While the estrogenic properties of certain pesticides have been established, associations between pesticide exposure and risk of breast cancer have been inconsistently observed. We investigated the relation between pesticide exposure and breast cancer risk using methods capable of objectively assessing exposure to specific pesticides occurring decades before diagnosis.A case–control study was conducted to evaluate the risk of postmenopausal breast cancer associated with historic pesticide exposure in California’s Central Valley, the most agriculturally productive region in the United States where pesticide drift poses a major source of nonoccupational exposure. Residential and occupational histories were linked to commercial pesticide reports and land use data to determine exposure to specific chemicals. Cases (N = 155) were recruited from a population-based cancer registry, and controls (N = 150) were obtained from tax assessor and Medicare list mailings.There was no association between breast cancer and exposure to a selected group of organochlorine pesticides thought to have synergistic endocrine-disrupting potential; however, breast cancer was three times as likely to occur among women exposed to chlorpyrifos compared with those not exposed, after adjusting for exposure to other pesticides including organochlorines .Organophosphate pesticides, such as chlorpyrifos, have rarely been evaluated in studies of breast cancer risk. Additional research is needed to confirm these findings and to better understand the underlying mechanisms given that chlorpyrifos has been detected in local air monitoring at levels of concern for residents living in the agricultural regions where it is used. Exposure to pesticides might contribute to breast cancer development, but estimating adult cancer risks associated with previous cumulative exposures presents methodologic challenges, requiring accurate measurements over many decades. We have constructed a comprehensive pesticide exposure assessment using historic pesticide data and geocoded location histories in a case–control study of pesticides and breast cancer. This study suggests that pesticides other than organochlorines, such as the organophosphate chlorpyrifos, may be important for breast cancer risk and that additional research is needed to improve etiologically relevant measures of exposure to protect people who are unknowingly exposed to these chemicals.3 While the estrogenic properties of some pesticides have been established,6 results from previous studies of pesticide exposure on breast cancer risk are conflicting: some studies show a positive association,11 while others are null.20Because lifetime estrogen exposure is a key factor in breast cancer development, exposure to endocrine-disrupting pesticides might contribute to breast cancer development.20 Aside from a cohort study which did find some associations for specific pesticides,13 the majority of these studies grouped together pesticides with varying toxicologic effects, likely leading to nondifferential exposure misclassification and reported null effects that may have obscured associations with specific chemicals. Other studies used ecologic designs,16 spatial regression,9 or proximity to aggregated pesticide data at only one residential location.20 Studies that use biomarkers to measure pesticide metabolites in serum samples taken near the time of cancer diagnosis may not reflect previous exposures that are most relevant for breast cancer etiology and do not reflect long-term exposure.21 There is a need for research methodologies that can reconstruct exposures occurring decades before diagnosis and evaluate pesticide-specific exposures on breast cancer risk.23Many previous epidemiologic studies relied upon self-reported exposure based on pesticide usage, occupation, or living on a farm.24 in California’s highest-ranking counties  for agricultural density and commercial pesticide use in the United States.25 In highly agricultural regions, pesticide drift from neighboring application sites presents a major source of nonoccupational exposure.30 We evaluated a group of structurally and toxicologically similar31 organochlorine pesticides with known estrogenic effects that are most likely related to breast carcinogenesis because of their ability to accumulate in adipose tissue and potential to act synergistically .36 We also assessed breast cancer risk from exposure to three commonly applied pesticides in the region  detected at levels of concern to human health in air monitoring conducted by the California Department of Pesticide Regulation (CDPR) in 2006 in a Fresno County farming community.37 Because no regulatory ambient air standards exist for most pesticides, CDPR developed health-based screening levels for 35 pesticides and found that diazinon exceeded its screening level, chlorpyrifos approached its screening level and was frequently detected, and 1,3-dichloropropene exceeded its cancer potency value.We conducted a case–control study of breast cancer risk from exposure to pesticides using a Geographical Information Systems (GIS)–based method that combines geocoded residential and occupational histories with state pesticide use reports and land use dataCases were recruited from among women with histologically confirmed breast cancer diagnosed in 2007–2008 in the counties of Fresno, Tulare, or Kern from the Cancer Registry of Central California (CRCC), 55–74 years of age, and of non-Hispanic white ethnicity. From 2011 to 2013, cases were recruited by telephone. Among the 328 eligible cases we attempted to recruit, 10 were deceased and four too ill, 123 refused to participate, and we were unable to contact 32. Cases were excluded if they were premenopausal (N = 0) because postmenopausal breast cancer is more likely of hormone-related origin, reported Hispanic ethnicity (N = 2), or had been diagnosed with ovarian, uterine, or other female reproductive cancers before their diagnosis of breast cancer (N = 2). To match control selection criteria (below), cases were excluded if they had not lived in California for at least 5 years (N = 0) or had Parkinson’s disease (N = 0). A total of 155 participants with breast cancer completed the study.40 Controls were recruited from Medicare listings, mailings to a random selection of tax assessor parcel addresses using Internet searches and marketing companies to identify contact information, and from 2009, additional participants were enrolled in person during home visits to randomly selected households. The overall response rate among controls was 46%.40Controls were obtained from another population-based case–control study we conducted in the same geographic area between 2001 and 2011 examining the risk of Parkinson’s disease. Controls lived in California for at least 5 years before the study were at least 35 years old, resided in Fresno, Tulare, and Kern counties, and did not have Parkinson’s disease. Details of control selection are provided elsewhere.From the controls enrolled in the Parkinson’s disease study, we selected postmenopausal women 55–74 years of age of non-Hispanic white ethnicity (N = 208). After excluding women who had been diagnosed with breast cancer (N = 20) ovarian, uterine, or other female reproductive cancers (N = 9), and women who had opted to complete a shortened questionnaire without lifetime residential and occupational histories (N = 29), there were 150 participants included as controls in these analyses.http://geoservices.tamu.edu) and manually resolved to rooftops or by using additional information from participants such as cross streets and landmarks to more precisely identify a location41 and improve accuracy of rural locations in particular.42 We noted the level of “certainty” of each geocoded location and considered addresses to have high geocode certainty if geocoded to the centroid of a building, parcel, nearest parcel, street, or street intersection. Addresses geocoded to the centroid of a zip code, city, county, or state, or those that were unable to be geocoded were considered to have low geocode certainty.All controls (N = 150) and the majority of cases (N = 111) were interviewed over the telephone, with an additional 44 cases opting to complete a mailed questionnaire with follow-up by telephone to clarify or complete responses. All study participants were mailed a timeline to complete their historic residential and occupational workplace information (addresses and dates) before their telephone interviews. All historic residential and occupational workplace addresses were geocoded using Texas A&M GeoServices geocoder  data,43 containing the name of the pesticide active ingredient, the pounds applied, the crop and acreage of the field to which it was applied, the application method, and the date and location. These data were enhanced with land use data from countywide surveys conducted every 6–10 years by California’s Department of Water Resources (DWR), Division of Planning and Local Assistance,44 to refine the geographical resolution to the crop level as previously reported.45Historic pesticide exposures were determined from our GIS-based method that combines state-reported pesticide use data, land use surveys, and geocoded addresses to provide estimates of pesticide exposure within a 500-m buffer around residential and occupational locations. These methods have been described in detail elsewhere.24 This buffer distance was chosen based on studies that found measurable concentrations of pesticides from commercial pesticide application detectable in household dust of neighboring homes.48Historic ambient exposure to specific pesticides of interest was calculated by summing the annual density  of applied pesticide within a 500-m buffer around each residential and occupational location.2). Neighborhood socioeconomic status (SES) was based on the residential address at the time of diagnosis for cases or at the time of interview for controls using income and occupation information obtained from the 1990 US census data at the block group level and categorized into a quintile score.49Telephone interviews were conducted to obtain the covariates age (in years), ever lived on a farm (yes or no), ever worked on a farm (yes or no), education (in years), age at menarche, age at menopause , number of births (including stillbirth), oral contraceptive use (in years), menopausal hormone therapy use (in years) by type , ever smoked , ever consumed alcohol at least once a week (yes or no), and vigorous physical activity (defined as the number of hours of strenuous or moderate activity per week). Weight (pounds) and height (feet and inches) at the time of diagnosis or interview were used to calculate body mass index  inside their homes, outdoors in their yards or gardens, on their pets, and whether they had ever hired a professional to spray or fumigate, as well as whether they had ever worked on a farm or with pesticides or fertilizers. To identify occupational pesticide exposure based on self-reported data, other studies have used job-exposure matrices,Unconditional logistic regression was used to estimate ambient pesticide exposure on the risk of breast cancer in postmenopausal women. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for study participants exposed to specific pesticides compared with those not exposed. An individual was considered exposed to a particular pesticide when the pounds per acre of applied pesticide within the buffer area were greater than zero during the period from 1974 until the year of diagnosis for cases and the year of interview for controls. We chose 1974 as the start of our exposure assessment to include all years with complete pesticide information recorded by the State. To account for time between exposure and the development of the disease, we conducted sensitivity analyses excluding 10 and 20 years before diagnosis.2), age at menarche , age at menopause , number of births , oral contraceptive use , menopausal hormone therapy use , and ever consume alcohol at least weekly (yes or no), as well as the number of years lived in Fresno, Tulare, and Kern counties during the exposure assessment time period (continuous). Other factors, including the year of diagnosis, education, ever smoked, vigorous physical activity, ever lived or worked on a farm, and ever personally applied pesticides inside their home, outdoors in their yard or garden, or on their pets, and ever hired a professional to spray or fumigate, were evaluated as potential confounders and were included in the final models if they changed the estimates by >10%. All analyses were conducted using SAS, version 9.3 software .All analyses were adjusted for established breast cancer risk factors including age (continuous), SES (quintiles 1 lowest to 5 highest), body mass index . Pesticide exposure could not be identified for addresses outside of California because the exposure model includes only California PUR data, but participants reported whether any non-California addresses were on farms. We assessed the influence of missing data due to recall and locations outside of California by examining the change in our risk estimates after excluding missing data and conducted sensitivity analyses that included only women who lived in California for at least 30 years between 1974 and their year of diagnosis or interview. We also looked the potential impact of migration in our study by evaluating the influence of demographic factors (age and SES) and disease status among women who moved during our exposure assessment to women who resided in California for at least 30 years and to women who resided in Fresno, Tulare, and Kern counties for at least 30 years.For the years residential histories had missing location data due to incomplete recall of addresses or addresses that could not be found, we imputed exposures using the average exposures during all years with data for each person.The institutional review boards at the California Health and Human Services Agency and the University of Southern California approved the study protocol for cases participating in this study. The institutional review board at the University of California, Los Angeles, approved the study protocol for controls used in this study. Informed consent was obtained for all participants.Cases and controls appeared similar in terms of established breast cancer risk factors such as age, socioeconomic status, education, body mass index, age at menarche, age at menopause, number of births, menopausal hormone therapy use, and vigorous physical activity Table . Cases w10 year = 2.78; 95% CI = 1.20, 6.43; and OR20 year = 3.13; 95% CI = 1.30, 7.52) and remained close to the null for exposure to organochlorines, diazinon, and 1,3-dichloropropene, after accounting for 10- and 20-year latency periods.When assessing ambient pesticide exposure, the prevalence of exposure at residences and workplaces for the selected group of organochlorines, chlorpyrifos, and diazinon were over 40% among cases and controls Table . After aAmong those reporting that they never lived on a farm, 43.9% of cases and 40.0% of controls were exposed to pesticide drift from one of the selected pesticides at their residence. Among those who reported never working on a farm, 71.6% of cases and 54.7% of controls were exposed to pesticide drift from one of the selected pesticides at their workplace.Excluding the subjects with substantial missing information in their residential histories did not qualitatively change our estimates. There were three cases and four controls with more than one third of their residential timelines missing. The majority of participants had all residential locations within California during the timeframe of interest (82.0% of cases and 72.7% of controls). Few participants reported that their non-California residences were on farms (three cases and five controls). Restricting analyses to include only those who had lived in California for at least 30 years during our exposure assessment time frame increased estimates for exposure to chlorpyrifos at both residences and workplaces , while estimates remained null for exposures to the other pesticides (data not shown). Participants who migrated out of California or migrated from Fresno, Tulare, and Kern counties during the time period of our exposure assessment did not differ from participants who did not migrate by demographic factors (age and SES) or by disease status (data not shown).This population-based study examined historic and chemical-specific effects of hormone-related pesticides that are plausibly related to breast cancer in a region of intense agricultural production. We did not observe an association between breast cancer risk and exposure to a group of organochlorines after adjusting for coexposures to other commonly applied pesticides. Conversely, we observed that breast cancer was three times as likely to occur with exposure to the organophosphate chlorpyrifos, one of the three pesticides detected in air monitoring studies at levels of concern to public health . The majority of epidemiologic studies involving pesticides and breast cancer risk have focused on organochlorines, but the increased risk of breast cancer with exposure to the organophosphate chlorpyrifos became stronger after adjusting for exposure to other pesticides including organochlorines.Previous studies examining exposure to organochlorines have not considered other kinds of pesticides such as chlorpyrifos that may be highly correlated with organochlorines and are driving the breast cancer association. When assessing ambient pesticide exposure, it is usually difficult to distinguish the effects of specific chemicals because applications may be correlated and people can be exposed to multiple chemicals (as is the case with other kinds of toxic air pollutants). For example, among the controls in our study, 54.0% were exposed to both the organochlorines and chlorpyrifos, 24.7% were unexposed to either, and only 21.3% were exposed to one but not the other. Although our chemical-specific exposure model evaluated risk from a group of organochlorine pesticides with potential for synergistic effects, more research is needed to understand possible correlations among applications of pesticides.7 The prevalence of pesticide exposure from ambient sources in this study was over four times the prevalence of self-reported pesticide usage by farmer’s wives, and self-reported exposures do not account for exposures that people may not be aware of but are routinely exposed to, which is often the case with pesticide drift. According to our GIS-based exposure model, among participants in our study who never lived on a farm, 40% or more lived in residences that were within 500 feet of commercial pesticide applications and over 50% were potentially exposed by proximity to their workplace locations.Findings from this study suggest that exposure to pesticides other than organochlorines may also affect breast cancer risk. These results support the findings from a large cohort study of farmers’ wives in the Agricultural Health Study, which reported that exposure to chlorpyrifos was one of the pesticides driving a possible breast cancer association  but did not find any associations with entire classifications of organochlorine or organophosphate pesticides.52 and antiandrogenic.54 It can affect hormone pathways as an aryl hydrocarbon receptor agonist55 and induce proliferation of estrogen-dependent breast cancer cells in vitro.56 At low doses, chlorpyrifos promotes mammary tumor development and alters mammary gland hormone balance in vivo.58 More toxicologic research is needed to understand its mechanistic potential with regard to breast cancer risk. The findings from this study, however, support the 2012 regulations that restrict aerial pesticide application of chlorpyrifos to reduce the potential for exposure through pesticide drift.59Evidence is mounting for pesticides besides established organochlorines to act as endocrine disruptors that can increase the risk of breast cancer, particularly for chlorpyrifos. Chlorpyrifos is weakly estrogenic61 Although chlorpyrifos and diazinon are both organophosphate pesticides, and as many women were exposed to diazinon as to chlorpyrifos in our study, we did not find an association between diazinon and breast cancer risk after adjusting for exposure to chlorpyrifos. Reasons for this could be due to differing toxicities because chlorpyrifos is cytotoxic at far lower concentrations than diazinon in vitro62 or due to different exposure potential indicated by the finding that commercial application of chlorpyrifos but not diazinon was significantly correlated with measurements in household dust, even though diazinon was used twice as often.28Both chlorpyrifos and diazinon were voluntarily phased out for residential uses in 2000 and 2001, but still used in agriculture.63We did not observe an association between breast cancer and 1,3-dichloropropene; however, the prevalence of exposure was low (14.2% of cases and 13.3% of controls exposed at both residences and workplaces). The pesticide 1,3-dichloropropene is a respiratory carcinogen, but its role as a breast carcinogen is unknown.19 Our risk estimates based on a single residential address at diagnosis or interview were null for all pesticides considered in these analyses, including exposure to chlorpyrifos , and may underestimate actual risk because they do not account for exposures occurring at workplaces. Only one previous study conducted in Cape Cod, MA, collected residential histories to assess GIS-based proximity to pesticide applications and found no associations; however, the prevalence of pesticide exposures was much lower than observed in our study, and exposures were grouped by land use type instead of by specific pesticide of interest.12Strengths of this study include GIS-based exposure design that constructs exposure occurring over decades based on individual residential and occupational histories, while controlling for established breast cancer risk factors. Previous studies have estimated pesticide exposure by proximity to applications or crops using addresses at the time of cancer diagnosis with limited information on potential confounders.64Exposure in this study was based on reported address histories rather than self-reported pesticide usage, reducing the potential for recall bias. A study conducted in Australia found that the association between breast cancer risk and self-reported “noticing of pesticide spray drift” was strongly confounded by participants’ belief in whether or not pesticides caused breast cancer .Our GIS-based method also reduces the potential for selection bias from differential participation as a result of concerns about exposures in the environment. Selection bias may still be a concern because cases were recruited from a population-based cancer registry while controls were obtained for another population-based study in the same location. Although we started interviewing controls earlier than cases, the median and distribution of dates of interviews are similar between cases and controls Table . A time It is important to note that breast cancer cases participating in this study are of surviving cases. Participant cases were similar in age and SES to the population-based registry from which they were recruited but were less likely to have late-stage disease (data not shown). If there is a dose–response effect of pesticide exposure on severity of disease, then we are more likely to have recruited women with lower exposure, which would have resulted in an underestimation of the effects presented here. The geocode certainty of the historic addresses was similar for cases and controls for the period of our exposure assessment from 1974 until the year of diagnosis or interview. There were 92.3% of cases and 96.7% of controls residing 50% or more of the years during the time period at residential addresses having high geocode certainty and 78.1% of cases and 83.3% of controls working 50% or more of the years during the time period at workplace addresses having high geocode certainty. This suggests that the certainty of the geocoding is not likely to account for difference in the estimated effects.Estimating adult cancer risks associated with previous cumulative exposures present methodologic challenges for epidemiologic studies because it requires accurate measurements over lifetime and decades. The GIS-based methods presented here likely reduce exposure misclassification compared with estimates based on self-reported pesticide use. We have constructed a more comprehensive exposure assessment using historic data than has been done previously; however, a larger study is needed to confirm the chemical-specific associations we report and to examine different levels of exposure as well as potential dose–response relations.This study suggests that pesticides other than organochlorines, such as chlorpyrifos, may be important for breast cancer risk and that additional research is needed to improve etiologically relevant measures of exposure to protect people who are unknowingly exposed to these chemicals in the air.


BRCA1/2 mutation carriers, pre-menopausal obesity is associated with early-onset breast cancer. Here we show that obese, pre-menopausal BRCA1/2 mutation carriers have increased levels of aromatase and inflammation in the breast, as occurs in post-menopausal women. In a prospective cohort study of 141 women with germline BRCA1 (n = 74) or BRCA2 (n = 67) mutations, leptin, and aromatase expression were higher in the breast tissue of obese versus lean individuals (P < 0.05). Obesity was associated with breast white adipose tissue inflammation, which correlated with breast aromatase levels (P < 0.01). Circulating C-reactive protein, interleukin-6, and leptin positively correlated with body mass index and breast aromatase levels, whereas negative correlations were observed for adiponectin and sex hormone-binding globulin (P < 0.05). These findings could help explain the increased risk of early-onset breast cancer in obese BRCA1/2 mutation carriers.Obesity is associated with an increased risk of breast cancer in post-menopausal women and decreased risk in pre-menopausal women. Conversely, in  BRCA1 and BRCA2 DNA repair enzymes are causally linked to an increased risk of several malignancies including breast and ovarian cancers1. Not all women with mutations in BRCA1 or BRCA2 develop breast cancer and little is known about the factors that influence penetrance, making it difficult to personalize decisions concerning risk-reducing interventions. Previous epidemiological studies have suggested that obesity prior to menopause is associated with elevated risk and earlier onset of breast cancer in BRCA mutation carriers3. However, in unselected, predominantly wild-type populations, obesity is associated with a decreased risk of pre-menopausal breast cancer8. The mechanisms through which obesity contributes to early-onset breast cancer in BRCA1/2 mutation carriers are incompletely understood.Mutant BRCA mutation carriers owing at least in part to the pro-proliferative and mutagenic effects of estrogens12. The biosynthesis of estrogens is catalyzed by aromatase. Aromatase is expressed in breast adipose stromal cells and can be induced by both adipokines such as leptin and pro-inflammatory mediators including IL-6 and prostaglandin E214. Previous studies that did not focus specifically on BRCA1/2 mutation carriers demonstrated that levels of aromatase in the breast positively correlate with body mass index (BMI), breast adipocyte size, and white adipose tissue inflammation (WATi) manifested as crown-like structures of the breast (CLS-B)16. Post-menopausal status is independently associated with breast WATi, which contributes to elevated breast cancer risk in this population16. Systemic metabo-inflammation, characterized by altered levels of metabolic and inflammatory factors in the blood, may also play a role in the pathogenesis of breast cancer in the obese. For example, reduced levels of sex hormone-binding globulin (SHBG) have been observed in obese women leading to elevated levels of free estrogens in blood17. Pre- or post-menopausal women with impairments in DNA repair due to germline BRCA1/2 mutations may be more suceptible to the pro-mutagenic and pro-proliferative effects of free estrogens, which could lead to increased breast cancer risk.It has been suggested that cancers occur at hormone-sensitive sites in BRCA mutation carriers are provided with few options to reduce their risk of developing breast cancer. Although prophylactic bilateral mastectomy and oophorectomy protect against breast cancer development18, many young women are reluctant to undergo risk-reducing surgery and opt for surveillance as a means to mitigate risk. Notably, pharmacologic therapy including tamoxifen and aromatase inhibitors may reduce the risk of breast cancer in BRCA mutation carriers, though these agents are associated with considerable toxicity20.Currently, women identified as BRCA mutation carriers, the effect of excess body fat on breast aromatase has not been examined in this high-risk population. Accordingly, a major objective of this study was to determine whether excess body fat or breast WATi are associated with increased aromatase expression in the breast microenvironment of mutation carriers. A second goal was to identify blood biomarkers that indicate elevated intra-breast aromatase levels.Despite evidence that estrogens are likely to play a role in the pathogenesis of breast cancer in BRCA1 mutations and 67 had BRCA2 mutations. Clinicopathological features are shown in Table BRCA1 and BRCA2 mutations were of a similar age, BMI, and menopausal status. A greater percentage of hormone receptor-positive breast cancer was found in women with BRCA2 than BRCA1 mutations (44.78% vs. 22.97%). By contrast, triple-negative breast cancer was more common among women with BRCA1 than BRCA2 mutations (28.38% vs. 7.46%).In all, 141 women who underwent mastectomy for breast cancer treatment or prevention were included in the study: 74 were positive for BRCA1 and BRCA2 mutations  in women with either BRCA1 or BRCA2 mutations. In patients with BRCA1 or BRCA2 mutations, reduced levels of circulating adiponectin and SHBG were detected in association with elevated BMI (P < 0.05). Next, we correlated circulating factors with a range of breast measurements including the severity of WATi (#CLS-B/cm2), adipocyte diameter, and aromatase mRNA levels  and adipocyte diameters correlated positively with blood levels of hsCRP, IL-6, and leptin, whereas an inverse relationship was detected for adiponectin and SHBG levels. Notably, blood levels of hsCRP, IL-6, leptin, and insulin positively correlated with breast aromatase mRNA levels in women with either BRCA1 or BRCA2 mutations  were systematically extracted from the electronic medical record by research staff and physicians, and independent data review was carried out for quality assurance. Height and weight were prospectively recorded prior to surgery and used to calculate BMI. Standard definitions were used to categorize BMI as under- or normal weight (BMI < 25), overweight (BMI 25.0–29.9), or obese (BMI ≥ 30). Menopause status was described as either pre-menopausal or post-menopausal based on National Comprehensive Cancer Network (NCCN) criteria32. In brief, women were classified as post-menopausal if they had bilateral oophorectomy or reported permanent cessation of menses for 12 or more months in the absence of chemotherapy or endocrine therapy. All data were reviewed for accuracy independently by research staff and a physician.This study was approved by the Institutional Review Boards of Memorial Sloan Kettering Cancer Center (MSKCC) and Weill Cornell Medicine . Informed consent was provided by women undergoing mastectomy at MSKCC and breast WAT specimens were obtained under a standard tissue acquisition protocol. Patients underwent mastectomy for treatment of breast cancer or to reduce the risk of breast cancer. To ensure adequate tissue for analysis, patients undergoing lumpectomy were excluded. Clinicopathological data . A 30 mL fasting blood sample was obtained preoperatively on the day of surgery. Blood was separated into serum and plasma by centrifugation within 3 h of collection and stored at −80 °C.33. The presence of WATi was defined by CLS-B, which is comprised of a dead or dying adipocyte surrounded by CD68-positive macrophages34. Breast adipose tissue from the mastectomy specimen was formalin-fixed and paraffin-embedded (FFPE). Five FFPE blocks were prepared and one section per FFPE block (5 µm thick and ~2 cm in diameter) was generated such that five sections were stained for CD68, a macrophage marker . Immunostained tissue sections were examined by the study pathologist (DG) using light microscopy to detect the presence or absence of CLS-B and record the number of CLS-B per slide. Digital photographs of each slide were generated and WAT area was measured with Image J Software . The severity of WATi was quantified as the number of CLS-B per square centimeter of WAT (#CLS-B/cm2).Consistent with established methods, the presence or absence of breast WATi was determined by histologic assessment11. The H&E sections were photographed at ×20 using an Olympus BX50 microscope and MicroFire digital camera (Optronics). Mean diameters were calculated using measurements from 30 individual adipocytes for each patient using the linear dimensional tool in the Canvas 11 Software .Two Hematoxylin and Eosin (H&E) sections were generated from FFPE breast tissue in order to measure adipocyte diameters as previously describedTanalysis protocol.Total RNA was isolated from human breast tissue using the RNeasy Mini Kit (Qiagen). RNA (2000 ng) was reverse transcribed using the qScript cDNA Synthesis Kit (QuantaBio), and the resulting cDNA used for real-time PCR amplification with Fast SYBR green PCR master mix on a 7500 HT real-time PCR system (Applied Biosystems). GAPDH was used as an endogenous normalization control. Commercial primers for LEP (QT00030261), and GAPDH (QT00079247) were purchased from Qiagen. In-house primers for aromatase (forward: 5′-CACATCCTCAATACCAGGTCC-3′ and reverse: 5′-CAGAGATCCAGACTCGCATG-3′) were synthesized by Sigma-Aldrich. Relative fold-induction was determined using the ΔΔCPlasma levels of leptin, adiponectin, hsCRP, SHBG, interleukin-6 , and insulin , were measured by enzyme-linked immunosorbent assay.P < 0.05. All statistical analyses were conducted using R software .To examine relationships between two continuous variables, the Spearman correlation was used. Differences in a continuous variable across multiple categories were examined using the non-parametric Kruskal–Wallis test. Levels of a circulating factor and levels of relative expression of a gene were log-transformed for improving clarity in data visualization. For all analyses, statistical significance was set at two-tailed Further information on experimental design is available in the Reporting Summary Checklist


P for heterogeneity = 0.03). Our results suggest that male smokers are at increased risk of left colon cancer and female smokers are at increased risk of right colon cancer. Our study also suggests that females who smoke may have a higher risk of rectal cancer due to smoking than their male counterparts.The purpose of this study was to examine whether the increased risk of colorectal cancer due to cigarette smoking differed by anatomical subsite or sex. We analyzed data from 188,052 participants aged 45–75 years (45% men) who were enrolled in the Multiethnic Cohort Study in 1993–1996. During a mean follow-up period of 16.7 years, we identified 4,879 incident cases of invasive colorectal adenocarcinoma. In multivariate Cox regression models, as compared with never smokers of the same sex, male ever smokers had a 39% higher risk (hazard ratio (HR) = 1.39, 95% confidence interval (CI): 1.16, 1.67) of cancer of the left  colon but not of the right  colon , while female ever smokers had a 20% higher risk  of cancer of the right colon but not of the left colon . Compared with male smokers, female smokers had a greater increase in risk of rectal cancer with number of pack-years of smoking ( CIconfidence intervalCRCcolorectal cancerHRhazard ratioMECMultiethnic CohortMHTmenopausal hormone therapyColorectal cancer (CRC) is the third most common cancer among men and women in the United States. There is substantial variation in tumor anatomical location by sex and age. Women (vs. men) and older (vs. younger) persons have higher risks of developing cancer of the right  colon , 2. SeveSmoking was established as a causal factor for CRC almost 10 years ago , 9. The In the United States, the prevalence of smoking during the 20th century differed for men and women. Men born between 1911 and 1930 had a smoking prevalence that peaked well above 60%; prevalence in the 1931–1940 birth cohort peaked at around 60%; and the 1941–1950 birth cohort was the last to reach a prevalence above 50%. For females, the 1921–1930 and 1931–1940 birth cohorts were the only two that reached a smoking prevalence above 40%. In 2000, the prevalence of smoking was 25.7% for men and 21.0% for women .In 1996, Giovannucci et al.  hypothesThe purpose of this study was to examine whether the increased risk of CRC due to cigarette smoking differed by tumor anatomical subsite or sex in the Multiethnic Cohort (MEC) Study.The MEC consists of more than 215,000 men and women who were aged 45–75 years and living in Los Angeles County, California, and the state of Hawaii at the time of cohort recruitment. It comprises mainly 5 racial/ethnic populations . Briefly, between 1993 and 1996, participants born between 1918 and 1948 enrolled in the study by completing a 26-page mailed questionnaire asking for detailed information on demographic factors, dietary habits, other lifestyle factors, prior medical conditions, and family history of common cancers. We identified potential participants through driver’s license files from state departments of motor vehicles, voter registration lists, and Health Care Financing Administration (Medicare) data files. The institutional review boards of the University of Hawaii and the University of Southern California approved the study protocol. The cohort has been previously described in detail .n = 13,987), had prior CRC reported on the baseline questionnaire  or in tumor registries (n = 301), had invalid diet information , or had missing information on smoking status  were excluded; this left 188,052 participants (54.8% women) and 4,879 cases with a mean follow-up duration of 16.7  years in the analytical cohort. The multivariable analysis used a complete-case approach, which further excluded subjects with missing data on any of the covariates , leaving 171,626 participants (53.5% women) and 4,430 CRC cases for these analyses.Of the more than 215,000 MEC Study participants, those who did not belong to one of the 5 main racial/ethnic groups ), physical activity , and, for women, age at menopause, type of menopause, and ever use of menopausal hormone therapy (MHT). Dietary intake during the previous year was assessed at baseline using a self-administered quantitative food frequency questionnaire with over 180 food items, including alcoholic beverages /height (m)everages . We calcInternational Classification of Diseases for Oncology, Third Edition, codes: C18.0–C18.5 for the right colon, C18.6–C18.7 for the left colon, and C19.9 and C20.9 for the rectum. We identified deaths through linkage with death certificate files in Hawaii and California and with the National Death Index. Ascertainment of CRC cases and deaths was complete through December 31, 2013. We calculated person-years from cohort entry to the date of CRC diagnosis, death, or the end of follow-up , whichever occurred first. CRC cases other than adenocarcinoma (n = 324) were censored at the date of diagnosis. Cases with tumors at more than 1 subsite were not included in subsite-specific analyses.We identified incident invasive adenocarcinoma of the colon and rectum through linkage with Surveillance, Epidemiology, and End Results Program cancer registries covering Hawaii and California. We classified CRC cases according to anatomical subsite using We calculated sex-specific CRC incidence rates per 100,000 person-years, truncated to ages 45–85 years and age-adjusted to the 2000 US standard population . SubsequIn the multivariate analyses, we included the following covariates: race/ethnicity , age at cohort entry , family history of CRC , history of colorectal polyps , body mass index , multivitamin use , nonsteroidal antiinflammatory drug use , physical activity (hours spent in vigorous work or sports per day), and MHT  for women.In addition, we also adjusted for the following dietary intakes: total energy , alcohol consumption (g/day), red meat , fiber , calcium (mg/day), folate , and vitamin D (IU/day). We modeled the dietary intake variables in the multivariate Cox models as continuous variables.Participants with missing data on covariates tended to be older (63.7 years vs. 59.5 years), female (69.1% vs. 53.5%), and never smokers (47.4% vs. 43.7%) and to have similar proportions of CRC cases (2.7% vs. 2.6%) compared with those with complete data. We present the results from the complete-case analyses throughout this paper. The proportional hazards assumption was tested using Schoenfeld residuals and was found to hold , 16.We conducted tests for linear trends by including an ordinal exposure variable with equally spaced scores in the models, using never smokers as the first category. We assessed heterogeneity in the CRC-smoking association by sex on the basis of Wald statistics for cross-product terms between sex and smoking trend variables. We calculated CRC-smoking associations with adjustment for MHT (which was set to 0 in men) and other covariates using sex as a covariate. The effect of the interaction between smoking and MHT in women on the risk of CRC was similarly tested. We performed the analyses using SAS, version 9.4 .For men and women, the annual age-adjusted incidence rates of CRC were 126.4 per 100,000 person-years and 91.8 per 100,000 person-years (truncated to ages 45–85 years), respectively. For men, the distribution of cases by anatomical subsite was 42.6% right colon, 29.2% left colon, 26.2% rectum, and 2.0% unknown. The corresponding numbers for women were 52.8%, 26.3%, 18.3% and 2.6%, respectively. At enrollment, 18.2% of men and 14.4% of women were current smokers. Altogether, 70% of men and 44% of women reported ever smoking.https://academic.oup.com/aje) shows the direct associations of smoking status and the 3 measures of smoking dose/duration  with multivariate-adjusted CRC risk overall for each sex . These associations did not differ by sex .Web Table 1 , with no sex differences .P-for-trend values < 0.001) but not cancer of the right colon . For women, compared with never smokers, ever smokers had a 20% higher risk of right colon cancer . We observed direct associations between the different measures of smoking exposure and right colon cancer  but not left colon cancer . There was a sex difference for all 3 measures of smoking dose/duration displayed .P-for-heterogeneity values = 0.02).Web Table 2 shows that among postmenopausal women, both ever and never users of MHT had statistically significantly higher smoking-related multivariate-adjusted risks of right colon cancer. Among MHT ever users, several of the corresponding risk estimates for left colon cancer were above unity but not statistically significant. The test for heterogeneity across MHT status for the association between smoking status  and left colon cancer risk was statistically significant . We found a greater increase in risk of rectal cancer with number of pack-years for women than for men (P for heterogeneity = 0.03).In the present prospective study, although women were smoking less than men, we found that the smoking-related increased risks for CRC and for colon cancer overall were similar for both sexes. However, we observed that this increase in colon cancer risk for men was confined to the left colon, and for women to the right colon. Additionally, among postmenopausal women, for both ever and never users of MHT, we found a smoking-related increase in the risk of right colon cancer. Furthermore, while the association between smoking and rectal cancer was present for both men and women, it was stronger for women.We had previously examined the sex-specific associations between smoking and colon  and rectAs we pointed out in the Introduction, women in the United States took up smoking in large numbers more recently than men , 10, 12.Differences in incidence patterns for lung cancer, a cancer driven largely by smoking behavior, between the United States and Norway provides insight into the distinct results observed by country. As a result of reductions in smoking prevalence that began decades earlier , 10, 12,In further support of this notion are the results from one meta-analysis including older cohorts  and thosThe remaining cohort studies included only women  or did nSmoking cigarettes causes exposure to a mixture of more than 7,000 toxic chemicals, including at least 70 known carcinogens that can affect nearly every organ system in the human body . CarcinoBRAF) mutation–positive subtypes of CRC, which indicates that epigenetic modification may be functionally involved in smoking-related colorectal carcinogenesis (Several reports have described biological mechanisms by which smoking may cause CRC , 31–36. ogenesis . These rogenesis . Our finIn the United States, males have had a higher incidence of lung cancer than of CRC for many decades, while females had higher CRC incidence than lung cancer incidence until the early 1990s. For males, the decrease in CRC incidence also started in the early 1990s and paralleled that of lung cancer incidence. For females, CRC incidence rates have also been decreasing steadily since the mid-1980s, while the decrease for lung cancer started later (CRC incidence rates for people aged ≥50 years peaked in 1985 in the United States . The chaOur study had several major strengths. We had a high proportion of ever smokers in both sexes. We obtained smoking histories at enrollment and, thus, they were unlikely to have been subject to recall bias. In the United States, hardly anyone starts to smoke after age 50 years, which is similar to our minimum enrollment age of 45 years . FurtherThe main limitation of our study is that despite having close to 5,000 incident CRC cases, the numbers of cases were relatively small for some subset analyses. The lack of statistical significance for the association with left colon cancer among ever users of MHT may have been a function of low statistical power rather than the strength of the association. We plan to examine whether there are similar sex differences regarding tumor location in each of the 5 racial/ethnic groups when we have a longer duration of follow-up. The latency interval between smoking initiation and CRC diagnosis is presumed to be several decades . In our We need more knowledge about the biology of the sex differences for CRC in general and for the smoking-related differences specifically. We strongly recommend that cohort studies examining risk factors for CRC do this by sex and subsite. In addition, animal studies need to include females, which is rarely done today . IncreasThe smoking-attributable proportion of CRC cases has started to emerge for women during the last decade and could, at least temporarily, be greater in women than men in some populations. We hypothesize that our study will be the first of many cohort studies demonstrating that the increased risks of both colon and rectal cancer due to cigarette smoking are greater in women than in men.In conclusion, our results suggest that women who smoke have an increased risk of right colon cancer, whereas men who smoke have an increased risk of left colon cancer. Women may also have a higher risk of rectal cancer due to smoking than men.kwaa005_Gram_Web_Material_FinalClick here for additional data file.


Hereditary non-polyposis colorectal cancer (HNPCC) and Hereditary Breast and Ovarian Cancer Syndrome (HBOC) are the most common hereditary cancer syndromes in which a genetic test is available. Potential risks associated with testing include psychological harm, emotional distress and insurance problems.The aim of the present study is to investigate determinants of distress in a sample of Italian subjects undergoing genetic counseling. Demographic information and psychological distress were assessed by using a self-reported questionnaire and the “Hospital Anxiety and Depression Scale” (HAD), before attending the first counseling session.p = 0.02) and HAD global score (p = 0.01) than healthy ones. There was no difference in HAD score between individuals testing for different syndromes (p = 0.3). In the affected subgroup, there was a significant linear correlation between the HAD anxiety score and how much subjects perceived their disease as hereditary (p = 0.01). Female and younger subjects had higher levels of anxiety (p = 0.05). Also healthy single subjects show more general distress (p = 0.02) than those with a partner.Of the all subjects referred for the first time to our Center (January 2012–June 2013), a total of 227 were eligible  for the survey, 134 (59%) were oncologic patients and of these, 116 received genetic test (36 for HNPCC and 80 for HBOC). The remaining 93 (41%) were healthy subjects referred for suspected familiar history and of this group, 65 subjects performed predictive test in a family with a known pathogenic mutation (53 for HBOC and 12 for HNPCC). Affected subjects had a significantly higher level of anxiety (Greater level of distress identified on females, single and younger subjects. In about 5–10% of breast or colorectal cancer patients, the onset to the disease is the result of a heritable mutation (germline mutation), in a cancer predisposition gene. At-risk individuals tend to develop benign and/or malignant tumors at earlier age than usual and they have increased risk of developing more than one primary tumor.In addition, the siblings and offspring of an affected patient have a 50% chance of inheriting the cancer-predisposing mutation segregating in the family, consistent in most cases with autosomal dominant inheritance .Actually genetic testing is available for several cancer predisposition genes and became part of clinical practice. Familial Adenomatous Polyposis (FAP), Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch Syndrome, and Hereditary Breast and Ovarian Cancer Syndrome (HBOC) are the most prevalent and investigated hereditary cancer syndromes , 3.. The psychological impact of genetic susceptibility testing for hereditary cancer is reported in the literature over the last two decades [Genetic testing is always supported by counseling, which consists of pedigree reconstruction, risk assessment and education, facilitation to genetic testing , 5. The  decades , 6–11. R decades .. Furthermore, any previous cancer experience could greatly influence individual risk perception, emotional state before testing and experience of coping with a positive test result [In fact, hereditary cancer genetic testing has relevance for the entire family system: relatives with mutation may become aware of having increased cancer risk whilst those non-carriers may feel responsible or guilty , 13. Furt result . This ist result –18. In at result  and the In this cross-sectional study, we collected data from Italian subjects referred for counselling to the Genetic Oncology Center of the Marche Region, from January 2012 to June 2013. Participants were self-referred, referred by a relative or by their general physician or by specialist . They were healthy subjects or cancer patients. Selection criteria were: age of 18 years or older, no previous genetic counseling/test, no diagnosis of psychiatric disorders, ability to give informed consent.Age, sex, marital status, education level, having children, lifestyle and habits were obtained through specific items in the questionnaire. Data about personal and relatives medical history were collected by the counsellor and recorded in the medical source documents.Prior to attending the genetic counseling sessions, all subjects were provided with verbal information about the study and asked for consent in writing, which they all did provide. Besides socio-demographic data, the questionnaire was made of several multi-choice items regarding attitudes towards and reasons for pursuing genetic test.Psychological distress was assessed by a self-reported questionnaire. This was administered pre-counseling intervention that included the Hospital Anxiety and Depression Scale (HADs). The HADS is a fourteen item scale that generates ordinal data. Seven of the items relate to anxiety and seven relate to depression. Each item has a choice of four answers with scores ranging from 0 (no distress) to 3 (maximum distress). The subscales score range from 0 to 21. On either subscale, scores 0–7 are considered “normal”, scores 8–10 represent “borderline” and scores of 11 or more represent a “pathological” situation . The HADZigmond and Snaith created this outcome measure specifically to avoid reliance on aspects of these conditions that are also common somatic symptoms of illness, for example fatigue and insomnia or hypersomnia .Variance analysis and Chi square test were used to evaluate sociodemographic and clinical features.Fisher exact tests were used to analyze demographic and clinical characteristics and HADs findings to estimate association between categorical variables. Linear regression was used to study continuous variables. A statistic significant level of 0.05 was chosen. Statistical analysis were performed with STATA MP.11.0 software.The survey was proposed to a total of 250 subjects and of these, 227 partially or totally completed the questionnaire before the first genetic counseling interview. There were 23 drop out patients and the reason was the reported absence of interest.P values are referring to first entry among the three variables The subjects were attending the Cancer Genetic Center both for HBOC and suspected hereditary colon cancer syndromes. The mean age of the counselees was 48.7 years (range18–83 years), 53 subjects were males, 174 were women. The majority are patients with a previous cancer , 93 were healthy subjects referred because of being part of a high risk family.The descriptive analysis of the sample is described in Tables Of all patients, 116 (64.1%) had a cancer medical history which was highly suspected for hereditary tumors, specifically 36 patients suspected for HNPCC and 80 patients for HBOC syndromes. A further 65 (35.9%) conselees were healthy subjects referred for a possible predictive test. The remnant were referral patient and healthy subjects without a highly influential family tree.p = 0.03). This result was not confirmed on the single scales of anxiety and depression, respectively, but there was the evidence of a trend in both scales, so subjects with cancer resulted generally more stressed than healthy ones (predictive test) (p = 0.08). Women reported a trend towards higher distress (p = 0.05) than men, while there were no significant differences based on age and marital status. However, there was no difference in HAD score between individuals testing for different syndromes (p = 0.3). In the predictive test subgroup, single subjects presented higher level of distress (p = 0.04) than those who had a partner, on the contrary there was no evidence of this trend in the affected probands subset. Having children seemed to correspond to a higher score of anxiety and general distress  and in the predictive test subgroup, it was associated with a pathological level of anxiety (p = 0.02); this result was not present in the affected subgroup.We measured a significant difference in cumulative HAD score between affected subjects and healthy individuals, the first ones presented higher distress at a borderline and/or pathological level if compared to healthy participants . Most of healthy subjects stated that the decision to undergo predictive testing had to be taken principally on their own, while patients with cancer considered genetic testing only in agreement with the oncologist/health care physician. This association resulted statistically significant (p < 0.01). Those who had received information about genetic counseling by health care professionals were more frequently subjects with cancer , and they show a higher education  and a higher level of HAD score than those who received information from other sources.Of 227 participants, 137  received information about genetic counseling from health care professionals , the remaining 87  received information from other sources .Among the 131 patients with cancer, 35 answered positively to the question “do you think your disease is hereditary?”, while 90 participants responded they did not know. One hundred twenty two answered to the question “how much do you perceive your disease as hereditary ranging from 0 to 10?” and the mean value of the answers was 6.9 ± 0.2. Interestingly there was a significant linear correlation between the HAD anxiety score and how much subjects perceived their disease as hereditary (p = 0.04).In 58 healthy subjects perceived to be at risk of a hereditary disease and 49 (72% of the sample) answered to the question “how much do you perceive your risk of a hereditary condition ranging from 0 to 10?” and the mean value of the answers was 7.2 ± 0.3. Also in this group there was a significant linear correlation between the HAD anxiety score and how much the risk was perceived as hereditary (p = 0.01).Regarding family relationship, those healthy subjects who thought their family relationships were bad had a higher level of anxiety then those who perceived them good (. Furthermore, evidence from systematic reviews illustrates that a genetic counseling intervention does not appear to increase distress and therefore could improve the accuracy of individual’s perceptions of their personal risk [.In most cases, the perceived likelihood for carrying a gene mutation decreased after testing and subjects referred for genetic counseling found the counseling process extremely helpful for future medical decision-making –18. Butonal risk , 24.Our results show that cancer patients seem to report a higher level of distress and worries than disease free subjects.It could be hypothesized that the diagnosis of a neoplasm often leads to psychological instability, although previous studies are equivocal about the effects of a cancer diagnosis on distress –27. HoweMaybe healthy subjects with an affected first degree relative may have already accepted that cancer runs in their family and this may have reduced the probability of experiencing a high anxiety level. In addition, we confirmed that subjects with higher distress were more frequently women and more often single, without an adequate social support or with bad family relationships , 30–32. Healthy subjects appeared less distressed, but those with children presented higher levels of anxiety than those childless. This is reasonable considering that disease free members of an at-risk family could experience greater distress because of the consequently increased risk for their children. Most of these subjects experienced a lot of suffering in their family as children or siblings of cancer patients and this suffering quite often modeled their infancy or adolescence .High risk subjects seem to consider their lifestyle and their habits irrelevant in a sort of “fatalistic” view of their personal genetic predisposition to cancer.We confirmed greater adherence to genetic testing on the advice of their physician. So that, for adequate referral, physicians should be fully aware of the criteria of genetic risk and the process of genetic counseling and testing , 34.Moreover, our results showed a positive correlation between HAD anxiety score and the perceived risk of hereditary predisposition “per se” both in cancer patients and in healthy individuals referred for a predictive test. Even if most subjects in both subgroups were not able to answer with certainty about the risk of inheritance, the majority of them reported they perceived it to be high.It is likely that due to an overestimation of the personal and familial hereditary risk the HAD anxiety score could be influenced by the uncertainty of the personal genetic predisposition. This result is probably due to the lack of information necessary to give a real perception of risk in the pre-counseling phase. It would be interesting to assess these items in a post-counseling phase. Our survey confirms other literature results about disparities in health care access, indeed highly educated subjects more often received information about the test and hereditary risk of cancer from health care professionals . HoweverThis study adds to the understanding of the variables that can be used to identify subjects at higher risk of psychological distress, in order to give them appropriate strategy of coping. Investigating if the subject had cancer before, if he/she has a partner or children or lives alone, can give some information on the possible distress perceived by the counselee and give the counselor some hints on how to set up the counseling phase in order to offer the best of support, especially to counselees with more worries or emotional distress. Further investigation also in the post-test counseling phase are warranted to reach more reliable conclusions.


Height and leg length were not associated with risk. In normal weight women (18.5 ≤ BMI < 25), women with central obesity (WC > 88 cm) had an increased risk compared to women with normal WC (OR = 3.60(1.47–8.79)). Residuals of WC over BMI showed positive associations when adjusted for BMI (OR per 10 cm = 1.38 (0.98–1.94)). Body shape at younger ages and body shape evolution were not associated with risk. No heterogeneity was observed by receptor status. In this population of Latin American premenopausal women, different fat distributions in adulthood were differentially associated with risk of breast cancer.Cumulating evidence in Caucasian women suggests a positive association between height and premenopausal breast cancer risk and a negative association with overall adiposity; however data from Latin America are scarce. We investigated the associations between excess adiposity, body shape evolution across life, and risk of premenopausal breast cancer among 406 cases (women aged 20–45) and 406 matched population-based controls from Chile, Colombia, Costa Rica, and Mexico. Negative associations between adult adiposity and breast cancer risk were observed in adjusted models (body mass index (BMI): Odds ratio (OR) per 1 kg/m While only 12.4% of the women diagnosed with breast cancer in 2018 were younger than 45 years old in high-income countries, this proportion reached 21% in Latin America1. It has been shown that only part of this difference can be attributed to the difference in the age structure of the population2, suggesting a possible role for specific etiologic factors. However, to date only a few studies have investigated risk factors for premenopausal breast cancer among women in Latin America3.Although breast cancer is the most commonly diagnosed cancer among women worldwide5 and decreases with excess adiposity in adulthood4, as well as during childhood/puberty, although with less consistent results7. These associations are observed mostly for hormone receptor positive tumors8. However, only one study has so far been conducted in women in Latin America9, which suggested associations similar to those in Caucasian women but did not examine associations by subtypes. Given the rapidly growing burden of obesity in Latin America, where the mean body mass index (BMI) in women increased from 23 kg/m2 in 1980 to 27 kg/m2 in 201610, assessing the association between obesity and breast cancer risk is crucial.Studies from Caucasian populations indicate that the risk of breast cancer in premenopausal women increases with heightIn this study, we investigated the associations between several anthropometric factors assessed during adulthood, and changes in body shape during the life course, with the risk of premenopausal breast cancer, overall and by subtypes. This study was designed within the framework of the PRECAMA project, an ongoing multicentric population-based case-control study initiated in four countries in Latin America, coordinated by the International Agency for Research on Cancer (IARC).13. At recruitment, information was collected on lifestyle, health and reproductive history. Diet was assessed using a standardized food-frequency questionnaire that included country-specific foods. Standardized and harmonized protocols were used to perform anthropometric measures and to collect biological samples  at the time of the interview. Immunohistochemistry analyses on tumor tissue for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin 5/6 (CK5/6) was performed centrally at the Fred Hutchinson Cancer Research Center (FHCRC) in Seattle. All participants gave written informed consent before enrolment, and the study protocols were approved by the institutional review boards of Chile , Colombia (Cancer Institute Las Americas and University of Antioquia), Costa Rica  and Center for Strategic Development and Information in Health and Social Security (CENDEISSS) of the Costa Rican Social Security Fund (CCSS)), Mexico , and the International Agency for Research on Cancer (IARC). All methods were performed in accordance with the relevant guidelines and regulations of these approvals.PRECAMA is an ongoing multicentric population-based case-control study based in Chile, Colombia, Costa Rica, Mexico, and Brazil (pilot phase)13. The inclusion criteria were: age 20–45 years; being resident for ≥3 years in the same city district; having an incident primary invasive BC with positive biopsy and clinical staging and having menstruated at least once in the past 12 months. The exclusion criteria were having severe chronic disease, limited ability to communicate, being pregnant or nursing and having previous diagnosis and treatment of cancer (except non-melanoma skin cancer). Cases were recruited before any treatment and after signing an informed consent to participate.Incident cases of primary invasive breast cancer were recruited from general or cancer-specific hospitals or private oncology institutes, encompassing populations with a wide range of socio-economic status13. Controls were matched to cases on age (±3 years), city district of residence, and health insurance institution. The current analysis includes 406 cases and 406 matched controls.Controls were selected from the general population residing in the same city district as the case for at least 3 years using a multilevel sampling frame, applying the same exclusion criteria as for the casesHistology sections from paraffin-embedded tumor biopsies obtained before any treatment or obtained during surgery if no adjuvant therapy was administered, were reviewed for diagnosis, tumor grade, lymph vascular invasion and stromal and lymphocyte response in a centralized laboratory in Seattle, USA . Tumor samples with more than 1% immunostained tumor cell nuclei were considered ER-positive (ER+) and PR-positive (PR+). For HER2, samples were considered positive if there was strong membrane immunostaining (3+) and negative otherwise. Triple-negative (TN) tumors were defined as ER−, PR-, and HER2-. Among the TN tumors, basal-like cancers were defined as ER−, PR−, HER2−, and EGFR-positive (EGFR+) and/or CK5/6-positive (CK5/6+).14. Height, sitting height, weight, and waist (WC) and hip circumferences (HC) were measured.At recruitment, trained clinicians conducted anthropometric measurements according to standardized protocols and following Lohman’s recommendations16 used in similar studies9, adapted from Sørensen et al.17. Women were invited to choose, among 6 drawings , the one that best represented their body shape between ages 6 to 11 (childhood), 12 to 18 (adolescence), 19 to 25, before their first pregnancy, from age 26 to one year before inclusion, and at inclusion.Body shape from childhood to recruitment was assessed in the lifestyle questionnaire using a validated scale of silhouette pictograms2)), and waist-to-hip ratio. Silhouettes at different ages were also considered. To ensure that the number of women in each category would be appropriate for the statistical analysis, silhouettes were grouped as follows: between ages 6 to 11, 12 to 18, 19 to 25, and before their first pregnancy, categories were 1, 2, 3, and ≥4; between age 26 and one year before inclusion and at inclusion, categories were 1–2, 3, 4, and ≥5.Main characteristics of the population were described using mean and standard deviation (continuous variables) or frequency . Anthropometric measures of interest were height, leg length (height minus sitting height), weight (kg), waist and hip circumferences, BMI (weight (kg)/height  by examining significance of the model coefficient estimates and the Bayesian Information criterion (BIC) of each model. The BICs of the four models obtained were compared using log Bayes factor, considering the minimum number of women assigned to each trajectory, and examining mean posterior group membership probabilities for each trajectory (expected to be greater than 0.80)19.Trajectories of body shape were estimated based on silhouettes between ages 6 and 11, 12 to 18, and 19 to 25 (before categorization). Silhouettes at older ages were not considered because not all women were parous and because of the large range of ages represented in each category. Using a group-based trajectory modelling approach (SAS Proc TRAJ)20) and WC circumference categories (≤/>88 cm22) was also examined in order to investigate the role of central obesity across different weight categories.Odds ratios (OR) for risk of breast cancer and their associated 95% confidence intervals (CI) were estimated using conditional logistic regression analyses. Anthropometric measures were examined as continuous variables and in tertiles (T), defined on the distribution of control participants. For tests of linear trend across tertiles, participants were assigned the median value in each tertile and the corresponding variables were modelled as a continuous term. For silhouettes , linear trends were tested by including the variables as a continuous term in the model. A combination of BMI categories  was initially used (crude model). To identify potential confounders, models of the variables of interest were adjusted separately for each potential confounder (continuous and tertiles), and estimates obtained were compared with estimates from unadjusted models. Only variables that changed the risk estimates by more than 10% were retained in the multivariate model. Variables that were ultimately included were: education (≤primary/secondary/>secondary), history of benign breast disease (yes/no), family history of breast cancer (yes/no), total of daily hours of physical activity (tertiles), number of full-term pregnancies (0/1/≥2), age at first full-term pregnancy (tertiles/nulliparous), cumulated duration of breastfeeding (</≥12 months), age at menarche </≥ 12 years old), smoking status (current/former/never), type 2 diabetes (yes/no), daily alcohol intake (tertiles/non-consumer). Except for height and leg length, models were additionally adjusted for height (tertiles). Lastly, we examined the association of WC and HC independently from overall obesity using a mutually adjusted model including BMI and residuals of WC and HC on BMI, calculated from two separate linear regression models with BMI as the independent variable and WC or HC as the dependent variable24.Missing values on potential confounders were imputed to the median (age at menarche and at first full-term pregnancy) or mode (number of full-term pregnancies) as they represented less than 5% of the observations on the population© 2017, SAS Institute Inc.).For cases with currently available information on immunohistochemistry (n = 287), we stratified analyses of anthropometric factors by estrogen receptor status, and triple-negative tumor. Additionally, we investigated whether the associations observed differed by size of the tumor at diagnosis . All statistical tests were two-sided. Analyses were conducted using SAS software for Windows . Average WHR was 0.87 for cases and controls  and never had children . Cases had a first full-term pregnancy at older ages  and breastfed for shorter periods their children , had more frequently a history of benign breast disease  and smoked less than controls  (Table The most frequently chosen silhouettes were silhouette 1 (60.1%) during childhood, silhouette 2 during adolescence (40.5%), from age 19 to 25 (40.5%) and before first pregnancy (40.3%), and silhouette 3 from age 26 to 1 year before interview (31.8%) and at interview (30.7%) , whereas negative associations were observed with weight , WC , HC , and BMI . After adjustment for potential confounders, all variables but height remained associated with breast cancer risk : OR per 10 cm = 0.81, 95% CI = 0.69–0.96; HC (continuous only): OR per 10 cm = 0.80, 95% CI = 0.67–0.95). No association was observed with leg length (data not shown). In normal weight women (18.5 ≤ BMI < 25 kg/m2) women with central obesity (WC > 88 cm) had an increased risk of breast cancer compared to women with normal WC . Since almost all obese women had a high WC, it was not possible to investigate the effect of central obesity in this category. Finally, in the mutually adjusted model based on residuals, positive associations were observed with HC  and WC .In the crude model . However, evidence for heterogeneity was weak .Associations of anthropometric factors and risk of breast cancer did not differ by breast cancer subtype Table , althougNo heterogeneity was observed for any of the anthropometric factors when stratifying the analyses by tumor size  .In this population of premenopausal women from 4 Latin American countries, weight, waist and hip circumference, and BMI were negatively associated with risk of breast cancer, as was body shape from age 26 to study enrollment. However, in normal weight women, women with central obesity had an increased risk of breast cancer compared to women with normal WC, and, in all women, when adjusting for BMI, HC and WC were positively associated with risk of breast cancer. Height, leg length and WHR at recruitment were not associated with risk of breast cancer, neither was body shape at young ages and trajectories of body shape evolution. Stratified analyses showed no evidence of heterogeneity by subtypes of breast cancer. These findings point towards a role for body size in premenopausal breast cancer development in Latin American women, as reported in different populations.2) women had a decreased risk of breast cancer compared with those having a BMI lower than 25 kg/m2 9, consistent with our findings. Among Hispanic premenopausal women in the US, two studies reported an negative association between BMI and breast cancer risk, only in ER+PR+ tumors25 or in ER− tumors26, while a third one showed no association27. Our findings are in line with the negative association between adiposity and premenopausal breast cancer that has been reported in other populations4, as well as for genetically predicted BMI28, but are not in favor of an association restricted to hormone-receptor positive tumors8, although sample size in subgroups is limited in this analysis. This negative association between overall adiposity and premenopausal breast cancer is not well understood, but some mechanisms have been suggested, such as a reduced exposure to estrogens resulting from anovulation and abnormal hormone profiles induced by excess adiposity30.The only study on anthropometry and premenopausal breast cancer in Latin America  showed that obese  persisted9. In contrast, our results showed a positive association with HC  and with WC  when adjusted for BMI. The interpretation of WC and residuals of WC is slightly different, since WC is a measure of central adiposity that correlates with overall adiposity as measured by BMI (which might induce collinearity issues when included in the same model), while residuals of WC should be interpreted as a measure of central adiposity independent of overall adiposity23. Residuals of HC, when adjusted for BMI, are an indicator of the gluteofemoral lean mass, fat accumulation, and bone structure of the pelvis23. These findings are consistent with the increased risk in normal-weight women with central obesity observed in this study. Therefore, our data suggest that, although overall adiposity is negatively associated with risk of breast cancer, central adiposity and gluteofemoral adiposity show a specific positive association with breast cancer. These results are in line with the positive association with WC and risk of premenopausal breast cancer (independent of BMI) reported among Hispanic women living in the US in the Breast Cancer Health Disparities Studies25. A positive association between WC and risk of cancer has already been observed in other populations when adjusting for BMI4 and in normal-weight women31, and high HC has also been associated with increased risk of breast cancer32, in particular ER-PR- tumors33. The interpretation of such opposite associations between different measures of adiposity remains complex. Strong correlations between WC and high insulin levels34 could explain the link between higher WC and increased breast cancer risk35, while the specificities of the gluteofemoral adipose tissue, for which HC is a marker36, such as secretion of leptin37, might be involved in the association with HC. However, a better understanding of the parameters measured by HC is needed for further interpretation of these findings. In particular, HC and WC are only proxies for body fat distribution, and techniques such as DEXA or impedance, which provide information on body composition and fat distribution, would be helpful in disentangling these associations.Evidence from the CAMA study suggested a negative association between WC, HC and WHR and risk of premenopausal breast cancer9. In our study, neither height nor leg length, a marker of pubertal growth38, were associated with risk of breast cancer. It is possible that the absence of association we observed resulted from a reduced variability in our sample population compared with women from Caucasian populations40.In contrast with observations in Caucasian women and in the CAMA study, we did not observe any association between height and breast cancer risk9 or Hispanic women41. Although a negative association between body size in childhood/adolescence and risk of breast cancer in young women has been frequently reported in Caucasian populations42, in our study as in the premenopausal women of the CAMA study9, body shape early in life was not associated with breast cancer risk. Interestingly, a study on early-life body shape in Hispanic women in the US43 showed that, while changes in silhouettes was not associated with risk of premenopausal breast cancer risk, an negative association was observed with changes in weight at ages 10, 15 and 20 (heavier/same/lighter than peers) and that this association tended to be stronger in US-born than in foreign-born women. Thus, the absence of associations in Latin American women could indicate that the influence of early-life body size on breast cancer risk varies between populations, or could also suggest that the silhouette scales, although validated in European15 and North American16 populations, might not be a tool fully adapted to other populations.The negative associations reported for larger body shape during adult life and at interview are in line with our findings on overall adiposity measured by BMI, and with results in Latin America9. These associations are likely to be driven by the positive associations with postmenopausal breast cancer. Although weight gain has been associated with postmenopausal breast cancer risk, the evidence is limited for premenopausal women4 and studies in Hispanics in the US have yielded inconsistent results44. In addition, our trajectories focused on change in body shape during childhood and adolescence, not adulthood, which makes comparison difficult with most existing studies.The three trajectories that we obtained between childhood and early adulthood are comparable with some of the five trajectories obtained from the data of the CAMA study . However, both pre and postmenopausal women were included when modelling the trajectories. Similarly to our work, no association was observed with any of the trajectories in premenopausal women, and, although in pre and postmenopausal women combined, positive associations were observed when considering a very lean and stable body shape at the three ages as a reference, the risk estimate were very close for these three groups16. Also, anthropometry does not provide in-depth information on fat distribution, as would DEXA, or on body composition, which is why bioelectrical impedance measures are being implemented in the recruitment process of PRECAMA in Brazil. In addition, because of the case-control design of the study, some of the associations observed could result from reverse causation. Our design might also have impacted the trajectories obtained in this work, which is why the trajectories described are specific to this study and should not be generalized. However, the recruitment of cases before the start of any treatment which may result in weight loss, as well as the consistency of the negative association observed when considering measures of current body size, silhouette at interview and silhouette up to one year before interview does not support this hypothesis. Also, the negative association observed with BMI did not vary by size of the tumor. Finally, the relatively small sample size because of the ongoing recruitment resulted in limited power in some subgroups in the stratified analyses, even if the study is the largest ongoing effort in this population.Our study is the largest ongoing multicentric population-based case-control study of premenopausal breast cancer in Latin America. Strengths of this work include the use of standardized anthropometric factors measured by trained clinicians on incident cases, the use of standardized tools and questionnaires across centers to collect information on various current and past lifestyle factors, and the centralization of immunohistochemistry analyses, thus limiting the inter-laboratory variability. Still, some limitations must be acknowledged, such as the subjective nature of body shape evaluation based on silhouettes, although this tool has been validated and has proven to be reliable for lifelong approaches of body sizeIn summary, our findings suggest that the negative association between adiposity and risk of breast cancer before menopause established in other populations is also observed in women in Latin America, regardless of breast cancer subtype. More research is needed to better understand the underlying mechanisms of this relationship, and to characterize the distribution of body fat in this understudied population of premenopausal Latin American women.