Experimental evidence suggests a protective role for circulating 25-hydroxyvitamin D (25(OH)D) in breast cancer development, but the results of epidemiological studies have been inconsistent.We conducted a case-control study nested within two prospective cohorts, the New York University Women's Health Study and the Northern Sweden Mammary Screening Cohort. Blood samples were collected at enrollment, and women were followed up for breast cancer ascertainment. In total, 1,585 incident breast cancer cases were individually-matched to 2,940 controls. Of these subjects, 678 cases and 1,208 controls contributed two repeat blood samples, at least one year apart. Circulating levels of 25(OH)D were measured, and multivariate odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression.ptrend = 0.30). The temporal reliability of 25(OH)D measured in repeat blood samples was high (intraclass correlation coefficients for season-adjusted 25(OH)D > 0.70). An inverse association between 25(OH)D levels and breast cancer risk was observed among women who were ≤ 45 years of age  or premenopausal at enrollment .No association was observed between circulating levels of 25(OH)D and overall breast cancer risk D levels were not associated with breast cancer risk overall, although we could not exclude the possibility of a protective effect in younger women. Recommendations regarding vitamin D supplementation should be based on considerations other than breast cancer prevention. Vitame breast . Circulae breast  and has east [2D . Vitamineast [2D ,7.2 [The results of epidemiologic studies examining the association between circulating 25(OH)D levels and breast cancer risk have been inconsistent. Seven prospective studies reported no association overall -14, wher2 .Recent reviews have concluded that there is insufficient evidence to recommend vitamin D supplementation for the prevention of breast cancer but that additional research in humans is needed ,18,19. TDescriptions of the NYUWHS and the NSMSC have been provided previously ,21. BrieFor the NYUWHS, incident cases of invasive breast cancer were identified by mailed questionnaires or follow-up telephone interviews every 2 to 4 years after 1991, supplemented by linkages to state cancer registries in New York, New Jersey, and Florida and the US National Death Index. Medical records were reviewed to confirm self-reported cases. Using a capture-recapture analysis, we estimated that combining active and cancer registry-based follow-up resulted in a breast cancer ascertainment rate of 95% . For theEach case was matched to two controls who were selected from the respective cohort by using incidence-density sampling. Matching factors included age at enrollment in the study (± 6 months), date of enrollment/first blood donation , and number  and dates of subsequent blood donations. For the NYUWHS, matching factors also included menopausal status  at enrollment and race/ethnicity . The vast majority of women in the NSMSC were Caucasian. Initially, women were not matched on menopausal status in this cohort; however, 88% of the cases had at least one control matching on this factor . In total, 2,940 controls were included in the final analysis .For 678 matched sets (413 in NYUWHS and 265 in NSMSC), two blood samples were analyzed for 25(OH)D. For participants who had donated more than two blood samples, the first and last samples collected before the relevant case's diagnosis were selected.3 and 25-hydroxyvitamin D2. All samples, including repeat samples, from a case and her matched controls were analyzed together in the same laboratory batch to minimize laboratory variability. Laboratory personnel were blinded to case-control status of the study samples. Samples from quality-control pools  were masked and inserted randomly in the batches. The intra- and inter-batch coefficients of variation (CVs) were 9.5% and 11.4%, respectively, for NYUWHS and 7.4% and 9.0%, respectively, for NSMSC.Circulating 25(OH)D was measured by Heartland Assays, Inc.  by using a direct competitive chemiluminescence immunoassay by using the DiaSorin LIAISON platform  . The assEstrone was measured by double-antibody radioimmunoassay with reagents from Diagnostic System Laboratories  at the Laboratory for Hormone Analyses at the International Agency for Research on Cancer, France, for post-menopausal women who were not using hormone replacement therapy (HRT). Intra- and inter-batch CVs were 6.7% and 12.6%, respectively ,25.We examined the temporal reliability of circulating 25(OH)D by using the intraclass correlation coefficient (ICC). In addition to calculating the overall ICC, we calculated ICCs according to time (years) between sample donations and according to season, for each cohort separately.2-transformed to reduce departure from the normal distribution and were included in the model in one of three ways. First, we computed season-adjusted residuals to take into account the known variations of 25(OH)D with season [Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations of subject characteristics and circulating 25(OH)D with risk of breast cancer. We conducted analyses separately for each cohort as well as combining them. Because there was no evidence of cohort heterogeneity, most results are presented for the combined cohorts. 25(OH)D concentrations were logh season . For each season . This reh season . These aIn each of the two cohorts, multivariate linear regression analyses were conducted among the controls to explore associations of potential confounders with 25(OH)D. BMI was found to be a negative predictor of 25(OH)D, whereas multivitamin supplement use and past use of HRT were positive predictors in both cohorts. Caucasian race and physical activity were also positive predictors of 25(OH)D in the NYUWHS, and alcohol consumption was a negative predictor in the NSMSC. Covariates in the final multivariate models included the following known breast cancer risk factors: age at menarche (continuous), age at first birth/parity , family history of breast cancer (no or yes), BMI (continuous), past HRT use (never or ever), and alcohol consumption. It is debatable whether to control for outdoor physical activity and multivitamin use, which have been associated with higher levels of circulating 25(OH)D ,28 sinceWe performed multiple imputation by using fully conditional specification  for the P value of less than 0.05 was considered statistically significant.We conducted stratified analyses by using conditional logistic regression for the following variables: age at enrollment, lag-time between blood donation and diagnosis, and estrogen receptor (ER) status. In order not to lose the matched sets in which a case and her controls were discordant, unconditional logistic regression, adjusted for cohort and age at blood sampling, was performed for the following variables: menopausal status, BMI, circulating estrone levels , and insulin-like growth factor 1 (IGF-I) levels. Tertiles were used for the IGF-1 analysis because of the limited number of women for whom IGF-1 had been measured (193 cases and 269 controls from the NYUWHS only). Finally, we performed an analysis limited to Caucasians (90% of subjects). All significance testing was two-sided, and a Descriptive statistics for the breast cancer cases and their matched controls are presented in Table n = 113 subjects) and 0.63 (95% CI = 0.51 to 0.74) for samples collected between 8 and 11.7 years apart (n = 106 subjects). For both cohorts, the ICC was substantially lower when one sample was donated in the winter and the other one in the summer months .For women who donated more than one blood sample, the average time between sample donations was 2.1 years in the NYUWHS and 4.4 years in the NSMSC. Overall, the temporal reliability of 25(OH)D was good  and improved for NSMSC when season-adjusted residuals were used   or within either cohort. Results with 25(OH)D on the continuous scale were similar. Adjusting for physical activity and multivitamin use, in addition to the other confounders, did not materially affect the results .Table Ptrend = 0.10 for concentrations of at least 75 versus less than 50 nmol/L), but no such association was observed in the NSMSC.In analyses using pre-specified categories of circulating 25(OH)D (not adjusted for season), we observed no association with breast cancer risk for samples taken either in the winter, when more than half of the subjects had levels below 50 nmol/L, or in the summer, when more of the subjects had levels more than 75 nmol/L Table . A suggeQ5-Q1 = 0.67, 95% CI = 0.48 to 0.92, Ptrend = 0.03) but not among those who were post-menopausal . A similar protective effect was observed for women who were not more than 45 years old at blood donation . There was no evidence of effect modification by ER status of the tumor, lag-time between blood sampling and diagnosis, BMI, or circulating estrone levels. Results of the analysis limited to Caucasians were similar to those of the analysis that included all subjects.Table P = 0.61).When we examined the association of 25(OH)D with breast cancer risk by IGF-1 levels at baseline, the ORs for the third tertile were 0.62 (95% CI = 0.30 to 1.28) in the below-median stratum and 0.79 (95% CI = 0.39 to 1.62) in the above-median stratum. The test for interaction between IGF-1 and 25(OH)D on the continuous scale was not statistically significant D and breast cancer risk overall. We observed an inverse association between 25(OH)D and breast cancer risk in the subgroups of women who were not more than 45 years old or pre-menopausal at blood donation, although the test for interaction was significant only for menopausal status. Because of substantial overlap, we were not able to sort out whether younger age or pre-menopausal status was driving the association.Epidemiologic studies on vitamin D and breast cancer risk have been reviewed recently ,32. TradPtrend = 0.51). The French E3N cohort, though, reported a significant inverse association in women who were younger (< 53 years old) at blood donation, results consistent with ours, and also observed a non-significant protective association in the smaller subgroup of women who were pre-menopausal at diagnosis. The investigators suggested that vitamin D may act by inhibiting the tumor growth-stimulating effects of IGF-1 [In regard to younger and pre-menopausal women, data from prospective studies are more limited since eligibility in some of the largest studies was restricted to older women -10. Amonof IGF-1 . BecauseA protective effect of vitamin D on breast cancer would also be expected to be stronger in pre-menopausal women if vitamin D acts by inhibiting estrogen-stimulated breast cell proliferation , since eToo few subjects had very high concentrations of 25(OH)D for us to be able to examine the association of concentrations of at least 100 nmol/L with breast cancer risk. However, the lack of dose response in the less than 50 to at least 75 nmol/L range Table  suggestsSeveral factors that are associated with breast cancer risk are also associated with circulating 25(OH)D and therefore could confound the 25(OH)D-breast cancer association . Dark skWe used residuals obtained by local regression to take into account seasonal variation. This method has been used only rarely in studies of 25(OH)D , althougStrengths of this study include its prospective design, inclusion of two cohorts with different diet and sun exposure, and large sample size. This is also the first study to include repeat blood samples on a large number of women. The repeat samples enabled us to assess temporal reproducibility and gave an indication of the potential impact of ignoring seasonal variation when studying the association of circulating vitamin D with disease risk. The lower ICCs observed when samples were collected in different seasons, compared with the same season, highlight the importance of taking season into account in the study design or analysis or both, as other studies have concluded . The ICCThis large prospective study does not support a relationship between circulating 25(OH)D and risk of breast cancer, except possibly in younger women. These results add to a growing body of evidence from prospective studies and randomized trials that suggests that higher vitamin D levels do not reduce breast cancer risk. Recommendations in regard to vitamin D supplementation should be based on considerations other than breast cancer prevention, such as bone health.25(OH)D: 25-hydroxyvitamin D; BMI: body mass index; CI: confidence interval; CV: coefficient of variation; ER: estrogen receptor; HRT: hormone replacement therapy; ICC: intraclass correlation coefficient; IGF-I: insulin-like growth factor 1; MET: metabolic equivalent of task; MSC: Mammary Screening Cohort; NSMSC: Northern Sweden Mammary Screening Cohort; NYUWHS: New York University Women's Health Study; OR: odds ratio; VDR: vitamin D receptor.The authors declare that they have no competing interests.SS interpreted the data and drafted the manuscript. YA performed the statistical analysis and critically reviewed the manuscript. PL, AA, YC, GH, EL, PT, and RS critically reviewed the manuscript. KK conceived of the study design, interpreted the data, and critically reviewed the manuscript. RH performed the 25(OH)D assay and critically reviewed the manuscript. TC interpreted the data and critically reviewed the manuscript. SR performed the estrone analyses and critically reviewed the manuscript. AZ-J secured funding, conceived of the study design, interpreted the data, and drafted the manuscript. All authors read and approved the final manuscript.


Breast cancer today has many established risk factors, both genetic and environmental, but these risk factors by themselves explain only part of the total cancer incidence. We have investigated potential interactions between certain known genetic and phenotypic risk factors, specifically nine single nucleotide polymorphisms (SNPs) and height, body mass index (BMI) and hormone replacement therapy (HRT).We analyzed samples from three different study populations: two prospectively followed Swedish cohorts and one Icelandic case–control study. Totally 2884 invasive breast cancer cases and 4508 controls were analysed in the study. Genotypes were determined using Mass spectrometry-Maldi-TOF and phenotypic variables were derived from measurements and/or questionnaires. Odds Ratios and 95% confidence intervals were calculated using unconditional logistic regression with the inclusion of an interaction term in the logistic regression model.One SNP (rs851987 in ESR1) tended to interact with height, with an increasingly protective effect of the major allele in taller women (p = 0.007) and rs13281615 (on 8q24) tended to confer risk only in non users of HRT (p-for interaction = 0.03). There were no significant interactions after correction for multiple testing.We conclude that much larger sample sets would be necessary to demonstrate interactions between low-risk genetic polymorphisms and the phenotypic variables height, BMI and HRT on the risk for breast cancer. However the present hypothesis-generating study has identified tendencies that would be of interest to evaluate for gene-environment interactions in independent materials. Genome wide association studies (GWAS), have discovered several new genetic polymorphisms affecting breast cancer risk -3. Even The phenotypic variables height, body mass index (BMI) and use of hormone replacement therapy (HRT) reflect to varying degrees genetic background and environmental exposure. Both height and BMI have previously been shown to associate with breast cancer ,7. IncreAlthough there have been investigations on gene-environment interactions in breast cancer , this arThe aim of this study was to investigate if height, BMI and HRT modify the genetic predisposition to breast cancer conferred by reported low-risk polymorphisms. For this purpose we had access to two well defined Swedish population based cohorts as well as an Icelandic hospital based case control study, altogether 7738 samples (3016 cases and 4722 controls).The samples originate from two Swedish independent population based cohorts; the Malmö Diet and Cancer Study (MDCS) from southern Sweden and the North Sweden Health and Disease Study (NSHDS), together comprising 2410 incident cases and 3829 controls. The third sample collection was an Icelandic population-based case control study including 866 cases and 948 controls. Written informed consent was retrieved from all women prior to donating their samples. All cohorts have been described previously  and are st of December 2007, 730 incident cases of invasive breast cancer were identified among MDCS participants. They were matched to 1460 controls from the same cohort according to sex, age (+/− 6 months), and date of sampling at baseline (+/− 2 months). Median age at breast cancer diagnosis was 65 years (range 45–84). Thirty-three cases and 65 controls were ≤50 years of age at time of diagnosis.The Malmö Diet and Cancer Study (MDCS) is a prospective cohort study initiated in 1991. Totally it comprises 17035 female residents of Malmö Sweden recruited between 1991 and 1996 ,13. By lThe MDCS and the present analyses were approved by the Ethical Committee at Lund University .st, 2008, 1680 prospective cases of invasive breast cancer  were identified. They were matched to 2314 controls by sex, age (+/− 6 months), and date of sampling at baseline (+/− 2 months), (474 cases and 606 controls ≤50 years of age). Information on HRT use was available for 1420 of these cases.The Northern Sweden Health and Disease Study (NSHDS) include the Västerbotten Intervention Program (VIP), and the Mammography Screening Program (MSP), initiated in 1985 and 1995 respectively. Participants in the VIP are screened at 40, 50 and 60 years of age and mammography screening and blood sampling is performed among women between 50 and 69 years of age . ThroughThe NSHDS and the present analyses were approved by the Ethical Committee at Umeå University (Dnr: 2010-147-132 and 07–141).st of December 2007. Totally 866 cases  and 948 controls  had DNA available and were eligible to us.The Icelandic samples were collected between 1998 and 2006 and represent 45–77% of all Icelandic women with invasive breast cancer diagnosed between 1957 and 2007. The rate of participation varied somewhat depending on the year of diagnosis and was highest between 1999 and 2003 (77%). Unmatched controls were collected between 2000 and 2004, either from women who participated in the population-based cervical or breast cancer screening program and found free of breast cancer or from older women in retirement homes who had not been diagnosed with breast cancer, to generally reflect the ages of the cases. By linkage to the Icelandic cancer registry in 2008 we identified cases diagnosed before 31The use of these samples was approved by the data protection law (200605037), and the Icelandic Science Ethics Committee (VSNb2006050001/03-16 and VSNb2005070008/03-16).Participants in both Swedish cohorts completed a questionnaire providing information about current medication at the time of recruitment. Participants in the NSHDS also provided information about height and weight while a trained nurse at the study centre measured height and weight, for participants in MDCS .2.The Icelandic women answered questions about height, weight and HRT use when they attended the Detection Cancer Clinic  at the Icelandic Cancer Society. The women answered questions at least every tenth year and the most recent answers were used in the study. For the Icelandic cases only data collected prior to breast cancer diagnosis was used. BMI for all participants was calculated as kg/mst 2007 were initially included in the study [All loci identified by GWAS to be associated with breast cancer and published before June 31he study . Two SNPhe study  were alsEleven SNPs, combined by the SEQUENOM MassARRAY® Designer software in a single multiplex assay were analyzed on a MALDI-TOF mass spectrometer (SEQUENOM MassArray) using standard iPLEX reagents and protocol (SEQUENOM) and 10 ng DNA as PCR template. Primer sets were from Metabion .SNPs rs2981582 and rs1045485 were analyzed by a separate TaqMan® “assay by design” genotyping assay on a 7900HT instrument, using Master mix No UNG from Applied Biosystems according to the manufacturer’s instructions. Reaction mixtures (6μL) containing 2 ng of DNA template, primers   were subjected to two minutes at 50°C and ten minutes at 95°C, followed by 50 PCR cycles of 95°C for 15 seconds and 60°C for one minute.Three percent of the samples from NSHDS and five percent of the Icelandic samples were included as blinded duplicates for quality control purposes.Individual samples producing results in < 80% of the assays were excluded prior to statistical analyses to eliminate samples with low-quality DNA. Genotype data from control samples were tested for consistency with Hardy-Weinberg equilibrium (HWE) using a χ² p-value cutpoint of 0.001. Unconditional logistic regression was used to measure the independent association between each genotype and breast cancer, with Odds ratios and 95% confidence intervals (CI) estimated for each genotype. Per allele OR (p-trend) was calculated using 0, 1 or 2 copies of the minor allele (a) as a continuous variable. OR and 95% CI were calculated between each phenotypic variable  and risk for breast cancer, these results were also age adjusted. Data was then stratified into tertiles according to height , and into subcategories of BMI according to the WHO guidelines . For HRT subjects data was stratified according to reported “non use” and “current use”. The current users were further divided into users of only estrogen or combined hormones. OR and 95%CI were calculated for each variable  and risk for breast cancer.A p-value for interaction was estimated for each pair of genotype/phenotype and a value of less than 0.05 was considered statistically significant. As adjustment for multiple comparisons this value was divided by the number of interaction analyses, according to Bonferroni, (8 SNPs x 3 =24) and the new significance threshold was 0.002. All results were adjusted for age and study population.Of the initial 7738 samples selected for the project 7392 (95.5%) were successfully retrieved and genotyped for ≥ 80% of the SNPs. All SNPs had a genotyping success rate > 94%, with an average of 98.0%. Results of all 200 analyses performed on duplicate samples were in 100% concordance.Per allele OR for each independent SNP is presented in Figure Three of the SNPs in TOX3  exhibited linkage (results not shown) as has previously been reported [Independent analysis of risk association with each phenotypic variable  within the entire study population revealed a significantly increased risk of breast cancer for individuals >162 cm compared to shorter women, this association was weakened following age adjustment. No statistical significant correlation between BMI and risk for breast cancer was found in this population. For current use vs. non-use of HRT, a significantly increased risk was seen for users, OR (95% CI) 1.24 (1.08-1.42), which remained after adjustment for age , one SNP (rs851987) in ESR1 had a p-interaction = 0.007 with height, with an increasingly protective effect of the major allele in taller women, but it did not pass the threshold for multiple comparisons p = 0.002)  was significant only in non users of HRT with a p-for interaction of 0.03, indicating borderline significance  (Table -5) compared to a per-allele OR (95%CI) of 1.08 (1.01-1.15) (p-trend = 0.03) in all users. The SNP is situated in region 8q24 that contains no known genes but is in close proximity to FAM84B (coding for a breast cancer membrane associated protein) and the proto-oncogene MYC. The 8q24 locus has previously been reported to associate with other types of cancer in addition to breast cancer [One SNP (rs13281615 in 8q4) first described by Easton et al. [t cancer  and to bt cancer .Since the first GWAS on breast cancer was published in 2007 several replication and interaction studies of varying sizes have been published -27. In 2Considering demographic traits, participants in the MDCS have a slightly higher socioeconomic status than the general population, but as this selection is the same for the study base from which cases and controls are derived, it should not affect the validity of our study . MDCS paOur evaluation of genetic predisposition for breast cancer in relation to three different environmental risk factors found no significant gene-environment interactions. We did find tendencies for certain SNPs to exert an effect on breast cancer risk only in women with certain phenotypes. In particular the potential interaction between height and rs851987 in ESR1 in relation to breast cancer risk could merit further investigation. However, independent studies with many more cases would be needed to verify this finding.The authors declare that they have no competing interests.SH had full access to all the data and in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. SH and JC are the principal investigators of the study and were responsible for the planning of the study. JD participated in the conception and design of the study and contributed with supervising, funding, and administrative support. MILI participated in designing the study. JEE and PL contributed samples to the study. SH and MILI performed all genetic analysis. SH analyzed the data . SH, JC, JD, SB and JM interpreted the data. SH performed the literature search. SH wrote; JC, JD, JM revised; MILI, SB, JEE, and PL reviewed the paper and contributed suggestions for improvement that were implemented. All authors approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/12/17/prepub


Spouses and partners (“partners”) of women at-risk for (“previvors”) and surviving with hereditary breast/ovarian cancer are a primary source of support within their families. Yet, little is known about partners’ needs for psychoeducational intervention to enhance their cancer risk knowledge, coping, and support role functioning.To determine the type and range of need for psychoeducational intervention among partners of hereditary breast cancer previving and surviving women, and to understand the potential role of the Internet and other communication channels in meeting that need.We conducted a secondary data analysis on partners’ needs that were originally assessed via an online community-based organization devoted to hereditary breast cancer. Partners’ demographic characteristics, need for psychoeducation, and likelihood of using various communication channels were assessed along with other constructs. Analyses examined commonly-occurring clusters of likely intervention use and by communication channel.2,132 = 9.15, P < .001).Partners (n =143) endorsed a moderately high level of need for psychoeducation and did so across multiple content areas . Factor analysis identified three commonly-preferred communication channels: 1) self-help materials, 2) online interactions, and 3) interpersonal interactions. A cluster analysis among these factors identified three groups of partners based on their likelihood of psychoeducational intervention use . In a covariate-adjusted MANOVA, moderate and high intervention users reported significantly greater need for psychoeducation compared to low users (FA majority of assessed partners perceived a need for psychoeducational interventions surrounding hereditary breast cancer risk. Internet-based, interactive resources may be an efficient mechanism to reach large numbers of partners with tailored content. Research is warranted to inform the design and deployment of these resources to ensure quality and high impact, and ultimately to examine ways to integrate these resources into clinical care. BRCA1 or BRCA2 (BRCA1/2) [BRCA1/2 mutation face up to an 85% lifetime risk for breast cancer and up to a 65% lifetime risk for ovarian cancer [BRCA1/2 mutation is identified in an individual, there is a 50% chance that first-degree relatives  have also inherited the mutation and may therefore face increased risks for cancer [BRCA1/2 mutation, breast cancer screening consists of mammography and breast magnetic resonance imaging starting at age 25 years [Every year in the United States, breast cancer is diagnosed in over 200,000 women : internaBRCA1/2) ,5. Womenn cancer ,5. Moreo25 years ,8. Breas25 years ,8. Proph25 years -11.BRCA1/2 mutations is a well-established component of the identification and management of hereditary breast and ovarian cancer syndrome among those who are at risk [BRCA1/2 test-related decisions are often discussed with their partners, and most women feel supported by their partners [Genetic counseling and testing for  at risk ,13. Thou at risk ,15. Indepartners ,17. Howepartners ,18,19. Bpartners .BRCA1/2 mutations and their family members, it is imperative that adjunctive models of psychoeducational support be offered outside of the health care setting to better meet the needs of women who are at risk of familial breast cancer and their partners. Psychoeducation, which is a well-known intervention model for providing informational and psychosocial support for chronically ill women and their partners [In light of this, it is critical that families facing the risk of hereditary breast and ovarian cancer be adequately supported and empowered, both medically and psychosocially, before and after learning about their disease risk -21. Givepartners -25, may The Internet is a primary resource for those seeking information and support about cancer ,27. InteBRCA1/2 genetic counseling and testing and options after testing, establishing and maintaining open communication within the partnership about hereditary breast cancer and related concerns, and performing supportive behaviors and managing stress and uncertainty in the face of previvors’ and survivors’ hereditary cancer risk and risk of cancer in the family [Partners of previvors and survivors, especially male partners, may prefer Internet-based resources to face-to-face psychoeducation because they offer anonymity surrounding sensitive topics and emotional experiences, and provide direct access to needed information -33. It ie family ,34. To dwww.facingourrisk.org) is the leading site specifically devoted to the community of hereditary breast cancer previvors and survivors. It is expected that the findings of this assessment would be used to inform the planning and development of new interactive and Internet-based psychoeducational interventions for this target population.To address this gap, we report on the results of an online community needs assessment conducted with this target population. Specifically, the assessment, which was conducted by Facing Our Risk of Cancer Empowered (FORCE), sought to describe the need for psychoeducational interventions that could be offered via the Internet and other communication channels among partners of hereditary breast cancer previvors and survivors. FORCE maintains in-person, telephone, and Web-based programs that provide information, peer support, resources, and a community tailored to individuals at high genetic risk. Its website 3 not-for-profit organization devoted to raising awareness about hereditary breast and ovarian cancer and BRCA1/2 genetic mutations or a family history of breast cancer, or who had a BRCA1/2-linked cancer. Respondents were asked to affirm the following statement prior to completing the survey:The needs assessment sample consisted of 143 partners who responded to the Web-based survey. The anonymous survey took approximately 10 minutes to complete, and no personally identifying information was collected. The survey was made available via the FORCE website homepage from November 2010 to February 2011. The heading for the survey targeted spouses or partners of women who were at risk for hereditary breast cancer, defined specifically as having Participation in this survey is limited to spouses and partners  of women with a BRCA mutation or family history of cancer. By continuing, you are agreeing with the above terms and volunteering to participate. If you do not wish to participate, please close your browser or exit at any time.The protocol for this secondary data analysis was reviewed and approved by the Institutional Review Board at Georgetown University.The demographic characteristics assessed were respondent age, gender, race/ethnicity, and highest level of education attained. In addition, 2 items assessed whether respondents had any children, and whether they had any female children.BRCA1/2 genetic mutation, and had surgery to remove her breasts  or ovaries , or for breast reconstruction. Based on these items, 3 variables were created to indicate whether each respondent’s partner had (1) a diagnosis of breast cancer, (2) received BRCA1/2 genetic testing, and (3) undergone any of the 3 surgery types we inquired about.Clinical characteristics assessed included whether respondents’ partners had a diagnosis of breast cancer, had been tested for a Because FORCE has a large and active online community, respondents’ use of technology was presumed to be moderately high. However, to evaluate this presumption among partners, Internet use was formally assessed using 2 items. The first item asked “How often do you go online to access the Internet?” with response options ranging on a 4-point Likert-type scale from never to very often (more than once/week). The second item asked “When you go online, where do you primarily access the Internet from?” with response options including home, work, Internet café, family members or friends’ home, and other. Email use was assessed by asking “Do you have an email address for your personal use?” Respondents were dichotomized as high Internet users if they accessed the Internet very often, did so from home, and had an email address for personal use; all other participants were categorized as low Internet users .We used 7 items to assess respondents’ preferences for psychoeducational content. From the perspective of partners, topics queried included understanding my role/knowing what to expect, communicating with my spouse/partner, helping my spouse/partner make decisions, communicating with adult relatives, communicating with children, intimacy after diagnosis or surgery, and speaking with other spouses/partners going through a similar situation. Response options for each item were yes, no, and I don’t know. Principal components factor analysis of these 7 items confirmed a single-factor solution . Items were analyzed individually and an overall content score was also created by summing responses to the 3 items where yes, I don’t know, and no were again assigned a value of 2, 1, or 0, respectively . As such, higher scores reflected stronger preferences for more psychoeducational content.We used 11 items to assess respondents’ likelihood of using psychoeducational resources offered through the following communication channels: regular mail, toll-free telephone line, email, national and local FORCE meetings, printed booklet or guide, periodic newsletter, expert teleconference or webinar, video or DVD, Web-based message board, and Web-based chat. Items were preceded by a statement instructing respondents to indicate how likely they would be the find the following resources and information for spouses or partners to be useful. Response options were based on a 7-point Likert-type scale with anchors at values of 1  and 7 (very likely).self-help subscale consisted of 4 items assessing the likelihood of using a printed booklet or guide, newsletter, and materials delivered via postal mail and email . The online interaction subscale consisted of 4 items assessing the likelihood of using resources offered via a Web-based message board or chat, embedded video or DVD, and expert webinar or teleconference . Finally, the interpersonal interaction subscale consisted of 3 items assessing the likelihood of using in-person resources offered via national and local FORCE meetings, as well as a telephone hotline .These 11 items were subsequently factor-analyzed to empirically derive subscales with eigenvalues >1: we identified 3 subscales. Subscale scores were then created by averaging responses to the items loading on each subscale to ensure that all subscales were based on a common underlying metric. The need for more resources or support, (2) wanted more resources or support, and (3) would use more resources or support. Response options for each item were yes, no, and I don’t know. An overall score was created by summing responses to the 3 items with assigned values of yes (2), I don’t know (1), and no (0) : higher scores reflect greater need.We used 3 items to assess respondents’ perceived need for psychoeducation. The items were introduced to respondents with a brief description of the informational resources that could be made available through FORCE, followed by items assessing whether respondents (1) perceived a Statistical analyses were conducted in several steps. First, we used descriptive statistics to characterize the study sample and describe their preferences and perceived needs for psychoeducation. Second, we subjected communication channel subscale scores  to a hierarchical cluster analysis to determine first-order groupings of respondents based on their self-reported likelihood of using resources offered through specific communication channels. We selected the unweighted pair group method using arithmetic averages, which defines clusters based on the average pairwise proximities between clusters of all pairs of observations ,39. ThisF tests, t tests, χ2 tests) examined relationships between demographic and clinical characteristics, and perceived need for psychoeducation, across user clusters. Finally, a multivariate analysis of variance (MANOVA) examined variability in need across users, adjusting for participant demographic characteristics  as covariates [Subsequently, bivariate tests . To account for missing data on these variables, we used a single regression-based imputation method, imputing predicted values based on demographic characteristics including age, gender, and race/ethnicity. This method has been shown to be adequate for imputing missing values when the proportion of participants with missing data is relatively low, as was the case for our sample [Prior to analyses, we studied patterns of missing data in focal variables, including preferred communication channels, psychoeducational content, and need for support. While data were missing for any one of these variables for only a few participants , Little’s  χ2 test r sample .BRCA1/2 genetic testing previously (91.6%) and some form of surgery (78.3%). Most respondents (55%) were categorized as high-level Internet users, and their need for psychoeducation was moderately high . Descriptive information for partners’ preferred psychoeducational content is displayed in Characteristics of the respondent sample (n = 143) are displayed in P < .05 using the Tukey post hoc adjustment, confirming cluster validity [The cluster analysis of communication channels identified 3 distinct groups of partners based on their likelihood of using psychoeducational interventions . The smavalidity .F2,142 = 13.3, P < .001).Participants in the low-, moderate-, and high-use clusters did not significantly differ based on demographics, family characteristics, clinical characteristics, or Internet use. Significant differences in partners’ perceived need for psychoeducation were evident across clusters. Specifically, those in the moderate-use and high-use clusters reported significantly greater need than did participants in the low-use cluster . Examination of pairwise comparisons of adjusted mean need across clusters indicated that partners in the low-use cluster  reported significantly less need than partners in the high-  and moderate-use  clusters: need did not differ significantly between partners in the high- and medium-use clusters (P = .32).Findings from the MANOVA indicate that, after adjusting for demographic covariates including age, gender, white race, and any children, significant variability existed in partners’ need for psychoeducation across clusters . With respect to content that would likely resonate with partners, all topics inquired about during the needs assessment received endorsement and particularly topics normalizing the partner’s role as an informed supporter of women facing hereditary cancer risk, with training in coping and communication skills. Interestingly, we were able to empirically derive 3 distinct groups of potential users of psychoeducational interventions based on their communication channel preferences, including those who may be the most and least likely to use self-help, as well as online and interpersonal interaction-based resources. It is expected that these results can inform planning and development of new interactive, Internet-based intervention tools that are specifically directed toward and designed to meet the psychoeducational needs of previvors’ and high risk survivors’ partners.Prior studies of the use of Internet-based information and support resources suggest that our findings may, in part, reflect the fact that the needs assessment sample comprised predominantly men. Partners endorsed a high need for psychoeducation, which is consistent with earlier research demonstrating that men are less likely than women to participate in in-person psychosocial cancer support groups, yet express levels of need for resources and support similar to those of women ,43. AddiOur findings also raise important considerations regarding the diversity of partners and the need for psychoeducation targeted toward special populations. While a majority of the sample were men, a significant minority (14%) of partners who responded to the online needs assessment were women. This suggests that when developing resources targeted to partners’ needs, one should take into consideration the diversity within this population, including same-sex couples. Evidence regarding the impact of breast cancer on same-sex couples and their need for psychoeducation is scarce -51. FurtBRCA1/2 mutation carriers have elevated risks for breast and prostate cancer, as well as other forms of the disease [Male breast cancer is rare and accounts for only a small proportion of hereditary breast cancer cases (<1%) ,53. Men  disease . Familie disease ,55. MaleInternet-based resources may be ideally suited to address the psychoeducational needs of partners as described herein. Internet-based tools could enable health professionals to effectively incorporate self-help materials that could be downloaded or printed from the Web, as well as leverage interactive multimedia aids such as videos, expert webinars, and online discussion forums to deliver content, provide support, and engage participants . MoreoveProfessionally facilitated, Internet-based psychoeducational interventions implemented in diverse populations , includiBRCA1/2 genetic counseling and testing services [Moving forward, it is critical to carefully investigate the ways in which Internet-based psychoeducation and support resources can be integrated into the delivery of health care services for hereditary breast cancer, such as services ,60. The services ,27, and services . Clinicaservices ,63.BRCA1/2 genetic testing or undergoing surgery).The findings reported herein should be interpreted in light of the limitations of this work. The original needs assessment data relied on a convenience sample of partners responding to an Internet-based survey of unknown denominator (reach). Moreover, our focus was on partners of previving and surviving women, and did not include partners of affected men. We caution against generalizing the findings to the broader population of partners. The potential influence of selection bias is also an important consideration, as partners completing the assessment may differ from those who did not. The assessment used self-reported measures of communication channels, perceived need for psychoeducation, and other key constructs. Due to the anonymous, Internet-based nature of the needs assessment, it was also not possible to verify reported clinical characteristics  and interactive, Internet-based resources seems promising. Additional research is needed to rigorously develop and evaluate Internet-based resources targeting partners. Medical Internet and health communication researchers should work closely with cancer genetic content experts to examine ways in which Internet-based resources can be offered to this target population.


This study assessed the effects of a pre-visit website with tailored information and question prompt sheet (QPS), named E-info geneca. All counselees completed a pre- and post-visit questionnaire and visits were videotaped. We studied effects on counselees' information recall, knowledge about breast cancer and heredity, fulfillment of needs, risk perception alignment, anxiety and perceived personal control, using multilevel regression analyses.A total of 197 counselees were randomized to receive usual care (UC) or UC plus E-info genen = 103) had higher levels of recall of information from the consultation : .04 to .60; P = .02; d = .17) and post-visit knowledge of breast cancer and heredity  than counselees in the UC group (n = 94). Also, intervention group counselees reported better fulfilment of information needs . The effects of the intervention were strongest for those counselees who did not receive an indication for DNA testing. Their recall scores showed a larger increase  and their anxiety levels dropped more in the intervention compared to the UC group . No intervention effects were found after the first visit on risk perception alignment or perceived personal control.Intent-to-treat analysis showed that counselees in the intervention group (This study shows that pre-counseling education, using tailored information technology, leads to more effective first visits for breast cancer genetic counseling, in particular for counselees who received no indication for DNA testing and, therefore, had no indication for a second visit. Future study should focus on the effects of a pre-visit website on the outcomes after a complete series of visits.ISRCTN82643064.Dutch Trial Register  Women who are diagnosed with breast cancer or have affected relatives often worry about their or their children's risk to (re)develop breast cancer. Therefore, they might be referred to genetic counseling. Breast cancer genetic counseling aims to educate individuals about their breast cancer risk to optimize risk management, increase personal control and decrease anxiety . HoweverGenerally, recall of information from medical visits is low. Kessels  reportedUnidirectional transfer of generic instead of tailored or counselee-specific information has long been the preferred method in cancer genetics, both in the consultation and in pre-visit information. However, counselee-specific information is expected to be more effective in reaching the aims of counseling by increasing the counselee's recall ,10. TailPrevious studies of computer-based education prior to genetic counseling were limited to assessment of counselees' knowledge about breast cancer and heredity -20. The ca [ca, counselees communicated more assertively by more often sharing their agenda, directing the flow of the visit and paraphrasing the counselor to check their understanding. As a result, counselors provided more counselee-specific information [This article describes an randomized controlled trial (RCT) focused on the effects of a pre-visit educational website, E-info geneca  to optimca . Additioca . These qormation . The curISRCTN82643064). The department of Medical Genetics enrolled counselees from February 2008 to April 2010. This department offers breast cancer genetic counseling according to the Dutch guidelines [BRCA1/2 gene mutation in a relative. All counselees who did not return the opt-out form were randomly assigned 1:1 to the usual care (UC) or intervention group (UC + website E-info geneca) by a secretary unaware of respondent's characteristics using sequentially numbered, sealed, opaque envelopes. UC comprised a brief standard pre-visit leaflet with information about the counseling procedure. Both UC and intervention group respondents received a login to access the web-based baseline questionnaire. Upon completion of the baseline questionnaire the intervention group respondents received a link to access the website E-info geneca. At the start of the consultation the counselor collected the informed consent form. The visits were videotaped. In the week after their visit, counselees again completed a web-based questionnaire. Because with 100 counselees in each group, there was an 80% power to detect effect sizes of d = .40 or higher, we aimed to include 200 counselees.This study was conducted at the department of Medical Genetics of the University Medical Center (UMC) Utrecht. The study was approved by the institutional medical ethical committee and is registered in the Dutch Trial Register ISRCTN843064. ThCounselors were clinical geneticists, residents in clinical genetics, genetic counselors or genetic counselors in training. All will be referred to as 'counselor'. Their age, gender and number of years of experience with cancer genetic counseling was assessed at the start of their participation in the study.Age, having children, personal and family cancer history and educational attainments were assessed in the baseline counselee questionnaire. All but the latter were derived from the medical file if missing. Additionally, whether or not there was an indication for DNA testing for the counselee or an affected family member was collected from the medical files.ca [Counselees' recall of information from the visit was assessed in the post-visit questionnaire with seven questions for cued recall. Each question prompted a topic of the consultation, for example, limitations of DNA testing and involving family members, and these topics were based on the counselees' information needs questionnaire Quote-geneca . Each quca .Coders first assessed whether the topic was discussed in the visit based on the videotapes. Second, each item recalled was compared with the specific items mentioned by the counselor ,7. The pRecall was coded by AA and a second coder recoded a random 10% of the visits. Coders were blinded to group allocation. Interrater reliability was assessed. Cohen's kappa for the recall per item averaged 0.70 (range 0.39 to 1.0). The intra class correlation (ICC) of the overall recall percentage was 0.89.The level of accurate knowledge about hereditary breast cancer was assessed using the validated Dutch hereditary breast cancer knowledge scale ,27. Respca) [ca includes four generic needs, which refer to what a counselor should do during counseling (25 items) and four cancer genetic information needs, which refer to receiving explanations on hereditary cancer (19 items), as identified by principal component analysis with good internal consistency [Pre-visit, counselees' needs were assessed with the QUality Of counselling Through counselees' Eyes scale for cancer genetic counseling (QUOTE-geneca) . At baseca) . The QUOsistency .Risk perception alignment was defined as the degree of agreement of the counselee's risk perception with the counseled risk . CounselPre- and post-visit anxiety was assessed with the validated Dutch state version of State-Trait Anxiety Inventory  ,31. ScorPre- and post-visit perceived control was assessed using the validated Dutch version of the Perceived Personal Control questionnaire (PPC) ,32. Scorca or other protocol irregularities. The few missing values (seven cases) on the baseline measures of educational attainment were imputed with the median. Post-visit, the number of missing data on the outcome variables ranged from 18 for knowledge and recall, 20 for fulfillment, 22 for perceived personal control, 25 for anxiety and 43 for risk perception. All counselees were included in analyses through use of repeated measures analysis which makes use of available data of all cases  [We analyzed respondents in the groups to which they were randomized regardless of whether they actually accessed E-info genenalysis) .P < .05. Furthermore, Cohen's effect sizes (d) were calculated [Intervention and UC group counselees were compared on socio-demographics, cancer history and levels of pre-visit measures using Chi-square tests and t-tests. To account for the multilevel structure of measurements (level 1) nested within counselees (level 2) nested within counselors (level 3), random effects multilevel regression analyses were conducted. This method corrects for the number of consultations per counselor . The perlculated .P = .11), disease status , family history of cancer  and referral pathway . A flowchart of the study and reasons for non-response are shown in Figure Few counselees were ineligible because of lack of internet or email access 24 of 371; 6.5%). The response was 58.6%. Half of the decliners gave a reason . Most decliners preferred that the visit not be videotaped . There were no significant differences between participants and decliners in age . Three were experienced 5 to 15 years) clinical geneticists  and five were clinical geneticists in training , see Table 5 years c2 X= 5.10; P = .02). There was no significant association between having (had) breast cancer and baseline knowledge . One counselee was affected with ovarian cancer. There were no significant baseline differences in knowledge, information needs, risk perception, anxiety and perceived personal control between the study groups . The effect size of the intervention was larger for the counselees who did not receive an indication for DNA testing  versus 59.94(25.09); d = .30). The educational level of the counselee was also a significant predictor . Additionally, counselees who were asked to return for a follow-up consultation recalled less than those who had only one consultation . And finally, counselees tended to have more recall when they had seen a genetic counselor (nurse) instead of a clinical geneticist  and less recall when they had seen a counselor who was still in training . This was not related to the total number of information items discussed .Less than half of all information items were recalled correctly Table . The tot2 X= 4.81; P = .03). The probability that hereditary breast cancer runs in the family was recalled best and information about risk reducing breast surgery and the emotional consequences of genetic counseling had the lowest recall scores.In almost all visits, the counselor provided information about the probability that hereditary breast cancer runs in the counselee's family and of the possibilities and limitations of DNA testing Table . The posP = .03; d = .28). Additionally, counselees in whose family there was an indication for DNA testing had more post-visit knowledge . In the intervention group, knowledge especially concerning inheritance and penetrance of BRCA1/2 mutations was enhanced . Intervention group counselees especially indicated higher fulfillment of their need for information about the determination and meaning of being a carrier of a mutation in one of the breast cancer genes  and about hereditary breast cancer than the UC group counselees . Counselees in both groups reported high fulfillment of generic needs .As shown in Table P = .79). Table Intervention condition was not associated with the alignment of the counselee's perception of her breast cancer risk with the counselor's risk estimation ). Other counselees underestimated these risks  and , respectively). The alignment of these risk perceptions with the counselors' estimations was not significantly associated with the intervention condition .Most counselees  also overestimated the risk of hereditary cancer running in their family, (average overestimation of 25.68 percentage points (SD = 17.84)) and their risk of being a carrier of a n = 49) had significantly lower anxiety scores in the intervention compared to the UC group when controlled for baseline values .For the whole group of counselees, post-visit anxiety was unrelated to the intervention condition Table . OverallP = .06).Perceived personal control was not significantly related to the intervention condition Table . Levels This study showed that a pre-visit educational website with QPS improves counselees' recall of information discussed in their first visit for breast cancer genetic counseling. As counselees' transfer of information from the visit to relatives has been described as a whisper game where the counselor's information fades out , our finIn 45-minute first consultations for breast cancer genetic counseling a large amount of information is transferred with the intention that the counselee will understand and remember this information so as to make decisions about DNA testing and to involve relatives ,38. ThisEffects of the intervention on anxiety were only found for counselees who did not receive an indication for DNA testing. The lack of improvements for those who received an indication for DNA testing is understandable as the indication itself and waiting for the test results can be a source of distress . For couThe pre-visit web-based education had no effect on alignment of counselees' risk perceptions with the counselors' estimation. An explanation for this lack of effect might be the fact that the risk perception, as opposed to factual recall of information, seems to be determined to a large extent by personal experiences of loss , identifAdditionally, there were no intervention effects on perceived personal control. Effects of web-based information alone might be limited to cognitive outcomes and might therefore not include perceived personal control ,45. In tAnalyses were performed with multilevel analysis to take differences in counselor styles into account. This study is the first to show the percentage of variance at the counselor level and this might provide insight into the extent to which differences in individual counseling styles affect genetic counseling outcomes. Although differences between counselors have not been studied before with appropriate methods, several authors have mentioned the need for this ,44. For The first visit for breast cancer genetic counseling has been described as an educational session in which large amounts of standard information are being transferred and the counselee's understanding is scarcely checked upon . This fiThere are some limitations. First, counselors were not blinded to group allocation as they received counselees' question sheet (QPS) and their responses to these questions were part of the intervention. Second, the response rate is moderate, but relatively high for studies using video recordings of genetic counseling visits ,38,53. IWhen counselees had prepared with a pre-visit website, they remembered more information from their first consultation for breast cancer genetic counseling and their information needs were better addressed. If adopted in practice, pre-counseling education could lead to more effective first visits for breast cancer genetic counseling due to improved cognitive outcomes. Counselees who need to transfer information to their family might better succeed as a result of increased recall of what was discussed with the counselor.B: beta; BC: breast cancer; CI: confidence interval; d: Cohen's effect size; GP: general practitioner; ICC: intra class correlation; M: mean; MRI: magnetic resonance imaging; N: number; P: probability; PPC: perceived personal control; QPS: question prompt sheet; QUOTE: Quality of counselling Through counselees' eyes questionnaire; RCT: randomized controlled trial; SD: standard deviation; STAI: State-Trait Anxiety Inventory; UC: usual care; UMC: University Medical Center.The authors declare that they have no competing interests.AA carried out the acquisition of data, performed the statistical analyses and drafted the manuscript. SvD conceived of the study, participated in its design and coordination and helped to interpret the data and helped to draft the manuscript. JB participated in the design of the study, helped to interpret the data and helped to revise the manuscript. MA participated in the study design and coordination, helped to interpret the data and helped to revise the manuscript. All authors read and approved the final manuscript.


POLG) gene, which encodes the catalytic subunit of enzyme responsible for directing mitochondrial DNA replication in humans, contains a polyglutamine tract encoded by CAG repeats of varying length. The length of the CAG repeat has been associated with the risk of testicular cancer, and other genomic variants that impact mitochondrial function have been linked to breast cancer risk in African-American (AA) women. We evaluated the potential role of germline POLG-CAG repeat variants in breast cancer risk in a sample of AA women (100 cases and 100 age-matched controls) who participated in the Women's Circle of Health Study, an ongoing multi-institutional, case-control study of breast cancer. Genotyping was done by fragment analysis in a blinded manner. Results from this small study suggest the possibility of an increased risk of breast cancer in women with minor CAG repeat variants of POLG, but no statistically significant differences in CAG repeat length were observed between cases and controls . Our study suggests that POLG-CAG repeat length is a potential risk factor for breast cancer that needs to be explored in larger population-based studies.The DNA polymerase-gamma ( Despite the overall higher incidence of breast cancer in European-American (EA) women as compared to African-American (AA) women, AA women are more likely to be diagnosed with breast cancer before age 40 and to have tumors with aggressive pathological characteristics, including high tumor grade, lack of expression of estrogen and progesterone receptors  and HER2 (triple-negative breast cancers), and additional features of basal-like breast cancer (ER−/PR−/HER2−/cy5/6+/EGFR+) POLG gene may result in depletion of mtDNA and confer breast cancer phenotype POLG consists of an exonuclease domain with three exonuclease motifs, I, II and III, and a polymerase domain with three polymerase motifs, A, B and C, along with an intervening linker region POLG affects its expression POLG-CAG repeat expansion with testicular cancer has also been reported POLG in breast cancer has not been investigated. The purpose of this study was to evaluate the association of POLG CAG repeat length with breast cancer risk in the Women's Circle of Health Study (WCHS), an epidemiological case-control study of breast cancer in AA and EA women.A recent study demonstrated that tumor-cell mitochondrial DNA copy (mtDNA) number correlates with tumor progression as well as patient prognosis and disease-free survival in breast cancer 2). All study subjects provided written informed consent for the study and use of biospecimens, and the study was approved by the Institutional Review Boards at Mount Sinai School of Medicine (MSSM) (coordinating site for NY enrollment and NYC controls), the local NYC hospital (for cases), The Cancer Institute of New Jersey (coordinating site for NJ enrollments) as well as at Roswell Park Cancer Institute IRB approval number I-120807.The data and samples from 100 AA women with breast cancer and 100 age-matched AA controls for this study were obtained from the Women's Circle of Health Study (WCHS), a case-control study specifically designed to investigate risk factors for early, aggressive breast cancer in AA women. The WCHS has been previously described POLG_F: 6FAM-tggatgtccaatgggttgtgc and POLG_R: aagccaggtgttctgactcc) were designed to amplify a 275-bp fragment encompassing CAG repeats present in exon 2 of POLG1. The amplification was carried out in a 25 ul reaction containing approximately 50 ng of template DNA, 0.6 uM of both forward and reverse primers, and 0.5 ul AccuPrime™ Taq DNA polymerase  using a TECHNE TC-412 (96×0.2 ml) thermocycler . The PCR conditions were as follows: initial denaturation at 94°C for 4 min in 26 amplification cycles at 94°C for 10 sec, annealing at 61°C for 30 sec and 68°C for 40 sec, followed by a final extension step at 68°C for 5 min.Genomic DNA was extracted from whole blood and evaluated for purity and concentration using a Nanodrop UV spectrophotometer and quantified on a spectrofluorometer  using the PicoGreen dsDNA quantification kit, as per the manufacturer's instructions .. Double-stranded DNA was quantitated using a PicoGreen-based fluorometric assay. Primers , according to the DNA fragment analysis protocol. DNA fragment data were collected and then visualized using GeneMapper® software. The data containing the genomic location (peak size) for all samples were exported as delimited text files and blindly analyzed for the number of CAG repeats in each sample. Assay reproducibility was tested using multiple runs of both positive and negative samples with different CAG repeat variants.POLG and breast cancer risk. All statistical analyses were computed with SAS 9.2 .Differences between categorical variables were assessed with the chi-square test, and continuous variables were compared using Student's t-test. All statistical tests were two-sided. Age at menarche and the number of children were divided into tertiles using cut-points based on the distribution of these variables among controls. Age at first pregnancy was categorized as nulliparous, ≤19, 20–24, 25–29, or ≥30. Menopausal status at the time of cancer diagnosis was determined during the interview and categorized as premenopausal, perimenopausal, or postmenopausal. Crude and multivariate unconditional logistic regression models adjusted for age, age at menarche, age at first full-term pregnancy, menopausal status , first-degree family history of breast cancer (yes/no) and BMI were used to calculate odds ratios (OR) and corresponding 95% confidence intervals (CI) as a measure of association between CAG repeats of mitochondrial DNA POLG-CAG repeat was absent. The common 10/10 CAG repeat occurred in 52 cases (55%) and 56 controls (60%); heterozygous 10/non-10 CAG repeats were observed in 36 cases (38%) and 31 controls (33%)  . The repls (33%) .Compared with 10/10 repeats, the crude odds ratios (ORs) for 10/11, 10/12 repeats, and all other repeats were 1.16 , 0.93 , and 1.31 , respectively . After aPOLG is the only polymerase known to be involved in human mitochondrial DNA replication. POLG contains CAG trinucleotide repeats in the coding region, and CAG repeat sequences are described to be highly unstable, leading to expansion or contraction of the repeat sequences POLG gene, we reported the CAG repeat expansion occurs in 20% of breast cancer patients POLG-CAG repeat-length and an increased risk of breast cancer in AA women.Mitochondrial POLG-CAG repeat length (r = −0.81) and the age of onset of disease in Friedreich's Ataxia (FRDA) patients The presence of expanded or contracted CAG repeats has been linked with several diseases, including the human polyglutamine diseases POLG-CAG repeat variants with cancer risk. Interestingly, a study of CAG repeat polymorphisms in the POLG gene in testicular cancer showed 36 (74%) wild-type homozygotes and 13 (26%) lacked one or both wild-type alleles, with the 10/11 variant in 10 patients and the 10/12, 10/6 and 11/11 variants in one patient each, suggesting that variants of the DNA POLG1 gene were more frequent in testicular cancer patients than in healthy men POLG may be a contributing factor in the pathogenesis of testicular seminoma POLG-CAG repeat variants in cancer compared the frequency of these variants in tumor tissue to the frequency in either surrounding normal tissue or blood samples. In contrast, our study evaluated germline polymorphisms in POLG in both breast cancer cases and in controls. Results of studies may differ depending on the DNA source due to additional clonal mutations that can occur in tumor cells.There are two additional reports associating POLG gene, which encodes mitochondrial DNA polymerase-gamma, in breast cancer risk. While our findings suggest a possible correlation between CAG repeat length and risk of breast cancer in AA women, the size of this genetic study sample was small, as evidenced by reduced power to demonstrate significant associations of known breast cancer risk factors such as age at menarche and family history of breast cancer with cancer risk in this study. Larger, preferably population-based, studies are needed in order to draw any firm conclusions about the role of POLG-CAG variants in breast cancer risk in AA or other populations.This study is the first to investigate the role of CAG repeat polymorphisms in the nuclear


To investigate urban-rural differences in the distribution of risk factors for breast cancer.We analyzed the data from the first round of the “Mamma” population based-screening program conducted in Croatia between 2007 and 2009 and self-reported questionnaire results for 924 patients with histologically verified breast cancer. Reproductive and anthropometric characteristics, family history of breast cancer, history of breast disease, and prior breast screening history were compared between participants from the city of Zagreb (n = 270) and participants from 13 counties with more than 50% of rural inhabitants (n = 654).P < 0.001 in all cases): no pregnancies (15% vs 7%), late age of first pregnancy (≥30 years) (10% vs 4%), and the most recent mammogram conducted 2-3 years ago (32% vs 14%). Women from rural counties were more often obese (41% vs 28%) and had early age of first live birth (<20 years) .The screen-detected breast cancer rate was 4.5 per 1000 mammographies in rural counties and 4.6 in the city of Zagreb, while the participation rate was 61% in rural counties and 59% in Zagreb. Women from Zagreb had significantly more characteristics associated with an increased risk of breast cancer (Identification of rural-urban differences in mammography use and their causes at the population level can be useful in designing and implementing interventions targeted at the reduction of inequalities and modifiable risk factors. Significant differences in breast cancer frequency have been identified in different socioeconomic groups, ethnic groups, and between urban and rural populations ,2. LivinRisk factors for breast cancer are mostly those related to the reproductive life of women ,10: menaIn Croatia, breast cancer is the leading cancer among women, amounting to 27% of new female cancer cases; moreover, the incidence rate in 2007 was 17% higher than in the previous year . In 2007Population-based screening for breast cancer is conducted through mammographic examination of all women of a specified age at prescribed time intervals. The implementation of population-based screening requires technical resources and trained personnel for double reading of mammograms, as well as a major media campaign .Within a more extensive study of breast cancer risk factors, this study investigated urban-rural differences in reproductive, anthropometric, and family history of breast cancer and personal history of breast disease among women aged 50-69 from 13 rural counties and the city of Zagreb who participated in the first round of population-based mammography screening in Croatia.Organized population-based screening program in Croatia started in November 2006 on a target population of women aged 50-69. Coordinators from the Public Health Institutes from 21 counties distributed the invitations and coordinated the program at the county level. A separate database was formed for each county and the central unit had access to each of these databases through a common server located at the Croatian Ministry of Health and Social Welfare. The program is centrally coordinated by the Croatian National Institute of Public Health and includes 81 mammography units and more than 200 radiologists. Double reading is obligatory; if the result is normal, women are sent a letter of invitation to another routine screening in 2 years. If the result is abnormal, women and their family physicians are informed about the need for further assessment .We used the Organisation for Economic Co-operation and Development definition of rural and urban from the Croatian Rural Development Strategy 2008-2013 of the Croatian Ministry of Agriculture, Fisheries, and Rural Development . Rural cThe first round of the “Mamma” screening program included 80 092 women aged 50-69 from the city of Zagreb and 184 425 from 13 rural counties. Participation in the screening program was free and based on an invitation. Of 204 352 women screened from 2007-2009, 924 were found to have breast cancer. Of these, 270 were from the city of Zagreb and 654 from rural counties: Bjelovarsko-bilogorska, 56; Brodsko-posavska, 64; Karlovačka, 42; Koprivničko-križevačka, 47; Krapinsko-zagorska, 41; Ličko-senjska, 16; Požeško-slavonska, 32; Sisačko-moslavačka, 45; Šibensko-kninska, 31; Virovitičko-podravska, 30; Vukovarsko-srijemska, 96; Zadarska, 70; and Zagrebačka, 84.Screening was performed in women who lacked breast physical examination abnormalities, including nipple discharge, lumps, or thickening. Women with previous breast cancer and women who had not answered the questions addressing the studied risk factors were excluded.Questionnaire. Women involved in the program were sent the questionnaire with an invitation letter to their home address. The invitation list was generated based on records of the Croatian Health Insurance Institute and the Ministry of Interior Affairs. The oldest women invited into the program were born between 1937 and 1941. The participants at the time of mammography completed the questionnaire and reported their age, age at menarche, number of pregnancies and deliveries, age at first live-birth, history and duration of breast feeding, use and duration of use of birth control pills , use and duration of hormone replacement therapy, menopausal status, age at menopause, personal or family history of breast cancer (defined as having first-degree or second-degree relative with breast cancer), breast symptoms , breast procedures, weight, height, and the time of their last mammogram. To increase the response rate, reminders were made by telephone and field nurses motivated women to attend the screening.2) and divided into three categories: lean weight (BMI≤25), overweight (25<BMI<30), and obesity (BMI≥30)  .The patients gave informed consent when they filled in the questionnaire. The study was approved by the Ethics Committee of the Medical School, University of Zagreb.2 test with Yates correction when necessary and two-sided t-tests to compare predictive variables for urban-rural differences , with specialized equipment and trained staff who performed screening and/or for further assessment in cases when an abnormality was detected. There were only slight rural-urban differences in the number of screening facilities per 10 000 invited women when all rural counties were considered, although there were great differences between individual rural counties, from 0.8 in Brodsko-posavska county to 3.9 in Ličko-senjska county .P < 0.001 for all comparisons). Compared with women from Zagreb, women from rural counties were more often obese – BMI>30  and had early age of first live birth (<20 years)  . CompareThe participation rate was 59% in the city of Zagreb and 61% in rural counties, ranging between 47% in Zagrebačka county and 78% in Bjelovarsko-bilogorska county . The numP < 0.001). They also had significantly greater median age of first live birth   .P = 0.455) (P = 0.044) and at menopause . Fifteen percent of urban women and 7% of rural women had never been pregnant  . There wP < 0.001) and there was no difference in height between urban and rural women  (P = 0.905)  .P = 0.039), and more rural women breast fed for longer than 12 months   .P = 0.455) (P = 0.003) but there was no difference in the history of oral contraceptive use . Significantly more rural women had early age of the first live birth (<20 years) . There was a significant urban-rural difference in having performed a mammogram in the last 2 years  (P = 0.011), and similar number of women from both groups had a second degree relative with breast cancer history   . The his< 0.001) . More ur= 0.493) .Our study investigated urban-rural differences in the distribution of risk factors for breast cancer among women participating in the first round of the population-based screening program in Croatia. We found that women from Zagreb had significantly more characteristics associated with Western lifestyle that increased the risk of breast cancer , includiPossible explanations for the less frequent use of preventive services in rural counties than in Zagreb include greater distances to medical facilities, less access to services, and lower socioeconomic status . Indeed,We found a similar level of screen-detected breast cancer rate per 1000 mammographies (4.6 vs 4.5) when all rural counties were considered, although our findings, as well as previous cancer incidence data in Croatia , showed After the first invitation round, the participation rate in Croatia stabilized at around 60%, which is lower than the 70% rate that the EU Guidelines for Quality Assurance in Mammography Screening recommend as acceptable and the 75% rate that they recommend as desirable . The covThere are no uniformly accepted definitions of rural and urban areas and this makes the comparison between studies difficult . Also, wContrary to our findings, Chelpin et al  found a On the other hand, Blair et al found substantial differences in the distribution of breast cancer and probable risk factors  between the urban San Francisco Bay Area and rural regions . IncreasOur study found a significantly higher proportion of obesity among postmenopausal rural women (BMI≥30), which is in accordance with previous results -39. ThisA family history of breast cancer has long been recognized as a risk factor for the disease, and the risk of developing breast cancer is increased 1.5- to 3-fold if a woman has mother or sister with breast cancer . Our stuOur findings suggest that mammographic screening has played a major role in the increase in incidence of breast cancer in Croatia , but theEpidemiological studies have consistently identified a number of breast cancer risk factors associated with increased exposure to endogenous estrogens ,43-47. OThis study has several strengths: population-based design, the response rate of 99%, and availability of information on many established and probable risk factors that may influence breast cancer, with no recall bias. However, one of its limitations is that the questionnaire did not distinguish among types of hormone replacement therapy. Also the data on socioeconomic status, such as education, income, type of occupation, and in some populations, ethnicity, and religion were missing . BesidesIn conclusion, our study identified several reproductive and anthropometric risk factors for breast cancer that are modifiable and can reduce inequalities between urban and rural areas. Although there are some effective programs that may reduce some of the preventable risk factors, the availability of these programs may need to be improved in several remote rural areas. Future studies should implement the Gail breast cancer risk prediction model ,54 to ex


BRCA genes, BRCA1 or BRCA2, the risk of developing breast or ovarian cancer is significantly increased. In recent years there has been increased penetrance of BRCA1 and BRCA2 associated breast cancer, prompting investigation into the role of modifiable risk factors in this group. Previous investigations into this topic have relied on participants recalling lifetime weight changes and subjective methods of recording physical activity. The influence of obesity-related biomarkers, which may explain the link between obesity, physical activity and breast cancer risk, has not been investigated prospectively in this group. This paper describes the design of a prospective cohort study investigating the role of predictive and modifiable risk factors for breast cancer in unaffected BRCA1 and BRCA2 gene mutation carriers.Breast cancer is the most common female cancer worldwide. The lifetime risk of a woman being diagnosed with breast cancer is approximately 12.5%. For women who carry the deleterious mutation in either of the Participants will be recruited from breast cancer family risk clinics and genetics clinics. Lifestyle risk factors that will be investigated will include body composition, metabolic syndrome and its components, physical activity and dietary intake. PBMC telomere length will be measured as a potential predictor of breast cancer occurrence. Measurements will be completed on entry to the study and repeated at two years and five years. Participants will also be followed annually by questionnaire to track changes in risk factor status and to record cancer occurrence. Data will be analysed using multiple regression models. The study has an accrual target of 352 participants.BRCA gene. Additionally, the study will attempt to identify potential blood biomarkers which may be predictive of breast cancer occurrence.The results from this study will provide valuable information regarding the role of modifiable lifestyle risk factors for breast cancer in women with a deleterious mutation in the  BRCA1 or BRCA2. Women who carry these mutations have up to an 80% risk of developing breast and up to a 60% risk of ovarian cancer   .Many biomarkers of carcinogenesis must be collected and stored prior to cancer occurrences, to ensure an unequivocal link between biomarker exposure and tumorgenesis . PreviouPeripheral blood mononuclear cells (PBMCs) will be isolated from venous blood by density centrifugation using Ficoll-Paque™ Plus . Genomic DNA will be extracted from PBMCs by standard procedures. Telomere length will be measured in extracted genomic DNA by quantative polymerase chain reaction (qPCR) using a method adapted from the one origanally described by Cawthon . BrieflyT [CT(telomere)/CT(single gene)]. Telomere length (expressed as a relative T/S ratio) will be normalized to the average T/S ratio of the reference sample.Genomic DNA samples (50 ng) will be amplified in a total reaction volume of 20 μl containing 2X Quantifast ™ SYBR green PCR master mix , 1 μl of forward and reverse primer  and 7 μl of DNase free water. For the telomere amplification PCR, 300 nM of each primer  will be used. The thermal cycling profile for the telomere amplification will be 30 cycles of amplification at 95°C for 15 s and at 56°C for 60 s. For the control gene amplification, 300nM of forward primer (36B4u: CAGCAAGTGGGAAGGTGTAATCC) and 500nM (36B4d: CCCATTCTATCATCAACGGGTACAA) of reverse primer will be used. The thermal cycling profile in this instance will be 35 cycles of amplification at 95°C for 15 s and at 56°C for 20 s and 72°C for 20 s. Both PCR reactions will require an initial denaturation step at 95°C for 15 min. Threshold cycle (Ct) values for each sample will be converted into nanograms of DNA using standard curves. Ct values from the telomere assay will be normalized to the single gene reference. The telomere length (x) from each sample will be calculated as the telomere to single copy gene ratio (T/S ratio) and will be based on the calculation of the ΔC-1) or drug therapy for lipid abnormalities; reduced HDL-C (<1.3 mmol.L-1) or drug therapy for lipid abnormalities; elevated blood pressure (systolic ≥130 mmHg and/or diastolic ≥85 mmHg) or antihypertensive medication; elevated fasting glucose (≥100 mg.dL-1) or glucose-lowering medication [The metabolic syndrome will be diagnosed in the presence of any three of the following: elevated waist circumference (≥80 cm); elevated TG . The RT3 activity monitor is a small, lightweight, battery operated device, designed to measure accelerations along three orthogonal planes . Participants will wear the monitor for seven days, during waking hours, following each active assessment . Participants will be provided with the monitor following completion of each assessment and will be given detailed written and verbal instruction on its use. Participants will also be provided with a stamped addressed envelope to return the monitor to the centre after one week (battery life of the monitor = 21 days). The validity  and reliPhysical activity during the preceding 12 months will be estimated using two quantitative history questionnaires, The Minnesota Leisure-Time Physical Activity Questionnaire (MLTPAQ) and the Tecumseh Occupational Physical Activity Questionnaire (TOPAQ). The MLTPAQ is an interview administered questionnaire, measuring leisure-time physical activities  while thFinally, the Godin Leisure-Time Exercise Questionnaire will be incorporated into the annual questionnaire. Participants will be required to complete this questionnaire independently, but will be informed to contact study personnel should issues arise. The Godin is a simple, self-administered, four-item questionnaire that is designed to measure an individual’s leisure-time activity during a typical week to provide a global impression of an individual’s activity status . The valBRCA1 and BRCA2 associated breast cancer, “current” intake will be assessed using food diaries and “habitual” or usual intake assessed by a food frequency questionnaire (FFQ).Dietary intake will be assessed using two different methods, in this prospective cohort study on modifiable risk factors for Participants will be asked to complete an open-ended estimated 3 day food diary following each of the three active assessments. Participants will be provided with the diary at the end of each assessment and receive detailed verbal and written instructions detailing how it should be completed. The diet-diary booklet contains clear instructions of how to complete the food diary, as well as a detailed good and poor example of food intake. The instructions indicate that the respondent should record the food brand, portion size, cooking methods and includes a prompt for the name and daily dose of any vitamin, mineral or food supplements taken each day. General questions, for example, on the type of milk, spreadable fat usually consumed, and salt use will be asked as part of a general food habits questionnaire. Participants will be given a prepaid envelope to return the completed diary. The energy intake: calculated BMR ratio  will be The FFQ used in this prospective study is a self-administered Willett FFQ adapted from the European Prospective Investigation of Cancer (EPIC) study  and was Information on cancer occurrence and potential confounding factors affecting breast cancer risk will be gathered using questionnaires which will be completed on an annual basis after participants have enrolled. The questionnaire aims to track changes in modifiable lifestyle risk factors , risk reducing procedures (prophylactic surgery), reproductive factors  or cancer occurrence.Sample size calculation: There is some evidence to suggest an association between BMI and breast cancer incidence in BRCA mutation carriers [-1 between cases and controls, with a relative risk of 2.08, 80% power and two-sided 5% significance level 141 participants would be required per group. However, a minimum sample size of 352 was calculated to allow for a 25% drop out rate due to the long-term nature of this study.carriers -21 and ecarriers . TherefoData will be analysed using the SPSS package for Windows. Baseline descriptive statistics will be presented as means (standard deviations) for normally distributed continuous data, medians  for non-normal data and as frequency (percentage) for categorical variables. Distributions will be checked for normality using the K-S test and non-normal data will be transformed using appropriate transformations. Differences in means of continuous variables , lipids , glucose, insulin, HBA1c, CRP, leptin, adiponectin, BMI, fat free mass, percentage body fat, muscle mass, percentage time in each domain of physical activity, energy intake, telomere length) will be compared across categories  using independent sample t-tests or ANOVA as appropriate for normally distributed variables and the Mann Whitney U test  for non-normally distributed data. Chi-squared analysis will be used to compare categorical variables across the above stated groups.Pearson or Spearmans correlation analysis will be conducted between body composition , physical activity and dietary intake and the following variables: insulin resistance, leptin, adiponectin, inflammatory markers and telomere length for normally and non-normally distributed data respectively. Variables found to be associated at p<0.10 will be examined further using separate multiple regression analyses with body composition, physical activity and dietary intake as the dependent variables.-2, 18.5-24.9 kg.m-2, 25-29.5 kg.m-2, 30-34.9 kg.m-2, 35-39.9 kg.m-2, >40 kg.m-2; waist circumference will be categorised as <80 cm, 80-87.9 cm, >88 cm; physical activity will be categorised as adherence to physical activity guidelines  and non-adherence to activity guidelines). Quartile cut-offs will be established for markers without pre-determined categories. Metabolic syndrome variables  will be standardized to z-score variables with mean=0, SD=1 and a composite z-score will be computed for the presence/absence of the metabolic syndrome. Confounding variables that will be considered in the analysis include: prophylactic surgery, hormone therapy use, parity, age at menarche, breastfeeding history, age at first birth, oral contraceptive use, smoking and age. All analyses will be stratified according to menopausal status.Cox proportional hazards regression models will be used to compute adjusted hazard ratios of time to breast cancer (event) with 95% confidence intervals. Data will be censored at the last available follow-up where the breast cancer status was recorded. Continuous variables will be categorised into known cut-points based on previous research . All measurements and data processing will be completed at the Trinity Centre for Health Sciences, St. James’s Hospital, Dublin, Ireland.BRCA1 and BRCA2 gene carriers. To date, no study has prospectively examined lifestyle risk factors in this group, nor have the biological mechanisms linking obesity and physical inactivity to breast cancer risk been investigated. This study will provide information regarding whether modifiable factors including body composition, the metabolic syndrome, physical activity and dietary intake can modulate breast cancer risk in BRCA mutation carriers, whether risk of breast cancer occurrence can be predicted in these individuals by PBMC telomere length and whether telomere length in these individuals is associated with various physical activity and lifestyle factors.The results of this prospective cohort study will provide valuable information regarding the risk reducing potential of modifiable risk factors for breast cancer in unaffected BMI: Body mass index; PCR: Polymerase chain reaction; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TG: Triglycerides; HBA1c: Glycosylated haemoglobin; CRP: C – reactive protein; cm: Centimetre; mmol.L-1: Millimoles per litre; mmHG: Millimetres mercury; mg.dL-1: Milligrams per decilitre; MLTPAQ: Minnesota leisure time physical activity questionnaire; TOPAQ: Tecumseh occupational physical activity questionnaire; MET-min/day: Metabolic equivalent minutes per day; Kg.m-2: Kilograms per metre squared; FFQ: Food frequency questionnaire; BMR: Basal metabolic rate.The authors declare that they have no competing interests.EG, JH, JOS and EC developed the idea for this study. EG was responsible for drafting the manuscript with contributions from SMcG and LH. EG is responsible for measurement and analysis of body composition, physical activity and metabolic syndrome outcomes. SMcG is responsible for DNA processing and analysis and for measurement of DNA telomere length. LH is responsible for assessment and analysis of dietary outcomes. KB provided statistical advice and contributed to the study design. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/138/prepub