A case-control study conducted in South Africa provides new estimates of the risk of specific cancers of the female reproductive system associated with use of injectable and oral contraceptives. Oral contraceptives are known to influence the risk of cancers of the female reproductive system. Evidence regarding the relationship between injectable contraceptives and these cancers is limited, especially in black South Africans, among whom injectable contraceptives are used more commonly than oral contraceptives.n = 1,664), cervical , ovarian (n = 182), and endometrial (n = 182) cancer, with self-reported contraceptive use in 1,492 control patients diagnosed with cancers with no known relationship to hormonal contraceptive use. We adjusted for potential confounding factors, including age, calendar year of diagnosis, education, smoking, alcohol, parity/age at first birth, and number of sexual partners. Among controls, 26% had used injectable and 20% had used oral contraceptives. For current and more recent users versus never users of oral or injectable contraceptives, the odds ratios (ORs) for breast cancer were significantly increased in users of oral and/or injectable contraceptives  and separately among those exclusively using oral  and exclusively using injectable  contraceptives; corresponding ORs for cervical cancer were 1.38 , 1.01 , and 1.58 . There was no significant increase in breast or cervical cancer risk among women ceasing hormonal contraceptive use ≥10 y previously . For durations of use ≥5 y versus never use, the ORs of ovarian cancer were 0.60  for oral and/or injectable contraceptive use and 0.07  for injectable use exclusively; corresponding ORs for endometrial cancer were 0.44  and 0.36 .We analysed data from a South African hospital-based case–control study of black females aged 18–79 y, comparing self-reported contraceptive use in patients with breast ; most injectable hormonal contraceptives contain only manmade progesterone preparations. Hormonal contraceptives, which prevent pregnancy by disrupting the cyclical changes in estrogen and progesterone levels that prepare the body for pregnancy, have revolutionized birth control since they first became available in the early 1960s. However, it is now known that taking the pill also influences women's risk of developing cancers of the female reproductive system. Current and recent users have an increased risk of developing breast and cervical cancer (the cervix is the structure that connects the womb to the vagina) compared to never users, although this increased risk quickly disappears when women stop taking the pill. By contrast, women who have used the pill have a reduced risk of developing ovarian cancer and cancer of the womb  compared to never users that increases with the duration of pill use and persists for many years after use ceases. These effects on reproductive system cancers are thought to occur because these cancers depend on naturally occurring sex hormones for their development and growth.Although the evidence that the pill influences the risk of developing cancers of the female reproductive system is extensive, much less is known about how injectable hormonal contraceptives affect cancer risk. In this hospital-based case–control study (a study that compares the characteristics of people with and without a specific condition), the researchers investigate the relationship between the use of oral and injectable hormonal contraceptives and cancers of the breast, cervix, ovary, and endometrium among black South African women. Injectable contraceptives have been used for longer in South Africa than elsewhere and are used more commonly than oral contraceptives among black South African women.As part of the Johannesburg Cancer Case Control Study, which recruits black patients attending Johannesburg public referral hospitals for cancer treatment, the researchers compared hormonal contraceptive use in women with breast, cervical, ovarian, or endometrial cancer with contraceptive use in women diagnosed with other cancers such as lung, colon, and rectal cancers, which are not known to be influenced by hormonal contraceptives. Among the controls, a quarter had used injectable contraceptives and a fifth had used oral contraceptives. After adjusting for other factors that might influence cancer risk such as age, smoking, and number of sexual partners, the odds ratio (OR) of breast cancer among current and recent users of oral and/or injectable contraceptives compared to never users was 1.66. That is, the risk of developing breast cancer among current and recent users of hormonal contraceptives was 1.66 times that among never users. For women using oral contraceptives exclusively or injectable contraceptives exclusively, the ORs of breast cancer were 1.57 and 1.83, respectively. There were also increases in cervical cancer risk among current and recent users of hormonal contraceptives compared to never users, but no significant increase in breast or cervical cancer risk among women who had ceased hormonal contraceptive use more than ten years previously. Finally, the use of hormonal contraceptives for more than five years reduced the risk of both ovarian and endometrial cancer.These findings indicate that, among black women in South Africa, the use of oral or injectable hormonal contraceptives is associated with a transiently increased risk of breast and cervical cancer, and that extended use of these contraceptives is associated with a reduced risk of ovarian and endometrial cancer. Moreover, they suggest that the effects of oral versus injectable contraceptives on cancer risk do not differ substantially, although for endometrial and ovarian cancer the small number of cases exposed to injectable contraceptives limits the accuracy of the risk estimates. Other limitations of this study include the possibility that the findings may be affected by uncontrolled confounding. That is, women who used hormonal contraceptives may have shared other unidentified characteristics that affected their cancer risk. Nevertheless, these findings provide new information about the effects of oral and injectable hormonal contraceptives on cancer risk that should help women make informed decisions about their choice of contraceptive method.http://dx.doi.org/10.1371/journal.pmed.1001182.Please access these web sites via the online version of this summary at breast cancer , cervical cancer, ovarian cancer, and endometrial cancer for patients and health professionals, and a fact sheet on oral contraceptives and cancer risk (in English and Spanish)The US National Cancer Institute provides information on breast cancer, cervical cancer, ovarian cancer, and endometrial cancer and information about the birth control pill and cancer riskCancer Research UK also provides information on Eyes on the Prize, an online support group for women who have had cancers of the female reproductive system, has personal stories; further personal stories about breast, cervical, and ovarian cancer are provided by the charity Healthtalkonline Hormonal contraceptives are among the most commonly used medications. Worldwide, in 2007, 9% of women aged 15–49 y were estimated to be using the oral contraceptive pill and 4% were using injectable contraceptives or implants Large-scale epidemiological evidence has shown that use of oral contraceptives significantly affects the risk of cancers of the liver and of the female reproductive system, specifically cancers of the breast, cervix uteri, ovary, and endometrium Injectable contraceptives are predominantly given as progestagen-only depot preparations—notably, depot medroxyprogesterone acetate —although a small proportion contain both oestrogen and progestagen In South Africa, injectable progestagen-only contraceptives have been used more commonly, and for longer durations, than anywhere else in the world The study was approved by the University of the Witwatersrand Human Research Ethics Committee .This study uses an established case–control design for cancer epidemiology studies done in resource-limited settings, where cases are individuals with the cancer of interest and controls are individuals with other cancers that are not associated with the exposure under investigation n = 52); other genital cancers ; Kaposi sarcoma (n = 278), because of its overwhelming association with HIV and because its preponderance among women in HIV-positive individuals suggests that some hormonal factors may be at play; and cancers of ill-defined, secondary, or unspecified sites (n = 157), because they could not be assigned to be cases or controls.The Johannesburg Cancer Case Control Study is a large ongoing case–control study recruiting self-defined black (not mixed race/ancestry) male and female cancer patients with all cancer types, conducted at the greater Johannesburg public referral hospitals that offer cancer treatment. Female patients recruited from 8 March 1995 to 31 December 2006 were included in the present analysis. Trained nurses used a standard questionnaire to interview newly diagnosed black cancer patients in their preferred language . Participants were interviewed as soon as possible (maximum 6 mo) after diagnosis, prior to receiving chemotherapy and/or radiotherapy . For all analyses, age at and calendar year of interview are taken as being age at and calendar year of diagnosis. Socio-demographic and behavioural information was solicited, including age, birthplace, residence, years of education, alcohol and tobacco use, reproductive history, and lifetime sexual history. In the large majority of cases the clinical diagnosis of cancer was supported by laboratory investigations giving microscopic verification. “Cases” for this study were women with a newly diagnosed invasive breast, cervical, ovarian, or endometrial cancer. Controls consisted of women diagnosed with cancer types that have no known relationship to oral or injectable contraception, based on data from the International Agency for Research on Cancer Women were asked separately about oral and injectable contraceptives: (1) Have you ever taken them? (2) If yes, how old were you when you started taking them? (3) If yes, how old were you when you stopped taking them? (4) If yes, how long in total did you take them? Women reported their age at starting and stopping contraceptives in whole years, while the reporting of duration could include months. To account for the time that the different contraceptives take to be eliminated from a woman's body, in recording of duration of use, 1 mo was added to the date of the last prescription for oral contraceptives, and 2 or 3 mo were added to the time of the last injection for injectable contraceptives, depending on the preparation used. Responses to question 1 indicated whether the patient had ever used oral and/or injectable contraceptives. Responses to question 3 combined with age at cancer diagnosis  provided the number of years since last use of oral and/or injectable contraceptives.Discrete categories were then created for users of oral contraceptives exclusively, users of injectable contraceptives exclusively, users of both oral and injectable contraceptives, and users of oral and/or injectable contraceptives. “Users of oral contraceptives exclusively” was defined as women who reported ever having used oral contraceptives but never having used injectable contraceptives. “Users of injectable contraceptives exclusively” was defined as women who reported ever having used injectable contraceptives but never having used oral contraceptives. “Users of both oral and injectable contraceptives” was defined as women who reported ever having used both oral and injectable contraceptives, and “users of oral and/or injectable contraceptives” was a combined group including women who reported having used either or both oral and injectable contraceptives. For these last two groups, time since last use was calculated as time since last reported use of either oral or injectable contraceptives, and duration of use was calculated by adding together the total amount of time that either oral or injectable use was reported. For each of the use categories, variables were then constructed as follows: ever use (ever versus never), total duration of use , and time since last use . The categories used in these variables were defined a priori and were broadly based on those used in prior analyses, for oral contraceptives, taking into account the sample size in the relevant categories Based on the most recent meta-analyses of the worldwide evidence, we hypothesised that recency of use of hormonal contraceptives would be most important for cancers of the breast and cervix, i.e., that increased risks would be seen in current and recent users For this study, oral contraceptives were assumed to be combined oestrogen-progestagen oral contraceptives, as progestagen-only pills distributed in South African public sector clinics are recommended only for breastfeeding women Evidence regarding the relationship of potential confounding factors to the outcomes of interest was obtained from the relevant published evidence, e.g., In order to investigate independently the effects of oral and injectable contraceptive use, the estimated odds ratios (ORs) for specific cancer types were presented for users of oral contraceptives exclusively, users of injectable contraceptives exclusively, users of both oral and injectable contraceptives, and users of oral and/or injectable contraceptives. Women who had never used either oral or injectable hormonal contraceptives were used as the reference group throughout. Cancer-specific multivariable unconditional binary logistic regression models were fitted to the data, and separate models were fitted for non-mutually exclusive categories of contraceptive use .p-values—referred to here as p(heterogeneity)—were used to compare the associations between oral contraceptive and injectable contraceptive use and risk of specific cancers, and to examine effects according to duration of use and time since last use.All multivariable unconditional logistic regression models were adjusted for age at diagnosis ; year of diagnosis ; years in full-time education ; smoking status (never versus ever); alcohol use ; parity/age at first birth ; area of residence ; province of birth ; and lifetime number of sexual partners  . Wald tests for heterogeneity and corresponding HIV infection is a potential confounder in the relationship between hormonal contraceptives and cervical cancer; data on HIV status, based on serological testing of blood collected at the time of interview, was available for 90% of study participants. We conducted sensitivity analyses of the effect of additional adjustment for HIV status in this relationship. However, because additional adjustment for HIV did not materially affect the OR estimates for hormonal contraceptives and cervical cancer see , and becThe estimated ORs for cervical cancer were not adjusted for history of cervical cancer screening (Pap smears) because this item was added to the study questionnaire in 2001 and is missing for 61% of participants. We did, however, conduct sensitivity analyses using the reduced complete-case datasets whilst additionally adjusting for cervical smear history in the analysis of cervical cancer, and we also performed analyses on the full dataset after multiple imputation of the unobserved Pap smear histories. Briefly, this involved imputing missing Pap smear histories (“never/this illness only” versus “yes before this illness”) of cervical cancer and control patients 20 times using a binary logistic imputation model. Predictors in the imputation model included all confounders included in the primary analysis models, time since last contraceptive use  and also the participant's disease status . The analysis models for the multiply imputed data contained the same covariates as those in the corresponding primary analysis models with additional adjustment for Pap smear history (“never/this illness only” versus “yes before this illness”).The effect of systematically excluding each control cancer type, in turn, from the control group on the estimated OR for the main exposure–outcome relationships of interest was also examined.All analyses were performed using Stata 11.0 software (StataCorp).n = 1,664), cervical , ovarian (n = 182), or endometrial (n = 182) cancer. There were 1,492 controls, comprising patients with other types of invasive cancers not known to be influenced by hormonal contraceptive use, as described in n = 301), colon and rectal cancer (n = 163), non-Hodgkin lymphoma (n = 125), and lung cancer (n = 118).For the study period, there was a total of 5,702 study participants with full information on the exposures and risk factors of interest. This sample included women with newly diagnosed invasive breast  in women who had used either oral or injectable contraceptives within the previous 10 y and did not differ significantly  in those ceasing use ≥10 y previously, compared to women who had never used hormonal contraceptives (p[heterogeneity] = 0.6). In women who had used either or both preparations, this elevated risk declined significantly with increasing time since last use of hormonal contraceptives (p = 0.004) but was not significantly related to duration of use (p = 0.4)  .p = 0.01), while those ceasing use ≥10 y previously had no significant difference in risk  (p[heterogeneity] = 0.2); the risk in recent users of injectable contraceptives exclusively was significantly increased, compared to women who had never used hormonal contraceptives  (p = 0.02), but was not significantly related to duration of use (p = 0.96)  . The mag= 0.004) . Risk di = 0.96) .p = 0.2) (p = 0.04) (p = 0.7) or by time since last use (p = 0.97) for oral and/or injectable contraceptives (p = 0.004) , compared with never users of hormonal contraceptives  . For tho = 0.04)  comparedceptives . The sam= 0.004)  and longceptives .p = 0.02); women who used these contraceptives for less than 5 y had no significant difference in risk  (p[heterogeneity] for duration = 0.007)  . The samAdditional adjustment for the number of previous Pap smears (using multiply imputed data) and for HIV status had no material effect on the OR for cervical cancer in relation to recent or past use of hormonal contraceptives see  and S2. This study was conducted among black women in South Africa. Use of injectable contraceptives was very common, with over one-quarter of the controls in the study reporting they had used them at some point in their lives. In contrast to most of the rest of the world, use of injectable contraceptives was more common than use of oral contraceptives, and was often for long durations.The study shows that the risk of breast cancer and cervical cancer is increased significantly among women who are current and recent users of oral and/or injectable contraceptives and, separately, among current and recent users of injectable contraceptives exclusively. The elevated risk diminishes following cessation of use, such that no significant increase in risk is present ≥10 y after ceasing use. Risk is not significantly related to duration of use and does not differ significantly between users of oral and injectable contraceptives.There were far fewer cases of ovarian and endometrial cancer  than breast and cervical cancer , thus limiting statistical power. Nevertheless, we found that women who had used oral and/or injectable contraceptives for a duration of ≥5 y had significantly reduced risks of both ovarian and endometrial cancer compared with women who had never used hormonal contraceptives.Our results show a significantly increased breast cancer risk in women who in the previous 10 y used injectable contraceptives exclusively  compared with women who have never used hormonal contraceptives, based on 186 exposed cases. These findings add substantially to the previously available evidence. The most comprehensive summary of worldwide evidence on breast cancer and use of progestagen-only injectable contraceptives was published by the Collaborative Group on Hormonal Factors in Breast Cancer in 1996 The pooled analysis on injectable contraceptive use and the risk of cervical cancer from the International Collaboration of Epidemiological Studies of Cervical Cancer Our finding of a significantly protective effect of the exclusive use of injectable contraceptives for ≥5 y on ovarian cancer is based on one exposed case only. However, our results are consistent with the non-significant results of the two previous studies we identified in this area, which together included only two cases exposed to injectable contraceptives exclusively The sum total of the worldwide evidence regarding endometrial cancer and injectable progestagen-only contraceptives is based on ten exposed cases; it broadly indicates a reduction in risk in users but is not able to distinguish reliably the effects of injectable contraceptives independent of previous combined oral contraceptive use The findings reported here for oral contraceptive use in relation to the risk of cancers of the breast, cervix, ovary, and endometrium are broadly compatible with the well-established relationships quantified to date The large numbers of women in this study, and the high prevalence of use of injectable contraceptives, means that this study is able to add to the existing evidence on the effects on cancer risk of progestagen-only injectable contraceptives: the dataset allowed us to examine risk separately in users of injectable contraceptives exclusively, particularly for breast and cervical cancer. The long duration of injectable contraceptive use in the study participants also means the risks related to prolonged use were able to be investigated. Although we were able to adjust for multiple potential confounding factors, and sensitivity analyses indicated robust findings in the face of additional adjustment, the possibility that results were affected by uncontrolled confounding cannot be excluded. Although the vast majority of the oral contraceptives investigated here are likely to be combined oral contraceptives, we cannot exclude the possibility that a small proportion comprises progestagen-only preparations. Similarly, a small proportion of the injectable contraceptives may be combined oestrogen-progestagen preparations.The Johannesburg Cancer Case Control Study has proven valuable for investigation of other exposures and outcomes and has been conducted efficiently for over 10 y in a resource-poor and logistically difficult setting This study was designed a priori to focus on black women who belong to a disadvantaged and under-researched community, but nonetheless represent around 79% of the South African population and 74% of the residents of Gauteng Province, where the study was based The hospitals that recruited for this study are the only public tertiary referral hospitals for medical oncology and radiation therapy in the study area. Current and historic referral patterns mean that the patients constituting cancer cases and cancer controls in this study should have similar probabilities of presentation at these hospitals and recruitment into the study, and this probability should not vary substantively according to exposure to hormonal contraceptives. Additional adjustment for socio-demographic factors, including education, urban/rural residence, and province of birth further safeguards against this.The cancer profile of the participants interviewed resembled the background distribution of histologically reported cancers in black women in South Africa in 2003, as reported to the pathology-based South African National Cancer Registry Oestrogens and progestagens exert different effects on different tissues, and the exact mechanisms underlying their ability to influence the risk of cancer are unclear. Combined oral contraceptives and injectable progestagens exert their main contraceptive effects via the suppression of ovulation, through the feedback inhibition of follicle stimulating hormone and luteinising hormone. Use of combined oral contraceptives suppresses endogenous oestradiol, but this is essentially replaced by exogenous oestradiol and, averaged over a cycle, the net exposure to oestrogen in women taking these preparations may not differ markedly from that in non-users Oestrogens are known to increase the rate of cell division within the ductal epithelium of the breast, and hence increase the probability of a mutation occurring or of promotion of an existing mutation; progesterone and progestagens may augment this effect The net effect on health of hormonal contraceptives includes their intended highly effective contraceptive properties and a range of side effects, some beneficial and some adverse. The evidence from this study, in the context of the evidence to date, indicates that the adverse effects of both oral and injectable hormonal contraceptives on breast and cervical cancer are transient, and risks in users return to those of never users within 10 y after stopping use. However, the exact time point at which the risk in users returns to that in never users is not known. Beneficial effects of both types of hormonal contraceptives on ovarian and endometrial cancers are predominantly in long-duration users.Figure S1Sensitivity of cervical cancer main results to the potential confounding effects of Pap smear frequency. Squares represent ORs, and horizontal lines indicate 95% CI. Diamonds represent the ORs and confidence intervals for the group comprising women from all three exposure categories immediately above.(TIF)Click here for additional data file.Figure S2Sensitivity of cervical cancer main results to the potential confounding effects of HIV status. Squares represent ORs, and horizontal lines indicate 95% CI. Diamonds represent the ORs and confidence intervals for the group comprising women from all three exposure categories immediately above.(TIF)Click here for additional data file.Figure S3Adjusted OR (95% CI) for breast cancer in relation to use of oral and/or injectable contraceptives, demonstrating the effect of removal of single specific cancer types from the control group. Adjusted for age at diagnosis, year of diagnosis, education, tobacco smoking, alcohol consumption, parity/age at first birth, number of sexual partners, urban/rural residence, and province of birth. Squares represent ORs, and horizontal lines indicate 95% CI.(TIF)Click here for additional data file.Figure S4Adjusted OR (95% CI) for cervical cancer in relation to use of oral and/or injectable contraceptives, demonstrating the effect of removal of single specific cancer types from the control group. Adjusted for age at diagnosis, year of diagnosis, education, tobacco smoking, alcohol consumption, parity/age at first birth, number of sexual partners, urban/rural residence, and province of birth. Squares represent ORs, and horizontal lines indicate 95% CI.(TIF)Click here for additional data file.Figure S5Adjusted OR (95% CI) for ovarian cancer in relation to use of oral and/or injectable contraceptives, demonstrating the effect of removal of single specific cancer types from the control group. Adjusted for age at diagnosis, year of diagnosis, education, tobacco smoking, alcohol consumption, parity/age at first birth, number of sexual partners, urban/rural residence, and province of birth. Squares represent ORs, and horizontal lines indicate 95% CI.(TIF)Click here for additional data file.Figure S6Adjusted OR (95% CI) for endometrial cancer in relation to use of oral and/or injectable contraceptives, demonstrating the effect of removal of single specific cancer types from the control group. Adjusted for age at diagnosis, year of diagnosis, education, tobacco smoking, alcohol consumption, parity/age at first birth, number of sexual partners, urban/rural residence, and province of birth. Squares represent ORs, and horizontal lines indicate 95% CI.(TIF)Click here for additional data file.


TGFB1) T29C and TGF β receptor type 1 (TGFBR1) 6A/9A polymorphisms have been implicated in the modulation of risk for breast cancer in Caucasian women. We analyzed these polymorphisms and combinations of their genotypes, in pre menopausal breast cancer patients (N = 182) and healthy women (N = 236) from western India as well as in breast cancer patients and healthy women from the Parsi community . Western Indian women were characterized by a higher frequency of TGFB1*C allele of the TGF β T29C polymorphism (0.48 vs 0.44) and a significantly lower frequency of TGFBR1*6A allele of the TGFBR1 6A/9A polymorphism  as compared to healthy Parsi women. A strong protective effect of TGFB1*29C allele was seen in younger western Indian women . Compared to healthy women, the strikingly higher frequencies of low or intermediate TGF β signalers in patients suggested a strong influence of the combination of these genotypes on the risk for breast cancer in Parsi women . The frequency of low signalers in Parsi healthy women, while comparable to that reported in Europeans and Americans, was three times higher than that in healthy women from western India . These observations, in conjunction with the low incidence rate of breast cancer in Indian women compared to White women, raise a possibility that the higher frequency of TGFB1*29C allele and lower frequency of TGFBR1*6A allele may represent important genetic determinants that together contribute to a lower risk of breast cancer in western Indian women.Transforming growth factor β1 ( Germline mutations in various cancer susceptibility genes can account for only around 10% of all breast cancer cases TGFB1 T29C  and TGFBR1 6A/9A remain the two most extensively studied polymorphisms. In TGFB1 T29C polymorphism, a replacement of Leucine by Proline at position 29 (TGFB1 codon10 T>C) has been shown to result in an increased secretion of the cytokine TGFBR1 gene, giving rise to the TGFBR1*6A allele TGFBR1*9A allele In breast cancer patients, TGFB129*C allele that is associated with higher production of the cytokine, would provide protection in the initial stages by virtue of its anti-proliferative action on mammary epithelial cells but would enhance the risk at later stages by promoting invasion as well as spread of the disease TGFB1*CC genotype at a higher risk of the disease TGFB1*CC genotype in older American women and pre menopausal Japanese women TGBR1 6A/9A polymorphism on the risk for breast cancer TGFBR1*6A allele et alWith regard to the risk for breast cancer, it has been proposed that the presence of TGFBR1 6A/9A gene polymorphisms in pre menopausal women from western India. To that effect the study was restricted to Marathi-speaking (Maharashtrian) subjects from Western India, living within 200 km radius around the state-capital city of Mumbai (who constitute 80% of the population of the state of Maharashtra). In parallel, we have studied these polymorphisms in breast cancer patients and healthy women from the Parsi (Zoroastrian) community. Parsis in India represent a geo-ethnically isolated community Population based data regarding these polymorphisms in Indian women remains limited TGFB1*CC) on the risk for breast cancer, our results reveal trends that are opposite to those reported in Caucasian women. The basis for these observations and their implications has been discussed with due supporting literature.The comparison of genotypes in healthy subjects from the two ethnically distinct communities studied, and the comparisons between patients and healthy subjects within each of the groups have revealed novel trends with significant implications. Further, in terms of the influence of high TGF beta1 producing genotype  pre menopausal women with confirmed diagnosis of breast cancer treated at the Tata Memorial Hospital during 1999–2005 were recruited within twelve to eighteen months of diagnosis. Lack of family history for malignant disorders could be confirmed for 75% of the cases. Two hundred and thirty six unrelated, healthy premenopausal Maharashtrian women without family history for cancer were recruited from the community. Parsi women with breast cancer (N = 48) comprised of those treated in various hospitals in the city. Mean post diagnosis duration for these patients was 4.8±4.2 yrs (Median  = 3), with sixty two percent of the patients recruited within five years of diagnosis. Healthy, unrelated Parsi women (N = 171) were recruited from the community. Eight Parsi patients as well as ten healthy women volunteers reported a strong family history for cancer.The mean age was lower in healthy Maharashtrian controls than in patients . The available data indicated comparable age at menarche and age at first child birth in the two groups . In ParsTGFB1 T29C polymorphism was performed by PCR–SSP based method, using primers described by Perrey et alInternational Histocompatibility Workshop Group (IHWG) reference panel were used to validate the PCR conditions and as controls in each experiment. Results were further confirmed by DNA sequencing in representative samples. Genomic DNA was amplified using primers, GAGGCCCTCCTACCTTTTG (F) and GCAGCTTGGACAGGATCT (R) as described in the SNP500 cancer database http://snp500cancer.nci.nih.gov/sequencing_assays.cfm?snp_id=TGFB1-01. The amplified products were sequenced by the standard method using big dye terminator kit (ABI) on 3100AVANT Genetic Analyzer (Applied Biosystems).Isolation of DNA for genotyping was carried out as described in our earlier report TGFBR1 6A/9A genotyping, exon 1 of TGFBR1 was amplified using primers described by Kaklamani et al2+, 10% DMSO, 0.1% BSA and 0.5 units of Taq polymerase (Fermentas) along with 1.0 µM primers. PCR conditions were as follows −95°C for 5 min, 35 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 5 min. PCR products (256/247 bp) were analyzed on 2% agarose gel stained with ethidium bromide. The genotypes were also confirmed by running the products on 10% PAGE and by sequencing of representative samples.For TGFB1*29T and TGFBR1*9A, were taken as reference for the respective polymorphisms. For Maharashtrian subjects, the risk was determined after adjusting for age, and was expressed as odds ratio with 95% confidence interval. In each case, the odds ratios were computed for dominant, additive and recessive models. Further, various combinations of genotypes of these two polymorphisms were categorized into high, intermediate and low signalers TGFB1*CC and TGFBR1*9A/9A genotype combination were identified as high signalers in accordance with the high TGF β levels and strong TGFBR1 activity associated with these genotypes. Individuals homozygous for the allele associated with either higher TGF β levels or with strong signaling (TGFB1*CC or TGFBR1*9A/9A) were grouped as intermediate signalers, and subjects with the remaining combinations constituted low signalers. The risk was estimated for the latter two groups with high signalers as reference. SPSS software (version 15.0) was used for statistical analysis. Power calculations were performed using Quanto Chi-square test was used to find out if the genotype frequencies in controls are in Hardy-Weinberg equilibrium and also to determine the significance of difference in genotype frequencies between different groups. For various comparisons, two sided p values were calculated using Fisher’s exact test. To estimate the risk for breast cancer, the frequencies of subjects homozygous for the wild type alleles, namely Two hundred and twenty four healthy controls (95%) and one hundred and sixty (88%) premenopausal breast cancer patients from the Maharashtrian community could be genotyped for the two polymorphisms studied. Similarly, one hundred and sixty (94%) healthy Parsis and forty three Parsi patients (95%) could be genotyped and included in the analysis. The genotype frequencies for the two polymorphisms studied were in agreement with Hardy-Weinberg equilibrium in healthy subjects from both the communities.TGFB1*29C) in healthy Maharashtrian women was higher compared to that in healthy Parsi women .The frequency of the variant allele  was noted in Maharashtrian subjects for the dominant model only  and a protective influence of the TGFB1*CC genotype recessively, were not statistically significant  as reported by two groups In Maharashtrian subjects, the observed frequencies for TGFBR1*6A allele in Maharashtrian women compared to that seen in Parsis [2.0% vs 6.8%, respectively; p<0.01; power-67%) is also in contrast to data from Whites, where the frequency of this allele varies between six and eleven percent TGFBR1*6A allele observed in Maharashtrian women in the present study is the lowest reported so far.The strikingly lower frequency of TGFB1*29C allele (45% power), and a reduced risk for the disease especially in the younger age group . These observations are in direct contrast with those from the Breast Cancer Association Consortium TGFBR1*6A allele to the observed effect of the TGFB1*29C allele on the risk for the disease in Maharashtrian women cannot be ruled out.Our findings in Maharashtrian premenopausal women suggest an association between the presence of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian patients. Of the one hundred and twenty two patients where the information was available, the hormone receptor negative tumor bearing subjects were characterized by a strikingly lower frequency of TGFB1*CC genotype compared to the hormone receptor positive group  with adequate power, sixteen thousand (dominant model) and forty six thousand (recessive model) patients and controls would have to be studied TGFB1*T29C polymorphism with the observed allele frequencies in the western Indian population, to detect a 15% change in the risk for breast cancer, nearly four thousand patients and an equal number of healthy controls would be needed to achieve a power of 80% (at α = 0.05). Similar computations for TGFBR1*6A allele indicate that for a fifteen percent increment in the risk for breast cancer The associations seen in our study however must be viewed with caution in view of the inadequate power of our study  & 4. TheTGFB1*29C allele and lower frequency of TGFBR1*6A allele in healthy Maharashtrian women was revealed when combinations of these genotypes were analyzed. Categorization of individuals into low, moderate and high TGF β1 signalers based on an approach described by Kaklamani et alvs 10.6%; p<0.01). A comparison of the frequencies of high, intermediate and low signalers among healthy subjects reported in literature  for breast cancer indicated for low and intermediate  in western Indian women suggested by our study and in White women by others In summary, the present study on Figure S1Analysis of association of TGFB1 T29C genotypes with hormone receptor status in Maharashtrian subjects. OR – Age adjusted odds ratios with 95% CI; n = 224; **p<0.01; *p = 0.05 for TGFB1*CC genotype.(TIF)Click here for additional data file.Table S1Distribution of various TGFB1 T29C genotypes with respect to ER/PR status.(DOC)Click here for additional data file.


The reasons for the increasing breast cancer incidence in indigenous Māori compared to non-Māori New Zealand women are unknown. The aim of this study was to assess the association of an index of combined healthy lifestyle behaviours with the risk of breast cancer in Māori and non-Māori women.A population-based case–control study was conducted, including breast cancer cases registered in New Zealand from 2005–2007. Controls were matched by ethnicity and 5-year age bands. A healthy lifestyle index score (HLIS) was generated for 1093 cases and 2118 controls, based on public health and cancer prevention recommendations. The HLIS was constructed from eleven factors . Equal weight was given to each factor. Logistic regression was used to estimate the associations between breast cancer and the HLIS for each ethnic group stratified by menopausal status.Among Māori, the mean HLIS was 5.00 (range 1–9); among non-Māori the mean was 5.43 (range 1.5-10.5). There was little evidence of an association between the HLIS and breast cancer for non-Māori women. Among postmenopausal Māori, those in the top HLIS tertile had a significantly lower odds of breast cancer  compared to those in the bottom tertile.These findings suggest that healthy lifestyle recommendations could be important for reducing breast cancer risk in postmenopausal Māori women. The burden of breast cancer is considerable in New Zealand; women have an age standardised incidence rate of 89.4 per 100,000 compared with 84.8 in Australia, and 76.0 in the USA . Māori aThe incidence of breast cancer in Māori women appears to be increasing faster than in other ethnic groups. The age standardised breast cancer rate for European/Other women rose from 114 to 170 per 100,000 women per year from 1981–86 to 2001–04. Over the same period the corresponding rates for Māori rose from 123 to 210 per 100,000 women , and theThere is considerable evidence regarding individual lifestyle factors and breast cancer risk -13. TherThe aim of this study was to assess the combined effect of healthy lifestyle behaviours on the risk of breast cancer, separately for Māori and non-Māori women. A “healthy lifestyle index” was developed, in which study participants were scored according to lifestyle behaviours and adherence to recognised public health and cancer prevention recommendations ,20-24 orThe New Zealand Breast Cancer Study, a population-based case–control study, was conducted to investigate risk factors for breast cancer among different ethnic groups in New Zealand. A detailed description of the study design and methods has previously been published , and thePacific study participants were excluded due to insufficient numbers for the current analysis . Thus, we present here results for Māori and non-Māori/non-Pacific (hereafter referred to as non-Māori) women only. We further excluded cases who completed questionnaires more than one year after the date of their diagnosis of breast cancer (n = 492), since many of the questions in the questionnaire asked participants about their behaviours one year previously. Participants with incomplete diet, lifestyle, and covariate information were also excluded (n = 375). After all exclusions, there were a total of 3211 participants (1093 cases and 2118 controls) included in analyses.All participants completed comprehensive questionnaires on health related behaviours including socio-demographic factors, diet, lifestyle, and reproductive and medical histories. Questions on exercise assessed the average frequency of leisure activities over the preceding year (Godin Leisure Time Exercise Questionnaire) ,27. DietWomen were classified as premenopausal if they had had a menstrual period in the last three months, or if their periods had stopped due to pregnancy/lactation, or use of hormonal birth control. Women were classified as postmenopausal if they reported not having a period in the last three months, and that this was due to natural menopause, surgical menopause involving bilaterial oophorectomy, or use of hormone replacement therapy (HRT). Women who did not fall into these categories, who reported surgical menopause without bilaterial oophoretoomy, and other or unknown reasons for menses cessation were classified in an ‘other amenorrhea’ category; we then assumed that those aged less than 49 years were premenopausal (n = 85) and those aged 49 years or more were postmenopausal (n = 350), based on data from New Zealand and the UK, which indicate 49 years as the median age at menopause for similar birth cohorts ,29.2, 25–29.9 kg/m2 (overweight), and 30 kg/m2 or higher (obese). Participants scored one point for each reported healthy behaviour derived from considering usual weekly patterns of consumption against recommendations [2; and cumulative breastfeeding for 6 months or more. Participants received 0.5 points in the intermediate categories of each health behaviour and 0 points for least healthy behaviours. For the analyses, the index score was categorised into tertiles.A healthy lifestyle index score was calculated for each participant based on public health and cancer prevention recommendations ,20-22,24ndations ,21. Thesndations , having Covariates included were age, parity, age at menarche, history of maternal breast cancer, oral contraceptive use, HRT use, diabetes, and socioeconomic position (SEP). The New Zealand Deprivation Index 2006  was usedDescriptive analyses were initially conducted to explore the variable values and summarise the data. To compare exposure distributions between cases and controls, chi-squared tests were used for categorical variables and Kruskal-Wallis for continuous variables. Logistic regression was used to estimate the association between breast cancer and the lifestyle index by each menopausal and ethnic group. The lifestyle index was assessed as a categorical variable, adjusted for age at menarche and age at diagnosis/interview as continuous variables, and all other covariates as categorical variables.Because of the low response rates in the control group, and evidence of differential non-response by deprivation quintile , we perfAll statistical analyses were performed using Stata version 11.2.Participants excluded due to missing information were compared to those with complete information; for both Māori and non-Māori, those with incomplete data were less likely to be in the most affluent category and to have ever taken oral contraception. There were 776 Māori women  and 2435 non-Māori women  included in the analyses. For cases, the mean time from diagnosis to interview was 247 days. The proportions of cases and controls by all components of the lifestyle index score for each ethnic group are shown in Table There were 987 women classified as premenopausal in the study, including 337 Māori and 650 non-Māori women. The remaining 2224 participants were classified as postmenopausal, including 439 Māori and 1785 non-Māori women. Table Among postmenopausal women, Māori cases were more likely to live in deprived areas than controls. Non-Māori cases were less likely to have used oral contraception, and live in affluent areas; and more likely to have maternal breast cancer than controls. Statistically significant differences were found between Māori and non-Māori controls for HRT, diabetes, deprivation, parity, age at menarche, and age.The associations between the healthy lifestyle index and breast cancer, adjusted for all covariates, are presented in Table Weighting controls for differential non-response by deprivation level did not materially alter our results. Compared to the bottom tertile of the healthy lifestyle index score among premenopausal Māori women, the OR for the top tertile decreased from 0.69 to 0.60 (95% CI 0.22 to 1.65). Among postmenopausal Māori women, the OR for the top scoring tertile remained unchanged 0.47 (95% CI 0.23 to 0.95).This study has found little evidence of an association of the healthy lifestyle index score with breast cancer in non-Māori women, but a moderate association in Māori women, with stronger evidence in those who were postmenopausal than in those who were premenopausal. These results were not explained by accounting for differential non-response in the controls by deprivation levels.While there are an increasing number of studies on combined lifestyle factors and disease outcomes -18, few The results presented here are novel; however, other factors should also be considered with their interpretation. The study had several limitations, including the potential selection and recall biases commonly associated with case–control studies involving patient interviews. We accounted for non-response in the control group using post-stratification weighting. Exclusion of cases who completed questionnaires after one-year post diagnosis was done to minimise recall bias and reverse causation. Many questions related to behaviours in the previous year i.e. pre-diagnosis and it is likely that those interviewed after one year would have changed behaviour patterns since their cancer diagnosis. Therefore to ensure that the exposures measured were not a direct result of the cancer diagnosis (reverse causation), these participants were excluded from this study.The study involved relatively small participant numbers. This restricted the levels of categorisation for some variables, and hence, the heterogeneity of the index as there was not enough data for finer stratification. Furthermore, small numbers in some strata, especially among premenopausal Māori women, limit the precision of the effect estimates. However, in light of the different risk profiles for breast cancer by menopausal status these groups were kept separate, rather than producing menopause-adjusted results.A further limitation was the level of detail available for some exposures, particularly for dietary information. Moreover, the index includes foods that could be inter-related. For example, reducing one’s intake of cheese could be compensated by either increasing consumption of fish  or red meat . The former would receive 2 points associated with healthy behaviours whereas the later would receive only 1 point on the index. The index could also be imprecise for any vegetarians in the study as there was no information available on alternative protein sources. However, the focus of the study was patterns of behaviours rather than specific individual exposures, and the intention was to capture the combined effect of multiple dietary choices and health behaviours. The index components were given equal weight as they were all considered to be indicators of healthy living rather than specific risk factors for breast cancer. The behaviours, or behaviour patterns, are an indication of general lifestyle choices, and measures are likely to reflect habitual exposures, rather than just at a single point in time ,33. As cWhile all of the components of the healthy lifestyle index score involve recommended health behaviours, for the dietary components, there is ‘limited’ or ‘suggestive’ evidence with regard to the specific risk associated with breast cancer. The World Cancer Research Fund/American Institute for Cancer Research states this to mean that the current evidence is too limited to permit a probable or convincing causal judgement, but that the evidence shows a generally consistent direction of effect .Based on the healthy lifestyle index score, the healthy behaviours most likely to be followed by participants were breastfeeding, and eating white meat; while the lowest scores were generated for fruit and vegetables, followed by exercise, and cheese consumption. The relative contribution of the components of the index score also differed by ethnic group. Fish consumption generated fewer points for non-Māori, while fruit and vegetables, smoking, and BMI generated fewer for Māori. Overall, our findings suggest that healthy lifestyle interventions could have potential for reducing ethnic inequalities in breast cancer.The method of cancer detection was not known for the cases in this study (screening or symptomatic); however, the tumour characteristics for the full cohort have been previously published , and theTwo previous studies have found protective effects for breast cancer associated with a healthy lifestyle, based on different index components. A Mexican case–control study found women in the highest quintile of the calculated healthy lifestyle index had significantly lower odds of developing breast cancer than their counterparts in the lowest quintile of the index  . Their iExamination of combined modifiable factors can be an effective way of translating cancer epidemiological findings into primary prevention programmes ,18,31. HEthnic differences have been found in the understanding and interpretation of currently used food labels in New Zealand ,44. DisaThese findings suggest that, at a population level, the promotion of healthy lifestyle interventions could have a positive impact on postmenopausal breast cancer risk in Māori women. This study, however, was limited by low numbers, and therefore further work in this area is needed to confirm whether targeted public health intervention programmes would be effective for addressing inequalities in breast cancer incidence.The authors declare that they have no competing interests.F McK participated in the design of the study, performed the statistical analysis, and drafted the manuscript. LEL participated in the study conception, design, and acquisition of data. MJ contributed to the interpretation of data and helped to draft the manuscript. RF participated in the study conception, design, and acquisition of data. NP contributed to the interpretation of data and helped to draft the manuscript. IR conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/12/prepub


Although a previous study found high risk of breast cancer in mothers of children with soft tissue sarcomas, breast cancer risks in mothers of sufferers of other childhood cancers largely remain unknown. The aetiology is not fully understood. The present study explored this excess by varying type of childhood solid cancer and formulated a hypothesis.Mothers of 2668 children with solid tumours included in the Manchester Children’s Tumour Registry, 1954–96, were traced and followed up to 31 December 2000 through the UK National Health Service Central Register. Standardized incidence ratio (SIR), p-values and 95% confidence intervals were calculated from age and calendar-year-specific female breast cancer incidence rates for England and Wales.There was a significant excess of breast cancer in mothers overall  mainly due to mothers of children with rhabdomyosarcoma (RMS) , skin cancer  and central nervous system tumours . Maternal breast cancer risk was associated with late age at birth of the index child, and male sex and young age at diagnosis in the index child. Risk was highest in the ten years, following the birth of the index. The pattern was seen most strongly in mothers of children with embryonal RMS.There are excesses of breast cancer in mothers of children with solid tumours in general and specifically in RMS, skin and central nervous system (CNS). There appears to be a temporal relationship between certain tumours in children and breast cancer in their mothers, suggesting an origin of their respective pregnancy. We propose a mother–foetal interaction mechanism to explain this association. Since a twofold increased risk of breast cancers among first degree relatives of children with soft tissue sarcomas was reported , 2, it hThe mothers of 2668 children with histologically confirmed solid tumours diagnosed younger than 15 years of age, who were included in the Manchester Children’s Tumour Registry (MCTR) between 1 January 1954 and 31 December 1996 were eligible. The ascertainment procedure and the follow-up procedure have been described in detail elsewhere , 12 and Expected numbers of breast cancers were calculated from serial age-specific female breast cancer incidence for England and Wales, from 1971 onwards, applied to the similarly defined arrays of person-years at risk generated by the data. National rates were used rather than north-west England regional rates to allow for migration of families out of the region during the lengthy follow-up period. All population data were supplied by the Office for National Statistics, London, UK. For earlier years at risk, the incidence rates for 1971 were used to calculate expected numbers. To calculate the risk of carcinoma of the breast, mothers entered the person-years at risk on the date of birth in the index child and left the person-years at risk on the date of diagnosis of carcinoma of the breast, date last known to be alive, date of embarkation from the UK, date of death, or the closing date of the study, whichever was the earlier. Two-sided p-values and 95% confidence intervals (CI) of standardized incidence ratios (SIRs) were computed on the assumption the observed numbers followed a Poisson distribution. All analyses have been carried out using Stata .Separate analyses were initially performed for groups of mothers defined by diagnostic group in the index child. In those groups, showing a statistically  significantly elevated SIR, the excesses were explored in more detail by comparing sub-groups defined by characteristics in the children and their mothers according to the following rationale. Germline mutations may lead to earlier ages of cancer onset. We hypothesized that early age of diagnosis in the index child may be a marker for breast cancer risk in the mothers. We therefore analysed breast cancer risks in selected sub-groups of mothers whose children were below the population median age for diagnosis of their respective tumours. Similarly, we conducted analyses of breast cancers by age at diagnosis in the mothers. Factors that may indicate mother–foetal interaction in pregnancy include differences in breast cancer risks in mothers of boys compared with girls  and a teThe mothers of 2345 (88%) were successfully traced and flagged at NHSCR. The reasons for non-trace were due to lack of name/date of birth, adoption, emigration, in prison and in the armed forces.There was no significant excess risk of carcinoma of the breast in mothers of children with osteosarcoma and chondrosarcoma. Therefore, previous findings based on an earlier series from the MCTR were not substantiated in this extended series .In view of our previous findings, mothers of children with embryonal RMS (eRMS) were analysed separately and showed a significant excess of breast cancer .The following diagnostic groups were selected for sub-group analyses of maternal breast cancer risk: eRMS, skin cancer and CNS tumours .For the individual diagnostic group of eRMS in the index, there were significant excesses of carcinoma of the breast in mothers of boys , breast cancer diagnosed under 50 years old , breast cancer in the ten years following birth of the index , and in mothers above 35 years old at birth of the index . In addition analyses by age at diagnosis in the index child showed that mothers of children under median age at diagnosis had a much higher SIR of 7.3  than mothers of children at the median age and above (SIR=0). This was due to mothers of children in the first age quartile at diagnosis . For skin cancer in the index child, there were significant excesses among mothers in all sub-groups. However, markedly higher SIRs were observed for mothers in the first ten years, following the birth of the index and aged 35 years or more at the birth of the index. The population median age at diagnosis of skin cancer is above 60 years. Therefore, no analyses by age of the index case were carried out since all index cases were exceptionally young and located at the extreme lower end of the age distribution. When melanoma and non-melanoma skin cancers were analysed separately, a similar pattern of results was obtained for both groups. For individual diagnostic group of CNS tumours in the index, non-significant SIRs above 1.5 were found for mothers of boys, mothers in the ten years following birth of the index, and mothers above 35 years old at birth of the index. PNET was the only CNS tumour sub-group with a population median age below 15. Analyses of breast cancer in mothers of children with PNET diagnosed below the median age of nine years showed O 8, SIR 1.9 and 95%CI 0.8–3.8.The study indicates that the excess risk of carcinoma of the breast among mothers of children with solid tumours is not uniform across all groups, but is associated with a small number of tumour types and patient characteristics.The observed pattern is seen most strongly in eRMS. There are higher risks in mothers of children with eRMS diagnosed at very young ages, mothers of boys and in the ten years following birth of the index, suggesting an origin of the respective pregnancy. A biologically feasible interpretation would be hormonal mother–foetal interaction in pregnancy in genetically susceptible individuals .This is suggested by higher risks of developing carcinoma of the breast in mothers in the first few years after birth of the index and higher risk in mothers of boys. It is likely that in these children the eRMS originates before birth. It is known that the foetus itself is actively involved in the production and regulation of oestrogen, a hormone that is an established aetiological factor in breast cancer.Risk factors for breast cancer, identified in population-based studies, include early menarche, late menopause, late first full-term pregnancy, and use of exogenous oestrogens. All these factors increase the amount of oestrogen to which the breast epithelium is exposed . To meetThe foetal adrenal cortex also undergoes a process of maturation involving apoptosis (programmed cell death), a basic process in tissue remodelling, to eventually produce the mature adrenal cortex. Apoptosis starts in the 14th week, increases with advancing gestation and is maximal during the first postnatal month .We hypothesize that a germline defect in the foetus in a gene involved in cell-cycle control, apoptosis and tissue development, results in abnormal levels of steroid hormones via aberrant functioning of the foetal adrenal cortex, due to failure of the normal process of apoptosis and maturation.This would explain the observed temporal relationship between pregnancy, birth of the index child, development of tumour in the child and development of breast cancer in the mother. One such gene is TP53.The differences in risks between mothers of boys and girls with eRMS might suggest that the relevant cancer-associated genes in male foetuses are more important in maintaining development and normal functioning of fast growing organs, such as the foetal adrenal cortex than in females. Male foetuses grow faster and males have higher average birth weight but shorter gestations than girls , 20.The striking effects of maternal age at birth of the index child may suggest that timing of pregnancy in mothers is also important. In older women, there may be a greater chance that precancerous changes are present in the breast epithelium. In these circumstances, carrying a foetus with a cancer predisposing mutation may trigger the process of carcinogenesis, and thus accelerate the development of breast cancer, following the birth of the index child. The effect would be even more marked in mothers who carried the same mutation.A practical implication of our model is that the highest risks and earliest ages at onset would be seen in mothers of babies who were mutation carriers and who themselves also carried the mutation. These mothers are predisposed and are also subjected to abnormal exposure to steroids produced by the foetal adrenal cortex, which had failed to mature (apoptosis) in the normal way. Mothers of babies who were mutation carriers would also be at increased risk even though they themselves do not carry the mutation. We would predict that carrier mothers of non-carrier babies and non-parous carriers would have a later age of onset (though still young) than carrier mothers of carrier babies.In this series of eRMS, there was one known case with a germline TP53 mutation, whose mother was also a mutation carrier. In addition, this case had a sister, 15 months older, who carried the mutation. The mother’s breast cancer was diagnosed at age 27 years, 19 months after the birth of the child. Four children with other tumours, ACC, astrocytoma and PNET (2 cases) carried germline TP53 mutations. Their mothers, who were also confirmed mutation carriers, all developed breast cancer seven years or less after the birth of the respective children.The striking excesses of breast cancer in mothers of children with eRMS in the ten years following birth of the index are unlikely to be completely explained by a transient increase in risk of breast cancer following the first pregnancy in mothers. First pregnancy has been reported to be associated with increased risk of breast cancer in the 15 years or less after birth , 22. HowThe observed breast cancer excess is not explained by these potential confounders relevant to the first birth. The results need not be seen as contradictory to the previous findings of transient increase in risks of breast cancer in the first pregnancy since the present study shows that the pregnancy is a crucial period in the development of breast cancer in women. However for the mothers in the present study, the pregnancy with the child who subsequently develops cancer appears to be of greater importance in the development of breast cancer than that with the first child and the striking effects in the mothers observed in the present study might reflect a different aetiology from mothers of children without cancer. The mechanism proposed above, which relates breast cancer risk to peculiarities of adrenal cortex development, might explain the marked excess.Because of the nature of this cancer-registry-based study, retrospective direct measurement of oestrogen levels in pregnancy with the index is not possible. Similarly, access to birth records which may contain other relevant details would require individual written consent, which would be impractical since the births date back to 1939 and many index patients and their mothers have died. Furthermore, it is likely that many records will have been destroyed. However, data on birth weight are available for children with RMS as part of previous family studies. Oestrogens are known to stimulate cell proliferation and represent an important determinant of foetal growth . BirthweWe compared the birthweights of the children with RMS whose mothers developed breast cancer, and index children whose mothers did not, with that of live-born children in the general population. Mean birthweights of index children of mothers with breast cancer and mothers without are 257 and 54 g more than that of the general population, respectively, . Mean birthweights of boys with RMS whose mothers did or did not develop breast cancer are 114 and 27g more than that of male babies in the general population, respectively. Mean birthweights of girls with RMS whose mothers did or did not develop breast cancer are 658 and 75 g more than that in the general population, respectively. There is a trend of decreasing birthweight among index cases whose mothers had breast cancer, index cases whose mothers did not have breast cancer and children in the population. This is consistent with the role of oestrogen in foetal development, and with our hypothesis relating to failure of normal maturation of the foetal adrenal cortex.Interestingly, the present study reported an excess of carcinoma of the breast in mothers of children with skin cancer (melanoma and non-melanoma combined) with the highest excess in the first ten years, following birth of the index (SIR 23.5). Skin cancer is typically an adult-onset cancer and is very rare in children. The median age for sporadic melanoma is 67 and 35 years for familial melanoma. Therefore, all childhood cases are unusual and may be associated with genetic susceptibility. It is known that germline mutations in CDKN2A are the most common cause of inherited susceptibility to melanoma. CDKN2A encodes two proteins via alternate reading frames (ARFs). The p14ARF product, acting via the p53 pathway, can induce cell-cycle arrest or apoptosis. In multi-case melanoma families with mutations affecting p14ARF specifically there appears to be increased risk of breast cancer . This isIn mothers of children with CNS tumours, the present study showed borderline significant excesses among mothers of boys and mothers diagnosed in the ten years following the birth of the index. This might suggest roles of BRCA2 as well as TP53. Biallelic BRCA2 germline mutation carriers do develop childhood brain cancers as recently reported , 10, altIn contrast to an earlier study , we did Considering childhood cancers are rare events, the sample size in our study is relatively large and allows us to carry out stratified statistical analyses. However, non-statistically significant results in certain diagnostic groups in children cannot completely rule out the possibility of increased breast cancer risk due to the aforementioned mechanism. Among other diagnostic groups, the highest point estimate of SIR for breast cancer is in mothers of children with adrenocortical carcinoma (ACC) although statistically not significant due to the small number. Of 12 mothers of children with ACC, two mothers developed breast cancer following birth of the child, compared with an expected number of 0.3. It is thought that ACC originates in the foetal adrenal cortex, that is an abnormality in the process of cell proliferation and programmed cell death during adrenal development. Furthermore, germline mutations in TP53 have been found among the majority of ACC cases in a number of studies , 30.In this population-based study, a near complete ascertainment of children with solid tumours in a well-defined geographical area was achieved, and the case children included in the study were representative of all the case children in the corresponding population series. Therefore, the present study is unlikely to be subject to ascertainment bias due to notification of severely affected families or the knowledge of family history of cancer in surviving cases only. Neither can the excesses be explained by the bias due to the follow-up procedure, because the majority of mothers were successfully traced and there are no reasons to believe that the causes of non-trace are influenced by occurrence of breast cancer.The study is free of reporting bias because all data were obtained from population registries rather than self-reports, that is both breast cancer in mothers and childhood cancers were derived from population-based data. However, another possible bias worth mentioning is the ‘healthy mother effect’, by analogy with the healthy worker effect, that is mothers may experience a lower breast cancer risk than those non-parous women in the general population.Childbearing has been demonstrated to be associated with decreased breast cancer risk in human and animal studies due, in part, to the pregnancy-specific hormone, chorionic gonadotropin (hCG) . The conIn conclusion, the study confirms and extends the previous findings of a temporal relationship between embryonal RMS in children and breast cancer in their mothers. We propose a hypothesis to explain these observations and suggest a possible mechanism. This mechanism may also be operating in mothers of children with other types of cancers. The present study points to pregnancy as the critical period, when mothers and children are physiologically linked. Germline mutations to cancer-associated genes, inherited or de novo might play an important role. Germline mutations might commonly target the p53 pathway affecting tissue remodelling in the foetal adrenal cortex, not just a single gene within that pathway. The pathway may be disrupted by a germline mutation within the TP53 gene itself, as well as p14ARF, BRCA1 and BRCA2, etc. Further studies of aetiology of breast cancer in mothers of children with cancer incorporating genetic/molecular measurements including mutation screening of cancer-associated genes will be useful in exploring this hypothesis. This is feasible as tissue banks and appropriate genetic techniques become available. The study may help to define periods of highest risk to enable health interventions aimed at reducing mortality from breast cancer in susceptible women.


PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer.Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC).A single-nucleotide polymorphism from the P=4.6 × 10−3), but did not reach significance in the BCAC replication study alone .Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, PIK3CA does not have a strong influence on the risk of breast cancerCommon germline variation in  PIK3CA) oncogene on chromosome 3q26 that encodes the p110α catalytic subunit of PI3K, are commonly observed in human cancers. Amplification and overexpression of PIK3CA results in increased production of the phosphatidylinositol-3,4,5-triphosphate second messenger, hyperactivation of the PI3K/AKT pathway, and stimulation of cellular transformation and tumour progression , gastric (4–25%), endometrial (36%), liver (36%), brain (27%) and breast (18–40%) tumours (Phosphatidylinositol-3 kinases (PI3Ks) constitute a lipid kinase family integral to signalling pathways that regulate many cancer-related processes, including cell proliferation, adhesion, apoptosis, survival and motility . Alteratgression . Somatic tumours .PIK3CA mutations have been consistently associated with ER-positive and human epidermal growth factor receptor-2 (HER2)-positive tumour status  have been described previously .The Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer case–control study and 26 case–control studies from Breast Cancer Association Consortium (BCAC) contributed data to these analyses  were selected (r2>0.80 in European–American genotype data from HapMap release 21). A total of 1741 Mayo Clinic samples  were genotyped on custom oligo pool assays at Illumina Corporation  using the Illumina GoldenGate assay. All SNPs had genotype call rates >95%. Concordance between duplicate samples was 100%. Genotyping of rs1607237 in CGEMS and BCAC was performed using a TaqMan allelic discrimination assay or the Sequenom platform  via standard protocols. Genotyping concordance was verified with internal duplicates and overall data quality was ensured using independent genotyping of 96 CEU samples by each genotyping center (Four haplotype-tagging single-nucleotide polymorphisms (SNPs) within The collection of pathology and tumour marker information for BCAC has been described previously . ER/PR sP⩾0.001). Single-nucleotide polymorphism associations were tested using unconditional logistic regression adjusting for age and state of residence in a log-additive model. We also calculated odds ratios (ORs) and 95% confidence intervals (CIs) separately for heterozygotes and rare homozygotes. The association between rs1607237 and breast cancer risk in stage 1 of the CGEMS GWAS was evaluated as previously described (P=0.10), and further adjustment for age did not change the results. Analyses of pathology-specific subsets of cases were conducted using polytomous regression with controls as the reference outcome, adjusting for study site.Evidence of departure from Hardy–Weinberg equilibrium (HWE) was assessed in controls using a goodness of fit test and none was observed . To provide a more stable estimate of risk in this population, 8429 additional CGEMS subjects were genotyped for rs1607237. In all 5436 cases and 5280 controls from stage 1 and 2 of CGEMS, rs1607237 was strongly associated with a decrease in breast cancer risk  or in the two Asian BCAC studies . Similarly, a significant association was observed when considering only women of European ancestry in the combined analysis , and two BCAC studies of Asian women . In addition, we explored differences in PIK3CA SNP associations in the combined data set by tumour subtype  in all studies combined and when considering only women of European ancestry in the combined studies . However, the association did not achieve significance in the large third stage involving only BCAC studies. Although the first two stages of our analysis suggest an association between PIK3CA and breast cancer risk, our inability to confirm this finding in the BCAC studies suggests that the result should be interpreted with caution.Here we report an association between inherited variation in the oncogene PIK3CA coding and promoter regions to better understand the relationship between rs1607237 and other variation in this region. Rs1607237 was not in strong linkage disequilibrium with two non-synonymous polymorphic variants in the coding region of PIK3CA, rs1051399 (r2=0.0060) and rs3729680 (r2=0.034), which had been genotyped in HapMap samples of European ancestry. However, an additional 18 non-synonymous variants were either not polymorphic or had not been genotyped in the HapMap samples, making inference about the relationship between rs1607237 and all variants of unknown significance in the PIK3CA coding region difficult. In addition, two PIK3CA promoter SNPs were in low LD with rs1607237 . However, it remains possible that PIK3CA promoter SNPs that were not captured in this study are related to breast cancer risk.We further explored the linkage disequilibrium patterns in the PIK3CA SNPs. Thus, it remains possible that evaluation of these variants in the larger BCAC cohort might detect associations with risk. While the effect of rs1607237 on risk is small, the association between inherited variation in this important oncogene and breast cancer risk does provide valuable biological insight into the development of this disease. Validation of rs1607237 in GWAS studies from other large collaborative groups and additional studies by BCAC with detailed pathology information are necessary to confirm this association. Functional evaluation of this variant is needed to fully understand the relationship between inherited PIK3CA variation and breast cancer risk.It is also important to note that the effect estimate for rs1607237 in the BCAC replication studies and in the overall BCAC, MCBCS and CGEMS studies is quite small (OR=0.97). This limits our statistical power to detect significant associations in these studies despite the large sample size, particularly in analyses utilising pathology information that is available for only a subset of subjects. Similarly, we had limited power to detect associations in the original MCBCS study with the three non-significant


Breast development and hormonal changes at puberty might affect breast cancer risk, but epidemiological analyses have focussed largely on age at menarche and not at other pubertal stages.We investigated associations between the timing of pubertal stages and breast cancer risk using data from a cohort study of 104,931 women . Pubertal variables were reported retrospectively at baseline. Breast cancer risk was analysed using Cox regression models with breast cancer diagnosis as the outcome of interest, attained age as the underlying time variable, and adjustment for potentially confounding variables.in situ) occurred. An increased breast cancer risk was associated with earlier thelarche , menarche , regular periods  and age reached adult height , and with increased time between thelarche and menarche , and shorter time between menarche and regular periods . These associations were generally similar when considered separately for premenopausal and postmenopausal breast cancer.During follow-up (mean = 4.1 years), 1094 breast cancers (including ductal carcinoma Breast duct development may be a time of heightened susceptibility to risk of carcinogenesis, and greater attention needs to be given to the relation of breast cancer risk to the different stages of puberty. Breast cancer is the most common invasive cancer among women world-wide . Timing Menarcheal age is a well-established breast cancer risk factor , with anWe, therefore, investigated the associations of age at various pubertal stages and the duration between these stages with breast cancer risk in the Breakthrough Generations Study (BGS), a large cohort study.The BGS is a UK cohort study focussed on breast cancer aetiology. It has received approval from the South Thames Multicentre Research Ethics Committee and all participants gave written informed consent. All women living in the UK and aged 16 years or older were eligible to join the study. Registered supporters of the charity Breakthrough Breast Cancer were invited, and other women joined either by self-referral or by nomination from an existing participant. Over 113,000 participants were recruited between 2003 and 2013. As a requirement of study entry, all BGS participants completed a postal recruitment questionnaire, which included detailed questions about known and possible breast cancer risk factors. Participants are followed-up approximately every two and a half years, using further postal questionnaires that update the recruitment information and reports of cancer diagnoses. Follow-up is based on time since recruitment; thus, at any given time different participants are at different stages of follow-up, because recruitment occurred over 10 years. At present, up to two rounds of follow-up questionnaires have been sent to participants, depending on when they were recruited. Further details of the cohort can be found elsewhere [N = 111,344). Of these, 6,399 women were diagnosed with invasive or in situ breast cancer and 14 had a prophylactic bilateral mastectomy before they joined the BGS, and therefore were excluded from the analysis. This left 104,931 subjects who formed the study cohort.BGS participants were potentially eligible for the current analyses if they joined the study before 1 April 2010, and thus had reached at least their first follow-up point  of reported cases, the diagnosis was confirmed by cancer registry data or pathology reports (95%) or from the participant’s general practitioner (4%); the remaining 1% were assumed to be true cases because they reported appropriate treatment for breast cancer.Incident invasive and The explanatory and confounding variables analysed were from self-reported data on the recruitment questionnaire. The explanatory variables were age at thelarche, age at menarche, age at regular periods, age when reached adult height, the intervals between these ages, and growth spurt in height between ages 7 and 11 years (as a proxy for peak growth). Age at regular periods was only analysed for women who indicated that their periods had become regular naturally rather than as a consequence of oral contraceptives or some other cause. All ages were reported to the last completed year. Information about height in childhood was collected as self-reported height relative to other girls of the same age, in five categories. Subjects were classed as having had a growth spurt between ages 7 and 11 years if their height compared with girls of a similar age increased by at least one category from age 7 to 11.The potential confounding variables analysed were age at recruitment , menopausal status , family history of breast cancer in a first-degree relative , adult height , age at first full-term pregnancy , and hormone replacement therapy (HRT) use . These variables were selected as they are breast cancer risk factors and, in exploratory analyses, affected the relationship between at least one of the explanatory variables and breast cancer risk. Adjustments for ethnicity and socio-economic status, (based on place of residence (Acorn scores )), were in situ breast cancer, death, emigration from the UK or loss to follow-up. The follow-up period was based on the date of the second follow-up questionnaire for the 49,880 women who joined the study before 1 July 2006, and on the date of the first follow-up questionnaire for the remaining women. Participants who were lost to questionnaire follow-up were flagged with the NHS Central Registers, if they had agreed to this. If flagged, they were then censored at 31 December 2010 because national cancer incidence data are currently incomplete after that date; otherwise, they were censored at loss to follow-up.The data were analysed using survival analysis methods. Subjects contributed person-time from recruitment into the BGS until the earliest of: end of follow-up period, diagnosis of invasive or in situ breast cancer diagnosis as the event of interest, and all other outcomes censored. The validity of the proportional hazards assumption was assessed for each model. The initial models were unadjusted but accounted for age, because it was the underlying time variable, and menopausal status, as time at risk was stratified by menopausal status . Also analyses were conducted separately for these two menopause categories as pre- and post-menopausal breast cancer have been shown to have different aetiologies to some extent [Cox regression models were fitted in Stata 10.1   with atte extent . Menopauin situ breast cancers as the outcome of interest in turn. Receptor status was known for too few women to allow analyses to be split by this factor.Sensitivity analyses were performed by repeating the analyses in various subgroups: women who were younger than age 60 years at recruitment, because recall might be better in these women; after exclusion of women who had a younger first-degree relative in the BGS because of potential correlation within pairs; and taking only invasive or Table in situ  breast cancer, 20 (0.02%) had a prophylactic bilateral mastectomy, 349 (0.3%) died, and 99,545 (94.9%) were followed to the end of their follow-up period. Of the remaining 3,924 (3.7%), 3,240 (3.1%) were followed to December 2010 by flagging, 423 (0.4) were censored at emigration and 261 (0.3%) were lost to follow-up.During follow-up , 1,094 (1.0%) women in the cohort were diagnosed with invasive  or Table in situ breast cancer by the duration between pubertal events. Breast cancer risk increased as the time between thelarche and menarche increased, but was only significant in those whose menarche occurred at least two years after the onset of thelarche ). Breast cancer risk also increased as the time between menarche and regular periods decreased, but again was only significant when the time between the two events was at least two years ). Breast cancer risk was not significantly associated with the time between thelarche and onset of regular periods, or between menarche and attaining adult height.Table in situ breast cancers were broadly similar with no significant differences.For premenopausal and postmenopausal breast cancer analysed separately  reported their absolute height at these ages because they or their parents had kept records. Based on these recorded data, the average increase in height from age 7 to 11 was 26.8 cm in the growth spurt group and 23.4 cm among participants who were not classed as having had a growth spurt (P <0.01 for difference), suggesting that our growth spurt measure was a reasonable proxy.Existing evidence suggests that rapid growth in childhood and adolescence may increase breast cancer risk ,27. In tIt is also of interest whether the timing of the growth spurt affects breast cancer risk. The age at peak growth is one year earlier than menarche on average  and was The timing of pubertal events is likely to be correlated to some extent, which may partly explain our findings and the fact that many of the effect sizes were similar across pubertal events. However, Table These analyses included comprehensive adjustment for potential confounders. While benign breast disease is an established risk factor for breast cancer, we did not adjust for it as it is unlikely to be a confounder of the relationship between puberty variables and breast cancer. It is possible that benign breast disease is on the same causal pathway and is a mediator of the relationship between pubertal variables and breast cancer risk, though evidence linking pubertal factors to benign breast disease is lacking.The pubertal variables that we analysed were reported retrospectively; therefore their accuracy should be considered. Whereas menarche is a well-defined single event that women can recall with reasonable accuracy , the onAnother limitation of the present analyses is that the average follow-up was only four years. Despite this, more than 1,000 breast cancers had occurred due to the size of the cohort. In addition to the large sample size, the other strengths of this study were its prospective nature, and that it examined several potential breast cancer risk factors for which there has been no or little existing research.While the only inclusion criteria for women to join the BGS were that they must be aged at least 16 years and living in the UK, there are indications that the study participants are on average a somewhat high-risk population - they tend to be of higher socioeconomic status and more often to have a family history of breast cancer than the UK general population . There sIn conclusion, the novel findings of this study are that earlier thelarche and a longer time between thelarche and menarche might increase breast cancer risk, as well as the already-investigated effects of early menarche, regular periods and the time between the two. This suggests that breast duct development may be a period of raised susceptibility to breast carcinogenesis, and that the association between puberty and breast cancer is complex and not best represented by a single event. Further research on the effects of pubertal stages other than age at menarche is needed to elucidate these relationships.BGS: Breakthrough generations study; HRT: Hormone replacement therapy.All authors declare that they have no competing interests.AJS and AA are Principal Investigators of the Breakthrough Generations Study and conceived the study. All authors have contributed towards the design and data collection of the Breakthrough Generations Study. DHB conceived these analyses and planned and performed them with input from MJS, MEJ, EM and JG. DHB wrote the first draft of the manuscript and all authors contributed to data interpretation and revisions of the manuscript. All authors have approved the final version of the manuscript.in situ breast cancer in relation to pubertal variables, by menopausal status at breast cancer incidence. Table S2. Risk of invasive or in situ breast cancer in relation to duration between pubertal events, by menopausal status at breast cancer incidence. Table S3. Sensitivity analyses of risk of invasive or in situ breast cancer in relation to pubertal variables. Table S4. Sensitivity analyses of risk of invasive or in situ breast cancer in relation to duration between pubertal events.Risk of invasive or Click here for file