P=0.03) as compared with those who perceived themselves as thin. Self-reported obesity compared with leanness at diagnosis was associated with a relative risk of 0.78 . Women who reported having been plump at age 10 years and overweight or obese at diagnosis had a relative risk of 0.75  as compared with those who reported being thin at age 10 years and at diagnosis. Findings for three related measures of body fatness suggested that obesity is associated with a reduced risk of premenopausal breast cancer. There was no association between sports participation and breast cancer risk in these premenopausal women. The relative risk for spending an average of more than 1 h per week in sports compared with less from ages 12 to 30 years was 1.00 .We examined the relationship between body fatness, sports participation and breast cancer risk in 1560 premenopausal cases and 1548 controls, from three related population-based case–control studies in the UK. Half of the women with breast cancer were aged less than 36 years at diagnosis. Women who perceived themselves as plump at age 10 years had a relative risk of 0.83 (95% confidence interval 0.69–0.99,  Obesity in adulthood has been related to a decreased risk of premenopausal breast cancer, but data on the relationship with body fatness in childhood and adolescence are limited , resultsWe report here on the relationship between premenopausal breast cancer, body fatness at age 10 years and in adulthood, and sports participation during puberty, late adolescence and early adulthood from three related UK case–control studies.This investigation includes data from three related UK population-based case–control studies of breast cancer of a similar design: cases were diagnosed between 1 January 1982 and 31 December 1985, below age 36 years in the first ; betweenCases were women with an incident invasive breast cancer, identified chiefly through the Regional Cancer Registries, and supplemented by hospital discharge records from Hospital Activity Analysis registers and patient lists kept at major treatment centres. Permission from hospital consultants and general practitioners was obtained before names were disclosed by the Cancer Registry and before women were contacted. Breast cancer was confirmed when case notes were reviewed in 97% of cases. Out of 1049, 838 and 635 eligible cases in the three studies of women less than 36, 36–45 and 46–54 years of age, 755 (72%), 644 (77%) and 535 (84%), respectively, were interviewed. Reasons for nonparticipation included consultants’ or general practitioners’ refusal , woman's own refusal , death  and relocation from the study area .For each case, one age-matched control was randomly selected from the list of the case's general practitioner. If a chosen control could not be interviewed, a further age-matched control was selected at random. Of the 755, 644 and 535 first eligible controls in the studies of women less than 36, 36–45 and 46–54 years of age, 675 (89%), 588 (91%) and 491 (92%), respectively, were interviewed. Reasons for first controls’ nonparticipation included refusal by the general practitioners  or the woman herself .Participants were interviewed in their homes using a standardised structured questionnaire, each case–control pair by the same trained interviewer, on average 2 years after diagnosis in each study. Women were asked to state whether they had been thin, average or plump at age 10 years, their weight at age 20 years and their weight and height at diagnosis . Participants were further asked about the number of hours spent per week in compulsory school sports at ages 12, 14, 16 and 18 years as well as the number of hours per week in all other sports at ages 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 and 50 years, as appropriate. They were also asked about their menstrual history and other risk factors for breast cancer. The information requested, the structure of the questionnaire and phrasing of the questions was similar in the three studies.−2, respectively, at age 20 or <22.5, 22.5–25.0 or >25.0 kg m−2, respectively, at diagnosis. The subjects were also crossclassified by (a) their body fatness at age 10 years and their BMI at age 20 years and (b) their body fatness at age 10 years and their BMI at diagnosis, according to the following nine categories: thin/thin; thin/average; thin/overweight; average/thin; average/average; average/overweight; plump/thin; plump/average and plump/overweight.Perceived body fatness at age 10 years was classified according to the women's responses as thin, average or plump. Body mass index (BMI) at age 20 years and at around the time of diagnosis was calculated as weight (kg) divided by the square of height (m); women were categorised as being thin, average or overweight if they had a BMI of <20.0, 20.0–22.5 or >22.5 kg mat around puberty (ages 12–14 years), late adolescence (ages 16–18 years), early adulthood (ages 20–30 years), at around diagnosis  and during all young ages (ages 12–30 years).We used the self-reported information on sports participation to create indices of the average number of hours per week of participation in compulsory school and other sports n=306), bilateral oophorectomy (n=32) or hysterectomy and were aged 48 years or older at diagnosis (n=154). All analyses were restricted to premenopausal women, with users of hormone replacement therapy in this group being further excluded (n=270). Thus, the final study group comprised 1560 cases and 1548 controls.Women were defined as postmenopausal if they reported having had natural menopause  and region of recruitment. Odds ratios as estimates of relative risks (RRs) and 95% confidence intervals (CIs) are reported. Where more than two groups were compared, variances were estimated by treating the relative risks as floating absolute risks (FARs). This approach yields floated standard errors and floated confidence intervals . Present−1), age at menarche , time from menarche to onset of regular cycles  and history of irregular menstrual cycles . Correspondingly, we also adjusted results of early participation in sports for perceived body fatness at age 10 years , BMI at age 20  years, BMI at diagnosis  years and menstrual characteristics as above.All estimates were routinely adjusted for parity and age at first birth , height , use of oral contraceptives , alcohol consumption , family history of breast cancer  and socioeconomic status . In addition to these standard factors, we further adjusted results for perceived body fatness at age 10 years, the number of hours of participation in sports at young ages and at diagnosis  quoted are two sided.All analyses were performed using Stata version 8.1 . All statistical significance levels (Characteristics of cases and controls are given in P=0.03)  . Women wP=0.03) .−2, respectively. Only 0.2 and 6.5% were obese (i.e. had a BMI ⩾30 kg m−2) at age 20 years and at diagnosis, respectively. The RR in the small subgroup of women who were obese at diagnosis was 0.78 (95% CI 0.56–1.06), as compared with those who had a BMI of less than 22.5 kg m−2. As compared with women who were under- or normal weight, those who reported to be overweight (i.e. had a BMI ⩾25 kg m−2) at age 20 years or at diagnosis were not at a significantly altered risk of breast cancer (RR=0.93 (95% CI 0.72–1.19) and 0.98 (95% CI 0.80–1.21), respectively).Neither BMI at age 20 years nor BMI at diagnosis, within the narrow BMI range under study, was significantly related to risk among premenopausal women . Among cThe average numbers of hours that controls reported engaging in sports, at ages 12–14, 16–18, 20–30 years, at all young ages (12–30 years) and at pseudodiagnosis were 4.7, 2.9, 1.3, 2.8 and 1.3 per week, respectively. Sports participation at these ages was not related to breast cancer risk . The RR Further adjustment of perceived body fatness at age 10 for participation in sport or age at menarche and other menstrual factors, and participation in sport for body fatness at different ages or menstrual factors, did not alter the risk estimates . SimilarThe observed relationships between breast cancer, perceived body fatness at age 10 years and participation in sports at ages 12–30 years did not vary significantly with levels of other characteristics  and 4. IP=0.03) compared with those who perceived themselves as thin. Self-reported obesity compared with leanness at diagnosis was associated with an RR of 0.78 , and women who reported having been both plump at age 10 years and overweight at diagnosis had an RR of 0.75  as compared with those thin at age 10 years and at diagnosis. We did not find a relationship between any level of BMI at age 20 years and risk, but few women reported having been obese or even overweight at that age. The modest evidence of an inverse association between body fatness at age 10 years and risk of breast cancer among premenopausal women in our data is consistent with other reports . Recent reviews concluded that physical activity both at young ages and in adulthood reduced the risk of breast cancer; We did not observe any association between sports participation at puberty, late adolescence, early adulthood or at diagnosis and breast cancer risk in premenopausal women. The RR of breast cancer associated with an average of more than an hour each week of sports participation from ages 12 to 30 years, as compared with less often, was 1.00 . However, sports participation could, however, be assumed to account for the major proportion of physical activity at young ages.This is the largest study of premenopausal breast cancer on body fatness and sports participation at young ages with an over 80% power to detect an RR of 0.77 or less for at least 4 h weekIn summary, this large study provides evidence of an inverse association between body fatness, but not physical activity, at young ages and the risk of breast cancer in premenopausal women.


Cyp19) in the development of this disease. Our genotyping of 348 cases with breast cancer and 145 controls  for a published tetranucleotide repeat polymorphism at intron 4 of the Cyp19 gene revealed the presence of six common and two rare alleles. Contingency table analysis revealed a significant difference in allelic distribution between cases and controls . The allele measuring 171 bp was over-represented in cases; of 14 individuals homozygous for this allele, 13 were cases. These individuals had a higher incidence of cancer in family members and an earlier age at diagnosis than other cases. In sequencing Cyp19's coding exons and regulatory regions, we discovered a perfect association between a silent polymorphism  and the high-risk genotype. Our conclusion is that constitutional genetic variation at the Cyp19 locus is associated with the risk of developing breast cancer, with the 171-bp allele serving as the high-risk allele. © 1999 Cancer Research CampaignThe activity of the aromatase enzyme, which converts androgens into oestrogens and has a major role in regulating oestrogen levels in the breast, is thought to be a contributing factor in the development of breast cancer. We undertook this study to assess the role of constitutional genetic variation in the human aromatase gene (


P=0.69).In a pooled analysis of two prospective studies with 35 004 Japanese women, green-tea intake was not associated with a lower risk of breast cancer (222 cases), the multivariate relative risk for women drinking ⩾5 cups compared with <1 cup per day being 0.84 (95% confidence interval 0.57–1.24, Trend  Although laboratory studies have suggested a protective effect of green tea on breast cancer risk, few epidemiological studies have examined the association. A case–control study of Asian Americans in the United States found a lower risk of breast cancer in association with green-tea intake  and 95% confidence interval (CI) of breast cancer according to green-tea consumption, using Cox's proportional-hazards regression with the adjustment for age and potential confounders. To obtain a summary measure of results between Cohorts 1 and 2, the general variance-based method was used to pool each RR and 95% CI. We repeated all analysis after excluding breast cancer cases diagnosed in the first 3 years of follow-up (38 in Cohort 1 and 40 in Cohort 2). We found no inverse association between green-tea intake and the risk of breast cancer, whether data for Cohorts 1 and 2 were combined or separated (P=1.00) among those consuming soybean soup less than daily (35 cases) and 0.81  among those consuming soybean soup daily (185 cases).When we performed stratified analyses according to variables used as potential confounders in the multivariate analysis, the association between the consumption of green tea and breast cancer risk was not substantially modified. In a stratified analysis according to soybean soup intake (less than daily and daily), the pooled multivariate RRs (95% CI) of breast cancer for women who drank five or more cups per day, as compared with women who drank less than one cup per day, were 0.95 . The corresponding risks for coffee were 0.78 (0.53–1.13) and 0.81 (0.55–1.18) (Trend P=0.44).We found no relation between breast cancer risk and the consumption of black tea and coffee. The pooled multivariate RRs (95% CI) compared with women who never drank black tea were 0.90 (0.64–1.27) for those drinking black tea occasionally and 1.44 (0.77–2.69) for those drinking one or more cups per day (Trend We found no association between green-tea consumption and breast cancer incidence among Japanese women who consumed green tea much more frequently than women in Western countries. The results agree with a prospective study (Two hospital-based cohort studies (We also found no relationship between the risk of breast cancer and the consumption of black tea or coffee. Our results are consistent with the judgment of World Cancer Research Fund that it is possible that black tea has no relationship with breast cancer risk and that it is convincing that coffee has no relationship with the risk of breast cancer .In conclusion, in a pooled analysis of two prospective cohort studies in rural northern Japan, we found no association between consumption of green tea, coffee or black tea and the risk of breast cancer.


Numerous studies and meta-analyses have shown that hormone replacement therapy (HRT) for menopausal symptoms increases the risk of developing breast cancer, estimated to be 2.3% for each year of use. The influence of different oestrogen–progestin regimens has still not been fully evaluated. Using longitudinal data from the population-based prescription database of the county of North Jutland, Denmark, and the Danish Cancer Registry, we examined the risk of developing breast cancer in relation to HRT in a cohort of 78 380 women aged 40–67 years from 1989 to 2002. A total of 1462 cases of breast cancer were identified during a mean follow-up of 10 years. Use of HRT did not increase the risk of breast cancer in women aged 40–49 years. Restricting the cohort to 48 812 women aged 50 years or more at entry, of whom 15 631 were HRT users, we found an increased risk associated with current use of HRT . The risk increased with increasing duration of use and decreased with time since last HRT prescription, reaching unity after 5 years. No material risk difference was observed among the various HRT-regimens. This population-based cohort study provides further confirmation that HRT increases the risk of developing breast cancer in women aged 50 years or more. Numerous studies have shown that hormone replacement therapy (HRT) with oestrogen with or without progestin for menopausal symptoms increases the risk of breast cancer. A collaborative reanalysis of data from 51 epidemiological studies of 52 705 women with breast cancer and 108 411 women without breast cancer demonstrated a 2.3% increase in breast cancer risk for each year of HRT use . The higWe have conducted a study on HRT use in relation to the risk of breast cancer using the population-based prescription database of North Jutland, Denmark, which allows for a complete non-self reported history of HRT exposure, including the specific type, to be derived for every woman in the county.The study was conducted within the population of North Jutland, a county with nearly 500 000 inhabitants, representing approximately 9% of the total Danish population. The National Health Service in Denmark provides tax supported health care for all inhabitants, guaranteeing free access to general practitioners, hospitals and public clinics, and refunds a variable proportion of the costs of medication prescribed by physicians through a computerised accounting system. In North Jutland, this accounting system also provides prescription data to the Pharmaco-Epidemiologic Prescription Database, which was initiated in 1989  and the From the files of the CPR, we identified 83 873 women who were 40–66 years of age at any time during the period 1 January 1989 to 31 December 2002 and resident in the county of North Jutland. These women were linked to the Danish Cancer Registry to identify cases of breast cancer occurring through 2002. The Danish Cancer Registry has recorded incident cases of cancer on a nation-wide basis since 1943 with accurate and virtually complete ascertainment , and sinThe remaining 82 429 women were linked to the Pharmaco-Epidemiologic Prescription Database. We excluded 1079 women who received prescriptions for sex hormones other than those used in HRT  including androgens, during 1989–2002, and 2970 women who had used systemic HRT before the age of 40 years. Among the remaining 78 380 women, we identified 17 466 who received at least two prescriptions for systemic HRT  from 1 January 1989 until 31 December 2002. In Denmark, there is normally no reimbursement for oral contraceptives, but we included women recorded with reimbursed prescriptions for oral contraceptives, since this indicates that the hormones were given for reasons other than contraception. Prescription of nonsystemic HRT was not judged as HRT exposure for the purposes of the present study.recency of use ;number of prescriptions ;levonorgestrel, norethisteron, norgestimat, desogestrel, gestoden), sequentially combined oestrogen–progesterone derived progestin (medroxyprogesteron), continuously combined oestrogen–testosterone derived progestin (norethisteron), tibolone, and progestins. Users contributed person-years to the appropriate group until they received a prescription for HRT from one of the other groups, and from that time on they contributed person-years to a category of mixed use.type of first prescription of HRT  for nonmelanoma skin cancer), date of death, of migration from North Jutland, or 31 December 2002, whichever came first. Since women with only one prescription may never have actually taken the drug, we classified such women as nonexposed. The follow-up time was stratified according to use of HRT in unexposed time (less than two prescriptions) and exposed time (two or more prescriptions) . The expWe computed rate ratios as the breast cancer incidence rate for HRT exposure divided by the breast cancer incidence rate for nonexposure (<two prescriptions). Age-specific as well as age-standardised  rate ratios were calculated. In addition, we performed analyses based on Cox proportional hazards models with age as the time scale. These analyses were adjusted for calendar-period as a time-dependent covariate with two levels (1989–1996 and 1997–2002) and number of children and age at first child obtained from the CPR as time-dependent linear variables. Tests were based on the likelihood ratio test statistics calculated from Cox's partial likelihood. Confidence intervals were based on Wald's test of the corresponding regression parameters, that is, on the log scale for the rate ratios. The statistical analyses were performed using SAS version 8.02. The proportional hazards assumption of the Cox models was tested by visual inspection of transformations of the survival function using standard techniques.In the cohort of 78 380 women we identified 1462 cases of breast cancer during a mean follow-up of 10 years (maximum 14 years). Other details of the cohort including year of entry, number of children, and age at first birth are presented in Women aged 40–49 years are likely to represent a mixture of pre-, peri-, and postmenopausal women and may have used HRT for a variety of reasons, not only for menopausal symptoms. To study HRT use mainly in postmenopausal women and having no information on menopausal status, we restricted the subsequent analyses to the person-time contributed by women after they had reached 50 years of age. With this restriction, the cohort was reduced to 48 812 women with 869 breast cancers during a mean follow-up of 7.6 years . HoweverOf the 48 812 women aged 50 years or more, 15 631 or 32% had filled two or more prescriptions for HRT. Among women age 50 or older, current use of HRT was associated with a significantly increased risk of breast cancer, with an RR of 1.61 (95% CI 1.38–1.88), adjusted for calendar period, number of children and age at first birth . The risElevated risks were observed for oestrogens alone, progestins alone, mixed use, and sequential preparations of oestrogen and testosterone derived progestin, while the risk was not increased for sequential preparations of oestrogen and progesterone derived progestin, continuous combined therapy, and for tibolone. However, each of the latter estimates was based on fewer than 15 cases. A total of 790 women had at least one prescription for tibolone, but since tibolone is mostly prescribed when more conventional HRT is tolerated poorly, tibolone use is therefore generally included in the category of mixed use. When the category of mixed use was split into those with at least one prescription for tibolone (eight breast cancers in 2492 person-years) and those without tibolone (132 breast cancers in 40 894 person-years), adjusted RR estimates were 1.37 (95% CI 0.68–2.75) and 1.51 (95% CI 1.25–1.84) respectively.The breast cancer findings of the present population-based cohort study are consistent with those of prior observational studies of HRT . If, as The strength of this study is that it was conducted in a well-defined geographical area for which standardised population-based information is available on both exposure and disease. The data on exposure provide a full history of prescriptions for HRT during follow-up, including specific information on HRT formulation. We are confident the exposure information is reasonably complete since it is very unusual for the study population to consult doctors or purchase prescription drugs outside their own county, and HRT is available in Denmark by prescription only. In a study of validity of self-reported use of HRT 95% of Danish nurses registered in the prescription database as being prescribed HRT, reported having complied with the treatment . Overall, among women aged 40–49 years, we did not detect an increased breast cancer risk associated with HRT use, which may well be due to the fact that these women represent a mixture of pre-, peri-, and postmenopausal states. Despite the lack of control for age at menopause, our risk estimate of RR=1.61 (95% CI 1.38–1.88) for current HRT use in women aged 50 years or more agrees well with that reported for postmenopausal women from the With regard to the different types of HRT, our results were similar to those reported in the Million Women Study and a recent Danish study (In conclusion, this study reports elevated risk estimates for breast cancer with HRT use in women over age 50 years, increasing with duration of use, and with no evidence of increased risk 5 years after stopping HRT. Risk did not differ markedly for treatment with oestrogen alone, progestins alone, or combinations thereof.


ASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn have been associated with risk of breast cancer and lung cancer. Haplotype carriers are defined as the homozygous carriers of RAI IVS1 A4364GA, ERCC1 Asn118AsnT and ASE-1 G-21AG. We aimed to evaluate whether the three polymorphisms and the haplotype are associated to risk of colorectal cancer, and investigated gene-environment associations between the polymorphisms and the haplotype and smoking status at enrolment, smoking duration, average smoking intensity and alcohol consumption, respectively, in relation to risk of colorectal cancer.Single nucleotide polymorphisms (SNPs) are the most frequent type of genetic variation in the human genome, and are of interest for the study of susceptibility to and protection from diseases. The haplotype at chromosome 19q13.2-3 encompassing the three SNPs Associations between the three individual polymorphisms, the haplotype and risk of colorectal cancer were examined, as well as gene-environment interaction, in a Danish case-cohort study including 405 cases and a comparison group of 810 persons. Incidence rate ratio (IRR) were estimated by the Cox proportional hazards model stratified according to gender, and two-sided 95% confidence intervals (CI) and p-values were calculated based on robust estimates of the variance-covariance matrix and Wald's test of the Cox regression parameter.No consistent associations between the three individual polymorphisms, the haplotype and risk of colorectal cancer were found. No statistically significant interactions between the genotypes and the lifestyle exposures smoking or alcohol consumption were observed.ASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn polymorphisms and the previously identified haplotype are not associated with risk of colorectal cancer. We found no evidence of gene-environment interaction between the three polymorphisms and the haplotype and smoking intensity and alcohol consumption, respectively, in relation to the risk of colorectal cancer.Our results suggest that the  ERCC1 (excision repair cross complementary group1), ASE-1  and RAI , strongly associated with risk of post-menopausal breast cancer [RAI IVS1 A4364G, ERCC1 Asn118Asn and ASE-1 G-21A, of which haplotype carriers are defined as the homozygous carriers of RAI IVS1 A4364GA, ERCC1 Asn118AsnT and ASE-1 G-21AG.SNPs in DNA repair genes have been associated to risk of several types of cancer, reviewed in . Combinit cancer  and lungt cancer , and a rt cancer . Thus, tRAI encodes a specific inhibitor of the RelA subunit (p65) in the NF-κB transcription factor [RAI expression is increased in both adenoma and adenocarcinoma tissue compared to tissue from normal colonic mucosa in the same person [RAI expression occurs very early in the malignant transformation. However, contradictory results have been published as to whether the increased expression levels inhibit or promote apoptosis [ERCC1 and in particular RAI are strongly associated with risk of basal cell carcinoma [subunit p5 in the n factor  and as an factor . The regn factor . Inhibitn factor . In human factor ,11. The n factor . It was e person  indicatipoptosis ,13. The arcinoma ,15.ERCC1 encodes a subunit of the nucleotide excision repair (NER) complex, which is required for the incision step of NER [ERCC1 Asn118Asn polymorphism [ERCC1 C8092A polymorphism as well, for lung cancer patients [p of NER . For colmorphism -19, and patients . These eASE-1 encodes a nucleolar protein, and is positioned in an anti-sense orientation to and overlaps with the gene for ERCC1. It is possibly involved with the RNA polymerase I transcription complex [ASE-1 and whether the overlapping between ERCC1 and ASE-1 gene has biological relevance are unknown. complex . The exaERCC1 Asn118Asn, ASE-1 G-21A and RAI IVS1 A4364G was strongly associated to risk of post-menopausal breast cancer [In studies of Caucasians, the haplotype encompassing the three SNPs: The mechanisms underlying the possible association between alcohol consumption and tobacco smoking and risk of colorectal cancer are unknown. Smoking has consistently been associated with increased risk for adenomas/polyps in colon and rectum . FollowiTo our knowledge only two studies have been reported on gene-environment interactions with the predefined haplotype: A Norwegian case-control study on colorectal adenomas and adenocarcinomas  and our RAI is an obvious candidate gene, since it has been shown to be required for induction of apoptosis [p53 gene [In the present study, we hypothesized that female homozygous carriers of the previously identified haplotype have higher risk of colorectal cancer compared to individuals not carrying the homozygous haplotype, . We expepoptosis , possiblp53 gene . FurtherIn 1993–1997, a total of 160,725 individuals were invited to participate in the Danish prospective "Diet, Cancer and Health" (DCH) study, the inclusion criteria being age 50–64 years, born in Denmark and with no prior cancer diagnosis at the time of inclusion. In total, 57,053 participants accepted the invitation and were enrolled in the cohort . At enroDNA was isolated from frozen blood samples as described . All genERCC1 Asn118Asn (C/T) (rs #3177700), ASE-1 G-21A (rs #967591) and RAI A4364G (rs #1970764) genotyping were as previously described [Primers and probes for C/T) rs #77700, ASASE-1), 62°C (RAI) and 63°C (ERCC1) for 60s.Five μL reactions contained 100 nM of each probe, 900 nM primers, 20–50 ng DNA and 1x Mastermix . The polymorphisms were analysed for 40 cycles at 15s at 94°C, and an elongation step performed at 62°C  and separately according to topology of the cancer .Smoking intensity was calculated as the average of the self-reported smoking intensity for the age intervals 20–29, 30–39, 40–49 and 50–59 years and reported smoking status, smoking duration and alcohol consumption refer to the time of enrolment into the cohort study. Former smokers were defined as the participants who have been smoking regularly, at least one cigarette, pipe, cheroot or cigar daily, for at least one year, but who were not smoking daily at the time of enrolment. Smoking intensity for former smokers were calculated as the smoking intensity for each age intervals subtracted the periods of not smoking.For each of the continuous variables, the hypothesis of a linear association was evaluated using a linear spline with three boundaries, placed at the quartiles among cases, as covariates in the Cox model . The linInteractions between the genotypes and each of the environmental factors, smoking status, smoking duration, average smoking intensity and alcohol consumption, were analysed by estimating separate associations for each genotype . We tested the null hypothesis that the effect of the environmental factor was the same for the three genotypes. All models on gene-environment interactions were adjusted for smoking status (ever/never) or alcohol intake (yes/no). Thus, the effect of smoking intensity and duration were estimated among smokers only, and the effect of alcohol intake was estimated among alcohol consumers only. In the Cox regression model, we stratified according to gender allowing separate underlying risk for men and women, respectively. Hence, adjustment for HRT use in the analyses affects the risk estimates for women only. We used SAS PROC PHREG (version 8.2) for the estimations.An 80% power required IRRs between 1.3 and 1.4 per variant allele for the three polymorphisms in the analyses of a linear trend at a significance level of 5%. A similar power required an IRR of 1.5 for the homozygous haplotype versus non-carriers of the homozygous haplotype. The power calculations were made on the basis of the allele frequencies and number of participants in the present study.Fifty-two percent of the cases were diagnosed with colon cancer, 45% with rectal cancer, and in 3% of the cases the topography was not specified. Among cases, 96% had a diagnosis of adenocarcinomas, 2% of carcinoid tumor and 2% of various other histological subtypes. The distribution of cancer location was comparable for men and women, data not shown. The median consumption of alcohol was comparable between cases and sub-cohort members Table . The proASE-1 G-21A (0.17), RAI IVS1 A4364G (0.19), and ERCC1 Asn118Asn (0.36) were similar to the frequencies of alleles found in previous studies of Caucasians [The genotype distributions were in Hardy-Weinberg equilibrium among the sub-cohort members for all three polymorphisms both before and after gender separation, results not shown. The allelic frequencies of the variant allele for ucasians ,31. The ASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn and colorectal cancer was not statistically significant different  between genders.Overall, there were no statistically significant associations between the three polymorphisms and risk of colorectal cancer, either among men or women Table . The assThere were no significant effects of the three polymorphisms and the haplotype in relation to risk of colorectal cancer in the topological sub-groups , and no statistically significant effects were observed of the three polymorphisms and the high risk haplotype in relation to risk of colorectal cancer in either of the two age groups (≤ 60 years and > 60 years), results not shown. Thus, further statistical analysis includes all cases regardless of location of the cancer and age when diagnosed with colorectal cancer. The effect of smoking status at enrolment, smoking duration, average smoking intensity and intake of alcohol on colorectal cancer risk did not differ between genders, results not shown. However, since we previously have found gender-specific gene-environment interactions in the recent lung cancer study, the interaction studies on the polymorphisms and the haplotype were stratified by gender.ASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn polymorphisms, respectively, and smoking intensity or alcohol consumption , was observed among Norwegian women carrying the homozygous haplotype compared to women not carrying the homozygous haplotype –5.04, wa. A possiASE-1 G-21A polymorphism. The effect may very possibly be a chance finding due to low numbers of cases and members of the sub-cohort. Thus, no convincing statistical significant interactions were observed between the three genotypes and alcohol consumption or the smoking variables related with risk of colorectal cancer.Smoking intensity increased the risk of colorectal cancer among homozygous carriers of the variant We observed no interaction between the haplotype and smoking intensity on risk of colorectal cancer. Our previous study on risk of lung cancer found a borderline significant interaction (P = 0.06) between high smoking intensity (>20 g tobacco/day) and the haplotype related with risk of lung cancer, with a 2.03-fold increased risk of lung cancer per additional 5 g tobacco/day among women carrying the haplotype, while no association were observed among female non-carriers of the haplotype with a similar smoking intensity . Among wAlcohol consumption did not have a significantly different effect on carriers and non-carriers of the homozygous haplotype (P > 0.13) among men and women, respectively, in relation to risk of colorectal cancer. However, we detected an adverse effect of alcohol among male carriers of the homozygous haplotype, with an IRR of 1.20 (CI: 1.00–1.43), per 10 g alcohol intake per day. In the Norwegian study of colorectal cancer, the alcohol consumption was lower among cases than in the present study, and approximately 40% of the cases were abstainers . No inteASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn polymorphisms and the previously identified haplotype were not associated with risk of colorectal cancer. No statistically significant interactions were observed between the high risk haplotype, the three polymorphisms and smoking intensity and alcohol consumption, respectively, in relation to the risk of colorectal cancer.Overall, the The author(s) declare that they have no competing interests.RDH isolated the DNA for analysis, genotyped the DNA samples, made the data analysis and drafted the manuscriptMS and ORN contributed with strategies for data analysisAT and KO brought the idea of the "Diet, Cancer and Health" study and organized itHW and UV contributed with idea and design of the present studyAll authors contributed to interpretation of the results and to the final manuscript.The pre-publication history for this paper can be accessed here:ERCC1 Asn118Asn, ASE-1 G-21A and RAI IVS1 A4364G genotype-specific effects of average smoking intensity and consumption of alcohol on the risk of colorectal cancer. The table presents the genotypic distribution of the three polymorphisms in the ERCC1, ASE-1 and RAI genes for both cases and controls, and the incidence rate ratios of colorectal cancer risk associated with the three polymorphisms.Table 4 Gene-environment interaction stratified by gender. Click here for file


As tamoxifen stimulates ovarian steroidogenesis in premenopausal women, induces ovulation and increases the incidence of benign ovarian cysts, there has been concern that it might also increase ovarian cancer risk in women treated premenopausally. In a national case–control study in Britain, treatment histories were collected for 158 cases of ovarian cancer after breast cancer diagnosed at ages under 55 years and 464 controls who had breast cancer at these ages without subsequent ovarian cancer. Risk of ovarian cancer was not raised for women overall who had taken tamoxifen (odds ratio (OR)=0.9, 95% confidence interval (CI) 0.6–1.3) or for those treated when premenopausal  or perimenopausal . There was also no relation of risk to daily dose, duration or cumulative dose of tamoxifen, or time since last use. There was, however, a significantly raised risk in relation to non-hormonal chemotherapy. The results suggest that tamoxifen treatment of premenopausal or perimenopausal women does not materially affect ovarian cancer risk, but that non-hormonal chemotherapy might increase risk. Tamoxifen, a non-steroidal anti-oestrogen, has been used successfully in the treatment of breast cancer since the early 1970s, and, more recently, has been investigated as a prophylactic agent in women at high risk of breast cancer. In premenopausal women, however, tamoxifen induces ovulation , stimulaWith the appropriate ethical approval, data were extracted from each of the population-based cancer registries in Britain except the Northern registry, on all women with breast cancer occurring from 1983 to 1996, or to the most recent year of available data, if earlier. The Northern registry, covering 5% of the population, was excluded because the requisite data were not available. The restriction to breast cancers incident since 1983 was because a pilot investigation had shown that tamoxifen use at premenopausal ages had been relatively uncommon before that year. The data extracted included information on second cancers. Subjects in the registry files were eligible as cases if they (i) had had a registered primary breast cancer diagnosed during 1983–1996 below age 55 years, (ii) at that time had had no previous or concurrent cancer, (iii) had had a registered primary ovarian cancer diagnosed during 1988–1996, at least 3 months after incidence of the breast cancer, with no other second malignancy, except non-melanoma skin cancer or breast cancer, occurring in the intervening period.The controls were women with breast cancer diagnosed during 1983–1996 below age 55, selected from the same computer files as the cases. Two controls were chosen per case, individually matched on (1) date of incidence of the primary breast cancer, within ±6 months, (2) age at incidence of the primary breast cancer, within ±6 months, (3) registry region of residence at diagnosis of the primary breast cancer, (4) survival without second cancer after breast cancer diagnosis for the same length of time as that from breast cancer diagnosis to ovarian cancer diagnosis in the matched case (‘the index duration’). Additionally, it was required that controls (5) had not had an oophorectomy by the end of their index duration after diagnosis of breast cancer and (6) had case-notes that included the period up to the end of the index duration. As the last two criteria could not be determined from the cancer registry file, we examined the case-notes of potential controls chosen on criteria (1–4), and if criterion (5) or (6) was not met (or the case-notes could not be found), a replacement control was chosen on criteria (1–4), with repetition as needed.For each case and control, we extracted data from the hospital case-notes on demography, the general practitioner, tamoxifen treatment , other treatments of the first primary cancer, abdominal and pelvic radiotherapy before the first primary cancer, date of diagnosis of the first and second cancers and histological type of the ovarian cancer. Treatment histories for cases were included up to the date of diagnosis of the second cancer. For matched controls, they were included for the equivalent duration from diagnosis of the first primary (i.e. for the ‘index duration’). For subjects for whom tamoxifen data in the hospital notes were incomplete, we contacted general practitioners to obtain more complete information.In parallel with the ovarian cancer study, case–control studies of risks of several other cancers after breast cancer treatment were conducted, using the same procedures and data extraction forms. The controls from these studies who had not had an oophorectomy met the general criteria to be controls for the ovarian cancer cases, and we therefore used them as controls in the present analyses, to maximise power. To include these controls, we created matching strata for each of the critical variables , and assessed the effects of tamoxifen and other risk factors on ovarian cancer risk by calculation of stratum-matched odds ratios (ORs), as estimates of relative risks (and referred to as such below). Odds ratios (ORs), 95% confidence intervals (CIs) for the ORs, and trend tests were calculated by conditional logistic regression . In addiBecause cancer registry data do not include menopausal status, we included in the study all women diagnosed at ages under 55, and after we had extracted data from the case-notes we divided the subjects for analysis into (i) women known to be premenopausal, or presumed to be so because they were aged under 45 at diagnosis of breast cancer and of unstated menopausal status and (ii) women stated to be postmenopausal or perimenopausal  because of the age range of the study), and (iii) women aged 45 and older of unstated menopausal status.We identified 162 patients with ovarian cancer who met the study criteria and for whom full case-notes were found. We also identified 47 provisionally eligible case patients from cancer registry records whose case-notes could not be located or who had insufficient information for the study. Not all of these patients would have been eligible if their notes had been located. The 47 excluded case patients were similar to the patients with full case-notes with respect to age but slightly more likely to have been diagnosed early in the study period. Of the 162 patients with full case-notes available, at least one stratum-matched control patient was found for 158 subjects, who formed the study cases.Full case-notes were available, and extracted, for 464 eligible controls in strata that contained any cases. We also identified 182 potential controls from cancer registry records whose full case-notes could not be found. Some of these 182 subjects would have been ineligible if the notes had been located and a record of oophorectomy found. These 182 subjects were similar to the 464 controls with available notes with regard to age and slightly more likely to have been diagnosed in the early years of the study.Most cases and controls lived in England . In 40% The OR for ovarian cancer after tamoxifen was 0.9 (0.6–1.3) for the subjects overall , 1.0 , but was significantly raised in those who had been given non-hormonal chemotherapy  and thisRisk was not significantly related to smoking history, whether parous, or use of hormone replacement therapy , but increased significantly with increasing weight (OR=1.31 (1.05–1.63) per 10 kg for all subjects; 1.51 (1.11–2.04) per 10 kg for premenopausal subjects) (not shown in table).Tamoxifen stimulates the ovary when used premenopausally, raising concern that it might increase the risk of ovarian cancer , and cloOvarian cancer after breast cancer is not common, and previous studies have been based on small numbers. One case–control study has been published of ovarian cancer risk after tamoxifen treatment, based on only 34 cases, of whom seven were premenopausal. This found no increase in ovarian cancer, or relation of risk to duration of treatment, but the CIs were wide . A canceOur data show no effect of tamoxifen on risk of ovarian cancer overall, no effect of dose or duration, and no relation to duration since last use. The CIs for the dose and duration relations exclude all but modest effects. Subanalyses by menopausal status, identifying this as so-stated or based on age, did not show raised risk for either premenopausal or perimenopausal women. The only significant relationship with tamoxifen use was for the category of unknown dose and duration of use. This is most unlikely to represent a real effect, and is probably an artefact of the difficulty of gaining detailed data for patients who have died several years ago, who tend to be cases rather than controls. In our case–control study of endometrial cancer risk after tamoxifen in the same population , for whiIn principle, any tamoxifen-related risks of ovarian cancer might be confounded by genetic factors, if breast cancers occurring in patients with BRCA1, BRCA2 or PeutzJeghers syndrome (which predispose to ovarian as well as breast cancer) were more, or less, likely to be treated with tamoxifen than other breast cancers. A greater proportion (above 90%) of tumours in BRCA1 patients are oestrogen receptor-negative than occurs in other breast cancers at the same ages , so the Although risks of ovarian cancer were not related to non-tamoxifen hormonal treatments for breast cancer or to radiotherapy, there was a significantly raised risk in relation to chemotherapy. On the basis of small numbers we could not attribute this to a specific treatment, risks being similar for CMF as for other (heterogeneous) non-hormonal chemotherapy. Published data on ovarian cancer risks after chemotherapy for breast cancer are based on small numbers of cases, but in an Italian cohort there was a significant excess of ovarian cancer after chemotherapy for breast cancer, most of which was CMF . Breast


Twenty index cancers  were diagnosed amongst these 741 women during a median follow-up of 6.8 years. Ultrasound results separated the women with CA125 elevation into two groups. Those with normal ovarian morphology had a cumulative risk (CR) of index cancer of 0.15%  0.02–1.12) which is similar to that of the entire population of 22 000 women . In contrast, women with abnormal ovarian morphology had a CR of 24% (15–37) and a significantly increased relative risk (RR) of 327 (156–683). Ultrasound can effectively separate post-menopausal women with raised CA125 levels into those with normal scan findings who are not at increased risk of index cancer and those with abnormal findings who are at substantially increased risk of index cancer. © 1999 Cancer Research CampaignWe have previously shown that, in asymptomatic post-menopausal women, serum CA125 elevation is associated with a 36-fold increase in risk of ovarian cancer. This study was undertaken to assess the value of pelvic ultrasound for further stratification of ovarian cancer risk. Of 22 000 post-menopausal women, aged ≥ 45 participating in an Ovarian Cancer Screening Trial, 741 with a CA125 ≥ 30 U ml


DCN) and lumican (LUM) genes may contribute to breast cancer.The stroma is the supportive framework of biologic tissue in the breast, consisting of various proteins such as the proteoglycans, decorin and lumican. Altered expression of decorin and lumican is associated with breast tumors. We hypothesized that genetic variation in the decorin ).We investigated associations of 14 common polymorphisms in the Ptrend ≤ 0.05). The association with LUM rs2268578, evaluated further in SEARCH, was positive, although the odds ratios (OR) were weaker and not statistically significant. ORs were 1.4  for heterozygotes and 2.2  for homozygotes in the Mayo Clinic sample, and were 1.1  for heterozygotes and 1.4  for homozygotes in the SEARCH sample. In combined analyses, the ORs were 1.1  for heterozygotes and 1.6  for homozygotes. Positive associations for this polymorphism were observed for estrogen receptor-positive tumors in both the Mayo Clinic sample  and the SEARCH sample . In combined analyses, the ORs were 1.1  for heterozygotes and 1.9  for homozygotes.In the Mayo Clinic sample, six polymorphisms were associated with breast cancer risk (LUM rs2268578 was associated with breast cancer in the Mayo Clinic study, particularly estrogen receptor-positive breast cancer, weaker and modest associations were observed in the SEARCH sample. These modest associations will require larger samples to adequately assess the importance of this polymorphism in breast cancer.Although  Stromal changes are well documented in breast tumors ,2 and inDCN) gene or the lumican (LUM) gene are viable, but show skin fragility, marked reductions in tensile strength and loosely packed collagen fibers with abnormal sheath diameters [The stroma is the supportive framework of biologic tissue consisting of an extensive extracellular matrix that supports cells, separates tissues and regulates intercellular communication. The extracellular matrix is composed of different proteins: decorin and lumican are members of the small leucine-rich proteoglycan family of proteins and are involved in matrix assembly and structure, and in the control of cell proliferation . Knockouiameters ,8 – impliameters .Recent evidence supports an anti-oncogenic role for decorin. Injection of decorin protein into mammary carcinoma rodent models resulted in a marked reduction in both primary tumor growth and metastatic spread compared with animals injected with vehicle alone . Low leva priori, that common genetic variation in the DCN and LUM genes is associated with breast cancer risk. We evaluated this hypothesis using data from a clinic-based case-control study of breast cancer, with follow-up in a large British case-control study.Given the role of the stroma in breast cancer ,2, the oThe Mayo Clinic Breast Cancer study is an Institutional Review Board-approved, ongoing clinic-based case-control study initiated in February 2001 at Mayo Clinic, Rochester, MN, USA. The study design has been presented previously ,17. ClinConsecutive cases were women aged 18 years or older with histologically confirmed primary invasive breast carcinoma who were recruited within 6 months of their date of diagnosis. Women with a history of cancer (excluding nonmelanoma skin cancer) were ineligible. Cases lived in the six-state region that defines Mayo Clinic's primary service population . Although Mayo Clinic is widely perceived to be a specialty tertiary care facility, it also provides primary care for over 500,000 individuals per year.Control individuals without prior history of cancer (other than nonmelanoma skin cancer) were frequency matched on age (5-year age category), race and six-state region of residence to cases. Controls were recruited from the outpatient practice of the Divisions of General Internal Medicine and Primary Care Internal Medicine at Mayo Clinic, where they were seen for routine medical examinations.Written informed consent was obtained from all participants. Case participation was 69% and control participation was 71%. The present investigation genotyped Caucasian women (99% of study participants) enrolled up to 30 June 2005, representing 798 cases and 843 controls.Both the cases and controls completed a self-administered questionnaire comprised of known or suspected breast cancer risk factors, and they provided blood samples from which genomic DNA was isolated using the Gentra AutoPure LS Purgene salting out methodology . Quantities of 250 ηg genomic DNA were adjusted to 50 ηg/μl before genotyping and were verified using the PicoGreen dsDNA quantitation kit . The samples were bar coded to ensure accurate and reliable sample processing and storage.DCN and LUM genes within 5 kb of the largest cDNA isoform (genome build 35) were selected from the Caucasian samples within the HapMap Consortium's release 21 [r2 ≥ 0.80. tagSNPs were selected from these bins that met the criteria for predicted likelihood of successful genotyping using the Illumina GoldenGate Assay™ quality score metrics . We also included all putative functional SNPs  with MAF ≥ 0.05 identified in Ensembl version 34 . Eight SNPs in DCN  and six SNPs in LUM  were identified and examined in the Mayo Clinic study based on these two methods of selection.All SNPs in the lease 21 . We appllease 21  to bin SDCN and LUM SNPs were assayed at Illumina Corporation  using the GoldenGate Assay™ on the Illumina BeadLab [The  BeadLab -22 as paStudies of Epidemiology and Risk Factors in Cancer Heredity (SEARCH), an ongoing population-based study of cases with invasive breast cancer ascertained through the Eastern Cancer Registration and Information Centre in England , was useDCN rs3138165 and LUM rs2268578, the SNPs with the most significant associations with risk in each gene, was performed in the SEARCH study. These SNPs were selected for their strength of associations  with breast cancer risk to genotype in the SEARCH study comprising 4,470 cases and 4,560 controls with a 5' nuclease assay (Taqman®) using the ABI PRISM 7900 HT Sequence Detection System according to manufacturer's instructions . Primers and probes were supplied directly by Applied Biosystems [Evaluation of the Mayo Clinic findings for osystems  as AssayGenotypes from the Mayo Clinic cases and controls were used to estimate allele frequencies. Among control subjects only, the genotypes were compared with those expected under Hardy-Weinberg equilibrium using a Pearson goodness-of-fit test; no departures were found.r2 values [Pairwise linkage disequilibrium between SNPs was estimated with 2 values  using Ha2 values . IndividHaplotype analyses were performed to identify whether the association with breast cancer risk could be informed further by phased combinations of alleles within each gene. Such combinations of alleles on a single chromosome can provide information about the possible presence of nearby breast cancer risk alleles that were not genotyped. Haplotype frequencies for each gene were estimated using all SNPs within the gene, and associations between individual haplotypes and breast cancer risk were evaluated compared with all other haplotypes combined. Haplotype frequencies for each gene were estimated using all SNPs within the gene, and a global haplotype score test of no association between haplotypes and breast cancer risk was evaluated at the gene level by the method proposed by Schaid and colleagues . When thIn the SEARCH study, age-adjusted single-SNP associations were conducted using unconditional logistic regression under one-copy/two-copy and ordinal genetic models as described above.LUM rs2268578 in cases with estrogen receptor (ER)-positive and ER-negative tumors compared with control subjects because of recent reports of differential lumican protein expression by tumor ER status [In secondary analyses in both the Mayo Clinic and SEARCH studies, we stratified the data to evaluate the risk of breast cancer attributable to R status ,28.DCN and LUM genes are associated with breast cancer risk, corrections for multiple testing were not performed. Two-tailed P ≤ 0.05 was considered statistically significant.Analyses were implemented using Haplo.stats , the SASDCN representing 21 individual SNPs and six tagSNPs in LUM representing 15 individual SNPs were identified from the HapMap . This redundancy resulted from genotyping all putative functional SNPs in addition to the tagSNPs; only data from one DCN tagSNP (rs3138165) are therefore shown in subsequent analyses. Further, another DCN tagSNP (rs3138268) had MAF = 0 among control subjects and was excluded from further analyses.Eight tagSNPs in The Mayo Clinic cases and controls were adequately matched on age and region of residence, but differed in distribution by menopausal status, postmenopausal hormone use, age at menarche and smoking Table . CovariaDCN and three SNPs in LUM were associated with breast cancer risk in the Mayo Clinic sample: results of the one-copy/two-copy genetic model and of the ordinal model are presented in Table DCN rs7441 , DCN rs516115  and DCN rs3138165 . In LUM, the associations with each copy of the minor allele were similar for LUM rs2268578 , LUM rs10859110  and LUM rs17018765 .In the individual SNP analyses, three SNPs in LUM (P = 0.01) showed evidence of a significant association across haplotypes for breast cancer risk  – accounting for 71% of all estimated haplotypes – was associated with decreased breast cancer risk (P = 0.01), while two separate haplotypes with 6% (haplotype 2 h) and 7% (haplotype 2i) frequency were associated with increased risk (P ≤ 0.03).At the gene level, only sk Table . Of nineDCN rs3138165 and LUM rs2268578 SNPs were genotyped in the SEARCH study. The association of DCN rs3138165 in the Mayo Clinic sample was not confirmed in the SEARCH study, whereas LUM rs2268578 was positively associated with breast cancer risk but the ORs attenuated and did not reach statistical significance  and the SEARCH study sample . When the data from the two studies were pooled in age-adjusted and study-adjusted models, LUM rs2268578 was associated with increased risk among heterozygotes  and homozygotes  (Table Ptrend = 0.004). These data suggest that LUM rs2268578 or a variant in strong linkage disequilibrium with rs2268578 may be a risk factor for breast cancer.The LUM rs2268578 were at higher risk, compared with control individuals, of ER-positive breast cancer in both the Mayo Clinic study  and the SEARCH study  (Table Ptrend = 0.0003) in the Mayo Clinic sample and 1.1  in the SEARCH sample. In pooled age-adjusted and study-adjusted analyses, LUM rs2268578 was associated with ER-positive tumors among heterozygotes  and homozygotes . The corresponding per-minor allele risk was 1.1 .Women with two copies compared with no copies of the minor allele in The number of ER-negative cases was small in both samples and associations with breast cancer were not evident Table . These dLUM rs2268578 and breast cancer risk in two large independent case-control studies, although the association in the SEARCH sample was attenuated and did not reach statistical significance. Two copies of the minor allele in LUM rs2268578 were associated with an average 60% increased risk of breast cancer compared with women with no copies, and the data suggest increased risk for ER-positive tumors. DCN rs3138165 was positively associated with breast cancer risk in the Mayo Clinic sample but the finding was not confirmed in the SEARCH sample. Haplotypes from each gene were also associated with breast cancer risk in the Mayo Clinic sample.We found a positive association between LUM rs2268578 with breast cancer risk in the SEARCH sample may be due to a lack of causal association of this SNP with breast cancer, or the results may be a more valid estimate of effect. It is not uncommon for replicated findings to report ORs for the variant homozygote that are weaker compared with the initial study's findings, a phenomenon known as winner's curse [The weaker association of 's curse . Further's curse  – as we post hoc evaluation we also compared our results for LUM rs2268578 with those from the Cancer Genetic Markers of Susceptibility (CGEMS) genome-wide association study of breast cancer risk among 1,145 postmenopausal breast cancer cases and 1,142 controls of European ancestry from the Nurses' Health Study [P2 df = 0.52), compared with no copies, of the minor allele were not at risk of breast cancer despite similar MAF = 0.12 among controls. The CGEMS data did not report ER-positive or ER-negative results. At MAF = 0.12, if the true OR for homozygotes is in the range 1.1 to 1.5, then a much larger study than SEARCH or CGEMS would be needed to have sufficient power to replicate the association at either the conventional (P ≤ 0.05) or the genome-wide (P ≤ 10-7) level of significance. It should be noted that, even in the combined Mayo Clinic and SEARCH samples, there were only 108 cases and 75 controls homozygous for the minor allele. Comparable information from CGEMS is not available. It remains possible that the distribution of other exposures – for example, 60% of Mayo Clinic cases did not use postmenopausal hormones versus <30% in the Nurses' Health Study [In th Study ,32. Whenth Study  – may alLUM is associated with breast cancer is based on a recent series of reports by Watson and colleagues of altered regulation of LUM in human breast tumors [in situ hybridization and western blot techniques, LUM mRNA levels were significantly higher (P < 0.0001) in stroma associated with breast carcinoma compared with stroma associated with adjacent normal tissue in the same woman [LUM intronic SNP rs2268578 or from a SNP in strong linkage disequilibrium with rs2268578 may be consistent with increased protein expression of lumican in the study by Leygue and colleagues [LUM SNP(s) is needed in order to provide insight into the effects on risk.The hypothesis that genetic variation in t tumors ,11. UsinDCN and LUM genes were observed with haplotype analyses in the Mayo Clinic sample. Of note, the three haplotypes in LUM that were associated with breast cancer risk had in common the G allele at position 2 (rs17714469) and a graduated change in the combination of alleles in the last three positions  from GGA (haplotype 2a), associated with decreased risk, to AAA (haplotype 2h) and AAG (haplotype 2g), each associated with increased risk. It is possible that the association between breast cancer risk and these inferred haplotypes may be attributable to LUM rs2268578 or a SNP in strong linkage disequilibrium with rs2268578 because this single SNP was also associated with risk in both the Mayo Clinic and SEARCH samples. Further association testing in this chromosomal region – based on genotypes from a denser marker set – is required, however, in order to fully understand the nature of the relationship.Associations with breast cancer risk for the LUM rs2268578 may be due to the positive association with ER-positive breast tumors in both the Mayo Clinic and SEARCH samples, although the analyses were underpowered and were not based on a priori hypotheses. The association between lumican expression and ER-positive breast tumors is supported by the findings from two studies [n = 99) compared with ER-negative tumors (n = 6) had lumican protein expression ≥ 25th percentile among 140 women with breast cancer (P = 0.002). Mackay and colleagues [LUM gene was the most highly upregulated by a factor, on average, of 2.9-fold following aromatase inhibitor treatment, and the DCN gene was upregulated by a factor of 2.3. Collectively, these data suggest a potential mechanistic link between LUM expression and ER-positive tumors, which requires further investigation.The increased risk of breast cancer from  studies ,28. Trou studies  found thLUM SNP by ER status. Furthermore, it remains possible that the other variants in DCN and LUM that were not selected for genotyping in SEARCH may also influence breast cancer risk.The strengths of the present study include the incorporation of a second, independent large sample of cases and controls to confirm initial findings. Our study populations were enrolled from defined regions, were of Caucasian ancestry and were less probably influenced by population stratification . This doLUM rs2268578 was associated with breast cancer in the Mayo Clinic study, particularly ER-positive breast cancer, weaker and modest associations were observed in the SEARCH sample. Evaluation of this SNP in a larger study (such as the Breast Cancer Association Consortium) along with functional studies will be needed to adequately assess the importance of this SNP in breast cancer.Although DCN: decorin gene; 2 df: two degrees of freedom; ER, estrogen receptor; LUM: lumican gene; MAF: minor allele frequency; SEARCH, Studies of Epidemiology and Risk Factors in Cancer Heredity; SNP: single nucleotide polymorphism; UTR: untranslated region.CGEMS: Cancer Genetic Markers of Susceptibility; The authors declare that they have no competing interests.LEK drafted the manuscript. LEK, FJC and CMV revised the manuscript for intellectual content. CMV conceived the study hypothesis and selected the genes. FJC, VSP, ELG, DNR, XW, JRC and CMV designed the Mayo Clinic study and acquired data. ZSF, RAV, VSP and CGS performed the statistical analyses. FJC and CMV were responsible for funding for the Mayo Clinic Study. AMD, PDPP and DFE were responsible for study design and funding for, and SA for genotyping in, the SEARCH study. All authors contributed to data interpretation, and read and approved the final manuscript.


We used data from 765 cases and 564 controls in the population-based Australian Breast Cancer Family Study to investigate whether, in women under the age of 40, the profile of risk factors differed between breast cancer subtypes defined by joint oestrogen and progesterone receptor status. As hypothesised, no significant differences were found. BRCA1 mutation tend to be ER− (It has been postulated that joint oestrogen (ER) and progesterone receptor (PR) status might define aetiologically distinct subtypes of breast cancer , and comprised all women living in the metropolitan areas of Melbourne and Sydney who were aged less than 40 years at the diagnosis of a histologically confirmed first primary cancer of the breast (ICD-9 174). Recruitment began with a letter to the attending doctor, requesting permission to approach the woman. If permission was granted, a letter to the woman sought her participation. Of 1208 eligible cases, 856 (71%) agreed to participate. Reasons for nonparticipation included: death ; refusal by the attending doctor (8%) or the woman (14%); nonresponse by the attending doctor (1%) or the woman (1%); and the woman having moved and unable to be located (3%).Potential controls were women aged less than 40 years and living in the metropolitan areas of Melbourne and Sydney, who were selected from the electoral roll  using proportional random sampling based on the expected age distribution of the cases. Of 913 eligible controls, 600 (66%) agreed to participate, 27% refused and 8% did not respond.Participation by cases and controls included a face-to-face interview in the subject's home. The two questionnaires covered: (a) demographic and ethnic background; height; weight; medical history; reproductive factors; and use of oral contraceptives, hormone replacement therapy, tobacco, and alcohol and (b) family history of breast and other cancers. In the period 1992–1995, participation was restricted to cases and controls who could speak English, while in the period 1996–1999 non-English speakers were also included.We obtained both ER and PR status of the tumour for 694 (81%) participating cases  – 87 through immunohistochemical testing  but also in the magnitude of their risk of breast cancer. ‘Asian descent’ was defined as having any grandparent with a southeast Asian ethnic background ; 50 cases and 36 controls were of ‘Asian descent’. One case of ‘Asian descent’ carried a mutation. Thus, 765 cases (618 with known ER/PR status) and 564 controls were used in the analyses below.Polytomous logistic regression models were used to estimate breast cancer risk in five groups defined by ER/PR status  in relation to the following known or suspected risk factors: family history of breast cancer reported in a first-degree relative ; height ; body mass index  or interview (controls)); age at menarche ; parous ; number of live births ; age at first live birth ; and whether oral contraceptives had ever been used . Cut-points were chosen to be consistent with P=0.3). Among the 564 controls, the mean age at recruitment was 33.7 years (s.d. 4.4). Cases with known and unknown ER/PR status were similar with respect to educational level (P=0.4), marital status (P=0.5), and whether or not they had been born in Australia (P=0.1).The mean age at diagnosis of the 765 cases was 34.9 years (s.d. 3.6), with no difference in age between the five groups defined by ER/PR status (P=0.1).Among the 618 tumours with known ER/PR status, the distribution of ER/PR subtypes was 53% ER+PR+, 6% ER+PR−, 13% ER−PR+, and 29% ER−PR−, and there was no difference in this distribution between tumours diagnosed in 1992–1995 and those diagnosed in 1996–1999 (P<0.9). The inclusion of women of ‘Asian descent’, and/or those with a BRCA1 or BRCA2 mutation, and the exclusion of women with unknown ER/PR status, made essentially no difference to this finding.BRCA1 or BRCA2, no evidence that the effects of any of the major established risk factors differ for breast cancers defined by joint ER and PR status. This lack of heterogeneity in disease in young women accords with the analysis of As hypothesised, this population-based study in non-Asian Australian women under the age of 40 years found, with or without excluding cases known to carry a germline mutation in P-values of marginal significance, and some comparisons lacked statements about the statistical significance of observed differences in odds ratios. A third study, of early onset disease, found, as we did, no clear support for aetiologically distinct subtypes (BRCA1 or BRCA2 mutation carriers nor women of Asian descent had been excluded from these studies.The Danish data support the suggestion that ER/PR receptor status might define aetiologically distinct subtypes of breast cancer in older women. While two other population-based studies of breast cancer have claimed evidence for some differences in risk factor profile according to joint ER/PR receptor status (Our null finding must be tempered by considerations of statistical power – we could have missed modest differences in risk factor profiles. Each of the previous studies (


Breast cancer is the third commonest cancer in Ugandan women. Women present late for breast cancer management which leads to high mortality rates. The objective of the study was to assess the knowledge, attitudes and practices of Ugandan women concerning breast cancer and mammography.This was a descriptive cross-sectional study where 100 women reporting to the Radiology department were interviewed. We used consecutive sampling.  Interviewer-administered questionnaires were used to collect opinions of the participants. For data analysis, answers were described as knowledge, attitude, practice and they were correlated with control variables through the chi-square. Bivariate and logistic regression analyses were also used.Most of the women (71%) had no idea about mammography. More than 50% did not know about risk factors for breast cancer. The attitude towards mammography was generally negative. Regarding seeking for mammography; level of literacy, occupation and marital status were significant on bivariate analysis, however only level of literacy and employment remained the significant independent variables on logistic regression analysis. The main barrier to mammography was mainly lack of information.Women in this study had inadequate knowledge and inappropriate practice related to mammography as a procedure for breast cancer investigation. Breast cancer is the third commonest cancer in women in Uganda after Kaposi’s sarcoma and cervical cancer , 2. BreaPrimary randomized controlled trials have shown the importance of mammography in early diagnosis of breast cancer in asymptomatic women and it has been effective in decreasing mortality especially in women aged 50-69 years with reductions of 20% to 35% -15. HoweThe primary factors that increase risk of breast cancer in women include certain inherited genetic mutations, a personal or family history of breast cancer, and biopsy-confirmed hyperplasia . Other fZincir reported that the percentage of women over the age of 40 having a professional breast investigation every year was 21.1% in the eastern region of Turkey . Annual Despite breast cancer being one of the few cancers that can be detected early before seeing symptoms using mammography, mammography is still only performed on a low proportion of the women population in Uganda. Despite the wealth of literature available globally documenting knowledge, attitudes and practices of women about breast cancer and mammography, there is still paucity of literature on the African experience in this area. The aforementioned gaps form the basis of the present study. Hindrances to accessing mammography services not only in Uganda or Africa, but also globally should be identified and then health care authorities should establish strategies to overcome them.The purpose of this study therefore, was to assess the knowledge, attitudes and practices of women about cancer and mammography as well as identify potential barriers hindering women from accessing mammography services. Hopefully, findings from this study will provide a starting point for health authorities to raise awareness amongst women about breast cancer and the role of mammography in breast cancer investigation. Due to operational costs, this study could neither be extended to all women reporting to the hospital nor to women elsewhere in Uganda. This coupled with no focus group discussions conducted with the study participants are potential limitations of the study. We could not conduct focus group discussions since all women in the study were outpatients and just trickle in the Radiology department it would be difficult to gather them. We suggest more similar studies to be conducted with other groups of women from other areas. Combined with the data from this study, it could provide a better understanding of the barriers faced by women when accessing a procedure such as mammography.The study was conducted at Mulago Hospital which is Uganda´s National Referral Hospital. It also acts as the teaching hospital for the College of Health Sciences, Makerere University. The hospital is located in the northern part of Kampala City, Uganda’s capital and has a bed capacity of 1,500.It was a descriptive cross-sectional study using interviewer-administered questionnaires. The questionnaires were administered in English and Luganda  covering the following domains: Demographics , knowledge, attitudes and practice (KAP) of women on breast cancer and mammography. To ensure validity and reliability of the data collected, the questionnaire was reviewed for information quality and legitimacy and any corrections arising were made. It was then pre-tested before the survey.  Questions asked sought knowledge, attitudes and practices on breast cancer, use of mammography, BSE as well as CBE. Women above 30 years of age reporting to the Radiology department during the study period and had consented were included in the study. Additionally, all women who reported to the radiology department specifically for mammography examination were also excluded because they already had some information about mammography. Correct and consistent answers regarding the value of mammography were considered adequate knowledge. Attitude was considered adequate if women were positive about mammography and supported their statements consistently. Practice was considered satisfactory if women correctly answered questions regarding the use of mammography and the desire of doing it. For women who had not had a mammogram, the main reasons for that were also sought.100 women reporting to the Radiology department during the study period were involved in the study. Consecutive sampling was used. This was done in order to give an opportunity to every woman who meets the inclusion criteria to participate in the study without any bias.A quiet place was chosen for the interviews. Responses were compiled, entered in the computer and stored on flash disks and kept securely in a locked place. Only the investigators had access to this information. Information in the computer was protected using passwords.Data was both quantitative and qualitative. Thematic analysis was used for qualitative data. It involved content analysis to extract the meanings of the participants, and also transcription. Raw data was proof-read and coded into categories of similar meaning. Categories were established, resulting into content themes. These themes summarized the meaning of the data which addressed the purpose of the study. Quantitative data went through cleanliness and consistency tests, frequency analysis and entered into SPSS version 10.0 statistical program. Age of participants wasexpressed as means of ± SD. Associations between socio-demographic characteristics and adequacy of KAP of mammography were determined using both bivariate and logistic regression analysis. A Chi-square was used to test statistical significance and a p-value of 0.05 was adopted for this study.Permission to carry out this study was granted by the Ethics and Research Committee of the College of Health Sciences, Makerere University. Participants provided written consent prior to beginning of the interview after a thorough explanation of what the study was all about by the interviewers. English and Luganda; a local language were used in the consent process, dialects understood by the participants. Verbal consent was obtained from those participants who could not write, also after a thorough explanation. The study strictly followed the principles of the Declaration of Helsinki.th decade age group. Majority had primary level of education.The socio-demographic characteristics of participants are summarized in Participants´ knowledge about the incidence and risk factors of breast cancer is provided in The women’s attitude towards mammography was negative. Most of them confused mammography with breast ultrasound.“I can never go back for those checks in machines because I do not see anything”, one woman commented.“Ka T.V ako nkamanyi, naye siyinza kukagendamu mirungi mingi kubanga nyinza okufuna cancer”, another women retorted, literally meaning that, “I know about ultrasound, but I cannot go for it frequently as I fear getting cancer” One lady who had some knowledge about mammography from a friend said:“I was told that machine presses your breasts so hard causing pain, so I better do BSE and go for CBE instead of going for those other things?”Majority of the women (62%) said that they were told that they could check their breasts at home monthly and also go for check up with the doctor once in a while.“I was told to check my breasts every month by the doctor, but he never told me anything else”, one lady responded.All women in this study had never had a mammogram. Majority reported that they did not undergo mammography because they had never had of it and its use in investigating breast cancer. The few women who had knowledge about mammography said that they had been told it was expensive and could not afford, hence not seeking for it. Regarding seeking for mammography; level of literacy, occupation and marital status were significant on bivariate analysis (p=0.003), however only level of literacy and employment remained the significant independent variables on logistic regression analysis (p=0.002). Women beyond primary level of education were about four times more likely to go for mammography than women below that level of education . Employed women were seven times more likely than the unemployed to go for mammography, . The impediment to go for mammography expressed by all women was the lack of knowledge about the role of mammography in breast cancer investigation and limited access to mammography services. However, some women had done other investigations like breast ultrasound (12 %). Majority (66%) reported indulging in BSE most frequently. The commonest reason cited for this was that it is convenient and does not cost anything. However, all women who had done breast ultrasound were worried about getting cancer from the scan if they did repeatedly, which also affected the seeking behaviour.“I did not go for the scan the last time the doctor told me because I feared getting cancer”, one lady said.The 3 methods recommended for detection of breast cancer are BSE, CBE by a health care professional, and mammography, the third being the most effective ,5,7,20. This study also showed that majority of the women did not know about risk factors for breast cancer. The reason for this is again due to lack of exposure to knowledge and facts about breast cancer. The low levels of education in majority of the women who participated in this study is another reason, in that the women may not know how to access accurate information about breast cancer. Like Aylin et al  found inAll women generally reported a negative attitude towards mammography. The limited knowledge about mammography probably contributed to the negative attitude since they did not know its exact role. However, the participants expressed willingness to change their attitude towards mammography provided they got adequate information regarding its role. This is partly due to the wide campaign about breast cancer and its effects not only in Uganda, but globally as well. Therefore, being an investigative procedure to detect breast cancer is probably the main reason for women having acceptance for it provided they get to know its benefits. Majority of women (75%) in this study feared that breast ultrasound would pose a potential risk to cancer. Conversely, Aylin et al found out in their study that majority of the women (72.5%) were comfortable with breast ultrasound without any fear . This shSome women who had done CBE reported embarrassment especially when being examined by a male doctor which changed their attitude towards breast screening procedures. This finding was also reported by Dibble et al  and SainMajority of the women frequently practiced BSE and occasionally sought for CBE, but did not go for mammography. It is thought that BSE makes women more aware of their breasts which in turn may lead to an earlier diagnosis of breast cancer . The ratThere may be several reasons for not undergoing mammography. The cost of mammography in Uganda and probably globally is high, particularly for a woman who does not have social security like most of the participants in this study. Although Dibble et al  have repParticipants in this study lacked adequate knowledge had poor attitude and inappropriate practice about mammography. The main barrier to mammography was lack of information about its role in early detection of breast cancer which improves survival. Awareness campaigns and subsidizing the costs for mammography by Ministry of Health would improve survival from breast cancer. Involving men as well in this awareness will also greatly improve the current situation. All this should be coupled with acquisition of mammography machines by regional referral hospitals to ease access to the service.The authors declared that they have no competing interests


Gonadotropin releasing hormone (GNRH1) triggers the release of follicle stimulating hormone and luteinizing hormone from the pituitary. Genetic variants in the gene encoding GNRH1 or its receptor may influence breast cancer risk by modulating production of ovarian steroid hormones. We studied the association between breast cancer risk and polymorphisms in genes that code for GNRH1 and its receptor (GNRHR) in the large National Cancer Institute Breast and Prostate Cancer Cohort Consortium (NCI-BPC3).GNRH1 and GNRHR in 95 invasive breast cancer cases. Resulting single nucleotide polymorphisms (SNPs) were genotyped and used to identify haplotype-tagging SNPs (htSNPS) in a panel of 349 healthy women. The htSNPs were genotyped in 5,603 invasive breast cancer cases and 7,480 controls from the Cancer Prevention Study-II (CPS-II), European Prospective Investigation on Cancer and Nutrition (EPIC), Multiethnic Cohort (MEC), Nurses' Health Study (NHS), and Women's Health Study (WHS). Circulating levels of sex steroids  were also measured in 4713 study subjects.We sequenced exons of GNRH1 and GNRHR genes, nor were there any statistically significant interactions with known breast cancer risk factors. Polymorphisms in these two genes were not strongly associated with circulating hormone levels.Breast cancer risk was not associated with any polymorphism or haplotype in the GNRH1 and GNRHR genes are not associated with risk of invasive breast cancer in Caucasians.Common variants of the  Exposure to steroid hormones (estrogens and androgens) is a risk factor for breast cancer. Increased exposure to estrogens, for instance by early menarche, late menopause, low parity and post-menopausal obesity, contributes to increased breast cancer risk (reviewed in ref. ). High cThe primary stimulus for production of estrogen and other ovarian steroid hormones is the release of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), from the anterior pituitary. These are released when gonadotropin-releasing hormone 1 (GNRH1), from the hypothalamus, binds to the gonadotropin-releasing hormone receptor (GNRHR) in the anterior pituitary. The resultant G-protein activation of a phosphatidylinositol-calcium second messenger system ultimately triggers the release of FSH and LH.GNRH1 activity is low during childhood but increases at puberty. GNRH1 production is pulsatile. In females, the GNRH1 pulse frequency varies during the menstrual cycle, with a large surge of GNRH1 just before ovulation. The size and frequency of the GNRH1 pulse, and feedback from androgens and estrogens, control production of LH and FSH .GNRH1 activity can be disrupted by hypothalamic-pituitary disease. Elevated prolactin levels decrease GNRH1 activity. In contrast, hyperinsulinemia increases pulse activity leading to disorderly LH and FSH activity, as seen in polycystic ovary syndrome .GNRH1 gene is located on chromosome 8p21.2. It spans about 5 kb and contains 3 exons. It encodes the GNRH1 precursor, which contains 92 amino acids and is processed to GNRH1, a decapeptide. The GNRHR gene is located on chromosome 4q13.2. Its genomic sequence covers about 19 kb and it includes 3 exons.The GNRH1 and GNRHR genes can harbor rare germline mutations which lead to idiopathic hypogonadotropic hypogonadism (IHH) or Kallmann syndrome  in both men and women [The GNRH1 and GNRHR could influence breast cancer risk by modifying production of FSH/LH and steroid hormones. We used a haplotype tagging approach to examine this hypothesis using cases and controls from the BPC3.We hypothesized that common, functional polymorphisms of The BPC3 has been described in detail elsewhere . BrieflyCases were identified in each cohort by self report with subsequent confirmation of the diagnosis from medical records or tumor registries, and/or linkage with population-based tumor registries (method of confirmation varied by cohort). Controls were matched to cases by ethnicity and age, and in some cohorts, additional criteria, such as country of residence in EPIC.Most of the subjects were Caucasians of European descent. One cohort (MEC) provided most of the non-Caucasian samples. In total, we genotyped 4,401 Caucasian cases and 5,966 controls, 329 Latino cases and 385 controls, 341 African American cases and 426 controls, 425 Japanese American cases and 418 controls, and 107 Native Hawaiian cases and 285 controls.Written informed consent was obtained from all subjects, and the project has been approved by the competent institutional review boards for each cohort.GNRH1 and GNRHR in a panel of 95 metastatic breast cancer cases from the MEC and EPIC. These included 19 cases from each ethnic group represented in the study . About 45 kb were surveyed for GNRH1 and about 56 kb for GNRHR. No non-synonymous or splice-site variants were identified in sequencing of the exons.We sequenced exons and intron/exon junctions of Based on the resequencing and SNPs available in dbSNP, we identified 17 SNPs in GNRH1 and 36 SNPs in GNRHR with minor allele frequency greater than 5% in any of the five ethnic groups or greater than 1% overall. These SNPs were genotyped in a reference panel of 349 healthy women ) at the Broad Institute  using the Sequenom  and Illumina  platforms.et al. [2H among Caucasians. Three htSNPs were selected for GNRH1  and seven for GNRHR.Haplotype tagging SNPs (htSNPs) were then selected using the method of Stram et al.  to maximhttp://www.uscnorris.com/mecgenetics/CohortGCKView.aspx. Sequence validation for each SNP assay was performed on samples from the SNP500 project http://snp500cancer.nci.nih.gov[Genotyping of htSNPs was performed in 3 laboratories  using a fluorescent 5' endonuclease assay and the ABI-PRISM 7900 for sequence detection (TaqMan). Initial quality control checks of the SNP assays were performed by the manufacturer ; an additional 500 test reactions were run at the University of Southern California. Characteristics for the 10 TaqMan assays are available on a public website i.nih.gov and 100%2), estrone (E1) and androstenedione (Δ4) were measured by direct double-antibody radioimmunoassays from DSL , while testosterone (T) was measured by direct radioimmunoassays from Immunotech . Measurements were performed on never thawed serum sample aliquots. Mean intrabatch and interbatch coefficients of variation were 5.8 and 13.1%, respectively, for E2 (at a concentration of 250 pmol/l), 10.2 and 12.6% for E1 (at 75 pmol/l), and 4.8 and 18.9% for Δ4 (at 1.40 nmol/l), 10.8 and 15.3% for T (at 1.40 nmol/l).Circulating serum hormones were measured at the International Agency for Research on Cancer for EPIC and MEC samples and at the Harvard School of Public Health for NHS samples, for a total of 4713 subjects . The different assays for hormone analyses were chosen on the basis of a previously published comparative validation study . EstradiGNRHR. To test the global null hypothesis of no association between variation in GNRH1/GNRHR haplotypes and htSNPs and risk of invasive breast cancer (or subtypes defined by receptor status), we used a likelihood ratio test comparing a model with additive effects for each common haplotype (treating the most common haplotype as the referent) to the intercept-only model.We used conditional multivariate logistic regression to estimate odds ratios (ORs) for invasive breast cancer in subjects with a linear (log-odds additive) scoring for 0, 1 or 2 copies of the minor allele of each SNP. We also used conditional logistic regression with additive scoring and the most common haplotype as the referent to estimate haplotype-specific ORs using an expectation-substitution approach to assign haplotypes based on the unphased genotype data and to account for uncertainty in assignment ,17. Haplvs. localized disease, and age at diagnosis (≤55 years vs. >55 years). We also investigated interactions between single SNPs or haplotypes and completion of a full term pregnancy (ever/never), age at first full term pregnancy , body mass index , height , smoking status (never/former/current smoker), and use of menopausal hormone therapy (ever/never). Other common risk factors, including family history of breast cancer, personal history of benign breast disease, and age at menopause were unavailable for large numbers of women, and therefore were not included in the models.We performed subgroup analyses stratifying by cohort, ethnicity, country within EPIC, estrogen receptor/progesterone receptor status, metastatic Relationships of genetic variants with serum hormone levels were estimated by standard regression models, adjusted for BMI, age, assay batch, ethnicity, and country within EPIC. These analyses were performed both using all the study subjects for whom hormone levels have been measured, and only the controls, who represent the populations giving rise to the cases.GNRH1 and GNRHR are shown in Figure GNRH1 consists of a single LD block, whereas GNRHR includes two LD blocks, one of them including exon 1 and the other exons 2 and 3. GNRH1 was tagged by 3 SNPs, which account for 94% of haplotype diversity. Block 1 of GNRHR was tagged by 3 SNPs and block 2 by 4 SNPs . Frequency of common haplotypes ranged between 19 and 35% in controls for GNRH1 and 5% and 52% for GNRHR.The genomic regions surrounding A total of 5,603 invasive breast cancer cases and 7,480 controls were available for genotyping from each of the participating cohorts. Samples not yielding a genotype were removed from individual SNP analyses, and samples not yielding at least one genotype were removed from haplotype analyses. Both between-center genotyping concordance and within-center blinded quality control concordance were above 99%. Genotype success rate among cases and controls in all cohorts was greater than 95%. No polymorphisms deviated from Hardy-Weinberg Equilibrium among the controls.GNRHR showed a nominally significant association (p = 0.04) with estradiol levels in post-menopausal women. Presence of the minor allele at this polymorphism was associated with an increase in estradiol level . However, this effect was entirely driven by the association observed in post-menopausal breast cancer cases within EPIC (p = 0.012 in this subgroup), and was not observed in the other subgroups. A few borderline associations were observed between hormone levels and SNPs in pre-menopausal women, however sample size of this group of subjects was considerably smaller, and all subjects derived from only one cohort (EPIC). No associations between polymorphisms and hormone levels remained significant after correction for multiple testing.Detailed results of associations between serum concentrations of steroid hormones and SNPs are presented in Additional file GNRH1 and breast cancer risk are presented in Table GNRHR are presented in Tables GNRH1; d.f. = 5, p = 0.897 for GNRHR block 1 and d.f. = 6, p = 0.967 for GNRHR block 2). Analyses were unadjusted  or adjusted for known breast cancer risk factors, but results did not show any difference.Results of association analyses between htSNPs of Analyses performed by stratifying cases by age at diagnosis (greater or lower than 55 years), localized or metastatic disease or estrogen/progesterone receptor status did not show significant differences. Stratification of subjects by cohort, country in EPIC or ethnicity in MEC showed only few results supported by p values ranging from 0.01 to 0.05, which were always based on a small number of subjects. For these, we performed heterogeneity tests, which in all cases were not statistically significant. For example, heterozygotes for SNP rs1812594 of GNRH1 had an odds ratio (OR) of 1.08  = 1.00–-1.16, p = 0.04). When we analyzed the results for each cohort, it resulted that the association was driven by EPIC data , and within EPIC the only significant result came from the Spanish sub-cohort , which is based on 62 cases and 79 controls heterozygous for this SNP. However, heterogeneity tests for this genotype were not statistically significant for either the entire study (p = 0.226) or within EPIC (p = 0.147). Nor were homozygotes for the less common allele at this SNP significantly associated with increased risk in any subgroup.No statistically significant interactions were observed between htSNPs or haplotypes and known breast cancer risk factors, including age at first full term pregnancy, number of pregnancies, never/ever menopausal hormone therapy, height, smoking status, or body mass index.This large, comprehensive study found no statistically significant associations between polymorphisms in the genes that code for GNRH1 or its receptor and either circulating ovarian sex hormones or breast cancer risk. An influence of SNPs in these two genes on breast cancer risk, mediated by altered levels of estrogens, was plausible, due to the known physiology of steroid hormone stimulation. If any common variants with functional relevance exist in the two candidate genes, our resequencing and haplotype tagging approaches should have detected its effect on hormone measurements and/or cancer risk. The null results are especially convincing because of the large sample size  and the extensive resequencing that preceded and informed the selection of htSNPs. The study has over 80% power to detect main effects of common polymorphisms  with relative risks of 1.2 or greater, and to investigate interactions between genetic variants and known environmental or lifestyle exposures .GNRH1 and GNRHR do not substantially affect breast cancer risk in Caucasians. Among the many tests performed in subgroups, some associations were supported by p values ranging from 0.01 and 0.05, yet these associations were driven by subgroups containing small numbers of cases, and are therefore compatible with chance. None of these subgroup findings remain statistically significant when adjusted for multiple hypothesis testing. Likewise, the weakly significant association we observed between estradiol levels in post-menopausal women and SNP rs2630488 of GNRHR derived from a single subgroup in one cohort, and is also likely to reflect chance.Based on these results, we conclude that common polymorphisms in A limitation of this study was the relatively small number of subjects from racial or ethnic groups other than Caucasian. The MEC provided most of the cases and controls in this regard, but none of these groups exceeded 425 breast cancer cases. This limitation is particularly relevant to African Americans, for whom additional SNPs would be needed to provide comparable coverage of common variants. Coverage is satisfactory for the other ethnic groups . This isGNRH1 and GNRHR confer large or even moderate breast cancer risks in Caucasians. We cannot exclude the possible existence of moderate risks due to polymorphisms of GNRH1 and GNRHR in non-Caucasian populations. Larger studies of non-Caucasians will be necessary to test this hypothesis.In conclusion, we can exclude the possibility that common polymorphisms in The authors declare that they have no competing interests.FC, RK, DGC, KDH and BEH made up the writing committee for this work, and were responsible for data analyses, manuscript preparation and editing. CLP performed the htSNP selection and contributed substantially to manuscript editing. LD, CAH, DOS, SC provided expertise in genotyping and results analyses, as well as manuscript editing. All other authors contributed substantially to sample collection and manuscript editing. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/257/prepubDetailed results of associations between serum concentrations of steroid hormones and SNPs.Click here for file