King M, Wieand S, Hale K, Lee M, Walsh T, Owens K, Tait J, Ford L, Dunn BK, Costantino J, Wickerham L, Wolmark N, Fisher B. Tamoxifen and Breast Cancer Incidence Among Women With Inherited Mutations in BRCA1 and BRCA2National Surgical Adjuvant Breast and Bowel Project (NSABP-P1) Breast Cancer Prevention Trial. JAMA. 2001;286(18):2251–2256. doi:10.1001/jama.286.18.2251
Author Affiliations: Departments and Medicine and Genomic Sciences (Drs King, Lee, Walsh, and Owens and Ms Hale) and Laboratory Medicine (Dr Tait), University of Washington, Seattle; and National Surgical Adjuvant Breast and Bowel Project, University of Pittsburgh, Pittsburgh, Pa (Drs Wieand, Costantino, Wickerham, Wolmark, and Fisher); and National Cancer Institute, Bethesda, Md (Drs Ford and Dunn).
Context Among cancer-free women aged 35 years or older, tamoxifen reduced the
incidence of estrogen receptor (ER)–positive but not ER-negative breast
cancer. The effect of tamoxifen on breast cancer incidence among women at
extremely high risk due to
inheritedBRCA1 or BRCA2mutations is unknown.
Objective To evaluate the effect of tamoxifen on incidence of breast cancer among
cancer-free women with
inherited BRCA1 or BRCA2 mutations.
Design, Setting, and Participants Genomic analysis of BRCA1 and BRCA2
for 288 women who developed breast cancer after entry into the
randomized, double-blind Breast Cancer Prevention Trial of the National Surgical
Adjuvant Breast and Bowel Project (between April 1, 1992, and September 30,
Main Outcome Measure Among women with BRCA1 or BRCA2 mutations, incidence of breast cancer among those who were receiving
tamoxifen vs incidence of breast cancer among those receiving placebo.
Results Of the 288 breast cancer cases, 19 (6.6%) inherited disease-predisposing BRCA1 or BRCA2 mutations. Of 8
patients with BRCA1 mutations, 5 received tamoxifen
and 3 received placebo (risk ratio, 1.67; 95% confidence interval, 0.32-10.70).
Of 11 patients with BRCA2 mutations, 3 received tamoxifen
and 8 received placebo (risk ratio, 0.38; 95% confidence interval, 0.06-1.56).
From 10 studies, including this one, 83% of BRCA1
breast tumors were ER-negative, whereas 76% of BRCA2
breast tumors were ER-positive.
Conclusion Tamoxifen reduced breast cancer incidence among healthy BRCA2 carriers by 62%, similar to the reduction in incidence of ER-positive
breast cancer among all women in the Breast Cancer Prevention Trial. In contrast,
tamoxifen use beginning at age 35 years or older did not reduce breast cancer
incidence among healthy women with inherited BRCA1
mutations. Whether tamoxifen use at a younger age would reduce breast cancer
incidence among healthy women with BRCA1 mutations
The randomized, double-blind, Breast Cancer Prevention Trial (BCPT)
was conducted between 1992 and 1998 by the National Surgical Adjuvant Breast
and Bowel Project (NSABP). The purpose of the BCPT was to determine whether
tamoxifen use by cancer-free, high-risk women significantly altered incidence
of invasive breast cancer.1 The result of the
BCPT, which involved 13 388 women, was that the incidence of invasive
breast cancer among those randomized to tamoxifen compared with those randomized
to placebo (risk ratio [RR]) was 0.51.
The reduction in breast cancer due to tamoxifen revealed by the BCPT
trial was consistent for women with no family history of breast cancer (RR,
0.46) and for
women with mothers, sisters, and/or daughters with breast cancer
(RR, 0.53). This observation led to the suggestion that tamoxifen might be
of use in reducing breast cancer risk among women with
in the breast cancer predisposing
genesBRCA1 or BRCA2.2- 5
Because the risk of breast cancer is much higher among women with BRCA1 or BRCA2 mutations compared with women
overall, an RR of 0.51 among mutation
carriers would convey a substantial
However, the estrogen-receptor (ER) profiles of BRCA1 and BRCA2 breast tumors introduced a complexity
into these considerations. In the breast, tamoxifen is an antiestrogen that
targets the ER. Therefore, precancerous changes in the breast that have lost
the cytoplasmic receptor (ie, tissues that are ER-negative) are not affected
by tamoxifen. In the BCPT, tamoxifen reduced the incidence of ER-positive
tumors, but had no effect on the incidence of ER-negative tumors. Tumors of
women with mutations in BRCA1 and BRCA2 differ in ER status and other biological features, in that BRCA1 tumors lack estrogen and progesterone receptors and
overexpress P53 more frequently, and also exhibit higher nuclear grade and
proliferative rates than do BRCA2 tumors or breast
tumors generally.6- 10
These differences raised the possibility that tamoxifen might be effective
in reducing breast cancer risk among women with BRCA2
mutations, but not among women with BRCA1 mutations.
To address these questions, we evaluated the effect of tamoxifen among
participants in the NSABP/BCPT with inherited BRCA1
or BRCA2 mutations. Women entered the BCPT trial
without knowledge of their inherited BRCA1 or BRCA2genotype since the genes had not been
cloned when the trial began. Participants provided peripheral blood samples at entry to
the BCPT trial, from which constitutional
could now be extracted and BRCA1 and BRCA2 sequenced. This
report presents the genetic analysis of BRCA1 and BRCA2 for the participants who developed invasive breast
cancer during the BCPT trial. Based on these invasive breast cancer cases,
we estimate RRs for breast cancer among women with BRCA1 or BRCA2 mutations associated with use of
tamoxifen vs placebo.
The complete eligibility requirements for participants in the BCPT trial
have been described previously.1 Briefly, eligibility
required that participants be 35 years or older with a 1.66% or greater risk
of breast cancer over the next 5 years, estimated by the model by Gail et
al11; have had lobular carcinoma in situ; or
be 60 years or older.
For our current analysis, cases were defined as all incident invasive
breast cancers occurring before the assigned treatment of BCPT participants
was revealed on April 1, 1998, and all cases reported to the NSABP headquarters
between April 1, 1998, and September 30, 1999. Because BCPT participants who
were randomized to placebo were offered the opportunity to use tamoxifen if
they chose to do so after April 1, 1998, this choice was recorded for each
case diagnosed after that date. By September 30, 1999, follow-up was available
for 13 195 participants. Complete 3-year follow-up was available for
80% of participants, complete 5-year follow-up was available for 65% of participants.
Median follow-up was 5.7 years.
When this study was initiated, the NSABP Biostatistical Center created
a data file that contained key clinical outcome and demographic information
for all participants who developed breast cancer. At that time, the NSABP
identification number of each patient was provided to the Northwest Lipid
Research Laboratory (Seattle, Wash) where buffy coat samples were stored,
and to the Molecular Diagnostics Laboratory (University of Washington, Seattle).
Buffy coats for each identified patient were provided to the Molecular Diagnostics
Laboratory, where DNA was extracted. Three aliquots (15 µg each) were
prepared and labeled with the NSABP identifier. Remaining DNA was returned
to the Northwest Lipid Research Laboratory. While the DNA was being extracted,
clinical data were provided to an independent statistician, who matched the
NSABP identification number with another identification number. Only the independent
statistician had access to the matched numbers. After the DNA was extracted,
the independent statistician took both identification numbers to the Molecular
Diagnostics Laboratory and replaced the sample labels containing NSABP identification
numbers with new labels. Immediately thereafter, the data set was returned
to NSABP, and the link between the 2 sets of identification numbers was destroyed.
Subsequently, 2 aliquots of DNA for each subject were provided to the King
Laboratory (University of Washington, Seattle) for genomic analysis, and the
third aliquot was held in the Molecular Diagnostics Laboratory. Thus, once
mutation analysis began, there was only 1 identification number for both the
DNA and the clinical data, and there was no longer any link between those
data and the original patient identification number.
Blood samples were obtained from BCPT participants at entry to the initial
study. At that time, buffy coats were isolated from the 10 mL of whole blood
and frozen at −70°. For the BRCA1/BRCA2 sequencing study, DNA was extracted using Purgene
DNA isolation kits (Gentra Systems, Minneapolis, Minn) and resuspended in
10 mmol buffer to a final concentration level of 20 µg/mL. DNA quality
was assessed by spectrophotometry and gel electrophoresis to monitor purity
and molecular weight.
DNA was gridded in 96-well format using a Hydra robot (Robbins Scientific,
Sunnyvale, Calif). DNA samples were amplified by
polymerase chain reaction (PCR)
using 78 pairs of M13-tagged PCR
primers. Each pair of primers was designed
to amplify an
exon and flanking
intronic sequence encompassing splice sites.
Larger exons were amplified in overlapping amplicons. When possible, primers
were chosen to avoid Alu sequences and regions that produced compressions
or slippage during cycle sequencing. Optimal PCR conditions allowing maximal
levels of product were determined empirically. A complete list of primers
and PCR conditions are available on request.
Sephacryl-purified PCR products were cycle-sequenced using BigDye (Applied
Biosystems, Foster City, Calif) primer sequencing chemistry following the
manufacturer's specifications and analyzed on ABI 377 DNA Sequencer (Applied
Biosystems). Amplicons of each sample were sequenced in both forward and reverse
directions. For each DNA sample, 24 133
nucleotidebase pairs of BRCA1 and BRCA2 were evaluated.
These included the 5589–base pair coding sequence, and 2752–base
pair flanking intronic sequences and regulatory regions of BRCA1, the 10 254–base pair coding sequence, and 5538–base
pair intronic and regulatory sequences of BRCA2.
All cases were fully screened for both genes.
Sequences from study samples were compared with consensus genomic sequences
of BRCA112 at GenBank
L78833 and of BRCA2 at GenBank Z74739 and GenBank
Z73359.13 Base pairs were identified with the
Phred and PolyPhred programs14- 16
and aligned using the Phrap program.17 We used
a modified version of the SeqHelp program18
in conjunction with Consed19 to display multiple
aligned forward and reverse sequences and to highlight variants noted by PolyPhred
analysis. Variants were confirmed first by sequencing the same exon again
from the original DNA sample, then by separating wild-type and mutant alleles
electrophoresis, cutting each band from the gel, and sequencing each
To assess the sensitivity and accuracy of the sequencing effort, 26
DNA samples with known BRCA1 or BRCA2 mutations were included in the study. These control samples were
obtained from laboratories not affiliated with this project. Control samples
were assigned identification numbers at the same time as samples from participants,
so that the genetic analysis group were blind to the identities and genotypes
of the controls. After samples were evaluated, the identification numbers
of the control samples were revealed. The control samples included 10 different
single nucleotide substitutions, leading variously to
or splice mutations, and 17 insertions or deletions of between 1 and 11 base
pairs. (One control carried 2 different mutations.) All control mutations
were successfully identified.
For the study participants, all mutations definitely predisposing to
breast cancer were included in this analysis. These were defined as protein-terminating
mutations anywhere in BRCA1 and in exons 2 through
26 of BRCA2, and missense mutations in the canonical
cysteine residues of the BRCA1 ring finger. Protein-terminating
mutations were defined as insertions or deletions leading to frameshifts,
nonsense mutations, and mutations in splice sites known to lead to frameshifts.20 Protein-terminating mutations in BRCA2 exon 2721 and missense mutations
and potential splice variants of uncertain significance in either gene were
identified but were not included in this analysis.
Randomization ensured that a woman with a BRCA1
or BRCA2 mutation was equally likely to receive tamoxifen
or placebo. Hence, if tamoxifen had no protective (or harmful) effect on cancer
incidence in women with a BRCA1 or BRCA2 mutation, then a woman with a mutation who developed cancer was
equally likely to have received tamoxifen or placebo. To test the hypothesis
that tamoxifen did not alter the incidence of breast cancer, it was only necessary
to find the mutation status of women who developed cancer and test the null
hypothesis that the proportion of these women who received tamoxifen (T) was consistent with the hypothesis (pT = 0.50). If the
observed proportion pT was less than 0.50, this was evidence that
tamoxifen was beneficial in these women. For example, under the null hypothesis,
the probability that 3 or fewer of the 11 women with cancer and BRCA2 mutations received tamoxifen could be expressed as the probability
of observing T of 3/11 or less when pT is equal
to 0.50, corresponding to a 1-sided P value of .11.
It would have been possible to condition on the proportion of women with follow-up
who received tamoxifen (6591/13 195) or on the proportion of person-years
of follow-up for women who received tamoxifen (32 959/65 860). However,
these numbers are so close to a pT of 0.50 that the 3 approaches
lead to identical results, as one would expect in such a large randomized
Similar reasoning applies to our presentation of relative risks. Denoting
the number of mutation carriers who received tamoxifen by MT and
the number of mutation carriers who received placebo by MP, we
assumed that MT/ MP is equal to 1 and that the number
of cases with a mutation was small relative to the number of participants
without a mutation. In that case, the relative risk is approximately equal
to T divided by (1 −T) and the 95%
confidence interval (CI) for the relative risk is (pL/[1 −
pL] to pU/[1 − pU]), in which pL to pU is the 95% CI for pT when T is observed. All CIs were computed using exact distributions.
As of September 30, 1999, 320 participants in the BCPT had developed
invasive breast cancer. For 13 participants, DNA was not available because
the participant either withdrew consent for additional involvement in the
BCPT after developing cancer or chose not to have her sample included in this
genetics study. For 19 participants, adequate DNA could not be obtained from
the stored buffy coat samples. Hence 90% (288/320) of cases are included in
Of the 288 breast cancer cases screened for BRCA1 and BRCA2, 19 (6.6%) carried inherited, disease-predisposing
mutations (Table 1). Sixteen different
definite cancer-associated mutations are distributed throughout the BRCA1 and BRCA2 sequences. Carrier
status of BRCA1 and BRCA2
was associated with a family history of breast cancer, especially if 2 or
more first-degree relatives were affected (Table 2). Also as expected, the proportion of patients with mutations
was higher for those diagnosed when they were younger than age 50 years compared
with those diagnosed at age 50 years or older (17% vs 3%, respectively). Mutation
frequencies among BCPT cases were similar to those of white breast cancer
patients in the population-based Carolina Breast Cancer Study (6.9%),22 higher than those detected in 2 population-based
series of young-onset patients,23,24
and slightly lower than those of incident series of Ashkenazi Jewish patients
Frequencies of invasive breast cancer among women with BRCA1 and BRCA2 mutations in the tamoxifen
and placebo groups are indicated in Table
3. Of 8 women with BRCA1 mutations who
developed breast cancer, 5 were in the tamoxifen group and 3 were in the placebo
group (RR, 1.67; 95% CI, 0.32-10.70). Of 11 women with BRCA2 mutations who developed breast cancer, 3 were in the tamoxifen
group and 8 in the placebo group (RR, 0.38; 95% CI, 0.06-1.56). Of the remaining
269 cases without BRCA1 or BRCA2 mutations, 87 were in the tamoxifen group and 182 in the placebo group
(RR, 0.48; 95% CI, 0.37-0.61). These analyses include participants of all
ancestries. Seven black participants developed invasive breast cancer during
the BCPT. Six of these participants had DNA available for sequencing. One
of these cases, who was in the tamoxifen group, carried a BRCA2 mutation.
Estrogen-receptor status of breast tumors of women with BRCA1 or BRCA2 mutations is indicated in Table 4. Among participants with BRCA1 mutations, 1 developed ER-positive breast cancer
and 6 developed ER-negative breast cancer. In contrast, among participants
with BRCA2 mutations, 6 developed ER-positive breast
cancer and 3 developed ER-negative breast cancer. Tumors of women with inherited BRCA1 mutations are more frequently ER-negative than are BRCA2 tumors or breast tumors generally, both in the BCPT
and in the population as a whole. When data were combined from several series
of breast cancer patients of known BRCA1 and BRCA2 genotype, 17% of BRCA1 tumors
were ER-positive vs 76% of BRCA2 tumors (Table 5).6,25,27- 33
Generally, the proportion of ER-positive tumors is lower among women who are
diagnosed at a younger age.34 Women with BRCA1 mutations tend to be diagnosed at a younger age.34 However, the association of ER status with age is
not sufficient to explain the low frequency of ER-positive tumors among women
with BRCA1 mutations.
Women with inherited BRCA1 and BRCA2 mutations are at increased risk for ovarian cancer.35,36
Tamoxifen use was not associated with any change in ovarian cancer incidence
in the BCPT as a whole. Among all women (regardless of genotype) who developed
breast cancer during the BCPT, 1 participant also developed ovarian cancer.
This participant carried a BRCA2 mutation and was
in the placebo group.
Healthy women with inherited cancer-predisposing BRCA1 or BRCA2 mutations face high risks of
breast and ovarian cancer. Prophylactic mastectomy significantly reduces breast
cancer risk among these women. Over 3 years of follow-up, invasive breast
cancer occurred in 8 of 63 women with inherited BRCA1
or BRCA2 mutations, who had opted for surveillance
alone, but in none of 76 women who underwent prophylactic mastectomy.37 Other data indicates that early prophylactic oophorectomy
reduces the risk of subsequent breast cancer among BRCA1 mutation carriers by approximately 50%.38
The critical question for our study was whether chemoprevention, specifically
prophylactic use of tamoxifen, would also reduce incidence of invasive breast
cancer among cancer-free women with inherited BRCA1
or BRCA2 mutations. Given the sample size of the
BCPT, inference from the genetic data alone cannot fully answer this question.
The observed risk ratio for BRCA2 of 0.38 would be
statistically significant if based on 24 rather than 11 cases, requiring a
trial of approximately twice the size. The consistency of genetic and biological
evidence favors tamoxifen for cancer-free women with BRCA2 mutations, but not for cancer-free women with BRCA1 mutations.
For women with BRCA1 mutations, an important
question remains unanswered. It is possible that early in the course of BRCA1 tumors, tamoxifen might still have a role to play.
If tamoxifen and oophorectomy are nearly equivalent, as they are in breast
cancer treatment, and if oophorectomy is performed before age 35 years and
is effective in reducing breast cancer incidence among women with BRCA1 mutations,38 then tamoxifen might
be effective in younger, cancer-free women with BRCA1
mutations. This question could best be addressed by a prospective randomized
trial involving a sufficient number of such women.
Finally, it is important to bear in mind that this study addressed the
efficacy of tamoxifen in reducing incidence of breast cancer among healthy
women with BRCA1 or BRCA2
mutations. The BCPT, and thus this genetics study, did not address treatment
with tamoxifen of existing breast cancer. For women with ER-positive breast
cancer, including ER-positive tumors of women with BRCA1 or BRCA2 mutations, tamoxifen has been shown
to be effective in reducing risk of contralateral breast cancer and recurrence
of disease.39- 41
In other words, it is well established that for ER-positive breast cancers,
tamoxifen is an effective treatment regardless of the patient's genotype.
On the other hand, this genetic analysis of the BCPT reveals that for women
who have not yet developed breast cancer, genotype at BRCA1 and BRCA2 has a major impact on the expected
effect of tamoxifen in reducing incidence of primary breast cancer.