Affiliations of authors: L. C. Hartmann, M. H. Frost (Division of Medical Oncology), T. A. Sellers, D. L. Sitta (Division of Clinical Epidemiology), D. J. Schaid, S. K. McDonnell (Division of Biostatistics), C. L. Soderberg, F. J. Couch, R. B. Jenkins (Department of Laboratory Medicine and Pathology), C. S. Grant, J. H. Donohue, J. E. Woods (Department of Surgery), C. W. Vockley (Department of Medical Genetics), Mayo Clinic and Foundation, Rochester, MN; T. S. Frank, A. Deffenbaugh, Myriad Genetic Laboratories, Salt Lake City, UT.
Correspondence to: Lynn C. Hartmann, M.D., Division of Medical Oncology, Mayo Clinic and Foundation, 200 First St., SW, Rochester, MN 55905 (e-mail: hartmann.lynn{at}mayo.edu).
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ABSTRACT |
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INTRODUCTION |
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At the time of our initial report, the BRCA1 and BRCA2 gene mutation status of the study participants was not known; thus, it was unclear whether or not prophylactic mastectomy was equally effective for women in this highest risk group. Such data are clearly needed to adequately inform women with BRCA1 or BRCA2 mutations about the effectiveness of their health management options.
Herein we report the effect of bilateral prophylactic mastectomy on breast cancer risk in that subset of women from our original high-risk group who have since been identified as carriers of alterations in BRCA1 or BRCA2.
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PATIENTS AND METHODS |
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Details regarding the identification of the cohort of women with a family history of breast cancer who had bilateral prophylactic mastectomy at the Mayo Clinic, Rochester, MN, have been published previously (4). The criteria used to identify the 214 high-risk women in that cohort have also been published previously (4). We used medical records, a study-specific questionnaire (4), and telephone interviews to obtain information about cancer risk factors and cancer occurrences after prophylactic mastectomy.
Screening Strategy for Mutations in BRCA1 and BRCA2 Genes
We requested a blood sample from all known living women (probands) in the high-risk group (n = 206). Because only three of these women had breast cancer, we also sought a blood specimen from a living affected relative (i.e., a relative with breast cancer) of each woman because an affected relative is more likely to have a BRCA1 or BRCA2 mutation than the unaffected proband herself. The probands and the affected relatives who agreed to participate in the BRCA1 and BRCA2 mutation screening were sent a blood specimen kit, which they took to a local laboratory where a phlebotomy was performed. The blood specimen was returned to the Mayo Clinic. If blood specimens were available for both a proband and her affected relative (n = 82), the affected relative's specimen was screened first. If that sample was negative for BRCA1 or BRCA2 mutations, the proband's sample was not screened. If a genetic alteration was detected in the relative, the proband's sample was then tested for the presence of the same alteration. If no blood specimen was available for an affected relative (n = 94), the proband's specimen was screened for BRCA1 or BRCA2 mutations. For the three women who developed breast cancer despite prophylactic mastectomy, one is still alive and provided a blood specimen. The other two are deceased. For one of the deceased women, her mother (who had ovarian cancer) provided a specimen. For the other, no specimen was available. All of the contact procedures, testing protocols, and consent forms were approved by the Institutional Review Board of the Mayo Clinic. All of the individuals who were tested signed a consent form.
BRCA1 and BRCA2 Screening Methods
Initially, our screening strategy was to analyze all specimens by direct sequence analysis. Later, we employed conformation-sensitive gel electrophoresis (CSGE), which detects sequence differences in double-stranded DNA fragments on the basis of their altered electrophoretic mobilities, as an initial screen, especially for samples from women who were less likely to be mutation carriers (5). For direct sequencing, dye primer sequence analysis, in both the forward and reverse directions, of 48 coding exons and adjacent splice junctions of BRCA1 and BRCA2 was performed at Myriad Genetic Laboratories (Salt Lake City, UT) on genomic DNA extracted from peripheral blood lymphocytes (6). For CSGE analyses (7), the 48 coding exons and adjacent splice junctions of BRCA1 and BRCA2 were amplified by polymerase chain reaction (PCR) from the same genomic DNA samples with the use of 72 previously described oligonucleotide primer sets (8,9). Where possible, genomic DNAs from patients containing known BRCA1 and BRCA2 mutations were PCR amplified and used as positive controls in CSGE analysis. A total of 20 such controls were available for different PCR products.
To prepare PCR products for CSGE analysis, we added 2 µL of nondenaturing loading dye to 8 µL of each PCR product, denatured the mixture by boiling it for 5 minutes, and then allowed the mixture to reanneal at 68 °C for 30 minutes. Samples were loaded onto 10% polyacrylamide gels (99 : 1 acrylamide : 1,4-bis(acroyl)piperazine (Fluka Chemical Corp., Milwaukee, WI) containing 10% ethylene glycol, 15% formamide, and 0.5x TTE (i.e., 44.4 mM Tris, 14.25 mM taurine, and 0.1 mM EDTA [pH 9.0]) (8,9). Gels were subjected to electrophoresis at 400 V overnight in 0.5x TTE, after which they were stained with ethidium bromide and photographed under UV illumination. DNA samples that were found to contain variants of BRCA1 or BRCA2 by CSGE were reamplified and sequenced directly in the molecular biology core facility of the Mayo Clinic (9,10) to identify sequence alterations. For the 176 probands tested, 137 were analyzed by direct sequencing and 39 were screened only by CSGE.
Categories of BRCA1 and BRCA2 Alterations
Based on documentation of deleterious mutations and polymorphisms in BRCA1 and BRCA2 (11,12), a mutation in BRCA1 was considered to be deleterious if it would result in the premature termination (truncation) of the protein product of BRCA1 at least 10 amino acids from the C-terminus. A mutation in BRCA2 was considered to be deleterious if it would result in the premature termination (truncation) of the protein product of BRCA2 at least 110 amino acids from the C-terminus. In addition, specific missense mutations and noncoding intervening sequence (intron) mutations were considered to be deleterious on the basis of data derived from linkage analysis of high-risk families, functional assays, and/or the demonstration of abnormal messenger RNA (mRNA) transcript processing (13). Genetic alterations that did not result in the truncation of either BRCA1 or BRCA2 were considered to be clinically inconsequential if they had been reported at a heterozygote frequency of approximately 2% in a suitable control population, if they were unlikely to affect the function of the protein or the integrity of the mRNA transcript encoded by the gene (including so-called "silent mutations"), or if clinical observations had strongly suggested that they were not associated with cancer risk (13).
The clinical significance of some identified genetic variants is not known; these mutations were considered to be uncertain. Such variants include both missense mutations and mutations occurring in analyzed intronic regions. The specific uncertain variants detected in our cohort are E143K and I1766S (in BRCA1) and S158L, S326R, D1280V, E2020K, Q2384K, V3079I, and L2653P (in BRCA2). All of the mutations in BRCA1 and BRCA2 discovered in the course of this study have been entered into the Breast Cancer Information Core (14).
Statistical Analyses
To predict the number of breast cancers expected in the BRCA1 and BRCA2 mutation carriers, we used two published penetrance models, a high-penetrance model by Easton et al. (1,3) and a lower penetrance model by Struewing et al. (2). To calculate the efficacy of prophylactic mastectomy, we then compared the number of breast cancers observed with the number expected. Both models considered the age of the women from the time of their prophylactic mastectomy to the last observation time, which, for this study, was the occurrence of breast cancer, a censored time of death, or the last follow-up. The conditional probability of breast cancer by a woman's age at her last observation time (t), knowing that she was free of disease at the age at which her mastectomy was performed (p), was computed as [F(t) F(p)]/[1 F(p)], where F is the cumulative penetrance. The cumulative penetrances, at 10-year age intervals, were abstracted from (1) and (3) for the high penetrance model and from (2) for the lower penetrance model. Linear interpolation was used to determine cumulative penetrance for ages within the 10-year intervals. The model by Easton et al. (1,3) reflects breast cancer occurrences in families with a large number of affected women; these families demonstrate genetic linkage to BRCA1 or BRCA2. For this model, the cumulative penetrances, beginning at age 20 years in 10-year intervals, are 0% (ages 2029 years), 3.2% (ages 3039 years), 19.1% (ages 4049 years), 50.8% (ages 5059 years), 54.2% (ages 6069 years), and 85% (ages 70 years). The results of Struewing et al. (2) are data derived from a study of Ashkenazi Jewish residents from the Washington, DC, area who were unselected for family history of cancer. Seventy-six percent of these study subjects had neither a personal history nor a family history of cancer. For this model, the cumulative penetrances, beginning at age 30 years in 10-year intervals, are 0% (ages 3039 years), 15% (ages 4049 years), 33% (ages 5059 years), 52% (ages 6069 years), 56% (ages 7079 years), and 60% (ages
80 years). Given the family history makeup of the women in our prophylactic mastectomy cohort, we anticipated that their cancer risk would be higher than that of the population of Struewing et al. We reasoned, however, that we could calculate a more comprehensive range of risk estimates for the women in our study by using both models. These age-specific risks were applied to the follow-up age for each BRCA1 and BRCA2 mutation carrier in our cohort. In this manner, we calculated an age-adjusted expected number of events. We calculated confidence intervals (CIs) by assuming that the number of events had a Poisson distribution.
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RESULTS |
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We obtained a blood sample suitable for screening mutations in BRCA1 and BRCA2 for 176 of the 214 high-risk women. For 82 of the 176 women, we also received a blood sample from an affected relative. Of the 82 affected relatives, 21 had a deleterious mutation and six had a mutation of uncertain clinical significance in either BRCA1 or BRCA2. When the corresponding probands were tested, we found that 10 of the 21 probands whose relatives had a deleterious mutation and three of the six probands whose relatives had an uncertain mutation had inherited the same mutation present in their relative. Among the 94 probands for whom no affected relative's specimen was available, eight had a deleterious mutation and five had mutations of uncertain clinical significance. Thus, we detected 18 deleterious mutations and eight mutations of uncertain clinical significance in the 176 high-risk probands who had undergone bilateral mastectomy and were screened for their BRCA1 and BRCA2 mutational status.
Table 1 lists the characteristics of the 18 probands who had deleterious mutations, the eight probands who had uncertain mutations, the 150 probands who were negative for a mutation, and the 38 probands for whom no blood specimen was available. The latter group included women who were deceased as well as those who did not provide a blood specimen. Altogether, eight of the 214 high-risk women from the original cohort have died. The cause of death for two of these women was breast cancer that developed after their prophylactic mastectomies. The causes of death for the other six women were ovarian cancer, pancreatic cancer, glioblastoma, heart disease, aortic aneurysm, and postoperative complications following neck surgery. The woman who died of ovarian cancer had a deleterious mutation in BRCA1. The BRCA1 and BRCA2 mutation status of the two women who died of postprophylactic mastectomy breast cancers is discussed below. The BRCA1 and BRCA2 mutation status for the five other deceased women is not known; however, none had evidence of breast cancer.
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None of the 26 women with a mutation in BRCA1 or BRCA2 has developed breast cancer during a median follow-up of 13.4 years (range, 5.828.5 years) after mastectomy. We previously reported that three of the 214 women in our cohort of women at high risk for breast cancer developed the disease after prophylactic mastectomy (4). Direct sequencing of a blood specimen obtained from one of these three women revealed no mutations in either BRCA1 or BRCA2. The other two women are deceased. For one, her mother (who had ovarian cancer) provided a blood specimen; no mutation in BRCA1 or BRCA2 was detected. From this result, we inferred that her daughter was not a carrier. The BRCA1 and BRCA2 mutation status of the third woman is unknown because no blood specimen from an affected relative was available for screening. As described in the next section, we calculated the efficacy of prophylactic mastectomy in two ways, by assuming that this individual was and was not a BRCA1 or BRCA2 mutation carrier.
Incidence of Breast Cancer After Bilateral Prophylactic Mastectomy in BRCA1 and BRCA2 Mutation Carriers: Expected Versus Observed
We determined the efficacy of prophylactic mastectomy in BRCA1 and BRCA2 mutation carriers by using two different penetrance models to predict the number of expected breast cancers in our group of carriers (Table 2). For each of the two models, we calculated the reduction in risk by including and by excluding the deceased woman with unknown BRCA1 and BRCA2 status who developed breast cancer after prophylactic mastectomy in the total number of BRCA1 and BRCA2 mutation carriers.
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The presence of multiple women with early-onset breast cancer and/or ovarian cancer in a family indicates an increased likelihood of a BRCA1 or BRCA2 mutation in that family (6). To provide additional information about the possible BRCA1 and BRCA2 mutation carrier status of the three women who developed breast cancer, we compared the number of affected members in their families with the number of affected members in the families of the women with deleterious and uncertain mutations in BRCA1 and BRCA2 (Table 3). The three women who developed breast cancer were not members of the most heavily affected families, whereas most of the BRCA1 and BRCA2 mutation carriers were. This additional analysis reveals a low likelihood that these three women were carriers of BRCA1 or BRCA2 mutations.
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DISCUSSION |
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We now report that none of the 26 carriers of a BRCA1 or BRCA2 mutation of our original group of 214 high-risk women who had bilateral prophylactic mastectomy at the Mayo Clinic has developed breast cancer after a median of 13.4 years of follow-up. Using published data for the likelihood of breast cancer in BRCA1 and BRCA2 mutation carriers, we would have expected six to nine breast cancers to develop in this group of women over the period of follow-up. Our results suggest that the risk of breast cancer is reduced by 89.5%100.0% in BRCA1 or BRCA2 mutation carriers after prophylactic mastectomy. Of note, Meijers-Heijboer et al. (15) recently reported on 76 BRCA1 or BRCA2 mutation carriers who had bilateral prophylactic mastectomy and 63 BRCA1 or BRCA2 mutation carriers who opted for regular surveillance. The actuarial mean 5-year incidence of breast cancer was 17% in the surveillance group. No breast cancers have occurred in the prophylactic mastectomy group after a mean follow-up of 2.9 years (15).
A major strength of our study is the long period of follow-up, i.e., 13.4 years (range, 5.828.5 years), for the 26 BRCA1 and BRCA2 mutation carriers. Moreover, these carriers were identified within a well-defined cohort who had surgery at a single institution, the Mayo Clinic, which used a standardized surgical approach. Although we did not have a blood sample for 38 women in the high-risk cohort of 214, our complete follow-up revealed that only one of those 38 women subsequently developed breast cancer. In our analyses of the efficacy of prophylactic mastectomy, we treated that woman as both a BRCA1 or BRCA2 mutation carrier and as a noncarrier, and we found that her BRCA1 or BRCA2 mutation status had little impact on the results. If even more of those 38 women had proven to be BRCA1 or BRCA2 mutation carriers, the expected number of breast cancers in our analysis would have been greater, improving the calculations of the procedure's efficacy.
We used both a high-risk model (model by Easton et al.) and a moderate-risk model (model by Struewing et al.) to analyze the efficacy of prophylactic mastectomy in the prevention of breast cancer for the women in our cohort. The data for the model by Easton et al. were derived from families with multiple women who had been diagnosed with early-onset breast cancer, similar to the women with deleterious BRCA1 and BRCA2 mutations in our study (Table 1). Thus, we anticipate that the breast cancer risk for the women with the deleterious mutations would more closely approximate the predictions based on the data of Easton et al. than those based on the model by Struewing et al. Although the calculations from the moderate-risk model by Struewing et al. have a lower range of predicted risk, the efficacy of prophylactic mastectomy was estimated to be similar for both risk models.
We anticipated finding more BRCA1 or BRCA2 mutation carriers in our high-risk cohort than we did. There are several explanations for this finding. First, as described in the "Results" section, 14 probands did not inherit the BRCA1 or BRCA2 mutation (11 deleterious and three uncertain) detected in their family member. Because these probands did not have cancer at the time of prophylactic mastectomy, they had only a 50% probability of having inherited a predisposing mutation if one was segregating in their family. Second, mutations in BRCA1 and BRCA2 that are identifiable by conventional techniques account for only 30%40% of hereditary breast cancer overall. However, that proportion varies considerably, depending on the constellation of cancers within a family. If both breast and ovarian cancers are present, mutations in BRCA1 and BRCA2 account for closer to 80%85% of hereditary breast cancer (16). In those families with only breast cancer, approximately 12% harbor a BRCA1 or BRCA2 mutation (16,17). Third, current testing strategies fail to detect some types of BRCA1 and BRCA2 mutations, such as larger genomic rearrangements and mutations in noncoding regions that influence gene expression and RNA stability (18,19). As tests for these types of BRCA1 and BRCA2 mutations become available, we will continue to analyze this cohort for those types of mutations.
From this analysis, we conclude that prophylactic mastectomy is as effective in BRCA1 and BRCA2 mutation carriers as it is in women of unknown BRCA1 or BRCA2 mutation status who are nonetheless considered to be at high risk for breast cancer on the basis of family history of breast cancer. Because these results are based on a median of 13.4 years of follow-up, it will be important to continue to observe this cohort of high-risk women for late occurrences of breast cancer.
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NOTES |
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Supported in part by grant DAMD 17-94-J-4216 from the Department of Defense and by Public Health Service grant R01CA80181 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We are indebted to the participants in the study for their help in addressing an important topic; to Dr. Susan Nayfield at the National Cancer Institute, Bethesda, MD, for suggestions regarding the study design; to Ms. Ann Harris and members of the Survey Research Center of the Mayo Clinic, Rochester, MN, for follow-up of the patients; to Ms. Tammy Greenwood of the Mayo Clinic for mutation analysis; to Mr. Randy Vrabel, Mr. John Hermans, and Mr. Jeff Slezak of the Mayo Clinic for data analysis; and to Ms. Bonny Reinmuth of the Mayo Clinic for assistance with the preparation of the manuscript.
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REFERENCES |
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Meijers-Heijboer H, van Geel B, van Putten WL, Henzen-Logmans SC, Seynaeve C, Menke-Pluymers MB, et al. Breast cancer after prophylactic bilateral mastectomy in women with a BRCA1 or BRCA2 mutation. N Engl J Med 2001;345:15964.
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Manuscript received March 5, 2001; revised August 29, 2001; accepted September 10, 2001.
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