There is sufficient evidence supporting a relationship between increased body mass index (BMI) and an increased risk for breast cancer among postmenopausal women. However, most studies have found a decreased risk for premenopausal breast cancer. This study was conducted to find out the different effects of BMI on the risk of breast cancer among premenopausal and postmenopausal women, and explore the potential factors that influence the associations.A dose-response meta-analysis with 3,318,796 participants from 31 articles was conducted. Cohort studies that included BMI and corresponding breast cancer risk were selected through various databases including PubMed, Medline, Web of Science, the China National Knowledge Infrastructure (CNKI) and Chinese Scientific Journals (VIP). Random effects models were used for analyzing the data.2) the RR in total postmenopausal women were1.03 (95% CI: 1.02–1.05) per 1 kg/m2 increment. However, no statistically significant association among total premenopausal women was detected. In subgroup analysis among European premenopausal women, the summary RR was 0.79(95%CI: 0.70–0.88). The non-linear relationship showed a negative non-linear association between BMI and breast cancer risk among European premenopausal women. When compared to the mean level of the normal BMI category, the RRs were 0.98 (95%CI: 0.96–1.00) per 1 kg/m2 increment, respectively.The summary relative risks (RRs) were 1.33 (95%CI: 1.20–1.48) and 0.94(95%CI: 0.80–1.11) among postmenopausal and premenopausal women, respectively. The dose-response meta-analysis indicated a positive non-linear association between BMI and breast cancer risk among postmenopausal women, and compared to the mean level of the normal BMI category  contains supplementary material, which is available to authorized users. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide. It accounts for 25% of all cancer cases and 15% of all cancer deaths among females [. However, studies among premenopausal women are inconsistent and unclear. Some studies have shown a decreased risk for premenopausal breast cancer [There is sufficient evidence supporting a relationship between increased body mass index (BMI) and an increased risk for breast cancer among postmenopausal women –5. Howevt cancer , while ot cancer , 8, 9. Tt cancer  currentlWe searched PubMed, Medline, Web of Science, and Chinese academic databases including the China National Knowledge Infrastructure (CNKI) and Chinese Scientific Journals (VIP) databases for publications on the associations between BMI and breast cancer in humans, through December 31, 2015. The keywords were (obesity OR obese OR adiposity OR fat OR fatness OR “body mass index” OR BMI OR “body size” OR weight OR overweight) AND (“breast cancer” OR “breast carcinoma” OR “breast neoplasm”). References from reviews and meta-analyses were also searched for additional publications.Original article publication.Study design was a prospective cohort study.Study had BMI categories of no fewer than three, with a relative risk (RR) or hazard ratio (HR), and corresponding 95% confidence interval (CI) for the breast cancer presented for each BMI category.The studies provided the cases and person-years number for each BMI category.Studies were included according to the following criteria:From the selected studies the information extracted was: first author’s last name; publication year; country of origin; duration of follow-up; intervals of each BMI category; number of cases and cohort size of each category; relative risks and the 95% CI for each BMI category; and variables adjusted for in the multivariable analysis. The results that were adjusted by the most possible confounding factors were extracted. If data were reported by age category, hormone replacement therapy (HRT) use or other factors in one publication, they were considered as different studies. Two investigators (Cai and Wang) independently retrieved the data. The disagreement was resolved by mutual discussion.1375 studies were duplicated or irrelevant to our study according to titles or abstracts.18 studies were reviews or meta-analyses.35 studies were case-control studies.29 studies were excluded because the data needed were not available.Figure Using the Newcastle-Ottawa Scale (NOS), the quality of the included studies was assessed independently by two reviewers (Cai and Wang). This scale ranges from 0 to 9 stars and awards four stars for selection of study participants, two stars for comparability of studies, and three stars for the adequate ascertainment of outcomes. Scores of 0–3, 4–6, and 7–9 are viewed as low, moderate, and high quality, respectively.Because the incidence of breast cancer is low, HR is mathematically similar to RR. The results were reported as RR. The associations between BMI and the risk of breast cancer were quantified by comparing the highest versus the lowest categories. For the dose-response meta-analysis, the dosage value corresponding to each BMI was the median of the upper and lower boundaries. For the studies that did not report a median, the midpoint of closed categories was used. In the case of open ended highest category or lowest category, we assumed that the boundary had the same amplitude as the adjacent category , 12.The least squares estimation was conducted to account for the correlation with the logRR estimates across the BMI , 14. Theχ2 test. The quantification of heterogeneity was assessed by the I2 statistic. An I2 above 50% indicated high heterogeneity. A random effect model was implemented. Publication bias was identified with the Begg’s rank correlation test and Egger’s regression test [P < 0.05, except where there was a publication bias or heterogeneity when P < 0.10.Heterogeneity among studies was assessed using the ion test , 17. Theion test  were estAdditional file Among the studies, 12 were conducted in Europe , 41, 45,P = 0.01, I2 = 49%). Both Begg’s regression test (P = 0.705) and Egger’s correlation test (P = 0.347) showed no publication bias. Another 26 studies reported a link between BMI and breast cancer risk among postmenopausal women, with a summary RR of 1.33(95%CI: 1.20–1.48) . The Begg’s correlation test (P = 0.113) and Egger’s regression test (P = 0.603) showed no publication bias for these studies, as shown in Table In this study, we selected the RRs corresponding to the highest BMI categories as the highest dose, and the RRs corresponding to the normal BMI categories as the lowest dose. Eighteen studies reported on the relationship of BMI with premenopausal breast cancer risk, the summary RR was 0.94(95%CI: 0.81–1.11) see Fig , and hetst P = 0.47 showedP = 0.68, I2 = 0). When subgroup analysis was done for different geographical locations, it showed significant associations between BMI and breast cancer in Asia  and America , but poor association in Europe . Interestingly, for premenopausal women, an inverse significant association between BMI and breast cancer was detected in Europe , but no significant associations in America  or Asia .The subgroup analyses are shown in Table Additionally, we conducted a subgroup analysis stratified by follow-up year, the number of cases in the studies with shorter follow-up is lower, and consequently the power is reduced. In postmenopausal women, there was a significant association between BMI and breast cancer risk when the follow-up was above 5 years, while no significant association was detected when the follow-up was below 5 years. In premenopausal, there was no significant association between BMI and breast cancer risk in both subgroups.2. In terms of premenopausal women, a dose-response analysis including 18 studies with 25 comparisons was conducted, the non-liner relationship between BMI and breast cancer risk showed no statistical significance among total premenopausal women (χ [P = 0.168). In the subgroup analysis of premenopausal women, high BMI showed a decreased breast cancer risk in European women, and it showed a 2.3% decreased breast cancer risk per 1 kg/m2 compared to 21.5 kg/m2. The detailed information of the dose-response meta-analysis results and charts of premenopausal and postmenopausal women as well as the subgroup analyses are shown in Table For postmenopausal women, 26 studies including 62 comparisons reported RRs for corresponding BMI categories. The dose-response meta-analysis indicated a positive non-linear association between BMI and breast cancer risk among postmenopausal women. When compared to the mean level of the normal BMI range, it showed a 3.4% increased breast cancer risk per 1 kg/mP = 0.046). After using a trim-and-fill analysis, no study showed bias, and the results were unchanged.No publication bias was observed for any of the studies or the subgroup studies, except for studies on postmenopausal women in Europe using the Egger’s test  to 0.98(95%CI: 0.84–1.15), and the result was not influenced by any single study. Similarly, the sensitivity analysis in postmenopausal women showed stable results, which ranged from 1.30 (95%CI: 1.17–1.45) to 1.36 (95%CI: 1.23–1.51).2 increment of BMI in postmenopausal women.In this meta-analysis, there was a significant positive association between BMI and breast cancer risk among postmenopausal women but no correlation among total premenopausal women. Non-linear dose-response analysis showed that the breast cancer risk increased by 3.4% for every 1 kg/mI2 = 0) when stratified by ER status, suggesting ER status may be an interactive factor for the relationship between BMI and breast cancer risk. The lastest WCRF 2017 report showed that in postmenopausal women BMI was significantly positively associated with ER+ breast cancer, but not ER– breast cancers, it was consistent with our study.Heterogeneity was observed among the studies included in our meta-analysis. The diversity most likely comes from differences in study design, sample source and size, reference categories, different BMI cut-offs, different weight distribution, length of follow up, racial difference, ER status, HRT use or other potential confounders. We conducted a subgroup analysis to explore the heterogeneity and potential factors influencing the associations. The heterogeneity was zero  between studies remained, and the relationship between BMI and BC risk was not statistically significant. In WCRF 2017 report, dose-response meta-analyses for postmenopausal breast cancer by geographical location showed a statistically significant increased risk in North American and European studies, and a stronger association in Asian studies. The AICR&WCRF 2017 report includes 19 cohort and case-control studies of Europe (I2 = 75%), while 9 cohort studies of Europe (I2 = 71%) were included in this study. Potential confounding factors such as the difference in fat distribution, life styles or a possible interaction effect might be the source of the high heterogeneity. It necessitates further studies in order to provide more insights into such effects among European women.When stratified by geographical location among postmenopausal women, a significant non-linear dose-response relationship between BMI and breast cancer risk existed in American and Asian women, but not among European women. These results were inconsistent with the previous report conducted by Xia Xiaoping, et al.  Xia showFor premenopausal women, the AICR&WCRF report summarized the epidemiologic evidence available up to 2017 and concluded that there is a substantial dose response relationship for the inverse association between BMI and risk of breast cancer among premenopausal women. However, the non-significant association of BMI with risk of premenopausal breast cancer observed in this study is not consistent with the WCRF report. This may be because the studies included in this study differed from those included in AICR&WCRF 2017 report. The studies included in this article were all cohort studies which were more convincing, however, the number of studies included in AICR&WCRF 2017 report were more than this study, and more recent studies may be included. Therefore, the sample source and size, different fat distribution, follow-up year, race, ER status, HRT use or other confounders are possibly the reasons of differences.2. In American premenopausal women, there was no association between BMI and breast cancer. These results are comparable to those observed in a systematic review by Amadou et al. [2, which was similar to the report of Amadou et al. These results suggest that ethnicity or geographical location may be a factor influencing the relationship between BMI and breast cancer risk, but this conclusion is speculative. In this meta-analysis, breast cancer risk could be influenced by overweight or obesity according to different geographical locations, irrespective of menopausal status. The difference may be related to different genetic susceptibility among different ethnic groups, as demonstrated for breast cancer susceptibility genes (BRCA1 or BRCA2) mutations previously [A lack of data prohibited many subgroup analyses from being carried out. However, stratification by geographical location demonstrated an inverse significant association between BMI and breast cancer among European premenopausal women but not among Asian and American premenopausal women. This conclusion is consistent with the AICR&WCRF 2017 report when stratified by geographical location. When compared to women with a normal BMI , women with a higher BMI were at a decreased risk of breast cancer risk by 2.3%. In Asian women, the results showed a positive non-linear relationship between BMI and breast cancer when BMI was above 25 kg/mu et al. , which eu et al. , in addieviously .BMI, adult weight gain, body weight and fat distribution may play a role in the prognosis of breast cancer separately or together in combination. IARC working group , 60 repoThe studies included in this meta-analysis were all cohort studies, with large sample sizes, and adequate subgroup analyses. Moreover, the confirmation of the breast cancer cases was done by diagnosis, hospital medical record or linkage data. Thus, the results were persuasive, and were less likely to suffer from recall bias and selection bias. However, some limitations also exist for this meta-analysis; the measurement of BMI was determined mostly by questionnaire investigation or self-reported, and people tended to report their weight lower and height higher than reality. The adjustments factors of each study were not the same, thus the RRs we extracted from the studies were possibly affected by potential confounders even after selecting the adjusted RRs. In addition, the categorization of BMI in a number of articles included in this study was not in accordance with the WHO standard, thus misclassifications may be caused when we conduct the highest versus the lowest BMI meta-analysis.s. In postmenopausal women, breast cancer risk increased along with increasing BMI. In premenopausal women, there was no signification between BMI and breast cancer. This is inconsistent with the 2017 report of AICR&WCRF which showed higher BMI is defined as probably decreasing breast cancer risk. However, most conclusions stratified by various factors in this study are consistent with AICR&WCRF.In conclusion, BMI had different effects on premenopausal and postmenopausal breast cancer riskSubgroup analyses suggested that the geographical location or genetic factors may influence the relationship between BMI and breast cancer. Further research is needed to provide more continuous updating insights.Additional file 1:Study characteristics of published cohort studies on body mass index(BMI) and breast cancer risk. (DOC 725 kb)Additional file 2:The related data and materials in this study. (ZIP 1785 kb)


HMGB1/RAGE) pathway to breast cancer, we genotyped six well‐defined polymorphisms in this pathway among 524 breast cancer patients and 518 cancer‐free controls from Heilongjiang province, China. There were no deviations from Hardy–Weinberg equilibrium for all polymorphisms. In single‐locus analysis, the frequency of rs1800624 polymorphism mutant A allele in RAGE gene was significantly higher in patients than in controls , with the carriers of rs1800624‐A allele being 1.51 times more likely to develop breast cancer relative to those with rs1800624‐GG genotype after adjustment . In HMGB1 gene, haplotype analysis did not reveal any significance, while in RAGE gene, haplotypes C‐T‐A and C‐A‐G  were significantly associated with an increased risk of breast cancer . In further genetic score analysis, per unit and quartile increments of unfavourable alleles were significantly associated with an increased risk of breast cancer after adjustment . Our findings altogether demonstrate a significant association between RAGE gene rs1800624 polymorphism and breast cancer risk, and more importantly a cumulative impact of multiple risk associated polymorphisms in HMGB1/RAGE pathway on breast carcinogenesis.Breast cancer constitutes an enormous burden in China. A strong familial clustering of breast cancer suggests a genetic component in its carcinogenesis. To examine the genetic predisposition of high mobility group box‐1/receptor for advanced glycation end products ( In recent two decades, breast cancer has escalated to an emerging epidemic and constitutes an enormous burden in China HMGB1 and RAGE, and examined their genetic predisposition to breast cancer in a large Han Chinese population from Heilongjiang province.HMGB1 is the acronym for high mobility group box‐1, a proangiogenic nuclear cytokine implicated in tumorigenesis, proliferation and metastasis This is a case–control association study. All study subjects were females of Han nationality, and they were enrolled from four local hospitals  in Heilongjiang province, China between January 2013 and August 2015. This study was approved by the Ethics Committee of Qiqihar Medical University. All study subjects signed written informed consent before agreeing to participate in this study according to the Declaration of Helsinki.Breast cancer patients were newly diagnosed, histopathologically confirmed or previously untreated. There were no restrictions on age, gender and cancer‐stage at enrolment.This study enrolled a total of 1042 female subjects, including 524 breast cancer patients and 518 cancer‐free controls.For breast cancer patients, data were recorded on the age of first onset, age of menarche, menopausal age, family history of cancer, tumour size (T1‐T4), histological grade (G1‐G3) and lymph node. For controls, age at enrolment and age of menarche were recorded, and they reported to have no prior history of any cancer types (except for non‐melanoma skin cancer).A blood heparinized sample was obtained from each study subject, and genomic DNA was extracted from leucocytes by the phenol‐cholesterol method according to a standard procedure.HMGB1 gene and three polymorphisms  in RAGE gene were selected for genotype determination and association analyses. These six polymorphisms were well defined and widely evaluated in association with a broad range of cancers Three polymorphisms  in http://seq.yeastgenome.org/cgi-bin/web-primer. For each allele, a specific probe was synthesized, and an additional common probe capped with 6‐carboxy‐fluorescein at the 3′ end and with horylated at the 5′ end was also synthesized. The ligation reaction mixture contained PCR product (2 μl), 10× Taq DNA ligase buffer (1 μl), each discriminating probe (1 μM) and Taq DNA ligase (5 U) in double‐distilled water to make a volume of 10 μl. The ligation conditions were 30 cycles of 30 sec. at 94°C and of 3 min. at 56°C. After that, 1 μl ligation product was mixed with 1 μl of ROX passive reference and 1 μl of loading buffer before being denatured at 95°C for 3 min. and chilled rapidly in ice water. The fluorescent products of ligase detection reaction were differentiated using ABI 3730XL sequencer .The genomic sequences of six examined polymorphisms were amplified by polymerase chain reaction (PCR), and their genotypes were further distinguished by ligase detection reaction method To test the validity and accuracy of this genotyping method, 48 DNA samples were randomly selected and genotyped for the second time by ligase detection reaction method, and reduplication results were 100% consistent. Genotyping was determined by laboratory workers in a manner blind to the case–control status and pertinent characteristics of study subjects.All examined polymorphisms were checked for adherence to Hardy–Weinberg equilibrium by the chi‐squared test to avoid population stratification or genotyping misclassification. The genotype/allele distributions between patients and controls were compared by the chi‐squared test or Fisher's exact test where appropriate. Besides overall comparisons, stratified analyses according to the median values of age and age of menarche among all subjects were also conducted for the genotype/allele distributions between the two groups. The risk prediction for breast cancer was quantified by Logistic regression analyses before and after controlling for confounding factors (age and age of menarche). Effect‐size estimates were expressed as odds ratio (OR) and its 95% confidence interval (95% CI).www.r-project.org/).Considering the fact that the impact of a single polymorphism might be small, the co‐occurrence of unfavourable alleles of multiple polymorphisms can enhance the risk for breast cancer. A genetic score is therefore created on the basis of the number of unfavourable alleles for each subject by assigning zero, one or two unfavourable alleles of each polymorphism and summing them up. Besides per score increment, total genetic score was also collapsed into quartiles, and risk prediction was quantified by Logistic regression analyses before and after controlling for confounding factors. In addition, haplotype analysis is proven to be more informative than studying the role of polymorphisms independently. Haplotype analysis was undertaken within each gene under a generalized linear model by using the HAPLO.STATS program before and after controlling for confounding factors. The HAPLO.STATS program was implemented in the R Project for Statistical Computing version 2.6.2 . The power to reject null association was calculated by the PS: Power and Sample Size Calculation software version 3.0  versus 53.76 years, P < 0.001). In contrast, the mean age of menarche was higher in patients than in controls . The mean menopausal age was 50.19 years in patients, and about 6% of patients had a positive family history of cancers. As for tumour size, there were 49.69%, 42.77%, 3.77% and 3.77% of patients having T1, T2, T3 and T4 respectively. With regard to tumour stage, the G2 (49.27%) and G3 (46.10%) stages accounted for the majority of breast cancer patients. A total of 42.47% of patients were detected with positive lymph node.There were 524 breast cancer patients and 518 controls in this study, and their baseline characteristics are listed and compared in Table RAGE gene differed significantly between patients and controls (P = 0.008), and the frequency of its mutant A allele was significantly higher in patients than in controls , even after the Bonferroni correction (P < 0.05/6). The power to reject the null hypothesis of no allelic difference for rs1800624 polymorphism between patients and controls was estimated to be 82.9%. In addition, for rs2249825 and rs1800625 polymorphisms, there was marginal significance in allele distributions between the two groups , and after the Bonferroni correction, no significance was found.The chi‐squared‐based goodness‐of‐fit test revealed that the genotype distributions of six examined polymorphisms did not deviate from Hardy–Weinberg equilibrium at a significance level of 5%. As shown in Table RAGE gene rs1800624 polymorphism among subjects aged <55 years (P = 0.005) and for HMGB1 gene rs2249825 polymorphism among subjects aged ≥55 years . Grouping subjects by age of menarche at a cut‐off value of 14 years (median) revealed only significant allelic association for RAGE gene rs1800624 polymorphism among subjects with age of menarche ≥14 years .In further stratified analyses by age at a cut‐off value of 55 years (median), the allelic association with breast cancer risk was strikingly significant for P = 0.001), even after the Bonferroni correction (P < 0.05/6). In addition for rs1800625 polymorphism, the mutant genotype conferred a marginally increased risk for breast cancer before and after adjusting for age and age of menarche, especially under the dominant model, that is, the odds of having breast cancer was 1.34  and 1.31  before and after adjustment. In contrast to rs2249825 polymorphism, the carriers of mutant genotype or allele had a reduced risk for breast cancer with marginal significance, which did not survive the Bonferroni correction.Given the small number of mutant homozygotes, only additive and dominant models were conducted for six examined polymorphisms Table . The sigHMGB1 (13q12) and RAGE (6p21.3) are located on different chromosomes, haplotype analysis is conducted separately , which was over‐represented in patients relative to controls  and was marginally associated with breast cancer risk . In RAGE gene, when compared with the reference haplotype T‐T‐G , two haplotypes, C‐T‐A and C‐A‐G, were over‐represented in patients, and were significantly associated with an increased risk of breast cancer even after adjusting for age and age of menarche .In theory, a haplotype is defined as the combination of multiple alleles on 1 chromosome. Haplotype analysis refers to the simultaneous analysis of multiple polymorphisms. Considering that the genes encoding ly Table . The mosP = 0.961), 3rd quartile  and 4th quartile  after adjusting for age and age of menarche. In addition, per unit and quartile increments of unfavourable genotypes were significantly associated with an increased risk of breast cancer after adjustment .As some examined polymorphisms were significantly or marginally associated with breast cancer, a genetic score analysis was performed to evaluate the cumulative impact of risk associated polymorphisms, and the results are summarized in Table RAGE gene rs1800624 polymorphism and breast cancer. More importantly, further haplotype and genetic score analyses suggested that there was a cumulative impact of multiple risk associated polymorphisms in this pathway on the development of breast cancer.In this case–control study, we examined the genetic predisposition of six well‐defined polymorphisms in HMGB1/RAGE pathway to breast cancer in a large Han Chinese population, and we observed a significant association between Currently, evidence for the implication of activated HMGB1/RAGE pathway in cell proliferation, angiogenesis and metastasis during breast cancer progression is rapidly accumulating RAGE gene was significantly associated with an increased risk of having breast cancer in Han Chinese, in contrast to the negative findings for this polymorphism in a recent study by Pan et al. among 1013 local residents of Qiqihar city P = 0.006) in this study, and the power to detect this significant association was around 80%. The association between rs1800624 polymorphism and breast cancer, albeit statistically significant, has to be interpreted with caution, and independent confirmation will be important. In addition, both this study and the study by Pan et al. RAGE gene, two haplotypes C‐T‐A and C‐A‐G , which differed only in the latter two loci, were observed to both confer an increased risk for breast cancer, a finding contradictory to the significant predominant role of rs1800624 polymorphism in single‐locus analysis. It is possible that the part played by rs2070600 polymorphism was not significant unless the coinheritance of the wild allele of rs1800624 polymorphism. To the best of our knowledge, this is the first study interrogating the combined association of HMGB1 and RAGE genes with the risk of breast cancer, and further validation in other ethnic groups is required.Candidate gene approach is a key research paradigm for unfolding the genetic underpinnings of complex diseases, including cancers Finally, the interpretation and extrapolation of our findings must consider several potential limitations. The first limitation is the retrospective case–control association design, and such design cannot reveal the possible cause‐effect between HMGB1/RAGE pathway and breast cancer RAGE gene rs1800624 polymorphism and breast cancer risk, and more importantly, there was a cumulative impact of multiple risk associated polymorphisms in this pathway on the development of breast cancer.Taken together, we through a genetic analysis of HMGB1/RAGE pathway observed a significant association between This study was financially supported by the Project of Department of Education of Heilongjiang Province (grant no. 12541903).None declared.Table S1 The genotype distributions and allele frequencies of six examined polymorphisms in HMGB1/RAGE pathway between breast cancer patients and controls by age at a cut‐off value of 55 years (median).Table S2 The genotype distributions and allele frequencies of six examined polymorphisms in HMGB1/RAGE pathway between breast cancer patients and controls by age of menarche at a cut‐off value of 14 years (median).Click here for additional data file.


Environmental Persistent Organic Pollutants (POPs) can alter the hormone homeostasis by mimicking, interfering or blocking the function of hormones; moreover POPs are hypothesized to modify the risk of breast cancer. The association between POPs and breast cancer has been widely studied but the conclusions are inconsistent. The present study examined the associations between serum levels of POPs and breast cancer with focus on the highly exposed Greenlandic Inuit population.The study design was a case-control study of Inuit women from Greenland. The participants were asked to complete a questionnaire with information on reproductive history and lifestyle and to provide a blood sample. The sampling was carried out in two time periods (2000–2003 and 2011–2014). The serum levels were determined of 14 polychlorinated biphenyls (PCBs), 11 organochlorine pesticides (OCPs), 16 perfluoroalkyl acids (PFAAs), 1 polybrominated biphenyl (PBB), and 9 polybrominated diphenyl ethers (PBDEs). Independent samples t-test was used to compare differences between cases and controls and odds ratios (OR) adjusted for identified confounders were obtained using logistic regression.The study population included 77 breast cancer cases and 84 controls. The majority of the measured compounds declined significantly from 2000 – 2003 to 2011–2014. However, for the perfluorinated carboxylic acids (PFCAs) an increase was observed. The serum levels were significantly higher in cases compared to controls for the majority of the compounds, and after adjusting for age the difference was maintained for ∑OCP, dichlorodiphenyldichloroethylene , ∑PFAA, ∑perfluorinated sulfonic acids (PFSA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS). For the lipophilic POPs, high serum levels (middel/highest vs. lowest tertile) of ∑PCB, ∑estrgoenicPCB, PCB99, PCB138, PCB153, PCB170, PCB170, and PCB183 was associated with breast cancer risk; for the amphiphilic PFAAs, high serum levels of ∑PFAA, ∑PFCA, ∑PFSA, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), PFHxS, and PFOS were associated with breast cancer risk.Significant, positive associations between breast cancer risk and PCBs and PFAAs were observed. The associations indicate that environmental exposure to POPs can be a factor increasing the risk for breast cancer in Inuit women.The online version of this article (doi:10.1186/s12940-017-0269-6) contains supplementary material, which is available to authorized users. Normal development, growth, and function of the mammary glands depend on a well functioning and balanced endocrine system. Thus, endocrine-related processes altering the hormone levels such as early age at menarche, nulliparity or delayed childbearing, late age at menopause, and use of hormone replacement therapy are established risk factors for breast cancer , 2. EnviEnvironmental pollutants, such as polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and perfluorinated alkylated acids (PFAAs), are reported potential endocrine disrupters . DespiteExisting studies on environmental exposure and breast cancer risk are mainly conducted in Europe and America where the majority of the population belongs to the Caucasian race and the exposure levels are relatively low. It has been indicated that susceptibility to environmental pollutants differs between different ethnic groups. In the meta-analysis by Zhang et al. 2015), geographic heterogeneity in the association of PCB groups with breast cancer risk was observed . In the , geograpWith the possible ethnic differences in mind, additional attention on breast cancer risk in ethnic populations is needed. In the present study, we assessed the influence of environmental pollutants on breast cancer risk in the highly exposed Greenlandic Inuit women.We used a case-control design. Participants were recruited during 2000–2003 and 2011–2014. The sampling has been described earlier by Bonefeld-Jørgensen et al.  for the Breast cancer cases were recruited at diagnosis at Dronning Ingrids Hospital in Nuuk, Greenland. The breast cancer diagnosis was confirmed by a positive histological sample. Controls for the participants recruited during 2000–2003 were selected from two cross-sectional studies on healthy persons with serum measurements on persistent organic pollutant (POP) in the same period , 19. TheAll participants had to be of Greenland Inuit descent, defined as having more than two grandparents born in Greenland.A total of 115 controls and 31 cases were enrolled during 2000–2003 and 66 cases and 62 controls were enrolled during 2011–2014. To optimise the similarity of the study populations from the two recruitment periods, the controls from 2000 to 2003 were reduced to one control per case. From the 115 enrolled controls we selected 31 controls based on age and geographical living area in Greenland.At enrollment the participants completed an assisted lifestyle questionnaire and provided a blood sample. For cases the blood samples were obtained before any treatment was initiated.Information about age, body mass index (BMI), smoking status, menopause status, number of full term pregnancies, and history of breastfeeding was obtained from questionnaires.Serum levels of lipophilic persistent organic pollutant (POP) from both recruitment periods were measures at Le Centre de Toxicologie du Québec . The POP measurements for the 2000–2003 collection are described elsewhere . The samPFAAs were determined in serum samples at Department of Environmental Science, Aarhus University and details of the method have been described previously , 20. In The plasma cotinine level was measured using Calbiotech Direct ELISA kit  in accordance with the manufacturer’s protocol. Briefly, 10 μL of standard or sample was pipetted into the well, 100 μL Enzyme Conjugate was added and the plate was shaken for 30 s followed by 60 min of incubation. The wells were washed six times with 300 μL distilled water after incubation before adding 100 μL substrate reagents. After 30 min of additional incubation, 100 μL stop solution was added and the absorbance was detected at 450 nm on an EL8000 Universal Microplate Reader . Samples with levels higher than 100 ng/mL were diluted and measured again to ensure that the measured level was within the range of the standard controls. The measurement was carried out in duplets.We report data on single compounds detected in more than 50% of the samples and analyses were also performed for the summed concentration of the compound groups: 1) ∑PCB: PCB 99, 101, 105, 118, 128, 138, 153, 156, 170, 180, 183, 187, 2) ∑PCB group 1 (estrogenic PCBs): PCB 101, 187 3) ∑PCB group 2 (anti-estrogenic and dioxin-like PCBs): PCB 105, 118, 128, 138, 156, 170, 4) ∑PCB group 3 (CYP1A1 and CYP2B inducing PCBs): PCB 99, 153, 180, 183, 5) ∑DL-PCBs (dioxin-like PCBs): PCB 105, 118, 156, 6) ∑OCP: p,p′-DDT, p,p′-DDE, mirex, β-HCH, HCB, cis- and trans-nonachlor, and oxychlordane, 7) ∑PFCA: PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA and PFTrA, 8) ∑PFSA: PFHxS, PFOS and PFOSA, and 9) ∑PFAA: ∑PFCA + ∑PFSA. For sum calculations only the compounds measured in both recruitment periods were included.Independent samples t-test was used to compare the demographical factors  and POP levels between cases and controls, and between estrogen-receptor-negative (ER-) and estrogen-receptor-positive (ER+) cases. The distribution of the variables was checked by Q-Q plots and when non-normal distribution was found, data was ln-transformed to improve the normality.2- test was used to test for distribution differences in age groups, BMI groups, self-reported smoking status, menopause status, and ever breastfeed (yes/no) between cases and controls.Pearson’s chiANCOVA was used for comparison of the variables between cases and controls with adjustment for age. The analyses were performed on ln-transformed data. The assumption of linear relationship between outcome and the covariates was tested by visual inspection of scatterplots. In the analyses there was checked for outliers, homogeneity of regression slopes and homoscedasticity and homogeneity of variances.To estimate the odds ratios (OR), unconditional logistic regression models were used and estimates were obtained under adjustment for potential confounders. The exposure levels were both considered as continuous and categorical tertiled variables, based on the distribution among the controls. Confounding by the variables of interest was assessed using the change-in-estimate approach through backward elimination . The chaCorrelations were calculated using Spearman’s rho.p ≤ 0.05.All estimates are reported with 95% confidence intervals (95% CI). The statistical analyses were produced using IBM SPSS Statistics 22 and the statistical significance level was set to 2, respectively. However, the distribution between normal weight, overweight and obese was not significantly different between cases and controls . Reproductive factors  did not differ between cases and controls. For the 2010–2014 recruitment period age at menche and age at menopause were also registred and no difference between cases and control were observed (data no shown).The self-reported smoking status and plasma cotinine level were similar in the two groups. Although, 20.0% of controls and 8.6% of cases reported that they had never been smokers. Self-reported smoking status was highly correlated with the measured cotinine level , three OCPs , and all flame retardants  were below the detection limit in more than 50% of the samples and excluded from the single compound analyses. Detection limits and frequencies are reported in Additional file The level of the tested lipophilic POPs (PCBs and OCPs) was significantly higher in cases than in controls except for PCB105, PCB156, p,p’DDT, and βHCH Table . Upon adFor all of the lipophilic POPs, the levels had significantly decreased from 2000 - 2003 to 2011–2014 Table .Table 3SSimilar odds ratio estimates for the lipophilic POP were observed for the two recruitment periods separately  were below the detection limit in more than 50% of the samples and thus excluded from the analyses of single compounds. Detection limits and frequencies are reported in Additional file The cases had significantly higher serum levels of the tested PFAAs compared to the controls except for PFNA and PFDoA Table . Upon adFor the two recruitment periods, the levels of ∑PFCA and the individual PFCAs (expect for PFOA) were significantly increased in the period 2011–2014 compared to the first period (2000–2003). In contrast, the levels of both the ∑PFSA and the individual PFSAs decreased significantly over time between the two recruitment periods, 2000–2003 and 2011–2014 Table . A notabThe analyses with continuous PFAA variables showed a significant, positive association between ∑PFAA, PFOA, ∑PFSA, PFHxS, and PFOS and breast cancer risk Table . In the Information on estrogen receptor status was available for 81.8% of the breast cancer cases. In the 63 cases with information on estrogen receptor status, 20.6% were ER- and 79.4% were ER+. The ∑OCP was significantly higher in ER- cases compared to ER+ cases (data not shown). When adjusting for age, there was no difference between ER- and ER+ cases  or moderately (38.3%) differentiated. The serum levels of the tested compounds did not differ statistically between the tumor grades, however the levels were consistently highest among the cases with moderately differentiated tumors Fig. .Fig. 2SeThe results of the present study suggest a positive association between breast cancer risk in Greenlandic Inuit women and the measured serum POP levels. We found that the breast cancer risk may be increased by exposure to PCBs and PFAAs, especially PFSA, and to a smaller extent by exposure to OCPs.The Greenlandic Inuit population is exposed to high concentrations of lipophilic POPs through their traditional food, especially through intake of marine mammals –24. Gene, in animals studies and ex vivo studies of complex serum mixtures extracted from Danish pregnant women [The association between exposure to environmental pollutants and breast cancer risk has been widely studied , 28, 29,nt women –40. Studnt women .Other mechanisms of POP influencing breast cancer risk have been proposed, such as, promoting development of obesity ultimately leading to development and/or progression of breast cancer , disruptIt is generally accepted that estrogen exposure alters the risk of breast cancer development and progression. The same mechanisms may alter breast cancer risk concerning POPs with estrogenic potential. However, when analyzing the PCBs by their potential mechanism of action as potentially estrogenic (Group 1), potentially antiestrogenic, immunotoxic, and dioxin-like (Group 2), and CYP1A and CYP2B inducers (Group 3) we did not observe any difference in odds ratios between the groups. The individual serum PCB levels were, however, strongly correlated and the similar odds ratio estimates may result from uncontrolled confounding that cannot be eliminated. For each of the three PCB groups we found an increased risk in the analyses with continuous exposure variables, although with odds ratios close to 1.00 Table . The ∑PCWhile several studies report on the association between breast cancer and PCBs, only a few have investigated associations with PFAAs. We found in a prospective Danish study an increased risk of the highest PFOSA quintile ) and the association were strongest among the young women (below 40 years of age) . In a prPolymorphisms in the cytochrome 450 system (CYP450) have been shown to interact with the effects of POPs on breast cancer risk , 46, 47.Breast cancer tumors can be classified as either ER- or ER+. ER- tumors are not influenced by estrogen and do not respond to hormone therapy treatment. ER+ tumors, respond to the hormone therapy and have a stronger association with estrogen-related factors such as early age at menarche, nulliparity or delayed childbearing, and postmenopausal obesity . We did Our study has several limitations. The study design may cause selection bias, since selecting a comparable control group can be difficult. The cases were slightly older and leaner than the controls, although frequencies in age and BMI groups did not differ. The differences might influence our results but both age and BMI were considered to be potential confounders in the analyses. Furthermore, some of the controls were hospital patients with nonmalignant abnormalities in the uterus, ovaries and breasts. This may have caused a bias, most likely toward the null and then the effect may be greater than reported in the present study.Our study determined the serum POP levels at the time of diagnosis. The body chemistry, metabolism etc. may have changed during disease development. The fact that the cases in our study were leaner than the controls may be due to disease-related weight loss, which is a symptom of advanced breast cancer. Weight loss has been reported to increase the lipid adjusted plasma concentrations of OCPs and PCBs , 51. InfOur study showed a positive association between breast cancer risk and most of the measured POPs. Although, associations are weak, they indicate that exposure to environmental pollutants may be a risk factor for breast cancer in the Greenlandic Inuit women. A case-control study nested within the existing Greenlandic cohorts with prospectively colledted samples in more relevant timeframes is warranted to further evaluate the effect of POPs.Additional file 1:Detection limits and frequencies of the measured compounds. (PDF 61 kb)Additional file 2:Odds ratio of breast cancer risk associated with lipophilic POPs stratified for recruitment period. (PDF 133 kb)Additional file 3:Odds ratio of breast cancer risk associated with PFAA stratified for recruitment period. (PDF 102 kb)


Breast cancer is the most prevalent form of cancer in Ethiopia of all female cancers. It is considered to be a progressive disease with a poor prognosis if detected late. Breast self-examination is an important prevention method of breast cancer. This study was aimed at assessing practice and associated factors of breast self-examination (BSE) among female Debre Berhan University students in Ethiopia. A cross-sectional study was conducted in 2015 among 420 using self-administrated questionnaire. Multistage sampling technique was used to select the study participants. Bivariate and multivariate logistic regression analysis were done. Majority of the study participants, 338 (84.5%), were between 20 and 24 years old with the mean age of 21.1 ± 1.65. Only 14 (3.5%) had family history of breast cancer. Two hundred fifty-six (64%) of the participants had heard about BSE and 30.25% had good knowledge about BSE. Mass media were the most common source of information about breast cancer. Few of the participants (28.3%) had performed BSE. Lack of knowledge on how to perform BSE was cited as the main reason for not practicing BSE. Knowing how to perform, when to perform, and position to perform BSE and having a perception that BSE is important and useful to detect breast cancer were significant predictors of practices of BSE. This study revealed that most of the participants had low knowledge and practice of BSE. Therefore, it important to develop health educational programs in the university to raise awareness about BSE and breast cancer so as to practice self-breast examination. Breast cancer is characterised by the uncontrolled growth of abnormal cells in the milk producing glands of the breast or in the passages (ducts) that deliver milk to the nipples . Breast Breast cancer is a public health problem. It attacks women in their most productive years of life but breast cancer can be cured with limited resources if detected early, but treating advanced stage disease is expensive and outcome is often poor . The mosAs many studies indicated the practices of BSE are low among university students. A study conducted in Cameroon among female undergraduate students in the University of Buea indicated that only 9.0% knew how to perform BSE, only 13.9% knew what to look for while performing BSE, and only 3% had performed BSE regularly. Furthermore, Lack of knowledge on BSE was cited as the main reason for not performing BSE . SimilarLimited knowledge about the realities of breast cancer and lack of knowledge about the importance of self-examination and how it is performed are the main barriers for not practicing BSE. However, the magnitude practices of BSE are limited in our country, particularly in the study area. Therefore, this study aimed to assess the magnitude of practices of BSE and associated factors among female DBU students.Institutional based cross-sectional study design was conducted on January 2015 in Debre Berhan University, which was established in 2007. The university is found in Debre Berhan Town, the capital city of North Showa Administrative Zone of Amhara Regional State. It has ten colleges with a total of 10551 regular undergraduate students. Of this 3959 are female students. In the town, there are three health centers and one government and one private hospitals.All regular undergraduate female students were included in the study. However, students who live outside the university (nondormitory) were excluded.The sample size (420) was calculated using single population proportion formula by Epi Info software based on the following assumption: 22.7% of university students who practice breast self-examination , 95% CI,A self-administrated questionnaire was used to collect data. The questionnaire was initially prepared in English by reviewing different literature –14 and tParticipant's knowledge towards BSE was measured by the total number of correct answers to 9 items on knowledge questions. A knowledge score was calculated for each participant based on the number of questions correctly answered in the knowledge section. A score of “1” was assigned to every correct answer and a score of “0” to incorrect responses. Knowledge questions were scored and pulled together. The overall knowledge score of the participants were categorized into three: poor, medium, and good knowledge. Participants who answer less than 50% of the knowledge questions were considered as having poor knowledge. Similarly, participants who answer 50–75% and greater than 75% were also classified as having medium and good knowledge about BSE, respectively . FurtherP < 0.05.After checking its completeness, the collected data was entered in Epi Info version 3.5.2 and then exported in to Statistical Package for Social Science (SPPS) version 20 for analysis. Frequency, mean, standard deviation, and percentages were used for descriptive statistics. Multivariable logistic regression analyses were used to identify independent predictors of BSE. Adjusted odds ratio (AOR) with 95% CI was used to measure the strength of association. Statistical significance was set at Ethical clearance was obtained from Debre Berhan University, College of Health Science Ethical Committee (chs/cbe/2/07). The data was collected after clear discussion with the study participants about the purpose and the procedures of the study and after obtaining informed consent from each respondent. Participants were assured that they would never face any problem for participating in the study. Privacy and confidentiality of the study participants were strictly maintained.A total of 420 female DBU students filled the questionnaire correctly which gives response rate of 94%. Most of the participant students (84.5%) were between 20 and 24 age categories with mean age of 21.1 ± 1.65 and single by marital status. Of the participants, 14 (3.5%) had family history of breast cancer .Two hundred fifty-six (64%) of the participants had heard of BSE and 143 (35.8%) of the participants knew how to perform BSE. Furthermore, 3 in 10 (30.5%) participants were aware when to perform BSE. The main sources of information were mass media (39.8%) and health professions (22.3%). Overall, 37.3%, 32.5%, and 30.25% of the study participants had poor (<50%), medium (50–75%), and good knowledge (>75) on BSE, respectively .Almost all of the study participants approved that BSE is important and useful to detect breast cancer and 89.9% of the participants also believed that early detection will increase the chance of long term survival. Moreover, 93% of the participant students believe that BSE is not out of the community social norm. Almost all (96.8%) of the study participants stated that they will go to health facilities if they had any symptoms of breast cancer .Only 113 (28.3%) of the participants had ever performed BSE one year preceding the study. Among the participants who practiced BSE 61.9% were practicing monthly. The main reason cited by participants for performing BSE was to know early if they have any change on their breast (64.6%). However, majority (71.7%) of the participants were not practicing BSE. The main reasons for not performing BSE were lack of knowledge on how to conduct BSE and not having any symptoms of breast cancer .All the independent factors were checked for having an association with practicing of BSE in bivariate logistic regression. College, year of study, having heard about breast cancer, knowing how to perform BSE, knowing when to perform BSE, knowing the three positions to perform BSE, and believing that BSE is important and useful to detect breast cancer were shown as having association with practicing of BSE. However, after controlling the confounding factors, variables remained as predictor factors of practicing BSE were knowing how to perform BSE , knowing when to perform BSE , knowing the three positions to perform BSE , and believing that BSE is important and useful to detect breast cancer  .Breast cancer is the most common cancer among women worldwide. The present study was conducted to determine the practice of BSE and associated factors among female DBU students.The result of the current study revealed that only 28.3% of the participants had performed BSE. This finding is almost similar to the results of study conducted in Nigeria  and MalaDespite the fact that 64% of the participants had heard about BSE, significant proportion of the participants had limited knowledge about BSE. Almost only 3 in 10 participants knew how to perform and when to perform BSE, respectively, which is similar to finding from Malaysia  but highIn the present study, the main sources of information about BSE were mass media (39.8%) followed by health professionals (22.3%). Similarly, studies from Ethiopia , NigeriaThe result of multivariable logistic regression indicated that there was no significant relation between practice of BSE and the sociodemographic variables. However, participants who know how to perform BSE were 11 times, participants who know when to perform were 3.5 times, participants who know the three positions to perform were 2.3 times and participants who perceive BSE as important and useful were 6.8 times more likely to practice BSE as compared to those who do not know. This may be explained by the fact that knowledge of BSE was recognized as a necessary precursor to student's adherence performance of BSE .Finding of this study should be interpreted with the following limitations. The study was cross-sectional, so causal conclusions cannot be drawn. The study was carried out in only students of Debre Berhan University and therefore might not be representative of other Universities of the country and young adults in general, and the practice of BSE may be different in other sectors of the population.The results of the current study show that knowledge and practices of BSE among female DBU students were poor. Awareness on how to perform, when to perform, and position to perform BSE and perceiving BSE as important and useful to detect breast cancer were the predictor factors of practice of BSE among the female university students. The university and other stakeholders should develop educational programs that can increase knowledge and practice of breast self-examination among young female university students.


Enhancer of zeste homolog 2 (EZH2) is a polycomb-group protein that is involved in stem cell renewal and carcinogenesis. In breast cancer, increased EZH2 expression is associated with aggressiveness and has been suggested to identify normal breast epithelium at increased risk of breast cancer development. However, the association between EZH2 expression in benign breast tissue and breast cancer risk has not previously been evaluated in a large prospective cohort.We examined the association between EZH2 protein expression and subsequent breast cancer risk using logistic regression in a nested case-control study of benign breast disease (BBD) and breast cancer within the Nurses’ Health Studies. EZH2 immunohistochemical expression in normal breast epithelium and stroma was evaluated by computational image analysis and its association with breast cancer risk was analyzed after adjusting for matching factors between cases and controls, the concomitant BBD diagnosis, and the Ki67 proliferation index.p value 0.026). Additionally, women with high EZH2 expression and low estrogen receptor (ER) expression had a 4-fold higher risk of breast cancer compared to women with low EZH2 and low ER expression .Women with a breast biopsy in which more than 20% of normal epithelial cells expressed EZH2 had a significantly increased risk of developing breast cancer (odds ratio (OR) 2.95, 95% confidence interval (CI) 1.11–7.84) compared to women with less than 10% EZH2 epithelial expression. The risk of developing breast cancer increased for each 5% increase in EZH2 expression  contains supplementary material, which is available to authorized users. BRCA1/2), family history, and mammographic density have major ability to predict breast cancer, with other factors such as reproductive history playing a smaller role in determining risk [Prior studies have elucidated a variety of clinical, epidemiologic, radiologic and histopathologic features associated with an increased risk of breast cancer. However, our understanding of breast cancer risk factors and carcinogenesis in the normal breast is still rudimentary, limiting our ability to predict breast cancer risk accurately. Currently, germline mutations in high penetrance cancer predisposition genes i.e. BRCA/2, familing risk . Among ting risk , 5. Addiing risk –8. HowevEnhancer of zeste homolog 2 (EZH2) is a core protein of the polycomb-repressive complex 2 (PRC2) that controls dimethylation and trimethylation of Lys27 of histone H3 (H3K27me3), a marker linked to transcriptional silencing. Due to its role in regulating fundamental cellular processes, such as cell cycle regulation, senescence and differentiation, EZH2 has been thought to contribute to malignant transformation. However, this view has been challenged and it has been suggested that high EZH2 is a consequence rather than a cause of cancer .in situ (DCIS) [More relevant to this study, the expression and prognostic significance of EZH2 has been extensively investigated and in breast cancer it is now generally accepted to be associated with a worse prognosis . Associau (DCIS) . More reu (DCIS) . Also EZu (DCIS) . Howeveru (DCIS) , 15. TheThis study is a nested case-control study of members of the Nurses’ Health Study (NHS) and Nurses’ Health Study II (NHS II) cohort with biopsy-confirmed BBD. The NHS is an ongoing prospective cohort study that began in 1976, when 121,700 female registered nurses between the ages of 30 and 55 years completed a mailed questionnaire. The NHS II consists of 116,609 female registered nurses who were between the ages of 25 and 42 years when the study began in 1989. In both cohorts, participants have been followed via biennial questionnaires that provide information on lifestyle factors (body mass index (BMI), reproductive history, postmenopausal hormone (PMH) use, and alcohol use) and incident disease [in situ were included in the study. To reduce potential reverse causation due to subclinical tissue change, women were excluded if they had evidence of in situ or invasive carcinoma at biopsy or reported a diagnosis of breast cancer within 6 months of their BBD biopsy. There was a median 9 years between BBD biopsy and cancer diagnosis among the cases.Details on the BBD diagnosis reporting on the questionnaires have been previously described , 6. BrieEligible controls were women who completed the questionnaire in the same year that the breast cancer case was reported and had a previous diagnosis of biopsy-confirmed BBD, but were free from breast cancer at the time of the case (index date). Using incidence density sampling, up to four controls were selected for each breast cancer case by age at index date, year of BBD biopsy, and time since BBD biopsy. Due to considerable missing information on the laterality of the carcinoma in the cases, this information was not considered in analysis. The study was approved by the Human Subjects Research Committee of Brigham and Women’s Hospital, Boston, Massachusetts. Completion of the self-administered questionnaire was presumed to imply informed consent.Eligible cases and controls were contacted for permission to obtain their BBD pathology records and biopsy specimens, and specimens were then obtained from hospital pathology departments when possible For each immunohistochemical (IHC) stain, a 5-μm paraffin section was cut from each TMA block and immunostained for EZH2  after appropriate processing. Immunostaining was performed in a single staining run on a Leica Bond automated stainer . Positive and negative controls were included in all staining runs. Details on the antigen retrieval conditions and quantification are provided in Additional file Immunostaining results of each core were interpreted using an automated computational image analysis system . EZH2 expression was recorded as the percentage of EZH2-expressing cells with nuclear expression as previously reported , 15. DueIn the cell/nuclear identification and stain evaluation module, we defined an intensity and size threshold for nucleus identification and introduced an intensity threshold for nuclear stain positivity. Based on these criteria, each cell was classified as either positive or negative. The classification as a continuous scale of positive EZH2 cells was reviewed by a pathologist (FB), blinded to outcome. The image analysis results were strongly correlated with a manual 2 and 3-tier scoring system  and their corresponding 95% confidence intervals (95% CIs). In the logistic regression analysis, we modeled EZH2 expression as both continuous and categorical variables . Cutoff points were set based on previous reports , 22 and Potential confounding by other established breast cancer risk factors was evaluated by examining their association with EZH2 expression, though no additional covariates were included in the final models. We performed a test for trend by conducting the Wald test when including the EZH2 expression category in the model as the continuous variable. Interactions between EZH2 expression and ER and Ki-67 expression were evaluated by dichotomizing the expression about the median level among controls and performing a likelihood ratio test for the product term. Polytomous logistic regression models were utilized to evaluate EZH2 expression as a predictor of subsequent breast tumor ER expression .Statistical significance in all the analyses was assessed at the level of 0.05. The analyses were performed using SAS software . All the analysis performed and reporting are compliant with the Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD) statement .r = 0.79, p < 0.001  and weakly correlated with p53  and AR expression . There was no correlation between EZH2 and ER or PR expression  of 1.89–12.46%. In stroma, the percentage of EZH2-positive cells ranged from 0 to 58.39% with a median of 5.86% and an IQR of 3.09–10.91%. The intraclass correlation coefficient (ICC) across cores was 0.69 (95% CI 0.63–0.74) in the epithelium and 0.64 (95% CI 0.58–0.70) in stroma, suggesting moderately low heterogeneity in EZH2 expression across the cores. Stromal and epithelial expression of EZH2 in normal breast tissue were moderately correlated  . EZH2 expression in stromal cells in normal breast tissue was not significantly associated with subsequent breast cancer risk. To compare with previous studies and facilitate clinical translation of the results and estimation of EZH2 expression by a trained pathologist, we also tested the association with categories of EZH2 expression , relative to women with <10% expression of EZH2. There was also a 1.22-fold increased risk of breast cancer per 5% increase in epithelial EZH2 expression .As the cases represented patients who developed both invasive and p = 0.017). Individuals with high epithelial EZH2 and lower ER expression had a fourfold increased risk of breast cancer development  compared with those who had low EZH2 and ER expression. There was no statistical association with co-expression of Ki67. However, the association between risk and EZH2 expression in cases with Ki67 below the median was close to statistical significance . The increased risk of EZH2 expression in cases with low ER expression and potentially in cases with low Ki67 expression suggests that the effect of EZH2 in breast carcinogenesis could be dependent on the biologic activity of estrogens and more pronounced in low proliferative states).Due to the recently reported association between proliferative activity in breast epithelium and breast cancer risk , 8, we nEZH2 overexpression had been associated with aggressive ER-negative IBC , 24 and This is the first study, to our knowledge, to directly examine the independent association between EZH2 expression in the normal breast and risk of breast cancer, in a large prospective study. We found that EZH2 expression in normal breast epithelium was associated with breast cancer risk, particularly when >20% of the epithelial cells expressed EZH2. Importantly, this association was independent of the two most important tissue-specific risk factors in this population, the concomitant BBD lesion present and the proliferative activity as determined by Ki67 expression.On its own, EZH2 expression in >20% of the normal breast epithelial cells was associated with a threefold increased risk of breast cancer, and a fourfold increased risk if there is concomitant low ER expression. When compared to other traditional risk factors, the magnitude of the increased risk associated with higher EZH2 expression is in between the increased risk associated with atypical hyperplasia and proliferative disease without atypia, and is well above that associated with a positive family history , 25. ThuTwo prior studies have analyzed EZH2 expression in normal breast and BBD lesions with the goal of establishing EZH2 as a potential marker of breast cancer risk , 15. DinIn our secondary analysis, we found that the expression of ER appeared to modulate the effect of EZH2 on breast cancer risk. When performing cross-classification of samples using EZH2 and ER expression, women with ER expression below the median in normal epithelium and concomitant EZH2 expression above the median displayed even higher risk of breast cancer than if only using EZH2 for classification. A similar pattern of effect modification of a risk biomarker and ER expression has also been recently reported. While Ki67 expression in normal breast epithelium was associated with breast cancer risk, when cross-classification with ER was performed, high Ki67 expression was no longer predictive of risk when high ER expression was also detected [Despite the similar pattern of association between risk of breast cancer and effect modification with ER, in our study there was only weak correlation between Ki67 and EZH2, and no significant change in the magnitude of the association between EZH2 and breast cancer risk when including Ki67 expression as a covariate in the model. While we cannot totally exclude the possibility that the ability of EZH2 expression to predict breast cancer is linked to cell proliferation, we favor the previously suggested hypothesis that its role as a predictor of breast cancer may depend on its effect on stem cell survival and alteration of DNA repair pathways , 26–30.We acknowledge a number of limitations in this study. First, as we used TMAs, our expression data may not be representative of the whole biopsy slide or breast, due to sampling variability. Despite a very good intraclass correlation coefficient for detection of EZH2 across biopsy cores, and thus low heterogeneity in this sample, we tried to reduce this potential problem of variability by including all patients with at least three TMA cores. Given the limited sample size, we had limited power to estimate the effect sizes, thus some of the results should be interpreted cautiously, in particular the analyses using cross-classification and the differential ER status of the different tumors. As in previous studies using this cohort, women were excluded if they did not provide biopsy specimens, did not have normal TDLUs, or if there were insufficient numbers of cells in the biopsy tissue blocks. However, our ability to obtain biopsy blocks did not significantly differ by breast cancer case and control status or by established breast cancer risk factors.Finally, the generalizability of the result could be limited to the population with concomitant BBD diagnosis in a breast biopsy, as normal breast tissue adjacent to a BBD may be different from that in women without BBD . HoweverIn conclusion, the currently available evidence suggests EZH2 expression in the normal breast can be used as a biomarker of breast cancer risk. In conjunction with previously reported breast cancer risk biomarkers by our group of investigators, such as IGF1R and Ki67, these results reinforce a new paradigm of predicting breast cancer risk by incorporating molecular markers [


In a previous paper, we had assumed that the risk of screen-detected breast cancer mostly reflects inherent risk, and the risk of whether a breast cancer is interval versus screen-detected mostly reflects risk of masking. We found that inherent risk was predicted by body mass index (BMI) and dense area (DA) or percent dense area (PDA), but not by non-dense area (NDA). Masking, however, was best predicted by PDA but not BMI. In this study, we aimed to investigate if these associations vary by tumor characteristics and mode of detection.We conducted a case-control study nested within the Melbourne Collaborative Cohort Study of 244 screen-detected cases matched to 700 controls and 148 interval cases matched to 446 controls. DA, NDA and PDA were measured using the Cumulus software. Tumor characteristics included size, grade, lymph node involvement, and ER, PR, and HER2 status. Conditional and unconditional logistic regression were applied as appropriate to estimate the Odds per Adjusted Standard Deviation (OPERA) adjusted for age and BMI, allowing the association with BMI to be a function of age at diagnosis.For screen-detected cancer, both DA and PDA were associated to an increased risk of tumors of large size (OPERA ~ 1.6) and positive lymph node involvement (OPERA ~ 1.8); no association was observed for BMI and NDA. For risk of interval versus screen-detected breast cancer, the association with risk for any of the three mammographic measures did not vary by tumor characteristics; an association was observed for BMI for positive lymph nodes (OPERA ~ 0.6). No associations were observed for tumor grade and ER, PR and HER2 status of tumor.Both DA and PDA were predictors of inherent risk of larger breast tumors and positive nodal status, whereas for each of the three mammographic density measures the association with risk of masking did not vary by tumor characteristics. This might raise the hypothesis that the risk of breast tumours with poorer prognosis, such as larger and node positive tumours, is intrinsically associated with increased mammographic density and not through delay of diagnosis due to masking. Mammographic density (MD) is a risk factor for breast cancer –5 and isIn a previous paper , we postVery few studies have analysed the association between MD and risk of breast cancer by tumor characteristics, separately for each detection mode –10. Of tFor screen-detected cancer, studies have observed that higher density was associated with increased risk of larger tumors , 10 and Here we have used the same case-control study nested within the Melbourne Collaborative Cohort Study (MCCS) analysed before , to inveThe MCCS is a prospective cohort study, which started recruiting participants from the Melbourne metropolitan area between 1990 and 1994. At study entry there were 41,514 participants  aged between 27 and 76 years. A nested case-control study was designed based on the subset of MCCS women who had been identified to have attended BreastScreen Victoria, a population-based screening program, through a record linkage conducted in 2009 . Cases were women who subsequently had a first diagnosis of invasive adenocarcinoma of the breast . Four controls were matched to each case by year of birth, year of entry into the MCCS and country of origin. The mammogram with craniocaudal view and closest to study entry was chosen for measurement. Screen-detected cases were identified at BreastScreen Victoria. Cases diagnosed within 2 years of a negative screen at BreastScreen Victoria were defined as interval cases. There were 244 screen-detected cases matched to 700 controls and 148 interval cases matched to 446 controls. Further details about the nested case-control study have been published elsewhere , 12.P < 0.0001; for PR, κ = 0.30, P < 0.0001; for HER2, κ = 0.32, P < 0.0001). Given the good agreement between the ER, PR, and HER2 data, when archival tumor tissue was not available, ER, PR, and HER2 status was assigned according to the histopathology reports held at the VCR.The Victorian Cancer Registry (VCR) reviewed the pathology reports and classified the cancers according to tumor size, tumor grade, lymph node involvement, and ER, PR, and HER2 status. The original diagnostic tumor slides were retrieved for 85% of the cases from pathology laboratories and reviewed by a single pathologist (C. McLean) who assessed ER, PR, and HER2 status using immunohistochemistry techniques . ER and Associations between the mammographic measures and risk were estimated in terms of odds per adjusted standard deviation (OPERA) according to models with different combinations of the variables, DA, PDA, NDA and BMI, as mentioned in our previous publication . Further0.2–1)/0.2, (PDA0.2–1)/0.2 and (NDA0.5–1)/0.5, respectively. Linear regression was applied on each transformed mammographic measure, adjusting for age at mammogram, BMI (standardized according to the controls) and all the matching variables, and the residuals were standardized to have zero mean and unit variance. Conditional logistic regression of case-control status was then applied separately for screen-detected and interval cancers and for each tumour characteristic. The type-specific OPERA estimates were obtained by fitting an interaction term between the standardised residuals and a set-specific variable equal to the tumour type of the matching case. Heterogeneity by tumor characteristics was assessed using likelihood ratio test. Age at mammogram was fitted as a potential confounder.Firstly, by applying the Box-Cox method for transforming variables to the mammographic measures of the control group, DA, PDA and NDA were transformed to  and reference age (age at diagnosis for the case and for her matched controls) and its significance was assessed using the likelihood ratio test. We have reported the risk estimates for BMI at ages 50 and 70 to show the predicted risks corresponding to the pre- and postmenopausal age groups.For the analyses of interval versus a screen-detected breast cancers, unconditional logistic regression was applied only to cases adjusted for age at mammogram. Association between BMI and risk of interval versus a screen-detected breast cancer did not depended on age at diagnosis.The Bayesian information criterion (BIC) and the area under the receiver operating characteristic curve (AUC) were used to test for relative goodness of fit. Differences between AUCs were tested using the De Long’s tests .Sensitivity analyses were conducted in which we further adjusted for potential confounders: BMI at age 18–21 years; age at menarche; parity and lactation; menopausal status; HRT use; OC use; alcohol consumption and energy intake; and the matching variables  using unconditional logistic regression. We further adjusted for family history of breast cancer. A sensitivity analysis was also conducted by excluding cases diagnosed within 2 years from the mammogram, and their matching controls.A more detailed explanation of the method used to derive OPERA has been given in our previous publication .P < 0.05 was considered to be nominally statistically significant.Statistical analyses were performed using Stata 12.1 . Two-sided P < 0.001), at study entry when covariates were measured , and at the mammogram closest to study entry . Interval cases had on average greater DA and PDA and lesser total breast area and NDA than screen-detected cases (P < 0.01). There was no significant difference in BMI and all the other confounders except for menopausal status and alcohol consumption between the two types of cases. Within the screen-detected cases there were a higher percentage of menopausal women (P = 0.02) and lower percentage of alcohol consumers (P < 0.01) at cohort entry than the interval cases.As shown in Table ER, PR and HER2 status was known for 95%, 94% and 93% of the cases, respectively. Within the cases with known ER, PR or HER2 status, 282 (76%) were ER+, 183 (50%) were PR+, and 114 (31%) were HER2+. Grade was known for 94% of the cases, which included 86 (23%) well differentiated, 156 (42%) moderately differentiated, and 126 (34%) poorly differentiated tumors. Lymph node involvement was known for 93% of the cases of whom 104 (28%) were node positive. Size of the tumor was known for 97% of the cases for which 101 (26%) were < 1 cm, 167 (44%) were between 1 to 2 cm and 114 (30%) were ≥ 2 cm.P < 0.01), PR- , poorly differentiated tumors , positive nodal status  and larger tumor size, ≥ 2 cm .Interval cases had more tumors with features predictive of poorer prognosis than screen-detected cases; ER- ; the risk was significant but lower for tumors of size 1–2 cm and not significant for smaller tumours of size lesser than 1 cm. DA and PDA also were positively associated with positive lymph nodes with risk gradients of about 90% and 110%, respectively, per adjusted SD under all models; whereas the risk associated to negative lymph nodes was lower . The model including only PDA gave the best fit when analysing tumor size  and lymph node involvement . BMI and NDA were not associated with the size of the tumor and nodal involvement under any model. None of the three mammographic measures and BMI were associated with the other tumor characteristics.Table Similar to risk of breast cancer overall, DA and PDA were positively associated with risk of screen-detected breast tumors of large size and positive lymph node involvement Table . But unlThe association between risk of interval cancer and DA, NDA and PDA did not vary by any of the tumor characteristics Table . Higher p = 0.02, 0.03 and 0.06).None of the three mammographic measures were associated with risk of interval versus screen-detected breast cancer by any of the tumor characteristics Table . BMI wasThe findings above were similar when we adjusted for all the confounders. No substantial differences in estimates were observed from the sensitivity analyses.As assumed in our previous paper , that riOur results are similar to those of a large study consisting of six pooled datasets, which also found DA and PDA to be associated with increased risk of larger tumors, but found only PDA to be positively associated with positive nodal status . Unlike Some studies analysing screen-detected breast cancer had also found MD to be associated with increased risk of larger tumors , 10 and We found no association between any of the mammographic measures and risk of interval breast cancer by tumor characteristics. Few studies had investigated risk by tumor characteristics separately for interval cancer. One study found greater MD to be associated with increased risk of ER+ interval cancer but it had not adjusted for BMI . AnotherWe found no association between all three mammographic measures and risk of interval versus screen-detected cancer by tumor characteristics. A study which had further categorised interval cancer into true interval, false negatives, minimal-sign cancers and occult tumors also found no association between PDA and risk of each of the interval cancer category mentioned above compared with screen-detected cancer for the tumor phenotype, luminal A, luminal B, HER2 and triple-negative . AnotherTo our knowledge, our study is the first to estimate the differential risk of developing breast cancer and risk of masking by tumor characteristics through investigating the concurrent associations with all three mammographic measures, DA, NDA and PDA stratified by detection mode. BMI has been measured at cohort entry and we had realistically allowed the BMI association to vary by age since BMI is known to have differential associations on breast cancer risk; BMI is negatively associated with breast cancer risk for premenopausal women and positively associated with breast cancer risk for postmenopausal women . A limitWe found that both DA and PDA predicted inherent risk of larger breast tumors and positive nodal status. There was no differential risk of masking by tumor characteristics associated with DA, NDA and PDA. None of the three mammographic measures were associated with differential nodal involvement for interval versus screen-detected cancer. Our finding suggest that the dense tissues of the breast play a role in faster growth rate of the tumor and in spreading of the tumor to the lymph nodes that would not be explained by delay of the diagnosis due to masking.


Endometrial biopsies are undertaken in premenopausal women with abnormal uterine bleeding but the risk of endometrial cancer or atypical hyperplasia is unclear.To conduct a systematic literature review to establish the risk of endometrial cancer and atypical hyperplasia in premenopausal women with abnormal uterine bleeding.Search of PubMed, Embase and the Cochrane Library from database inception to August 2015.Studies reporting rates of endometrial cancer and/or atypical hyperplasia in women with premenopausal abnormal uterine bleeding.CI were estimated using logistic regression with robust standard errors to account for clustering by study.Data were independently extracted by two reviewers and cross‐checked. For each outcome, the risk and a 95% CI 0.23–0.48%, n = 29 059; 97 cases) and risk of endometrial cancer or atypical hyperplasia was 1.31% . Risk of endometrial cancer was lower in women with heavy menstrual bleeding (HMB)  compared with inter‐menstrual bleeding (IMB) . Of five studies reporting the rate of atypical hyperplasia in women with HMB, none identified any cases.Sixty‐five articles contributed to the analysis. Risk of endometrial cancer was 0.33% (95% IMB and older age may be indicators for further investigation. Further research into the risks associated with age and the cumulative risk of co‐morbidities is needed.The risk of endometrial cancer or atypical hyperplasia in premenopausal women with abnormal uterine bleeding is low. Premenopausal women with abnormal uterine bleeding should first undergo conventional medical management. Where this fails, the presence of Contrary to practice, premenopausal women with heavy periods or inter‐menstrual bleeding rarely require biopsy. Contrary to practice, premenopausal women with heavy periods or inter‐menstrual bleeding rarely require biopsy. Endometrial cancer has a world‐wide incidence of 9 per 100 000 women, with a 1% lifetime risk.13,11 In some, such as the UK National Institute for Health and Care Excellence (NICE) guideline, it is recommended that biopsy should only be undertaken when conventional medical management has failed,Endometrial cancer most commonly presents with post‐menopausal bleeding (PMB).In the UK, despite the NICE guideline, it is unclear whether all premenopausal women with abnormal uterine bleeding complete conventional management before referral for biopsy, or whether women considered ‘high risk’ are directly referred. Although guidelines, including the UK NICE guideline, were underpinned by some research evidence, none appears to have been based on a comprehensive literature review and there has been no other systematic review examining the risk of endometrial malignancy in this group. The aim of this work was therefore to conduct a systematic review to establish the risk of endometrial cancer and atypical hyperplasia in women with abnormal uterine bleeding and to make some judgment about the relative validity of current guidelines.A search was performed in PubMed, Embase and the Cochrane library from database inception to August 2015 with terms related to abnormal uterine bleeding and terms for endometrial cancer or indications or methods for investigation (Appendix S1).Prospective or retrospective studies of patients with abnormal uterine bleeding, where endometrial cancer or atypical hyperplasia was an outcome, were included in the review. Studies in populations of exclusively post‐menopausal women, and studies of mixed populations of pre and post‐menopausal women where the data could not be separated, were excluded. Study selection was undertaken by one reviewer.Information on study and patient characteristics and numbers of individuals with endometrial cancer and atypical hyperplasia were extracted from included studies. Data was extracted independently by two reviewers, with any disagreements resolved by consensus or reference to a third reviewer. Quality assessment was conducted by one reviewer, examining the internal and external validity of risk estimates. Internal validity was judged by the accuracy of the method used to investigate for the presence of malignancy. Where histological testing was conducted for all women, internal validity was judged to be good. In studies where a pathological diagnosis was not present for all women, internal validity was also potentially good where investigators appeared to have implemented adequate referral/treatment pathways. External validity was assessed as the applicability of the study sample to the population of this review i.e. women presenting in primary care with abnormal uterine bleeding. Where studies included women who appeared to have been included in the study because of high suspected risk, these were judged as less applicable, with low external validity.The risk was calculated using logistic regression with robust standard errors to account for clustering by study. Analyses were conducted for rates of (i) endometrial cancer and (ii) endometrial cancer and atypical hyperplasia combined. Sensitivity analysis was conducted, including only those studies judged to have good internal and external validity. The risk of endometrial cancer was also estimated within subgroups of studies defined by location (Western/non‐Western), study design (prospective/retrospective), and size . Where studies reported data separately for premenopausal women of different ages, these were included in an additional analysis estimating the risk of endometrial cancer in women aged <40, 40–50 and >50 years.n = 29 059 premenopausal women with abnormal uterine bleeding) were included and their characteristics and study risk of endometrial cancer are shown in Table S1. Studies were conducted in Europe (n = 32), North America (n = 6), Australasia (n = 2) and non‐Western countries (n = 25). Most were in populations of women undergoing investigation exclusively for abnormal bleeding, but some included women undergoing investigation for other indications (data for women with bleeding problems was extracted separately). The mean age in the majority of studies was between 40 and 50 years.Of 2736 original articles retrieved, 125 were obtained as full papers; 60 were excluded as indicated in Figure n = 37), including women who had been investigated with invasive testing such as endometrial biopsy or dilation and curettage. The reason for further investigation in these populations was not often reported but where it was, the reasons were suspected risk factors such as obesity, anovulation or previous oligomenorrhoea,The internal validity of studies was generally considered to be good. In most, the majority of women had undergone histopathological testing and it appeared likely that the majority of cases of malignancy were identified. Fifty‐three studies were judged to have good internal validity, but 12 were considered to be potentially less reliable. The external validity of studies was generally considered to be lower. Many were retrospective (n = 65), the risk was low, at 0.33% . When data for subpopulations of premenopausal women were separated, the risk of endometrial cancer was lower for women with HMB 0.11%  than for women with IMB 0.52% .Risk of endometrial cancer according to menstrual status, based on aggregated numbers of events and individuals across studies, is shown in Table n = 15 772, 207 cases). Only five studies reported rates of atypical hyperplasia separately for women with HMB and all reported that there were no cases of atypical hyperplasia. No studies reported rates of atypical hyperplasia separately for women with IMB.Rates for the risk of endometrial cancer or atypical hyperplasia are also shown in Table n = 2401; 8 cases), 40–50 years 0.51%  and >50 years 1.04% . However, for the risk of endometrial cancer or atypical hyperplasia, the increase with age group was significant: <40 years 0.81 , 40–50 years 1.99%  and >50 years 14.12% .Twelve studies reported rates of endometrial cancer and four studies reported rates of both endometrial cancer and atypical hyperplasia in specific age groups Figure . For endn = 29) the estimated risk of endometrial cancer in all premenopausal women was 0.28% , suggesting that the findings from the main analysis were robust. In the subgroup analysis, estimates of risk were similar in subgroups defined by study size (<100 versus ≥100), design (prospective versus retrospective) or location (Westernised versus non‐Westernised) .In the sensitivity analysis Table , includiThis review aimed to estimate the risk of endometrial cancer and atypical hyperplasia in premenopausal women presenting with abnormal uterine bleeding and to make some judgement about whether current guidelines are appropriate. Given the large numbers of women presenting in primary care, and the potential for complications, anxiety and wasted healthcare resources in undertaking unnecessary biopsies, estimating this risk is important. NICE use a > 3% cancer risk to underpin screening guidance.The risk of endometrial cancer in women with HMB was particularly low  and no cases of atypical hyperplasia were observed in studies of women with HMB. The risk of endometrial cancer was slightly higher in women with IMB  and IMB may represent a symptom of endometrial cancer. The number of cases of atypical hyperplasia was not reported separately for women with IMB and it was therefore not possible to assess the overall endometrial cancer/atypical hyperplasia risk. However, if it is assumed that risk in women with HMB is lower than that of the whole premenopausal group, it may be assumed that the risk in women with IMB is likely to be greater than the whole premenopausal group, i.e. women with IMB will have a greater than 1.33% risk of endometrial cancer/atypical hyperplasia. Therefore, for women with persistent IMB who are not responding to medical management, referral for further testing may be appropriate.There were some data regarding the association of age with risk of endometrial cancer and atypical hyperplasia. There was an increase in risk for women aged 40–50 years compared with <40 years that was significant when considering endometrial cancer and atypical hyperplasia together. Although the numbers of studies contributing to these analyses was very small  it appears likely that there is some increase in risk associated with increased age during the 40s. The NICE guideline recommends age as a criterion for biopsy only after all conventional medical management has failed.The current review did not find data on risks associated with co‐morbidities but we reviewed the literature. Mutations related to HNPCC, or Lynch syndrome, are associated with a high lifetime risk of endometrial cancer (12–44%);For other risk factors, the association appears less pronounced. A recent meta‐analysis showed PCOS to be a significant risk factor for endometrial cancer (odds ratio 2.79),Caution is needed in relating these findings as studies are of general populations of women, and not specifically in women with bleeding problems. It is therefore unclear whether the relative risks would be the same. It is also unclear to what extent risks factors are independent or whether they confound one another. It appears unlikely that the risks are cumulative and treating them as such is likely to overestimate total relative risk.However, even without these cautions, given the low risk of endometrial cancer, the absolute risks are likely to be low. For example, considering BMI, for a woman presenting with HMB, her risk of endometrial cancer increases from 0.11%  for suspicion of abnormalities or pathology. They are therefore unlikely to reflect typical populations of women presenting in primary care. Women undergoing invasive testing for suspected pathology may have a higher incidence compared with general populations of women presenting with abnormal uterine bleeding and these studies may thus overestimate the risk of endometrial cancer. The findings of the review are therefore likely to be conservative, further supporting the view that premenopausal abnormal uterine bleeding confers a low risk for endometrial cancer or atypical hyperplasia.Another limitation of the review was the difficulty in searching for studies. Most studies did not set out to determine rates of endometrial cancer as a primary outcome and results are incidentally reported. It is therefore possible that some studies were missed. However, a search strategy was used where endometrial cancer was not specified as a required outcome, and it appears likely that the majority of studies with relevant data were obtained.There generally appears to be a low risk of endometrial cancer in premenopausal women and much current guidance appears over‐cautious. The majority of women in studies were aged >40 years and overall risk of endometrial cancer was only 0.33%. When cases of atypical hyperplasia were also considered, the rate was still well below the NICE 3% threshold for cancer referrals.HMB conferred a particularly low risk and although studies suggest that some co‐morbidities increase the risk of endometrial cancer, given the very low baseline risk associated with HMB, the absolute risk in the presence of co‐morbidities is likely to still be low. Guidance recommending direct referral for biopsy in premenopausal women >40 yearsThe risk associated with IMB appears to be slightly higher. It is unclear whether the cumulative risk of endometrial cancer and atypical hyperplasia would reach the 3% risk NICE recommend for cancer referral but, where medical management fails, referral of women with IMB may be justified.The UK NICE guidanceThis review demonstrates that the risk of endometrial cancer or atypical hyperplasia in premenopausal women with abnormal uterine bleeding is low. Consequently, this group of women should first undergo conventional medical management. In women who have failed medical management, the presence of persistent IMB and older age may be indicators for further investigation. The review suggests that practice needs to change, where referral for biopsy in premenopausal women is only in selected cases. Further research is needed to better understand the effect of age and the cumulative effect of co‐morbidities.None declared. Completed disclosure of interests form available to view online as supporting information.MP and RL conceived the study. MP, PM and RM collected the data. SS analysed the data. MP, RL, GH and AP interpreted the data. MP, RL and RM drafted the manuscript. All authors commented on drafts of the manuscript and approved the final version.The work was undertaken under the auspices of the Centre for Diet and Activity Research (CEDAR), a UKCRC Public Health Research Centre of Excellence which is funded by the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the National Institute for Health Research, and the Wellcome Trust.Figure S1. Subgroup analysis.Click here for additional data file.Table S1. Study characteristics and prevalence of endometrial cancer in premenopausal women.Click here for additional data file.Appendix S1. Search strategies.Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.