Breast cancer is a leading cause of death amongst women, several studies have shown significant association between alcohol consumption and breast cancer. The aim of this overview is to highlight some of the mechanisms by which alcohol consumption could increase the risk of developing breast cancer. Using online Medline search engine, article containing details about mechanisms which explain the link between alcohol and breast cancer were examined. A number of mechanisms were found by which alcohol could increase the risk of breast cancer, alcohol's interaction and effect on oestrogen secretion; number of oestrogen receptors; the generation of acetaldehyde and hydroxyl free radicals; cells migration and metastasis; secretion of IGF1 and interaction with HRT and folate metabolism. In conclusion, it is essential for clinicians to understand these mechanisms and inform patients of the link between alcohol and breast cancer.Breast cancer; Alcohol; Mechanisms Breast cancer is now the most common cancer in the UK. In 2005 more than 45,500 women were diagnosed with breast cancer, that’s around 125 women a day [As the concern for breast cancer is increasing, studies have been conducted in all the different aspects of women’s lives trying to find out what could reduce the risk of breast cancer, and what could increase it. One of those important aspects is alcohol consumption.A significant association between alcohol intake and breast cancer has been found, with an increase of risk of 7% for each additional 10 grams of alcohol consumed on a daily basis .The aim of this overview is to highlight some of the mechanisms by which alcohol consumption could increase the risk of developing breast cancer.Ethanol metabolism is often considered to be the predominant factor in the onset of tissue damage, notably through the formation of acetaldehyde. Ethanol metabolism occurs predominantly in the liver, although a variety of other tissues have a significant capacity for ethanol metabolism. Acetaldehyde is the primary product of ethanol oxidation, irrespective of whether it is mediated by aldehyde dehydrogenase (ADH), cytochrome P-450 isoforms, or catalase.Acetaldehyde is itself oxidised predominantly by means of a highly efficient aldehyde dehydrogenase (ALDH2), localised in the mitochondrial matrix. As a result, both cellular and circulating concentrations of acetaldehyde are maintained in the low micromolar range despite elevated blood ethanol concentrations.The rate of both ethanol oxidation and acetaldehyde oxidation is determined by the rate of NADH oxidation through mitochondrial electron transport. This activity depends on the supply of other substrates, the demand for ATP in the cell, and the supply of oxygen.An inadequate electron transport activity would result not only in an inefficient removal of ethanol and acetaldehyde and a highly reduced state of both cytosolic and mitochondrial nicotinamide adenine dinucleotide (NAD), but also promote the overflow of electrons passing through the mitochondrial electron transport chain into the formation of reactive oxygen species (ROS), notably superoxide .Reporting drinking habits can sometimes be difficult, and so the patient tends not to mention that they drink alcohol regularly or in some cases, the clinician doesn’t even ask.It is widely reported that women often drink less and have a lower prevalence of drinking problems than men, but this is starting to change. As drinking levels in women begin to approach those in men, rates of drinking problems in women are likely to overtake those of men, as women have greater physiological sensitivity to the effects of alcohol .This physiological sensitivity is probably due to women's lower total body water, gender differences in alcohol metabolism, and effects of alcohol on postmenopausal oestrogen levels. Mortality and breast cancer are increased in women who report drinking more than two drinks daily .Many women have read studies linking hormone replacement therapy (HRT) use with increased breast cancer incidence. It’s due to this that they may avoid HRT. The link between alcohol use and breast cancer risk, in contrast, is at least as strong as that of (HRT), yet little publicity has reached practising physicians or patients . Much ofTwo types of research link alcohol and cancer. Epidemiological research has shown a dose-dependent association between alcohol consumption and breast cancer; as alcohol consumption increases, so does risk of developing certain cancers. More tenuous results have come from research into the mechanism by which alcohol could contribute to cancer development. Smith et al  found thModerate consumption of alcohol (0.07 g/kg) by women who use hormone replacement therapy was found to elevate oestradiol (high levels are risk factors for breast cancer) level beyond exceeding the ovulatory range for four hours .For postmenopausal women even less than one drink a day was associated with up to a 30% increase in breast cancer mortality compared to non-drinkers .In 2004 and their review G. Poschl and H. K. Seitz  found thAnalyses included 58 515 women with invasive breast cancer and 95 067 controls from 53 studies. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, . Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32  for an intake of 35-44 g per day alcohol, and 1.46  for ≥ 45 g per day alcohol.It is interesting to note that the relationship between smoking and breast cancer was substantially confounded by the effect of alcohol. When analyses were restricted to 22 255 women with breast cancer and 40 832 controls who reported drinking no alcohol, smoking was not associated with breast cancer . If the observed relationship for alcohol is causal, these results suggest that about 4% of the breast cancers in developed countries are attributable to alcohol [Oestrogen is a hormone largely produced (in women) by ovaries. When ovarian production of oestrogen ends, adipose cells are important sites for the conversion of androgenic precursors from the adrenal gland to oestrogen. Moderate alcohol consumption was found to be associated with elevating oestrogen levels in the blood .Oestrogen has many biological effects; amongst them is a proliferative action. Many lines of breast cancer cells depend on oestrogen and progesterone in growth. That means that the breast cells would be exposed to higher levels of oestrogen if women consumed alcohol. This may in turn trigger the cells, which are oestrogen sensitive in these women, to become cancerous.Alcohol consumption-related increases in oestrogen levels may in turn be partially responsible for the associated decreased risk for coronary artery disease and osteoporosis, as well as for increased risk for breast cancer .Alcohol could increase plasma oestrogen levels either by promoting the induction of aromatases, which can convert androgens to estrogens, or by impairing the metabolism of oestrogen in liver, resulting in oestrogen accumulation in the circulation .This association between alcohol and high oestrogen levels was supported by findings in premenopausal women, as well as postmenopausal .Chronic alcohol intake is associated with ovarian failure, infertility and early menopause, i.e. alcohol has a damaging effect on the female reproductive system. Thus, in premenopausal women, the alcohol effect could be via an effect on the pituitary-ovarian axis .Another suggested mechanism is ethanol effect on level and activity oestrogen receptors on the human breast cancer cells. The oestrogen receptors are members of the steroid hormone receptor family, which are nuclear receptors. They are capable of eliciting a variety of cell responses and influencing signal transduction pathways both in ligand-dependent and ligand-independent manners.Not all cancer cells have oestrogen receptor. An interesting study was carried by Keith et al  to deterPositive results for cells with oestrogen receptors included: increase in cell proliferation, increased cAMP production, enhanced DNA synthesis, and increased number of oestrogen receptors.The elevated level of cAMP was suggested as a mechanism for the effects of ethanol on cell proliferation, but the results did not support this, as cAMP levels were not consistent with cell proliferation. Bearing in mind that the ethanol-cAMP relationship is tissue specific and so depends on the tissue examined.Another proposed mechanism was through affecting oestrogen receptors numbers. Despite the fact that total cell numbers increased at ethanol levels lower than those needed to elevate receptors numbers, the other component for ethanol effect (increasing the receptor activity) is still a possibility.From this study it might appear that ethanol only affects human cancer cells with oestrogen receptors and not the others. But, there is recent evidence that ethanol intake by women may increase the risk of developing breast tumours with oestrogen or oestrogen and progesterone receptors .6-methyl guanine demethylase, which is known to be critical for the repair of O6-methyl guanine lesions in DNA caused by N-nitrosodimethylamine and of other equivalent alterations resulting from other alkylating carcinogens [Another possibility is that alcohol is converted to carcinogenic substances inside the human body. Oxidation of ethanol to acetaldehyde and the generation of free radicals were studied by Castro et al . Acetaldcinogens 2-), hydroxyl radical (OH), and hydrogen peroxide (H2O2).The enzymes involved in oxidation of acetaldehyde to acetate are xanthine dehydrogenase (XDh) and xanthine oxidase (XO). Both XOR and AOX can generate reactive oxygen species (ROS), e.g. superoxide anion . The study  found thIn addition for being carcinogenic, and helping in promoting tumour growth via oestrogen levels, alcohol is being investigated for its role in metastasis. Metastasis is the process of tumour cells detaching from primary site and spreading to secondary sites within the human body. It is essential for the cells to detach themselves form the original tissue, be able to attach to extracellular matrix, degradation of extracellular structures and then invade the tissue and the surrounding vessels.Cells use specific interaction molecules to maintain tissue integrity. Two major groups of these are the E-cadherin (transmembrane glycoprotein) and catenins  . In a stFor all the other necessary steps for metastasis, it was found that alcohol has a promoting effect of cell adhesion to basement membrane extract and stimulation of cell invasion and migration.Increasing the motility of cells is essential for cell migration. To examine this, a monolayer of cells was scratched with a sterile tip (a minor wound was created). With alcohol presence, cell flattening and spreading was significantly enhanced along the edges of the wound, compared to untreated samples.The human breast cancer cells were incubated with Matrigel basement membrane extract. At alcohol concentration of 80mM, adhesion of the cancer cells was significantly enhanced. 95% of cells were attached compared to only 65% in absence of alcohol.The invasion step is a complex one; it involves secreting enzymes to dissolve the tissue material followed by increased motility. This was tested on invasion of Matrigel basement membrane extract. When 40 and 80 mM of alcohol were present, the invasion increased by approximately 23% and 52% compared with untreated control, respectively.Even from a protective prospective, alcohol had a negative effect. BRCA1 (breast cancer susceptibility gene 1) is a human gene that is associated with suppression of breast carcinoma . It is oAnother set of studies found that women using the hormone replacement therapy (HRT) and drinking alcohol were at higher risk of developing breast cancer. The extent of impact reached a 3-fold increase in circulating oestradiol on alcohol ingestion (0.7 g/kg/day), for women using HRT . GapsturHRT is the administration of the female hormones oestrogen and progesterone. The use of hormone replacement therapy is highly effective for improving the quality of life of women suffering from acute symptoms of menopause, such as hot flashes, night sweats, insomnia, increased fatigue and irritability. It is also thought that HRT provides some long-term protection against cardiovascular disease and osteoporosis.The explanation for the study findings is very important. When no explanation could be found, it discourages from emphasising the link between a factor and the studied condition. The same thing happened with alcohol and breast cancer, as no biological explanation could be found for any association, and thus alcohol was not mentioned in some studies that explored HRT and breast cancer. Mills et al  describeThe findings that brought alcohol as the major factor to increase the risk of breast cancer, and not oestrogen supplements (and the fact that there has been no studies that disagreed with those findings), led Zumoff  to come From mammography screening, a peak incidence for duct carcinoma in situ (DCIS) of the breast is seen at the age of 45-50 years followed by a steady decline . It suggThis observation may suggest that mechanisms of breast cancer associated with alcohol are likely to be different in premenopausal from postmenopausal. In postmenopausal disease, it may involve a promoting role of hyperinsulinaemia.One of the most known consequences of chronic alcohol consumption is liver cirrhosis. Nearly all patients with liver cirrhosis are insulin resistant, β-cells respond to this resistant by increasing insulin levels (hyperinsulinaemia) .In a review by Stoll , the eleThe liver is the major source of circulating IGF1 and at least six IGF-binding proteins (IGFBPs). The bioavailability of circulating IGF1 is regulated by the different IGFBPs. Whereas IGFBP1 regulates IGF1 availability in response to rapid changes in insulin levels, over 90% of circulating IGF1 is bound to IGFBP3, the latter controls long-term adaptive change to long-term hyperinsulinaemia.In normal subjects, the effect of insulin is to suppress free IGF1 and IGFBP1 concentrations, but IGFBP3 concentrations are stimulated. But in patients with alcoholic cirrhosis, hyperinsulinaemia decreases IGFBP3 concentrations, resulting in increased IGF1 bioactivity, which stimulates insulin-like growth factor 1 receptor IGF1R activity in mammary tissue cells.IGF1 has been shown to induce proliferative activity in cells that have undergone transformation . AlthougAll the above is through the effect of IGF1, but insulin itself may have a role in mammary carcinoma. Most breast cancer specimens show both IGF1 and insulin receptors. Over-expression of insulin receptors (IR) has been noted in human breast cancer cells and this may make them more responsive to insulin stimulation .Another possible route for IGF1 to affect human breast cancer cells is via its interaction with oestrogen. Both oestrogen and IGF1 are potent mitogens for most human breast cancer cell lines, interaction between their receptors is likely to be involved in modulating the progress of mammary carcinogenesis . In mostThe above evidences for IGF1 effects on breast cancer cells, and the how its levels are indirectly affected by alcohol (via hyperinsulinaemia), all that led Stoll  to come Whilst IGF1 might synergies with oestrogen in a mitogenic effect on mammary epithelium and at the same time prevent apoptosis, IGFBP3 might limit the bioavailability of IGF1 and at the same time increase apoptosis independently of IGF1.The important link between alcohol and folate is that alcohol is a folate antagonist.This action of alcohol was investigated by Zhang et al  in a stuThe study found that the risk for breast cancer was higher in women who consumed at least 15g/day of alcohol with low folate intake. High intake of folate had a reducing effect on breast cancer; with at least 600 microgram/day of folate, the risk of breast cancer was reduced by 0.45%, compared with 150-299 microgram/day.The association between alcohol and breast cancer was observed again; women (with folate intake >300 microgram/day) consuming >15 g/day of alcohol had an 5% increased risk of breast cancer compared with < 15 g/day.It has long been known that folate can help prevent certain spinal cord birth defects, which is why it is recommended as a supplement for women of child-bearing age.The protective role of folate from cancer has been studied by Shrubsole et al . The stu6 and B12) have important roles in folate metabolism. Vitamin B6 is coenzyme in the conversion of folate to the form used in DNA synthesis, and vitamin B12 facilitates methyl transfer.Folate is a B vitamin found naturally in many foods, including dry beans, peas, and leafy green vegetables, such as spinach. It is essential for regenerating methionine, which is the methyl donor for DNA methylation and for purines and pyramidines required for DNA synthesis. Folate co-factors (vitamin BThe body is able to absorb folic acid (used in fortifying foods and in supplements) about twice as easily as the folate found naturally in foods. The recommended daily intake of folate for adults is at least 400 micrograms (ug).The study evaluated the association between folate and breast cancer in a population who lived in Shanghai, China. It was important that the study is not conducted in a western country. The reason for this is fortification of food. Since 1998, folic acid fortification of cereal food has been mandated in the USA. Therefore studies in North America might lack the appropriate food composition; thus it would not be possible to assess the exact folate intake of the population.The Shanghai population included women who have diet composed of unfortified and unprocessed food. And they rarely take vitamin supplements that might result in miscalculations of total folate intake. Also in the population, few women drink regularly. This is significant to evaluate the effect of folate, independently from other factors.The study found that high intake of folate was associated with 0.38 % reduction of breast cancer. Folate co-factors intake was assessed as well. It was found that women with high levels of folate co-factors intake had a 0.53% reduction of breast cancer.Although the inverse association (between folate intake and breast cancer) was more evident in postmenopausal women, similar pattern of association was found in premenopausal women.This led Shrubsole et al  to come The alcohol intervention with folate occurs at several aspects of folate transport and metabolism, disrupting its supply to tissues. They include: intestinal absorption, transport to tissue, storage and release by liver .Thus, alcohol could be taken as a factor that reduces folate supply to tissues; this in its turn results in disrupted gene expression and DNA breakage, which are major risk factors for cancer development.Breast cancer is the most common cause of cancer death in women world-wide. Epidemiological research has shown a dose-dependent association between alcohol consumption and breast cancer. His overview highlighted the mechanisms by which alcohol consumption could increase the risk of developing breast cancer.If the observed relationship for alcohol is causal, results suggest that about 4% of the breast cancers in developed countries are attributable to alcohol. Clinicians need to inform women of the link between alcohol and breast cancer, since the use of alcohol is under-reported and rarely explored in depth-even in epidemiological research. The relationship between alcohol and breast cancer should be stressed even for women who do not admit to alcohol use, given the poor rate of intake estimation in most clinical settings.


Myeloperoxidase (MPO) is a metabolic/oxidative lysosomal enzyme secreted by reactive neutrophils at the sites of inflamed organs and tissues during phagocytosis. MPO has been either directly or indirectly linked to neoplasia, which is a well-established risk factor for many types of cancer. A large number of studies have reported the role of MPO G-463A polymorphism regarding breast-cancer risk. However, the published findings are inconsistent. Therefore, we conducted a meta-analysis to determine more precise estimations for the relationship. Eligible studies were identified by searching several electronic databases for relevant reports published before June 2012. According to the inclusion criteria and exclusion criteria, a total of five eligible studies were included in the pooled analyses. When the five eligible studies concerning MPO G-463A polymorphism were pooled into this meta-analysis, there was no evidence found for a significant association between MPO G-463A polymorphism and breast-cancer risk in any genetic model. We also categorized by ethnicity (Caucasian or Asian) for subgroup analysis; according to this subgroup analysis, we found no significant association between MPO G-463A polymorphism and breast-cancer risk in any genetic model. However, in the stratified analysis for the premenopausal group, women carrying the AA genotype were found to have a significantly reduced risk . Under the recessive model, there was a significant association between MPO G-463A polymorphism and breast-cancer risk . We conclude that MPO-G463A polymorphism might not be a good predictor of breast-cancer risk, though menopausal status modified women’s risk of developing breast cancer. Breast cancer is the most common type of malignant neoplasm in women worldwide, and its incidence is increasing in both developed and developing countries Myeloperoxidase (MPO) is a metabolic/oxidative lysosomal enzyme secreted by reactive neutrophils at the sites of inflamed organs and tissues during phagocytosis Several studies have shown the possible involvement of MPO in the pathogenesis of breast cancer; however, the conclusions are inconsistent. To our knowledge, G-463A is the most extensively studied polymorphism in the MPO gene with respect to breast cancer susceptibility. We therefore undertook a meta-analysis to evaluate the association between MPO G-463A polymorphism and breast cancer risk.Eligible studies were identified by means of an electronic search of PubMed, Elsevier ScienceDirect, EMBASE, and EBSCO for relevant reports published before June 2012 with the following search terms: ‘‘myeloperoxidase’’ or ‘‘MPO’’ combined with ‘‘breast cancer.’’ All eligible studies were examined carefully. Review articles and references cited in the retrieved articles were manually obtained to find additional eligible studies.The following criteria were used for the study selection: the study (1) evaluated the association between MPO G-463A polymorphism and the risk of breast cancer; (2) used a case-control design; (3) included a full-text article; (4) offered the size of the sample and sufficient data (genotype distributions of both cases and controls were available) for estimating an odds ratio (OR) with a 95% confidence interval (CI) or information for helping infer the results in the papers; and (5) used the English language. Exclusion criteria were as follows: the study (1) used only case-group data, (2) included no available genotype frequency, and (3) used overlapping data published by the same first author.Based on the inclusion and exclusion criteria, the following data were extracted for each study: the first author’s surname, year of publication, ethnicity of the subjects, enrollment criteria of case and control, genotyping methods, study size, and genotype distribution in cases and controls . Two authors from the present study (Qin and Deng) independently and carefully collected the data. For conflicting evaluation, these two authors carried out discussions until a consensus was reached. If they could not reach a consensus, disagreement was adjusted by a third author (Zeng).Three reviewers  independently evaluated the quality of selected studies by scoring according to a set of predetermined criteria , which wThe strength of association between MPOs and breast cancer risk was measured by ORs with 95% CIs. The pooled ORs were calculated, including (1) the GA genotype versus the GG genotype, (2) the AA genotype versus the GG genotype, (3) GG+GA genotypes versus the AA genotype (the dominant model), and (4) the GG genotype versus GA+AA genotypes (the recessive model). Heterogeneity between and within groups was checked by using the Q statistic. A value of p≥0.10 for the Q-test indicated there was no heterogeneity among studies, so the pooled OR estimate from each study was calculated by the fixed-effects model; otherwise, the random-effects model was used http://snp500cancer.nci.nih.gov). Four clinical treatment studies were excluded Five eligible studies were included in the pooled analyses  [18]–[22In the five studies of MPO G-463A polymorphism, we identified 2996 cases and 3738 controls. Overall, the results showed no significant association between MPO G-463A polymorphism and breast cancer risk  . In the The premenopausal  and postmenopausal  groups came from three and two studies, respectively. In the premenopausal group, women carrying the AA genotype were found to have a significantly reduced risk of breast cancer , and we found a significant association between MPO G-463A polymorphism and breast cancer risk with the recessive model  . In the A single study involved in the meta-analysis was deleted each time to reflect the influence of the individual data set to the pooled ORs. The results suggested that no individual study significantly affected the pooled ORs. Sensitivity analysis was performed after excluding HWE-violating studies, and the corresponding pooled ORs were not materially altered, indicating that our results are statistically robust .www.cochranehandbook.org), which states that the test for publication bias yields unreliable results when less than 10 studies are included in a meta-analysis, the negative results in our meta-analysis are possibly because the number of publications we assessed were too few to determine statistical significance.Both Begg’s funnel plot and Egger’s test were performed in order to assess the publication bias of the literature. As shown in 2O2 during infection but also in the metabolic activation of a number of procarcinogens MPO is a member of the mammalian heme peroxidase enzyme family and is a key component of the phagocyte oxygen-dependent intracellular microbicidal system, playing an important role in innate immune responses Unlike studies that have noted an association between the MPO A/A genotype and a reduced risk of breast cancer, we did not find an association between MPO G-463A polymorphism and breast cancer risk; however, consistent with the findings of Lin et al. We have read with great interest the recent meta-analysis reported by Pabalan et al However, there were also several limitations to our study. First, our meta-analysis had a limited statistical power for subgroup analysis, and the precision of our estimates needs to be assessed in consideration of the small numbers. Our analysis of premenopausal women only included two studies, both of which were of Caucasian women. Clarifying whether ethnicity has a biological influence on cancer susceptibility, therefore, requires more subjects and different ethnicities. Second, as positive associations have a greater chance of publication than true negatives, many studies remain unpublished and were therefore not included in this meta-analysis. Finally, due to a lack of original data, such as age, alcohol consumption, and other factors, we could not perform a more precise calculation of adjusted ORs, which limited our analysis of potential gene-environment interactions.In summary, our results indicated that MPO-G463A polymorphism might not be a good predictor of breast cancer risk, while menopausal status modifies a woman’s risk of breast cancer. Premenopausal women carrying the AA genotype were found to have a decreased risk of breast cancer. However, given the limited data, more studies are needed to clarify the role of MPO in breast carcinogenesis.Text S1Checklist(DOC)Click here for additional data file.


The Internet can increase access to psychosocial care for breast cancer survivors through online support groups. This study will test a novel prosocial online group that emphasizes both opportunities for getting and giving help. Based on the helper therapy principle, it is hypothesized that the addition of structured helping opportunities and coaching on how to help others online will increase the psychological benefits of a standard online group.A two-armed randomized controlled trial with pretest and posttest. Non-metastatic breast cancer survivors with elevated psychological distress will be randomized to either a standard facilitated online group or to a prosocial facilitated online group, which combines online exchanges of support with structured helping opportunities  and coaching on how best to give support to others. Validated and reliable measures will be administered to women approximately one month before and after the interventions. Self-esteem, positive affect, and sense of belonging will be tested as potential mediators of the primary outcomes of depressive/anxious symptoms and sense of purpose in life.This study will test an innovative approach to maximizing the psychological benefits of cancer online support groups. The theory-based prosocial online support group intervention model is sustainable, because it can be implemented by private non-profit or other organizations, such as cancer centers, which mostly offer face-to-face support groups with limited patient reach.NCT01396174ClinicalTrials.gov:  Ongoing and timely access to relevant information and social support can improve psychological outcomes in breast cancer patients , but sucDepression and anxiety symptoms are highly prevalent among breast cancer survivors  and ofteDozens of interventions have been designed to reduce distress in cancer survivors, but the evidence on their efficacy and acceptability is weak ,27. MoreTo date, the evidence on the efficacy of cancer OSGs is limited. Winzelberg et al.  randomizreceiving social support [providing social support also confers benefits [The current study will develop and evaluate a novel OSG intervention that theoretically should boost the benefits observed in standard facilitated OSGs ,29. Most support , but thebenefits ,31-33. Sbenefits ,31. In gbenefits . Howeverbenefits  or longibenefits  are rareThe trial also aims to identify potential mechanisms underlying the psychological benefits of helping. Based on the literature and Midlarsky's  conceptuThe major objective of the "Life Beyond Cancer" trial is to test the efficacy, acceptability, and feasibility of a novel OSG for distressed breast cancer survivors that provides structured helping opportunities and coaching on how best to give support to others. The trial targets distressed breast cancer survivors because they will likely benefit more than non-distressed patients from psychosocial interventions . The stuHypothesis 1: Relative to women in the standard OSG (S-OSG) condition, women in the prosocial OSG (P-OSG) condition will have a lower mean level of depression/anxiety symptoms at posttest.Hypothesis 2: Relative to women in the S-OSG condition, women in the P-OSG condition will have a significantly greater mean level of sense of purpose in life, self-esteem, positive affect, and sense of belonging at posttest.Hypothesis 3: Positive increases (posttest-pretest) in mean level of self-esteem, positive affect, and sense of belonging will partially mediate the effects of the P-OSG intervention on mean level of depression/anxiety symptoms and sense of purpose at posttest.Hypothesis 4: Positive increases (posttest-pretest) in mean level of sense of purpose will partially mediate the effects of the P-OSG intervention on mean level of depression/anxiety symptoms at posttest.Hypothesis 5: Given the equal level of attention across conditions, we hypothesize that there will be no significant between group differences in retention (percent completing study) and mean level of satisfaction with the intervention.A two-group randomized controlled trial design with pretest and posttest measures will be used to address the study aims. Distressed breast cancer survivors will be randomized equally to one of two groups: (a) P-OSG, a facilitated OSG with structured helping opportunities and coaching on how to recognize and be responsive to others needs for help or (b) S-OSG, a comparison standard facilitated OSG. Data will be collected approximately one month before and after the intervention. The study design is guided by the CONSORT criteria . The stuEligibility inclusion criteria are: diagnosed with stage I or II breast cancer within the preceding 36 months; age 21 to 65 years , access to and ability to use a computer and the Internet, able to understand the informed consent form, fluent in spoken English, and meet the screening criteria for psychological distress. Women will qualify as distressed if they fall above the "non-case" cut-point (≥ 8) on either the depression or anxiety subscale of the Hospital Anxiety and Depression Scales (HADS ). This aThe sampling frame will be generated in conjunction with a State Cancer Tumor Registry and will target women meeting the age and diagnostic criteria. Advance letters will be sent to potential participants. Respondents will be screened for eligibility using standardized measures administered over the phone. Eligible participants will be consented. Non-eligible participants will be sent a resource list.Trained research assistants will collect data via structured telephone interviews. A random subset of interviews will be monitored and reviewed for quality control. Interviewers will be blind to participants' intervention condition. Within one month following end of treatment, participants will be re-interviewed to collect follow-up data. Again, a random subset of interviews will be reviewed for quality control and interviewers will remain blind to participants' condition.Because age is related to level of Internet use  and to lFacilitators will be graduate-level, trained health professionals. Trained researchers will use treatment fidelity checklists to evaluate facilitators' adherence to study protocols. Co-investigators will conduct supervision sessions with facilitators to review treatment fidelity and to address any problems.All major study measures have established reliability and validity. The primary outcome measure will be the HADS, which has been used extensively and validated with breast cancer and other cancer patient populations . The HADMeasures of potential covariates include background variables  and other potential confounding variables [e.g., Social Provisions Scale  to measuFinally, as a manipulation check, we will assess if the P-OSG intervention increased helping behaviors relative to the S-OSG. Research assistants will code instances of online support provisions using Bambina's Support OnLine (SOL) coding scheme . SOL canThe S-OSG comparison condition is based on an empirically validated facilitated breast cancer OSG ,29. The The weekly topics, which were drawn from research on breast cancer survivorship e.g., -69, incl, incl69,The P-OSG intervention includes elements of the S-OSG, but with critical modifications and key additions. First, participants will receive a tip sheet that describes how to recognize others' needs for different kinds of support and how to be helpful in an online environment. Second, the facilitator will post reminders at the beginning of each session on the importance of responding to one another's needs for help. Third, participants will be asked to commit to a goal of offering informational or emotional support to group members each week during the live synchronous sessions or later using the discussion board, email or chat. Fourth, prior to each week's meeting, participants will receive an email describing the topical problem to be discussed . Their "homework" is to prepare 1-2 sentences on how their experiences with the problem might help others to cope with the problem. If they have not had the problem, they will be asked to write 1-2 sentences on how their approach to coping with other life problems might be useful to group members coping with the particular problem of the week. Fifth, the facilitator will encourage helping by highlighting direct and indirect requests for support by group members, will praise those who offer help , and will encourage additional help .Members of the P-OSG also will be asked to select one of the six weekly topics to prepare a blog about the topic to share with the group. The goal will be to write about their experience as a way of helping other breast cancer survivors who have gone through a similar experience. The writing instructions will emphasize that participants should not worry about grammar or spelling and that if they wish the research staff can help to edit their writing for grammar or spelling before posting. Participants will be asked to blog about any effective ways they have developed to cope with a cancer-related problem. The exercise will emphasize that people often have experiential knowledge that is valuable to others, but can only be accessed if there is sharing. Bloggers will be told that the exercise is designed to help other patients to understand the different consequences of breast cancer and its treatment, as well as how they might best manage or address these consequences. Instructions also will explain that the bloggers do not necessarily have to provide answers or solutions to the problems they write about, they simply need to share their experience. Reading others similar experiences can help to normalize the experience or make readers feel less alone. We recognize that some participants might be resistant to sharing their writings with others, either because they do not feel it will be valuable or they feel it is too personal. We will offer individuals the opportunity to have their blog posted anonymously, or only post select information, or for us to paraphrase what they have written in a list of comments comprised of input from multiple blogs. All blogs will be fact checked by research staff to avoid posting misinformation.Finally, in addition to promoting helping within the group, the P-OSG will promote helping outside of the group through an outreach activity called the "Breast Cancer Awareness Ambassadors" program. Women in the P-OSG group will be asked to increase breast cancer awareness by sharing a "Breast Cancer and You" fact sheet and "Mammograms Save Lives" e-Cards, both developed by the Centers for Disease Control and Prevention, with women in their social network who are 40+ years old.Descriptive statistics will be generated for the total sample and the treatment groups for each time point. Distributional properties will be analyzed to determine if variance stabilizing or normalizing transformations should be applied. Outliers will be assessed and checked for accuracy. Non-response and attrition must be addressed in longitudinal research . TherefoHypotheses 1 and 2: Post-test differences between P-OSG and S-OSG in primary and secondary outcomes will be tested using ANCOVA, with intervention group as the predictor and appropriate pre-test scores as covariates. Intent-to-treat analyses will be used. Variance associated with cohort membership will be estimated via intraclass correlation coefficients. If necessary cohort can be entered into the ANCOVA models as a random effect or fixed-effects analyses can be performed if there is evidence for unobserved heterogeneity. Group-level analyses can be performed on aggregate data if necessary.Hypotheses 3 and 4: Mediation will be tested in a series of ANCOVA models using standard statistical software or structural equation modeling software. Hypothesis tests will be based on bootstrapped standard errors .Hypothesis 5: Acceptability will be assessed by recruitment and retention rates, reasons for refusal/dropout, and level of participants' ratings of satisfaction and helpfulness of different intervention elements. Fisher's exact test will be used to evaluate differences between conditions for categorical outcomes; t-tests or non-parametric equivalents can be performed to compare ordinal or continuous outcomes.The feasibility of manipulating online helping behavior will be assessed by comparing mean levels of observed helping behaviors (using SOL data) enacted by participants in the P-OSG versus S-OSG and participants' willingness and ability to generate a "helping blog" and engage in "outreach" activities in the P-OSG condition. Additional analyses will be conducted to confirm that treatment fidelity was equivalent across conditions and to explore dose-response relations by examining whether level of participation  moderates intervention effects.Because this is a preliminary study of the effect of the novel P-OSG intervention, we are interested in estimating effect sizes of the P-OSG intervention group. More specifically, we are interested in gaining an estimate of the true treatment effect size (which is independent of sample size) and to establish a plausible confidence interval around this effect size. Thus, we present information regarding the aim of accuracy in parameter estimation (AIPE)  as well w) within which an effect size can be bracketed with a specified probability, independent of the magnitude of the observed effect size. It is easiest to interpret in the metric of standardized regression coefficients, i.e., β ± w. For these analyses, AIPE will depend on (a) the number of predictors, (b) variance in predictor of interest accounted for by other predictors, N = 180 , we expect to be able to bracket effect sizes for treatment effects within ± .11 to ± .15 depending on model R2. Effect sizes for mediators will be estimated with somewhat less precision but within ± .12 to ± .22AIPE identifies a range  for specific pre-post correlations (r01) by the formula: 01 = .9) to .42 (r01 = 0).Power to identify post-test differences between P-OSG and S-OSG controlling for pre-test values and group membership depends on the correlation between pre-test and post-test scores. Minimum detectable effect size In terms of clinical practice, we believe that the current lack of conclusive evidence on the efficacy of OSGs has stymied the broader implementation and dissemination of facilitated OSGs. The majority of National Cancer Institute-designated cancer centers in the United States, as well as community-based hospitals and cancer centers, primarily offer face-to-face supportive care, which is costly and has limited patient reach. If successful, results of the proposed trial could stimulate broader implementation of facilitated OSGs and offer an innovative approach to implementing OSGs that emphasizes prosocial behavior within the group and outside the group. Specifically, the proposed prosocial OSG model offers an alternative to the dominant self-help OSG, which lacks facilitation and emphasizes emotional expression over mutual exchange of help. Importantly, the prosocial OSG provides a model for practice that can be easily adopted by organizations that offer psychosocial care to cancer survivors.The following abbreviations were used: AIPE: accuracy in parameter estimation; ANCOVA: Analysis of covariance; FIML: full information maximum likelihood; MI: multiple imputation; OLS: ordinary least squares; OSG: 0nline support group; P-OSG: prosocial online support group; S-OSG: standard online support group.The authors declare that they have no competing interests.SJL, JB, ML, MG developed the study concept and aims. AD assisted with the analytic plan. SJL drafted the manuscript. SL and JB will implement the protocol. SL will oversee enrollment and data collection. JB, ML and MG will oversee facilitator training and monitoring. AD will assist with sampling, randomization procedures and data analysis. All authors contributed to and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/379/prepub


Vitamin D has been recognized for its immune-modulating properties. We have previously found that levels of 25OHD, and cytokines including IL5, IFNα2, and TNFα, are also associated with estrogen receptor (ER) negative breast cancer in younger women. Thus, we hypothesized that there may be interactions between vitamin D and the immune system in influencing breast cancer ER status, which was tested in 490 women with incident breast cancer. There was no correlation of the levels of 25OHD with any cytokine, and their associations with tumor ER negative status were independent of each other. However, premenopausal women with low 25OHD and high TNFα levels had the highest likelihood of having ER negative cancer , with evidence of synergy between the two . There were similar synergistic associations between 25OHD and IL5, and several IFNα2 to Th2 cytokine ratios. This is the first study to provide evidence of interactions between vitamin D and the immune system in relation to breast cancer ER status, which may inform combinational use of vitamin D and anti-inflammatory drugs for cancer prevention and therapy. There is a rich body of evidence supporting vitamin D as a potent immunomodulator ,2,3,4. VGiven the evidence of potential immuno-modulating activities of vitamin D, it is plausible to speculate that there may be interactions between vitamin D and the immune system relevant to the risk of breast cancer. It has been shown in prostate cancer cell lines that the combination of calcitriol with the non-steroidal anti-inflammatory drugs (NSAIDs), including naproxen and ibuprofen, can achieve the same magnitude of cancer cell growth inhibition at 1/2–1/10 the concentrations of the drugs used as a single agent [vs. ER− cancer. In our previous study, we found that high blood levels of 25-hydroxyvitamin D (25OHD) were associated with a lower risk of breast cancer, particularly risk of the estrogen receptor (ER)-negative and triple-negative cancer subtype, in premenopausal women [A number of epidemiologic studies have examined the role of vitamin D in breast cancer etiology with inconclusive results. One reason for the inconsistent findings might be cancer heterogeneity—the association of a risk factor with cancer may differ by cancer subtypes. For breast cancer, estrogen receptor (ER) is one of a few most important prognostic and predictive biomarkers for breast cancer. It is likely that ER-positive and ER-negative breast cancer follows distinct etiology pathways. We hypothesized that vitamin D is related to breast cancer in an ER-status specific way, which were examined by comparing ER+ al women . Similaral women . AlthougAmong 490 women, newly-diagnosed with invasive breast cancer in our previous studies of 25OHD and cytokines, we examined the inter-dependence of the associations of 25OHD and cytokines and further, their combinational associations with breast cancer ER status.Detailed description of the patient population has been published elsewhere ,12. BrieBlood samples were collected, prior to surgery and any adjuvant treatment, in phlebotomy when specimens for clinical measures are drawn, transported to the laboratory through a pneumatic tube system, and processed within one hour of blood draw. Specimens were maintained in liquid nitrogen until analysis. Serum 25-hydroxyvitamin D (25OHD) concentrations were measured by the immunochemiluminometric assay on the DiaSorin Liasion automated instrument performed by Heartland Assays . The coefficient of variation (CV) from laboratory technical QCs was 8.8%. A panel of 27 cytokines was measured based on plasma samples by Luminex xMAP immune-bead array assays  performed by the RPCI Flow Cytometry Core. Intra-plate CVs from laboratory technical QCs ranged from 1.4% to 7.5% and inter-plate CVs ranged from 2.7% to 11.9%. To reduce the burden of multiple testing, only cytokines and ratios which had been significantly associated with breast cancer ER status in our previous study were included in this analysis; these were IL5, IFNα2, TNFα, six IFNα2 to Th2 cytokines ratios , and four Th1 cytokine to IL5 ratios . vs. ER positive), each analyte or cytokine ratio was dichotomized at the median (high vs. low) and examined in relation to ER status using unconditional logistic regression. Odds ratio (OR) and 95% confidence intervals (CI) were derived with adjustment for age at diagnosis, specimen storage time, season of blood draw, timing of blood draw in relation to receipt of treatment, and American Joint Committee on Cancer (AJCC) breast cancer stage. Further adjustment with other potential covariates, including breast cancer family history, body mass index, tumor grade, and smoking status did not significantly alter the estimates, and were thus not included. Correlation between levels of 25OHD and cytokines/ratios was tested using Spearman correlation with adjustment for the same set of covariates. To test whether the associations of 25OHD and cytokines on cancer ER status were independent from each other, mutual adjustment in the logistic regression models with the same covariates was performed. Descriptive characteristics of the patient population were summarized using mean and standard deviation for continuous variables and count and percent for categorical variables. To examine the associations of 25OHD and cytokines on breast cancer ER status (ER negative 11 − OR10 − OR01 + 1), where OR11, OR10 and OR01 were odds ratios associated with low 25OHD high cytokine (highest risk group), low 25OHD low cytokine (sub-high risk group), and high 25OHD and high cytokine (sub-high risk group), respectively. Confidence intervals and p-values for RERI were computed by published SAS program by Lundberg and Andersson [p-value <0.05 was considered statistically significant for main effects. Given the relatively limited sample size, a p-value for interaction <0.20 was considered statistically significant. All analyses were performed in SAS 9.3.To examine the combinational associations of 25OHD and cytokines on breast cancer ER status, a composite variable between 25OHD and each cytokine/ratio with four levels was created  and examined in relation to ER status using similar logistic regression models as described above, with women at high 25OHD and low cytokine levels as the reference group . For the four Th1 cytokine to IL5 ratios, where IL5 was used as a denominator , the high and low risk categories were reversed as the low level of each ratio was associated with increased risk. Additive interactions were estimated using the relative excess risk due to interaction  and only 19% of women were considered vitamin D sufficient (serum 25ODH ≥30 ng/mL). Most of the women had early stage breast cancer (stage I-IIIA), 63% of tumors had high histological grade, and 23% were ER negative. Descriptive characteristics of the 490 patients with invasive breast cancer are summarized in Spearman partial correlations between serum levels of 25OHD and plasma levels of selected cytokines and ratios with adjustment for covariates are shown in vs. ER positive cancer. Lower ratios of Th1 cytokines to IL5, including IL12p70/IL5, IFNγ/IL5, CXCL10/IL5, and TNFα/IL5, were associated with higher odds of ER negative disease. To examine whether the associations of 25OHD and cytokines/ratios were independent from each other, we mutually adjusted for each other in the same multivariate models, and the associations remained essentially unchanged (data not shown). As we previously reported ,12 and ip-values for additive and multiplicative interactions. The most notable synergistic association was between 25OHD and TNFα. Compared to the lowest risk group (high 25OHD and low TNFα levels), patients in the highest risk group (low 25OHD and high TNFα levels) had more than 7-fold increased odds of ER negative vs. ER positive cancer . The estimated RERI was 5.46 (95% CI = −1.84–12.76); p for additive interaction was 0.14 and p for multiplicative interaction was 0.09. Similar synergistic associations were also found between 25OHD and IL5, as well as seven cytokine ratios, including IFNα2/IL4, IFNα2/IL10, IFNα2/CCL2, IFNα2/TNFα, IL12p70/IL5, IFNγ/IL5, CXCL10/IL5, and TNFα/IL5, although tests for additive or multiplicative interactions were not significant (p for additive interaction =0.63 and p for multiplicative interaction =0.10). Similar antagonistic associations were also found between 25OHD and IFNα2/CCL7 and IFNα2/CCL11.Because the main effects of 25OHD and cytokines on ER status were found only in premenopausal patients, their combinational associations were analyzed in this subgroup only. The ORs and 95% CIs shown in nificant . We alsoIn this study, we examined the combinational associations of serum levels of 25OHD with plasma levels of three cytokines, IFNα2, TNFα and IL5, as well as ten cytokine ratios, on ER status of breast cancer. There was no correlation between blood levels of 25OHD and any of the cytokines/ratios, and the main effects of 25OHD and cytokines on breast cancer ER status, primarily in premenopausal women, were independent of each other. However, when considered together, premenopausal women with low 25OHD and high TNFα had the highest likelihood of having ER negative breast cancer, which was higher than the addition of main effects of each. Similar synergistic associations were also found for IL5 and several cytokine ratios. On the contrary, there was some evidence for antagonistic associations between 25OHD and IFNα2 on ER status, which was also found for two cytokine ratios where IFNα2 was the numerator. These findings, although preliminary due to the limited sample size and retrospective measurement of the analytes, provide the first evidence for extensive interactions between vitamin D and immune factors, which may influence breast cancer risk and aggressive characteristics. 3 had no effect on circulating levels of TNFα [The strongest synergistic association on breast cancer ER status was found between 25OHD and TNFα. As a pro-inflammatory cytokine from macrophages and tumor microenvironment, TNFα has been implicated in cancer promotion and progression , possibl of TNFα ,24. In ovs. ER positive cancer, is inconsistent with the anti-tumor properties of IL5. It is possible that IL5-driven immune surveillance targets more strongly against ER positive cancer cells and thus the higher ratio of ER negative vs. ER positive odds in patients with high IL5 levels. Alternatively, this observation could be due to a stronger stimulation of IL5 response by ER negative cancer cells. Nonetheless, these observational findings need to be further investigated in prospective studies. Similarly to the case of TNFα, we also found synergistic associations between 25OHD and IL5 on breast cancer ER status in premenopausal patients. IL5 is produced by Th-2 lymphocytes and plays an important role in B-cell differentiation and eosinophilopoiesis ,27. In-d3 supplementation did not increase circulating IL5 concentrations [Although vitamin D is known to enhance Th2 immune responses, the relationship between vitamin D and IL5 in human has been ambiguous. In two small trials among ambulatory or obese adults, vitamin Dtrations ,30, consIFNA gene has been observed in acute leukemia and glioma cases [In addition to the above synergistic associations, we also found some evidence of antagonistic associations between 25OHD and IFNα2, as well as IFNα2 to Th2 cytokine ratios, on breast cancer ER status in premenopausal patients. IFNα2 belongs to the large family of interferon alpha, which is Th1 cytokine in response primarily to viral infection. Deletion of ma cases , and impma cases . Althougin vitro [The synergistic associations between vitamin D and cytokines on breast cancer aggressive characteristics may have important implications to breast cancer prevention and prognosis. Both vitamin D and NSAIDs have been studied separately in relation to reduced breast cancer risk and superior prognosis ,37,38,39in vitro . Our stuvs. luminal A subtype. However, when examining the combinational associations between 25ODH and cytokines, the numbers of triple-negative breast cancer patients were rather limited, especially after stratifying by menopausal status. Although associations for triple negative breast cancer were similar to those reported here for ER status, that women with low 25OHD and high cytokine were most likely to have triple negative breast cancer vs. luminal A subtype (data not shown), the confidence intervals of the ORs were very wide; thus, we reported only the results of ER status. A limitation of our study is that blood samples were drawn at the time of diagnosis, and may not necessarily reflect etiologic events but rather, could be the result of disease onset or progression. Although the average time from diagnosis to the time of blood draw was relatively short (27 days), and the majority of blood samples were collected before any treatment (92%), we still cannot exclude a possibility of reverse causal relationships between blood analytes and breast cancer aggressive characteristics. However, exclusion of the samples collected after the initiation of treatment had little impact on the results. Another limitation of our study is the relatively small number of patients included in the analysis, particularly in subgroup analyses among premenopausal women. As a result, none of the interaction tests reached statistical significance when multiple comparison error was considered. Due to the concern of multiple testing, we refrained from analyzing the interactions between 25OHD and all cytokines and their ratios examined in our previous study, but instead focused on those shown significant main effects. However, it is possible that significant interactions exist even without significant main effects. In our previous analyses of the main effects of 25OHD and cytokines, we examined both ER status and triple-negative subtype In women diagnosed with invasive breast cancer, we found no correlation of serum 25OHD levels with plasma levels of cytokines/ratios, and associations of 25OHD and cytokines with ER status were independent of each other. When 25OHD and cytokines/ratios were combined in our analyses, synergistic associations on ER status were found between 25OHD and TNFα and IL5, as well as several cytokine ratios, in premenopausal breast cancer patients. Caveats of a limited sample size and measurement of blood levels 25OHD and cytokines at the time of diagnosis should be considered when interpreting our results. Our study provides the first evidence of potential interactions between 25OHD and the immune system in modulating breast cancer aggressive characteristics, which may warrant further investigation in a future large prospective study.


Prior research supports an association between endogenous sex steroids and breast cancer among postmenopausal women; the association is less clear among premenopausal women.We evaluated the associations between estrogens, androgens, progesterone and sex hormone binding globulin (SHBG) and breast cancer in a nested case-control study in the Nurses' Health Study II. Between 1996 and 1999, 29,611 participants provided blood samples; 18,521 provided samples timed in early follicular and mid-luteal phases of the menstrual cycle. A total of 634 women, premenopausal at blood collection, developed breast cancer between 1999 and 2009 and were matched to 1,264 controls . We used conditional logistic regression controlling for breast cancer risk factors for overall analyses; unconditional logistic regression additionally controlling for matching factors was used for subgroup analyses.th vs. 1st quintile odds ratio (OR): 1.7 : 1.0 to 2.9), Ptrend = 0.02). Luteal estrone, free estradiol and progesterone were not associated with risk. Androgens were suggestively or significantly associated with risk when the sample was restricted to invasive tumors , Ptrend = 0.23) and ER+/PR+ disease (testosterone: OR: 1.7 (1.1 to 2.6) Ptrend = 0.10; dehydroepiandrosterone sulfate (DHEAS) OR: 1.3 (0.8 to 2.0) Ptrend = 0.05). SHBG was not associated with breast cancer risk. The results varied by menopausal status at diagnosis, with follicular estradiol suggestively positively associated with breast cancers in women premenopausal at diagnosis (OR: 1.1 (0.9 to 1.3) and significantly inversely associated with postmenopausal disease (OR: 0.6 (0.4 to 0.9); Pheterogeneity < 0.01).In analyses of premenopausal estrogens including breast cancers diagnosed both before and after menopause, there was no association between follicular estradiol, estrone and free estradiol and risk of either total or invasive breast cancer. Luteal estradiol was positively associated with estrogen receptor positive (ER+)/progesterone receptor positive (PR+) cancers (5Androgens were associated with modestly increased risk of breast cancer in this population, with stronger associations for invasive and ER+/PR+ disease. Luteal phase estradiol levels were suggestively associated with ER+/PR+ tumors but no other strong associations were observed with estrogens. Associations with follicular phase estrogens may vary by menopausal status at diagnosis, but case numbers were limited. Additional studies to confirm the role of premenopausal hormones in the etiology of both premenopausal and postmenopausal breast cancer are needed. Experimental data support an association between estrogens and breast cancer risk, and suggest that androgens also may alter risk. Estrogens are hypothesized to act through cell proliferation, decreased apoptosis and possibly DNA damage . The rolIn epidemiologic studies, circulating sex steroid hormones are well confirmed to increase risk of postmenopausal breast cancer , but datGiven the limitations of the prior literature, we conducted a nested case-control study in the Nurses' Health Study II (NHSII) to examine associations among breast cancer, overall and by hormone receptor subtype, and plasma estrogens and progesterone by menstrual cycle phase, as well as androgens and sex hormone binding globulin (SHBG). The current analyses include six additional years of follow-up and more than double the number of cases compared to our initial evaluations ,14.n = 29,611), a subset of the study population, ages 32 to 54 y, provided blood and urine samples.The NHSII began in 1989 when 116,430 female registered nurses, ages 25 to 42 y, completed a mailed questionnaire. The participants have been followed biennially to update exposure information and ascertain disease diagnoses. Between 1996 and 1999 the blood cohort  provided blood samples timed within the early follicular  and mid-luteal  phases of the menstrual cycle; the remainder provided single untimed samples. Participants were mailed a sample collection kit with detailed instructions and a questionnaire. Women providing timed samples separated, then froze the follicular plasma 8 to 24 hours after collection, and stored this sample in their home freezer to be shipped with the luteal phase sample. After collection of the luteal sample, or untimed sample, samples were sent with an ice pack via overnight courier to our laboratory where they were processed and have been subsequently stored in continuously monitored liquid nitrogen freezers at ≤ -130°C. Approximately 93% of samples were received within 26 hours of collection. The stability of sex steroids using these blood collection methods has been previously validated [Women who had not used oral contraceptives, been pregnant or breastfed within the past six months  were confirmed through medical record review; the remaining 27 cases (4.3%) were verbally confirmed by the nurse but medical records were not available. Given the high confirmation rate by medical record for breast cancer in this cohort (99%), all cases are included in this analysis. Among the 634 cases, 428 were invasive and 295 were ER+/PR+. A total of 425 women were premenopausal at diagnosis, while 144 were postmenopausal at diagnosis (timed and untimed samples).Participants report disease status on the biennial NHSII questionnaires. Cases included in this analysis were premenopausal at blood collection, reported no prior cancer diagnosis, and were diagnosed with breast cancer through the 2009 biennial questionnaire; 634 breast cancer cases were identified. Breast cancer cases were confirmed through medical record review by a study physician; invasive vs. Cases were diagnosed an average of 6.2 years after blood draw (range 1 month to 13 years). Each case was matched to two controls on age at blood draw (± 2 years), menopausal status at the time of the case's diagnosis, race/ethnicity , luteal day of menstrual cycle , and for each blood collection, fasting status , month and year (± 2 months) and time of day of blood collection (± 2 hours).R, Abbott Diagnostics, Chicago, IL). Batch 5 of DHEAS was assayed at the Mayo Clinic by a solid-phase, chemiluminescent enzyme immunoassay . Masked replicate quality control samples (10% of the samples) were included in each batch to assess coefficients of variation (CVs). Average CVs within batch ranged from 5% for DHEAS to 14% for SHBG, with the exception of progesterone for which the CV was 17% in one batch (CVs for progesterone 7 to 15% in remaining batches). Free estradiol and free testosterone were calculated using the formula described by Södergard et al. [Case-control sets, as well as follicular and luteal samples from the same person, were assayed together. Samples were ordered randomly within a set, and laboratories were masked to both case-control status and repeated samples within woman. Samples were assayed for estrogens and testosterone in five batches at either Quest Diagnostics  by radioimmunoassay preceded by organic extraction and celite chromatography (batches 1 and 2) -24 or thd et al. .2), parity , age at first birth , family history of breast cancer , history of benign breast disease .Lifestyle factors and other exposures were collected on the biennial questionnaire, as well as a questionnaire completed at the time of blood collection. Covariates included in this analysis were (year of data collection): age at menarche (1989), height and weight at age 18 , kg/m2 scale for each hormone by menstrual phase using the extreme Studentized deviate many-outlier procedure [2 transformation.Statistical outliers were identified on the logrocedure , with asrocedure , taking P-values were calculated with the Wald statistic. Heterogeneity by hormone receptor subtype was evaluated using polytomous logistic regression [We used conditional logistic regression to estimate relative risks (RR) and 95% confidence intervals (95% CI) in analyses including all cancers. For subgroup and stratified analyses, we used unconditional logistic regression controlling for matching factors. Multivariable models were adjusted for BMI at age 18, age at menarche, age at first birth and parity, family history of breast cancer, and history of benign breast disease. Tests for trend were conducted using quantile medians and gression , using tgression , to corrgression ,30. We aP < 0.05 was used to define statistical significance. Analyses were conducted in SAS v. 9.2 .All statistical tests are two-sided; Cases and controls were similar with regards to most breast cancer risk factors Table , includiin situ or ER+/PR+ subgroup . After exclusion of women anovulatory in the cycle of blood collection, the OR was somewhat attenuated and no longer statistically significant . No significant associations were observed for luteal estrone, free estradiol or progesterone overall or by subgroup.There were no associations between follicular estradiol, estrone and free estradiol and breast cancer risk overall, or by invasive, up Table . Luteal th vs. 1st OR: 1.4, 95% CI: 1.0 to 2.0, Ptrend = 0.23) and ER+/PR+ disease  . Tests for trend suggested a positive association between DHEAS and risk of ER+/PR+ breast cancer (Ptrend = 0.05), but the effect estimates did not reach statistical significance . SHBG was not associated with breast cancer risk.Testosterone was not associated with breast cancer overall but was associated with invasive  . Associations for follicular free estradiol were similar to those for total estradiol; follicular estrone was not associated with disease in pre- or postmenopausal women. Associations with a doubling of luteal estradiol and free estradiol were somewhat stronger in premenopausal disease as compared to postmenopausal disease . However, findings for luteal estradiol were attenuated after restricting them to women with ovulatory cycles at blood collection  and the positive association in premenopausal women was attenuated after exclusion of women with anovulatory cycles.To examine the importance of menopausal status at diagnosis, we repeated the analyses stratifying by menopausal status at diagnosis . For example, for invasive breast cancer, premenopausal testosterone was not associated with premenopausal disease , but was significantly associated with postmenopausal disease  with a similar pattern for ER+/PR+ cancers, as well as for free testosterone.The associations between premenopausal androgens and breast cancer generally appeared stronger among women who were postmenopausal at diagnosis, but these differences by menopausal status were not statistically significant . Given the positive association between exogenous hormone use and SHBG in this population (P < 0.001) we conducted sub-analyses for SHBG and free testosterone excluding these women; results were similar to the overall results. Further, with only a few exceptions, the associations did not vary significantly by BMI at blood collection, duration of past oral contraceptive (OC) use, menstrual cycle characteristics , or age at blood draw. For follicular free estradiol, no significant association was observed with BMI < 25  and an inverse association was observed for women with BMI ≥ 25 . Similarly, there was no association between progesterone and breast cancer risk in women age < 45 at blood draw  and a suggestively inverse association in women ≥ 45 .Results were similar when we excluded cases diagnosed within two years of blood draw. A small number of women providing untimed samples reported exogenous hormone use at blood collection (Phet > 0.05). For follicular estrone, there was a suggestive positive association for ER+/PR+ disease  and an inverse association for ER-/PR- disease  (Phet = 0.01), although these results were limited by a small number of ER-/PR- cases (60 timed samples).Estradiol, testosterone and DHEAS appeared more strongly associated with ER+/PR+ tumors, as compared to overall associations Tables , 3, 4. HWe conducted analyses cross-classifying follicular and luteal phase estradiol and no clear pattern emerged. For example, for invasive breast cancer ORs were 1.7 (95% CI: 0.8 to 1.8) for high follicular/low luteal estradiol, 1.5 (95% CI: 1.0 to 2.3) for low follicular/high luteal estradiol, and 1.3 (95% CI: 0.9 to 1.9) for high estradiol in both phases, all relative to low estradiol in both phases.We corrected the hormone/breast cancer associations for within-person variability and laboratory measurement error using our prior reproducibility study results ,20. ResuIn this prospective study of premenopausal plasma sex steroids and SHBG and breast cancer risk, we found positive associations between androgens and risk of both invasive and ER+/PR+ breast cancer, as well as luteal estradiol and risk of ER+/PR+ tumors. Several of the associations varied by menopausal status at breast cancer diagnosis. Total and free estradiol levels in the follicular phase were positively, but not significantly, associated with premenopausal invasive and ER+/PR+ disease but significantly inversely associated with postmenopausal disease. Testosterone levels also appeared more strongly associated with postmenopausal breast cancer, although these differences were not statistically different. We observed no significant associations when evaluating all cancers combined.The positive associations between endogenous estrogens and androgens and postmenopausal breast cancer risk are well established in prior epidemiologic studies ,31-33, aet al. [n = 285) in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, found no association with invasive breast cancer risk for either estrone  or estradiol . A prior analysis in a subset of the population included in the current analysis [n = 197 cases; n = 192 premenopausal at diagnosis), found a significant positive association between follicular estradiol with breast cancer overall , that appeared somewhat stronger for invasive and ER+/PR+ disease. Results in the present study, with the addition of 317 timed cases, are attenuated relative to our prior findings, though we continue to see the suggestion of a weak positive association for follicular estradiol and invasive breast cancer diagnosed in premenopausal women. In our prior analysis, we saw no clear association between luteal estradiol and breast cancer; results in the current analysis are suggestive of an association between luteal estradiol and invasive and ER+/PR+ tumors.There is an inherent complexity in measuring estrogens in premenopausal women given the variation in estrogen levels across the menstrual cycle, and prior studies have accounted for this in different ways. The eight prior prospective studies -7,9-13 eet al. , in the n = 370 cases), significant positive associations with breast cancer risk were observed for both testosterone  and DHEAS  [n = 356 cases) [Ptrend = 0.03) and free testosterone , though a weaker, non-significant positive association was observed with DHEAS . In our initial report in the NHSII, positive associations were seen for luteal testosterone , free testosterone  [Ptrend = 0.08) [The association between circulating premenopausal androgens and breast cancer risk has been investigated in seven prospective studies ,10-12,15 = 0.14) , and DHEin situ, hormone receptor status, grade, stage), and risk factor status . Data from our prior reproducibility study [In this updated analysis, with six more years of follow-up, we observed an attenuation of our previously published results, particularly for follicular estradiol. Cases included in the previous analysis were similar to the new cases with regards to case characteristics  is associated with reduced proliferation in ER+ tumors  and postPtrend = 0.23; postmenopausal, OR: 1.7, 95% CI: 1.1 to 2.6, Ptrend = 0.02), although this difference also was not statistically significant (P for interaction > 0.15) [th vs. 1st quartile, overall results for testosterone, OR: 3.3, 95% CI: 1.5 to 7.5, Ptrend = 0.006; among women ≥ 55 at diagnosis, OR: 4.5, 95% CI: 1.6 to 13.0, Ptrend = 0.009) [Our androgen/breast cancer associations appeared stronger in women postmenopausal at diagnosis, although the differences by menopausal status were not statistically significant. This has been evaluated in few other cohorts. In the NYUWHS, the association between testosterone and breast cancer risk was suggestively stronger in women postmenopausal at diagnosis  . Clearly= 0.009) ,43, and Among prior prospective assessments of premenopausal progesterone levels and breast cancer risk, three ,8,10 of Our study has both strengths and limitations. This is the largest prospective study to date, although we had limited power in analyses stratified by menopausal status at diagnosis . Our samples were carefully timed in the menstrual cycle. We used highly specific and sensitive assays, and laboratory CVs were excellent. Additionally, we are among the first to present data for premenopausal hormones and breast cancer by tumor hormone receptor status. Although we are limited to samples collected during one menstrual cycle, a prior reproducibility study provides evidence of reasonable stability across a three-year period (follicular estradiol: 0.38 to DHEAS: 0.86), except for luteal progesterone (ICC = 0.29) . We alsoIn summary, this large, prospective study of circulating premenopausal hormones and breast cancer risk provides some evidence for moderate associations between plasma hormones and breast cancer risk. We provide suggestive evidence of a role for premenopausal estrogens and androgens in postmenopausal disease, with premenopausal estrogens inversely and premenopausal androgens positively associated with postmenopausal breast cancer. However, further work is needed to explore the relationships between premenopausal hormone levels and postmenopausal disease.BMI: body mass index; CI: confidence interval; CVs: coefficients of variation; DHEAS: dehydroepiandrosterone sulfate; EPIC: European Prospective Investigation into Cancer and Nutrition cohort; ER+: estrogen receptor positive; ICC: intraclass correlation coefficients; NHSII: Nurses' Health Study II; NYUWHS: New York University Women's Health Study; OR: odds ratio; PMH: postmenopausal hormones; PR+: progesterone receptor positive; RR: relative risk; SHBG: sex hormone binding globulinThe authors state that they have no competing interests.RTF analyzed and interpreted the data and wrote the manuscript. AHE conceived of the study design, acquired the data, contributed to the interpretation and analysis of data, and critically reviewed the manuscript. DS provided statistical expertise, contributed to the interpretation and analysis of data, and critically reviewed the manuscript. RLB contributed to the interpretation of data and critically reviewed the manuscript. WCW and SEH conceived of the study design, secured funding, acquired the data, contributed to the interpretation and analysis of data, and critically reviewed the manuscript. All authors read and approved the final manuscript.Table S1. Quartiles of premenopausal sex steroids and SHBG and breast cancer risk, by menopausal status at diagnosis: Nurses' Health Study II, 1999 to 2009.Click here for file


The association between striking life events, an important stress and acute anxiety disorder, and the occurrence of primary breast cancer is unclear. The current meta-analysis was designed to assess the relationship between striking life events and primary breast cancer incidence in women.breast cancer, breast tumor, cancer of breast, mammary carcinoma, life events, life change events, case–control studies, case-base studies, cohort study, and cohort analysis and identified a total of 307 papers published from January 1995 to April 2012. Following evaluation of methodological quality with the Downs & Black criteria, seven case–control or cohort studies were selected and the association between striking life events and primary breast cancer incidence in women was measured using random effect or fixed-effect odds ratios combined with 95% confidence interval.Systematic computerized searching of the PubMed, ScienceDirect, Embase, and BMJ databases with the combinations of controlled descriptors from Mesh, including P = 0.003), indicating that women with striking life events were at 1.5-fold greater risk of developing breast cancer. The pooled OR for severe striking life events and breast cancer was 2.07 (95% CI 1.06 - 4.03), indicating that women with severe striking life events were at 2-fold greater risk of developing breast cancer.The seven studies included in the final meta-analysis included 99,807 women. A meta-analysis showed that the pooled OR for striking life events and breast cancer was 1.51 (95% CI 1.15 - 1.97, The current meta-analysis showed significant evidence for a positive association between striking life events and primary breast cancer incidence in women. Primary breast cancer is one of the main public health problems worldwide. Over 1.3 million women are diagnosed annually with primary breast cancer and approximately 458,000 will die from the disease ,3, with An important stress disorder, called striking life events, has been classified as an acute anxiety disorder. This disorder is characterized by aversive anguishing experiences and physiological responses that develop after exposure to stressful life events, including change in marital status, such as separation, divorce, or widowhood; death of a spouse, child, or close relative; a friend’s illness; personal health problems; and change in financial status. This disorder has short-term features, distinguishing it from chronic or delayed-onset stress disorder -13. A prConflicting results regarding the association between stressful life events and breast cancer may be due to differences in subject population, number of subjects, study type, and sample type. These findings suggested the need for a meta-analysis examining the relationship between striking life events and primary breast cancer incidence in women.A systematic review and meta-analysis of primary cohort and case–control studies addressed whether women exposed to stressful life events are at increased risk of developing breast cancer. Hence, the objective was to evaluate the association between striking life events and primary breast cancer in women. The use of human materials was approved by the Peking Union Medical College Hospital Medical Ethics Committee (No.S-406).breast cancer, breast tumor, cancer of breast, mammary carcinoma, life events, life change events, case–control studies, case-base studies, cohort study, and cohort analysis. The reference lists of the retrieved articles were also reviewed to identify additional articles missed by this search.Eligible studies were identified by systematic computerized searching of the PubMed, Science Direct, Embase, and BMJ databases for relevant reports published from January 1995 to April 2012. The database search strategy used combinations of controlled descriptors from Mesh, including The following inclusion criteria were adopted for the studies: (a) type of study – prospective and historical cohort and case–control studies; (b) type of sample – women aged 20 years or older at first occurrence of breast cancer in cohort studies and women with the appearance of first breast cancer in case–control studies; (c) mean follow-up time – a minimum of ten years in cohort studies, with no limit between exposure and diagnosis in case–control studies; (d) type of variables – studies in which the stress variable was measured quantitatively, using a numerical scale, questionnaire, or checklist, with stress assessed by measuring the frequency of exposure and intensity of the event; (e) statistical type and analysis – studies that calculated relative risk (RR) for the first episode of breast cancer in relation to the stress variable, adjusting for confounding factors, including age, use of oral contraceptives, any type of hormone replacement, menopause, alcohol intake, smoking, socioeconomic status, and family history of breast cancer.Studies were excluded if: (a) the articles which not had English version; (b) the articles addressed life style and daily stress; (c) stress was assessed in women with a psychiatric history; or (d) breast cancer recurrence or other diseases of the breast were measured. In addition, review articles and editorials were excluded.The article titles and abstracts were initially evaluated by three reviewers to verify that each primary study addressed the underlying question of the systematic review. The abstracts were grouped into selected versus not selected. The selected articles were retrieved, read in full, and screened for those indexed in more than one source or in another language.In the next phase, data from the selected studies were assigned to an instrument to verify whether they met the inclusion and exclusion criteria, with discrepancies resolved by discussion and consensus. Studies lacking a consensus for inclusion were analyzed by a fourth reviewer.Data from the case–control and cohort studies were assigned to a structured form, which included the last name of the first author, the year of publication, country of origin, type of study, adjustment for confounding factors, and odds ratios (ORs) and 95% confidence interval (CI). The data were reviewed by the four reviewers.P < 0.05 as described [p < 0.05.Statistical analysis was performed preferentially using Cochrane Review Manager Software (version 5.1). For categorical variables, weighted risk ratios and their 95% CIs were calculated using RevMan 5.1 software . Resultsescribed . A fixedThe search strategy identified 307 titles and abstracts. Of these, 284 were excluded after reading the titles and abstracts. Our inclusion and exclusion criteria were applied to the remaining 23 articles describing case–control and cohort studies. A higher intensity of psychological events resulting from severe, major life, stressful, and overall life events were described and classified to calculate the ORs in these articles.Of the 23 articles, seven, containing sufficient data, were included in our meta-analysis Table . Most ofOf the seven studies included in our meta-analysis, four were case–control studies -20 and tThe remaining 16 identified articles not included in our meta-analysis were examined. Risk factors related to psychiatric, psychological, and social disorders have been described . In addiORs for primary breast cancer occurrence related to striking life events are shown in Table p < 0.00001, Figure p = 0.003). As shown in forest plots, rhombus shapes that represent the studied variable appeared on the right side of invalid line, suggesting the studied variable was a strong pathogenic factor. Interestingly, the rhombus shape suggested that the variable had not been characterized. The OR was far from the midline and differed markedly from other studies. The weight ratio depended on the model used for analysis, with a minimum weight box displayed in the forest plots. The maximal weight box did not represent those reported previously and included the highest number of samples , although others had difference perspectives . Although both were prospective cohort studies, subject age was limited from 50 to 79 years, with no specific age limitations.The consistency of the seven studies was poor and varied markedly , suggesting that individuals with severe striking life events would be at two-fold greater risk of developing breast cancer than individuals without these severe striking life events . A forest plot showed a diamond shape, with striking life events on the right side of the invalid line, suggesting that striking life events were strongly associated with the incidence of primary breast cancer. However, although our results indicated that striking life events were positively associated with breast cancer occurrence, the OR was not high and the lower limit of the 95% CI was only 1.15.More importantly, our meta-analysis found that women with a severe degree of striking life events had an OR of 2.07 (95% CI 1.06 - 4.03) of developing breast cancer, suggesting that more severe striking life events contribute to a higher risk of primary breast cancer in women. Our findings suggest that psychological treatment of striking life events may reduce breast cancer occurrence. Discrepancies over the definition of striking life events would change the association between these events and the risk of developing breast cancer.Although studies have yielded contradictory results on the association between stress and breast cancer development, our results confirm that high-intensity stress has a borderline association with the development of breast cancer. However, relative to the findings in most of studies that stress can increase the risk of breast cancer, whether those women who had the most aggressive form of breast cancer also had the highest stress levels was unclear, and there is no real way to tell how much stress the women were under before their diagnosis of breast cancer. Obviously, based on that it’s not clear what’s driving the association between stress and breast cancer development, future studies are necessary to elucidate this relationship.YL and CW contributed equally to this work. Both designed the study, collected and analyzed data, and wrote the manuscript. YZ, XH, and LP participated in the collecting and analyzing data. GS and KW performed statistical analyses. QS conceived the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.


BRCA1 or BRCA2 which however, explain only small number of breast and ovarian cancer cases. Data suggest that majority breast and ovarian cancers are caused by low or moderate penetrance gene mutations. Identification of such mutations provides cancer risk assessment and will help in prophylactic, early diagnosis and treatment.Breast and ovarian cancers belong to the most common malignancies diagnosed in women. The major inherited susceptibilities to breast and ovarian cancers are germline mutations in either Breast Cancer Association Consortium (BCAC), Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and Ovarian Cancer Association Consortium (OCAC) which are forums of investigators from centers over the world, including International Hereditary Cancer Center in Szczecin. The aim of these consortia’s is to combine data from many studies, to provide a reliable assessment of the breast and ovarian cancer risks associated with different genetic and environmental factors, and to identify potential modifiers of cancer risk in carriers of BRCA1 and BRCA2 mutation.In 2005 three international multidisciplinary consortia have been initiated: BCAC includes 54 centers (table rs table  over thehttp://www.srl.cam.ac.uk/consortia/bcac/pubs/pubs.html).Currently, BCAC database includs demographic, clinical and epidemiological data from 73,000 breast cancer patients and 80,000 unaffected women. Up to now, 125 genetic alterations (SNPs) localized in different genes have been examined. These SNPs have been selected based on its positive association with breast cancer risk detected in preliminary analyses or Genome-Wide Association Study (GWAS). Results have been presented in 12 manuscripts published in high impact journals, e.g. J Natl Cancer Inst, Nature and Nat Genet .Results have been presented in 9 manuscripts published in high impact journals, e.g. Am. J Hum Genet, Hum Mol Genet (Six SNPs have been found to be associated with breast cancer risk table .OCAC includes 24 centers over the world and focuses on identification of genes that may be related to the risk of ovarian cancer (table er table .http://www.srl.cam.ac.uk/consortia/ocac/pubs/pubs.html).Currently, OCAC database includs demographic, clinical and epidemiological data from 22,000 ovarian cancer patients and 18,000 unaffected women. Several genetic alterations (SNPs) localized in different genes have been examined. Results have been presented in 13 manuscripts published in high impact journals, e.g. Nat Genet, Cancer Res, In J Cancer (Seven SNPs have been found to be associated with ovarian cancer risk overall or by clinical and pathological characteristics table .