U.S. patent application number 10/639595 was filed with the patent office on 2004-02-26 for isolated ductal fluid sample.
This patent application is currently assigned to Cytyc Health Corporation. Invention is credited to Hung, David.
Application Number | 20040038281 10/639595 |
Document ID | / |
Family ID | 25179846 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038281 |
Kind Code |
A1 |
Hung, David |
February 26, 2004 |
Isolated ductal fluid sample
Abstract
A sample for diagnosis of breast cancer can be prepared by
isolating a ductal fluid sample from one duct of a breast of a
patient. The isolated ductal fluid is not mixed with ductal fluid
from any other duct of the breast. Generally the target duct is not
spontaneously discharging. The isolated ductal fluid sample can be
examined to determine the presence or absence of a marker
associated with cancer or pre-cancer. An isolated ductal fluid
sample not mixed with ductal fluid from any other duct of the
breast permits identification of the duct which is diseased and
provides increased sensitivity for existing diagnostic and analytic
techniques.
Inventors: |
Hung, David; (Belmont,
CA) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Cytyc Health Corporation
Boxborough
MA
|
Family ID: |
25179846 |
Appl. No.: |
10/639595 |
Filed: |
August 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10639595 |
Aug 13, 2003 |
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09800970 |
Mar 8, 2001 |
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6642009 |
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09800970 |
Mar 8, 2001 |
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09625399 |
Jul 26, 2000 |
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6610484 |
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09625399 |
Jul 26, 2000 |
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09502404 |
Feb 10, 2000 |
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6642010 |
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09502404 |
Feb 10, 2000 |
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09313463 |
May 17, 1999 |
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6638727 |
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09800970 |
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09473510 |
Dec 28, 1999 |
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6413228 |
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60166100 |
Nov 17, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
A61K 31/4196 20130101;
A61K 31/566 20130101; A61M 2210/1007 20130101; A61K 31/352
20130101; A61B 10/0045 20130101; A61K 31/437 20130101; C12Q 1/6806
20130101; A61M 3/0262 20130101; A61K 31/4535 20130101; A61M 3/0279
20130101; A61K 31/138 20130101; C12Q 1/6886 20130101; A61K 31/00
20130101; G01N 33/57415 20130101; A61B 10/0041 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 preparing a sample for use in diagnosis of breast
cancer or pre-cancer comprising: isolating a ductal fluid sample
from one duct of a breast of a patient, said isolated ductal fluid
not mixed with ductal fluid from any other duct of the breast.
2. A method as in claim 1, further comprising: examining the
isolated ductal fluid sample to determine the presence or absence
of a marker.
3. A method as in claim 1, wherein the duct from which the ductal
fluid is isolated is not spontaneously discharging ductal
fluid.
4. A method as in claim 2, wherein the marker is selected from the
group consisting of: lysophosphatidic acid, a lysophospholipid,
paladin, a portion of palladin, a nucleic acid encoding a
polypeptide comprising at least a portion of paladin, Lg, a portion
of Lg, a nucleic acid encoding a polypeptide comprising at least a
portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding a
polypeptide comprising at least a portion of E2F1, T1A12/mac 25, a
portion of T1A12/mac 25, a nucleic acid encoding a polypeptide
comprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a
portion of MAGUKIZO-1, a nucleic acid encoding a polypeptide
comprising at least a portion of MAGUK/ZO-1, repressor of estrogen
receptor activity (REA), a portion of REA, a nucleic acid encoding
a polypeptide comprising at least a portion of REA, prothymosin
alpha (PTA), a portion of PTA, a nucleic acid encoding a
polypeptide comprising at least a portion of PTA, c-raf kinase, a
portion of c-raf kinase, a nucleic acid encoding a polypeptide
comprising at least a portion of c-raf kinase, CD66a, a portion of
CD66a, a nucleic acid encoding a polypeptide comprising at least a
portion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding
a polypeptide comprising at least a portion of KL-1, cell adhesion
molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acid
encoding a polypeptide comprising at least a portion of CAM 5.2,
leptin, a portion of leptin, a nucleic acid encoding a polypeptide
comprising at least a portion of leptin, Bcl-2 gene product, at
least a portion of Bcl-2 gene product or polypeptide, a nucleic
acid encoding a polypeptide encoding at least a portion of Bcl-2
gene product, nuclear matrix 23(nm23), a portion of nm23, a nucleic
acid encoding a polypeptide comprising at least a portion of nm23,
an apotosis-related protein, a portion of said protein, a nucleic
acid encoding a polypeptide comprising at least a portion of the
apoptosis-related protein, lipocalin NGAL, a portion of lipocalin
NGAL, a nucleic acid encoding a polypeptide comprising at least a
portion of lipocalin NGAL, complement regulatory protein CD46, a
portion of CD46, a nucleic acid encoding at least a portion of
CD46, complement regulatory protein CD59, a portion of CD59, a
nucleic acid encoding at least a portion of CD59, a nucleic acid
encoding a portion of an FHIT gene, loss of heterozygosity at an
FRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid
encoding at least a portion of MRP-1/CD9, KAI1/CD82, a portion of
KAI1/CD82, a nucleic acid encoding at least a portion of KAI1/CD82,
a Fibroblast Growth Factor (FGF), a portion of FGF, a nucleic acid
encoding a polypeptide comprising at least a portion of an FGF,
Vascular Epithelial Growth Factor (VEGF), at least a portion of
VEGF, a nucleic acid encoding at least a portion of VEGF,
Insulin-like Growth Factor -1 (IGF-1), at least a portion of IGF-1,
a nucleic acid encoding at least a portion of IGF-1, tumor
amplified kinase STK15 (also BTAK and aurora2), at least a portion
of STK15, a nucleic acid encoding a polypeptide comprising at least
a portion of STK15, TMS-1, a portion of TMS-1, a nucleic acid
encoding a polypeptide comprising at least a portion of TMS-1,
maspin, at least a portion of maspin, a nucleic acid encoding a
polypeptide comprising at least a portion of maspin, at least a
portion of breast cancer associated (BRCA) gene, and at least a
portion of a BRCA gene product; CDw60 protein, a portion of CDw60
protein or polypeptide, a nucleic acid encoding at least a portion
of a CDw60 protein or polypeptide, mammary expressed enzymes
including cytochrome P450s, catechol-Omethyltransferase, epoxide
hydrolase, peroxidases, glutathione S-transferases,
N-acetyltransferases, or sulfotransferases, a nucleic acid encoding
at least a portion of a mammary expressed enzyme, Kallikrein 6
(zyme/protease M/neurosin) protein or polypeptide (hK6), a nucleic
acid encoding at least a portion of an hK6 protein or polypeptide;
and mammastatin protein or polypeptide, a nucleic acid encoding at
least a portion of a mammastatin protein or polypeptide.
5. A method as in claim 1, further comprising: examining the
isolated ductal fluid to determine absorption of a molecule by
abnormal cells in the fluid.
6. A method as in claim 5, wherein the molecule comprises
iodide.
7. A method as in claim 1, further comprising: examining the
isolated ductal fluid for a loss of heterozygozity.
8. A method as in claim 1, further comprising examining the
isolated ductal fluid for the presence of two or more markers.
9. A method as in claim 1, further comprising examining the
isolated ductal fluid for the absence of two or more markers.
10. A method as in claim 1, further comprising examining the ductal
fluid for the presence of at least one marker and the absence of at
least one marker.
11. A method as in claim 1, further comprising analyzing collected
ductal epithelial cells by cytology.
12. A method as in claim 9, wherein the markers are selected from
the group consisting of DNA content, p53 gene or gene product, and
G-actin or a nucleic acid encoding a polypeptide comprising at
least a portion of G-actin.
13. An isolated ductal fluid sample collected from a breast duct in
a breast, said isolated ductal fluid not mixed with ductal fluid
from any other breast duct.
14. An isolated ductal fluid sample as in claim 13, wherein the
sample comprises a marker for analysis.
15. An isolated ductal fluid sample as in claim 14, wherein the
analysis comprises determining the presence or absence of the
marker.
16. An isolated ductal fluid sample as in claim 13, a portion of
said isolated ductal fluid not spontaneously discharging from the
breast duct.
17. An isolated ductal fluid sample as in claim 13, wherein the
sample comprises 10 or more ductal epithelial cells.
18. An isolated ductal fluid sample as in claim 13, wherein the
sample comprises at least one ductal epithelial cells clump.
19. An isolated ductal fluid sample as in claim 18, wherein the
clump comprises 5 or more ductal epithelial cells.
20. A method for analyzing breast markers or epithelial cells,
comprising: determining the presence or absence of a marker in an
isolated ductal fluid sample collected from a breast duct in a
breast, said isolated ductal fluid not mixed with ductal fluid from
any other breast duct.
21. A method as in claim 20, wherein the duct from which the ductal
fluid is isolated is not spontaneously discharging ductal
fluid.
22. A method as in claim 20, wherein the marker is selected from
the group consisting of: lysophosphatidic acid, a lysophospholipid,
paladin, a portion of palladin, a nucleic acid encoding a
polypeptide comprising at least a portion of paladin, Lg, a portion
of Lg, a nucleic acid encoding a polypeptide comprising at least a
portion of Lg, E2F1, a portion of E2F1, a nucleic acid encoding a
polypeptide comprising at least a portion of E2F1, T1A12/mac 25, a
portion of T1A12/mac 25, a nucleic acid encoding a polypeptide
comprising at least a portion of T1A12/mac 25, MAGUK/ZO-1, a
portion of MAGUK/ZO-1, a nucleic acid encoding a polypeptide
comprising at least a portion of MAGUK/ZO-1, repressor of estrogen
receptor activity (REA), a portion of REA, a nucleic acid encoding
a polypeptide comprising at least a portion of REA, prothymosin
alpha (PTA), a portion of PTA, a nucleic acid encoding a
polypeptide comprising at least a portion of PTA, c-raf kinase, a
portion of c-raf kinase, a nucleic acid encoding a polypeptide
comprising at least a portion of c-raf kinase, CD66a, a portion of
CD66a, a nucleic acid encoding a polypeptide comprising at least a
portion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding
a polypeptide comprising at least a portion of KL-1, cell adhesion
molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acid
encoding a polypeptide comprising at least a portion of CAM 5.2,
leptin, a portion of leptin, a nucleic acid encoding a polypeptide
comprising at least a portion of leptin, Bcl-2 gene product, at
least a portion of Bcl-2 gene product or polypeptide, a nucleic
acid encoding a polypeptide encoding at least a portion of Bcl-2
gene product, nuclear matrix 23(nm23), a portion of nm23, a nucleic
acid encoding a polypeptide comprising at least a portion of nm23,
an apotosis-related protein, a portion of said protein, a nucleic
acid encoding a polypeptide comprising at least a portion of the
apoptosis-related protein, lipocalin NGAL, a portion of lipocalin
NGAL, a nucleic acid encoding a polypeptide comprising at least a
portion of lipocalin NGAL, complement regulatory protein CD46, a
portion of CD46, a nucleic acid encoding at least a portion of
CD46, complement regulatory protein CD59, a portion of CD59, a
nucleic acid encoding at least a portion of CD59, a nucleic acid
encoding a portion of an FHIT gene, loss of heterozygosity at an
FRA3B site, MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid
encoding at least a portion of MRP-1/CD9, KAI1/CD82, a portion of
KAI1/CD82, a nucleic acid encoding at least a portion of KAI1/CD82,
a Fibroblast Growth Factor (FGF), a portion of FGF, a nucleic acid
encoding a polypeptide comprising at least a portion of an FGF,
Vascular Epithelial Growth Factor (VEGF), at least a portion of
VEGF, a nucleic acid encoding at least a portion of VEGF,
Insulin-like Growth Factor -1 (IGF-1), at least a portion of IGF-1,
a nucleic acid encoding at least a portion of IGF-1, tumor
amplified kinase STK15 (also BTAK and aurora2), at least a portion
of STK15, a nucleic acid encoding a polypeptide comprising at least
a portion of STK15, TMS-1, a portion of TMS-1, a nucleic acid
encoding a polypeptide comprising at least a portion of TMS-1,
maspin, at least a portion of maspin, a nucleic acid encoding a
polypeptide comprising at least a portion of maspin, at least a
portion of breast cancer associated (BRCA) gene, and at least a
portion of a BRCA gene product; CDw60 protein, a portion of CDw60
protein or polypeptide, a nucleic acid encoding at least a portion
of a CDw60 protein or polypeptide, mammary expressed enzymes
including cytochrome P450s, catechol-O-methyltransferase, epoxide
hydrolase, peroxidases, glutathione S-transferases,
N-acetyltransferases, or sulfotransferases, a nucleic acid encoding
at least a portion of a mammary expressed enzyme, Kallikrein 6
(zyme/protease M/neurosin) protein or polypeptide (hK6), a nucleic
acid encoding at least a portion of an hK6 protein or polypeptide;
and mammastatin protein or polypeptide, a nucleic acid encoding at
least a portion of a mammastatin protein or polypeptide.
23. A method as in claim 20, wherein the marker is absorption of a
molecule by abnormal cells in the fluid.
24. A method as in claim 23, wherein the molecule comprises
iodide.
25. A method as in claim 20, wherein the marker is a loss of
heterozygozity.
26. A method as in claim 20, wherein the presence of two or more
markers is determined.
27. A method as in claim 20, wherein the absence of two or more
markers is determined.
28. A method as in claim 20 wherein the presence of at least one
marker and the absence of at least one marker is determined.
29. A method as in claim 20 wherein the marker determined is
cytology of ductal epithelial cells.
30. A method as in claim 20, wherein the marker is selected from
the group consisting of DNA content, p53 gene or gene product, and
G-actin or a nucleic acid encoding a polypeptide comprising at
least a portion of G-actin.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
no. 09/625,399 filed Jul. 26, 2000, which is a continuation-in-part
of application no. 09/502,404, filed on Feb. 10, 2000, which was a
continuation-in-part of application no. 09/313,463, filed on May
17, 1999. This application is also a continuation-in-part of
application no. 09/473,510, filed on Dec. 28, 1999. This
application also claims the benefit under 37 CFR 1.78 of
provisional application 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] 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 for identifying patients at risk for breast cancer.
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 (Jan/Feb 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. Further, an intraductal aspiration
method for cytodiagnosis in situations of spontaneous nipple
discharge (Hou et al, Acta Cytologica 2000 v. 44:1029-1034)
describes use of intraductal aspiration to collect specimens from
spontaneously discharging ducts in order to make a
cytodiagnosis.
[0003] Breast cancer is believed to originate in the lining of a
single breast milk duct; and additionally the human breast is
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.sup.th, 1973, The
Medical Post, article entitled "New tests may speed breast cancer
detection". 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 ductoscope 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 collect the
ductal fluid from within the duct and so facilitate duct-specific
analysis.
SUMMARY OF THE INVENTION
[0004] It is an object of the invention to provide a method for
preparing a sample for use in diagnosis of breast cancer or
pre-cancer.
[0005] It is another object of the invention to provide an isolated
ductal fluid sample suitable for analyzing breast cancer and
pre-cancer.
[0006] It is yet another object of the invention to provide a
method for analyzing breast markers or epithelial cells.
[0007] These and other objects of the invention are provided by one
or more of the emobidments described below. In one embodiment a
method is provided for preparing a sample for use in the diagnosis
of breast cancer or pre-cancer. A ductal fluid sample is isolated
from one duct of a breast of a patient. The isolated ductal fluid
is not mixed with ductal fluid from any other duct of the
breast.
[0008] According to another emobidment of the invention an isolated
ductal fluid sample is provided. The sample is collected from a
breast duct in a breast. The isolated ductal fluid is not mixed
with ductal fluid from any other breast duct.
[0009] According to still another embodiment of the invention a
method is provided for analyzing breast markers or epithelial
cells. The presence or absence of a marker in an isolated ductal
fluid sample is determined. The sample is collected from a breast
duct in a breast. The isolated ductal fluid not mixed with ductal
fluid from any other breast duct.
[0010] The present invention thus provides the art with improved
samples and sampling techniques for diagnosing and prognosing
breast cancer and pre-cancer.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0011] The following preferred embodiments and examples are offered
by way of illustration and not by way of limitation.
[0012] The invention comprises an isolated ductal fluid sample
collected from a breast duct in a breast, the fluid not mixed with
ductal fluid from any other breast duct. The isolated ductal fluid
sample can be a sample from a non-discharging breast duct. A
non-discharging duct is a breast duct that is not spontaneously
discharging fluid or material, i.e., a duct which is not leaking
fluid to the nipple surface. Spontaneously discharging ducts
discharge fluid of various coloration. The spontaneous discharge
itself is a warning sign usually requiring further investigation,
such as, mammography, ductoscopy, and/or galactography. The present
invention provides an isolated ductal fluid sample from a
non-discharging duct, i.e., a ductal fluid and/or material sample,
a portion of which would not otherwise have contacted the nipple
surface. However, the isolated ductal fluid sample may also be from
a discharging duct, provided the sample collected is not mixed with
ductal fluid from any other duct.
[0013] The isolated ductal fluid sample can be examined for the
presence of a marker, the absence of a marker, or the state or
quality of a particular marker. The markers can comprise those
detailed herein and related markers that indicate the status or
condition of the breast. The marker status can be used to identify
pre-cancer or cancer of the breast. The ductal fluid sample is
collected from one duct of a breast of a patient. Ductal fluids may
be collected from multiple ducts of a breast or from ducts in both
breasts of a patient, e.g., in sequence, provided the fluid and
material from each duct is kept separate for analysis from the
other ducts. The ducts are also marked or otherwise identified so
that follow-up and/or treatment of a duct that indicates the need
for treatment can be conducted. The ductal fluid sample when
collected or provided is not mixed with ductal fluid from any other
duct of the breast.
[0014] The number of epithelial cells in a ductal fluid sample may
range, for example, from a few to a hundred, to several hundred, to
several thousand, and up to tens of thousands, e.g., 20,000 to
100,000 or more cells. At least ten epithelial cells are required
to designate an isolated sample as adequate for analysis of the
cells. An isolated ductal fluid sample can have 10 or more cells
for analysis, and possibly a single clump of cells or more than one
clump. A clump comprises a plurality of cells, generally at least
about 4 to about 6 cells are in a clump, and the clump can comprise
more cells than 6. Samples with one or more clumps can also include
individual cells that are distinct from the clump(s). Thus, an
isolated sample retrieved by infusing fluid into the duct and
collecting the infused fluid mixed with the ductal fluid can
provide multiple cells and one or more cell clumps for analysis.
The advantage of cell clumps is that the clumping provides a
framework for analysis of cell-cell interaction or a cell-to-cell
relationship that in turn provides information about the status of
the cells themselves. The invention provides the a ductal fluid
sample comprising sufficient ductal epithelial cells from a breast
duct for an analysis of the breast in which the duct is located.
Insufficient ductal epithelial cells in a sample means that a
cytological analysis of those cells can not be performed, or that
the accuracy of the cytological analysis is compromised. The method
of the invention and the composition provide samples from single
breast ducts that can be analyzed because the samples so isolated
contain sufficient material for an adequate analysis to be made.
Ductal samples can comprise markers present in the fluid in
addition to cells, i.e., molecules present in the cells collected
and/or in the extracellular material retrieved from the breast
duct. An advantage provided by the invention is that many more
cells than have been previously collected are collectable and an
accurate cytological analysis can therefore be made of the
sample.
[0015] Relatively undisrupted cells and clumps can be analyzed to
provide information on the cellular status in the breast duct from
which the sample was collected. Further, collection of the ductal
fluid from the breast duct provides enough cells and/or other
material from the duct to provide a useful analysis of the
condition of the breast. This is largely due to the fact that
collection of the ductal fluid, cells and other material by
infusing saline or other biocompatible wash fluid and collecting
the wash fluid mixed with the ductal material results in collection
of sufficient fluid and material for analysis. Suction may be
applied to the lumen in the duct to facilitate collection of the
ductal fluid and material once the duct has been filled with wash
fluid (in order to prevent collapse of the ductal walls);
reinfusion of wash fluid can follow in order to prevent collapse of
the ductal walls and provide the opportunity for a second or
subsequent intraductal aspiration and/or retrieval. Squeezing and
massaging the breast may also be used in concert with infusion and
collection procedures. The amount of material that is sufficient
for analysis in a sample is at least one ductal epithelial clump up
to at least 10 ductal epithelial clumps or more--each clump having
from at least 4 to 6 ductal epithelial cells. For example, the
sample from a non-discharging or discharging breast duct may have
at least from 10 to 20 ductal epithelial cells, 20 to 50, 50 to
100, 100 to 1000, 1000 to 10,000, 10,000 to 50,000, or 50,000 to
100,000 ductal epithelial cells. The ductal epithelial cells may be
present either individually or in clumps or both. Insufficient
samples might include samples with less than 10 epithelial cells.
An evaluation of insufficiency may be contingent on whether the
cells are clumped or not.
[0016] The method of the invention is preparing a sample for use in
diagnosis of breast cancer or pre-cancer comprising isolating a
ductal fluid sample from one duct of a breast of a patient. The
isolated ductal fluid is not mixed with ductal fluid from any other
duct of the breast. The method can further include examining the
isolated ductal fluid sample to determine the presence or absence
of a marker. The duct from which the ductal fluid is isolated can
be a duct that is not spontaneously discharging fluid. The marker
for analysis can be selected from any known and useful markers for
a breast condition, including pre-cancer and/or cancer markers, and
further optionally including markers listed herein.
[0017] The isolated fluid sample can be examined to determine the
presence of a marker. The presence of any marker, the absence of
any marker, or the quality or state of any marker can be analyzed
or examined. Particularly, the markers listed herein, and related
forms or species of the markers listed herein are contemplated. The
presence, absence or state of more than one marker can be examined.
Markers can be examined in conjunction with ductal epithelial cell
cytological analysis. The markers can be, for example,
intracellular, nuclear, cytoplasmic, cell-surface, secreted, or
extracellular markers. The markers can include any markers
described in co-owned, co-pending, parent applications 09/625,399
filed Jul. 26, 2000, application no.09/502,404, filed on Feb. 10,
2000, and application no. 09/313,463, filed on May 17, 1999, hereby
incorporated by reference in their entirety.
[0018] Examination of the ductal fluid for a marker can comprise
determining absorption of a marker molecule by abnormal cells in
the fluid. For example, absorption of iodide or a like molecule by
cells in a ductal fluid sample can be measured. Examination of the
ductal fluid for a marker can comprise analysis or examination of a
quality and/or state of nucleic acid for such characteristic
changes from the normal state as, for example, a loss of
heterozygozity. Examination of the ductal fluid can comprise
examining the fluid for the absence of a marker; especially where
the marker is present in normal ductal fluid in a predetermined
quantity in the population, and standards are set for benchmarks
indicating a particular condition in the breast (ie., pre-cancer or
cancer, or their various sub-categories). Examination of the ductal
fluid can comprise examining the ductal fluid for the presence or
absence of two or more markers, including examining two or more
markers for their state or quality, and including absorption of a
marker, or loss of heterozygosity in the DNA of the cells retrieved
from the isolated sample.
[0019] For example, the markers in this latter case can comprise
DNA content, p53 gene or gene product, and G-actin or a nucleic
acid encoding a polypeptide comprising at least a portion of
G-actin. Thus, for example, examining the ductal fluid can comprise
examining the fluid for the presence of at least one marker and the
absence of at least one marker, e.g., examining the ductal fluid
for the presence of an oncogene or its gene product, and examining
the ductal fluid for the absence of a tumor suppressor molecule
normally present in a given range or quantity in normal breast duct
fluid or breast tissue. As an example, the ductal fluid can be
examined for markers comprising such parameters as DNA content of
the ductal epithelial cells, the absence or lowered levels of p53
gene or its gene product, and the presence of G-actin protein,
polypeptide, or portion thereof, or a nucleic acid encoding a
G-actin protein or polypeptide or portion thereof, e.g., as
described in Rao et al, Cancer Epid, Biomarkers & Prevention,
1993 v. 7:1027-1033.
[0020] Preparing an isolated ductal fluid sample can comprise
accessing the duct with a ductal access tool and collecting the
ductal fluid sample while the tool remains in the duct. Having the
tool remain in the duct for fluid infusion and fluid collection
also ensures that the ductal fluid and ductal material collected
are collected from a single duct not mixed with fluid or material
from any other duct. Wash fluid infusion is used in the cases where
the duct is not spontaneously discharging fluid so that the duct is
filled or partially filled, the wash fluid mixes with ductal fluid
and ductal contents, and retrieval of the mixed fluids comprises
retrieval of a sample having sufficient cells and/or other markers
for analysis of the condition of the duct from which the sample is
taken. Previously the usefulness of ductal fluid retrieved by other
means has been hampered by insufficient material or cells for
analysis in the retrieved samples and/or not being able to identify
the specific duct to which abnormal cells or other findings can be
attributed. Since most breast cancers begin in a single, isolated,
milk duct of a breast, the identification of a specific duct as
abnormal (i.e., cancerous or pre-cancerous) is extremely useful,
especially in concert with sufficient information from the isolated
fluid sample in order to make a diagnosis. Collection of the
isolated fluid sample can be facilitated by fluid infusion into the
duct and collection of the wash fluid that has been infused mixed
with ductal fluid and other ductal contents including ductal
epithelial cells and other markers. The collection through the
accessing lumen is facilitated after wash fluid infusion by a
number of techniques that can be used together or separately and
which are not limited to squeezing the breast, massaging the
breast, applying negative pressure on the lumen to pull-up fluid
into the lumen and/or collection receptacle, and using an additive
in the wash fluid that delays or inhibits absorption of the fluid
into the ductal walls (thereby keeping more fluid in the duct to be
retrieved). Many of these techniques and tools for practicing these
techniques are described in co-owned U.S. Ser. No. 09/067,661, U.S.
Ser. No. 09/301,058, PCT US99/09141, U.S. Ser. No. 09/313,463, U.S.
Ser. No. 09/473,510, PCT US99/31086 herein incorporated by
reference in their entirety.
[0021] 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 09/067,661,
09/301,058, PCT US99/09141, 60/122,076, 09/313,463, 60/143,359, and
U.S. Ser. No. 09/473,510, all incorporated by reference in their
entirety. 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. The duct
can be flushed by infusing saline into the duct until resistance is
met, applying pressure and/or squeezing the breast, e.g.,
particularly at the base of the breast, and capturing the fluid
that moves up through the duct after the pressure is applied.
Flushing can continue by infusing more saline and applying more
pressure.
[0022] In order to retrieve cells and ductal material sufficient
for analysis of a single non-discharging breast duct and a
corresponding diagnosis, a non-discharging duct can be accessed by
a tool capable of infusing wash fluid and also capable of
collecting the ductal fluid mixed with wash fluid while the tool
remains in the duct as described herein. Thus, 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
fluid and ductal fluid from the duct without removal of the tool.
Thus, by the method of the invention, the tool remains indwelling
while wash fluid is infused and wash fluid mixed with ductal fluid
(comprising cells and cellular material, etc.) is collected. The
fluid from the breast duct can contain ductal epithelial cells,
including cells of a stage considered to be pre-cancerous or
cancerous as described, and may also contain various molecules
either connected to the cells or separate from them that may be
used as markers. Either presence or absence or decrease or increase
relative to a normal or benign control amount can indicate cancer
or pre-cancer.
[0023] 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 be accomplished by obtaining the sample
from the breast or by 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. The isolated fluid is
collected by some available technique including, for example,
ductal lavage of a single duct. In general, collection of isolated
ductal fluid not mixed with ductal fluid from another duct of the
breast can be accomplished by accessing the duct with a breast duct
access tool that infuses fluid and collects ductal fluid mixed with
the infused fluid, while the tool remains in the duct. Also, the
collection tube can be marked and the duct can be marked so that
the analysis of the fluid is traceable to one duct which can be
re-identified and re-accessed if appropriate.
[0024] The marker may be a nucleic acid or protein form of the
marker. For example, the marker may be "X", and either a nucleic
acid sequence encoding at least a portion of X, or a gene product
at least a portion of protein or polypeptide X can be determined or
measured. The marker may also be a non-nucleic acid or a non-amino
acid molecule, such as, for example, a small organic molecule, a
lipid, a fat, a biologically formed organic acid or base, a
carbohydrate or other sugar type molecule, a polymer type molecule
or a portion of such molecule, a moiety that characterizes a
marker, for example a side chain on a marker, etc.
[0025] Thus, for example, the method of providing an isolated
ductal fluid sample, and examining the sample for one or more
markers can comprise examining the sample for the presence,
absence, or relative level of any one or more of the following
markers:
[0026] 1. lysophosphatidic acid (LPA) or a lysophospholipid, or a
receptor of lysophosphatidic acid, e.g., as described in Goetzl et
al, Cancer Res 1999 Sep 15 v.59: 4732-7, Xu et al, Biochem J 1995
v.309: 933-40, and Contos et al, Mol Pharmacol 2000 v.58:
1188-1196;
[0027] 2. palladin, a portion of palladin, or a nucleic acid
encoding a polypeptide comprising at least a portion of paladin,
e.g., as described in Reuters Health News Aug. 7, 2000, and Parast
and Otey, J Cell Biol 2000 Aug. 7 v. 150:643-56;
[0028] 3. Lg, a portion of Lg, or a nucleic acid encoding a
polypeptide comprising at least a portion of Lg, e.g., as described
in Ranganathan et al, J Steroid Biochem Mol Biol 1999 v.70:
151-8;
[0029] 4. E2F1, a portion of E2F1, or a nucleic acid encoding a
polypeptide comprising at least a portion of E2F1, e.g., as
described in Klein-Szanto et al Cancer Epid Biomarkers &
Prevention 2000 v. 9: 395-401;
[0030] 5. TIA 12/mac 25, a portion of TIA 12/mac 25, or a nucleic
acid encoding a polypeptide comprising at least a portion of
T1A12/mac 25, e.g., as described in Burger et al Oncogene 1998 v.
16:2459-67;
[0031] 6. MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid
encoding a polypeptide comprising at least a portion of MAGUK/ZO-1,
e.g., as described in Hoover et al, Am J Pathol 1998 v.153:
1767-73;
[0032] 7. Repressor of estrogen receptor activity (REA), a portion
of REA, a nucleic acid encoding a polypeptide comprising at least a
portion of REA, e.g., as described in Simon et al, Cancer Res 2000
v. 60:2796-9;
[0033] 8. prothymosin alpha (PTA), a portion of PTA, a nucleic acid
encoding a polypeptide comprising at least a portion of PTA, e.g.,
as described in Domineguez et al, Br J of Cancer 2000 v.
82:584-590; and Magdalena et al, Br J Cancer 2000 v.
82:584-590;
[0034] 9. TNF-related apoptosis-inducing ligand (TRAIL), a nucleic
acid encoding a polypeptide comprising at least a portion of TRAIL,
e.g., as described in Griffith et al, J lmmunol 2000 v.
165:2886-94; and Herrnring et al Histochem Cell Biol 2000 v.
113:189-94;
[0035] 10. BU101 protein, a nucleic acid encoding a polypeptide
comprising at least a portion of BU0101, e.g., as described in WO
98/07857;
[0036] 11. c-raf kinase, a portion of c-raf kinase, a nucleic acid
encoding a polypeptide comprising at least a portion of c-raf
kinase, e.g., as described in E1-Ashry et al, Oncogene 1997 v.15:
423-35, and Callans et al, Ann Surg. Oncol 1995 v.2: 38-42;
[0037] 12. CD66a, a portion of CD66a, a nucleic acid encoding a
polypeptide comprising at least a portion of CD66a, e.g., as
described in Huang et al Anticancer Res. 1998 v.18: 3203-12;
[0038] 13. KL-1, a portion of KL-1, a nucleic acid encoding a
polypeptide comprising at least a portion of KL-1, e.g., as
described in Okumura et al, Jpn J Clin Oncol 1998 v.28: 480-5;
[0039] 14. cell adhesion molecule 5.2 (CAM 5.2), a portion of CAM
5.2, a nucleic acid encoding a polypeptide comprising at least a
portion of CAM 5.2, e.g., as described in Okumura et al, Jpn J Clin
Oncol 1998 v.28: 480-5;
[0040] 15. leptin, a portion of leptin, a nucleic acid encoding a
polypeptide comprising at least a portion of leptin, e.g., as
described in Tessitore et al, Int J Mol Med 2000 v.5: 421-6;
[0041] 16. Bcl-2 gene product, at least a portion of Bcl-2 gene
product or polypeptide, a nucleic acid encoding a polypeptide
encoding at least a portion of Bcl-2 gene product, e.g., as
described in Krajewski et al Endocr Relat Cancer 1999 v.6: 29-40,
and Castiglione et al Anticancer Res. 1999 v.19:4555-63,
Simony-Lafontaine et al, 2000 v.82: 1958-66;
[0042] 17. nuclear matrix 23(nm23), a portion of nm23, a nucleic
acid encoding a polypeptide comprising at least a portion of nm23,
e.g., as described in Vazquez-Ramirez et al, Pathol Res Pract 2000
v.196: 553-9, Cipollini et al, Cancer Genet Cytogenet 2000 v.
121:181-5;
[0043] 18. an apotosis-related protein, a portion of said protein,
a nucleic acid encoding a polypeptide comprising at least a portion
of the apoptosis-related protein, e.g., as described in Dowsett et
al, Endocr Relat Cancer 1999 v.6: 25-8:
[0044] 19. lipocalin NGAL, a portion of lipocalin NGAL, a nucleic
acid encoding a polypeptide comprising at least a portion of
lipocalin NGAL, e.g., as described in Stoesz et al, Int J Cancer
1998 v.79: 565-72;
[0045] 20. thymosin beta-15, a portion of thymosin beta-15, a
nucleic acid encoding a polypeptide comprising at least a portion
of thymosin bet-15, e.g., as described in U.S. Pat. No. 5,663,071
and WO 97/48805;
[0046] 21. tumor amplified kinase STK15 (also BTAK and aurora2), at
least a portion of STK15, a nucleic acid encoding at least a
portion of STK15; e.g., as described in Zhou et al, Nat Genet 1998
v.20:189-93;
[0047] 22. complement regulatory protein CD46, a portion of CD46, a
nucleic acid encoding at least a portion of CD46; as described
e.g., in Thorsteinsson et al APMIS 1998 v.106:869-78;
[0048] 23. complement regulatory protein CD59, a portion of CD59, a
nucleic acid encoding at least a portion of CD59; as described
e.g., in Thorsteinsson et al APMIS 1998 v.106:869-78;
[0049] 24. a nucleic acid encoding a portion of an FHIT gene, e.g.,
as described in Huiping et al, Eur J Cancer 2000 v.36: 1552-7,
Gatalica et al, Cancer 2000 v.88: 1378-83, Ahmadian et al, Cancer
Res 1997 v. 57:3664-8, and Campiglio et al, Cancer Res 1999 v.
59:3866-9;
[0050] 25. loss of heterozygosity (LOH), e.g., as described in
Linginger et al, Mod Patbol 1998 v. 11:1151-9; and Larson et al, Am
J Pathol 1998 v. 152:1591-8;
[0051] 26. LOH at an FRA3B site, e.g., as described in Ahmadian et
al, Cancer Res 1997 v. 57:3664-8;
[0052] 27. MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid
encoding at least a portion of MRP-1/CD9, e.g., as described in van
den Heuvel-Eibrink et al, Int J Clin Pharmacol Ther 2000
v.38:94-110, Huang et al Am J Pathol 1998 v 0.153: 973-83, Miyake
et al Cancer Res 1996 v.56:1244-9, and Miyake et al Cancer Res.
1995 v.55: 4127-31;
[0053] 28. KAI1/CD82, a portion of KAI1/CD82, a nucleic acid
encoding at least a portion of KAI1/CD82, e.g., as described in
Huang et al Am J Pathol 1998 v.153: 973-83;
[0054] 29. TMS-1, a portion of TMS-1, a nucleic acid encoding a
polypeptide comprising at least a portion of TMS-1, for example as
described in McConnell and Vertino, Cancer Res. 2000 Nov.
15;60(22):6243-7; Conway et al, Cancer Res 2000 Nov.
15;60(22):6236-42; and Grossman et al, J Exp Biol 2000 v.203:
447-57;
[0055] 30. at least a portion of breast cancer associated gene
(BRCA), e.g., as described in Seances et al, Soc. Biol Fil 1998 v.
192:35-40, and Deng and Brodie, Bioessays 2000 v.22: 728-37;
[0056] 31. absorption of a marker (e.g., iodide), e.g., as
described in De La Vieja et al, Physiol Rev 2000 v.80: 1083-105,
Tazebay et al, Nat Med 2000 v. 6:871-8;
[0057] 32. Fibroblast growth factor (FGF) protein, a portion of an
FGF protein or polypeptide, a nucleic acid encoding at least a
portion of an FGF protein or polypeptide, e.g., as described in
Femig et al, Cancer Treat Res 1991 v.53: 47-78, and De Benedetti
and Harris, Int J Biochem Cell Biol 1999 v.31: 59-72;
[0058] 33. Vascular endothelial growth factor (VEGF) protein, a
portion of a VEGF protein or polypeptide, a nucleic acid encoding
at least a portion of a VEGF protein or polypeptide e.g., as
described in Gasparini, Oncologist 2000; 5 suppl 1:37-44;
[0059] 34. Insulin-like growth factor -1 (IGF-1 protein, a portion
of an IGF-1 protein or polypeptide, a nucleic acid encoding at
least a portion of an IGF-1 protein or polypeptide e.g., as
described in Pollack, Eur J Cancer 2000 v. 36:1224-8;
[0060] 35. Maspin protein, a portion of a maspin protein or
polypeptide, a nucleic acid encoding at least a portion of a maspin
protein or polypeptide, e.g., as described in Sager et al, Adv Exp
Med Biol 1997 v.425: 77-88.
[0061] 36. CDw60 protein, a portion of CDw60 protein or
polypeptide, a nucleic acid encoding at least a portion of a CDw60
protein or polypeptide, e.g., as described in Gocht et al,
Histochem J 2000 Jul; 32(7):447-56.
[0062] 37. Mammary expressed enzymes (e.g., cytochrome P450s,
catechol-O-methyltransferase, epoxide hydrolase, peroxidases,
glutathione S-transferases, N-acetyltransferases, and
sulfotransferases) a nucleic acid encoding at least a portion of a
mammary expressed enzyme, e.g., as described in Williams and
Phillips, Cancer Res 2000 Sep 1:60(17):4667-77.
[0063] 38. Mammastatin protein or polypeptide (47 kD and/or 65 kD),
a nucleic acid encoding at least a portion of a mammastatin protein
or polypeptide, e.g., as described in Ervin et al, Science 1989;
244(4912); 1585-7.
[0064] 39. Kallikrein 6 (zyme/protease M/neurosin) protein or
polypeptide (hK6), a nucleic acid encoding at least a portion of an
hK6 protein or polypeptide, e.g., as described in Diamandis et al,
Clin Biochem 2000 Oct; 33(7):579-583; Diamandis et al, Clin Biochem
2000 Jul; 33(5):36975; Yousef et al, Genomics 2000 Nov.
1;69(3):331-41.
[0065] 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
pre-cancerous or cancerous or having another noncancerous
abnormality), pre-cancerous (i.e., comprising hyperplasia, a
typical ductal hyperplasia (ADH) or low grade ductal carcinoma in
situ (LG-DCIS)) or cancerous (ie., 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 pre-cancerous
or cancerous condition in the cells.
[0066] 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 distinguishing
between any two 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.
[0067] Analysis of cell contents may serve to establish similar
staging as established by morphology, capturing generally a
progression of a pre-cancerous 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
lumenal ductal epithelial cells not from the TDLU including, e.g.,
hyperplasia, atypia, in situ carcinoma, and invasive carcinoma.
[0068] Once the wash fluid has 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. These
materials may be found in the cell, on the cell surface or as
material secreted from the cell and found in fluid outside the
cell. These materials may be synthesized by a cell, or may be
otherwise present in the fluid from the duct, e.g., as by-products
or degradation products of molecules in the body. Cytology, or any
other suitable method for analyzing the condition of the cells can
be used to examine whole cells. Other markers present in the
cellular material, ductal fluid, 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 techniques for distinguishing and
identifying biological molecules obtained from biological material.
Examining the ductal fluid sample can also be performed to
determine the presence of a marker comprising, 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, or a
complex of a plurality of molecules. Detection and analysis of
these classifications of markers can be accomplished, for example,
using standard assays for determining the presence of a particular
marker or marker classification and/or 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).
[0069] Examining the ductal fluid sample can comprise determining
the presence of a marker comprising RNA, DNA, protein, polypeptide,
or peptide form of a marker such as lysophosphatidic acid, a
lysophospholipid, paladin, a portion of palladin, a nucleic acid
encoding a polypeptide comprising at least a portion of paladin,
Lg, a portion of Lg, a nucleic acid encoding a polypeptide
comprising at least a portion of Lg, E2F1, a portion of E2F1, a
nucleic acid encoding a polypeptide comprising at least a portion
of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, a nucleic acid
encoding a polypeptide comprising at least a portion of T1A12/mac
25, MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic acid encoding a
polypeptide comprising at least a portion of MAGUK/ZO-1, repressor
of estrogen receptor activity (REA), a portion of REA, a nucleic
acid encoding a polypeptide comprising at least a portion of REA,
prothymosin alpha (PTA), a portion of PTA, a nucleic acid encoding
a polypeptide comprising at least a portion of PTA, c-raf kinase, a
portion of c-raf kinase, a nucleic acid encoding a polypeptide
comprising at least a portion of c-raf kinase, CD66a, a portion of
CD66a, a nucleic acid encoding a polypeptide comprising at least a
portion of CD66a, KL-1, a portion of KL-1, a nucleic acid encoding
a polypeptide comprising at least a portion of KL-1, cell adhesion
molecule 5.2 (CAM 5.2), a portion of CAM 5.2, a nucleic acid
encoding a polypeptide comprising at least a portion of CAM 5.2,
leptin, a portion of leptin, a nucleic acid encoding a polypeptide
comprising at least a portion of leptin, Bcl-2 gene product, at
least a portion of Bcl-2 gene product or polypeptide, a nucleic
acid encoding a polypeptide encoding at least a portion of Bcl-2
gene product, nuclear matrix 23(nm23), a portion of nm23, a nucleic
acid encoding a polypeptide comprising at least a portion of nm23,
an apotosis-related protein, a portion of said protein, a nucleic
acid encoding a polypeptide comprising at least a portion of the
apoptosis-related protein, comprises lipocalin NGAL, a portion of
lipocalin NGAL, a nucleic acid encoding a polypeptide comprising at
least a portion of lipocalin NGAL, a nucleic acid encoding a
portion of an FHIT gene, loss of heterozygosity at an FRA3B site,
MRP-1/CD9, a portion of MRP-1/CD9, a nucleic acid encoding at least
a portion of MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a
nucleic acid encoding at least a portion of KAI1/CD82, at least a
portion of breast cancer associated gene, TMS-1, a portion of
TMS-1, a nucleic acid encoding a polypeptide comprising at least a
portion of TMS-1; at least a portion of breast cancer associated
gene (BRCA); absorption of a marker (e.g., iodide). fibroblast
growth factor (FGF) protein, a portion of an FGF protein or
polypeptide, a nucleic acid encoding at least a portion of an FGF
protein or polypeptide, vascular endothelial growth factor (VEGF)
protein, a portion of a VEGF protein or polypeptide, a nucleic acid
encoding at least a portion of a VEGF protein or polypeptide,
insulin-like growth factor -1 (IGF-1 protein, a portion of an IGF-1
protein or polypeptide, a nucleic acid encoding at least a portion
of an IGF-1 protein or polypeptide; maspin protein, a portion of a
maspin protein or polypeptide, a nucleic acid encoding at least a
portion of a maspin protein or polypeptide, CDw60 protein, a
portion of CDw60 protein or polypeptide, a nucleic acid encoding at
least a portion of a CDw60 protein or polypeptide, mammary
expressed enzymes (e.g., cytochrome P450s,
catechol-O-methyltransferase, epoxide hydrolase, peroxidases,
glutathione S-transferases, N-acetyltransferases, and
sulfotransferases) a nucleic acid encoding at least a portion of a
mammary expressed enzyme, mammastatin protein or polypeptide (47 kD
and/or 65 kD), a nucleic acid encoding at least a portion of
mammastatin protein or polypeptide; kallikrein 6 (zyme/protease
M/neurosin) protein or polypeptide (hK6), and a nucleic encoding at
least a portion of an hK6 protein or polypeptide.
[0070] 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. Increased or decreased amounts relative to such normal
controls can also be determined. The normal control can be
determined relative to the particular patient, or relative to a
patient population. 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. A tumor suppressor, e.g., mammastatin
may be used as a marker where a reduction in the marker identifies
a cancerous or pre-cancerous condition in the breast.
[0071] Chromosomal abnormalities in ductal epithelial cells can
also provide information and act as a marker to identify cancer or
pre-cancer 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.
[0072] 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.
[0073] 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
procedures 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). 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.
[0074] In general, markers can be categorized nonexclusively, and
often in overlapping categories, for example, protein expression,
mRNA expression, post-translational change in a protein and/or DNA
change in a gene may all be used in concert or separately for the
same or a plurality of markers to make a diagnosis.
[0075] Cytology, or any other suitable method for analyzing the
condition of the cells can be used to examine whole cells. 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 techniques
for distinguishing and identifying biological molecules obtained
from biological material.
[0076] 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 WH & King EB, 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
cytological examination of abnormal cells can also be conducted as
described in Sartorius etal (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 and
observing under a light microscope. The cells can be studied for
atypical 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: Nov. 199S, "Cytopathology of the Breast" ISBN
0891893806; and Feldman PS, American Society of Clinical Pathology,
Nov. 1984, "Fine Needle Aspiration Cytology and Its Clinical
Applications: Breast and Lung" ISBN 0891891846.
[0077] 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.
[0078] The invention also provides systems for preparing a sample
for use in diagnosis of breast cancer or pre-cancer, the system
comprising a tool to retrieve ductal fluid from a breast duct and
instructions for use to isolate a ductal fluid sample from a duct,
particularly a non-spontaneously discharging breast duct in order
to determine the presence of one or more markers. Materials and
instructions may also be included in the system to determine the
presence or absence of a marker in the isolated ductal fluid.
Materials may be included to make a cytodiagnosis of collected
ductal epithelial cells. A cytological reading of the ductal
epithelial cells collected with the infused wash fluid is one type
of marker which can be used for diagnosing a condition in a breast
duct. Instructions in the kit or system can include guidance for
interpreting cytological data and/or other marker data in order to
make a diagnosis. The systems or kits may 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 specific duct (so that the duct can be
accessed later for treatment and/or further monitoring). Methods
for identifying the non-spontaneously discharging duct may also be
included in the system or kit. The instructions in the systems or
kits can include directions according to the methods of identifying
breast cancer or pre-cancer described herein, and possibly
including any marker or markers or marker classification group or
groups that could be useful and/or are described herein. The system
or kit can include assay reagents for detecting the marker or
markers. The system or kit may comprise a panel of reagents for
detecting a plurality of markers either simultaneously or
sequentially, or some other practical combination of testing
modalities. The system or kit can also include indexes and
parameters for making a diagnosis, depending on the marker or
markers. The system or kit can include a container for the contents
of the system or kit.
EXAMPLES
[0079] Retrieval of Ductal Fluid and Analysis of Markers in the
Fluid
[0080] A patient is prepared for a ductal access procedure. Using a
ductal access tool, a duct on each breast is infused with
sufficient wash fluid, and the wash fluid mixed with ductal fluid
is collected separately from each accessed duct. The fluid in each
duct that is accessed is analyzed for the presence, absence or
relative levels (as compared to a predetermined normal level) of
one or more of the following markers using standard techniques:
lysophosphatidic acid, a lysophospholipid, paladin, a portion of
palladin, a nucleic acid encoding a polypeptide comprising at least
a portion of paladin, Lg, a portion of Lg, a nucleic acid encoding
a polypeptide comprising at least a portion of Lg, E2F1, a portion
of E2F1, a nucleic acid encoding a polypeptide comprising at least
a portion of E2F1, T1A12/mac 25, a portion of T1A12/mac 25, a
nucleic acid encoding a polypeptide comprising at least a portion
of T1A12/mac 25, MAGUK/ZO-1, a portion of MAGUK/ZO-1, a nucleic
acid encoding a polypeptide comprising at least a portion of
MAGUK/ZO-1, repressor of estrogen receptor activity (REA), a
portion of REA, a nucleic acid encoding a polypeptide comprising at
least a portion of REA, prothymosin alpha (PTA), a portion of PTA.
a nucleic acid encoding a polypeptide comprising at least a portion
of PTA, TNF-related apoptosis-inducing ligand (TRAIL), a nucleic
acid encoding a polypeptide comprising at least a portion of TRAIL,
BU101 protein, a nucleic acid encoding a polypeptide comprising at
least a portion of BU101 , c-raf kinase, a portion of c-raf kinase,
a nucleic acid encoding a polypeptide comprising at least a portion
of c-raf kinase, CD66a, a portion of CD66a, a nucleic acid encoding
a polypeptide comprising at least a portion of CD66a, KL-1, a
portion of KL-1, a nucleic acid encoding a polypeptide comprising
at least a portion of KL-1, cell adhesion molecule 5.2 (CAM 5.2), a
portion of CAM 5.2, a nucleic acid encoding a polypeptide
comprising at least a portion of CAM 5.2, leptin, a portion of
leptin, a nucleic acid encoding a polypeptide comprising at least a
portion of leptin, Bcl-2 gene product, at least a portion of Bcl-2
gene product or polypeptide, a nucleic acid encoding a polypeptide
encoding at least a portion of Bcl-2 gene product, nuclear matrix
23(nm23), a portion of nm23, a nucleic acid encoding a polypeptide
comprising at least a portion of nm23, an apotosis-related protein,
a portion of said protein, a nucleic acid encoding a polypeptide
comprising at least a portion of the apoptosis-related protein,
comprises lipocalin NGAL, a portion of lipocalin NGAL, a nucleic
acid encoding a polypeptide comprising at least a portion of
lipocalin NGAL, thymosin beta-15, a portion of thymosin beta-15, a
nucleic acid encoding a polypeptide comprising at least a portion
of thymosin beta-15, a nucleic acid encoding a portion of an FHIT
gene, loss of heterozygosity at an FRA3B site, MRP-1/CD9, a portion
of MRP-1/CD9, a nucleic acid encoding at least a portion of
MRP-1/CD9, KAI1/CD82, a portion of KAI1/CD82, a nucleic acid
encoding at least a portion of KAI1/CD82, at least a portion of
breast cancer associated gene, TMS-1, a portion of TMS-1, a nucleic
acid encoding a polypeptide comprising at least a portion of TMS-1;
at least a portion of breast cancer associated gene (BRCA);
absorption of a marker (e.g., iodide). fibroblast growth factor
(FGF) protein, a portion of an FGF protein or polypeptide, a
nucleic acid encoding at least a portion of an FGF protein or
polypeptide, vascular endothelial growth factor (VEGF) protein, a
portion of a VEGF protein or polypeptide, a nucleic acid encoding
at least a portion of a VEGF protein or polypeptide, insulin-like
growth factor -1 (IGF-1 protein, a portion of an IGF-1 protein or
polypeptide, a nucleic acid encoding at least a portion of an IGF-1
protein or polypeptide; maspin protein, a portion of a maspin
protein or polypeptide, a nucleic acid encoding at least a portion
of a maspin protein or polypeptide, CDw60 protein, a portion of
CDw60 protein or polypeptide, a nucleic acid encoding at least a
portion of a CDw60 protein or polypeptide, mammary expressed
enzymes (e.g., cytochrome P450s, catechol-O-methyltransferase,
epoxide hydrolase, peroxidases, glutathione S-transferases,
N-acetyltransferases, and sulfotransferases) a nucleic acid
encoding at least a portion of a mammary expressed enzyme,
mammastatin protein or polypeptide (e.g., 47 kD and/or 65 kD), a
nucleic acid encoding a mammastatin protein or polypeptide;
kallikrein 6 (zyme/protease M/neurosin) protein or polypeptide
(hK6), and a nucleic acid encoding at least a portion of an hK6
protein or polypeptide. Cytodiagnosis of a sample of the collected
ductal epithelial cells, as individual cells and as cells in clumps
is also made to support any other marker data from the collected
fluid and material.
[0081] 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.
* * * * *