P≤0.04). Higher levels of the pro-inflammatory marker COX-2 were also associated with lower prevalence of predominant type 1/no type 3 lobules in the breast, an indicator of complete involution, in age-adjusted analysis (P = 0.017). Higher tissue levels of inflammatory markers, mainly the pro-inflammatory ones, are associated with less involuted breasts and may consequently be associated with an increased risk of developing breast cancer.Increased levels of pro-inflammatory markers and decreased levels of anti-inflammatory markers in the breast tissue can result in local inflammation. We aimed to investigate whether local inflammation in the breast tissue is associated with age-related lobular involution, a process inversely related to breast cancer risk. Levels of eleven pro- and anti-inflammatory markers were assessed by immunohistochemistry in normal breast tissue obtained from 164 pre- and postmenopausal breast cancer patients. Involution status of the breast (degree of lobular involution and the predominant lobule type) was microscopically assessed in normal breast tissue on hematoxylin-eosin stained mastectomy slides. Multivariate generalized linear models were used to assess the associations. In age-adjusted analyses, higher levels of pro-inflammatory markers IL-6, TNF-α, CRP, COX-2, leptin, SAA1 and IL-8; and anti-inflammatory marker IL-10, were inversely associated with the prevalence of complete lobular involution (all  Many cellular processes controlling normal breast maturation-regression have also been found to be involved in cancer development. The immune system has been linked to normal mammary gland development as well as breast carcinogenesis –3. ContiDifferent breast maturation-regression stages were firstly described by Russo and Russo . During P-trend = 0.009) among 73 postmenopausal women diagnosed with benign breast diseases in multivariate analysis [P-trend = 0.03, P-trend = 0.04 and P-trend = 0.04, respectively) [P-trend = 0.04 and P-trend = 0.02, respectively) among 92 postmenopausal women undergoing diagnostic breast biopsy [The ARLI was found to be associated with lower breast cancer (BC) risk. Women having involuted breasts have lower BC risk compared to those with partial or no involution –12. The analysis . Moreovectively) . Of notet biopsy .Interestingly, a study found that lobulitis, defined as the presence of immune cells, namely helper T cells (CD4), cytotoxic T cells (CD8), B cells (CD20) and leukocytes (CD45), found in lobules, varied by the degree of lobular involution assessed in 109 lobules in the normal breast tissue of healthy women . To our Selection of the study population has been described elsewhere . BrieflyLaterality and location of BC were identified by review of pathology reports. Information on known and suspected BC risk factors were collected during personal and telephone interviews; anthropometric measures , reproductive data , history of breastfeeding, use of hormonal contraceptives or hormonal replacement therapy, breast biopsies, smoking, regular use of multivitamins or anti-inflammatory drugs, alcohol intake and family history of BC. Postmenopausal women were defined as those who had natural or surgically induced amenorrhea for 12 months or more , or those aged 53 or 55 years or more for smokers and non-smokers, respectively, and had hysterectomy without bilateral oophorectomy. Other women were considered premenopausal. Menopausal status could not be determined in eleven women aged <53 years. They were added to premenopausal women (n = 71) in further analyses as their ARLI were similar to that of premenopausal women.® Tissue Microarray Technology, Estigen, Sun Prairie, WI, USA).For all participants, two pathologists (BT and SJ), blinded to women’s information, identified zones of morphologically normal terminal ductal lobular units (TDLUs) on the hematoxylin-eosin (H&E) stained slides located at >1.0 cm from the tumor. Next, six cores (1.0 mm in diameter each) were extracted from the corresponding zones on the formalin-fixed paraffin embedded (FFPE) mastectomy blocks and randomly arrayed on recipient paraffin tissue microarray (TMA) blocks. Every participant was represented twice on a TMA block. Furthermore, each of the three BC cell lines  was placed in duplicate on every TMA block to serve as internal positive controls during the immunohistochemistry staining. In addition, a TMA block containing normal , inflammatory (colon) and malignant  human tissues, was constructed to serve as an external control. All TMAs were constructed using the tissue punch arrayer  were performed. The first and last sections from each block were H&E stained for histological examination. Other sections were stained according to standardized immunohistochemistry protocol for eleven pro- and anti-inflammatory markers. The choice of inflammatory markers was based on 1) their expression in normal breast tissue in detectable amount, 2) their documented effect on mammary cells behaviour , and 3) For all markers, the intensity and extent of immunostaining of the epithelial component of the cores present on TMA slides were visually evaluated by one blinded reader (MH) For COX-2, immunostaining was assessed separately in both the epithelium and stroma. Staining intensity was scored 0–3 corresponding to negative, weak, medium or strong staining, respectively. The extent was expressed as the proportion of positively stained cells, scoring 0, 1, 2, or 3 for 0%, 1–9%, 10–50%, and >50% of cells stained positive, respectively. The immunostaining was individually evaluated in each of the 1–6 cores judged interpretable on the TMA stained slides for one woman. Then, the median of the intensity and the extent of all cores were estimated (representative of different categories of intensity and extent of immunostaining are provided in The study pathologists (BT and SJ) selected one H&E stained slide containing normal breast tissue located at least 1.0 cm from the tumor for each woman. Involution status verified on a single tissue specimen represents the involution status of both breasts .We considered two measures of the ARLI previously shown to be associated with BC risk: the degree of lobular involution, a qualitative measure, and the predominant lobule type, a quantitative measure , 11, 20.Our objective was to evaluate whether the breast involution status, either the degree of lobular involution or the predominant lobule type, varied by levels of inflammatory proteins in normal breast tissue. So, we considered inflammatory marker expression using a two-category quick score, a pro-inflammatory or an anti-inflammatory score as the explanatory variables. The low expression category of the two-category quick score was considered as the referent category. The pro- or anti-inflammatory scores were assessed as continuous variables. The dependent variables were the dichotomous categories for the breast involution status. We defined having involuted breast; either complete lobular involution or predominant type 1/no type 3 lobules as our outcome of interest.P-value was based on score test for difference in prevalence.We hypothesized that higher tissue levels of pro-inflammatory markers would be associated with less involuted breasts , whereas, higher expression levels of anti-inflammatory markers would be associated with completely involuted breasts . Univariate and multivariate generalized linear models were used to estimate the prevalence ratio and corresponding 95% CIs of our outcome of interest. P value less than 0.05 was considered significant. All statistical tests were carried out using the SAS software system 9.3 .We initially evaluated the potential confounding effects of age at mastectomy (years) as it was consistently associated with the ARLI across studies , 20, 21.P = 0.177 and r = -0.009, P = 0.904 for the degree of lobular involution and the predominant lobule type, respectively). For BC risk factors, older age  and higher waist circumference  were correlated with higher prevalence of complete lobular involution and predominant type1/no type 3 lobules in univariate analysis. Postmenopausal status was correlated with higher prevalence of complete lobular involution and predominant type1/no type 3 lobules in univariate analysis . Postmenopausal women were more likely to have completely involuted breasts; 56 women (68.3%) had complete lobular involution and 51 women (62.2%) had predominant type 1/no type 3 lobules. More than half of women having complete involution  and those having predominant type 1/no type 3 lobules  had a waist circumference ≥86 cm, the median waist circumference for our study population. In addition, the two measures of the ARLI were moderately correlated as previously reported  [P = 0.798 and r = 0.010, P = 0.895 for the degree of lobular involution and the predominant lobule type, respectively). Similarly, no correlation was observed between the use of hormone replacement therapy and the ARLI in age-adjusted analyses .Characteristics of the study population are listed in P = 0.001, P = 0.0002, P = 0.004, P = 0.040, P = 0.037, P<0.0001, P = 0.001, P = 0.20 and P = 0.024 for IL-6, TNF-α, CRP, COX-2 , COX-2 , leptin, SAA1, STAT3 and IL-8, respectively)  . SimilarSimilar associations were observed among pre- and postmenopausal women when assessed separately Tables  and 3. AP<0.0001), pre- (P<0.0001) and postmenopausal women (P = 0.008); and the prevalence of predominant type1/no type 3 lobules among all (P = 0.022) and premenopausal women (P = 0.019) in the age-adjusted models.When considering the pro-inflammatory score, we observed inverse associations with the prevalence of complete lobular involution among all women  . No significant associations were observed for the anti-inflammatory score with the prevalence of predominant type 1/no type 3 lobules .Contrary to our hypothesis, the levels of IL-10 was inversely associated with the prevalence of complete lobular involution among all and postmenopausal women in the age-adjusted models (ctively) . FurtherIn this study, we report for the first time, significant inverse associations between the levels of inflammatory markers in breast tissue, mainly the pro-inflammatory ones, and the ARLI. In addition, the expression of all pro-inflammatory markers combined together was inversely associated with the prevalence of complete lobular involution and the predominant type 1/no type 3 lobules.To our knowledge, we are the first to assess the association between the protein levels of inflammatory markers in normal breast tissue and the ARLI. Our findings are consistent with the previous observation that pro-inflammatory markers can promote sustained proliferation of mammary cells, DNA damage, angiogenesis, and induce an increase in local estrogen levels , 22–24. 2 [In previously published studies, the ARLI expressed either as the degree of lobular involution , 20; the2 ; the lob2 ; or the 2  has beenDespite the consistent association between the ARLI and reduced BC risk, the association between BC risk factors and the ARLI remains understudied. In our study, there was no correlation between parity and the two measures of the ARLI considered in our study. This is in line with previous studies reporting that neither the number of acini/lobule  or the p+ and CD4+ T lymphocytes. On the other hand, IL-10 can also promote BC development by reducing the macrophage-antigen presenting capacity and inhibiting the production of many cytokines involved in the immunosurveillance process [IL-10 is a pleiotropic immunosuppressive cytokine that can suppress the secretion of pro-inflammatory markers such as IL-6, TNF-α and IL-8. In our study, IL-10 was inversely associated with the prevalence of complete lobular involution particularly among postmenopausal women. The unexpected inverse association with the degree of lobular involution may be explained by its complex and controversial role in BC development. It is suggested that IL-10 can suppress the tumor development through the activation of natural killer, CD8 process . Not sur process .2 and median TDLU span was associated with PMD  among 226 premenopausal women diagnosed with benign breast disease in multivariate analyses [P-interaction<0.0001) [Of interest, we have demonstrated associations between levels of inflammatory markers in normal breast tissue and the percent mammographic density (PMD), a known BC risk factor, in this population . Women hanalyses . Of inteThe strength of this study relies on the rich collection of epidemiological data available for study participants, the evaluation of the expression of multiple pro- and anti-inflammatory markers in normal breast tissue rather than their circulating levels, the application of TMA technique and the evaluation of all markers by one blinded reader. It was previously shown that circulating levels of inflammatory markers do not reflect their protein levels in breast tissue , 38. Of P<0.001) [P = 0.377 and r = -0.110, P = 0.164 for the degree of lobular involution and the predominant lobule type, respectively).The main limitation of this study is the relatively small sample size that should be kept in mind when interpreting the results. Nevertheless, we were still able to detect statistically significant associations. Another limitation was the assessment of the ARLI in normal breast tissue obtained from BC patients, which may not be completely representative of normal breast tissue. However, there was no difference in types of lobules assessed in normal breast tissue located at >5.0 mm from the tumor and that obtained from women having benign breast diseases . MoreoveP<0.001) . In factP<0.001) . In our P<0.001) . In our In summary, our findings suggest that the pro-inflammatory markers expressed by mammary cells may influence breast carcinogenesis by affecting the ARLI. Our findings have the potential to generate hypothesis about early events in BC development that provide the basis for future studies. Given the exploratory nature of this study, the limited sample size and the use of normal breast tissue obtained from women diagnosed with BC, larger studies are needed to elucidate these findings and to investigate whether the expression of inflammatory markers in breast tissue affects the age of onset and the rate of the ARLI.


Breast cancer (BC) is the leading cause of cancer death in women. Adipokines, and other inflammation molecules linked to adiposity, are suspected to be involved in breast carcinogenesis, however prospective findings are inconclusive. In a prospective nested case-control study within the EPIC-Varese cohort, we used conditional logistic regression to estimate rate ratios (RRs) for BC, with 95% confidence intervals (CI), in relation to plasma levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), interleukin-6, leptin, and adiponectin, controlling for BC risk factors. After a median 14.9 years, 351 BC cases were identified and matched to 351 controls. No marker was significantly associated with BC risk overall. Significant interactions between menopausal status and CRP, leptin, and adiponectin were found. Among postmenopausal women, high CRP was significantly associated with increased BC risk, and high adiponectin with significantly reduced risk. Among premenopausal women, high TNF-α was associated with significantly increased risk, and high leptin with reduced risk; interleukin-6 was associated with increased risk only in a continuous model. These findings constitute further evidence that inflammation plays a role in breast cancer. Interventions to lower CRP, TNF-α, and interleukin-6 and increase adiponectin levels may contribute to preventing BC. It is now clear that chronic inflammation is associated with several human cancers and that pro-inflammatory cytokines and other immunomodulatory molecules can be produced by cells in cancerous tissue to favor tumor growth, infiltration and metastasis3. C-reactive protein (CRP), an acute-phase protein of hepatic origin that is a sensitive yet nonspecific marker of the inflammatory response, has been associated with breast cancer risk in some7 but not all studies6. Several adipokines (immunomodulatory proteins produced by adipose and other tissues) are also suspected to play a role in breast carcinogenesis8. In particular, altered levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), adiponectin, and leptin have important roles in promoting inflammation in obesity and chronic inflammatory diseases9, and may play a role in carcinogenesis10. However prospective studies on markers of inflammation and breast cancer risk have produced conflicting results13.As long ago as 1863, Rudolf Virchow proposed that cancers originate at sites of chronic inflammationWe carried out a case-control study to prospectively assess whether pre-diagnostic levels of CRP, TNF-α, IL-6, leptin, and adiponectin in plasma are associated with risk of developing breast cancer.Baseline characteristics of cases and controls, by tertiles of plasma level of inflammatory biomarkers, are shown in Table Table Among postmenopausal women, high levels (third tertile) of CRP were associated with significantly increased risk  compared to the first tertile, fully-adjusted model; while high levels (third tertile) of adiponectin were associated with significantly reduced risk  compared to the first tertile, fully-adjusted model. None of the other biomarkers was significantly associated with breast cancer risk in postmenopausal women.Among premenopausal women, high TNF-α was associated with significantly increased breast cancer risk in the tertile model only (P trend = 0.017); and high IL-6 was associated with increased risk in the continuous model only . By contrast, high plasma leptin was associated with significantly reduced risk, both in the tertile-based model  and for a 1 standard deviation increase in leptin . None of the other biomarkers was significantly associated with breast cancer risk premenopausal women.In this nested case-control study, none of the inflammatory biomarkers analyzed was associated with breast cancer risk in the overall population. However, there were significant interactions between menopausal status and levels of CRP, leptin, and adiponectin. Among postmenopausal women, high CRP was associated with increased breast cancer risk, and high adiponectin was associated with decreased risk. And among premenopausal women, high TNF-α and IL-6 were associated with increased risk, and high leptin was associated with decreased risk.14. The first4, which examined 12 prospective studies, found that risk increased significantly by 7% overall and by 6% in postmenopausal women, for each doubling of CRP concentration. The other study14 analyzed 15 cohort and case-control studies, and included premenopausal women, finding that risk increased by 16% for each natural log unit increase in CRP; however when postmenopausal and premenopausal women were analyzed separately, the risk increase was significant only in postmenopausal women.Our finding of a direct association between plasma CRP and risk of postmenopausal breast cancer is in line with the findings of two recently published meta-analyses16. The lack of association between CRP and breast cancer risk in premenopausal women suggests that inflammation plays little or no role in premenopausal breast cancer.CRP is an established systemic marker of inflammation, being produced by the liver in response to cytokines (including IL-6 and TNF-α) produced by cells in inflamed tissue18: three found no significant association between TNF-α and breast cancer; while a fourth case-control study on postmenopausal women nested in the Malmö Diet and Cancer Cohort19 found an association between high TNF-α and reduced breast cancer risk.However this conclusion is opposed by our finding that high TNF-α was associated with increased breast cancer risk in premenopausal women. Our finding, in fact, contrasts with the results of the only four prospective studies we are aware of to have investigated TNF-α and breast cancer risk21. However, when produced chronically, TNF-α seems to act as a tumor promoter, contributing to the tissue remodeling and stromal development necessary for tumor growth and spread21. Recent data suggest that TNF-α is involved in carcinogenesis at least in part because it activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (reviewed in22), which is responsible for inducing the expression of genes associated with cell proliferation, apoptosis, inflammation, metastasis, and angiogenesis23. TNF-α can also stimulate the activity of the inducible nitric oxide synthase (iNOS), which is implicated in cellular changes leading to malignancy .TNF-α is a major mediator of inflammation: its induction, for example by tissue damage, induces a cascade of other inflammatory cytokines, chemokines, growth factors and endothelial adhesins which recruit and activate a range of cells at the site of tissue damage to promote healing25. High TNF-α could therefore promote breast cancer by this mechanism, which is likely to be more important in premenopause − before the fall in estrogen synthesis heralded by the menopause.Although our finding that TNF-α is associated with increased breast cancer risk in premenopausal women is not supported by previous studies, the association could be real since TNF-α has been found to stimulate the enzymes of estrogen synthesis22 to promote a cellular microenvironment that may promote cancer growth. However, no association between increased IL-6 levels and breast cancer risk has been reported in previous studies, specifically the case-cohort study nested within the Women’s Health Initiative Observational Study12, the British Women’s Heart and Health Study cohort study and Caerphilly Cohort26, and the Health Aging and Body Composition prospective cohort study17.We found that plasma IL-6 was associated with increased breast cancer risk among premenopausal women, but only in the continuous model. IL-6 has been reported to activate Janus kinase (JAK) and signal transducer and activator of transcription 3 (STAT3) pathwayset al.27 on premenopausal women. By contrast the case-control study of Touvier et al.13 found no association between leptin and breast cancer risk. Other prospective studies that investigated plasma leptin and breast cancer mainly involved postmenopausal women, and found no association28 or a direct association29. Leptin is thought to be involved in promoting breast cancer in obese women by stimulating the conversion of aromatizable androgens (androstenedione and dehydroepiandrosterone) to estradiol. This occurs not only in adipose but also in breast epithelial cells, particularly when levels of circulating estrogens decline, as they do in postmenopausal women31. This mechanism might explain why high leptin was associated with non-significantly increased breast cancer risk in our postmenopausal women, even after adjusting for BMI. However, in premenopausal women, high leptin may lower breast cancer risk, since leptin is involved in the regulation of ovarian folliculogenesis32 and at high levels may reduce follicular estradiol secretion33. Very high leptin levels have been reported in women with chronic anovulation34, a condition that may be associated with reduced breast cancer risk35.The inverse association we found between plasma leptin and breast cancer risk in premenopausal women is supported by the findings of a case-control study by Harris 36, and found no association of adiponectin with breast cancer risk overall, but decreased risk in postmenopausal women. A 2014 meta-analysis37 which examined 15 observational studies (6 prospective and 9 case-control studies), found a 5% reduction in overall breast cancer risk for 3 μg/ml increments in adiponectin, but no significant associations in post- or premenopausal women examined separately. A 2015 case-cohort study on postmenopausal women found no association between plasma adiponectin and breast cancer risk12. A 2016 meta-analysis of 107 epidemiological studies38 found that circulating adiponectin levels were lower in patients with various cancers than controls. However, other studies have found that increased adiponectin levels correlate with cancer progression (reviewed in39); and in patients with viral infections or chronic inflammation, increased levels of adiponectin predict cancer development39. Obesity is protective against breast cancer in premenopause but increases breast cancer risk in postmenopause. Adiponectin levels are low in obesity, so a presumed cancer-promoting effect of low adiponectin in premenopause may be masked by concomitant and protective obesity. In postmenopause, obesity is not protective so low levels of adiponectin may be “freed” to exert a cancer promoting effect, possibly explaining our finding that high adiponectin was associated with lowered breast cancer risk in postmenopausal women only40.As regards adiponectin, we found decreased breast cancer risk with increasing levels in postmenopausal women, in agreement with the findings of a 2013 meta-analysis that examined 17 observational studies (4 nested case-control studies and 9 case-control studies)41.Strengths of our study are its prospective design, relatively large sample size, and availability of detailed information on lifestyle that made it possible to control for confounding effects. A limitation is that we assessed variables at baseline only and do not know to what extent they may have changed subsequently. Limited data indicate that adipokine levels in a single blood sample are useful biomarkers of inflammation in population-based studies42. Another possible limitation is that samples were collected, stored at −196 °C, and analyzed up to 20 years later. There may have been differential decay of the analytes over that period44. However, unless analyte decay varied with initial concentration (which seems unlikely), this will not bias analyte-risk associations.Another limitation is that the relation between the circulating levels of the biomarkers we examined and their activity in breast or adipose is unknown, and it is possible that plasma levels may be a poor surrogate for local activity. For example leptin and adiponectin seem to function primarily in a paracrine manner, so circulating levels are unlikely to reflect biological activity in the breastTo conclude, the findings of this case-control study nested in the EPIC-Varese cohort suggest that high levels of CRP and low levels of adiponectin may increase the risk of postmenopausal breast cancer, while high levels of TNF-α and IL-6, and low levels of leptin may increase breast cancer risk among premenopausal women. Further research is required to elucidate the mechanisms by which leptin can influence the etiology of premenopausal breast cancer; interventions to lower CRP and increase adiponectin levels might help reduce the risk of developing postmenopausal breast cancer, while interventions to lower TNF-α and IL-6 levels might help reduce the risk of developing premenopausal breast cancer.The case-control study was nested within the 9378 women, resident in Varese Province, northern Italy, who were recruited in 1992-1997 (age 35–69 years) to the European Prospective Investigation into Cancer and Nutrition (EPIC)-Varese study, and gave blood samples on recruitment.45.At baseline, detailed information was collected on reproductive and medical history, physical activity, alcohol consumption, smoking, education, and socioeconomic variables, using a standardized lifestyle questionnaire. Diet over the previous year was investigated using a food frequency questionnaire specifically developed to capture local dietary habits. Weight, height, and blood pressure were measured, and a 30 mL fasting blood sample was collected. The blood samples were divided into 0.5 mL aliquots of plasma, serum, red blood cells, and buffy coat, on the day of collection, and stored in liquid nitrogen at −196 °CThe study protocol was approved by the ethics committee of the Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. At baseline, participants signed a written informed consent to use clinical data for research. Consent forms were stored with barcode ID for subject identification. The ethics committee approved this consent procedure. The study protocol and informed consent procedure met the requirements of Italian legislation and the Declaration of Helsinki of 1975, as revised in 2008.The women were followed-up to December 31, 2009 (median 14.9 years), through the Varese section of the Lombardy Cancer Registry, characterized by high data completeness and quality. A total of 362 new breast cancers was identified.For each case, one matched control was chosen, using an incidence density sampling protocol, from appropriate risk sets consisting of cohort members alive and free of cancer at the time of diagnosis of the index case. Matching criteria were age at recruitment (±5 years), date of recruitment (±180 days), menopausal status , and analysis of inflammatory markers in the same batch.46. All the analyses were performed in duplicated and results with a intra-assay %CV > 20% were discarded. The contents of each well were read by Bio-Plex 100 System array reader , which identifies and quantifies each analyte based on bead color and fluorescent signal intensity. Instrument calibration procedure was performed daily by Bio-Plex Calibration Kit (Bio-Rad) for optimal performance and reproducibility of results. The data were processed using Bio-Plex Manager software (version 6.1) using five-parametric curve fitting and converted to pg/ml.Plasma samples were analyzed using Luminex multiplex technology, which determines multiple analytes in a single microwell plate, using antibody kits purchased from Bio-Rad  or Merck (CRP)All kits supplied lyophilized standards that were reconstituted and diluted at 7 serial concentrations following manufacturer’s instructions (standard curves). Standards included all recombinant proteins tested and were considered as positive controls for the procedure. Standard diluent buffers alone were used as negative controls.2, 25- < 30 kg/m2, ≥30 kg/m2), and a fully-adjusted model, with the following additional covariates: age at menarche , parity , oral contraceptive use , education , smoking status , and alcohol consumption (continuous).Plasma levels of inflammatory molecules were grouped into tertiles based on the distribution in controls. Baseline characteristics of cases and controls, according to tertiles of plasma inflammatory biomarkers, were summarized as means and standard deviations (continuous variables) or frequencies . Conditional logistic regression models were used to estimate rate ratios (RRs) for breast cancer with 95% confidence intervals (CIs), with lowest tertile as reference. The significance of linear trends was assessed by treating each tertile as a continuous variable in the model and performing the Wald test. RRs were also calculated for 1 standard deviation increments in plasma concentration as a continuous variable. We ran a minimally adjusted model, adjusted for age (continuous) and BMI .We excluded seven cases and their matched controls because a plasma sample was not available for the case or the control. We excluded four additional cases and controls because confounder variables were missing for the case or control. The analyses were therefore performed on 351 cases and 351 matched controls, total 702 women – 334 postmenopausal, 360 premenopausal, and 8 perimenopausal. All statistical tests were two-sided, differences were considered significant for The data that support the findings of this study are held by the corresponding author, however their availability is restricted: for ethical reasons, the Ethical Committee does not allow open/public sharing of data pertaining to individuals. However aggregated data are available to other researchers, upon request.


Endometrial cancer is the fourth most common cancer in European women. The major risk factors for endometrial cancer are related to the exposure of endometrium to estrogens not opposed to progestogens, that can lead to a chronic endometrial inflammation. Diet may play a role in cancer risk by modulating chronic inflammation.In the framework of a case-control study, we recruited 297 women with newly diagnosed endometrial cancer and 307 controls from Northern Italy. Using logistic regression, we investigated the role of fruit and vegetable intake, adherence to the Mediterranean diet (MD), and the dietary inflammatory index (DII) in endometrial cancer risk.Women in the highest quintile of vegetable intake had a statistically significantly lower endometrial cancer risk . Women with high adherence to the MD had a risk of endometrial cancer that was about half that of women with low adherence to the MD . A protective effect was detected for all the lower quintiles of DII, with the highest protective effect seen for the lowest quintile .These results suggest that high vegetable intake, adherence to the MD, and a low DII are related to a lower endometrial cancer risk, with several putative connected biological mechanisms that strengthen the biological plausibility of this association.The online version of this article (10.1186/s12885-017-3754-y) contains supplementary material, which is available to authorized users. Endometrial cancer is the fourth most common cancer in European women,  with aboFurthermore, the hormonal regulation of the growth and shedding of the endometrial mucosa during the menstrual cycle is associated with endometrial inflammation, . which cEndometrial cancer cases were recruited at the Turin University Gynaecological Hospital, where about 70% of endometrial cancers occurring in the Piedmont Region of Northwest Italy area are treated. We recruited 297 women who lived in the Piedmont Region (age 40–74 years) and were newly diagnosed with histologically confirmed endometrial cancer.N = 98) [N = 209). Both control groups were made up of women residing the Piedmont Region who had not undergone hysterectomy. The two control groups were comparable with respect to the distribution of major confounding factors (data not shown).Two sets of controls were recruited: i) a random sample of females age 40–74 years) from the Turin centre of the European Prospective Investigation into Cancer and Nutrition  and University of Turin ethical committee and all subjects enrolled in the study signed an informed consent form.The information analyzed in this study was collected using questionnaires from EPIC Italy: a lifestyle questionnaire and a validated food frequency questionnaire (FFQ). Age, age at menarche, parity, oral contraceptive use, menopausal status, use of hormone replacement therapy, body mass index (BMI), physical activity , education, tobacco smoking status, diet and education was taken from the EPIC Italy lifestyle questionnaire . Women wA trained interviewer administered the questionnaires to cases and hospital-based controls during a face-to-face interview. During the questionnaire interviews, measures of weight, height, and waist and hip circumferences were taken for all subjects, and before the beginning of any cancer treatment among cases.Information on the EPIC-Turin population controls was taken from the same EPIC Italy questionnaires. These questionnaires were administered in an identical manner, and the same anthropometric measures were taken, but these steps were completed at the time of enrolment in the EPIC Study.The dietary habits of the women included in the study were summarised in two indices: an MD index and a DII index. The MD index was constructed based on women’s adherence to MD as per Trichopoulou et al.,  using foN-3 fatty acid, n-6 fatty acid, protein, PUFA, riboflavin, saturated fat, thiamin, vitamin A, vitamin B6, vitamin C, vitamin D, vitamin E, and zinc), and three available food items , and we weighted their intake using the overall inflammatory effect scores as computed by Shivappa et al [A DII was then derived based on the original DII by Cavicchia et al  and its pa et al   or the frequency and percentage for quantitative or qualitative variables, respectively. The intake of fruits and vegetables was divided into quintiles of consumption (using the distribution of controls).We used the Wilcoxon rank sum test with continuity correction and the Chi-squared test to determine differences in general factors and in food and nutrient groups between cases and controls. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were computed using unconditional logistic regression models, both univariate and multivariate, adjusting for age, age at menarche, parity, oral contraceptive use, menopausal status, use of hormone replacement therapy, BMI, physical activity, education, smoking status, and total energy intake.2), overweight women , and obese women , and sensitivity analyses were performed among the two control groups to exclude discrepancies in the results obtained for these groups. All the analyses were performed using SAS V9.2 package .Subgroup analyses were also carried out among normal weight women  and controls (N = 307) were comparable with respect to age, with a mean age at interview of 61.49 (SD 7.48) years for cases and 60.40 (SD 7.72) years for controls. The group of endometrial cancer cases included more nulliparous women (15.41% vs 5.61%) when compared to controls, as well as fewer patients with a parity ≥2 (6.45% vs 21.05%). Among endometrial cancer cases there was a higher percentage of women with lower education (40.94% with primary school or less vs 27.27% for controls) and a slightly higher percentage of never-smokers (67.99% vs 61.15%). Among controls there was a lower percentage of postmenopausal women (83.89% vs 93.93% for cases), a lower mean age at menarche , and a lower mean BMI . Parity , menopausal status , BMI (p < 0.0001), and education  showed the most evident differences (Table Cases N = 97 and cop < 0.0001) lower vegetable intake among cases  with respect to controls  and a less pronounced, lower fruit intake , while no association was found with alcohol intake or total energy intake , and a possible protective effect of high fruit intake was suggested . Both moderate  and high  adherence to the MD resulted in a reduction in endometrial cancer risk of about 50%. A borderline trend  of increasing risk for increasing DII was found, with highly significant results obtained for the 2nd, 3rd, and 4th quintiles with respect to the 1st quintile  and other pro-inflammatory cytokines (such as IL1 receptor antagonist) were found to be positively associated with endometrial cancer –23.Consumption of pro-inflammatory foods  seems to increase CRP levels . This caThe present study has some limitations that are inherent to the case-control design. Potential selection and information bias should be considered. The participation rate for both cases and controls was higher than 95%, and we excluded all women with diagnoses that could be related to known risk factors for endometrial cancer from the control group, as well as patients with previous hysterectomy. Recall bias was possible due to the case-control design; however the hypothesis of a dietary aetiology for endometrial cancer is not well known in the general population. The comparability of recall between cases and the hospital-based controls was improved by interviewing them in a hospital setting. In spite of these limitations, the study has some strengths, such as accurate exposure assessment with a validated questionnaire, and the possibility to adjust the analysis for several known confounders.In conclusion, the present case-control study provided some evidence that high vegetable intake, adherence to the MD, and a low DII are related to a lower endometrial cancer risk, with several putative connected biological mechanisms that strengthen the biological plausibility of this association.


Over 40% of women undergoing breast screening have mammographically dense breasts. Elevated mammographic breast density (MBD) is an established breast cancer risk factor and is known to mask tumors within the dense tissue. However, the association of MBD with high risk benign breast disease (BBD) is unknown.We analyzed data for 3400 women diagnosed with pathologically confirmed BBD in the Mayo Clinic BBD cohort from 1985–2001, with a clinical MBD measure  or Breast Imaging Reporting and Data Systems (BI-RADS) density) and expert pathology review. Risk factor information was collected from medical records and questionnaires. MBD was dichotomized as dense  or non-dense . Associations of clinical and histologic characteristics with MBD were examined using logistic regression analysis to estimate odds ratios (ORs) with 95% confidence intervals (CIs).Of 3400 women in the study, 2163 (64%) had dense breasts. Adjusting for age and body mass index (BMI), there were positive associations of dense breasts with use of hormone therapy (HT), lack of lobular involution, presence of atypical lobular hyperplasia (ALH), histologic fibrosis, columnar cell hyperplasia/flat epithelia atypia (CCH/FEA), sclerosing adenosis (SA), cyst, usual ductal hyperplasia, and calcifications. In fully adjusted multivariate models, HT , ALH , lack of lobular involution , fibrosis  and CCH/FEA  remained significantly associated with high MBD.Our findings support an association between high risk BBD and high MBD, suggesting that risks associated with the latter may act early in breast carcinogenesis.The online version of this article (doi:10.1186/s13058-017-0922-6) contains supplementary material, which is available to authorized users. Mammographic breast density (MBD) reflects the proportion of the breast composed of fibroglandular tissue and is an established risk factor for breast cancer (BC) , 2. WomePrevious reports provide insight into the association between MBD and breast cancer, by studying the histology underlying dense tissue –11. ThesHere, we report a comprehensive evaluation of the histologic features of benign breast disease in dense and non-dense breasts. In addition, we also investigate the differential association of fibrosis and fat with lobular involution in dense breasts.The Mayo BBD cohort has been described in detail previously and includes 13,441 women, 18 to 85 years of age at biopsy, diagnosed with BBD at Mayo Clinic in Rochester, Minnesota, between 1967 and 2001 and with no prior history of breast cancer [Of note, all the women in the Mayo BBD cohort had breast biopsies that showed benign findings that were concordant with imaging. If imaging and pathology results were discordant, an excisional biopsy was performed and in the event of malignancy, the patients were excluded. Hence, there were no prevalent cancers in this study.All of the study procedures and contact materials were approved by the Mayo Clinic Institutional Review Board. Patients consenting to research authorization allow medical records, images, and cancer data to be used for research (93% response rate). Only patients who provided this consent were included in the current study.MBD was available from the Mayo Clinic medical record starting in 1985. From 1985 to 1996, MBD was assessed clinically by radiologists using the parenchymal pattern (PP) consisting of four categories, and based on extent and type of density Fig. : N1  4th edition classification Fig. : 1  or non-dense (PP N1 or P1 or BI-RADS a or b).All histologic assessments of benign breast tissue were performed by one expert breast pathologist masked to the patient’s MBD and BBD reports. The pathologist (DV) assessed the overall histologic impression of the benign breast lesion as non-proliferative disease (NP), proliferative disease without atypia (PDWA), and atypical hyperplasia . In addition, specific characteristics of the tissue, including columnar cell hyperplasia (CCH), flat epithelial atypia (FEA), sclerosing adenosis (SA), cyst, usual ductal hyperplasia (UDH), calcification, fibroadenoma, intra-ductal papilloma, radial scar, duct ectasia, and mucocele-like lesions, were recorded as previously described , 25, 26.Data were summarized using frequencies and percentages for categorical variables, and means and standard deviations for continuous variables. The association of the frequency of missing data with patient characteristics was assessed using the chi-square test for categorical variables and Wilcoxon rank sum test for continuous variables. Multivariate stepwise logistic regression analysis was then used to determine independent predictors of missingness after accounting for the effects of other variables.p < 0.05), were then included in a final, multivariate model. Histologic impression was not included in this final model because it is defined in large part based on the individual histologic characteristics that were examined. Primary analyses were of associations with MBD in the entire group of subjects. Secondary analyses included re-examination of the associations within strata defined by ever vs. never using HT, premenopausal vs. postmenopausal at biopsy, and type of density measure (BI-RADS vs. PP). All statistical tests were two-sided, and all analyses were carried out using the SAS software system .We examined associations between having a dense breast and demographic, clinical, and histologic characteristics using logistic regression analysis. The following characteristics were examined: age at biopsy, body mass index (BMI) at biopsy, ever use of HT, extent of lobular involution, and presence vs. absence of ADH or ALH, fibrosis, SA, CCH/FEA, cysts, UDH, calcifications, fibroadenoma, intra-ductal papilloma, radial scars, duct ectasia, and mucocele-like lesions. Because of our a priori interest, we also examined associations beween dense breasts and combinations of the extent of lobular involution and fibrosis . For each variable described above, we first fit a series of age-adjusted and BMI-adjusted models, examining associations between each of the characteristics of interest and dense vs. non-dense MBD. All such variables found to be statistically significant . With multivariate adjustments, age, BMI, HT, ALH, lobular involution, fibrosis, and CCH/FEA were statistically significantly associated with dense breasts (p ≤ 0.05). Presence of fibrosis was associated with a 2-fold increased risk of having dense breasts  and no involution with a 1.6-fold increased risk relative to complete involution . CCH/FEA was positively associated with dense breasts  (Table p = 0.05).For the 3400 eligible women, the median age at biopsy was 53 years, with 13% aged < 40 years, 53% between 40 and 59 years, and 34% ≥ 60 years. Median BMI was 26, and 61% were current or past HT users. In this sample, 56% of women had non-proliferative disease, 38% had PDWA, and 6% had AH. Further, 64% had dense breasts . Associations between MBD and histologic characteristics are presented in Table p < 0.05 for all combinations). In particular, after adjustment for age and BMI, women with no involution and with fibrosis were more than three times as likely to have dense breasts as those with complete involution and no fibrosis . These associations were attenuated somewhat, but still remained highly significant after multivariate adjustment. There was no statistically significant interaction between involution and fibrosis (p > 0.2 in models adjusted for age and BMI and multivariate models), indicating that the marginal effects of each variable on the odds of having dense breasts are not modified by the other.Associations between MBD and combinations of lobular involution and fibrosis are presented in Table p = 0.036), but was null for PP measures . However, the power was limited for these comparisons.Results were similar when examining associations within subsets of women who had ever vs. never taken HT and premenopausal vs. postmenopausal status (data not shown). Results were also similar when examining the associations in a subset of women with BI-RADS  would actually have a positive association, and involution with greater proportions of fat (fat replacement) would have an inverse association. Although both fibrosis and involution were both strongly associated with density, we found no evidence of effect modification; specifically, the association between fibrosis and density was similar across all levels of involution. The greatest risk of dense breasts was among women with no involution and fibrosis in the breast tissue. Together, these findings suggest the possibility that stromal factors may influence breast epithelium, thereby predisposing it to changes leading to malignancy in women with dense breasts.p = 0.9) but did detect a borderline significant association between ALH and dense breasts (p = 0.05). Since ours is the first report to examine the type of atypical hyperplasia with dense breasts, this finding warrants further evaluation in future studies.Approximately 40% of women who have had screening mammography have dense breasts classified as BI-RADS c and d . In our p = 0.04) [We also noted a positive association between MBD and CCH/FEA. In the literature, columnar cell lesions have been reported to include changes such as proliferation of columnar shaped epithelial cells within enlarged TDLUs and have been classified with or without atypia , 31. Tur = 0.04) . They al = 0.04) . FurtherOur study of benign breast characteristics and breast density is the largest and most comprehensive analysis to date and based on a well-established cohort of BBD. It confirms and extends prior work in this area and is the first study to examine the interaction between involution and fibrosis. Further, this study utilized two different but validated measures of breast density and had similar findings. As the study was performed among women with BBD, the study results are generalizable to the sizable population of women who have had benign breast biopsies who have tissue available to evaluate benign characteristics. Women with BBD are considered at elevated risk compared with the general population . In thisOur study had additional limitations. We acknowledge the occurrence of inter-observer variability in diagnosing BBD microscopically . HoweverIn examining the association between benign breast changes and MBD, we report that in addition to the known association between MBD with fibrosis and lack of lobular involution, women with dense breasts may be more likely to have some high-risk lesions such as ALH and CCH/FEA compared to women with non-dense breasts. Continued research on mammary epithelial changes and of molecular markers in dense tissue is needed to shed further light on MBD so that targeted efforts on reducing breast cancer risk factors can occur.Additional file 1: Table S1.Distributions of subject characteristics by study inclusion among women enrolled in the BBD study from 1985 to 2001. (DOCX 21 kb)Additional file 2: Table S2.Association between mammographic breast density and benign breast findings for the 728 women with BI-RADS density measures. (DOCX 20 kb)Additional file 3: Table S3.Association between mammographic breast density and benign breast findings for the 2672 women with parenchymal pattern density measures. (DOCX 20 kb)


Radon and its decay products, a source of ionizing radiation, are primarily inhaled and can deliver a radiation dose to breast tissue, where they may continue to decay and emit DNA damage-inducing particles. Few studies have examined the relationship between radon and breast cancer.The Nurses’ Health Study II (NHSII) includes U.S. female registered nurses who completed biennial questionnaires since 1989. Self-reported breast cancer was confirmed from medical records. County-level radon exposures were linked with geocoded residential addresses updated throughout follow-up. Time-varying Cox regression models adjusted for established breast cancer risk factors were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs).3) had a suggested elevated risk of ER−/PR- breast cancer compared to women in the lowest quintile (<27.0 Bq/m3) . No association was observed for ER+/PR+ breast cancer.From 1989 to 2013, 3966 invasive breast cancer cases occurred among 112,639 participants. Increasing radon exposure was not associated with breast cancer risk overall . However, women in the highest quintile of exposure  contains supplementary material, which is available to authorized users. Internaommended . Radon dommended , 15.3 is 0.42 mSv and 0.02–0.15 mSv (depending on blood clearance rates), respectively, as compared to 1.2 mSv and 35.8–159 mSv for the lung [Radon and radon decay products have been predicted to deliver radiation doses to breast tissue , 16. Altthe lung .Molecular and cellular studies have demonstrated that ionizing radiation emitted from the radioactive decay of radon and its decay products, primarily alpha particles, can cause cytogenetic damage, chromosome aberrations, and gene mutations . Animal Although there is biological plausibility that radon exposure could influence breast carcinogenesis, few epidemiologic studies have been conducted. Increased breast cancer incidence was observed among former female employees of a Missouri school with elevated radon levels . EcologiThe Nurses’ Health Study II (NHSII) is an ongoing prospective cohort study of 116,429 U.S. female registered nurses aged 25–42 years at baseline in 1989. Although participants originally resided in California, Connecticut, Indiana, Iowa, Kentucky, Massachusetts, Michigan, Missouri, New York, North Carolina, Ohio, Pennsylvania, South Carolina, and Texas, as of the mid-1990’s, participants currently reside in all 50 states and the District of Columbia. Self-administered questionnaires are completed biennially acquiring information regarding incident disease, medical history, diet, lifestyle factors, and health behaviors. Response rates for each questionnaire cycle are ≥90%. We excluded women at baseline who were missing exposure information, with prior diagnoses of other cancers (except non-melanoma skin cancer), missing menopausal status, or who resided outside of the contiguous U.S. After exclusions, 112,639 women were included in the analysis. This study was approved by the Institutional Review Board of Brigham and Women’s Hospital; participants provided implied consent through returning questionnaires.Invasive and in situ breast cancer cases were identified through self-report on biennial questionnaires. Deaths were reported by family members, the U.S. Postal Service, or ascertained from the National Death Index. A medical record review was conducted to confirm breast cancer cases and abstract information regarding tumor characteristics. As 99% of breast cancer cases were confirmed via medical record review, self-reported cases without medical record confirmation were also included in the analysis. Hormone receptor status was based on tissue microarrays (TMAs) constructed at the Dana-Farber/Harvard Cancer Center Tissue Microarray Core Facility. Three 0.6 mm diameter cores from tumor tissue samples were inserted into TMA blocks. Immunohistochemical staining for markers including estrogen receptor (ER) and progesterone receptor (PR) was performed on 5 μm paraffin sections cut from TMA blocks. Immunostained TMA sections were reviewed under a microscope and visually scored for ER and PR positivity as determined by any nuclear staining (≥1%) , 29. Tum3), and estimating exposure using the University of Pittsburgh database of average radon levels . The University of Pittsburgh database was created using approximately 272,000 residential radon measurements in 1217 counties from the University of Pittsburgh Radon Project (PITT), 40,000 EPA residential measurements in 39 states, and state-based studies in Florida, Idaho, Iowa, New Hampshire, New Jersey, New York, Ohio, South Carolina, and Utah [Participant residential addresses, biennially updated beginning in 1989, were geocoded Fig.  in a geoand Utah .Fig. 1NuThe following information collected from biennial questionnaires  were evaluated as potential confounders and/or effect modifiers, known to be established or suspected breast cancer risk factors: age, race, family history of breast cancer, personal history of biopsy-confirmed benign breast disease (BBD), age at menarche, parity and age at first birth, lactation, oral contraceptive (OC) use, menopausal status and hormone use , screening mammography, height, body mass index (BMI) at age 18, change in BMI since age 18, smoking status, diet scored by the Alternate Healthy Eating Index (AHEI), physical activity, alcohol consumption at age 15 and 18, adult alcohol consumption, and individual-level socioeconomic status (SES) .2.5) and coarse particulate matter (PM2.5–10) air pollution were determined using geocoded residential addresses. PM2.5 and PM2.5–10 were predicted using validated GIS-based spatiotemporal exposure models [In addition, area-level SES , region of residence, population density, county-level housing characteristics , and percentage of the population using well water from the U.S. Census Bureau, and fine (PMe models . The mis3). Tests for trend were calculated using the median value of each quintile of exposure. Cubic regression splines were used to test for deviations from linearity. All models were stratified by age and questionnaire period. Potential confounding was evaluated by adding each covariate (or group of covariates) to the model and noting its impact on the effect estimate for radon exposure . Three models were assessed: a basic model minimally adjusted for age and race; a parsimonious model additionally adjusted for area- and individual-level SES and region of residence; and a fully adjusted model additionally controlling for established breast cancer risk factors , PM2.5 air pollution, and population density. Results from shared frailty Cox models with a frailty term for county, which modifies the hazard multiplicatively and assigns each woman in a county  the same level of frailty, were compared to results from Cox models without a frailty term for county to determine the impact of within-county clustering [Person-time accrued from June 1989 until the end of follow-up in May 2013, incidence of invasive breast cancer or other cancer , date of death, or loss to follow-up, whichever occurred first. Time-varying Cox regression models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between risk of incident invasive breast cancer and cumulative average radon exposure based on quintiles or continuous radon exposure per interquartile range (IQR) increase 37.3 Bq/m.3 Bq/m3.2.5, PM2.5–10, population density, urbanicity, and region of residence  using partial likelihood ratio tests. All statistical analyses were conducted using SAS .We explored potential effect modification by race, menopausal status, age at first birth, parity, lactation, screening mammography, BMI, smoking status, Census tract median home value, Census tract median income, individual-level income, PMTable n = 2373 ER+; n = 585 ER-; n = 2074 ER+/PR+; n = 513 ER−/PR-; n = 285 ER+/PR-; n = 293 ER−/PR−/HER2-). Increasing radon exposure was not associated with invasive breast cancer risk overall , ER+ cases, ER+/PR+ cases, or ER+/PR- cases in multivariable models (Table p for trend = 0.01) and ER−/PR−/HER2- breast cancer (p for trend = 0.003) and a suggestive association for risk of ER- breast cancer (p for trend = 0.06). However, these associations were attenuated in fully adjusted models, primarily due to confounding by area-level socioeconomic status  had a suggested elevated risk of ER- , ER−/PR- breast cancer , and ER−/PR−/HER2- breast cancer  compared to women in the lowest quintile (<27.0 Bq/m3) in fully adjusted models. Similar results were observed when examining radon exposure continuously (Table p > 0.05). Similar results were observed when using the University of Pittsburgh radon metric (results not shown), assessing baseline (1989) exposure (results not shown), defining exposure using the EPA action level of ≥148 Bq/m3 , where among those residing in the Western U.S., women in the highest tertile of radon exposure (≥57.4 Bq/m3) had a 47% increased risk  of invasive breast cancer compared to women in the lowest tertile (<33.3 Bq/m3) .There was a statistically significant interaction between radon exposure and region of residence  or in the West (−0.11 and −0.08). Further, although urban and rural differences in radon levels have been observed in previous research, partially attributed to differences in housing characteristics  and dwelling habits , we did not observe evidence of effect modification by urbanicity [There was a statistically significant interaction between radon exposure and region of residence, where higher radon exposure was significantly associated with an elevated risk of invasive breast cancer in the Western U.S. This interaction was not explained by differences in screening mammography practices, population density, urbanicity, residential mobility, PM air pollution, or area-level percentage of the population using well water, housing type, and year the housing was built. Radon was not strongly correlated with PMbanicity –56. The banicity . Among Nbanicity . Howeverbanicity , 60. RegThis analysis includes several important limitations. The radon exposure metric was available at a county-level spatial resolution, which may not reflect individual-level exposure. Household radon levels may vary within a county due to differences in housing characteristics, geology, and remediation. As counties are large geographic units often inhabited by many participants, we conducted sensitivity analyses accounting for within-county clustering using shared frailty models (frailty term for county). HRs from Cox models with and without frailty terms were similar. Exposure measurement errors are also likely due to errors in the radon exposure model’s failure to account for time-activity patterns by not having information available on amount of time spent at home, and lack of information on exposure to non-residential sources of radon including the workplace . HoweverStrengths of this study include a long follow-up period of over 20 years allowing for a large number of breast cancer cases to accrue. To the best of our knowledge, this is the first prospective study assessing radon exposure and breast cancer risk. We objectively assessed radon exposure using a metric created from short- and long-term radon monitoring surveys, predictive of cancer risk in previous epidemiologic studies examining cancers . Using tWe observed suggestive positive associations between environmental radon exposure and ER-, ER−/PR-, and ER−/PR−/HER2- invasive breast cancer in a large prospective study of U.S. women, but no association overall, with ER+, ER+/PR+, or ER+/PR- invasive breast cancer. Further research is needed to clarify the association between radon exposure and invasive breast cancer risk, with a focus on hormone receptor-negative tumors, and to determine potential biological mechanisms.


An earlier age at onset of breast development and longer time between pubertal stages has been implicated in breast cancer risk. It is not clear whether associations of breast cancer risk with puberty or predictors of onset of puberty, such as weight and height, are mediated via mammographic density, an important risk factor for breast cancer.We investigated whether childhood body size and pubertal timing and tempo, collected by questionnaire, are associated with percentage and absolute area mammographic density at ages 47–73 years in 1105 women recruited to a prospective study.p trend <0.001), and positively associated with absolute non-dense area. Greater height at age 7, but not age 11, was associated with lower percentage density (p trend = 0.016). Later age at menarche and age at when regular periods were established was associated with increased density, but additional adjustment for childhood weight attenuated the association. A longer interval between thelarche and menarche, and between thelarche and regular periods, was associated with increased dense area, even after adjusting for childhood weight , and was independent of age at pubertal onset.After controlling for adult adiposity, weight at ages 7 and 11 years was strongly significantly inversely associated with percentage and absolute dense area  contains supplementary material, which is available to authorized users. Breast cancer is the most common type of cancer in women, and the incidence has been increasing . The disWhile earlier menarche is an established risk factor for breast cancer, we recently reported that other pubertal stages also contribute to the risk, based on data from a large prospective cohort study. Earlier breast development (thelarche), and a longer interval between thelarche and menarche were independently associated with a 20–30% increase in breast cancer risk. Risk was also increased in women in whom menses became regular and adult height was reached at an earlier age .Whether pubertal associations with breast cancer risk are mediated through mammographic density is unclear. Breast tissue composition has been hypothesised to be determined by genetic factors and growth and development in early life . During Childhood height and adiposity are established predictors of pubertal onset  to the date of entry to the cohort study at screening ages 47–73 years were selected for this analysis. Percentage mammographic density and absolute dense and non-dense area (in centimetres squared) was determined using Cumulus software . Images The baseline questionnaire included information on weight and height relative to peers at age 7 and 11 years, in five categories . It also included information on age at first breast development, at menarche, at establishment of regular cycles and at reaching adult height, based on which the time intervals between stages were computed, and on other breast cancer risk factors including adult height and weight, which were used to compute the participant’s body mass index (BMI). Information on follow-up questionnaires was used to update exposures, where applicable, for women in whom mammography was conducted after they had completed the baseline questionnaire.2 dense area and 110 cm2 non-dense area, respectively, so that the effect estimates could be directly compared between variables, because the absolute difference would otherwise depend on the average of the density parameter in the reference group. The statistical package Stata 14.0 was used throughout [p values are two-sided.We analysed mammographic density parameters in relation to pubertal factors and childhood body size with a linear regression model using density parameters which were square-root-transformed to ensure the normality of the residuals. We derived absolute differences in density parameters between categories of explanatory factors so that effect estimates could be presented as percentage point differences for percent density and in centimetres squared for dense and non-dense area. This was done by back-transforming the coefficients relative to a predetermined reference level of 25% density, 30 cmroughout . All repAnalyses were adjusted for age at mammography and other mammographic density risk factors possibly associated with childhood body size or pubertal onset: age at first birth and parity, duration of oral contraceptive use, alcohol consumption and physical activity level, menopausal status and, in postmenopausal women, time since menopause and postmenopausal use of oestrogen and progestogen hormone therapy.In the literature, analyses of percentage density with respect to breast cancer risk are conventionally adjusted for BMI, as the same percentage density for a woman with high BMI does not represent the same amount of dense tissue  than in a woman with low BMI. However, for our analyses of determinants of density, given the correlation between BMI and childhood weight, adjustment for BMI could result in over-adjustment of the association between childhood body size, puberty and density. We therefore conducted the analyses with and without adjusting for BMI, as recommended elsewhere . We also2 for absolute dense area and 112.9 cm2 for non-dense area. Numbers of subjects per category of body size and pubertal factor are provided in Additional file Mammograms were retrieved for 81.6% of subjects who were within screening ages 47–73 years at the time of the baseline questionnaire, with the main reasons for non-retrieval being that films were no longer held at the screening centre or lack of detail on the questionnaire to locate the screening centre. A total of 1105 subjects were included in the analysis: their mean age at mammography was 58.9 years, and 80.1% were postmenopausal at the time of the mammogram (Table r = 0.74), but weak correlation between other stages , menarche (p trend = 0.037), or total breast area (p trend = 0.023) in a model accounting for adult and childhood adiposity. Likewise, the association with the time from thelarche to establishment of regular periods remained significant after controlling for age at thelarche (p trend = 0.035) or became borderline significant after controlling for age at the establishment of regular cycles (p trend = 0.060) or for total breast area (p trend = 0.048) (not shown). Density was not associated with the interval between age at menarche and establishment of regular cycles (Additional file There was evidence of association between a longer time interval between thelarche and menarche and increased mammographic dense area and between a longer time interval from thelarche to establishment of regular periods and increased mammographic dense area, even after adjusting for childhood adiposity Table . There wThis is, to our knowledge, the first study to investigate pubertal stages other than age at menarche with respect to quantitatively assessed adult breast density. We found evidence of association between later onset of pubertal stages, in particular age at menarche and age at establishment of regular cycles, and increased mammographic density. This study also showed that girls who were heavier than their peers in childhood had significantly lower mammographic density in adulthood, even after adjusting for adult adiposity, which correlates with childhood adiposity. As expected, increased childhood weight predicted earlier pubertal onset, and we found that the positive association of delayed puberty with density appeared to be driven by childhood weight. However, we observed a tendency for increased mammographic dense area in women reporting longer intervals between thelarche and menarche, and between thelarche and regular cycles, which was independent of the effect of age at onset and it is of interest that a prolonged pubertal tempo has also been implicated in breast cancer risk in a previous publication from our study .An inverse association of childhood weight with adult mammographic density is supported by most, but not all, previous studies . A revieOur study suggested an inverse association between percentage density and height at age 7 years and no association between percentage density and height at age 11 years, contradicting the two previous studies of similar design, in which there was higher percentage density in those who reported to have been taller than their peers in childhood , 18. OurBreast density has been hypothesised to represent the cumulative exposure of tissue to hormones and growth factors that stimulate cell division and it has been proposed that tissue composition reflects such exposures at young ages during the greatest susceptibility of the breast according to the Pike model , 34. TheBody adiposity is a strong predictor of pubertal onset, possibly mediated by leptin. Age at thelarche normally indicates gonadotropin-driven ovarian oestrogen production, but it has been postulated that breast development in obese girls is a consequence of aromatisation from adrenal androgen precursors to oestrogens in adipose tissue, which might explain the fact that early onset of breast development appears to be compensated by slower progression to menarche . IncreasAfter controlling for adult adiposity, we found that women with later onset of first or regular menses had higher mammographic density than those with early onset. Our finding is broadly in line with previous studies that after controlling for adiposity, have shown significant positive association , 14–16 bOur analyses suggest that previously reported associations between breast cancer risk and earlier thelarche, menarche, regular periods or age that adult height is reached , are unlOur study has the strength that subjects were selected from a prospective study with comprehensive information on breast cancer risk factors. A limitation is that the pubertal and weight variables that we collected were self-reported. Also, BMI was assessed at baseline and was not available at the exact time of mammography, and we were unable to collect exact weights in childhood and our proxy variables of weight in childhood relative to peers and the variable for growth spurt are therefore relatively crude measures. The accuracy of reporting of age at menarche and body size in childhood is thought to be reasonably good , but recAdult mammographic density was inversely associated with weight compared to peers at ages 7 and 11 years, and was not independently associated with age at onset of pubertal stages. The role of a prolonged duration between breast development and onset of first or regular menses on breast density needs investigation in future studies.


FSCN1 single nucleotide polymorphisms (SNPs) and the risk or prognosis of breast cancer. We report on the association between 6 SNPs of the FSCN1 gene  and breast cancer susceptibility as well as clinical outcomes in 316 patients with breast cancer and in 222 healthy controls. Carriers of the AC or AC + CC allele of the variant rs56156320 were at greater risk of breast cancer compared with wild-type (AA) carriers. Moreover, carriers of at least one G allele in rs3801004 were likely to progress to stage III/IV disease and lymph node metastasis. Individuals with at least one T allele at FSCN1 SNP rs2966447 were at higher risk of developing pathologic grade G3 disease. Furthermore, individuals bearing the C/C haplotype at SNPs rs56156320 and rs3801004 had nearly twice the risk of breast cancer. Our results indicate that genetic variations in the FSCN1 gene may serve as an important predictor of early-stage breast cancer.Breast cancer is a major cause of cancer mortality worldwide. Fascin-1 (FSCN1) is an actin-binding protein found in mammalian cells, including endothelial, neuronal and mesenchymal cells. FSCN1 overexpression has been indicated in breast cancer patients. However, scant information is available regarding the association between  Risk factors associated with breast cancer in women include her age, family history, reproductive and gynecologic factors, and lifestyle factors including alcohol consumption and lack of physical activity, amongst others2. Women who are at high risk of breast cancer may be advised to maintain their mammography screening schedule, undergo genetic testing, or commence chemoprevention.Breast cancer is associated with high mortality. Over a million women worldwide are diagnosed with breast cancer every year, and over 500,000 succumb to the disease2. Researchers have therefore explored genetic variation associated with breast cancer risk, hoping that single nucleotide polymorphism (SNP) genotyping will more accurately stratify breast cancer risk and guide disease management. Emerging reports indicate an association between SNPs in certain genes and susceptibility to breast cancer, as well as clinicopathologic status. Besides the recognized BRCA1 and BRCA2 mutations that markedly increase the risk of developing breast cancer4, a number of additional low- and moderate-risk susceptibility variants have been identified, including caspase-8 (CASP8), an enzyme involved in apoptosis5.Current statistical models for estimating breast cancer risk have limited sensitivity and specificity7. Upregulation of FSCN1 has been indicated in various cancer cell types such as stomach, colon, lung, ovary and breast12. Overexpression of FSCN1 significantly promotes colon cancer cell migration and metastasis13, while knockdown of FSCN1 in cellular models diminishes cell motility and tumor metastasis in prostate cancer14 and oral squamous cancer15. Several cytokines, including interleukin-6 and oncostatin M, control fascin expression through the activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway in breast cancer cells16. Recent evidence suggests that aberrant STAT3 signaling promotes breast tumor progression by deregulating the expression of downstream target genes that control angiogenesis, including nuclear factor kappaB (NF-κB) and hypoxia-inducible factor 1 (HIF-1), increasing their binding to the fascin gene promoter to induce its expression17. Interestingly, a highly significant correlation has been observed between fascin expression and decreased overall survival in African American women with triple-negative breast cancer (TNBC)18. Similarly, we have previously described how strong positive FSCN1 expression can be used as a diagnostic marker of TNBC in Chinese women19. However, any association between FSCN1 SNPs with breast cancer risk and prognosis remains to be clarified. We therefore conducted a case-control study to evaluate the role of 6 FSCN1 SNPs in breast cancer susceptibility and clinicopathological features in a cohort of Chinese Han individuals.Fascin-1 (FSCN1), an actin-binding protein found in mammalian cells including endothelial, neuronal and mesenchymal cells, is low or absent in normal epithelial cellsp < 0.05), but not for alcohol consumption (p > 0.05). Most patients (76.9%) had stage I/II breast cancer; 23.1% had stage III/IV disease . In both patients and controls, most of those with the rs56156320 SNP, the rs2966447 SNP, or the rs852479 SNP were homozygous for the AA genotype, while most of those with the rs8772 SNP, the rs3801004 SNP, or the rs1640233 SNP were homozygous for CC . There were no significant differences between cases and controls in regard to the frequency of rs8772, rs3801004, rs2966447, rs852479 and rs1640233 polymorphisms  at the rs3801004 SNP were at more than twice the risk of developing stage III/IV disease  and lymph node metastasis  , which was therefore selected as the reference. The C-C FSCN1 haplotype significantly increased the risk for developing breast cancer by almost 2-fold compared with the reference group A-C (p < 0.05) . No significant differences were observed between patients and healthy controls in the frequencies of the rs8772, rs3801004, rs2966447, rs852479 and rs1640233 polymorphisms. The polymorphisms in the 3ʹ-flanking region of a gene can control gene expression28. Data from the GTEx database demonstrated that variant C at rs56156320 showed a trend for increased expression of FSCN1, compared with the wild-type AA homozygous genotypes. This result confirms our SNP data and indicates that the FSCN1 rs56156320 SNP may control the expression of FSCN1.Breast cancer is the most commonly diagnosed neoplasm and the third leading cause of cancer-associated mortality in the United States, with 22.2 mortalities per 100,000 women associated with breast cancer each year. The 5-year relative survival rate for breast cancer has gradually increased since the early 1990s; between 2007 and 2011 it was ~89.2%. The prognosis of patients with breast cancer is critically dependent on the disease stage at the time of diagnosis. Therefore, it is important to increase screening rates and genetic testing for hereditary breast cancer, to increase the chances of early diagnosisFSCN1 rs3801004 polymorphism had a higher risk of developing stage III/IV disease and lymph node metastasis. Similarly, the FSCN1 rs2966447 polymorphism was also associated with a higher risk of developing pathological grade (G3) disease. Interestingly, the rs2966447 SNP was associated with a higher risk of developing stage III/IV disease and lymph node metastasis in the Luminal A subgroup, as well as a higher risk of pathologic grade (G3) disease in the Luminal B subgroup. It is established that overexpression of the FSCN1 gene is implicated in the development and metastasis of breast cancer29. In addition, FSCN1 is involved in the chemotherapeutic resistance of breast cancer cells30. However, more research is required to determine whether an association exists among advanced-stage disease, FSCN1 expression levels and FSCN1 genotype, and clarification is needed in regard to the effects of the FSCN1 genotype on breast cancer risk.This study found that breast cancer patients with the 31. We evaluated the impacts of different haplotype combinations of 2 FSCN1 SNPs (rs56156320 and rs3801004) upon the risk of breast cancer and found that the CC haplotype was associated with a higher risk for breast cancer. It is possible that the FSCN1 CC haplotype is in linkage disequilibrium with other functional polymorphisms that are responsible for susceptibility to breast cancer. On the other hand, we also found that rs56156320 and rs3801004 showed 96% linkage disequilibrium. Furthermore, rs852479 and rs2966447 as well as rs3801004 and rs1640233 expressed 98% and 92% linkage disequilibrium, respectively. These results suggest that these FSCN1 haplotypes play an important role in breast cancer development.Linkage disequilibrium is expressed across the human genome. Thus, loci can be used as genetic markers to locate adjacent variants that participate in the detection and treatment of disease. Haplotype analyses can provide data on the genetic contribution to disease susceptibilityFSCN1 gene variants and the risk of breast cancer. We show that the FSCN1 rs56156320 polymorphisms significantly increase the risk of breast cancer progression among Chinese Han females. This study is the first to report a correlation between FSCN1 polymorphisms and breast cancer risk. FSCN1 may serve as a predictive marker for breast cancer therapy.In conclusion, our results demonstrate an association between 32. Breast cancer cases were categorized by estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki‐67 status, into 4 subtypes: Luminal A ; Luminal B ; HER2‐enriched ; or TNBC . A standardized questionnaire and the electronic medical record system were searched for demographic data on age, sex, smoking history, and alcohol consumption.Between 2014 and 2016, we collected 318 blood specimens from patients (cases) who had been diagnosed with breast cancer at Dongyang People’s Hospital. The control group consisted of 222 healthy participants without a history of cancer. All participants provided written informed consent, and the study was approved by the Ethics Committee of Dongyang People’s Hospital. This study’s protocol was approved by the Ethics Committee of Dongyang People’s Hospital and all experiments were performed in accordance with relevant guidelines and regulations. Pathohistologic diagnosis followed the World Health Organization classification of breast tumors and tumors were graded using the Scarff-Bloom-Richardson method2 (pH 7.8) and stored at −20 °C until it was subjected to quantitative polymerase chain reaction (PCR) analysis. Six FSCN1 SNPs  were examined with the use of a commercially available TaqMan SNP genotyping assay , according to the manufacturer’s protocols34.Total genomic DNA was isolated from whole blood specimens using QIAamp DNA blood mini kits , as per the manufacturer’s instructions. The DNA was dissolved in Tris-EDTA (TE) buffer composed of 10 mM Tris-HCl containing 1 mM EDTA•Na35. We conducted an investigation of expression quantitative trait loci (eQTLs), to determine the functional role of phenotype-associated SNPs.Genotype-Tissue Expression (GTEx) data were used to identify correlations between SNPs and levels of FSCN1 expression36. A p value of <0.05 was considered statistically significant. Data were analyzed using SAS statistical software .The genotype distribution of each SNP was analyzed for Hardy–Weinberg equilibrium and confirmed by Chi-square analysis. Demographic characteristics were compared between patients and controls using the Mann–Whitney U-test and Fisher’s exact test. Associations between genotypes, breast cancer risk and clinicopathologic characteristics were estimated using adjusted odds ratios (AORs) and 95% confidence intervals (CIs), after controlling for other covariates. Significant differences in haplotype frequencies between cases and controls were analyzed using Haploview, according to the software package