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, wherein an increase
in the level of bFGF in the test sample, as compared to samples
from subjects not having breast cancer, indicates breast cancer
or the high risk of breast cancer in the 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 oxytoxin.
8. The method of claim 1, wherein the step of measuring bFGF in
the test sample is performed by using a diagnostic kit comprising
reagents to measure bFGF in the test sample.
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
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 344:82-86
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. 86:356-361
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
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
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 cancer.
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
TABLE 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).
TABLE 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 subjects.
Additional Correlation of bFGF Levels with Breast Cancer in Human
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 3)
TABLE 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.
TABLE 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.