The risk of sporadic colorectal cancer is mainly associated with lifestyle factors and may be modulated by several genetic factors of low penetrance. Genetic variants represented by single nucleotide polymorphisms in genes encoding key players in the adenoma carcinoma sequence may contribute to variation in susceptibility to colorectal cancer. In this study, we aimed to evaluate whether the recently identified haplotype encompassing genes of DNA repair and apoptosis, is associated with increased risk of colorectal adenomas and carcinomas.ERCC1, ASE-1 and RAI, and risk of colorectal adenomas and carcinomas in a Norwegian cohort. Odds ratio (OR) and 95% confidence interval (CI) were estimated by binary logistic regression model adjusting for age and gender.We used a case-control study design  to test the association between polymorphisms in the chromosomal region 19q13.2-3, encompassing the genes ASE-1 polymorphism was associated with an increased risk of adenomas, OR of 1.39 (95% CI 1.06–1.81), which upon stratification was apparent among women only, OR of 1.66 (95% CI 1.15–2.39). The RAI polymorphism showed a trend towards risk reduction for both adenomas  and carcinomas  among women, although not significant. Women who were homozygous carriers of the high risk haplotype had an increased risk of colorectal cancer, OR of 2.19 (95% CI 0.95–5.04) compared to all non-carriers although the estimate was not statistically significant.The We found no evidence that the studied polymorphisms were associated with risk of adenomas or colorectal cancer among men, but we found weak indications that the chromosomal region may influence risk of colorectal cancer and adenoma development in women. Colorectal cancer (CRC) still is one of the leading causes of cancer deaths in developed countries and adenomas are the predominant precursors of this disease .The risk of sporadic CRC is mainly associated with lifestyle factors and may be modulated by several genetic factors of low penetrance ,3. GenetERCC1, ASE-1 and RAI was identified as a high-risk haplotype, defined as homozygous carriers of a haplotype consisting of ERCC1 Asn118AsnA, ASE-1 G-21AG and RAI IVS1 A4364GA. This haplotype consisting of three marker SNPs, is shown to be strongly associated with increased risk of skin cancer [Many published candidate gene association studies have assessed cancer risk by examining a single SNP per gene or a single locus at a time analysis approach. It has been suggested that use of haplotypes in association studies may have increased power over single-allele studies . Haplotyn cancer , breast n cancer  when comERCC1 gene encodes a subunit of the nucleotide excision repair complex required for the incision step of nucleotide excision repair (NER) [ERCC1 may play a role in carcinogenesis.The ir (NER) ,10. SincASE-1 gene (Anti Sense ERCC1) also called CD3EAP , encodes a nucleolar protein localized to fibrillar centers of the nucleolus and to the nucleolus organizer region of mitotic chromosomes [The omosomes . The mouomosomes .RAI (RelA-associated Inhibitor) also called iASSP and PPP1R13L, is an inhibitor of the RelA subunit of the transcription factor NF-κB [The gene In the present paper, we aimed to evaluate whether the recently identified haplotype encompassing genes of DNA repair and apoptosis, is associated with increased risk of colorectal adenomas and carcinomas in a homogenous Norwegian cohort. In addition we investigated whether polymorphisms in these genes alone contributed to colorectal adenoma and carcinoma risk. To the best of our knowledge this high-risk haplotype has not previously been studied in relation to colorectal adenomas or carcinomas. Studying colorectal adenomas in addition to colorectal cancer may give information about the risk factors in the earlier stages of carcinogenesisThe KAM cohort  is based on the screening group of the Norwegian Colorectal Cancer Prevention study (The NORCCAP study) in the county of Telemark , and a sThe KAM biobank consists of blood and tissue samples from 160 CRC cases , 1044 individuals identified with adenomas in the large intestine  and 400 controls, defined as individuals with normal findings at flexible sigmoidoscopy screening. Adenoma cases and controls were all drawn from the NORCCAP study. All of the participants completed a questionnaire on demographics, health status, dietary, and smoking habits, alcohol consumption, physical exercise and occupation. The questionnaire contained information on a family history of adenomas and carcinomas, and the included cases and controls had no known personal history of genetic predisposition. The adenocarcinomas were collected prior to chemo- or radiotherapy treatment. Two specialist histopathologists examined the histology of the adenomas independently in order to determine the tumor stage as mild, moderate or severe dysplasia. They reached the same conclusion in all cases. The KAM study is approved by the Regional Ethics Committee and the Data Inspectorate.In the present study we analyzed available blood samples from 156 cases with adenocarcinoma, 981 cases with adenomas, (i.e. 227 high-risk and 754 low-risk adenomas) and 399 controls. The 53 hyperplastic polyps were not included in the analysis. Diminished numbers of available cases in the KAM biobank and analyzed cases are due to loss of samples during preparation and/or lack of information from questionnaires. A high-risk adenoma is defined as an adenoma measuring = 10 mm in diameter and/or with villous components and/or showing severe dysplasia . The disGenomic DNA was isolated from blood samples according to standard procedures  with minERCC1 Asn118Asn (rs#3177700) was determined on a light cycler as described [ASE-1 G-21A (rs#967591) was determined using the primers 5'-TCT GCA ACC TGG TGC GAG-3', 5'-CCT TTC TCC TTC CAC CAA CG-3' and the probes G-allele: 5'-FAM- AGG GTT GCC TGA GGT GTG GGT CC-BHQ, A-allele: Yakima Yellow- AGG GTT ACC TGA GGT GTG GGT CC-BHQ-3'. The reactions were run for 40 cycles at 15 s at 94°C, 60 s at 62°C.RAI IVS1 A4364G (rs#1970764) was determined as described [Genotypes were determined on an ABI 7500 using endpoint readings or on a light cycler . escribed .ASE-1 G-MiniTab Statistical Software, Release 13.1 Xtra (Minitab Inc.) and SPSS  12.0.1 for Windows were used for the statistic calculations. Odds ratios (OR) and 95 % confidence intervals (CI) were calculated using binary logistic regression to assess the relationship between each polymorphism and the colorectal adenoma or carcinoma cases. When, estimating OR for carriers of the haplotype the comparison group was all the other remaining individuals, individuals who were not homozygous carriers of the high-risk haplotype. We have previously shown that neither known heterozygotes nor possible heterozygotes are at increased risk of breast cancer. This strongly suggests that the haplotype is detecting a recessive marker. We have therefore chosen only to look at homozygous carriers' vs. the rest. All data are adjusted for age and gender.Smoking dose and alcohol intake were also included in the adjustment in the logistic regression analyses, but the results did not differ from those adjusted only for age and gender. Including smoking and alcohol parameters resulted in lower numbers of cases and controls due to missing data in the questionnaires. Due to this we choose to use the data adjusted only for age and gender.ERCC1 polymorphism  and a 92 % chance for the ASE-1 polymorphism  and the RAI polymorphism .With 156 CRC cases and 399 controls we have a 74 % chance (at the 5 % level) to detect an OR of 2 for the RAI, ASE-1 and ERCC1 genes for both cases and controls are shown in Table 2 [see ERCC1 Asn118Asn p = 0.945, ASE-1 G-21A p = 0.757 and RAI IVS1 A4364G p = 0.673), and the distributions of the alleles are in agreement with those found in other Scandinavian populations [The genotypic distribution of the three polymorphisms in the le 2 see . The genulations  also preRAI polymorphism showed a trend towards risk reduction for both adenomas and carcinomas in women. The risk estimate was lowest but not statistically significant for carcinoma. For adenomas the result was borderline statistically significant, OR of 0.70 (95% CI 0.49–1.01).The ASE-1 polymorphism was associated with an increased risk of adenomas, OR of 1.39 (95% CI 1.06–1.81), which upon stratification was apparent among women only, OR of 1.66 (95% CI 1.15–2.39). No association was found for carcinoma cases.The ERCC1 polymorphism and risk of colorectal adenomas and carcinomas.There was no significant association between the Women who were homozygous carriers of the high risk haplotype had an increased risk of colorectal cancer, OR of 2.19 (95% CI 0.95–5.04) compared to all non-carriers although the estimate was not statistically significant. Among men an inverse association between the high-risk haplotype and colorectal cancer was found, although the association was not statistically significant. No association was found with adenoma cases.We have also tested the gene-environment interaction between the haplotype and the three polymorphisms, and cigarette smoking and alcohol intake, but they showed no significant association with colorectal adenoma or carcinoma (results not shown).In this case-control study we investigated whether carriers of a specific haplotype, encompassing genes of DNA repair and apoptosis located on chromosome 19q13.2-3, were at increased risk of colorectal adenomas and carcinomas. In addition we investigated whether polymorphisms in these genes alone contributed to colorectal adenoma and carcinoma risk.et al. [The high-risk haplotype has previously been shown to be associated with increased risk in young or middle-aged persons. For basal cell carcinoma, effects were only seen for the age group below 50 years ,19. For et al.  have fouRAI is shown to be the strongest determinant of the haplotype [RAI which increases the protein level of RAI, resulting in a defective apoptosis trigger. It has previously been observed that the polymorphism in RAI and the high risk haplotype may be associated with cancer risk in women only [RAI, we have consistently found protective effect of the variant allele. For lung cancer, however, it was found that the variant allele was protective among women , while no protective effect was observed for men . Here, we see the same pattern. Among women, carriers of the variant allele of RAI IVS1 A4364G were at reduced risk of both colorectal cancer  and adenomas . Vogel et al. [The polymorphism in aplotype ,21. RAI aplotype , but itsaplotype . Up reguaplotype ,23. Prevmen only -8. For tl et al.  have shoet al. [Very little is known about the function of ASE-1. Whitehead et al.  identifiet al. . Involveet al. . We founThe CRC cases and controls have not been matched by age which may affect the results in a study. However, the controls were recruited from the same cohort as the adenoma and carcinoma cases. The adenoma cases and controls are from 50 to 64 years of age. Further, our controls have been screened and found polyp free and the risk of any of them having colon cancer at the time of inclusion is not very likely.RAI are consistent with previous findings [Taking the number of statistical tests performed and the p-values we cannot exclude that our findings are due to chance. The relatively small numbers of the CRC cases limited our power to detect and association with carcinomas. However, the findings for findings -8.ASE-1 polymorphism was associated with an increased risk of adenomas in women. The RAI polymorphism showed a trend towards risk reduction for both adenomas and carcinomas in women, although not significant. There was no significant association between the ERCC1 polymorphism and risk of colorectal adenomas and carcinomas. In conclusion we found no evidence that the studied polymorphisms were associated with risk of adenomas or colorectal cancer among men, but we found weak indications that the chromosomal region may influence risk of colorectal cancer and adenoma development in women.The high-risk haplotype is not associated with increased risk of adenomas or colorectal cancer prior to gender stratification. When stratifying for gender, we did detect an effect in women and an inverse effect in men in relation to CRC cases, although not significant. Of the high-risk haplotype markers, the The author(s) declare that they have no competing interests.CFS prepared samples for analysis and contributed with the data analysis. She prepared the first draft of the paper. MS prepared samples for analysis and contributed with the data analysis. She also contributed to the manuscript. ILH participated in collection and quality control of the questionnaires. UBV, HW and BAN genotyped the DNA samples. EHK brought the idea of the KAM study and organized it. All authors discussed the results, contributed to interpretation of the results and the final manuscript.The pre-publication history for this paper can be accessed here:RAI, ASE-1 and ERCC1 genotypes and high-risk haplotype, and development of colorectal carcinomas and adenomasTable 2: Distributions of . The table presents the genotypic distribution of the three polymorphisms in the RAI, ASE-1, and ERCC1 genes for both cases and controls, and the estimates of relative risk associated with the three polymorphisms as well as the predefined haplotype.Click here for file


Celecoxib inhibits PGE2 production in cancerous tissue. We previously reported that PGE2 levels in nipple aspirate fluid (NAF) and plasma were not decreased in women at increased breast cancer risk who received celecoxib 200 mg twice daily (bid). The endpoints of the current study were to determine if a short course of celecoxib 400 mg bid would decrease PGE2 levels in women 1) at increased breast cancer risk, and 2) with established breast cancer.NAF and plasma samples were collected before, 2 weeks after taking celecoxib 400 mg bid, and two weeks after washout from 26 women who were at increased breast cancer risk. From 13 women with newly diagnosed breast cancer, NAF from the incident breast and plasma were collected before and on average 2 weeks after taking celecoxib. Additionally, in nine of the 13 women with breast cancer, NAF was collected from the contralateral breast.No consistent change in NAF or plasma PGE2 levels was noted in high risk premenopausal women. NAF PGE2 levels decreased after celecoxib administration in postmenopausal high risk women (p = 0.02), and in both the NAF (p = 0.02) and plasma (p = 0.03) of women with breast cancer.Celecoxib 400 mg bid taken on average for 2 weeks significantly decreased NAF, but not plasma, PGE2 levels in postmenopausal high risk women, and decreased both NAF and plasma PGE2 levels in women with newly diagnosed breast cancer. PGE2 levels may predict celecoxib breast cancer prevention and treatment efficacy. Our observations are preliminary, and larger studies to confirm and extend these findings are warranted. Eligibility for enrollment in the first National Surgical Adjuvant Breast and Bowel Project (NSABP) breast cancer prevention trial required that a participant have a > 1.66% projected 5 year probability of developing IBC [Women at increased breast cancer risk include those with a strong family history of breast cancer, those with a history of ductal carcinoma 2 is produced from arachidonic acid by either COX-1 or -2. PGE2 has tumor and cell growth promoting activity [2 levels above 15 ng/g appear to have a significantly worse survival rate than those with levels ≤ 15 ng/g [2 than benign breast tumors or normal breast tissue [Cyclooxygenase (COX)-1 and COX-2 may be present in breast tumors to catalyze the conversion of arachidonic acid to prostaglandins, prostacyclins, or thromboxanes. While COX-1 expression is constitutive, COX-2 is inducible . Prostagactivity . Women wt tissue . A retrot tissue .Nonsteroidal antiinflammatory drugs (NSAIDs), including aspirin, indomethacin and ibuprofen, inhibit both COX-1 and COX-2. Inhibition of COX-1 leads to a number of adverse effects, including gastrointestinal ulcers and renal toxicity . Recent 2 levels in the breast, as measured both in NAF and in plasma, providing both organ specific and systemic information [2 is concentrated in NAF compared to matched plasma, but that celecoxib 200 mg bid administered for two weeks did not significantly decrease PGE2 concentrations in NAF or plasma.We are currently able to collect breast nipple aspirate fluid (NAF) from 95% of nonlactating adult females ages 18 to 80 with the use of a modified breast pump . Using oormation . We obse2 levels in women at high risk for, or with newly diagnosed breast cancer. We observed that celecoxib 400 mg bid lowered PGE2 levels in the NAF and plasma of women with breast cancer, and in the NAF of high risk postmenopausal women.Subjects with familial adenomatous polyposis (FAP) who received celecoxib for six months at 400 mg bid, but not those receiving 100 mg bid, demonstrated a significant reduction, compared to placebo, in mean polyp number . ConsequWomen were provided an Institutional Review Board approved protocol and required to give written informed consent in order to enroll in the study. Subjects evaluated had to be ≥ 18 years old and be at increased breast cancer risk, based on the subject having either a Gail model risk of developing IBC in a 5 year period of > 1.66%, or previously treated DCIS or IBC (now finished with treatment and free of disease).Pregnant and lactating women were not eligible. Women could not have been currently on NSAIDs, aspirin, a COX-2 inhibitor, warfarin, or have taken such a medication within two weeks of enrollment. Subjects could not have a significant history of peptic ulcer disease, upper gastrointestinal bleeding, asthma, or be allergic to sulfonamides or NSAIDs. A complete blood count, serum electrolytes and liver panel had to be within normal limits. Subjects were recruited from the Breast Evaluation Clinics at the University of Missouri-Columbia.Celecoxib was taken daily for 14 days by women at increased risk, and for 10 to 24 (median 14) days by women with recently diagnosed breast cancer. Compliance was assessed through the count of returned pills. All subjects were required to have taken at least 80% of the prescribed medication. While the goal was to have women at increased risk and women with cancer take celecoxib for the same time period, variability in the operative date required some flexibility in the latter group.2 values were assigned to the cancer group. For all subjects, NAF was collected from the same breast for each of the three visits. A detailed account of the NAF collection technique has been reported [For all women, both those with cancer and those at increased risk, NAF was analyzed from only one breast. If one breast contained cancer and the other did not, only NAF from the breast with cancer was analyzed. For serum collected from women with cancer, PGEreported ,13.Baseline NAF and blood collection were performed prior to the ingestion of celecoxib. For women at increased risk, washout NAF and blood samples were collected 14 days after stopping celecoxib. Thus, subjects at increased risk were asked to provide three NAF and three plasma samples , whereas subjects with recently diagnosed cancer provided two NAF and two plasma samples. Each subject granted permission for us to contact them yearly to determine if they had developed new or recurrent breast cancer.NAF samples were collected into capillary tubes and stored at -80°C until analysis. Eight mL of blood were also collected from the subject in a tube containing heparin, the blood spun for 10 min at 1600 rpm, and the plasma fraction decanted and stored at -80°C until analysis. All women had NAF and plasma collected within 12 hrs of their last dose of celecoxib. The half life of the medication is 11.5 hrs.2, due to its established link to cancer growth. NAF and plasma samples were analyzed by immunoassay for their PGE2 content. NAF and plasma samples were analyzed as per the manufacturer's instructions . The kit uses a monoclonal antibody to PGE2 to competitively bind the PGE2 in the standard or sample. Briefly, samples were diluted in 100 μL assay buffer supplied by the manufacturer, pipetted into appropriate wells, incubated for 18–24 hrs at 4°C, washed, substrate solution added, followed by 1 hr incubation, and absorbance measured at 405 nm.The biomarker chosen for analysis was PGE2. A linear regression equation was created from standards of known PGE2 concentration, and PGE2 concentrations of unknown samples fit to the standard curve regression equation, corrected for aliquot volume and expressed as nanograms of PGE2/mL of original sample. The goodness of fit of the standard curve, R2, for NAF samples was 0.999. The goodness of fit was similar for the plasma samples.For NAF and plasma analyses, a standard curve was prepared using serial dilutions of PGEMedian values of continuous variables were computed for the various groups of subjects. Due to the potential non-normality of the data, ranking procedures were used for all analyses with continuous variables. The Wilcoxon Rank Sum Test was used to compare independent groups. Examples of these comparisons include comparing pre- and postmenopausal women, comparing the cancer to the high risk group, etc. The Wilcoxon Signed Ranks Test was used to make within group comparisons such as comparing pretreatment to posttreatment, pretreatment to washout, etc.2 before and after treatment in the samples.Subjects were enrolled from May 2003 to December 2004. Complete recruitment data were available for all subjects screened in 2004, during which the majority of the subjects analyzed in the study were recruited. In 2004, 158 women were screened, of which 27 were eligible, enrolled and were evaluable. Reasons for ineligibility included use of nonallowed medication, medical history, and blood screen abnormalities. All eligible subjects were evaluable, which required successful collection of NAF or plasma and detection of PGENAF and plasma samples were collected before, 2 weeks after taking celecoxib 400 mg bid, and two weeks after washout from 26 women who were at high risk for developing breast cancer. NAF was successfully collected from the same breast at for 95% 113/120) of subject visits. From 13 women with newly diagnosed cancer, NAF from the incident breast and plasma were collected before and on average 2 weeks after taking celecoxib 400 mg bid. Additionally, in nine of the 13 women with breast cancer, NAF was collected from the contralateral breast. Thus, we collected 26 NAF and 26 plasma samples from the 26 women at increased risk of breast cancer, plus 9 NAF samples from breasts contralateral to a breast with cancer, for a total of 35 NAF samples from breasts at increased cancer risk. We collected NAF from 12/13 subjects from the breast containing cancer, as well as plasma in the 13 subjects with cancer . Changes in individual subjects after treatment are illustrated in Figure We evaluated all NAF samples for PGE2 concentrations in plasma were determined in all samples . Changes in individual subjects after treatment are illustrated in Figure PGEes Table . Similar2 levels (Tables 2 levels in the NAF (p = 0.02) but not the plasma of postmenopausal high risk women. Changes in individual subjects after treatment and after washout are illustrated in Figure 2 levels in premenopausal women.We next determined if menopausal status influenced the effect of celecoxib on PGEs Tables  and 5. C2 levels were significantly higher in pre- than in postmenopausal high risk women  in pre- than in postmenpausal women  and after washout (p = 0.003), NAF PGE and afteen Table . No diff2 were compared before and after treatment with celecoxib. In the high risk (cancer) groups, median concentrations of PGE2 in NAF were 27.9 (16.4), 63.7 (13.9), and 48.1 (not applicable) times higher than in corresponding plasma before celecoxib, after celecoxib, and after washout. The differences in the ratios of NAF to serum PGE2 levels before vs. after treatment, before vs. after washout, and after treatment vs. after washout were significant in neither the high risk nor the cancer group.When both NAF and plasma were available from the same subject, relative concentrations of PGEIn the high risk group, there were 11 subjects who experienced side effects from celecoxib, four of whom dropped out. In the other 7 subjects, the side effects resolved spontaneously. In the four who dropped out, the side effects  all resolved shortly after stopping celecoxib. Of the 13 subjects with recently diagnosed cancer who received celecoxib, one had insomnia which resolved spontaneously. There were no dropouts in the cancer group.2 in NAF or plasma of women at risk for breast cancer or in women with known cancer. This study builds on our initial study, which found that celecoxib 200 mg bid did not alter PGE2 levels in NAF or plasma under normal, nonstressed conditions.We carried out experiments to investigate whether the COX-2 inhibitor celecoxib at a dose of 400 mg bid could significantly affect endogenous levels of PGE2 levels significantly decreased in the NAF, but not in the plasma, of postmenopausal women at increased risk of breast cancer. Second, PGE2 levels significantly decreased in both the NAF and plasma of celecoxib-treated women with newly diagnosed breast cancer.In the current study, we observed two important findings. First, PGE2 levels significantly decreased in post- but not premenopausal high risk women. We did not routinely record whether premenopausal subjects started celecoxib in the first or second half of their menstrual cycle. We have this information for 10 women in the high risk group and one woman for the cancer group. We did not find a significant difference in PGE2 response to celecoxib in either the high risk or cancer group based on when the subject started medication in the first or second half of her cycle, although sample size limits the reliability of these findings.It is unclear why PGE2 was concentrated in NAF, we determined its relative concentration in NAF vs. plasma in both high risk women and in women with newly diagnosed breast cancer. In both groups, PGE2 was concentrated in NAF relative to plasma. The NAF/plasma relative concentration was numerically, although not significantly, lower in women with cancer than in those at increased risk.In order to assess if PGE2 in NAF vs. plasma differed in the current study compared to our earlier study [2 in human bodily fluids by other investigators [The relative concentration of PGEer study . There atigators . Third, COX-2 expression has been evaluated in preclinical models and in clinical breast specimens. In a dimethylbenzanthracene (DMBA) rat model of breast cancer, celecoxib was found to dramatically reduce the incidence, multiplicity, and volume of breast tumors relative to control . While C2 is measurable and is concentrated in NAF compared to plasma. Plasma concentrations of PGE2 in women with a predisposition to breast cancer are within the normally accepted basal range (less than 1 nanogram/mL). Celecoxib 400 mg bid significantly decreased PGE2 levels in women with newly diagnosed breast cancer in both NAF and plasma, and in the NAF of high risk postmenopausal women, consistent with clinical studies evaluating its effects on colon and duodenal polyps. We acknowledge that additional studies, with a placebo control, are required to determine whether monitoring NAF eicosanoid concentrations will be of value in describing the development and progression of breast cancer, or in monitoring the effect of candidate chemopreventive agents such as celecoxib.PGEin situ; DMBA: dimethylbenzanthracene; FAP: Familial Adenomatous Polyposis; IBC: invasive breast cancer; NAF-nipple aspirate fluid; NSABP: National Surgical Adjuvant Breast and Bowel Project; NSAIDs: nonsteroidal anti-inflammatory drugs; PG: prostaglandinADH: atypical ductal hyperplasia; DCIS: ductal carcinoma The author(s) declare that they have no competing interests.ERS designed the study, enrolled subjects, and performed the majority of manuscript preparation. WQ conducted all PGE2 analyses. JTF assisted with manuscript preparation and critique. LS enrolled subjects and entered data. JEH performed the statistical analyses.The pre-publication history for this paper can be accessed here:


The association between coffee consumption and the incidence rate of breast cancer has been analysed in 152 incident cases of breast cancer that developed among 14,593 Norwegian women during a mean follow-up of 12 years. At the time of inquiry they were between 35 and 51 years of age, and at the end of follow-up between 46 and 63. There was an overall weak negative association between daily intake of coffee and risk of breast cancer, which was not statistically significant. However, the association with coffee varied, depending on the body mass index (BMI) of the women. In the lean  there was an inverse relation between coffee intake and risk of breast cancer . In this group, women who reported drinking 5 cups or more per day had an age-adjusted IRR of 0.5  compared to women who had 2 cups or less. In women with Quetelet's index equal to or greater than 24 there was a positive relation between coffee intake and breast cancer risk , where the corresponding age-adjusted IRR was 2.1 . This interaction effect between coffee intake and BMI was statistically significant . In summary, the results of this study suggest that coffee consumption reduces the risk of breast cancer in lean women, whereas coffee might have the opposite effect in relatively obese women.


In a retrospective cohort study, the mothers and sisters of 740 breast cancer patients aged under 36 at diagnosis have been studied for mortality and cancer incidence. Significantly increased breast cancer mortality was observed below age 60 , but not at older ages . The cumulative breast cancer incidence in the relatives was 3.6% by age 50, 7.6% by age 60 and 11.6% by age 70. They also suffered excess mortality below age 60 for cancers of reproductive sites  and lung , but not for other sites . This large population-based cohort study provides further confirmation of genetic susceptibility to breast cancer at young ages.


BRCA1, BRCA2, and ATM can cause a substantial increase in risk of breast cancer. However, these mutations are rare in the general population, and account for little of the incidence of sporadic breast cancer in the general population. Therefore, research has been focused on examining associations between common polymorphisms and breast cancer risk. To date, few associations have been described. This has led to the hypothesis that breast cancer is a complex disease, whereby a constellation of very low penetrance alleles need to be carried to present a risk phenotype. Polymorphisms in the manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX-1) genes have been proposed as low penetrance alleles, and have not been clearly associated with breast cancer. We investigated whether variants at both polymorphisms, while not independently associated with breast cancer risk, could influence breast cancer risk when considered together.Germ-line mutations in genes such as MnSOD  and GPX-1  were genotyped via TaqMan assay. Disease risk was evaluated using logistic regression.A case-control study nested within the Nurses' Health Study was performed comparing 1262 women diagnosed with breast cancer to 1533 disease free women. The MnSOD and the Leu198Leu genotype of GPX-1.While neither allele alone shows any change in breast cancer risk, an increase in the risk of breast cancer  is observed in individuals who carry both the Ala16Ala genotype of GPX-1 and MnSOD genes are associated with an increased risk of breast cancer.Polymorphisms in the  Both manganese superoxide dismutase and glutathione peroxidase act as antioxidant enzymes ,2. Non-sFew polymorphisms have shown statistically significant associations with breast cancer risk to date. One explanation for this lack of association could be that common polymorphisms do not have a large enough effect on the function of any particular gene to be responsible for cancer development alone. Rather, polymorphisms in several biologically related genes which slightly modify the function of each individual gene, may combine to explain the genetic component of breast cancer risk. Additionally, lifestyle factors could also play a role in disease etiology, either independently or in combination with genetic variants.MnSOD  and GPX-1  genes, and found no association between these individual polymorphisms and breast cancer risk [GPX-1 which is less responsive to selenium [MnSOD which is associated with increased urinary DNA adduct levels [We have previously genotyped non-synonymous polymorphisms in the cer risk . Howeverselenium , and theMnSOD  and GPX-1  polymorphisms were performed by the 5' nuclease assay (TaqMan) on the ABI Prism 7900HT Sequence Detection System . TaqMan primers, probes, and conditions for genotyping assays are available on request from the authors. Our study included a total of 1262 incident breast cancer cases diagnosed after blood draw up to June 1, 2000, and 1533 matched controls, all drawn from 32,826 women in the Nurses' Health Study who gave a blood sample in 1989–90. Controls were randomly selected participants who were free of diagnosed cancer (except non-melanoma skin cancer), and matched to cases based on age, menopausal status, recent post-menopausal hormone use, and time, day, and month of blood collection. A detailed description of this study population has been previously reported [Genotyping assays for the We used SAS v9.1  for all statistical analyses. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression, controlling for matching factors, age at menopause, age at menarche, age at first birth and parity, body mass index at age 18, weight gain since age 18, history of benign breast disease, and family history of breast cancer using PROC LOGISTIC. Interactions were tested by likelihood ratio tests comparing the model with main effects for each polymorphism to the model including indicator variables for the cross-tabulation of the two polymorphisms. All p-values reported are two-sided. Power calculations were carried out using Quanto [MnSOD and GPX-1 genotypes and breast cancer risk. The distribution of the combined genotypes shown is not statistically different from that expected, given the respective genotype frequencies (Chi-squared = 3.14 with 1 d.f p = 0.08). Neither of these two SNPs individually increases breast cancer risk. Given that these two genes are active on the pathway of detoxification of reactive oxygen species (ROS) from O- to H2O2 (MnSOD), and further to H2O (GPX-1) and these polymorphisms influence the efficiency of this detoxification, we hypothesized that risk could be detected when the two SNPs are combined. In these analyses, the combination of the two polymorphisms is associated with significantly increased breast cancer risk. Individuals homozygous for the Ala16 allele of MnSOD and Leu198 allele of GPX-1 have a 1.87 fold increase in breast cancer risk compared to Val16 and Pro198 carriers, and the p-value for the interaction between these genotypes is 0.03. Further in vitro or in vivo work should be carried out to examine the differences in ROS levels in cells containing variant GPX-1 and MnSOD alleles.Table To date, few such interactions have been detected and reported in the literature. Of major concern to studies examining the interaction between multiple exposures (either gene × gene or gene × environment) on disease risk is study size. In order to have sufficient power to detect such associations, large data sets are necessary. Our study has 80% power to detect an odds ratio of 1.85 for an interaction between the recessive models for two polymorphisms such as those reported here, where neither polymorphism alone increases risk. In the present analyses, we benefit from the relatively high prevalence of the two polymorphisms. If the prevalence of one or both polymorphisms was lower, very large studies would be needed to detect an interaction similar to the one we observed. Of obvious interest would be to examine more genes along the same pathway, as well as non-genetic variables such as plasma antioxidant levels or lifestyle factors which affect oxidative stress such as cigarette smoking, and consumption of alcohol, as well as dietary antioxidants or supplements. However, including further variables would necessitate even larger sample size. Future work on polymorphisms in genes along biologically related pathways, and the inclusion of gene × environment interactions, will necessitate further development of statistical methods. Currently accepted methods to model the association between large numbers of variables (both genetic and non-genetic) and disease risk are plagued with problems of sparse data, as well as model choice and uncertainty. When methods robust to these problems are developed, further examination of the antioxidant activity pathway via multivariate analyses of gene × gene and gene × environment interactions can be examined.GPX-1 and MnSOD genes are associated with an increased risk of breast cancer.Polymorphisms in the MnSOD: Manganese superoxide dismutaseGPX-1: Glutathione peroxidase 1OR: Odds RatioCI: Confidence IntervalThe author(s) declare that they have no competing interests.DGC performed statistical analyses and wrote the manuscript with RMT. DJH designed and obtained funding for the study, and provided editorial support. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Breast cancer appears to be a disease of both the developing and developed worlds. Among Turkish women, breast cancer is the second leading cause of cancer-related deaths. The aims of this cross-sectional study were to determine levels of knowledge about breast cancer and to evaluate health beliefs concerning the model that promotes breast self- examination (BSE) and mammography in a group of women aged 20–64 in a rural area of western Turkey.244 women were recruited by means of cluster sampling in this study. The questionnaire consisted of sociodemographic variables, a risk factors and signs of breast cancer form and the adapted version of Champion's Health Belief Model Scale (CHBMS). Bivariate correlation analysis, Chi square test, Mann-Whitney U test and logistic regression analysis were performed throughout the data analysis.The mean age of the women was 37.7 ± 13.7. 49.2% of women were primary school graduates, 67.6% were married. Although 76.6% of the women in this study reported that they had heard or read about breast cancer, our study revealed that only 56.1% of them had sufficient knowledge of breast cancer, half of whom had acquired the information from health professionals.Level of breast cancer knowledge was the only variable significantly associated with the BSE and mammography practice . BSE performers among the study group were more likely to be women who exhibited higher confidence and perceived greater benefits from BSE practice, and those who perceived fewer barriers to BSE performance and possessed knowledge of breast cancer.By using the CHBMS constructs for assessment, primary health care providers can more easily understand the beliefs that influence women's BSE and mammography practice. Breast cancer appears to be a disease of both the developing and developed worlds. It is the leading type of cancer in women. Among Turkish women, breast cancer represents 24.1% of all cancers seen in women and is the second leading cause of cancer-related deaths. About 2390 new cases of breast cancer were diagnosed in 1999 in Turkey [Early detection and prompt treatment offer the greatest chance of long-term survival . MammogrWomen in their 20 s and 30 s should have a CBE as part of a periodic (regular) health examination by health professionals preferably every 3 years. After the age of 40, women should have a CBE every year, as recommended by the ACS .Annual mammography is considered the most valuable tool for detecting breast cancer in the earliest possible stages, before the cancer has metastasized and when interventions are most effective and least invasive and debilitating. The decline in breast cancer mortality has been largely attributed to regular mammography screening practice . The ACSThe Health Belief Model (HBM) was one of the first models to adapt theories from the behavioural sciences in order to examine health related problems. It is still one of the most widely recognised and used models in health behaviour applications. This model was originally introduced by a group of psychologists in the 1950's to help explain why people would or would not use available preventive services, such as chest x-rays for tuberculosis screening and immunisations for influenza .Many investigators studying beliefs related to cancer screening practices have used the HBM as a theoretical framework to study breast cancer screening behaviour such as BSE or mammography screening ,16.The HBM has frequently been applied to breast cancer screening . The modAccording to the HBM, a woman who perceives that she is susceptible to breast cancer and that breast cancer is a serious disease would be more likely to perform regular breast examinations. Similarly, a woman who perceives more benefits from and fewer barriers to BSE would be more likely to practice BSE. A woman who has an internal cue (body perception) or who has been exposed to an external cue  would also more readily adopt BSE, as would a woman who wants to improve her health and who is confident of positive results ,19.The HBM consists of 6 concepts: (1) perceived susceptibility to an illness, (2) perceived seriousness of the illness, (3) perceived benefits for the presumed action, (4) perceived barriers for the presumed action, (5) confidence in one's ability, and (6) health motivation. Behaviour is also a result of the belief that a certain action will benefit the individual and that this benefit will outweigh any barriers .The investigation of attitudinal components of health-related behaviour has been important. If attitudes related to health behaviour can be identified, health protection interventions for attitudinal change can be developed, and an increase in desirable health behaviour would result .Despite being one of the few cancers which is able to be detected in its preclinical stage, BSE and mammography are still only practised by a low proportion of the population in our country, and this anomaly forms the basis of this study. The aims of this study were to determine knowledge levels about breast cancer, and to evaluate the health belief model about BSE and mammography in a group of 20–64 year-old women in Muradiye-Manisa, a rural area of western Turkey.In this cross-sectional study, the data was collected from the rural area of Manisa, a city located in the western region of Turkey. The total study population consisted of 1,829 women aged between 20–64 years living in the Muradiye Health District in Manisa. The aim of the study was to reach at least 236 women with a 99.9% confidence level and 0.50 prevalence by using the Epi info 2000 statistical software. Cluster sampling method was performed proportional to the percentage of the three neighbourhood populations in Muradiye District. A total of 24 clusters were selected. It was decided that each cluster would consist of 10 houses and the first house of the cluster was selected randomly. After the first initially selected house, every subsequent forth house was selected . For the women who were absent, the visits were repeated once. And if they weren't found on these second visits either, the neighbouring house was included in the study. As a result, 244 women were interviewed. When the visits were made to the selected houses, it was found that, in some houses, more than one female occupant aged 20–64  resided at the given address. The response rate of the study surpassed 100%, since all of the women aged 20–64 living in the selected houses were incorporated in the study. Although the advancement in age is a risk factor in breast cancer, women over 65 were not included in this study, due to a perceived risk of inadequate perception. Four trained intern doctors from the medical faculty collected the data in face-to-face interviews between January and February 2005. They made clear the confidentiality, benefits, risks, and future implications of the research. Data was then collected from those who verbally consented to participate. The study was ethically approved by the Manisa Province Health Directorate.The questionnaire comprised of sociodemographic variables, a form regarding risk factors and signs of breast cancer and the measurement of the health belief model of breast cancer see . SociodeThe subjects were also asked if they had knowledge of breast cancer and if there were family members and/or friends with breast cancer histories. 24 questions were comprised to determine the individuals' level of knowledge of breast cancer; 15 of which were about risk factors and 9 about the signs and symptoms of breast cancer. The answers were 'true', 'false' and 'don't know'. The knowledge score was computed by totalling the number of correct answers for all 24 questions. The knowledge score was re-coded into dichotomous variables by taking the mean value as the cut off value to evaluate knowledge levels, coded sufficient = 1 and insufficient = 2. Univariete risk analysis was performed with the demographic, socioeconomic variables and knowledge levels. Logistic regression analysis was used to evaluate the significant variables with 95% CI  found in univariete analysis.CHBSM was also applied to the subjects. The Health Belief Model Scale was developed in 1984 and was revised in later works by Champion ,22. It wBivariate correlation analysis, Chi square test, Mann-Whitney U test and logistic regression analysis were performed in the data analysis, by using SPSS v10.0 statistical package.The women's mean age was 37.7 ± 13.7. 49.2% of women were primary school graduates, 67.6% were currently married, 61.1% had a sufficient income level and 91.0% were housewives  reported that they had heard or read about breast cancer, but only 56.1% of them had sufficient knowledge of it. TV/ radio programs were identified as the main source of information on breast cancer by 39.3% of the participants. Health professionals were mentioned as a source of information by 23.4% of the sample.According to logistic regression analysis; the odds of having insufficient knowledge about breast cancer was 2.2 (1.2–4.0) times higher in women who lived in extended families than in nuclear families, 3.7 (1.7–7.9) times higher in women who had never had a CBE than in women had had a CBE and 5.2 (2.5–10.8) times higher in women with no family/friends history of breast cancer than in women with family/friends breast cancer history  times higher in women who lived in extended families than in nuclear families, 3.7 (1.7–7.9) times higher in women who had never had a CBE than in women had had a CBE. A better understanding of the fore mentioned is made possible when taking in to account the knowledge that a majority of the women who live in extended families are more likely to be illiterate and to have a more traditional lifestyle. Champion and Miller have indicated that sociodemographic variables may influence attitudinal variables and thus indirectly influence behavioural patterns. Experiential and demographic variables have several direct paths to attitudinal variables and potential indirect paths to BSE behaviour . In thisAs in studies of Black American, Australian, Hispanic American, Jordanian, Chinese, Vietnamese, and Asian Indian women, the results of this study indicate that the revised CHBMS is a useful framework to identify factors that influence BSE for Turkish women ,29,33,34In this study seriousness, susceptibility and motivation were not significant in explaining BSE performance on a regular basis, but increased confidence, BSE-benefit and reduced BSE-barriers were significantly associated with it. In other studies with Chinese, Jordanian and U.S. women, seriousness has been continuously reported as a non-significant predictor of BSE ,35-37. TMotivation was found to be the significant factor for mammography practice in this study. This finding was parallel to the results of Holm and Eun-Hyun ,38. In BBenefits were a significant variable predicting BSE and mammography performance. It was supported by the findings of American studies that have reported where women who perceived more benefits from BSE behaviour were more likely to perform BSE ,19,38,39Confidence was found to be a significant factor for both BSE and mammography practice in this study. The confidence levels of women who perform BSE on a regular basis, and have recommended mammograms, were much higher than among non-practitioners. Research on the relationship between confidence and breast cancer screening has been reported by several researchers with varying degrees of attention to measurement issues. One research group found a positive relationship between confidence and BSE . In a stOur results indicate that an increase in BSE practice and recommendations for mammograms may be achieved through enhancement of breast cancer awareness and possibly by reducing barriers. By using the CHBMS constructs for assessment, primary health care providers can gain an understanding of the beliefs that influence women's BSE and mammography practice. That of the 76.6% of the sample that reported ever hearing or reading about breast cancer, the majority (39.3%) mentioned TV/radio as their main source of information underscores the potential effectiveness of the media in modifying health behaviour. Health professionals were mentioned as a source of information by 23.4% of the sample. So, the role of health care workers as an information source in breast cancer should be increased. Further research is recommended using a larger sample size with women in rural and urban areas, including the cost-effectiveness of designing and implementing preventive care.The author(s) declare that they have no competing interests.PED conceived of the study, performed the statistical analysis, and drafted the manuscript. DÖ participated in the design of the study and performed the statistical analysis and helped to draft the manuscript. BÖ participated in the design of the study and helped to draft the manuscript. GH, FA, SÇ, GÇ helped acquisition of data and participated in the design of the study. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:QuestionnaireClick here for file


Thyroid cancer incidence has been increasing in many countries, whereas mortality has been falling due to better survival. Radiation is the best-established risk factor and there has been concern that recent rises in incidence might be related to fallout radiation from atmospheric nuclear weapon tests. We examined thyroid cancer time trends and geographical distribution in England and Wales and possible interpretations of these. During 1962-84, there were significant increases in incidence (P < 0.001) in each sex at ages under 45. Cohort analysis by single year of birth showed an overall increase in incidence risks in women aged 0-44 born since 1920, with a sudden rise in risk for the birth years 1952-55 followed by a lower risk for the more recent cohorts. In men, there was an overall increase in risk at ages 0-44 in successive birth cohorts, but the pattern was irregular. In each sex, the risk in persons aged 45 and over decreased slightly in successive generations. Geographically, highest incidence risks were in countries in North and Mid Wales, in which the risk was almost twice that in the rest of the country. This pattern was present only at ages 45 and over and was most clear in rural areas. The peak of thyroid cancer risk in women born in 1952-55 is consistent with a carcinogenic effect of fallout radiation, since these women were children in the late 1950s and early 1960s when fallout radiation was greatest in England and Wales. The focus of high thyroid cancer risks in Wales was in areas with high levels of fallout radiation. However, thyroid cancer risks in Wales were not high for more recent cohorts , and a focus on high risk of benign thyroid diseases was present in Wales well before nuclear weapons existed. The distributions of these benign thyroid diseases, or of factors causing them, seem more likely than fallout to explain the high risk areas for thyroid cancer in the country.


This prospective study of 63,090 Norwegian women with 124 cases of thyroid cancer diagnosed during 1961-1989 revealed no strong associations with reproductive factors. Late last birth was related to increased risk, whereas no association was noted with parity. A long reproductive period was related to increased risk of papillary carcinomas, whereas a decreased risk of follicular carcinomas and other adenocarcinomas was observed in women with early menarche and late menopause. The risk of thyroid cancer was significantly increased among women in the occupational category 'fishing, ships officers and crew'. Our results are consistent with a modest effect of certain reproductive factors upon thyroid cancer development.


BRCA1/2 mutations in order to create a biorepository for use in identifying biomarkers of breast cancer risk.The aim of this study was to collect serial samples of nipple aspirate (NA) and ductal lavage (DL) fluid from women with germline BRCA1 or BRCA2 mutations  were scheduled for six-monthly NA, DL and venesection. DL was attempted for all NA fluid-yielding (FY) and any non-FY ducts that could be located at each visit.Between March 2003 and February 2005, 52 women with germline P = 0.026). Eighty-five percent of women had successful DL. Success was most likely in women with FY ducts (FY 94% versus non-FY 71% (P = 0.049). DL samples were more likely to be cellular if collected from FY ducts . Total cell counts were associated with FY status . Four women (8%) had ducts with severe atypia with or without additional ducts with mild epithelial atypia; seven others had ducts with mild atypia alone (11/52 (21%) in total). Median total cell count was greater from ducts with atypia  than those with no atypia .Twenty-seven (52%) women were postmenopausal, predominantly (19/27) from risk reducing bilateral salpingo-oophorectomy (BSO). FY ducts were identified in 60% of all women, 76% of premenopausal women versus 44% of postmenopausal (It is feasible to collect serial NA and DL samples from women at high genetic risk of breast cancer, and we are creating a unique, prospective collection of ductal samples that have the potential to be used for discovery of biomarkers of breast cancer risk and evaluate the ongoing effects of risk reducing BSO. DL cellular atypia was not predictive of a current breast cancer and longer follow up is needed to determine whether atypia is an additional marker of future breast cancer risk in this population already at high genetic risk of breast cancer. BRCA1 or BRCA2 mutations are associated with a markedly increased lifetime risk of developing breast cancer [Germline BRCA1/2 genetic status is not known. Although published reports of NA and FNA have included women at high risk of breast cancer, the proportion of these women who carry a BRCA1/2 mutation is unknown and no conclusions can be drawn regarding the utility of these findings specifically in BRCA1/2 mutation carriers.There is a well established association between atypical ductal epithelium identified by histological biopsy , nipple Ductal lavage (DL) can also be used to access the ductal epithelium  and promBRCA1/2 mutations with the aim of establishing a biorepository of samples to be used to identify biomarkers of breast cancer risk. Our long-term goal is to examine these samples for ductal epithelial atypia, candidate gene methylation frequency and candidate gene expression by RT-PCR and DNA micro-arrays in addition to ascertaining their proteomic profiles. This report describes our initial experience, with the primary objective being to investigate the practicality of establishing this collection and the nature of the DL samples collected.Investigations into the potential role of DL as a breast cancer risk-assessment tool have commenced. Epithelial atypia identified by DL is assigned the same clinical significance in the Gail model of risk as atypia in histological tissue sections for determining eligibility for the Study of Tamoxifen and Raloxifene (STAR) trial . It has BRCA1 or BRCA2 mutation were eligible for the study. Eligible women were invited to participate by letter following permission from their breast specialist. Reasons for exclusion included pregnancy, lactation within the last 12 months, previous subareolar or other surgery that could disrupt the ductal systems, breast implant or breast cancer on the side of the proposed lavage. The study was approved by each Hospital Research Ethics Committee and all participating subjects provided written, informed consent.Between March 2003 and February 2005, eligible women were recruited from three familial cancer centres , which they had attended for the purpose of genetic testing. Women aged between 25 and 65 years with at least one breast unaffected by cancer and carrying a deleterious germline Women were scheduled to attend at six-monthly intervals for NA and DL from each breast eligible for the study as well as venesection (for the collection of serum and genomic DNA) for a maximum of three years. Follow up for 10 years following completion of the collection of DL samples with regards breast cancer development is planned. For premenopausal women, we would have preferred to collect samples at the same point in each menstrual cycle but as this proved to be impracticable, details of last menstrual period were obtained for each collection visit. All women were advised to continue with the breast cancer surveillance programme prescribed for them by their breast specialist prior to their participation in this study. At a minimum, this included a recommendation to perform monthly breast self-examination, six-monthly clinical breast examination by their specialist and annual mammography.After a clinical breast examination, NAF was collected from each breast using the technique as previously described . All NAFDL was performed with the FirstCyte™ DL catheter  essentially as described  with minAll women completed a 'tolerability questionnaire' immediately after the procedure, scoring the intensity of sensation perceived during the lavage procedure on a visual analogue scale of 0  to 10 (the worst pain imaginable) and comparing the intensity of sensation to that experienced during mammography.Between 10 and 12 ml of DL fluid was recovered from each duct; initially 2 ml was separated into 30 ml Cytolyt™ solution (Cytyc Corporation) for transport to the laboratory for cytological processing and the remainder placed on ice. A significant proportion of our initial samples were considered to have inadequate cellular material for cytological diagnosis (ICMD). Consequently, from September 2003, half the recovered DL fluid was used for cytological analysis. The remaining lavage fluid was temporarily placed on ice and processed within 2 h into cell pellets of approximately 10,000 cells/pellet and supernatants stored separately at -80°C. Total cell counts were obtained using a haemocytometer and Trypan Blue at the start of the handling process.® technique [DL samples were processed for cytological examination using the ThinPrepechnique , stainedechnique . BrieflyBRCA1/2 mutation carriers registered on the study prior to February 2005 were included in the analysis. Baseline characteristics of these women were summarised in addition to the characteristics of their breasts and cannulated ducts.All data were recorded on study specific case record forms and entered into a database created using Microsoft Access software . Data consistency checks were made at the time of data entry and subsequently at the time of statistical analysis. All P values are two-sided. Statistical analysis was performed using StatXact version 6.0 .Fisher's exact test was used to compare the proportion of NAF-producing breasts, successful cannulations and ICMD ducts, according to both menopausal status and previous history of breast cancer, parity and breast feeding. The nonparametric Mann-Whitney test was used to compare total cell counts  according to menopausal status, NAF-producing status, previous history of breast cancer, parity and breast feeding, development of breast cancer during the study and the presence of DL cytologic atypia. No adjustments have been made for multiple comparisons and all BRCA1/2 mutations (Table per se on NA and DL. Eighty-five percent were parous and seventy-three percent had breast-fed their children.We recruited 52 women with P = 0.004) but not related to a previous history of contralateral breast cancer (Table P = 0.008) but not related to menopausal status, previous history of contralateral breast cancer or lactation or parity . Three women (6%) had not yet commenced mammographic screening. Of the remainder, 33% of the women thought that the procedure was comparable to the discomfort experienced by mammography, while 33% reported that the procedure caused more discomfort and 29% less discomfort than mammography.P = 0.037; Table P = 0.002). So far, we have found no statistical difference in total counts between the small number (n = 5) of women who developed breast cancer during the study versus women who did not.DL samples were more likely to have adequate cellular material for diagnosis if they were collected from FY ducts ; the features of their cancers and associated DL samples are summarised in Table Five women, all with BRCA1 and BRCA2 genes. We have demonstrated that it is both acceptable and feasible to collect these samples and we have established a prospective collection of samples with ongoing epidemiological data that will be available for future breast cancer biomarker discovery.This is the first published study, to our knowledge, to describe the findings from serial NA and DL in a cohort of women with germline mutations in We found that we could obtain ductal fluid by NA in 60% of our cohort overall, but that NAF was more often obtained from premenopausal women. Our overall figures are less successful than reported by most (48% to 99%) ,7,23. Onet al. [Half of the women in our cohort were postmenopausal despite the median age of only 43 years, predominantly because of risk-reducing BSO . Seventy-six percent of the premenopausal women had FY ducts in contrast to forty-four percent of postmenopausal women. Similar low rates of FY ducts in ovariectomised women have been observed previously; BL King, Yale, found FY ducts in only 23% of women with oophorectomy with or without selective estrogen receptor modulator (SERM) use . Conversely, Kurian The ability to re-cannulate the same duct on repeated occasions was less successful than we had anticipated Fig. . FrequenIf the DL sample had adequate material for cytological diagnosis (non-ICMD samples), the cellularity was comparable to previous reports . In contet al. [et al. study were scored as mildly, rather than severely, atypical, as were most of ours, and it is the differentiation between benign and mildly atypical that is the most debated in cytological scoring.Four women (8%) were identified with severe cytological atypia (in at least one duct on at least one occasion) and 7/52 (13%) with mild cytological atypia, which is consistent with the results from the multicentre DL study  [Five women were diagnosed with breast cancer during our study; one had only mild cytological atypia while the others had no atypia in their DL samples. These women are likely to have had an established malignancy at study entry, although this was not detected by pre-study assessment. Our results are consistent with previous reports that have suggested that both NAF and DL fluid have low sensitivity for the identification of an established malignancy. For NAF, only a low percentage of specimens contain severely atypical or malignant cells, even when the aspirate is obtained from a known cancer-bearing breast . Twoo 2003,) ) of DL fWe believe that it is important to be aware of the poor correlation between DL atypia and current breast cancer and that the significance of detecting DL atypia in a high-risk woman is unknown. The finding of no atypia should not reassure a high-risk woman that no malignancy is present and she should be encouraged to continue with regular breast self examination, clinical breast examination and mammography. The finding of atypia requires investigation to exclude malignancy, but we do not consider that there is sufficient evidence currently to consider DL atypia as an additional risk factor in support of prophylactic mastectomy in an otherwise high-risk population.BRCA1/2 mutation carriers each in the UK and US are being recruited using a similar protocol, which will permit a combined analysis of results in the future. In the first two years of our study, five women have developed breast cancer, consistent with published rates of breast cancers in this high-risk group [BRCA1/2 mutations.Our results demonstrate that DL is an acceptable, minimally invasive method of repeatedly accessing the breast ductal environment, although it requires further validation before it can be established as a breast cancer risk assessment tool, particularly in the high-risk population. It does not appear to be a sensitive breast cancer screening method. We are creating a prospective repository of biologic samples that have the potential to be used to identify biomarkers of breast cancer risk in a high-risk population. Two further cohorts of 50 sk group . If simiBSO = bilateral salpingo-oophorectomy; DL = ductal lavage; FNA = fine needle aspiration; FY = fluid yielding; ICMD = inadequate cellular material for diagnosis; NA = nipple aspiration; NAF = nipple aspirate fluid.The authors declare that they have no competing interests.


Nipple aspiration is a noninvasive technique for obtaining breast fluids from the duct openings of the nipple for the evaluation of abnormalities associated with breast cancer. Nipple aspirate fluid (NAF) can be elicited from 48 to 94% of healthy women, and its production has been linked to an increased relative risk for breast cancer development. NAF production has been used in studies to guide the selection of ducts for ductal lavage, a procedure in which ducts are cannulated and flushed with saline to collect cells. In a previous multicenter trial to evaluate intraductal approaches in women at high-risk for breast cancer, NAF production was observed in 84% of the subjects. However, we observed a significantly lower proportion of fluid-yielding subjects in a similar series of high-risk women. The purpose of the present study was to identify variables associated with this reduction.BRCA1 or BRCA2 germline mutation) to identify ductal orifices for lavage procedures. Lavage was performed on all fluid-yielding ducts and on nine non-fluid-yielding ducts.Nipple aspiration was performed on 33 high-risk women . Reduced NAF yields were associated with postmenopausal status (P = 0.02), BRCA germline mutations (P = 0.004), and risk reduction therapies, including bilateral salpingo-oophorectomy (BSO) and/or selective estrogen receptor modulators . All nine (100%) of the ductal lavage specimens collected from non-fluidyielding ducts were acellular, in comparison with 3 of 13 specimens from fluid-yielding ducts (P < .001).Fluid-yielding ducts were identified in 12 of 33 (36%) of the subjects in the present series, compared with 16 of 19 (84%) of the subjects undergoing identical procedures at our facility during a multicenter trial (BRCA-positive women, many of whom had undergone BSO and/or were using SERMs. Our data suggest that endocrine mechanisms associated with these risk-reducing therapies may be related to patterns of diminished breast fluid production.Analysis of high-risk women in the present series revealed patterns of reduced NAF production and ductal lavage cellularity compared with a previous multicenter trial. The present series included more  Nipple aspiration is a noninvasive suction technique used to obtain breast fluid from the duct openings at the nipple. The procedure elicits droplet formation from one or more ducts in 48 to 94% of healthy, nonlactating women, and these fluids often contain exfoliated ductal epithelial cells -10. The P = 0.001) when we performed the procedure on a similar series of 33 high-risk women after the conclusion of the trial. Here we present data suggesting that the reduced NAF yields observed in the second series of subjects may be related to the use of risk reduction therapies including selective estrogen receptor modulators  and/or oophorectomy.More recently the ductal lavage technique, which employs a microcatheter to cannulate an individual duct opening and flush the associated ductal system with saline, has been used to collect higher numbers of exfoliated cells. In a multicenter trial that we participated in to evaluate this approach, nipple aspiration was performed on all subjects for the purpose of locating fluid-yielding ducts (presumed to contain higher numbers of cells) before the lavage procedure . Fluid-yBRCA1 or BRCA2), who were asymptomatic at enrollment, were recruited through the Yale Cancer Center Genetics Counseling Shared Resource and through referrals from several regional breast clinical practices. The enrollment criteria were identical to those of the multicenter trial that we participated in, with the exception that women taking SERMs were admitted to the present study, whereas they had been excluded from the multicenter trial. To expand our analysis of certain variables, we contacted all 19 of the multicenter trial subjects who had participated at our center by letter to request oophorectomy history, which had not been collected at the time of the trial. We received responses from 10 subjects, all of whom are included in a subset of the analyses in this report.Subject participation for this study was approved by the Yale Human Investigation Committee (protocol no. 11401), and informed consent was obtained in all cases. Thirty-three high-risk women , and then subjected to nipple aspiration using a FirstCyte™ Aspirator  to identify the location of fluid-producing duct orifices. A subject was classified as a fluid yielder if one or more drops of fluid were obtained from one or more ducts. Each productive duct was cannulated with a FirstCyte™ DL Catheter (Cytyc Corporation) and lavaged with 10 to 20 ml of sterile saline . In addition, nine non-fluid-yielding ducts were cannulated and lavaged.Cytomorphologic evaluation was performed by a board-certified cytopathologist (DLR) specifically trained in ductal lavage interpretation. Specifically, lavage samples were graded on a four-point scale on the basis of the epithelial cell morphology . Specimens lacking sufficient epithelial cells for classification were categorized as ICMD (insufficient cells to make a diagnosis).2 test. The proportions of fluid-yielding women were compared as follows: first, between the multicenter trial and the present series of 33 subjects; second, between subsets of the 33 subjects in the present series; and third, between subsets of the pooled group of 43 subjects.Frequencies were compared using the two-tailed Fisher exact test and the χP = 0.001; Table BRCA1/2 germline mutation and risk reduction therapy status. In the multicenter trial, fewer than 1% of the women were BRCA-positive, whereas in the present trial 16 of 33 (48.5%) of the subjects had BRCA germline mutations (P < 0.001). Another difference is that the multicenter trial excluded women who were taking SERMs, whereas in the current study, 12 of 33 (36.4%) of the participants were taking SERMs . There was also a higher, but statistically insignificant, proportion of postmenopausal women in the present series (P < 0.10). A substantial number of these women (7 of 23) had undergone surgically induced menopause before the age of 49 years as the result of bilateral salpingo-oophorectomy (BSO). The proportion of women in the multicenter trial who had undergone oophorectomy was not documented. Two of the 10 trial participants who responded to our letters reported a history of oophorectomy at the time of trial enrollment. Five women in the present series were employing both forms of risk reduction at the time of study enrollment.In the present series, the overall proportion of women with fluid-yielding ducts was 12 of 33 (36%), compared with 427 of 507 (84%) for the multicenter trial at large, and 16 of 19 (84%) for the Yale trial site , and in 11 of 17 (65%) BRCA-negative women versus 2 of 16 (13%) of BRCA-positive women (P = 0.004). Fluid-yielding ducts were identified in 8 of 11 (73%) women not employing any form of risk reduction, versus 5 of 22 (23%) women who had undergone oophorectomy and/or were taking SERMs at the time of enrollment into the study (P = 0.009). To study the independent contributions of these variables to NAF reduction, we performed a set of analyses on a pooled set of subjects including the 33 subjects from the present series plus a subset of the multicenter trial subjects for whom we had retrospectively obtained oophorectomy status. To separate the contributions of age, menopause and surgically induced menopause resulting from oophorectomy, we first stratified the 43 women into two groups: those aged 49 years and below, and those aged 50 years and above. NAF production within each group was then analyzed as a function of BRCA mutation or risk reduction therapy status. These comparisons are shown in Table BRCA germline mutations (P = 0.008) and risk reduction strategies (P = 0.0003) among women below the age of 50 years. When BRCA-negative women above and below the age of 50 years were considered separately, there were significantly lower proportions of fluid-yielders among the women on risk reduction therapy . No significant association was observed between NAF production and previous breast cancer history (P = 0.468).Within the present series of 33 subjects, NAF production varied according to menopausal status, us Table . NAF wasP = 0.00046). The proportion of ICMD in the multicenter trial was 84 of 383 (22%) [In the multicenter ductal lavage trial  only NAFP < 0.001) [P < 0.001) suggests that the overall decline observed in the present series is associated with additional variables. The subject population in the present study differed in having more women with BRCA germline mutations, many of whom had undergone BSO and/or were using SERMs. Seven of 23 postmenopausal women in this series were less than 49 years old and had undergone surgically induced menopause as the result of BSO. When NAF production was analyzed among the 33 subjects in relation to these variables, we found it to be significantly reduced in association with BRCA mutations and risk reduction therapies (Table BRCA-negative women (P = 0.045) and a similar, but insignificant, trend for BRCA-positive women.The overall percentage of fluid-yielding subjects in the present series 36%) was relatively low in comparison with those reported in other studies (48 to 94%) 6% was re-10,14 anBRCA germline mutations [BRCA1/2 mutations. The overall NAF rate in this study was 48%. Although no direct correlation between fluid production and risk reduction therapies was observed, there were significantly fewer fluid-yielding subjects among women with a previous history of breast or ovarian cancer. The authors suggested therapy-related anti-hormonal mechanisms including chemotherapy-induced amenorrhea as possible explanations for the trend. Mitchell and colleagues, who performed intraductal approaches on a series of 52 women with germline BRCA1/2 mutations, observed NAF production in 60% of all subjects . Although no correlation between NAF production and previous cancer was noted in this study, significantly reduced levels of NAF secretion and ductal lavage cellularity were seen in association with natural and surgically induced menopause. Although we cannulated and lavaged only nine dry ducts, all of them were also characterized as acellular. Kurian and colleagues [Although NAF and ductal lavage have been evaluated in women at high risk for breast cancer development, there have been only a handful of studies describing the application of intraductal approaches to high-risk women with utations ,18. The BRCA1 and BRCA2 mutation carriers [BRCA1 mutation carriers, and although most BRCA1 tumors are estrogen receptor negative, both oophorectomy and tamoxifen have been shown to protect against the development or recurrence of breast cancer in this group [The risk-reducing benefits of SERMs and oophorectomy for breast cancer patients and women at high risk for breast cancer are well documented. Clinical trials have demonstrated that tamoxifen significantly decreases recurrence rates in breast cancer patients and markedly reduces the incidence of invasive and noninvasive breast cancer development in high-risk women -26. Breacarriers -33. Our is group ,30,32,33The biologic regulation and significance of breast duct fluid production in nonlactating women are not currently understood. Our data showing that risk reduction therapies are associated with diminished breast fluid secretion complement previous epidemiologic studies showing that NAF production is linked to increased relative risk for breast cancer development -13. The Because NAF production and cytologic atypia are both associated with increased relative risk for breast cancer development, it is tempting to speculate that NAFs reflect the secretory activity of proliferative ductal epithelial cells. However, several recent studies have generated paradoxical results concerning the relationship between breast fluid, cytologic atypia, and breast cancer risk. These studies have revealed the presence of cytologic atypia in non-fluid-yielding ducts and in NAF-negative women ,17,18,35BRCA-positive women, many of whom had undergone BSO and/or were using SERMs. Our data suggest that endocrine mechanisms associated with these risk-reducing therapies may be related to patterns of diminished breast fluid production. Although NAF production does not seem to be obligatorily linked to cytologic atypia, it may be a general indicator of endogenous reproductive hormone levels that are widely implicated in breast cancer development. More importantly, NAF seems to reflect the impact of the systemic hormonal milieu on the breast tissue itself. Thus, its production during the NAF procedure could potentially provide additional information to a woman's risk profile, and could serve as a valuable endpoint in chemoprevention trials.An analysis of high-risk women in the present series revealed patterns of reduced NAF production and ductal lavage cellularity in comparison with a previous multicenter trial. The present series included more BRCA = breast cancer predisposition gene; BSO = bilateral salpingo-oophorectomy; ICMD = insufficient cells to make a diagnosis; NAF = nipple aspirate fluid; SERM = selective estrogen receptor modulator.The authors declare that they have no competing interests.SH and BH performed the NAF and ductal lavage procedures. EM recruited subjects and performed informed consent. DR provided cytologic evaluation. JD performed statistical analyses. BK designed and coordinated the study, performed statistical analyses, and wrote the manuscript. All authors read and approved the final manuscript.


TCF7L2 expression modifies Wnt signaling and mediates oncogenic effects through the upregulation of c-MYC and cyclin D. Genetic alterations in TCF7L2 may therefore affect cancer risk. Recently, TCF7L2 variants, including the microsatellite marker DG10S478 and the nearly perfectly linked SNP rs12233372, were identified to associate with type 2 diabetes.The transcription factor 7-like 2 (TCF7L2) is a critical component of the Wnt/β-catenin pathway. Aberrant TCF7L2 rs12255372 variant on familial breast cancer (BC) risk by means of TaqMan allelic discrimination, analyzing BRCA1/2 mutation-negative index patients of 592 German BC families and 735 control individuals.We investigated the effect of the P = 0.04), and the Cochran-Armitage test for trend revealed an allele dose-dependent association of rs12255372 with BC risk (Ptrend = 0.04).The T allele of rs12255372 showed an association with borderline significance (OR = 1.19, 95% C.I. = 1.01-1.42, TCF7L2 rs12255372 on the risk of familial BC.Our results suggest a possible influence of  TCF7L2 alias TCF-4) gene product, a HMG box transcription factor, is part of the Wnt/β-catenin signaling cascade, one of the key mechanisms of development and growth regulation in the cell [et al. [TCF7L2 as a novel candidate gene for type 2 diabetes and reported an association of the microsatellite marker DG10S478 within intron 3. The associated tetranucleotid repeat was shown to have six alleles, namely alleles 0, 4, 8, 12, 16 and 20. The combined non-zero alleles of DG10S478 (referred to as X) were associated with an increased risk in three independent populations . The composite at-risk allele X was nearly perfectly correlated with the T allele of the single nucleotide polymorphism (SNP) rs12255372, while the G allele was linked to allele 0. Consequently, the rs12255372 T allele showed association with type 2 diabetes as well [The transcription factor 7-like 2 . This supports the biological significance and clinical relevance of the Wnt/β-catenin signaling pathway in carcinogenesis [There is accumulating evidence that aberrant activation of the Wnt/β-catenin pathway has oncogenic effects ,6. Wnt sogenesis -9.Given these facts, we investigated the effect of the rs12255372 polymorphism on familial BC risk.TCF7L2 rs12255372 in 592 German familial BC cases and 735 control individuals. The BC cases comprised unrelated female index patients  without mutations in the high-penetrance genes BRCA1 and BRCA2. Mutations in the open reading frame of BRCA1 and BRCA2 were excluded by applying denaturing high performance liquid chromatography (DHPLC) on all exons, followed by direct sequencing of conspicuous exons. Cases were collected during the years 1996–2005 through the Institute of Human Genetics , the Department of Gynaecology and Obstetrics  and the Department of Medical Genetics . According to the German Consortium for Hereditary Breast and Ovarian Cancer, BC cases were divided into six categories based on family history: (A1) families with two or more breast cancer cases including at least two cases with onset below the age of 50 years (39.0%); (A2) families with at least one male breast cancer case (0.7%); (B) families with at least one breast cancer and one ovarian cancer case (18.1%); (C) families with at least two breast cancer cases including one case diagnosed before the age of 50 years (25.5%); (D) families with at least two breast cancer cases diagnosed after the age of 50 years (8.4%) and (E) single cases of breast cancer diagnosed before the age of 35 years (8.3%) [TCF7L2 rs12255372 analysis comprised one index case per family. DNA of further family members to evaluate segregation of the variant with BC risk was not available.We analyzed s (8.3%) ,11. The The corresponding control series consisted of healthy, unrelated and ethnically matched blood donors  sharing the ethnic background with the patients. They were recruited in 2004 and 2005 by the Institute of Transfusion Medicine and Immunology . The study was approved by the Ethics Committee of the University of Heidelberg , and written informed consent was obtained from all individuals.et al. investigated five SNPs located within a 92.1 kb linkage disequilibrium (LD) block, encompassing parts of introns 3 and 4 and the whole of exon 4 [2 = 0.95) for genotyping.Grant TCF7L2 rs12255372 was performed by TaqMan allelic discrimination as described before [Genotyping of P values were computed by unconditional logistic regression using the Statistical Analysis System software . Hardy-Weinberg equilibrium test was undertaken using Pearson's goodness-of-fit chi-square test with one degree of freedom. Power calculation was carried out with the power and sample size software PS [Genotype-specific odds ratios (OR), 95% confidence intervals (95% C.I.) and tware PS . With thTCF7L2 rs12255372 T allele frequency of the controls (German Caucasians) was in accordance with those published in previous studies  [P = 0.64, Table P = 0.04, Table Ptrend = 0.04, Table The casians) , and theBRCA1/2 mutation-negative familial BC cases were included in order to avoid effects caused by these high-penetrance susceptibility genes. Our study comprised individuals selected for familial BC, since the power of an association study based on cases with a family history of the disease is considerably higher compared to a study using unselected cases [The strengths of the present study on BC risk are based on a large sample size and a homogeneous study cohort of a single ethnic group. Only ed cases ,15.TCF7L2 as a candidate gene for cancer susceptibility, suggesting a prominent role of TCF7L2 variants in human cancers, especially in BC. According to Duval et al. [TCF7L2 stimulates its transcriptional activity synergistically with APC/β-catenin gene alterations. Moreover, it is predicted to show a reduced binding of the C-terminal binding protein (CtBP) which hence loses its capability to repress TCF7L2 activity [TCF7L2 transcriptional activation, leading to uncontrolled target gene expression. Along the lines of Grant et al. [This is the first study to investigate l et al. , mutant activity . Both hyTCF7L2 variants may contribute to the risk of familial BC. Regarding the borderline significance level of our results, confirmation in an independent BC cohort is essential. Moreover, it would be of interest to estimate their impact on further types of human cancer.In summary, our data suggest that TCF7L2 rs12255372 variant on the risk of familial BC.Our data suggest a possible influence of the BC – breast cancerCtBP – C-terminal binding proteinDHPLC – denaturing high performance liquid chromatography95% C.I. – 95% confidence intervalOR – odds ratioSNP – single nucleotide polymorphismTCF7L2 – transcription factor 7-like 2TCF-4 – HMG box transcription factor 4The author(s) declare that they have no competing interests.BRCA1/2 mutation screening. BF conducted the experiments, performed data acquisition and interpretation, and drafted the manuscript. All authors read and approved the final version of the submitted manuscript.BB designed and coordinated the study and reviewed the manuscript. KSS participated in SNP genotyping. KH participated in the study coordination and revised the manuscript. AM, RKS, CS, BW, MK and CRB collected DNA samples and were responsible for the The pre-publication history for this paper can be accessed here: