Bilateral Oophorectomy and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Bilateral oophorectomy and breast cancer risk in BRCA1 and BRCA2 mutation carriers

Joanne Kotsopoulos1, Tomasz Huzarski2, Jacek Gronwald2, Christian F. Singer3, Pal

Moller4, Henry T. Lynch5, Susan Armel6, Beth Karlan7, William D. Foulkes8, Susan L.

Neuhausen9, Leigha Senter10, Nadine Tung11, Jeffrey N. Weitzel12, Andrea Eisen13,

Kelly Metcalfe14, Charis Eng15, Tuya Pal16, Gareth Evans17, Ping Sun1, Jan Lubinski2,

Steven A. Narod1,* and the Hereditary Breast Cancer Clinical Study Group

Affiliations of Authors:

1. Women’s College Research Institute, Women’s College Hospital, Toronto, ON,

Canada

2. International Hereditary Cancer Center, Department of Genetics and Pathology,

Pomeranian Medical University, Szczecin, Poland

3. Department of Obstetrics and Gynecology and Comprehensive Cancer Center,

Medical University of Vienna, Vienna, Austria

4. Inherited Cancer Research Group, The Norwegian Radium Hospital, Department

for Medical Genetics; and Department of Tumor Biology, Institute of Cancer

Research, The Norwegian Radium Hospital; Oslo University Hospital, Oslo,

Norway

5. Department of Preventive Medicine and Public Health, Creighton University

School of Medicine, Omaha, NE, USA

6. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology,

University of Toronto, ON, Canada

7. Gynecology Oncology, Cedars Sinai Medical Center, Los Angeles, CA, USA

8. Program in Cancer Genetics, Department of Oncology and Human Genetics,

McGill University, Montréal, QC, Canada

9. Department of Population Sciences, Beckman Research Institute of City of Hope,

Duarte, CA, USA

10. Division of Human Genetics, The Ohio State University Medical Center,

Comprehensive Cancer Center, Columbus, OH, USA

11. Beth Israel Deaconess Medical Center, Boston, MA, USA

12. City of Hope National Medical Center, Duarte, CA, USA

13. Toronto-Sunnybrook Regional Cancer Center, 2075 Bayview Ave., Toronto, ON,

Canada

14. Faculty of Nursing, University of Toronto, Toronto, ON, Canada

15. Genomic Medicine Institute and Center for Personalized Genetic Healthcare,

Cleveland Clinic, Cleveland, OH, USA

16. Moffitt Cancer Center, Departments of Cancer Epidemiology, Biostatistics,

Anatomic Pathology, and Experimental Therapeutics, Tampa, FL, USA

17. Genomic Medicine, Manchester Academic Health Science Centre, The University

of Manchester, Manchester, UK

Other members of the Hereditary Breast Cancer Clinical Study Group: Carrie

Snyder, Aletta Poll, Sonia Nanda, Ellen Warner, Wendy Meschino, Barry Rosen,

292929222 Rochelle Demsky, Peter Ainsworth, Karen Panabaker, Gillian Mitchell, Melanie

Taylor, Fergus Couch, Siranoush Manoukian, Christine Rappaport, Barbara Pasini,

Mary B. Daly, Olufunmilayo Olopade, Linda Steele, Howard Saal, Taya Fallen, Marie

Wood, Wendy McKinnon, Dawna Gilchrist, Edmond Lemire, Albert E. Chudley, Kim

Serfas, Kevin Sweet, Louise Bordeleau, Christine Elser, Seema Panchal, Dana

Zakalik, Susan T. Vadaparamdil, Ophira Ginsburg, Stephanie Hurst, Carey A.

Cullinane, Robert E. Reilly, Joanne L. Blum, Theodora Ross, Caitlin Mauer, Ava

Kwong, Cezary Cybulski, Jeanna McCuaig, Judy Garber, Charmaine Kim-Sing, Sofia

Merajver, Eitan Friedman, Daniel Rayson, David Euhus, Claudine Isaacs, and Anna

Jakubowska.

Correspondence should be addressed to:

Dr. Steven A. Narod

Women’s College Research Institute, Women’s College Hospital

76 Grenville Street, 6th Floor

Toronto, Ontario

Canada

M5S 1B2

Tel: (416) 3513675 FAX: (416) 3513767. Email [email protected]

Running title: Oophorectomy and breast cancer in BRCA mutation carriers.

Keywords: BRCA1, breast cancer, oophorectomy

Abstract

Introduction: Whether or not bilateral oophorectomy reduces breast cancer risk among women with a BRCA1 or BRCA2 mutation continues to be a matter of debate. Thus, we undertook a prospective analysis of bilateral oophorectomy and breast cancer risk in

BRCA mutation carriers.

Methods: Eligible subjects included women with no history of cancer prior to study enrollment, with both breasts intact, and who had information on oophorectomy status (n

= 3,720). Women were followed biennially until either the date of the last follow-up questionnaire, date of breast cancer diagnosis, date of ovarian cancer diagnosis, date of prophylactic bilateral mastectomy or date of death. Cox proportional hazards model was used to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) of invasive breast cancer associated with a bilateral oophorectomy (coded as a time-dependent variable).

Results: Over a mean follow-up of 5.8 years (0-21.18), 371 new first primary breast

cancers were diagnosed in the entire cohort. Among women with either a BRCA1 or

BRCA2 mutation, oophorectomy was not associated with the risk of breast cancer

compared to women who did not undergo an oophorectomy (multivariate HR = 0.85;

95%CI 0.66-1.10; P = 0.22). The HR associated with oophorectomy was 0.95 (95%CI

0.72-1.27) for BRCA1 and was 0.64 (95%CI 0.36-1.16) for BRCA2 mutation carriers.

In an analysis stratified by age at diagnosis, the effect of oophorectomy was strongest

292929444 for breast diagnosed prior to age 50 (HR = 0.15; 95%CI 0.04-0.53) for BRCA2

mutation carriers, particularly estrogen receptor-positive tumors (HR = 0.09; 95%CI

0.01-0.74), but was not associated with risk prior to age 50 among BRCA1 mutation

carriers (HR = 1.04; 95%CI 0.72-1.50).

Discussion: Findings from this large prospective study support a role of oophorectomy for the prevention of breast cancer in BRCA2 but not BRCA1 mutation carriers.

Introduction

Whether or not bilateral oophorectomy reduces breast cancer risk among women with a

BRCA1 or BRCA2 mutation continues to be a matter of debate (1). Recently, a prospective study from the Netherlands, which included 822 BRCA mutation carriers with a median follow-up of three years, showed no protective effect of oophorectomy on breast cancer risk (hazard ratio [HR] = 1.0; 95%CI 0.67-1.77)(1). The authors concluded that previous reports of a beneficial effect of oophorectomy for breast cancer risk reduction were spurious and could be attributed to various biases. The null effect reported in the Netherlands contrasts with the results of a meta-analysis by Rebbeck et al., who reported a 51% (95%CI 0.37-0.65) highly significant reduction in breast cancer risk with bilateral oophorectomy for both BRCA1 and BRCA2 mutation carriers (2). The meta-analysis included both case-control and prospective studies, with individual sample sizes ranging from 597 to 988 women with BRCA mutations. In a case-control study of

1,439 pairs of BRCA mutation carriers, we reported a significant 54% reduction in breast cancer risk associated with oophorectomy (95%CI 0.32-0.65) (3).

Oophorectomy is routinely recommended to healthy women with a BRCA mutation at age

35 or thereafter to prevent cancers of the ovary, fallopian tube or peritoneum and to reduce all-cause mortality (4, 5). Also, among BRCA carriers with a previous diagnosis of breast cancer, oophorectomy significantly reduces breast cancer mortality and prevents contralateral breast cancer (6, 7). Given the negative consequences associated with surgical menopause, including climacteric symptoms, as well as possible adverse effects

292929666 on cognition, bone density and cardiac health, it is important that we clarify the impact of oophorectomy (and timing of surgery) on breast cancer risk (8).

A well-designed, prospective study is needed to help clarify the role of oophorectomy on breast cancer risk in high-risk women. Given the concerns regarding the methods of the case-control studies conducted to date, we undertook a prospective analysis of bilateral oophorectomy and breast cancer risk in 3,720 BRCA mutation carriers with no history of cancer prior to study enrollment. Subjects were observed from the date of ascertainment until the date of last follow-up. We also evaluated the association of oophorectomy and breast cancer risk by BRCA mutation type, by age at diagnosis and by estrogen receptor status of the tumor.

Materials and Methods

Study Population

Eligible study subjects included women who were enrolled in a prospective cohort

study of deleterious BRCA1 and BRCA2 mutation carriers and were identified from 78

participating centers in 12 countries. All subjects had sought testing for BRCA1 and

BRCA2 mutations because of a personal or family history of breast and/or ovarian

cancer. All study subjects (with the exception of some women from the University of

Utah and the University of California Irvine centers) received genetic counselling.

The institutional review boards of the host institutions approved the study. All

subjects provided written informed consent. Mutation detection was performed using

a range of techniques, but all nucleotide sequences were confirmed by direct

sequencing of DNA.

Data Collection

All study subjects completed a baseline questionnaire at the individual center at the

time of a clinic appointment or at their home at a later date. Follow-up questionnaires

were completed every two years thereafter. These were either mailed to each study

participant to complete and return, or were administered over the phone by a genetic

counsellor or research assistant at each center. The questionnaires requested

information on family and personal history of cancer, and reproductive and medical

histories, including preventive oophorectomy and mastectomy. Women were

classified as having a bilateral oophorectomy if both ovaries were removed (with or

without fallopian tubes or uterus intact). Women with a unilateral oophorectomy were

292929888 included in the no oophorectomy group. Information on incident breast cancers,

including hormone receptor status, was collected from the follow-up questionnaires

and pathology records were reviewed. For this analysis, incident breast cancers

consisted of first primary invasive breast cancers.

Study Subjects Available for Analysis

Women were eligible for the study if they completed at least one follow-up

questionnaire.

Of the 12,794 women who were initially eligible, we excluded women who had a prior diagnosis of breast cancer (n = 6,307), ovarian cancer (n = 1,546) or other cancer (n =

625), who had undergone a prophylactic bilateral mastectomy at baseline (n = 342), who were missing information on mastectomy status (n = 221) or who were missing age at menarche (n = 5). After these exclusions, a total of 3,748 subjects qualified for inclusion in the analysis.

Statistical Analysis

We estimated the annual breast cancer incidence (%) by calculating the total person-years for the entire cohort and the number of incident cases in the entire follow-up period.

Incidence (%) was estimated as the ratio of the number of breast cancer cases to the total number of person-years.

We used a Cox proportional hazards model to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) of breast cancer associated with a bilateral oophorectomy.

Bilateral oophorectomy was coded as a time-dependent variable. If a women had a bilateral oophorectomy after the completion of the baseline questionnaire (or at any point in the follow-up) the exposure was updated to reflect the change in oophorectomy status.

Participants were followed from the date of completion of the baseline questionnaire until either the: 1) date of completion of the last follow-up questionnaire, 2) date of breast cancer diagnosis, 3) date of prophylactic bilateral mastectomy, 4) date of ovarian cancer diagnosis or 5) date of death. In the multivariate models, we adjusted for age at baseline

(<40, 40-49, ≥50 years), family history of breast cancer (0, 1, 2, ≥3 affected first-degree relatives), country of residence (Poland, Canada, United States, other) oral contraceptive use (ever/never), age at menarche (≤12, 13, ≥14), parity (0, 1, 2, 3, ≥4) and breastfeeding

(ever/never). We also performed analyses stratified by BRCA mutation type, estrogen receptor status of the tumor and excluding women with an oophorectomy at or prior to the baseline questionnaire, as well as analyses censoring at different ages.

All analyses were performed using the SAS statistical package, version 9.1.3 (SAS

Institute, Cary, NC, USA). All P values were based on two-sided tests and were considered statistically significant if P ≤ 0.05.

292929101010 Results

There were 3,720 women with a BRCA1 or BRCA2 mutation included in the current analysis. The baseline characteristics are summarized in Table 1; 1,559 women had a bilateral oophorectomy either before (n = 866) or after (n = 693) study enrollment and

2,189 women did not have an oophorectomy. Many of the variables differed between the two exposure groups but this was likely a reflection of the mean age at study entry in the oophorectomy group (33.5 vs. 46.2 years; P < 0.0001). On average, compared to women with both ovaries intact, women with an oophorectomy were older, parous, have breastfed, used oral contraceptives, and have a stronger family history of breast cancer (P < 0.0001).

Over a mean follow-up of 5.8 years (0-21.18), 371 new first primary breast cancers were diagnosed in the entire cohort; 222 (10.1%) were among women in the no oophorectomy group and 149 (9.6%) in the oophorectomy group (P = 0.56). The total number of person-years, number of incident breast cancers and annual incidence among all the study subjects combined, and stratified by age, oophorectomy status and

BRCA mutation type, are displayed in Table 2. Among all women, the annual incidence of breast cancer was 1.7%, with similar estimates in women with (1.9%) and without an oophorectomy (1.6%). Among women with either a BRCA1 or BRCA2 mutation, oophorectomy was not associated with the risk of breast cancer compared to women who did not undergo an oophorectomy (Table 3). The univariate HR was 1.16

(95%CI 0.94-1.44; P = 0.16) and the multivariate HR was 0.85 (95%CI 0.66-1.10; P =

0.22).

We stratified the cohort by BRCA mutation status. The annual incidence was 1.7% for

BRCA1 and 1.6% for BRCA2 mutation carriers (Table 2). The HR associated with

oophorectomy was 0.95 (95%CI 0.72-1.27) for BRCA1 and was 0.64 (95%CI 0.36-

1.16) for BRCA2 mutation carriers (Table 4). After stratifying by age at diagnosis,

oophorectomy was not associated with the risk of breast cancer prior to age 50 among

women with a BRCA1 mutation (HR = 1.04; 95%CI 0.72-1.50). In contrast,

oophorectomy was associated with a significant 85% reduction in breast cancer

diagnosed prior to age 50 among women with a BRCA2 mutation (HR = 0.15; 95%CI

0.04-0.53), but did not protect against breast cancer diagnosed between the ages of 50

and 70 years (HR = 1.22; 95%CI 0.37-4.07). We had information on estrogen receptor

(ER) status for 41 of the 63 BRCA2 cases. The protective effect of oophorectomy on

breast cancer diagnosed prior to age 50 among ER-positive cases was very strong (HR

= 0.09; 95%CI 0.01-0.74). There was not enough data to evaluate the relationship for

ER-negative cases.

In a secondary analysis, we excluded women who had an oophorectomy prior to

baseline (n = 2,882). This did not substantially alter the findings (HR = 1.02; 95%CI

0.74-1.14). When stratified by BRCA mutation status, the risk estimates were 1.13

(85%CI 0.80-1.61) for BRCA1 and 0.67 (95%C 0.29-1.54) for BRCA2 mutation

carriers.

292929121212 Discussion

The current study is the largest prospective analysis conducted to date of bilateral oophorectomy and breast cancer risk among women with an inherited BRCA1 or BRCA2 mutation. In the study of 3,720 eligible participants, we identified 371 incident cancers.

After a mean follow-up of 5.8 years, we found no significant overall association between oophorectomy and breast cancer risk (RR = 0.85; 95%CI 0.66-1.10). However, we did observe a significant protective effect of oophorectomy on breast cancer risk prior to age

50 among women with a BRCA2 mutation (RR = 0.15; 95%CI 0.04-0.53; P = 0.003).

Although these findings were based on a post hoc analysis, the large sample size and long follow-up of our study, along with the biological plausibility of risk reduction in BRCA2 mutation carriers with predominantly ER-positive and progesterone receptor (PR) positive tumours, make it unlikely that our findings were due to chance.

There have been four prior prospective evaluations of oophorectomy and breast cancer risk in BRCA mutation carriers (1, 5, 9, 10). In the most recent, which included 822 unaffected BRCA mutation carriers, there were 89 incident breast cancers after a mean follow-up of 5.5 years in the oophorectomy group, Heemskerk-Gerristen and colleagues found no effect of oophorectomy on breast cancer risk (HR = 1.09; 95%CI 0.67-1.77) (1).

Similar to our findings, the relationship between oophorectomy and risk was strong for

BRCA2 (HR = 0.54; 95%CI 0.17-1.66) but not for BRCA1 and mutation carriers (HR =

1.21; 95%CI 0.72-2.06). The BRCA2 association did not achieve statistical significance, likely due to the small number incident cancers in this subgroup (n = 14).

Among 988 unaffected BRCA1 and BRCA2 mutation carriers enrolled in the EMBRACE study, with 64 incident breast cancers, and an average of 3.4 years of follow-up,

Mavaddat et al., reported a borderline significant protective effect of oophorectomy on breast cancer risk overall (HR = 0.62; 95%CI 0.35-1.09). For women with a BRCA1 mutation, the HR was 0.52 (95%CI 0.24-1.13) and for BRCA2 mutation carriers the HR was 0.79 (95%CI 0.35-1.80). There was a significant 61% reduction in risk if performed prior to age 45 (95%CI 0.17-0.87) but not after age 45 (HR= 1.14; 95%CI 0.50-2.61)

(10). The risk reduction was similar in carriers of either mutation. This analysis was strengthened by the inclusion of oophorectomy as a time-dependent variable to avoid immortal-time bias but was limited by the small number of incident cancers.

In a multi-center prospective analysis of 1,370 BRCA mutation carriers enrolled in the

PROSE study, 262 incident cancers and 4.7 years of follow-up, Domcheck et al., reported a significant protective effect of salpingo-oophorectomy on breast cancer risk overall

(HR = 0.53; 95%CI 0.37-0.79) with an HR of 0.63 (95%CI 0.41-0.96) for BRCA1 and

0.36 (95%CI 0.16-0.82) for BRCA2 mutation carriers (5). The risk reducing effect was greater if oophorectomy was performed prior to age 50 (HR = 0.51; 95%CI 0.32-0.82). It is not clear if the follow-up in this study was initiated at the date of genetic testing.

In the publication including 597 BRCA mutation carriers, 47 incident cancers and 3 years of follow-up, Kauff et al., reported a significant 47% reduction in risk overall (95%CI

0.29-0.96) that was strongest for BRCA2 (HR = 0.28; 95%CI 0.08-0.92) compared with

BRCA1 (HR = 0.61; 95%CI 0.30-1.22) mutation carriers, although both estimates were

292929141414 below unity (9). This study included women with and without a personal history of breast cancer and did not evaluate age at oophorectomy.

These earlier publications were limited by small sample sizes, relatively short follow-up, and in some cases, the inclusion of prevalent cases diagnosed prior to the date of ascertainment. In the current study, we included a large number of unaffected BRCA mutation carriers (n = 3,720) who were followed from the date of ascertainment (i.e., genetic testing) and we treated oophorectomy as a time-dependent exposure (11). The relatively long follow-up period (5.8 years) allowed us to assess short and long term effects of oophorectomy. We had 371 incident cancers, the largest of any study conducted to date.

Pathologic and etiologic differences between BRCA1- and BRCA2-associated breast

cancers may help explain the differing effects of oophorectomy on risk (12).

Tumours from women with a germline BRCA1 mutation are typically estrogen

receptor (ER) negative and progesterone receptor (PR) negative, while those from

women with a BRCA2 mutation are usually ER positive and PR positive and likely

more responsive to stimulation by sex hormones (12). We had information on

hormone-receptor status for 75% of the breast cancer cases in the study. Women

with a BRCA2 mutation were more likely to develop an ER positive (80%) cancer

compared with BRCA1 mutation carriers (27%). Among BRCA2 mutation carriers,

we found a stronger protective effect of oophorectomy for premenopausal/ER-

positive breast cancers. Thus, it appears that the effect of oophorectomy was on

hormone-receptor positive vs. negative tumours. Thus, it is biologically plausible

that surgical removal of the endogenous source of sex hormones would differentially

impact risk. It is somewhat surprising that oophorectomy does not influence

BRCA1-associated breast cancer given that we and others have demonstrated a

stronger role of reproductive factors and exogenous hormone exposure in BRCA1 but

not BRCA2 mutation carriers (13-15). Furthermore, we recently showed that one to

three years of tamoxifen protects against contralateral breast cancer in women with a

BRCA1 mutation (16). Although, the same protective effect of tamoxifen has also

been shown for women with a BRCA2 mutation (17, 18). In contrast, we recently

showed no adverse effect of HRT use on risk after natural or surgical menopause

among women with a BRCA1 mutation {Kotsopoulos, 2016 #13064}(submitted).

292929161616 Our findings have important clinical implications. BRCA mutation carriers face high lifetime risks of developing breast, ovarian and contralateral breast cancer (10).

Although our results show no effect of oophorectomy on BRCA1-associated breast cancer, bilateral salpingo-oophorectomy should continue to be recommended at age 35 for women with a BRCA1 mutation and age 40 for women with a BRCA2 mutation (or after childbearing is complete). This is based on the ages at which the annual rates for ovarian cancer start to rise (19). The protective role of oophorectomy diagnosed prior to age 50 further supports recommendations at age 40 for BRCA2 mutation carriers.

Among women in the general population, surgical menopause is associated with a reduction in breast cancer risk, likely through a reduction in the lifetime exposure to circulating ovarian hormones (20-22). However, surgical menopause (particularly at a young age and if without HRT) is not without its consequences. Data from observational studies and randomized controlled-trials in the general population have shown that oophorectomy has a significant adverse effect on mortality and other outcomes (23, 24).

In contrast, Finch and colleagues recently showed that oophorectomy was associated with a highly significant 77% reduction in all-cause mortality in women with a BRCA1 or

BRCA2 mutation (19). Of importance for BRCA mutation carriers with a prior breast cancer diagnosis is that oophorectomy is associated with a significant reduction in the risk of contralateral breast cancer (RR = 0.53; 95%CI 0.34-0.84), as well as, a significant reduction in breast cancer mortality (HR = 0.46; 95%CI 0.27-0.79) (66, 7). In addition, we have recently shown that a short-course of HRT does not increase the risk of breast cancer among BRCA1 mutation carriers {Kotsopoulos, 2016 #13064}(submitted).

Whether this is also true for women with a BRCA2 mutation is not yet known. There is clearly a need to balance the benefits and harms of surgical menopause in this population with substantially elevated lifetime risks of both breast and ovarian cancer.

Ours is the largest prospective study to date evaluating the magnitude of protection associated with an oophorectomy reported among BRCA mutation carriers. With 3,720 eligible women and a mean follow-up of 5.8 years, we were well powered overall and in our subgroup analyses. We avoided potential biases by excluding women with any cancer diagnosis at the time of the enrollment, by using the date of baseline questionnaire completion as the start of follow-up and by considering oophorectomy as a time- dependent variable.

In summary, findings from the current study support a role of oophorectomy for the prevention of breast cancer in BRCA2 mutation carriers but not BRCA1 mutation carriers.

Given the high lifetime risks of ovarian cancer in carriers of either BRCA mutation, the optimal age for bilateral salpingo-oophorectomy is still age 35 to prevent ovarian cancer and to reduce overall mortality. Furthermore, the evidence collectively supports an important role of oophorectomy in decreasing mortality among both affected and unaffected women with a BRCA1 or BRCA2 mutation. Management of women with early menopause and the long-term effects of surgical menopause is of importance and will depend on personal history of cancer and the timing of surgery. Our recent findings that HRT does not impact BRCA1-associated breast cancer support a short course of exogenous hormones to help mitigate the effects of surgical menopause in these women.

292929181818 19 20

Competing Interests

The authors declare that they have no conflict of interest.

Acknowledgements

We would also like to acknowledge the study coordinators Marcia Llacuachaqui, Farah

Shoukat, and Alejandra Ragone, as well as the students and staff Ellen MacDougall,

Zoella Pasta, Nida Mian, Jennifer Ng, Sarah Chin, Hamida Begum, Harmeet Chaudhary, and Yaminee Charavanapavan who helped with the data collection and data entry.

Joanne Kotsopoulos is the recipient of a Cancer Care Ontario Research Chair in

Population Studies and a Canadian Cancer Society Career Development Award in

Prevention. Charis Eng is the recipient of the Sondra J. and Stephen R. Hardis Chair of

Cancer Genomic Medicine at the Cleveland Clinic and of the ACS Clinical Research

Professorship. Susan L. Neuhausen is partially supported by the Morris and Horowitz

Endowed Professorship and her work was supported by a grant from the NIH,

R01CA74415. Steven A. Narod is the recipient of a Tier I Canada Research Chair. This study was supported by a Canadian Cancer Society Research Institute grant (703058).

Patients were recruited for study from the City of Hope Clinical Cancer Genomics

Community Research Network, supported in part by Award Number RC4CA153828 (PI:

J. Weitzel) from the National Cancer Institute and the Office of the Director, National

Institutes of Health. Siranoush Manoukian is affiliated with the Fondazione IRCCS

Istituto Nazionale dei Tumori (INT), Milan, Italy.

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292929242424 Table 1. Characteristics of BRCA mutation carriers with or without a bilateral oophorectomy Variable No oophorectomy Oophorectomy P (n = 2,189) (n = 1,559) Year of birth, mean (range) 1971.2 (1911-93) 1957.5 (1908-82) <0.0001 Age at oophorectomy, mean (range) n/a 46.3 (13-78) n/a Age at baseline, n (%) <40 1,682 (76.8%) 390 (25.0%) 40-49 304 (13.8%) 645 (41.4%) ≥50 203 (9.3%) 524 (33.6%) <0.0001 Mean (range), years 33.5 (13-85) 46.2 (21-88) <0.0001

Mean follow-up, years (range) 5.42 (0-21.18) 6.34 (0-19.50) <0.0001

Breast cancer No 1,967 (89.9%) 1,410 (90.4%) Yes 222 (10.1%) 149 (9.6%) 0.56 Age at diagnosis, mean (range) 42.7 (25-75) 52.6 (33-81) <0.0001

BRCA mutation BRCA1 1,795 (82.7%) 1,192 (77.0%) BRCA2 376 (17.3%) 357 (23.1%) <0.0001 BRCA1 and BRCA2 or missing 18 10

Parity, n (%) Nulliparous 910 (41.6%) 189 (12.1%) 1 444 (20.3%) 217 (13.9%) 2 493 (22.5%) 656 (42.1%) 3 217 (9.9%) 350 (22.5%) ≥4 106 (4.8%) 139 (8.9%) <0.0001 Mean (range) 1.2 (0-10) 2.1 (0-8) <0.0001 Missing 19 8

Breastfeeding, n (%) Never 1,065 (51.5%) 387 (28.7%) Ever, < 12 months 568 (27.4%) 541 (40.0%) Ever, ≥ 12 months 439 (21.2%) 423 (31.3%) <0.0001 Mean (range), months 6.9 (0-101) 10.3 (0-14) <0.0001 Missing 114 208

Oral contraceptive use at baseline, n (%) Never 857 (39.3%) 509 (33.0%) Ever 1,322 (60.7%) 1,035 (67.0%) <0.0001 Missing 10 15

Family history of breast cancer, n (%)1 0 815 (44.1%) 562 (45.8%) 1 905 (49.0%) 478 (38.9%) 2 99 (5.4%) 158 (12.9%) <0.0001 ≥ 3 29 (1.6%) 30 (2.4%) Mean (range) 0.6 (0-4) 0.7 (0-4) 0.003 Missing 341 331

Age at menarche, n (%) ≤11 228 (10.8%) 200 (13.3%) 12 489 (23.1%) 359 (23.8%)

13 620 (29.3%) 405 (26.9%) ≥14 780 (36.9%) 544 (36.1%) 0.08 Mean age 13.1 (9-19) 13.1 (9-28) 0.47 Missing 71 51

Country of residence, n (%) Poland 1,161 (53.0%) 450 (28.9%) Canada 438 (20.9%) 488 (31.3%) Other 231 (10.6%) 282 (18.1%) USA 359 (16.4%) 339 (21.7%) <0.0001

292929262626 Table 2. Person-years, number of incident breast cancers and incidence of breast cancer among all women and stratified by oophorectomy status and BRCA mutation status Person-years Number of events Incidence (n) (%) BRCA1 and BRCA2 mutation carriers (n = 3,720) All 21757 371 1.71 No oophorectomy 13910 222 1.60 Oophorectomy 7846 149 1.90

BRCA1 mutation carriers (n = 2,987) All 17684 307 1.74 No oophorectomy 11460 180 1.57 Oophorectomy 6224 127 2.04

BRCA2 mutation carriers (n = 733) All 2362 41 1.74 No oophorectomy 1578 22 1.39 Oophorectomy 3940 63 1.60

Censored at age 501 BRCA1 mutation carriers (n = 2,481) All 12984 201 1.55 No oophorectomy 10187 146 1.43 Oophorectomy 2797 55 1.97

BRCA2 mutation carriers (n = 519) All 2320 35 1.51 No oophorectomy 1747 32 1.83 Oophorectomy 572 3 0.52 1Follow-up was censored at age 50; thus, women with an age at baseline of ≤50 years were excluded from this analysis. Women with both a BRCA1 and BRCA2 mutation were coded as missing.

Table 3. Bilateral oophorectomy and risk of breast cancer in BRCA1 and BRCA2 mutation carriers Univariate P Multivariate1 P HR (95%CI) HR (95%CI) Oophorectomy2 No 1.00 (reference) 1.00 (reference) Yes 1.16 (0.94-1.44) 0.16 0.85 (0.66-1.10) 0.22

Age at baseline, years <40 1.00 (reference) 1.00 (reference) 40-49 1.51 (1.20-1.92) 0.0006 1.54 (1.17-2.00) 0.002 50 + 1.55 (1.20-2.02) 0.001 1.65 (1.19-2.28) 0.003

Family history of breast cancer3 0 1.00 (reference) 1.00 (reference) 1 1.38 (1.08-1.77) 0.009 1.39 (1.09-1.78) 0.009 ≥2 1.58 (1.11-2.25) 0.01 1.38 (0.96-1.99) 0.08

Oral contraceptive use Never 1.00 (reference) 1.00 (reference) Ever 1.02 (0.84-1.28) 0.76 1.13 (0.88-1.44) 0.35

BRCA mutation BRCA1 1.00 (reference) 1.00 (reference) BRCA2 0.94 (0.72-1.24) 0.67 0.92 (0.67-1.25) 0.59

Country of residence Poland 1.00 (reference) 1.00 (reference) Canada 0.94 (0.73-1.22) 0.64 0.85 (0.61-1.18) 0.34 Other 1.13 (0.84-1.52) 0.41 0.84 (0.50-1.39) 0.49 United States 1.10 (0.82-1.48) 0.52 1.03 (0.73-1.44) 0.88 1Additionally adjusted for age at menarche (≤12, 13, ≥14), parity (0, 1, 2, 3, ≥4) and breastfeeding (ever/never). 2Oophorectomy was coded as a time-dependent covariate. 3Number of affected first-degree relatives.

292929282828 Table 4. Bilateral oophorectomy and risk of breast cancer, stratified by BRCA mutation status and by age at diagnosis Univariate P Multivariate1 P HR (95%CI) HR (95%CI) All women BRCA1 mutation carriers Oophorectomy2 No 1.00 (reference) 1.0 (reference) Yes 1.25 (1.00-1.58) 0.06 0.95 (0.72-1.27) 0.73

BRCA2 mutation carriers Oophorectomy No 1.00 (reference) 1.00 (reference) Yes 0.82 (0.49-1.39) 0.47 0.64 (0.36-1.16) 0.14

Breast cancer diagnosed prior to age 503 BRCA1 mutation carriers Oophorectomy 1.00 (reference) 1.00 (reference) No 1.32 (0.97-1.81) 0.08 1.04 (0.72-1.50) 0.83 Yes

BRCA2 mutation carriers Oophorectomy No 1.00 (reference) 1.00 (reference) Yes 0.30 (0.09-0.98) 0.05 0.15 (0.04-0.53) 0.003 1Adjusted for age at baseline (<40, 40-49, ≥50 years), family history of breast cancer (0, 1, 2, ≥3 affected first-degree relatives), country of residence (Poland, Canada, United States, other) oral contraceptive use (ever/never), age at menarche (≤12, 13, ≥14), parity (0, 1, 2, 3, ≥4) and breastfeeding (ever/never). 2Oophorectomy was coded as a time-dependent covariate. 3Follow-up was censored at age 50; thus, women with an age at baseline of ≤50 years were excluded from this analysis.