U.S. patent application number 10/622743 was filed with the patent office on 2004-01-29 for identifying material from a breast duct.
This patent application is currently assigned to Cytyc Health Corporation. Invention is credited to Hung, David T..
Application Number | 20040018546 10/622743 |
Document ID | / |
Family ID | 24505894 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018546 |
Kind Code |
A1 |
Hung, David T. |
January 29, 2004 |
Identifying material from a breast duct
Abstract
Methods and systems for identifying material from a breast duct
using one or more markers that can be identified in ductal fluid
retrieved from the breast are provided.
Inventors: |
Hung, David T.; (Belmont,
CA) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Cytyc Health Corporation
Boxborough
MA
01719
|
Family ID: |
24505894 |
Appl. No.: |
10/622743 |
Filed: |
July 21, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10622743 |
Jul 21, 2003 |
|
|
|
09625399 |
Jul 26, 2000 |
|
|
|
6610484 |
|
|
|
|
09625399 |
Jul 26, 2000 |
|
|
|
09502404 |
Feb 10, 2000 |
|
|
|
6642010 |
|
|
|
|
09502404 |
Feb 10, 2000 |
|
|
|
09313463 |
May 17, 1999 |
|
|
|
6638727 |
|
|
|
|
09625399 |
|
|
|
|
09473510 |
Dec 28, 1999 |
|
|
|
6413228 |
|
|
|
|
60117281 |
Jan 26, 1999 |
|
|
|
60166100 |
Nov 17, 1999 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
A61K 31/437 20130101;
C12Q 1/6806 20130101; A61K 31/4535 20130101; A61K 31/4196 20130101;
A61B 10/0041 20130101; A61K 31/00 20130101; A61M 3/0262 20130101;
G01N 33/57484 20130101; A61K 31/566 20130101; A61K 31/138 20130101;
G01N 33/57415 20130101; A61M 2210/1007 20130101; C12Q 1/6886
20130101; A61K 31/352 20130101; A61M 3/0279 20130101; G01N 33/57492
20130101; A61K 31/138 20130101; A61K 2300/00 20130101; A61K 31/352
20130101; A61K 2300/00 20130101; A61K 31/4196 20130101; A61K
2300/00 20130101; A61K 31/437 20130101; A61K 2300/00 20130101; A61K
31/4535 20130101; A61K 2300/00 20130101; A61K 31/566 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
435/6 ;
435/7.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What is claimed is:
1. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker
comprising a protein, a polypeptide, a peptide, a nucleic acid, a
polynucleotide, an mRNA, a small organic molecule, a lipid, a fat,
a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide,
a chromosomal abnormality, a whole cell having a marker molecule, a
particle, a secreted molecule, an intracellular molecule, and a
complex of a plurality of molecules.
2. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker
comprising RNA, DNA, protein, polypeptide, or peptide form of a
marker selected from the group consisting of a receptor, a ligand,
a protein factor, an antigen, an antibody, an enzyme, a soluble
protein, a cytosolic protein, a cytoplasmic protein, a tumor
suppressor, a cell surface antigen, a phospholipid, a lipoprotein,
a hormone responsive protein, a differentiation associated antigen,
a proliferation associated antigen, a metastasis associated
antigen, an integral membrane protein, a protein that participates
in an apoptosis pathway, a protein that participates in a
transcriptional activation pathway, a cell adhesion molecule, an
extracellular matrix protein, a proteolipid, a cytokine, a basement
membrane protein, a mucin-type glycoprotein, a histone, a
ribonucleoprotein, a sialic acid, a bone matrix protein, a
carbohydrate antigen, a nuclear protein, a nuclear phosphoprotein,
a proto-oncogene, an oncogene, an apolipoprotein, a serine
protease, a tumor rejection antigen, a surfactant protein, a cell
death protein, a zinc endoprotease, and a trefoil gene.
3. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker
comprising RNA, DNA, protein, polypeptide, or peptide form of a
marker selected from the group consisting of a chemokine, a lectin,
an integrin, a selectin, a keratin, an interleukin, a taxin, a
ferritin, a lipocalin, a laminin, a cyclin, a relaxin, a nuclein, a
caspase, a melanoma-associated antigen, a macrophage inflammatory
protein, a gap junction protein, a calcium binding protein, an
actin binding protein, a phospholipid binding protein, a heat shock
protein, a cell cycle protein, an activator of tyrosine and
tryptophan hydroxylase, a member of the tumor necrosis factor
family of proteins, a member of the transforming growth factor
alpha family of proteins, a member of the transforming growth
factor beta family of proteins, a member of the Bcl2 family of
proteins, a Bcl2-interacting protein, a Bcl2-associated protein, a
member of the vasopressin/oxytocin family of proteins, and a member
of the CCAAT/enhancer binding protein family of proteins.
4. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker wherein
the marker is an enzyme and the enzyme comprises an RNA, DNA,
protein, polypeptide, or peptide form of an enzyme selected from
the group consisting of a phosphorylase, a phosphatase, a
decarboxylase, an isoenzyme, a kinase, a protease, a nuclease, a
peptidase, a protease, a DNase, an RNase, an aminopeptidase, a
topoisomerase, a phosphodiesterase, an aromatase, a cyclooxygenase,
a hydroxylase, a dehydrogenase, a metalloproteinase, a telomerase,
a reductase, a synthase, an elastase, a tyrosinase, a transferase,
and a cyclase.
5. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker wherein
the marker is a receptor and the receptor comprises an RNA, DNA,
protein, polypeptide, or peptide form of a receptor selected from
the group consisting of a steroid hormone receptor, a growth factor
receptor, a kinase receptor, a G-protein linked receptor, a TNF
family receptor, a tyrosine kinase receptor, a vasopressin
receptor, an oxytocin receptor, and a serine protease receptor.
6. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker wherein
the marker is a protein factor and the factor comprises an RNA,
DNA, protein, polypeptide, or peptide form of a factor selected
from the group consisting of a growth factor, a proteolytic factor,
a stromal cell factor, an epithelial cell factor, an angiogenesis
factor, an epithelial cell factor, an angiogenic factor, and a
colony stimulating factor.
7. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from one duct of a breast of a patient, said fluid not mixed
with ductal fluid from any other duct of the breast; examining the
ductal fluid sample to determine the presence of a marker wherein
the marker is an inhibitor and the inhibitor comprises an RNA, DNA,
protein, polypeptide, or peptide form of an inhibitor selected from
the group consisting of an inhibitor of a cyclin, an inhibitor of a
cyclin complex, a serpin, an inhibitor of proteolytic degredation,
a tissue inhibitor of a metalloprotease, and an angiogenesis
inhibitor.
8. A method of identifying a patient having breast cancer or breast
precancer, said method comprising: providing a ductal fluid sample
from one duct of a breast of a patient, said fluid not mixed with
ductal fluid from any other duct of the breast; examining the
ductal fluid sampie to determine the presence of a marker
comprising a protein, a polypeptide, a peptide, a nucleic acid, a
polynucleotide, an mRNA, a small organic molecule, a lipid, a fat,
a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide,
a chromosomal abnormality, a whole cell having a marker molecule, a
particle, a secreted molecule, an intracellular molecule, and a
complex of a plurality of molecules; wherein the marker is capable
of differentiating between any two of cytological categories
consisting of normal, abnormal, hyperplasia, atypia, ductal
carcinoma, ductal carcinoma in situ (DCIS), ductal carcinoma in
situ--low grade (DCIS-LG), ductal carcinoma in situ--high grade
(DCIS-HG), invasive carcinoma, a typical mild changes, a typical
marked changes, a typical ductal hyperplasia (ADH), insufficient
cellular material for diagnosis, and sufficient cellular material
for diagnosis.
9. A method as in any of claims 1-7 further comprising analyzing
the ductal fluid for abnormal cytology.
10. A method as in any of claims 1-8 wherein the ductal fluid is
retrieved by placing a ductal access tool in the duct and infusing
fluid into the duct through the tool and retrieving from the
accessed duct through the tool a portion of the infused fluid mixed
with ductal fluid.
11. A method as in any of claims 1-8 wherein the method is repeated
for more than one duct on a breast.
12. A method as in any of claims 1-8 wherein the method is repeated
for a plurality of ducts on a breast.
13. A method for identifying a patient having breast cancer or
breast precancer, said method comprising: providing a ductal fluid
sample from at least one duct of a breast of the patient; and
examining the ductal fluid sample to determine the presence of a
marker comprising an expression product of a gene encoding a
nuclear matrix protein.
14. A method as in claim 13, wherein the expression product
comprises a nucleic acid or a polypeptide.
15. A method as in claim 13, wherein the expression product
comprises RNA.
16. A method as in claim 13, wherein the expression product
comprises a protein or a part of a protein.
17. A method as in claim 13, wherein the nuclear matrix protein is
selected from the group consisting of lamin A, lamin B, lamin C, a
peripheral matrix protein, nuclear mitotic spindle apparatus
protein (NuMA), topoisomerase II, and an internal nuclear matrix
protein.
18. A method as in claim 13, wherein the expression product is a
polypeptide and examining comprises contacting the polypeptide
marker with an antibody that specifically binds a portion or the
polypeptide.
19. A method as in claim 13, wherein the expression product is a
nucleic acid and examining comprises detecting the presence of the
nucleic acid.
20. A method as in claim 19, wherein detecting the presence of the
nucleic acid comprises amplifying the nucleic acid.
21. A method as in claim 13, wherein providing the ductal fluid
sample comprises obtaining the sample from the breast.
22. A method as in claim 13, wherein providing the ductal fluid
sample comprises receiving a sample which had been previously
obtained.
23. A method as in claim 13, wherein the fluid was obtained by
nipple aspiration of the milk ducts.
24. A method as in claim 13, wherein the fluid sample was obtained
by washing the ductal lumen and retrieving fluid and cells from the
lumen.
25. A method as in claim 13, wherein the fluid collected is from a
single duct.
26. A method as in claim 13, wherein the fluid is collected from a
plurality of ducts.
27. A system for diagnosing breast cancer or precancer comprising a
tool to retrieve ductal fluid from a breast duct and instructions
for use to determine the presence of a marker identified in any of
claims 1-8.
28. A system for diagnosing breast cancer or precancer comprising a
tool to retrieve ductal fluid from a breast duct and instructions
for use to determine the presence of a marker identified in any of
claims 13-26.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/502,404, filed on Feb. 10, 2000, which was a
continuation-in-part of application Ser. No. 09/313,463, filed on
May 17, 1999, which claimed the benefit of provisional application
No. 60/117,281, filed on Jan. 26, 1999. This application is also a
continuation-in-part of application Ser. No. 09/473,510, filed on
Dec. 28, 1999. This application also claims the benefit under 37
CFR 1.78 of provisional application No. 60/166,100 filed on Nov.
17, 1999. The full disclosures of each of the prior applications
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The field of this invention is methods and systems for
detecting breast cancer and breast precancer in humans.
[0004] 2. Description of the Background Art
[0005] For several decades significant members of the medical
community dedicated to studying breast cancer have believed and
shown that the cytological analysis of cells retrieved from nipple
discharge from the breast milk ducts can provide valuable
information leading to an identifying patients at risk for breast
cancer. Indeed Papanicolaou himself contributed to the genesis of
such a possibility of a "Pap" smear for breast cancer by analyzing
the cells contained in nipple discharge. See Papanicolaou et al,
"Exfoliative Cytology of the Human Mammary Gland and Its Value in
the Diagnosis of Cancer and Other Diseases of the Breast" Cancer
(1958) March/April 377-409. See also Petrakis, "Physiological,
biochemical, and cytological aspects of nipple aspirate fluid",
Breast Cancer Research and Treatment 1986; 8:7-19; Petrakis,
"Studies on the epidemiology and natural history of benign breast
disease and breast cancer using nipple aspirate fluid" Cancer
Epidemiology, Biomarkers and Prevention (January/February 1993)
2:3-10; Petrakis, "Nipple Aspirate Fluid in epidemiological studies
of breast disease", Epidemiologic Reviews (1993) 15:188-195. More
recently, markers have also been detected in nipple fluid. See
Sauter et al, "Nipple aspirate fluid: a promising non-invasive
method to identify cellular markers of breast cancer risk", British
Journal of Cancer 76(4):494-501 (1997). The detection of CEA in
fluids obtained by a nipple blot is described in Imayama et al.
(1996) Cancer 78: 1229-1234.
[0006] Breast cancer is believed to originate in the lining of a
single breast milk duct in the breast; and additionally human
breasts are believed to contain from 6 to 9 of these ducts. See
Sartorius, JAMA 224 (6): 823-827 (1973). Sartorius describes use of
hair-like single lumen catheters that are inserted into breast
ducts using an operating microscope and the ducts were flushed with
saline solution as described in Cassels, D Mar. 20, 1973, The
Medical Post, article entitled "New tests may speed breast cancer
detection". Sartorius et al, Contrast ductography for recognition
and localization of benign and malignant breast lesions: an
improved technique. pp. 281-300. In: Logan WW, ed. Breast Carcinoma
New York, Wiley, 1977. After the fluid was infused, the catheter
was removed because it was too small to collect the fluid, the
breast was squeezed and fluid that oozed onto the nipple surface
was removed from the surface by a capillary tube. Similarly, Love
and Barsky, "Breast-duct endoscopy to study stages of cancerous
breast disease", Lancet 348(9033):997-999, 1996 describes
cannulating breast ducts with a single lumen catheter and infusing
a small amount of saline, removing the catheter and squeezing to
collect the fluid that returns on the nipple surface. The use of a
rigid 1.2 mm ductscope to identify intraductal papillomas in women
with nipple discharge is described in Makita et al (1991) Breast
Cancer Res Treat 18: 179-188. It would be advantageous to develop
methods and devices to collect the ductal fluid from within the
duct.
[0007] Galactography, or contrast ductography has for years located
breast ducts based on spontaneous nipple discharge, infused the
ducts (using cannulas for this purpose) with contrast dye
solutions, and taken x-ray pictures to determine the source of the
discharge within the duct. See generally, The Breast: Comprehensive
Management of Benign and Malignant Breast Diseases, Bland and
Copeland eds. W. B. Saunders Co. Philadelphia Pa. 1991 pages
61-67.
[0008] Nuclear matrix proteins are implicated in bladder, colon,
prostate, breast and other cancers, and have been put forth by
Matritech, Inc (Newton, Mass. 02460) as part of a kit for testing
for bladder cancer using body fluid. For testing for breast cancer,
a blood test has been developed using antibodies to nuclear matrix
proteins (see website for Matritech, Inc. http:/www.matritech.com)
The blood and body fluid tests are promoted as being capable of
early detection of the cancers they seek to identify. In addition,
profiles and differential patterns of expression of nuclear matrix
proteins have been detected for several different cancers (Fey and
Penman 1986, Stuurman 1990, and Getzenberg 1990).
[0009] Matritech, Inc. has patented various aspects of proteins and
nucleic acids of nuclear matrix proteins as well as kits for
testing for their presence in order to identify cancer in U.S. Pat.
Nos. 5,965,376, 5,914,238, 5,882,876, 5,858,683, 5,840,503,
5,830,677, 5,783,403, 5,780,596, 5,698,439, 5686,562, and
5,547,928. Specifically, Matritech has patented claims in U.S. Pat.
No. 5,914,238 to a method for diagnosing breast cancer in a patient
comprising detecting the presence of a breast cancer-associated
protein in a tissue or body fluid obtained from the patient. The
breast cancer-associated protein has a molecular weight of about
32,500 or 33,000 Daltons and an isoelectric point of about 6.82,
and has a continuous amino acid sequence from several amino acid
sequences disclosed in the application. The nuclear matrix protein
is detected by polyclonal or monoclonal antibodies or by PCR
amplification of an expression product of the target gene. The
patient sample in the examples is breast tissue samples, although
the possibility of testing blood or body fluid is claimed and
mentioned in the specification. Testing breast duct fluid is not
described.
SUMMARY OF THE INVENTION
[0010] An object of the invention is to identify a patient having
breast cancer or breast precancer. In accordance with this object,
is provided a method comprising providing a ductal fluid sample
from one duct of a breast of a patient, the fluid not mixed with
ductal fluid from any other duct of the breast, and examining the
ductal fluid sample to determine the presence of a marker
comprising a protein, a polypeptide, a peptide, a nucleic acid, a
polynucleotide, an mRNA, a small organic molecule, a lipid, a fat,
a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide,
a chromosomal abnormality, a whole cell having a marker molecule, a
particle, a secreted molecule, an intracellular molecule, and a
complex of a plurality of molecules. In accordance with this object
is also provided methods for determining markers which can identify
a patient having breast cancer or precancer by examining the ductal
fluid sample to determine the presence of a marker comprising RNA,
DNA, protein, polypeptide, or peptide form of the marker. The
invention also includes a method of identifying a patient having
breast cancer or breast precancer, said method comprising providing
a ductal fluid sample from one duct of a breast of a patient, said
fluid not mixed with ductal fluid from any other duct of the
breast, examining the ductal fluid sample to determine the presence
of a marker comprising a protein, a polypeptide, a peptide, a
nucleic acid, a polynucleotide, an mRNA, a small organic molecule,
a lipid, a fat, a glycoprotein, a glycopeptide, a carbohydrate, an
oligosaccharide, a chromosomal abnormality, a whole cell having a
marker molecule, a particle, a secreted molecule, an intracellular
molecule, and a complex of a plurality of molecules; wherein the
marker is capable of differentiating between any two of cytological
categories consisting of normal, abnormal, hyperplasia, atypia,
ductal carcinoma, ductal carcinoma in situ (DCIS), ductal carcinoma
in situ--low grade (DCIS-LG), ductal carcinoma in situ--high grade
(DCIS-HG), invasive carcinoma, a typical mild changes, a typical
marked changes, a typical ductal hyperplasia (ADH), insufficient
cellular material for diagnosis, and sufficient cellular material
for diagnosis.
[0011] Any of the methods can comprise further analyzing the ductal
fluid for abnormal cytology. Any of the methods can comprise that
the ductal fluid is retrieved by placing a ductal access tool in
the duct and infusing fluid into the duct through the tool and
retrieving from the accessed duct through the tool a portion of the
infused fluid mixed with ductal fluid. The method can be repeated
for more than one duct on a breast; for example, the method can be
practiced comprising a plurality of ducts on a breast.
[0012] The invention provides additionally, a method for
identifying a patient having breast cancer or breast precancer, by
providing a ductal fluid sample from at least one duct of a breast
of the patient; and examining the ductal fluid sample to determine
the presence of a marker comprising an expression product of a gene
encoding a nuclear matrix protein. The expression product can
comprise a nucleic acid or a polypeptide. The expression product
can comprise RNA, or a protein or a part of a protein. The nuclear
matrix protein can be lamin A, lamin B, lamin C, a peripheral
matrix protein, nuclear mitotic spindle apparatus protein (NuMA),
topoisomerase II, or an internal nuclear matrix protein. The
expression product can be a polypeptide and examining can comprise
contacting the polypeptide marker with an antibody that
specifically binds a portion of the polypeptide. The expression
product can be a nucleic acid and examining can comprise detecting
the presence of the nucleic acid. Detecting the presence of the
nucleic acid can comprise amplifying the nucleic acid. Providing
the ductal fluid sample can comprise obtaining the sample from the
breast. Providing the ductal fluid sample can comprise receiving a
sample that had been previously obtained. The fluid can be obtained
by nipple aspiration of the milk ducts. The fluid sample can be
obtained by washing the ductal lumen and retrieving fluid and cells
from the lumen. The fluid collected can be from a single duct. The
fluid can be collected from a plurality of ducts.
[0013] The invention also provides systems for diagnosing breast
cancer or precancer comprising a tool to retrieve ductal fluid from
a breast duct and instructions for use to determine the presence of
a marker.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0014] The following preferred embodiments and examples are offered
by way of illustration and not by way of limitation.
[0015] The method of the invention provides a method of screening
women for breast cancer or precancer comprising providing a ductal
fluid sample from at least one duct of a breast of the patient; and
detecting an increased level of a marker wherein an increased level
of one or more markers indicates an increased risk for breast
cancer or precancer.
[0016] The method is practiced by providing a ductal fluid sample
from at least one duct of a breast of the patient. Providing the
ductal fluid sample can comprise obtaining the sample from the
breast. Providing the ductal fluid sample can also comprise
receiving a sample that had been previously obtained. For example,
a laboratory can receive a ductal fluid sample from a patient or a
practitioner, and the laboratory can be directed to make an
analysis of the sample. Where the fluid is obtained from the
breast, the fluid sample can be obtained e.g. by nipple aspiration
of the milk ducts or by ductal lavage of at least one breast milk
duct. When fluid is collected by nipple aspiration, or by ductal
lavage, the fluid can be collected from a single duct. For example
the duct and the collection tube can be marked so that the analysis
of the fluid is traceable to one duct.
[0017] By the procedure of ductal lavage, ductal epithelial cells
that line the walls of the ductal lumen are washed out of the duct.
Lavage or wash fluid is infused into the duct, and the lavage fluid
mixed with ductal fluid is collected. Lavage is described in
copending and co-owned applications including Ser. No. 09/067,661,
09/301,058, PCT US99/09141, No. 60/122,076, 09/313,463, 60/143,359,
and U.S. application Ser. No. 09/473,510, all incorporated by
reference in their entirety. In some cases suction can be applied
to the tool accessing the ductal lumen in order to retrieve a
maximum amount of cells and/or fluid. Lavage or wash fluid can be
infused into the duct, and collected. Suction can be applied to the
tool accessing the ductal lumen in order to retrieve a maximum
amount of cells and/or fluid.
[0018] Access of a breast duct can be facilitated as described in
e.g. Love & Barsky, (1996) Lancet 348: 997-999, Makita et al
(1991) Breast Cancer Res Treat 18: 179-188, or Okazaki et al (1991)
Jpn J. Clin. Oncol. 21:188-193. Alternatively, ductal fluid can be
retrieved by a medical tool, e.g. a catheter or a cannula placed
into the duct to infuse wash fluid to retrieve a mixture of wash
and ductal fluids. The fluid from the breast duct can contain
ductal epithelial cells, including cells of a stage considered to
be precancerous or cancerous.
[0019] Nipple aspiration of breast ductal fluid is achieved by
using vacuum pressure. Nipple aspiration techniques are also
described and claimed in co-pending and co-owned patent application
U.S. patent application Ser. No. 09/438,219, herein incorporated by
reference in their entirety. Nipple aspirate fluid can be retrieved
as described in e.g. Goodson W H & King E B, Chapter 4:
Discharges and Secretions of the Nipple, The Breast: Comprehensive
Management of Benign and Malignant Diseases (1998).sub.2nd Ed. vol
2, Bland & Kirby eds. W. B. Saunders Co, Philadelphia, Pa. pp.
51-74; Wrensch et al., (1992) American Journal of Epidemiology.
135(2):130-41; and Sauter et al (1997) British Journal of Cancer.
76(4):494-501. Ductal lavage is described in copending patent
application U.S. Ser. No. 09/067,661 filed Apr. 28, 1998. Cells of
the lesion can be retrieved by collecting the ductal fluid that
contains some of these cells, e.g. by aspirating the nipple to
obtain nipple aspirate fluid, e.g. as described in Petrakis (1993)
Cancer Epidem. Biomarker Prev. 2:3-10, Petrakis (1986) Breast
Cancer Res. Treat 8: 7-19, Wrensch et al (1992) Am. J. Epidem.
135:130-141, Wrensch et al (1990) Breast Cancer Res Treat 15:
39-21, and Wrensch et al (1989) Cancer Res. 49: 2168-2174. Also
fluid secretions from the nipple can be collected as they
spontaneously appear on the nipple surface. In order to collect the
fluid not mixed with ductal fluid from other ducts, a practitioner
carefully watches for the signs of fluid and retrieves the fluid
from the nipple surface near the orifice before it has a chance to
mix with fluid from any other orifice.
[0020] The ductal fluid may be analyzed in situ, i.e. inside the
breast and inside the breast duct, e.g. where a particular marker
can be introduced into the duct and can be identified from within
the breast. In situ testing within the duct is also considered a
non-invasive means of examining the ductal epithelial cells. Ductal
epithelial cells that are examined by the method of the invention
can be examined in situ (i.e. in the duct; e.g. where a marker can
bind the cells or a component of the cells in the duct and be
identified from within the breast by a tag attached to the marker),
or after the ductal epithelial cells have been removed from the
breast of the patient by non-invasive means, e.g. as just
described. Methods of in situ analysis can include use of such
molecular biology tools, methods, and materials as described in
e.g. U.S. Pat. Nos. 5,169,774, 5,720,937, 5,677,171, 5,720,954,
5,725,856, 5,770,195, and 5,772,997. Markers to breast cancer and
breast precancer described elsewhere and herein may also be used
for an in situ analysis of the breast duct.
[0021] The ductal fluid is examined to detect the presence of
precancerous or cancerous ductal epithelial cells. The fluid sample
(comprising ductal epithelial cells) can be analyzed by any
effective means for identifying breast precancer or cancer,
including e.g. cytological analysis of the cells retrieved or
identified. Examination of the ductal epithelial cells can be
accomplished by examining useful indicators such as, e.g. the
morphology of the cells or the cellular contents. The cellular
contents can include, e.g. protein, nucleic acid, particles,
complexes or other biochemical or molecular markers in the cells.
Cell morphology can serve to establish whether the ductal
epithelial cells are normal (i.e. not precancerous or cancerous or
having another noncancerous abnormality), precancerous (i.e.
comprising hyperplasia, a typical ductal hyperplasia (ADH) or low
grade ductal carcinoma in situ (LG-DCIS)) or cancerous (i.e.
comprising high grade ductal carcinoma in situ (HG-DCIS), or
invasive carcinoma).
[0022] Analysis of cell contents may serve to establish similar
staging as established by morphology, capturing generally a
progression of a precancerous or cancerous condition in the cells.
Thus the ductal epithelial cells may be analyzed for other markers,
e.g. protein markers, nucleic acid markers, particles, complexes,
or biochemical or molecular markers in the cells or on the cell
surfaces or secreted by the cell or for any marker providing
evidence of neoplasia. The ductal epithelial cell can be derived
from any part of the breast milk duct, including, e.g. the ductal
lumen and/or the terminal ductal lobular unit (TDLU). Cells derived
from the TDLU may also have similar stages as found in other
luminal ductal epithelial cells not from the TDLU including, e.g.
hyperplasia, atypia, in situ carcinoma, and invasive carcinoma.
[0023] Once the wash fluid had been infused in the duct and the
wash fluid and ductal fluid is collected from a breast duct, the
cellular material can be separated and can be examined. The
cellular material can include, e.g. substances selected from the
group consisting of whole cells, cellular debris, proteins, nucleic
acids, polypeptides, glycoproteins, lipids, fats, glycoproteins,
small organic molecules, metabolites, and macromolecules. Cytology,
or any other suitable method for analyzing the condition of the
cells can examine whole cells. Other markers present in the
cellular material, ductal fluid generally, or other material
obtained from the breast duct can be analyzed as is appropriate for
the marker being sought, including e.g. binding assays,
immunohistochemistry, or using other analytical technology for
distinguishing and identifying biological molecules obtained from
biological material.
[0024] Identifying a patient having breast cancer or breast
precancer can be accomplished by removing breast duct fluid from
the patient and analyzing the fluid comprising ductal contents for
markers that may indicate a cancerous or precancerous condition in
the breast. Providing a ductal fluid sample from one duct of a
breast of a patient includes that the fluid is not mixed with
ductal fluid from any other duct of the breast. The method is
practiced by providing a ductal fluid sample from at least one duct
of a breast of the patient. Providing the ductal fluid sample can
comprise obtaining the sample from the breast. Providing the ductal
fluid sample can also comprise receiving a sample that had been
previously obtained. For example, a laboratory can receive a ductal
fluid sample from a patient or a practitioner, and the laboratory
can be directed to make an analysis of the sample. Where the fluid
is obtained from the breast, the fluid sample can be obtained e.g.
by nipple aspiration of the milk ducts or by ductal lavage of at
least one breast milk duct. When fluid is collected by nipple
aspiration, or by ductal lavage, the fluid can be collected from a
single duct. For example the duct and the collection tube can be
marked so that the analysis of the fluid is traceable to one
duct.
[0025] The ductal fluid can be retrieved by placing a ductal access
tool in the duct and infusing fluid into the duct through the tool
and retrieving from the accessed duct through the tool a portion of
the infused fluid mixed with ductal fluid. The process may be
repeated for more than one duct on a breast, and/or the process can
be repeated for a plurality of ducts on a breast. Either sequential
or simultaneous access of the duct on a breast can be used.
[0026] The next step in the method after the fluid is collected is
examining the ductal fluid sample to determine the presence of a
marker comprising a protein, a polypeptide, a peptide, a nucleic
acid, a polynucleotide, an mRNA, a small organic molecule, a lipid,
a fat, a glycoprotein, a glycopeptide, a carbohydrate, an
oligosaccharide, a chromosomal abnormality, a whole cell having a
marker molecule, a particle, a secreted molecule, an intracellular
molecule, and a complex of a plurality of molecules.
[0027] Examining the ductal fluid sample can comprise determining
the presence of a marker comprising RNA, DNA, protein, polypeptide,
or peptide form of a marker selected from the group consisting of a
receptor, a ligand, a protein factor, an antigen, an antibody, an
enzyme, a soluble protein, a cytosolic protein, a cytoplasmic
protein, a tumor suppressor, a cell surface antigen, a
phospholipid, a lipoprotein, a hormone responsive protein, a
differentiation associated antigen, a proliferation associated
antigen, a metastasis associated antigen, an integral membrane
protein, a protein that participates in an apostasis pathway, a
protein that participates in a transcriptional activation pathway,
a cell adhesion molecule, an extracellular matrix protein, a
proteolipid, a cytokine, a basement membrane protein, a mucin-type
glycoprotein, a histone, a ribonucleoprotein, a sialic acid, a bone
matrix protein, a carbohydrate antigen, a nuclear protein, a
nuclear phosphoprotein, a proto-oncogene, an oncogene, an
apolipoprotein, a serine protease, a tumor rejection antigen, a
surfactant protein, a cell death protein, a zinc endoprotease, and
a trefoil gene.
[0028] Examining the ductal fluid sample can comprise determining
the presence of a marker comprising RNA, DNA, protein, polypeptide,
or peptide form of a marker selected from the group consisting of a
chemokine, a lectin, an integrin, a selectin, a keratin, an
interleukin, a taxin, a ferritin, a lipocalin, a laminin, a cyclin,
a relaxin, a nuclein, a caspase, a melanoma-associated antigen, a
macrophage inflammatory protein, a gap junction protein, a calcium
binding protein, an actin binding protein, a phospholipid binding
protein, a heat shock protein, a cell cycle protein, an activator
of tyrosine and tryptophan hydroxylase, a member of the tumor
necrosis factor family of proteins, a member of the transforming
growth factor alpha family of proteins, a member of the
transforming growth factor beta family of proteins, a member of the
Bcl2 family of proteins, a Bcl2-interacting protein, a
Bcl2-associated protein, a member of the vasopressin/oxytocin
family of proteins, and a member of the CCAAT/enhancer binding
protein family of proteins.
[0029] Examining the ductal fluid sample can comprise determining
the presence of a marker wherein the marker is an enzyme and the
enzyme comprises an RNA, DNA, protein, polypeptide, or peptide form
of an enzyme selected from the group consisting of a phosphorylase,
a phosphatase, a decarboxylase, an isoenzyme, a kinase, a protease,
a nuclease, a peptidase, a protease, a DNase, an RNase, an
aminopeptidase, a topoisomerase, a phosphodiesterase, an aromatase,
a cyclooxygenase, a hydroxylase, a dehydrogenase, a
metalloproteinase, a telomerase, a reductase, a synthase, an
elastase, a tyrosinase, a transferase, and a cyclase.
[0030] Examining the ductal fluid sample can comprise determining
the presence of a marker wherein the marker is a receptor and the
receptor comprises an RNA, DNA, protein, polypeptide, or peptide
form of a receptor selected from the group consisting of a steroid
hormone receptor, a growth factor receptor, a kinase receptor, a
G-protein linked receptor, a TNF family receptor, a tyrosine kinase
receptor, a vasopressin receptor, an oxytocin receptor, and a
serine protease receptor.
[0031] Examining the ductal fluid sample can comprise determining
the presence of a marker wherein the marker is a protein factor and
the factor comprises an RNA, DNA, protein, polypeptide, or peptide
form of a factor selected from the group consisting of a growth
factor, a proteolytic factor, a stromal cell factor, an epithelial
cell factor, an angiogenesis factor, an epithelial cell factor, an
angiogenic factor, and a colony stimulating factor.
[0032] Examining the ductal fluid sample can comprise determining
the presence of a marker wherein the marker is an inhibitor and the
inhibitor comprises an RNA, DNA, protein, polypeptide, or peptide
form of an inhibitor selected from the group consisting of an
inhibitor of a cyclin, an inhibitor of a cyclin complex, a serpin,
an inhibitor of proteolytic degredation, a tissue inhibitor of a
metalloprotease, and an angiogenesis inhibitor.
[0033] Examining the ductal fluid can comprise identifying a level
or quality of at least one marker comprising an expression product
of a gene encoding a nuclear matrix protein.
[0034] A level of the marker can be a presence relative to a normal
control or an absence relative to a normal control of a given
marker. The normal control can be determined relative to the
particular patient, or relative to a patient population.
[0035] In addition, the quality of the marker can be assessed. A
quality of a marker can be such changes as DNA mutation, or a
quantity of mutations, a deterioration of chromosomal quality or
quantity, degradation of a protein, or a change in quantity of a
nucleic acid or chromosome. A quality can be an erosion of a
molecule, particle, molecule or organelle with respect to a normal
quality.
[0036] Once the wash fluid had been infused in the duct and the
wash fluid and ductal fluid is collected from a breast duct, the
cellular material can be separated and can be examined. The
cellular material can include, e.g. substances selected from the
group consisting of whole cells, cellular debris, proteins, nucleic
acids, polypeptides, glycoproteins, lipids, fats, glycoproteins,
small organic molecules, metabolites, and macromolecules. Examining
the ductal fluid sample to determine the presence of a marker
comprising a protein, a polypeptide, a peptide, a nucleic acid, a
polynucleotide, an mRNA, a small organic molecule, a lipid, a fat,
a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide,
a chromosomal abnormality, a whole cell having a marker molecule, a
particle, a secreted molecule, an intracellular molecule, and a
complex of a plurality of molecules. Detection and analysis of
these classifications of markers can be accomplished as described
below, using standard assays for determining the presence of
markers or marker classifications listed, for example as described
in Sambrook et al., Molecular Cloning: A Laboratory Manual,
2.sup.nd Ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
1989).
[0037] Cytology, or any other suitable method for analyzing the
condition of the cells can examine whole cells. Other markers
present in the cellular material, ductal fluid generally, or other
material obtained from the breast duct can be analyzed as is
appropriate for the marker being sought, including e.g. binding
assays, immunohistochemistry, or using other analytical technology
for distinguishing and identifying biological molecules obtained
from biological material.
[0038] Intracellular components, either secreted or non-secreted
and which are found in the ductal fluid may be tested as well. For
example, ring-shaped particles which comprise protein, DNA, and RNA
can be identified using an assay and/or a binding immogen as
described in U.S. Pat. Nos. 5,635,605, and 5,459,035, and EP
465,715, including (as described) an affinity chromatography medium
specific for proteins with particular characteristics (in the case
of the ring-shaped particle a dinucleotide fold) and a standard
immunoassay proceeding as described. In addition, complexes of
proteins or other molecules may be identified. For example,
antibodies may be used to bind complexes, e.g. extracellular matrix
complexes in order to identify such a complex that is considered a
breast cancer marker. The process of identification of such
complexes is described in WO 96/12192.
[0039] Exemplary markers are described in Masood S., (Prediction of
recurrence for advanced breast cancer. Traditional and contemporary
pathologic and molecular markers) Surgical Oncology Clinics of
North America. 4(4):601-32, 1995; Lopez-Guerrero et al (1999) J.
Hematother 8(1):53-61; Maijumdar and Diamandis (1999) Br J Cancer
79(9-10):1594-602; Balleine et al (1999) Br J Cancer 79
(9-10):1564-71; Houston et al (1999) Br J Cancer 79(7-8):1220-6;
Nikolic-Vukosavljevic et al (1998) Tumori 84(6):691-4; Maguire et
al (1998) Int J Biol Markers 13(3): 139-44; Steams et al (1998)
Breast Cancer Res Treat 52(1-3):239-59; Eiriksdottir et al (1998)
Eur J Cancer 34(13):2076-81, and U.S. Pat. No. 5,169,774. Many
known breast cancer markers are discussed and described in readily
available medical textbooks on breast cancer. In addition, several
markers can be identified and analyzed in the same sample, e.g.
Fabian et al 1993 J. Cellular Biochemistry 17G:153-16 and Fabian et
al 1994 Breast Cancer Res Treat 30(3):263-74 looking at estrogen
receptor (ER), epidermal growth factor receptor (EGFR), mutant p53,
HER-2 neu by immunohistochemistry and aneuploidy by image analysis
in fine needle aspirates. Methods described therein can be
practiced by analogy to analysis of ductal fluid contents,
particularly ductal epithelial cells retrieved by nipple aspiration
and/or by ductal lavage techniques.
[0040] Chromosomal abnormalities in ductal epithelial cells can
also provide information and act as a marker to identify cancer or
precancer as described in Mark et al (1999) Cancer Genet Cytogenet
108:26-31; Lundlin and Mertens (1998) Breast Cancer Res Treat
51:1-15; Newsham (1998) Am J Pathol 153:5-9; Larson et al (1998) Am
J Pathol 152:1591-8; Adelaide et al (1998) Genes Chromosomes Cancer
22:186-99; Fejzo et al (1998) Gene Chromosome Cancer 22:105-113;
Dietrich et al (1998) Hum Pathol 12: 1379-82; Cavalli et al (1997)
Hereditas 126:261-8; Adeyinka et al (1997) Cancer Genet Cytogenet
97:119-21; Afify and Mark (1997) Cancer Genet Cytogenet 97:101-5;
Brenner and Aldaz (1997) Prog Clin Biol Res 396: 63-82; Mark et al
(1997) Ann Clin Lab Sci 27:47-56; and Fabian et al 1993 J. Cellular
Biochemistry 17G:153-16.
[0041] Other breast cancer markers can be detected as described in
Springer, G. F. et al, Dao et al, Eds, Tumor Markers and Their
Significance in the Management of Breast Cancer, pp.47-70, New
York; A. R. Liss, 1986. In addition to some markers discussed
and/or articles or books cited on breast cancer and breast
precancer markers, including markers listed in Porter-Jordan and
Lippman, "Overview of the biological markers of breast cancer",
Hematology/Oncology Clinics of North America vol. 8 (1):73-100,
1994), the following cancer markers are listed here as exemplary
and may be used as well as other markers to analyze the condition
of a breast duct, including analysis of the ductal contents
(including fluid and cells). Standard assay procedures for
identifying the markers can be used, including antibodies or other
binding partners, labels, stains, pattern analysis (for cells and
cell components), and in general any other chemical or visual
identification techniques.
[0042] Exemplary markers that are presently being studied by
researchers directing their research to breast cancer include, for
example, carcinoma embryonic antigen (CEA), prostate specific
antigen (PSA) Erb B2 antigen, gross cystic disease fluid protein-15
(GCDFP-15), and lactose dehydrogenase (LDH). For CEA see Imayama et
al, Cancer 1996, 78(6):1229-34; Inaji et al, Cancer
1987,60(12):3008-13; Mori Int Conger Seer 1989, 807:211-8; Inaji,
et al. An To Kagaku Ryoho 1991, 18(2):313-7; Yayoi, et al Gan To
Kagaku Ryoho 1994, 21 Suppl 2:133-9; Mori, et al Jpn J Clin Oncol
1989,19(4):373-9; Foretova, et al Proc Annu Meet Am Soc Clin Oncol
1995,14:A11; and Nishiguchi, et al Rinsho Byori 1992,40(1):67-72.
For PSA see Foretova and Garber Lancet 1996,347(9015):1631; Sauter
et al, Cancer Epidemiology, Biomarkers & Prevention.
5(12):967-70, 1996; Sauter and Daly (1996)Proc Annu Meet Am Assoc
Cancer Res 37:A1458; and Foretova and Garber (1996) Proc Annu Meet
Am Assoc Cancer Res 37:A1446. For Erb B2 see Motomura (1995) Breast
Cancer Res and Treat 33:89-92; and Inaji et al (1993) Tumour Biol
14: 271-8. For GCDFP-15 see Petrakis et al (1994) Proc Annu Meet Am
Assoc Cancer Res 35:A1698. For LDH see Mannello et al (1995) Cancer
76:152-4; and Kawamoto (1994) Cancer 73:1836-41.
[0043] Generally markers can be, for example, a protein, a
polypeptide, a peptide, a nucleic acid, a polynucleotide, an mRNA,
a small organic molecule, a lipid, a fat, a glycoprotein, a
glycopeptide, a carbohydrate, an oligosaccharide, a chromosomal
abnormality, a whole cell having a marker molecule, a particle, a
secreted molecule, an intracellular molecule, and a complex of a
plurality of molecules. These markers can be detected by detecting
an RNA, DNA, protein, polypeptide, or peptide form of a marker
selected from the group consisting of, for example, a chemokine, a
lectin, an integrin, a selectin, a keratin, an interleukin, a
taxin, a ferritin, a lipocalin, a laminin, a cyclin, a relaxin, a
nuclein, a caspase, a melanoma-associated antigen, a macrophage
inflammatory protein, a gap junction protein, a calcium binding
protein, an actin binding protein, a phospholipid binding protein,
a heat shock protein, a cell cycle protein, an activator of
tyrosine and tryptophan hydroxylase, a member of the tumor necrosis
factor family of proteins, a member of the transforming growth
factor alpha family of proteins, a member of the transforming
growth factor beta family of proteins, a member of the Bcl2 family
of proteins, a Bcl2-interacting protein, a Bcl2-associated protein,
a member of the vasopressin/oxytocin family of proteins, and a
member of the CCAAT/enhancer binding protein family of proteins; or
selected from the group consisting of a chemokine, a lectin, an
integrin, a selectin, a keratin, an interleukin, a taxin, a
ferritin, a lipocalin, a laminin, a cyclin, a relaxin, a nuclein, a
caspase, a melanoma-associated antigen, a macrophage inflammatory
protein, a gap junction protein, a calcium binding protein, an
actin binding protein, a phospholipid binding protein, a heat shock
protein, a cell cycle protein, an activator of tyrosine and
tryptophan hydroxylase, a member of the tumor necrosis factor
family of proteins, a member of the transforming growth factor
alpha family of proteins, a member of the transforming growth
factor beta family of proteins, a member of the Bcl2 family of
proteins, a Bcl2-interacting protein, a Bcl2-associated protein, a
member of the vasopressin/oxytocin family of proteins, and a member
of the CCAAT/enhancer binding protein family of proteins. An
example of a peptide marker is fibrinogen degredation peptide,
which can be assayed as described in WO 98/55872. Another marker
that may be detected, e.g., by ELISA tests on the ductal fluid is
called DR70.TM.. DR70.TM. may be sought from ductal fluid
essentially as described in Wu et al., J. Immunoassay, 1998 vol.
19; no. 1:63-72.
[0044] The marker may also or alternatively be an enzyme and the
enzyme can comprise an RNA, DNA, protein, polypeptide, or peptide
form of an enzyme such as for example, a phosphorylase, a
phosphatase, a decarboxylase, an isoenzyme, a kinase, a protease, a
nuclease, a peptidase, a protease, a DNase, an RNase, an
aminopeptidase, a topoisomerase, a phosphodiesterase, an aromatase,
a cyclooxygenase, a hydroxylase, a dehydrogenase, a
metalloproteinase, a telomerase, a reductase, a synthase, an
elastase, a tyrosinase, a transferase, or a cyclase.
[0045] The marker may also or alternatively be a receptor and the
receptor can comprise an RNA, DNA, protein, polypeptide, or peptide
form of a receptor such as for example a steroid hormone receptor,
a growth factor receptor, a kinase receptor, a G-protein linked
receptor, a TNF family receptor, a tyrosine kinase receptor, a
vasopressin receptor, an oxytocin receptor, and a serine protease
receptor.
[0046] The marker may also or alternatively be a protein factor and
the protein factor can comprise an RNA, DNA, protein, polypeptide,
or peptide form of a protein factor such as for example a growth
factor, a proteolytic factor, a stromal cell factor, an epithelial
cell factor, an angiogenesis factor, an epithelial cell factor, an
angiogenic factor, or a colony stimulating factor.
[0047] The marker may also or alternatively be an inhibitor and the
inhibitor can comprise an RNA, DNA, protein, polypeptide, or
peptide form of an inhibitor such as for example an inhibitor of a
cyclin, an inhibitor of a cyclin complex, a serpin, an inhibitor of
proteolytic degredation, a tissue inhibitor of a metalloprotease,
and an angiogenesis inhibitor.
[0048] The different categories of markers are tested differently
depending on the category and possibly also on the location of the
marker in the cell (for example, a cell surface protein might be
detected differently than a cytoplasmic or nuclear protein).
Typically, assays comprising one or more of binding, coloration,
precipitation, affinity column selection, in-situ binding, solution
phase binding, nucleic acid probe labeling, protein probe labeling,
polypeptide probe labeling, peptide probe labeling, and/or a
combination or variation of these processes can be used. Standard
procedure for conducting such assays generally (e.g. ELISA, RNA or
DNA probe hybridization, and other binding or other detection
assays) are described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2.sup.nd Ed. (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. 1989).
[0049] More specifically, examples of detection of particular
markers or classes of markers are described in Table I below that
lists exemplary markers and cites references in which those markers
are detected. For markers not specifically listed, broad categories
such as e.g. proteins, lipids, RNA transcripts, glycoproteins, and
other categories can be detected for other specific markers in the
same fashion as specific markers in a same or similar category.
Some of these specific markers belonging to broader categories are
listed in Table I.
[0050] In addition to some markers discussed and/or articles or
books cited on breast cancer and breast precancer markers, the
following cancer markers are listed here as exemplary and may be
used as well as other markers to analyze the condition of a breast
duct. Standard assay procedures for identifying the markers can be
used, including antibodies or other binding partners, labels,
stains, pattern analysis (for cells and cell components), and in
general any other chemical or visual identification techniques.
[0051] The following are exemplary potential markers for such
identification and analysis: cathepsins (including cathepsin D);
maspin, fas, fas ligand, tissue inhibitor of matrix
metalloproteinas-1 (TIMP-1); chemokines (both C--C and C--X--C type
chemokines); collagenases, metalloproteinases, TIMP's, cathepsins,
disrupted basement membrane epitopes, stromolysin-3; cytokeratins
(e.g. keratin 14, BI, KA1, KA4 and 312C8-1); estrogen and
progesterone receptors (or any androgen or other steroid receptor);
growth factor receptors for members of the fibroblast growth family
(FGF) including FGF1-18, vascular endothelial growth factor (VEGF),
insulin-like growth factor-1 (IGF-I), IGF-II, platelet-derived
growth factor (PDGF), keratinocyte growth factor (KGF), and
epithelial growth factor (EGF); placental growth factor (PLGF),
hepatocyte growth factor (HGF), tumor necrosis factor (TNF),
transforming growth factor (TGF) both alpha and beta forms, and
angiopoietin, for example; heat shock proteins (HSP) (e.g. HSP27)
27 (HSP27); ErB type 1 tyrosine kinase receptors (e.g. Her2 (an EGF
receptor) or any ligand or receptor of the ErbB family of ligands
and receptors); integrins, selectins, cadherins, for example (i.e.
alpha and beta 3 integrin); keratin-14; known cancer antigens
including, for example Ki-67, Ki-S1, p53, nm23, bcl-2, p21 ras,
cyclins, and pS2; thrombin receptor activating peptide; urokinase,
urokinase-type plasminogen activator (UPA), plasmin antiplasmin;
UPA receptor (UPAR), fibrinogen, plasmin activator inhibitor-1 and
2 (PAI-1 and 2); telomerase; antibodies to tumor associated
antigen-72 (TAG-72) (e.g. B72.3, B6.2, and TKH2); carcinoembryonic
antigen (CEA) (see e.g. EP 319,686); prostate specific antigen
(PSA); gross cystic disease fluid protein-15 (GCDFP-15); lactose
dehydrogenase (LDH); chromosomal abnormalities (e.g. aneuploidy or
other abnormalities); S1 protein; alkaline phosphatase; myosin;
sialyl Tn (STn) glycopeptide (e.g. TAG-72); Tn glycopeptide; and
nuclear matrix proteins (as described in provisional patent
application filed 11-17-99 docket no. PDH 99-029, herein
incorporated by reference in its entirety).
[0052] Other breast cancer markers include, e.g. alanine
aminopeptidase, alpha 6 integrin, alpha-lactalbumin, AN43 antigen
(BB 1), annexin 1, anti-Her2, anti-pS3, Bad, BAG-1, Bak, Bax,
BCA225, Bcl-2, Bcl-x, beta 1-6 branched oligosaccharides, beta-2
microglobulin (BMG), Bfl-1, bone sialoprotein (BSP), C/EBP
beta-LIP, Ca 1 antigen, CA27.29, CA M26, CA M29, CA125, CA15.3,
CA195, CA19-9, CA50, CA549, Cadherin-11, calcitonin receptor (CTR),
cathepsin B, cathepsin L, CD 105, CD24, CD34 (pan-endothelial
marker), CD44, CEA, c-met, c-myc, Cox-1, Cox-2, CPP32, cyclic
nucleotide phosphodiesterase, cyclin E, DNA topoisomerase II-alpha,
DNA topoisomerase II-beta, EGF, EGFR, E-selectin, fast
homoarginine-sensitive alkaline phosphatase (FHAP), fatty acid
synthase, ferritin, GCDFP-15/BRST-2, Her-2 (extracellular), h-mtsl
(S100A4), hsc73, hsp70, hsp90alpha, hsp90beta, ID1, ID3,
interleukin-1 beta, keratin 8, keratin 18, keratin 19, laminin,
laminin receptor (MluC5), leucine aminopeptidase (LAP), lipid-bound
sialic acid (LSA), MAGE-1, MAGE-2, MAGE-3, Man6-P glycoproteins,
MCA, Mcl-1, metallothionein (MT), MKP-1, MMP-2, MMP-9, MSA, MUC-2,
MUC-3, MUC-6, Nm23, omithine decarboxylase (ODC), osteopontin
(OPN), P114 (MAR binding protein), P120 (a nucleolar proliferation
antigen), p125FAK, p330d/CENP-F (a marker for cell proliferation),
PAI-2, Pepsinogen C. placental alkaline phosphatase (PLAP),
platelet factor 4 (angiogenic marker), protein kinase C (PKC), PSA,
pyrimidine nucleoside phosphorylase, ras p21, reduced glutathione
(GSH), retinoid X receptor alpha, ribosomal S2 protein,
sialyltransferase, Stromelysin-1 (MMP-3), surfactant proteins A,
surfactant proteins B, TAG-12, TFF-1, TFF-3 (also called ITF, hP1.B
and is another trefoil protein besides pS2), thrombin,
thrombomodulin, thymidine phosphorylase (TP), thymosin beta 15,
tissue cytosol ferritins, tissue polypeptide antigen (TPA), TPS
(antigen for M3 antibody), uPAI, VEGF-B, VEGF-C, VEGF-R1, VEGF-R2,
and VEGF-R3.
[0053] In general, markers can be categorized nonexclusively, and
often in overlapping categories as follows: 1. Markers that are
detected or detectable by virtue of protein expression or
overexpression (detection may occur, e.g. by immunohistochemistry
or in situ hybridization); 2. Markers that are detected or
detectable by virtue of mRNA expression or overexpression
(detection may occur, e.g. by differential display techniques); 3.
Markers that are detected or detectable by virtue of a post
translational change in a protein, e.g. a phosphorylation of the
protein, a ubiquitination, a famesylation, methylation, or other
modification to the protein that can be detected, e.g. by
antibodies specific to the post translational modification; and 4.
Markers may also be detected based on alteration of a gene, for
example methylation of the gene, for example, methylation of the
retinoic acid receptor beta-2 gene (RARbeta-2), as described in
Widschwenedter et al., J. Natl Cancer Inst. 2000 92
(10):826-32.
[0054] Genes that are overexpressed in breast cancer and can be
found by differential display include, e.g. Claudin-7,
zinc-alpha-2-glycoprotein, apolipoprotein B, B94, EST (R08988),
thrombospondin (THBS1), FGF-1, NGAL-Lipocalin 2, EST (N77731),
BS247 (Abbott labs WO 99/22027), AIB-1. Genes that are identifiable
by tyrosine phosphorylation changes include, e.g. Erb-B2 and EGFR.
Genes that are identifiable by gene methylation include e.g.
14-3-3, SPR1, cyclin D2, GST-pi, and estrogen. Markers that are
absent in tumors and are thus termed tumor suppressor markers and
when absent or lower than normal levels indicate a tumorogenic
condition, include, e.g. mammastatin, maspin, retinoic acid
receptor-beta 2, and BRMS1 (a metastasis-suppressor gene). See,
Cancer Res 2000 60: 2764-2769.
[0055] Accordingly, markers such as the following can sought in
ductal fluid, e.g. proteins that are overexpressed, mRNA
transcripts that are over expressed, and proteins comprising post
translational modifications. For example, the following markers can
be identified to distinguish a cancer or precancer cell from a
normal cell. Proteins that are overexpressed can include e.g.
Stromelysin-3, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP),
Matrix Metalloproteinase-3 (MMP-3), Placental Isoferrintin (p43),
Nuclear Matrix Protein (NMP22), NM-200.4 specific antigen, Vascular
Endothelial Growth Factor (VEGF), Endoglin (CD105), Telomerase,
ErbB-2, ErbB-3, Carcinoembryonic Antigen (CEA), Heat Shock
protein-27 (HSP-27), Breast Cancer-specific Gene (BCSG),
Plasminogen Activator Inhibitor (PAI-1), Urokinase Plasminogene
Activator (uPA), Urokinase Plasminogene Activator Receptor (uPAR),
Colony Stimulating Factor-1 (CSF-1), Colony Stimulating Factor-1
receptor (fins), Annexin 1, Vasopressin, the CC Chemokine Regulated
on Activation Normal T cell Expressed and Secreted (RANTES), 44-3A6
specific antigen, A-80 specific antigen, MUC-1, H23 specific
antigen, 83 D4 specific antigen, SP-2 specific antigen, 323/A3
specific antigen, tumor associated antigen-72 (TAG-72), and MBE6
specific antigen.
[0056] Other breast cancer markers detected by any means including
e.g. protein expression, mRNA expression, or post translational
modification can include e.g. (listed alphabetically) alanine
aminopeptidase, alpha 6 integrin, alpha-lactalbumin, AN43, p53,
Bcl2-antagonist of cell death (Bad), Bcl2-associated athanogene
(BAG-1), Bcl2-antagonist/killer 1 (Bak), Bcl2-associated X protein
(Bax), Breast cancer antigen 225 (BCA225), B-cell CLL/lymphoma 2
(Bcl-2), Bcl2-like 1 (Bcl-x), beta 1-6 branched oligosaccharides,
beta-2 microglobulin (BMG), Bcl2 related protein A1 (Bfl-1), bone
sialoprotein (BSP), CCAAT/enhancer-binding protein liver-enriched
inhibitory protein (C/EBPbeta-LIP), Carcinoma Antigen 1 (Ca 1),
Carcinoma Antigen 27.29 (CA 27.29), Carcinoma Antigen M26 (CA M26),
Carcinoma Antigen M29 (CA M29), Carcinoma Antigen 125 (CA125),
Carcinoma Antigen 15.3 (CA15.3), Carcinoma Antigen 195 (CA195),
Carcinoma Antigen 19-9 (CA19-9), Carcinoma Antigen 50 (CA50),
Carcinoma Antigen 549 (CA549), Cadherin-11, calcitonin receptor
(CTR), cathepsin B, cathepsin L, Endoglin (CD105), CD24, CD34
(pan-endothelial marker), CD44, c-met/hepatocyte growth factor
receptor, c-myc, cyclooxygenase-1 (Cox-1), cyclooxygenase-2
(Cox-2), caspase-3 (CPP32), Cyclic nucleotide phosphodiesterase,
cyclin E, DNA topoisomerase II-alpha, DNA topoisomerase II-beta,
EGF, EGF receptor, E-selectin, fast homoarginine-sensitive alkaline
phosphatase (FHAP), fatty acid synthase, ferritin, gross cystic
disease fluid protein (GCDFP-15/BRST-2), metastasis-associated
h-mtsl (S100A4), heat shock cognate protein-73 (hsc73), heat shock
protein-70 (hsp70), heat shock protein-90 alpha (hsp90alpha), heat
shock protein-90 beta (hsp90beta), inhibitors of differentiation-1
(ID1), inhibitors of differentiation-3 (ID3), interleukin-1 beta,
Keratin 8, Keratin 18, Keratin 19, Laminin, Laminin receptor
(MLuC5), Leucine Aminopeptidase (LAP), lipid-bound sialic acid
(LSA), Melanoma antigen-1 (MAGE-1), Melanoma antigen-2 (MAGE-2),
Melanoma antigen-3 (MAGE-3), Man6-P glycoproteins, Mucin-like
carcinoma associated antigen (MCA), myeloid cell leukemia-1
(Mcl-1), metallothionein (MT), mitogen-activated protein kinase
phosphatase-1 (MKP-1), Matrix Metalloproteinase-2 (MMP-2), Matrix
Metalloproteinase-9 (MMP-9), mammary serum antigen (MSA), breast
cancer mucin-2 (MUC-2), breast cancer mucin-3 (MUC-3), breast
cancer mucin-6 (MUC-6), Nm23 nucleoside diphosphate kinase,
ornithine decarboxylase (ODC), osteopontin (OPN), P114 (MAR binding
protein), P120 (a nucleolar proliferation antigen), focal adhesion
kinase p125FAK, nuclear autoantigen p330d/CENP-F, plasminogen
activator inhibitor-2 (PAI-2), Pepsinogen C, placental alkaline
phosphatase (PLAP), Platelet factor 4 (angiogenic marker), protein
kinase C (PKC), prostate specific antigen (PSA), pyrimidine
nucleoside phosphorylase, ras p21, reduced glutathione (GSH),
retinoid X receptor alpha, ribosomal S2 protein, sialyltransferase,
Stromelysin-1 (MMP-3), surfactant proteins A, surfactant proteins
B, tumor associated antigen-12 (TAG-12), trefoil gene TFF1, trefoil
gene TFF3/ITF/hP1.B, Thrombin, Thrombomodulin, thymidine
phosphorylase (TP), thymosin beta 15, tissue cytosol ferritins,
tissue polypeptide antigen (TPA), tissue polypeptide specific
antigen (TPS), Vascular Endothelial Growth Factor -B (VEGF-B),
Vascular Endothelial Growth Factor-C (VEGF-C), Vascular Endothelial
Growth Factor receptor-1 (VEGFR1), Vascular Endothelial Growth
Factor receptor-2 (VEGFR2), and Vascular Endothelial Growth Factor
receptor-3 (VEGFR3).
[0057] With respect specifically to nuclear matrix proteins the
process is similar as for other markers: the ductal fluid can be
examined to determine the presence of a marker comprising an
expression product of a gene encoding a nuclear matrix protein. The
expression product can comprise a nucleic acid or a polypeptide.
The expression product can comprises an RNA. The expression product
can comprise a protein or a part of a protein. The nuclear matrix
protein can be selected from the group consisting of lamin A, lamin
B, lamin C, a peripheral matrix protein, nuclear mitotic spindle
apparatus protein (NuMA), topoisomerase II, and an internal nuclear
matrix protein. The expression product can be a polypeptide and
examining the polypeptide can comprise contacting the polypeptide
marker with an antibody that specifically binds a portion of the
polypeptide.
[0058] The expression product can be a nucleic acid and examining
it can comprise detecting the presence of the nucleic acid.
Detecting the presence of the nucleic acid can comprise amplifying
the nucleic acid, e.g. by PCR. For example, the ductal fluid can be
tested for the presence of NMP66, a nuclear matrix protein specific
for breast cancer. The advantage of testing for NMP molecules by
retrieval of ductal fluid is that the sensitivity is increased, and
the likelihood of early detection of ductal carcinoma in situ is
increased by measuring a local concentration of the marker and not
a concentration derived from the whole body. This advantage is also
aided by the opportunity that ductal fluid and cell retrieval via a
known and identified duct provides the opportunity to treat the
duct or ductal system and not necessarily the breast. This may be
important especially where precancer (such as ductal carcinoma in
situ) is detected, and such detection does not necessarily warrant
either a systemic treatment, nor a treatment directed to active,
invasive cancer. Detection of breast cancer specific NMPs is
described, e.g., in U.S. Pat. No. 5,914,238.
[0059] Once the ductal fluid is located or isolated, the fluid can
be tested for the presence of one or more expression products of
genes encoding nuclear matrix proteins (e.g. either an RNA or a
polypeptide) in order to evaluate a presence of cancerous or
precancerous cells in the duct. Such tests can typically be
antibody or nucleic acid amplification tests that are commonly
performed in the art of marker detection. The gene products
identified can be any nuclear matrix protein gene product,
including nuclear matrix gene products specific to malignancy in
the breast, and possibly including e.g. lamin A, lamin B, lamin C,
a peripheral matrix protein, nuclear mitotic spindle apparatus
protein (NuMA), topoisomerase II, or an internal nuclear matrix
protein. Assays, kits and methods described in U.S. Pat. Nos.
5,965,376, 5,914,238, 5,882,876, 5,858,683, 5,840,503, 5,830,677,
5,783,403, 5,780,596, 5,698,439, 5686,562, or 5,547,928 can be
adapted and applied to testing ductal fluid samples.
[0060] Once the ductal fluid is analyzed for one or more markers,
the fluid may also be analyzed cytologically to determine the
cytological status of the ductal epithelial cells and other cells.
Cytological assays that can be performed on the cells retrieved
from a duct or from nipple aspirate can include e.g. assays
described in King et al, J Nat'l Cancer Inst (1983) 71:1115-21,
Wrensch et al. (1992) Am. J. Epidem. 135: 130-141, Papanicolaou et
al, (1958) Cancer, 11:377-409 and Goodson W H & King E B,
Chapter 4: Discharges and Secretions of the Nipple, THE BREAST:
COMPREHENSIVE MANAGEMENT OF BENIGN AND MALIGNANT DISEASES (1998)
2.sup.nd Ed. vol 2, Bland & Kirby eds. W. B. Saunders Co,
Philadelphia, Pa. pp. 51-74. For example, as described in Goodson
and King (page 60) a typical hyperplasia presents as having
cellular abnormalities, increased coarseness of the chromatin, and
tendency for more single cells as well as groups of cells. With
regard to carcinoma in situ, Papanicolaou et al, described cellular
abnormalities, e.g. nuclear abnormalities diagnosed by cytology of
fluid from nipple secretions containing ductal cells. The cytology
of abnormal cells can also be conducted as described in Sartorius
et al (1977) J. Natl Cancer Inst 59: 1073-1080. and King et al,
(1983) JNCI 71(6) 1115-1121. Atypia and carcinoma in situ are
widely characterized pathologically, as described in Page et al,
(1998) Mod Pathol 11(2): 120-8. The ductal fluid can be analyzed by
cytological techniques by placing some of the fluid on a slide with
a standard cytological stain using a light microscope. The cells
can be studied for a typical growth patterns in individual cells
and clusters of cells using published methods, including Mouriquand
J, (1993) S Karger Pub, "Diagnosis of Non-Palpable Breast Lesions:
Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic
and Prognostic Implications of Cytology" (ISBN 3805557477); Kline
TS and IK, Pub Igaku-Shoin Medical ""Breast: Guides to Clinical
Aspiration Biopsy" (LSBN 0896401596; Masood, American Society of
Clinical Pathology: November 199S, "Cytopathology of the Breast"
ISBN 0891893806; and Feldman P S, American Society of Clinical
Pathology, November 1984, "Fine Needle Aspiration Cytology and Its
Clinical Applications: Breast and Lung" ISBN 0891891846.
[0061] Other references that discuss cytological analysis and which
give guidance to an analysis of ductal epithelial cells derived
from ductal fluid include Silverman et al, (Can FNA biopsy separate
a typical hyperplasia, carcinoma in situ, and invasive carcinoma of
the breast?: Cytomorphologic criteria and limitations in diagnosis,
Diagnostic Cytopathology) 9(6):713-28, 1993; Masood et al,
(Immunohistochemical differentiation of a typical hyperplasia vs.
carcinoma in situ of the breast) Cancer Detection & Prevention.
16(4):225-35, 1992; Masood et al, (Cytologic differentiation
between proliferative and nonproliferative breast disease in
mammographically guided fine-needle aspirates) Diagnostic
Cytopathology.7(6):581-90, 1991; Masood S., (Occult breast lesions
and aspiration biopsy: a new challenge) Diagnostic Cytopathology.
9(6):613-4, 1993; Masood S., (Prognostic factors in breast cancer:
use of cytologic preparations) Diagnostic Cytopathology.
13(5):388-95, 1995; Novak and Masood, (Nuclear grooves in
fine-needle aspiration biopsies of breast lesions: do they have any
significance?) Diagnostic Cytopathology. 18(5):333-7, 1998; Sidawy
et al, (Interobserver variability in the classification of
proliferative breast lesions by fine-needle aspiration: results of
the Papanicolaou Society of Cytopathology Study) Diagnostic
Cytopathology. 18(2):150-65, 1998; Masood et al, (Automation in
cytology: a survey conducted by the New Technology Task Force,
Papanicolaou Society of Cytopathology) Diagnostic Cytopathology.
18(1):47-55, 1998; and Frykberg and Masood Copeland EM 3d. Bland
KI., (Ductal carcinoma in situ of the breast) Surgery, Gynecology
& Obstetrics 177(4):425-40, 1993.
[0062] As discussed, the cells collected can comprise ductal
epithelial cells and the ductal fluid collected can comprise
molecular and cellular material. The collected cells and fluid and
fluid components can be analyzed, e.g. as described or suggested
herein. Fluid collected from the milk ducts, can include
constituents of biological fluids, e.g. those typically found in
breast duct fluid, e.g. water, cells, cellular markers, molecular
markers, nucleic acids, proteins, cellular debris, salts, particles
or organic molecules. These constituents can be analyzed by any
appropriate method depending on the marker and the diagnostic
purpose. In addition, any of the cells of the duct can be analyzed
for morphological abnormalities in cell components, including, e.g.
morphological abnormalities of the nucleus, cytoplasm, Golgi
apparatus or other parts of a cell. Cell morphology can serve to
establish whether the ductal epithelial cells are normal (i.e. not
precancerous or cancerous or having another noncancerous
abnormality), precancerous (i.e. comprising hyperplasia, a typical
ductal hyperplasia (ADH) or low grade ductal carcinoma in situ
(LG-DCIS)) or cancerous (i.e. comprising high grade ductal
carcinoma in situ (HG-DCIS), or invasive carcinoma). Analysis of
cell contents may serve to establish similar staging as established
by morphology, capturing generally a progression of a precancerous
or cancerous condition in the cells.
[0063] Once the ductal fluid sample is retrieved from the breast it
is examined for the presence of a marker such as, for example a
protein, a polypeptide, a peptide, a nucleic acid, a
polynucleotide, an mRNA, a small organic molecule, a lipid, a fat,
a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide,
and a chromosomal abnormality, a whole cell having a marker
molecule, a particle, a secreted molecule, an intracellular
molecule, and a complex of a plurality of molecules as described
above. In addition, the marker may be capable of differentiating
between any two of cytological categories consisting of normal,
abnormal, hyperplasia, atypia, ductal carcinoma, ductal carcinoma
in situ (DCIS), ductal carcinoma in situ--low grade (DCIS-LG),
ductal carcinoma in situ--high grade (DCIS-HG), invasive carcinoma,
a typical mild changes, a typical marked changes, a typical ductal
hyperplasia (ADH), insufficient cellular material for diagnosis,
and sufficient cellular material for diagnosis. These categories
classify the epithelial cells cytologically, and these
classifications may indicate either cancer or its precursors, or
absence of cancer indicia. The marker may be capable of
differentiating between any two of cytological categories
consisting of normal, abnormal, hyperplasia, atypia, ductal
carcinoma, ductal carcinoma in situ (DCIS), ductal carcinoma in
situ--low grade (DCIS-LG), ductal carcinoma in situ--high grade
(DCIS-HG), invasive carcinoma, a typical mild changes, a typical
marked changes, a typical ductal hyperplasia (ADH), insufficient
cellular material for diagnosis, and sufficient cellular material
for diagnosis.
[0064] For example, the number of epithelial cells in ductal lavage
samples may range from none to several thousand. At least ten
epithelial cells are required to designate a sample as adequate.
Benign duct cells may be present singly, in monolayer sheets, or in
tight clusters, using one to two cell layers thick. The cells are
small with small nuclei (in a range from about 8 to about 12 .mu.m
in diameter). The nuclear to cytoplasmic ration may appear to be
high depending on the orientation of the cells in clusters. Single
benign duct cells are often difficult to identify, often appearing
similar to surrounding lymphocytes or histiocytes. Duct cells may
be recognized by the columnar shape of their cytoplasm, or by the
presence of discreet small vacuoles in the cytoplasm. The smooth,
discrete cytoplasmic border may also help to distinguish duct
cells. Benign duct cells are more easily recognized when they occur
in groupings. Cohesive groups, as opposed to looser clusters, are
more suggestive of epithelial origin. Benign groups are one or two
cell layers in thickness, and are composed of cells which are
uniform in size. The cell nuclei are also uniform in size, and are
regularly round to oval in shape. Markers that may be identified in
addition to cytological notations, may assist a diagnosis by
confirming a cytological reading and or adding additional
information to any noteworthy subcategory within the category of
benign.
[0065] The cytological category including a typical epithelial
cells, with mild changes, includes duct cells from proliferative
conditions including hyperplasia. The cells may occur singly, in
cohesive multilayered and complex groups, and in monolayers. The
groups may show an increase in the number of cell layers, which can
be appreciated by focusing through the groups. Duct groupings also
may show increased overlap, with nuclear crowding. The cells may be
minimally enlarged, and may show moderate increases in nuclear
size, in a range from 12 to 16 .mu.m in diameter. Slight
anisonucleosis may be present among cells in groups. Nucleoli are
often present. Markers found in the ductal fluid may assist to
identify a typical cells or a typical cells with mild changes, or
may confirm such cytological identification.
[0066] Atypical cells can also include a typical cells with marked
changes. More marked changes are often associated with a typical
hyperplasia and low grade ductal carcinoma in situ (DCIS). Enlarged
duct cells may be present, showing more marked nuclear increase and
variation in size and shape. Single cells are enlarged, with the
cytoplasm in some cases abundant, nuclear-to-cytoplasmic rations
may actually appear decreased. Chromatin may appear coarse, with
mild abnormality in distribution. Nucleoli may be larger, multiple,
and more prominent. Nuclei in groups may appear to be overlapping.
Mitotic figures may be seen. Markers found in the ductal fluid may
assist to identify a typical cells with marked changes, or may
confirm such cytological identification.
[0067] Malignant epithelial cells include duct cells from high
grade breast carcinoma and exhibit common features of malignancy.
More single cells are present, as cell cohesiveness is lost. Loose
clusters of epithelial cells are present, along with the more usual
tight groups of cells. Cell and nuclear enlargement may be marked.
High nuclear to cytoplasmic ratios may be present in some cases.
However, some high grade specimens often have lots of cytoplasm in
a portion of the tumor cells, resulting in low or variable
nuclear-to-cytoplasmic ratios. Nuclear membranes are often
irregular, and chromatin is clumped, hyperchromatic, and unevenly
dispersed. Nucleoli are often large and conspicuous and may be
multiple. Marked variation among the cells can be seen in terms of
cell and nuclear size. Accompanying these changes is often a
background of necrotic debris. Microcalcifications may be seen in
the background material. These may appear as dense material with
smooth borders and concentric layers, or may be dystrophic,
amorphous in nature. Markers found in the ductal fluid may assist
to identify malignant cells, aspects of malignant indicia, or may
confirm such cytological identification. Markers may also help to
stage the malignancy or provide other valuable information which
might aid in directing a detailed diagnosis and/or viable treatment
options.
[0068] Other cytological criteria and processes related to ductal
fluid analysis are described in Barret et al, Acta Cytol
1976;20:174-180; Goodson et al, Discharges and Secretions of the
Nipple, THE BREAST: COMPREHENSIVE MANAGEMENT OF BENIGN AND
MALIGNANT DISEASES, Second Edition, Vol. 1, Chapter 4, page 1; King
et al, Cytometry 1984; 5: 124-130; King et al, A.J.C.P. 1975; 64:
739-748; King et al, A.J.C.P. 1975; 64: 728-738; King et al,
Cytopathology of Abnormal Mammary Duct Epithelium, Prevention and
Detection of Cancer, Part II, Detection, vol 2 Cancer detection in
specific sites, 1976; King et al, J Natl Cancer Inst, 1983; 71:
1115-1121; Kjellgren et al, Acta Cytol 1964; 8: 216-217; Masood et
al, The Breast Journal 1999; 5:1-2; Papanicolaou et al, Cancer
1958; 377-409; Petrakis et al, Cancer Epidemiology, Biomarkers and
Prevention 1993; 2:3-10; Ringrose et al, Acta Cytol 1966;
10:373-375; Sartorius et al, NCI 1977; 59:1073-1080; Sauter et al,
British J. Cancer 1997; 76(4):494-501; Wrensch et al, Amer J. Epid.
1992; 135: 130-141.
[0069] The invention also provides systems for diagnosing breast
cancer or precancer comprising a tool to retrieve ductal fluid from
a breast duct and instructions for use to determine the presence of
a marker identified. The systems can comprise kits which include a
ductal access tool, for example in order to retrieve the ductal
fluid, e.g. especially where it is preferred that the ductal fluid
be identified as coming from a particular duct (so that the duct
can be accessed later for treatment or further monitoring). The
instructions in the kits can include directions according to the
methods of identifying breast cancer or precancer described herein,
and including any marker or markers or marker classification group
or groups that are described herein. The kit or system can include
assay reagents for detecting the marker or markers. The kit may
comprise a panel for testing a plurality of markers either
simultaneously or sequentially, or some practical combination of
testing modalities. The kit can also include indexes and parameters
for making a diagnosis, depending on the marker or markers. The kit
can include a container for the kit contents.
[0070] The following table supplies some exemplary markers and
marker categories and publications that identify particular
identification methods for that category of marker and/or for the
specific marker as well.
1TABLE I NOTES (further description; marker forms and # CATEGORY
SPECIFIC EXAMPLES identification contexts; assays) 1 Integral
membrane protein Claudin Kubota et al, Curr Biol. 1999; 9(18):
1035-8 (e.g. claudins 1, 2, or 3) Furuse et al, J Cell Biol. 1998;
141(7): 1539-50 2 Glycoprotein Zinc-alpha-2-glycoprotein
Lopez-Boado et al, Breast Cancer Res. Treat 1994; Hormone
responsive protein 29(3): 247-58 Cytosolic protein Diez-Itza et al,
Eur. J. Cancer 1993; 29A(9): 1256-60 Chaubert and Hurlimann, Arch
Pathol Lab Med 1992; 116(11): 1181-8 3 Aspartyl proteinase Gross
cystic disease fluid Chaubert and Hurlimann, Arch Pathol Lab Med
1992; Differentiation associated protein-15 kD (GCDFP-15) 116(11):
1181-8 protein Caputo et al, J Biol Chem, 2000; 275(11): 7935-41 4
Lipoprotein Apolipoprotein Lane et al, Breast Cancer Res Treat
1995; 34(2): 161-9 5 Peptide CD36-binding peptide Carron et al,
Biochem Biophys Res Comm 2000 270(3): 1124-1127 6 Ligand
Thrombospondin-1 Carron et al, Biochem Biophys Res Comm 2000
Extracellular matrix protein 270(3): 1124-1127 Murphy-Ullrich and
Poczatek, Cytokine Growth Factor Rev 2000; 11(1-2): 59-69 7 Growth
factor Fibroblast growth factor Femig et al, Biochem Biophys Res
Comm 2000; 267(3): 70-6 8 Lipocalin Neu-related Zerega et al, Eur J
Cell Biol 2000; 79(3): 165-72 lipocalin/neutophil gelatinase-
associated lipocalin (NRL/NGAL) 9 Hormone responsive protein
Amplified in breast cancer-1 Eng et al, J Biol Chem 1998; 273(43):
28371-7 (AIB-1) 10 Hormone responsive protein Transcriptional
intermediary Eng et al, J Biol Chem 1998; 273(43): 28371-7 factor-1
and -2 (TIF-1 & -2) 11 Transferase Glutathione S-transferase pi
Huang et al, Oncol Rep 2000; 7(3): 609-13 (GST-pi) 12
Differentiation associated SPR-1 Anisowicz et al, Mol Med 1999;
5(8): 526-41 protein 13 Differentiation associated HME-1 (25 kd)
Prasad et al, Cell Growth Differ 1992; 3(8): 507-13 protein 14-3-3
sigma protein USPN 5,776,676 (nucleic acid) Activator of tyrosine
and stratifin USPN 4,707,438 (immunoassay) tryptophan hydroxylase
Isobe et al, J. Mol Biol 1991 217(1): 125-32 USPN 5,597,719 (Raf
interaction) USPN 5,424,191 (polypeptide) 14-3-3 sigma accession
no.: AFO29082 Laronga et al, J Biol Chem, 2000: Apr. 14 (no page
numbers available) 14 Proto-oncogene Cyclin D1 Bukholm et al,
Virchows Arch 1998; 433(3): 223-8 Cyclin Sweeney et al, Oncogene
1997; 14(11): 1329-40 Buckley et al, Oncogene 1993; 8(8): 2127-33
Weinstat-Saslow et al, Nat Med 1995; 1(12): 1257-60 Barnes and
Gillett, Breast Cancer Res Treat 1998; 52(1- 3): 1-15 Reed et al,
Virchows Arch 1999; 435(2): 116-24 15 Growth factor Vascular
endothelial growth Toi et al, Clinical Cancer Res. 1995; 1: 961-964
factor (VEGF) Lichtenbeld et al, Int J Cancer 1998; 77(3): 445-9
Vascular permiability factor Guidi et al, Cancer 1997; 80(10):
1945-53 (VPF) Locopo et al, Breast Cancer Res Treat 1998; 52(1- 3):
159-73 Wright et al, Exp Mol Pathol 1997; 64(1): 41-51 Yoshiji et
al, Cancer Res 1996; 56(9): 2013-6 Brown et al, Human Pathol 1995;
26(1): 86-91 16 Receptor Vascular endothelial growth Yoshiji et al,
Cancer Res 1996; 56(9): 2013-6 factor receptor (VEGF-R) flt-1 17
Proteolytic factor Cathepsins D and L Harbeck et al, Int J Markers
2000 15(1): 79-83 Ioachim et al, Anticancer Res 1998; 18(3A):
1665-70 18 Tumor suppressor Maspin Domann et al, Int J Cancer 2000;
85(6): 805-10 Angiogenesis inhibitor USPN 5,470,970 Zou et al, J
Biol Science, 2000; 275(9): 6051-4 Zhang et al, Nat Med 2000; 6(2):
196-9 19 Receptor Fas Mottolese et al, Int J Cancer 2000 89(2):
127-32 Tumor necrosis family Apo-1 Reimer et al, Cancer Res 2000;
60(4): 822-8 Apoptosis pathway protein CD95 Cell death protein 20
Ligand Fas ligand (fasL) Mottolese et al, Int J Cancer 2000 89(2):
127-32 Tumor necrosis family Reimer et al, Cancer Res 2000; 60(4):
822-8 Apoptosis pathway protein Cell death protein 21 Inhibitor
Tissue inhibitor of Brummer et al, Virchows Arch 1999; 435(6):
566-73 metalloproteinase 1 and 2 Remacle et al, Int J Cancer 2000;
89(2): 118-21 (TIMP-1, TIMP-2) Luparello et al, Breast Cancer Res
Treat 1999; 54(3): 235-44 22 Chemokine CC chemokine regulated on
Luboshits et al, Cancer Res. 1999; 59(18): 4681-7 activation normal
T cell Prest et al, Clin Exp Metastasis, 1999; 17(5): 389-96
expressed and secreted Luboshits et al, Cancer Res. 1999; 59:
4681-4687 (RANTES) 23 Chemokine Macrophage inflammatory Tedla et
al, Cytokine 1999; 11(7): 531-40 protein 1 alpha and 2 beta Prest
et al, Clin Exp Metastasis, 1999; 17(5): 389-96 (MIP-1 alpha, MIP-2
beta) 24 Cell adhesion molecule E-cadherin and N-cadherin Hazan et
al, J Cell Biol 2000; 148(4): 779-90 Boterberg et al, Cell Adhes
Comm, 2000; 7(4): 299-310 25 Basement membrane protein LH39
(antibody recognizing Kakolyris et al, Br J Cancer 2000; 82(4):
844-51 Antibody the lamina lucida of mature small veins and
capillaries 26 Basement membrane protein Laminin Sidhom and Imam,
Int Clin Lab Res, 1999; 29(1): 26-9 Ioachim et al, Anticancer Res
1998; 18(3A): 1665-70 27 Cell adhesion molecule Integrin beta 1
Berry et al, Eur J Surg Oncol, 2000; 26(1): 25-9 28 Cell adhesion
molecule E-selectin Hebbar et al, Int J Biol Markers, 2000; 15(1):
15-21 29 Catenin (E, alpha, beta, Bukholm et al, J Pathol, 2000;
190(1): 15-9 gamma) 30 Receptor Thrombin receptor Henrikson et al,
Br J Cancer, 1999; 79(3-4): 401-6 31 Protease Urokinase plasminogen
Stephens et al, Breast Cancer Res Treat, 1998; 52(1-3): activator
(uPA) 99-111 32 Serpin Serine protease inhibitors Wojtukiewicz et
al, Haemostasis 1998; 28(1): 7-13 (SERPINS) - alpha-1-
antichymotrypsin, alpha-1- antirypsin, alpha2- macroglobulin,
antithrombinIII, C1 inhbitor, alpha2-antiplasmin 33 Tumor
suppressor Mammastatin (polypeptides Ervin et al, Science 1989;
244(4912): 1585-7 Tissue specific inhibitor 47 kD and 65 kD) 34
Protein Cytokeratin Bae et al, J Korean Med Sci 2000; 15(2): 194-8
Nucleic Acid 35 Sialic acid Lipid bound sialic acid (LSA) Raval et
al, Int J Biol Markers, 1997; 12(2): 61-7 Romppanen et al,
Anticancer Res, 1997; 17(2B): 1249-53 36 DNase Alkaline DNase (ADA)
Raval et al, Int J Biol Markers, 1997; 12(2): 61-7 37
Ribonucleoprotein Telomerase Mokbel et al, Eur J Surg Oncol 2000;
26(1): 30-3 Enzyme 38 Serine protease Prostate specific antigen
Black et al, Breast Cancer Res Treat, 2000; 59(1): 1-14 (PSA) Black
et al, Clin Cancer Res, 2000; 6(2): 467-73 39 Antibody Antibody
specific for myosin Chiavegato et al, Virchows Arch 1995; 426(1):
77-86 smooth muscle heavy chain (SM1, SM2); Antibody specific for
myosin non-muscle (NM-MyHC) 40 Proliferation associated Endoglin
(EDG, CD105) Matsuno et al, Clin Cancer Res, 1999; 5(2): 371-82
antigen 41 Nuclear protein 108 kD, 53 kD, 48 kD nuclear Brys et al,
Cytobios 2000; 101 (397): 87-94 polypeptides 42 Cytoplasmic protein
36 kD cytoplasmic Brys et al, Cytobios 2000; 101 (397): 87-94
polypeptide 43 Transforming growth factor Transforming growth
factor Humphreys and Hennighausen, Oncogene 2000; 19(8) alpha
family of proteins alpha (TGF-alpha) Bourhis et al, Int J Cancer
1997; 71(1): 42-8 Yoshiji et al, Cancer Res 1996; 56(9): 2013-6 44
Transforming growth factor Transforming growth factor Yoshiji et
al, Cancer Res 1996; 56(9): 2013-6 beta family of proteins beta
(TGF-beta) Pawlina et al, Mol Cell Endocrinol 1990; 72(1): 55-61 45
Protein factor Colony stimulating factor 1 Sapi et al, Proc Soc Exp
Biol Med 1999; 220(1): 1-8 Hormone responsive protein (CSF-1) Tang
et al, J. of Cellular Biochemistry 1992; 50: 350-356 M-CSF Sapi et
al, J Soc Gynecol Investig 1998; 5(2): 94-101 46 Receptor Colony
stimulating factor Sapi et al, Proc Soc Exp Biol Med 1999; 220(1):
1-8 Hormonre responsive protein receptor (CSF-1R) Sapi et al,
Cancer Res 1999; 59(21): 5578-85 Maher et al, Clin Cancer Res 1998;
4(8): 1851-6 Sapi et al, Oncogene 1995; 10(3): 529-42 Tang et al,
J. of Cellular Biochemistry 1992; 50: 350-356 Sapi et al, J Soc
Gynecol Investig 1998; 5(2): 94-101 Flick et al, Oncogene 1997;
14(21): 2553-61 47 Proto-oncogene c-fms Sapi et al, Proc Soc Exp
Biol Med 1999: 220(1): 1-8 Sapi et al, Cancer Res 1999; 59(21):
5578-85 Maher et al, Clin Cancer Res 1998; 4(8): 1851-6 Sapi et al,
Oncogene 1995; 10(3): 529-42 48 Phospholipid binding protein
Annexin-1 Pencil and Toth, Clin Exp Metastasis 1998; 16(2): 113-21
Actin binding protein Ahn et al, Clin Exp Metastasis 1997; 15(2):
151-6 Calcium binding protein 49 Receptor Vasopressin and Oxytocin
Zingg, Baillieres Clin Endocrinol Metab 1996; 10(1): 75- G-protein
linked receptor receptors 96 50 Vasopressin: arginine North et al,
Breast Cancer Res Treat 1995; 34(3): 229-5 vasopressin (VP), North
et al, Breast Cancer Res Treat 1995; 34: 229-235 provasopression
(ProVP), vaopressin-associated human glycopepeptide (VAG) 51
Oxytocin (OT), oxytocin North et al, Breast Cancer Res Treat 1995;
34(3): 229-5 associated human neurophysin (OT-HNP) 52 Peptidase
Alanine aminopeptidase Severini et al, Cancer Biochem Biophys 1991;
12(3): 199- (AAP) 204 53 Antigen Tissue polypeptide antigen
Severini et al, Cancer Biochem Biophys 1991; 12(3): 199- (TPA or
TPS) 204 Antigen recognized by M3 Aydiner et al, Acta Oncol 1994;
33(2): 181-6 antibody 54 Lactalbumin Alpha-lactalbumin Simickova et
al, Neoplasma 1991; 38(4): 407-13 55 Bcl2 family member Bcl-2
Schorr et al, J Mammary Gland Biol Neoplasia, 1999; Proto-oncogene
4(2): 153-64 Knowlton et al, J Surg Res, 1998; 76(1): 22-6 Veronese
et al, Int J Cancer, 1998; 79(1): 13-8 Olopade et al, Cancer J Sci
Am, 1997; 3(4): 230-7 Zhang, et al, Clin Cancer Res 1997; 3(12):
2329-35 56 Bcl2 family member Bax Schorr et al, J Mammary Gland
Biol Neoplasia, 1999; 4(2): 153-64 Knowlton et al, J Surg Res,
1998; 76(1): 22-6 Veronese et al, Int J Cancer, 1998; 79(1): 13-8
Olopade et al, Cancer J Sci Am, 1997; 3(4): 230-7 57 CCAAT/enhancer
binding C/EBP Rosen et al, Biochem Soc Symp 1998; 63: 101-13
protein family (C/EBP) 58 Bone matrix protein Bone sialoprotein
(BSP) Castronovo and Bellahcene, Int J Oncol 1998; 12(2): 305-8 59
Metastasis associated protein CA-15-3 Aydiner et al, Acta Oncol
1994; 33(2): 181-6 Carbohydrate antigen Guarner et al, Arch Med Res
1997; 28(4): 523-6 60 Metastasis associated protein Beta-2
microglobulin (BMG) Aydiner et al, Acta Oncol 1994; 33(2): 181-6 61
Metastasis associated protein ferritin Aydiner et al, Acta Oncol
1994; 33(2): 181-6 62 Glycoprotein P-glycoprotein (MDR1 or Sikic,
Ann Oncol 1999; 10 suppl 6: 149-53 MRP gene expression Kroger et
al, Cancer Treat Rev 1999; 25(5): 279-91 product) 63 CCAAT/enhancer
binding ICBP90 (89, 758 kD) Hopfner et al, Cancer Res 2000; 60(1):
121-8 protein family (C/EBP) 64 Enzyme Aromatase (CYP19)
Brueggemeier et al, Cancer Lett 1999; 140(1-2): 27-35 65 Enzyme
Cyclo-oxygenase 1, 2 (COX- Brueggemeier et al, Cancer Lett 1999;
140(1-2): 27-35 1, COX-2) Liu and Rose, Cancer Res 1996; 56(22):
5125-7 Prostoglandin endoperoxide synthase 66 Antigen PGE2
Brueggemeier et al, Cancer Lett 1999; 140(1-2): 27-35 67 Hormone
responsive protein Hormone induced gene-1 Russo and Russo, J Cell
Biochem Suppl 2000; 34: 1-6 (HI-1) 68 Heat shock protein BAG-1
(Hsp70/Hsc70) Krajewski et al, Endocr Relat Cancer 1999; 6(1):
29-40 69 Bcl2 family of proteins Bcl-X(L) Krajewski et al, Endocr
Relat Cancer 1999; 6(1): 29-40 70 Caspase Caspase-3 Krajewski et
al, Endocr Relat Cancer 1999; 6(1): 29-40 71 Antigen Melanoma
associated Fujie et al, Ann Oncol 1997; 8(4): 369-72 Tumor
rejection antigen antigen-1, -3 (MAGE-1, -3) Toso et al, Cancer Res
1996; 56(1): 16-20 MZ2-E (antigen) Brassaur et al, Int J Cancer
1992; 52(5): 839-41 MZ2-D (antigen) Gaugler et al, J Exp Med 1994;
179(3): 921-30 72 Antigen SART-1 (800 kD antigen) Kawamoto et al,
Int J Cancer 1999; 80(1): 64-7 Tumor rejection antigen 73 Inhibitor
P16 (INK4, MTS-1) Jaffrain-Rea et al, Clin Endo 1999; 51: 317-325
Inhibitor or cyclin D-CDK4 complex 74 Nuclein Breast cancer
specific gene-1 Ji et al, Cancer Res 1997; 57(4): 759-64 (BCSG-1)
Lavedan et al, Hum Genet 1998; 103(1): 106-12 gamma-synuclein
(SNCG) Jia et al, Cancer Res. 1999; 59(3): 742-7 SNC-gamma Ji et
al, Cancer Res 1997; 57: 759-764 75 Peptide Fibrinogen degredation
WO 98/55872 peptide 76 Gap junction protein Connexin 26 Jamieson et
al, J Pathol 1998; 184(1): 37-43 77 Gap junction protein Connexin
43 Jamieson et al, J Pathol 1998; 184(1): 37-43 78 Fibronectin
Ioachim et al, Anticancer Res 1998; 18(3A): 1665-70 79 Inhibitor
Relaxin Pawlina et al, Mol Cell Endocrinol 1990; 72(1): 55-61 80
Growth factor Fibroblast growth factor - Yoshiji et al, Cancer Res
1996; 56(9): 2013-6 basic (FGFb) 81 Antigen Carcinoembryonic
antigen Kuhajda et al, Cancer 1983; 52: 1257-1264 Dedifferentiation
marker (CEA) Schmitt and Andrade, J Clin Pathol 1995; 48(1): 53-6
Mangili et al, Cancer 1996; 78(11): 2334-9 82 Differentiation
marker Human milk fat globulin DePotter et al, Pathol Res Pract
1988; 183(3): 271-6 (HMFG) 83 Phosphoprotein p53 Poller et al, Br J
Cancer 1992; 66: 583-588 Tumor suppressor Poller et al, Hum Pathol
1993; 24(5): 463-8 Zhang, et al, Clin Cancer Res 1997; 3(12):
2329-35 Schmitt et al, J Pathol 1995; 176(3): 233-41 Rajan et al,
1997; 42(3): 283-90 Rudas et al, Eur J Cancer 1997; 33(1): 39-44
Lisboa et al, Virchows Arch 1997; 431(6): 375-81 Done et al, Cancer
Res. 1998; 58(4): 785-9 Naidu et al, Anticancer Res. 1998; 18(1A):
65-70 Jerry et al, Oncogene 2000; 19(8): 1052-8 Ioachim et al,
Anticancer Res 1998; 18(3A): 1665-70 Midulla et al, Anticancer Res
1999; 19(5B): 4033-7 84 Receptor Epidermal growth factor Poller et
al, Br J Cancer 1992; 66: 583-588 receptor Poller et al, Hum Pathol
1993; 24(5): 463-8 Ioachim et al, Anticancer Res 1998; 18(3A):
1665-70 85 Oncogene c-erbB-2 Poller et al, Br J Cancer 1992; 66:
583-588 Oncoprotein Poller et al, Hum Pathol 1993; 24(5): 463-8
Lodata et al, Mod Pathol. 1990; 3(4): 449-54 Ioachim et al,
Anticancer Res 1998; 18(3A): 1665-70 Zaretsky et al, FEBS 1990;
265(1-2): 46-50 Mangili et al, Cancer 1996; 78(11): 2334-9 86
Oncogene c-erbB-3 Naidu et al, Br. J. Cancer 1998; 78(10): 1385-90
Oncoprotein 87 Antigen Her-2/neu Allred et al, Hum Pathol. 1992;
23(9): 974-9 Storm et al, Ann Surg Oncol 1995; 2(1): 43-8 88 Heat
shock protein Heat shock protein - 27 Storm et al, Ann Surg Oncol
1995; 2(1): 43-8 89 Protease Urokinase plasminogen Kennedy et al,
British J. of Cancer 1998; 77(10): 1638- activator (uPA) 1641
Bianchi et al, Cancer Res. 1994; 54: 861-866 Grondahl et al, Cancer
Res. 1993; 53: 2513-2521 90 Receptor Urokinase plasminogen Bianchi
et al, Cancer Res. 1994; 54: 861-866 activator receptor (uPAR) 91
Proteolytic factor Urokinase-type plasminogen Grondahl et al,
Cancer Res. 1993; 53: 2513-2521 inhibitor activator (uPA) inhibitor
type Harbeck et al, Int J Markers 2000 15(1): 79-83 1 (PAI-1)
Bianchi et al, Int J. Cancer 1995; 60(5): 597-603 Harbeck et al,
Breast Cancer Res Treat 1999; 54(2): 147- 57 92 Whole cell having a
marker Oncofetal ferritin bearing Moroz et al, Cancer 1989; 64(3):
691-7 molecule lymphocytes (FBL) 93 Ferritin Oncofetal ferritin
Moroz et al, Cancer 1989; 64(3): 691-7 Antigen Placental
isoferritin (p43) Auberbach et al, Abstracts and Proceedings from
10.sup.th (PLF) European Cancer Conference Sep. 12, 1999 to Sep.
16, 1999, Vienna Austria; abstract 589 Rosen et al, Breast Cancer
Res Treat 1992; 24(1): 17-26 Reinerova et al, Neoplasma 1996;
43(6): 363-6 Rosen et al, Cancer Lett 1991; 59(2): 145-51 Stierer
et al, Breast Cancer Res Treat 1991; 19(3): 283-8 Rosen et al,
Cancer Lett 1992; 67(1): 35-45 94 Protein Type IV
collagen Ioachim et al, Anticancer Res 1998; 18(3A): 1665-70 95
Proliferation associated Ki-67 Ioachim et al, Anticancer Res 1998;
18(3A): 1665-70 antigen 96 Proliferation associated PCNA Ioachim et
al, Anticancer Res 1998; 18(3A): 1665-70 antigen 97 Zinc
endoprotease Matrix metalloproteinase 1, 2, Brummer et al, Virchows
Arch 1999; 435(6): 566-73 Collagenases and 3 Jpn J Cancer Res 1999;
90: 516-522 Gelatinases (MMP-1, MMP-2, MMP-3) Polette et al, Clin
Exp Metastasis 1997; 15: 157-163 Stromelysins Collagenases (MMP-1,
Ueno et al, Cancer Res 1997; 57: 2055-2060 Matrilysin MMP-8,
MMP-13) Nakopoulou et al, Hum Pathol. 1999; 30(4): 436-442
Metalloelastase Gelatinases (MMP-2, MMP- Lee et al, Clin Exp
Metastasis 1996 14(6): 512-9 9) Polette et al, Invasion Metastasis
1993; 13(1): 31-7 Stromelysins (MMP-3, MMP- Ioachim et al,
Anticancer Res 1998; 18(3A): 1665-70 10) Su et al, Hybridoma 1995;
14(4): 383-390 Matrilysin (MMP-7) Rha et al, Breast Cancer Res
Treat 1997; 43(2): 175-81 Metalloelastase (MMP-12) MMP-14 98
Stromelysin Stromelysin-3 (ST3) Hahnel et al, Int J Cancer 1993;
55(5): 771-4 Stromelysin-3 (MMP-11) Hahnel et al, Int. J Cancer
1994; 58(2): 157-60 Kawami et al, Anticancer Res 1993; 13(6A):
2319-23 Brummer et al, Virchows Arch 1999; 435(6): 566-73 Jpn J
Cancer Res 1999; 90: 516-522 Polette et al, Clin Exp Metastasis
1997; 15: 157-163 Ueno et al, Cancer Res 1997; 57: 2055-2060
Nakopoulou et al, Hum Pathol. 1999; 30(4): 436-442 Lee et al, Clin
Exp Metastasis 1996 14(6): 512-9 Polette et al, Invasion Metastasis
1993; 13(1): 31-7 Ioachim et al, Anticancer Res 1998; 18(3A):
1665-70 Engel et al, Int J Cancer 1994; 58(6): 830-5 Su et al,
Hybridoma 1995; 14(4): 383-390 Santavicca et al, Int J Cancer 1995;
64: 336-341 Basset et al, Nature 1990; 348: 699-704 99 Zinc
endoprotease MMP-2 Talvensaari-Mattila et al, Cancer 1998; 83(6):
1153-62 collagenase 72 kD Kawami et al, Anticancer Res 1993;
13(6A): 2319-23 type IV collagenase Su et al, Hybridoma 1995;
14(4): 383-390 gelatinase 100 Kinase Nm23 Hahnel et al, Int. J
Cancer 1994; 58(2): 157-60 Nucleoside diphosphate kinase 101
Antibody Mab 44-3A6 (detecting a 40 Duda et al, Tumor Biol 1991;
12: 254-260 Antigen kD cell surface protein on adenocarcinomas) 102
Antibody Mab A-80 (detecting mucin Shin et al, APMIS 1989; 97(12):
1053-260 Antigen type glycoprotein; tumor Eriksson et al, Human
Pathol 1992; 23(12): 1366-1372 associated cytoplasmic Castagna et
al, Path Res Pract. 1992; 188: 1002-1008 mucin-type glycoprotein)
MM 1-80 Polymorphic epithelial mucin (PEM) 103 Antibody Mab to DF3
(detecting tumor Kufe et al, Hybridoma 1984; 3(3): 223-32 Antigen
associated antigen 290 kD Ohuchi et al, JNCI (1987); 79(1): 109-115
cell surface protein in breast Szpak et al, Ada Cytol. 1984; 28(4):
356-67 carcinoma cells; CA15.3 antigen) 104 Antibody H23 breast
tumor associated Zaretsky et al, FEBS 1990; 265(1-2): 46-50 Antigen
antigen Keydar et al, Proc. Natl. Acad Sci USA 1989; 86: 1362- Gene
17.5 1366 Stein et al, Int J Cancer 1991; 47(2): 163-9 105 Protein
pS2 Zaretsky et al, FEBS 1990; 265(1-2): 46-50 106 Antibody
Antibody B72.3 Stein et al, Int J Cancer 1991; 47(2): 163-9 Antigen
Tumor associated Thor et al, Cancer Res 1986; 46: 3118-3124
glycoprotein-72 (TAG-72) Prey et al, Human Pathol 1991; 22(6):
598-602 Oncofegal antigen tumor Contegiacomo et al, Eur J Cancer
1994; 30A(6): 813-820 associated glycoprotein - 72 Shousha et al, J
Clin Pathol. 1990; 43(12): 1026-8 Mangili et al, Cancer 1996;
78(11): 2334-9 Szpak et al, Acta Cytol. 1984; 28(4): 356-67 Muraro
et al, Cancer Res 1988; 48(16): 4588-96 Stein et al, Int J Cancer
1991; 47(2): 163-9 Lottich et al, Breast Cancer Res Treat 1985;
6(1): 49-56 Nuti et al, Int J Cancer 1982; 29(5): 539-45 Tavassoli
et al, Am J Surg Pathol. 1990; 14(2): 128-33 Thor et al, Sem Oncol
1986; 13(4): 393-401 107 Antigen Tn-associated antigen Konska et
al, Int J Oncol 1998; 12(2): 361-7 108 N-acetyl-lactosamine Konska
et al, Int J Oncol 1998; 12(2): 361-7 109 Lectin Lectin Konska et
al, Int J Oncol 1998; 12(2): 361-7 110 Receptor Lectin receptor
Konska et al, Int J Oncol 1998; 12(2): 361-7 111 Antibody Mab
detecting 83D4 anttgen Pancino et al, Hybridoma 1990; 9(4): 389-395
Antigen Beuzelin-Yuraut et al, J Clin Pathol 1995; 48: 433-437 112
Antigen SP-2 (90 kD antigen) Iacobelli et al, Cancer Res. 1986; 46:
3005-3010 Proteolipid 113 Antibody Mab 323/A3 (detecting Mr Edwards
et al, Cancer Res 1986; 46(3): 1306-17 Antigen 43 kD glycoprotein)
Courtney et al, Cancer Lett 1991; 57(2): 115-9 114 Antigen Ca-1
Courtney et al, Br J Cancer Suppl 1990; 10: 92-5 115 Antibody
T-antigen Teramoto et al, Cancer 1982; 50: 241-249 Antigen MBE6
antibody 116 Receptor c-met tyrosine kinase receptor Nakopoulou et
al, Histopathology 2000; 36(4): 313-25 117 Growth Factor Hepatocyte
growth factor Nakopoulou et al, Histopathology 2000; 36(4): 313-25
Ligand (HGF) Qiao et al, Cell Growth Diff 2000; 11(2): 123-33 118
Ligand Angiopoietin-1 Huang et al, Blood 2000; 95(6): 1993-9
Nucleic Acid 119 Nucleic acid Nm23 Guo et al, Chung Hua I Hsueh I
Chuan Hsueh Tsa Chih Protein 2000; 17(2): 91-93 Midulla et al,
Anticancer Res 1999; 19(5B): 4033-7 120 Protein Ki67 Midulla et al,
Anticancer Res 1999; 19(5B): 4033-7 121 Protein P21 Reed et al,
Virchows Arch 1999; 435(2): 116-24 122 Protein P27 Reed et al,
Virchows Arch 1999; 435(2): 116-24 123 Protein TKH1 Kjeldsen et al,
Cancer Res 1988; 48(8): 2214-20 Antibody TKH2 124 Antigen
Sialosyl-Tn Kjeldsen et al, Cancer Res 1988; 48(8): 2214-20
Disaccharide 125 Enzyme Lactate Dehydrogenase Kher et al, Indian J
Pathol Microbiol 1997; 40(3): 321-6 (LDH) 126 Enzyme Myosin light
chain kinase Nguyen et al, J Cell Biol 1999; 146(1): 149-64 127
Tumor suppressor beta2 microglobulin Carmon et al, Int J Cancer
2000; 85(3): 391-7 128 Protein MUC1 Carmon et al, Int J Cancer
2000; 85(3): 391-7 Peptide
EXAMPLES
[0071] 1. Retrieval of Ductal Fluid and Analysis of Markers in the
Fluid
[0072] A patient is prepared for a ductal lavage, using a ductal
access tool and a duct on each breast is lavaged and the ductal
fluid collected separately from each accessed duct. The fluid in
each duct that is accessed is analyzed for nuclear matrix proteins,
maspin, claudin 7, telomerase, basic FGF, fibrinogen degradation
peptide, and CSF-1 receptor using standard techniques.
[0073] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims
* * * * *
References