whichever occurred first.From the original study population, we excluded women with missing data on number of nevi . For analyses exploring family history of breast cancer, we excluded from the original study population women with missing data on number of nevi  and those with missing data on family history of breast cancer (n = 728). The final study populations for these analyses included 63,629 women for history of BBD and 96,056 women for family history of breast cancer.For the analyses exploring number of nevi in relation to history of BBD, we further excluded women with non-biopsied BBD  were ascertained among the 89,902 included women. phomogeneity = 0.27 in Model 2); however, the HR for number of nevi in invasive cases was statistically significant in the model adjusted only for age (Model 1). The 10-y absolute risk of invasive breast cancer increased from 3,749 per 100,000 women without nevi to 4,124  per 100,000 women with “very many” nevi.In age-adjusted models, women in the highest category of number of nevi had a significantly higher breast cancer risk  compared to those in the lowest . The assAlthough “none” was the obvious reference category to explore number of nevi, it was the smallest category in our cohort, and it could be argued that this could inflate error estimates. However, since the “none” group was not small per se—it included over 9,000 participants—and since our findings were not substantially modified when testing different reference groups for this variable (“none/a few”: pinteraction = 0.27) or family history of breast cancer (pinteraction = 0.97) for the risk of breast cancer. We also detected no significant interaction of number of nevi with skin color (pinteraction = 0.73), physical activity at inclusion (pinteraction = 0.17), UV dose in county of birth (pinteraction = 0.07), or UV dose in county of residence at inclusion (pinteraction = 0.06). Since the p-values for the last two factors were close to statistical significance, we conducted stratified analyses according to the median of UV dose in county of birth . The association was restricted to premenopausal women, in whom the relation remained even in the multivariable model   , corresp = 0.03)  or ER/PR = 0.03)  yielded ptrend<0.0001) and family history of breast cancer in first-degree relatives .Associations between number of nevi and BBD and family history of breast cancer are reported in In the present study, we found a modest association between number of nevi and overall breast cancer risk, which was restricted to premenopausal women. In these women, the highest versus lowest category of number of nevi was associated with a HR of 1.34 for breast cancer . A high number of nevi was also associated with the risk of biopsy-confirmed BBD and with family history of breast cancer in first-degree relatives. While adjustment for personal history of BBD and family history of breast cancer reduced the association between number of nevi and overall breast cancer risk, it did not substantially modify the association with premenopausal breast cancer risk.While a causal relationship between number of nevi and breast disease risk seems unlikely, our observed associations between number of nevi and risk of premenopausal breast cancer, history of BBD, and family history of breast cancer may suggest at least two potential mechanisms.One potential mechanism is that these relations could reflect a common hormonal influence on nevi and breast diseases. The fact that significant associations were restricted to premenopausal breast cancer and that number of nevi was associated with both BBD and breast cancer risk are consistent with this hypothesis, although we failed to find a stronger association with ER+ than with ER− tumors, possibly because of a lack of power in subgroup analyses. Consistent with this hypothesis, melanocytes are known to express estrogen and androgen receptors CDKN2A mutations CDKN2A, a tumor suppressor gene encoding cyclin-dependent kinase inhibitors known to be frequently mutated or suppressed in a number of cancers. This gene has been identified as a high penetrance susceptibility gene for melanoma, with germline mutations occurring in 20%–40% of families with three or more melanoma cases CDKN2A and CDKN2B at 9p21, rs1011970, was reported to be associated with breast cancer in a recent genome-wide scan A second potential mechanism is that nevi and breast diseases could share genetic factors, which is consistent with our observed association between number of nevi and family history of breast cancer. Interestingly, several studies have reported associations between melanoma and breast cancer, finding either a higher risk of breast cancer following melanoma diagnosis CDKN2A codes for two proteins, p14 and p16 However, because it is unclear whether the associations we found reflect common hormonal, genetic, or environmental pathways, more research is warranted to understand their underlying biological mechanisms.Strengths of our study include the large sample size and prospective design of the E3N cohort; we also had detailed data on breast cancer cases, personal history of BBD, and family history of breast cancer.The main limitation regarded self-report of nevi number, and use of a qualitative scale instead of counts. Repeatability studies of number of nevi indeed show a moderate reliability Another limitation is that race information was not available in our cohort, as this type of question is not acceptable to French ethical committees. However, while E3N women did report their skin color, we detected no significant interaction in our findings according to this factor. Further, while the large sample size of our cohort makes it possible to detect small associations, our findings regarding breast cancer risk were of modest effect size and sensitive to adjustment, particularly for history of BBD and family history of breast cancer, for which we found an independent association with number of nevi. Also, given the number of interaction tests that we performed, some of our findings could be attributable to chance and should therefore be interpreted with caution. However, while a multiple testing issue can arise when a high number of hypothesis-free tests are performed Another limitation was that history of BBD was self-reported and could not always be confirmed through biopsy reports, which may have introduced some degree of misclassification. However, when we studied BBD as a potential confounder, our results were unchanged whether analyses included all BBDs or biopsied BBDs only, and when we studied history of BBD as an outcome, restricting the analyses to biopsy-confirmed BBD likely reduced this bias.Another potential bias could arise from a complex relationship between UV exposure, number of nevi, and vitamin D levels. Indeed, exposure to UVB rays results in higher vitamin D synthesis, and normal versus insufficient or deficient vitamin D levels in adulthood have been associated with a reduced breast cancer risk In conclusion, these data from a large prospective cohort study suggest associations between number of nevi and risk of premenopausal breast cancer, history of BBD, and family history of breast cancer. Because associations were modest and the results for breast cancer were sensitive to adjustment, we mostly consider our findings in terms of enhancement of our knowledge of pathophysiological mechanisms. More research is warranted before these findings could possibly be used in diagnosis or screening scores for breast cancer. If confirmed, these findings may suggest that nevi could be associated with other markers of breast cancer risk, such as mammographic density, which should warrant a specific study.Table S1Baseline characteristics of the study population according to the availability of data on number of nevi.(DOCX)Click here for additional data file.Table S2Hazard ratios and 95% confidence intervals for number of nevi  in relation to the risk of breast cancer, E3N cohort .(DOCX)Click here for additional data file.Table S3Hazard ratios and 95% confidence intervals for number of nevi  in relation to the risk of breast cancer, E3N cohort .(DOCX)Click here for additional data file.Table S4Hazard ratios and 95% confidence intervals for number of nevi in relation to the risk of breast cancer, stratified by mean UV dose in county of birth, E3N cohort .(DOCX)Click here for additional data file.Table S5Hazard ratios and 95% confidence intervals for number of nevi in relation to the risk of breast cancer, stratified by mean UV dose in county of residence at baseline, E3N cohort .(DOCX)Click here for additional data file.


Folate has been shown to play a complex but unclear role in carcinogenesis, with some studies showing that low folate intake protects against early carcinogenesis while high folate intake promotes advanced carcinogenesis. Other studies have shown that high folate is associated with decreased breast cancer risk and overall survival, yet others found no such association.This study therefore sought to determine the association between red blood folate levels and breast cancer among women seen at a tertiary Ugandan hospital.A case control study was conducted where female patients with a histological diagnosis of breast cancer were recruited as cases, and females without cancer attending other surgical clinics as controls. Demographics and social behavior data were collected and 5mls of blood drawn for laboratory testing of red blood cell (RBC) folate, serum vitamin B12 and RBC count. Ethical approval was obtained.P = 0.290. Ninety participants (63%) had low RBC folate and 53 participants (37%) had normal RBC folate. Thirty five (45%) of the women from a rural setting had normal folate levels compared to 18(28%) women from an urban setting.In this study, a total of 145 women were recruited as 72 cases and 73 controls. The odds of having breast cancer among women with normal folate levels compared to those with low folate levels were 1.4 (95% CI 0.7 to 2.9) There was no significant association found between RBC folate and breast cancer among this group of women in Uganda. Most studies have looked at high dietary folate food as a protective factor against breast cancer and most of these studies were carried out in countries where either folate fortification is performed or the incomes and nutritional status of participants are higher than in Uganda.This was a hospital-based case control study. The study was carried out at the Breast Clinic at Mulago National Referral and Teaching Hospital of Makerere, University College of Health Sciences, Uganda.Mulago Hospital is located in Kampala, the capital city of Uganda. Patients seen in Mulago Hospital are referred from all over the country; however a significant number of patients are self-referred.The study included patients attending the Breast Clinic with histologically confirmed breast cancer and not receiving chemotherapy.Patients who were 18 years of age and above were included because breast cancer is rare below this age. Secondly, 18 years is the minimum age in Uganda at which written and informed consent is obtained.Patients who were pregnant, taking folic acid or multivitamin supplementations containing folic acid, taking anticonvulsants, and patients with a history of menorrhagia (menstruation lasting more than seven days) were excluded.The control patients were without breast cancer or any other cancer . They were recruited from surgical outpatient clinics other than the Breast Clinic. Excluded from the control group were pregnant women, participants taking folic acid supplementation or multivitamins containing folic acid, patients taking anticonvulsants, or those with a history of menorrhagia , residence , use of hormonal contraception, tobacco use versus no tobacco usage, alcohol consumption, hemoglobin level, and serum vitamin B12 level.The purpose of the study, the assessment, and sample collection procedures were carefully explained to each patient individually. Participants were allowed to ask questions freely to ensure that they understood the intention of the study.Screening for suitability included history taking and physical examination.Written informed consent was obtained from those who satisfied the inclusion criteria and they were subsequently enrolled in the study on a consecutive basis. Those who did not meet the inclusion criteria as well as those who declined to participate in this study were offered the care that had been scheduled.Under aseptic conditions, 5mls of blood from each participant was drawn from the antecubital vein. Following this, 3mls were put into a red top vacutainer and 2mls were put into a purple top vacutainer containing ethylenediaminetetraacetic acid (EDTA). The samples were transported to the lab for processing at below room temperature and within two hours. Blood samples were stored in a freezer at -20°C to ensure that they were available in case the need for re-examination arose in the near future.Samples were processed by using the COBAS e601 analyzer  and two lab technologists processed the samples.Interviews were conducted using a pretested questionnaire and laboratory results were recorded. The laboratory result forms were safely and confidentially stored.P values were two tailed and P values of <0.05 were considered to indicate statistical significance. Categorical data was displayed as frequencies and percentages.Data was double entered using Epidata version 3.1 . The data was checked for range and consistency. Data was transferred to STATA version 11.0  and normal folate (471to 1289 ng/ml). The outcome variable was breast cancer which was treated as a binary variable: breast cancer cases and controls. All continuous variables were arranged into categories to allow usage of the Chi squared test.2), normal (18.5 to 24.9 kg/m2), overweight (25 to 29.9 kg/m2), and obese (30 kg/m plus2). Parity was categorized as nulliparous (zero), low parity (one to four), and mutiparity (five and above). Menopausal status was binary; pre-menopausal and post-menopausal. Menopause was defined as age at which the participant ceased menstruation plus one year.Age was grouped into five categories: 18 to 39, 40 to 49, 50 to 59, 60 to 69, and 70 and above. Social and cultural behavioral factors which were assumed to influence participant’s dietary patterns were mainly determined by looking at residence, which were categorized as rural versus urban. Body Mass Index was categorized as underweight  guidelines for non-pregnant women over the age of 15 years; severe 12, BMI, alcohol, tobacco use and contraceptive use).The other variables were categorized according to whether the participant was identified with the variable or not. Chi-square and logistic regression were used to examine the association between RBC folate levels and breast cancer. Bivalent association between other factors and having breast cancer was examined and presented as odds ratio, and multivariate logistic regression was used to look for potential confounders  were processed.Samples were kept in a freezer  at -20°C in the clinical chemistry laboratory for future re-evaluation if need arose.Institutional ethical approval was obtained from the Ethics and Research Committees of Makerere University College of Health Sciences. Written informed consent was obtained from each study participant.Seventy two cases of breast cancer and 73 controls were enrolled in the study from January 2012 to May 2012. Two cases were not included in the analysis due to missing laboratory results. Study participants were compared for study variables and results are shown in Table The mean age for cases was 51 years (range 23 to 87) and the mean age for controls was 52 years (range 23 to 80).Only 14(10%) of women reported tobacco use, 6(4%) reported smoking, 2(2%) reported use of a pipe and 6(4%) reported chewing tobacco. The average frequency of use of tobacco per day was 2.3 times ranging from one to four times a week.The average age of menarche was 14 years (range 9 to 20). The mean ages at menarche among cases and controls were 14 (range 10 to 20) and 14 years (range 10 to 19), respectively.The overall average age at menopause was 48 years (38 to 66). Among cases and controls, the average was 48 (40 to 66) and 49(38 to 55) years respectively.The number of participants that reported using contraceptives was 52 (36%) with an average duration of contraceptive use of 4 years (range 1 to 15). Of these, 26 (37%) of cases reported the use of oral contraceptives compared to 26 (36%) of controls.The mean RBC folate level was 430 ng/ml; range (105 ng/ml to 857 ng/ml) as shown in Figure The average vitamin B12 level among women was 445 pg/ml (50 to 2000) with cases showing an average of 492 ng/dl (50 to 2000) and controls an average of 401 ng/ml (58 to 1114). Figure The mean hemoglobin level was 13 g/dl (6.6 g/dl to 16.7 g/dl), with a mean of 12.7 g/dl (6.6 to 16.3) and 13.7 g/dl (9.2 to 16.7 g/dl) among cases and control respectively. See Figure Among cases, 6 participants were nulliparous (8%), 23 participants were of low parity (32%) and 43 were multiparous (60%). Among controls, 1 participant was nulliparous (1%), 19 participants had low parity (26%) and 53 were multiparous (73%).Among the cases, one participant was underweight (1%), 38 participants had normal weight (53%), 18 participants were overweight (25%) and 15 participants were obese (21%). Among controls, 4 participants were underweight 6%, 29 participants had normal weight (40%), 25 participants were overweight (34%) and 15 participants were obese (21%).Among the cases, 43 (60%) reported alcohol use compared to 29 (40%) who reported no alcohol usage. Among the controls, 30 (41%) reported alcohol usage compared to 43 (59%) who reported no alcohol usage.Among the cases, 41 (59%) were from rural areas and 29 (41%) were from urban areas. Among the controls, 38 (52%) were from rural areas compared to 35 (49%) who were from urban areas.Contraceptive use and area of residence were the only characteristics associated with RBC folate levels Table . Thirty P value 0.291. Multivariate logistic regression showed that none of the potential confounding factors examined affected the association between folate levels and breast cancer.There was no association seen between RBC folate levels and breast cancer among study subjects after controlling for confounding factors. Using logistic regression, the odds of having breast cancer among women with normal folate levels compared to those with low folate levels were 1.4 (95% CI 0.7 to 2.9), 2), showed that alcohol use was associated with having breast cancer(Table P = 0.026). Details of the bivariate association of others factors with having breast cancer are shown in Table Examination of the crude association between other study variables using Pearson’s chi-squared test  Folate plays a very important biochemical role in DNA synthesis, repair and methylation. Folate Cytosine methylation produces 5-methylcytosine, which is important for chromosome inactivation. It has therefore been hypothesized that high intake of folate may reduce the risk of human cancers, including breast cancer.et al.in 1999 and Rossi et al. in 2006[Though not statistically significant, our findings suggest that females with low folate are less likely to have breast cancer when compared with females who have normal folate levels. This may mean that the observed higher RBC folate among cases may be associated with breast cancer. However, the observation of lower RBC folate among controls may also mean that low folate is protective against early breast carcinogenesis. These observations may lend themselves to other studies that have suggested that high folate is associated with breast cancer events, and that low folate is protective against breast cancer. This dual modulatory effect of folate on cancer has been observed by other epidemiologic, clinical, and animal studies. The obs in 2006, 11. The in 2006, 13. How in 2006, 9.We observed that females in our study had lower RBC folate than those from other countries, including China, Mexico and Australia where most of the studies on the association between RBC folate and breast cancer have been conducted, 12–15, Both the mean and range of RBC folate levels was slightly higher among cases, although this observation was not statistically significant. It is possible that the reason for this disparity lies in the fact that the breast cancer patients seen are prevalent rather than incident cases, and dietary changes due to nutritional improvement following illness may occur during the course of the disease which may affect RBC folate levels. On the other hand, since the difference among cases and controls is not statistically significant, the observed difference may not merit further scrutiny.Confounding factors that were studied were not found to have an effect on the association between RBC folate and breast cancer. These included age, residence, alcohol use, BMI, tobacco use, hormonal contraceptive use, menopausal status, vitamin B12 levels, hemoglobin level, parity, and age at menarche and menopause.This study showed that 43% of study participants from rural areas were either overweight or obese compared to 62% overweight or obese participants from urban areas. This observation shows that living in urban settings is associated with either overweight or obese BMIs when compared to leaving in rural areas. Participants of a normal weight were found to differ considerably based on area, with 51% of participants coming from rural areas compared to only 34% coming from urban areas. Social and dietary habits may be the contributing factor, as participants from an urban area are more exposed to processed foodstuffs and drinks which may be rich in fats and calories, compared to participants from rural areas whose diet may mainly be based on cereals and fresh foods.The average age of underweight participants was 64,that of normal weight participants was 52, that of overweight participants was 49, and that of obese participants was 52. Although underweight is observed to be associated with older age, other age categories were not associated with BMI. The observed low BMI among elderly participants may be due to the reduced general health status that is associated with this age group.In this study, we used RBC folate levels instead of serum folate levels, 17 giveThis study used the RBC folate cutoffs from the Australian population (471 ng/ml). Though this was found to be higher than the average RBC folate of the study controls, an earlier study observed that the status of folate levels among healthy young individuals was not different from that in Australia.It was not possible to study only incident cases as patients are likely to seek care from other sources before they present at the national referral hospital, therefore the pre-morbid folate status may have been altered by interventions such as herbal medication (which may be rich in folate).Dietary history was not obtained because of lack of a contextually appropriate tool. This would have provided a more accurate pre-morbid folate exposure.Vitamin B6 (pyridoxine) was not studied yet it is an obligate cofactor, together with vitamin B12, for the conversion to the form of folate used for DNA synthesis and methylation.Both the cases and controls may not have been true representatives of the general population given the fact that this was a hospital-based study, although this hospital is the only public institution with free comprehensive breast cancer services in the country and the majority of patients depend on free health care.There could have been recall bias, for other variables such as age, contraceptive use though the main predictor variable was a biomarker which is not influenced by what a participant may recall.There was no association found between RBC folate levels and breast cancer.


There are an increasing number of total body irradiation (TBI) survivors. UK haematology and oncology units follow international guidelines and advise that they should have annual mammography from age 40.Blood in 2008 based on 3,337 women from 83 centres, who had survived at least 5 years after transplant [A literature review found a single large study published in ansplant . The radThe study found that: overall breast cancer risk was moderately increased, by 2.2 (95% CI = 1.7 to 2.9); risk was concentrated in younger patients, those treated in their teens and twenties; and the standardised incidence of those presenting after 20 years was also 10-fold that expected.There are similarities between the risks of TBI and mantle radiotherapy, cancers occurring if irradiated when young and cumulative risk increasing disproportionately with length of follow up. More work needs to be done comparing the radiation dose to the breast and relative breast cancer risk of TBI and mantle radiotherapy. If this shows that the dose and risk are similar, then TBI patients are most likely to benefit from similar breast imaging follow up.