This invention relates to a method of determining, in a subject
having active breast gross cystic disease, the risk of future breast
cancer development. The level of glycoprotein GCDFP-15 in the plasma
is measured at the time of cyst aspiration in order to provide an
indication as to the likelihood of future breast cancer development.
1. A method of determining the risk of breast cancer development
in a subject with active breast gross cystic disease having between
one and nine cysts, comprising determining the level of GCDFP-15
in the plasma of said subject at the time of aspirating said cysts,
an increased level of GCDFP-15 in the plasma indicating an increased
risk of breast cancer development in said subject.
2. The method of claim 1 wherein said increased level of GCDFP-15
is greater than 40 ng GCDFP-15 per ml plasma.
3. The method of claim 2 wherein said increased risk of breast
cancer development is 4.2 times greater than the risk of breast
cancer development in a normal subject not having active breast
gross cystic disease.
4. The method of claim 1 wherein said breast cancer is breast carcinoma.
5. The method of claim 1 wherein said subject has had no prior
6. A method of determining the risk of breast cancer development
in a subject with active breast gross cystic disease having ten
or more cysts, comprising determining the level of GCDFP-15 in the
plasma of said subject at the time of aspirating said cysts, an
increased level of GCDFP-15 indicating an increased risk of breast
cancer development in said subject.
7. The method of claim 6 wherein said increased level of GCDFP-15
is greater than 40 ng GCDFP-15 per ml plasma.
8. The method of claim 7 wherein said increased risk of breast
cancer development is 7.1 times greater than the risk of breast
cancer development in a normal subject not having active breast
gross cystic disease.
9. The method of claim 6 wherein said breast cancer is breast carcinoma.
10. The method of claim 6 wherein said subject has had no prior
FIELD OF THE INVENTION
This invention relates to a method of assessing the risk of breast
cancer development. More specifically, this invention relates to
a method of determining the risk of future breast cancer development
in a subject with active breast gross cystic disease. The level
of glycoprotein GCDFP-15 in the plasma of said subject is measured
in order to provide a prognosis for breast carcinoma development.
BACKGROUND OF THE INVENTION
Human breast gross cystic disease is a benign breast condition
common in adult middle-aged women (Haagensen, Diseases of the Breast,
3rd ed., WB Saunders Publ., pp. 250-266 (1986)). Gross cysts evolve
from microcystic apocrine metaplasia, which occurs in the terminal
ductal lobular units of the breast (Wellings et al., Hum. Path.,
18:381-386 (1987)). The cause of apocrine microcyst transformation
into macrocysts is unknown. However, there is evidence that such
transformation is a hormonally mediated event. Gross cystic disease
first appears in women in their 20's and increases in frequency
up to the 40-50 age range, and then essentially disappears as a
disease process after menopause (Haagensen, 1986, supra).
Breast gross cystic disease is confirmed by aspiration of cyst
fluid. It has been found that as a population, women who have gross
cystic disease have an increased risk of developing breast carcinoma
(see Jones et al., Brit. J. Surg., 67:669-671 (1980); Harrington
et al., Breast, 7:13-17 (1980); Ciatto et al., Eur. J. Cancer, 26:555-557
(1990); Bundred et al., Brit. J. Cancer, 64:953-955 (1991); and
Bodian et al., Cancer Det. Prev., 16:7-15 (1992)). The fluid contained
within breast gross cysts has been analyzed for a number of components,
and has been found to be a unique secretion. The major component
proteins have been identified (Haagensen et al., J. Natl. Cancer
Inst., 62:239-247 (1979)). One of these component proteins, termed
GCDFP-15 for a 15K dalton monomer sized glycoprotein, has been investigated
as a marker protein with regard to its circulating blood levels
in various clinical situations (see Haagensen et al., 1979, supra;
Haagensen et al., 1986, supra, pp. 474-500; and Haagensen et al.,
Ann. N.Y. Acad. Sci., 586:161-173 (1990)). GCDFP-15 blood levels
in "normal"post-menopausal women averages 17 ng/ml.
GCDFP-15 protein has been well characterized with regard to its
amino acid structure and the location of its gene on chromosome
7 (see Haagensen et al., 1986, supra; and Myal et al., Somatic and
Mol. Genetics, 15:256-270 (1989)). GCDFP-15 is a normal constituent
protein of all apocrine gland cells (normal and metaplastic), and
of some exocrine glands with apocrine features (serous cells of
mandibular salivary glands and the minor salivary glands in the
major bronchi) (see Haagensen et al., 1986, supra; Mazoujian et
al., Am. J. Pathol., 110:105-112 (1983)). The biological purpose
of apocrine cells secreting GCDFP-15 remains obscure. Recent data
has shown that breast cell lines exposed to GCDFP-15 in the culture
medium results in an enhancement in their growth rate (Cassoni et
al., Int. J. Cancer, 60:216-220 (1995)). The protein does have a
binding affinity for fibrinogen and is transported in blood bound
to fibrinogen (Haagensen et al., 1990, supra). However, no enzymatic
effect of GCDFP-15 on fibrinogen has been determined, thus far,
and its purpose of binding to fibrinogen has not been established.
The secretion of GCDFP-15 has been shown to be regulated by various
hormones both in vivo and in vitro (see Chalbos et al., Cancer Res.,
47:2787-2792 (1987); Simard et al., Mol. Endocrinology, 3:694-702
(1989); Dejardin et al., J. Mol. Endocrinology, 7:105-112 (1991);
and Haagensen et al., Br. Cancer Res. Treat., 23:77-86 (1992)).
Androgens markedly enhance the excretion of GCDFP-15 from both the
T47D and ZR75 breast cancer cell lines. The secretory effects of
androgens on GCDFP-15 have been shown to be due to increased mRNA
synthesis as a primary mechanism (Simard et al., 1989, supra). Differences
have been observed between the T47D cells and the ZR75 cells with
regard to growth effects of various steroids compared to secretion
effects on GCDFP-15. In the ZR75 cell line, a positive correlation
has been demonstrated between increased GCDFP-15 secretion and decreased
growth rates (Simard et al., 1989, supra). This has been shown in
both directions, with androgens slowing growth while increasing
GCDFP-15 secretion and estrogens enhancing growth while decreasing
In contrast, with the T47D cell line, androgens have been shown
to enhance GCDFP-15 secretion while not appreciably effecting the
growth rate (Haagensen et al., 1992, supra). Progestins slow T47D
growth rate, while enhancing GCDFP-15 secretion. However, RU486,
an antiprogestin, also slowed growth in the T47D cell line while
markedly inhibiting GCDFP-15 secretion (Haagensen et al., 1992,
supra). Thus, the interaction of various steroids on breast cancer
cell lines with regard to GCDFP-15 secretion is complex, but does
appear to have androgen-modulated enhancement as a central effect.
SUMMARY OF THE INVENTION
This invention is directed to a method of determining the risk
of breast cancer development in a subject with active breast gross
cystic disease wherein said subject has at least one aspirated cyst.
At the time of cyst aspiration, the level of GCDFP-15 in the plasma
is measured. An increased level of GCDFP-15 in the plasma indicates
an increased risk of breast cancer development.
A subject having between one and nine aspirated cysts, and having
a GCDFP-15 level of greater than 40 ng GCDFP-15 per ml of plasma
at the time of cyst aspiration, has a 4.2 times greater risk of
developing breast cancer than a normal subject not having active
breast gross cystic disease. A subject having ten or more aspirated
cysts, and having a GCDFP-15 level of greater than 40 ng GCDFP-15
per ml of plasma at the time of cyst aspiration, has a 7.1 times
greater risk of developing breast cancer than a normal subject not
having active breast gross cystic disease .
DETAILED DESCRIPTION OF THE INVENTION
135 patients with active breast gross cystic disease were studied
for GCDFP-15 production and breast cancer development. These patients
provided 7 ml samples of peripheral blood for measurement of GCDFP-15
at the time of cyst aspiration. None of these subjects had prior
breast cancer. Active gross cystic disease (GCD) was confirmed by
aspiration of breast cyst fluid.
Follow-up reports of breast carcinoma at later dates were confirmed
by obtaining copies of pathology reports. Follow-up time for each
patient was measured from the date of first blood sample obtained
at the time of active GCD, to the date of diagnosis of breast cancer
(12 patients), death (2 patients), or last contact (121 patients),
whichever came first. The average follow-up time was 10.1 years.
This group of 135 patients contributed 214 blood samples at the
time of breast cyst aspiration. One hundred contributed a single
sample and 35 contributed from 2 to 7 samples, taken at the time
of aspiration for GCD.
Blood samples were collected in EDTA anticoagulated vacutainer
tubes and were processed to plasma, then frozen at 70.degree. C.
until analysis. The analysis of the plasma level of GCDFP-15 was
performed by a two stage RIA (see Haagensen et al., 1979, supra;
Haagensen et al., 1986, supra; and Haagensen et al., Proc. Am. Assoc.
Clin. Chem., 26:980 (1980)). Briefly, purified GCDFP-15 was radiolabeled
with .sup.125 I using Iodogen as described by Freker et al., Biochem.
Biophys. Res. Comm., 40:849-857 (1978)). Specific activity obtained
was approximately 20 uCi/.mu.g protein. For the two stage RIA, 100
.mu.l of a specific rabbit anti-GCDFP-15 antiserum (diluted at 1/5000
in 0.01 M sodium azide containing 1 mg/ml bovine serum albumin)
was added to 50 .mu.l of the test plasma sample in an assay tube
containing 800 .mu.l of assay buffer (0.01M sodium azide with 1
mg/ml BSA). The reaction was allowed to incubate at room temperature
overnight. The next morning, 100 .mu.l of a solution of the .sup.125
I GCDFP-15 diluted to contain approximately 4 ng of the labeled
protein was added. The assay tubes were gently vortexed, and then
incubated for four hours at room temperature. The assay was terminated
by the addition of 500 .mu.l of a 1% solution of goat-anti-rabbit
antibody attached to a solid support matrix of Kynar (see Haagensen
et al., 1980, supra; and Newman et al., Clin. Chem., 35:1743-1746
(1989)). The assay tubes were reacted for ten minutes and then centrifuged
at 2000 rpm for 15 minutes. The supernatants were decanted and the
pellets were counted in a Packard-Prias gamma counter.
An inhibition curve was developed from 0 to 5 ng of GCDFP-15, with
the 5 ng point measuring approximately 10% of added counts. This
standard curve measured from 0 to 100 ng/ml of GCDFP-15 for a 50
.mu.l plasma sample. Plasma samples with blood levels of GCDFP-15
above 100 ng/ml were diluted in horse plasma and then reassayed
in 50 .mu.l aliquots. All plasma samples were assayed in duplicate
with any duplicate sample having a variation of greater than .+-.2%
being reassayed. This assay has a sensitivity of .+-.5 ng/ml of
GCDFP-15 and an interassay CV of .+-.7%.
For data analysis, the plasma levels were grouped into three categories:
normal (less than 40 ng GCDFP-15/ml plasma), midly elevated (40-49
ng/ml), and markedly elevated (50 ng/ml or greater). These ranges
were chosen empirically on the basis of prior work on GCDFP-15 blood
levels in a large sample of normal women (see Haagensen et al.,
1986, supra; and Haagensen et al., 1990; supra. The total numbers
of breast gross cysts confirmed by aspiration for the study group
of 135 patients with GCD are shown in Table 1, below.
TABLE 1 ______________________________________ Breast Gross Cystic
Disease Confirmed by Aspiration of Cyst Fluid Number of Number of
Aspirated Cysts Patients in Study ______________________________________
1 13 (9.6%) 2-9 59 (43.7%) .gtoreq.10 63 (46.7%) 135 (100%) ______________________________________
The relationship between the total number of aspirated cysts and
GCDFP-15 blood level is shown in Table 2, below. In order to maintain
comparability among patients, patients are categorized according
to their initial value of GCDFP-15. There is a slight, but not statistically
significant, inverse association between number of aspirated cysts
and blood level of GCDFP-15. As can be seen in Table 2, among women
with only one aspirated cyst, 77% had GCDFP-15 plasma levels in
the normal range and 23% had elevated levels. GCDFP-15 plasma levels
were in the normal range in 59% of women with 10 or more aspirated
cysts, and were elevated in the remaining 41%.
TABLE 2 ______________________________________ Total Number of
Aspirated Breast Cysts Versus Plasma Level of GCDFP-15 in the First
Blood Sample Concurrent with Aspiration Percentage of Patients with
GCDFP-15 Plasma Levels Number of Number of in ng/ml Aspirated Cysts
Patients <40 40-49 .gtoreq.50 Total ______________________________________
1 13 77% 0 23% 100% 2-9 59 66% 14% 20% 100% .gtoreq.10 63 59% 12%
29% 100% Total 135 64% 12% 24% 100% ______________________________________
Among the study group of 135 women, 12 developed initial carcinoma
of the breast during the follow-up period. Ten of the cancers developed
in women who had contributed a single blood sample, and two in women
who had contributed serial (several) samples. The elapsed time between
entering this study and developing carcinoma ranged from less than
one year to 15 years.
The correlation between elevated plasma levels of GCDFP-15 and
increased risk of developing breast cancer is shown in Table 3,
below. As part of this analysis, the follow-up for the 35 patients
with serial blood samples was allocated to blood level groups according
to their highest last-known value. For example, one patient contributed
a blood sample at age 40, which had a GCDFP-15 plasma level of 46,
and another blood sample was contributed two years later, with a
plasma level of 77. At the time of last contact, there was no known
cancer. Two years of this patient's follow-up showed mildly elevated
blood levels, and 9 years showed markedly elevated blood levels.
Eight of the 35 patients who provided serial blood levels changed
categories in this way.
TABLE 3 __________________________________________________________________________
Relative Risk of Developing Breast Cancer As Determined by Highest
Prior Plasma Level of GCDFP-15 By Total Number of Aspirated Breast
Cysts Plasma Level Numb. Numb. Numb. Numb. of GCDFP-15 Pts Br.Ca.
Relative Risk Pts Br.Ca. Relative Risk __________________________________________________________________________
<40 ng/ml 49 2 1.8 37 2 2.0 40-49 ng/ml 9 1 6.5 11 2 7.1 4.2
7.1 .ltoreq.50 ng/ml 16 1 3.1 21 4 7.2 Study Group 74 4 2.5 69 8
4.4 Total __________________________________________________________________________
The risk of developing breast cancer is increased among women with
several aspirated cysts. Therefore, data concerning cancer risk
are shown separately according to whether or not the patient had
a history of 10 or more breast cyst aspirations. In considering
risk of breast cancer, it is also important to control for differences
among groups in follow-up time, ages and calendar years of exposure.
This was accomplished by calculating the relative risks of developing
breast cancer for each group, that is, the ratios of the number
of women in the group who developed breast cancer, to the number
that would have been expected of normal women according to incidence
rates from the Connecticut Tumor Registry for comparable ages and
calendar years of exposure.
Table 3 shows that regardless of the total number of aspirated
breast cysts, women with elevated levels of GCDFP-15 develop breast
cancer at substantially higher rates than do women with normal blood
levels of GCDFP-15. For women with fewer than 10 aspirated breast
cysts, the relative risk among those with elevated blood levels
of GCDFP-15 was 4.2, as compared to a relative risk of 1.8 for women
with normal GCDFP-15 range blood levels. The difference in relative
risks was more marked among women who had 10 or more aspirated breast
cysts: 7.1 for those with elevated blood levels of GCDFP-15, versus
2.0 for those with normal GCDFP-15 range blood levels.
The last row of Table 3 shows the overall relative risk of developing
breast cancer among women in the group of 135 women studied. For
those women with fewer than 10 aspirated breast cysts, the risk
is 2.5 times higher than for normal women. For those women with
10 or more aspirated breast cysts, the risk is 4.4 times higher
than for normal women. These results correlate with breast cancer
development risks determined by cyst aspiration studies performed
in a study of 1770 patients with GCD (see Bodian et al., Cancer
Det. Prev., 16:7-15 (1992)).
The data presented herein indicate that blood levels of GCDFP-15
in women with active breast cystic disease distinguish subgroups
with differing degrees of risk for developing breast carcinoma.
Women with active breast gross cystic disease, as demonstrated by
breast cyst aspiration, who also had an elevated GCDFP-15 blood
level above 40 ng/ml, were at an increased relative risk for breast
cancer development (Table 3). The degree of this increase in relative
risk related to the degree of active gross cystic disease clinically
in that it was higher in women who had developed 10 or more breast
cysts. In this subgroup the increase in relative risk was elevated
7.1 fold, and was similar in degree to the increase in relative
risk seen with the occurrence of atypical epithelial hyperplasia
(Dupont et al., New England J. Med., 312:146-151 (1985)), or with
lobular carcinoma in situ (Bodian et al., Epidemiological Reviews,
In Press (1995)). Since breast gross cystic disease is clinically
much more common than either atypical epithelial hyperplasia or
lobular carcinoma in situ (see Bodian et al., 1992, supra; and Bodian
et al., Cancer, 71:3896-3907 (1993)), the measurement of GCDFP-15
blood levels is an important evaluation step in breast cancer risk
It is likely that GCDFP-15 blood level reflects both a secretory
signal to apocrine metaplastic breast tissue and the amount of apocrine
tissue present which can respond to the secretory signal. Both of
these changes, jointly, appear to correlate with an increased susceptibility
for breast cancer development. The use of blood levels of GCDFP-15
to reflect risk for breast cancer development opens new pathways
into better understanding of this disease process and methods to
alter this risk.
Although the invention herein has been described with reference
to particular embodiments, it is to be understood that these embodiments
are merely illustrative of various aspects of the invention. Thus,
it is to be understood that numerous modifications may be made in
the illustrative embodiments and other arrangements may be devised
without departing from the spirit and scope of the invention.