TP53 tumor suppressor gene at the codon 72 codes proline instead of arginine causes an altered p53 protein expression and has been found to be associated with an elevated risk of various cancer; especially breast and lung cancer. As the previous case-control studies on the South Asian population have shown controversial results, we performed a meta-analysis to evaluate a precise estimation of the relationship between the TP53 Arg72Pro polymorphism with breast and lung cancer. A transversion missense polymorphism of theA total of 12 related studies on the South Asian population have been included through comprehensive database searching. Six studies were selected for breast cancer meta-analysis involving 950 cases and 882 controls; the other six studies were for lung cancer meta-analysis including 975 cases and 1397 controls. The results have been determined by using the Review Manager (RevMan) 5.3. Additionally, the stability of our analysis was assessed by heterogeneity, publication bias analysis and sensitivity testing. A significantly increased risk of breast cancer was found in Pro allele (Pro vs. Arg), co-dominant model 2 (Pro/Pro vs. Arg/Arg), dominant model (Pro/Pro + Arg/Pro vs. Arg/Arg). In case of lung cancer, significantly increased risk was found in the allele, co-dominant 1, co-dominant 2, co-dominant 3, dominant, and recessive models. No association with other genetic models with breast and lung cancer risk was found in the South Asian population. TP53 Arg72Pro polymorphism is a risk factor for the development of breast cancer and lung cancer in the South Asian population.Our results indicate that  Cancer is one of the most severe human diseases with a high rate of morbidity results from uncontrolled, abnormal cellular proliferation due to genetic mutation. The global cancer rate is rapidly rising and in 2018, new cancer cases rose to 18.1 million, with 9.6 million deaths. Lung cancer and female breast cancer are the leading types of cancers worldwide in terms of the number of new cases and deaths. In 2018, approximately 2.1 million diagnoses were estimated for both types of cancers, contributing about 11.6% of the total cancer incidence burden . According to Globocan 2018, among the total number of cancer cases, 13.6% was breast cancer (ranked 1) and 8% was lung cancer (ranked 2) cases in the WHO Southeast Asia region .TP53 is one of the major tumor suppressor genes that provides a critical barrier to the development of cancer by blocking cellular proliferation or eliminating cancer cells. Mutation of the TP53 gene interferes with its tumor suppression function and has a critical role in the formation of human cancer, whereas a mutant TP53 gene is relatively higher in cancer patients than in the control population . The majority of TP53 mutations are transversion missense mutations that result in the formation of different amino acids rather than the normal one due to purine and pyrimidine nucleotide substitution. The position of mutation lies within the part of the gene that encodes the site-specific DNA-binding domain that causes false transcription and failure in tumor suppression. Mutant p53 proteins are generally very stable in cancer cells than the normal p53 protein and can be detected easily by immunohistochemistry Positive regulation of tumor suppressor genes inhibits the formation of a tumor by halting various mechanisms during the cell cycle. TP53 missense mutations are found during cancer development in the highly mutable region of the human gene – CpG region, where the frequencies of cysteine and guanine base are comparatively higher. The region contains essential transcription factor binding site that in turn controls structural and chemical contact with the combined effect of p53 protein and specific DNA sequences that develop specific p53 response elements. The most extensively studied SNP (rs1042522) is a transversion polymorphism of G nucleotide to C nucleotide in codon 72 in exon 4 at the second position, leading to the formation of proline rather than arginine (Pro72 protein variants). The gene product of Arg72 variant is more efficient in apoptotic activity than the mutant Pro72 variant of TP53 gene. The allelic frequencies of TP53 codon 72 differ among different ethnic populations of the world that changes cancer susceptibility in these populations .About 30% of TP53 codon 72 rs1042522 polymorphism with breast and lung cancer risk, but results have been contradictory and no clear evidence has been found. The conflicting results may arise from insufficient data, small sample size or different study designs. A single study is less potential to make a reliable outcome due to these limitations .Numerous studies have conducted research over the association of TP53 codon 72 polymorphism and its relation to breast cancer or lung cancer, mostly on the Caucasian and Asian population, including Japanese , Chinese  and Taiwanese  regions. But no previous meta-analysis has been conducted on the South Asian population, including Bangladeshi, Indian, Pakistani population, although the prevalence of lung cancer and breast cancer is extremely high in South Asia . A high degree of variation in the genetic profile occurs due to geographic and ethnic variation that changes the prevalence of the disease in different regions of the world . Even in the same ethnicity, two different studies may show the opposite results . The ancestry of the Indian subcontinent population is unique and admixture of the population was observed from other parts of the world .Moreover, several meta-analyses have been conducted on TP53 codon 72 polymorphism in the South Asian population. To evaluate the potential correlation comprehensively between the TP53 Pro72Arg polymorphism with the lung and breast cancer risk in South Asian population, and to improve our understanding for a more precise characterization of the Pro72Arg polymorphism, we designed a systematic review and meta-analysis of 12 candidate studies conducted on the South Asian population.For this reason, it was necessary to find out the relation between lung cancer and breast cancer with MethodologyLiterature search for identification of relevant studies TP53 codon 72 Arg>Pro polymorphism with lung or breast cancer conducting on the South Asian population 3) studies providing information regarding genotype and allelic frequencies of both cases and controls, odds ratio with 95% confidence interval (CI) values. The exclusion criteria were: 1) studies published in an animal model or cell line 2) studies having only the data of cases 3) lacking any genotype frequency for TP53 codon 72 Arg>Pro polymorphism 4) studies conducted on the other ethnic groups except the South Asian population.A systemic comprehensive search was performed in Pubmed, Web of Science, Scopus and Google scholar. Two independent researchers searched the publications up to September 2019 using either single or combination of given following keywords- “Arg72Pro polymorphism”, “p53 codon 72 rs1042522 polymorphism”, “p53 and lung cancer or breast cancer”. The summary of the process of selecting eligible studies was presented in Data extractionTwo researchers (SF and MSI) independently collected the first author’s name, year of publication, sample size, the ethnicity of the cases and controls, genotype and allele frequencies for each case and control.Statistical analysisRevMan (Version 5.3) software (http://www.cochrane.org/revman) was used to conduct this meta-analysis. All of the data in the studies are dichotomous data. The outcome has been expressed as odds ratios (ORs) with 95% confidence intervals (CIs) to assess the association between the polymorphisms and cancer. OR>1 indicates increased risk and <1 indicates reduced risk.TP53 polymorphisms and the risk of breast or lung cancer. For each type of cancer to find the association with polymorphism, the ORs were calculated for co-dominant, dominant, recessive, overdominant, and allele genetic models. Heterogeneity in the forest plot also assessed using the I2 statistic and the Cochrane chi-square Q-test. The significance level of heterogeneity is p<0.10 and I2>50%. The fixed-effects model (the Mantel-Haenszel method) was applied for meta-analysis when heterogeneity was not significant. The random-effects model was used to estimate pooled ORs when heterogeneity values were significant. Z-test was performed to evaluate the significance of the pooled OR and p<0.05 was considered to be statistically significant . Publication bias was appraised using Begg’s funnel plot. The degree of asymmetry of Begg’s funnel plot was measured using Egger’s linear regression test and the level of significance of publication bias was p<0.05. The sensitivity analysis was done to examine the influence of each study on the pooled results by removing studies one by one. The significance level of publication bias was p<0.05. Chi-square tests of control group data was performed for each study to determine the Hardy-Weinberg equilibrium (HWE). The HWE p values are shown in Study characteristics TP53 codon 72 Arg/Pro polymorphism with lung cancer and breast cancer in the South Asian population based on the inclusion criteria . Among these, six studies evaluated the association for breast cancer (950 cases and 882 controls) and the other six studies for lung cancer . All the studies were performed in the various ethnic groups of South Asia. A total of 12 case-control studies were included in this meta-analysis to find out the association of Association of TP53 codon 72 Arg>Pro polymorphism with breast and lung cancer72 Arg>Pro polymorphism was mentioned in The association between breast and lung cancer with Association of TP53 codon 72 Arg>Pro polymorphism with breast cancerTP53 codon 72 polymorphism, we pooled together six studies related to the breast cancer (950 cases and 882 controls). As shown in -5), dominant , and co-dominant 2  genetic models. Though co-dominant models 1 and 3  and recessive model  showed increased risk, these models were not statistically significant to develop breast cancer. Overdominant model , on the contrary, showed decreased susceptibility for the breast cancer that was not statistically significant. To measure the association of breast cancer with Association of TP53 codon 72 Arg>Pro polymorphism with lung cancer-4), co-dominant model 1 , co-dominant model 2 , co-dominant model 3 , dominant model  and the recessive  of TP53 codon 72 polymorphism were significantly associated with lung cancer development. No statistically significant association was found with overdominant model .Through the meta-analysis of six studies related to lung cancer including 975 cases and 1397 controls, it was found that (Heterogeneity and Publication bias2> 50) in case of breast cancer. Whereas co-dominant model 2 (Pro/Pro vs. Arg/Arg), dominant model (Pro/Pro + Arg/Pro vs. Arg/Arg), recessive model (Pro/Pro vs. Arg/Pro + Arg/Arg) and allele model (Pro vs. Arg), showed heterogeneity in case of lung cancer (Heterogeneity was checked and the co-dominant model 1 (Arg/Pro vs. Arg/Arg) co-dominant model 3 (Pro/Pro vs. Arg/Pro), overdominant model (Arg/Pro vs. Arg/Arg + Pro/Pro) and recessive model (Pro/Pro vs. Arg/Pro + Arg/Arg) showed heterogeneity . Excess presence of Proline will change the overlapping tendency of TAD2 as well as tumor suppression activity. During cancer development, mutation of the TP53 gene not only results in the ablation of wild type p53 tumor-suppressor activity but also gives rise to a mutant protein that encompasses oncogenic functions . TP53 Arg72Pro (rs1042522 ) polymorphism occurs at exon 4 among the 11 exons of codon 72 of the p53 tumor suppressor gene located on chromosome 17p13. The polymorphism causes the transition of CGC to CCC, leading to a differently functioning protein with a proline-rich domain at codon 72 (Arg72Pro). Studies have reported that the codon 72 polymorphism is associated with a risk for the development of cancer .The TP53 codon 72 polymorphism with lung and breast cancer, yet the outcome was conflicting. A number of studies have reported that there is a significant association between TP53 codon 72 polymorphism and breast cancer risk  and other studies showed that there is no significant association . To overcome this conflicting situation, we performed a meta-analysis to evaluate the association between TP53 Arg72Pro polymorphism and breast cancer risk in the South Asian population by analyzing six studies with a total of 950 cases and 882 controls. The polymorphism showed significant risk association in terms of three genetic models . The other three genetic models  also showed a higher risk, but the result was not statistically significant. According to the result, TP53 codon 72 polymorphism is a substantial risk factor for the development of breast cancer in the South Asian population.Many previous studies suggested that there may be a significant risk association of TP53 codon 72 polymorphism is susceptible to cause lung cancer, according to many previous studies . The polymorphism of p53 codon 72 Arg/Pro also has been demonstrated to modify the risk for lung cancer among South Asians in many previous case-control studies. Several previous studies demonstrated that the p53 codon 72 Arg/Pro polymorphism was not significantly associated with lung cancer susceptibility . On the contrary, the p53 codon 72 polymorphism was confirmed to be related to an elevated risk of lung cancer in other studies Chua et al., 2010; Hossain et al., 2017; Hiraki et al., 2003; Sakiyama et al., 2005; Zhang et al., 2006; Jung et al., 2008). Different conflicting results may be results from the insufficient sample size, variable genotyping method or different study design. To evaluate the actual association between TP53 codon 72 polymorphism and lung cancer in the South Asian population, we performed a meta-analysis based on 6 articles with a total of 975 cases and 1397 controls. The polymorphism showed a significantly increased risk of lung cancer in various genetic models. Allele, co-dominant genetic models 1-3, dominant model and recessive showed a significantly increased risk for the development of lung cancer (p<0.05). The overdominant model showed no risk for the development of lung cancer in the South Asian population. Similarly, Our study findings have some limitations, although we systemically searched for eligible publications. Firstly, only six studies of breast cancer and six studies of lung cancer were included as no other relevant studies were found. Secondly, only the South Asian cases and controls were included in the study. Therefore, our pooled results may not be extrapolated to other ethnic groups. Despite these limitations, this is the first meta-analysis of the South Asian population to evaluate the association of p53Arg72Pro polymorphism with breast and lung cancer. We performed the heterogeneity, sensitivity and publication bias analyses that are strengths of this meta-analysis.In conclusion, this combined meta-analysis indicates that the TP53 codon 72 polymorphism increases the risk of both breast cancer and lung cancer in the South Asian population.


Our study provides valuable evidence for adding dietary acid load scores to dietary guidelines for breast cancer survivors and developing specific guidelines for past smokers among these survivors.Current dietary guidelines do not consider cancer survivors’ and past smokers’ low capacity to regulate their acid–base balance. People with a low capacity to regulate their acid–base balance are more susceptible to acid-producing diets. We studied a cohort of 2950 early stage breast cancer survivors who provided dietary information at baseline and during follow-up. We assessed the intakes of acid-producing diets via two commonly used dietary acid load scores: potential renal acid load (PRAL) and net endogenous acid production (NEAP). We assessed past smoking intensity by pack-years of smoking. After an average of 7.3 years of follow-up, there were 295 total deaths, 249 breast cancer-specific deaths, and 490 cases of recurrent breast cancer. Increased intakes of dietary acid load and pack-years of smoking were each independently and jointly associated with increased total mortality and breast cancer-specific mortality; tests for trends and overall associations were statistically significant for NEAP and marginally significant for PRAL. Compared to women in the lowest tertile of NEAP and pack-year of smoking = 0, women in the highest tertile of NEAP and pack-years of smoking >15 had the greatest increased risk of total mortality . Further, dietary acid scores were associated with increased breast cancer recurrence among women with pack-years of smoking >0 but not in those with pack-years of smoking = 0 ( Cancer survivors, especially breast cancer survivors, experience accelerated aging due to the detrimental effects of cancer treatments and cancer cells as well as some additional existing risk factors among them ,2,3; thuDietary acid load may play important roles in breast cancer prognosis and may interact with past smoking intensity in the development of worse prognoses in breast cancer survivors. The proper balance between the acids and bases  in the human body is crucial for normal physiologic functions and cellular metabolism ,8,9. AciWe will leverage a large cohort of breast cancer survivors, the Women’s Healthy Eating and Living (WHEL) study, to conduct the current study. The range of acid-producing diets in this cohort is wider than that of the typical American diet , which eThis study leverages an existing prospective cohort, the WHEL study, comprising mainly early stage  breast cancer survivors. Between 1995 and 2000, the WHEL study enrolled 3088 women within 4 years of diagnosis. The WHEL study was initially a multi-site randomized trial including several sites in the U.S. . The trial was designed to test whether a diet low in fat and rich in vegetables, fruit, and fiber improved breast cancer prognosis. Extensive details regarding inclusion and exclusion criteria can be found in previous publications [The initial study used several ways to identify potential participants by using tumor registries as well as communicating and distributing information to community oncologists. Participants completed a run-in period so investigators could identify who might have difficulties adhering to the study requirements. This cohort had five clinical visits in different years, while baseline, year 1, and year 4 had more complete information related to diet. The WHEL study developed an adherence score. The WHEL Feasibility Study indicated that more than 80% of participants reached an adherence score of 450 (full adherence score was 600). This initial trial provided telephone counseling and group cooking classes to the intervention group. Extensive communication programs were implemented to build support and motivate participants to complete the program. For instance, participants could receive an airline ticket as an incentive based on the points earned. Nutritional summaries were also provided to participants after the dietary assessments ,21.The intervention did not significantly change breast cancer prognosis after an average of 7.3 years of follow-up. Therefore, the present study considered and analyzed the study sample as a single cohort while controlling the initial trial assignment. For this analysis, we excluded women who were current smokers at baseline; as a result, the analytical cohort comprised 2950 women. The Institutional Review Board (IRB) at the University of California at San Diego approved the original study. All subjects provided written informed consent. The de-identified data were provided by the principal investigator of the WHEL study. The current study was an ancillary study using the de-identified data from the WHEL study; thus, the exempt IRB was approved by the San Diego State University IRB committee (protocol number: Temp-1286).At baseline, year 1, and year 4, dietary intakes were assessed by four prescheduled, 24-h dietary recalls collected by telephone on random days over a 3-week period: two on the weekends and two during weekdays. Dietary assessors used the multi-pass, software-driven recall protocol of the Nutritional Data System software .In terms of the assessment of acid-producing diets, two commonly used dietary acid load scores were used to estimate dietary acid load in epidemiological studies: the potential renal acid load (PRAL) score and the net endogenous acid production (NEAP) score. The PRAL score considers the intestinal absorption rates for contributing nutrient ionic balances for protein, potassium, calcium, and magnesium and the dissociation of phosphate at pH 7.4 . FrassetPRAL (mEq/day) = (0.49 × protein (g/day)) + (0.037 × phosphorus (mg/day)) − (0.021 × potassium (mg/day)) − (0.026 × magnesium (mg/day)) − )NEAP (mEq/day) = (54.5 × protein (g/day)/potassium (mEq/day)) − 10.2This study used both scores for dietary acid assessment because they reflect slightly different nutritional intakes and biological mechanisms. A negative PRAL value reflects an alkaline-forming potential; a positive value reflects an acid-forming potential . For NEAA brief smoking history questionnaire was administered to participants at baseline. The questionnaire included age of smoking initiation and cessation, duration of smoking, and the number of cigarettes/day. We classified a lifetime history of <100 cigarettes as never smoking. Former smokers reported having quit at this baseline survey. All ever smokers reported their intensity of smoking (cigarettes/day) and the number of years they smoked regularly. Pack-years exposure was determined by multiplying duration of smoking by intensity. One pack-year is equal to smoking one pack per day for one year or two packs per day for half a year for 4–6% . The primary outcome of this study is total mortality, breast cancer-specific mortality, and breast cancer recurrence. At the close of the study in June 2006, vital status was known for 96% of the participants. Information on death and recurrence from participants was ascertained via confirmation interviews, periodic reviews (including with a family member), and oncologists’ reviews of the medical record and/or death certificate. In addition, both the Social Security and the National Death Index were searched using the Social Security number, name, and date of birth. Causes of death were coded using the International Classification of Diseases, 9th Revision (ICD-9) codes. All breast cancer deaths or recurrences were confirmed by the study’s oncologist. Breast cancer recurrence was defined as the combination outcome of breast cancer recurrence  or new primary breast cancer. Carcinoma in situ was not counted as a study outcome. Survival was assessed as the time from study entry to death or the most recent available review of the Social Security Death Index (updated until 2009). Follow-up time was censored at the earlier of (a) time of the last documented staff contact date or (b) study completion (June 2006) for participants without an event . Approximately four percent of study participants were lost during follow-up and these were censored at the date of last contact.Demographic characteristics and health status, including a series of comorbid conditions , were self-reported.Variables abstracted from patient records included initial cancer diagnosis and treatment. Specific variables abstracted included tumor stage, size, hormone receptor status, and use of radiation, chemotherapy, and/or post treatment anti-estrogens use. Physical activity levels were assessed using an adapted validated questionnaire from the Women’s Health Initiative . Physicat-test and analysis of variance (ANOVA) for normally distributed continuous variables, Wilcoxon rank-sum test and Kruskal–Wallis test for non-normally distributed continuous variables, and χ2 test for categorical variables. Differences in baseline characteristics across breast cancer prognosis or across baseline dietary acid load were evaluated using a We used Cox proportional hazard models to assess the association of dietary acid load with total mortality, breast cancer-specific mortality, and breast cancer recurrence. Censored times for participants who did not die were calculated from the study entry to lost to follow-up or to the end of the follow-up period, whichever came first. Event time was calculated with respect to each outcome as follows. For all-cause mortality, time was calculated from the study entry to the time of death. For breast cancer-specific mortality, time was calculated from the study entry to breast cancer-specific death; death from other causes was treated as a competing risk. For recurrent breast cancer, time was calculated from the study entry to diagnosis with recurrent breast cancer, and death was treated as a competing risk.As previously introduced, dietary acid load was characterized by PRAL and NEAP scores. Repeated measures of PRAL and NEAP at years 0, 1, and 4 were analyzed as time-varying covariates. PRAL and NEAP scores were classified into quartiles using the average intakes at years 0, 1, and 4 to set up the cut-point for each quartile. We classified baseline pack-years of smoking into three categories . We controlled the following covariates based on a priori assumption: age at diagnosis, race/ethnicity, education level, intervention group, menopausal status, total calorie intake, alcohol intake, smoking status, pack-years, physical activity, body mass index (BMI), education level, tumor stage, tumor size, estrogen and progesterone receptor status, type of anti-estrogen therapy, radiotherapy, chemotherapy, study site, and medical comorbidities. Among these covariates, time-varying covariates included BMI, physical activity, smoking status, total calorie intake, alcohol intake, and types of anti-estrogen therapy. We used baseline data for other covariates. p-value for the interaction term in a model that also included the main effects. We further evaluated both joint impacts of dietary acid load and past smoking intensity on outcomes and effect modification by past smoking intensity. To evaluate joint impacts, dietary acid load was categorized by tertile to improve the stability of point estimates. Women with the lowest tertile of dietary acid load and pack-years of smoking = 0 were treated as the reference group. To evaluate the effect modification by smoking intensity, we conducted stratified analyses by two pack-years of smoking strata (=0 and >0). To assess whether a significant interaction occurred between dietary acid load and pack-years of smoking, we used the Wald The proportional hazards assumption was examined and satisfied in all Cox proportional hazard regression models by testing the significance of the product terms for our variable of interest and log time. All analyses were conducted using SAS version 9.2 . p-values were <0.05 for these comparisons.After a median 7.3 years of follow-up, 295 deaths and 249 breast cancer-specific deaths as well as 490 breast cancer recurrences were reported in the cohort . Comparep-values were <0.01 for these comparisons.As shown in p-values for trends were <0.05 for both outcomes. The corresponding HRs for PRAL were similar but marginally significant. Pack-years of smoking was positively and statistically significantly associated with the three outcomes. As shown in p-values for trends were <0.01 for both PRAL and NEAP. We also observed that the positive associations between dietary acid load and total mortality were stronger in the highest category of pack-years of smoking (>15) than the lower two categories of pack-years of smoking (0 and 0–15). Similar patterns were observed for breast cancer-specific mortality and recurrence; however, the magnitudes and significance were attenuated for recurrence .We found statistically significant joint associations of dietary acid load and past smoking intensity with total mortality, breast cancer-specific mortality, and breast cancer recurrence see . Both dip-values for interactions were 0.1 for total mortality, 0.03 for breast cancer-specific mortality, and 0.01 for breast cancer recurrence).In In these comprehensive analyses of a cohort of breast cancer survivors, increased dietary acid load and past smoking intensity were both independently and jointly associated with increased total mortality and breast cancer-specific mortality. We also found an increased risk of breast cancer recurrence among women with pack-years of smoking >0.Our study is the first to highlight the importance of the independent and joint impacts of dietary acid load and past smoking intensity on total mortality, breast-cancer specific mortality and recurrence among early stage breast cancer survivors. Previous prospective studies have demonstrated that the dietary acid load or higher metabolic acid load (measured by lower serum bicarbonate and overnight fasting urine pH) had a positive or U-shaped relationship with total mortality or cardiovascular mortality but not with cancer-associated mortality ,29,30. TWe propose the following to help explain why NEAP was found to be a better predictor of breast cancer prognosis than PRAL in our study. Both PRAL and NEAP have their own advantages and disadvantages. NEAP is an approximate of renal net acid excretion (RNAE) and was found to account for 70% of the variation of RNAE . It is aSeveral mechanisms can explain why past smokers are more susceptible to dietary acid load than never smokers. Quitting smoking can avoid further damage but does not remove past damages by smoking ,52; smokThe original intervention was a low-fat, high-fruit, and -vegetable dietary intervention, not an intervention aiming to change dietary acid load scores, though we did find that dietary acid load scores were significantly lower in the intervention group than the control group. One may question why dietary acid load was found to have a significant impact on prognosis whereas the original intervention did not have a significant impact on prognosis. There are several possible reasons for this. Dietary acid load scores were greatly reduced in the intervention group when compared to the control group; however, 17% and 53% from the intervention group still had high NEAP scores (higher than median) at year 1 and 4, respectively, as compared to 54% and 63% from the control group. Such a mismatch can significantly attenuate the association if it were analyzed as a clinical trial. In addition to dietary acid load, types of fat and intakes of refined carbohydrates were not controlled in the original WHEL intervention. As we know, diets with high intakes of trans fatty acids and glycemic load can increase inflammation and hyperglycemia ,58, whicThis study has several strengths. It is the first large prospective cohort study investigating the independent and joint associations of dietary acid load and past smoking intensity with breast cancer prognosis among breast cancer survivors. Four 24-h recalls during each visit  were the unique advantages of this cohort but were rarely conducted in other cohorts. Such advantages enable us to assess dietary acid load more accurately and examine its longitudinal relationships with prognosis outcomes. As this study was originally a trial of high-vegetable, high-fruit, and low-fat intake interventions, we observed a wider range of dietary acid load than other cohorts. This study assessed pack-years of smoking, which can better evaluate past smoking intensity than smoking status. The large sample size provided us with sufficient power to adjust for multiple covariates. However, this study also has limitations. This cohort’s follow-up time was relatively short, and this cohort was comprised predominantly of White women, which will not allow us to examine long-term impacts or generalize our results to other ethnic groups. There was also the possibility of volunteer bias, as the women in the WHEL cohort were highly motivated, health-conscious breast cancer survivors. HER2 status was a not available for the WHEL participants, which may have resulted in residual confounding. WHEL participants were enrolled up to 4 years after their initial diagnosis; thus, the study may not represent breast cancer patients who recur shortly after diagnosis.Current dietary guidelines, such as the American Cancer Society’s (ACS) dietary guidelines for breast cancer survivors, do not include dietary acid load and specific dietary guidelines for former smokers ,64. Incr


Genetic mutations and polymorphisms play an important role in the transformation of primary cells to malignant cells as it may lead to disturbance of vital pathways regulating cell cycle, DNA damage repair, and apoptosis. In this study, we genotyped single nucleotide polymorphisms (SNPs) which were predicted to affect certain pathways and to increase the risk of breast cancer. SNPs  were then genotyped by TaqMan genotyping assay and the allele and genotype distribution was compared. The study included 81 Saudi breast cancer patients and 100 matching healthy controls from the Eastern Province in Saudi Arabia. The following SNPs  was T=0.17, A=0.28, A=0.22, and G=0.16 respectively. The G allele of the SNP rs3168891 was significantly associated with increased breast cancer risk (P = 0.00001) while the T allele of the same locus was associated with reduced risk of breast cancer in both heterozygous and homozygous states. The T allele of SNP rs2229714 which is located in the RPS6KA1 gene was also significantly associated with the increased risk of breast cancer. However, the rs2899849 SNP located in the Integrin beta-1 (ITGB1) gene was not associated with the increased risk of breast cancer in our study population. Haplotype analysis revealed the presence of three risk haplotypes that increases the risk of breast cancer . The minor allele frequency of the following We showed that three, previously untested, SNPs are associated with increased risk of breast cancer in our population. This may be added to the list of factors involved in breast cancer risk assessment studies. The benefit and the utility of the in-silico prediction of disease risk factors and their genetic association had been demonstrated in this study, yet the predicted risk alleles have to be tested in clinical studies. BRCA1 and BRCA2 among others . Genetic mutations and polymorphisms in the human genome could also be linked to the risk of sporadic breast cancer especially in those genes related to cell development and differentiation. One of the early attempts to investigate the correlation between p21 and bcl2 SNPs with susceptibility to breast cancer showed that p21 was more commonly expressed in breast cancer patients and bcl2 polymorphism was likely to be associated with breast cancer risk . Interestingly, hundreds of SNPs have been associated with breast cancer risk so far . The latest Global Cancer statistics indicates that breast cancer was expected to account for a quarter of cancer cases amongst women in 2018 . Genetic predisposition plays a crucial role in breast cancer development. Up to 10% of breast cancer cases can be attributed to hereditary factors comprising personal or family history of breast or ovarian cancer and inherited gene mutations such as Genome-wide association studies (GWAS) facilitated the identification of a large number of breast cancer susceptibility alleles with a much lower size effect . Although GWAS studies have shed light on the discovery and validation of novel disease-causing gene mutations and polymorphisms, it remains a challenge to prove the link between the identified risk allele and the biological mechanism of its involvement in the disease process . RPS6KA1, ITGB1, and MBIP genes, which modify the growth hormone signaling, PTEN pathway, and the mitogen-activated protein kinase (MAPK) pathways respectively .Since the transformation of breast epithelial cells to malignant cells involves the disturbance of vital pathways regulating cell cycle, DNA damage repair and apoptosis, Lee et al. designed software to identify the causal SNP from GWAS and their candidate affected pathway in an attempt to link the causal SNPs to a hypothetical mechanism of the disease . They identified several candidate SNPs among which four are with very strong putative biological mechanisms located in BRCA1 gene and the VEGF -2578AA genotype were found to be associated with increased risk of breast cancer in among Saudi females . While other allele variants such as the progesterone receptor gene mutations and the microRNA-423 T allele were associated with advanced stage and metastasis of breast cancer . A previous study from Saudi Arabia showed that breast cancer was the ninth prominent cause of death in women . In contrast to the global decrease in Breast cancer mortality, the incidence of breast cancer has shown a steady incline in Saudi Arabia, with the Eastern province reporting the highest incidence in the country . Previous studies have found an association of breast cancer with multiple variants and SNPs among the Saudi population. A mutation variant within the 3’-untranslated region (3’-UTR) of the rs3168891, rs2899849, rs2230394, rs2229714) and breast cancer risk among the Saudi population. These SNPs have been previously identified in GWAS in the following genes RPS6KA1 (rs2229714), ITGB1 (rs2230394), and MBIP (rs3168891 and rs2899849) . These SNPs are expected to play a role in pathways that are vital for cell survival and development. We sought in this study to identify the correlation between SNPs  in Alkhobar for interventional treatment or follow up. The targeted control group was Saudi females over the age of 40 years with no history of breast cancer or familial history of breast cancer up to second-grade relatives.Informed consent was obtained from all patients and control individuals. The ethical approval for the study was obtained from the Institutional Review Board at Imam Abdulrahman Bin Faisal University (number IRB-UGS- 2017-01-092).DNA extractionoC till the time of analysis.Blood samples were collected in 3ml EDTA vacutainer tubes. DNA was then extracted using DNA extraction kit from Qiagen according to the instructions . The quality and quantity of extracted DNA were measured by NanoDrop 2000 spectrophotometer. DNA was stored at -20SNP genotypingFour SNPs  were selected from the publication which described and used the prediction software ICSNPathway to predict the pathway associated SNPs that may affect the risk of breast cancer from genome-wide association studies . The targeted SNPs were genotyped using TaqMan SNP genotyping Assay . The real time-polymerase chain reaction (RT-PCR) was performed in 25ul reactions and was run on the thermocycler 7500Fast from applied Biosystems  according to the manufacturer’s instruction. Statistical analysisSPSS software version 24 was used to calculate the Chi-square. Logistic regression was used to estimate the association between SNPs and the risk of breast cancer at a confidence interval of 95%. All studied SNPs were found to be consistent with Hardy-Weinberg equilibrium. Linkage disequilibrium (LD) and the risk of the possible haplotypes were analyzed using the HaploView software version 4.2. A P value of less than 0.05 was considered significant.Study populationEighty-one breast cancer female patients and one hundred control female individuals were recruited to the study. The age range for the patients was 26-79 years (median 50) at the time of diagnosis and for the control was 40-78 years (median 51). Patients’ diagnostic parameters are summarized in Association between the studied SNPs and Breast cancerrs2899849 SNP was associated with risk of breast cancer only in the homozygous state   while thus state . No assot cancer . The T at cancer .Association of studied SNPs with patient clinical parametersThere was no significant statistical association between any of the studied SNPs and other biometric parameters such as age at diagnosis, stage or grade of cancer, or the expression of hormone receptors. Haplotype analysisMBIP1 gene but not with any of the other SNPs.Analysis of the haplotypes in the study revealed that three haplotypes are associated with increased breast cancer risk in our study . The haprs1799950 SNP which is located in the BRCA1 gene . The TP53 gene, a tumor suppressor gene, is one of the most commonly downregulated genes among cancers in humans, including breast cancer . A study from Riyad revealed a significant association between R72P located in exon 4 of the TP53 gene and the early start of breast cancer among the Saudi population . In particular, the RP genotype was found to be protecting against breast cancer . However, all studies focus on genes involved in DNA repair machinery, while the transformation of breast epithelial cells to malignant cells may involve the disturbance of vital pathways regulating cell cycle and apoptosis. Lee et al. designed software called “ICSNPathway” to identify the causal SNP from GWAS and their candidate affected pathway in an attempt to link the causal SNPs to a hypothetical mechanism of the disease biology . They identified several candidate SNPs among which four are with very strong putative biological mechanisms located in RPS6KA1, ITGB1, and MBIP genes, which modify the growth hormone signaling, PTEN pathway, and the mitogen-activated protein kinase (MAPK) pathways respectively . Breast cancer is emerging as a common female cancer in Saudi Arabia, with a higher incidence reported in the Eastern Province. Multiple studies investigated the association of specific SNPs with breast cancer risk. A significant association was found between breast cancer and the GG genotype of the SNPs; rs3168891, rs2899849, rs2230394, and rs2229714 predicted by the ICSNPathway software with breast cancer risk in Saudi patients. It is important to note that none of these SNPs were previously described to be associated with the risk of cancer. This current study focuses on the correlation of the following  SNPs rs3168891 and rs2899849 are located adjacent to each other in the MBIP gene, which affects the MAPK pathway. Both SNPs were found to be associated with breast cancer in our study on both allele and genotype levels. The association was stronger with rs3168891. The association with rs2899849 seems to be influenced by rs3168891 because they are in linkage disequilibrium (LD) (Theium (LD) . MAPK paSNP rs2229714 was also significantly associated with breast cancer in our study. This SNP is located in the ribosomal protein S6 kinase A1 gene (RPS6KA1), which encodes a serine/threonine kinase. The RPS6KA1 is one of the S6 kinase family of proteins . The ribosomal S6 kinase family of proteins is the downstream targets of the MAPK pathway effectors and in their turn phosphorylate several regulatory proteins involved in cell growth and differentiation . Currently, several members of this family are considered promising targets for antiviral therapy . Furthermore, these proteins are implicated in growth hormone signaling which plays an important role in breast normal physiology through driving the differentiation of ductal epithelium and normal proliferation secretory functions of lobular epithelial cells and disruption of these functions would promote tumor formation .The PTEN and has04360 pathways. Both pathways are expected to play an important role in breast cancer . However, this SNP seems not to play an important role in the genetic predisposition of breast cancer in our population. Haplotype analysis showed that the most frequent haplotype in the study population indeed consists of the risk alleles of the three SNPs associated with breast cancer in the study (The rs2230394 was not associated with breast cancer in our study population. This SNP is located in the gene encoding integrin subunit beta 1 (ITGB1) and was expected to affect the he study .In conclusion, breast cancer is emerging as an alarming health burden in Saudi Arabia. Extensive studies should be directed towards identifying the factors that predispose to its increased incidence and discovering the pertinent risk factors. We showed that three previously untested SNPs are associated with breast cancer in our population. This may be added to the long list of factors involved in breast cancer risk assessment studies. The benefit of the utility of the software in the prediction of disease risk factors and the genetic association had been demonstrated in this study, yet, on a cautionary note, the predicted risk alleles have to be tested in clinical studies.


Perinatal factors have been associated with some adult health outcomes, but have not been well studied in young-onset breast cancer. We aimed to evaluate the association between young-onset breast cancer and perinatal exposures and to explore etiologic heterogeneity in the relationship between associated perinatal factors and estrogen receptor status of the tumor.We addressed this in a sister-matched case-control study. Cases were women who had been diagnosed with ductal carcinoma in situ or invasive breast cancer before the age of 50. Each case had a sister control who was free of breast cancer up to the same age at which her case sister developed the disease. The factors considered were self-reported and included the mother’s preeclampsia in that pregnancy, mother’s smoking in that pregnancy, gestational hypertension, prenatal diethylstilbestrol use, and gestational diabetes, as well as low birth weight (less than 5.5 pounds), high birth weight (greater than 8.8 pounds), short gestational length (less than 38 completed weeks), and being breastfed or being fed soy formula.In conditional logistic regression analyses, high birth weight  and preeclampsia  were positively associated with risk. The association with preeclampsia was stronger when the analysis was restricted to invasive breast cancer . We also used case-only analyses to assess etiologic heterogeneity for estrogen receptor (ER)-positive versus estrogen receptor-negative cancer. Women who were born to a preeclamptic pregnancy and later developed young-onset breast cancer were at increased odds for the ER-negative type .These results suggest that being born to a preeclamptic pregnancy may increase risk for young-onset breast cancer, especially for the ER-negative subtype. Breast cancer is common in the United States (US), with approximately one in eight women diagnosed during their lifetime . Inciden. He proposed that prenatal exposure to endogenous or exogenous hormones might influence the trajectory of breast cancer development later in life, implying that a critical window for carcinogenesis of the breast might begin during prenatal life. For example, intrauterine exposure to insulin-like growth factors has been proposed to have a role in breast cancer risk [According to a provocative hypothesis proposed by Trichopoulos in 1990, some breast cancers may etiologically originate in utero . He propcer risk . EvaluatOne example of this is preeclampsia, which may be associated with high maternal concentrations of androgens and low concentrations of estrogen , 7. BecaOther perinatal factors, such as birth weight and birth length, might reflect in utero exposure to growth hormones . Most ofEvaluating the role of rare perinatal factors such as preeclampsia and twin membership on young-onset breast cancer requires a large sample of women. Many of the previous studies of perinatal factors and breast cancer risk have used registry data , 27, 28 Participants in The Two Sister Study were enrolled between 2008 and 2010. Participants were eligible as cases if they had been diagnosed with breast cancer before 50 years of age and within the previous 4 years and had one or more unaffected sisters who had enrolled in the Sister Study (2003–2009). Sister Study participants had to be 35–74 years of age at the time of enrollment and had to live in the United States or Puerto Rico. Participants in the Sister Study had reported on their sister’s earlier breast cancer diagnosis at the time of their enrollment, and the list of families to target for recruitment into the Two Sister Study was based on that information. The sisters with a previous breast cancer diagnosis who appeared to be eligible were contacted through their participating sister, who was asked to send their affected sister a letter inviting their participation. Case sisters who then contacted the study and were found to be eligible were administered the same computer-assisted telephone interviews used in the Sister Study and another questionnaire about their breast cancer diagnosis. Their consent was requested for retrieval of their breast cancer medical record, which was used to collect pathological data on tumor characteristics (including estrogen receptor (ER) status). When medical records were not provided, we relied on self-report. We were able to obtain medical records or pathology reports to confirm the diagnosis for 88.7% breast cancer cases, and we relied on self-report for the remaining 11.3%. Very high positive predictive values for self-reported breast cancer (> 99.0%) have been reported for incident breast cancer cases in the Sister Study .Because of the sampling design, controls were often older than their case sister. To address this potential source of bias, we restricted our controls to participants who had remained disease-free (breast cancer) up to the age at which their case sister was diagnosed with breast cancer, so that either could have been the case. The sample for the Two Sister Study was augmented by recruiting additional incident young-onset cases that were diagnosed during follow-up of the Sister Study cohort through September 2016, netting an additional 243 cases, many of whom also had an unaffected Sister Study sister who was also being followed. Those that did not have a control sister were nonetheless included in the case-only analyses to study etiologic heterogeneity. In total, 1759 cases and 1672 sister controls  were enrolled in the Two Sister Study.Perinatal factors obtained via questionnaire at study enrollment included participant’s birth weight, used to identify those with high birth weight (≥ 8.8 pounds) or low birth weight (≤ 5.5 pounds); participant’s gestational age at birth, used to categorize them as less than 38 weeks or not; and whether their gestation was complicated by diethylstilbestrol exposure, preeclampsia, eclampsia, gestational hypertension, or gestational diabetes. We dichotomized gestational age into < 38 weeks or ≥ 38 weeks because the question women had been asked was as follows: Were you born within 1 week of your mother’s due date, more than 1 week before her due date, or more than 1 week after her due date? If the participant reported that she was born early or late, she was further asked the number of weeks . We also ascertained some postnatal exposures: having been breastfed as an infant and history of soy formula consumption during infancy.Data on perinatal and postnatal exposures were from a “Family History” questionnaire mailed to all participants at enrollment. Participants were encouraged to speak to their mothers (or other family members) to improve accuracy and most questions allowed qualified answers . In addition, we carried out a validation study (manuscript in preparation) in which mothers of participants who were under 60 at enrollment were asked to complete the same questionnaire. Agreement was good for most variables, with kappas ranging from 0.5 to 0.9, but mother’s data was also retrospectively reported. Examiners retrieved these questionnaires from Sister Study participants when conducting home visits for blood collection. Because most cases were identified retrospectively and had no home visit, they had to mail in their questionnaire. The cases consequently had more missing data than controls.Data on participants’ demographics  and height and weight  were obtained at enrollment.N = 2717, 1250 cases and 1467 controls) and stratifying by ER status.We provide descriptive statistics for cases and controls separately for each pre- and perinatal factor. Since there was a small fraction of data missing for some of the covariates and exposures of interest, we conducted a complete case analysis . We usedWe also conducted case-only analysis to explore etiologic heterogeneity in the relationship between perinatal factors and the ER status of the breast tumor . The casTable In general, we observed weak associations between perinatal factors and breast cancer Table . We exclTable In the Two Sister Study, we observed that maternal preeclampsia and high birth weight showed some evidence of a positive association with young-onset breast cancer. We observed a weak inverse association between having been breastfed during infancy and young-onset breast cancer. Additionally, women born from pregnancies with preeclampsia or gestational hypertension who did develop young-onset breast cancer appeared to be at increased odds for the difficult-to-treat ER-negative subtype. Restricting attention to invasive breast cancer strengthened those associations.Among the studies that have assessed the relationship between birth weight and young-onset breast cancer –23, 33, It has been proposed that the biological mechanism that underlies this birth weight association encompasses a complex interplay between the influences of growth factors and steroid hormones on mammary cells and epigenetic changes . MaternaGestational length has been studied in association with risk of breast cancer , 44. MatInconsistent results have been reported in previous studies assessing the association between pre-eclampsia and daughter’s breast cancer risk , 12, 14.The results from our study are subject to some limitations. First, exposures were self-reported, which could have led to misclassification of the exposures. Also, limited numbers with the exposure prevented us from assessing gestational diabetes as a potential exposure.Our results are strengthened by our study design, and a large number of young-onset breast cancer cases contributing to a case-only analysis. We had sufficient numbers to evaluate the association of perinatal factors with ER status among women affected by young-onset breast cancer. Finally, the sister-matched design helped us control for unmeasured confounding.In summary, in the present study, we observed small and imprecisely estimated positive associations between two prenatal exposures (high birth weight and being gestated in a preeclamptic pregnancy) and young-onset breast cancer. These results provide indirect support for the hypothesis that prenatal exposures affect the subsequent risk of breast cancer in young women. In addition, our results suggest that being born to a preeclamptic pregnancy may increase risk for young-onset breast cancer, especially for the ER-negative subtype. Future epidemiological research on the connection between maternal hypertensive disorders, birth weight, and young-onset breast cancer is warranted to help us gain a better understanding of the etiology of the disease.Additional file 1.


Breast cancer is the most common cancer among women in Malaysia, and the incidence of 31.1 per 100,000 population is comparatively higher than other Southeast Asian countries. Diagnosis tends to occur at later stages which may be due, partly, to inadequate knowledge about warning signs and symptoms. Therefore, this study investigated the validity and reliability of a UK-developed measure in the context of assessing women’s awareness of breast cancer in Malaysia. This study aimed to translate, adapt and validate the internationally recognised Breast Cancer Awareness Measure (B-CAM) into the Malay language. The original B-CAM (Cancer Research UK) was forward and backward translated and content validation was ascertained. Face validity (n=30), test-retest reliability (n=50) and the internal consistency of the B-CAM-M  were assessed in a community sample of adults (n=251) in 2018. The translated B-CAM-M was validated by an expert panel. The Item-Content Validity Index ranged from .83 to 1.00. The results from the survey (n=251) indicated that the B-CAM-M was well received by Malay-speaking women across the main ethnic groups . Cronbach alpha scores for the knowledge about breast cancer symptoms (0.83) and the barriers to healthcare seeking items (0.75) were high. Test-retest reliability  with 50 randomly selected participants from the community survey produced intra-class correlations ranging from 0.39 to 0.69. The Malay-version, the B-CAM-M, is a culturally acceptable, valid and reliable assessment tool with which to measure breast cancer awareness among Malay-speaking women.  Breast cancer is the commonest cancer among women worldwide . Similarly, it is the leading cause of cancer among women in Malaysia with an incidence of 31.1 per 100,000 population . The incidence in Malaysia is higher compared to neighbouring Southeast Asia countries such as Laos, Thailand, Cambodia, Myanmar and Viet Nam . Breast cancer is a curable disease if diagnosed at an early stage. However, breast cancer is often diagnosed late, due to lack of awareness, denial, negative beliefs about cancer, reliance on alternative medicine and unawareness about breast cancer screening programs . There is a need to raise awareness about breast cancer symptoms in Malaysia in order to counter delayed diagnosis. Cancer awareness campaigns intend to transform the attitudes and behaviours of the public and to change their perspectives on traditional and social norms . A number of campaigns and awareness raising events have been organised over the years in Malaysia, however, systematic evaluation of these awareness raising efforts is lacking . There is a need to create a robust evaluation framework to assess cancer awareness in Malaysia. However, a validated assessment tool for use in Malaysia is lacking. A validated tool in Malay language would help to investigate women’s awareness about signs and symptoms and risk factors for breast cancer and the utilization of screening programs as well as contribute to the evaluation of the effectiveness of interventions. The Breast Cancer Awareness Measure (B-CAM) is the internationally recognised measure that was developed  to measure breast cancer awareness in the UK . Since then, it has been translated and validated in Arabic, Persian and Swahili . ObjectiveThis study aimed to translate, adapt and validate the internationally recognised B-CAM for use in Malaysia. In addition, findings regarding breast cancer awareness in Malaysia as assessed with B-CAM-M  affords potential to draw meaningful international comparison with other countries.The questionnaire validation was based on the original B-CAM developed by Cancer Research UK, King’s College London and University College London . The permission for the translation and cultural adaptation of B-CAM was granted by the developer (CRUK). Ethical approval was obtained from the University of Malaya Medical Centre (UMMC) Ethics Committee (Ref No. 2016126-4668) and Melaka-Manipal Medical College, Research Ethics Committee. Study design and participantsThe study was conducted in Melaka city, a historical and the fifth largest city in Malaysia. Data was collected between April and May 2018. The sample size of 250 participants was estimated by using the formula of N/p ≥10   focusing on the symptoms and barrier domain items. Participants were recruited by using quota sampling with an equal number of women from Malay, Chinese and Indian ethnic backgrounds. Inclusion criteria comprised: women who were aged 40 years and above who could answer independently and who provided written consent. Women who were diagnosed with breast cancer or who were working in health care were excluded. Adaptation of the B-CAM questionnaire The B-CAM Toolkit was validated in the UK . An expert panel comprising public health specialists (n=2), an expert in instrument development (n=1) and translation and language specialists (n=2) reviewed critically the B-CAM and then adapted and modified the content and response format to be culturally sensitive and appropriate to Malaysians and the local health care system. In addition, the experts  amended the content so that it would be acceptable and clear to the ethnic groups in Malaysia. All seven domains of the B-CAM were translated into the Malay language except ‘Knowledge of the NHS Breast Screening Programme’ domain, as this was not applicable to the Malaysian health care system. Breast cancer screening guidelines in Malaysia recommend annual clinical breast examination (CBE) for women aged 40 years or older and biennial mammogram for women aged between 50 and 74 years . Therefore, the following two items were added to the modified version in line with the Malaysia breast cancer screening program ‘Were your breasts examined by trained health professionals such as doctors or nurses within one year?’ and ‘Have you had a breast cancer screening (mammogram) in the last two years?’. In the risk factor domain, the following risk factors were explained: one alcohol unit, having children early on in life, early menarche, late menopause and moderate physical activity. The other domains were translated into Malay without changing the original items. Linguistic validity The English version of the B-CAM was translated to Malay by using the forward and backward translation method following the World Health Organisation (WHO) guideline . The professional translator translated the conceptual equivalent of the questions by using culturally acceptable Malay words and phrases. Three rounds of harmonization checked the content, concepts and discrepancies between the original English version and the forward-translated Malay version. Firstly, the translated Malay version was reviewed by the five-person independent bilingual expert panel. Secondly, a group discussion was conducted with eight bilingual experts. Lastly, harmonisation was completed via appraisals that were conducted by a bilingual expert and an occupational therapist educator. The final Malay version was backward translated to English by a bilingual academician. The backward translated English version of the questions and the original English versions were checked for conceptual and cultural equivalence. Content validity Content validity was examined in consultation with the expert panel. The content validation experts were independent and different from those experts who participated in the linguistic validation. The English and Malay versions of the questionnaire were assessed by six bilingual experts who were oncologist (n= 1), a psychiatric doctor (n=1) and primary health care physicians (n= 4) and the Item-Content Validity Index (I-CVI) was calculated based on their ratings. The items which achieved I-CVI of ≥ .85 were included in the questionnaire. Face validityFace validation was carried out with 30 participants, 10 in each ethnic group . At the end of the interview the participants were asked questions regarding the quality, readability and understanding the questionnaire. Test-retest reliabilityReproducibility of the B-CAM-M was assessed through the test-retest method. The sample size was calculated with alpha of 0.05 and power 90%, to detect the ICC value of .30 targeting for three awareness scores, while considering 25% of attrition rate  The test-retest reliability was conducted with 50 randomly selected participants from the community survey. For the second visit, the enumerators called the selected participants and arranged appointments. These participants were interviewed for the second time by using the same questionnaire with an interval of 2 weeks after the first visit. Scoring and statistical analysisThe B-CAM-M included 11 items about warning signs and symptoms of breast cancer. Nine out of 11 items asked about symptoms other than lumps. Participants who responded “Yes” to five or more questions about non-lump symptoms were considered to be “aware of the warning signs” of breast cancer ; and participants who responded that, “50 year-old women are more likely to get breast cancer in the next year” were considered to be aware of age-related risk. Reported breast self-examination once a week or once a month was considered to denote awareness of the value of self-examination for any changes in the breast. Therefore, the total awareness score ranged from 0 to 3 . The data were analysed using PASW (version 18) statistical software. Descriptive analysis was carried out for the participants’ demographic variables. The content validation was assessed by computing the Item- Content Validity Index (I-CVI). Reliability analysis was conducted by the test-retest method by computing the intraclass correlation (ICC) and internal consistency by Cronbach’s alpha. Socio-demographic characteristicsA total of 281 participants  were recruited for this study. The ethnic distribution of participants in the community survey (n=251) was 33.9% Malay, 33.5% Chinese and 32.6% Indian . ApproxiAdaptation and Linguistic validation Regarding knowledge about signs and symptoms items, the experts advised use of the word “ketulan” rather than “benjolan” for the most accurate translation of ‘breast lump’. The English phrase of “puckering or dimpling of the breast skin”was amended to read as “kedutan atau kulit mengelupas di kulit payudara” in order to be appropriate and accurate in Malay language. In the risk factors domain, “Having children later on in life or not at all” was amended to read as “Melahirkan anak pada usia yang lanjut atau tiada pernah melahirkan anak”. Content validation According to the protocol, we invited nine bilingual experts who were public health specialist, oncologists and clinicians. However, only six experts responded and provided their opinions about the items for content validation. The I-CVI was calculated based on the rating for each item that was provided by the experts. The I-CVI of all 50 items in this questionnaire ranged between 0.83 - 1.00. Face validation Thirty participants, ten from each ethnic group, were interview for the face validation. The participants responded that all the questions were understandable, acceptable in Malaysian culture and they had no objections to answer all the questions. Some participants noted that “Worried about wasting the doctor’s time” might not be a barrier in this context as the community believed that devoting time for a patient was a doctor’s responsibility. Distribution of responses to the B-CAM-M A total of 251 participants were recruited for the face-to-face interview and asked to complete the B-CAM-M. The majority of participant were aware of lump as a symptom (90%), discharge (80.5%) and pain (75.3%). ‘Worrying about what the doctor might find may stop me from going to the doctor’ was the commonest barrier reported (37.4%). Approximately half of participants admitted that they performed regular breast self-examination, i.e. once a week or once a month (52.2 %) .Test-retest reliability Open-ended questions were excluded from the test-retest reliability analysis. In the individual item analysis, 19 items had an ICC value of < 0.4, 15 items had an ICC value between 0.41 - 0.69 and four items had an ICC value >0.7. The composite score for breast cancer awareness was calculated for the participants and the ICC value for test-retest reliability analysis was 0.79 . Internal consistencyThe internal consistency was assessed by calculating Cronbach’s alpha. The alpha coefficient for the items regarding knowledge about breast cancer symptoms was 0.83 and 0.75 for items regarding barriers to healthcare seeking . This study developed a tool to measure awareness on breast cancer adopting a tool used in UK, and adapting to suit the local culture and context. The validation process showed that the majority of items in the original B-CAM were applicable to Malaysians suitably translated and adapted and only a small number of items, specifically referring to the local health care system were changed. A rigorous linguistic validation amended the initial forward-translated measure to become a more accurate and culturally acceptable instrument. Since Malaysia is interwoven with a multicultural and multilingual community, input from Malay Chinese and Malay Indian during the process of linguistic validation were ascertained in order to prepare an acceptable and understandable questionnaire tool for different ethnic groups. Bahasa Malaysia (BM) is the official language in Malaysia, and therefore the translation process was focused on the BM. Meanwhile, the Malay terms were carefully checked and revised by the experts to maintain the accuracy of the meaning that is conveyed in the original B-CAM and to avoid the cultural sensitivity.The translated B-CAM-M was reviewed by an expert panel of oncologist, psychiatric doctor and primary health care physicians, and they provided ratings regarding, for example, the relevancy of the items and advice about how to establish content validity. According to Lynn (1986), an item scored .85 was considered to be acceptable for inclusion in a questionnaire . All 50 items in the modified and translated B-CAM indicated that they met this criterion and that they were relevant to be included in the tool. During the face validation process, the women’s responses and very low rate of missing data proved that the B-CAM-M questions were clear and that there was no objection, culturally or otherwise, to answering the questions. The team decided to retain the item about ‘wasting a doctor’s time’ in order to test its value in the community survey. The analysis of the community survey data revealed that the item had the lowest score among all the barrier items. The internal reliability of the B-CAM-M was measured by Cronbach’s alpha, and the findings suggested that there was high internal consistency between the items regarding knowledge about breast cancer symptoms (.83). This finding is in keeping with previous translation and validation studies of B-CAM in Arabic , and Persian languages  . Test-retest reliability was checked by conducting a second-time interview with 50 participants after two weeks. According to Fleiss (1986), the ICC value of .40 to .75 is considered to be “fair to good” . Some items from the domains of symptoms, risk factors and barriers had ICC values lower than 0.4 and discrepancies mainly occurred with respect to participants’ knowledge and barriers for health care seeking. In comparison, a higher ICC value of 0.84 was reported for warning signs in the Persian version . A possible explanation for the lower ICC value might be related to the fact that although participants were not informed that they would be asked the same questions, some participants may have searched for information about breast cancer from the media and websites due to their interest and curiosity being stimulated by the first interview. During the validation process of the original B-CAM, Linsell et al reported lower ICC values ranging from 0.28 – 0.68 as a reason for the improvement in scoring the second time of applying the questionnaire . None the less, the overall knowledge score, age-related risk score and breast self-examination score of B-CAM-M had moderate to good ICC (0.39 to 0.69). Therefore, all the items were maintained in the questionnaire after the analysis. Among the Malay population, the Bahasa Malaysia can also be different because of dialects in different states. Our validation study was conducted with Malay, Indian and Chinese ethnic groups in Malaysia, and therefore indicates that the questionnaire measure appears to be acceptable across the different Malay-speaking ethnic groups. The instrument was considered to be easy to understand though more than 70% of participants completed secondary education and, so, it is unclear how well the questionnaire would work with people who have not completed secondary education or who have low levels of literacy. However, the questionnaire measure appeared to work well with people across all income brackets including low-income earners.A limitation of this study was that the participants were recruited by quota sampling method. However, quota sampling provided to recruit approximately equal number of participants from the three major ethnic groups  in Malaysia. In conclusion, the B-CAM-M is culturally acceptable and valid among the Malay-speaking different ethnic groups in Malaysia. This tool may be used to provide a valid assessment of breast cancer awareness in the Malaysian context. Understanding breast cancer awareness is important for guiding public health authorities and policy makers to implement effective strategies to improve awareness of symptoms and utilisation of breast cancer screening services. In addition, this tool will be useful for the evaluation of future awareness raising interventions and other public health programs. Implications for PracticeThe culturally acceptable, valid and reliable B-CAM-M tool could help to assess the breast cancer awareness level in the community and to evaluate the effectiveness of awareness raising interventions.


To clarify the limitations of mammography screening for women with dense breasts, we examined breast density and its effects on screening results. We performed a cross-sectional, observational study on women who underwent mammography. Data from the National Cancer Screening Program(NCSP) from 2009 to 2013 were used. The study population consisted of participants with high breast density. We used a logistic regression analysis to evaluate the relationships between breast density and reproductive factors and screening results according to menopause status. High breast density was reported for 57.5% of all participants . Screening results indicated breast density of <25%, 25-50%, 51-75%, and ≥76% for 16.4%, 26.3%, 37.8%, and 19.5%, respectively, of participants. According to the screening results, high breast density was correlated with high deferment and recall rates. Reproductive factors, especially parity, breastfeeding, and use of oral contraceptives, had consistent effects on screening results of premenopausal and postmenopausal women. Regardless of menopausal status, age, early onset of menarche (15 years or younger), fewer live births (≤1 birth), and previous benign breast disease were correlated with increased breast density. In postmenopausal women, early-onset menopause and longer-term hormone replacement therapy (≥2 years) also independently increased breast density.Breast density influenced screening results, which could increase the rate of recall. Breast density was also influenced by reproductive factors, with patterns similar to those of breast cancer risk, regardless of menopausal status. We need to identify high-risk women with high density who would probably benefit from supplemental breast cancer screening. Breast density refers to the percentage of dense tissue in the entire breast. Depending on its density, fibroglandular mammary tissue has different characteristics and results in different compositions on radiographs . Breast density is an independent predictor of breast cancer risk. Based on studies of Caucasian women living in Western countries, women with more than 75% dense tissue are at four- to six-times higher risk of breast cancer than those without dense breast tissue . Although more Asian women than Western women have dense breasts, the incidence of breast cancer is relatively lower among Asian women.Bae et al., (2016) reported an association between breast density on mammography and breast cancer risk for Asian women, regardless of menopausal status. However, Rajaram et al., (2017) suggested that premenopausal Asian and Caucasian women are at similar risk of breast cancer regardless of different breast density. Furthermore, several reproductive and lifestyle factors that influence the risk of breast cancer have been consistently associated with breast density .In Korea, the National Breast Cancer Screening Program (NBSCP) conducts mammography for women older than 40 years biannually. Although the rate of participation has not increased since 2016, the rate of advanced breast cancer detection has not decreased. The false-negative rate of cancer detection by mammography is an unresolved problem; however, the early detection rate of cancer has increased . Furthermore, South Korea has a low birth rate. Most women marry at a later age, resulting in lower childbirth rates .To maintain the participation of Korean women in breast cancer screening and to improve its efficiency, it is necessary to clarify how factors for Korean could influence the sensitivity of breast screening including breast density. It is also necessary to clarify how breast density affects breast cancer screening and further stratified screening strategies for women at high risk or average- risk with dense breasts. This topic have been discussed in Asian country where the incidence of breast cancer has been rapidly increasing, but large-scaled population based study in these area were needed. Therefore, we conducted a cross-sectional analysis of the Korean National Screening Program from 2009 to 2013 to determine the distribution of breast density and its correlation with reproductive factors and screening results.Study populationThis cross-sectional observational study was conducted using data maintained by the NCSP during 2009 to 2013. The NCSP recommends that all Korean women 40 years and older should undergo mammography biannually. The baseline cohort comprised 9,469,234 women 40 years and older who underwent screening by the NCSP between 2009 and 2013. We excluded 5907 participants with a previous diagnosis of breast cancer and 6,051,915 participants with unknown breast density according to mammography. Therefore, a total of 3,417,319 Korean women aged 40 years or older were included, and their 7,265,584 available screening results were analyzed.The current study collected data from the NCSP database, which included information regarding participant demographics and screening results. Written informed consent was received from participants for the collection of their screening results. We collected data regularly from the NHIS. The need for informed consent for this specific study was waived because the NCSP database is quite large. After obtaining permission from the Ministry of Health and Welfare, the investigators used data maintained and de-identified by the NHIS. The study was approved by the Institutional Review Board of Inje University Haeundae Paik Hospital in Korea (IRB no. 2017-07-639-009).Categorization of mammographic density and screening resultsThe most commonly used tool for assessing breast density is the breast imaging reporting and data system (BI-RADS). Level one indicates a breast comprising almost entirely fatty tissue with 5-24% tissue density. Level two indicates breast tissue comprising scattered areas with 25-49% density and fatty tissue. Level three indicates heterogenous density with 50-75% tissue density and areas of non-dense tissue. Finally, level four indicates ≥75% tissue density with very little to no fatty tissue and is designated as extremely dense. Women with heterogeneously dense or extremely dense breast tissue are considered to have high breast density .Screening results were sent to participants and classified into four qualitative categories: normal, benign breast disease, suspected malignancy, and deferred. A percentage of MDMD was not included in the report of screening results. However, the report indicated the limitation caused by breast density and indicated that participants with more than 50% dense breast tissue should revisit the screening center for further examination, even if the results were normal or if they had any breast cancer symptoms such as breast pain, lump, or nipple discharge. Women with suspected malignancy and those who were deferred were also encouraged to undergo further screening. Selected reproductive risk factorsThe NCSP also collected other breast cancer risk factors from the self-reports of participants. Collected data included current history of benign breast disease , parity , duration of breastfeeding , age at menarche , duration of oral contraceptive use , age at menopause , and hormonal replacement therapy use .Statistical analysisBecause the associations between reproductive risk factors and screening results varied according to the menopausal status, and because PMD was lower for postmenopausal women compared to premenopausal women, all analyses were conducted separately for premenopausal and postmenopausal women . The chi-square test was used to evaluate the correlation between screening results and personal characteristics. Logistic regression was conducted to determine personal risk factors for breast density. Analyses were conducted using SAS version 9.4 , and results were considered statistically significant if P ≤ 0.05.Characteristics of participantsAmong 7,265,584 screening results, 16.4%, 26.3%, 37.8%, and 19.5% had level one, level two, level three, and level four breast density, respectively. More than 50% breast density was present in 57.3% of women Suppl. .Screening results and recall rates according to breast density2 = 262,313; P < 0.001)  . Recall < 0.001) .Associations between reproductive factors and screening resultsBreast density and reproductive risk factors influenced the screening results, but the patterns of influence were different. Older age decreased the deferment rate instead of increasing the normal and suspected malignancy rates. Nulliparous women tended to be recalled because of a relatively high rate of deferment or suspected malignancy. Longer breastfeeding durations (≥12 months) increased the proportion of participants with normal results and suspected malignancy compared to women who did not breastfeed . Parity,Breast density and reproductive factors for premenopausal and postmenopausal womenFor premenopausal participants, younger age at screening was correlated with high breast density. Early onset of menarche (15 years or younger), nulliparity, and absence of previous benign breast disease was correlated with high breast density. Never breastfeeding was correlated with high breast density. The multivariate analysis indicated that all these reproductive factors were independently correlated with breast density . For posDuring our retrospective observational study performed at NCSP, we found that breast density influenced screening results and could increase the recall rate. Breast density was also influenced by reproductive factors, with patterns similar to those of breast cancer risk. This finding was consistent regardless of menopausal status.Parity was significantly and inversely associated with the percentage of collagen in the breast. Smaller breast size was reported to be associated with more collagen and glandular tissue .HRT, such as combination estrogen and progesterone, is known to increase breast density, but estrogen therapy alone does not significantly increase breast density. Previous reports have found a positive correlation between breast density and HRT that resembles the well-studied relationship between HRT and breast cancer risk .In interventional trials, breast density has been proposed as a potential surrogate marker of breast cancer risk . However, the extent to which reproductive factors influence breast cancer risk through their effects on breast density and the extent to which they influence breast cancer risk through other pathways are unknown . Additionally, the underlying mechanisms of the positive association between breast density and the risk of breast cancer remain to be elucidated.Racial differences have been considered an important factor when determining breast density. Asian women tend to have higher breast density and African American women tend to have lower breast density than Caucasian women; furthermore, breast density was found to be significantly higher in Chinese women .Supplemental imaging modalities have been used to improve examination sensitivity because they can detect breast cancer at an early stage on the basis of the mass shape, even in the dense parenchyma of premenopausal women. Some observational studies have questioned which supplemental ultrasonography method can increase the frequency of interval cancers in women with dense breasts . Two randomized clinical trials using supplemental imaging have demonstrated a significant reduction in interval breast cancer rates. The Japan Strategic Anti-cancer Randomized Trial (J-START) investigated the efficacy of adjunctive ultrasonography, thereby contributing to the understanding of the efficacy of adjunctive ultrasonography for breast cancer screening of women 40 to 49 years by reporting that adjunctive ultrasonography increases the sensitivity and early detection rates of cancers. The Dense Tissue and Early Breast Neoplasm Screening Trial reported fewer cases of interval breast cancer with biennial supplemental magnetic resonance imaging plus mammography than with mammography alone for women 50 to 74 years of age with extremely dense breasts . Studies have not evaluated whether supplemental imaging reduces advanced breast cancer rates or breast cancer mortality. Another modality, digital breast tomosynthesis (DBT), reduced the recall rate significantly for women with dense breasts, indicating that all women may benefit from improved screening with DBT regardless of age or breast density . After implementation of DBT for screening with digital mammography, the invasive cancer detection rate increased from 2.9 to 4.1 per 1,000 participants, a relative increase of 41%, whereas the detection rate of in situ cancer was unchanged for 1.4 per 1,000 participants.Melniknow et al. identified one randomized controlled trial that compared the potential danger of different breast density notifications for a control group; however, they reported that there have been no studies of the potential danger of assigning different breast density classifications to sequential examinations . The dangers of supplemental imaging for women with dense breasts include higher recall rates and higher biopsy rates. Long-term follow-up is needed to assess whether a combined approach could reduce the frequency of advanced breast cancers and breast cancer mortality. This approach has not been conducted in Korea, and most participants ignored the screening results because of low sensitivity or had higher recall rates and biopsy rates.Nelson et al., (2012) found that the only risk factor to confer a two-fold higher relative risk among women 40 to 49 years old was family history in a first-degree relative or extremely dense breasts. Also, they have proposed risk-based screening, such as selective screening of women 40 to 49 years old who are at increased risk of breast cancer, as a means of improving the benefit-to-risk ratio using a meta-analysis. However, a recent retrospective study suggested that 88% and 86% of women with screening-detected breast cancer did not have a very strong family history of breast cancer or extremely dense breast tissue, respectively . Among patients with screening-detected malignancies, 76% did not have a very strong family history or extremely dense breasts . Kerlikowske et al., (2019) reported the dangers of frequent screening and supplemental imaging and suggested that women aged 50 to 74 years undergoing screening mammography should be informed of their clinical automated BI-RADS breast density and their overall breast cancer risk to determine whether they have dense breasts and are at low risk of breast cancer so they can consider routine biennial screening. Conversely, women with dense breasts and those at high risk of breast cancer due to strong risk factors or those with dense or non-dense breasts and very strong risk factors may want to consider annual screening and supplemental imaging.In Korea, because of the lower cost of ultrasonography, widespread use of private insurance, and confidence in ultrasonography, most women tend to supplement breast examinations with ultrasonography. Supplemental testing could consistently find additional breast cancers not identified by mammography, but it could also increase false-positive results. Additionally, no studies have examined the impact of supplemental screening on breast cancer recurrence rates or mortality for women with dense breasts in Korea.The present study is the first report about MD with breast screening mammography from NCSP data. Almost 57.3% of Korean women had more than 50% breast density by mammography. Because a high rate of high breast density was found in the NCSP, there occurred two major issues. First, high breast density decreases the detection sensitivity of mammography screening and increases the recall rate in NCSP. Second, breast density was probably known as an independent risk factor for breast cancer, and it needs to evaluate who should be notified about their MD. In Korea, high breast density is an issue when attempting to detect breast cancer, it has started to be known as an independent risk factor of breast cancer for these women from several studies . Furthermore, when adjusted for age according to unpublished data, breast density was found to have a significant effect on breast screening results and the breast cancer diagnosis. However, we did not disclose these data because data from the NHIS were categorized and not continuous; therefore, they could not be verified.In conclusion, high breast density was found in 57.5% of the study participants. Breast density influenced the screening results, which could lead to increased recall rates. Breast density was also influenced by reproductive factors, with patterns similar to those of breast cancer risk, regardless of menopausal status. It is possible that breast density could be a limitation of breast cancer screening in Korea. We need to identify high-risk women who would probably benefit from supplemental breast cancer screening using population-based screening cohort. Furthermore, women with both normal mammography results and dense breasts need to be discussed to undergo supplemental testing according to their risk of breast cancer in a clinical practice.


Background parenchymal enhancement (BPE) of normal tissue at breast magnetic resonance imaging is suggested to be an independent risk factor for breast cancer. Its association with established risk factors for breast cancer is not fully investigated.To study the association between BPE and risk factors for breast cancer in a healthy, non-high-risk screening population.We measured BPE and mammographic density and used data from self-reported questionnaires in 214 healthy women aged 43–74 years. We estimated odds ratios for the univariable association between BPE and risk factors. We then fitted an adjusted model using logistic regression to evaluate associations between BPE (high vs. low) and risk factors, including mammographic breast density.P = 0.002) and BMI (P = 0.03). We did find a significant association between systemic progesterone medication and BPE, but due to small numbers, the results should be interpreted with caution. The adjusted odds ratio for high BPE was 3.1 among women with density D (compared to B) and 2.1 for density C (compared to B). However, the association between high BPE and density was not statistically significant. We did not find statistically significant associations with any other risk factors.The majority of women had low BPE (84%). In a multivariable model, we found statistically significant associations between BPE and age (Our study confirmed the known association of BPE with age and BMI. Although our results show a higher likelihood for high BPE with increasing levels of mammographic density, the association was not statistically significant. The American College of Radiology Breast Imaging-Reporting and Data System (BI-RADS) Atlas 5th Edition classifies BPE into four categories  according to the proportion of fibro-glandular tissue that enhances. BPE usually has a symmetric distribution between the two breasts and has slow early and persistent delayed kinetic features . BPE is 3There have been conflicting results regarding whether BPE is an independent risk factor for breast cancer. Several studies of high-risk women, with a cross-sectional study design, have shown increased odds of having cancer with increasing levels of BPE –13. A stThe association between BPE and other risk factors for breast cancer has not been fully investigated. A risk factor of particular relevance is mammographic density, which measures the amount of radio-opaque fibro-glandular tissue on the mammogram. It is well established that women with the highest level of mammographic density have an increased risk of developing breast cancer compared to women with the lowest level of density –19. All 21The lack of MRI examinations in a healthy cohort with average risk of breast cancer causes difficulties in the interpretation and generalizability of previous results. The unique feature of our study is that the participants were recruited from a cohort study of healthy women, none of whom were known to be high risk. Additionally, we had unique access to information on many risk factors for breast cancer through this prospective cohort study. We performed MRI on a subset of 214 asymptomatic participants. We used these MRI examinations to measure the degree of BPE and studied its association with other established risk factors for breast cancer.Between the years 2014 and 2016, 1727 participants aged 40–74 years attending the national, population-based biennial screening program at Södersjukhuset Hospital, Stockholm, Sweden were recruited to participate in a study to test the accuracy of a three-dimensional infrared imaging (3DIRI) prototype ,26. WomeOf 1727 women, 219 (13%) had a negative mammography and a positive 3DIRI result. These 219 women underwent an MRI examination for verification. Five (2%) women had an MRI-detected malignancy. The remaining 214 women, with no detected malignancy, were followed up for one year and none of them had a diagnosis of breast cancer. These women comprise the study population for the present study.Digital mammography examinations were performed using Philips microdose system  Amsterdam with breasts examined in the craniocaudal and mediolateral oblique views. All examinations were performed at a single clinic and images were stored in the clinic’s local archive.DCE-MRI of the breast was performed according to the guidelines by the European Society of Breast Imaging . In premScores were based on the first post-contrast subtracted fat-suppressed images and the software generated maximum intensity projection (MIP) images. In cases of BPE asymmetry between the left and right breasts, the higher level of BPE was recorded. The degree of BPE was estimated by visual assessment into two categories: low BPE  and mild (b)) and high BPE (BI-RADS lexicon 5th edition categories moderate (c) and marked (d)) .Density was categorized by visual assessment according to the breast density BI-RADS lexicon 5th edition four composition categories: A = the breasts are entirely fatty; B = there are scattered areas of fibro-glandular density; C = the breasts are heterogeneously dense which may obscure small masses; and D = the breasts are extremely dense which lowers the sensitivity of mammography. If there was a difference in the density of the two breasts, the higher level of density was recorded.Density measurements on the previous mammogram for determination of eligibility for participation in the study was performed using the automated software program Volpara .Two radiologists with 8 and 20 years of experience in mammography and 1 year of experience in breast MRI, and one radiology resident with considerable experience in mammography and MRI, performed BPE and density assessments. The final score was decided in consensus.Data on age, menopausal status, height, and weight were collected from the case report file, and had been self-reported only a few days before the MRI examinations.Information on ongoing hormone replacement therapy  or use of systemic contraception containing progesterone was gathered from the case report file data and medical records. In six cases where up-to-date information was not available in our records, the information was collected by contacting the women by telephone.Data on remaining covariates were retrieved from the cohort study database. The data were collected two years before MRI examinations through a self-reported questionnaire.Continuous variables such as age, BMI, age at first birth, age at menarche, parity, intake of alcohol, and physical activity were arranged into categories.All explanatory variables were modelled as categorical variables. We first estimated odds ratios (OR) for the univariable associations between BPE and each of the explanatory variables. We then fitted an adjusted logistic regression model to evaluate associations between BPE (classified as high vs. low) and density, age, local estrogen treatment, systemic estrogen treatment (tablets and patches), systemic progestogen treatment (combination hormone replacement therapy as well as contraceptives), BMI, age at menarche, age at first birth, parity, oral contraceptive use, family history of breast cancer, alcohol, smoking, and physical activity. We used likelihood ratio tests to assess the statistical significance of each categorical variable.Stata version 14.1  was used for all statistical analysis.A total of 214 women  were included in the study. The main characteristics of the study participants are listed in 2, women with BMI > 30 kg/m2 were 4.8 times more likely to have high degree of BPE (95% CI = 1.2–19.4) in the unadjusted model and 12.6 times more likely after adjusting (95% CI = 1.9–82.7). We found a trend for increasing likelihood of having high BPE with increasing levels of density both in the adjusted and the unadjusted models with ORs of 1.7 and 2.1, respectively for BIRADS category C compared to B and 3.3 and 3.1, respectively, for BIRADS category D compared to B. The P value, however, was above the 0.05 threshold (0.07 in the unadjusted model and 0.23 in the adjusted model). We did not find any significant associations between high BPE and the remaining risk factors for breast cancer  and measurement of apparent diffusion coefficient (ADC) values. A recently published meta-analysis shows that ADC measurements have a pooled sensitivity of 89% and specificity of 82% for breast cancer . KawamurIn conclusion, the likelihood of having high levels of BPE is increased with young age, high BMI, and progesterone medication. We did not find a statistically significant association between BPE and mammographic density although we found a trend of increasing likelihood for having high level of BPE with increasing levels of density categories.