Epidemiological studies have been used to show associations between modifiable lifestyle habits and the incidence of breast cancer. Among such factors, a history of alcohol use has been reported in multiple studies and meta-analyses over the past decades. However, associative epidemiological studies that were interpreted as evidence that even moderate alcohol consumption increases breast cancer incidence have been controversial. In this review, we consider the literature on the relationship between moderate or heavy alcohol use, both in possible biological mechanisms and in variations in susceptibility due to genetic or epigenetic factors. We argue that there is a need to incorporate additional approaches to move beyond the associations that are reported in traditional epidemiological analyses and incorporate information on molecular pathologic signatures as a requirement to posit causal inferences. In particular, we point to the efforts of the transdisciplinary field of molecular pathological epidemiology (MPE) to evaluate possible causal relationships, if any, of alcohol consumption and breast cancer. A wider application of the principles of MPE to this field would constitute a giant step that could enhance our understanding of breast cancer and multiple modifiable risk factors, a step that would be particularly suited to the era of “personalized medicine”. Breast cancer presents as solid tumors that originate in breast tissue due to deleterious mutations in different cell types, most commonly originating in ductal epithelium. As is true for most cancers, there are multiple subtypes, which have different etiologies and require different treatments. Breast cancer remains a global concern notwithstanding the 2015 National Cancer Institute fact sheet ; and genetics (inherited changes in certain genes and family history of breast cancer) .Various modifiable lifestyle risk factors have been identified, including dietary habits , smoking, exposure to radiation or synthetic estrogens, physical inactivity, use of hormone-replacement therapy (HRT) or diethylstilbestrol, obesity, diabetes, breast implants, epigenetic factors, viruses , occupational exposure to chemicals , exposure to pesticides and air pollution, and even night-shift work. Needless to say, other risk factors may exist that have not yet been fully identified. For example, a higher relative abundance of bacteria that have the ability to cause DNA damage, and a decrease in some anticarcinogenic lactic acid bacteria were detected in breast cancer patients, which raises the question of the role of the mammary microbiome in modulating the risk of breast cancer development .The risk attributable to modifiable and non-modifiable factors was calculated in a German population-based, case-control study of postmenopausal breast cancer . The attIn the following sections we will briefly discuss genetic and epigenetic risk factors to highlight the complexity of breast cancer etiology and to point to the impact of alcohol use as a modifiable risk factor in specific genetic and epigenetic contexts. A better understanding of such gene-environment interactions may help identify potential biological pathways and shed light on breast cancer etiology.The incidence of breast cancer in first-degree relatives of women with the disease is about twice that of the general population . Thus, tBreast cancer susceptibility genes can be categorized as high-, moderate-, or low-penetrance based on the relative risk of breast cancer associated with these genes’ mutations. These susceptibility genes were discussed by Apostolou and Fostira in 2013 , and 84 BRCA1, BRCA2, p53, PTEN, STK11, and CDH1 are associated with a relative risk of breast cancer development higher than 5.High-penetrance genes, such as BRCA1 and BRCA2, were identified as major risk factors for breast cancer [BRCA1 gene mutations, resulted in discordant phenotypes: One had breast cancer twice in 27 years while her MZ twin remained healthy [BRCA1-associated cancers are more likely to present specific characteristics like the absence of ER, PR, and HER2 receptors [BRCA2-related cancers, which usually express ER and PR. Examination of the role of BRCA1 and BRCA2 in the DNA damage response has led to the identification of several breast cancer susceptibility genes such as FALB2, CHEK2, BRIP1, all of which interact directly or indirectly with BRCA1 or BRCA2 [BRCA gene mutation [Mutations in two prominent susceptibility genes, t cancer ,18. Howe healthy . BRCA1-aeceptors ,21, unlior BRCA2 . Other smutation ,24.PALB2  [CHEK2) [ATM) [BRCA1-interacting protein C-terminal helicase 1 (BRIP1) [FALB2, BARD1, BRIP1, RAD51C, and RAD51D [Moderate-penetrance genes confer a relative increase in the risk of breast cancer between 1.5 and 5 . GWAS hah BRCA2) ; checkpo [CHEK2) ; ataxia 2) [ATM) ; and BRC (BRIP1) . Studiesd RAD51D .FGFR2); TOX high mobility group box family member 3 (TNRC9); mitogen-activated protein kinase kinase kinase 1 (MAP3K1); and lymphocyte-specific protein 1 (LSP1).Low-penetrance genes are associated with a relative increase in the risk of breast cancer of about 1.5. Four loci were identified that exhibited consistent association with breast cancer and contain plausible causative genes : fibroblTERT) [ERS1) [FGFR2) are more specific for ER-positive breast cancer [The susceptibility to breast cancer is polygenic; i.e., conferred by a large number of loci, each with limited contribution to breast cancer risk . Some ofTERT)  and ethy) [ERS1) ; while ot cancer . FGFR2, TOX3, STXBP4, SLC4A7, LSP1, 2q35, and 5p12) [LGR6, MDM4, CASP8, 2q35, 2p24.1, TERT-rs10069690, ESR1, TOX3, 19p13.1, RALY), and identified 15 additional breast cancer loci  [Additional susceptibility loci have been identified for breast cancer in several recent studies. For example, a 2014 study showed association between breast cancer risk and seven breast cancer susceptibility loci (nd 5p12) . A genomnd MKL1) . In addind MKL1) . A 2014 nd MKL1) . By usinnd MKL1) .PPM1D  [MTHFR (methylenetetrahydrofolate reductase), Ala222Val gene polymorphisms [APOBEC3B  overexpression [Other risk factors reported for breast cancer include mutations in the p53-inducible protein phosphatase ent, 1D) , MTHFR  could reduce breast cancer risk. Further studies confirmed this protective effect and showed that the per-allele odds ratio is 0.88 for the CASP8 D302H (rs1045485) [Studies ,45 condu1045485) . BRCA1, BRCA2, p53, PTEN, ATM, CHEK2, HER2) in breast carcinogenesis.The above discussion demonstrates the critical role of mutations in tumor suppressors and oncogenes in the nuclear genome , mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms. In breast cancer, mitochondrial function is severely impaired . One earBRCA1, BRCA2, p53, ATM, or PTEN) or early, life-transforming events that affect the immature mammary cells, whereas later-onset breast cancer may be associated with environmental or lifestyle factors [The incidence of invasive breast cancer or its precursor lesion, ductal carcinoma in situ (DCIS), increases at an exponential rate until menopause, followed by a slower rate of increase, supporting the notion that breast cancer biology is age dependent. Early-onset breast cancer, therefore, could largely represent inherited mutations  and the Fanconi anemia DNA-repair pathway (Fancd2 knockout) display susceptibility to the toxic effects of ethanol; the Fanconi anemia pathway prevents aldehyde lesions from degenerating into DSBs . A critiAs described above, mutations in oncogenes and tumor-suppressor genes, and chromosomal abnormalities result in specific gene expression profiles that are characteristic of distinct types of breast cancer and their responses to treatment. These genes are involved in the regulation of cellular homeostasis, including cell proliferation, DNA repair, and survival. However, mammary stem cells undergoing differentiation to primitive progenitor cells that give rise to luminal and myoepithelial progenitors, which in turn differentiate into epithelial subtypes, are under epigenetic control. Epigenetic mechanisms, which result in changes in gene expression patterns without altering DNA sequence, contribute to the mammary gland’s developmental phases from fetal to menopause, as well as in breast carcinogenesis, by changing gene expression patterns for cell differentiation, proliferation and apoptosis. Two major classes of epigenetic modulation include DNA methylation and covalent modifications of histones that determine DNA accessibility. In addition, a multitude of additional mechanisms exist that determine the fate of mRNA, e.g., through differential splicing, or by altering mRNA stability or translation, including through effects of non-coding RNAs such as microRNA (miRNA) and various long non-coding RNAs (lncRNAs), as well as other mRNA modification mechanism . Below, DNA methylation involves the transfer of a methyl group from the global methyl donor S-adenosyl methionine (SAM)—predominantly by a set of core DNA methyltransferases (DNMTs)—onto the 5′-position of the cytosine residue in the CpG islands .SAM is generated in the methionine cycle by condensing ATP with methionine, which itself is generated by re-methylation of homocysteine . This prBRCA1, VHL and p16Ink4a) leads to the inactivation of these cancer-preventing proteins. Hypermethylation of numerous genes, whose biological functions include hormone regulation ; DNA repair ; cell cycle regulation ; apoptosis ; cell-growth inhibition ; angiogenesis (maspin and THBS1); invasion ; and metastasis , has been identified in breast tumors [The CpG islands, which exist in about 70% of gene promoters, are generally unmethylated ; their mt tumors .BRCA1 expression is suppressed by a combination of gene mutation and DNA hypermethylation [ESR1, the promoter of which shows a higher degree of methylation in ER− vs. ER+ tumors [ESR1 gene results in a lack of expression of ER, which is reversed by 5-aza-2′deoxycytidine, a DNMT inhibitor [Furthermore, DNA hypermethylation results in aberrant regulation of the Wnt pathway in breast cancer , and BRChylation . ERα is + tumors . This hynhibitor . LINE) promotes chromosomal translocation, deletion, duplication, and genomic instability [ID4, urokinase, N-cadherin, β-catenin, and annexin A4), which are associated with tumor metastasis and drug resistance to endocrine therapy [DNA hypomethylation can also be a factor in breast carcinogenesis . For exatability . This eptability . In addi therapy . There is large literature indicating that alcohol consumption can affect the DNA methylation state, both in analyses on human patients and in studies on experimental animals. Some of these changes may be associated with a suppression of the availability of the methyl donor SAM through effects on the methionine cycle . For insChronic heavy alcohol use can also affect the availability of the folate derivative THF as a methyl donor for methionine synthesis. There is considerable evidence that chronic alcohol use can impair folate uptake and cause folate deficiency, which is likely to affect folate methylation pathways . Thus, lIn addition, there is evidence that a woman’s genotype for the MTHFR variant modulates the effect of alcohol consumption on breast cancer risk. For example, women with the TT genotype are at a higher risk of breast cancer than those with other genotypes. In postmenopausal women, the breast cancer risk was increased in women with the C677T MTHFR variant who had high lifetime daily alcohol intake, suggesting that folate metabolism has an impact on cancer development . DespiteHistone modification represents another epigenetic mechanism that influences chromatin remodeling during stem and/or progenitor cell differentiation. The histone “tails” that extend from the nucleosome contain lysine residues with ε-amino groups that invariably undergo post-translational modifications, which mainly include methylation, acetylation, phosphorylation, and ubiquitination. Histone tails can enhance or inhibit the accessibility of transcription factors to target loci. Histone acetylation by acetyl transferases (HATs); deacetylation by deacetylases (HDACs); methylation by methyltransferases (HMTs); or demethylation  further modify chromatin structure. Acetylation of lysine residues in histones results in an open chromatin structure (euchromatin) which allows gene transcription. On the other hand, deacetylation of these lysine residues results in the formation of closed chromatin (heterochromatin) and repression of gene transcription . MethylaAs is true for DNA methylation, histone modification reactions also depend on the availability of an adequate supply of methyl donors or acetyl donors. Interference with the operation of the methionine cycle and the supply of folate and THF could equally limit the rate of histone methylation at a global level. Similarly, the supply of acetyl CoA for histone acetylation may be altered by the availability of this metabolic intermediate. Interestingly, although acetyl CoA is normally derived predominantly from mitochondrial metabolism, which can limit its availability for nuclear acetylation reactions, alcohol metabolism generates abundant acetate as a metabolic product that is largely released from the liver to enter the circulation. Other tissues have the capacity to take up acetate and activate it to acetyl CoA through the cytosolic/nuclear acetyl CoA synthetase-2 (ACSS2). This makes acetyl CoA available systemically during ethanol metabolism and potentially influences histone (or other protein) acetylation in multiple tissues ,89. To wAlcohol use, or other environmental factors can also alter the activity and/or expression of the multiple classes of histone-modifying proteins and their associated factors. As an example, persistent exposure of mammary gland stem and progenitor cells to different environmental factors such as xenoestrogens  alters their epigenetic reprogramming during epithelial differentiation . This isEpigenetic mechanisms acting through mRNA modifications: There is substantial emerging evidence for an impact of alcohol intake on epigenetic regulation through modification of mRNA through splicing factors or by the impact of microRNAs (miRNAs), lncRNAs, or other non-coding RNAs or RNA binding proteins that affect mRNA stability and structure . Like prThere has been considerable evidence that miRNAs and other non-coding RNAs play important roles in epigenetic regulation relevant to cancer . DiffereUnlike genetic mutations, epigenetic alterations are potentially reversible under the right circumstances, and therefore are targets for medications development for cancer treatment. Effective treatment for cancer includes the use of DNMT inhibitors and HDAC inhibitors . HoweverSimilar to other types of cancer, breast cancer initiates and further progresses at the molecular level when specific hallmarks of cancer are acquired. Those hallmarks include evading growth suppressors, sustaining proliferative signaling, avoiding immune destruction, deregulating cellular energetics, resisting cell death, enabling replicative immortality, acquiring genome instability, inducing tumor-promoting inflammation, inducing angiogenesis, and activating invasion and metastasis . A betteIn summary, multiple alcohol effects on specific pathways relevant for carcinogenesis have been observed, both in human patients and in experimental animal studies. These studies do not exclusively fit into a model where one mechanism  is responsible for all the effects of alcohol, or where one type of breast cancer would be particularly susceptible to alcohol use. However, to the extent acetaldehyde is the culprit (in specific circumstances), it generally requires high levels of alcohol use and/or a deficiency in the ability to metabolize acetaldehyde. To what extent acetaldehyde accumulation occurs in breast tissue may not be immediately obvious, since ethanol metabolism is predominantly hepatic. More indirect consequences of alcohol use, on estrogen levels, or the effects of estrogen, are another set of pathways by which alcohol could act more broadly, including in breast tissue. Thus, a broader perspective may be needed. Epigenetic mechanisms are likely to be affected through other mechanisms than oxidative ethanol metabolism and may be altered in multiple tissues. However, there is little specific understanding of the impact alcohol has on these regulatory mechanisms and, in particular there is no information on dose dependency and temporal consequences of these actions. Despite these insights, however, the overall lack of mechanistic understanding of these regulatory events is a major impediment in exploring causal factors that are relevant in human patients.Chronic heavy alcohol consumption  and binge drinking  are risky behaviors that could result in various pathological conditions, including cancer. Some epidemiological studies have suggested that even moderate alcohol consumption can increase the risk of breast cancer, although by a small extent . SeveralExamination of There seems to be quite a significant discordance between molecular and epidemiological data. For example, some molecular studies have ascribed the carcinogenic effect of ethanol, among other things, to estrogens, which is quite plausible since alcohol increases circulating estrogen levels in humans . Most epAnother area of discrepancy between epidemiology and molecular biology is the role of acetaldehyde. While acetaldehyde is postulated to play a role in breast cancer, three epidemiological studies have examined the role of ALDH2*2  in the development of breast cancer and reported no association between ALDH2*2 and risk of breast cancer . FurtherThe central theme of observational epidemiology is studying conditions that are identified as being associated with one or more specific factors, and importantly, to assess likely causal relationships that could be used to inform health policy recommendations and suggest clinical interventions. When it comes to non-communicable diseases, major limitations of such studies include non-replicable and non-causal findings . Indeed,Several meta-analyses have examined the association between alcohol consumption and breast cancer risk see . While sMeta-analyses collect and summarize information from many single observational studies which highlight improbably large effects despite weak evidence. The effect sizes usually shrink in meta-analyses, and some meta-analyses may control for potential confounding factors . NonetheThere are numerous challenging issues concerning meta-analysis of observational epidemiological studies. These issues include: different study design ; variation in definition of various confounders, and in confounders included in various studies; selection bias in meta-analysis by including studies favoring certain outcomes, excluding non-English articles, or by abstracting incorrect data—the basic premise of meta-analysis being to average out errors, a slight change in selection criteria can get a different result; and under-reporting of alcohol consumed in observational studies. In fact, in a recent commentary, Ioannidis  echoed tmoderate drinking is not moderate according to US dietary guidelines for alcohol consumption [A 2017 meta-analysis  based onsumption .Persistent methodological problems continue to plague the field of observational epidemiology, which may account for some of the inconsistency and uncertainty reflected in these studies. These include measurement error and confounding factors. Measurement error is especially problematic in alcohol research, which depends on self-report of alcohol consumption, an imperfect measure that more often than not leads to underestimates, particularly for moderate drinkers . AdditioNumerous observational studies have associated alcohol consumption with breast cancer. While heavy alcohol consumption may increase the risk of breast cancer, linking alcohol consumption—especially in moderate amounts—to cancer, based solely on observational studies, is rarely definitive. It is intrinsically difficult to adequately account for confounding factors, including other lifestyle factors that are causally linked to carcinogenesis, and significant inaccuracies in estimates of long-term alcohol drinking history. The concept of causality is central to the development of interventions designed to reduce exposure to cancer-causing risk factors. Numerous causal risk factors have been linked to cancers  only after the accumulation of sufficient evidence derived from epidemiological studies, clinical research, and mechanistic studies in laboratory animals . The fraWhen confounding factors are present in a conventional epidemiological study but cannot be controlled for, Mendelian randomization is a widely used approach to address some of the problems of weak observational studies. Mendelian randomization combines genomic and epidemiologic methods to obtain more unbiased estimates of causal associations and offers a potential way to correct, in part, for the difficulties introduced by confounding factors and measurement errors.A Mendelian randomization study published in 2007 by Smith and colleagues  using daIn alcohol research, no Mendelian randomization studies, to our knowledge, were performed that aimed to assess the relationship between alcohol use and breast cancer. However, alcohol-related studies using Mendelian randomization in other contexts have been based on the fact that genes involved in alcohol metabolism are polymorphic, and different allelic variants result in alcohol-metabolizing enzymes with various activities that can serve as proxies for different degrees of alcohol consumption. In other words, for polymorphic genes, allelic variants can produce physiological and/or biochemical states that encourage different levels of drinking and therefore reflect various levels of alcohol exposure at the population level. marginal structural models were introduced by VanderWeele et al. [. Agent-based modeling is an apt way of tackling independence of causal effects and non-interference by simulating counterfactual outcomes. The goal is to make causal inferences as independent of theoretical opinions and expert judgement as possible [Etiologies of complex diseases such as cancer are characterized by inference and multiple interacting causal effects . Many ste et al.  in 2010.e et al. . Agent-bpossible . To whatWith the National Institutes of Health currently encouraging “precision medicine” to speed the movement of basic discoveries from the lab to the clinic , there aThe premise of classical epidemiology is that patients with the same diagnosis  have similar causes and disease progression despite variations in molecular pathology. Because each tumor possesses its own unique characteristics, such as molecular makeup, microenvironment, and communications within and between cancer and host cells, “it is essential that epidemiologic research rely on modern molecular classification of disease.” . TherefoModern epidemiology is starting to incorporate genomic medicine and biomedical sciences, which is contributing a lot to the evolving field of molecular pathological epidemiology (MPE). The concept of MPE was developed in 2010 by Ogino and Stampfer . These iFor example, studies on colorectal cancer—a suitable disease model for the immunology-MPE approach, given rich microbiota and immune tissues of intestines, and the well-established carcinogenic role of intestinal inflammation—provided insights into immunomodulating effects of aspirin, vitamin D, inflammatory diets, and omega-3 polyunsaturated fatty acids . PreviouThe transdisciplinary field of MPE “can advance biomedical and health research by linking exposure to molecular pathologic signatures, enhancing causal inference, and identifying potential biomarkers for clinical impact.” . The fieMPE enables researchers to provide evidence about how endogenous and exogenous factors may interact and contribute to tumor evolution, and about the diversity of tumor phenotypes . The conMolecular classification plays a pivotal role in MPE, with the underlying premise that breast cancers that share similar features are likely also to possess similar etiologies and behave in a similar manner in their progression and their response to treatment. Classically, etiologic heterogeneity of tumor subtypes is accomplished in case-control studies by comparing the odds ratios of candidate risk factors between the tumor subcategories and a common control group. This approach is incomplete, since many genetic risk factors for cancer have not been identified. Unfortunately, hitherto the MPE approach has not been used to evaluate the impact of alcohol use as a risk factor for breast cancer.The discussion presented in this review has aimed to highlight both the broad scope of possible molecular mechanisms that may contribute to the onset or progression of cancers of the breast, as well as our ignorance of the detailed underlying processes and their regulation that dictates the outcome. Some of these potential processes influenced by alcohol use are likely to require relatively high levels of drinking and prolonged exposure. However, other mechanisms, such as those mediated by various epigenetic controls, are as yet poorly characterized and, as a result, remain unpredictable. The survey of the multiple epidemiological studies and meta-analyses also highlight the substantial variability that exists in the relative risk estimates, which are particularly difficult to interpret when light to moderate drinking frequencies are considered.What is the reason to be so concerned about differentiating between potential cancer risk at high and moderate or low levels of drinking? Why is it important to have an understanding of the causative mechanisms of breast cancer and, more particularly, whether alcohol intake had anything to do with increasing the risk of specific subtypes of breast cancer? Alcohol has a social function that is important in many people’s lives. For the vast majority of drinkers this social function is associated with occasional or moderate drinking. The relative risk of negative consequences, including the relative risk of developing breast cancer, is therefore an issue that has to be weighed against these positives. Most observational epidemiological studies are in agreement that heavy drinking carries significant and diverse negative consequences and should be actively avoided. However, the negative consequences of light or moderate drinking are minimal, even in the majority of studies that show a detectable odds ratio for cancer or other diseases at these lower levels. The important question in this context is then whether a particular individual carries a higher risk than the general population as a consequence of specific susceptibilities that may vary from one person to another. It may be important, therefore, to be able to advise individual patients on specific vulnerabilities, either in their susceptibility to potential damaging effects of alcohol  or with significant risk factors for developing breast cancer, BRCA1 or BRCA2 mutations the impact of which may increase their susceptibility to other potential risk factors. In addition, women with a positive family history of breast cancer, or are using contraceptive pills or hormone replacement therapy may be at higher risk.A better understanding of the mechanistic basis of these susceptibilities then becomes a critical element. For this it is essential to obtain a more detailed knowledge of the molecular mechanisms by which alcohol use can impact cell function over prolonged periods. Such insights would enable the development of biomarkers that are mechanism-based and that, if well understood, could give guidance both to a physician who is asked to advise a patient, and to the individual patient who has to weigh these decisions. It would also facilitate the development of early detection tools that would help distinguish relatively innocuous indicators from more significant malignancies, and, of course, assist in the development of more individually targeted treatment modalities, the ultimate goal of “personalized medicine”. This is the promise of an approach such as that proposed under the umbrella of MPE. Breast cancer is a dreadful disease that can be treated effectively if caught early. In view of variable characteristics of different subtypes of breast cancer, the various susceptibilities of women to different risk factors and extents of exposure, as well as their various genetic susceptibilities, determining the molecular pathologic signatures is essential to facilitate better causal inference, and identify potential biomarkers for clinical impact and response to treatment. This endeavor will benefit greatly from the application of a multidisciplinary effort such as MPE, which encompasses various medical fields, including immunology, in addition to observational epidemiological studies. MPE facilitates the realization of precision medicine . In the


Epidemiologic evidence suggests that certain dietary patterns were associated with breast cancer risk, but the results have been inconclusive. We assessed the associations between different dietary patterns and the risk of breast cancer by conducting a meta-analysis of observational studies.Relevant articles were searched in PubMed, Embase, and Cochrane library databases through September 2017. Multivariable-adjusted relative risks (RRs) and 95% confidence intervals (CIs) comparing the highest and lowest categories of Western and prudent dietary patterns were combined by using the random-effects meta-analyses.We identified 32 eligible articles including 14 cohort and 18 case-control studies (34 Western and 35 prudent studies). The pooled analyses found that a Western dietary pattern was associated with a 14% increased risk , whereas a prudent dietary pattern was associated with an 18% reduced risk of breast cancer . In addition, sub-group analyses showed that the positive association between a Western dietary pattern and breast cancer risk was significant among postmenopausal , but not premenopausal women , and significant for hormone receptor-positive tumors , but not receptor-negative tumors . In contrast, the inverse association between a prudent dietary pattern and breast cancer was significant in premenopausal , but not postmenopausal women , and significant for both hormone receptor-positive and receptor-negative tumors.The results of the current meta-analysis suggest a possible increased risk of breast cancer associated with a Western dietary pattern and a reduced risk with a prudent dietary pattern. Large-scale cohort studies with a high quality need to be conducted to further confirm the findings of the current meta-analysis. As dietary patterns are modifiable, these findings may provide viable strategies for breast cancer prevention through changes in dietary intake.The online version of this article (10.1186/s13058-019-1096-1) contains supplementary material, which is available to authorized users. Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women in both developed and developing countries. Globally, the incidence rate of breast cancer has been rising rapidly over the past few decades . Most ofA substantial number of epidemiological studies have examined the associations between individual foods and the risk of breast cancer. High intakes of red meat, animal fats, and refined carbohydrates have been shown to be associated with an increased risk –6, wheren = 43,285 vs. 19,908 cases) than in the previous meta-analysis. Considering the inconsistent results in the current evidence and the insufficient statistical power of the previous meta-analysis due to the small number of studies and breast cancer cases, we conducted an updated meta-analysis of observational studies to review and summarize the epidemiologic evidence on the association between dietary patterns and the risk of breast cancer. We further examined these associations by study designs and characteristics of study populations.Numerous epidemiological studies have assessed the associations between different dietary patterns and the risk of breast cancer. Some studies have found a positive association between a Western dietary pattern and breast cancer risk , 11, andWe followed the PRISMA standard guidelines to perform the meta-analyses of observation studies and write the manuscript according to the PRISMA checklist  it was a case-control or a cohort study; (2) the exposure of interest was the most common dietary consumption pattern derived from factor analysis and/or principal component analysis. To reduce the heterogeneity across the studies, only the dietary patterns with similar factor loadings of foods were selected. For example, the Western or Western-like dietary pattern with high loadings of foods including red and/or processed meats, high-fat dairy products, potatoes, and sweets was selected as a representative unhealthy dietary pattern, whereas a prudent or similar dietary pattern with high loadings of foods such as fruits, vegetables, fish, whole grains, and low-fat dairy products was considered as a healthy dietary pattern; (3) the outcome of interest was incident breast cancer cases excluding recurrent cases; all incident breast cancer cases were diagnosed and verified by pathological biopsies or other standard methods, with controls/non-cases being females without breast cancer; all breast cancer types were included such as in situ or invasive cancer; (4) the relative risks (RRs), hazards ratios (HRs), or odds ratios (ORs) and the corresponding 95% CI for the highest compared with the lowest category of dietary patterns were reported.Two reviewers independently screened the titles and abstracts of the searched papers and excluded the articles which did not meet the above-described inclusion criteria. For those that were difficult to determine their eligibility, a full-text assessment was conducted. All disputes, if any, were resolved by discussion.We initially identified 2517 potentially relevant articles from the databases, and 370 records were excluded because they were duplicates. After title and abstract review, 2080 articles were further excluded. After reviewing the full text of the remaining 67 articles, 35 papers were excluded due to the following reasons: one article was not an observational study ; eight sTwo reviewers independently abstracted data on study characteristics and results by using a standardized data collection form. Discrepancies in data extraction between the reviewers were resolved by consensus. Data extracted included the following: first author’s last name; year of publication; location of the study; study design; sample size; average age of participants; dietary assessment methods; dietary patterns; RRs, HRs, and ORs with the corresponding 95% CIs from the fully adjusted model for the highest compared with the lowest category of dietary patterns; and potential confounders adjusted in the multivariate analysis.We also systematically assessed the study quality. Briefly, a nine-score system on the basis of the Newcastle Ottawa Scale (NOS) was used to assess the quality of the included studies. Each study was evaluated on three broad criteria: (1) the proper selection of study population, (2) the comparability of the study groups, and (3) the ascertainment of the exposure or outcome of interest. Two reviewers independently assessed the quality of each study. Disagreements were resolved through discussion to reach a consensus. Studies scored greater or equal to 7, out of a maximum 9 points, were considered to be high-quality studies.P < 0.10 level of significance. We also calculated the I2 statistic, which describes the total variation across studies attributable to heterogeneity rather than chance; an I2 value greater than 50% indicates at least moderate heterogeneity [RRs were used as a common measure of the association between dietary patterns and the risk of breast cancer across studies. HRs, ORs, or incidence rate ratios (IRRs) were directly considered as estimates of RR. To calculate summary RR and its 95% CI, we pooled the results by using random-effects meta-analysis. The random-effects analysis was chosen a priori because of the anticipated clinical and methodological heterogeneity and because it is considered more conservative than the fixed-effects analysis, as it accounts for both within- and between-study heterogeneity . Heterogogeneity .Because participant characteristics and confounder adjustment differed across the studies, which may result in different associations between dietary patterns and breast cancer risk among the studies, we further conducted stratified analyses to explore possible sources of heterogeneity and to examine the influence of various inclusion criteria on the overall risk estimate. Pre-specified subgroup analyses by menopausal status, hormone receptor status, study design, number of breast cancer cases, number of adjusted variables, quality scores of studies, and with or without adjustment for certain risk factors were performed to assess whether these variables modify the overall risk estimate. We also conducted a sensitivity analysis to investigate the influence of each individual study or a group of studies, for example, studies that used diet history questionnaires, or with the low quality score, on the overall risk estimate by removing one study or a group of studies at a time.P value < 0.05 was considered statistically significant, except where otherwise specified.Potential publication bias was assessed by visual inspection of Begg’s funnel plots in which the log RRs were plotted against their standard errors (SEs). We also performed Begg’s rank correlation test and Egger’s linear regression test , 56. AllThe characteristics of the included 32 articles are presented in Table Table P = 0.017), with significant heterogeneity . The positive association was only significant in case-control studies  with significant heterogeneity , but not in cohort studies  with no evidence of significant heterogeneity .The multivariable-adjusted RRs for each study and the combined RR for the highest compared with the lowest categories of a Western dietary pattern are shown in Fig. P < 0.001), with large heterogeneity  , with large heterogeneity . In addition, an inverse association between prudent dietary and breast cancer risk was also observed in cohort studies , with no heterogeneity .Overall, the combined RR of breast cancer for the highest compared with the lowest category of a prudent pattern was 0.82  Fig. . A signiP = 0.058) . However, a significantly increased risk of breast cancer was found among postmenopausal women  . In contrast, a significant inverse association comparing the highest to the lowest category of prudent dietary patterns and breast cancer risk was observed among premenopausal women  and/or progesterone (PR+) breast tumors  and the prudent dietary pattern was significantly associated with a reduced risk of breast cancer , respectively. In addition, when the five studies [Sensitivity analyses were also conducted to determine whether the results would change when one study was removed at a time. The results were fairly robust. The summary estimates ranged from 1.08  to 1.16  for the Western dietary pattern . Although there was marginally significant publication bias for prudent dietary pattern studies , no evidence of publication bias was observed after using the trim-and-fill method , and the association remained significant .Although the funnel plot was slightly asymmetric, after using the trim-and-fill method, visual inspection of Begg’s funnel plot did not identify substantial asymmetry  but not in cohort studies (2% increase), suggesting that recall bias might at least partially explain the discrepant results in different study designs. The prudent dietary pattern, comparing high vs. low groups, was associated with a reduced risk  of breast cancer in both case-control (30% decreased risk) and cohort studies (11% decrease). Furthermore, results of a Western dietary pattern are null in study designs with greater covariate adjustment and higher study quality, suggesting that the observed association may be limited to study designs with more limitations. Other stratified-analyses showed that the positive association between a Western dietary pattern and breast cancer risk was statistically significant among postmenopausal women, but not significant among premenopausal women. In contrast, the inverse association between a prudent dietary pattern and the risk of breast cancer was significant among premenopausal women, but not significant among postmenopausal women. In addition, the Western dietary pattern was significantly associated with an increased risk of ER+ and/or PR+, but not ER− and/or PR− breast tumors. In comparison, the prudent dietary pattern was significantly associated with a lowered risk of both ER+ and/or PR+ and ER− and/or PR− tumors.n = 43,285 vs. 19,908 cases) than in the previous meta-analysis, the current study had greater statistical power to detect significant associations.In 2007, the WCRF report concluded that there was insufficient evidence to make a judgment about the relationship between dietary patterns and the risk of breast cancer . SubsequBecause estrogens have long been hypothesized to play an essential role in breast cancer development and the source and metabolic pathway of estrogens are different between premenopausal and postmenopausal women , the etiHormone receptor status is an important diagnostic and prognostic characteristic of breast tumor and, therefore, merits consideration. Among the studies which examined the dietary patterns and breast cancer association by hormone receptor status , 76–78, Our study had several strengths. With a larger number of studies and breast cancer cases than the previous meta-analysis, our meta-analysis had more statistical power to detect a significant association between the Western dietary pattern and breast cancer risk and to calculate a more reliable estimate for the prudent dietary pattern and breast cancer association. In addition, to the best of our knowledge, this is the first meta-analysis to analyze the dietary pattern and breast cancer risk by menopausal status and hormone receptor status. We also carried out sensitivity analyses to show that the results were fairly robust.Potential limitations of this study should also be considered. First, the quality of meta-analyses is largely dependent on the quality of the original studies included in the meta-analyses. The current meta-analysis included 18 case-control studies and 14 cohort studies. Therefore, the possibility of recall bias related to differential recalls of dietary intake between cases and controls and control selection bias in case-control studies cannot be completely ruled out. Due to a lack of significance in association between a Western dietary pattern and breast cancer in cohort studies, the significant association in case-control studies may not be a true association as a result of recall bias. Second, unmeasured and uncontrolled confounding is always of a concern in observational studies, although most included studies adjusted a large number of factors which may potentially confound the dietary patterns and breast cancer association. However, not all potential confounders were adjusted for in every study, such as breast density  and histIn summary, our meta-analysis provides potential evidence of a possible positive association between the Western dietary pattern and an inverse association between the prudent dietary pattern and breast cancer risk. However, the results should be interpreted with caution, as the observed positive association may be limited to study designs with more limitations. Subgroup analyses found that these associations differed by menopausal status and hormone receptor status. As diet is potentially modifiable, the findings may have important implications to promote a prudent dietary pattern for breast cancer prevention.Additional file 1:PRISMA checklist. (DOC 71 kb)Additional file 2:Figure S1. Sensitivity analysis of the associations between a Western dietary pattern and the risk of breast cancer by eliminating one study at a time. (JPG 190 kb)Additional file 3:Figure S2. Sensitivity analysis of the associations between a prudent dietary pattern and the risk of breast cancer by eliminating one study at a time. (JPG 190 kb)Additional file 4:Figure S3. Funnel plots of associations between a Western dietary pattern and risk of breast cancer. (JPG 34 kb)Additional file 5:Figure S4. Funnel plots of associations between a prudent dietary pattern and risk of breast cancer. On the left is the plot before using the trim-and-fill method, and on the right is the plot after using the trim-and-fill method. The boxes represent the filled studies. (JPG 82 kb)


Telomeres and telomerase play key roles in the chromosomal maintenance and stability. Recent epidemiological studies have shown that longer telomeres are associated with increased risk of several cancer types. However, epidemiological data for telomere length and risk of breast cancer are sparse.We prospectively studied the association between telomere length and risk of breast cancer in 14,305 middle-aged or older Chinese women of the Singapore Chinese Health Study including 442 incident breast cancer cases after 12.3 years of follow-up. Relative telomere length in peripheral blood leukocytes was quantified using a validated monochrome multiple quantitative polymerase chain reaction method. The Cox proportional hazard regression method was used to estimate hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) for breast cancer associated with longer telomeres after adjustment for potential confounders.Ptrend = 0.006); the highest quartile of telomere length was associated with a statistically significant 47% higher risk of breast cancer compared with the lowest quartile of telomere length after the adjustment for age and other known risk factors for breast cancer .Longer telomeres were significantly associated with higher risk of breast cancer in a dose-dependent manner  contains supplementary material, which is available to authorized users. Breast cancer is the most common cancer for women. Worldwide, 2.4 million new cases of breast cancer and 534,000 deaths from breast cancer occurred in 2015 . New preTelomeres are tandemly repeated sequences of TTAGGG located at the distal ends of linear chromosomes . They plIn the last decade, a growing number of studies have examined the association between telomere length and the risk of cancer. To date, 8 retrospective case-control studies –20 and 5The present study was based on the data from the Singapore Chinese Health Study, a population-based prospective cohort study with original aims of investigating the role of diet, environmental exposures, and genetic factors on the etiology of cancer and other chronic diseases among Chinese in Singapore. The Institutional Review Boards of the National University of Singapore and the University of Pittsburgh approved the study. Detailed information on study design and methods has been described elsewhere . BrieflyThe first follow-up interview was conducted in 1999–2003 to all surviving cohort participants. A total of 52,326 (90.6%) surviving participants completed the follow-up interview that updated information on cigarette smoking, alcohol drinking, history of respiratory diseases and other medical conditions, medication use, current body weight and height, and current menopausal status (for women).°C for future analyses.Blood and urine samples were initially collected from a 3% random sample of cohort participants which began in April 1994. We expanded the biospecimen collection to all surviving cohort participants who consented for blood and urine donation at the end of the first follow-up interview. By the end of 2005, 28,346 (57%) of all eligible participants provided blood specimens. Blood components  were then stored at − 80 n = 20,998) in terms of body mass index, alcohol consumption, cigarette smoking, age when period became regular, family history of breast cancer, and number of hours slept per day  to a single copy gene number for albumin (S) in the experimental sample to a standardized reference sample values using multiplexed quantitative polymerase chain reaction (PCR) method developed by Cawthon . The median time interval from the date of blood collection to the date of breast cancer diagnosis was 6.3 years (range from < 1 month to 18.4 years).Table r = − 0.24; P ≤ 0.0001) and accounted for 5.5% of the telomere length variation (P < 0.0001). After adjustment for age and father dialect as the cohort recruitment criteria factors, a greater number of live births, postmenopausal status, Hokkien dialect, and shorter duration of sleep were significantly or borderline significantly associated with longer telomeres.The mean relative telomere lengths by observed or potential risk factors for breast cancer are shown in Table P = 0.012). Longer telomeres were significantly associated with a higher risk of breast cancer in a dose-dependent manner after adjustment for age and other potential confounders (Ptrend = 0.006) (Table 2) (Table Ptrend > 0.30).Overall mean relative telomere length measure was slightly higher in breast cancer cases  than those without breast cancer mean 1.04, SD 0.23) . The corresponding figure for women with five or more years of follow-up was 1.35 (0.95–1.92) (Ptrend = 0.12). However, P for heterogeneity in comparison of the two hazard ratios was not statistically significant (P = 0.47). As shown in Additional file P for heterogeneity = 0.32).We performed a sensitivity analysis by dividing breast cancer cases with various number of years from blood draw to cancer diagnosis to evaluate if the underlying subclinical disease progression had any impact on telomere length and so on the association between telomere length and risk of breast cancer Table . For womWe investigated the association between white blood cell telomere length and breast cancer risk in a prospective study of 14,305 middle-aged or older Chinse women in Singapore. Our results showed that women in the top quarter of telomere length had a statistically significant 47% higher risk of breast cancer than women in the bottom quarter of telomere length after adjustment for age and other potential confounders. The positive association between telomere length and risk of breast cancer was in a dose-dependent manner.We used peripheral blood leukocytes as the surrogate for breast tissue, which may have different telomere length. Studies have shown that telomere lengths varied considerably across different tissue types but were strongly correlated with one another within a person –37. In aFour prior case-cohort or nested case-control studies within prospective cohorts also reported mixed results. Three of the four studies in the USA and the UK found no significant association between telomere length and risk of breast cancer incidence , 22, 24.To date, two Mendelian randomization studies , 42 haveLittle is known about the potential biological mechanisms through which telomere influences breast cancer risk. In general, cells with very short telomeres may induce replicative senescence or apoptosis, which suppress the proliferative potential of a cell and, thus, lend support to a tumor-suppressor activity , 43. On Several strengths of the current study are noteworthy. This is the first prospective cohort study conducted in an ethnically homogenous population in Eastern Asia. In addition, exposure assessment  was performed on samples from all cohort women who provided blood prior to the diagnosis of breast cancer; thus, reverse causality bias is not a concern in our findings. The study also took advantage of a long duration of follow-up. Finally, the Singapore population-based Cancer Registry, as a nationwide program, has been recording cancer cases for more than the last 50 years, and therefore, breast cancer case ascertainment should be essentially complete . There aIn conclusion, the results of this study show that longer prediagnostic telomere length in blood leukocytes is significantly associated with an enhanced risk of breast cancer in a stepwise manner among a predominantly postmenopausal Asian women population. Our findings provide further evidence that leukocyte telomere length has the potential to act as a moderate risk factor for breast cancer. Whether telomere length can be implemented as a biomarker for breast cancer risk prediction warrants replication in additional large prospective cohort studies with diverse populations.Additional file 1:Tables S1. Distributions of participants’ characteristics for women who donated blood samples for telomere length measurement and women who did not provide blood samples for telomere length measurement, the Singapore Chinese Health Study. Table S2. Associations between relative telomere length and risk of breast cancer stratified by length of follow-up, Singapore Chinese Health Study. (DOCX 21 kb)


Two common polymorphisms in the MTHFR gene, C677T and A1298C, are associated with reduced enzyme activity and may be associated with breast cancer susceptibility.We performed a case-control study to investigate the association between the two SNPs in the MTHFR gene and risk of breast cancer. In total, 58 breast cancer patients and 58 unaffected controls were enrolled in the study. Polymerase chain reaction/restriction fragment length polymorphism technique (PCR-RFLP) was conducted to determine the genotypes.No significant differences were found in the genotypes of the two polymorphisms of the MTHFR gene between cases and controls. The OR and 95% CI for the 677CC, 677CT and 677TT genotypes were 1.00, 0.95 (0.39-2.31) and 0.87 (0.27-2.80), respectively; those of the 1298AA, 1298AC and 1298CC genotypes were 1.00, 0.59 (0.26-1.36) and 0.78 (1.32-4.66) respectively. Furthermore, it has been shown in patients with breast cancer a risk of presenting with an aggressive biophenotype about twice or three times higher in the presence of the C677T and A1298C polymorphisms, respectively. Finally, the A1298Cpolymorphism is significantly associated with increased recurrence risk of lymph node-positive breast cancer.Our study has not shown a significant association between MTHFR gene polymorphisms and breast cancer risk. However, it highlighted the key-role played by the presence of mutant alleles for both polymorphisms in increasing the risk of developing more aggressive phenotypes; moreover, specifically in A1298C, it might also lead to a higher risk of developing lymph node metastasis. The Methyleletetrahydrofolate reductase (MTHFR) gene is located on chromosome 1, it has 11 exons Two functional and well characterized SNPs in the MTHFR gene, C677T rs 1801133) and A1298C (rs 1801131), have been associated with decreased enzyme activity and increased levels of plasma homocysteine 01133 andThe present study aims firstly at evaluating the role of C677T and A1298C polymorphisms as risk factors in a small cohort of healthy women and breast cancer patients. In the literature, study findings on such polymorphisms are discordant, due to the fact that they are strongly influenced by differences among specific ethnic groups. For what concerns the Sardinian area, these polymorphisms have never been studied: thus regardless the results found, a higher number of cases will have to be analysed in the future. Secondly, the study aims at associating data obtained by molecular subtypes from diagnosed tumours to related clinical parameters in order to evaluate the potential role of the two polymorphisms, then using the latter as prognostic and predictive factors in breast cancer disease development.A total of 116 women were evaluated in this case-control study: 58 breast cancer patients enrolled at the Medical Oncology Unit, Sassari University Hospital, Sassari and 58 healthy women without history of cancer enrolled by the local branch of Lega Italiana Lotta contro i Tumori (LILT), Sassari .All women taking part to the study were asked to fill in a questionnaire regarding all known risk factors for breast cancer development such as age, body weight, reproductive-hormone profile, cancer familiarity, smoking habits, alcohol, lifestyle and diet. The questionnaire was administered to healthy women at Sassari LILT local office, while women affected by breast cancer were interviewed at the Medical Oncology Unit, Sassari University Hospital.In addition, venous blood samples were collected from both patient's groups.Each part of the questionnaire has been accurately and thoroughly explained during the meetings held to enrol healthy women so as to transform these meetings into learning opportunities aimed at prevention; probably due also to this approach, a study adherence over 90% has been achieved. in situ, with age between 36 to 85 years. The overall adherence to the study has been approximately 75%.On the other hand, the cancer patients enrolled were diagnosed with infiltrating ductal carcinoma and some cases presented with ductal carcinoma This experimental study was approved by ASL Sassari Bioethical Committee and written informed consent was obtained from each participant, according to Italian legislation.The MTHFR polymorphisms were analyzed by a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. For each analyzed polymorphism, 10% of the total samples were randomly retested with 100% concordance.All participants were requested to provide 3 ml peripheral whole blood, which was collected in K3-EDTA, aliquoted and stored at -20°C.Genomic DNA was isolated from 200 μl of whole blood, using QIAmp DNA Blood Mini Kit  following manufacturer's instructions.MTHFR C677T. PCR was performed with a final volume of 20 μl of reaction mixture containing 100 ng DNA, 1x PCR buffer, 1,5 mM of MgCl2, 0,2 mM of each dNTP, 1,25 units of Taq Gold DNA polymerase  and 0,6 mM of each primer: forward 5I-TGAAGGAGAAGGTGTCTGCGGGA-3I and reverse primer 3I-AGGACGGTGCGGTGAGAGTG-5I, yielding a 201-bp fragment The PCR products were digested with HinfI  in a volume of 20μl for 3 hours at 37°C, electrophoresed in a 3% Metaphor Agarose gel and visualized by ethidium bromide staining. The C→T valine variant creates a HinfI restriction enzyme site and produces fragments of 178-bp and 23-bp  of exon 7 of the gene and converts a glutamic acid into alanine, cancelling the cutting site for the enzyme MboII. PCR was performed with the final volume of 25 μl of reaction mixture containing 150 ng DNA, 5x PCR buffer, 0,2 mM of each dNTP, 2,5 units of Q5 Hot Start High-Fidelity DNA Polymerase  and 0,6 mM of each primer: forward 5I-CTTTGGGGAGCTGAAGGACTACTAC-3I and reverse primer 3I-CACTTTGTGACCATTCCGGTTTG-5I, yielding a 163-bp fragment The PCR products were digested with MboII  in a volume of 25 μl for 3h at 37°C, electrophoresed in a 3% Metaphor agarose gel and visualized by ethidium bromide staining. The 1298C allele abolishing one of the MboII restriction site, therefore digestion of the 163 bp PCR product of the 1298A allele yields 5 fragment of 56, 31, 30, 28 and 18 bp, whereas the 1298C allele resulting in 4 PCR bands of 84, 31, 30 and 18 bp  than healthy controls , their BMI is also higher , a lower number of nulliparae  and a reduced use of oral contraceptives . No statistically significant differences are seen between the two groups for what concerns the onset of menstruation, familiarity of breast cancer and cigarettes and alcohol consumption.The composition of the two analysed samples and data related to the most important variables analysed are shown in Table For what concerns epidemiological and molecular analysis results, Table  in situ tumour  and by Luminal B Her2 negative . Less recurrent are those characterized by more aggressive biophenotypes .Among patients enrolled in our study, it can be observed that the most recurrent molecular biophenotype (data not shown) is Luminal A , followed by Moreover, an association between each polymorphism and variable considered important for prognostic and/or predictive purposes has been analysed. The association with the biophenotype is reported in Table Cases with metastasis were 46% of the total number of breast cancer. The risk of lymph node metastasis resulted significantly increased of 2,5 times for each mutant allele in A1298C polymorphism  prevalence among patients with a CC homozygous mutant allele of A1298C polymorphism has been observed (Figure 5 prevaleBreast cancer is a complex and heterogeneous disease with a multifactorial etiology. Several studies have found an association between the C677T and A1298C polymorphisms and an increase in the risk of various types of cancer including pancreas, esophagus, stomach, colon, lung and, breast cancer nullipara status and use of oral contraception. Instead, no significant differences were observed for what concerns age of first menstruation, cigarette and alcohol consumption, and breast cancer familiarity. This result, apparently in contrast to what should have been expected, is probably related to the method used to enrol the control group. The latter was in fact formed by women consecutively enrolled who voluntarily accessed the LILT local office for preventive counselling and investigation. Women from any socioeconomic status might access to LILT, but there might be a self-selection bias as women with cancer familiarity are probably more sensitive to the issue; thus they might be more used to undergo periodic medical check-ups at LILT, being more likely to be selected as controls. This consideration might be confirmed by the extremely reduced absence of a difference in tumour familiarity prevalence observed between the two groups.The present study was conducted on the basis of a detailed questionnaire on different known risk factors, including the patient's diet composition. The descriptive analysis on the two compared groups highlighted significant differences concerning some variables such as age, BMI, Based on epidemiological and statistical analysis conducted on the different MTHFR polymorphic patterns between the two examined groups, no relevant data emerged to confirm an association of C677T and A1298C with a higher breast cancer risk. With the aim to shed light on the link between the two aforementioned polymorphisms and the breast cancer etiology in our area, a prosecution of the study has been foreseen, including the collection of a larger number of samples compared to the present study, as well an extra control group enrolled taking into consideration the self-selection bias.For what concerns the biophenotype of tumours observed in the study cases, the pattern does not differ from what expected based on literature. Therefore, the sample well represents the cancer population subjected to the study. The risk inferences are thus consistent. As a consequence, the result of associating the biophenotype and the polymorphisms highlights the risk of presenting with an aggressive biophenotype which is two or three fold higher in patients with polymorphisms C677T and A1298C, respectively. In relation to the risk of having a lymph node metastasis, the analysis based on unconditional logistic regression shows that the A1298C polymorphism results highly and strongly associated to such a risk, showing a positivity that is likely to increase approximately 2,5 times in each mutant allele. For what concerns other variables potentially associated to breast cancer risk, the present study shows an association between overweight and homozygous CC mutant allele of A1298C polymorphism.Based on this observation and discussion, to the best of our knowledge we can declare that the current study represents the first observational analysis on the association between breast cancer and C677T and A1298C polymorphisms of MTHFR gene in Northern Sardinian female population.The study can be considered the pathfinder for a wider research project and preliminary results here reported strongly indicate the need to carry on the research through increasing the number of subjects analysed in both groups. Increasing the sample and including a second control group will lead to higher effectiveness so as to analyse potential associations and or interactions with other behavioural variables emerging from the questionnaire.In conclusion, although our study has not ultimately confirmed the association between MTHFR gene polymorphism and breast cancer risk, it clearly shows the crucial key-role of mutant allele presence of both polymorphisms in relation to the risk of developing more aggressive biophenotypes and, just in the case of A1298C, an increased risk to develop lymph node metastasis.The biological plausibility of the association of MTHFR polymorphisms with the risk of developing more aggressive biophenotypes is supported by numerous evidences that the deregulation of DNA methylation, de novo synthesis of purines and pyrimidine nucleoside as well as maintaining DNA integrity by MTFHR polymorphic variants, which determine conformational modifications leading to isoenzymes with altered functional activity, may contribute to malignant transformation However the chance that significant association is really true should be better investigated Therefore, a higher number of cases analysed might ultimately define the role played by different polymorphisms on cancer risk and might, on one hand, enable to better define the prognosis of observed cases and on the other hand, it might allow the identification of tools for early diagnosis to be stratified on differentiated risk profiles in order to increase the efficacy of screening campaigns.In conclusion, although with the limitations described, our results suggest a biological role of the MTHFR enzyme and its polymorphisms in the progression of breast cancer, related to the association of MTHFR polymorphisms with aggressive biophenotype and lymph node metastasis. Extensive and independent study should be warranted to evaluate the biological mechanisms of this association.


Oral contraceptives (OCs) use has been linked to increased risk of breast cancer (BC) in several reports from the world. Limited number of similar studies have been conducted in the Middle Eastern female population. This study aimed to explore any possible correlation between the contemporary and duration of OCs use among Jordanian women and the risk of breast cancer.A case control study was conducted in 450 Jordanian women (225 as cases and 225 as controls), aged 18 to 65. Chi-square test was used to study the association between risk of breast cancer and different factors. Mann Whitney-U test was employed to evaluate the relation between time-dependent risk factor and breast cancer.p = 0.002), while the duration of OCs use was not associated with the increased risk of breast cancer (p > 0.05). In addition, other factors demonstrated significant association with the increased risk of breast cancer such as age at puberty, age at menopause, previous pregnancies, menopausal status, and family history of cancer.Our results indicated that regular use of OCs exhibited association with increased risk of breast cancer  contains supplementary material, which is available to authorized users. Breast cancer (BC) is the most prevalent type of cancer affecting females in both the developed and developing countries . WorldwiThe significant increased concern of breast cancer, worldwide and in Jordan, may be attributed to the prolonged life expectancy and the adoption of the western lifestyle with its related risk factors . GeneticGlobally, around 140 million women worldwide utilize oral contraceptives; this portion approximately accounts for 13% of women between 15 and 49 years old  should be evaluated.This study is the first report in Jordan to investigate the correlation between the contemporary and duration of use of OCs among Jordanian women and the risk of breast cancer. In addition to regular OCs use, significant differences in correlation of breast cancer were found between the case and the control groups, especially for the following risk factors: age at puberty, age at menopause, previous pregnancies, menopausal status, and family history of cancer. Future studies should include a larger sample size in multi-center setting all over the Kingdom. Also, the relationship between OCs use and specific subtypes of breast cancer [Estrogen Receptor-Positive


BRCA1 breast cancer development. Furthermore, lower circulating RANKL has been reported among women with a BRCA mutation compared to non-carriers; however, there have been no reports of plasma RANKL levels and subsequent breast cancer risk. We prospectively evaluated the relationship between plasma RANKL and breast cancer risk among women with a BRCA1 or BRCA2 mutation.Aberrant progesterone/receptor activator of nuclear factor κβ (RANK) signaling has been implicated in BRCA mutation carriers. Women were stratified into high vs. low RANKL based on the median levels of the cohort (5.24 pg/ml). Kaplan-Meier survival analysis was used to estimate the cumulative incidence of breast cancer by baseline plasma RANKL and cox proportional hazards models were used to estimate the adjusted hazard ratios (HRs) and 95% confidence intervals (CI) for the association between plasma RANKL and risk.An enzyme-linked immunosorbent assay was used to quantify plasma RANKL levels in 184 P-log-rank = 0.85). There was no significant association between plasma RANKL levels and breast cancer risk .Over a mean follow-up of 6.3 years (0.02-19.24), 15 incident breast cancers were identified. The eight-year cumulative incidence was 10% in the low RANKL group and 12% in the high RANKL group (BRCA mutation carriers. Pending validation in a larger sample, these findings suggest that RANKL is likely not a biomarker of breast cancer risk among BRCA mutation carriers.These findings suggest that circulating RANKL levels are not associated with breast cancer among  Brca1-mammary carcinogenesis suggesting that this may be a novel target for prevention in women at a high risk of developing breast cancer due to an inherited BRCA1 mutation [et al., reported significantly lower circulating levels of osteoprotegerin (OPG) and RANKL, as well as higher circulating progesterone levels, among premenopausal BRCA mutation carriers compared to non-carrier controls indicating possible dysregulation of sex hormones and RANK signaling in this population [The receptor activator of nuclear factor κB (RANK), its cytokine ligand (RANKL) and the soluble receptor osteoprotegerin (OPG) are proteins of the tumor necrosis factor (TNF) and TNF receptor superfamily , 2. Theymutation , 9. Furtpulation .BRCA mutation carriers [BRCA1 or BRCA2 mutation.We recently published a significant inverse relationship between plasma OPG levels and breast cancer risk in a cohort of 206 carriers . Our fincarriers . Among wcarriers . To our BRCA mutation face high lifetime risks of breast and ovarian cancer [BRCA mutation, along with the important role of RANK signaling in BRCA1-breast cancer development, it is necessary to evaluate whether levels of circulating RANKL also predict cancer risk. Thus, the goal of this study was to prospectively evaluate whether plasma RANKL levels are associated with the risk of breast cancer among women with a BRCA1 or BRCA2 mutation.Women with an inherited n cancer . Prophyln cancer , 15. TheBRCA1 or BRCA2 mutation included in the current study. Among all the women combined, the mean plasma RANKL levels were 5.54 pg/ml (range 1.78-14.97). Table P = 0.01). As a result, the length of storage of the blood sample prior to quantification of RANKL was also significantly longer among women with high vs. low RANKL levels . All other baseline characteristics were similar between the two groups including reproductive factors, oophorectomy, menopausal status and BMI.There was a total of 184 women with a P log-rank = 0.85) . The results did not change substantially in the multivariate model .There were 15 incident breast cancer cases identified over the mean follow-up period of 6.3 years 0.02-19.24); seven cases (47%) among women in the low RANKL group and 8 (53%) among women in the high RANKL group. The eight-year cumulative incidence was 10% in the low RANKL group and 12% in the high RANKL group Both a plasma OPG and RANKL level was available for 176 of the women in the current study. There was no significant correlation between circulating OPG and RANKL levels  [To our knowledge, this represents the first report of plasma RANKL and breast cancer specifically in this high-risk population. In contrast, there have been two recent similar reports conducted among women from the general population , 17. In et al., evaluated circulating RANKL (and RANKL/OPG) with breast cancer risk [P-trend = 0.20) but not ER-negative (ER–) disease . Results were similar with the RANKL/OPG ratio. The increased risk with both RANKL and RANKL/OPG ratio was limited to women with ER+ disease diagnosed after age 50. In this report, the inter-batch CVs was 21.7%, which is substantially higher than the acceptable value of 15% indicative of laboratory precision, and thus, the results should be interpreted with caution. Given the limited amount of evidence and lack of dose-response, it is currently not clear if RANKL is a biomarker for women without an inherited BRCA mutation.In a case-control analysis nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort which included 1,976 incident invasive breast cancers and the same number of controls, Sarink cer risk . In thiset al., observed a modest inverse correlation between circulating OPG and RANKL in 458 premenopausal controls . Importantly, Widschwendter et al., demonstrated that both RANKL and OPG levels were aberrant in BRCA mutation carriers implicating both proteins in BRCA-breast cancer pathogenesis. However, in their study, changes in RANKL levels within the breast were not observed in circulating RANKL levels. Conversely, decreases in OPG levels within the breast were reflected by subsequent decreases in circulating OPG. This finding suggested that a correlation between circulating OPG and RANKL may not exist. Further investigation is needed regarding potential correlations between OPG and RANKL at the tissue level.Circulating levels of RANKL and OPG were not correlated in our study. In general, little is known regarding the correlation between circulating OPG and RANKL in healthy women. This is not unexpected considering the wide array of hormones and cytokines that are involved in regulating RANK signaling . A potenBRCA-associated breast cancer, it is not entirely surprising that we found no association between circulating RANKL and risk. In the initial publication by Widschwendter and colleagues, the authors reported significantly lower circulating levels of both RANKL and OPG among the BRCA mutation carriers compared to the non-carrier controls [With respect to controls . Interescontrols .BRCA1-breast cancer development [Brca1 mouse models resulted in the expansion of mammary stem cells (MaSCs) and mammary tumorigenesis [BRCA1 mutation carriers have an expanded pool of RANK+ luminal progenitor cells [BRCA1 mutation [RANK+ luminal progenitor population, Widschwendter et al., demonstrated that BRCA mutation carriers have 121% higher levels of circulating progesterone compared to non-carriers – this may lead to increased activation of progesterone-mediated RANK signaling [BRCA chemoprevention.Emerging experimental and epidemiologic data strongly implicate progesterone-mediated RANK signaling in elopment , 9. Two igenesis , 9. Of por cells . These cmutation , 22. In ignaling . CollectBRCA mutation carriers with an average follow-up of 6.5 years, we recently reported a significant inverse relationship between plasma OPG levels and breast cancer risk [BRCA mutation and our team is validating these findings in a larger cohort of BRCA mutation carriers. Similar to what has been reported among women in the general population, we confirmed a potentially harmful role of progesterone-containing hormone replacement therapy (HRT) [BRCA1 mutation carriers, we reported cumulative incidence of breast cancer of 22% among women who used estrogen plus progesterone HRT compared to 12% among women who used estrogen-alone HRT (P-log rank=0.04).While experimental data strongly support a role of RANKL in breast carcinogenesis, the action of RANKL can be inhibited by OPG, the endogenous decoy receptor for RANKL that antagonizes RANK signaling . In a prcer risk . Women wpy (HRT) . In thisBRCA1 mutation carriers face extremely high lifetime risks of breast cancer estimated to be 60% by age 70, although penetrance estimates up to 85% have been reported [reported –27. Propreported , 29 and reported . Despitereported . Indeed,BRCA mutation type; however, the latter was not a significant predictor of RANKL levels in the cox proportional hazards model. We only assessed plasma RANKL at one time point using blood which was collected, on average, 12.64 years (range 3.44-22.03) previously and may not reflect changes in RANKL concentrations over time. In a reproducibility study, which included 221 women enrolled in the EPIC cohort with blood collected at baseline and 14-15 years after recruitment, Fortner et al., reported spearman correlation coefficients of 0.60 and 0.38 for RANKL after one year and 14 years, respectively [Our study was not without limitations. First, was the relatively small sample size which did not allow for stratified analyses by various factors including ectively . CorrespBRCA breast cancer risk suggests that circulating RANKL may not be a reliable biomarker of breast cancer risk in this high-risk population, although this requires confirmation in a larger sample of women. Despite this, the current findings reinforce our earlier notion that OPG may serve as a biomarker of breast cancer risk among women with a BRCA mutation, identifying those at the highest risk of developing disease. Inherently lower levels of circulating OPG in mutation carriers, which reflects less inhibition of RANK signaling, has been shown in experimental and epidemiologic studies to be important for BRCA-associated breast cancer development. The integration of circulating OPG levels into existing risk prediction models may enable the identification of those women who might benefit most from chemoprevention with RANKL blockade.The lack of an association between RANKL and BRCA1 or BRCA2 mutation carriers and who had a blood sample available were potentially eligible for inclusion. These women sought testing for BRCA mutations because of a personal or family history of breast and/or ovarian cancer. Mutation detection was conducted using a range of techniques, but all nucleotide sequences were confirmed by direct sequencing of DNA. The study was approved by the institutional ethics review boards of the host institutions and all study subjects provided written informed consent. For the current prospective analysis, we only included women who received genetic counselling and testing at Women’s College Hospital .This study population has previously been described , 16. BriEach subject completed a baseline questionnaire at the time of a clinic appointment or at their home. The baseline questionnaire collected detailed information on family or personal history of cancer, reproductive and medical histories, as well as information on selected lifestyle factors. Follow-up questionnaires were completed every two years thereafter to update information on relevant exposures and to ascertain incident disease. Blood samples were collected at the time of genetic testing by venipuncture, processed and separated into plasma and DNA and stored at -80°C and 4°C respectively.Women were eligible for inclusion in the current study if they were between the ages of 18-70 years at the time of enrolment and completed at least one follow-up questionnaire. Of the 743 women who were initially eligible, we excluded those who had a previous history of breast, ovarian or other cancer (n=293), those who had previously undergone a prophylactic bilateral mastectomy (n=16), and those who did not have a baseline plasma sample available (n=250). After these exclusions, a total of 184 participants were available for the current analysis.Plasma RANKL was quantified using a commercial ELISA kit from ALPCO  according to the manufacturer’s protocol. All plasma samples were run in duplicate. The RANKL concentration (pg/ml) was calculated as the average of duplicate samples  and subsequently converted to a total RANKL concentration upon comparison to RANKL standards provided in the kit. A common quality control (QC) sample was included on every plate. The average intra-assay coefficient of variation (CV) was approximately 2.7%. This was calculated using the mean CV of duplicate samples within each plate. The average inter-assay CV was 3.4%. This was calculated using the common QC sample run on each plate.t-test and Ӽ2 test. Participants were followed from date of the baseline questionnaire until either date of: 1) breast cancer, 2) prophylactic mastectomy, 2) ovarian cancer, 3) death or 4) completion of last follow-up questionnaire. The follow-up period of this analysis was from the date of baseline questionnaire until November 25th, 2017. Kaplan-Meier survival analysis was used to estimate the eight-year cumulative incidence of breast cancer in women with high vs. low RANKL levels and compared using a log-rank test. Cox proportional hazards models to estimate the hazard ratio (HR) and 95% confidence interval (CI) associated with plasma RANKL levels adjusted for age at blood draw (continuous). The multivariate analysis was further adjusted for date of blood draw (continuous), BRCA mutation type (BRCA1 or BRCA2), oophorectomy , breastfeeding , oral contraceptive use , and parity . All analyses were conducted using SAS version 9.4 software . All P values were 2-sided and were considered statistically significant if P ≤0.05.Women were categorized into high or low plasma RANKL based on the median levels in the entire cohort (≤5.24 and >5.24 pg/ml). Baseline characteristics of the women with high vs. low RANKL levels were compared using the student’s