Providing lifestyle advice at cancer screening may help reduce the cancer burden attributable to health-related behaviour. We examined determinants of willingness to receive advice about several behavioural cancer risk factors.n = 1221) completed items on willingness to receive lifestyle advice. Sociodemographic, psychological  and behavioural factors were used to predict interest in advice about diet, weight, physical activity, smoking and alcohol consumption.A population-based sample of English adults eligible for cancer screening (p < 0.01). Non-White ethnicity was associated with interest in advice about diet, physical activity and smoking . Willingness to receive advice about diet, weight, physical activity and alcohol consumption increased with greater recognition of cancer risk factors .Two thirds (62–67%) reported interest in advice about diet, weight, and physical activity; 17% were willing to receive advice about smoking, and 32% about alcohol consumption. Willingness to receive advice was higher in those not adhering to guidelines for weight, physical activity, smoking and alcohol consumption (all Willingness to receive lifestyle advice at cancer screening was high, suggesting this context may provide an opportunity to support behaviour change. Increasing awareness of cancer risk factors may facilitate interest in lifestyle advice. We have previously shown that three quarters of people intending to attend English breast, cervical and bowel screening programmes would be in favour of receiving lifestyle advice around the time of cancer screening, even if they received an abnormal result . Interventions delivered at the time of breast and bowel cancer screening have reported adequate uptake and positive health behaviour change.10 One concern about delivering lifestyle advice in the cancer screening context is the potential negative effect it may have on screening uptake; 6–9% reported advice would be a deterrent in our previous study . Understanding the sociodemographic, psychological and behavioural determinants of interest in healthy lifestyle advice at cancer screening may aid the development of effective interventions which minimise negative effects on screening uptake.The cancer screening context could provide ‘teachable moments’ for the delivery of behaviour change advice and interventions.12 Uptake of Flexible Sigmoidoscopy is lower (33%) in the most deprived areas, compared with the least deprived areas (53%).11 In addition, ethnic minority groups are less likely to participate in cancer screening than white populations.15 Similar sociodemographic variation has been observed for other cancer related health behaviours including smoking and diet.17 Less is known about whether there is also variation in receptivity to different topics of lifestyle advice at cancer screening. Some research has found that interest in advice at breast screening was higher in women who were older, less educated and overweight.18 However, other research has found that receptivity to advice at cervical screening may be greater among more educated and non-White populations . One lifestyle intervention evaluated within a bowel screening setting reported high levels of recruitment and retention from deprived groups, and found no differences in outcomes between more and less deprived groups.19There is sociodemographic variation in the uptake of English cancer screening programmes.20 There is evidence to suggest changing perceptions of risk may affect intentions and behaviour.21 Risk perceptions are a key component of the Teachable Moment Heuristic.22 The model states that for behaviour change to occur following the teachable moment, perceived risk must increase. It has also been suggested that to capitalise on the teachable moment, people must be aware of the risk factors for cancer and how they relate to their own behaviour.8 A survey of British adults found variation in the recognition of different risk factors for cancer.23 Recognition was high for smoking (98%), but lower for other important risk factors such as overweight (62%) and fruit and vegetable consumption (48%). Awareness of the link between lifestyle and cancer could be a determinant of interest in lifestyle advice at cancer screening.8 Furthermore, receptivity to lifestyle advice may increase if people are given convincing evidence showing a link between lifestyle and cancer-related outcomes.24Risk perceptions are commonplace in theories attempting to understand and change health behaviour.This research aimed to gauge public interest in advice about five different aspects of lifestyle at cancer screening: diet, weight, physical activity, smoking, and alcohol consumption. In addition, this research aimed to identify sociodemographic, psychological  and behavioural predictors of interest in each of the lifestyle advice topics.Cross-sectional population-representative data were collected as part of the Attitudes, Behaviour and Cancer UK Survey (ABACUS). This survey aims to explore the determinants of early detection and prevention behaviours related to cancer. Data were collected as part of an omnibus survey, conducted by market research company Taylor Nelson Sofres (TNS) in April and May 2016 using home-based computer-assisted personal interviews.n = 121), people who did not meet age requirements (n = 471), and people who did not intend to attend a cancer screening programme in the future (n = 235), there was a final sample of 1221.A nationally representative sample of 2048 English adults (aged 18–70) was recruited using stratified random location sampling, based on 2011 Census data and Postcode Address File data. Quotas were set for gender, working status and presence of children in the home. This research included a sub-sample of men and women currently eligible to participate in breast, bowel or cervical screening, and people approaching the age of eligibility. We included people approaching the age of eligibility for cancer screening for two reasons. Firstly, if lifestyle advice is routinely offered alongside cancer screening it is important to sample potential future attenders as well as people currently eligible to attend. Secondly, the narrow age range of patients invited to take part in flexible sigmoidoscopy and delays in the roll out of this screening programme would have limited our sample. Participants were included in the analysis if they intended to take part in at least one of the cancer screening programmes. Taking into account recent changes to cancer screening programmes, people classified as currently eligible for cancer screening were women aged 25–70, and men aged 60–70, and people classified as approaching eligibility were women aged 18–24 and men aged 45–59. Three cancer screening programmes operate in England. Women are invited to attend cervical screening between the ages of 25–64, and breast screening between the ages of 50–70. Men and women are invited to take part in Faecal Occult Blood testing (FOBT) between the ages of 60–74, and a one off Flexible Sigmoidoscopy (FS) aged 55. The upper age limit for FOBT eligibility has recently increased from 69 to 74, and FS was not available in all parts of England at the time of this survey. After excluding participants with a diagnosis of cancer  were recorded. For analyses, ethnicity was categorised into White and non-White. Educational attainment was categorised into ‘degree level or above’ and ‘education below degree level’, based on the item ‘what is the highest level of educational qualification you have obtained’.Intention to participate in cancer screening was assessed separately for each of the four cancer screening programmes . Four response options were offered , which were dichotomised into yes and no. A composite item was created which identified participants intending to take part in at least one of the screening programmes in the future.25). Participants were presented with 11 risk factors for cancer including smoking, exposure to another person’s cigarette smoke, alcohol consumption, fruit and vegetable consumption, red and processed meat consumption, overweight, childhood sunburn, age over 70 years, having a close relative with cancer, infection with HPV, and physical inactivity. Five response options were provided, which were categorised into correct (agree / strongly agree) or incorrect (strongly disagree / disagree / not sure). For each participant, the number of risk factors that they correctly identified was combined giving each participant a score out of 11.Knowledge of cancer risk factors was assessed using an 11-item scale from the Cancer Research UK Cancer Awareness Measure (CRUK CAM27 Five response options were categorised into lower (much lower / a little lower), the same (about the same), and higher (a little higher / much higher).Comparative cancer risk perception was assessed using the item ‘How would you rate your chances of getting cancer, compared with other men / women your age?’, adapted from existing measures.28‘Over the past month, how many portions of fruit / vegetables did you usually eat?’ Responses options were: less than 1 per week, 1 per week, 2–3 per week, 4–6 per week, 1 per day, 2 per day, 3 or more per day. Values ≥1 or more per day for each item were added together to create a composite measure of fruit and vegetable consumption. Participants consuming five or more portions of fruit and vegetables per day were classified as meeting guidelines. Body Mass Index  was calculated from self-reported height and weight. Implausible BMI data were excluded (BMI < 14 /> 50). BMI was dichotomised to ≥25 (overweight) vs <25 (not overweight). A single item was used to assess levels of physical activity ‘In the past week on how many days have you done a total of 30 minutes or more of physical activity, which was enough to raise your breathing rate?’.30 Participants taking part in 30 min of physical activity on five or more days per week were classified as meeting guidelines.31 Smoking status was assessed using a single item; ‘Do you smoke at all nowadays?’. Participants were categorised as smokers  or non-smokers . Alcohol consumption items were adapted from the AUDIT-C questionnaire.33 ‘In a typical week, on how many days do you have a drink containing alcohol?’ and ‘How many units of alcohol do you drink on a typical day when you are drinking?’ Participants consuming 14 units or less per week were classified as meeting guidelines for alcohol consumption.Fruit and vegetable consumption was assessed using two items;The following questions were asked of participants intending to take part in at least one screening programme in the future; ‘At cancer screening / If you were to attend cancer screening in the future, how interested would you be in any information or advice to… Help you have a healthy diet / Help you maintain a healthy weight / Help you increase your physical activity / Help you stop smoking / Help you reduce your alcohol consumption?’. Five response options were collapsed into ‘interested’ (a little interested / somewhat interested / very interested) and ‘not interested’ .p < 0.010 was used to adjust for multiple testing.Five adjusted logistic regression models explored sociodemographic, psychological and behavioural predictors of interest in each of the categories of lifestyle advice among people intending to participate in cancer screening in the future. The simultaneous entry method was used and each model included variables which have been associated with health behaviours and cancer screening participation: age, gender, ethnicity, educational attainment, screening eligibility, cancer risk factor recognition, comparative cancer risk, and current health behaviours. We applied survey weights calculated by the market research company to adjust for response bias . Sample characteristics are presented unweighted and weighted, univariate analyses are presented weighted, multivariate analyses are presented unweighted. Participants with a previous diagnosis of cancer and those who did not intend to take part in screening were excluded from analyses. Data were analysed using Stata SE 14. An alpha level of n = 901) were female, which reflects the inclusion criteria based on screening eligibility. The majority  were White. One third of the sample  were educated to degree level or above. Three quarters of the sample  were currently eligible to participate in at least one cancer screening programme.Of the 2048 adults included in the ABACUS, 1221 were included in this analysis (Table n = 1028), and consumed fewer than 14 alcoholic units per week . Half of the sample had a BMI of 25 or lower . Around one third reported meeting guidelines for fruit and vegetable consumption  and physical activity . Most of the sample reported their risk of developing cancer to be the same as others of their age and sex . On average, people were able to recognise 5.9 (SD 2.7) of the 11 cancer risk factors. For unweighted sample characteristics, see Table The majority of the sample did not smoke , weight  or physical activity  during future cancer screening appointments (Table n = 199), and a third of the sample were interested in information about alcohol consumption .Two thirds of participants were interested in receiving advice about diet  . Smokers had greater odds of interest in smoking cessation advice (59.7%) compared with non-smokers . Participants who exceeded 14 alcoholic units per week were more likely to report interest in advice about alcohol consumption (49.3%) when compared with participants meeting alcohol consumption guidelines . Analyses were re-run excluding those participants who felt the advice would not be applicable to them, and results were broadly unchanged. However, for interest in PA and alcohol, meeting the guidelines was not as strongly associated, and for interest in dietary advice, those who perceived themselves to be at lower risk were more likely to be interested in advice.Participants whose health behaviour fell short of recommendations were more likely to be interested in advice about weight, physical activity, smoking and alcohol consumption Table ; Fig. 1.p = 0.007). Non-White participants were more likely to be interested in dietary and physical activity advice. Interest in dietary advice was expressed by 80.5% of non-White participants, compared with 65.5% of White participants . Similarly, 78.9% of non-White participants expressed interest in advice about physical activity compared with 59.2% of White participants . There were no associations between gender or education and interest in any of the five topics of advice.The odds of reporting interest in advice about alcohol decreased with increasing age , weight , physical activity , and alcohol consumption  increased. We also explored whether knowledge of individual risk factors were associated with interest in information about their corresponding topics of advice. People who recognised low fruit and vegetable consumption as a risk factor for cancer were more likely to want dietary advice at cancer screening . Recognition of alcohol consumption as a risk factor for cancer was associated with interest in advice about alcohol consumption . Recognition of overweight , low physical activity , and smoking  were not associated with interest in their respective topics of advice. Comparative cancer risk perceptions were not strongly associated with interest in any of the topics of lifestyle advice.Cancer risk factor awareness was positively associated with interest in advice about most lifestyle topics. With each additional cancer risk factor recognised, the odds of being willing to receive advice about diet . One concern about delivering lifestyle advice at cancer screening is that screening non-attenders will not be reached.38 Tailoring may be particularly important for less prevalent behaviours, such as smoking and excessive alcohol consumption.Current health behaviour was associated with interest in advice about physical activity, weight, smoking and alcohol consumption at cancer screening. This suggests that a tailored approach to intervention design, which takes current behaviour into consideration, may be best in this setting. Tailored advice has been delivered within the context of colorectal cancer screening, and has been successful at increasing reported fruit and vegetable consumption but did not increase physical activity or reduce alcohol consumption.39 Reassuringly, three quarters of overweight participants in our sample were willing to receive advice about weight as part of cancer screening services. This is supports the finding that advice about weight in a primary care setting is considered appropriate and helpful by the majority of patients, and that interest in diet and weight advice at breast screening is greater among overweight women.40Previous research suggests that health care professionals may be reluctant to deliver advice about topics such as weight, fearing that this advice may cause distress to patients.27 Further research is needed to understand whether other aspects of risk, such as affective or experiential risk perceptions are associated with interest in lifestyle advice at cancer screening.41 Risk factor awareness was positively associated with willingness to receive advice about diet, weight, physical activity, and alcohol consumption. Previous research has highlighted the importance of providing convincing evidence about the link between lifestyle and cancer risk within cancer screening settings.24 This suggests that interventions delivered during the cancer screening process may benefit from increasing awareness of cancer risk factors.Cancer risk factor awareness and risk perceptions were explored as potential determinants of interest in advice at cancer screening. Risk perceptions were not strongly associated with interest in any of the topics of advice. However, a single item measure of comparative cancer risk was used.42 and willingness to receive lifestyle advice may differ between people who intend to participate in screening, and people who actually participate. Interest in lifestyle advice is high across breast, bowel and cervical screening programmes . However, in this research participants were asked to rate their interest in lifestyle advice at cancer screening in general, not for each cancer screening programme individually. Therefore it is not known whether interest in different topics of advice may vary between screening modalities. A further limitation to this research is the dichotomisation of responses to items assessing interest in each of the topics of advice. This involved grouping participants who answered “not at all interested” and “not applicable”. We reanalysed the data excluding participants who responded “not applicable” and results were broadly unchanged.This research has limitations. As stated in previous research conducted with this sample, participants were not recruited from screening settings so questions relating to the delivery of lifestyle advice at cancer screening were hypothetical . The well-documented intention-behaviour gap means it is likely that a number of people intending to participate in cancer screening will not take up the offer when invited,5 The health behaviours of screening attenders have been found to differ from non-attenders, which may also be the case for the screening intenders within our sample.35 In addition, survey respondents may also be healthier than the general population.43 However, measures of health behaviour used in this study may also impact the findings of this research. All health behaviour measures were self-reported, which may be subject to a number of biases.44. For example, we used fruit and vegetable consumption as a proxy of diet, however the accuracy of self-reported dietary data has been questioned.45 These self-reported measures were then used to gauge whether people were meeting guidelines. To classify people as meeting guidelines for alcohol consumption we used a cut off of 14 units, however, it is recognised that any alcohol consumption can increase cancer risk.46 Willingness to receive advice about alcohol consumption may vary depending on the leniency of the recommendation used.Our sample appear to be more health conscious than would be expected in the general population. For example, around half of our sample had a BMI > 25, compared with 61% of English adults in the general population.In conclusion, the majority of people who intend to participate in cancer screening would be willing to receive advice about common cancer risk factors at this time. Advice appears to be acceptable to people who do not meet recommendations for health behaviours, and to non-White participants. Increasing cancer risk factor awareness and tailoring advice to current behaviour may provide an important basis for the development of interventions within the cancer screening setting.


XRCC4 c.1394G>T SNP and breast cancer development among Filipinos. The identification of cancer-associated single nucleotide polymorphisms (SNP) and mutation genes is a promising approach in recognizing individuals who are at risk of developing cancer. Hence, this study was conducted to determine the association between XRCC4 c.1394G>T SNP was performed on breast cancer patients (n=103) and their age- and sex- matched clinically healthy controls (n=103) by polymerase chain reaction – restriction fragment length polymorphism. Genotyping for XRCC4 c.1394G>T was observed between the breast cancer cases and controls. Carriers of the XRCC4 c.1394 G>T genotype were observed to have significantly higher risk of developing breast cancer compared to individuals with T/T genotype . XRCC4 c.1394G>T combined with passive smoking may also significantly increase risk of breast cancer .Significant difference in genotype (p=0.007) and allele (p=0.003) frequencies in XRCC4 c. 1394G>T may be associated with breast cancer development among Filipinos.  Breast cancer remains the leading cause of cancer among women worldwide . Epidemiological studies show that breast cancer is a multifactorial disease associated with exposure to ionizing radiation, tobacco smoking, alcohol consumption, and use of hormones or oral contraceptives . Furthermore, it is widely established that breast cancer could result from a series of genetic alterations that disrupt the normal mechanism which controls cell proliferation, differentiation, apoptosis, and genomic stability . To counterbalance the detrimental effects of DNA damage, cells have established a dedicated DNA repair system, which can be classified into few distinct mechanisms based on the degree of lesion in the DNA .The most biologically perilous types of DNA damage are called the double strand breaks (DSBs). A single unrepaired double strand break is reasonable to induce cell death. These events have been linked with the progress of many types of cancer or other genomic defects . The two important mechanisms in DSBs are (1) homologous recombination (HR) and (2) non-homologous end-joining (NHEJ). The two repair systems require a different necessity for a homologous template DNA and also the fidelity of DSB repair. HR-directed repair is an error-free mechanism because it utilizes the genetic information confined in the undamaged sister chromatid as a template . On the other hand, NHEJ is error-prone and involves elimination of DSBs by direct ligation of the broken ends. In all phases of the cell cycle, NHEJ is the principal pathway in mammalian cells while HR is constrained to the late S and G2 phases .XRCC4) gene are associated with breast cancer pathogenesis . The XRCC4 gene which encodes for XRCC4 protein is involved in non-homologous end joining (NHEJ) mechanism critical in double strand break repair . The XRCC4 protein helps in repairing the DNA double-strand breaks by stimulating the ligation of non-complementary and complementary DNA ends using XRCC4 ligases . Furthermore, this protein plays a pivotal role in the completion of VDJ recombination in order to generate antigen receptors that can collectively recognize different types of molecules .Several studies have shown that some genetic variants of DNA repair genes, such as the X-ray cross-complementing group 4 . Furthermore, numerous studies have reported that SNP in the XRCC4 gene is associated with risk of developing breast , skin , gastric , liver , and oral cancer . However, it must be noted that their findings were also race-specific. Hence, this study is the first to present the possible association of XRCC4 (c.1394G>T) polymorphism with the risk of breast cancer among Filipinos.Meta-analyses have shown the association of polymorphisms in Study PopulationFilipino patients, 18 years old and above, with histologically confirmed breast cancer, either newly diagnosed, receiving treatment, or in remission, seen at the University of Santo Tomas Hospital (USTH), Manila between December 2015 to April 2016, were recruited for the study. The cancer patients (cases) were age- (±2 years) and sex-matched with volunteer controls who were assessed by a physician collaborator to be clinically healthy and not suspected to have any type of malignancy. Both cases and controls were asked to accomplish through interview a standardized questionnaire inquiring on their risk factors . Five (5) mL peripheral blood collected from the participants were immediately stored at -80°C until molecular analysis. Clinical data of the cases  were retrieved from clinical records and histopathology reports. Ethical clearance was obtained from the Institutional Review Board (Protocol Reference No. IRB-MD-09-2015-133) of USTH and all participants gave their written informed consent. Genotyping XRCC4 c.1394G>T was analyzed by polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP), and further processed as described  but with minor modifications with the annealing temperature and duration of digestion.Genomic DNA was isolated from the blood samples of all study participants using ReliaPrep™ Blood gDNA Miniprep System  following the manufacturer’s protocol. All samples were genotyped in triplicate with consistent results to confirm that the genotypes have no mixture of germline and somatic mutations. The genotype at the polymorphic locus of HincII at 37°C. The digested amplicons were subjected to 12% polyacrylamide gel electrophoresis for 60 min at 100V and viewed under a UV transilluminator. Expected fragment sizes were as follows: T/T: 300 bp; G/G: 200, 100; G/T: 300, 200, and 100 .A single PCR reaction comprised of a 20 μl total volume of 10 μl of PCR master mix , 0.5 μl each of 0.25 μM forward and reverse primers, 7 μl nuclease free water, and 2 μl DNA (89 ng/μl). Target gene was amplified using the primers 5’-GATGCGAACTCAAGATACTGA-3’ (forward) and 5’-TGTAAAGCCAGTACTCAAATT-3’ (reverse) under the following conditions: initial cycle at 94°C for 5 min; 40 cycles of 94°C for 30 s, 52.8°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. PCR products were digested overnight with Statistical AnalysisXRCC4 SNP in the control subjects from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi-square test or Fisher’s exact test (when the expected number in any cell was less than five) were used to compare the distribution of the XRCC4 genotypes between cases and controls. Epidemiologic risk factors associated with the genotypes were estimated as odds ratio (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. Data was recognized as significant when the statistical p-value was less than 0.05. All statistical tests were performed using SAS, Version 9.1.3  on two-sided probabilities.To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequency of A total of 206 samples  were included in this study. Majority of the cases were 41 to 50 years old, had either well-differentiated (G1) or moderately-differentiated (G2) tumor, and were in their early stage of cancer  at the time of diagnosis .XRCC4 c.1394G>T were significantly different between breast cancer cases and healthy controls. It appears that G/T genotype  is associated with breast cancer among Filipinos  or T/T  genotype, had increased risk for breast cancer (Results show that genotypic (p=0.007) and allelic (p=0.003) distribution pattern of ilipinos . Moreovet cancer . Meanwhit cancer . BRCA1 and BRCA2  with breast cancer among Filipino women. In contrast, variants of the GSTM1 and GSTT1 genes were not found to be risk factors for breast cancer development among the same population . Thus, identifying genes associated with cancer development continues to be a major goal of current research. Previous studies have associated the trinucleotide repeat polymorphism (CAGn) in the exon 1 of the androgen receptor gene (AR)  and mutations in XRCC4, which plays a role in both non-homologous end joining and completion of the V(D)J recombination, is among the genes that have been associated with various types of cancer . The XRCC4 c.1394G>T, a mutation which lies at the coding region of the XRCC4 gene, might cause an alteration in the amino acid sequence of the XRCC4 protein . This alteration might affect the function of XRCC4 protein in stabilizing the DNA ligase IV which is a crucial part in double strand break repair .G/T genotype of XRCC4 c.1394G>T is associated with breast cancer in selected Filipino population. Similarly, XRCC4 c.1394G>T G/T genotype has been associated with breast, gastric, and prostate cancers among Taiwanese  and colorectal cancer among Iranians . In contrast, the XRCC4 c.1394G>T G/T genotype was not associated with urinary bladder cancer among the Indians. Besides, the G/G genotype was associated with reduced risk for urothelial bladder in this population . In the current study, the T/T variant of XRCC4 c.1394G>T was associated with reduced risk for breast cancer. This study shows that heterozygous XRCC4 c.1394G>T with clinical and epidemiologic risk factors. Among cases carrying the G/T genotype, those exposed to passive smoke had higher risk of breast cancer compared to active smokers. Inhaled side stream smoke, the main component of second-hand smoke, is about four times more toxic than active smoking as it contains about 69 known carcinogens and more particulate matter pollution that get into the air . The present study also considered the combined effect of the genotype variants of XRCC4 c.1394G>T with breast cancer development among selected Filipinos. Results of this study contribute to existing evidence supporting the hypothesis that polymorphisms in the XRCC4 c.1394G>T gene may influence the functioning of the DNA repair pathway. Despite the small number of participants in this study, the strict age- and sex-match design adds to the reliability of the findings. However, related future studies should screen for other polymorphisms in the XRCC4 gene and other DNA repair genes, and to determine their combined effects with lifestyle risk factors. Furthermore, participants from other parts of the country should be included to accurately represent the Filipino population. To the researchers’ knowledge, this is the first report on the association of This study was supported by research grants from the Department of Science and Technology – Science Education Institute (DOST – SEI) and the Commission on Higher Education (CHED) of the Philippines.The authors declare no conflict of interest


BCAM) questionnaire.Enhancing awareness level about breast cancer is a pivotal strategy for reducing breast cancer burden. There is no fully validated Persian instrument for evaluating breast cancer awareness. This study aimed at investigating the validity and reliability of the Persian version of the Breast Cancer Awareness Measure  coefficient. Construct validity was investigated using both exploratory and confirmatory factor analyses as well as Latent class analysis (LCA), and discriminant validity using ROC curve. Convergent validity was assessed using phi and eta correlation coefficients. Ceiling and floor effects, SE of measurement (SEM), and smallest detectable change (SDC) were also determined.This methodological cross‐sectional study was conducted among 1078 Persian language women , which selected from different parts of Isfahan city using multistage cluster random sampling method. Translation of BCAM showed excellent test‐retest reliability (ICC = 0.841) and internal consistency . Most of the computed kappa coefficients were in the range moderate to very good (0.47‐0.81). Medical/clinical experts had higher levels of breast cancer awareness than general women, indicating good discriminant validity (Area under the curve [AUC]) of 0.822 . Construct validity evaluation by exploratory factor analysis (EFA) led to extraction of two factors from 11 items (“breast shape changes” and “breast pain and lump”), and the confirmatory factor analysis (CFA) confirmed the adequacy of extracted construct from EFA. Latent class analysis for evaluating of construct validity led to extracting three classes from participants  in terms of awareness levels about early warning signs of breast cancer. All item‐scale correlation coefficients exceeded the set value of 0.40, indicating satisfactory convergent validity. No ceiling and floor effects were detected. SEM and SDC were found to be 0.85 and 2.36, respectively.Persian version of BCAM is a reliable and valid instrument for monitoring levels of breast cancer awareness in general women population, also it can be used for evaluating the impacts of interventions attempting to raise breast cancer awareness.The Persian version of  All medical/clinical experts were randomly selected among female physicians/nurses/midwives from three major hospitals affiliated with Isfahan University of Medical Sciences . General women were selected from different parts of Isfahan city  using multistage cluster random sampling method. Eligibility criteria for participating in this study were as at least 18 years old, able to read and write Persian, and should be permanent resident of Isfahan. Those participants who did not answer to main questions (items of BCAM questionnaire regarding breast cancer awareness measures) were excluded. Eligible women were invited to participate in the study by face‐to‐face invitation. Interviews were performed by trained interviewers. After obtaining oral consent to participate in the study, we requested from participants to complete the BCAM questionnaire. The study was approved by the bioethics committee of the Isfahan University of Medical Sciences (Project Number: 194126).2.2P = 0.001).UK Cancer Research center developed and validated an awareness measure specific to breast cancer awareness which called BCAM.2.3In accordance with the guidelines recommended by Beaton et al,2.4In this study, psychometric characteristics of the Persian version of BCAM, including reliability  and validity , were evaluated.2.5We investigated two aspects of reliability, that is, test‐retest reliability and internal consistency. In order to test the extent to which the measure was repeatable (the stability of the measurement over time), we recruited 50 women aged 18 years old and over. The women were asked to complete the BCAM questionnaire at two separate days with a 7‐day interval. Test‐retest reliability was assessed separately for each item using the unweighted kappa statistic .α coefficient .Internal consistency was evaluated using Cronbach's 2.62.6.1P < 0.05).df [relative chi‐square], root mean square error of approximation [RMSEA], comparative fit index [CFI], and Tucker‐Lewis index [TLI]) for evaluating the goodness of fit model. Relative chi‐square <5.00, a CFI and TLI value of >0.90, and a RMSEA value of <0.08 were considered as acceptable model fit.The factor structure of the BCAM was explored using the exploratory factor analysis (EFA). Factor‐item loadings values greater than 0.40 were considered as satisfactory for assigning an item to a specific factor. The data viability for factorability was guided through Kaiser‐Meyer‐Olkin (KMO) measure of sample adequacy  and Bartlett's test of Sphericity . In other words, the level of “awareness” about breast cancer warning signs was considered as a latent construct and it was evaluated using LCA. LCA is a parallel approach or counterpart, with factor analysis, but it applicable for categorical variables.2.6.2t test. In addition, receiver operating characteristic (ROC) curve along with the sensitivity and specificity values was used to gauge the ability of the total score of warning signs subscale of BCAM to discriminate between general women and medical/clinical experts. We also assessed the BCAM ability to discriminate between participants with “personal and family history of breast problems” and general women in terms of awareness levels about breast cancer warning signs using chi‐square test or independent Student's t test and ROC curve.Discriminant validity was assessed based on the BCAM ability to discriminate between general women and medical/clinical experts in terms of awareness levels about breast cancer warning signs. The validity of the measure is supported if distribution of the BCAM items is significantly different across two groups. We distributed the BCAM questionnaire to 965 general women and 113 medical/clinical experts and compared their responses. We tested difference in the distribution of correct answer to each item and total score of correct answer to all items between two groups using chi‐square test or independent Student's 2.6.3As both the items and the underlying structure of the BCAM questionnaire (extracted latent classes based on items) were discrete variables, the phi correlation coefficient was used to examine item‐scale correlations, corrected for overlap . We also used eta correlation coefficient corrected for overlap to assess correlation between each item and the total score of items . Item convergent validity should be at least 0.40.2.6.4Ceiling and floor effects were assessed on the first administration of the BCAM to determine content validity. Ceiling/floor effects were considered to be present if more than 15% of the participants achieved the highest or lowest possible scores.2.6.5SD×1−r.1.96×2×SEM.In this study, quantitative and qualitative variables were expressed as mean (SD) and number (percent), respectively. Additional data about age, education level, marital status, job status, personal history of breast problems, family history of breast cancer, and sources of gaining awareness about breast cancer were also collected. The determinants of the awareness level about breast cancer warning signs were evaluated using “latent class regression” analysis (LCR). In the LCR, covariates were included to describe both the formation of the latent classes and how they may be differently measured by the observed indicators. The prediction of latent class membership is obtained by multinomial regression of latent class variable on covariates. The standard error of measurement (SEM) was measured as 33.1A total of 1078 women participated in this validation study, including 965 (89.5%) general women and 113 (10.5%) medical/clinical experts. The mean (SD) age was 36.5 (11.65) and 34.28 (7.14) years old for general women and medical/clinical experts, respectively. The majority of the participants were married (73% for both groups). Approximately half (50.2%) of the women had academic level of education, and only 28.5% were employed. Personal history of breast problems was reported by 159 (16.6%) and 12 (10.8%) of general women and medical/clinical experts, respectively. Among women with the breast problems, history of breast cancer was reported by 7/159 and 1/12 of general women and medical/clinical experts, respectively. In addition, family history of breast cancer was reported by 198 (20.9%) and 19 (16.8%) of general women and medical/clinical experts, respectively.  statistics over a week interval in a subsample of 50 women. Results of the test‐retest reliability of the BCAM are shown in Table The ICC coefficient for the total score of warning signs subscale suggests strong test‐retest reliability ; also all items loaded significantly on their respective factors . EFA with Varimax rotation extracted two factors from the warning signs subscale of BCAM which were labeled as “breast shape changes” and “breast pain and lump” accounting for 26.98% and 23.67% of total variance, respectively. A KMO value of 0.896 and Results of LCA on the 11 items of the warning signs subscale showed that model with three classes has highest quality of fit to the data . The entropy was 0.88 suggesting that individuals are correctly classified by our fitted model. Table 3.5Table 3.6All item‐scale correlations based on phi's coefficients exceeded the set value of 0.40, indicating satisfactory convergent validity Table . Item 7 3.7Ten percent of the participants achieved the minimum score of 0, and 14% of the respondents reached the maximum score of 11. These percentages were below the cutoff point of 15%, suggesting no floor and ceiling effect.4In current study, the psychometric properties  of the Persian version of BCAM were evaluated. To the best of our knowledge, this questionnaire, which is self‐report and quick to complete, is the first fully validated instrument to assess knowledge of breast cancer symptoms and age‐related risk and reported frequency of breast checking in Iranian women. The results showed that the Persian version of BCAM has excellent test‐retest reliability and internal consistency. Medical/clinical experts had higher levels of breast cancer awareness than general women and patients than general women, indicating good discriminant validity. Applying latent class analysis for evaluating of construct validity led to three classes  in terms of awareness levels about early warning signs of breast cancer. The instrument showed satisfactory convergent validity, because all item‐scale correlation coefficients exceeded the cut‐off 0.40. No ceiling and floor effects were found. The main determinants of awareness level about early warning signs of breast cancer were educational level, marital status, family history of breast cancer, and age.In the present study, the ICC index, as a measure of test‐retest reliability, for the total score of warning signs subscale of the BCAM was 0.84 which is acceptable. Previously reported ICC for the total score of signs and symptoms subscale of Thai version was 0.786.The SEM as a measure of absolute reliability, and the SDC, as a real change greater than the measurement error, were not evaluated for the original English versionAccording to our results, the Persian version of BCAM well discriminated general women and medical/clinical experts; in which knowledge level about warning signs, age‐related risk and breast checking were all significantly higher in the medical/clinical experts. This result indicated acceptable discriminant validity for this instrument. The result of Linsell et al'sIn the current study, the evaluation of construct validity BCAM‐Persian by EFA led to extraction of two factors as “breast shape changes” and “breast pain and lump,” and the adequacy of extracted construct confirmed by CFA. The construct validity based on factor analysis was not evaluated in the original English versionIn the current study, the most effective determinants of awareness levels about early warning signs of breast cancer were educational level, marital status, family history of breast cancer, and age, respectively. The highlighted role of educational status on awareness level in the present study was in agreement with previous studies.5It is important to recognize some strengths and limitations of the current study. In contrast to the original questionnaire, which is only had been validated in older women (67‐73 years), our instrument was developed among a general women population (older than 18 years old). In addition, this study was carried out on a relatively large sample of the general women in the Isfahan city which is one of the cities located in central of Iran with high incidence rate of breast cancer.6In conclusion, the Persian version of BCAM questionnaire showed good psychometric properties in terms of test‐retest reliability, internal consistency, convergent, and discriminant validity, suggesting it will have utility in assessing breast cancer awareness about early warning signs in Persian language women. It is a self‐report questionnaire, inexpensive, and quick to complete for monitoring levels of breast cancer awareness, and evaluating the impacts of interventions attempting to raise breast cancer awareness. The results of the present study also indicated that studied population has a three‐class latent structure  in terms of knowledge levels about early warning signs of breast cancer more predominant by moderate and particularly low levels. The women with higher educational attainments, and family history of breast cancer, had higher levels of awareness, emphasizing on the need for raising awareness about breast cancer among Iranian women as an effective way to overcome increasing trend and burden of breast cancer in the population.The authors declare that they have no conflict of interest.


Late diagnosis of prostate cancer is common in low and middle income countries and contributes to high morbidity and mortality of the disease. Utilization of prostate cancer screening services plays a major role in prevention of adverse outcomes. However, there is limited information on the knowledge about, the perceived risk of, and the utilization of prostate cancer screening in Tanzania.  To determine knowledge and perceived risk of prostate cancer, and the utilization of prostate cancer screening services, and associated factors, among men in Dar es Salaam, Tanzania.  A population-based cross-sectional study involving men aged 40 years and above living in Dar es Salaam was conducted between May and August, 2018.  Participants were recruited through multistage random sampling and took part in structured face-to-face interviews. Categorical variables were summarized using proportions while continuous variables were summarized as medians and inter-quarterly range (IQR). Chi square test was used to compare differences between proportions, and logistic regression modelling was used to determine factors associated with utilization of prostate cancer screening. Both crude and adjusted odds ratios (OR), with corresponding 95% confidence intervals, are reported. All analyses were two-tailed and the significance level set at 5%. n = 125) perceived the risk of prostate cancer to be low. Only 30 respondents (7.7%) had ever been screened for prostate cancer. Utilization of prostate cancer screening services was independently associated with age above 60 years , monthly income above 305 US Dollars , the perceived risk of prostate cancer  and knowledge about prostate cancer .  A total of 388 men with a median age of 53 years (IQR 44–55) participated. Half (52.1%) had poor knowledge about prostate cancer and prostate cancer screening. A third (32.3%,  Knowledge about prostate cancer and prostate cancer screening services was low among men in Dar es Salaam with a third perceiving themselves to be at no risk for the disease. Utilization of screening services was low and associated with low income, younger age, low perceived risk of prostate cancer and low knowledge about the disease. Intervention measures aiming to increase knowledge about prostate cancer and screening services, and affordable provision of services, are urgently called for. Breast cancer in women and prostate cancer in men have now become the most commonly diagnosed cancers in many Sub-Saharan African countries. Prostate cancer is an adenocarcinoma of the male prostate and is one of the most common cancers in the world and a leading cause of cancer related deaths among men globally –3. WhileWhile screening for prostate cancer using the prostate-specific antigen blood test (PSA) and digital rectal examination (DRE) are effective available measures for early detection of disease, utilization of these services ranges from 0% to 15% in Africa , 7–10. AVarious factors associated with utilization of prostate cancer screening have been documented both in developed and developing countries. A recent study reported that African and African American men were less likely than European and European American men to seek prostate cancer-screening as a direct or indirect consequence of financial barriers, lack of health insurance, and/or poor health-seeking behaviour . StudiesRecently there have been a number of public campaigns to promote prostate cancer awareness in Tanzania, but the utilization of prostate cancer screening services has not been evaluated. The present study therefore aimed to examine the level of knowledge about prostate cancer, perceived risk of the disease, and utilization of prostate cancer screening services, and their associated factors, among men in Dar es Salaam in order to inform ongoing and new intervention measures.A population-based cross-sectional study was conducted from May to August 2018 among men aged 40 years and above who were residents of Dar es Salaam (had an address in the city and had lived in it for more than one year). Based on the level of knowledge of prostate cancer and prostate cancer screening reported in a study from Kenya , sample Multistage cluster sampling was used to recruit participants for this study. We first randomly selected two of Dar es Salaam's five administrative municipalities (Kinondoni and Ubungo). From each of these, two wards were selected randomly , from each of which two streets were randomly selected, from each of which two administrative cells were randomly selected. A list of households with eligible men was obtained from the ward office and served as our sampling frame within these administrative cells. Eligible participants were recruited through house to house visit during the evening.A questionnaire was followed during structured face-to-face interviews to collect data on socio-demographic characteristics of the respondents, their knowledge about and perceived risk of prostate cancer, and their utilization of screening services. A set of questions were used to measure knowledge of prostate cancer and pretested to assess their face validity prior to actual data collection. All questions were developed in English and then translated into Swahili, the language used during the interview.Data were entered into and analysed in the Statistical Packages for the Social Sciences (SPSS) version 20.0. Categorical variables were summarized using proportions while continuous variables were summarized as medians and inter-quarterly range (IQR). Knowledge (8 questions) and internal consistency was examined using Cronbach's alpha (0.856). Chi square test was used to compare differences between proportions and both bivariate and multivariate logistic regression modelling was used to determine independent factors influencing the utilization of prostate cancer screening services. Both crude odds ratios (OR) and adjusted odds ratios (AOR) were calculated and presented with corresponding 95% confidence intervals. All analyses were two-tailed and the significance level set at 5%.Ethical approval for this research was obtained from MUHAS Ethical Research Committee (MU/PGS/SAEC/VOL.IX/56) and the permission to conduct the study was thereafter requested from local authorities in Kinondoni and Ubungo municipalities. All study participants provided written informed consent prior to the interview. Confidentiality was maintained and no names were recorded in any questionnaire. Interviews were conducted in privacy.About 400 men were approached to consent and participate in the study-a total of 388 (97%) men with a median age of 53 years (IQR: 44–55) living in Ubungo and Kindondoni municipality of Dar es Salaam participated in the study. Majority were married (85.1%) and about a third had completed secondary education with only 1% reporting no formal education. Nearly half (45.6%) were employed whereas 39.9% engaged in business and 10.8% were retired. The median monthly earning among respondents was 305 US Dollars (IQR = 174–348 US Dollars) .n = 186) of the study participants were knowledgable about prostate cancer and prostate cancer screening method  while knowing someone currently was reported by 17.3% (n = 67). The majority of participants  knew that prostate cancer is a disease that affects men, nearly two thirds  knew that the disease is severe, and half  knew that the disease is curable if detected early. While only 27.3% (n = 106) had knowledge about the symptoms of prostate cancer, 62.4% (n = 242) knew about the screening methods (PSA and/or DRE).n = 95) said men are at high risk, 62% (n = 240) said they are at some risk and 23.4% (n = 52) that they are at no risk at all. With regards to the interviewee's individual  risk of prostate cancer, 13.7% (n = 53) felt they were at high risk, half  thought they were at some risk whereas about a third  assumed that they were at no risk of developing prostate cancer . Participants who were above 60 years, who had a monthly income above 305 US Dollars, who were knowledgable about prostate cancer and who perceived themselves to be at risk of prostate cancer, were more likely to have utilized screening services in the bivariate analysis  have poor knowledge about prostate cancer. Similar findings have recently been reported among men in Nigeria, South Africa and black men in the Caribbean, America and Africa , 18, 19.Perceived risk of disease has been associated with intention to seek health services including cancer screening services , 15. A tUtilization of screening services for prostate cancer was uncommon among participants in this study. Only 8% of men aged 40 years and above, reported to have ever been screened for prostate cancer. Studies in Nigeria, Kenya and elsewhere in Africa have also found alarmingly low use of screening services for prostate cancer. Low utilization of prostate cancer screening have been associated with poor knowledge about prostate cancer and the screening methods in black populations in America and Africa , 23, 24.In this study, low utilization of prostate cancer screening services were associated with age, monthly income, and knowledge about prostate cancer and screening services. The association between service utilization and older age may be a result of prolonged exposure to awareness campaigns on prostate cancer but also increased risk and symptoms of prostate cancer with age that may prompt health seeking. Old age has also been associated with decreased sexual activity and increased risk of prostate cancer hence seeking screening services . SimilarHigher income was associated with increased utilization of prostate cancer screening services in this study. Access to health services including screening for cancer has been associated with socio-economic status in several studies worldwide. Education level and income are highly correlated and people who are educated not only have higher odds of better income but also can have access to health education messages, internalize them and act on them. Moreover, income has also been associated with access to health services through increased ability to pay in areas with low coverage of health insurance like Tanzania –30. ThouAs described earlier, perceived disease risk and knowledge are strong predictors of access to health services. In this study, we found a strong association between the perceived risk of prostate cancer and knowledge of prostate cancer and screening services with utilization of services. It is therefore of paramount importance to scale up ongoing prostate cancer campaigns among males above 40 years in the country.The findings presented here should be interpreted in light of a number of limitations. Firstly, the cross sectional nature of the design limits the ability to make causal inference for factors identified to be associated with prostate cancer screening. However, most factors described have also been published in other studies utilizing various designs and have consistently been associated with service utilization. Secondly, the sample size was calculated to estimate awareness of the screening method and its power to estimate relatively low rate of use is limited. Therefore our estimates of utilization of prostate cancer screening services should be interpreted with caution; thirdly, screening for prostate cancer involves a sensitive procedure particulary if DRE was used. This may have resulted in underestimation of our proportion of utilization due to desirability bias. Efforts were made to build good rapport during interviews and we believe that the accuracy of our estimates may have been strengthened by this, given that these estimates were similar to estimates from elsewhere in Africa.This study revealed that the level of knowledge about prostate cancer and screening services is alarmingly low among men in Dar es Salaam. Utilization of screening services for prostate cancer are rare in this population and associated with poor perceived disease risk, low income, lack of knowledge and younger age. Intervention measures aiming at increasing knowledge about prostate cancer and screening services need to be scaled up alongside ensuring that screening services are freely available.


P < 5.0 × 10−8 with nine previously reported loci and two novel loci that include rs9862599 on 3q13.11 (ALCAM) and rs75286142 on 21q22.12 (CLIC6-RUNX1). Validation study was carried out with 981 breast cancer cases and 1,394 controls from the Aichi Cancer Center. Pathway analyses of GWAS signals identified association of dopamine receptor medicated signaling and protein amino acid deacetylation with breast cancer. Weighted genetic risk score showed that individuals who were categorized in the highest risk group are approximately 3.7 times more likely to develop breast cancer compared to individuals in the lowest risk group. This well-powered GWAS is a representative study to identify SNPs that are associated with breast cancer in the Japanese population.Genome-wide association studies (GWAS) have successfully identified about 70 genomic loci associated with breast cancer. Owing to the complexity of linkage disequilibrium and environmental exposures in different populations, it is essential to perform regional GWAS for better risk prediction. This study aimed to investigate the genetic architecture and to assess common genetic risk model of breast cancer with 6,669 breast cancer patients and 21,930 female controls in the Japanese population. This GWAS identified 11 genomic loci that surpass genome-wide significance threshold of  Breast cancer is also the fifth leading cause of cancer death among women in Japan, with an estimated death of 14,285 in the year of 2017. Despite better 5-year survival rates for breast cancer compared to other malignancies, the age-adjusted incidence and mortality rate in Japan has increased steadily since the 1970s. Hence, predictive genetic markers and early detection screening methods to identify individuals at risk of breast cancer are crucial to reduce breast-cancer associated death.Breast cancer is the most common malignancy among women worldwide. Based on the report of Cancer Statistics in Japan 2018BRCA1 and BRCA2 genes, moderate effect size genes  as well as common variants conferring small effect sizes7. A total of 28 genome-wide association studies (GWAS) showing association with breast cancer risk have been published8. These studies successfully identified common variants in 70 genetic loci from diverse worldwide populations: Europe (70 loci), East Asians (8 loci), Africans (3 loci), Latinos (2 loci) and Ashkenazi Jews (1 loci)35. Risk loci and risk variants differ across different populations due to several possible reasons. These include insufficient statistical power in individual studies, complexity in linkage disequilibrium, and differences in allele frequencies as well as environmental exposure. Notably, studies have indicated the importance of carrying out regional GWAS to identify specific genetic risk factors that are associated with complex disease, which could facilitate better risk assessment in the regional clinical settings36. Our group has published two breast cancer GWAS: The first reported association of chromosome 10q26 (FGFR2) and 16q12 (TOX-LOC643714) to breast cancer while the second showed association of 3q25.1 (SIAH2) with hormonal-positive breast cancer in the Japanese population38. The size of our current study is three times that of our previous two studies38. To the best of our knowledge, it is the largest and most well-powered GWAS to date that aims to investigate the genetic architecture as well as to assess the common genetic risk model of breast cancer in the Japanese population.Breast cancer is a complex polygenic disease with diverse risk factors that include lifestyle and genetic mutations. Common mutations linked to breast cancer include highly-penetrant Two sets of GWAS were performed in this study with samples obtained from Phase I-II and Phase III Biobank Japan that consist of 6,669 breast cases and 21,930 female controls.39  of the test statistic for the GWAS of Phase I-II and Phase III were 1.202 and 1.019, respectively  against chromosome location . Subsequently, principal component analysis (PCA) was performed to assess population substructure of the sample populations. PCA revealed that case and control subjects that participate in this study were clustered into two major clusters, the mainland (Hondo) cluster and the Ryuukyu (southern island of Japan) clusterP-value threshold of 5.0 × 10−8 to be associated with breast cancer. These loci included 2q33.1 (TRAK2-ALS2CR11), 3q13.11 (ALCAM), 3q25.1 (SIAH2), 5p12 (FGF10-MRPS30), 5q11.2 (MAP3K1), 6q25.1 (CCDC170-ESR1), 10q26.13 (FGFR2), 12p11.22 (PTHLH), 12q24.21 (UBA52P7), 16q12.1 (2 independent SNPs on TOX3-CASC16) and 21q22.12 (CLIC6-RUNX1) shown in Table ALCAM) and 21q22.12 (CLIC6-RUNX1) are novel associated loci that surpassed genome-wide significance threshold . Despite this, the 12 SNPs showed similar effect size trends with Phase I-II and III cohorts. In addition, the inclusion of the replication cohort improved association P-values in the meta-analysis combining GWAS and replication studies  and rs75286142  imply an established link between these SNPs and breast cancer susceptibility  from Panther database that encompasses with genes mostly from the CLIC, EPB41 and FRMD families , which mostly consist of genes from the SIRT family  and higher expression of ALS2CR12 in breast mammary tissue  and higher expression of ALS2CR12  SNPs as well. eQTL was detected for rs2540431-linked SNPs rs2714486 and rs2540334 , rs9862599 (0.18249), rs1838337 (0.11120), rs7701466 (0.10993), rs79160707 (0.18772), rs6900157 (0.17682), rs2912778 (0.19709), rs805583 (0.16442), rs10744856 (0.12644), rs3803662 (0.12069), rs4784227 (0.18744) and rs75286142 (0.15262). The risk score groups were divided into 5 categories, odds ratio of each category increased concordantly to the level of risk score. Individuals (4% of cases and 2% of controls) in group 5, who carry the most risk alleles, have approximately 3.7 times higher risk to develop when comparing group 1 as reference with AUC of 0.593 , is a serine/threonine kinase that involved in the mitogen-activated protein kinase (MAPK) pathway that involves Ras, Raf, Mek, and Erk. MAPK cascade is known to be an important pathway for cancer cell survival, dissemination, and resistance to drug therapyALCAM gene. ALCAM encode for Activated Leukocyte Cell Adhesion Molecule, which is a glycoprotein that binds to T-cell differentiation antigen CD6 as well as plays a role in the process of cell adhesion and migration48. Decreased expression of ALCAM protein is reported as an indicator of poor prognosis in breast cancer49. The second novel SNP, rs75286142, is located on CLIC6-RUNX1. CLIC6 is one of the family members of chloride intracellular channels and CLIC6 expression profile was shown to be altered in breast cancer50. Runx1, a transcription factor, regulates various physiological processes that include cell proliferation, survival, differentiation and cell cycle progression. Importantly, RUNX1 somatic mutations were found in ER+, luminal subtype of breast cancer and indicate a tumor suppressor role for RUNX152. Further in-silico or functional analysis should be carried out to further investigate the effect of the identified SNP to ALCAM and RUNX1 gene as well as the crosstalk in between germline variations and somatic mutations in these breast cancer-associated genes.The first novel SNP, rs9862599, identified to be suggestively associated with breast cancer from this GWAS is located on chromosome 3q13.11 near to the 5′ end of 53. For instance, the expression of CLIC4 transcript is regulated by p53 and tumor necrosis factor α as well as related to Myc-induced apoptosis55. Additionally, CLIC1 protein levels were detected to increase in multiple cancers53. The second associated pathway, protein amino acid deacetylation, consist of SIRT and HDAC families. Sirtuins (SIRT1-7) play a significant role in cancer by regulating cancer-associated metabolism, modifying tumor microenvironment and affecting the response to genomic instability56. In breast cancer, besides SIRT6 that shows to have increased expression and act as oncogene, SIRT1, SIRT2, SIRT3 and SIRT4 exhibits to have reduced expression and act as tumor suppressor genes56. Although SNPs in these gene sets showed only moderate association with breast cancer, gene-set enrichment P-value indicated their cumulative association might be of significant for further investigations.Pathway analysis identified two pathways, dopamine receptor mediated signaling pathway and protein amino acid deacetylation, to be associated with breast cancer in this GWAS study. Dopamine receptor mediated signaling pathway consists of Chloride intracellular ionic channels family (CLIC1-6), Proteins of the 4.1 family (EPB41), Serine/threonine-protein phosphatase family (PPP1C) and FERM Domain Containing (FRDM) family. Among these gene sets, there are substantial reports about the involvement of the CLIC protein in tumorigenic process57. In addition to its central role in apoptosis, CASP8 also plays a number of non-apoptotic roles in cells, namely promoting activation NFκB signaling, regulating autophagy and altering endosomal trafficking, and enhancing cellular adhesion and migration57. The role of CASP8 varies and is highly dependent on cellular context57. Based on the in silico GTEx eQTL analysis, the risk allele of rs2714486-A and rs2540334-T is correlated with lower expression of CASP8. GWAS data show that both risk alleles are more frequent in breast cancer patients compared to healthy controls. All factors considered, our data suggests that CASP8 plays an apoptotic role in breast cancer, suppressing tumor malignancy. The ALS2CR12 gene product is a structural component of the sperm flagellum58. With no previous reports linking it to breast cancer, this makes ALSCR12 a weak candidate for breast cancer susceptibility.As an apical caspase, CASP8 functions to initiate a caspase cascade upon receipt of apoptosis signaling from a death receptor–ligand interactionThe AUC-value from wGRS analysis by utilizing 12 SNPs is 0.593, which indicates the current prediction model required further improvement by identifying additional markers that are associated with breast cancer susceptibility. Nevertheless, this well-powered GWAS is a representative study for the Japanese population to identify common genetic variations that are associated with breast cancer.59. In the Phase I-II study, there were 2412 estrogen receptor (ER+), 2010 progesterone receptor (PR+) and 2059 human epidermal growth factor receptor (HER+) breast cancer patients. In the Phase III study, there were 998 ER+, 790 PR+ and 1143 HER+ breast cancer patients. As for control, genotyping information of 16,496 and 5,434 female individuals who do not have cancer history from population-based cohorts of the Tohoku Medical Megabank organization (ToMMo) (http://www.megabank.tohoku.ac.jp/english/), Iwate Tohoku Medical Megabank Organization (IMM), Japan Multi-Institutional Collaborative Cohort (J-MICC) Study and the Japan Public Health Center-based Prospective (JPHC) Study60, and a hospital-based cohort of Biobank Japan were collected, respectively.Breast cancer case samples for the discovery study were recruited from the Biobank Japan . Biobank Japan collaboratively collects and stores DNA and serum samples throughout Japan. For the discovery set, a total of 5,272 and 1,397 breast cancer patients were recruited from Phase I-II and Phase III Biobank Japan Project, respectivelyTo validate the associations identified from GWAS, a total of 981 breast cancer cases and 1,394 females were collected from Aichi Cancer Centre. The detailed sample demographic and clinical parameters are summarized in Supplementary Table All participating studies obtained informed consent from all participants by following the protocols approved by their institutional ethical committees before enrollment. The ethical committees from the Institute of Medical Science, the University of Tokyo, RIKEN Center for Integrative Medical Sciences, Tohoku Medical Megabank organization, Iwate Medical University, National Cancer Center, and Aichi Cancer Center have approved this project.61. The top two principal components that could distinguish the clusters were used to produce scatter plot for the evaluation of distribution  by using EIGENSTRAT software (ver3.0) was carried out to compare the distribution of principal component scores between samples with four major reference population obtained from the HapMap Database that consist of Europeans (represented by Utah Residents (CEPH) with Northern and Western European Ancestry, CEU), Africans  and East Asian P-value versus expected P-value to evaluate potential population substructure  values were calculated to evaluate the deviation of the GWAS distribution from the null distribution. Since inflation factor scales with sample size and considering our large sample size, λ1000 was also calculated. Calculation was done as previously described62. We used Haploview 4.2 to visualize all SNP association P-values in a Manhattan plot, expressed as –log10 against chromosome position  software65. Association study of the imputed genotype dosage was carried out by using mach2dat (ver1.2.4)66. Stringent imputation quality (R2) threshold was applied by excluding SNPs with R2 < 0.9 for further studies. To combine Phase I-II GWAS and Phase III GWAS, whole-genome meta-analysis was carried out by using Inverse-variance meta-analysis method. The schematic study workflow is summarized in Fig. P-value < 0.05 considered a valid association.Statistical analysis for both case-control GWAS, Phase I-II and Phase III, were performed by using logistic regression analysis by incorporating associated principal components as covariates. Quantile-quantile plot (Q-Q plot) of each GWAS was constructed between observed To select SNPs for validation study, logistic regression analysis was performed by including the effects of primary associated SNPs of a genomic locus in order to exclude SNPs that have the similar effects and to identify SNPs that are independently associated with breast cancer from the primary associated SNPs.Pmeta < 1.0 × 10−5 that are not published previously to be associated with breast cancer were selected for validation study by using an independent samples group of 981 breast cases and 1,394 controls from the Aichi Cancer Centre, Japan. Genotyping of the SNPs were performed by using Multiplex Invader Assay. Considering the multiple testing for validation study, Bonferroni correction threshold at P < 5.00 × 10−3 was applied.A total of 22 SNPs with 62.To evaluate the combined effects of discovery Phase I-II, Phase III GWAS and validation study, meta-analysis was performed using weighted inverse-variance67 software was used to assess potential pathways that are associated with breast cancer by using GWAS and 1000 G imputed dataset (RSQ > 0.9). In brief, gene boundary between 110 kb upstream of the gene’s most extreme transcript start site and 40 kb downstream to the gene’s most extreme transcript end site was set. This boundary was suggested by a comprehensive study of putative functional regulatory element (cis-eQTLs) using expression data from human lymphoblastoid cell line. After assigning SNPs within the gene boundary, cumulative P-value of the SNPs within individual genes were calculated after correcting for confounders such as gene size, variant number and LD properties using step-wise multiple linear regression analysis. Gene-set enrichment analysis P-value was calculated by referring to a total of 3,217 gene-sets from Gene ontology, KEGG, PANTHER, Biocarta and Reactome databases. False discovery rate (FDR < 0.05) was used to evaluate the significance of associations of the pathway with breast cancer.Meta-Analysis Gene-set Enrichment of variaNT Associations To assess expression quantitative trait loci (eQTL), correlation between SNP genotypes and expression of the nearby genes in a genomic locus was evaluated from the GTEx portal V8  focusing specifically on breast-mammary tissue. The eQTL analysis was expanded to include all 12 genome-wide level linked variants (LD > = 0.8 ASN 1 K genomes) due to potential differences in the database SNP repository.P < 5.0 × 10−8 from the whole-genome meta-analysis of Phase I + II and Phase III GWAS were utilized to establish the model. These SNPs include rs2540431 on chromosome 2q33.1, rs9862599 on 3q13.11, rs1838337 on 3q25.1, rs7701466 on 5p12, rs79160707 on 5q11.2, rs6900157 on 6q25.1, rs2912778 on 10q26.13, rs805583 on 12p11.22, rs10744856 on 12q24.21, rs3803662 and rs4784227 on 16q12.1, rs75286142 on 21q22.12. The estimate (weight) of each associated SNP was evaluated by multivariate logistic regression analysis after incorporating 12 SNPs into the model. The cumulative risk scores were calculated by multiplying the weight (estimate) of the SNPs with the number of risk alleles (0/1/2) of the SNPs carried by each of the individual, subsequently the sum of the scores were taken across the number of SNPs in the model. The risk scores were then classified into four different categories that derived from mean and standard deviation (SD); group 1, < mean-1SD; group 2, mean-1SD to mean; group 3 mean to mean + 1 SD; group 4, mean + 1 SD to mean + 2 SD and group 5 > mean + 2 SD. Odds ratio and 95% confidence interval was evaluated by using group 1 as reference. Validation of this model was carried out by genotyping the 12 SNPs using the sample groups from Aichi Cancer Centre. Similar categorization was performed to evaluate the validity of this model. Lastly, receiving operating characteristic (ROC) curve was plotted to observe how well this model could be used as prediction model for breast cancer.wGRS analysis was carried out to evaluate the cumulative effects of genetic variants associated with breast cancer risk. A total of 12 SNPs that are with Supp Table 1, Supp Table 2, Supp Table 3, Supp Table 4, Supp Table 5, Supp Table 6Supplementary Information


The relationship between mitochondrial DNA (mtDNA) and breast cancer has been frequently examined, particularly in European populations. However, studies reporting associations between mtDNA haplogroups and breast cancer risk have had a few shortcomings including small sample sizes, failure to account for population stratification and performing inadequate statistical tests. In this study we investigated the association of mtDNA haplogroups of European origin with several breast cancer risk factors in mothers and children of the Avon Longitudinal Study of Parents and Children (ALSPAC), a birth cohort that enrolled over 14,000 pregnant women in the Southwest region of the UK. Risk factor data were obtained from questionnaires, clinic visits and blood measurements. Information on over 40 independent breast cancer risk factor-related variables was available for up to 7781 mothers and children with mtDNA haplogroup data in ALSPAC. Linear and logistic regression models adjusted for age, sex and population stratification principal components were evaluated. After correction for multiple testing we found no evidence of association of European mtDNA haplogroups with any of the breast cancer risk factors analysed. Mitochondrial DNA haplogroups are unlikely to underlie susceptibility to breast cancer that occurs via the risk factors examined in this study of a population of European ancestry. The relationship between mitochondrial DNA (mtDNA) and breast cancer has been frequently explored. Carcinogenesis has been associated with oxidative stress, with mitochondria acting as a major source of production of reactive oxygen species (ROS) . AdditioSince mtDNA is maternally inherited, it has been suggested that it could underlie the observation that having a mother diagnosed with breast cancer increases a woman’s risk of the disease.During evolution mtDNA mutations have segregated and clustered in groups of related haplotypes or haplogroups  that differ prominently in frequencies across continents ,7. The gNine major mtDNA haplogroups have been identified in Europeans . Haplogroup H is the most frequent, though the frequencies of individual clades vary within Europe .The association of mtDNA variants and haplogroups with cancer and other diseases has been extensively explored in the European population. For instance, there are reports on breast cancer ,11, prosIn order to elucidate the putative role of mtDNA in breast cancer we investigated the distribution of European mtDNA haplogroups across a range of well-known and possible risk factors for breast cancer in mothers and children of the Avon Longitudinal Study of Parents and Children (ALSPAC). If mtDNA variation plays a role in breast cancer susceptibility it may do so via risk factors that lie in the causal pathway to disease. Thus, we might be able to detect an association of mtDNA haplogroups with these exposures with less bias in a large and substantially homogeneous cohort like ALSPAC.http://www.bristol.ac.uk/alspac/). Hormone level measurements  were reported earlier [http://www.bris.ac.uk/alspac/researchers/data-access/data-dictionary/.The Avon Longitudinal Study of Parents and Children (ALSPAC) is a prospective, population-based birth cohort study that recruited 14,541 pregnant women residing in Avon County, United Kingdom, with an expected delivery date between 1 April 1991 and 31 December 1992. From these initial pregnancies, there were a total of 14,676 fetuses, resulting in 14,062 live births and 13,988 children who were alive at one year of age. When the oldest children were approximately seven years of age, an attempt was made to bolster the initial sample with eligible cases who had failed to join the study originally. The total sample size for analyses using any data collected after the age of seven is therefore 15,247 pregnancies, resulting in 15,458 fetuses, of which 14,775 were live births and 14,701 were alive at one year of age. Data were gathered through self-completed questionnaires or assessment at research clinics at regular intervals. The study is described in detail elsewhere ,26 . Written informed consent was obtained from all participants in the study.Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees  have been associated with both pre- and postmenopausal breast cancer. Conversely, factors that reduce the risk of breast cancer include increased physical activity, and breastfeeding, whereas being overweight or obese acts as a protective factor in premenopausal women.Other established risk factors are older age, White ethnicity, family history of breast cancer, a prior diagnosis of cancer, early menarche, late natural menopause, nulliparity, first pregnancy after the age of 30, hormone replacement therapy, use of oral contraceptives, high bone mineral density, diabetes mellitus and exposure to radiation such as X-rays.Possible breast cancer risk factors include smoking and night shift work. In contrast, a healthy diet and regularly taking aspirin or other non-steroidal anti-inflammatory drugs are considered protective factors.We also tested women having had reproductive surgery (hysterectomy and/or oophorectomy), and using non-oral hormonal contraceptives, which may affect breast cancer risk by altering hormone concentrations, and serum levels of sex hormone binding globulin (SHBG), testosterone, androstenedione, dehydroepiandrosterone-sulfate (DHEAS), insulin like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein 3 (IGFBP-3).More detailed information on the association of the risk factors considered with breast cancer can be found in the literature ,36,37,38Data on most risk and protective factors were available in ALSPAC from mothers, children or both.Details on the continuous and categorical variables examined in mothers and children are given in In some cases, usually when numbers were low, we created new variables that reflected whether the individual had ever experienced the activity or shown the trait of interest by combining the different instances where it was assessed. We did this for smoking, having diabetes, having had cancer, having undergone a hysterectomy and/or an oophorectomy, doing night shift work, taking oral contraceptives, using non-oral hormonal contraceptives, receiving hormone replacement therapy, taking aspirin, being a biological parent, undertaking physical activity and getting X-rays. The variable age of menarche in the children was determined by integrating all the information provided by the mother and the child .Genotyping methods for mitochondrial and nuclear DNA polymorphisms in ALSPAC have been previously described . HaplogrSamples with a quality score of more than 80% were included in the analysis , J, K, T, U, other European ; South Asian = M + subclade R5; East/Southeast Asian = A + C + D; and African = L. The ‘other European’ group consisted of haplogroups that were present in less than 3% of the sample.We restricted our analysis to all individuals who were of European genomic ancestry 2 individuals) or self-identified as White (if data on genomic ancestry was missing) and carried a European mtDNA haplogroup. There was information on 7781 mtDNA haplogroups in our working dataset.We used linear regression to investigate the association of continuous variables with mtDNA haplogroups and chi-squared tests to examine differences in categorical variables. Adjustment for confounders was carried out using linear and logistic regression models with continuous and categorical variables, respectively. When categorical variables exhibited more than two ordered categories ordinal logistic regression was run, also adjusted for confounders. Confounders introduced in the models were age, sex, gestational age and the top 10 principal components accounting for population stratification, where appropriate.We checked that the residuals of the linear regression of each continuous variable on mtDNA haplogroups were normally distributed. Only residuals for DHEAS levels in children showed a markedly non-normal distribution, and therefore the variable was natural log-transformed .p-value cut-off was 0.001 in both cases.Similarly to Howe et al. , we usedWe tested whether there was residual population stratification due to mtDNA clustering within our working dataset of European/White individuals by plotting the top two principal components by mitochondrial lineage as reported by Erzurumluoglu et al. for Y chromosome haplogroups in ALSPAC .All analyses were performed with the statistical package Stata 14 .http://bioinformatics.cesga.es/mitpower/) [Statistical power was calculated with mitPower using binary variables, as this approach has not been developed for continuous or ordinal variables yet (tpower/) .Mitochondrial DNA haplogroups found in mothers and children of ALSPAC that were used in this study are shown in It is interesting to note, although not completely unexpected , that dep = 0.15).No differences were identified in the distribution of mtDNA haplogroups by sex of the child , those ORs become ~1.3 to 2.1. This calculation excludes the variable ‘having diabetes’ in children as there were only 26 subjects with the disease and mtDNA data in ALSPAC.Given the sample sizes available for the binary variables, the haplogroup frequencies and the number of haplogroups included in the analysis, we had 80% power to detect odds ratios (OR) of ~1.2 (ever smoked) to 1.7  at an α level of 0.05 if considering HV as the risk haplogroup. Under the same conditions, at the significance level corrected for multiple testing (p ≤ 0.05). Most of these associations involved body composition variables such as body mass index (BMI), weight, height and bone mineral density , which were seen in mothers as well as children and at various instances across the lifetime. A few earlier studies have shown an association of mtDNA haplogroups with obesity and obesity-related traits ,49,50, wWhilst we did not have a replication cohort to confirm our findings, the analysis was run in two groups of individuals, albeit related, each with a different set of phenotypes. Further investigation is needed to strengthen the results presented here; however, these could prove useful to generate hypotheses for future, more powerful studies.Well-established and possible breast cancer risk factors were not found to be associated with mtDNA haplogroups in ALSPAC, a cohort of predominantly European ancestry. This study can serve as the basis for a further detailed analysis of the influence of mtDNA variation on nutritional, anthropometric and lifestyle exposures that underlie the susceptibility to breast cancer and other cancer types.