This study investigated the association between intake of common alcoholic and non-alcoholic beverages and breast cancer risk among Japanese women. This study included 33,396 Japanese women aged 40–79 years from 24 areas in Japan from the Collaborative Cohort study. During the follow-up period (≥20 years), 245 incidents or mortal breast cancers were documented. Multivariable logistic regression analysis was performed to assess the independent association between breast cancer risk and the intake of Japanese green tea, coffee, and alcohol. Japanese green tea was the most commonly consumed non-alcoholic beverage (81.6% of participants), followed by coffee (34.7%) and alcohol (23.6%). No significant associations were identified between the intake of green tea and coffee with breast cancer risk . Alcohol intake was associated with significant breast cancer risk , and even infrequent alcohol consumption (<1 times/week) was associated with substantially increased breast cancer risk . Alcohol type, especially, wine and whisky intake tended to be marginally associated with breast cancer risk . Alcohol consumption would be associated with increased breast cancer risk. However, intake of green tea or coffee does not appear to be associated with increased breast cancer risk.  Breast cancer is the most common female malignancy and results in substantial morbidity and mortality worldwide . Compared with the United States and Europe countries, Japan have relatively low level of breast cancer mortality rates previously, however it has been increasing rapidly . This constant increase in breast cancer incidence was acknowledged as an important public health concern in Japan. While drinking habits were considered as prominent lifestyle risk factor of breast cancer, therefore it worth further investigation. Green tea is the most commonly consumed and been a routine diet beverage among Japanese. Epicatechin derivatives in green tea, has been hypothesized to have notable anti-carcinogenic effects. It was demonstrated in an animal experiments that drinking green tea can reduce the risk of developing breast cancer . Contrasting viewpoints reported on association between green tea intake and breast cancer risk, lead to uncertain conclusions . Methodological issues, such as differences in study design and variations in exposure, may underlie this inconsistency.While coffee is known as the most consumed beverage worldwide, the lowest intake has been reported among people in Eastern Asia . However, over the past 50 years, the Japanese population has been adapting to Western lifestyles in terms of dietary intake , resulting in high rate of coffee consumption. Knowing that, coffee contains many components, including caffeine and polyphenols, it may play a dual role as a carcinogen (due to caffeine) by enhancing cell proliferation and as a chemopreventive agent (due to polyphenols) with antioxidative and weak estrogenic properties . A study conducted among premenopausal Japanese women showed that intake of caffeine-containing beverages was associated with the risk of developing breast cancer . In contrast, a dose-response meta-analysis on prospective cohort studies reported coffee consumption was associated with decreased risk of postmenopausal breast cancer . Nevertheless, epidemiological studies outcomes examined the associations between coffee consumption and breast cancer risk, the results remain inconclusive .Alcohol consumption is a well-established risk factor of breast cancer, also supported by a meta-analysis including 53 studies . Previous Japanese cohort studies reported no significant relationship between alcohol consumption and breast cancer risk , but most recent cohort studies have revealed the higher alcohol consumption associated with increased breast cancer risk . This inconsistency in the results may be related to the amount of alcohol consumption: Japanese women consume a low amount of alcohol compared to Western women, the difference stemming from a Japanese social stigma surrounding alcohol consumption . Moreover, Japanese women consume particular kinds of alcoholic drinks, such as sake and shochu;the former is known as Japanese wine, made by fermenting rice and the latter is a spirit distilled from rice, barley, sweet potatoes, etc. However, so far there is no evidence on the association of type of alcoholic beverages consumed and breast cancer risk. Since green tea, coffee, and alcohol are important dietary exposures in the Japanese population, any association between the consumption of these beverages and breast cancer risk could have major public health impacts. Therefore, this study aimed to investigate the associations between the consumption of common alcoholic and non-alcoholic beverages and breast cancer risk among Japanese women using the data from a large prospective cohort study in Japan. Study population and assessmentThis study used the database of the Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC study), which is a nationwide, multicenter, population-based, prospective cohort study to evaluate the risk of cancer incidence and mortality, sponsored by the Ministry of Education, Science, Sports and Culture of Japan. Details of the study have been described elsewhere . Briefly, the JACC study was initiated in 1988, and the enrolment of subjects continued for two years until 1990. The original cohort included 110,585  participants aged 40–79 years who were recruited in 45 areas throughout Japan and underwent general health check-ups periodically, provided by the Japanese municipalities . Study participant’s health details were updated until 2009, unless they had moved out from their domicile or developed one of the endpoints determined in advance.Exposure assessmentInformation on beverage intake and other lifestyle factors was obtained using a self-administered questionnaire. For green tea and coffee, the participants were asked to report the frequency and amount of intake . For alcohol consumption, subjects were asked to report their drinking status . For current drinkers, the frequency of alcohol consumption , amount of alcohol per drink, and types of alcoholic beverages  they preferred were further interrogated. Follow-up and ascertainment of breast cancer occurrenceThe study outcome was incidence of breast cancer as well as deaths from breast cancer without previous breast cancer documentation. Data on cancer incidence, such as date of diagnosis and primary site, were collected only in 24 of the 45 study areas through prefectural population-based cancer registries or by reviewing the records of local major hospitals. The vital status of the participants was confirmed by reviewing death certificates with the permission of the Director General of the Prime Minister’s Office until the end of 2009, except for certain areas where follow-ups were discontinued. Both breast cancer death and incidence were treated as incidence because some participants had death records without incidence records.. Statistical AnalysisPotential risk factors were dichotomized to ensure sufficient statistical power. As for missing values, mean values were imputed for continuous variables, and the most likely categories were applied for categorical variables.2 vs ≥25 kg/m2), green vegetable intake (< 3 times/week vs ≥ 3 times/week), red meat intake (< 3 times/week vs ≥ 3 times/week) , physical activity , smoking habits (non-smoker vs smoker), parity (parous vs nulliparous), family history of breast cancer (no vs yes), and age at menarche (< 15 years old vs ≥15 years old). Age at menopause was not included in the model because the menopausal state was highly dependent on age. Logistic regression analyses were performed to estimate odd ratios (ORs) and their 95% confidence intervals (CIs) for the incidence of breast cancer according to the categories of the exposure variable and to adjust for confounding variables. Univariable analyses were performed to screen for potential independent predictors of breast cancer. In the subsequent multivariable analysis, established or biologically essential and statistically significant risk factors were adjusted in the model to investigate the independent association of each factor with the outcome. We considered the following variables to be potential confounders: age at baseline (40-59 vs 60-79 years old), education level (low<19 years old vs high≥19 years old), body mass index  SPSS statistics version 22.0. All tests of significance were two-tailed and p values of <0.05 or 95% CIs for ORs not including 1 were considered statistically significant. The present study included 40,466 women aged 40–79 years from 24 areas where cancer incidence or mortality were reported. After excluding 250 women with a past history of breast cancer, 1,879 dropouts, and 4,941 women who gave no information on drinking status, the remaining 33,396 women were included in the analysis. During the follow-up from 1988 through 2009, a total of 255 incident cases of breast cancer were documented. Several established risk factors, i.e., high education level, body mass index ≥25, nulliparity, and family history of breast cancer, were significantly associated with breast cancer (not shown in table). The associations between the type of beverage and breast cancer risk are shown in Since there was increased breast cancer risk for alcohol consumption, we further focused on the frequency of alcohol intake . CompareFor type of alcohol intake on breast cancer risk, we calculated the ORs for the restricted set of alcohol consumers who had reported the usual type of alcoholic beverage consumed  . No signThe absolute risk of consumption for the various beverages was calculated as 0.01% (95% CI -0.13–0.33) for green tea, -0.09% (95% CI -0.28–0.11) for coffee, and 0.29% (95% CI 0.06–0.53) for alcoholic beverages for this cohort. Thus, alcohol intake yielded a small but significant absolute risk of breast cancer. For alcoholic beverages, the absolute risk was calculated as 0.42% (95% CI -0.00–0.88) for sake, 0.28% (95% CI -0.03–0.59) for beer, 1.11% (95% CI 0.17–2.05) for wine, and 1.05% (95% CI 0.11–1.99) for whiskey.This large prospective cohort study revealed intake of alcoholic beverages significantly increased breast cancer risk among Japanese women. The increased risk was observed for low-frequency intake (<1 time per week). Among alcoholic drinkers, wine and whiskey consumers presented with marginally increased breast cancer risk compared with non-drinkers. However, intake of Japanese green tea and coffee was not significantly associated with breast cancer risk. Regarding green tea, our findings supported the results of five other prospective cohort studies, including three Japanese studies , that reported no association between intake of green tea and breast cancer risk. Even though the large proportion of daily green tea drinkers (80%) weakened the statistical power, our study further strengthens past evidences . Finding of coffee intake in our study seems to be compatible with those of other cohort studies conducted in Western settings . A previous meta-analysis demonstrated no association between breast cancer risk and coffee consumption , while two other meta-analyses reported statistically significant inverse associations between coffee consumption and breast cancer risk, depending on the women’s menopausal status  and hormonal sensitivity of the tumors . However, our study could not examine these factors due to insufficient of data. There are few studies carried out in the regions of the world where consumption of green tea dominates over coffee, such as Japan, lending great importance to our findings. Alcohol consumption significantly increased breast cancer risk in this study, but the absolute risk was small. Subsequent to the report of the World Cancer Report Fund in 2007, three cohort studies in Japan also demonstrated positive associations between alcohol consumption and risk of breast cancer , in line with our findings. In addition, our results were also consistent with a large European cohort study and a meta-analysis conducted in Western populations , although drinking patterns and types and amounts of alcohol intake largely differed from those of Japanese women. Our findings can be explained by the actions of estrogen and alcohol metabolism on breast cancer. Alcohol consumption substantially increases estrogen concentrations, and estrogens have a carcinogenic effect on breast cancer . Alcohol metabolites, such as acetaldehyde and lipid peroxides-reactive oxygen species, are also known as carcinogens , which may increase breast cancer risk. Furthermore, drinking patterns such as heavy episodic or binge drinking, leading to larger amounts of alcohol consumed in shorter periods of time , are known to elevate the levels of endogenous estrogen and lead to an accumulation of carcinogenic derivatives . There is a possibility that the majority of binge drinkers in our cohort study were classified into the group with intake <1 times/week. In this study, an attempt was made to calculate the amount of alcohol intake (g/ml) in each frequency category; however, a lot of data were missing for amount of alcohol consumed, especially in the low frequency group, so we could not accurately determine the amounts of alcohol consumed by the low-frequency drinking group.As concerns the types of alcoholic beverages consumed, the habitual intake of sake and beer had no association with the risk for breast cancer, whereas that of wine and whiskey showed marginal increases in breast cancer risk, albeit not statistically significant. Additionally, we observed a small significant absolute risk in both wine and whiskey. The amount of ethanol consumed was similar between the different types of alcoholic beverages, except for whisky, which contributed more to ethanol intake than other types of alcoholic beverages. Several previous studies  reported significant associations for beer, wine and liquor with breast cancer, while others did not . Since the amount of ethanol intake was not very different with respect to type of alcoholic beverage, the differences in drinking styles, such as time of drinking, consumption of aperitifs or night caps, and type of food consumption (side dishes) accompanying the specific alcoholic beverages, may affect subsequent health status. Since wine and whiskey were not common beverages when the study participants were recruited, it is not surprising that only few participants reported consumption of these beverages, preventing us from drawing firm conclusions.The key strength of our study was the relatively large number of participants involved and the adjustments made for most specific potential confounders. Since, as only few studies have investigated the type of alcohol consumed in relation to breast cancer risk among Asians, particularly Japanese women, our study adds further evidence in this area.This study has several limitations. First, the data on beverage intake were based on self-reporting; hence, measurement biases were inevitable. Second, we could not update the exposure data during the follow-up period; the types of beverages consumed and drinking patterns would have changed during the 20-year follow-up period, which could attenuate the true associations. Third, data on the amounts of alcohol consumed were unavailable, especially for the group of low-frequency alcohol consumption. We should generalize our findings with caution, and further research is warranted to overcome these limitations. In conclusion, in a population-based, prospective cohort study in Japan, alcohol intake increased breast cancer risk, even when consumed at a low frequency. However, both green tea and coffee intake did not appear to be associated with breast cancer risk.


Background: In 2018, the global estimate of newly diagnosed breast cancer cases among women totaled 2.1 million. The economic and social burden that breast cancer places on societies has propelled research that analyzes the role of modifiable risk factors as the primary prevention methods. Healthy behavior changes, moderated alcohol intake, healthy body weight, and regular physical activity may decrease the risk of breast cancer among women. This review aimed to synthesize evidence on the cost-effectiveness of lifestyle-related interventions for the primary prevention of breast cancer in order to answer the question on whether implementing interventions focused on behavior changes are worth the value for money.Methods: A rapid review was performed using search terms developed by the research team. The articles were retrieved from MEDLINE and the Tufts Medical Center Cost-Effectiveness Analysis Registry, with an additional web search in Google and Google Scholar. Comparisons were performed on the cost-effectiveness ratio per quality-adjusted life-year between the interventions using a league table, and the likelihood of cost-effective interventions for breast cancer primary prevention was analyzed.Results: Six studies were selected. The median cost-effectiveness ratio (in 2018 USD) was $24,973, and 80% of the interventions had a ratio below the $50,000 threshold. The low-fat-diet program for postmenopausal women was cost-effective at a societal level, and the physical activity interventions, such as the Be Active Program in the UK, had the best cost saving results. A total of 11 of the 25 interventions ranked either as highly or very highly likely to be cost-effective for breast cancer primary preventions.Conclusion: Although the review had some limitations due to using only a few studies, it showed evidence that diet-related and physical-activity-related interventions for the primary prevention of breast cancer were cost-effective. Many of the cost-effective interventions aimed to reduce the risk of non-communicable diseases alongside breast cancer. Breast cancer has been ranked as the leading cause of cancer deaths in over 100 countries, accounting for 11.6% of all cancer deaths worldwide . In 2018In recent years, the role of modifiable health behaviors in cancer prevention has been extensively studied –9. AssocTo reduce the risk of breast cancer, primary prevention measures can focus on women who adopt healthy behaviors such as maintaining a normal weight, breastfeeding, minimizing alcohol consumption, eating a balanced diet, reducing stress, and decreasing the use of long-term hormone replacement therapy –14. OverThe control of breast cancer through both early detection and primary prevention is of high priority in order to decrease the incidence and the premature mortality among women and to reduce the economic losses worldwide , 15. It Our study aimed to review and synthesize the evidence on the cost-effectiveness of lifestyle-related interventions for the primary prevention of breast cancer. The objective of this review was to provide up-to-date evidence on the cost-effectiveness of the breast cancer prevention interventions focused on healthy weight programs, balanced diet interventions, physical activity (PA) programs, limited alcohol consumption interventions, and tobacco cessation programs. A rapid review approach, which aims to systematically synthesize the available evidence within a “limited time and resource framework,” was adopted to summarize the relevant information –23.Systematic reviews provide a rigorous and reproducible method to collect and summarize the available current evidence in the literature. They require very intensive resources and time to be conducted. They often fail to answer the research question when no or little relevant evidence is available. Rapid reviews have emerged as an alternative to address this issue. They are a novel form of systematic review which aim to produce faster and relevant evidence following the same methodological steps of a systematic review . They arA pre-specified review protocol was developed and followed for all of the methods . The Preferred Reporting Items for Systematic Review (PRISMA) guidelines were used to report our findings .www.cearegistry.org), was searched from 2014 to 2017 since a systematic review performed by Winn et al. summarized evidence on the cost-utility analysis of cancer prevention and treatment with studies dated up to 2013  framework to identify the relevant terms: P: breast cancer, I: primary prevention, and O: cost-benefit analysis. The complete MEDLINE search strategy is presented in To be included, the studies had to fulfill the PICO framework:Populations: Adult women aged 16 years and older with no diagnosed breast cancer.Interventions: Studies considering lifestyle-related primary prevention interventions such as dietary interventions, weight-loss-related interventions, PA interventions or physical exercise programs, alcohol consumption reduction interventions, and/or tobacco use reduction programs. The interventions were identified and informed based on international literature and previous studies –29. StudComparators: Women without interventions, women with standard care or status quo, such as usual diet or current practice for PA, also called “usual care.”Outcomes: The primary outcomes of the cost-effectiveness analysis were the costs and the quality-adjusted life-years (QALYs) or the disability-adjusted life-years (DALYs) and the incremental cost-effectiveness ratio (ICER) that considers the change in the costs and the effects of interventions on breast cancer, including other non-communicable diseases (NCDs) or not, compared to the status quo.Study design: We applied no restriction on the type of study eligible for this review. We excluded any reports without results. We did not consider published letters or comments to be included.Only the articles published in English were considered for this review.https://www.covidence.org). The title/abstracts and the full text were screened by two reviewers (JPR and MB). One reviewer (MB) screened all of the abstracts and the full text of the relevant references. A second reviewer (JPR) double-checked 15%  of the abstracts and inspected all of the full text of the rejected articles (185/191) to ensure that no relevant study was excluded. Disagreements were resolved after discussion.All search results were imported and de-duplicated using Covidence Software (Two reviewers (MB JR) extracted data from the studies included. The data extraction form was piloted and modified as required based on the feedback from the team. The data were extracted from all of the studies included using a standardized template to capture optimal information. The extracted data about the general information of the published studies was collected in an EXCEL spreadsheet.https://www.crd.york.ac.uk/). Of note is the fact that since there is no standardized method to critically appraise the quality of the studies included in a systematic review, we considered the guidelines recommended in the health economic evaluation as the most appropriate for our rapid review.The quality of the selected studies was assessed  using the guidelines recommended by Drummond and Jefferson for cost-effectiveness analysis studies . The quahttps://www.bls.gov/cpi/data.htm), as was done in previous studies , irrelevant population (n = 52), and irrelevant outcome measure (n = 14). One full text was not accessible. Six studies were considered for the qualitative analysis. Also, we found one protocol which analyzes the impact and the cost-effectiveness of the lifestyle interventions for breast cancer, but the results of this study will not be published until the end of 2019  and diet (n = 2) (The six studies included were published between 2007 and 2014. All of the studies were conducted in high-income countries (HICs): two studies were from the USA, and one study each was from Australia, Belgium, Netherlands, and UK . Two typ (n = 2) –41.Breast cancer was the primary focus of prevention, along with ovarian cancer only, in Bós et al. who analyzed the cost-effectiveness of a low-fat diet on these two cancers . In fiveAll of the PA-related studies were carried out in a community setting, except for one study which combined PA and diet in a secondary care setting. There were three types of study designs: hypothetical cohorts, closed cohorts of a given population, and randomized control trials (RCTs). The adult populations with ages from 16 to 30, as well as the populations aged 50 and above, were the most commonly targeted groups , 39, 41.n = 5) or cost-utility analyses (n = 1) based on Markov models (n = 6). The model inputs  were derived from RCTs (n = 3), from literature (n = 4), and from national databases (n = 3). A natural experiment was used in Frew et al.  or an incremental cost per utility ratio (ICUR) (n = 1). The additional costs per QALY gained were estimated in most studies. Only (n = 5). The time horizon used in the studies varied from 5 years to the lifetime horizon of the population studied. Different time horizons were used in the sensitivity analysis. In all studies but one, the cost-effectiveness analysis was presented from the perspective of the society, and in half of the studies, both the society and the healthcare payer perspectives were included. Society WTP thresholds are presented  reported in the four studies, of which ICER/QALY was estimated and for which the price-year was available, was $24,973 , 39–41. The low-fat-diet program for postmenopausal women, which is the sole study focusing only on breast cancer and ovarian cancer, was cost-effective from a societal perspective . When loPA interventions targeting five major NCDs, including breast cancer, were ranked first in terms of their cost-effectiveness . SpecifiA total of 11 out of 25 interventions were assessed as likely to be cost-effective for the primary prevention of breast cancer, and their likelihood levels of cost-effectiveness were ranked as very high or high . The incThis rapid review shows evidence of the cost-effectiveness of the diet-related interventions on breast cancer and ovarian cancer as well as the PA-related programs on breast cancer and other major NCDs. Our review also included interventions that addressed breast cancer alongside other NCDs, such as coronary heart disease, stroke, diabetes, and colorectal cancer. Only one study differed from that approach, focusing only on two gynecological cancers . The benEstimating the cost-effectiveness of the lifestyle-related interventions only for breast cancer vs. the cost-effectiveness of these interventions for all NCDs would likely result in higher ICERs since, for the same change in costs, the differences in QALYs for breast cancer alone, in the denominator of the ICER, might be smaller. However, the favorable cost-effectiveness ratios of diet and PA-related interventions for all NCDs would remain below $50,000 per QALY for breast cancer alone. Despite our communication with the authors of these studies, we were not able to get the ICERs for breast cancer alone. For the low-fat-diet interventions, based on personal communication from Bós, favorable ICERs were found for breast cancer alone, and all were below the $50,000 threshold . The priThe long-term effects of PA interventions have been shown to make the primary prevention interventions cost-effective, which is very sensitive to the time horizon in the economic evaluation. The longer the time, the lower the cost-effectiveness ratio will be. Time is needed to observe the potential outcomes of a primary prevention. Overall, the benefits would be greater in the long term than in the short term. Of the seven interventions assessed in the USA by Roux et al., six of them were cost-effective over a 40-years time horizon . Some inTo our knowledge, this rapid review is the first review of its kind that focused on the lifestyle prevention interventions such as healthy weight programs, nutrition and balanced diet interventions, PA programs, limited alcohol consumption interventions, and tobacco cessation programs, excluding a previous study based on breast cancer preventions that found limited evidence of the effectiveness of primary prevention interventions . A benefOur study had several limitations. Firstly, the number of studies that could be included was limited. Only two types of interventions were identified: physical activity (in five studies) and diet (in two studies). The small number of interventions did not permit the differentiation of the primary-prevention-related impact of intervention on breast cancer. More studies might be required to reach such an impact of public health interventions. The lack of sufficient evidence on the primary prevention interventions in reducing breast cancer might hinder the economic evaluations of lifestyle-related interventions. Also, it might be a result of our rapid review strategy and the limited number of databases searched. However, similar limitations were observed in previous systematic reviews in a number of studies retrieved . SecondlThere are further limitations. While physical inactivity, excess weight, and unhealthy diet are significant threats to worldwide populations, our cost-effectiveness estimates were limited to HICs only , 47, 48.The rapid review of the six primary prevention studies highlighted that the use of PA programs and low-fat-diet interventions among particular subgroups of women had high cost-effectiveness. Many of the cost-effective interventions aimed to reduce the risk of NCDs alongside breast cancer, allowing public health professionals to use a holistic program addressing multiple aspects of a woman's health. Societies have invested in primary prevention drug therapies and surgical procedures for breast cancer, and the same investment can be made in the lifestyle interventions targeting breast cancer. We intend that a future systematic review will help in identifying the additional cost-effectiveness of lifestyle-related primary prevention of breast cancer.MB, J-PR, and KB contributed to conceptualization and design. MB and JR collected and assembled information. All authors contributed to data analysis and interpretation, contributed to manuscript writing, and agree to be accountable of all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rheumatoid arthritis (RA) is one of the chronic autoimmune diseases that affects about 0.5 to 1.0% of the general population worldwide. The main symptom of RA is the destruction of the synovial joint, leading to a reduced quality of life and increased mortality. RA may be accompanied by several comorbidities, on which several studies have been conducted on the association between RA and breast cancer. However, the association between RA and breast cancer has shown different directions and has not been clearly established. In this study, we tried to determine whether RA had a causal effect on breast cancer using Mendelian randomization (MR) analysis, but causal evidence was not found. Therefore, additional studies are needed to determine whether RA patients are at high risk of breast cancer, based on large-scale cohorts to validate these results.Previous studies have been reported that the association between rheumatoid arthritis (RA) and breast cancer remains inconclusive. A two-sample Mendelian randomization (MR) analysis can reveal the potential causal association between exposure and outcome. A two-sample MR analysis using the penalized robust inverse variance weighted (PRIVW) method was performed to analyze the association between RA and breast cancer risk based on the summary statistics of six genome-wide association studies (GWAS) targeting RA in an East Asian population along with summary statistics of the BioBank Japan (BBJ), Breast Cancer Association Consortium (BCAC), and Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) targeting breast cancer. We found that the direction of the effect of RA on breast cancer varied among GWAS-summary data from BBJ, BCAC, and CIMBA. Significant horizontal pleiotropy based on a penalized robust MR-Egger regression was observed only for BBJ and CIMBA BRCA2 carriers. As the results of the two-sample MR analyses were inconsistent, the causal association between RA and breast cancer was inconclusive. The biological mechanisms explaining the relationship between RA and breast cancer were unclear in Asian as well as in Caucasians. Further studies using large-scale patient cohorts are required for the validation of these results. To date, the association between rheumatoid arthritis (RA) and cancer risk is unclear. The association of RA with some type of cancers, such as lymphoma and skin cancer, has been reported ,2,3. A mAlthough the underlying biological mechanism explaining the association between RA and cancer remains unclear, the biological pathways associated with the development and prognosis of breast cancer and RA share several components ,16,17,18Several studies conducted to determine the association between breast cancer and RA reported inter-racial differences in their results. This may be due to differences in the incidence of breast cancer   and diffThe Mendelian randomization (MR) method may be a useful tool to determine the association between diseases and exposure from independent observational studies based on the concept of solving the temporal relation in causality by using the germline variants as instrumental variables (IVs) to shift the exposure time of the main risk factor to the subject’s birth time or earlier . Summary statistics of a genome-wide association studies (GWAS) conducted using the Biobank Japan (BBJ) , Breast p-values, beta coefficients, standard errors (SEs), risk alleles, and risk allele frequencies for the association between single nucleotide polymorphisms (SNPs) and RA in previous GWAS papers and for the association between SNPs and breast cancer risk in the public GWAS summary statistics from the BBJ, BCAC, and CIMBA are shown in The summary statistics including Results of the penalized robust inverse variance weighted (PRIVW) analysis for causal inference of RA on breast cancer risk in two-sample MR are shown in P-values for the intercept terms in penalized robust MR Egger regression were 0.31, 0.59, 0.04, and ≤0.001 for BCAC, CIMBA BRCA1 carriers, BBJ, and CIMBA BRCA2 carriers, respectively.No Significant horizontal pleiotropy based on PRIVW method was observed in overall breast cancer and hereditary breast cancer in BCAC and BRCA1 carriers. In this study, we used two-sample MR using PRIVW analysis to determine the association between RA and breast cancer using six GWAS targeting RA in an East Asian population and the summary results from the BBJ project, BCAC, and CIMBA targeting breast cancer. A previous study suggested the possibility that the direction of the effect of RA on breast cancer may vary in different populations ,10,11,12To confirm the association between RA and breast cancer, we focused on GWAS conducted in both the East Asian and European populations. Although the results of the two-sample MR analysis based on the BBJ, BCAC, and BRCA1 carriers from CIMBA were statistically significant, these findings were inconsistent with the GWAS summary statistics. The direction of association between RA and breast cancer seems to differ among Western and Asian populations, and there may be several possible explanations for this. First, the causal association between RA and breast cancer remains inconclusive due to the inconsistency of results among different databases. The other explanation is that our analysis only investigated the differences in the causal association between RA and breast cancer between Western (BCAC) and Asian ancestries (BBJ). Previous meta-analyses conducted to determine the association between RA and breast cancer risk found that RA increased the risk of breast cancer in non-Caucasians, whereas it decreased the risk in Caucasians . In thisIn the BRCA2 carriers from CIMBA, as the horizontal pleiotropy was significant and the PRIVW result was insignificant, confounders were not completely excluded, thus, the causality could not be investigated.To investigate whether RA-associated SNPs were also associated with other phenotypes or not, we reviewed RA-associated SNPs in the GWAS catalog . Table SThe MR result of BRCA1 carriers from CIMBA suggested that RA is associated with a reduced risk of breast cancer, although most of the participants were Caucasian. Breast cancer from the BRCA1 carriers is a kind of early-onset, and many BRCA1 mutations cause breast cancer before age 45 . TherefoAlthough the exact biological mechanism for the increased or decreased risk of breast cancer in RA patients is still unclear, several biological pathways may play a role. The mammalian target of rapamycin, which is known to be involved in the development of breast cancer  is knownThe incidence pattern of breast cancer in the Asian population is known to be significantly different from that of western populations , suggestThere are some limitations to our results that need to be considered. Even though two-sample MR can be performed when the exposure of interest and the outcome are not simultaneously measured within one dataset, full datasets such as large-scale patient cohorts such as discovery and external validation sets are needed for a comprehensive understanding of causality, considering potential confounding factors. In other words, two-sample MR analysis is an indirect observation as compared to large-scale epidemiological studies, such as patient cohort analysis. The consistency of our results should be confirmed from various data sources. Moreover, not all genome-wide significant SNPs that predicted RA were available in the breast cancer GWAS we used. In addition, we included the results of a GWAS targeting RA in an East Asian population, but we used the summary results of the BCAC and CIMBA targeting breast cancer in European populations. Although summary statistics of the SNPs significantly associated with the exposure are required in MR analysis, BCAC and CIMBA have not opened the GWAS summary statistics to the public yet. Although there is a possibility that genome-wide significant SNPs associated with RA can differ according to the onset of RA, our study could not consider the variation of the RA onset. Since SNPs were obtained from six previous GWAS studies, we had a difficulty obtaining the information on the onset of RA.The summary statistics of GWAS for RA were extracted from six studies in the Korean and Japanese population ,48,49,50p-value of SNPs was 5 × 10−8 in the discovery dataset. The study of Hu et al. (2011), also conducted GWAS on Koreans with 100 RA cases and 600 controls for 300,909 SNPs. Based on an independent case-control sample consisting of 578 RA cases and 711 controls, replication analysis was performed. A total of eight SNPs were selected with genome-wide significant p-value 1 × 10−5 [p-value of SNPs was 5 × 10−8 [p-values from 5 × 10−8 to 5 × 10−5 [p-value of SNPs was 5 × 10−8 [p-value < 1 × 10−3 were selected as candidates for further studies [Briefly, the study of Freudenberg et al. (2011) performed GWAS on the Korean population to identify susceptibility loci for RA, based on 801 RA cases and 757 controls for 441,398 SNPs. Among them, 79 SNPs were replicated by an independent European population, consisting of 718 RA cases and 719 controls . The gen1 × 10−5 . The stu5 × 10−8 . The stu5 × 10−5 . The stu5 × 10−8 . The stu studies . Five ofGWAS was conducted from BBJ, based on about 159,000 participants of Japanese ancestry . Among pGWAS from the BCAC was conducted with the largest breast cancer. The processes of genotyping, quality control, and imputation in BCAC are already provided in more details elsewhere . The BCA2 ≤0.3 and minor allele frequency (MAF) ≤0.005 were excluded in further analyses. SNPs with a call rate under 95% were also removed.The CIMBA consists of 15,252 BRCA1 and 8211 BRCA2 carriers, of whom 12,127 participants with breast cancer (7797 BRCA1 and 4330 BRCA2 carriers). The procedures of genotyping and quality control have already been provided in more details elsewhere . Shortlyp-values below 1 × 10−5, considering statistically significant. The beta coefficients and SEs of these SNPs were searched from the summary results of the following GWAS data sources: BBJ [SNPs associated with RA in the East Asian population were identified from the summary statistics of six GWAS ,48,49,50ces: BBJ , BCAC [2ces: BBJ , and CIMces: BBJ . SNPs wiiX) of interest has a causal effect (GXβ) on the outcome (iY) by genetic variants as IVs, which are strongly associated with exposure  the genetic variants considered as IVs should be strongly associated with exposure, (2) the genetic variants referred as to IVs should not be associated with any confounding factors, and (3) the genetic variants as IVs should only have an effect on the risk of the outcome via exposure.p-value below 0.05 were considered statistically significant, and the intercept term from penalized robust MR Egger regression with a p-value below 0.05 were considered statistically significant in horizontal pleiotropy. Statistical analyses were conducted using the R 3.6  statistical software and Mendelian randomization package [To evaluate whether the MR analysis follows the above three assumptions, we used a maximum likelihood estimation with Cochran’s Q heterogeneity tests to evaluate the possibility of horizontal pleiotropy ,60 and t package .Even though the two-sample MR method is useful to evaluate the association between exposure and outcome by excluding the effects of other confounders, the causal inference between RA and breast cancer remains unclear. Once large-scale GWAS of RA effects are publicly available, the same method can be used to examine RA causally related to breast cancer and the relationship between RA and breast cancer. Additional research should include GWAS targeting both RA and breast cancer conducted in large-scale patient cohorts such as a nationwide patient cohort, using health insurance claim data and MR analyses to evaluate the correlation between RA and other cancers using GWAS summary statistics.


WT1 and CA10, and breast cancer risk remain unknown.Studies have shown that abnormal changes of specific-gene DNA methylation in leukocytes may be associated with an elevated risk of cancer. However, associations between the methylation of the zinc-related genes, WT1 and CA10 was analyzed by methylation-sensitive high-resolution-melting (MS-HRM) in a case-control study with female subjects (N = 959). Logistic regression was used to analyze the associations, and propensity score (PS) method was used to adjust confounders.The methylation of WT1 hypermethylation was associated with an increased risk of breast cancer, with an odds ratio (OR) of 3.07 . Subgroup analyses showed that WT1 hypermethylation was specifically associated with an elevated risk of luminal A subtype  and luminal B subtype . CA10 hypermethylation was associated with an increased risk of luminal B subtype .The results showed that WT1 methylation in leukocytes is significantly associated with an increased risk of breast cancer. The hypermethylation of WT1 is associated with an increased risk of luminal subtypes of breast cancer, and the hypermethylation of CA10 is associated with an increased risk of luminal B subtype of breast cancer.The results of the present study suggest that the hypermethylation of  Breast cancer is one of the most common malignancies in women worldwide  and presWT1) is a tumor suppressor gene which involved in human cell growth and differentiation. WT1 has been reported to be significantly different methylated in the tissues of hepatocellular carcinoma [WT1 aberrant methylation may lead to a reduction or absence of WT1 expression, which results in the overexpression of the insulin-like growth factor I receptor (IGF 1R) and insulin-like growth factor II (IGF II), thereby promoting breast cancer process [WT1 and CA10 hypermethylation were significantly different between breast cancer tumor tissues and non-malignant tissues [Wilm’s Tumor gene  or ductal carcinoma in situ (DCIS), other types of breast cancer  were excluded from our study. Controls were recruited from patients admitted to the Orthopedic and Ophthalmology Department of the Second Affiliated Hospital of Harbin Medical University and volunteers from the Xiangfang community of Harbin within the same period. All controls were also female. In addition, all control participants were asked about their disease history in a questionnaire, and individuals who reported a history of any cancer were excluded from our final subjects. Finally, 402 female breast cancer cases and 557 female controls were included in our study. Blood sample (5 mL) was collected from each participant and then stored at − 80 °C.We investigated the relationship between All subjects were interviewed face-to-face by trained investigators with normalized questioning methods. The questionnaire was adopted from the study by Shu et al. , and incCA10 and WT1 and breast cancer risk. The blood samples were also collected and other anthropometric measurements were taken. The sample selection criteria and the methods were reported by Riboli et al. [The study was validated with the GEO-GSE51032  dataset with a nested case control study design to analyze the association between the methylation of i et al. . We extrDNA was extracted from peripheral blood samples using a commercial DNA extraction kit . The concentration and the purity of DNA were assessed using a Nanodrop 2000 Spectrophotometer . Genomic DNA was bisulfite-modified with an EpiTect Bisulfite kit . Bisulfite DNA was normalized to a concentration of 20 ng/mL and was stored at − 20 °C for the following experiment. DNA extraction and DNA sodium bisulfite modification were performed according to the manufacturers’ instructions.WT1 and CA10 with the LightCycler 480 system  equipped with Gene Scanning software (version 2.0). The primers were adopted from a published study [WT1 and CA10  and 0.94 (P < 0.01), respectively  to evaluate the methylation of ed study . We used version .0. The ply Table .Fig. 2TFour subtypes of breast cancer were defined as luminal A, luminal B, HER-2 enriched and triple negative breast cancer (TNBC) by immunohistochemical analysis based on previously validated clinicopathological criteria .2) tests. For missing values in the environmental factors, we applied multiple imputation to generate possible values. To measure the association between methylation of WT1, CA10 and breast cancer risk and different molecular types breast cancer, we used univariate and multivariate unconditional logistic regression analyses to estimate the crude and adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs). For our case-control study, we used 0% methylation as a cutoff for both WT1 and CA10. We used receiver operating characteristic curve (ROC) to calculate the cut-off value of β for the validation dataset. We also applied the propensity score (PS) method to adjust covariates , in which the study outcome served as the dependent variable and PS served as the confounding variable. Kappa values were calculated to analyze the consistency between same samples in different runs. All two-sided P values < 0.05 were considered statistically significant. Data were analyzed by using SPSS v.24.0 .For the distribution of basic demographic characteristics, continuous variables such as age were analyzed by two-sample t-tests, and categorical variables were analyzed by chi-square  and 3.07 , respectively. CA10 methylation was statistically significant associated with breast cancer in the multivariable adjustment with an OR of 1.53 , but was only marginally associated with breast cancer after PS adjustment, with an OR of 1.35  (Table WT1 methylation was associated with breast cancer risk in both the younger (< 60-years-old) and older (≥60-years-old) groups, with ORs of 2.64  and 4.72 , respectively. CA10 methylation was associated with breast cancer risk in younger age group (< 60-years-old) before PS adjustment, with OR of 1.56 ; However, the association was not statistically significant after PS adjustment , and PS adjusted OR of 2.62 . WT1 methylation was also significantly associated with the risk of luminal B subtype breast cancer with ORs of 2.49  and 3.23  after multivariable and PS adjustment. However, WT1 methylation was not significantly associated with the risk of HER-2 enriched and TNBC subtypes  and 1.80 , respectively. However, CA10 methylation had no significant associations with the risk of luminal A, HER-2 enriched and TNBC subtypes after the adjustment of PS. The association between the methylation of WT1, CA10 and other clinicopathological characteristics of breast cancer patients were analyzed are showed in Table The associations between WT1 and CA10 sequences . However, the average methylation level of Cg14054928 and Cg20405017, which are located within the targeted CA10 sequence, was not statistically significant associated breast cancer risk   with a nested case control study design and found the significant association between WT1 methylation and breast cancer risk, with two CpG probes inside our sequence, with OR of 1.88.t tissue . WT1 meter cells . Our invCA10 can undergo methylation during breast carcinogenesis in tumor tissue [CA10 was reported to be hypermethylated among a panel of genes in urine, which may contribute to the highly accurate and early detection of bladder cancer [CA10 methylation in leukocyte DNA was marginally associated with an elevated breast cancer risk after PS adjustment. The amplified sequence contained 7 CpGs and located in the second exon of CA10. The external validation dataset of GEO-GSE51032 only included 2 CpG probes and did not exhibit a significant association between CA10 hypermethylation and breast cancer risk.A previous study showed r tissue . CA10 war cancer . The resWT1 and CA10 associated with the alteration of their expression. Therefore, we investigated the correlations between methylation probes and expression using TCGA (http://cancergenome.nih.gov/) and Mexpress (https://mexpress.be/) databases. The results showed that WT1 hypermethylation was also negatively correlated with its expression , and CA10 hypermethylation was negatively related to its mRNA expression as well . Although discounted by different sample-derived DNA, the significant negative correlations between WT1, CA10 methylation and gene expression were consistent with our study and indicated promising potential in breast cancer risk assessment.To further investigate the functional relevance of the observed associations, it would be important to test whether methylation of the specific CpGs in WT1 methylation level with both MS-HRM and pyrosequencing, and the results were highly correlated between these two methods [In our previous study, we tested the accuracy of MS-HRM by assessing the  methods . However methods . The hig methods .WT1, CA10 could not be analyzed. Third, selection bias may have occurred, since we recruited the breast cancer patient subjects at the Tumor Hospital of Harbin Medical University, which might not be representative of the distribution of breast cancer patients to some extent.The limitations of this study should be taken into consideration before drawing a conclusions: first, as in all retrospective analyses, our study may have some recall bias when collecting information on environmental factors. Second, the sample size of our study is not large enough for subgroup analysis, including the subgroup analyses of low frequency environmental factors, such as smoking behavior, therefore their associations with DNA methylation of WT1, CA10 in blood leukocytes may be associated with the risk of breast cancer. Associations between WT1 methylation and the risk of luminal subtypes of breast cancer and between CA10 methylation and the risk of luminal B subtype breast cancer were also observed.In summary, the results of our study suggested that methylation of Additional file1 Table S1. Demographic variables and questionnaire-derived variables of participants before and after multiple imputation in this study.Additional file2 Table S2. Primer sequences and reaction condition for methylation-sensitive high-resolution melting analysis.Additional file3 Table S3. Reaction system for methylation-sensitive high-resolution melting analysis of WT1 and CA10.Additional file4 Table S4. Result of methylation-sensitive high-resolution melting analysis for the same samples in different runs.Additional file5 Table S5. The methylation of WT1 and CA10 and clinicopathological characteristics in breast cancer patients.


Large breast adipocyte size was significantly associated with iIBC risk (odds ratio (OR) 2.75, 95% confidence interval (95% CI) = 1.25–6.05). High cyclooxygenase (COX)-2 protein expression in the DCIS cells was also associated with subsequent iIBC (OR 3.70 (95% CI = 1.59–8.64). DCIS with both high COX-2 expression and large breast adipocytes was associated with a 12-fold higher risk  for subsequent iIBC compared with women with smaller adipocyte size and low COX-2 expression. Large breast adipocytes combined with high COX-2 expression in DCIS is associated with a high risk of subsequent iIBC. Besides COX-2, adipocyte size has the potential to improve clinical management in patients diagnosed with primary DCIS.Although ductal carcinoma in situ (DCIS) is a non-obligate precursor to ipsilateral invasive breast cancer (iIBC), most DCIS lesions remain indolent. Hence, overdiagnosis and overtreatment of DCIS is a major concern. There is an urgent need for prognostic markers that can distinguish harmless from potentially hazardous DCIS. We hypothesised that features of the breast adipose tissue may be associated with risk of subsequent iIBC. We performed a case–control study nested in a population-based DCIS cohort, consisting of 2658 women diagnosed with primary DCIS between 1989 and 2005, uniformly treated with breast conserving surgery (BCS) alone. We assessed breast adipose features with digital pathology (HALO In Western countries, DCIS comprises 15–30% of all newly diagnosed breast neoplasms6. DCIS is generally treated with surgery, often followed by radiotherapy and sometimes adjuvant hormonal therapy. The majority of patients with primary DCIS, however, will not develop a subsequent ipsilateral invasive breast cancer (iIBC)10, suggesting overdiagnosis and overtreatment. Clinicians urgently need prognostic biomarkers to distinguish hazardous from indolent DCIS for adequate risk stratification. Previous studies identified several clinicopathological prognostic factors for subsequent in situ or IBC recurrences24. A previous study in our Dutch nationwide DCIS cohort revealed human epidermal growth factor receptor 2 (HER2) overexpression, high cyclooxygenase (COX)-2 protein expression and the presence of periductal fibrosis as promising markers for predicting subsequent iIBC after primary DCIS13.Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer (IBC). DCIS incidence has increased manifold since the implementation of population-based mammographic breast cancer screening25 and is associated with an increased risk for developing postmenopausal IBC31 and poorer outcome in pre- and postmenopausal breast cancer33. Obesity at initial DCIS diagnosis has also been associated with increased risk of second breast cancers34.Obesity prevalence is rapidly increasing35. Increased adiposity is associated with (breast) adipocyte hypertrophy and low-grade inflammation of white adipose tissue (WAT), resulting from hypertrophic dying adipocytes encircled by macrophages forming crown-like structures (CLS)37. Although present in the majority of obese and overweight individuals38, enlarged adipocytes and WAT inflammation have also been found in the breast tissue of women with a normal BMI39. Apart from chronic inflammation of WAT, hyperadiposity commonly leads to altered local steroid hormone biosynthesis and disruptions in adipokine levels and insulin metabolism43. The altered microenvironment of (mammary) hyperadiposity has been associated with increased breast cancer risk44.Body mass index (BMI) alone is not sufficient for assessing adipose-tissue-related risk of IBC, as some postmenopausal women with higher body fat level have an elevated risk for IBC, despite of a normal BMIAlthough adipose tissue is a major component of the mammary gland, its potential role in development of subsequent iIBC after primary DCIS has received little attention. We hypothesised that breast adipocyte hypertrophy in DCIS is associated with a risk of subsequent iIBC.n = 108) are women with specimen histology showing pure DCIS that predates diagnosis of iIBC. Controls (n = 168) are women with pure DCIS that have not developed subsequent iIBC and are matched for age of DCIS diagnosis (± 0 to 6 months). Median time from primary DCIS diagnosis to iIBC was 5.8 years (range 0.5–19.2). Clinical characteristics were comparable across cases and controls  alone were included in this case–control study Fig. . Cases n = 08 are woP = 0.10) and positive margin status (P = 0.10) were somewhat more common among cases compared to controls. Both margin status and lesion size were not always reliably reported in these older patient series and for 16% and 67%, respectively, these data were missing.Histologic grade was comparable between cases and controls. Grade 1 was found in 13.9% of cases and 13.1% of controls, grade 2 in 56.5% of cases and 61.9% of controls and grade 3 in 29.6% of cases versus 25.0% of controls  3.70, 95% confidence interval (95% CI) = 1.59–8.64, th percentile (adipocyte diameter75th) and adipocyte area at the 75th percentile (adipocyte area75th) were assessed with digital pathology among all patients (see Methods). Median percentage of breast adipose tissue was 65% (interquartile range (IQR) 47–77%). A median number of 2514 adipocytes was measured per patient with digital pathology (IQR 1313–5419). Median adipocyte diameter at the 75th percentile (adipocyte diameter75th) was 86 µm (IQR 77–94) and median adipocyte area at the 75th percentile (adipocyte area75th) was 6174 µm2 (IQR 5165–7604), respectively. Figure Breast WAT features including relative amount (percentage) of adipose tissue, adipocyte diameter at the 75P < 0.001, Fig. P < 0.001) and larger adipocyte size (P < 0.001) than the age group of <45 years.Higher relative area of breast adipose tissue and larger adipocyte size were significantly associated with higher age P < 0.00, Fig. 1.2 was 1.4 . The number of CLS per 10 cm2 correlated with adipocyte area75th  and a low (<5 CLS/10 cm2) CLS count  for whom at least five paraffin blocks were available. Median number of CLS per 10 cm75th (range 47–118 µm), the OR for a subsequent iIBC increased with 25% (95% CI = 1.02–1.54), while for each 103 µm2 increase in adipocyte area75th (range 2016–11569 µm2) the OR for subsequent iIBC increased with 17% (95% CI = 1.01–1.35). Women with an adipocyte area75th within the highest quartile had a 2.75-fold (95% CI = 1.25–6.05) increased risk for subsequent iIBC compared to women with an adipocyte area75th within the lowest quartile. When compared to women with an adipocyte area75th within the lowest three quartiles, women with an adipocyte area75th of the highest quartile had a 2.08 times (95% CI = 1.16–3.74) increased risk for subsequent iIBC. Relative area of breast adipose tissue was not associated with subsequent iIBC risk.Breast adipocyte size at primary DCIS diagnosis was positively associated with risk for a subsequent iIBC Table . For eac75th remained associated with iIBC risk when adjusted the presence of periductal fibrosis, Her2 status and COX-2 expression in DCIS cells (Table 75th remained an independent predictor of subsequent iIBC. Women with an adipocyte area75th within the highest quartile had a 3.05-fold (95% CI = 1.32–7.05) increased risk for subsequent iIBC compared to women with an adipocyte area75th within the lowest quartile, while COX-2 was an independent predictor for iIBC in the presence of adipocyte area75th . Compared to women with an adipocyte area75th within quartiles 1–3, women with an adipocyte area75th within the highest quartile had a 2.34-fold increased risk for subsequent iIBC . The risk of subsequent iIBC in DCIS patients with high COX-2 expression and an adipocyte area75th within the highest quartile was 12 times higher than in DCIS patients with low COX-2 expression and an smaller (quartile 1–3) adipocyte area75th  had a 10- and 15-year cumulative incidence of 22.7% and 28.7%, respectively  and low COX-2 expression had an estimated 10- and 15-year iIBC cumulative incidence of 2.0% and 2.6%, respectively. Within our study population, a combination of large adipocyte size and high COX-2 expression (area75thq4/COX-2high) was present in 25.9% of cases and 14.3% of controls  have a 12-fold increased risk for a subsequent iIBC, resulting in an estimated 10- and 15-year cumulative iIBC incidence of 22.7% and 28.7%, respectively. Women with smaller breast adipocytes  in combination with low COX-2 have a 10- and 15-year cumulative iIBC incidence of 2.0% and 3.9%, respectively, which is similar to the 10- and 15-year breast cancer incidence in the general population. However, it should be noted that the DCIS patients in this lowest risk category have been treated . Taken together, besides COX-2 expression, breast adipocyte size appears to be promising for clinical management in primary DCIS.In this study we investigated mammary adiposity in relation to risk of subsequent iIBC after primary DCIS. We show that the presence of large breast adipocytes in primary DCIS is associated with a risk for subsequent iIBC. DCIS with high COX-2 expression, a cytoplasmic enzyme involved in prostaglandin synthesis, was also associated with iIBC risk, as we previously showed36. As DCIS incidence sharply rises during menopause and is highest in the postmenopausal group , future research may focus on the role of adipocytes in the pathogenesis of iIBC after primary DCIS in the context of the menopausal transition.Several other important findings have emerged from this study. Firstly, we found that women with DCIS in the age group of <45 years on average have the smallest adipocytes and the lowest percentage of mammary adipose tissue among all age groups. DCIS patients in the age group >55 years  have the largest adipocytes and highest relative area of adipose tissue. The association between breast adipocyte characteristics and age underlines that matching DCIS cases and controls for age is essential. Iyengar et al. found a correlation between breast adipocyte size, CLS and postmenopausal status among breast cancer patients that underwent mastectomy2. Number of breast CLS and BMI previously has been shown to correlate with breast adipocyte diameter in IBC45. Carter et al. found that a high CLS count (>5 CLS/sample) was associated with subsequent IBC  in biopsies of 172 patients with benign breast disease44. In our study, number of CLS/10 cm2 was not associated with subsequent iIBC in 56 DCIS patients, although the analysis had low power to detect such an association.Secondly, we found that breast adipocyte size correlates with adipose inflammation expressed as the number of CLS per 10 cm47, resulting in the induction of aromatase, the rate-limiting enzyme for oestrogen biosynthesis48. The subsequent locally produced oestrogen may be a key driver to ER-positive IBC. In the present study, however, ER status alone was not a predictor of iIBC risk and did not significantly change the risk estimate when added to a model containing adipocyte area75th and COX-2. The study potentially lacked power to show significant interaction. Larger studies and mechanistic studies are needed to investigate the relationship between WAT inflammation, COX-2, ER status and outcome after primary DCIS.WAT inflammation has been associated with elevated secretion of proinflammatory mediators and adipokines such as COX-2, TNF-α, IL-1ß and leptin35. Furthermore, a second study of Iyengar et al. in women with a normal BMI undergoing mastectomy for breast cancer treatment or risk reduction showed WAT inflammation in 39% of women39. Since WAT inflammation was associated with larger breast adipocytes, higher circulating leptin levels and increased aromatase in breast tissue, breast adipocyte size may be more accurate for breast cancer risk assessment than BMI alone39.Our study presented some limitations. Firstly, BMI was not available from the old patient records from 1989–2004 in our study. However, a study of Iyengar et al. indicates that BMI is not always an appropriate proxy for breast cancer risk in postmenopausal women, because women with increased body fat as assessed by dual-energy X-ray absorptiometry (DXA) are at elevated risk of breast cancer despite of a normal BMISecondly, in 35% of the DCIS lesions, breast adipocytes were too severely damaged and therefore we were unable to assess adipocyte size in these patients. In addition, analyses of CLS were limited by the relatively low number of DCIS patients with ≥5 available formalin-fixed paraffin-embedded (FFPE) blocks. In order to increase the number of DCIS specimen with intact and sufficient breast adipose tissue, future studies on breast adipocytes could benefit from adaptations in the surgical breast pathology pipeline.Thirdly, data on lesion size was often missing (67% of patients). Furthermore, patients with a positive margin status typically undergo a re-excision lumpectomy or mastectomy according to Dutch guidelines. The final margin status after re-excision was not reliably registered between 1989 and 2004. Among the patients with known lesion size and margin status, no significant differences were observed between cases and controls.A major strength of our study is the population-based nested case–control design allowing for the study of a high number of subsequent iIBC with long-term follow-up. Treatment of DCIS with BCS alone was more commonly practiced between 1989 and 2004. All DCIS patients received a uniform treatment of BCS alone, without radiotherapy or endocrine therapy that could impact subsequent iIBC risk. Furthermore, the population-based nature and the fact that the patient-material was derived from 58 different pathology laboratories, makes our results potentially more generalisable.The study is further strengthened by the use of digital pathology, enabling tissue segmentation and the automatic evaluation of the diameter and area of high numbers of intact adipocytes. The measurement of the adipocyte area rather than its diameter approximates the three-dimensional nature of an adipocyte in real life more accurately, as compared to manual measurements.75th and COX-2 are promising prognostic markers for prediction of iIBC risk in primary DCIS patients. Patients with an adipocyte area75thq4/COX-2high DCIS lesions when treated with BCS alone, had a 12-fold increased risk for subsequent iIBC compared to adipocyte area75thq1-3/COX-2low DCIS lesions, and thus may benefit from more careful monitoring during follow-up.In conclusion, breast adipocyte area49, after which our findings could be used to improve clinical management at primary DCIS diagnosis. Further studies are needed to unravel the exact underlying mechanisms behind the notable associations of mammary adiposity, adipocyte inflammation and anthropometric factors. The acquired insights may be used to reduce risk of subsequent iIBC after primary DCIS.As a next step, our findings need to be confirmed in independent cohorts in which not only subsequent iIBC but also contralateral iIBC risk is considered13. Women with primary DCIS that developed iIBC were defined as cases. Controls correspond to women with primary DCIS that did not develop iIBC for at least the same follow-up duration as time to iIBC in the corresponding cases. Cases and controls were matched for age at DCIS diagnosis (±0 to 6 months). Cases and controls were not matched for year of diagnosis. None of the patients developed subsequent contralateral IBC while on study. FFPE tissue blocks and pathology reports were obtained from 58 pathology laboratories in the Netherlands13. Margin status and lesion size were not routinely registered in the pathology reports, especially not in the older cases of our retrospective series.We conducted a nested case–control study within a nationwide population-based cohort of 2658 DCIS women diagnosed in the Netherlands with primary DCIS between 1 January 1989 and 31 December 2004, and a median follow-up of 12.0 years (IQR 9.0–15.3). Women were uniformly treated with BCS without radiotherapy and/or anti-hormonal therapy. A detailed description of this cohort was published previously50 and the international Guideline on Good Clinical Practice. The study also meets the General Data Protection Regulation (GDPR) criteria that came into effect on 25 May 2019.The study was approved by the review boards of the Netherlands Cancer Registry and the Dutch National Pathology Automated Archive (PALGA). The secondary use of tissue and data in this study is covered by an opt-out regimen conform Dutch regulations, the Code of Conduct of Federa-COREONAfter sample selection, intact adipose tissue was assessed in haematoxylin and eosin (H&E) slides from 108 cases and 168 controls. In each case–control set, a case was matched with at least 1 control.® image analysis software . For each patient, one representative H&E slide had previously been selected for histopathological assessment and immunohistochemistry (IHC) analysis of DCIS. For the measurement of adipocyte size, areas of intact adipocytes at a distance of ≥500 µm from a DCIS duct were annotated by two experienced pathologists (M.M.A. and J.S.), blinded for case or control status. DCIS patients without intact breast adipocytes (adipocytes with intact membranes) were excluded.Breast adipose tissue was characterised by digital pathology using HALORepresentative whole slides were scanned with a 20x objective using a Leica Aperio AT2 . To assess the relative amount of breast adipose tissue, we trained a supervised machine learning algorithm (random forest classifier) to recognise adipose, stromal and epithelial compartments from which the corresponding tissue surface and percentage were calculated.Adipocyte diameter (µm) was digitally assessed with the HALO vacuole module v2.1 and calculated as follows: the algorithm of the HALO vacuole module calculated the centroid of the object. Subsequently, 18 diameters were passed through the centroid at different angles taken at 10-degree increments, from which the median was taken. The algorithm settings included conditions on the roundness and the regularity of the contours of the measured objects in order to discriminate between intact adipocytes and in-between areas. The same algorithm settings were used for all patients.36. To guarantee the measurement of true and intact adipocytes, we systematically determined adipocyte size at the 75th percentile of the adipocyte population of each patient as the unique value characterising adipocyte size of that patient. Taken together, digital breast adipose characterisation resulted in percentage of adipose tissue, adipocyte diameter at the 75th percentile (adipocyte diameter75th) and adipocyte surface area at the 75th percentile (adipocyte area75th) for each patient.Measurements with a diameter under 30 µm were not considered in our analysis in order to discriminate mature adipocytes from artefacts13. Antibodies are listed in Supplementary Table H&E slides, histopathological data as well as ER, PR, HER2 and COX-2 IHC data were already available as previously describedCD68 IHC analysis of 56 FFPE DCIS samples was performed on a BenchMark Ultra autostainer. Briefly, paraffin sections were cut at 3 µm, heated at 75 °C for 28 min and deparaffinized in the instrument with EZ prep solution . Heat-induced antigen retrieval was carried out using Cell Conditioning 1  for 32 min at 95 °C. CD68 was detected using clone KP1 . Bound antibody was detected using the OptiView DAB Detection Kit . Slides were counterstained with Haematoxylin and Bluing Reagent . IHC stained slides were scanned using an Aperio AT2 Slide Scanner .52. At least five tissue blocks were available in a subset of 56 DCIS patients (26 cases and 30 controls). Number of CLS per 10 cm2 of adipose tissue as a measure of adipocyte inflammation was assessed by an experienced pathologist (M.M.A.).A CLS was defined as an adipocyte surrounded by a rim of CD68-positive macrophages. In order to investigate sufficient amounts of breast adipose tissue, one section of CD68 IHC data were generated on five different FFPE blocks per patient2 adipose tissue with subsequent iIBC risk after primary DCIS. ORs were calculated as estimates of relative risks (RR); while 95% confidence intervals (95% CI) were Wald-based and P-values were likelihood ratio-based. We subsequently assessed whether adipocyte size remained an independent predictor of subsequent iIBC risk when accounting for the association of other histopathological  and immunohistochemical features  with iIBC risk. Margin status and lesion size were excluded from the multivariable models because they were not always reliably reported in these older patient series and part of data was missing. Variables showing association with IBC risk at a P-value ≤ 0.1 were included in a multivariable model. Model fit was assessed by likelihood ratio tests or Akaike information criterion, in case of non-nested models. We also evaluated whether variables with a main effect in multivariable analysis modified the association of adipocyte size with subsequent iIBC risk by adding an interaction-term to our model.Conditional logistic regression was used to estimate associations of adipocyte size, relative area of adipose tissue and number of CLS/10 cmn = 249). The Shapiro-Wilk test was used to assess departure from the normality assumption. Relative area of adipose tissue was compared between age categories using the non-parametric Mann-Whitney U test and the Kruskal-Wallis test. For comparisons of adipocyte size with age the student’s t-test and one-way ANOVA test was used. Correlation of the number of CLS/10 cm2 of adipose tissue and adipocyte size was assessed by the Spearman’s rho test.BMI and menopausal status were not available. The following age categories were used to approximate menopausal status: <45 years , ≥45–55 years  and ≥55 years . Both relative area of breast adipose tissue and adipocyte size were correlated with age using these age categories as a proxy for menopausal status in all unique patients , using 5-year band for age 1-year band for calendar year.We estimated the expected cumulative incidence of breast cancer for our population from the expected breast cancer incidence and expected all-cause mortality based on the Hakulinen methodP-values < 0.05 were considered statistically significant. Analyses were performed using Stata/SE , software packages SPSS for Windows version 25.0  and Graph Pad Prism 7.0 . Beeswarm plots to represent adipocyte size distribution of individual patients were performed in R  using ggplot2 and dplyr packages.Statistical analyses were two-sided and Further information on research design is available in the Supplemental material.Reporting Summary Checklist.


GSTP1) is one of the common type of the GSTs family. This gene has several genetic polymorphisms that the rs1695 and rs1138272 are the most common variations in this gene. This study aimed to examine the association of these genetic variations with breast cancer risk which was followed by bioinformatics analysis. The glutathione S transferases P1  and allelic  genetic comparisons. This despite the fact that the rs1138272 polymorphism was not associated with breast cancer risk. Our bioinformatics analysis based on WALTZ output showed that the rs1695 polymorphism reduces the amyloid propensity of the GSTP1 enzyme (dWALTZ= -228.00). Based on our findings, the rs1695 genetic variation is a genetic risk factor for breast cancer and it could be considered as a biomarker for screening of susceptible women. According to the studies, breast cancer has been recently one of the commonest types of cancer with continuous prevalence throughout the world. Moreover, this cancer is the main cause of cancer-associated deaths among women . Considering the previous investigations, an annual diagnosis of approximately 1.15 million patients with breast cancer diagnosed has been reported, with the maximum occurrence of breast cancer in the USA and Europe . However, breast cancer prevalence is quickly increasing in the People’s Republic of China . Also, in Iranian women, breast cancer is considered the most common malignancies especially in younger women . π gene encodes the GSTP1 enzyme is chiefly observed in the heart, spleen, and lung tissues. The GST class π enzyme is also expressed in breast cancer tissue .The pathologic factors of breast cancer are not well understood, though some reports referred to the genetic and environmental parameters as the causes of breast cancer . One of the significant factors of the genetic susceptibility to cancer is the inherited differences in the potential of the xenobiotic-metabolizing enzymes. Moreover, glutathione S-transferases (GSTs) have been considered the phase II enzymes that contribute to detoxifying various toxic and potentially carcinogenic compositions . Researchers recognized 5 classes of GST enzymes in human . A separate gene or gene family encodes each of the classes. Then, the allelic variants for each gene can cause less efficient or absent enzymatic detoxification, thereby increasing susceptibility to cancer; however, we have no accurate knowledge of the biochemical procedures. One of the most common members of GSTs is the GSTP1 which has a well-known genetic variation entitled rs1695 single nucleotide polymorphism (SNP). This gene is located on chromosome 11 (11q13) and the mentioned SNP causes an amino acid substitution has been described at codon 105 , resulting in the production of a functionally changed enzyme . Another common genetic variation for GSTP1 is the rs1138272 which results in Ala to Val substitution at codon 114 . The GST class GSTP1 common polymorphisms . Nonetheless, these investigations did not lead to a firm conclusion. On the other hand, there are no sufficient studies investigating the association of these single nucleotide polymorphisms with breast cancer risk. Hence, this case-control study has been done to provide a relatively reliable result which is followed by a bioinformatics approach.Most investigations found a relationship between breast cancer and Case-control studySubjectsAn attempt was made to design the project and the paper according to the rules of the STREGA (Table S1). In this case-control study, based on the number of available samples, 100 women with the mean age of 44.26±6.80 years with only sporadic breast cancer and 100 healthy women with the mean age of 45.48±6.08 years were enrolled. We chose the controls and cases from the women referring to the Pasteur pathobiology and genetics laboratory and also Rohani hospital . Diagnosing breast cancer has been proved by histological examinations for case participants. Moreover, the controls have been chosen from healthy women referring to the same hospital for routine examinations. Notably, each control participant lacked a history of oncological diseases. In addition, 3 mL of blood has been obtained from each subject. Besides, the present research has been performed according to the principles proposed in the Declaration of Helsinki.GSTP1 polymorphisms genotypingGSTP1 gene. According to the research design, a commercial Kit  has been used to extract genomic DNA from the blood samples. Then, the PCR strategy has been used to amplify the GSTP1 fragment containing rs1695 and rs1138272 polymorphisms. Moreover, PCR has been amplified in a final volume of 30 μl. Its mixture consisted of 1×PCR buffer, 50 ng of template DNA, 0.5 μL dNTPs mix, 2.5 μM MgCl2, and 2.5 U of Taq polymerase, and 0.35 μM each of forward and reverse primers (F: 5’-CTCTCATCCTTCCACGCACATCC-3’ and R: 5’-CTGCACCCTGACCCAAGAAGGG-3’ for rs1695 and F: 5’-ACAGGATTTGGTACTAGCCT-3’ and R: 5’-AGTGCCTTCACATAGTCATCCTTG-3’ for rs1138272). All PCR reagents were ordered from CinnaGen Company . PCR procedure was done in an Eppendorf thermal cycler . Notably, PCR amplification started with an early denaturation at 94°C for five minutes and then 33 cycles with 94°C (45 sec), 60°C (45 sec) and 72°C (45 sec) have proceeded. In addition, the final extension has been done at 72°C for 10 min, and thus the cycle ended to maintain at 4°C. The PCR products were treated with BsmAI restriction enzyme  for rs1695 and AciI  for rs1138272 according to manufacturer’s instruction. Finally, the PCR-RFLP products have been electrophoresed on 1.5 % agarose stained with Green Viewer™. UV transillumination has been used to visualize the amplicon bands. For rs1695 SNP, the samples with one band (363-bp) on 2.0% agarose gel had genotype AA while the samples with two bands (226- and 137-bp) on agarose gel had genotype GG. Therefore, the sample containing three mentioned fragments  were considered as heterozygote genotype AG. Regarding rs1138272 polymorphism, the fragment with allele C was digested to two fragments (143- and 27-bp) and allele T has no restriction site for the enzyme (170-bp) that was detected on 3.0% agarose gel.In this study, we evaluated two common SNPs in Statistical analysesAccording to the research design, the Chi-squared test has been applied for evaluating the Hardy-Weinberg equilibrium (HWE). Moreover, a p-value less of than 0.05 has been considered as a significant deviation from the HWE. Then, in the present case-control study, we computed OR with 95% CI for all genotypes and alleles in the cases and controls. Additionally, a chi-square test has been used to evaluate the differences between cases and controls . Then, a two-tailed p-value<0.05 has been regarded as statistically significant. The SPSS 20  has been used to run the statistical analysis.In silico analysisGSTP1 structure by the SNPeffects web server. For this purpose, at first, the entire sequence of the GSTP1 gene was obtained from NCBI. Then the coding sequence of the gene was deduced and then was translated to amino acid sequence by ExPASy bioinformatics webserver. The location of the rs1695 variation was determined on codon 105 . This sequence was introduced to SNPeffects online web server and then the influence of rs1695 SNP on aggregation propensity (TANGO), amyloid propensity (WALTZ) and chaperone binding (LIMBO) was evaluated .In this study, we evaluated the molecular effects of rs1695 polymorphism on the Genetic association outcomesFor, rs1695 polymorphism, our data from genotypes distribution showed no deviation from Hardy-Weinberg equilibrium in our study for both case and control groups. The distribution of alleles and genotypes of rs1695 polymorphism are summarized in For, rs1138272 polymorphism, our data from genotypes distribution showed no deviation from Hardy-Weinberg equilibrium for both case and control groups. The distribution of alleles and genotypes of rs1138272 polymorphism are summarized in In silico analysis GSTP1 function by SNPeffects online software. Our data revealed that based on the TANGO model, the Ile105Val SNP does not influence the aggregation tendency of our studied protein as well as LIMBO analysis, the mentioned SNP does not affect the chaperone binding tendency of GSTP1 protein. But, based on WALTZ output, we found that the Ile105Val polymorphism decreases the amyloid propensity of the GSTP1 enzyme. WALTZ algorithm specifically and accurately predicts amyloid-forming areas in the sequences of protein. The overall WALTZ value for CSTP1 estimated 906.54 and mutations/variations could elevate (dWALTZ >50), reduce (dWALTZ <-50) or not influence amyloid propensity (dWALTZ between -50 and 50). In our analysis, dWALTZ was calculated -228.00 which means that the polymorphism reduces the amyloid propensity of the GSTP1. In We evaluated the effects of Ile105Val single nucleotide polymorphism on the GSTP1 with breast cancer risk. This enzyme is a member of enzymes family that has an essential role in detoxification by catalyzing the conjugation of various electrophilic and hydrophobic compounds with reduced glutathione. Our data revealed that Ile105Val variation as an exonic SNP could alter the risk of breast cancer. In detail, the genotype homozygote GG is associated with the risk of breast cancer. Also, allele analysis revealed a true association of allele G and breast cancer susceptibility. However, we did not find any significant association between rs1138272 polymorphism and breast cancer risk. Some studies were investigating the association of the GSTP1 polymorphisms with breast cancer risk. For example, Samson et al., (2007) reported a non-significant elevation in the risk of breast cancer was observed among women who had the GSTP1 Val/Val genotype . While, Ge et al., (2013) reported a positive association between GSTP1-Ile105Val polymorphism and breast cancer risk . The dissimilar results from different studies may be due to environmental, geographic, race, and other factors.It has been found that allelic variability at a single locus could not explain the etiology of a majority of the common cancers. Besides, a main load of cancer in the general population likely is the result of the complicated interaction of several environmental and genetic parameters during the time. However, perception of the interactions of endogenous physiology, xenobiotic exposure, and genetic variability at numerous loci would provide information about the cancer etiology and detect people at the greater risks of progressing cancer. In this study, we evaluated the association of two common genetic variations  in the GSTP1 is a gene that is related to DNA repair, and keeps DNA from an impairment, controls detoxification and metabolism, so preventing tumor incidence. The GSTP1 gene methylation often shows tumors progression, including breast cancer or unfavorable prognosis . Consistent with the role of methylation of GSTP1 in the tumor’s progression, a study showed significantly increased gene methylation of GSTP1 in breast cancer cells, which was positively associated with tumor size and TNM stage, and negatively associated with the expression of ER/PR . This evidence could elucidate the main role of disrupted GSTP1 in breast susceptibility. This could explain the role of key single nucleotide polymorphisms in the pathogenesis of GSTP1. Genetic variations based on their positions in a gene could alter the gene function . The SNPs on the promoter of a gene may alter the gene expression however the SNPs in the intron regions could alter the production of mature mRNA by interfering with the splicing process . But, the missense mutations could alter the structure and function of proteins  what may be true for GSTP1-Ile105Val genetic variation. Evaluation of the impacts of genetic variations by biological experiments is a very difficult process and evaluation of these impacts could be much easier with the in silico tools . In this study, also we employed the SNPeffects bioinformatics tool to evaluate the molecular effects of Ile105Val SNP on the GSTP1 gene and we found that this polymorphism decreases the amyloid propensity of GSTP1 protein and therefore, the pathogenic effect of Ile105Val may arise from this issue.Notably, the conjugation or addition of the aliphatic-aromatic heterocyclic radicals, epoxide, or arene oxide to glutathione is catalyzed by the mentioned enzymes . Moreover, the conjugation reaction at the electrophilic center of such compositions happens at the sulfur atom of the glutathione molecule . Therefore, the molecules function as the glutathione peroxidase; however, they did not need a selenium co-factor for performing the conjugation reaction . Also, GSTP1, recently, some reviews  illustrated the protein structure, inducibility, enzymology, and level of expression  of GSTTl and GSTM1. They provided a summary of GSTTl or GSTM1 contribution to the metabolisms of and induction by multiple popular or uncertain carcinogenic compounds. The compounds are benzo-(a)pyrene, styrene-7,8-oxide, and trans-stilbene oxide by GSTM1 and epoxybutanes, halomethanes, ethylene oxide, and methyl bromide by GSTT1. According to the above list, we do not observe a single class of chemical compositions, which has been related to the GSTM1 or GSTT induction or metabolism . Finally, it has been found that GSTTl and GSTM1 contribute to the metabolisms of multiple xenobiotics like the chemotherapeutic factors, environmental carcinogens, and reactive oxygen samples . Besides, it seems that GSTM1 distinctly involves the susceptibility to cancer including breast cancer due to its possible distinctive substrate qualities.In addition to GSTP1-Ile105Val polymorphism could be a genetic risk factor for breast cancer. Based on this theory, the mentioned polymorphism could be considered as a biomarker for screening of susceptible women. However, there are some limitations in our study which should be mentioned. At first, we did not evaluate the influence of gene-gene and gene-environmental factors. Also, the small sample size of our study could be considered as the second limitation of our study. Therefore, evaluation of this polymorphism in larger sample size with regard to the aforementioned interactions could result in more accurate outcomes.Our study showed that