SUPPORTING ONLINE MATERIAL

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Jasmin Pearson
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1 SUPPORTING ONLINE MATERIAL Materials and methods. Oncologists and surgeons from 12 major cancer centers in the greater New York metropolitan area referred eligible breast cancer patients to the project. Eligible patients were identified by physicians or genetic counselors by review of hospital tumor registries or (for private practitioners) by review of patient files. Patients diagnosed with invasive breast cancer, within 12 months of chart review and between September 1996 and December 2000, and of Ashkenazi Jewish ancestry were eligible for the project. Ancestry was determined for most patients from their physicians personal acquaintance and in the remaining cases from patients names. We recognize that relying on names to indicate ancestry may introduce errors of both inclusion and exclusion. Physician permission was obtained for each patient before notices of the study were sent to patients. Approximately 1400 patients expressed interest in the study, each of whom was contacted by a genetic counselor. For those who confirmed their interest, pre-test genetic counseling was scheduled and carried out. The 1021 patients who chose to participate provided informed consent, a blood sample for genetic testing (10 ml drawn into ACD by venipuncture), family history of cancer for genealogy construction, a questionnaire addressing non-genetic risk factors for breast cancer, and access to pathology reports and materials verifying her cancer diagnosis. Patients could elect either of two forms of participation in the project. Completely anonymous participation involved providing a blood sample, pathology information and completing the family history and questionnaire but providing no name or contact information to the NYBCS. Alternatively, full participation involved providing one's name and contact information, with the expectation of receiving results of BRCA1 and BRCA2 testing in the context of genetic counseling. Of the 1021 probands, 32 chose anonymous participation and 989 full participation. The proportion of patients invited to participate who ultimately enrolled in the study varied somewhat among centers, leading to the concern that family history might have differentially influenced study participation at different sites. Had sites differed in this way, the proportions of probands found to carry mutations in BRCA1 or BRCA2 might differ by site as well. Therefore after probands were genotyped, we compared prevalences of the three mutations among participants from each site (Supplementary Table 1). Sites did not differ from each other or from the overall proportion of probands (0.103) with BRCA1 or BRCA2 mutations. The most extreme outlier was the site with the fewest participants (8), two of whom carried BRCA1.185delAG. This proportion (0.25) did not deviate significantly from the other sites, nor were the family histories of these two probands unusual among mutation carriers. We subsequently contacted a sample of patients from multiple sites who had received letters of invitation but not expressed an interest in the study. The most frequent reasons for declining participation were concern for insurance discrimination and distance from one s residence to the center. Genotyping was carried out by automated DNA sequencing at the University of Washington (MCK laboratory), which is licensed by the New York State Health Department and by CLIA to carry out clinical genetic testing. Of the 1021 probands, 1018 were female and three were male. DNA samples from 10 participants were inadequate and the patients too ill to be sampled again. The three male probands were wildtype at all three sites. Genotypes were determined for 1008 female probands. Each of the 989 probands requesting full participation was contacted by her genetic counselor and learned her test results in the context of post-test counseling

2 Supplementary Table 1. Prevalences of BRCA1 and BRCA2 mutations by NYBCS participating clinical site Site Index cases in NYBCS BRCA1 185delAG BRCA1 5382insC BRCA2 6174delT Any of 3 mutations Proportion of index cases with mutation Std. error (proportion) Albert Einstein-Montefiore Medical Center Beth Israel Medical Center Columbia Presbyterian Medical Center Englewood Hospital and Medical Center Greenwich Hospital of Yale Cancer Center Hackensack University Medical Center Memorial-Sloan Kettering Cancer Center North Shore University Hospital New York University Medical Center Private practitioners Stamford Hospital Strang Cancer Prevention Center White Plains Hospital Center TOTAL From the probands with mutations, permission was sought to contact living siblings and parents, to access pathology records and specimens of relatives who had died of cancer, and subsequently to contact aunts, uncles, cousins, and grandparents from the lineage carrying the mutation. We enrolled and tested informative relatives from 84 of the 104 families with mutations. Twenty families were not available for molecular analysis. The reasons for unavailability were as follows: no surviving informative relatives (10 families), proband did not want her parents to know she carried a mutation (8), proband died of her breast cancer prior to follow-up (1), proband requested anonymous participation so did not learn her own result (1). Prior to genetic testing, probands of all 20 of these families had provided complete pedigree information. All these families were small and fit our criteria for low incidence families; namely, no cases of breast or ovarian cancer in the nuclear family of the proband. The disproportionate number of small, low incidence families among those not enrolled for follow-up was an important rationale for our separate analysis of low incidence families who were tested. We subsequently determined (text and Supplementary Table 2C) that risk of breast cancer among mutation carriers in low incidence families was the same as the risk in other families. Genetic analysis was carried out for more than 700 adult male and female relatives (siblings, parents, aunts, uncles, cousins and grandparents) of 104 mutation-carrier probands. Genotypes were determined by sequencing DNA from blood samples provided by the relative, by extracting DNA from surgical specimens archived by pathology departments, or by reconstructing genotypes from those of surviving children. Of relatives with confirmed genotypes, 212 were females older than 30 years with BRCA1 or BRCA2 mutations. These 212 female carriers were the subjects for the Kaplan-Meier risk analysis. Risks were calculated by censoring age for affected women at age of diagnosis and age for unaffected women at age of most recent follow-up, age at prophylactic surgery, or age at death. We undertook four tests to evaluate the possibility of biased ascertainment of probands with more severe cancer histories. First, we compared family cancer histories of the participating probands vs. a sample of 63 non-participants drawn from all eligible patients who declined to participate in the study. Of these 63 non-participants, 38 (60%) had at least one relative and 12 (19%) had at least three relatives with breast or ovarian cancer whereas among the 1008 participating probands, 597 (59%) had at least one and 101 (10%) had at least three affected relatives. Clearly participants did not have more severe family histories than the sample of eligible patients who declined participation

3 Next we calculated risk of breast cancer among all female relatives with mutations except for mothers. We reasoned that breast cancer patients whose mothers had also developed breast cancer might have been more likely to participate. These participants would not have known whether they inherited BRCA1 or BRCA2 mutations, but they might have been more motivated to find out. Excluding mothers from the analysis, breast cancer risks to relatives with mutations were 24% by age 40, 59% by age 60, and 88% by age 80 (Supplementary Table 2B). High risks of breast cancer among women with mutations were not due to inadvertent selection of families with mothers with inherited disease. Supplementary Table 2. Cumulative risk of breast cancer (standard errors) among female relatives with BRCA1 or BRCA2 mutations. Probands were excluded from the risk analysis. Risk by age A. All female relatives with mutations B. Mothers excluded C. Only "low incidence" families (.01) 0.03 (.01) 0.01 (.01) (.02) 0.10 (.02) 0.11 (.03) (.03) 0.24 (.04) 0.20 (.04) (.03) 0.34 (.04) 0.27 (.04) (.04) 0.45 (.04) 0.37 (.04) (.04) 0.56 (.05) 0.47 (.05) (.04) 0.59 (.05) 0.51 (.05) (.04) 0.67 (.05) 0.53 (.05) (.04) 0.69 (.05) 0.62 (.05) (.04) 0.81 (.06) 0.71 (.06) (.05) 0.88 (.06) 0.78 (.06) In order to test whether our sample included a subset of truly low-risk families, we evaluated risk among mutation carriers from families with no cancer in the proband s nuclear family. There are two possible explanations for lack of cancer in the nuclear family of a patient with a BRCA1 or BRCA2 mutation. (1) The family may have truly low penetrance of the BRCA1 or BRCA2 mutation, with female mutation carriers remaining unaffected. (2) Alternatively, the family may include few female mutation carriers, because the family is small and/or there is a preponderance of males and/or normal (rather than mutant) BRCA1 and BRCA2 alleles segregated by chance to female relatives. It is important to note that the only means of distinguishing these alternatives is by direct testing of female relatives. Therefore breast cancer incidence in these families was evaluated for second, third, and fourth degree relatives (grandmothers, aunts, and cousins) with confirmed BRCA1 or BRCA2 mutations. Breast cancer risks to mutation carriers among these distant relatives were 20% by age 40, 51% by age 60, and 78% by age 80 (Supplementary Table 2C). There was no difference in risk between relatives with mutations in these families and relatives with mutations in other families. Finally, we determined whether older, cancer-free women with BRCA1 or BRCA2 mutations clustered in a subset of families. That is, could we identify low risk families by tracing relationships among older women with mutations who remained cancer free? Eleven female relatives with BRCA1 or BRCA2 mutations attained age 65 without developing breast or ovarian cancer. These 11 women were from 11 different families, clearly not evidence for clustering. Potential modification of BRCA1 and BRCA2 risks by environmental and lifestyle factors. In order to determine if any environmental or lifestyle factors were associated with cancer risk due to BRCA1 and BRCA2 mutations, we used Cox regression analyses to evaluate the effects of such factors on age at breast cancer diagnosis among probands (Supplementary Table 3). All probands developed breast cancer; their cancer qualified them as index cases (probands) in the NYBCS. Our logic was that among breast cancer patients, factors associated with older onset of breast cancer might be protective, whereas factors associated with younger onset of breast cancer might exacerbate risk. Information on environmental exposures, reproductive history, and lifestyle - 3 -

4 choices was provided by probands in written questionnaires (questionnaire available on request to J.B.M.). Analyses were carried out for all NYBCS participants as a group and after stratifying by BRCA1 and BRCA2 genotype. So as to disentangle effects of individual factors from the overall effects of many correlated temporal changes in lifestyle, we adjusted all analyses for birth decade of the proband. We understand that this is conservative. P-values were adjusted for multiple comparisons. Exercise and weight. The associations of exercise and body weight with age at breast cancer onset are discussed in the text. Probands were asked to assess their level of exercise at various points in their lives, as well as answer multiple questions about specific activities (walking, bicycle riding, sports, dance, and so on). Probands who had undertaken active exercise as young girls had delayed age at breast cancer onset. Probands who were active or very active as teenagers engaged in a wide range of sports and personal exercise. Teenage exercise influenced age at onset protectively among the NYBCS probands both as a whole (P=0.025) and specifically among women with BRCA1 or BRCA2 mutations (P=0.034). Normal weight at age 21, rather than being overweight, was also associated with protective delay in breast cancer onset (P=0.017). This effect is not surprising given the strong correlation between physical exercise and healthy body weight in this cohort (P = 0.016). Conversely, heavier weight at age 21 was associated with earlier breast cancer onset among women with BRCA1 or BRCA2 mutations (P=0.012). Note that weight at age 21 was measured as a continuous scale (i.e. body mass index), so a small relative hazard (RH) for each body mass unit corresponds to a major effect between normal weight and overweight. Reproductive and hormonal history. Among women generally, pregnancy (especially young pregnancy) is protective against breast cancer. Hence it is not surprising that pregnancy was associated with later cancer onset among NYBCS probands (P=0.009). The protective effect has approximately the same magnitude among probands with or without BRCA1 or BRCA2 mutations but is significant only in the much larger group who are wildtype at both genes (P=0.014). Hormone replacement therapy is known to be associated with increased breast cancer incidence, yielding a relative hazard of 1.26 (95% CI ) in the Women s Health initiative randomized trial (30). Similarly, hormone replacement therapy may have a complex influence on age at breast cancer onset among NYBCS probands. The association of HRT with earlier onset appears similar among wildtype probands (RH=1.27) and women with BRCA mutations (RH=1.40), although significant only among the larger group of women; i.e. those without mutations (P=0.036). However, among women who reported HRT use, breast tumors were diagnosed at slightly earlier stage. Therefore it is possible that earlier age of breast cancer onset among women using HRT reflects both more assiduous medical scrutiny and a biological effect on tumor development. Our questionnaire was not adequate to distinguish these alternatives. Other exposures. Although alcohol consumption has been reported to be associated with breast cancer risk, there was no association of alcohol consumption and age at breast cancer onset among the NYBCS probands. However, alcohol consumption levels in this population are very modest and thus likely to be below any threshold of influence. Among probands wildtype at BRCA1 and BRCA2, cigarette smoking was associated with earlier cancer onset; the magnitude of the association was the same among probands with BRCA mutation, though the effect was not significant in the latter group. Previous studies have generally detected either no association between cigarette smoking and breast cancer or a modest protective effect, probably attributable to the influence of smoking in hastening menopause

5 Supplementary Table 3 mutation status.. Association of possible modifiers with age at breast cancer diagnosis among all probands and by Characteristic reported by participant Genotype of participant All probands Wildtype BRCA mutation RR P-value RR P-value RR P-value Childhood, adolescence, and young adult experience Birthweight (of proband herself) First menstrual period age vs < 12 years First menstrual period age 16+ vs < 12 years Weight at menarche: normal weight vs. overweight Physical activity as teenager: active or very active vs. inactive Weight adjusted for height at age Menstrual periods ages 18-34: irregular vs. regular Reproductive history (born < 1970) Ever pregnant vs. never pregnant Age at first pregnancy Number of live births Ever vs. never breastfed children Total months breastfeeding all children Spontaneous miscarriage Spontaneous miscarriage before first live birth Induced abortion Induced abortion before first live birth No pregnancy after trying at least a year Use of fertility medication to become pregnant Contraceptive use (born 1940 or later) Oral contraceptives Depoprovera Experience at menopause Age at natural menopause (if menopause prior to cancer dx) Hormore replacement therapy (born < 1945) Alcohol consumption: low or moderate vs. never Cigarette smoke Ever vs. never smoked cigarettes Second hand exposure at home as child Second hand exposure at home as adult Second hand exposure at work Environmental exposures in work environment or at home X-ray or other radioactive materials Transformers or capacitors Ionizing radiation Cut flowers Cotton Pesticides Based on 976 female probands with complete questionnaires All analyses are Cox regressions adjusted for decade of birth RH = relative risk; wildtype = probands with no mutation at the three BRCA1 and BRCA2 sites tested - 5 -

6 Potential modification of BRCA1 and BRCA2 risk by other genes. Evidence that penetrance of BRCA1 and BRCA2 may be influenced by genotypes at other loci has been very well reviewed recently (31). We genotyped several of these potential modifier loci in the NYBCS cohort. RAD51 promoter. Breast cancer risk among women with BRCA2 mutations may be exacerbated by a variant genotype (135G>C) in the promoter region of the DNA repair gene RAD51 (32, 33). We tested whether RAD51 genotype influences age at onset among the NYBCS probands with BRCA2 mutations. The 135C allele appeared in only one of 37 probands with BRCA2 mutations, but the breast cancer of the RAD51 mutation carrier was diagnosed at the youngest age of all BRCA2 carriers in the cohort. The effect was not significant, but the high relative hazard (7.09) associated with the 135C allele is consistent with previous data. CHEK2.1100delC. An inherited mutation in the cell cycle gene CHEK2 (RAD53) increases breast cancer risk two-fold among women and ten-fold among men (34, 35). The mutation is 1100delC, which abrogates the kinase activity of the CHEK2 protein. The relatively high frequency of the CHEK2 1100delC mutation in Sweden, Denmark, and Finland suggests this allele is likely to have originated in the Nordic region. The CHEK2 mutation 1100delC plays a very small role in the NYBCS. Among NYBCS probands wildtype for BRCA1 and BRCA2, 4/905 (0.004) carried the CHEK2.1100delC mutation. Prevalence of the mutation was slightly higher among NYBCS probands with at least one relative with breast or ovarian cancer (3/538 = 0.006). There were no carriers of CHEK2.1100delC among 18 NYBCS families with male breast cancer. CHEK2.1100delC is significantly rarer among Ashkenazi Jewish familial breast cancer patients (3/538=0.006) than among familial breast cancer patients of other European ancestries (30/718=0.042; P = ).. Prevalence of CHEK2.1100delC among healthy Ashkenazi controls has been estimated at (5/1665) (36). Therefore an estimate of relative risk associated with CHEK2.1100delC in Ashkenazi families with at least two breast cancer cases is 1.9, 95% CI (0.5, 7.7), approximately the same as in other European populations, although not statistically significant among the Ashkenazim given the very low prevalence of the mutation. Androgen receptor (AR). Transactivation of the androgen receptor (AR) varies with the length of the polymorphic polyglutamine (CAG) repeat at the N-terminus of the protein, with longer alleles associated with lower transactivation activity (37). BRCA1 is a co-activator of the androgen receptor (38). Exceptionally long alleles of the CAG repeat have been associated with higher breast cancer risk, both among women with BRCA1 mutations and generally, although an association has not been consistently observed (39-41). Exacerbation of risk associated with longer alleles is consistent with the hypothesis that reduced androgen activity increases breast cancer risk. Among BRCA2 mutation carriers in the NYBCS, exceptionally long AR alleles with >29 repeats were associated with increased breast cancer risk (RH=2.48, P=0.05). The effect was similar but not significant for relatives with BRCA1 mutations (RH=1.40, P=0.52). Among probands with either BRCA1 or BRCA2 mutations, median risk (i.e. the age at which 50% of women in the group developed breast cancer) was six years younger for relatives with alleles >29 repeats. Bloom s syndrome (BLM). Heterozygosity for an Ashkenazi Jewish allele of the Bloom's syndrome helicase is associated with increased risk of colon cancer (42). Among the breast cancer probands, frequency of heterozygosity for BLM.2281del6ins7 mutation was 10/1008 (0.98%), within the % range of frequencies reported among controls (43). Age at breast cancer diagnosis did not differ for probands with the BLM mutation (52.2 years) vs. probands wildtype at BLM (52.9 years; P = 0.85). Fanconi anemia (FANCC). Fanconi anemia types B and D2 are caused by homozygosity or compound heterozygosity for mild mutations of BRCA2 (44). We tested a related question: whether breast cacner risk was associated with heterozygosity for an allele of Fanconi anemia C (FANCC) found in the Ashkenazi Jewish population. Frequency of heterozygosity for FANCC ivs4(+4)a>t was 13/1008 (1.29%) among breast cancer probands, not significantly different than - 6 -

7 the % frequencies among controls (43). Age at breast cancer diagnosis did not differ for probands with the FANCC mutation (57.1 years) vs. probands wildtype at FANCC (52.8 years; P = 0.21). Supplementary references 30. J. E.Rossouw et al., J.A.M.A. 288, 321 (2002). 31. S. A. Narod, Nat. Rev. Ca. 2, 113 (2002). 32. E. Levy-Lahad et al., Proc. Natl. Acad. Sci. U.S.A. 98, 3232 (2001). 33. W.W. Wang et al., Ca. Epid. Biom. Prev 10, 955 (2001). 34. H. Meijers-Heijboer et al., Nat Genet. 31, 55 (2002). 35. P. Vahteristo et al., Am. J. Hum. Genet. 71, 432 (2002). 36. K. Offit et al., BMC Med. Genet. 4, 1 (2003) 37. N. L. Chamberlain, E. D. Driver, R. L. Miesfeld. Nucleic Acids Res. 22, 3181 (1994). 38. J. J. Park et al., Cancer Res. 60, 5946 (2000). 39. Y. Giguere et al., Cancer Res. 61, 5869 (2001). 40. L. Kadouri et al., Br. J. Cancer 85, 36 (2001). 41. T. R. Rebbeck et al., Am. J. Hum. Genet. 64, 1371 (1999). 42. S. B. Gruber et al., Science. 297, 2013 (2002). 43. L. Peleg et al., Isr. Med. Assoc. J. 4, 95 (2002). 44. N. G. Howlett et al., Science. 297, 606 (2002). This project was approved by the Institutional Review Boards of the 12 collaborating clinical centers as well as the University of Washington (laboratory and statistical center) and Sarah Lawrence College (coordinating center for genetic counseling)

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