Ile655Val single nucleotide polymorphism (SNP) and susceptibility to early-onset breast cancer. Human epidermal growth factor receptor 2 (HER2) plays an important role in the development and progression of breast cancer. To understand the precise association, this meta-analysis was conducted to estimate the association between HER2Ile655Val SNP and risk of breast cancer. Adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to appraise the association under the additive model , dominant model , and recessive model . A comprehensive database retrieval from PubMed, Embase, Web of Science and Google Scholar was pooled to investigate links between the HER2Ile655Val SNP is associated with an increased risk of breast cancer in an additive and dominant model. In the subgroup analysis with age stratification, a significant association between the HER2 codon 655 SNP and the risk of breast cancer was found in young women in an additive, dominant, and recessive model; conversely, no significant associations were indicated in older women. In the breast cancer subgroup, HER2Ile655Val SNP was significantly associated with younger age women with breast cancer in the dominant model. In contrast, no association between the HER2 codon 655 SNP and age was found in control populations. Seventeen relevant studies with 11,749 cases and 8,105 controls were finally included. We found that HER2Ile655Val SNP might be a susceptibility factor that favours early-onset breast cancer.Our findings suggest that the Val allele in HER2 codon 655 SNP is strongly associated with breast cancer susceptibility in the young female population and is also significantly associated with younger age in women with breast cancer. HER2 Breast cancer is the most common cancer among women, with an increasing incidence in most countries, representing a public health threat . In the United States, breast cancer caused 42,000 deaths in 2017 . Clearly, there is a link with ageing, especially among women aged 45 to 65, and it is increasing among younger women . Breast cancer under the age of 40 accounts accounts for 3-7% of all breast cancer cases in women . In a recent analysis, breast cancer patients aged under 30 and 30-39 years were found to have a significantly lower survival rate than those aged 40-49 and 50-59 .Human epidermal growth factor receptor 2 (HER2), also known as c-erbB2 and neu, is located on human chromosome 17q21 and is responsible for encoding a 185-kDa cross-membrane glycoprotein receptor. HER2 belongs to the ErbB family of growth factor receptors with intrinsic tyrosine kinase activity. The members of this family take the form of homodimer and heterodimer when activated via cell growth, specifically chemical and invasion . HER2 overexpression is seen in breast cancer, gastric cancer, and ovarian cancer . HER2 targeted therapies have significantly enhanced the clinical outcome for HER2-positive breast cancer patients . Targets in downstream or resistant pathways of particular interest in HER2-positive breast cancer include mTOR, PI3K, IGF-1R, Akt, HSP90, and VEGF that allow cell development, survival, and differentiation . Ile655Val SNP has been identified in the transmembrane domain-coding region of the HER2 gene at codon 655, encoding either isoleucine (Ile: ATC) or valine  . Substitution of these two amino acids can alter the hydrophobicity of proteins, affecting the shape stability of the regions in the protein . Fleishman et al. found that substitution of Val for Ile in this position of the transmembrane region will destabilize the formation of active HER2 dimers, leading to reduced receptor activation and tyrosine kinase activity, even under conditions of HER2 overexpression . The association between the HER2Ile655Val SNP and the risk of breast cancer has been widely investigated in populations worldwide . Single nucleotide polymorphisms (SNPs) residing in regulatory or functionally relevant gene regions may affect protein function . HER2Ile655Val SNP and the risk of early-onset breast cancer has also been investigated; however, these results are inconclusive and controversial. Several articles have shown the association of HER2Ile655Val SNP with an increased risk of early-onset breast cancer in Chinese, Australian, and Taiwanese women . Nonetheless, the association has not been observed in other studies . In the present meta-analysis, we aimed to obtain a more reliable estimate of the association between HER2Ile655Val SNP and susceptibility to early-onset breast cancer.The association between HER2Search strategy and identification of relevant studiesWe systematically searched electronic databases including PubMed, Embase, Web of Science, and Google Scholar to identify all relevant articles with either single or combined search terms: “human epidermal growth factor receptor 2”, “Her2”, “ERBB2”, “neu”, “EGFP2”, “single nucleotide polymorphism”, “SNP”, “polymorphism”, “Ile655Val”, “rs1136201”, “Herceptin receptor polymorphism” and “breast cancer”. Records were included up to 1 January 2020 and screened based on title and abstract with duplicates removed. Full-text papers were independently reviewed and selected to identify the relevant articles for the meta-analysis by three of the authors .Criteria for inclusion and exclusionIle655Val SNP and breast cancer risk; (b) included allelic or genotype frequencies; (c) provided results with age stratification; (d) represented original data; (e) used a cohort study design, and (f) were written in English. Papers were excluded when they: (a) described a review, case report or conference abstract; (b) did not contain original data or were irrelevant to the current analysis; (c) did not provide genotypic distribution or allele frequency data.Studies that fulfilled the inclusion criteria were considered eligible for this meta-analysis. Studies were selected when they: (a) evaluated the association between HER2Data Extraction Ile655Val was assessed using Hardy-Weinberg equilibrium. The extracted data elements included the year of publication, ethnicity, number of cases and control samples, cut-off age, genotype distribution, allele frequency for each case, control, and age stratification groups. The genotype distribution of HER2Statistical analysisIle655Val SNP and susceptibility to breast cancer. The heterogeneity study was described using the Higgins I2 metric. An I2 value of 0% was considered to mean no observed heterogeneity, while a value greater than 50% was considered substantial heterogeneity.The meta-analysis was performed using Stata SE version 13.1 software . Random-effects models were used to synthesise the association between the HER2The strength of association was assessed by calculating adjusted odds ratios (ORs) with 95% confidence intervals (CIs). Pooled ORs were performed for the allele genetic (additive) model , dominant model , and recessive model , respectively. The results of all included studies were summarised using a conventional forest plot. Publication bias was estimated using funnel plot asymmetry and Egger’s linear regression test. A P-value < 0.05 was considered as representative of statistical significance.Subgroup analysisIle655Val SNP and breast cancer susceptibility in young and advanced age populations, subgroup analysis was performed by calculating adjusted ORs comparing cases and controls. To investigate the association between HER2 codon 655 polymorphism and the age of onset, subgroup analysis was performed by calculating adjusted ORs comparing older and younger participants in each breast cancer patient and control group.We employed subgroup analysis by age stratification (young and advanced age) and by study participants (breast cancer patients and control population). To investigate the association between HER2Characterisation of eligible studiesIle655Val SNP and the risk of breast cancer. Finally, 17 observational studies that met the inclusion criteria were selected for the meta-analysis . The detailed steps in the literature selection process are shown in The initial search identified 989 potential records from electronic databases, and 378 full-text articles were assessed for eligibility. Of these, 40 articles addressed HER2Ile655Val genotype in the control groups from the studies was consistent with Hardy-Weinberg equilibrium.The general characteristics of the studies in the present analysis are shown in Association between HER2Ile655Val SNP and breast cancer susceptibility in worldwide populationsIle655Val SNP and the risk of breast cancer in worldwide populations is shown in 2=61.0%; n=16). There was a 21% significant increase in the risk of breast cancer in subjects who were Val carriers  . The recessive model HER2 codon 655 was not associated with the risk of breast cancer . There was publication bias in the studies .The association between HER2HER2Ile655Val SNP is associated with increased breast cancer susceptibility in young women, but not in older women2=67.3%; n=6), dominant model , and recessive model . There was a significant 27% increase in the risk of breast cancer in young female Val carriers . Publication bias was found in the studies    . In contHER2Ile655Val SNP is associated with increased early-onset breast cancer susceptibilityIle655Val SNP and the age at onset of breast cancer. Ile655Val SNP polymorphism with the age of onset was found in the subgroup of breast cancer women under an additive model  and a recessive model . There was no publication bias in the studies . Furthermore, subgroup meta-analysis of the control population indicated no significant association between HER2Ile655Val SNP and the age of participants was found in control women under all genetic models  . Riaz et al., (2016) conducted a meta-analysis research and concluded that HER2Ile655Val SNP is significantly correlated with six major types of cancer , suggesting that carriers of the Val allele and Val/Val genotype may be linked with an elevated risk of these cancers. This meta-analysis found that HER2Ile655Val SNP is associated significantly with an increased risk of breast cancer, particularly in young women. Our findings are consistent with results from previous meta-analyses by Tao et al., (2009), Lu et al., (2010), Wang et al., (2013), Chen et al., (2014), and Krishna et al., (2018). However, a meta-analysis by Ma et al., (2011) revealed that HER2Ile655Val SNP is not associated with breast cancer susceptibility. Our results further demonstrate the potential contribution of this single nucleotide polymorphism to the oncogenesis of breast cancer. Several studies have independently discovered the association between HER2HER2 gene and the onset of breast cancer . Additionally, Millikan et al., (2003) and Tommasi et al., (2007) reported that there was a strong association between the variant allele 655Val and breast cancer in younger women when combined with family history . However, other published data reveal the opposite results, with no significant correlation between the HER2Ile655Val genotype and the risk of early-onset breast cancer in patients 40 to 50 years . We conducted the present meta-analysis, which collected data from 17 age-stratified articles with a cut-off age value from 40 to 55 years old (46.53 ± 3.84). We found that in young women, HER2 codon 655 polymorphism was strongly and significantly associated with breast cancer in all genetic models , which was in contrast to the results from a subgroup of older women. In the breast cancer population, the dominant model of HER2Ile655Val SNP was shown to be significantly associated with younger age. Therefore, the high presence of the Val allele in codon 655 of the HER2 gene might be an explanation for the increasing frequency of younger age onset of breast cancer. Prior studies independently found a correlation between the high presence of the Val allele in the codon 655 of the Ile655Val SNP, a non-polar to non-polar amino acid mutation, has been investigated in previous studies. Using computational exploration, Fleishman et al., (2002) proposed that the transmembrane region of the HER2 homodimer can exist in two stable conformations, either in an active or inactive form. The dimer mediated by the C-terminal dimerisation motif is more stable than the dimer formed by the N-terminal motif. The authors found that substitution of Val for Ile in this position of the transmembrane region will destabilise the formation of active HER2 dimers mediated by the N-terminal dimerisation motif and lead to reduced receptor activation and tyrosine kinase activity. However, the presence of the Val allele could reinforce the stabilisation of the receptor’s active state, which results in augmentation of autophosphorylation, hyper-active tyrosine kinase, and cellular proliferation . Bocharov and colleagues researched the spatial structure of the dimeric transmembrane domain of the HER2 protein. They found that the Ile655Val variant can excessively stabilise the ErbB2 active dimeric state due to substitution of the bulk side chain of Ile with the smaller Val, thus allowing tighter TM helix packing . In another experiment, Tanaka et al. assessed the role of amino acid substitutions in the conformational stability of human lysozyme protein via thermodynamic analysis at high temperature and very low pH. They showed that in constructed isoleucine to valine mutants, the stability of mutant proteins was reduced compared to that of the wild-type protein .The molecular mechanism of HER2REG1A, CK20, and MAP3K8 gene expression was shown to be related to early-onset colorectal cancer formation . Using PPI network analysis, Zhao et al., (2019) suggested that early-onset colorectal cancer is associated with vascular smooth muscle contraction signalling pathway. They also identified seven hub genes, namely, ACTA2, ACTG2, MYH11, CALD1, MYL9, TPM2, and LMOD1, along with this signalling pathway. Recently, using weighted gene co-expression network analysis and other analysis methods, Mo et al. identified seven genes  associated with the development and prognosis of early-onset colorectal cancer. These genes may serve as novel biomarkers for the diagnosis of early-onset colorectal cancer . In prostate cancer, a study conducted by Weischenfeldt et al. found the genomic alteration landscapes of early-onset prostate cancer compared to older-onset cancer. They discovered that early-onset prostate cancer possesses a higher frequency of balanced structural rearrangements, with a specific abundance of androgen-regulated ETS gene fusions and concluded that ETS fusion genes are signs of early-onset prostate cancer . Furthermore, Gerhauser et al., (2018) demonstrated the role of androgen receptor-driven rearrangements, an early APOBEC-driven mutational mechanism, and ESRP1 gene duplication that contributed to the pathogenesis seen in early-onset prostate cancer. Nevertheless, the molecular mechanism involved in the pathogenesis of early-onset breast cancer is to date poorly understood and needs to be studied further.Molecular mechanisms of the early onset state have been studied in certain types of cancer. Several candidate genes and signalling pathways have been found in the early onset of colorectal cancer. Ile655Val SNP and an increased risk of breast cancer. In addition, we propose that HER2Ile655Val SNP might be considered as a susceptibility factor for early-onset breast cancer. Further molecular studies are required to reveal the mechanism of this correlation. In conclusion, our analyses showed an association between HER2


Candidate endophenotypes should be systematically assessed against five criteria: (i) the endophenotype is associated with disease in the population; (ii) the endophenotype is heritable; (iii) within families, endophenotype and disease co-segregate; (iv) the endophenotype found in affected family members is found in non-affected family members at a higher rate than in the general population and (v) the endophenotype is primarily state independent . This study assesses the suitability of mammographic breast density as an endophenotype for breast cancer. Formally establishing a trait as a disease endophenotype confirms that the trait and endophenotype share a biological basis, thereby enabling genetic dissection of an endophenotype to inform disease risk. As breast density can be measured for any woman who has had a mammogram, studies investigating the genetic architecture of breast density could identify breast cancer risk variants that act through effects on this trait.Mammographic breast density (MBD) is a strong and highly heritable predictor of breast cancer risk and a biomarker for the disease. This study systematically assesses MBD as an endophenotype for breast cancer—a quantitative trait that is heritable and genetically correlated with disease risk. Using data from the family-based kConFab Study and the 1994/1995 cross-sectional Busselton Health Study, participants were divided into three status groups—cases, relatives of cases and controls. Participant’s mammograms were used to measure absolute dense area (DA) and percentage dense area (PDA). To address each endophenotype criterion, linear mixed models and heritability analysis were conducted. Both measures of MBD were significantly associated with breast cancer risk in two independent samples. These measures were also highly heritable. Meta-analyses of both studies showed that MBD measures were higher in cases compared to relatives  and in relatives compared to controls . This study formally demonstrates, for the first time, that MBD is an endophenotype for breast cancer. BRCA1/2, identified by linkage/positional cloning in breast cancer families more than 20 years ago, remain the single largest known genetic risk factor for breast cancer, accounting for ~30% of excess familial risk [Breast cancer is the second most commonly diagnosed cancer in Australia and the most commonly diagnosed cancer in females, with a lifetime prevalence of 1 in 7 [Mammographic breast density (MBD) is a strong ,5 and hiCandidate endophenotypes should be systematically assessed against five endophenotype criteria:  (i) the This study aims to assess the suitability of MBD measures as endophenotypes for breast cancer. As it is accepted that MBD is state independent of breast cancer  , we inveTwo study populations were used to assess the suitability of mammographic density measures as endophenotypes for breast cancer—kConFab and the Busselton Health Study (BHS).n = 405 participants from 183 families (plus friends) with 114 cancers).kConFab  has been collecting genetic, epidemiological, medical and psychosocial data from families with a strong history of breast cancer since 1997 and has accumulated data on more than 1400 multigenerational, multicase kindreds [Ethics approval was obtained from the Western Australian Department of Health Human Research Ethics Committee (#RGS0000002834) and the University of Western Australia Human Research Ethics Office (#RA/4/1/9183).Busselton is a rural, historically stable community ~230 km south of Perth, WA; predominantly of British (Anglo-Saxon) expatriate origin. The BHS is one of the longest-running international epidemiological research programs, with repeated cross-sectional surveys of adults undertaken between 1966 and 2007. The recruitment and data collection of participants from the BHS have previously been described in detail . In 1994Informed consent was granted from all participants in the 1994/1995 survey and ethics was obtained by the University of Western Australia Human Research Ethics Committee (#RA4/1/6694). The current study was approved by the Western Australian Department of Health Human Research Ethics Committee (#RGS0000002801).The participants for both studies were divided into three status groups: cases, unaffected relatives of cases (henceforward relatives) and controls. For the kConFab participants, information collected about each participant’s families was used to create pedigrees and assign each participant to a status group. For BHS participants, allocation to a status group was determined using the genetic relatedness matrix (GRM) generated from genome-wide SNP genotype data. BHS participants were assigned either as a case if they had a WA Cancer Registry-confirmed breast cancer, as a relative if they were not a case and the GRM estimated them to have a relatedness of greater than 0.0875 to a case , or finally as a control if they were not related to a case and were not a case themselves . Within Cranio-caudal film mammograms were retrieved from BreastScreen WA, digitized and measured by author JS using the Cumulus software . Where mammograms from multiple screening visits were available, the pre-diagnosis mammogram closest to the diagnosis date was selected for cases. For relatives and controls, the mammogram closest to the epidemiological data collection date was selected. If a relative or control had no epidemiological data, the earliest mammogram available was selected.The MBD measurements included absolute dense area and percentage dense area. Percentage dense area could not be measured for six participant’s mammograms from the kConFab study as the images had bad edges and thus total breast size could not be measured. Mammograms were measured twice for 10% of participants to assess reliability. Intraclass correlation coefficients for absolute dense area and percentage dense area were 0.98 in the kConFab Study and 0.99 in the BHS.Analyses were conducted in R version 3.6.3  and GenoWe assessed the association of absolute dense area and percentage breast density with breast cancer by testing for differences between cases and combined relatives and controls. Generalised linear mixed models, with a binomial distribution with logit link function (R package GMMAT ) was useNarrow-sense heritability is the proportion of the variability of the phenotype that can be attributed to additive genetic variation. Heritability estimation for absolute dense area software . Square Linear mixed models and estimated marginal means were used to assess whether the density measures differed across the three status groups: cases, relatives and controls. This is akin to testing genetic correlation, and in the presence of non-traditional family structures within these samples, comparisons of means between the three status groups was considered the most statistically powerful method to assess this. A square root transformation was applied to the MBD measures to normalise the distributions and models were adjusted for age at mammogram, BMI, time between mammogram and BMI collection, and the GRM, plus number of live births and menopause status for BHS. As before, estimates on the boundary space were assessed and are presented without GRM adjustment. A meta-analysis of the results from both studies was conducted using the R library ‘meta’.The final number of participants within the kConFab study was 323  and 1587  for the BHS. Descriptive statistics are shown in 2 was associated with an increased odds of breast cancer of 1.015 , compared to women with no breast cancer (relatives and controls). A smaller effect size  was observed within the BHS. Similarly, an increase of 1% in percent dense area was associated with an increased odds of breast cancer within kConFab , with a smaller increase in the BHS .The estimated heritability of absolute dense area and percentage dense area were both significant in both studies and were higher in kConFab  and percentage dense area  among relatives compared to controls. However, the evidence was weak.We have systematically assessed, for the first time, whether breast density is an endophenotype for breast cancer using five endophenotype criteria. The results of this study show that mammographic breast density (absolute and percent dense area measures) meets most of the criteria for being an endophenotype for breast cancer. Using two independent samples with a combined sample size of 1910, we provide evidence that (i) MBD is associated with breast cancer, (ii) MBD measures are heritable, (iii) within families, MBD and breast cancer co-segregate, and (iv) MBD measures within relatives of breast cancer cases are higher than in the general population.We found that both MBD—dense area and percent dense area—were positively associated with breast cancer risk, independent of age and BMI. These findings replicate well-established knowledge that MBD is an independent risk factor for breast cancer risk ,5, and o2 = 0.39, h2 = 0.30, respectively). Heritability estimates within kConFab were calculated using known familial relationships, while estimates within the BHS were calculated using the SNP-based relatedness estimates (and therefore represent the variation due only to the SNPs). The lower estimates within the BHS compared to kConFab are therefore likely due to the fact that total heritability  is assessed in the kConFab, whereas in the BHS the heritability estimate only reflects genetic variation captured by SNPs.Consistent with the literature, we estimated the heritability of dense area and percent dense area to be 0.59 and 0.68, respectively, within the kConFab study. We have previously reported heritability estimates between 0.6 and 0.67 for percent dense area [In our meta-analysis between the kConFab and BHS studies, we identified higher breast density in breast cancer cases, intermediate density in relatives of cases, and lower density in controls for both MBD measures. Evidence for these differences was strong comparing cases and relatives but was limited when comparing the relatives and controls. As the relatives of cases within the kConFab study may be more likely to have genetic variants predisposing them to breast cancer, the estimates involving relatives may be subject to selection bias. The differences in estimates involving relatives between kConFab and BHS might be due to both breast cancer and MBD having a greater genetic contribution  in the kConFab sample. The pooled marginal mean estimates were not significantly different , but did show an increase across each category, in line with cases having higher density, relatives with intermediate density and controls with the lowest density. These associations suggest that there is a genetic component in common between breast cancer and MBD measures.p-value = 0.008 and BHS: rhoG = 0.946, p-value < 0.001).This study has a number of strengths. First, we had access to genetic, mammography, and breast cancer case status from two epidemiological studies, representing 1910 women, consisting of 180 cases, 251 relatives of cases, and 1479 controls. The addition of family members within these studies allowed us to test the criteria among relatives of controls which is often not available in population-based or case–control cohorts. Second, the use of two studies also allowed us to validate our findings in an independent cohort, which is integral to genetic studies of this type. Third, we assessed the endophenotype criteria using two measures of MBD, percent and absolute dense area, and observed consistent results for both phenotypes. This is consistent with strong genetic correlation between percent and absolute dense area , and thow , genetic investigations of MDB may potentially identify novel risk variants for breast cancer and help identify novel breast cancer mechanisms. Improved understanding of these genetic associations could also inform future research towards tailored screening programs and prevention strategies.


No significant association with breast cancer risk was observed in premenopausal women. Greater adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations may reduce breast cancer risk in this black urban population of Soweto. Adherence thereof should be encouraged and form a part of cost-effective breast cancer prevention guidelines.Breast cancer prevention is of great importance to reduce high incidence in South Africa. This study aimed to investigate adherence to the 2018 World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) Cancer Prevention Recommendations and the association with breast cancer risk in black urban women from Soweto, South Africa. A total of 396 breast cancer cases and 396 population-based controls from the South African Breast Cancer study (SABC) matched on age and demographic settings were included. Validated questionnaires were used to collect dietary and epidemiological data. To assess adherence to these recommendations, an eight-point adherence score was developed, using tertiles among controls for scoring each recommendation  with zero indicating the lowest adherence to the recommendations. OR and 95 % CI were estimated using multivariate logistic regression models to analyse associations between the WCRF/AICR score and breast cancer risk. Greater adherence (>4·5  In South Africa, breast cancer has an age-standardised rate of 49 per 100 000 women and a mortality rate of 16·3 per 100 000 in 2018. Considering the emergence of the ongoing nutrition transition in Southern African countries, this burden is prospected to double by 2030 if no preventive actions are taken,4. Access to early detection and treatment of breast cancer are limited and contribute to high breast cancer incidence and mortality rates in South Africa. Primary prevention guidelines are therefore urgently required to aid in the reduction of breast cancer incidence rates and the burden on the public healthcare system. Although policy and guidelines exist in South Africa for early detection and referral of patients with breast cancer symptoms, primary prevention guidelines have not yet been developed,7. As an important first step in guiding the development of primary breast cancer prevention guidelines for South Africa, evidence of lifestyle (including dietary) factors in association with breast cancer risk in this population is needed.Breast cancer is the most commonly diagnosed cancer in women across the globe and the most common incident cancer in South African women. Adherence to the WCRF and AICR recommendations, for instance, high consumption of fruit, vegetables and whole grains, being active and low consumption of processed meat and alcoholic beverages, reduces the risk of developing cancer, including breast cancer, and other non-communicable diseases–18. When these Recommendations were revised in 2018, concern was drawn to the different scoring algorithms used to assess adherence to the 2007 WCRF/AICR Cancer Prevention Recommendations. Therefore, a standardised scoring algorithm , AICR and WCRF International, WCRF/AICR Continuous Update Project Expert Panel and international researchers) was developed to assess adherence to the updated recommendations and to enhance comparability between studies,19. Evidence used to compile the NCI-led standardised scoring algorithm is predominantly based on results of studies compiled in high-income countries.In 2007, experts from the World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR) developed a public health message to promote cancer prevention in the form of ten Cancer Prevention Recommendations focusing on diet, body weight and physical activity. However, little is known about the impact of adhering to these recommendations in low-and-middle-income populations such as black urban South African women.Greater adherence to the 2018 WCRF/AICR’s Cancer Prevention Recommendations, using the NCI-led standardised scoring algorithm, has shown inverse associations with breast cancer risk in high-income countriesPromotion of adherence to the 2018 WCRF/AICR’s Cancer Prevention Recommendations may contribute in establishing affordable breast cancer prevention guidelines in South Africa. Therefore, this study aimed to investigate the association between higher adherence to the 2018 WCRF/AICR’s Cancer Prevention Recommendations and breast cancer risk in black urban women from Soweto, South Africa. Adherence to individual recommendations of the 2018 WCRF/AICR Cancer Prevention Recommendations will also be investigated.n 396, controls n 396) conducted among black urban women from the greater Soweto population, in South Africa, was used to conduct this study,21. Breast cancer case participants (between the ages 27 and 86 years) were newly diagnosed breast cancer patients from the Chris Hani Baragwanath Academic hospital in Soweto, Gauteng, who were enrolled in the study as soon as possible after their cancer diagnoses before receiving any cancer treatments. The stage at breast cancer was clinically assessed at diagnoses and coded according to the tumour-node-metastasis classification. Controls (between the ages 26 and 88 years) were healthy (no illness or being pregnant) and unrelated to breast cancer cases with no history of cancer diagnoses and matched by age (±5 years) and area of residence. Participants were recruited from December 2014 to June 2017. The sample size had a sufficient power of 80 % (when type II error rate = 10 %) for OR ≥ 1·5 and type I error set at 5 %.The database from the South African Breast Cancer (SABC) study, a population-based, case–control study . Permission to conduct research at Chris Hani Baragwanath academic hospital was obtained from the Gauteng Province Medical Advisory Committee. All subjects gave written informed consent prior to participation.–25. The QFFQ consisted of 145 food items reported by recently published literature as frequently consumed staple foods and foods less frequently consumed. The dietary intake frequency included the amount of times foods were consumed per d/week per month or never. Life-size colour photographs of thirty-seven foods (in three portion sizes) were displayed in the food portion picture booklet. Participants were asked about their habitual dietary intake over the past month. Daily intakes of the different foods included in the QFFQ were calculated via a stepwise approach. At first, consumption frequencies were converted into number of days per month. Then, the amount of each serving consumed  was converted into g using standardised tables to convert household measurements into g. Finally, the daily consumption was calculated by multiplying the frequencies of consumption (d/month) with the portion sizes divided over 30 d. The daily energy and nutrient intakes were determined by multiplying the daily intake of each food item (as consumed) by the nutrient and energy content (per 100 g), derived from the South African Food Composition tables, and then adding the contribution from all food items together.A reproducible and validated culture-specific quantified FFQ (QFFQ)  was used together with food portion pictures, food models and household utensils. All questionnaires used to obtain the anthropometry and lifestyle information were validated and proven reproducible in studies conducted in South Africa and elsewhere–30.Trained fieldworkers and investigators conducted face-to-face interviews. Detailed information on demographic and socio-economic status such as ethnicity, history of health, family history of breast cancer, reproductive risk factors , family history of cancer, breast health , smoking habits and physical activity  was collected. Weight, height, sitting height and waist circumference were measured according to Lohman’s laws. ,19.The SABC study database was used to calculate an adherence score to the 2018 WCRF/AICR’s Cancer Prevention Recommendations and to assess adherence to individual recommendations of the 2018 WCRF/AICR’s Cancer Prevention RecommendationsEach of the participants’ adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations was calculated for overall adherence as well as each individual recommendation of the 2018 WCRF/AICR Cancer Prevention Recommendations. Adherence points were calculated as follows: one (1) point when adhering to the recommendation; 0·5 points for partial adherence and lastly, zero (0) points for non-adherence to the recommendation. Recommendations regarding cancer survivors and using supplementation to prevent cancer were not applicable to this study and were therefore excluded from the adherence score. A maximum overall adherence score of eight was therefore possible, where zero (0) indicates the lowest and eight (8) the highest adherence to the overall score. Each individual recommendation contributed equally to the total adherence score. The recommendations, ‘be a healthy weight’ and ‘eat wholegrains, fruit, vegetables and beans’, had two sub-recommendations that were scored individually and divided by two to determine an average score . The recommendation on breast-feeding was only applied to parous women .S1). Thus, using the predefined NCI cut-offs may result in insufficient statistical power to assess adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations and breast cancer risk in our study due to this (rather overweight and obese) population group. Therefore, the authors decided to use an alternative approach to score the individuals  for their level of adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations by using data-driven tertiles derived from the controls (33rd and 66th percentiles). Recommendations based on the data-driven tertiles in the current study population will be referred to as ‘adapted WCRF/AICR Cancer Prevention Recommendations’  were highly skewed  and Wilcoxon Signed-Rank test for skewed data with a skewness value <−0·5 and >0·5  for continuous variables. Paired χ2 test was used for categorical variables . Specifications of the WHO were used to calculate BMI, using measured height and weight (kg/m2).A total of 399 breast cancer cases and 399 controls were recruited in the SABC study. Of those, three breast cancer cases and three matched controls were excluded due to missing dietary information. Descriptive analyses were performed, and differences between breast cancer cases and controls were assessed using a paired sample v. post) and obesity (limited to BMI ≥ 30 kg/m2 due to sample size).To determine the association between breast cancer risk and adherence to both the 2018 WCRF/AICR and adapted WCRF/AICR Cancer Prevention Recommendations , conditional logistic regression models were used to compute OR and associated 95 % CI. Adherence scores  were stratified by hormonal breast cancer receptor subtypes (ER+ and PR+), menopausal status and obesity. Since breast cancer cases and controls were not matched on menopausal or obesity status, unconditional logistic regression was used when adherence  analyses were stratified by menopausal status (pre v. lowest), age at menarche (continuous), full-term pregnancy (yes/no), age at first pregnancy (<24 v. >24 years of age), age at menopause (<48 v. >48 years of age), parity (≤three children v. >three children), duration of exclusive breast-feeding (months), use of exogenous hormones , or hormone replacement therapy after menopause), family history of breast cancer (yes/no), HIV status (positive v. negative), total energy intake in kJ (continuous) and under-reporting . Under-reporting (13·1 % of breast cancer cases and 11·6 % of controls) and over-reporting (24·0 % of breast cancer cases and 27 % of controls) cut-off points were calculated using the Goldberg and Black principle to determine over- and under-reporting of energy intake,32. The Goldberg and Black principle is based on the following idea: if weight is stable, then energy expenditure equals energy intake,32.The following confounders were examined for adherence to the overall and individual recommendations of the 2018 WCRF/AICR Cancer Prevention Recommendations (as well as the adapted WCRF/AICR Cancer Prevention Recommendations): ethnicity , individual income , level of education , smoking (smokers and non-smokers), height , habitual physical activity per d (active and less active), ever breast-feeding (yes/no) and alcohol consumption (continuous). These additional confounders were only included in the analysis when not part of the individual recommendation itself.S2 and S3). These analyses were run as sensitivity analyses because predefined cut-points used in the NCI-led standardised scoring algorithm in our population (2018 WCRF/AICR Cancer Prevention Recommendations) resulted in highly skewed adherence categories  of the 2018 WCRF/AICR Cancer Prevention Recommendations.Although this study mainly focuses on the results of scoring systems based on data-driven analysis of the adapted 2018 WCRF/AICR Cancer Prevention Recommendations , sensitisd 13·8) cm) compared with controls (95·8 (sd 13·7) cm) and were often less HIV-positive (16·5 %) than controls (22·6 %). Controls had higher percentage of alcohol drinkers, higher consumption of ethanol (g/d), higher intakes of fruit, non-starchy vegetables, red-and-processed meat and energy-dense, fast foods than cases. Hormone-responsive breast cancers, ER+ (75·2 %) and PR+ (75·2 %) were the dominant breast cancer subtypes, while triple-negative breast cancer accounted for 16·1 % .v. lowest adherence (0–3·25) to the adapted WCRF/AICR Cancer Prevention Recommendations showed a significant inverse association with breast cancer risk overall  as well as in postmenopausal women , in ER+, in PR+ breast cancer’s  and in obese women . Furthermore, per one-point increment adherence to the adapted WCRF/AICRS’s Cancer Prevention Recommendations showed an inverse association with breast cancer risk overall  and in participants with ER+ and PR+ breast cancers . No significant association with breast cancer risk was observed in premenopausal women . Sensitivity analyses were conducted by excluding under- and over-reporters (according to the Goldberg and Black principle),32 and HIV-positive cases in the analysis (separately) but did not alter the outcome (results not shown).Adherence to the overall adapted WCRF/AICR’s Cancer Prevention Recommendations is presented in P = 0·010), in postmenopausal women , in participants with ER+ breast cancers  and in obese women . Higher consumption of fresh fruit and non-starchy vegetables showed an inverse association with breast cancer risk overall  and in postmenopausal women , while lower fast-food consumption showed an inverse association with breast cancer risk overall . Greater adherence to the recommendation on ‘limiting alcohol consumption’ showed a positive association with premenopausal breast cancer risk .Adherence to individual recommendations of the adapted WCRF/AICR Cancer Prevention Recommendations, using data-driven tertiles, is presented in S1), only 42 % of controls and 31 % of breast cancer cases consumed more than 400 g of fruit and non-starchy vegetables per day. As for adherence to the recommendation on limiting consumption of fast foods and other processed foods high in fat, refined starches or sugars, only 43 % of cases and 37 % of controls adhered to this recommendation . Furthermore, over 80 % of both breast cancer case and control participants did not adhere to the recommendation on ‘being a healthy weight’ (BMI < 25 kg/m2), while only 5 % of control and 6 % of case participants adhered to the recommendation on ‘be physically active ’.In contrast, when looking at the adherence to individual recommendations of the 2018 WCRF/AICR Cancer Prevention Recommendations  and increasing fresh fruit and vegetable intakes .v. lowest adherence) overall  and per one-point increment adherence to the recommendations . Similar results were observed in a cohort study that was conducted in two Swedish populations, showing that higher adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations was inversely associated with overall cancer risk  and per one-point increment adherence to the score . These studies did not differentiate between menopausal status, breast cancer receptor subtypes and obesity. The overall results from this study, using an adapted scoring algorithm, are similar to these findings mentioned above, even though there was a significant difference in the population under study.Studies investigating the relationship between adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations and breast cancer risk are still scarce, especially in non-Caucasian populations. One of the first case–control studies  investigating this relationship showed a significant inverse association with breast cancer risk (highest 2). Similar low adherence to this recommendation was observed in an American cohort study . However, more than 60 % of breast cancer cases and controls in the European Prospective Investigation into Cancer and Nutrition cohort study adhered to the individual recommendation of ‘Be a healthy weight’. Similar high adherence to this recommendation was observed in a Moroccan-matched case–control study (1516 cases and 1516 controls), a Spanish cohort study (n 10 930) and the MCC case–control study conducted in Spain (1343 breast cancer cases),36. In addition, this study showed concerning low levels of adherence (5 % of controls and 6 % of breast cancer cases) to the 2018 individual WCRF/AICR Cancer Prevention Recommendation to ‘be physically active’ . The case–control study of Turati et al. (2020) conducted in Italy and Switzerland showed higher adherence to this particular individual recommendation (30·1 % of controls and 23·5 % of breast cancer cases). Low adherence to these particular recommendations is concerning. Physical inactivity may increase the risk of being overweight or obese and being overweight or obese may increase postmenopausal breast cancer risk.When adherence to individual recommendations of the 2018 WCRF/AICR Cancer Prevention Recommendations (using the NCI-led predefined cut-off points) was first analysed, highly skewed distributions  were observed in the majority of the individual recommendations. For instance, over 80 % of both breast cancer case and control participants did not adhere to the individual 2018 WCRF/AICR Cancer Prevention Recommendation of ‘being a healthy weight’  have too little participants, to obtain sufficient statistical power, given the sample size. Thus, using predefined NCI-led cut-offs may not reflect true associations with adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations and breast cancer risk in this study population. Therefore, data-driven tertiles were used to determine more evenly distributed cut-points to assess adherence to an adapted version of the 2018 WCRF/AICR Cancer Prevention Recommendations. However, results on adherence to the adapted WCRF/AICR Cancer Prevention Recommendations should be interpreted with caution as the adapted score only refers to a partially healthier lifestyle for this non-Caucasian population (while yet not adhering to the WCRF/AICR’s recommendations), as such requiring further investigation.. Thus, lower-income populations may not be able to afford or prepare healthier foods as recommended by the WCRF/AICR. This is worrisome as results of this study indicated that higher adherence to the adapted WCRF/AICR Cancer Prevention Recommendations may protect against the development of breast cancer in this population.In this study, adherence to the overall adapted WCRF/AICR Cancer Prevention Recommendations (an adherence score >4·5–8) was low in both breast cancer case and control participants, and the vast majority of our study participants (over 80 % in both case and control participants) were not following a healthy lifestyle. Low adherence to the adapted WCRF/AICR Cancer Prevention Recommendations may be attributed to not only the income levels of this population but also the high availability of ultraprocessed foods. In South Africa, a diet rich in wholegrains, fruit and vegetables can cost up to 69 % more than a diet consisting of ultraprocessed, known to be energy-dense and nutrient-poor foods et al. (2020), which showed that greater consumption of wholegrains, fruits and vegetables and greater limitation of fast food and other processed foods high in fat, refined starches or sugars were associated with a reduction in breast cancer risk overall . However, when adherence was compared with the 2018 WCRF/AICR Cancer Prevention Recommendations, low adherence was noted in both breast cancer cases and controls for ‘consuming fruit and vegetables (>400 g/d)’ and ‘limit consumption of fast foods and other processed foods high in fat, refined starches or sugars . The low consumption of fruit and vegetables and higher consumption of ultraprocessed foods may partially be explained by their cost, availability and convenience,39. Due to rapid economic growth and urbanisation, the ongoing nutrition transition in South Africa contributes to more obesogenic food environments and displacement of whole foods with ultraprocessed foods–43. Intervention is therefore required to improve accessibility, availability and affordability of healthier (nutrient-dense) foods such as fruit and vegetables to enhance healthier lifestyles of the South African population.In particular, higher adherence to the adapted individual recommendations of ‘consuming fruit and vegetables’ (>543 g/d) and ‘limit consumption of fast foods and other processed foods high in fat, refined starches or sugars’  showed strong inverse associations with breast cancer risk in this study. Similar results were observed in the case–control study of Turati Only two of the adapted individual WCRF/AICR Cancer Prevention Recommendations, consuming fruit and vegetables and limit consumption of fast foods and processed foods high in added sugar, fat and refined starches, showed significant inverse associations with breast cancer risk in this study. Yet, adherence to the overall adapted WCRF/AICR Cancer Prevention Recommendations showed a strong inverse association with breast cancer risk overall, in postmenopausal women, in women with ER+ and PR+ breast cancers and in obese women. This highlights the importance of following an overall healthy lifestyle pattern, instead of adhering only individual WCRF/AICR Cancer Prevention Recommendations for cancer prevention. The WCRF/AICR Cancer Prevention Recommendations on diet, body weight and physical activity should therefore be promoted as a major tool for cancer prevention.2) compared with having higher BMI (>34·7 kg/m2) did not show any significant association with breast cancer risk in our study. However, strong evidence shows that greater body weight increases postmenopausal breast cancer risk. In this study, almost half of the breast cancer cases were diagnosed in later stages of breast cancer (stage III/IV), which could cause involuntary weight loss due to advanced cancer symptoms,45. Thus, breast cancer cases with late-stage cancer diagnoses may have lost weight prior participation in the study and may therefore not reflect their usual weight. In addition, the way fat is distributed in the body may also influence breast cancer risk. The positive association between breast cancer risk and a smaller waist circumference should be interpreted with caution and requires further investigation.Interestingly, smaller waist circumferences (<92 cm) were positively associated with breast cancer risk, while having a lower BMI  together with low quantities of alcohol (ethanol) consumption among alcohol consumers (cases = 5·4 g and controls = 4·6 g). Low alcohol intakes and consumers of alcohol, and low socio-economic status together with the small sample size may contribute to the unexpected results. The positive association between adhering to alcohol recommendations and breast cancer risk in this study should be interpreted with caution and requires further investigation.Greater adherence to the adapted recommendation ‘limiting alcohol consumption’ showed a positive association with premenopausal breast cancer risk in this study. Yet, robust evidence shows that alcohol consumption (no identified threshold) increases both pre-and-postmenopausal breast cancer risk, especially in higher-income countriesThe sample size of this study is rather limited  but provides much-needed evidence to consider in breast cancer prevention policies in South Africa. No physical examination  was performed to determine whether control participants were breast cancer-free and could have been asymptomatic or undiagnosed. Dietary intake was measured over the past month when the habitual dietary intake of case participants could have changed due to illness. Seasonal variability of foods may have influenced usual dietary intakes. The nature of the study design is more prone to information bias since dietary data collection relies on the memory of participants. Also, the homogeneity of the study population for some of the individual WCRF/AICR recommendations may be considered as a potential limitation as no associations can be found when most of the people are sharing the same characteristic.This study had improved statistical precision due to the population-based and matched case–control study design . Cases were recruited prior to any breast cancer treatment; questionnaires used to obtain data were proven to be validated and data used in the analysis were standardised. In addition, sensitivity analyses excluding under- and over-reporters and HIV-positive cases did not modify our results. This study provides quantitative evidence for assessing adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations in low-and-middle-income populations where cancer research is lacking. In addition, this study included two complementary approaches to investigate associations between breast cancer risk and adherence to the 2018 WCRF/AICR Cancer Prevention Recommendations, namely the fixed cut-off approach as well as a data-driven approach in response to the very skewed distributions that were found for some of the individual WCRF/AICR Cancer Prevention Recommendation.In conclusion, greater adherence to the adapted WCRF/AICR’s Cancer Prevention Recommendations is associated with a reduced breast cancer risk in this black urban female population. In addition, higher consumption of fruit and vegetables and reduced consumption of fast/processed foods may play a key role in reducing breast cancer risk in this urban population. Considering the very low number of participants adhering to the individual 2018 WCRF/AICR Cancer Prevention Recommendations on being at a healthy weight and being physically active, adherence to these recommendations should be encouraged to promote health. Last, in an effort to promote the overall health of all South Africans, intervention is required to improve accessibility, availability and affordability of healthier foods such as fresh fruits and vegetables in South Africa.


Recent studies comparing canine mammary tumors (CMTs) and human breast cancers have revealed remarkable tumor similarities, identifying shared expression profiles and acquired mutations. CMTs can also provide a model of inherited breast cancer susceptibility in humans; thus, we investigated breed-specific whole genome sequencing (WGS) data in search for novel CMT risk factors that could subsequently explain inherited breast cancer risk in humans.WGS was carried out on five CMT-affected Gold Retrievers from a large pedigree of 18 CMT-affected dogs. Protein truncating variants (PTVs) detected in all five samples (within human orthlogs) were validated and then genotyped in the 13 remaining CMT-affected Golden Retrievers. Allele frequencies were compared to canine controls. Subsequently, human blood-derived exomes from The Cancer Genome Atlas breast cancer cases were analyzed and allele frequencies were compared to Exome Variant Server ethnic-matched controls.Carcinoembryonic Antigen-related Cell Adhesion Molecule 24 (CEACAM24) c.247dupG;p. was the only validated variant and had a frequency of 66.7% amongst the 18 Golden Retrievers with CMT. This was significant compared to the European Variation Archive  and non-Golden Retriever American Kennel Club breeds . With no direct ortholog of CEACAM24 in humans but high homology to all CEACAM gene family proteins, all human CEACAM genes were investigated for PTVs. A total of six and sixteen rare PTVs were identified in African and European American breast cancer cases, respectively. Single variant assessment revealed five PTVs associated with breast cancer risk. Gene-based aggregation analyses revealed that rare PTVs in CEACAM6, CEACAM7, and CEACAM8 are associated with European American breast cancer risk, and rare PTVs in CEACAM7 are associated with breast cancer risk in African Americans. Ultimately, rare PTVs in the entire CEACAM gene family are associated with breast cancer risk in both European and African Americans with respective p-values of 1.75 × 10–13 and 1.87 × 10–04.CEACAM mutations and breast cancer risk, and potentially implicates the whole gene family in genetic risk. Precisely how these mutations contribute to breast cancer needs to be determined; especially considering our current knowledge on the role that the CEACAM gene family plays in tumor development, progression, and metastasis.This study reports the first association of inherited  Breast cancer is a serious health concern. Amongst both sexes, it globally ranks as the second most commonly diagnosed type of cancer and the second leading cause of cancer-related deaths, accounting for ∼2.1 million new diagnoses and 626,679 deaths in 2018 . WorldwiCarcinoembryonic Antigen-related Cell Adhesion Molecule 24 (CEACAM24) protein-truncating variant (PTV) in a Golden Retriever CMT pedigree, which ultimately revealed that rare PTVs in the CEACAM gene family are associated with breast cancer risk in humans. Aberrant expression of many CEACAM genes have previously been associated with tumorigenesis, and CEACAM gene products are recognized as clinically-relevant tumor markers  was the only validated variant. Following validation, the 13 remaining CMT-affected Golden Retrievers underwent PCR and Sanger sequencing to determine their mutation status.As previously described by 1 was initially used to note the allele frequency of CEACAM24 c.247dupG;p.. The European Variation Archive provides high quality WGS variant calls of over 200 dogs from multiple breeds (breed and sex information was unknown). The data was obtained through Ensembl by accessing the canine gene’s “Variant table” under “Genetic Variation”; for a particular variant, “Population genetics” information was given, including European Variation Archive allele frequencies . CEACAM1, CEACAM23, and CEACAM30 did not have variant information available in Ensembl for European Variation Archive data.As a convenient, publically available, online canine genetic variant repository, the European Variation Archivequencies . FurtherCEACAM24 c.247dupG;p.. This included DNA from Golden Retrievers (n = 87), as well as 13 other breeds, including Petit Basset des Griffon (n = 10), Gordon Setter (n = 8), Australian Cattle Dog (n = 10), Siberian Husky (n = 10), Dalmatian (n = 10), Irish Setter (n = 9), Welsh Pembroke Corgi (n = 10), Standard Schnauzer (n = 10), Newfoundland (n = 10), Keeshond (n = 10), Great Dane (n = 8), Doberman Pinscher (n = 10), and Boxer (n = 10). PCR and Sanger sequencing were carried out to determine CEACAM24 c.247dupG;p. genotypes of each dog.Through the CHIC repository, blood or buccal-swab derived DNA from purebred, American Kennel Club registered dogs were randomly selected and obtained to determine the frequency of CEACAM24 c.247dupG;p. allele frequencies, p-values were generated using the Fisher’s Exact Test in R (v 3.5.1), comparing allele differences in Golden Retriever to control dogs, including both European Variation Archive and CHIC DNA samples.Upon determining EMBOSS water alignment  was carrCEACAM24 in humans, all human CEACAM family genes were investigated for rare PTVs in The Cancer Genome Atlas (TCGA) breast cancer cohort. Investigating inherited risk, only blood-derived exomes of breast cancer cases were analyzed. Overall, whole-exome binary sequence alignment mapping (BAM) files were downloaded using the Genomic Data Commons (GDC) Data Portal Repository through approved research project #10805. To acquire the samples, the specific filters under the “Cases” category included: Project (TCGA-BRCA), Samples Sample Type , and Race (“Black or African American” and “White”). The samples were further filtered under the “Files” category, including Experimental Strategy (WXS) and Data Format (BAM). A total of 170 sample files were obtained for African Americans and 650 for European Americans. These files were downloaded using the GDC Data Transfer Tool (version 1.2.0). Only individuals with known ages of breast cancer onset were used in this study; as a result, one European American and two African American BAM files were removed from further bioinformatics processing and statistical analysis.Due to the homogeneity of the CEACAM gene family and no direct ortholog of dog CEACAM1 , CEACAM3 (NM_001815 at chr19:41796587-41811554), CEACAM4 , CEACAM5 , CEACAM6 , CEACAM7 , CEACAM8 (NM_001816 at chr19: 42580243-42594924), CEACAM16 , CEACAM18 , CEACAM19 , CEACAM20 , and CEACAM21  were then extracted from the ethnic specific VCF files and annotated using ANNOVAR (version June2017). Variants were filtered to include rare PTVs with ethnic-specific minor allele frequencies of <1% in Exome Variant Server  Exome Sequencing Project)  best practices pipeline . Base quProject) .p-values for each aggregation test. To accommodate for the one-sided nature of the Fisher exact test p-values, compliments of p-values in the opposite direction were used in the calculations for the Fisher’s method aggregation analyses.Using the Fisher’s exact test  in R v . The R tMutalyzer was used to determine the effect of frame-shifting and non-sense variants on the coded protein . Human sCEACAM24 ) was determined to be a true positive upon validation  was subsequently determined in different American Kennel Club breeds  allele frequency ranged from 0 to 80% in the assessed breeds  abolishes the extracellular region, the transmembrane domain, and part of the cytoplasmic region, including the Ig V-set domain , was associated with breast cancer risk in both ethnicities  in Golden Retrievers with CMT and subsequently determined that rare PTVs in the entire CEACAM gene family were associated with inherited breast cancer risk in humans. We previously described a large Golden Retriever pedigree with segregating CMT, carried out WGS on five selected Golden Retriever cases, and highlighted variants in orthologs of human breast cancer susceptibility genes  was the only validated variant and had an allele frequency of 66.7% amongst the 18 CMT-affected dogs. Despite not being recognized as a breed highly affected by CMT, Golden Retrievers have a higher prevalence of cancer compared to many dog breeds with 65% of Golden Retrievers in the United States succumbing to the disease  allele frequency and cancer mortality rate are very similar.Utilizing a comparative oncology approach, our team identified ty genes . In this disease . The GolCEACAM24 c.247dupG;p. in the European Variant Archive was 17.3%, which corresponded to a p-value of 1.52 × 10–8 when compared to our CMT-affected Golden Retrievers from the United States. However, in addition to not knowing breed-specific information in the European Variant Archive, genetic bottlenecks upon importation to the United States need to be acknowledged. Thus, comparing allele frequencies to a United States dog population with known breed status was important, which can be determined through American Kennel Club registration. Overall, CEACAM24 c.247dupG;p. appears to be common in Golden Retrievers in the United States with an allele frequency of 67.8%, which is not significantly different from the CMT-affected Golden Retriever cases. However, that allele frequency was determined by screening 87 Golden Retrievers from the CHIC repository with unknown disease diagnoses and age at sample submission. This is not ideal for canine cancer studies; older dogs (> than 8 years of age) with unaffected CMT-status are recommended  truly is a high-frequency allele in Golden Retrievers due to a genetic bottleneck in the United States, it can explain why 65% of Golden Retrievers succumb to cancer  allele frequency of 22.4% was revealed, which was significantly different from CMT-affected Golden Retriever cases. Noting the small sample sizes of each breed, over half of the assessed breeds showed no presence of the variant. However, some breeds contained the variant at higher levels; most notably, Petit Basset Griffon Vendeen, Gordon Setter, Australian Cattle Dog, Siberian Husky, and Dalmatian. Petit Basset Griffon Vendeen, which had the highest allele frequency, has a cancer mortality rate of 33%  abolishes the extracellular region, the transmembrane domain, and part of the cytoplasmic region, including the Ig V-set domain; thus, it is presumed to be a loss-of-function mutation. According to Ensembl, no other stop gain or frame-shifting variants have been identified in dog CEACAM genes. However, one splicing mutation in CEACAM28 (c.1415-2A > G) was identified, which had a 34% allele frequency within the European Variation Archive. The CEACAM gene family is present in many mammalian species but has evolved in a highly species-specific manner, heavily influenced by pathogen/host coevolution , which was detected in 10.8 and 4.5% of European and African American cases, respectively, was absent in all EVS controls. It severely truncates the 265 amino acid proteins and results in a loss of the cytoplasmic region, as well as a large portion of the extracellular region, including disruption of the Ig-like and Ig V-set domains. It is likely a loss-of-function mutation  intron 5 or removing exon 6, the last non-coding exon, where many microRNA binding sites are located  were associated with African American breast cancer. These mutations were not detected in European American cases or controls, and are very rare in the general African American population. They were detected in significantly more African American breast cancer cases compared to ethnic-matched controls, suggesting their involvement in African American breast cancer risk. However, gene-based aggregation analyses did not support CEACAM4 as a breast cancer risk gene. Larger African American breast cancer cohorts will need to be studied to validate these findings. Interestingly, in a study of parous women with and without breast cancer, CEACAM4 has been reported to be up-regulated in normal breast compared to breast tumor samples  for the Protection of Human Subjects in Research. The patients/participants provided their written informed consent to participate in this study. Ethical review and approval was not required for the animal study because this research did not require ORC – Animal Care & Use (IACUC) approval since only dog DNA was studied upon receipt from the CHIC repository.AH and NM wrote the manuscript and performed variant and statistical analyses. AH and IM performed PCR for validation and determining mutational frequency. AH performed bioinformatic processing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Branched chain amino acids (BCAAs) are essential amino acids commonthroughout the US diet. Although circulating BCAAs have been implicated ininsulin resistance and some obesity-related cancers, the relationshipbetween dietary intake of BCAAs and incident breast cancer is unknown. Wesought to evaluate the association between long-term dietary intakes ofBCAAs and invasive breast cancer risk.Our analyses included 196 161 women from the Nurses’ Health Study andNurses’ Health Study II longitudinal cohorts. Average intakes oftotal and individual BCAAs  were estimated fromrepeated diet questionnaires and incident self-reported breast cancer caseswere confirmed via medical record review. Cox proportional hazards models,adjusted for reproductive history, lifestyle, body mass index, and otherbreast cancer risk factors, were used to estimate hazard ratios and95% confidence intervals.Ptrend= .20). Findings were consistent across molecular subtypes andaccording to type 2 diabetes diagnosis and body mass index categories.We observed 10 046 incident cases of breast cancer over a median of20.8 years of follow-up. No associations between dietary intakes oftotal or individual BCAAs with breast cancer risk were observed. Comparedwith women in the bottom quintile of BCAA intake, the hazard ratio of breastcancer for those in the top quintile was 1.05 (95% confidenceinterval = 0.98 to 1.12; 2-sided Dietary intakes of BCAAs are not likely a risk factor for breast cancer. Branched chain amino acids  are essential aminoacids derived solely from diet and from a wide range animal and vegetable proteins,including processed meats, fish, poultry, dairy, beans, and some grains. The metabolismof BCAAs as a building block in protein synthesis is well characterized, contributing toa wide range of physiologic functions throughout the human body . HoweverPrevious mechanistic evidence from basic human and animal studies are largely consistent,demonstrating BCAAs have the ability to impair insulin action and signaling in skeletalmuscle through upregulation of the mTOR pathway . Breast Therefore, we evaluated dietary intakes of BCAAs in relation to breast cancer risk in 2large prospective cohorts of US women. The NHS and NHS II have repeated measures of dietspanning several decades of follow-up. Further, incident breast cancer cases areconfirmed, and detailed information on tumor characteristics is collected. Wehypothesized that dietary intakes of BCAAs confer a modest increased risk of incidentbreast cancer.We conducted a prospective longitudinal analysis of dietary BCAA intake inrelation to incident breast cancer risk in the NHS and NHS II cohorts. The NHSand NHS II were established in 1976 and 1989 with 121 701 and 116 429 femalenurses, respectively. NHS participants were aged 30-55 years and NHS IIparticipants were 25-42 years at study baseline. Baseline questionnaireswere administered in both cohorts to establish medical and reproductive history,lifestyle characteristics, and other factors. Questionnaires update thisinformation on a biennial basis. A semiquantitative food frequency questionnaire(FFQ) was also administered to the NHS with 61 items in 1980 and NHS II with 116items in 1991, which were expanded to include 152 items. The FFQs areadministered to update usual diet approximately every 4 years. The studyprotocol was approved by the institutional review boards of the Brigham andWomen’s Hospital and Harvard T.H. Chan School of Public Health as well asthose of participating registries as required.r = 0.56 for animal protein;r = 0.66 for vegetable protein  . Deaths ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2),cytokeratins (CK5/6), epidermal growth factor receptor (EGFR), and tumor gradewere used to classify tumors into molecular subtypes. Cases that were ER+and/or PR+ and HER2− and histologic grade 1 and 2 were classifiedas luminal A cancers; cases that were either: 1) ER+ and/or PR+and HER2+, or 2) ER+ and/or PR+, HER2−, andhistologic grade 3 were classified as luminal B; cases that were ER−,PR−, and HER2+ were classified as HER2 enriched; and cases thatwere negative for ER, PR, and HER2 and positive for CK 5/6 and/or EGFR werecategorized as basal-like. Cases that lacked expression of all 5 markers wereconsidered “unclassified” .2. Weight change was estimated as thedifference between current body weight and weight at age 18 years  in kg/m18 years . Race or18 years . Total p18 years . Partici18 years . Confirm18 years  and the18 years .Dietary intakes of isoleucine, leucine, and valine were derived as grams per day,ln-transformed, then adjusted for total energy intake using the residual method. We anal2), body weight change since age 18 years ,family history of breast cancer , history of benign breast disease, oral contraceptive use , age at menarche, menopausal status andHT use , age at natural menopause(continuous), parity and age at first birth , totalduration of breastfeeding , alcoholconsumption , smoking status , totalphysical activity , total energy intake, and Alternative Health Eating Index dietary pattern score2010 diet quality score (quartiles). We created missing indicator categories formissing covariate data (≤5.5%). We modeled the medians of BCAAquintiles as a continuous variable to examine linear trends in the relationshipof BCAAs and breast cancer.We performed Cox proportional hazards regression models to estimate theassociations between quintiles of total and individual BCAAs with incidentbreast cancer risk. The bottom quintile (lowest intake) served as the referencegroup. We used quintiles for analysis a priori to examine the dose-responserelationship and visualize potential for nonlinear associations. Categories alsoconstrain influences of outlier data. Follow-up time began at the return of thefirst eligible FFQ until incident breast cancer diagnosis, death, or date of thelast questionnaire returned through end of follow-up , whichever came first. Cohort data were pooled for the combinedanalyses. In the multivariable models, age was the underlying time scale,stratified by calendar year and cohort, and we adjusted for cancer risk factors,including height (continuous), race and ethnicity (non-Hispanic White vs other),BMI at age 18 years  vs overweight orobesity (BMI ≥ 25 kg/m2) to evaluateeffect modification by these breast cancer risk factors. Statuses for thesecharacteristics were updated over follow-up. We hypothesized that BCAAs mayconfer an elevated risk of breast cancer through those susceptible to insulinresistance and type 2 diabetes; thus, BCAAs may be positively associated amongthose with type 2 diabetes or obesity. We created interaction terms betweenmedian scores of BCAA quintiles and effect modifiers and used likelihood ratiotests to evaluate heterogeneity.We conducted subgroup analyses by type 2 diabetes , premenopausal vspostmenopausal, and BMI category of normal, and/or greater than 70 FFQ items left blank  over20.8 years median follow-up (3 644 137 person-years);85.5% of these cases had data available for tumor ER status . Tumortissue samples were available from 3231 breast cancer cases . Of theinvasive tumors on tissue microarrays, 2858 could be classified into the luminal A, luminal B, HER2-enriched, basal-like, or unclassified subtypes. Unclassified tumors wereexcluded from further analyses given the relatively small number of cases.Ptrend = .66) for the age-adjusted model and1.05  for the multivariable-adjusted model. Associations were similar forthe individual BCAAs, with the multivariable models indicating no association withbreast cancer risk comparing the 5th and 1st quintiles for isoleucine , leucineHR = 1.04 , and valineHR = 1.04 . These findings were consistentbetween NHS and NHS II cohorts.In the age-adjusted model, there was no relationship between the long-term cumulativeaverage of total dietary BCAA intake with breast cancer risk, which persisted in themultivariable-adjusted model. In Ptrend = .03). Although trends were notstatistically significant in other subtypes, the point estimates were generallysimilar across luminal B, HER2, and basal-like tumors.We conducted secondary analyses to examine BCAA intake by breast cancer molecularsubtypes. There was no statistically significant association between intakes oftotal or individual BCAAs according to subtypes of estrogen or progesterone receptorstatus, luminal B, basal-like, or HER2-enriched cases . There wPinteraction = .98), BMIabove or below 25.0 kg/m2(Pinteraction = .97), or postmenopausalstatus (Pinteraction = .11)  and the Breast Cancer Research Foundation.Role of the funders: Funders had no role in design of the study; thecollection, analysis, and interpretation of the data; the writing of the manuscript;and the decision to submit the manuscript for publication.Disclosures: None.Author contributions: Statistical analysis: Dt, BC, AHE; Manuscriptdraft: DT; Manuscript edits: DT, BC, RT, JM, FH, WW, AHE; Funding and study design:RM, JM, WW, AHE.Acknowledgements: We would like to thank the participants and staff ofthe Nurses’ Health Studies for their valuable contributions as well as thefollowing state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL,GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA,RI, SC, TN, TX, VA, WA, WY.Disclaimer: The authors assume full responsibility for analyses andinterpretation of these data.http://www.nurseshealthstudy.org/researchers.The datasets analyzed during the current study are available by application byfollowing the instructions here: pkab032_Supplementary_DataClick here for additional data file.


Breast and prostate cancers are among the most commonly diagnosed cancers worldwide, and together represented almost 20% of all new cancer diagnoses in 2020. For both cancers, the primary treatment options are surgical resection and sex hormone deprivation therapy, highlighting the initial dependence of these malignancies on the activity of both endogenous and exogenous hormones. Cancer cell phenotype and patient prognosis is not only determined by the collection of specific gene mutations, but through the interaction and influence of a wide range of different local and systemic components. While genetic risk factors that contribute to the development of these cancers are well understood, increasing epidemiological evidence link modifiable lifestyle factors such as physical exercise, diet and weight management, to drivers of disease progression such as inflammation, transcriptional activity, and altered biochemical signaling pathways. As a result of this significant impact, it is estimated that up to 50% of cancer cases in developed countries could be prevented with changes to lifestyle and environmental factors. While epidemiological studies of modifiable risk factors and research of the biological mechanisms exist mostly independently, this review will discuss how advances in our understanding of the metabolic, protein and transcriptional pathways altered by modifiable lifestyle factors impact cancer cell physiology to influence breast and prostate cancer risk and prognosis. Breast and prostate cancers are among the most commonly diagnosed cancers worldwide, representing 19% of all new cancer diagnoses and 10.7% of cancer-related deaths in 2020 alone . The patModifiable risk factors encompasses both lifestyle choices and environmental exposures. These include physical exercise, diet, weight management, tobacco intake, exposure to environmental pollutants and infections . These fThe response to lifestyle and environmental cues occurs initially at the metabolic and hormonal level, which can dynamically alter gene expression through epigenetic and transcriptional mechanisms . While fIn addition to metabolic disruptions, ongoing overnutrition results in adipose tissue accumulation. Adipose tissue is known to be a source of estrogen production, particularly in postmenopausal women whose ovaries are no longer the major estrogen source . PostmenAdipose tissue, a major consequence of an unhealthy diet and a sedentary lifestyle, is comprised of adipocytes, adipose stem cells, endothelial cells, immune cells and fibroblasts. Adipose tissue can secrete a range of hormones, growth factors and cytokines, termed adipokines . The balin vitro and in vivo studies highlighting the benefits of fermented foods in breast and prostate cancer, but these findings are yet to be confirmed in the clinic , can also trigger inflammation. SFAs induce toll like receptor (TLR) activation, particularly TLR4 , with ace clinic .It is well established that the role of reactive oxygen species (ROS) is paradoxical, in that it has the potential to be beneficial and detrimental to the progression of tumors, depending on the balance of antioxidants . For simIn a pre-malignant context, increased ROS levels provide the opportunity for driver somatic mutations to occur, which during malignancy can drive phenotypes such as cell proliferation  and epitP21 and BRCA1, allowing the proliferation and growth of cancer cells  and Priority-driven Collaborative Cancer Research Scheme (grant #1130499), funded by the National Breast Cancer Foundation Australia with the assistance of Cancer Australia awarded to MN.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N = 276) with high-risk  and moderate high-risk (ATM and CHEK2) predisposition to breast cancer in a 48-month follow up. The estimated MRI detection rate in the entire group is 1.75% (18 per 1000 MRI tests). For the high-risk group, the estimated rate is 2.98% (30 per 1000 MRI tests). Many women discovered their genetic risk at an age much older (average age of the high-risk group was 48 years) than the age recommended to initiate enhanced screening (age 20 to 25 years). In total, 4 of the 11 primary breast cancers detected were identified by screening MRI within the first month after initial visit, which were not detected by previous mammography, suggesting the benefit of initiating MRI immediately after the discovery of genetic risk. Breast screening findings for women with Lynch syndrome and neurofibromatosis type 1 were also included in this report.For women with genetic risk of breast cancer, the addition of screening breast MRI to mammography has become a standard. The order and interval of annual imaging can be variable among providers. To evaluate the clinical implications related to the timing, we conducted a chart review on a cohort of women ( Several genetic risk factors are known to predispose women to breast cancer. Enhanced screening and preventive mastectomy have been employed to improve the outcome based on the levels of risk ,3,4,5,6.BRCA1 and BRCA2 mutation women carriers begin screening at age 25 and no women undergo preventive mastectomy, salpingo-oophorectomy or chemoprevention [BRCA1 PV carriers, the first screening breast MRI is recommended to begin at age 25. Regardless of the age to initiate screening, some providers choose to begin mammography followed by MRI 6 months later.To identify interval cancer between two rounds of annual screening, many high-risk cancer surveillance clinics adopt a schedule of annual mammogram and annual breast MRI, alternating every 6 months in women over the age of 30. In women at elevated risk under age 30, annual mammography is not routinely performed. The health insurance reimbursement schedule also adheres strictly to an interval of 12 months or longer for each screening imaging. When advanced breast screening is first initiated for an individual newly tested positive for a germline high risk PV, some providers attempt to adhere to the schedule aforementioned, start with a baseline mammography or continue the annual mammography, followed by a breast MRI 6 months after the most recent mammography. The recommendation to alternate mammogram and MRI every 6 months came from data generated by a computer simulation model using an assumed cohort in idealist scenario that all evention ,10. Howeevention . For exaATM, BRCA1, BRCA2, CHEK2, CDH1, PALB2, PTEN, STK11 and TP53. NF1 PV carriers are also regarded with increased risk between age 30 and 50 years. Yearly screening with mammography and MRI is recommended for these patients [At least nine genes are known to be associated with significantly increased lifetime risk of breast cancer in women: Giving the superiority of MRI, we suspect postponing the MRI 6 months later may delay the diagnosis of breast cancer undetectable by mammography. To investigate, we conducted a retrospective chart review in all women visited MCC GH high-risk surveillance clinic for screening. We recorded the age, type of genetic PVs, previous history of cancer, the time of screening imaging and the time of cancer diagnosis.BRCA1, BRCA2, CDH1, PALB2, PTEN, STK11, and TP53) or moderate-high risk (ATM and CHEK2) of breast cancer. For example, for women carrying BRCA1 and BRCA2 PVs, annual bilateral (B/L) breast MRI started at age 25 years old until age 30, then annual B/L screening mammogram was added, alternating or together with annual MRI. For carriers with TP53 PV, annual MRI started at age 20 until age 30, then annual mammogram was added, alternating or together with MRI. For carriers with PTEN, PALB2 and CDH1 PV, annual mammogram and MRI both started at age 30. For carriers with ATM and CHEK2 PV, both imaging started at age 40 or earlier if indicated by family history  [NF1 PV, both imaging started at age 30. At age 50, annual mammography continued, but MRI was no longer performed. GH clinic coordinates and accommodates the patients’ wishes that imaging be conducted in a preferred diagnostic imaging center, at separate visits or same visit. Depending on the cost or convenience, the patients may choose to have the imaging perform in the cancer center or in a diagnostic center in the community.The screening and surveillance schedule in GH follows the guidelines published by National Comprehensive Cancer Network (NCCN): annual screening mammogram and breast MRI were performed for individuals who carry PVs or LPVs in genes conferring high risk  [For women without a breast risk conferred by genetic PV, they were evaluated for the lifetime risk at the time of initial visit in GH, utilizing the online Tyrer–Cuzick (TC) Model Breast Cancer Risk Evaluation Tool version 8.0) by International Breast Cancer Intervention Study (IBIS) (er 2021) . Followi.0 by Inter 2021) ,13. Tyreer 2021) .A chart review was conducted for patients who attended screening and surveillance in MCC GH clinic in a 48-months period from March 2017 to February 2021.ATM, BRCA1, BRCA2, CHEK2, CDH1, PALB2, PTEN and TP53) and women carriers of mismatch repair (MMR) gene group . Among all individuals cared in GH, 441 women carry at least one PV in the eight genes associated with increased risk for breast cancer , 26 women carrying PVs in more than one gene were categorized under the gene with the highest breast risk, for example, a woman with PVs in BRCA2 and CHEK2 was counted under BRCA2, a woman with PVs in ATM and PMS2 was counted under ATM .Medical history collected during clinic visits were analyzed . BetweenThree additional recurrent/metastatic breast cancers were palpated in this period of time either by the patient or by the care provider, which accounted for 1.5% (3/202) of the breast risk women who have had a previous diagnosis of breast cancer .Lynch syndrome female PV carriers  were followed and the breast risk of majority of them were not high enough to warrant MRI screening. Their risk was estimated based on Tyrer–Cuzick model using personal and family history. Ninety percent (85/94) had both breasts or one remaining breast. Seven underwent breast biopsy after abnormal screening imaging in this time period, three primary breast cancers were discovered by routine mammogram screening   Scheme . AverageIn the breast risk PV group  , the breast cancer rate was similar to Lynch syndrome group, 3.4% (10/294). However, the average age at diagnosis in the breast PV group was younger, 46.2 years (Median age = 43 years). In addition, three of the women in breast risk PV group had a previous diagnosis of primary breast cancer , 6 (12%) of which have had a previous diagnosis of breast cancer, seven underwent breast biopsy, no breast cancer was detected in this period. Nine additional women who carry other genetic mutations underwent breast biopsy. No breast cancer was detected in this period.BRCA1 and BRCA2 possess a 50% to 80% lifetime risk of developing breast cancer [TP53, 58% for PALB2, 85% for PTEN, and 42% for CDH1 [BRCA1 and BRCA2 PV carriers, it is accepted that women carrying these high-risk genetic changes will benefit from bilateral preventive mastectomy, even though there are no specific data generated directly from TP53, PALB2, PTEN, or CDH1 PV carriers [BRCA1 and BRCA2 PV carriers are also known to have excessive risk to develop contralateral cancer 20 years after their initial cancer diagnosis [ATM and CHEK2 confers greater than 30% lifetime risk for breast cancer, but lower than that of BRCA1 and BRCA2, thus are regarded as moderate-high risk. Therefore, risk reduction mastectomy is generally not recommended for ATM and CHEK2 PV carriers [Germline genetic pathogenic variants (PVs) in hereditary cancer predisposition genes are responsible for 5% to 10% of diagnosed breast cancers. PVs in t cancer . Severalfor CDH1 ,17,18,19carriers . BRCA1 aiagnosis . The cumiagnosis . After aiagnosis . PVs of carriers . Screenicarriers . This stBRCA1 and BRCA2 group, 2.98% (30 per 1000 MRI) for high risk group , and 0.83% (8 per 1000 MRI) for moderate risk group . These rates of detection are comparable to previously published studies [Breast MRI has an estimated detection rate of 1.75%  for primary tumor in this group of women who carry genetic PVs conferring moderate or high risk for breast cancer. When breaking down into the high risk and moderate risk groups, the estimated rate was 2.28% (23 per 1000 MRI) for  studies ,26,27.Based on the experience of this clinic, we have observed that majority of the individuals were recently tested positive for a genetic breast cancer risk and just began advanced breast screening by MRI at an age that has far exceeded the recommended age to begin MRI. For the high-risk group, the average age initially presented in GH clinic was 48 years . Majority of them (approximately 90%) were recently found out they are mutation carriers or made the decision to begin high-risk screening within the past year.In addition, a number of breast cancer cases (four in five) were diagnosed by their first screening breast MRI within the first month of their initial clinical visit, with three of the four tumors being already at a locally advanced stage. Furthermore, these cancers were not detected by a concurrent or a prior mammography within 1 year preceding the MRI. This finding cautioned the practice that some providers delaying the first screening breast MRI for the purpose of maintaining 6 months interval between a mammography and breast MRI. Breast MRI has an absolute advantage of detecting breast cancer over mammogram. It should be initiated as the first screening modality when a woman is found out to carry a breast risk PV and has passed the age recommended to begin high-risk screening.BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 .For individuals whose genetic PVs do not significantly increase breast risk, it is equally important to ensure the adherence to routine screening recommended for general population, including screening mammography. Three examples of such cancer were detected by screening mammogram in case 14, 15 and 16. When caring for this high-risk population, we have noticed that neglecting routine cancer screening by the patients is quite common. Underutilizing breast screening is not unique for this population. Based on 2017 United States health statistics, only 67% of the women 50 years and over had a mammogram within the past 2 years . Many individuals with genetic risk for multiple cancer types or have had personal or family history of multiple cancers, are often emotionally, physically and financially overwhelmed. They often experience screening fatigue. Many focuses on the type of cancer they perceive as the most dangerous, while neglecting screening for other types which they perceive as not as important. For example, women carrying MMR gene PVs may neglect routine breast cancer screening because it has not been seen in her relatives or it is not listed as an increased risk.When caring individuals with genetic predisposition to cancers, it is beneficial to have a holistic approach to consider the individual as a whole. In addition to the genetic risk PVs, we should consider risk of all organs, environmental exposures, family histories and any cancer related personal history, such as histology, stage, type of treatment and remission status.Women with high-risk genetic predisposition to breast cancer often become aware about the risk at an age much older than the age recommended to begin enhanced screening with MRI. For women carrying high-risk genetic variants, it is likely beneficial to begin breast MRI immediately after the discovery of genetic high-risk, instead of beginning with mammogram, then followed by MRI 6 months later. In addition, despite the extensive screening utilizing MRI, some early-stage breast cancers were missed, highlighting the recommendations to undergo risk reduction mastectomy for women with high-risk PVs.