Endogenous Estrogen, Androgen, and Progesterone
Concentrations and Breast Cancer Risk Among
Stacey A. Missmer, A. Heather Eliassen, Robert L. Barbieri, Susan E.
Background: Levels of endogenous hormones have been as-
sociated with the risk of breast cancer among postmeno-
pausal women. Little research, however, has investigated the
association between hormone levels and tumor receptor sta-
tus or invasive versus in situ tumor status. Nor has the
relation between breast cancer risk and postmenopausal
progesterone levels been investigated. We prospectively in-
vestigated these relations in a case-control study nested
within the Nurses’ Health Study. Methods: Blood samples
were prospectively collected during 1989 and 1990. Among
eligible postmenopausal women, 322 cases of breast cancer
(264 invasive, 41 in situ, 153 estrogen receptor [ER]-positive
and progesterone receptor [PR]-positive [ER?/PR?], and 39
ER-negative and PR-negative [ER–/PR–] disease) were re-
ported through June 30, 1998. For each case subject, two
control subjects (n ? 643) were matched on age and blood
collection (by month and time of day). Endogenous hormone
levels were measured in blood plasma. We used conditional
and unconditional logistic regression analyses to assess asso-
ciations and to control for established breast cancer risk
factors. Results: We observed a statistically significant direct
association between breast cancer risk and the level of both
estrogens and androgens, but we did not find any (by year)
statistically significant associations between this risk and the
level of progesterone or sex hormone binding globulin. When
we restricted the analysis to case subjects with ER?/PR?
tumors and compared the highest with the lowest fourths of
plasma hormone concentration, we observed an increased
risk of breast cancer associated with estradiol (relative risk
[RR] ? 3.3, 95% confidence interval [CI] ? 2.0 to 5.4),
testosterone (RR ? 2.0, 95% CI ? 1.2 to 3.4), androstenedi-
one (RR ? 2.5, 95% CI ? 1.4 to 4.3), and dehydroepiandro-
sterone sulfate (RR ? 2.3, 95% CI ? 1.3 to 4.1). In addition,
all hormones tended to be associated most strongly with in
situ disease. Conclusion: Circulating levels of sex steroid
hormones may be most strongly associated with risk of ER?/
PR?breast tumors. [J Natl Cancer Inst 2004;96:1856–65]
Epidemiologic data now provide strong evidence for an in-
fluence of plasma steroid hormones on the risk of breast cancer
in postmenopausal women (1)—a long proposed, but previously
poorly supported, hypothesis. The associations between the risk
of breast cancer and the level of estrogens and androgens (with
relative risks [RRs] for breast cancer ranging from 2.0 to 2.5
when comparing the top 20% with the bottom 20% of hormone
levels) are strong compared with those of most other breast
cancer risk factors. However, few studies have investigated
these associations as stratified by tumor receptor status or by
invasive versus in situ disease. In addition, studies of the effect of
postmenopausal hormone use suggest that formulations containing
estrogen and progestin are associated with a greater increase in
influence of endogenous progesterone levels remains unknown.
Affiliations of authors: Channing Laboratory, Department of Medicine,
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA
(SAM, AHE, SEH); Department of Epidemiology, Harvard School of Public
Health, Boston, MA (SAM, AHE, SEH); Department of Obstetrics, Gynecology,
and Reproductive Medicine, Brigham and Women’s Hospital and Harvard
Medical School, Boston, MA (SAM, RLB).
Correspondence to: Dr. Stacey Missmer, Channing Laboratory, Brigham and
Women’s Hospital, 181 Longwood Avenue, Boston, MA 02115 (e-mail:
See “Notes” following “References.”
Journal of the National Cancer Institute, Vol. 96, No. 24, © Oxford University
Press 2004, all rights reserved.
1856 ARTICLESJournal of the National Cancer Institute, Vol. 96, No. 24, December 15, 2004
by guest on February 19, 2013
Within the large, prospective Nurses’ Health Study cohort,
we previously investigated (6) the relation between endogenous
estrogens and androgens and breast cancer risk among post-
menopausal women (156 cases of breast cancer with follow-up
from 1990 through 1994) and found. To explore the association
between endogenous hormone levels and breast cancer risk in
greater detail than was previously possible, we conducted a
second nested case-control study that extends the follow-up
through 1998 and increases the total number of incident cases of
breast cancer to 322. We evaluated the associations between
endogenous hormone levels and breast cancer risk overall and
assessed whether the associations varied by stratification by
other breast cancer risk factors, by tumor receptor status, or by
invasive versus in situ disease.
MATERIALS AND METHODS
The Nurses’ Health Study cohort was established in 1976
when 121 700 female registered nurses, 30–55 years of age,
completed and returned a mailed questionnaire. The cohort
continues to be followed every 2 years by questionnaire to
update exposure status and to identify cases of newly diagnosed
disease. Data have been collected on most known breast cancer
risk factors including height, weight, age at menarche and meno-
pause, age at first birth, postmenopausal hormone use, and
family history of breast cancer.
During 1989 and 1990, blood samples were collected from
32 826 cohort members, who were 43–69 years of age at blood
collection and formed the blood cohort. Details regarding the
blood collection methods have been previously published (6,7).
Briefly, each woman arranged to have her blood drawn and then
shipped, via overnight courier and with an ice-pack, to our
laboratory, where it was processed and separated into plasma,
red blood cell, and white blood cell components. Samples have
been stored in continuously monitored liquid nitrogen freezers
since collection. As of 1998, the follow-up rate among the
women who provided blood samples was 99%.
Both case and control subjects in this analysis are women
who, at blood collection, were postmenopausal and had not used
postmenopausal hormones for at least 3 months. Of the blood
cohort, 11 169 women met these criteria; case and control sub-
jects were selected from this sub-cohort. We defined a post-
menopausal participant in this study as a woman who reported
having a natural menopause or a bilateral oophorectomy or as a
woman who reported having a hysterectomy with either one or
both ovaries remaining when she was 56 years old (if a non-
smoker) or 54 years old (if a current smoker), ages at which
natural menopause had occurred in 90% of these respective
Case subjects in this analysis are women with no reported
cancer diagnosis (other than non-melanoma skin cancer) before
blood collection and who were diagnosed with breast cancer
after blood collection but before June 1, 1998. Overall, 322 cases
of breast cancer (264 invasive, 41 in situ, 153 estrogen receptor
(ER)- and progesterone receptor (PR)-positive [ER?/PR?], and
39 ER-negative and PR-negative [ER–/PR–] disease) were re-
ported from among the 11 169 women eligible at baseline. All
cases of breast cancer were confirmed by and tumor details
(receptor status and invasive versus in situ tumors) were ob-
tained from a medical record review, with one exception. A
single nurse confirmed the diagnosis of breast cancer, but the
medical record was unavailable. Because of the high confirma-
tion rate upon medical record review (99%) in the Nurses’
Health Study, we kept this case subject in the analysis. However,
17 cases were not included in the invasive versus in situ case
sub-analyses because the pathology report was unclear as to
whether the tumor was invasive or because the information was
missing. Time from blood collection to diagnosis ranged from
less than 1 month to 106 months (median ? 52 months; 5th
percentile-95thpercentile ? 4–96 months). Two control subjects
(total n ? 643) were matched per case subject by age (year),
month of blood collection, time of day that blood was drawn
(?2 hours), and fasting status at the time of blood collection
(?10 hours since a meal versus ?10 hours or unknown).
Ninety-four percent of control matches were exact; the most
relaxed matches were within ?6 years of age, ?14 months of
blood collection from case subjects, and ?11 hours for time of
blood collection. The study was approved by the Committee on
the Use of Human Subjects in Research at the Brigham and
Analyses were conducted by three different laboratories. For
estrone, estradiol, androstenedione, testosterone, dehydroepi-
androsterone (DHEA), and dehydroepiandrosterone sulfate
(DHEAS), all batches were assayed at Quest Diagnostic’s
Nichols Institute (San Juan Capistrano, CA). For estrone sulfate,
the first batch was assayed at the University of Massachusetts
Medical Center’s Longcope Steroid Radioimmunoassay Lab-
oratory (Worcester); the remaining batches were assayed at
Nichols. The first two batches of sex hormone-binding globulin
(SHBG) were assayed at the Longcope Laboratory; the third and
fourth batches were assayed at Massachusetts General Hospi-
tal’s Reproductive Endocrinology Unit Laboratory (Boston). All
batches of progesterone were assayed at the same time at Quest
Hormone assay methods have been described previously in
detail (6). Endogenous hormone levels were measured in blood
plasma. In brief, samples were extracted with a mixture of
hexane and ethyl acetate (4:1, vol/vol) and applied to a celite
column, the steroids were eluted from the column (celite in
ethylene glycol), and the fractions were subjected to radioim-
munoassay (8–12). DHEAS was assayed by radioimmunoassay
without a prior separation step (13). To quantify estrone sulfate
levels, estrone was first extracted from the plasma, and then the
estrone sulfate bond was enzymatically cleaved to release es-
trone, which was then extracted from the plasma by an organic
solvent and was subjected to chromatography and then radioim-
munoassay (14). Free and percent free estradiol were calculated
by the law of mass action according to the method described by
Sodergard et al. (15).
All case–control–control triplet samples were assayed to-
gether; the samples were ordered randomly within a triplet and
labeled so that the laboratory could not identify the case–control
status. Although all members of a triplet were analyzed at the
same time, the triplets were analyzed in up to five different
batches (sent in 1992, 1993, 1996, 1998, and 2001). To assess
laboratory precision, replicates of 10% of all samples assayed
were randomly interspersed and labeled to preclude their iden-
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Support for this project was from NIH grants P01 CA-87969, CA-49449, P50
CA-089393, and DAMD-17–02–1–0692. Dr. Missmer was partially supported
by Training grant in cancer epidemiology 5 T32 CA090001–281 from the
National Cancer Institute.
We thank Victor Pontes, Helena Judge Ellis, and Todd Reid for their
expert assistance and Drs. Eric Winer and Myles Brown for their thoughtful
suggestions. In addition, Drs. Graham Colditz, Bernard Rosner, and Walter
Willett who contributed helpful critical comments. We also thank the
participants of the Nurses’ Health Study for their longstanding
Manuscript received May 10, 2004; revised September 28, 2004; accepted
October 21, 2004.
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