Methods and kits for detecting breast cancer or a high risk of
breast cancer by measuring bFGF in nipple fluid from subjects compare
the levels of bFGF in samples from test subjects with the levels
of bFGF in subjects not having breast cancer, where increased levels
of bFGF in test subjects indicate the presence of breast cancer,
or a high risk of breast cancer, in the test subjects.
What is claimed is:
1. A method for diagnosing breast cancer, or a high risk of breast
cancer, in a subject, comprising measuring basic fibroblast growth
factor (bFGF) in a test sample of nipple fluid obtained from the
subject, and comparing the level of bFGF in the test sample with
samples from subjects not having breast cancer, an increase in the
level of bFGF in the test sample, as compared to samples from subjects
not having breast cancer, indicating breast cancer or the high risk
of breast cancer in the test subject.
2. The method of claim 1, wherein bFGF in the sample is measured
using anti-bFGF antibody.
3. The method of claim 1, wherein the amount of bFGF in the sample
of nipple fluid obtained from subjects not having breast cancer
is less than 200 pg/ml.
4. The method of claim 1, further comprising the step of detecting
at least one additional cancer marker.
5. The method of claim 4, wherein said additional cancer marker
is an angiogenic factor.
6. The method of claim 1, further comprising the step of administering
a substance to enhance the flow of nipple fluid from the subject.
7. The method of claim 6, wherein the substance is oxytocin.
8. A method for determining the progress of breast cancer, or of
treatment of breast cancer in a subject, comprising measuring the
level of bFGF in test samples of nipple fluid from a subject taken
over successive time intervals, and comparing the level of bFGF
in the test samples between time intervals to determine whether
the level of bFGF has increased or decreased in said samples.
9. A diagnostic kit for detecting breast cancer or a high risk
of breast cancer in a subject comprising reagents to measure bFGF
in a sample of nipple fluid from a subject.
10. The diagnostic kit of claim 9, wherein said reagents include
an anti-bFGF antibody.
11. The diagnostic kit of claim 9, wherein said kit further comprises
an agent to inhibit degradation of bFGF in the sample.
 This application is based on a provisional application U.S.
Ser. No. 60/217,372, filed Jul. 11, 2000, the contents of which
are hereby incorporated by reference, in their entirety into this
 Throughout this application, various publications are referenced.
The disclosures of these publications in their entireties, are hereby
incorporated by reference into this application, in order to more
fully describe the state of the art, as known to those skilled therein,
as of the date of invention, described and claimed herein.
FIELD OF THE INVENTION
 The present invention relates to methods for diagnosing
the presence of breast cancer, and more particularly to the measurement
of basic fibroblast growth factor (bFGF) in nipple fluid, to detect
breast cancer in a subject.
BACKGROUND OF THE INVENTION
 Breast cancer is the most frequently diagnosed cancer in
American women. It is the leading cause of death in young women
under 50 years of age, and is the second most common cause of cancer
death among American women. The key to increasing survival is early
diagnosis. Early detection through screening mammography saves lives.
However, mammography misses as much as 20% of the breast cancer
in premenopausal women and 10% in older women. Breast biopsies resulting
from abnormal mammograms confirm cancer in only 10-20% of the cases.
 While intense effort has been invested in refining the resolution
and interpretation of mammography, little has been devoted to the
search for tumor markers that may assist in detecting minute amounts
of breast cancer. Current serum tumor markers for breast cancer
are only useful in cases of widespread disease. (Harris J R, Morrow
M, Norton L. Malignant tumors of the breast. In: DeVita V T, Hellman
S, Rosenberg S A, eds., In Cancer: principles and practice of oncology.
Philadelphia: Lippincott-Raven, (1997).
 Recently, there has been increased interest in the possible
use of angiogenic factors as tumor markers. In the past few decades,
researchers have become increasingly interested in the observation
that tumor growth and metastasis are accompanied by significant
new blood vessel formation, i.e. angiogenesis. Angiogenic factors
have been found associated with several solid tumors such as retinoblastomas
and osteosarcomas. Studies have shown that angiogenic factors can
be significantly elevated in the serum and urine of breast cancer
patients. The levels of certain angiogenic factors have been shown
to correlate with the disease stage of the tumor. (Nguyen M., Invest
New Drug (1997)).
 Approximately 15 angiogenic peptides have been identified
and sequenced, including basic fibroblast growth factor (bFGF).
(Folkman J, In The molecular basis of cancer, Mendelsohn et al.,
(eds), W B Saunders, pp 206-232 (1995)). These angiogenic molecules
are either released by the tumor cells themselves, or mobilized
from extracellular matrix and/or released by host cells, such as
macrophages recruited into the tumor.
 bFGF, one of the most potent angiogenic factors, has been
reported to be widely distributed among normal and neoplastic tissue
(Folman J, sura). bFGF is a member of a family of heparin binding
growth factors found in a variety of normal and neoplastic tissues.
A method for detecting and measuring bFGF using a sandwich immunoassay
method is described in U.S. Pat. No. 5,187,062 to Sato et al. A
sensitive assay for the detection of bFGF in bodily fluids was not
reported until 1991 (Watanabe et al., Biochem. Biophys. Res. Comm.
175:229-235, (1991)), with the first clinical use reported by Fujimoto
et al., Biochem. Biophys. Res. Comm. 180:386-392 (1991)). bFGF was
elevated in serum of patients with renal cell carcinoma, but was
not detected in the urine of these patients. Only 6% of 235 patients
with breast cancer had detectable bFGF (>39 pg/ml) using the
Watanabe bioassay (Watanabe et al., (Abstract) Molec. Biol. Cell
3S:234a (1992)). An elevated level of bFGF has been found in the
urine of patients with a variety of tumors including kidney, bladder,
prostate, testicular, breast, colon, lung, brain, ovarian, sarcoma
and lymphoma (Nguyen et al., J. Natl. Cancer Inst. 86:356-361 (1994)).
 Improvements in the ELISA used for detecting FGF have permitted
improved detection of bFGF in urine from subjects with bladder tumors
(O'Brien et al., Br. J. Urol. 76:311-314 (1995)), Wilms' tumors
(Lin et al., Clin. Cancer Res. 1:327-331 (1995)) and in serum of
patients suffering from cervical cancer (Sliutz et al., Cancer Lett.
94:227-231 (1995)). Takei et al. (Clin. Chem. 40:1980-1981 (1994))
measured serum bFGF in patients with breast cancer and found significant
elevations in all stages of disease.
 The level of bFGF (basic fibroblast growth factor) has been
shown to correlate with the disease stage of the tumor. (Nguyen
M, Watanabe H, Budson A, Richie J, Hayes D, Folkman J, J Natl Cancer
Inst., 86: 356-61 (1994)). However, thus far, use of bFGF in urine
or serum samples cannot be used as a screening tool, since there
is significant overlap in levels of bFGF between normal subjects
and cancer patients. (Nguyen et al., J. Natl. Cancer Inst., supra,
and Nguyen M., Invest. New Drug., 15: 29-37 (1997)).
 bFGF has also been detected in the cerebrospinal fluid (CSF)
of patients with brain tumors but not in controls; the level of
bFGF correlated with mitogenic activity in CSF in vitro and with
density of microvessels in histological sections (Li et al., Lancet
 Angiogenic factors may be useful as markers of therapeutic
efficacy and to assess an individual cancer patient's prognosis.
Previously elevated urine bFGF levels have been shown to decrease
into the normal range following complete surgical removal of tumors.
Patients with progressive disease had increased bFGF levels detected
after repeat urine samples. (Nguyen et al., J. Natl. Cancer Inst.
 Breast cancer arises from the epithelial cells that line
the ductal/lobular systems of the milk ducts suggesting that examination
of this ductal system or its secretions might reveal signs of early
cancer. Breast fluid contains immunoglobulins, proteins, lipids,
cholesterol, fatty acids, lactose and hormones including prolactin,
growth hormone-like protein, EGF and TGF.alpha., calcitonin and
insulin-like growth factor (IGF) (Rose, Cancer Det. Prev., 16:43-51
(1992); and Gann et al., Cancer Epidemiol., Biomarkers & Prev.,
6:421-8 (1997)). Breast fluid is typically prevented from escaping
from the nipple because the nipple ducts are occluded by constricting
bands of smooth muscle, viscous and dried secretions and keratinized
epithelium (Petrakis, Epidemiol. Rev. 15:188-195 (1993)).
 Patients' nipple fluid (nipple aspirate fluid or "NAF")
has not been extensively investigated as a possible source for breast
cancer diagnostic purposes. Factors associated with the success
of obtaining NAF include age, (subjects within the age range of
30 to 50 years), subjects having early onset of menarche, subjects
of non-Asian race, and subjects with prior lactation. (Wrensch et
al., Breast Cancer Res. Treatm. 15:39-51 (1990)).
 Prior studies have attempted to detect cancer cells in NAF,
but technical difficulties have included a paucity of cancer cells,
probably because the cancer obstructs the ducts, and difficulty
in distinguishing cancer cells from dyplastic cells. (Wrensch et
al., Am. J. Epidemiol., 137:829-33 (1993)). Previous studies with
nipple fluid CEA (carcinoembryonic antigen) and PSA (prostate specific
antigen) showed significant overlap between the study groups. (Foretova
L, Garber J E, Sadowsky N L, Verselis S J, Joseph D M, Andrade A
F F, Gudrais P G, Fairclough D, Li F P. Carcinoembryonic antigen
in breast nipple aspirate fluid. Cancer Epidemiol Biomark Prevent,
7: 195-8 (1998); and Sauter E R, Daly M, Linahan K, Ehya H, Engstrom
P F, Bonney G, Ross E A, Yu H, Diamandis E. Prostate-specific antigen
levels in nipple aspirate fluid correlate with breast cancer risk.
Cancer Epidemiol Biomark Prevent, 5: 967-70 (1996)).
 There remains a need for improved diagnostic methods for
breast cancer that are better able to distinguish between normal
subjects and those having breast cancer.
SUMMARY OF THE INVENTION
 Accordingly, the present invention provides improved methods
for diagnosing breast cancer or a high risk of breast cancer, by
measuring bFGF levels in nipple fluid obtained from human subjects.
The method employs a bioassay for quantitation of bFGF in nipple
fluid using, for example, anti-bFGF antibody, and comparing the
levels of bFGF in test subjects with levels in subjects not having
cancer, an increase in the level of bFGF in the test sample, as
compared to samples form subjects not having cancer, indicating
breast cancer, or high risk of breast cancer. The methods include
diagnostic kits for measuring bFGF levels in nipple fluid.
DETAILED DESCRIPTION OF THE INVENTION
 The present invention is based on the discovery of measurable
amounts of bFGF in breast fluid, and provides methods for detecting,
quantifying and comparing levels of bFGF in a sample of nipple fluid
from a subject. The subject may be a human or an animal.
 Concentrations of bFGF are determined in the sample of nipple
fluid from a test subject and are compared to concentrations of
bFGF present in samples taken from multiple human subjects known
to be free of breast cancer ("normals"). In the examples,
infra, levels of bFGF were increased in subjects having breast cancer
or at high risk of having breast cancer as compared to levels of
bFGF in samples taken from the breasts of normal subjects. Values
for levels of bFGF from samples from subjects diagnosed with breast
cancer, or at risk for breast cancer, did not overlap with levels
from normal subjects.
 Concentrations of bFGF may also be determined over successive
time intervals to determine the progress of breast cancer in a subject,
or to determine the efficacy of therapeutic intervention. In addition,
the method of the invention can include the detection of other cancer
markers, such as other angiogenic factors including, but not limited
to, acidic FGF (aFGF), vascular endothelial growth vector (VEGF),
epidermal growth factor (EGF), transforming growth factor-alpha
and beta (TGF-.alpha. and TGF-.beta.), hepatocyte growth factor
(HGF), tumor necrosis factor-alpha (TNF-.alpha.), interleukin-8
(IL-8), granulocyte colony stimulating factor (G-CSF), E-selectin,
angiogenin, platelet-derived endothelial cell growth factor (PD-ECGF),
placental growth factor and pleiotrophin, angiogenic inhibitors
such as thrombospondin, TIMP, angiostatin, endostatin, platelet
factor 4, maspin. In addition, other cancer markers may be detected,
including, but not limited to CA125, Tac, soluble IL2 receptor alpha,
mCSF, OVX1, CEA, PSA, CA15-3 and CA19.9.
 Preferably, levels of bFGF are measured in a subject prior
to significant trauma such as surgery or chemotherapy, because of
the release of growth factors following such events, and the possibility
of duct blockage from scarring, hematomas or seromas. Also, due
to the elevation of bFGF in breast milk, sample results may be best
in subjects that have ceased lactating for at least 1 year.
 It is preferred to obtain sufficient nipple fluid, for example
from 10 .mu.l to 1 ml. to promote sampling from all regions of the
breast to enhance detection of cancer. In addition, since bFGF is
a labile peptide, it is preferred to assay as soon as possible after
sampling, and without repeated freezing, and thawing of the sample.
 Since nipple fluid is typically not spontaneously discharged,
samples are preferably obtained in a manner that optimizes the yield
of fluid, yet preserves the comfort of the human subject. For example,
a warm compress may be applied to the breast of the subject in an
upright (seated) position. To encourage fluid removal, keratin plugs
that may be present blocking the ducts in the nipple may be removed,
for example using dilute (5 to 15%) salicylic acid. Alternatively,
or in addition, drugs such as oxytocin, salagen or prolactin may
be given to encourage fluid flow. For example, nasal oxytocin can
be used to relax the constricting bands of smooth muscle in the
breast to enhance flow of nipple fluid for sampling. Nasal oxytocin
has been approved by the FDA since 1982 and has been used safely
in lactating as well as in non-lactating women (Cheales-Siebenaler,
J. Hum. Lact. 15:41-43 (1999); Renfrew et al., Cochrane Database
Syst. Rev. 2:CD000156, (2000)). If necessary, sufficient breast
fluid from a subject for assay may be obtained using assistive means
such as a ductoscope or microcatheter.
 Concentrations of bFGF in nipple fluid may be measured using
various known techniques. In the example, infra, an ELISA assay
(R & D Systems Inc., Minneapolis, Minn.) using anti-bFGF antibodies
is used. However, other assay methods employing reagents that can
bind to and/or detect bFGF may be used, including radioimmunoassays
or chemiluminescent assays.
 A model has been developed for predicting breast cancer
risk (Gail et al., J. Natl. Cancer Inst. 81:1879-1886 (1989). A
woman's risk factors are translated into an overall risk score by
multiplying her relative risks from several categories (age at menarche,
number of breast biopsies, family history and age at first live
birth). This risk score is then multiplied by an adjusted population
risk of breast cancer to determine the individual risk of breast
 The methods of the invention may be performed using pre-packaged
diagnostic kits. Such kits include reagents, such as monoclonal
antibodies, for assaying bFGF levels in nipple fluid samples, and
reagents for detecting the binding of antibodies to bFGF present
in the sample, as well as agents such as sucralfate to inhibit degradation
of bFGF. The kits may also include an apparatus or container, such
as a microplate or dip stick, for conducting the methods of the
invention, as well as suitable instructions for carrying out the
methods of the invention.
 The following examples are presented to demonstrate the
methods of the present invention and to assist one of ordinary skill
in using the same. The examples are not intended in any way to otherwise
limit the scope of the disclosure of the protection granted by Letters
Patent granted hereon.
 Correlation of bFGF Levels with Breast Cancer
 bFGF was measured using an ELISA assay (R&D Systems
Inc., Minneapolis, Minn.) in the nipple fluid from ten (10) control
breasts of human subjects (not identified as having breast cancer,
"normals"), four (4) lactating breasts, and ten (10) stage
1 or 2 breasts identified by surgery as having breast cancer.
 While the patient was awake, nipple fluid (1-2 drops) was
collected with a breast pump which is commonly used by women during
lactation. Both normal and cancer patients underwent biopsy. Up
to four attempts were typically made in order to elicit nipple fluid.
The nipple fluid was stored in a freezer (-20.degree. C.). The data
obtained from the analysis of nipple fluid was correlated with the
pathologic diagnosis obtained from the surgical specimens.
 The ELISA assay was performed as described by the manufacturer
for cell supemate, serum and plasma. Briefly, the assay uses E.
coli-expressed recombinant human bFGF and antibodies raised against
the recombinant factor. Anti-bFGF monoclonal antibody is pre-coated
onto a microplate. Standards and samples are pipetted into the wells
and any bFGF present is bound by the immobilized antibody. After
washing away unbound substances, an enzyme-linked anti-bFGF antibody
is added to the wells. Following a wash to remove unbound antibody-enzyme
reagent, a substrate solution is added to the wells and color develops
in proportion to the amount of bFGF bound in the initial step. The
color development is stopped and the intensity of the color (optical
density of each well) is measured within 30 minutes using a microplate
reader set to 450 nm.
 Duplicate readings for each standard, control and sample
were averaged, and the average zero standard optical density was
subtracted. The optical density for the standards versus the concentration
of standards was plotted and a best curve drawn. The bFGF concentration
of each test sample was determined by locating the absorbance value
on the y-axis of the plot and extending a horizontal line to the
standard curve. The corresponding bFGF concentration is read off
the x-axis. The minimum detectable level of bFGF using this assay
is typically less than 3 pg/ml. The results of the assay are shown
in (Table 1).
1TABLE 1 Nipple Fluid bFGF Concentrations (pg/ml) Normal Lactating
Cancer Breasts Breasts Breasts 190 1,670 7,470 nd*(9) 1,150 2,490
990 2,390 450 2,240 860 670 590 310 150 nd nd*: not detected
 Nipple fluid was obtained from the 24 breasts out of a total
attempt on 30 breasts (80% success rate). The detection limit was
100 pg/ml. Control nipple fluids had significantly lower levels
of bFGF in comparison to cancer nipple fluids (19.+-.19 pg/ml versus
1,717.+-.706 pg/ml, p=0.027, student's t-test). It is striking that
there was very little overlap in the values of bFGF measured in
these two groups. The one cancer patient with undetectable levels
of bFGF in the nipple fluid already had surgical resection of her
cancer at the time of this study. Lactating nipple fluids contained
a significant amount of bFGF (1,065.+-.251 pg/ml).
 Levels of another potent angiogenic factor VEGF (vascular
endothelial growth factor) were also measured in nipple fluids using
the same protocol as above for bFGF. There were no significant differences
between the three groups: 106,500.+-.19,000 pg/ml in control breasts,
92,400.+-.19,100 pg/ml in cancer breasts, and 46,100.+-.17,800 pg/ml
in lactating breasts (p is not significant). These results suggest
that VEGF in nipple fluids may not be useful in diagnosing breast
 In addition, the assay was performed on nipple fluid obtained
by spontaneous discharge (no pump used).
2TABLE 2 Nipple Fluid bFGF Concentrations (pg/ml) Normal High Risk*
Breasts Breasts 110 1510 150 503 93 1675 62 371 155 525 93 168 nd**
(13) High Risk* = no breast cancer diagnosed, but at high risk as
identified by family history or personal history of breast cancer
nd** = not detected
 These data demonstrate increased levels of bFGF in the nipple
fluid from subjects identified as having breast cancer, or at high
risk for breast cancer, as compared to levels of bFGF in normal
 Additional Correlation of bFGF Levels with Breast Cancer
in Human Subjects
 Additional human subjects were tested for bFGF levels, as
described above in Example I, supra. 42 patients had benign breast
lesions and 20 patients were diagnosed with breast cancer (Table
3TABLE 3 Nipple fluid bFGF (pg/ml) and patients' diagnoses BFGF
Value Benign Cases Malignant Cases Total Cases <100 36 (85.7%)
1 (5%) 37 100-200 4 (9.5%) 4 (20%) 8 201-300 0 1 (5%) 1 301-500
0 3 (15%) 3 501-800 2 (4.8%) 4 (20%) 6 801-1000 0 2 (10%) 2 >1000
0 5 (25%) 5 Total 42 (100%) 20 (100%) 62
 Table 4 shows the results if a level of 100 pg/ml of bFGF
is used as a cut-off for detection.
4TABLE 4 bFGF Benign Malignant Total Undetected (bFGF < 100)
36 (85.7%) 1 (5%) 37 Detected (bFGF .gtoreq. 100) 6 (14.3%) 19 (95%)
25 Total 42 (100%) 20 (100%) 62 (Chi-square test exact p-value <
 From these results, the estimated sensitivity of bFGF (the
probability of having positive bFGF values for breast cancer patients),
is 95% (1/20). The estimated specificity of bFGF (the probability
of having negative bFGF values for normal subjects) is 85.7% (36/42).
The true positive rate (the probability of having cancer for patients
with positive bFGF levels) is 76% (19/25). The true negative rate
(the probability of not having cancer for patients with negative
bFGF levels) is 97.3% (36/37).
 The above Examples demonstrate that measurement of bFGF
in nipple fluid has the potential of being a useful diagnostic tool
for breast cancer in human subjects.
 As will be apparent to those skilled in the art in which
the invention is addressed, the present invention may be embodied
in forms other than those specifically disclosed above without departing
from the spirit or potential characteristics of the invention. Particular
embodiments of the present invention described above are therefore
to be considered in all respects as illustrative and not restrictive.
The scope of the present invention is as set forth in the appended
claims and equivalents thereof rather than being limited to the
examples contained in the foregoing description.