Coffee contains caffeine and diterpenes that were associated with decreased breast cancer risk, but results remained inconsistent. The study purpose was to investigate the associations between coffee products and breast cancer risk among Hong Kong Chinese women. We conducted a hospital-based case-control study in three public hospitals. 2169 Chinese women aged 24–84 years old were interviewed using a standardized questionnaire with questions asking types, cups and duration on coffee drinking. We used unconditional multivariate logistic regression to calculate the adjusted odds ratio (AOR) and 95% confidence interval (95% CI) for breast cancer risk with different coffee products. 238 (20.6%) cases and 179 (17.7%) controls are habitual coffee drinkers. No association was found between overall coffee drinking and breast cancer risk. Compared to the non-habitual coffee drinkers, women who consumed instant coffee  were significantly associated with an increased breast cancer risk. Women who drank brewed coffee  were negatively associated with breast cancer risk. A positive association between instant coffee and breast cancer risk was observed, contradicted to the outcomes of drinking brewed coffee. Larger studies are warranted to ascertain the role of different types of coffee products in breast cancer risk. In Hong Kong, coffee consumption is becoming more popular among local Chinese people. Statistics from Hong Kong Statista showed that the revenue of the coffee market increased by more than twice from 119 million USD in 2010 to 279 million USD in 2018, especially for that of the instant coffee2. Coffee consumption was classified as “possibly carcinogenic to humans” in 1991 but it has now been downgraded to “not classifiable as to carcinogenicity” in 1996 by the World Health Organization’s International Agency for Research on Cancer3. The association between coffee consumption and the risk of developing breast cancer has been investigated since the 1970s, but the evidence from epidemiological studies remains controversial9. Additionally, the composition of coffee is different among a variety of coffee products, such as instant coffee and brewed coffee, so these products may contribute differently to breast cancer risk.Globally, breast cancer is the most common cancer in women, and their dietary habits are among the most important factors contributing to the etiology of breast cancer10. A prospective cohort study with 22 years of follow-up in Boston also demonstrated an inverse association of caffeine consumption, and the association was stronger with estrogen-receptor positive and progesterone-receptor positive breast cancer than that with the estrogen-receptor negative and progesterone-receptor negative breast cancer4. This inverse association may be caused by different coffee compounds and chemicals including caffeine, diterpene and polyphenols, which may influence breast cancer mechanisms. Coffee is the major dietary source of caffeine, and previous in vitro studies suggested that caffeine may bind the estrogen hormones through increased levels of sex hormone-binding globulin among post-menopausal women11. Thus, caffeine consumption may lower the levels of circulating sex hormones, thereby reduces the risk of breast cancer.A recent meta-analysis of 13 prospective studies did not show any significant association between coffee consumption and the risk of breast cancer, but an inverse relationship was found among postmenopausal women : 0.82–0.99)2. The toxicity of instant coffee in the Hong Kong market was tested by the Vitargent  Biotechnology Limited who followed the international standards 12. They found that more than 30% of instant coffee samples failed to achieve the standard in one or more of the following criteria regarding the acute toxicity, chronic toxicity and banned ingredients of instant coffee products 12. However, there currently remains a lack of knowledge on the relationship between different coffee products and breast cancer risk in women. It is also important to note that the differences in coffee habits between Asian and Western countries. The highest level of coffee consumption in most of the studies conducted in Western countries was ≥4 cups per day, whereas the highest level in Asian countries was only ≥1 cup(s) per day2, thus the coffee consumption in Western countries and its contribution to breast cancer are not applicable to the Hong Kong population. To address this information, this study aims to investigate the associations between different coffee products (mainly focusing on instant coffee and brewed coffee) and breast cancer risk among Hong Kong Chinese women.Different coffee products may also contain various levels of sugars and food additives, which may affect the rising trend of breast cancer in different ways. A majority of coffee consumption in Hong Kong is from instant coffee, while brewed coffee is the most popular choice in Western countriesOne thousand one hundred and fifty-six newly diagnosed breast cancer cases and 1,013 controls were recruited in this study with a response rate of 88.4% and 89.5%, respectively. As shown in Table The distribution of specific coffee products and the associations with breast cancer is summarized in Table Results of multivariate logistic regressions for the duration of specific coffee products are presented in Table Sensitivity analyses were conducted by further including history of hypertension and diabetes mellitus into the multivariate logistic models. As shown in Supplement We performed further stratified analyses according to menopausal status Table . Among pThis hospital-based case-control study demonstrated the different associations between brewed coffee and instant coffee with breast cancer risk. Women who habitually consumed brewed coffee were associated with 52% lower in the risk of breast cancer, and the inverse association was even predominant among postmenopausal women. In contrast, instant coffee consumption was positively associated with the risk of breast cancer, and a further higher risk was observed among habitual two-in-one or three-in-one coffee drinkers who were pre-menopausal. These findings add additional evidence to the current literature on the association between coffee products and the risk of breast cancer.RAR-Beta’s promoter and methylation13. Another mechanism of this negative association between brewed coffee and breast cancer risk may be related to a high level of caffeine intake which was evidently to be associated with a lower level of circulating estrogen14. A high level of circulating estrogen was found to be associated with a significantly increased risk of breast cancer15. Caffeine consumption may reduce total luteal cells and free estradiol level in premenopausal women14 but increase the levels of sex hormone-binding globulin in postmenopausal women11. In postmenopausal women, the ovaries were not the major source of estrogen, as the estradiol may be produced in a number of extragonadal sites16. Sex hormone-binding globulin helps to bind the estradiol and testosterone from number of extragonadal sites which decreases the levels of bioavailable estradiol and testosterone11, and the levels of sex hormone-binding globulin were associated with decreased risk of breast cancer among postmenopausal women17. Previous epidemiological study also observed an association of caffeine with higher levels of sex hormone-binding globulin and low levels of free testosterone14, which explains an inverse association between coffee intake and breast cancer risk through the possible pathway of estrogen among postmenopausal11. By contrast, the principal source of estrogen is produced in ovaries among premenopausal women whose estrogen levels are relatively higher than the postmenopausal women16, which explains the reduction of estrogen levels induced by the intake of coffee may not affect much on the risk of breast cancer for the premenopausal women. As a result, brewed coffee consumption may decrease the risk of breast cancer via reducing the levels of estrogen among postmenopausal women but not significant in premenopausal women. Consistent with some of the previous studies10, our study also demonstrated a significantly negative association between brewed coffee consumption habits and breast cancer risk in the postmenopausal women. However, one recent meta-analysis study reported a similarly pooled negative association of caffeinated and decaffeinated coffee with breast cancer risk5. These negative associations from the meta-analysis may suggest other potential coffee protective substances in coffee or the potential protective effects, which only affects post-menopausal women.We found that the duration and number of cups of brewed coffee consumption were negatively associated with the risk of breast cancer among Hong Kong Chinese women. Women who drank more than one cup of brewed coffee per day and drank for more than ten years were inversely associated with 60% breast cancer risk, respectively. One possible mechanism of this association may be related to the rich polyphenols  in coffee, which may inhibit the breast cancer related gene 18. Higher levels of two specific coffee diterpenes, cafestol and kahweol are more evident in boiled coffee than in filtered coffee18. These two diterpenes produced the biochemical effects such as induction of conjugating enzymes and increased protein expression in cellular antioxidant defense, which may result in a reduction of the genotoxicity of several carcinogens, such as 7,12-Dimethylbenz(a)anthracene, benzopyrene and 2-amino-1-methyl-6-phenylimidazopyridine20. The anti-carcinogenic activity of cafestrol and kahweol reduces the expression and inhibits enzymatic activity of phase I enzyme for carcinogen activation, and induces phase II enzymes in carcinogen detoxification19. Kahweol increased the production of reactive oxygen species and their cytotoxicity in breast cancer cells, which enhances apoptosis of breast tumor cells20. One prospective cohort study conducted in Vasterbotten reports a significantly decreased risk of breast cancer among women who habitually drink boiled coffee, but a weaker positive association was suggested in women with habitual filtered coffee drinking7. Among our participants who have habitual restaurant coffee drinking habits, approximately 90% of them drank their coffee in Hong Kong-style diners (cha chaan teng) or Hong Kong-style fast food restaurants. Such restaurants serve boiled coffee, which may be a plausible explanation of the stronger association with breast cancer observed in restaurant coffee than that of the homemade coffee.Another putative mechanism for the inverse association of brewed coffee consumption and breast cancer risk was the exposure of diterpene. The levels of diterpene vary among different brewing methods21. In addition, habitual instant coffee drinkers tend to have lower serum levels of high density lipoprotein (HDL) cholesterol22. One population-based study in Troms reported that low serum HDL cholesterol was associated with increased levels of estradiol concentration23, which may further increase the risk of breast cancer among the instant coffee drinkers.Intriguingly, there is a positive association of instant coffee consumption with the risk of breast cancer, and a clear exposure– response relationship of cups of instant coffee was revealed with breast cancer risk. These instant coffees contain less coffee but more additives, such as non-diary creamer, sugar and stabilizers to maintain the coffee texture, taste and smell. However, these additives may also contain hydrogenated fats and/or trans-saturated fats that may increase breast cancer risk24, indicating a good comparability between our cases and the general population. Controls recruited from the hospitals may not well represent the general population; however, we recruited controls with a variety of disease types from the same hospitals that were not related to any type of breast diseases. Previous hospital-based case-control studies provide evidence that a similar magnitude of risk was obtained between using the general population controls and hospital controls with a variety of disease types25. Thus, the potential selection bias, if presents in this study, should not be a major issue.To the best of our knowledge, this is the first study reporting the association between different coffee products (i.e. instant coffee and brewed coffee) and breast cancer risk in Hong Kong Chinese women. Information on coffee consumption habits were amassed, such as the type of coffee products, the number of cups and years consumed. Results of our study propose that instant coffee may be a novel breast cancer risk factor. We recruited our participants from three large public hospitals, covering around 8.5% of those who had breast cancer in Hong Kong’s population, with a high response rate (88.4%). We further re-interviewed 158 cases and 153 controls at least one month after the initial recruitment and showed good overall test-retest reliability for tea drinking (Kappa consistency rate of 83%). However, the major limitations of our study do exist. We did not collect the information regarding the preparation method of coffee , in which the content of compounds and chemicals including caffeine may vary. Misclassification of the concentration of caffeine and other components in the different coffee product may also be a concern, as we do not have direct measurements of each content. Furthermore, the issue of chance cannot be entirely excluded in subgroup analysis for the associations between different coffee products and breast cancer, since the 95% CI was relatively broad. Despite the mechanism for instant coffee on the risk of breast cancer is not yet clear and the significant results of these associations could be due by chance, the consistently positive dose-response relationships between daily amount of consumption or duration of instant coffee drinking with breast cancer risk increase the plausibility of our findings. Generally, the concern of the recall and/or interviewer bias cannot be eliminated in case-control studies, and thus we used a standardized approach to interview all participants and interviewed the controls within 6 month after the cases were recruited, which in turn reduced the potential recall and interview bias. In addition, we compared the findings between cases recruited from Department of Survey (cases were interviewed before biopsy) with those from Department of Clinical Oncology (the diagnosis of breast cancer had been known), and they were similar which further supports that the potential recall and/or interveiw bias of our study should not be a main concern. Selection bias of breast cases may be a concern as all cases of our study came from three hospitals. We found a similar proportion of incident breast cancer in different age groups to those obtained from the Hong Kong Cancer Registry from 2011 to 2016 (i.e. the last updated year)In summary, this study revealed that breast cancer among Chinese women in Hong Kong was inversely associated with the consumption of brewed coffee but positively related to the intake of instant coffee. Nevertheless, it should be noted that breast cancer cases in this study showed a low prevalence of habitual coffee consumption. Future larger analytic studies with different population subsets and types of coffee products are warranted.26. We conducted a hospital-based case-control study in three Hong Kong public hospitals from November 2011 to January 2018. Trained interviewers conducted a face-to-face interviews with a standardized questionnaire and obtained a written informed consent form. Eligible cases were Chinese women aged 24–84 years-old who were newly diagnosed as having primary breast cancer  within three months prior to the interview. We matched each case with one control by 5-year age group who did not have any conditions pertaining to breast cancer, and interviewed them within 6 month after the cases were recruited. We recruited our controls with a broad spectrum of diagnoses from the Department of Medicine or Surgery from the same hospital of the cases came from. We excluded cases and controls with prior history of physician-diagnosed cancer in any site. This study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committees and the Kowloon West Cluster, and was strictly compliance with local law and the Declaration of Helsinki.Details of the study design and subject characteristics have been described elsewhereA standardized questionnaire gathered information on socio-demographic characteristics, tobacco smoking, alcohol drinking, dietary habits such as tea and coffee consumption patterns, hormone-related factors including menopausal status, anthropometric risk factors, history of chronic diseases including breast diseases, family cancer history, physical activity, and occupational history including shift work.We asked each participant to report their coffee consumption patterns. Participants who drank any type of coffee product at least once a week over 5 years prior to our recruitment time frame were identified as habitual coffee drinkers, whereas those who did not have such a habit were defined as non-habitual coffee drinkers. We further requested the habitual coffee drinkers to provide information on the coffee product that they consumed , number of cups (defined as 250 ml per time) and years of coffee consumption. The category of ‘instant coffee’ refers to the coffee that is soluble and ready to drink. For instance, three-in-one instant coffee is the mixture of soluble coffee, non-dairy creamer and sugar. Brewed coffee refers to the coffee that requires different brewing processes before consumption, such as being boiled and filtered. For the participants who have switched their habitual consumption type of coffee , the recent type of coffee comsumption were defined as their habitual consumption types; if the participants’ habits of recent type of coffee comsuption were less than 5 years, they were then defined as the previous type coffee drinkers.Independent t-tests and chi-square test were performed to compare the differences between cases and controls for the continuous and categorical variables, respectively. We estimated the adjusted odds ratios (AOR) and 95% confidence interval (95% CI) for the associations between different coffee products and breast cancer with an unconditional multivariate logistic regression model. The reference group was defined as the participants who did not have a habitual coffee consumption . We adjusted for potential confounders including the participant’s age, educational attainment, family breast cancer history, previous history of benign breast diseases, menopausal status, body mass index (BMI), shift work, smoking habits, alcohol and tea drinking consumption, and deep fried food and green vegetable consumption. Further stratified analyses were conducted according to their menopausal status. Postmenopausal status was determined if the participants reported their menstrual periods stopped more than a year; otherwise, pre-menupausal status was defined. All statistical analyses were conducted with SPSS 20.0 for Windows , and two-sided p-value less than 0.05 was considered statistically significant.Distribution of specific coffee product among habitual coffee consumption and its association with breast cancer risk


By identifying pregnancy-related risk factors for endometrial neoplasia, women’s risk of developing this disease after childbirth can be predicted and high-risk women can be screened for early detection.Study data from women who gave birth in Korea in 2007 were collected from the Korea National Health Insurance (KNHI) claims database between 2007 and 2015. The adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for the development of endometrial neoplasia were estimated by multivariate Cox proportional hazards models.Data from 386,614 women were collected for this study. By 2015, 3,370 women from the initial cohort had been diagnosed with endometrial neoplasia secondary to delivery. Multivariate Cox proportional hazards regression revealed that preeclampsia , advanced maternal age , multifetal pregnancy , multiparity , cesarean section  and delivery of a large-for-gestational-age infant  were independent risk factors for future endometrial neoplasia. The risk for endometrial neoplasia increased as the number of risk factors increased .This study showed that six pregnancy-related factors—advanced maternal age, multiparity, multifetal pregnancy, cesarean section, delivery of a large-for-gestational-age infant, and preeclampsia—are positively correlated with future development of endometrial neoplasia, including endometrial hyperplasia or cancer. Close observation and surveillance are warranted to enable early diagnosis of endometrial diseases, including endometrial cancer after pregnancy in high-risk women. However, due to unavailability of clinical information, many clinical/epidemiological factors can become confounders. Further research is needed on factors associated with the risk of endometrial neoplasia. Pregnancy can affect the endometrium in a variety of ways. During pregnancy, the size and number of glands and blood vessels in the endometrium increase significantly. The vascular space is fused and interconnected to form the placenta, which supplies oxygen and nutrients to the embryo and fetus . PreviouEndometrial cancer is the most common cancer of the female genital tract worldwide , 11. RisSince 2000, the various health insurance systems in South Korea have been merged into a single system run by the National Health Insurance Service (NHIS). Consequently, most people living in South Korea are currently insured by the NHIS. Our study data were collected from the Korea National Health Insurance (KNHI) claims database from 2007–2015. In Korea, 97% of the population is obligated to enroll in the KNHI program; the remaining 3% are under the Medical Aid program. Therefore, the KNHI claims database contains information on all claims for approximately 50 million Koreans; nearly all information about disease incidence can be obtained from this centralized database, with the exception of procedures not covered by insurance, such as cosmetic surgery.A flowchart of patient enrollment is shown in Women diagnosed with endometrial neoplasia after their delivery were identified according to principal or secondary diagnosis by searching for relevant ICD-10 codes. Women were classified as having endometrial neoplasia if they were newly diagnosed with endometrial neoplasia  from delivery to December 31, 2015. The timing of each patient’s initial diagnosis was confirmed by the lack of a diagnosis for endometrial neoplasia before pregnancy.Pregnancy characteristics such as maternal age, primiparity, multifetal pregnancy, C/S, preeclampsia, postpartum hemorrhage, placental abruption, placenta previa, and uterine arterial embolization as identified by ICD-10 codes were evaluated using the KNHI claims database. Information about neonatal outcomes, specifically preterm birth, neonatal birthweight and gender, were analyzed using data from NHSP-IC. Preterm birth was defined as gestational age <37 weeks. Low birth weight (LBW) was defined as birth weight <2,500 g, and LGA as birth weight >4,000 g.t-test for continuous variables and the χ2 test for categorical variables. Cox proportional hazards models were used to estimate the adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for the development of endometrial neoplasia. All tests were two-tailed, and p-values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS version 18 .Continuous and categorical variables were expressed as mean ± SD and percentages, respectively. Clinical characteristics were compared using the Data from 386,614 women who gave birth in Korea from January 1, 2007 to December 31, 2007 were collected in this study. By 2015, 3,370 of these women had been diagnosed with endometrial neoplasia. In women with endometrial neoplasia, advanced maternal age, multiparity, multifetal pregnancy, C/S, preterm birth, neonatal birth weight, LBW, LGA, preeclampsia, postpartum hemorrhage, and placental abruption were more common than in women without endometrial diseases. The median age at birth was 30.75 years in patients with endometrial neoplasia and 29.99 years in patients without endometrial neoplasia. The proportion of women who developed endometrial neoplasia was two-fold greater in women who had multifetal pregnancies than in women without multifetal pregnancies. The proportions of preterm birth, low birth weight, and preeclampsia were more than 1.5 times greater in women who developed endometrial tumors after childbirth. However, no statistically significant difference was found between the two groups with respect to neonatal sex, placenta previa, and uterine artery embolization.This study showed that six pregnancy-related factors—advanced maternal age, multiparity, multifetal pregnancy, C/S, LGA, and preeclampsia—are positively correlated with future development of endometrial neoplasia, including endometrial hyperplasia or cancer.The most interesting finding in this study is that a history of preeclampsia is an important risk factor for endometrial neoplasia. Several studies have reported that the risk of breast cancer is reduced in women who have had preeclampsia , 17. HowThe inconsistent associations between preeclampsia and endometrial neoplasia reported in previous studies may be attributed to the different risk factors of two disease entities. The risk factors are different depending on ethnicity or race, suggesting that a history of preeclampsia might enhance the incidence of endometrial neoplasia in certain populations . GeneticThe pathophysiology of preeclampsia has not been clearly elucidated, but preeclampsia is thought to begin in the placenta and may potentially have long-term impact on the endometrium. Preeclampsia may arise due to an imbalance of angiogenic and antiangiogenic factors , which may affect endometrial vascular development or function during pregnancy. Most previous studies have focused on the antiangiogenic properties of sFlt-1 to treat cancer, while sFlt-1 was detected in colorectal and breast cancer tissue in some studies , 22. GivIn this study, advanced maternal age at birth was a risk factor for endometrial neoplasia. Many previous studies have shown that the risk of endometrial cancer decreases with increasing maternal age at delivery, while some studies have shown that the risk of endometrial cancer increases with increasing maternal age at delivery . The conIn this study, the incidence of endometrial neoplasia was found to increase in women who had multifetal pregnancies. This finding is similar to the results of a Swedish cohort study in which multiple births were associated with an increased risk of endometrial cancer  . The mecIn this study, C/S was one of the risk factors for endometrial neoplasia. The association of C/S with endometrial cancer has rarely been studied. However, neo-angiogenesis with VEGF expression occurs within the cesarean section scar site of the uterus, which is thought to act as an endometrial cancer implant . Other sEndometrial neoplasia was also increased in multiparous women in this study. These results are in contrast to those of many prior studies which reported having given birth to be a protective factor for endometrial cancer, and that the risk of endometrial cancer steadily decreases with increasing parity , 13, 41.It is possible that confounders such as obesity, oral contraceptive use, low SES and low maternal health status affected the outcomes in our study. Previous studies found positive associations between parity and obesity (38–40). In addition, it has been suggested that nulliparous women are more likely to take oral contraceptives that may have protective effects against endometrial cancer, and the tendency toward low SES, physical activity, and low maternal health status in obese patients may have affected the outcomes reported in some previous studies –46.Similar to multifetal pregnancies, LGA also increased the risk of developing endometrial neoplasia. Placental weight is known to be lower with underweight babies and higher with overweight and LGA babies , 48. PlaIn this study, six risk factors were associated with endometrial neoplasia, and the greater the number of risk factors reported, the greater the risk. Patients with three or more risk factors were twice as likely to have an endometrial neoplasia. Although the risk factors identified in this study cannot be determined to have been the cause of the endometrial neoplasia reported in our study population, including endometrial hyperplasia and cancer, there was an increased risk of endometrial neoplasia in the women with these factors. The results of this study suggest evaluation of the risk of endometrial neoplasia immediately after delivery and close follow-up of high-risk patients may be helpful in early detection of endometrial cancer. In particular, women with three or more risk factors are at greater risk of developing endometrial neoplasia. Therefore, in such women, annual transvaginal ultrasonography and endometrial biopsy of suspicious lesions may be helpful in the prevention of endometrial cancer. In addition, efforts to reduce modifiable risk factors, such as C/S rate and multifetal pregnancies, through the use of ART may reduce the incidence of subsequent endometrial cancers. However, additional studies are needed to determine whether close follow-up and reduction of modifiable risk factors can effectively prevent endometrial neoplasia.The study has several limitations. First, although six pregnancy related factors have been shown to increase the risk of endometrial neoplasia, none of them  have a statistically significant association with the risk of endometrial cancer. In this cohort, the sample size of cancer patients may not have been sufficient to show statistical significance, because the number of cancer patients among the 3,340 patients with endometrial neoplasia was only 75 (2.5%). Although six pregnancy-related factors do not increase the risk of cancer, atypical hyperplasia or endometrial carcinoma in situ can be considered precancerous lesions with a high malignancy potential. The 2014 World Health Organization (WHO) classified endometrial hyperplasia into two categories; hyperplasia without atypia, and atypical hyperplasia/endometrial intraepithelial neoplasia –55. AddiSecond, the study can be criticized for lack of clinical information in the HIRA database. Potential confounding variables including obesity, diabetes, hypertension, medication, and SES could not be controlled for due to unavailability of clinical information. However, the risk of cancer increased as the number of risk factors increased, although potential confounding variables might have affected the outcome.Nonetheless, this study had several advantages. First, it is a large-scale study with a 10-year follow-up period. Second, it is the first study to examine the relationships between pregnancy-related risk factors and endometrial neoplasia. Finally, there was no loss to follow-up due to the nature of the data.In this study, we confirmed the relationships between six pregnancy-related factors—advanced maternal age, multi-parity, multifetal pregnancy, cesarean section, delivery of a large-for-gestational-age infant and preeclampsia—and the occurrence of endometrial neoplasia  after pregnancy. Our results indicate close observation and surveillance may assist with the prevention and early diagnosis of endometrial neoplasia after pregnancy in high-risk women. However, there was no information on the many clinical factors such as BMI that could affect the outcome, and the pregnancy-related factors associated with endometrial cancer risk were not identified. Further studies on the relationship between pregnancy and future development of endometrial cancer will be needed.


African American women are substantially underrepresented in breast cancer genetic research studies and clinical trials, yet they are more likely to die from breast cancer. Lack of trust in the medical community is a major barrier preventing the successful recruitment of African Americans into research studies. When considering the city of Memphis, TN, where the percentage of African Americans is significantly higher than the national average and it has a high rate of breast cancer mortality inequities among African American women, we evaluated the feasibility of utilizing a community-based participatory (CBPR) approach for recruiting African American women into a breast cancer genetic study, called the Sistas Taking A Stand for Breast Cancer Research (STAR) study. From June 2016 and December 2017, African American women age 18 and above were recruited to provide a 2 mL saliva specimen and complete a health questionnaire. A total of 364 African American women provided a saliva sample and completed the health questionnaire. Greater than 85% agreed to be contacted for future studies. Educational workshops on the importance of participating in cancer genetic research studies, followed by question and answer sessions, were most successful in recruitment. Overall, the participants expressed a strong interest and a willingness to participate in the STAR study. Our findings highlight the importance of implementing a CBPR approach that provides an educational component detailing the importance of participating in cancer genetic research studies and that includes prominent community advocates to build trust within the community. Breast cancer is a major health concern in the United States. This year alone, more than 260,000 women will be diagnosed with breast cancer in the United States (US) and more than 40,000 of those women will die from the disease . AlthougMultiple risk factors across the breast cancer spectrum have been identified to explain the racial disparity in breast cancer mortality, including demographic and biological factors ,4,5,6. FDespite an increase in the number of genetic research studies examining the biological determinants of breast cancer mortality disparities in African American women ,11,12,13The city of Memphis, TN, where the percentage of African Americans is significantly higher than the national average, has a significantly high rate of breast cancer mortality disparities among African American women ,29,30. IIn order to better understand the biological determinants of breast cancer mortality inequities among African American women in Memphis, we hypothesized that establishing partnerships with prominent community breast cancer advocates and educating the Memphis community on the importance of participating in our cancer genetic research study would promote the successful recruitment and retainment of African American women in our STAR study. The objective of this study was to evaluate the feasibility of utilizing a CBPR approach for recruiting and retaining African American women in Memphis in the STAR study. In this paper, we describe how we utilized a CBPR approach to recruit African American women for our STAR breast cancer genetics research study.The STAR study is a community-based participatory genetic research study that was initiated in the autumn of 2016. The primary objective of the STAR study is to educate, recruit, and retain African American women participants throughout the Memphis community in our cancer genetic research study. Efforts to recruit participants in the STAR study involved a group of racially diverse team members who had a strong presence in the Memphis community. Team members were identified through word of mouth, interviews on local news coverage, and through interactions at the monthly Memphis Breast Cancer Consortium-Common Table Health Alliance meetings. Specifically, team members included academic professionals at the University of Tennessee Health Science Center and Southwest Tennessee Community College with cancer research experience, breast oncologists, and a research oncology nurse at the West Cancer Center, and a prominent leader and founder of a local breast cancer support group who was instrumental in providing suggestions and comments on the study logo, culturally sensitive educational materials, and strategies to recruit African American women in the STAR study. The research team also included non-paid undergraduate and graduate student volunteers. All team members completed study specific training that addressed consent procedures, saliva sample collection, quality control procedures, confidentiality, and data security protection. All team members also completed human subjects’ protection training that is required by the University of Tennessee Health Science Center Institutional Review Board. Written informed consent was obtained from all participants before they participated in the STAR study. Team members did not participate in the STAR study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of the University of Tennessee Health Science Center, Memphis, TN. IRB protocol 16-04551-XP was approved to recruit and consent volunteers at community-based outreach events, and IRB protocol 16-04502-XP was used to recruit and consent breast cancer patients that were being treated at the West Cancer Center.Women who self-reported as being African American aged 18 years or older and who were able to: (1) provide written informed consent, (2) complete a three-page health questionnaire, and (3) provide a 2-mL saliva specimen were eligible to enroll in the STAR study.The study and consent process was explained to the volunteer prior to enrollment. STAR study staff explained to volunteers that they would not be provided with personalized research results. Women who indicated an interest in participating were further asked to read and sign the IRB approved written consent form. The consent form also contained a brief introduction and basic description of breast cancer genetic research. During the consent process, the volunteer was also given the opportunity to indicate their willingness to follow up contact within two years to be informed of research opportunities and to ask about any new developments in their health. Participants who agreed to follow up contact provided their name, mailing address, e-mail address, and/or phone number on their consent form. Participants were not excluded from the study if they were not willing to be contacted in the future.After the participants provided written consent, participants were provided with a confidential three-page self-report questionnaire. The questionnaire consisted of demographics, which included information on participants’ reproductive history, diet and lifestyle factors, family history of cancer, and history of genetic testing.®•OG-500 DNA Self-Collection Kit  and was stored in a locked cabinet at room temperature. The average time for participants to obtain written informed consent, complete the health questionnaire, and collect a saliva specimen was approximately 8–10 min.After participants provided written consent and completed the health questionnaire, participants were asked to provide a 2-mL saliva sample. The 2 mL saliva sample was collected using OrageneEstablishment of collaborations and partnerships with key leaders who have a strong presence in the Memphis community was critical to engage volunteers and educate them about the importance of participating in the STAR study. Volunteers were recruited at several venues and events in Memphis, TN between June 2016 and December 2017. The venues and events included the West Cancer Center, Sista Strut 3K Breast Cancer Walk annual event, Benjamin L. Hooks Central Library, and ‘Carin and Sharin’ breast cancer support group. Participants at the Breast Cancer Walk received a bottle of water with the STAR logo on it soon after providing a saliva specimen and completing the health questionnaire. The recruitment and consenting of breast cancer patients who were being treated at the West Cancer Center was conducted in close collaboration with breast oncologists and a research nurse at the West Cancer Center in Memphis, TN.Workshops to engage and educate volunteers on the importance of participating in cancer genetic research were presented at the Live! Memphis Annual Breast Cancer Summit, Southwest Tennessee Community College, and the monthly Carin and Sharin breast cancer support group meeting. The Carin and Sharin breast cancer support group, led by Mrs. Gwendolyn Brown, is an organization that was developed to address the breast cancer mortality disparities among socio-economically disadvantaged, inner city, African American women in Memphis, TN ,30. CultBy contrast, due to the nature of the event, educational workshops were not provided to volunteers at the Sista Strut 3K Breast Cancer Walk or patients being treated for breast cancer at the West Cancer Center.p-values were two-sided and considered significant at the alpha 0.05 level. All statistical analyses were performed using GraphPad Prism, version 7 software .A total of 364 African American women participated in the STAR study by providing a saliva sample and completing the health questionnaire. Volunteers were recruited and consented at several venues and events in Memphis, TN between June 2016 and December 2017. Records of participants’ consent forms that provided the option to be contacted after two years were systematically collected by the study PI and were used to calculate the follow-up recruitment rates. Demographic characteristics and selected risk factors for breast cancer were compared between cases and controls using T-tests for continuous variables and Chi-square tests for categorical data. Variables were dichotomized in which women who reported any alcohol use or tobacco use were assigned a value of “1” and women who did not report consuming alcohol, were assigned a “0”. Data were expressed as the mean ± standard deviation. All Our initial goal was to recruit a total of 250 African American women for the STAR study between June 2016 and December 2017. We exceeded our goal by recruiting a total of 364 African American women who provided both a saliva sample and completed the health questionnaire. The total number of participants consisted of 94 breast cancer cases, including breast cancer survivors, and 270 healthy women controls . Women wp < 0.0001).A table summarizing the demographic characteristics of the STAR study participants is shown in We also assessed whether behaviors and lifestyle factors that are known to increase the risk of breast cancer development, specifically obesity, weekly alcohol consumption, and tobacco use ever were higher in women with breast cancer as compared to controls. To determine the relationship between obesity, alcohol consumption, or tobacco use and breast cancer status, we included the following questions in the health questionnaire: “How tall are you?”, What is your weight?”, “Do you smoke cigarettes or vapor?”, “How often do you smoke cigarettes or vapor?”, and “How many alcoholic drinks  do you currently drink weekly?” Interestingly, the percentage of women who consumed alcohol was significantly higher among controls versus breast cancer cases. Although obesity was a leading comorbidity among all participants, regardless of disease state, obesity status was not significantly different between cases and controls. Furthermore, less than 10% of cases and 25% of controls used tobacco. There was no significant difference between cases and controls for menopausal status and family history of cancer.Between June 2016 and December 2017, we consented 364 study participants at a total of six community outreach Memphis events and venues. Our most successful recruitment events were those where we provided both educational resources and workshops on the importance of participating in cancer genetics research . Specifin = 80) consented to follow up contact in two years. Similarly, 89% of healthy (control) participants (n = 241) agreed to follow up contact. Among breast cancer cases, 90% of participants who attended the educational workshops provided written consent to be follow up contacted. Similarly, close to 90% of cases and controls who participated at the Sista Strut 5K Walk or were treated for breast cancer at the West Cancer Center (clinic) provided written consent to follow up contact.More than 88% of STAR study participants provided written consent and contact information  to be contacted in two years by STAR study support staff to obtain any new information about their health and new research opportunities . ApproxiAlthough participants understood that they would not receive any research results, most participants expressed a strong interest and a willingness to participate in the STAR study, as previously described . Most paIn this study, we evaluated the feasibility of utilizing a CBPR approach for recruiting and obtaining a saliva sample from African American women in Memphis for our breast cancer genetic research study: the STAR study. We exceeded our target goal of 250 participants during the recruitment period by recruiting 364 participants who provided a saliva specimen and completed a health questionnaire. Approximately 90% of the women in our study agreed to follow up contact to obtain information about their health.It is well documented that a lack of trust in the medical community is a major reason why African Americans decline to participate in research studies ,34,35,36Two major factors that contributed to the successful recruitment of African American women in the STAR study included: (1) the development of partnerships with prominent breast cancer advocates who had a strong presence in the Memphis community and (2) incorporation of culturally appropriate educational workshops on the importance of participating in breast cancer genetic research studies. Approaches that use a CBPR approach have yielded similar results to our research findings ,43,44. IOur study has limitations. We were unable to determine how many women had as an initial interest in participating in our study due to limited study staff. Therefore, we were unable to calculate the recruitment rates. This was especially evident at the Sista Strut event that had over 5000 women in attendance. However, we were able to determine the percentage of participants who agreed to follow up contact. Additionally, our CBPR approach was limited to African American women who attended our recruitment events throughout the Memphis community. However, based on previous studies, it is plausible that our CBPR approach would yield similar recruitment results to other populations and underserved communities that may be difficult to recruit from.A major advantage of our study is the population under study. African American women with breast cancer in Memphis, TN suffer disproportionately from higher breast cancer mortality rates when compared to their non-Hispanic white counterparts ,29,30. ICBPR efforts to enhance participation in cancer genetics research have the potential to achieve breast cancer health equity. CBPR efforts can improve health outcomes in breast cancer screening, incidence, mortality, survivorship, and treatment of breast cancer ,52. The In conclusion, our findings highlight the importance of implementing a CBPR approach that includes key community leadership needed to build trust with the local community and that provides educational resources on the importance of participating in cancer genetic research studies. Establishment of collaborations and partnerships with breast cancer advocates and key leaders in the Memphis community was essential for engaging, educating, and successfully recruiting participants for the STAR study. Workshops that educate African American women about strategies to prevent breast cancer development, combined with increasing their knowledge of the importance of participating in breast cancer research studies, was most successful for recruiting African American women in the STAR study. Overall, African American women in Memphis, TN are willing and wanting to participate in cancer genetics research studies. When considering the long history of racial inequality and discrimination that has marred Memphis for many years, future research efforts that incorporate culturally sensitive educational tools and interventions that will engage and support the African American community should also be considered when recruiting for genetic research studies.


Cancer is characterized by genetic and molecular aberrations whose number and complexity increase dramatically as cells progress along the spectrum of carcinogenesis. The pharmacologic application of agents in the context of a lower burden of dysregulated cellular processes constitutes an efficient strategy to enhance therapeutic efficacy, and underlies the rationale for using cancer prevention agents in high-risk populations. A longstanding barrier to implementing this strategy is that the risk in the general population is low for any given cancer, many people would have to be treated in order to benefit a few. Therefore, identifying and treating high-risk individuals will improve the risk: benefit ratio. Currently, risk is defined by considering a relatively low number of factors. A strategy that considers multiple factors has the ability to define a much-higher-risk cohort than the general population. This article will review the rationale for evaluating multiple risk factors so as to identify individuals at highest risk. It will use breast and lung cancer as examples, will describe currently available risk assessment tools, and will discuss ongoing efforts to expand the impact of this approach. The high potential of this strategy to provide a way forward for developing cancer prevention therapy will be highlighted. Cancer is a disease that develops through a multistep process of carcinogenesis. In the case of advanced cancer, for example metastatic breast cancer, its presence can easily be detected in a given person through widely available technology. However, at this advanced stage, there exists a high number of aberrant genetic, molecular and functional defects that are present within a given individual’s cancer. This underlies the fact that in this situation it is not currently possible to inhibit eventual disease progression and resultant death, even with the application of multiple therapies. In contrast, early in the process of carcinogenesis, the number of aberrant defects that exist within cells is far fewer, and such at risk cells in fact appear morphologically normal. As such, they constitute early events that increase risk for developing cancer in the future, but exist within individuals who do not yet have any clinical evidence of cancer. From a pharmacologic standpoint, such a situation allows one to apply therapy in the context of a lower burden of targets that need to be modulated in order to achieve a given therapeutic outcome. These factors constitute a core rationale for preventing the development of clinical cancer by treating at risk individuals with cancer prevention drugs. An example is the use of single agent tamoxifen, a selective estrogen receptor modulator (SERM), that is able to decrease the chance of a women of ever developing breast cancer by fifty percent among high risk women , 2. ThisHowever, several practical barriers impede the attainment of this potential. They include the need to treat many people for long periods of time in order to benefit only a few. Further, there is low tolerance for toxicity, and with any drug, toxicity increases with length of administration. In the case of breast cancer prevention, only 20% of eligible women opt for SERM therapy because of side effects, real or perceived .A logical strategy to begin addressing these barriers involves identifying individuals at higher risk. This creates a more favorable risk: benefit ratio for therapeutic intervention. Further, it decreases the time it takes to assess agent efficacy. Historically, approaches to defining risk have relied upon very few factors, sometimes only age and gender. However, multiple factors have been associated with a given cancer. A strategy that takes into consideration multiple factors therefore has the potential to define risk-enriched cohorts.Genetic factors account for a portion of individuals’ risk, while demographic and environmental factors substantially contribute. Simultaneously incorporating a combination of genetic factors, personal factors and environmental factors has the potential to more precisely characterize an individuals’ risk, compared to existing approaches. In addition, and importantly, there is a rapidly evolving set of biosensors that provide readouts reflective of factors that either provide measures of early carcinogenic changes, or drivers thereof, thereby having the potential to further refine risk. They are largely driven by emerging technologies that can be applied at the population level and are financially sustainable. Incorporation of all these factors has the ability to identify those whose risk is far greater than that of the general population. The act of targeting screening, counseling and intervention strategies, inclusive of therapeutic, to individuals at the highest risk has the potential to optimally benefit individual outcomes. Further, the diminished clinical readout timeframe will improve the efficiency of testing interventions, inclusive of therapeutic, thereby advancing the field by increasing efficiency.This review will discuss cancer risk assessment models that take into account multiple factors, will discuss their design, will focus on personalized risk assessment, will focus on lung and breast cancer as examples and will discuss recent advances and future directions. Breast and lung cancer represent the world’s more common types of newly diagnosed cancer , and forhttps://www.bcsc-research.org/). The general statistical workflow is illustrated in P < 0.0001 for inclusion of covariates, in order to eliminate the concern that small effects can be statistically significant at P < 0.05 due to the very large sample size . Appropriate data sets need to be available which contain data on the primary endpoint, known and potential risk factors. Because the cancer incidence is rare in the general population, a data set required for development of a risk prediction model is usually very large . In a typical setting, data from a cohort study are used, which contains information on baseline risk factors, outcome (diagnosis of cancer), vital status and follow-up time. In order to evaluate the model performance, data are randomly divided into two sets: a training set and a validation set. The training set is used to develop a model, while the validation set is used to evaluate the model’s performance. In general, the model is developed using a multivariable regression model such as logistic regression for a binary outcome or Cox proportional hazard regression for a censored outcome. The standard variable selection methods  can be used to develop a parsimonious model. In particular, LASSO (least absolute shrinkage and selection operator) is popularly used as a variable selection procedure for high dimensional data such as genome-wide association studies (GWAS) to identify a subset of single nucleotide polymorphisms (SNPs) that are predictive of the cancer risk. The goodness-of-fit of the continuous model was often tested using Hosmer-Lemeshow method. The estimated regression coefficients are then used to generate a predicted risk or probability of the outcome for each subject. More recently machine learning algorithms are used to develop a prediction model, such as random forest and neural network , especiaA major limitation for development of a risk prediction model is the availability of data, particularly information related to risk factors. In many cancer types, large cohort study data are unavailable. Instead, case-control study data with the appropriate outcome and risk factor information may be available and can be used to develop a prediction model. The Gail model  addresseOf highest importance to consider in the context of prevention therapy, one should also note that different risk profiles may give rise to the same probability of cancer incidence, but the biological underpinnings that underlie the different risk factors may in fact be different. Further, this difference in biology may inform a difference in prevention therapy strategy. For example, a 50-year-old woman with a family history of breast cancer and no other risk factor may have a similar probability of a breast cancer as a 50-year-old woman with an extremely dense breast but without a family history of breast cancer. The predicted risk or probability itself may not inform an optimum intervention strategy for each individual; a thorough risk profile and understanding of the potential underlying biological mechanism may be required to develop a personalized prevention therapy strategy.We increasingly need to consider information available from assay methodologies that serve as biosensors, which in turn stem from our rapidly evolving understanding of cancer biology, rather than from evaluation of a population-based database, to be potentially incorporated into a risk prediction model. For initial investigation, a prevention clinical trial study design is a commonly used method. In general, a prevention therapy trial requires a large sample size and longer trial duration because of the very low event rate. For example, the largest prevention trial among women-the Women’s Health Initiative, enrolled 161,808 women and were followed for over 25 years , 10. NowIn 2019, there will be an estimated 268,600 new cases and 41,760 deaths attributed to female breast cancer in the United States , making In the past two decades, several breast cancer risk prediction models have been developed to evaluate a woman’s potential risk for this disease. In addition to the National Cancer Institute (NCI)’s summary on risk assessment models on breast cancer , three oThe Pike model was developed in 1983 using life-table analysis to estimate the breast cancer risk among breast cancer patients’ mothers and sisters . This moThe Gail model was developed in 1989 by Dr. Mitchell Gail  by combiin situ (DCIS) or lobular carcinoma in situ (LCIS), BRCA1/2 mutation status, current age, biopsy displaying atypical hyperplasia and race were added into the model.The earliest Gail Model took into account of age at menarche, age at the time of first live birth, the number of first degree relatives with breast cancer, the number of breast biopsies; later, history of breast cancer, ductal carcinoma The Gail model was also developed based on estrogen receptor (ER) status in postmenopausal women. Chlebowski et al. utilized data from the WHI cohort and compared the model’s performance for ER-positive and ER-negative breast cancer and showed that the Gail model could identify populations at increased risk for ER+ but not ER- cancer .Other extensions of Gail model include modification made by Clause et al. to consider women with first degree family history of ovarian cancer , and by The performance of the Gail breast cancer risk prediction model among the non-white population remains understudied in the US, though there have been some attempts to tailor it to specific non-white population . One modFinally, it is well recognized that breast cancer patterns vary between countries. Multiple extension models of the Gail model have therefore been tailored to different counties, including Japan , Korea , Tyrer and Cuzick et al. developed a breast cancer risk prediction model  developed the risk calculator to estimate both 5-year and 10-year breast cancer risk . For preWith the development of GWAS, there has been increasing interest in adding SNPs identified from GWAS to breast cancer risk prediction models. Supplementary Table 1 summarizes the risk prediction models for breast cancer incorporating SNPs in the models.Wachold et al. evaluated 10 common genetic variants that, when added to traditional risk factors, were used to predict breast cancer risk . The resDite et al. conducted age group stratified analyses among cases and controls aged 35–49 years in the Australian Breast Cancer Family Registry to evaluate whether young age groups would have better performance than older age group . ThroughJupe et al. developed a polyfactorial risk model (PFRM) to predict sporadic breast cancer risk among a Caucasian population . This moMcCarthy et al. evaluated the performance of the BCRAT and the combined BCRAT+SNPs model using a cohort of African American and white women . AgreemeVan Veen et al. evaluated a panel of 18 SNPs (SNP18) in combination with classic risk assessed by the Tyrer-Cuzick model . This stAntoniou et al. developed a hypergeometric polygenic BOADICEA model for populations with a known family history of breast or ovarian cancer by incorporating BRCA1 and BRCA2 mutations, as well as the joint multiplicative effects of a polygenic component of multiple genes of small effect . Later, In addition to the use of breast cancer or BRCA1/2 mutation status as the primary variable, Crooke et al. developed a model for looking at the kinetic effect of genetic variants of the enzymes CYP1A1, CYP1B1, and COMT as well as phenotype factors on the production of the main carcinogenic estrogen metabolite 4-hydroxyestradiol (4-OHE(2)), expressed as an AUC metric (4-OHE(2)-AUC) . This moP < .001) to the BCSC model and improved discriminatory accuracy  with LDCT , 79. ThiThe potential application of a robust multifactorial risk assessment tool for lung cancer risk could be well suited to identify ideal candidates for lung cancer screening programs. Taken a step further, a robust tool would similarly identify strong candidates for prevention therapy strategy, in a similar vein to the above proposed strategies with regards to breast cancer.New biosensor technology is increasingly able to identify high-risk populations, narrowing down prevention trial sample size. For example, special imaging is a fast developing field for early diagnosis. The basic principle of spectral analysis is that the epithelial surface exposed to the cohesive laser will reflect a unique spectral pattern. The nature of the pattern is determined by the intracellular nanoscale macromolecular structure, especially chromatin. Chromatin structure is one of the earliest and most common changes that can drive cancer. In upper respiratory tracts, including lung and several organ types, all of these organs are at risk of “field carcinogenic” effects . Assess Cancer is not predicted by a single factor. A combination of non-clinical, clinical, and genetic risk factors together provide a more comprehensive and accurate assessment of risk for each individual. Efficient prevention therapy strategies will need to rely on such comprehensive risk assessment tools for targeted intervention and for effective cancer prevention strategies that are both sustainable and acceptable. The landscape of biomarker technology is extremely dynamic, with many promising new candidates, covering a spectrum of operating characteristics. Therefore, the development of risk prediction models for prevention therapy will need to evolve in a rational manner in order to incorporate the many factors that contribute to risk. Future prevention therapy trials will increasingly rely on the continued development of increasingly robust risk assessment tools to not only quantify risk, but to also determine the biological basis of that risk, and thus the type of intervention strategy.


This study aimed to investigate the association of these SNPs/haplotypes with breast cancer susceptibility in an Iranian population. We conducted a case-control study of 1072 blood samples (505 breast cancer patients and 567 controls). Genotyping of rs4784227-CASC16 and rs4782447-ACSF3 SNPs was carried out by ARMS-PCR. Moreover, statistical analysis was done using SPSS version 20.0 , PHASE v 2.1 and SNP analyser 2.0. There was a strongly significant statistical association between alleles and genotypes of rs4784227-CASC16 with breast cancer risk in our study population (p<0.05). Moreover, a significant association was demonstrated between TA haplotype and breast cancer risk ; P-value=0.025). In this respect, although we did not observe a statistically significant association between rs4782447-ACSF3 with breast cancer susceptibility, the combination of the effects of rs4784227-CASC16 and rs4782447-ACSF3 SNPs may also affect the risk. This is in line with other studies suggesting these SNPs as risk-associated polymorphisms which may lead to a change in the affinity of FOXA1, as a distal enhancer, to TOX3 and thus change in TOX3 expression, which can eventually affect the risk of breast cancer. TOX3 and FOXA1 proteins are believed to be involved in the susceptibility of breast cancer.  Numeroal., 2013). TOX3 cal., 2013; Tajbakhal., 2013, 2019[31al., 2013). Changeal., 2013). In thial., 2005). Importal., 2005). The coal., 2012). A TOX3al., 2012; Seksenyal., 2012). TOX3/LOC643714 locus that may change the affinity of FOXA1 to TOX3. Moreover, the Encyclopedia of DNA Elements (ENCODE) is indicating about 80 % of the non-coding DNA may be functional . In breal., 2007; Long etal., 2007; Ruiz-Naal., 2007; Stacey al., 2007; Udler eal., 2007). Moreovoll, 2012). UndersFollowing approval by the ethics committee of Mashhad University of Medical Sciences (IR.MUMS.fm.REC.1394.399), 1072 blood specimens were collected from 567 healthy controls and 505 patients. A written informed consent form was signed by all individuals. A questionnaire was used to collect demographic information. 10 ml of whole peripheral blood was collected from each individual and divided into tubes having sterile ethylene diamine tetra acetic acid (EDTA) for DNA extraction. DNA extraction was completed using salting out technique and was quantified at a wavelength of 260 nm and 280 nm through BioTek™ Epoch™ Microplate Spectrophotometer  and also by gel electrophoresis. TOX3/LOC643714 in breast cancer. Moreover, we tried to select SNPs that are not located in strong linkage disequilibrium (LD) to prevent redundancy in genotyping.In the present study, target SNPs were determined using available SNP public databases, and also related published articles. These articles have investigated non-coding SNPs that may change the affinity of FOXA1 to rs4782447-ACSF3 and rs4784227-CASC16 SNPs, ARMS-PCR was used. PCR amplifications for rs4782447-ACSF3 and rs4784227-CASC16 have been carried out in a 10 μl final volume per reaction containing three µl Taq 2x master mix , one µl of each primer (10 µM) and 100 ng DNA. The primers used for detection of rs4782447-ACSF3 and rs4784227-CASC16 SNPs are listed in Table 1rs4784227-CASC16 was the same as rs4782477 with a different annealing temperature of 71 °C. The DNA fragments of PCR products were detected using electrophoresis in 2 % agarose gel.To determine the genotype frequency of 2 distribution. The association between breast cancer, risk factors and alleles/genotypes were assessed using binary logistic regression, which estimated Odds ratios (ORs) as well as 95% confidence intervals (CIs). For all analyses, a P-value=0<0.05 was considered statistically significant. Logistic regression was also used to measure the associations of risk factors using different genetic models. SPSS 20.0  and also SNP analyser 2 software  assumption was investigated using the Pearson χal., 2008) were usvalues less than 0.05 were considered as statistically significant difference. Haplotypes were assembled from genotype data using PHASE program and SNP analyser 2 software  was also found in age, age of menarche (year), age of menopause (year) and age of first and last gestation. Moreover, clinical characteristics of the target population presenting that most patients had invasive ductal carcinoma with ER+, PR+ and HER2-  status . The association of haplotypes and risk factors were evaluated by crosstab program in SPSS 20. There was no association between haplotypes and the risk factors of breast cancer in all samples. Furthermore, with two SNPs, we constructed four haplotypes Table 7. A signi16q locus including rs4784227-CASC16 and rs4782447-ACSF3 and their haplotypes with the risk of breast cancer and risk factors in Iranian population. There was an association between rs4784227-CASC16 with the risk of breast cancer. However, there was no association between rs4784227-CASC16 and rs4782447-ACSF3 and risk factors using different analysis models. Furthermore, there was a significant association between AT haplotype and risk of breast cancer that indicated the combination of haplotype and its effects may influence the risk of breast cancer in the populations. In other hand, the effect of the AT haplotype may be due to the more pronounced effect of rs4784227-CASC16.In the present study we evaluated the association of two related SNPs in rs4784227-CASC16 were TT (14.9 % in cases and 10.5 % in controls); and CT (41.8 % in cases and 38.7 % in controls). There was a significant association between TT and CT genotypes with the risk of breast cancer. Furthermore, in our study, risk allele frequency  was 0.39 in patients. A similar case-control study in Iran represented significant association between CT-rs4784227-CASC16 and the risk of breast cancer . Additionally, in Iranian and Korean populations the risk allele frequency for T- rs4784227-CASC16 allele was 0.26 and 0.24 to 0.29, respectively . The fr16q12 SNPs and nearby regions are located in the introns of a non-protein coding gene. As such, it has been recommended that risky alleles may modulate gene expression by changing the enhancers activity . In thial., 2010). It indal., 2012; Meyer aal., 2012). Thus,  rs4782447-ACSF3 SNP leads to the FOXA1 binding sequence change and consequently may increase the affinity of FOXA1 interacting to the TOX3 gene promoter . Furthe Figure 1. It has oll, 2012). Since oll, 2012) suggestTOX3 and FOXA1 is rs4784227-CASC16 SNP, located 18.4 Kb upstream of the TOX3 gene  is less observed at the T-rs4784227-CASC16 compared to the C allele ; moreoval., 2008). The Gral., 2012). It shors4784227-CASC16 has been associated with the expression of RB transcriptional corepressor like 2 (RBL2) protein as a regulatory sequence of the RBL2 gene, and may also affect the risk of breast cancer . In con, the expression of TOX3 has been correlated with breast cancer and is important in revealing biological mechanisms, which makes a bridge between pathways and diseases. More functional researches may help increase our understanding of the exact biological features of breast cancer. CollectivelyZahra Farjami, Susan Darroudi and Seyed Hasan Ayati contributed equally as second authors.Mohammad Mahdi Kushyar and Alireza Pasdar  contributed equally as corresponding authors.The authors declare that they have no conflicts of interest.


P = 0.020) and estrogen receptor (ER)-positive breast cancer (OR 1.14 [1.05–1.24] P = 0.004). Genetically raised HDL-cholesterol is associated with higher risk of ER-positive breast cancer (OR 1.13 [1.01–1.26] P = 0.037). HDL-cholesterol-raising variants in the gene encoding the target of CETP inhibitors are associated with higher risk of breast cancer (OR 1.07 [1.03–1.11] P = 0.001) and ER-positive breast cancer (OR 1.08 [1.03–1.13] P = 0.001). LDL-cholesterol-lowering variants mimicking PCSK9 inhibitors are associated (P = 0.014) with lower breast cancer risk. We find no effects related to the statin and ezetimibe target genes. The possible risk-promoting effects of raised LDL-cholesterol and CETP-mediated raised HDL-cholesterol have implications for breast cancer prevention and clinical trials.Observational studies have reported inconsistent associations between circulating lipids and breast cancer risk. Using results from >400,000 participants in two-sample Mendelian randomization, we show that genetically raised LDL-cholesterol is associated with higher risk of breast cancer (odds ratio, OR, per standard deviation, 1.09, 95% confidence interval, 1.02–1.18,  The link between circulating lipids and breast cancer risk is complex. Here, the authors utilise data from more than 400,000 participants in two-sample Mendelian randomization to assess the link between blood lipids and breast cancer risk, and they find risk-promoting effects of raised LDL-cholesterol and CETP-mediated raised HDL-cholesterol. Cardiovascular diseases and breast cancer share many metabolic risk factors, including diet, obesity and physical activity3. The role of lipids, particularly in estrogen receptor-positive (ER-positive) breast cancer, is well known, but causal pathways have been difficult to disentangle4. Observational studies on associations between blood lipids and breast cancer have yielded equivocal results, with suggestive associations between raised triglyceride and high-density lipoprotein-cholesterol (HDL-cholesterol) and lower risk of breast cancer6, that have, however, not been confirmed by other studies7 and may depend on menopausal status8. No evidence for an association between low-density lipoprotein-cholesterol (LDL-cholesterol)9 or use of statins (widely prescribed LDL-cholesterol-lowering drugs) and breast cancer has been detected12, although in women with breast cancer, statins may be associated with lower recurrence risk13 and reduced breast cancer-specific mortality14.Breast cancer affects up to 1 in 8 women during their lifetime and is the second leading cause of death among women in the Western world15. Kobberø Lauridsen et al.16 found no association between genetic variants in the NPC1L1 gene (encoding the target of ezetimibe) and cancer in ~67,000 persons. No concerns have been raised about associations between proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors and cancer risk17.The effects of other lipid-modifying drugs on breast cancer are less well studied. No association with cancer occurrence was reported in the largest trial of an HDL-cholesterol-increasing cholesteryl ester transfer protein (CETP) inhibitor18. Parental genetic variants are randomly inherited, and MR uses variants that are associated with an exposure as instruments to test for associations with an outcome. This concept is analogous to randomized designs and minimizes bias from confounding and reverse causation. It can also predict drug effects by using mutations in drug target genes as instruments19. MR makes several assumptions that are often difficult to ascertain, including the absence of genetic effects on the outcome that are independent of the exposure 20. It can thus only provide preliminary evidence of causality that may inform subsequent intervention studies, drug monitoring and public health approaches21.Limited follow-up time in clinical trials and confounding or reverse causation in observational studies render conclusions about causality uncertain. In epidemiologic settings, Mendelian randomization (MR) has been developed to assess causality23. Orho-Melander et al.24 studied ~16,000 women and found suggestive effects of raised HDL-cholesterol and reduced triglycerides on increased breast cancer risk that did not, however, reach nominal significance. In the same study, the LDL-lowering allele of a variant in the statin target gene HMGCR was associated with lower risk of breast cancer while an LDL-cholesterol genetic score was not, suggesting an LDL-independent mechanism. To our knowledge, MR has not been applied to study effects of lipids on breast cancer risk in the largest available genetic datasets from the Global Lipid Genetics Consortium (GLGC)25 and the 2017 release of the Breast Cancer Association Consortium (BCAC)26 of over 180,000 participants each.MR studies have demonstrated, for instance, an inverse association between genetically predicted obesity and risk of breast cancerCETP-mediated raised HDL-cholesterol that may have implications for breast cancer prevention.In this study, we use two-sample MR in the largest available genetic datasets to assess causal associations between circulating LDL-cholesterol, HDL-cholesterol, triglycerides and variants in genes encoding lipid-modifying drug targets on the risk of total breast cancer, ER-positive and ER-negative breast cancer. We find possible risk-increasing effects of raised LDL-cholesterol and P < 0.001).Figure P = 0.055). Genetically raised HDL-cholesterol was associated with breast cancer risk (P = 0.003) and ER-positive disease risk (P = 0.002). Raised triglycerides were negatively associated with all three outcomes. There was evidence of heterogeneity in all analyses . Following exclusion of pleiotropic variants in restrictive MR (Supplementary Table P = 0.017) and ER-positive breast cancer  with consistent estimates across the Egger and median methods but evidence of remaining heterogeneity . Raised HDL-cholesterol had no clear association with breast cancer risk  with significant remaining heterogeneity . There was evidence of an effect of raised HDL-cholesterol on increased ER-positive breast cancer risk . Triglycerides were not associated with any of the outcomes (P > 0.4).In comprehensive MR  and ER-positive breast cancer  and no association with ER-negative disease (P = 0.577). Raised HDL-cholesterol had no nominally significant association with either breast cancer risk  or ER-negative disease , but appeared associated with increased risk of ER-positive disease . Genetically predicted triglyceride levels were not associated with any of the outcomes. Application of MR-PRESSO to the comprehensive selection without exclusion of variants associated with other lipids produced causal estimates in the same direction, but there was significant (P < 0.05) remaining heterogeneity after exclusion of outliers in all cases  but not with ER-positive  or ER-negative breast cancer  at the nominal significance level  and ER-positive breast cancer risk , although in both cases, MR Egger was suggestive of a null association . The associations with ER-negative breast cancer risk did not reach nominal significance . Estimates for NPC1L1 were suggestive of risk-increasing effects of raised LDL-cholesterol on breast cancer and ER-positive breast cancer risk in inverse variance-weighted analysis, but MR Egger estimates were in the opposite direction, casting doubt on the validity of MR assumptions in this case. LDL-cholesterol-raising variants in HGMCR had a suggestive association with breast cancer risk  total breast cancer risk. The inverse association between triglycerides and breast cancer risk in comprehensive MR was abolished after excluding variants associated with other lipids, implying that triglyceride levels do not affect breast cancer risk independently. This result broadly concurs with observational studies that reported either absent or weak inverse associations27.The risk-increasing effect of genetically raised LDL-cholesterol on total and ER-positive breast cancer contrasts with observational studies that reported inconsistent results generally suggesting a null association, with marked heterogeneity depending on study design, menopausal status and body mass index of participants28, inverse associations in post-menopausal women6 and unidirectional associations in pre-menopausal women29. The effect of HDL-cholesterol on breast cancer risk in our study concurs with a non-significant association in an earlier genetic study24, and the absence of a stronger effect in our summary-level data may relate to lack of power and an inability to stratify participants by menopausal status. Another explanation could be different effects depending on the metabolic health of the individual, as in vivo studies found evidence that oxidation status of HDL-cholesterol in a normo- or hyperlipidaemic context may determine its effects on the promotion of breast cancer metastasis30.Genetically raised HDL-cholesterol was associated with increased risk of ER-positive breast cancer. Observational studies have reported inconsistent results on associations between HDL and breast cancer, including null effects31, and observational studies in women have hitherto not been able to consistently define potential effects. Our study provides genetic evidence of a harmful association between raised LDL-cholesterol and breast cancer occurrence, as well as a suggestive harmful effect of raised HDL-cholesterol. A deficit of our study is the inability to stratify women by menopausal status. The endocrine changes of the menopause likely affect plasma lipid composition and the interaction with breast tissue. For instance, a meta-analysis of observational studies found that an inverse association between HDL-cholesterol and breast cancer was only present in post- but not in premenopausal women6. Differing effects depending on menopausal status are further suggested by findings that genetically predicted obesity is inversely associated breast cancer risk, which contrasts with a positive association between obesity and postmenopausal breast cancer risk in observational studies22. Large biobanks such as the UK Biobank may in the future allow to further dissect the suggestive causal effects of HDL-cholesterol on breast cancer discovered in our study.Taken together, laboratory studies have demonstrated that lipoprotein fractions affect breast cancer growth both directly and as precursors for cholesterol metabolitesCETP was associated with raised total and ER-positive breast cancer risk, but only two of the 11 variants were individually associated with breast cancer at the nominal significance level and MR Egger implied absent effects  and the short follow-up of up to 4.1 years pose limitations on the detection of potential cancer-related effects. Table Raised HDL-cholesterol due to genetic variants in PCSK9 was associated with risk-reducing effects on breast cancer occurrence in our study. A 2017 review of clinical studies comparing PCSK9 inhibitors to placebo found no association with risk of any cancer, although the direction of association  does not exclude a possibly protective effect19. A similar non-significant protective association with cancer risk was found in a phenome-wide association study of genetic variants in PCSK9 in the UK Biobank sample32.Lowering of LDL-cholesterol due to variants in P = 0.086) of variants in the gene encoding the target of statins could point to a true signal that our study was underpowered to detect or that may differ in subgroups  that we were not able to assess. The possible risk-reducing effect of statin mimicry in MR chimes with observational studies that reported either null or protective associations with breast cancer risk and mortality14. Future studies with genetic and drug exposure data that allow analysis in subgroups should address any possible effects of statins on breast cancer.The potential, but not nominally significant effect  and pleiotropy (MR Egger), but remaining sources of bias such as canalization cannot be ruled out. Finally, we could not assess the influence of menopausal status and our results only apply to women of European ethnicity.Strengths of our study include the use of the largest available summary genetic datasets and extensive diagnostics to evaluate the validity of MR assumptions and limit the potential for bias from pleiotropy. Limitations include our inability to replicate results in independent datasets, concerns about pleiotropy from (un)measured confounders, possible weak instrument bias and lack of power for drug analyses. A bias toward the null because of Winner’s curse25 and from BCAC for risk of breast cancer 26. Both studies included rigorous quality control, imputation to the 1000 Genomes Project panel and adjustments for age and population structure. The studies have existing ethical permissions from their respective institutional review boards and include participant informed consent. The outcomes in the present study were risk of any breast cancer, ER-positive and ER-negative breast cancer as defined in BCAC26.Genome-wide association study results in persons of mostly European ancestry were obtained from the GLGC  for plasma lipidsn = 94,595) or on the Metabochip array  with imputation to the 1000 Genomes Project reference were studied. In most included studies, blood lipid concentrations had been measured after an >8 h fast. Participants on lipid-lowering medications were excluded. Traits were adjusted for age, age-squared, sex and principle components, as well as quantile-normalized within each cohort. For genetic association analysis by linear regression, lipid levels were inverse normal-transformed and cohort-wise results combined in fixed effect meta-analysis25.Persons of European ancestry from 47 studies genotyped with different genome-wide association study arrays . Genotypes were imputed to ~21 million variants using the 1000 Genomes Project (Phase 3) reference panel. Variants with minor allele frequency <0.5% and imputation quality score <0.3 were excluded resulting in ~11.8 million variants for logistic regression analysis adjusted for genetic principle components and country. Results were combined in fixed-effect meta-analysis with results from the Collaborative Oncological Gene-environment Study  and 11 other breast cancer genome-wide association studies . The current study uses summary results from women of European ancestry36. We constructed two genetic instruments for each lipid. First, we selected all genome-wide significant (P < 5 × 10−8) variants associated with each lipid for comprehensive MR . Second, to reduce possible pleiotropic effects we excluded in each selection those variants that were associated at P < 0.001 with any of the other two lipids for restrictive MR .We extracted association statistics for LDL-cholesterol, HDL-cholesterol and triglycerides in GLGC for 185 genetic variants in 157 loci previously demonstrated to be associated with at least one lipid fractionP-value for lipid association in GLGC and iteratively selected in the order of increasing P-value provided they were in low linkage disequilibrium (r2 < 0.4) with variants selected in preceding steps. We obtained pairwise linkage disequilibrium based on Phase 3 (Version 5) of the 1000 Genomes Project combined European reference sample via LDlink37. We used associations with HDL-cholesterol to construct the genetic instrument for CETP and associations with LDL-cholesterol to construct instruments for HMGCR (encodes the target of statins), PCSK9, NPC1L1 (encodes the target of ezetimibe) and LDLR. LDLR does not mimic a drug target but is a common site of mutations causing familial hypercholesterolemia (OMIM 606945) and was included to assess the role of the LDL receptor pathway.To assess potential causal effects of changes in lipid levels due to pharmacological intervention, we selected polymorphisms within ±100 base pairs of genes encoding drug targets that were genome-wide significantly associated with the target lipid and in low linkage disequilibrium with each other. Variants were ranked by r2 > 0.8) was available. Genetic effects were aligned to the lipid-increasing allele and alignment checked by comparing the minor allele frequencies reported by BCAC and GLGC. Palindromic variants that could not be unambiguously aligned and multi-allelic variants with different effect and reference alleles in BCAC and GLCL were removed.One variant (rs4332136) was not available and excluded as no proxy variant , we implemented the inverse variance-weighted and Egger methods with explicit modelling of correlations between genetic variants according to the method suggested Burgess et al.41 as implemented in the MendelianRandomization software in R42. To assess heterogeneity between individual genetic variants’ estimates, we used the Egger intercept test43, the Q′ heterogeneity statistic44 and the MR pleiotropy residual sum and outlier (MR-PRESSO)45 test. The Q′ statistic uses modified 2nd order weights that are a derivation of a Taylor series expansion and take into account uncertainty in both numerator and denominator of the instrumental variable ratio 44. The MR-PRESSO framework relies on the regression of variant-outcome associations on variant-exposure associations and implements a global heterogeneity test by comparing the observed distance  of all variants to the regression line with the distance expected under the null hypothesis of no pleiotropy45. In case of evidence of horizontal pleiotropy, the test compares individual variants’ expected and observed distributions to identify outlier variants. We used an implementation of MR-PRESSO in R (https://github.com/rondolab/MR-PRESSO) with default parameters to (i) test for global heterogeneity; (ii) if significant at P < 0.05 identify and remove outliers; and (iii) retest to evaluate if outlier removal had resolved heterogeneity. We consider as results causal estimates that agree in direction and magnitude across MR methods, pass nominal significance in inverse variance-weighted MR, and do not show evidence of bias from horizontal pleiotropy in heterogeneity tests. Analyses were carried out with the MendelianRandomization42, TwoSampleMR and MR-PRESSO45 packages in R version 3.3.2 (2016-10-31).We used inverse variance-weighted, Egger and weighted median MR to assess causal effects of lipid fractions. The inverse variance-weighted method regresses genetic associations with the outcome on associations with lipid levels and fixes the intercept at zero. In the absence of directional pleiotropy, it provides robust causal estimateshttp://cnsgenomics.com/shiny/mRnd/) for post-hoc power calculations. At an alpha level of 0.05, we estimated 80% power to detect causal effects on breast cancer risk per standard deviation increment in lipid level of OR 1.06 (LDL-cholesterol), OR 1.07 (HDL-cholesterol) and OR 1.07 (triglycerides). The corresponding estimates for ER-positive breast cancer were OR 1.06 (LDL-cholesterol), OR 1.07 (HDL-cholesterol) and OR 1.08 (triglycerides); and the estimates for ER-negative breast cancer were OR 1.10 (LDL-cholesterol), OR 1.11 (HDL-cholesterol) and OR 1.12 (triglycerides), respectively.We used mRnd (46. A careful comparison of the samples included BCAC and GLGC showed one common cohort (EPIC), which accounted for 2.9% of cases and 3.3% of control persons in BCAC, and for 1.7% (1.0% if only considering women) of participants in GLGC. Based on a simulation study of the association between sample overlap and the degree of bias in instrumental variable analysis46, this degree of overlap (<5%) is unlikely to influence results in a meaningful way.Participant overlap between the samples used to estimate genetic associations with the exposure and the outcome, respectively, in two-sample MR can bias resultsThe analysis code in R is available on request and all data displayed in figures are available in Supplementary Tables Supplementary InformationPeer Review File