Although women with breast cancer tend to have a greater proportion of their circulating oestradiol non-protein bound and albumin bound, and less SHBG-bound, than controls, it remains uncertain whether this has an aetiological role or is an effect of the tumour. Oestradiol and its binding to serum proteins was investigated: (a) in relation to risk factors for breast cancer in a normal population; (b) in women with proliferative benign breast disease as a risk group for breast cancer, and women with non-proliferative benign breast disease as a low risk group, as well as breast cancer patients. The strongest associations were with body mass index; the greater the body mass the greater the bioavailability of oestradiol. Changes in relation to age at menarche and menopause could have been a function of body mass. An interesting change with age was noted with a fall in bioavailability over the menopausal years. There was no relationship apparent for parity, age at first full term pregnancy, family history or country of birth. Similar differences in oestradiol binding between cases and controls were seen for patients with breast cancer, benign epithelial hyperplasia and fibrocystic disease without proliferative changes, but these were not significant. This study provides limited support for the concept that oestradiol binding has an aetiological role in the development of breast cancer.


HER2 proto-oncogene in human cancer cells has been associated with a poor prognosis, and survival improves with therapy targeting the HER2 gene. Animal studies and protein modeling suggest that the Ile655Val polymorphism located in the transmembrane domain of the HER2 protein might influence breast cancer development by altering the efficiency of homodimerization.Overexpression of the N = 1,094) and population controls of a similar age (N = 976) were interviewed during 2001 to 2003 regarding their risk factors for breast cancer. By using DNA collected from buccal samples mailed by the participants, the HER2 Ile655Val polymorphism was evaluated with the Applied Biosystems allelic discrimination assay. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated by logistic regression adjusted for numerous breast cancer risk factors. Analysis was restricted to women with self-reported European descent.To investigate this genetic polymorphism, incident cases of invasive breast cancer (2 or more), and cases diagnosed with non-localized breast cancer .Prevalence of the Val/Val genotype was 5.6% in cases and 7.1% in controls. In comparison with the Ile/Ile genotype, the Ile/Val genotype was not significantly associated with breast cancer risk , whereas the Val/Val genotype was associated with a reduced risk . This inverse association seemed strongest in older women , women without a family history of breast cancer , postmenopausal women with greater body mass index (OR 0.43, 95% CI 0.20 to 0.91 for a body mass index of 25.3 kg/mHER2 Ile655Val polymorphism and risk of invasive breast cancer, most other studies of this single-nucleotide polymorphism suggest an overall null association. Any further study of this polymorphism should involve sample populations with complete risk factor information and sufficient power to evaluate gene-environment interactions between the HER2 polymorphism and factors such as age and family history of breast cancer.Although results from our population-based case-control study show an inverse association between the  HER2/neu, also called c-erbB-2) belongs to a family of receptors involved in the tyrosine kinase-mediated regulation of normal breast tissue growth and development [HER2 amplification or overexpression is fairly common – present in 20 to 30% of human breast cancers – and is a significant predictor of response to therapy, prognosis, and overall survival [HER2 is also a target for therapy. Antibody therapy with trastuzumab, which binds the extracellular portion of HER2, has been associated with improved patient outcomes including survival [The proto-oncogene human epidermal growth factor receptor 2  at codon 655 indicates a guanine-to-adenine substitution  in the transmembrane domain-coding region of the ER2 gene . This SNER2 gene -6, althoER2 gene -9.HER2 Ile655Val polymorphism and breast cancer risk in a population-based case-control study of midwestern United States women.Epidemiologic studies of the association between the Ile655Val polymorphism and breast cancer risk have generally shown null associations, with risk estimates below unity ,5,10,11 in situ) aged 20 to 69 years were identified though the Wisconsin statewide tumor registry. Controls were randomly sampled from driver's license files (ages 20 to 64 years) and Medicare beneficiary lists (ages 65 to 69 years); controls were frequency-matched in 5-year intervals to have a similar age distribution to that of the cases. All participants were required to have an available telephone number, and controls who self-reported a personal history of breast cancer were not eligible. Before April 2003, when changes in federal law affected the willingness of physicians to acknowledge their care of our eligible participants, physicians (identified on the tumor registry reports) were contacted before case enrollment to obtain information that might contraindicate study participation, such as dementia. All cases and controls were contacted by mail before receiving an interviewer's call. The 35-minute structured telephone interview elicited complete reproductive and menstrual histories, exogenous hormone use, smoking history, recent alcohol use and recreational physical activity, lifetime occupational and residential history, and exposure to indoor and outdoor chemicals. Information regarding the women's personal and family history of cancer was obtained at the end of the interview to maintain interviewer blinding. During April 2001 to January 2004, 77% of eligible cases  and 70% of eligible controls  participated in the telephone interview. The major reasons for nonparticipation were refusal , death before the interview , and inability to locate . Before April 2003, physicians refused participation for 2% of cases.As part of a continuing epidemiologic study, we recruited population-based cases of incident invasive breast cancer as well as community controls across Wisconsin in accordance with a protocol approved by the University of Wisconsin Health Sciences Human Subjects Committee. Invasive breast cancer cases  and 1,727 controls (81%). Genomic DNA was extracted by using the Gentra Systems DNA extraction reagents and protocol. DNA was resuspended in sterile water. Samples contained an average yield of 29.3 μg of DNA.HER2 Ile655Val polymorphism with the Applied Biosystems allelic discrimination assay-by-design (no. 185078430). The primers and labeled oligonucleotide probes for this reaction were as follows: forward, 5'-CCTGACCCTGGCTTCCG-3' ; reverse, 5'-ACCAGCAGAATGCCAACCA-3' ; VIC probe (detects T), 5'-ACGTCCATCATCTC-3' ; FAM probe (detects C), 5'-CCATCGTCTCTGCG-3'. Samples were cycled with conditions recommended by ABI. Fluorescence was detected with the ABI 7700 and genotypes were called manually with the detection software for this instrument. Genotyping failed for 45 subjects (2%). For quality control, DNA from 79 subjects who had submitted two independent samples were genotyped; 100% (79 of 79) had identical genotypes for the two samples. HER2 genotype was obtained for the 1,098 invasive breast cancer cases and 991 controls with European descent who had mailed their mouthwash samples to study staff by 30 June 2003. Because of the small number of women with non-European descent  and the low prevalence of the HER2 Val/Val genotype in Asian and African populations, these women were not genotyped.The laboratory staff were blinded to the identity and disease status of the subjects. Samples were genotyped for the Only exposure status before an assigned reference date was used in this analysis. For cases, this was the date of breast cancer diagnosis. For comparability, control subjects were assigned a reference date corresponding to the average time from diagnosis to interview for the case group (about 1 year). The reference age was defined as the age at the reference date. Menopausal status was defined as postmenopausal if the subject reported natural menopause or bilateral oophorectomy before the reference date. Women reporting hysterectomy alone were classified as postmenopausal if their reference age was greater than or equal to the 90th centile of age at natural menopause for the control group (54 years for smokers and 56 years for nonsmokers). Menopausal status was considered to be unknown for women with hysterectomy without bilateral oophorectomy if their reference age was between 42 and 54 years (or 56 years for nonsmokers).Pentry = 0.20, Premoval = 0.30). After forward stepwise regression had been performed, covariates remaining in the model were: family history of breast cancer in a mother, daughter, or sister , recent alcohol consumption (four categories), parity (four categories), menopausal status and age at menopause , hormone replacement therapy use , age at menarche , height at age 25 years (continuous), weight at age 18 years (continuous) and weight change since age 18 years (five categories). Covariates that did not remain in the final model included age at first birth, education, and income. Women with unknown recent alcohol consumption, hormone replacement therapy use, or height at age 25 years were not included in the analysis , so that 1,094 cases and 976 controls remained in the analysis. Interactions with genotype in relation to breast cancer risk were evaluated by including a cross-product term in the regression model and measuring the change in the log-likelihood.Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were obtained from multivariable conditional logistic regression models stratified on age. Covariates for the models were chosen by forward stepwise regression . Participants in this analysis were similar to nonparticipants in body mass index  and family history of breast cancer , although control participants were somewhat older  and more likely to have attended college than nonparticipant controls . Among cases, participants in this analysis did not differ significantly from nonparticipants in age  but were slightly more likely to have attended college .Breast cancer cases were more likely than controls to report a positive family history of breast cancer, to drink modest amounts of alcohol, to have lower parity, to report menopause at later ages, to have a younger age at menarche, and to report taller adult height Table . Cases iP = 0.96) and the control group (P = 0.28) were consistent with Hardy-Weinberg equilibrium.The Ile allele frequency was similar for cases and controls , and the Val allele frequency was about 25% ; 58.2% of cases and 56.5% of controls were homozygous for the Ile allele, 36.2% of cases and 36.5% of controls were heterozygous, and 5.6% of cases and 7.1% of controls were homozygous for the Val allele , cigarette smoking status (P = 0.66), adult height (P = 0.78), recent alcohol intake (P = 0.83), parity (P = 0.81), or age at menopause (P = 0.41) (data not shown).Although no interactions between the ps Table . In partHER2 valine alleles at codon 655 for some subgroups of women, including women older than 55 years of age and women without a family history of breast cancer. Three other studies – one study of Asian women [HER2 Val allele, although the estimates from these three other studies were not statistically significant.We observed a 40 to 50% decreased risk of breast cancer associated with the inheritance of two an women  and two an women  – have aHER2 polymorphism in younger women because of small numbers. Only 4 controls and 12 cases in our study were 45 years of age or younger, reported a positive family history of breast cancer, and also had the Val/Val or Val/Ile genotype .Our null results for younger women and women with a positive family history of breast cancer do not concur with findings by Montgomery and colleagues , which sHER2 Ile655Val polymorphism in relation to breast cancer risk found a very high risk  of the Val/Val versus Ile/Ile genotype [The first study of the genotype . In thatHER2 Ile655Val polymorphism have used a case-control design. Only one study population was a prospective cohort [HER2 valine allele might be associated with a twofold to eightfold increased risk of breast cancer. As with the Millikan study [Most studies of the e cohort . Two othe cohort ,16. Usinan study , these iHER2 Ile655Val polymorphism had insufficient power to evaluate interactions between the SNP and subgroups according to risk factors such as age and family history of breast cancer. Limited power is a common problem in studies of genetic polymorphisms. Sample size for only one other study was larger than the case and control enrollment in our own study [HER2 Ile655Val genotype according to racial descent is warranted.Many studies of the wn study . Prevalewn study ,17 – furHER2 gene is probably not confounded with the variables that might influence a woman's participation in our epidemiologic study [HER2 polymorphism in our case and control groups was consistent with Hardy-Weinberg equilibrium, which suggests that any genotyping errors were not substantial. Duplicate genotyping of 79 samples was also reassuring, achieving 100% concordance.Potential limitations might have influenced our findings. Although participation in our study was excellent for a population-based case-control study, certain subgroups might have been under-represented because participation probably declines with increasing age, decreasing attained education, and other factors. However, genetic inheritance with the ic study . The disHER2 gene leads to the development of mammary adenocarcinomas [The mechanism through which this SNP might influence breast cancer risk is unclear, although studies in transgenic mice have demonstrated that activation or overexpression of the rcinomas -21. The rcinomas -25. In hrcinomas .HER2 Ile655Val polymorphism is associated with a reduced risk of breast cancer in comparison with the Ile/Ile genotype for some women. Although the sample size in our study was relatively large compared with other studies published so far, the inconsistency of the findings across all studies argues against a strong relation with breast cancer risk. Future large studies of the HER2 polymorphism might clarify this putative gene-environment interaction. However, given the promise of innovative and more comprehensive approaches to genomic and proteomic studies of breast cancer risk, focusing on this SNP without consideration of the role of other genes and polymorphisms may not be warranted.These data from our sample population of white women from the midwestern United States suggest that the Val/Val genotype of the CI = confidence interval; OR = odds ratio; SNP = single-nucleotide polymorphism.The author(s) declare that they have no competing interests.SEN performed the processing and genotyping of the samples and drafted the manuscript. MNG and ATD jointly conceived of and designed the study, obtained funding, and drafted the manuscript. JMH performed the statistical analysis and assisted with manuscript preparation. All authors read and approved the final manuscript.


Detection of cytologic atypia in nipple aspirate fluid (NAF) has been shown to be a predictor of risk for development of breast carcinoma. Manual collection of NAF for cytologic evaluation varies widely in terms of efficacy, ease of use, and patient acceptance. We investigated a new automated device for the non-invasive collection of NAF in the office setting.A multi-center prospective observational clinical trial involving asymptomatic women designed to assess fluid production, adequacy, safety and patient acceptance of the HALO NAF Collection System . Cytologic evaluation of all NAF samples was performed using previously described classification categories.500 healthy women were successfully enrolled. Thirty-eight percent (190/500) produced fluid and 187 were available for cytologic analysis. Cytologic classification of fluid producers showed 50% (93/187) Category 0 , 38% (71/187) Category I , 10% (18/187) Category II , 3% (5/187) Category III  and none were Category IV . Overall, 19% of the subjects produced NAF with adequate cellularity and 1% were found to have cytologic atypia.The HALO system is a simple, safe, rapid, automated method for standardized collection of NAF which is acceptable to patients. Cytologic assessment of HALO-collected NAF showed the ability to detect benign and pre-neoplastic ductal epithelial cells from asymptomatic volunteers. The majority of breast cancers originate in the epithelium lining the milk ducts. It is believed that most breast cancers are slow growing and progress from precancerous cells, which have cellular and nuclear changes that can be identified microscopically. Finding microscopic evidence of ductal epithelial atypia/atypical ductal hyperplasia (ADH) has been shown in previous epidemiologic studies to be a predictor of future breast cancer development in an individual woman. -10 This Nipple fluid or secretions, usually aspirated from the breast ducts, is a protein rich material termed nipple aspirate fluid (NAF) which can be microscopically examined for the presence of atypical ductal epithelial cells. Nipple fluid can be obtained from many women, with reports of NAF production ranging from 25%  to more Breast cancer risk assessment using breast fluid cytology has been suggested to have a role in risk stratification and clinical decision making for women who are at high risk for breast cancer development. Ductal lavage is for clinical use in high-risk women and involves identification and cannulation of one or more fluid-yielding duct(s) then rinsing each with saline, collecting and analyzing the lavaged fluid. The finding of atypical cells could potentially influence a woman's decision for more aggressive surveillance or chemoprevention. Women with atypical ductal hyperplasia in the Breast Cancer Prevention Trial showed an 86% risk reduction with tamoxifen chemoprevention .We analyzed cytologic features of samples obtained during a pilot study using a new suction-based automated mechanical device for the non-invasive collection of NAF in the office setting. This article reports the results from a multi-center prospective observational clinical trial involving asymptomatic women designed to evaluate fluid production, adequacy, safety, patient acceptance and ability to detect atypical breast epithelial cells.The study sponsor was Neomatrix, LLC . The study design and execution was the responsibility of the study sponsor. The author's institution (ARUP Laboratories) provided contracted pathology services for the study sponsor, on a usual and customary fee-for-service basis. Study administration expenses at the parent institution (University of Utah) were paid by the study sponsor. No direct compensation was made to the manuscript authors.The study was conducted over a one year period and no preparatory phase was incorporated. The participant enrollment sites were obstetric and gynecology clinic practices located in Avon, CT., Farmington, CT., and Baton Rouge, LA. There was a single set-up instructional visit that took less than one hour and was performed prior to initiation of the investigation. The only variation in collection rates was seen between centers. One site had a lower NAF collection rate initially. The HALO System at this site was evaluated and the equipment was found to be operating outside of its specified performance parameters. The equipment was replaced and the variability in NAF collection rates between centers was no longer observed. During the study, enrollment was stopped for approximately 6 months in order to make some minor design modifications to enhance equipment performance.The study population included only asymptomatic, non-pregnant, non-lactating women with no history of breast cancer, breast surgery (e.g. breast augmentation or breast reduction), or nipple piercing who were asked to volunteer as part of a prospective multi-center observational study..Participants were required to be at least 18 years old and there was no upper age limit. Written informed consent was obtained from all subjects before enrollment in the study. The study protocol was approved for all participating collection centers by Biomedical Research Institute of America , an independent Institutional Review Board (Protocol PP-01) and the University of Utah (IRB #11588) Institutional Review Board. All rules and regulations concerning biomedical studies with human subjects were followed. A standardized questionnaire was completed for all participants that included medical history, current medications, family history, obstetric and gynecologic history, and breast health history including any previous biopsies and mammogram results. Gail model 5-year risk profiles were calculated for each woman over the age of 35 After obtaining written informed consent and completing a study questionnaire, a clinical breast exam was performed. The subject was wearing a front-opening examination gown and seated in a comfortable position. Each breast was cleaned using an alcohol wipe. Abrasive cleanser was not used. The HALO NAF Collection System  Fig  has adjuAny collected NAF was transferred from the nipple or sample collection cup(s) to a vial of fixative  using a pipette if necessary. If fluid was obtained from either one or both breasts, all samples were combined into a single sample preservative vial. Only one attempt was made to obtain NAF in the five minute session, and if no NAF was produced by either breast, the participant was considered to be a non-producer.All samples were shipped to a single reference laboratory . Microscopic slides were prepared from the entire NAF sample using a Millipore filter technique , which was chosen due to the low cellularity of the specimens. The filter preps were stained with the modified Papanicolaou stain technique.Each slide was reviewed by one of a group of three cytopathologists with experience examining breast cytologic specimens, including ductal lavage, and who were trained in the NAF cytologic categories prior to beginning the study. All difficult or borderline cases were resolved by reviewing cases at a multi-headed microscope. The slides were classified according to the most severe abnormality detected using one of five categories based upon increasing nuclear abnormality. The classification system used categories developed by King et al Table . CategorEnrollment site investigators and study coordinators filled out case report forms recording relevant information about the subjects' medical history, eligibility, study procedures, adverse events, and any available follow-up care. All clinical sites were monitored and all study data, including final cytology results, where collected by the study sponsor, NeoMatrix.Adverse events were noted immediately after the procedure as well as at the four to eight week post-procedure survey. The participant was asked to rate her comfort level immediately following the procedure using a visual analog scale of 1 to 10, with one being most comfortable and 10 being least comfortable. Participants were also contacted four to eight weeks after the procedure to assess satisfaction with the procedure. Women with cytologic atypia or worse (Class III or IV) were referred to their regular physicians who determined the appropriate follow-up care. A standard protocol for following patients with atypia was not included as part of this investigation. This follow-up care may have included further assessment of the patient via imaging, biopsy in some cases, risk counseling, and increased surveillance.2 test of association. The difference between values was considered significant at P < 0.05.The result of NAF producers and non-producers were expressed as raw numbers for each demographic category. Comparison between two groups was performed using the χst degree family history produced NAF, 46% of subjects with one 1st degree relative with breast cancer, and 75% of subjects who had more than one 1st degree relative with breast cancer (p <= 0.10). Forty-two percent of women with a lactation history produced NAF while 34% of women who never lactated were fluid producers (p < 0.10). Overall, 14% had at least one first degree relative with cancer and 11% had a history of a previous breast biopsy. In summary, none of the differences between fluid producers and non-producers with regards to any of the listed demographics was statistically significant.Five hundred (n = 500) participants were successfully enrolled. Overall characteristics of the study participants are summarized in Table Three of the 190 specimens collected from the fluid producers had a container leak during specimen transport and therefore could not be analyzed, with the remaining 187 available for evaluation. The final cytology results are summarized in Table A total of 419/500 (84%) women were surveyed for procedure acceptance four to eight weeks after their procedure. The average comfort assessment rating immediate post-procedure was 5.0 on a scale of 1–10 (one being most comfortable) and 4.2 at the four to eight week telephone/mail post-procedure survey . Comparison of Gail risk (>1.7% vs. <1.7%) and cytology category results, for the 190 women assessed, showed no significant difference (p = 0.68).The five women found to have Category III changes were referred for further breast care by their regular physicians. One of these women initially had a benign breast biopsy but was subsequently noted to have ductal carcinoma in situ (DCIS) on a follow-up repeat breast biopsy six months later done as part of an increased surveillance plan as determined by her physician. A second woman had negative bilateral biopsies and the third woman with surgical follow-up had a biopsy that showed lobular carcinoma in situ (LCIS). One of the five Category III women had negative follow-up ultrasound imaging and is being closely followed with mammography every six months. The last of the Category III women, a 24-year-old Caucasian woman with a strong family history of breast cancer in her mother, maternal grandmother and aunt , had negative initial imaging but was subsequently found to have a lump by clinical breast exam. Follow-up ultrasound imaging was again negative. It was recommended that this woman undergo genetic counseling. Follow-up information was not obtained for the Category 0, I or II women and further follow-up of the Category III subjects was not part of this protocol. The Category III women are being followed as high-risk individuals per their physicians' standard protocol outside of this investigation. Only one of these Category III women was identified as being high-risk prior to the NAF collection, using the Gail risk profile calculated at enrollment.Many studies have shown that finding atypical hyperplasia of the breast ductal epithelium is associated with an increased risk of subsequent development of breast cancer -7. WrensOverall, the HALO NAF collection procedure was found to be acceptable by the women studied, rapid, and posed little physical risk. This device has an advantage over other methods of NAF collection in that it is automatic and easy to use, thereby removing most clinician variability. It is also less invasive than other methods of sampling breast epithelium in asymptomatic women, such as ductal lavage and fine needle aspiration.Criticisms of using NAF to detect cellular abnormalities include the observation that fewer adequately cellular specimens are obtained than with ductal lavage. Dooley et al compared NAF and DL specimens  and founDespite these limitations, the ease and convenience of this method of obtaining breast ductal epithelial cells might make it a more acceptable option for women who are undergoing NAF collection. As shown in this study, participant acceptance of the procedure was adequate with an average initial comfort assessment rating of 5.0 on a scale of 1–10. Dooley et al  reportedNo prospective studies have been done to date that assess the negative predictive value of ductal cytology in asymptomatic or high risk women. Nipple aspirate fluid assessment is not a diagnostic test for breast cancer. NAF production and cytologic assessment may be used in conjunction with the Gail model for risk prediction and the automated HALO system may facilitate NAF collection in the office setting.While clinical follow-up care of patients with abnormal breast fluid cytology is not standardized, Shaughnessy et al  publisheNAF specimens have important applications in addition to the cytologic assessments that are described in this paper. A study by Chagpar et al  showed tThe HALO procedure has been shown to be a feasible way to obtain NAF samples for cytologic assessment and it appears to be a safe, rapid, fairly well-tolerated, and non-invasive procedure. While fluid production and adequacy rates may not be as high as reported in manual NAF collection series, there may be advantages to using the HALO System over manual collection techniques. The collection cycle is automated, thus removing any clinician variability and allowing women to be consistently, objectively screened routinely to assess any NAF changes. The system is user-friendly, requires minimal training and can be performed by clinical staff. The system is designed for bilateral, simultaneous collection using heat, compression, and suction combined in a single five minute cycle. Further prospective studies with long-term clinical follow-up are necessary to determine the clinical significance of non-producers vs. producers, insufficient samples and other cytologic categories found on NAF samples collected with the HALO System.The author(s) declare that they have no competing interests.All authors contributing equally to this work. Dr. Proctor is a pathology resident. Dr. Proctor tabulated the final study data and wrote the draft of the manuscript. Ms. Rowe was the cytology data coordinator and research and development scientist. Dr. Bentz was Director of Cytopathology, and responsible for review of study samples. All authors read and approved this manuscript.The pre-publication history for this paper can be accessed here:


CYP17 can influence the degree of exposure of breast tissues to oestrogen, the interaction between polymorphisms in this gene and hormonal risk factors is of particular interest. We attempted to replicate the findings of studies assessing such interactions with the -34T→C polymorphism.Because CYP17 genotyping data were derived from a large Australian population-based case-control-family study of 1,284 breast cancer cases and 679 controls. Crude and adjusted odds ratio (OR) estimates and 95% confidence intervals (CIs) were calculated by unconditional logistic regression analyses.Risk factor and CYP17 genotype and breast cancer overall. Premenopausal controls with A2/A2 genotype had a later age at menarche (P < 0.01). The only associations near statistical significance were that postmenopausal women with A1/A1 (wild-type) genotype had an increased risk of breast cancer if they had ever used hormone replacement therapy  and if they had menopause after age 47 years . We found no associations in common with any other studies, and no evidence for interactions.We found no associations between the CYP17 genotype, although the experiment did not have sufficient statistical power to detect small main effects and modest effects in subgroups. Associations found only in subgroup analyses based on relatively small numbers require cautious interpretation without confirmation by other studies. This emphasizes the need for replication in multiple and large population-based studies to provide convincing evidence for gene–environment interactions.We observed no evidence of effect modification of reproductive risk factors by  CYP17 on chromosome 10q24.3, which encodes the enzyme cytochrome P450c17α . P450c17α functions at two different points in the steroid biosynthesis pathway; the 17α hydroxylase activity can convert progesterone to 17α-hydroxyprogesterone, and the 17/20 lyase function may further convert 17α-hydroxyprogesterone to androstenedione (the precursor of both oestrone and testosterone) [The association between exposure to endogenous and exogenous steroid hormones and breast cancer risk is well established . ConsequCYP17 has been extensively studied [A1, and the variant C allele as A2. The C allele creates an additional Sp1-type (CCACC box) promoter site, and although it was initially suggested to increase expression of the gene [CYP17 -34T→C polymorphism might influence endogenous steroid hormone levels [One common polymorphism in  studied -23. It ithe gene , a subsethe gene . There ie levels ,24-31, ae levels . A recene levels .A2) variant, when comparing allele frequencies, or genotypes defined by these alleles under a dominant or recessive model. Results were consistently null in different ethnic groups [Although a few studies have found evidence for an association between this polymorphism and risk of breast cancer ,9,19,23,c groups .CYP17 may influence the degree of exposure of breast epithelial cells to oestrogen, the possibility that the effects of different hormonal risk factors is dependent on different CYP17 genotype is of particular interest. Some studies have suggested that CYP17 genotype is associated with hormonal risk factors, and/or that the association between breast cancer and hormonal risk factors depends on CYP17 genotypes. That is, CYP17 genotype may be an effect modifier. The hormonal risk factors examined in this manner have included age at menarche, age at first birth, use of oral contraceptives, age at menopause, and hormonal replacement therapy. So far, studies examining these gene–environment interactions or effect modifications have generally been small and have reported conflicting results [CYP17 variant was significantly associated with earlier age at menarche in only two of eight reports, and an effect of later age at menarche (at least 13 years) limited to women with the wild-type T (A1) homozygous genotype was evident in only 4 of 11 reports. Moreover, as alluded to in a recent review of studies of the CYP17 polymorphisms and hormone levels [As  results ,27,35-37e levels , the pubCYP17 genetic polymorphism and the risk of breast cancer before the age of 40 years [We previously published Australian data on the overall relationship between this 40 years . In the 40 years ,19,24,35The Australian Breast Cancer Family Study (ABCFS) is a population-based case-control-family study of breast cancer before the age of 60 years -41. SampBRCA1 or BRCA2, and these subjects have been excluded from the analyses.For the purpose of these analyses, subjects were restricted to the women who identified themselves as being white/Caucasian (details in ). MolecuCYP17 -34T→C polymorphism was measured in DNA extracted from cases and controls with the use of the AIB1 Prism 7700 Sequence Detection System. DNA was extracted from peripheral blood cells by using salt extraction methods for those recruited before 1995 [As described in detail previously , the CYPore 1995  and withA2) variant [As one purpose of this study was to try to replicate the effect modification effects previously reported in the literature that are consistent with an increased exposure to endogenous oestrogen associated with genotypes defined by the C  and 95% confidence intervals (CIs) were calculated with and without adjustment for measured risk factors. Logistic regression was also used to assess, in controls, the associations of hormone-related risk factors with genotype after adjusting for the above potential confounders. The statistical significance of interaction terms was assessed by the likelihood ratio test. All analyses were conducted with Stata version 8.0. All statistical tests were two-sided and the P values quoted are nominal; that is, no attempt was made to adjust for multiple comparisons, either in terms of the number of covariates or in terms of the number of modes of inheritance being considered.The Hardy–Weinberg equilibrium assumption was assessed by comparing the genotype frequencies with those expected on the basis of the observed allele frequencies and random mating by using the Pearson χA visual comparison between the results of the ABCFS and those of the WNYBCS was conducted by plotting the corresponding log OR estimates from each study against one another, using R version 1.6.2. The size of the points was proportional to the average of the inverse of standard errors of the two studies for that particular risk factor's estimate, so that larger points were those for which there was more precision. A positive correlation between the data points would be evidence for replication of findings.CYP17, including 1,572 premenopausal women (mean age 38.3 years) and 391 postmenopausal women (mean age 52.3 years). There was no evidence of deviation from Hardy–Weinberg equilibrium in controls (P = 0.95), but in cases there was marginally significant evidence of such deviation, with a deficiency of heterozygotes . The C (A2) allele frequency in cases was 0.38 (SEM 0.001) in cases, and 0.37 (SEM 0.01) in controls (P for difference = 0.8).Analyses were conducted for the 1,284 cases and 679 controls genotyped for CYP17 genotypes under codominant inheritance, among either postmenopausal or premenopausal women, with or without adjusting for covariates. For dominant inheritance, the adjusted OR estimates were 0.94 (95% CI 0.76 to 1.18) and 1.31 (95% CI 0.84 to 2.05) among premenopausal and postmenopausal women, respectively. For recessive inheritance, the adjusted OR estimates were 1.15 (95% CI 0.85 to 1.56) and 1.12 (95% CI 0.60 to 2.09) among premenopausal and postmenopausal women, respectively. We also examined risk for women stratified by report of family history, because our previous examination of a subset of the data found evidence for increased risk associated with the CC (A2/A2) genotype among women reporting a first-degree or second-degree family history of breast cancer. The OR for the CC (A2/A2) genotype was 1.10 (95% CI 0.81 to 1.49) for no family history and 2.46 (95% CI 0.70 to 8.60) for family history under a codominant model of inheritance, and 1.13  for no family history and 2.37  for family history under a recessive model of inheritance.Table BRCA1 or BRCA2 germline mutation; because these mutations are associated with at least a 10-fold increased breast cancer risk [CYP17 genotype could have a modifying effect on their disease risk; however, the frequencies of the TT (A1/A1), TC (A1/A2) and CC (A2/A2) genotypes were 41% (n = 17), 41% (17) and 18% (7), respectively, in carriers, similar to those of 40% (513), 44% (565), and 16% (206) observed in the non-carrier cases (P = 0.95). The C (A2) allele frequency was 0.38 in both carriers and non-carriers.We excluded the 41 Caucasian cases with a known cer risk , for morCYP17 genotypes defined by the polymorphism, under a dominant model, in relation to reproductive and hormonally related risk factors, and family history status based on first-degree relatives, stratified by menopause status. The only significant association was between age at menarche and CYP17 genotype in the premenopausal women (P = 0.002), such that controls with the TT (A1/A1) genotype were more likely to have an age at menarche of less than 13 years. There was no evidence that women with a T (A1) allele were more likely to have used HRT. The strengths of the estimated associations between genotype and risk factor were little changed by also adjusting for potential confounders.Table P = 0.02). This effect was significant among women homozygous for the T (A1) allele , but not significant for women with at least one C (A2) allele ; the two estimates were not significantly different from one another (P = 0.7). There was at best marginally significant evidence that later age at menopause was associated with an increased breast cancer risk in women homozygous for the T (A1) allele , but not in women with at least one C (A2) allele, and the difference in risk estimate by genotype was also not statistically significant (P = 0.06).Table CYP17 genotype presented in Table A1/A1 genotype, but evidence was weak in the present study and not significant in the WNYBCS [We compared the adjusted OR estimates of each risk factor and breast cancer risk for each e WNYBCS . Other se WNYBCS  or age ae WNYBCS , or riske WNYBCS , and nonCYP17 genotype defined by the – 34 promoter region T→C nucleotide-substitution polymorphism and risk of breast cancer overall, or within premenopausal or postmenopausal women, whether the genotype be defined under a codominant or a recessive mode of inheritance. Specifically, we failed to confirm our own positive finding [A2/A2) genotype in women reporting a positive family history.Consistent with most previous studies and a recent meta-analysis was our failure to find any evidence for an association between the  finding  of a sigCYP17 genotypes and hormone-related risk factors in controls, the only significant finding was an increased risk for older age at menarche among premenopausal women with the C (A2) allele. This finding is not consistent with previous positive reports, or with the hypothesis that the C (A2) allele is associated with increased endogenous oestrogen levels and an earlier age at menarche. The point estimates for the effects on breast cancer risk of later age at menopause and HRT use were stronger among women carrying the TT (A1/A1) genotype, but the interaction terms were not statistically significant.When we examined associations between CYP17 genotype on hormonal risk factors have found varying results. For example, the CYP17 variant was associated with earlier age at menarche in only two of eight reports [Other studies investigating the influence of  reports ,27,36,37 reports ,9,11,21, reports  and with reports .CYP17 genotypes have also been conflicting. The most consistent association reported was an effect modification of age at menarche. Four studies have presented evidence that the protective effect for a later age at menarche (at least 13 years) was mainly limited to women with the wild-type homozygous genotype [CYP17 genotype were in opposing directions from two studies reporting such results [Results from previous studies on the effect modification of reproductive risk factors by genotype ,9,11,19,genotype ,19, and at least  years wagenotype ,21,27,36CYP17 genotype groups, these must be interpreted with caution. We considered both premenopausal and postmenopausal women, seven risk factors, and two genotype groups, so by chance alone we would expect to find a few significant results even if there were no real effects.Our larger study found no association between breast cancer and age at menarche or age at first birth within any of the variant genotypes, either overall or within postmenopausal or premenopausal women. Although we have found at best marginally significant associations between breast cancer risk and both HRT use and age at menopausal status within some CYP17 variant in modulating endogenous oestrogen levels and associated breast cancer risk factors is the results of a recent study of 1,975 postmenopausal women, which found no association between CYP17 genotypes defined by several polymorphisms and mean levels of sex hormones, in particular oestradiol, oestrone and sex-hormone-binding globulin [CYP17 polymorphisms, at least among postmenopausal women, although there is a possibility that there might exist other variants and haplotypes associated with hormone levels and risk of breast cancer.Perhaps more convincing evidence against a role of this globulin . This sua priori hypotheses about the likely existence and direction of interactions with risk factors. However, the few positive associations with serum hormone levels reported in the literature would suggest that the C (A2) variant would be associated with increased endogenous hormone levels, and we have observed significant effects both contradicting and in support of such an association. In addition, in this and other similar studies, there are multiple tests being conducted; the quoted P values are only nominal and should be interpreted accordingly. Consequently, we cannot claim with confidence that any of our 'significant' findings represent true effects. As there seems to be no overall effect of CYP17 genotype on breast cancer risk, finding any true interactions with breast cancer risk factors (should they exist) will require massive individual studies, or pooling of studies. The marginally significant deviation from Hardy–Weinberg equilibrium is unlikely to be due to genotyping error because cases and controls were genotyped at the same time on the same PCR plates, and thus any genotyping bias (and deviation from Hardy–Weinberg equilibrium) would be expected to be seen equally in both cases and controls, but this was not so. Furthermore, our PCR success rate was more than 99.5% for both cases and controls, and results were fully concordant for a subset of 168 duplicate DNAs for which PCR was successful.Although we have conducted a relatively large study, there are some limitations. Because the functionality, if any, of the polymorphism we have studied is not well established, it was not possible to specify A2/A2 (CC) genotype  [Despite early enthusiasm and numerous reports of positive associations between genotypes, risk factors and breast cancer risk from small studies, few of these have held up under further scrutiny through being reproduced in larger studies. One educational example is of the association between breast cancer and a protein-truncating variant in CHK2 carried by 2.1% of women. Even in a study of more than 10,000 cases and 9,000 controls reporting a 2.3-fold increased risk for the CHK2 mutation and a 1.4-fold increased risk of family history, no nominally significant interactions were found between CHK2 genotypes and family history (P = 0.1) . Future ABCFS = Australian Breast Cancer Family Study; CI = confidence interval; HRT = hormone replacement therapy; OR = odds ratio; PCR = polymerase chain reaction; WNYBCS = Western New York Breast Cancer Study.The author(s) declare that they have no competing interests.J-HC conducted the analyses and led the manuscript preparation. DMG led the literature review and interpretation of the data. XC conducted the genotyping assays. GSD was responsible for data management. MAJ, RLM and JLH were responsible for development and supervision of the statistical analyses. MCS was responsible for the management of laboratory staff and biospecimens and participated in the quality control of the genotyping and data cleaning. MREMcC, GGG and JLH initiated the ABCFS and have been instrumental in the ongoing execution of these studies. GC-T initiated and obtained funding for the genotyping component of this study, and was involved in the supervision of laboratory work. ABS was responsible for designing and supervising the genotyping and laboratory work, genotype data cleaning, literature review and manuscript preparation. All authors read and approved the final manuscript.


Breast cancers of different histology have different clinical and prognostic features. There are also indications of differences in aetiology. We therefore evaluated the risk of the three most common histological subtypes in relation to menopausal hormone therapy and other breast cancer risk factors.We used a population-based case-control study of breast cancer to evaluate menopausal hormone therapy and other breast cancer risk factors for risk by histological subtype. Women aged 50 to 74 years, diagnosed with invasive ductal , lobular (n = 308) or tubular (n = 93) breast cancer in Sweden in 1993 to 1995 were compared with 3,065 age-frequency matched controls randomly selected from the population. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for ductal, lobular, and tubular cancer.Women who had used medium potency estrogen alone were at increased risks of both ductal and lobular cancer. Medium potency estrogen-progestin was associated with increased risks for all subtypes, but the estimates for lobular and tubular cancer were higher compared with ductal cancer. We found OR 5.6 (95% CI 3.2–9.7) for lobular cancer, OR 6.5 (95% CI 2.8–14.9) for tubular cancer and OR 2.3 (95% CI 1.6–3.3) for ductal cancer with ≥5 years use of medium potency estrogen-progestin therapy. Low potency oral estrogen (mainly estriol) appeared to be associated with an increased risk for lobular cancer, but the association was strongest for short-term use. Reproductive and anthropometric factors, smoking, and past use of oral contraceptives were mostly similarly related to the risks of the three breast cancer subtypes. Recent alcohol consumption of > 10 g alcohol/day was associated with increased risk only for tubular cancer .Menopausal hormone therapy was associated with increased risks for breast cancer of both ductal and lobular subtype, and medium potency estrogen-progestin therapy was more strongly associated with lobular compared with ductal cancer. We also found medium potency estrogen-progestin therapy and alcohol to be strongly associated with tubular cancer. With some exceptions, most other risk factors seemed to be similarly associated with the three subtypes of breast cancer. Use of menopausal hormone therapy has been shown to increase the risk of breast cancer, and datWe report results on these relationships from a large Swedish case-control study. The types of estrogens and gestagens used for menopausal hormone therapy differ between countries and, to our knowledge, this is the first study outside the United States to report in detail on these associations.This study is an extension of a case-control study among all Swedish residents born in Sweden and aged 50 to 74 years between 1 October 1993 and 31 March 1995 -21. The Controls were frequency matched by the expected age distribution among cases and identified through the Swedish National Population Register holding data on national registration number, name, address, and place of birth of all Swedish residents. The response rate among controls was 82% . Women previously diagnosed with invasive cancer (other than non-melanoma skin cancer) were excluded from the study (112 cases and 91 controls). Menopause was defined as the age at last menstrual period or age at bilateral oophorectomy, if one year or more prior to data collection. Premenopausal women (198 cases and 152 controls), women below the age of 55 years with unknown age at menopause considered premenopausal (202 cases and 101 controls), and women with missing information on body mass index  or age at first birth (5 cases and 1 control) were excluded.In a second phase of the study, we retrieved information about histology and various other tumour characteristics from the medical records of all participating cases. Following a decision of the ethical review board of the University of Lund, written informed consent to retrieve this information was sought from cases in that region (n = 563), among whom 58 women did not provide informed consent. The medical records for 31 study participants could not be found.Information from the medical records led us to exclude a further 58 cases with non-invasive breast cancer, 35 cases with previous cancer, one case with a cancer diagnosis other than breast cancer, and 19 cases diagnosed before or after the study period. The final study comprised 2,643 breast cancer cases and 3,065 controls.Data on sociodemographic, anthropometric, reproductive and menstrual factors, use of oral contraceptives, medical history, lifetime physical activity and smoking habits, as well as recent (one year before data collection) dietary habits and alcohol use were collected by means of a postal questionnaire. Detailed information on use of menopausal hormone therapy, including timing and type of hormones for each treatment episode, was also requested and a colour chart displaying all preparations ever marketed in Sweden was included with the questionnaire to aid recall. In addition, approximately 50% of both cases and controls were contacted by telephone to complete missing or ambiguous responses, mainly on use of menopausal hormone therapy. Of all eligible controls, 14% did not return the questionnaire, but agreed to a telephone interview covering the most important items, including use of menopausal hormone therapy.We obtained information on tumour histology from the pathology report in the medical record. Histology was classified after the invasive component as ductal , lobular (n = 308), tubular , mixed lobulo-ductal (n = 58), medullary (n = 29), mucinous (n = 61), papillary (n = 10), adenocarcinoma not otherwise specified (n = 122), or unspecified tumours (n = 38). Information on histology was missing in 36 cases because the medical record was not identified, or the woman had not had a breast cancer operation. We analysed ductal, lobular, and tubular cases and all other cases were excluded from the analyses. Adenocarcinoma not otherwise specified was not analysed as we considered it an undefined subtype, and the other histology subtypes were too few to be analysed separately.p-value < 0.05 was considered a significant difference.We used unconditional logistic regression to estimate odds ratios (ORs) with associated 95% confidence intervals (95% CIs) separately for ductal, lobular, and tubular cancer cases compared with controls. To formally test whether effect estimates were different for lobular or tubular cancer compared with ductal cancer we fitted models comparing lobular or tubular cases with ductal cases. For the tests comparing lobular or tubular cancer with ductal cancer, a two-sided Use of menopausal hormone therapy was categorized according to type as medium potency estrogen alone (mainly estradiol or conjugated estrogens), medium potency estrogen with progestin, progestin alone (without concomitant estrogen), low potency oral estrogen , or local estrogen  for each treatment episode. Medium potency estrogen-progestin therapy was also classified according to regimen, that is, as sequential (progestin taken less than 16 days per 28 days) or continuous use (19 or more days per 28 days). We censored all exposure after a reference date, defined in cases as date of diagnosis minus 3 months, and in controls as the date of questionnaire arrival minus mean time from diagnosis to questionnaire arrival in cases, minus an additional 3 months.We studied the effect of ever use, duration and recency of use. Analyses of the associations between use of specific types of menopausal hormone therapy and the risk of breast cancer were performed both for non-exclusive use , and restricted to exclusive users of that particular type, with never users of any menopausal hormone therapy as the reference group. No more than five women reported exclusive current use of progestin alone therapy, so we were unable to evaluate this therapy. Only 31 women had simultaneously used progestin with low potency oral estrogen or local estrogen, so this combination was not analysed separately, but these women were excluded from all analyses on exclusive use.In the analyses on menopausal hormone therapy, women with an unknown age at menopause were excluded (296 cases and 301 controls) to avoid bias leading to underestimation of the associations as first pointed out by Pike and colleagues .2) one year before data collection, height, socioeconomic status, smoking, and ever use of medium potency estrogen-progestin therapy. Confounding was defined as changing the point estimates more than 10% from the age-adjusted values. Only covariates confounding the effect of at least one of the exposures in the same table were included in the models. In addition, age at first birth and parity were adjusted for each other.We adjusted all estimates for age. We also assessed the potential confounding effects of a range of other variables, including number of births, age at first birth, age at menopause, surgical menopause , menopausal symptoms, BMI (weight(kg)/height (m)The analyses of the influence of age at menopause on the risk of different breast cancer subtypes were restricted to women with a known age at menopause that had not used menopausal hormone therapy apart from local estrogen before menopause in order to reduce confounding. The influence of BMI one year before data collection and the risks of different breast cancer subtypes was evaluated only among never users of menopausal hormone therapy apart from local estrogen – as BMI and menopausal hormone therapy are known to interact biologically to cause breast cancer . This inControls were slightly older than cases Table . SurgicaUse of medium potency estrogen alone was similarly associated with increased risks of ductal and lobular cancer Table . Due to Use of medium potency estrogen-progestin was associated with increased risks of all three subtypes of breast cancer, with the highest risks among ≥5 years and current exclusive use Table . These rp = 0.36).Non-exclusive users of both sequential and continuous medium potency estrogen-progestin had a median duration treatment of four years, compared with two years for exclusive users (data not shown). This was reflected in the higher point estimates for non-exclusive compared with exclusive ever use for lobular and tubular breast cancer Table . ExclusiNon-exclusive and exclusive ever use of low potency oral estrogen was associated with an increased risk for lobular  but not ductal  or tubular  breast cancer . Previous operation due to benign breast disease was associated with slightly higher risk for lobular  compared with ductal  cancer. The OR for tubular cancer was near unity, but not statistically different from ductal cancer. Use of oral contraceptives  was not associated with any of the three histological subtypes of breast cancer among these postmenopausal women  than with ductal  breast cancer , and detailed information on use of menopasual hormone therapy and on other breast cancer risk factors. Still, for lobular and even more for tubular cancer, the number of cases is small, and chance variation may play an important role. Another possible limitation is that exposure information was self-reported and collected retrospectively. We cannot rule out the possibility that some women have reported use of medium potency estrogen alone while they actually have used medium potency estrogen-progestin. Such misclassification would possibly explain our strong associations between medium potency estrogen alone therapy and breast cancer, but the similar risks for both ductal and lobular cancer differ from what we found for medium potency estrogen-progestin therapy. Also, the concordance between self-reported use of menopausal hormone therapy and information from medical records has been shown to be satisfactorily high ,40, and Another limitation is that several pathologists at different laboratories did the histological classifications, which may have resulted in non-differential misclassification, possibly diluting the associations.Many risks are assessed in this study, but we believe it is more relevant to discuss the results in relation to previous results and biological credibility than to adjust for multiple comparisons in the statistical analyses.Most risk factors seem to affect ductal, lobular and tubular breast cancer similarly, but there is accumulating evidence for medium potency estrogen-progestin therapy as a stronger risk factor for lobular than ductal breast cancer. We also found significant associations between medium potency estrogen alone therapy and both ductal and lobular cancer, and between medium potency estrogen-progestin therapy and ductal breast cancer. Our findings of an association between low potency oral estrogen and lobular breast cancer, as well as the strong associations between tubular cancer and both medium potency estrogen-progestin therapy and alcohol intake is notable but has yet to be confirmed or refuted by other studies.BMI = body mass index; CI = confidence interval; OR = odds ratio.The authors declare that they have no competing interests.CM and PH identified the initial hypothesis. LR, CM and PH were involved in data collection. LR, EL and PD performed the statistical analyses. LR, CM, SW, PH and PD interpreted the data. LR and EL drafted the manuscript, and CM, SW, PH and PD revised the manuscript. All authors read and approved the final version of the manuscript.


Alcohol consumption is a well-established risk factor for breast cancer. Some studies have suggested that the risk of breast cancer associated with alcohol consumption is greater for women with a history of benign breast disease (BBD). We hypothesized that among women with biopsy-confirmed BBD, recent alcohol consumption would increase the risk of breast cancer in women with proliferative breast disease to a greater extent than in women with nonproliferative breast disease.n = 282) were women diagnosed with incident breast cancer, with a prior biopsy-confirmed breast disease. The controls  were participants with a previous BBD biopsy, but without a diagnosis of breast cancer. Pathologists reviewed benign breast biopsy slides in a blinded fashion and classified the BBD as nonproliferative, proliferative without atypia, or atypical hyperplasia, according to standard criteria.We conducted a nested case–control study in the Nurses' Health Study I and II. The cases (P for interactio n = 0.20).Women with nonproliferative breast disease consuming ≥ 15 g of alcohol per day had a nonsignificant 67% increased risk of breast cancer  compared with nondrinkers. There was no evidence that recent alcohol consumption increased the risk of breast cancer to a greater extent in women with proliferative BBD than among women with nonproliferative BBD (a priori hypothesis, there was no evidence that recent alcohol consumption increased the risk of breast cancer to a greater extent among women with proliferative BBD than among women with nonproliferative BBD.Contrary to our  Alcohol consumption is a well-established risk factor for breast cancer. Among those who drink, recent alcohol consumption of two or more drinks a day is associated with an approximately 30% increased risk of breast cancer compared with nondrinkers -3. A pooData from epidemiologic studies also suggest that alcohol may influence early stages as well as later stages of breast carcinogenesis. The primary evidence that alcohol may have an effect on early stages in the carcinogenic process comes largely from studies of alcohol consumption and mammographic density. Breast density is a strong predictor of breast cancer risk and is considered to be an early biomarker of breast cancer -9. In moA few studies have suggested that the increased risk of breast cancer associated with alcohol consumption is greater for women with a history of BBD ,17, althThe Nurses' Health Study (NHS I) cohort was initiated in 1976, when 121,700 US registered nurses ages 30 to 55 returned an initial questionnaire. The Nurses' Health Study II (NHS II) is also an ongoing cohort study of over 116,000 US female nurses who were 25 to 42 years of age in 1989 when the study was initiated. Every 2 years, information on reproductive variables, body mass index, exogenous hormone use, and disease outcomes is collected in both cohorts. Semiquantitative food frequency questionnaires were included as part of the biennial questionnaire in 1980, 1984, 1986, and 1990 in NHS I, and in 1991 in NHS II. Alcohol consumption was also assessed on the baseline 1989 questionnaire in NHS II. The methods developed to follow up participants and confirm incident cancers and death in the Nurses' Health Study have been described previously in detail elsewhere  and haveBeginning with the initial NHS I questionnaire in 1976, participants have been asked on every biennial questionnaire to report any diagnosis of fibrocystic disease or other BBD. Early questionnaires  asked whether the respondent had ever been diagnosed as having 'fibrocystic disease' or 'other BBD' and whether she had been hospitalized with this diagnosis. Beginning in 1982, the NHS I questionnaires sought specific details of a history of biopsy-confirmed BBD. The initial 1989 NHS II questionnaire and all subsequent biennial questionnaires also asked participants to report any diagnosis of BBD and to indicate if it was confirmed by biopsy or aspiration.We conducted a case–control study nested within the subcohort of women with a biopsy-confirmed BBD. Incident breast cancer cases in both cohorts were identified through the nurses' own reports and were confirmed by review of medical records. Cases in the study are cases of breast cancer diagnosed by 1 June 1994 (NHS I) or 1 June 1995 (NHS II) with a previous confirmed BBD biopsy and available pathology specimens (histologic sections and/or tissue blocks). Controls are women who did not have a diagnosis of breast cancer when the case was diagnosed and also had a previous biopsy-confirmed BBD and available pathology specimens. Controls were matched to cases on year of birth and year of biopsy. Attempts were made to identify four matched controls for each case, although this was not always possible. This study was approved by the Committee on Human Subjects at Brigham and Women's Hospital.n = 2), report of breast cancer either before study start or after the 1994/1995 follow-up cycle (n = 10), and date of benign breast diagnosis was within six months of the breast cancer diagnosis date (cases) or index date (controls) (n = 144).We identified incident confirmed breast cancer cases diagnosed after the return of the initial questionnaire through the 1994/1995 follow-up cycle and controls that also reported a previous biopsy-confirmed BBD. A total of 1,080 cases were originally identified for this study, and 4,353 controls matched on age and year of benign breast biopsy were selected. After selection, 2.1% were found to be no longer eligible and were excluded. Seventy-five percent of eligible participants confirmed their BBD biopsy and granted permission to review their pathology slides. In response to our slide requests, we received pathology specimens for 55% of the study subjects. The primary reason given by pathology departments for not sending slides or tissue blocks was that the specimens had been destroyed or were no longer available (33%). There were no significant differences in the success of obtaining slides for breast cancer cases and controls. A detailed description of the steps of specimen collection and exclusions of selected cases and controls during the collection process are included in Table in situ (n = 25) or invasive carcinoma (n = 4). These women were excluded from further analysis. There were 353 breast cancer cases and 1,495 controls with pathology slides available from their BBD biopsy.Slides were coded by a research assistant and submitted to one of two collaborating pathologists  in a blinded fashion. The pathologists independently reviewed the BBD biopsy slides. Any slide identified as having either questionable atypia or atypia was jointly reviewed by the two pathologists. For each set of slides reviewed, a detailed work sheet was completed quantifying the extent of proliferative and atypical changes, morphologic features, and other details. BBDs were classified according to the Page classification system  into oneInformation on alcohol consumption was obtained from semiquantitative food frequency questionnaires. In NHS I, questions regarding alcohol consumption were asked in 1980, 1984, 1986, and 1990. Women were asked about their average consumption of beer, wine, and liquor separately in the prior year. One drink was considered equal to one can or bottle of beer, a 4-ounce glass of wine, or one drink or shot of liquor. Participants were asked to select from the following categories: almost never, 1 to 3 per month, 1 per week, 2 to 4 per week, 5 to 6 per week, 1 per day, 2 to 3 per day, 4 to 6 per day, 6 or more per day. Similarly, women in NHS II answered questions on consumption of alcohol in the 1989 and 1991 questionnaires. In 1991, the NHS II alcohol questions were expanded to include red wine, white wine, light beer, regular beer, and liquor. Total alcohol consumption per questionnaire cycle was calculated by adding the alcohol contributions from beer, wine, and liquor. Grams of ethanol per day were calculated based on the following equivalents of 12.8 g for regular beer, 11.3 g for light beer, 11.0 g for wine, and 14.0 g for liquor. Alcohol consumption in NHS I has been shown to be valid and highly reproducible in repeated assessments .n = 36 cases, 129 controls). If alcohol consumption was missing from the questionnaire before the index date, the exposure from the preceding cycle was assigned. The Spearman correlation between reported alcohol consumption in questionnaire cycles was 0.80 (P<0.0001) or greater for all consecutive cycles. Women with missing alcohol exposure data from two cycles preceding the index date were excluded from the analyses . Thus, there were 282 patients with breast cancer and 1,223 controls eligible for this study with both BBD pathology and detailed alcohol exposure preceding their index date.Women were assigned the alcohol exposure from the cycle preceding the diagnosis of breast cancer. Cases and controls in NHS I with diagnosis (or index) dates preceding the initial food frequency questionnaire administered in 1980 were excluded from the analysis (2); age at menarche ; age at first birth/parity ; duration of postmenopausal hormone use ; and menopausal status/type of menopause .Odds ratios (ORs) and 95% confidence intervals (CIs) were determined using logistic regression models controlling for matching factors  and follow-up time using the SAS software package . Follow-up time was defined as the time from BBD diagnosis to breast cancer diagnosis date (cases) or index date (controls). Information on potential confounding variables was obtained from responses on biennial questionnaires. For each 2-year cycle of case–control selection, covariate information was determined from the responses on questionnaires immediately preceding the cycle in which the breast cancer case was diagnosed. In addition to matching factors and follow-up interval, multivariate analyses were adjusted for the following confounders and breast cancer risk factors: first-degree family history of breast cancer (yes/no); quartiles of body mass index  than women with nonproliferative benign histology Table . Women wOverall, women with proliferative breast disease without atypia had a 50% greater risk of breast cancer than women with nonproliferative disease (OR = 1.54) Table . Women wIn this nested case–control study, there was no increased risk of breast cancer among women drinking ≥ 15 g of alcohol per day compared with nondrinkers .The combined effects of alcohol consumption and category of BBD histology are presented in Table P = 0.20).Among women with nonproliferative BBD, there was a 10% increased risk of breast cancer per 5 g increase in daily alcohol consumption . Among women with proliferative BBD without atypia, there was no increased risk of breast cancer associated with a 5-g increase in daily alcohol consumption . Similarly, among women with proliferative BBD with atypia, there was no evidence that increasing alcohol consumption increases risk: a 5-g increase in alcohol consumption was associated with a nonsignificant 12% reduction in breast cancer risk . The interaction between alcohol consumption and BBD category was not statistically significant (n = 232 cases) showed similar results. We were limited in power to adequately assess this relation according to menopausal status and estrogen receptor status of the cancer.Analyses limited to invasive breast cancers  [P = 0.23) [Previous epidemiologic studies have examined the effect of alcohol consumption among women with a self-report of BBD, with inconsistent results ,16-18. I = 0.23) .Benign breast conditions are a heterogeneous group of diseases and therefore may not all respond to alcohol exposure in the same manner. Although all of the women in this study had a biopsy removing their benign lesion, we were operating under the assumption that the BBD is a marker of susceptibility and the remaining breast tissue may have a similar susceptibly to the effects of alcohol later in life. Nonproliferative breast diseases comprise a large proportion of reported BBD biopsies. The inconsistencies observed between our study and previous studies may be due to the heterogeneous nature of BBD and variability in distribution of BBD types between different studies.One proposed mechanism by which alcohol may influence breast cancer risk is by increasing circulating estradiol levels, as has been observed in controlled feeding studies in both premenopausal and postmenopausal women ,7. A seca priori hypothesis and suggests that recent alcohol consumption does not contribute additional risk to women with proliferative breast disease. One explanation may be that women with proliferative breast diseases are further along on the continuum to breast cancer, and are already at an elevated risk of breast cancer, which is no longer affected by alcohol. In contrast, women with nonproliferative breast disease are not as far along the pathway to breast cancer and specific exposures such as alcohol may exhibit harmful effects and influence the risk of breast cancer.The current study failed to support our a priori hypothesis and they observed no elevated risk of breast cancer among women with proliferative breast diseases according to current use of hormones or duration of use, again suggesting that these women may be at such an increased risk of breast cancer that the additional effect of exogenous hormone use is minimal or none.Byrne and colleagues conducted a similar study in the Nurses' Health Study to examine the effects of postmenopausal hormone use among women with BBD . SimilarIn Figure in situ. If all breast cancers arise from benign lesions, the results of this study, in conjunction with other data, imply that there may be a narrow window of time when alcohol consumption affects breast cancer risk. An alternative explanation may be that there are multiple pathways to breast cancer and pathways involving proliferative benign breast lesions may not be influenced by alcohol consumption. A third possibility is that the magnitude of risk associated with benign proliferative lesions, and in particular atypical hyperplasia, is so much greater than the effect of alcohol that we were underpowered to detect more subtle increases in risk attributable to alcohol in this study.The progression from tumor initiation to breast cancer is not well defined. One hypothesis is that the pathway from normal tissue to breast cancer arises from a series of preinvasive lesions: benign proliferative changes, atypical hyperplasia, and carcinoma Although this is one of the largest studies of its kind, the number of women with proliferative breast disease and high alcohol consumption was small. We were underpowered to examine this relation in greater detail with regard to menopausal status and estrogen receptor status of tumors. A potential concern of the study is that the final study population represents 37% of those originally selected. The study protocol required collection of pathologic specimens in order to have unified review of histologic sections. The major limitation of this is that many hospitals routinely destroy specimens after 5 or 10 years. As a result, many potential cases and controls were excluded, which could result in potential selection bias. There were no significant differences in reasons for loss comparing cases to controls, indicating that any differences that do occur are likely to be due to chance.In this study, we observed that women with proliferative BBD experienced no additional risk of breast cancer with increased alcohol consumption. It remains unclear whether alcohol acts early, acts late, or has multiple effects on breast cancer risk. Future studies with greater numbers should examine menopausal and hormone receptor status of the cancers in order to provide additional information on both timing of relevant exposure and biologic mechanisms.BBD = benign breast disease; CI = confidence interval; NHS  = Nurses' Health Study ; OR = odds ratio; RR = relative risk.The author(s) declare that they have no competing interests.RMT conducted the analysis and wrote the manuscript. CB participated in the concept design and data collection. HJB participated in the writing and editing of the manuscript. BR provided statistical support. SJS participated in the concept and design, data collection, and manuscript editing. JLC participated in the concept and design, data collection, and manuscript editing. GAC provided funding for the study and participated in the concept and design, data collection, and manuscript editing. All authors read and approved the final manuscript.


Hormone concentrations in blood and total 12 h urine values were compared between 40 post-menopausal women with breast cancer and 40 control women in a study which carefully controlled for the possible confounding effects of age, weight and pregnancy history by individually matching cases and controls on these factors. Breast cancer cases had received only surgical treatment for their localised disease, which was diagnosed from 1 to 9 years before hormonal evaluation. Cases had 15% higher serum oestradiol levels (P = 0.02), 40% more urinary oestradiol (P = 0.03) and 44% more urinary oestriol (P = 0.04) than control women. Cases also had higher levels of serum and urinary oestrone, but these differences were not statistically significant. The percentages of serum oestradiol not bound to albumin or sex-hormone binding globulin did not differ between cases and controls, nor were there statistically significant differences in the serum levels of prolactin, sex-hormone binding globulin or dehydroepiandrosterone sulphate. These results provide further support for the hypothesis that breast cancer risk is determined in part by post-menopausal serum oestrogen concentration.


Epidemiologic and laboratory investigations suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive effects against breast cancer due to their activity against cyclooxygenase-2 (COX-2), the rate-limiting enzyme of the prostaglandin cascade.We conducted a case control study of breast cancer designed to compare effects of selective and non-selective COX-2 inhibitors. A total of 323 incident breast cancer patients were ascertained from the James Cancer Hospital, Columbus, Ohio, during 2003–2004 and compared with 649 cancer free controls matched to the cases at a 2:1 ratio on age, race, and county of residence. Data on the past and current use of prescription and over the counter medications and breast cancer risk factors were ascertained using a standardized risk factor questionnaire. Effects of COX-2 inhibiting agents were quantified by calculating odds ratios (OR) and 95% confidence intervals.Results showed significant risk reductions for selective COX-2 inhibitors as a group , regular aspirin , and ibuprofen or naproxen . Acetaminophen, a compound with negligible COX-2 activity and low dose aspirin (81 mg) produced no significant change in the risk of breast cancer.Selective COX-2 inhibitors (celecoxib and rofecoxib) were only recently approved for use in 1999, and rofecoxib (Vioxx) was withdrawn from the marketplace in 2004. Nevertheless, even in the short window of exposure to these compounds, the selective COX-2 inhibitors produced a significant (71%) reduction in the risk of breast cancer, underscoring their strong potential for breast cancer chemoprevention. The recent recall of rofecoxib (Vioxx) from the marketplace due its alleged association with increased risk for cardiovascular disease has severely compromised further testing of all selective cyclooxygenase-2 (COX-2) inhibitors in the chemoprevention and therapy of cancer. Despite compelling evidence that COX-2 inhibitors have powerful anti-cancer effects, several clinical trials designed to evaluate these compounds in the chemoprevention and therapy of neoplasms have been discontinued or suspended ,2.increases have been observed with use of rofecoxib and celecoxib in clinical trials that were designed to evaluate their potential for treating arthritis or reducing colonic polyp recurrence [decreases have been observed in observational studies that were designed to evaluate effects of these same compounds on cardiovascular diseases [Both the magnitude and the direction of effect of selective COX-2 blockers on the risk of cardiovascular disease is the subject of controversy. Risk currence -5, wherediseases -8. Stilldiseases ,10.Among American women, breast cancer is the most frequently diagnosed malignancy and second leading cause of cancer death . DespiteTwo selective COX-2 inhibitors, celecoxib (Celebrex) and rofecoxib (Vioxx), were approved for the treatment of arthritis by the United States Food and Drug Administration (FDA) in 1999. Until the recall of Vioxx in September, 2004, these two compounds plus other selective COX-2 inhibitors valdecoxib (Bextra) and meloxicam (Mobic) were widely utilized in the United States for pain relief and treatment of osteoarthritis and rheumatoid arthritis. The time period between approval of Celebrex to the recall of Vioxx provides an approximate six-year window for evaluation of exposure to such compounds by a case control approach. The current case control study was designed to test the chemopreventive value of selective COX-2 blockade against human breast cancer.We studied 323 cases of invasive breast cancer with histological verification based upon review of the pathology records, and 649 group-matched controls with no personal history of cancer and no current breast disease based on screening mammography. Cases were sequentially ascertained for interview at the time of their diagnosis during 2003 through September, 2004 at The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (CHRI), Columbus, Ohio. There were no refusals to participate among cases. The controls were ascertained from the mammography service of the cancer hospital during the same time period and frequency matched to the cases at a rate of 2:1 by five-year age interval, race, and place (county) of residence. Controls were sequentially ascertained for each matching category resulting in a stratified random sample. Among women eligible for participation, 95% completed the questionnaire.Critical information on exposure to NSAIDs and other factors were obtained utilizing a standardized risk factor questionnaire. The questionnaires were administered in person by trained medical personnel prior to definitive surgery or treatment for the cases and at the time of screening mammography for controls. The data variables collected consisted of demographic characteristics, height, weight, menstrual and pregnancy history, family history of breast and ovarian cancer, comprehensive information on cigarette smoking, alcohol intake, pre-existing medical conditions , and medication history including over the counter (OTC) and prescription NSAIDs, and exogenous hormones. Regarding selective COX-2 inhibitors and other NSAIDs, the use pattern , and the type,  were recorded. Data on the related analgesic, acetaminophen were collected for comparison with selective COX-2 inhibitors and other NSAIDs.Case-control differences in means and frequencies were checked for statistical significance by t-tests and chi square tests, respectively. Effects of the selective COX-2 inhibitors as a group were quantified by estimating odds ratios and their 95% confidence intervals. Odds ratios were adjusted for age and classical breast cancer risk factors  by logistic regression analysis ,14. AdjuPertinent characteristics of the cases and controls are given in Table Table Table This is the first observation of a significant risk reduction in human breast cancer due to intake of selective COX-2 inhibitors. Standard daily dosages of celecoxib (200 mg) or rofecoxib (25 mg) taken for two or more years were associated with a 71% reduction in breast cancer risk. Comparator NSAIDs with non-selective COX-2 activity  also produced significant risk reductions, although their observed effects were not as strong as selective compounds. In contrast, neither acetaminophen nor 81 mg (baby) aspirin changed the risk of breast cancer.arom genetic expression and local estrogen biosynthesis [In general, NSAIDs inhibit cyclooyxgenase which is the key rate-limiting enzyme of prostaglandin biosynthesis -17. Moleynthesis -25. The Enthusiasm for the use of selective COX-2 blocking agents in the chemoprevention of breast cancer and other malignancies has been tempered by reports of adverse effects on the cardiovascular system leading to the recall of popular anti-arthritic compounds, rofecoxib (Vioxx) and valdecoxib (Bextra). However, such studies involved supra-therapeutic dosages given over long periods of time without consideration of body size or individual differences in metabolism .We observed a significant reduction in the risk of human breast cancer due to intake of selective COX-2 inhibitors. Chemopreventive effects against breast cancer were associated with recommended daily doses of celecoxib (median dose = 200 mg) or rofecoxib (median dose = 25 mg) for an average duration of 3.6 years. Notably, selective COX-2 inhibitors (celecoxib and rofecoxib) were only recently approved for use in 1999, and rofecoxib (Vioxx) was withdrawn from the marketplace in 2004. Nevertheless, even in the short window of exposure to these compounds, the selective COX-2 inhibitors produced a significant (71%) reduction in the risk of breast cancer, underscoring their strong potential for breast cancer chemoprevention.This research was supported in part by a grant from Pfizer, New York, NY, and grant P30 CA16058 from the National Cancer Institute, Bethesda, MD.REH designed and directed the study. JBD coordinated data collection and quality control, and assisted in the interpretation of results. GAA assisted in the analysis and interpretation of results.The pre-publication history for this paper can be accessed here:


Lumbar bone mineral density (BMD) determination by dual photon absorptiometry was used to study the influence of adjuvant chemotherapy for premenopausal breast cancer on the risk of premature osteoporosis. Six cycles of combination chemotherapy caused ovarian failure in 31 of 44 (71%) women, amenorrhoea mostly already beginning during treatment. In contrast, only seven of 44 (16%) women, who were pair-matched for age and year of breast cancer surgery and had not been treated with chemotherapy, were post-menopausal at the time of measurement. The mean interval after breast surgery was 3.5 years. The significantly decreased BMD in the treated group (1.17 compared to 1.29 g cm-2) could only be explained by the high incidence of menopause in these women, which on average occurred 10 years prematurely. Extrapolation of these findings suggests that adjuvant chemotherapy may precipitate osteoporotic fractures by some 10 years in a considerable proportion of women cured of premenopausal breast cancer.


The c.1-34T>C 5' promoter region polymorphism in cytochrome P450c17 (CYP17), a key enzyme in the biosynthesis of estrogen, has been associated with breast cancer risk, but most previous studies have been relatively small.CYP17 c.1-34T>C polymorphism and calculated odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression models.We genotyped 1,544 incident cases of primary breast cancer and 1,502 population controls, all postmenopausal Swedish women, for the CYP17 c.1-34T>C and breast cancer risk, OR 1.0 (95% CI 0.8–1.3) for the A2/A2 (CC) carriers compared to the A1/A1 (TT) carriers, regardless of histopathology. We detected an interaction between CYP17 c.1-34T>C and age at menarche (P = 0.026) but regarded that as a chance finding as no dose-response pattern was evident. Other breast cancer risk factors, including menopausal hormone use and diabetes mellitus, did not modify the overall results.No overall association was found between CYP17 c.1-34T>C has a role in breast cancer etiology, overall or in combination with established non-genetic breast cancer risk factors.It is unlikely that  Estrogen is a central factor in the etiology of breast cancer . One of CYP17 (c.1-34T>C) has been suggested to create an additional binding site for the transcription factor Sp1 [CYP17 polymorphism and advanced breast cancer.A single T  to C  base change in the 5' promoter region of P17 c.1-3T>C has bctor Sp1 -8 but otctor Sp1 -21. A rector Sp1  but thisctor Sp1 , who fouCYP17 c.1-34T>C and breast cancer risk among Swedish women. We also explored potential interaction between genotype and menopausal hormone use and diabetes mellitus as well as age at menarche, age at menopause, age at first birth, parity and body mass index.In a population-based study about causes of postmenopausal breast cancer, which included 1,544 cases and 1,502 controls, we evaluated the association between This nation-wide case-control study included all Swedish women, 50 to 74 years of age and resident in Sweden between October 1993 and March 1995. During that period, all incident cases of primary invasive breast cancer were identified at diagnosis through the six regional cancer registries in Sweden, to which reporting of all malignant tumors is mandatory. Controls that matched the cases in 5-year age strata were randomly selected from the Swedish Registry of Total Population. Of the eligible cases and controls, 3,345 (84%) and 3,454 (82%), respectively, participated in the study by providing detailed information via questionnaires about menopausal hormone use, reproductive history, and other lifestyle factors. Results from the founding study have been published -28.in situ of the cervix or non-melanoma skin cancer) were eligible for the present study. Premenopausal women and those with a previous malignancy were excluded. From the eligible participants, we randomly selected 1,500 cases and 1,500 age-frequency (in 5-year groups) matched controls. We were interested in exploring the interaction between CYP17 c.1-34T>C and menopausal hormones and diabetes mellitus as both are breast cancer risk factors, possibly acting through increased levels of exogenous and endogenous estrogens, respectively. Thus, in order to increase statistical power in the subgroup analyses, we further selected all remaining eligible women (191 cases and 108 controls) that had used menopausal hormones (estrogen only or any combination of estrogen and progestin) for at least 4 years and all women (110 cases and 104 controls) with self-reported diabetes mellitus. In addition, 345 eligible controls from the founding study, originally selected for a parallel endometrial cancer study but who fulfilled the inclusion criteria to the breast cancer study, were added to our control sample. In total, we selected 1,801 cases and 2,057 controls.Only postmenopausal women with a first primary breast cancer without any previous malignancy  for cases and controls were 73% and 61%, respectively.We isolated DNA from 3 ml of whole blood using a Wizard Genomic DNA Purification Kit  according to the manufacturer's instructions. From non-malignant cells in paraffin-embedded tissue, we extracted DNA using a standard phenol/chloroform/isoamyl alcohol protocol . The stuCYP17 c.1-34T>C (rs743572) genotyping: Multiplex fluorescent solid-phase minisequencing [We used two methods for quencing ; and dynquencing . Resultsquencing . Twenty-CYP17 genotype, estimates of the main effect of CYP17 could be biased. We did not expect confounding by other known breast cancer risk factors as they most likely would be intermediates between the genetic variant and breast cancer. We nevertheless included important breast cancer risk factors as co-variates in the models and assessed changes in the estimates of association between CYP17 genotype and breast cancer to investigate if there were any indications of such interrelations. Breast cancer risk factors other than menopausal hormone use that were analyzed for gene-environment interaction were: Age at menarche  and age at menopause  ; age at first birth ; parity ; and body mass index one year prior to diagnosis . The last three were categorized according to cut-offs that in previous studies have been shown to be informative with regard to the variables' influence on breast cancer risk. The likelihood ratio test and the Wald test statistic were used to test for interaction. We performed all analyses using the SAS system .Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression models with the A1/A1 genotype as the reference group. We conditioned the models on age (due to matching) and on duration of menopausal hormone use and self-reported diabetes mellitus (due to our over-sampling scheme). The conditioning variables were thus age (5-year age groups), menopausal estrogen only use , menopausal estrogen and progestin use  and self-reported diabetes mellitus (yes or no). We chose to condition because in the presence of interaction between any of the selection variables and CYP17 c.1-34T>C genotypes from 1,544 breast cancer cases and 1,502 controls. Among the controls, genotype frequencies were similar to previously published frequencies in Caucasian populations [We obtained ulations -11,20,21We reproduced previously established breast cancer risk factor associations in this study population ,25,33  have recently been reviewed [CYP17 genotype may be associated with use of menopausal hormones, an important risk factor for breast cancer, but results have been inconsistent [A possible mechanism for the carriers . Studiesreviewed . Increasreviewed ,32 as wereviewed ,40, but reviewed  and one reviewed . Resultsreviewed ,18,43-45reviewed ,21,46,47nsistent ,21,48-50We identified an interaction with age at menarche, but considered it unlikely that the A2/A2 genotype would increase breast cancer risk in women with age at menarche less than 13 years without also moderately increasing risk in women with age at menarche between 12 and 14 years. Instead, the A2/A2 genotype decreased risk in the latter group, which is difficult to explain biologically. A similar pattern was seen for the interaction with age at menopause. To our knowledge, other groups have not reported comparable findings and we therefore believe the results to be caused by chance alone.CYP17 c.1-34T>C does not appear to have any major influence on breast cancer risk. Despite this negative finding, it would be prudent to investigate common variants covering the entire CYP17 gene before concluding that this gene has no role in breast cancer etiology.CI = confidence interval; CYP17 = cytochrome P450c17; DASH = dynamic allele-specific hybridization; OR = odds ratio; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism.The authors declare that they have no competing interests.Kristjana Einarsdóttir wrote the article and performed the statistical analyses. Keith Humphreys and Gudrun Jonasdottir advised on the statistical methods. Tove Rylander-Rudqvist and Susanne Ahlberg performed the genetic analyses. Magnus Ingelman-Sundberg and Ingemar Persson conceived and designed the study. Elisabete Weiderpass oversaw the project progress and took part in interpretation and writing the article. Jianjun Liu and Kee Seng Chia critically revised the manuscript. Per Hall was involved in drafting the article, assisted in interpretation of data, and gave final approval of the version to be published. Sara Wedrén contributed to conception and design of the project, coordinated sample collection, was involved in writing the article, assisted in analysis and interpretation of data and gave final approval for publication. All authors read and approved the final manuscript.Microsoft Word document detailing the PCR reaction conditions as well as primer and probe sequences used to perform the minisequencing and DASH methods.Click here for file