U.S. patent application number 10/798084 was filed with the patent office on 2004-08-05 for novel method of diagnosing, monitoring, staging, imaging and treating breast cancer.
Invention is credited to Recipon, Herve, Salceda, Susana, Sun, Yongming.
Application Number | 20040152144 10/798084 |
Document ID | / |
Family ID | 32179301 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152144 |
Kind Code |
A1 |
Sun, Yongming ; et
al. |
August 5, 2004 |
Novel method of diagnosing, monitoring, staging, imaging and
treating breast cancer
Abstract
The present invention provides a new method for detecting,
diagnosing, monitoring, staging, prognosticating, imaging and
treating breast cancer.
Inventors: |
Sun, Yongming; (San Jose,
CA) ; Salceda, Susana; (San Jose, CA) ;
Recipon, Herve; (San Francisco, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 East Main Street
Marlton
NJ
08053
US
|
Family ID: |
32179301 |
Appl. No.: |
10/798084 |
Filed: |
March 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10798084 |
Mar 11, 2004 |
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09664249 |
Sep 18, 2000 |
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6730477 |
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09664249 |
Sep 18, 2000 |
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PCT/US99/16811 |
Jul 22, 1999 |
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60095232 |
Aug 4, 1998 |
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Current U.S.
Class: |
435/7.23 ;
435/6.14 |
Current CPC
Class: |
C12Q 2600/112 20130101;
G01N 33/57484 20130101; A61K 51/1051 20130101; C12Q 2600/136
20130101; A61K 49/16 20130101; A61K 2039/505 20130101; C12Q 1/6886
20130101; C12Q 2600/106 20130101; C07K 16/3015 20130101; G01N
33/57415 20130101 |
Class at
Publication: |
435/007.23 ;
435/006 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What is claimed is:
1. A method for diagnosing the presence of breast cancer in a
patient comprising: (a) measuring levels of BSG in cells, tissues
or bodily fluids in said patient; and (b) comparing measured levels
of BSG with levels of BSG in cells, tissues or bodily fluids from a
normal human control, wherein a change in measured levels of BSG in
the patient versus normal human control is associated with the
presence of breast cancer.
2. A method of diagnosing metastatic breast cancer in a patient
having breast cancer comprising: (a) identifying a patient having
breast cancer that is not known to have metastasized; (b) measuring
levels of BSG in a sample of cells, tissues, or bodily fluid from
said patient; and (c) comparing the measured BSG levels with levels
of BSG in cells, tissue, or bodily fluid type of a normal human
control, wherein a change in measured BSG levels in the patient
versus the normal human control is associated with a cancer which
has metastasized.
3. A method of staging breast cancer in a patient comprising: (a)
identifying a patient having breast cancer; (b) measuring levels of
BSG in a sample of cells, tissues, or bodily fluid from said
patient for BSG; and (c) comparing measured BSG levels with levels
of BSG in cells, tissues, or bodily fluid type of a normal human
control sample, wherein a change in measured BSG levels in said
patient versus the normal human control is associated with a cancer
which is progressing or regressing or in remission.
4. A method of monitoring breast cancer in a patient having breast
cancer for the onset of metastasis comprising: (a) identifying a
patient having breast cancer that is not known to have
metastasized; (b) periodically measuring BSG levels in a sample of
cells, tissues, or bodily fluid from said patient; and (c)
comparing the measured BSG levels with levels of BSG in cells,
tissues, or bodily fluid type of a normal human control, wherein a
change in BSG levels in the patient versus the normal human control
is associated with a cancer which has metastasized.
5. A method of monitoring the change in stage of breast cancer in a
patient having breast cancer comprising: (a) identifying a patient
having breast cancer; (b) periodically measuring BSG levels in a
sample of cells, tissues, or bodily fluid from said patient; and
(c) comparing the measured BSG levels with levels of BSG in cells,
tissues, or bodily fluid type of a normal human control, wherein a
change in measured BSG levels in the patient versus the normal
human control is associated with a cancer which is progressing in
stage, which is regressing in stage, or in remission.
6. The method of claim 1, 2, 3, 4 or 5 wherein the change
associated with the presence, metastasis or progression of breast
cancer in said patient is an increase in measured BSG levels in the
patient and the BSG comprises Mam001 (SEQ ID NO:2), Mam004 (SEQ ID
NO:4 or SEQ ID NO:10) or Mam005 (SEQ ID NO:3).
7. The method of claim 1, 2, 3, 4 or 5 wherein the change
associated with the presence, metastasis or progression of breast
cancer in said patient is a decrease in measured BSG levels in the
patient and the BSG comprises Mam002 (SEQ ID NO:1).
8. The method of claim 3 or 5 wherein the change associated with
the regression or remission of breast cancer in said patient is a
decrease in measured BSG levels in the patient and the BSG
comprises Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4 or SEQ ID
NO:10) or Mam005 (SEQ ID NO:3).
9. The method of claim 3 or 5 wherein the change associated with
the regression or remission of breast cancer in said patient is an
increase in measured BSG levels in the patient and the BSG
comprises Mam002 (SEQ ID NO:1).
10. A method of identifying potential therapeutic agents for use in
imaging and treating breast cancer comprising screening molecules
for an ability to bind to BSG or alter expression of BSG relative
to BSG in the absence of the agent wherein the ability of a
molecule to bind to BSG or alter expression of BSG is indicative of
the molecule being useful in imaging and treating breast
cancer.
11. An antibody which specifically binds a BSG wherein said BSG
comprises Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4 or SEQ ID
NO:10) or Mam005 (SEQ ID NO:3).
12. A method of imaging breast cancer in a patient comprising
administering to the patient an antibody of claim 11.
13. The method of claim 11 wherein said antibody is labeled with
paramagnetic ions or a radioisotope.
14. A method of treating breast cancer in a patient comprising
administering to the patient an antibody of claim 11.
15. The method of claim 14 wherein the antibody is conjugated to a
cytotoxic agent.
16. A method of treating breast cancer in a patient comprising
administering to the patient a molecule which modulates expression
or activity of BSG.
17. The method of claim 16 wherein the molecule downregulates
expression or activity of BSG comprising Mam001 (SEQ ID NO:2),
Mam004 (SEQ ID NO:4 or SEQ ID NO:10) or Mam005 (SEQ ID NO:3).
18. The method of claim 16 wherein the molecule upregulates
expression or activity of BSG comprising Mam002 (SEQ ID NO:1).
19. A method of inducing an immune response against a target cell
expressing BSG comprising delivering to a human patient an
immunogenically stimulatory amount of a BSG protein so that an
immune response is mounted against the target cell.
Description
INTRODUCTION
[0001] This application is a continuation in part of
PCT/US99/16811, filed Jul. 22, 1999, which claims the benefit of
priority from U.S. provisional application Serial No. 60/095,232,
filed Aug. 4, 1998.
FIELD OF THE INVENTION
[0002] This invention relates, in part, to newly developed assays
for detecting, diagnosing, monitoring, staging, prognosticating,
imaging and treating cancers, particularly breast cancer.
BACKGROUND OF THE INVENTION
[0003] One of every nine American women will develop breast cancer
sometime during her life based on a lifespan of 85 years. Annually,
over 180,000 women in the United States will be diagnosed with
breast cancer and approximately 46,000 will die of the disease.
[0004] Every woman is at risk for breast cancer. A woman's chances
of developing breast cancer increase as she grows older; 80 percent
of all cancers are found in women over the age of 50. There are
also several risk factors that can increase a woman's chances of
developing cancer. A woman may be at increased risk if she has a
family history of the disease, if she had her first child after the
age of 30 or has no children, or if she began menstruating
early.
[0005] However, more than 70 percent of women who develop breast
cancer have no known risk factors. Less than 10 percent of breast
cancer cases are thought to be related to the BRCA1 gene discovered
in 1994. Researchers are now investigating the role other factors
such as nutrition, alcohol, exercise, smoking, and oral
contraceptives may play in cancer prevention.
[0006] As with many other cancers, the best chance for successful
treatment occurs when breast cancer is found early. Mammograms,
special x-rays of the breast, can detect more than 90 percent of
all breast cancers. If breast cancer is found early, the chance of
cure is greater than 90 percent. Treatment options include surgery,
chemotherapy, and radiation therapy depending on the stage of the
cancer.
[0007] Procedures used for detecting, diagnosing, monitoring,
staging, prognosticating and imaging breast cancer are of critical
importance to the outcome of the patient. Patients diagnosed with
early breast cancer generally have a much greater five-year
survival rate as compared to the survival rate for patients
diagnosed with distant metastasized breast cancer. New diagnostic
methods which are more sensitive and specific for detecting early
breast cancer are clearly needed.
[0008] Breast cancer patients are closely monitored following
initial therapy and during adjuvant therapy to determine response
to therapy and to detect persistent or recurrent disease of
metastasis. There is clearly a need for a breast cancer marker
which is more sensitive and specific in detecting breast cancer and
its recurrence and progression.
[0009] Another important step in managing breast cancer is to
determine the stage of the patient's disease. Stage determination
has potential prognostic value and provides criteria for designing
optimal therapy. Generally, pathological staging of breast cancer
is preferable over clinical staging because the former gives a more
accurate prognosis. However, clinical staging would be preferred
were it at least as accurate as pathological staging because it
does not depend on an invasive procedure to obtain tissue for
pathological evaluation. Staging of breast cancer would be improved
by detecting new markers in cells, tissues, or bodily fluids which
could differentiate between different stages of invasion.
[0010] In the present invention methods are provided for detecting,
diagnosing, monitoring, staging, prognosticating, imaging and
treating breast cancer via breast specific genes referred to herein
as BSGs. For purposes of the present invention, BSG refers, among
other things, to native proteins expressed by the gene comprising
the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or
9. An exemplary BSG protein sequence is depicted in SEQ ID NO:10.
By "BSG" it is also meant herein polynucleotides which, due to
degeneracy in genetic coding, comprise variations in nucleotide
sequence as compared to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, but
which still encode the same protein. In the alternative, what is
meant by BSG as used herein, means the native mRNA encoded by the
gene comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3,
4, 5, 6, 7, 8 or 9, levels of the gene comprising the
polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9,
or levels of a polynucleotide which is capable of hybridizing under
stringent conditions to the antisense sequence of SEQ ID NO: 1, 2,
3, 4, 5, 6, 7, 8 or 9.
[0011] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in the art
from the following description. It should be understood, however,
that the following description and the specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
SUMMARY OF THE INVENTION
[0012] Toward these ends, and others, it is an object of the
present invention to provide a method for diagnosing the presence
of breast cancer by analyzing for changes in levels of BSG in
cells, tissues or bodily fluids compared with levels of BSG in
preferably the same cells, tissues, or bodily fluid type of a
normal human control, wherein a change in levels of BSG in the
patient versus the normal human control is associated with breast
cancer.
[0013] Further provided is a method of diagnosing metastatic breast
cancer in a patient having such cancer which is not known to have
metastasized by identifying a human patient suspected of having
breast cancer that has metastasized; analyzing a sample of cells,
tissues, or bodily fluid from such patient for BSG; comparing the
BSG levels in such cells, tissues, or bodily fluid with levels of
BSG in preferably the same cells, tissues, or bodily fluid type of
a normal human control, wherein a change in BSG levels in the
patient versus the normal human control is associated with a cancer
which has metastasized.
[0014] Also provided by the invention is a method of staging breast
cancer in a human which has such cancer by identifying a human
patient having such cancer; analyzing a sample of cells, tissues,
or bodily fluid from such patient for BSG; comparing BSG levels in
such cells, tissues, or bodily fluid with levels of BSG in
preferably the same cells, tissues, or bodily fluid type of a
normal human control sample, wherein a change in BSG levels in the
patient versus the normal human control is associated with a cancer
which is progressing or regressing or in remission.
[0015] Further provided is a method of monitoring breast cancer in
a human having such cancer for the onset of metastasis. The method
comprises identifying a human patient having such cancer that is
not known to have metastasized; periodically analyzing a sample of
cells, tissues, or bodily fluid from such patient for BSG;
comparing the BSG levels in such cells, tissue, or bodily fluid
with levels of BSG in preferably the same cells, tissues, or bodily
fluid type of a normal human control sample, wherein a change in
BSG levels in the patient versus the normal human control is
associated with a cancer which has metastasized.
[0016] Further provided is a method of monitoring the change in
stage of breast cancer in a human having such cancer by looking at
levels of BSG in a human having such cancer. The method comprises
identifying a human patient having such cancer; periodically
analyzing a sample of cells, tissues, or bodily fluid from such
patient for BSG; comparing the BSG levels in such cells, tissue, or
bodily fluid with levels of BSG in preferably the same cells,
tissues, or bodily fluid type of a normal human control sample,
wherein a change in BSG levels in the patient versus the normal
human control is associated with a cancer which is progressing or
regressing or in remission.
[0017] Further provided are methods of designing new therapeutic
agents targeted to a BSG for use in imaging and treating breast
cancer. For example, in one embodiment, therapeutic agents such as
antibodies targeted against BSG or fragments of such antibodies can
be used to treat, detect or image localization of BSG in a patient
for the purpose of detecting or diagnosing a disease or condition.
In this embodiment, a difference in the amount of labeled antibody
detected as compared to normal tissue would be indicative of tumor
metastases or growth. Such antibodies can be polyclonal,
monoclonal, or omniclonal or prepared by molecular biology
techniques. The term "antibody", as used herein and throughout the
instant specification is also meant to include aptamers and
single-stranded oligonucleotides such as those derived from an in
vitro evolution protocol referred to as SELEX and well known to
those skilled in the art. Antibodies can be labeled with a variety
of detectable and therapeutic labels including, but not limited to,
radioisotopes and paramagnetic metals. Therapeutic agents such as
small molecules and antibodies which modulate the concentration
and/or activity of BSG can also be used in the treatment of
diseases characterized by altered expression of BSG. Such agents
can be readily identified in accordance with teachings herein.
[0018] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in the art
from the following description. It should be understood, however,
that the following description and the specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
DESCRIPTION OF THE INVENTION
[0019] The present invention relates to diagnostic assays and
methods, both quantitative and qualitative for detecting,
diagnosing, monitoring, staging, prognosticating and imaging
cancers by comparing levels of BSG with those of BSG in a normal
human control. For purposes of the present invention, BSG refers,
among other things, to native proteins expressed by the gene
comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5,
6, 7, 8 or 9. An exemplary BSG protein sequence is depicted in SEQ
ID NO:10. By "BSG" it is also meant herein polynucleotides which,
due to degeneracy in genetic coding, comprise variations in
nucleotide sequence as compared to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,
8 or 9, but which still encode the same protein. In the
alternative, what is meant by BSG as used herein, means the native
mRNA encoded by the gene comprising the polynucleotide sequence of
SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, levels of the gene
comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5,
6, 7, 8 or 9, or levels of a polynucleotide which is capable of
hybridizing under stringent conditions to the antisense sequence of
SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9. Such levels are preferably
measured in at least one of, cells, tissues and/or bodily fluids,
including determination of normal and abnormal levels. Thus, for
instance, a diagnostic assay in accordance with the invention for
measuring changes in levels of any one of the BSG proteins compared
to normal control bodily fluids, cells, or tissue samples may be
used to diagnose the presence of cancers, including breast cancer.
By "change" it is meant either an increase or decrease in levels of
the BSG. For example, for BSGs such as Mam001 (SEQ ID NO:2), Mam004
(SEQ ID NO:4/SEQ ID NO:10) and Mam005 (SEQ ID NO:3), an increase in
levels as compared to normal human controls is associated with
breast cancer, metastasis and progression of the cancer, while a
decrease in levels is association with regression and/or remission.
For the BSG Mam002 (SEQ ID NO:1), a decrease in levels as compared
to normal human controls is associated with breast cancer,
metastasis and progression while an increase is associated with
regression and/or remission. Any of the 9 BSGs may be measured
alone in the methods of the invention, or all together or any
combination of the nine.
[0020] All the methods of the present invention may optionally
include measuring the levels of other cancer markers as well as
BSG. Other cancer markers, in addition to BSG, such as BRCA1 are
known to those of skill in the art.
[0021] Diagnostic Assays
[0022] The present invention provides methods for diagnosing the
presence of breast cancer by analyzing for changes in levels of BSG
in cells, tissues or bodily fluids compared with levels of BSG in
cells, tissues or bodily fluids of preferably the same type from a
normal human control. As demonstrated herein an increase in levels
of BSGs such as Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4/SEQ ID
No:10 or Mam005 (SEQ ID NO:3) in the patient versus the normal
human control is associated with the presence of breast cancer,
while a decrease in levels of BSGs such as Mam002 (SEQ ID NO:1) in
the patient versus the normal human control is associated with the
presence of breast cancer.
[0023] Without limiting the instant invention, typically, for a
quantitative diagnostic assay a positive result indicating the
patient being tested has cancer is one in which cells, tissues, or
bodily fluid levels of the cancer marker, such as BSG, are at least
two times higher or lower, and most preferably are at least five
times higher or lower, than in preferably the same cells, tissues,
or bodily fluid of a normal human control.
[0024] The present invention also provides a method of diagnosing
metastatic breast cancer in a patient having breast cancer which
has not yet metastasized for the onset of metastasis. In the method
of the present invention, a human cancer patient suspected of
having breast cancer which may have metastasized (but which was not
previously known to have metastasized) is identified. This is
accomplished by a variety of means known to those of skill in the
art. For example, in the case of breast cancer, patients are
typically diagnosed with breast cancer following traditional
detection methods.
[0025] In the present invention, determining the presence of BSG
level in cells, tissues, or bodily fluid, is particularly useful
for discriminating between breast cancer which has not metastasized
and breast cancer which has metastasized. Existing techniques have
difficulty discriminating between breast cancer which has
metastasized and breast cancer which has not metastasized and
proper treatment selection is often dependent upon such
knowledge.
[0026] In the present invention, the cancer marker levels measured
in such cells, tissues, or bodily fluid is BSG, and are compared
with levels of BSG in preferably the same cells, tissue, or bodily
fluid type of a normal human control. That is, if the cancer marker
being observed is just BSG in serum, this level is preferably
compared with the level of BSG in serum of a normal human patient.
An increase in BSGs such as Mam001 (SEQ ID NO:2), Mam004 (SEQ ID
NO:4/SEQ ID NO:10) or Mam005 (SEQ ID NO:3). in the patient versus
the normal human control is associated with breast cancer which has
metastasized while a decrease in BSGs such as Mam002 (SEQ ID NO:1)
in the patient versus the normal human control is associated with
breast cancer which has metastasized.
[0027] Without limiting the instant invention, typically, for a
quantitative diagnostic assay a positive result indicating the
cancer in the patient being tested or monitored has metastasized is
one in which cells, tissues, or bodily fluid levels of the cancer
marker, such as BSG, are at least two times higher or lower, and
most preferably are at least five times higher or lower, than in
preferably the same cells, tissues, or bodily fluid of a normal
patient.
[0028] Normal human control as used herein includes a human patient
without cancer and/or non cancerous samples from the patient; in
the methods for diagnosing or monitoring for metastasis, normal
human control preferably comprises samples from a human patient
that is determined by reliable methods to have breast cancer which
has not metastasized.
[0029] Staging
[0030] The invention also provides a method of staging breast
cancer in a human patient. The method comprises identifying a human
patient having such cancer; analyzing a sample of cells, tissues,
or bodily fluid from such patient for BSG. Then, the method
compares BSG levels in such cells, tissues, or bodily fluid with
levels of BSG in preferably the same cells, tissues, or bodily
fluid type of a normal human control sample, wherein an increase in
levels of BSGs such as Mam001 (SEQ ID NO:2), Mam004 (SEQ ID
NO:4/SEQ ID NO:10) or Mam005 (SEQ ID NO:3) or a decrease in levels
of BSGs such as Mam002 (SEQ ID NO:1) in the patient versus the
normal human control is associated with a cancer which is
progressing and a decrease in levels of BSGs such as Mam001 (SEQ ID
NO:2), Mam004 (SEQ ID NO:4/SEQ ID NO:10) or Mam005 (SEQ ID
NO:3)(but generally still increased over true normal levels) or an
increase in levels of BSGs such as Mam002 (SEQ ID NO:1) (but
generally still decreased as compared to normal levels) is
associated with a cancer which is regressing or in remission.
[0031] Monitoring
[0032] Further provided is a method of monitoring breast cancer in
a human having such cancer for the onset of metastasis. The method
comprises identifying a human patient having such cancer that is
not known to have metastasized; periodically analyzing a sample of
cells, tissues, or bodily fluid from such patient for BSG;
comparing the BSG levels in such cells, tissue, or bodily fluid
with levels of BSG in preferably the same cells, tissues, or bodily
fluid type of a normal human control sample, wherein an increase in
levels of BSGs such as Mam001 (SEQ ID NO:2), Mam004 (SEQ ID
NO:4/SEQ ID NO:10) or Mam005 (SEQ ID NO:3) or a decrease in levels
of BSGs such as Mam002 (SEQ ID NO:1) in the patient versus the
normal human control is associated with a cancer which has
metastasized. In this method, normal human control samples may also
include prior patient samples.
[0033] Further provided by this invention is a method of monitoring
the change in stage of breast cancer in a human having such cancer.
The method comprises identifying a human patient having such
cancer; periodically analyzing a sample of cells, tissues, or
bodily fluid from such patient for BSG; comparing the BSG levels in
such cells, tissue, or bodily fluid with levels of BSG in
preferably the same cells, tissues, or bodily fluid type of a
normal human control sample, wherein an increase in levels of BSGs
such as Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4/SEQ ID NO:10) or
Mam005 (SEQ ID NO:3) or a decrease in levels of BSGs such as Mam002
(SEQ ID NO:1) in the patient versus the normal human control is
associated with a cancer which is progressing in stage and a
decrease in the levels of BSGs such as Mam001 (SEQ ID NO:2), Mam004
(SEQ ID NO:4/SEQ ID NO:10) or Mam005 (SEQ ID NO:3) or an increase
in levels of BSGs such as Mam002 (SEQ ID NO:1) is associated with a
cancer which is regressing in stage or in remission.
[0034] Monitoring such patient for onset of metastasis is periodic
and preferably done on a quarterly basis. However, this may be more
or less frequent depending on the cancer, the particular patient,
and the stage of the cancer.
[0035] Prognostic Testing and Clinical Trial Monitoring
[0036] The methods described herein can further be utilized as
prognostic assays to identify subjects having or at risk of
developing a disease or disorder associated with altered levels of
BSG. The present invention provides a method in which a test sample
is obtained from a human patient and BSG is detected. The presence
of higher levels of Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4/SEQ
ID NO:10) or Mam005 (SEQ ID NO:3) or lower levels of Mam002 (SEQ ID
NO:1) as compared to normal human controls is diagnostic for the
human patient being at risk for developing cancer, particularly
breast cancer.
[0037] The effectiveness of therapeutic agents to alter expression
or activity of the BSGs of the invention can also be monitored by
analyzing levels of expression of the BSGs in a human patient in
clinical trials or in in vitro screening assays such as in human
cells. In this way, the gene expression pattern can serve as a
marker, indicative of the physiological response of the human
patient, or cells as the case may be, to the agent being
tested.
[0038] Detection of genetic lesions or mutations
[0039] The methods of the present invention can also be used to
detect genetic lesions or mutations in BSG, thereby determining if
a human with the genetic lesion is at risk for breast cancer or has
breast cancer. Genetic lesions can be detected, for example, by
ascertaining the existence of a deletion and/or addition and/or
substitution of one or more nucleotides from the BSGs of this
invention, a chromosomal rearrangement of BSG, aberrant
modification of BSG (such as of the methylation pattern of the
genomic DNA), the presence of a non-wild type splicing pattern of a
mRNA transcript of BSG, allelic loss of BSG, and/or inappropriate
post-translational modification of BSG protein. Methods to detect
such lesions in a BSG of this invention are known to those of skill
in the art.
[0040] Assay Techniques
[0041] Assay techniques that can be used to determine levels of
gene expression, such as BSG of the present invention, in a sample
derived from a host are well-known to those of skill in the art.
Such assay methods include, without limitation, radioimmunoassays,
reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry
assays, in situ hybridization assays, competitive-binding assays,
Western Blot analyses, ELISA assays and proteomic approaches,
two-dimensional gel electrophoresis (2D electrophoresis) and
non-gel based approaches such as mass spectrometry or protein
interaction profiling. Among these, ELISAs are frequently preferred
to diagnose a gene's expressed protein in biological fluids.
[0042] An ELISA assay initially comprises preparing an antibody, if
not readily available from a commercial source, specific to BSG,
preferably a monoclonal antibody. In addition a reporter antibody
generally is prepared which binds specifically to BSG. The reporter
antibody is attached to a detectable reagent such as radioactive,
fluorescent or enzymatic reagent, for example horseradish
peroxidase enzyme or alkaline phosphatase.
[0043] To carry out the ELISA, antibody specific to BSG is
incubated on a solid support, e.g. a polystyrene dish, that binds
the antibody. Any free protein binding sites on the dish are then
covered by incubating with a non-specific protein such as bovine
serum albumin. Next, the sample to be analyzed is incubated in the
dish, during which time BSG binds to the specific antibody attached
to the polystyrene dish. Unbound sample is washed out with buffer.
A reporter antibody specifically directed to BSG and linked to
horseradish peroxidase is placed in the dish resulting in binding
of the reporter antibody to any monoclonal antibody bound to BSG.
Unattached reporter antibody is then washed out. Reagents for
peroxidase activity, including a colorimetric substrate are then
added to the dish. Immobilized peroxidase, linked to BSG
antibodies, produces a colored reaction product. The amount of
color developed in a given time period is proportional to the
amount of BSG protein present in the sample. Quantitative results
typically are obtained by reference to a standard curve.
[0044] A competition assay may be employed wherein antibodies
specific to BSG attached to a solid support and labeled BSG and a
sample derived from the host are passed over the solid support and
the amount of label detected attached to the solid support can be
correlated to a quantity of BSG in the sample.
[0045] Using all or a portion of a nucleic acid sequence of BSG of
the present invention as a hybridization probe, nucleic acid
methods can also be used to detect levels of BSG mRNA as a marker
for breast cancer. Polymerase chain reaction (PCR) and other
nucleic acid methods, such as ligase chain reaction (LCR) and
nucleic acid sequence based amplification (NASBA), can be used to
detect cells for diagnosis and monitoring of various malignancies.
For example, reverse-transcriptase PCR (RT-PCR) is a powerful
technique which can be used to detect the presence of a specific
mRNA population in a complex mixture of thousands of other mRNA
species. In RT-PCR, an mRNA species is first reverse transcribed to
complementary DNA (cDNA) with use of the enzyme reverse
transcriptase; the cDNA is then amplified as in a standard PCR
reaction. RT-PCR can thus reveal by amplification the presence of a
single species of mRNA. Accordingly, if the mRNA is highly specific
for the cell that produces it, RT-PCR can be used to identify the
presence and/or absence of a specific type of cell.
[0046] Hybridization to clones or oligonucleotides arrayed on a
solid support (i.e. gridding) can be used to both detect the
expression of and quantitate the level of expression of that gene.
In this approach, all or a portion of a cDNA encoding the BSG is
fixed to a substrate. The substrate may be of any suitable type
including, but not limited to, glass, nitrocellulose, nylon or
plastic. At least a portion of the DNA encoding the BSG is attached
to the substrate and then incubated with the analyte, which may be
RNA or a complementary DNA (cDNA) copy of the RNA, isolated from
the tissue of interest. Hybridization between the substrate bound
DNA and the analyte can be detected and quantitated by several
means including, but not limited to, radioactive labeling or
fluorescence labeling of the analyte or a secondary molecule
designed to detect the hybrid. Quantitation of the level of gene
expression can be done by comparison of the intensity of the signal
from the analyte compared with that determined from known
standards. The standards can be obtained by in vitro transcription
of the target gene, quantitating the yield, and then using that
material to generate a standard curve.
[0047] Of the proteomic approaches, 2D electrophoresis is a
technique well known to those skilled in the art. Isolation of
individual proteins from a sample such as serum is accomplished
using sequential separation of proteins by different
characteristics usually on polyacrylamide gels. First, proteins are
separated by size using an electric current. The current acts
uniformly on all proteins, so smaller proteins move farther on the
gel than larger proteins. The second dimension applies a current
perpendicular to the first and separates proteins not on the basis
of size but on the specific electric charge carried by each
protein. Since no two proteins with different sequences are
identical on the basis of both size and charge, the result of a 2D
separation is a square gel in which each protein occupies a unique
spot. Analysis of the spots with chemical or antibody probes, or
subsequent protein microsequencing can reveal the relative
abundance of a given protein and the identity of the proteins in
the sample.
[0048] The above tests can be carried out on samples derived from a
variety of cells, bodily fluids and/or tissue extracts such as
homogenates or solubilized tissue obtained from a patient. Tissue
extracts are obtained routinely from tissue biopsy and autopsy
material. Bodily fluids useful in the present invention include
blood, urine, saliva or any other bodily secretion or derivative
thereof. By blood it is meant to include whole blood, plasma, serum
or any derivative of blood.
[0049] In Vivo Targeting of BSG/Breast Cancer Therapy
[0050] Identification of BSGs is also useful in the rational design
of new therapeutics for imaging and treating cancers, and in
particular breast cancer. For example, in one embodiment,
antibodies which specifically bind to BSG can be raised and used in
vivo in patients suspected of suffering from breast cancer.
Antibodies which specifically bind BSG can be injected into a
patient suspected of having breast cancer for diagnostic and/or
therapeutic purposes. Thus, another aspect of the present invention
provides for a method for preventing the onset and treatment of
breast cancer in a human patient in need of such treatment by
administering to the patient an effective amount of antibody. By
"effective amount" it is meant the amount or concentration of
antibody needed to bind to the target antigens expressed on the
tumor to cause tumor shrinkage for surgical removal, or
disappearance of the tumor. The binding of the antibody to an
overexpressed BSG is believed to cause the death of the cancer cell
expressing such BSG. The preparation and use of antibodies for in
vivo diagnosis and treatment is well known in the art. For example,
antibody-chelators labeled with Indium-111 have been described for
use in the radioimmunoscintographic imaging of carcinoembryonic
antigen expressing tumors (Sumerdon et. al. Nucl. Med. Biol. 1990
17:247-254). In particular, these antibody-chelators have been used
in detecting tumors in patients suspected of having recurrent
colorectal cancer (Griffin et al. J. Clin. Onc. 1991 9:631-640).
Antibodies with paramagnetic ions as labels for use in magnetic
resonance imaging have also been described (Lauffer, R. B. Magnetic
Resonance in Medicine 1991 22:339-342). Antibodies directed against
a BSG can be used in a similar manner. Labeled antibodies which
specifically bind BSGs can be injected into patients suspected of
having breast cancer for the purpose of diagnosing or staging of
the disease status of the patient. The label used will be selected
in accordance with the imaging modality to be used. For example,
radioactive labels such as Indium-111, Technetium-99m or Iodine-131
can be used for planar scans or single photon emission computed
tomography (SPECT). Positron emitting labels such as Fluorine-19
can be used in positron emission tomography. Paramagnetic ions such
as Gadlinium (III) or Manganese (II) can be used in magnetic
resonance imaging (MRI). Presence of the label, as compared to
imaging of normal tissue, permits determination of the spread of
the cancer. The amount of label within an organ or tissue also
allows determination of the presence or absence of cancer in that
organ or tissue.
[0051] Antibodies which can be used in in vivo methods include
polyclonal, monoclonal and omniclonal antibodies and antibodies
prepared via molecular biology techniques. Antibody fragments and
aptamers and single-stranded oligonucleotides such as those derived
from an in vitro evolution protocol referred to as SELEX and well
known to those skilled in the art can also be used.
[0052] Screening Assays
[0053] The present invention also provides methods for identifying
modulators which bind to BSG protein or have a modulatory effect on
the expression or activity of BSG protein. Modulators which
decrease the expression or activity of BSG proteins such as Mam001
(SEQ ID NO:2), Mam004 (SEQ ID NO:4/SEQ ID NO:10) and Mam005 (SEQ ID
NO:3) or increase the expression or activity of the BSG Mam002 (SEQ
ID NO:1) are believed to be useful in treating breast cancer. Such
screening assays are known to those of skill in the art and
include, without limitation, cell-based assays and cell free
assays.
[0054] Small molecules predicted via computer imaging to
specifically bind to regions of BSG can also be designed,
synthesized and tested for use in the imaging and treatment of
breast cancer. Further, libraries of molecules can be screened for
potential anticancer agents by assessing the ability of the
molecule to bind to the BSGs identified herein. Molecules
identified in the library as being capable of binding to BSG are
key candidates for further evaluation for use in the treatment of
breast cancer. In a preferred embodiment, these molecules will
downregulate expression and/or activity of BSGs such as Mam001 (SEQ
ID NO:2), Mam004 (SEQ ID NO:4/SEQ ID NO:10) and Mam005 (SEQ ID
NO:3) and/or upregulate expression and/or activity of the BSG
Mam002 (SEQ ID NO:1) in cells.
[0055] Adoptive Immunotherapy and Vaccines
[0056] Adoptive immunotherapy of cancer refers to a therapeutic
approach in which immune cells with an antitumor reactivity are
administered to a tumor-bearing host, with the aim that the cells
mediate either directly or indirectly, the regression of an
established tumor. Transfusion of lymphocytes, particularly T
lymphocytes, falls into this category and investigators at the
National Cancer Institute (NCI) have used autologous reinfusion of
peripheral blood lymphocytes or tumor-infiltrating lymphocytes
(TIL), T cell cultures from biopsies of subcutaneous lymph nodules,
to treat several human cancers (Rosenberg, S. A., U.S. Pat. No.
4,690,914, issued Sep. 1, 1987; Rosenberg, S. A., et al., 1988, N.
England J. Med. 319:1676-1680).
[0057] The present invention relates to compositions and methods of
adoptive immunotherapy for the prevention and/or treatment of
primary and metastatic breast cancer in humans using macrophages
sensitized to the antigenic BSG molecules, with or without
non-covalent complexes of heat shock protein (hsp). Antigenicity or
immunogenicity of the BSG is readily confirmed by the ability of
the BSG protein or a fragment thereof to raise antibodies or
educate naive effector cells, which in turn lyse target cells
expressing the antigen (or epitope).
[0058] Cancer cells are, by definition, abnormal and contain
proteins which should be recognized by the immune system as foreign
since they are not present in normal tissues. However, the immune
system often seems to ignore this abnormality and fails to attack
tumors. The foreign BSG proteins that are produced by the cancer
cells can be used to reveal their presence. The BSG is broken into
short fragments, called tumor antigens, which are displayed on the
surface of the cell. These tumor antigens are held or presented on
the cell surface by molecules called MHC, of which there are two
types: class I and II. Tumor antigens in association with MHC class
I molecules are recognized by cytotoxic T cells while antigen-MHC
class II complexes are recognized by a second subset of T cells
called helper cells. These cells secrete cytokines which slow or
stop tumor growth and help another type of white blood cell, B
cells, to make antibodies against the tumor cells.
[0059] In adoptive immunotherapy, T cells or other antigen
presenting cells (APCs) are stimulated outside the body (ex vivo),
using the tumor specific BSG antigen. The stimulated cells are then
reinfused into the patient where they attack the cancerous cells.
Research has shown that using both cytotoxic and helper T cells is
far more effective than using either subset alone. Additionally,
the BSG antigen may be complexed with heat shock proteins to
stimulate the APCs as described in U.S. Pat. No. 5,985,270.
[0060] The APCs can be selected from among those antigen presenting
cells known in the art including, but not limited to, macrophages,
dendritic cells, B lymphocytes, and a combination thereof, and are
preferably macrophages. In a preferred use, wherein cells are
autologous to the individual, autologous immune cells such as
lymphocytes, macrophages or other APCs are used to circumvent the
issue of whom to select as the donor of the immune cells for
adoptive transfer. Another problem circumvented by use of
autologous immune cells is graft versus host disease which can be
fatal if unsuccessfully treated.
[0061] In adoptive immunotherapy with gene therapy, DNA of the BSG
can be introduced into effector cells similarly as in conventional
gene therapy. This can enhance the cytotoxicity of the effector
cells to tumor cells as they have been manipulated to produce the
antigenic protein resulting in improvement of the adoptive
immunotherapy.
[0062] BSG antigens of this invention are also useful as components
of breast cancer vaccines. The vaccine comprises an immunogenically
stimulatory amount of a BSG antigen. Immunogenically stimulatory
amount refers to that amount of antigen that is able to invoke the
desired immune response in the recipient for the amelioration, or
treatment of breast cancer. Effective amounts may be determined
empirically by standard procedures well known to those skilled in
the art.
[0063] The BSG antigen may be provided in any one of a number of
vaccine formulations which are designed to induce the desired type
of immune response, e.g., antibody and/or cell mediated. Such
formulations are known in the art and include, but are not limited
to, formulations such as those described in U.S. Pat. No.
5,585,103. Vaccine formulations of the present invention used to
stimulate immune responses can also include pharmaceutically
acceptable adjuvants.
EXAMPLES
[0064] The present invention is further described by the following
examples. The examples are provided solely to illustrate the
invention by reference to specific embodiments. These
exemplifications, while illustrating certain specific aspects of
the invention, do not portray the limitations or circumscribe the
scope of the disclosed invention.
Example 1
[0065] Identification of BSGs were carried out by a systematic
analysis of data in the LIFESEQ database available from Incyte
Pharmaceuticals, Palo Alto, Calif., using the data mining Cancer
Leads Automatic Search Package (CLASP) developed by diaDexus LLC,
Santa Clara, Calif.
[0066] The CLASP performs the following steps:
[0067] Selection of highly expressed organ specific genes based on
the abundance level of the corresponding EST in the targeted organ
versus all the other organs.
[0068] Analysis of the expression level of each highly expressed
organ specific genes in normal, tumor tissue, disease tissue and
tissue libraries associated with tumor or disease. Selection of the
candidates demonstrating component ESTs were exclusively or more
frequently found in tumor libraries.
[0069] CLASP allows the identification of highly expressed organ
and cancer specific genes useful in the diagnosis of breast
cancer.
1TABLE 1 BSGs Sequences SEQ ID NO: LS Clone ID LSA Gene ID 1
2740238 (Mam002) 242151 2 1730886 (Mam001) 238469 3 y155b03
(Mam005) 348845 4 2613064 (Mam004) 27052 5 894184 221086 6 2299454
27681 7 2258254 248176 8 789767 156580 9 1213903 219737
[0070] The following example was carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. Routine
molecular biology techniques of the following example can be
carried out as described in standard laboratory manuals, such as
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
Example 2
[0071] Relative Quantitation of Gene Expression
[0072] Real-time quantitative PCR with fluorescent Taqman probes is
a quantitative detection system utilizing the 5'-3' nuclease
activity of Taq DNA polymerase. The method uses an internal
fluorescent oligonucleotide probe (Taqman) labeled with a 5'
reporter dye and a downstream, 3' quencher dye. During PCR, the
5'-3' nuclease activity of Taq DNA polymerase releases the
reporter, whose fluorescence can then be detected by the laser
detector of the Model 7700 Sequence Detection System (PE Applied
Biosystems, Foster City, Calif., USA).
[0073] Amplification of an endogenous control was used to
standardize the amount of sample RNA added to the reaction and
normalize for Reverse Transcriptase (RT) efficiency. Either
cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or
18S ribosomal RNA (rRNA) was used as this endogenous control. To
calculate relative Quantitation between all the samples studied,
the target RNA levels for one sample were used as the basis for
comparative results (calibrator) Quantitation relative to the
"calibrator" can be obtained using the standard curve method or the
comparative method (User Bulletin #2: ABI PRISM 7700 Sequence
Detection System). To evaluate the tissue distribution, and the
level of breast specific markers (BSM) Mam001 (SEQ ID NO:2), Mam002
(SEQ ID NO:1), Mam004 (SEQ ID NO:4/SEQ ID NO:10) and Mam005 (SEQ ID
NO:3) in normal and cancer tissue, total RNA was extracted from
cancer and matched normal adjacent tissues (NAT) and from unmatched
cancer and normal tissues. Subsequently, first strand cDNA was
prepared with reverse transcriptase and the polymerase chain
reaction carried out using primers and Taqman probes specific to
each of Mam001 (SEQ ID NO:2), Mam002 (SEQ ID NO:1), Mam004 (SEQ ID
NO:4/SEQ ID NO:10) and Mam005 (SEQ ID NO:3) respectively. The
results are obtained using the ABI PRISM 7700 Sequence Detector.
The numbers are relative levels of expression of Mam001 (SEQ ID
NO:2), Mam002 (SEQ ID NO:1), Mam004 (SEQ ID NO:4/SEQ ID NO:10) and
Mam005 (SEQ ID NO:3) compared to their respective calibrators.
[0074] Measurement of SEQ ID NO:2; Clone ID:1730886; Gene ID:
238469 (Mam001)
[0075] The numbers depicted in Table 2 are relative levels of
expression in 12 normal tissues of Mam001 (SEQ ID NO:2) compared to
testis (calibrator). These RNA samples were obtained commercially
and were generated by pooling samples from a particular tissue from
different individuals.
2TABLE 2 Relative levels of Mam001 (SEQ ID NO: 2) Expression in
Pooled Samples Tissue NORMAL Brain 0 Heart 0 Kidney 0 Liver 0 Lung
0 Mammary 6 Prostate 0 Muscle 0 Small Intestine 0 Testis 1 Thymus 0
Uterus 0
[0076] The relative levels of expression in Table 2 show that
Mam001 (SEQ ID NO:2) mRNA expression is detected in the pool of
normal mammary and in testis but not in the other 10 normal tissue
pools analyzed. These results demonstrate that Mam001 (SEQ ID NO:2)
mRNA expression is highly specific for mammary tissue and is also
found in testis. Expression in a male specific tissue is not
relevant in detecting cancer in female specific tissues
[0077] The tissues shown in Table 2 are pooled samples from
different individuals. The tissues shown in Table 3 were obtained
from individuals and are not pooled. Hence the values for mRNA
expression levels shown in Table 2 cannot be directly compared to
the values shown in Table 3.
[0078] The numbers depicted in Table 3 are relative levels of
expression of Mam001 (SEQ ID NO:2) compared to testis (calibrator),
in 24 pairs of matching samples. Each matching pair contains the
cancer sample for a particular tissue and the normal adjacent
tissue (NAT) sample for that same tissue from the same
individual.
3TABLE 3 Relative levels of Mam001 (SEQ ID NO: 2) Expression in
Individual Samples Matching Sample ID Tissue Cancer Normal. Mam
47XP Mammary Gland 0 0 Mam A06X Mammary Gland 23 1 Mam B011X
Mammary Gland 0 5 Mam 603X/C034 Mammary Gland 0 2.10 Mam 162X
Mammary Gland 1.96 0.15 Mam 42DN Mammary Gland 0.38 1.27 Mam S079
Mammary Gland 0.34 0.36 Mam S123 Mammary Gland 0.03 0.87 Mam S516
Mammary Gland 0.43 0.53 Mam S699 Mammary Gland 0.40 0.66 Mam S997
Mammary Gland 0.41 0.51 Sto AC44 Stomach 0 0 TST 39X Testis 0 0 Cln
SG45 Colon 0 0 Cln TX01 Colon 0 0 Cvx NK23 Cervix 0 0 Cvx NK24
Cervix 0 0 Endo 3AX Endometrium 0 0 Endo 4XA Endometrium 0 0 Endo
5XA Endometrium 0 0 Kid 11XD Kidney 0 0 Kid 5XD Kidney 0 0 Lng C20X
Lung 0 0 Lng SQ56 Lung 0 0
[0079] Among 48 samples in Table 3 representing 8 different tissues
expression is seen only in mammary tissues. These results confirm
the tissue specificity results obtained with normal samples shown
in Table 2. Table 2 and Table 3 represent a combined total of 60
samples in 16 human tissue types. Thirty-six samples representing
14 different tissue types excluding breast and testis had no
detected Mam001 (SEQ ID NO:2) mRNA (Table 2 and 3). Other than
breast tissue, Mam001 (SEQ ID NO:2) is detected only in one other
tissue type (Testis) and then only in the pooled tissue sample
(Table 2) but not in the matched testis cancer samples (Table
3).
[0080] Comparisons of the level of mRNA expression in breast cancer
samples and the normal adjacent tissue from the same individuals
are shown in Table 3. Mam001 (SEQ ID NO:2) is expressed at higher
levels in 2 of 11 breast cancer tissues (Mam A06X and Mam 162X)
compared with the corresponding normal adjacent tissue. The level
of Mam001 (SEQ ID NO:2) expression is lower in breast cancer
compared to normal adjacent tissue in four matched samples (Mam
B011X, Mam 603X/CO34, Mam 42DN and Mam S123). No expression was
detected in one set of matched samples (Mam 47XP). Equivalent
levels or very similar levels of expression were detected in four
other matched samples. (Mam S079, Mam S516, Mam S699 and Mam S997).
However increasing cancer mass might in these cases result in an
overall increase in the total amount of expression.
[0081] The high level of tissue specificity and increased or
equivalent expression in 6 of 11 individuals is demonstrative of
Mam001 (SEQ ID NO:2) being a diagnostic marker for detection of
mammary cancer cells using mRNA.
[0082] Measurement of SEQ ID NO:1; Clone ID: 2740238; Gene ID
242151 (Mam002)
[0083] The numbers depicted in Table 5 are relative levels of
expression in 12 normal tissues of Mam002 (SEQ ID NO:1) compared to
Thymus (calibrator). These RNA samples were obtained commercially
and were generated by pooling samples from a particular tissue from
different individuals.
4TABLE 4 Relative levels of Mam002 (SEQ ID NO: 1) Expression in
Pooled Samples Tissue NORMAL Brain 0.03 Heart 0.01 Kidney 0 Liver 0
Lung 0.06 Mammary 289.01 Muscle 0 Prostate 0.31 Small Int. 0 Testis
0.08 Thymus 1.00 Uterus 0
[0084] The relative levels of expression in Table 4 show that
Mam002 (SEQ ID NO:1) mRNA expression is detected at a high level in
the pool of normal mammary but at very low levels in the other 11
normal tissue pools analyzed. These results demonstrate that Mam002
(SEQ ID NO:1) mRNA expression is highly specific for mammary
tissue.
[0085] The tissues shown in Table 4 are pooled samples from
different individuals. The tissues shown in Table 5 were obtained
from individuals and are not pooled. Hence the values for mRNA
expression levels shown in Table 4 cannot be directly compared to
the values shown in Table 5.
[0086] The numbers depicted in Table 5 are relative levels of
expression of Mam002 (SEQ ID NO:1) compared to thymus (calibrator)
in 27 pairs of matching samples. Each matching pair contains the
cancer sample for a particular tissue and the normal adjacent
tissue (NAT) sample for that same tissue from the same individual.
In addition 2 unmatched mammary samples from normal tissues and one
unmatched ovarian cancer and one normal (non-cancerous) ovary were
also tested.
5TABLE 5 Relative levels of Mam002 (SEQ ID NO: 1) Expression in
Individual Samples Sample ID Tissue Cancer Matching Normal Mam 12X
Mammary 7.2 69 Gland Mam 42DN Mammary 1051 2075 Gland Mam 59X
Mammary 7.0 15.5 Gland Mam A06X Mammary 1655 1781 Gland Mam B011X
Mammary 32.1 2311 Gland Mam S127 Mammary 1.73 0 Gland Mam S516
Mammary 9.72 69.95 Gland Mam S699 Mammary 83.46 75.65 Gland Mam
S854 Mammary 133.23 836.56 Gland Mam S967 Mammary 59.77 188.28
Gland Mam S997 Mammary 94.14 73.64 Gland Mam 162X Mammary 674.0
31.1 Gland Mam C012 Mammary N/A N/A 11379.3 Gland Mam C034 Mammary
N/A N/A 3502.6 Gland Mam S079 Mammary 11772.5 903.5 Gland Mam S123
Mammary 3.4 170.5 Gland Ovr 103X ovary 0 0 Ovr 1118 ovary 0.13 N/A
Ovr 35GA Ovary N/A N/A 0.13 Utr 23XU Uterus 5.6 0 Utr 135XO Uterus
0 0 Cvx NK24 Cervix 0.9 1.4 End 4XA Endometrium 32.2 0 Cln AS43
Colon 2.3 0 Cln AS45 Colon 0 0 Cln RC01 Colon 0.2 0 Lng AC90 Lung 0
2.0 Lng LC109 Lung 0 0.6 Lng SC32 Lung 0.8 0 Sto AC93 Stomach 0 0
Tst 39X Testis 1.97 0
[0087] Among 58 samples in Table 5' representing 9 different
tissues, the highest expression is seen in mammary tissues. Amongst
the non-breast tissues which show expression, only one sample (End
4XA) has expression comparable to that seen in the majority of the
breast samples tested. This sample is endometrial tissue, which is
a female specific tissue. These results confirm the tissue
specificity results obtained with normal samples shown in Table 4.
Table 4 and Table 5 represent a combined total of 70 samples in 17
human tissue types. Twenty-two samples representing 11 different
tissue types excluding breast had no detected Mam002 (SEQ ID NO:1)
mRNA (Table 4 and Table 5).
[0088] Comparisons of the level of mRNA expression in breast cancer
samples and the normal adjacent tissue from the same individuals
are shown in Table 5. Mam002 (SEQ ID NO:1) is expressed at higher
levels in 3 of 13 matched breast cancer tissues (Samples Mam S127,
Mam 162X and Mam S079) compared with the corresponding normal
adjacent tissue. The level of Mam002 (SEQ ID NO:1) expression is
lower in breast cancer compared to normal adjacent tissue in eight
individuals (Mam 12X, Mam 42DN, Mam 59X, Mam B011X, Mam S516, Mam
S854, Mam S967, and Mam S123). Equivalent levels or very similar
levels of expression were detected in three other matched samples
(Samples Mam A06X, Mam S699 and Mam S997).
[0089] The high level of tissue specificity is demonstrative of
Mam002 (SEQ ID NO:1) being a diagnostic marker for detection of
mammary cancer cells using mRNA. Breast tissue is the only
significant source of this gene's expression so far detected. Eight
of 13 matched samples have lower levels of expression in cancer
than normal adjacent tissue. Thus, decreased expression of this
gene appears to be diagnostic of cancer presence.
[0090] Measurement of SEQ ID NO:4; Clone ID: 2613064; Gene ID:
27052 (Mam004)
[0091] The numbers depicted in Table 6 are relative levels of
expression in 12 normal tissues of Mam004 (SEQ ID NO:4) compared to
mammary (calibrator). These RNA samples were obtained commercially
and were generated by pooling samples from a particular tissue from
different individuals.
6TABLE 6 Relative levels of Mam004 (SEQ ID NO: 4) Expression in
Pooled Samples Tissue NORMAL Brain 0.059 Heart 0.131 Kidney 0.018
Liver 0 Lung 0.478 Mammary 1.000 Prostate 0.459 Muscle 0.003 Small
Intestine 0.048 Testis 0.130 Thymus 0.030 Uterus 0.071
[0092] The relative levels of expression in Table 6 show that
Mam004 (SEQ ID NO:4) mRNA expression is detected in the pool of
normal mammary and also in other tissues including lung, prostate,
testis and heart. These results demonstrate that although more
highly expressed in normal breast tissue Mam004(SEQ ID NO:4) mRNA
expression is not specific for mammary gland.
[0093] The tissues shown in Table 6 are pooled samples from
different individuals. The tissues shown in Table 7 were obtained
from individuals and are not pooled. Hence the values for mRNA
expression levels shown in Table 6 cannot be directly compared to
the values shown in Table 7.
[0094] The numbers depicted in Table 7 are relative levels of
expression of Mam004 (SEQ ID NO:4) compared to mammary
(calibrator), in 23 pairs of matching samples. Each matching pair
contains the cancer sample for a particular tissue and the normal
adjacent tissue (NAT) sample for that same tissue from the same
individual.
7TABLE 7 Relative levels of Mam004 (SEQ ID NO: 4) Expression in
Individual Samples Sample ID Tissue Cancer Matching Mam 12B Mammary
Gland 0 0 Mam 12X Mammary Gland 13.454 0 Mam 603X Mammary Gland
30.484 0 Mam 59X Mammary Gland 1.306 0 Mam 162X Mammary Gland 0.71
0.04 Mam 42DN Mammary Gland 0.25 2.17 Mam S079 Mammary Gland 42.18
0.47 Mam S123 Mammary Gland 0.01 0 Mam S516 Mammary Gland 1.17 0.41
Mam S699 Mammary Gland 0.11 0.55 Mam S997 Mammary Gland 10.43 1.29
Sto AC44 Stomach 0.61 0 Cln SG45 Colon 0.04 0 Cln TX01 Colon 0 0
Cvx NK23 Cervix 0 0 Cvx NK24 Cervix 0 0 Endo 3AX Endometrium 0 0
Endo 4XA Endometrium 0 0 Endo 5XA Endometrium 0 2.73 Kid 11XD
Kidney 0 0 Kid 5XD Kidney 0 2.63 Lng C20X Lung 0 0 Lng SQ56 Lung
10.37 0
[0095] Among 46 samples in Table 7 representing 7 different tissues
expression is highest in breast tissues particularly cancers.
Expression comparable to that seen in breast samples is also seen
in 1 of 4 lung samples (Sample 23), 1 of 4 kidney samples (Sample
21) and 1 of 6 endometrial samples (Sample 19). Table 6 and Table 7
represent a combined total of 58 samples in 16 human tissue types.
Twenty samples representing 7 different tissue types excluding
breast had no detected Mam004 (SEQ ID NO:4) mRNA (Table 6 and Table
7).
[0096] Comparisons of the level of mRNA expression in breast cancer
samples and the normal adjacent tissue from the same individuals
are shown in Table 7. Mam004 (SEQ ID NO:4) is expressed at higher
levels in 8 of 11 breast cancer tissues (Mam 12X, Mam 603X, Mam
59X, Mam 162X, Mam S079, Mam S123, Mam S516 and Mam S997) compared
with the corresponding normal adjacent tissue. The level of Mam004
(SEQ ID NO:4) expression is lower in breast cancer compared to
normal adjacent tissue in two matched samples (Mam 42DN and Mam
S699). No expression was detected in one matched sample (Mam
12B).
[0097] Elevated expression in the majority of matched cancer
samples compared to normal adjacent tissue is indicative of Mam004
(SEQ ID NO:4) being a diagnostic marker for detection of mammary
cancer cells using mRNA.
[0098] Measurement of SEQ ID NO:3; Clone ID:y155b03; Gene ID:
348845 (Mam005)
[0099] The numbers depicted in Table 8 are relative levels of
expression in 12 normal tissues of Mam005 (SEQ ID NO:3) compared to
testis (calibrator). These RNA samples were obtained commercially
and were generated by pooling samples from a particular tissue from
different individuals.
8TABLE 8 Relative levels of Mam005 (SEQ ID NO: 3) Expression in
Pooled Samples Tissue NORMAL Brain 0 Heart 0.0002 Kidney 0.0001
Liver 0 Lung 0 Mammary 5.4076 Muscle 0 Prostate 0 Small Intestine 0
Testis 1 Thymus 0 Uterus 0
[0100] The relative levels of expression in Table 8 show that
Mam005 (SEQ ID NO:3) mRNA expression is detected in the pool of
normal mammary and in testis but is not present at significant
levels in the other 10 normal tissue pools analyzed. These results
demonstrate that Mam005 (SEQ ID NO:3) mRNA expression is highly
specific for mammary tissue and is also found in testis. Expression
in a male specific tissue is not relevant in detecting cancer in
female specific tissues.
[0101] The tissues shown in Table 8 are pooled samples from
different individuals. The tissues shown in Table 9 were obtained
from individuals and are not pooled. Hence the values for mRNA
expression levels shown in Table 8 cannot be directly compared to
the values shown in Table 9.
[0102] The numbers depicted in Table 9 are relative levels of
expression of Mam005 (SEQ ID NO:3) compared to testis (calibrator),
in 46 pairs of matching samples. Each matching pair contains the
cancer sample for a particular tissue and the normal adjacent
tissue sample for that same tissue from the same individual. In
addition 2 unmatched mammary samples from normal tissues and one
unmatched ovarian cancer and one normal (non-cancerous) ovary were
also tested.
9TABLE 9 Relative levels of Mam005 (SEQ ID NO: 3) Expression in
Individual Samples Sample ID Tissue Cancer Matching Normal Mam 12X
Mammary 0.33 0.71 Gland Mam 42DN Mammary 0.22 0.63 Gland Mam 59X
Mammary 0.03 0.23 Gland Mam A06X Mammary 70.77 0.56 Gland Mam B011X
Mammary 0.03 1.52 Gland Mam 162X Mammary 0.43 0.09 Gland Mam C012
Mammary N/A N/A 1.6 Gland Mam C034 Mammary N/A N/A 2.9 Gland Mam
S079 Mammary 0.22 0.13 Gland Mam S123 Mammary 0.01 0.23 Gland Mam
S127 Mammary 0 0.28 Gland Mam S516 Mammary 0.15 0.05 Gland Mam S699
Mammary 0.21 0.42 Gland Mam S854 Mammary 1.12 0.54 Gland Mam S967
Mammary 30.61 0.54 Gland Mam S997 Mammary 0.40 0.22 Gland Mam 14DN
Mammary 0.07 0 Gland Mam 699F Mammary 0.01 0.09 Gland Mam S621
Mammary 1.82 0 Gland Mam S918 Mammary 6.89 1.06 Gland Cln CM67
Colon 0 0 Cln DC19 Colon 0 0 Cln AS43 Colon 0 0 Cln AS45 Colon 0 0
Cln RC01 Colon 0.0012 0.0003 Lng AC90 Lung 0 0 Lng LC109 Lung 0 0
Lng SQ32 Lung 0 0 Lng SQ43 Lung 0 0 Ovr 103X Ovary 0 0 Ovr 1118
Ovary 0 N/A Ovr A084 Ovary 0 0 Ovr G021 Ovary 0 0 Ovr 35GA Ovary
N/A N/A 0 Cvx NK23 Cervix 0 0 Cvx NK24 Cervix 0 0 Endo 3AX
Endometrium 0 0 Endo 4XA Endometrium 0 0 Sto 758S Stomach 0 0 Sto
AC44 Stomach 0 0 Sto AC93 Stomach 0 0 Tst 39X Testis 0.01 0.01 Utr
85XU Uterus 0 0 Utr 135XO Uterus 0 0 Utr 23XU Uterus 0 0 Kid 124D
Kidney 0 0 Lvr 15XA Liver 0 0 Pan CO44 Pancreas 0 0 Skn 448S Skin 0
0 SmInt 21XA Small 0 0 Intestines
[0103] Among 96 samples in Table 9 representing 14 different
tissues significant expression is seen only in breast tissues.
These results confirm the tissue specificity results obtained with
normal samples shown in Table 8. Table 8 and Table 9 represent a
combined total of 108 samples in 18 human tissue types. Sixty-seven
samples representing 16 different tissue types excluding breast and
testis had either no or very low levels of detected Mam005 (SEQ ID
NO:3) mRNA (Table 8 and Table 9).
[0104] Comparisons of the level of mRNA expression in breast cancer
samples and the normal adjacent tissue from the same individuals
are shown in Table 9. Mam005 (SEQ ID NO:3) is expressed at higher
levels in 10 of 18 cancer and normal adjacent tissue samples (Mam
A06X, Mam 162X, Mam S079, Mam S516, Mam S854, Mam S967, Mam S997,
Mam 14DN, Mam S621, and Mam S918) compared with the corresponding
normal adjacent tissue. The level of Mam005 (SEQ ID NO:3)
expression is lower in breast cancer compared to normal adjacent
tissue in eight cancer and normal adjacent tissue samples (Mam 12X,
Mam 42DN, Mam 59X, Mam B011X, Mam S123, Mam S127, Mam S699 and Mam
699F). No expression was detected in two matching samples.
[0105] The high level of tissue specificity and overexpression in
10 of 18 matched cancer and normal adjacent tissue samples is
indicative of Mam005 (SEQ ID NO:3) being a diagnostic marker for
detection of mammary cancer cells using mRNA.
Sequence CWU 1
1
10 1 544 DNA Homo sapiens unsure (505)..(506) a, c, g or t 1
ctagtctcga gtctagagcg ccttgccttc tcttaggctt tgaagcattt ttgtctgtgc
60 tccctgatct tcatgtcacc accatgaagt tcttagcagt cctggtactc
ttgggagttt 120 ccatctttct ggtctctgcc cagaatccga caacagctgc
tccagctgac acgtatccag 180 ctactggtcc tgctgatgat gaagcccctg
atgctgaaac cactgctgct gcaaccactg 240 cgaccactgc tgctcctacc
actgcaacca ccgctgcttc taccactgct cgtaaagaca 300 ttccagtttt
acccaaatgg gttggggatc tcccgaatgg tagagtgtgt ccctgagatg 360
gaatcagctt gagtcttctg caattggtca caactattca tgcttcctgt gatttcatcc
420 aactacttac cttgcctacg atatcccctt tatctctaat cagtttattt
tctttcaaat 480 aaaaaataac tatgagcaac taaannaaan aaaaaaaaaa
naaaaannaa naannaaaan 540 naga 544 2 1066 DNA Homo sapiens unsure
(729)..(813) a, c, g or t 2 gttgaccagt ggtcatgcca ctgcctgttg
atttgttgaa aatattgttt acacgtatgt 60 tcttgttact gattgtcaga
aagctggttt tgagactgca gcttggacta aattcagtca 120 tctggctgtc
tggggaagca tgctgaccag tctggtgttc tttggcatct actcagccat 180
ctggtccacc attctcattg ccccaaatat gagaggacag aagaatggta ccggtactgc
240 caatggagat ggaggaagga gacagaaaga aacagagccc agaccctagg
gaccaccagc 300 atttgcagaa tggataaaca gccttcttcc taacaaagga
agcacagcaa ctgtgatcct 360 gagctgtgca cacttctggt tgggattatt
tctggtttct acttcctgtt tgaagatgtg 420 gcatggagag tgaacaagct
gctgcccacc acctggcatc acagccccag aactcagcta 480 tttccatggg
accacagcat ctcatctctg ggctgagcca gaaagacccc tactgaagtc 540
cagaggcact tttctgaaag gctctgcttt gacctgaagt attttatcta tcctcagtct
600 caggacactg ttgatggaat taaggccaag cacatctgca aaaaagacat
tgctggagga 660 ggtgcaaaga gctggaaacc aagtctccag tcctgggaaa
agcagtggta tggaaaagca 720 atggaaagnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnncatagca ccaatgacct gaagagcctt 840 gttgaaggaa
gactccatct gatgactcag agcaagtatt ttttagtgtg ttattgttat 900
tagcagaaag agggccataa aatacatggg gcaagctgaa tatatcttag gcaaaagaag
960 aaaatattca aattcttatg ttattttatc taattatttt atctcttttt
gtgtgtgact 1020 tataatgtgt gtattgtatt aataaaagta tataaacatg tagttt
1066 3 4197 DNA Homo sapiens 3 ggcacgagac aactcatgct aggaggccag
tcctagcatc accttatgtt gaaaatctta 60 ccaatagtct gtgtcaacag
aatacttatt ttagaagaaa aattcatgat ttcttcctga 120 agcctacaga
cataaaataa cagtgtgaag aattacttgt tcacgaattg cataaagctg 180
cacaggattc ccatctaccc tgatgatgca gcagacatca ttcaatccaa ccagaatctc
240 gctctgtcac tcaggctgga gtgcagtggc gcaatctcgg ctcactgcaa
actctgcctc 300 ccaggttcac gccattctcc tgccacagcc tcccgagtag
ctgggactac aggcgcccgc 360 caccaagcac agctaagttt tttatttata
gtagagacgg ggtttcactg tgttagccag 420 gatggtctcg atctcctgac
ctcgtgatct gcctgcctcg gcctcccaaa gtgccgggat 480 tacaggcgtg
agccaccgcg ccgggcctga tttcagtttc ttccagccct tcctattgtt 540
aacatggggg ttgtgttgaa gaatataaag ttacaaagtc aaggaagtag gaaacatttt
600 tacaagtatt atgtagccat cttggtgggg ctgtggtgag gtaggctgca
aatgattctc 660 ctatttcttt ccctgagttc agaacatagg aattagattg
atagacatca acatacccgc 720 tttattgctg actcatgaca actaatggga
agacatggct cagatgtgca gccacagtga 780 gcttctgaac atttcttctc
agactaagct cttacacaca gttgcagttg aaagaaagaa 840 ttgcttgaca
tggccacagg agcaggcagc ttcctgcaga catgacagtc aacgcaaact 900
catgtcactg tgggcagaca catgtttgca aagagactca gagccaaaca agcacactca
960 atgtgctttg cccaaattta cccattaggt aaatcttccc tcctcccaag
aagaaagtgg 1020 agagagcatg agtcctcaca tggaaacttg aagtcaggga
aatgaaggct caccaattat 1080 ttgtgcatgg gtttaagttt tccttgaaat
taagttcagg tttgtctttg tgtgtaccaa 1140 ttaatgacaa gaggttagat
agaagtatgc tagatggcaa agagaaatat gttttgtgtc 1200 ttcaattttg
ctaaaaataa cccagaacat ggataattca tttattaatt gattttggta 1260
agccaagtcc tatttggaga aaattaatag tttttctaaa aaagaatttt ctcaatatca
1320 cctggcatga taacattttt ctccttcgag ttcctttttc tggagtttaa
caaacttgtt 1380 ctttacaaat agattatatt gactacctct cactgatgtt
atgatattag tttctattgc 1440 ttactttgta tttctaattt taggattcac
aatttagctg gagaactatt ttttaacctg 1500 ttgcacctaa acatgattga
gctagaagac agttttacca tatgcatgca ttttctctga 1560 gttatatttt
aaaatctata catttctcct aaatatggag gaaatcactg gcatcaaatg 1620
ccagtctcag acggaagacc taaagcccat ttctggcctg gagctacttg gctttgtgac
1680 ctatggtgag gcataagtgc tctgagtttg tgttgcctct tttgtaaaat
gagggtttga 1740 cttaatcagt gattttcata gcttaaaatt tttttgaaga
acagaacttt ttttaaaaac 1800 agttagatgc aaccatatta tataaaacag
aacagataca agtagagcta acttgctaaa 1860 gaaaggatgg aggctctgaa
gctgtgactt cattatccct taatactgct atgtcctctg 1920 tagtacctta
gatttctatg ggacatcgtt taaaaactat tgtttatgcg agagccttgc 1980
taatttccta aaaattgtgg atacattttt tctcccatgt ataattttct caccttctat
2040 ttaaaaagaa aaaaaaagtc agtgtagtat ttacatattt taccctataa
ggagctaaca 2100 taacttttga tttagtgtta ttcataaaat taggttagca
gtttattaac cttttgtatt 2160 tgctctggca atgtttaata tctcataagc
tatacacacc tcgaagccat caatgacaac 2220 cttttcttgc tgaatagaac
agtgattgat gtcatgaaga caattttatc tccttttgcc 2280 ttccataatt
tgtaccaggt tatataatag tataacactg ccaaggagcg gattatctca 2340
tcttcatcct gtaattccag tgtttgtcac gtggttgttg aataaatgaa taaagaatga
2400 gaaaaccaga agctctgata cataatcata atgataatta tttcaatgca
caactacggg 2460 tggtgctgaa ctagaatcta tattttctga aactggctcc
tctaggatct actaatgatt 2520 taaatctaaa agatgaagtt agtaaagcat
cagaaaaaaa aggtaaacaa attgctcctg 2580 tggagatgat tggcatcaca
tggtgttttg agctgataca cccaacactt gagctcactg 2640 caacagtacc
agattttcac cgctatgcct cctttcactc tgggagtctt ccagaggtct 2700
tgcactcggg agagcatgct caggtttccc cagctctaca aaatcaccca gaatgccaaa
2760 gacttcaaca caagggtaaa taaggttgat ctcagaattg tcacctcaaa
aaggccctgc 2820 cttccactgt tcagttctgg tcatctgcct atgagatatc
tgaagcttga aagagaacac 2880 ttgaaaatca ctgagaccgt gactcccatc
ccagcacaca cagcaagcca aagtccacac 2940 catggaaacc gattcctcat
cttttaagaa taccatatgg atacttatat ataggcatga 3000 attaagcaac
taggcctttc aacagttttg gagaaggcca tttcccactt ttaaaataaa 3060
taatgctcct ataagatcag atactgtgtt gaccagtggt catgccactg cctgttgatt
3120 tgttgaaaat attgtttaca cgtatgttct tgttactgat tgtcagaaag
ctggttttga 3180 gactgcagct tggactaaat tcagtcatct ggctgtctgg
ggaagcatgc tgaccagtct 3240 ggtgttcttt ggcatctact cagccatctg
gtccaccatt ctcattgccc caaatatgag 3300 aggacagatt aacaatggta
ccagcacgtg cagaaaagaa agagtctccg cttgtctttg 3360 tctgattctc
ctgtcctctc catggaagtt acattttctg taaaggatga gctgaaaatt 3420
ctcctggtcg ttgccagttg aacttctgct gtgctctggg aaggcattct cactctgttt
3480 atgttgtcta agtgcagaca tggatgtgca ggtttgctag aacctcctga
ggatgtgcaa 3540 tggttctgtt catgcctgaa tcagttcttt tgggagtgga
cattctttct ctccctcatg 3600 cacagcctca ggcacatggc ttgagctatg
gcggcacgca gtatggccat cacccaggta 3660 caccccttcc ctaagaagag
gctcttcagg ttacactcgg gtactgttgt tatctggctt 3720 attgtccata
ggatcaacat agagtcctga ggtcagttca aaccatcaaa ccagggatgt 3780
tacttattat ttgaaaactt ctttggaaag ataatcttgg gttgttcagt gggaccagtc
3840 tttgacgggc aaatctccag aatacatggg gtcagttctc tcaggttcag
gaagcatgta 3900 atctctctaa gattcattaa ttaaaaaaaa aagacacatg
catagaaaaa tagaacaaaa 3960 tggaaactct ttattggata cctactatgg
gttatgtgcc agggtttcct aatcatttgg 4020 ggacatgtgt gtataaacaa
aaccaggcta tgtggccagg cagtgtgtgg ctcacacctg 4080 taatcccagt
gcttagggaa gccaagttgc aaggatcgct tgaaaccagg agttcgagac 4140
tagcctgggc aacatagtga gacccggtct ctgcaaaaaa aaaaaaaaaa aaaaaaa 4197
4 1560 DNA Homo sapiens 4 agctcaatac ggaacatatt ctcagtcctc
ctctggtcta caaagcctgt gatttcttgt 60 ctatggacag aacgtctggt
ttaatctaca ggaacccata acttcctgaa gctttatgct 120 taacagtgac
aacgtgagtc agttgaattt tattgtgttt cagtccgtag agtattagct 180
acagaaacct ttccattgcc atactgagaa actggcagca ggcagtgtgc ctacaggtct
240 acaaagaaac ttcagatcat cttcttgagg gaaagaagct gaagtgctac
ataagatgct 300 tgtgcttcat aactctcaga agctgcagat tctgtataaa
tccttagaaa agagcatccc 360 tgaatccata aaggtatatg gcgccatttt
caacataaaa gataaaaacc ctttcaacat 420 ggaggtgctg gtagatgcct
ggccagatta ccagatcgtc attacccggc ctcagaaaca 480 ggagatgaaa
gatgaccagg atcattatac caacacttac cacatcttca ccaaagctcc 540
tgacaaatta gaggaagtcc tgtcatactc caatgtaatc agctgggagc aaactttgca
600 gatccaaggt tgccaagagg gcttggatga agcaataaga aaggttgcaa
cttcaaaatc 660 agtgcaggta gattacatga aaaccatcct ctttataccg
gaattaccaa agaaacacaa 720 gacctcaagt aatgacaaga tggagttatt
tgaagtggat gatgataaca aggaaggaaa 780 cttttcaaac atgttcttag
atgcttcaca tgcaggtctt gtgaatgaac actgggcctt 840 tgggaaaaat
gagaggagct tgaaatatat tgaacgctgc ctccaggatt ttctaggatt 900
tggtgtgctg ggtccagagg gccagcttgt ctcttggatt gtgatggaac agtcctgtga
960 gttgagaatg ggttatactg tccccaaata cagacaccaa ggcaacatgt
tgcaaattgg 1020 ttatcatctt gaaaagtatc tttctcagaa agaaatccca
ttttatttcc atgtggcaga 1080 taataatgag aaaagcctac aggcactgaa
caatttgggg tttaagattt gtccttgtgg 1140 ctggcatcag tggaaatgca
cccccaagaa atattgttga ttgattccac tgtccatttc 1200 aaatctttct
tatcagtaaa aaaacattaa ttcaaacaca agcattgtga tctacattag 1260
cacaaaatgc aactgattat ctaggatctg tgtattactt aagctcaccc ttaacagttt
1320 taccttcctt ctcctctgta ttcttacaga aaattagaag ctcaatttta
tggtctcata 1380 atttccttta tgacagacat ctcagaatta aaatcaccca
aagccaatca ttagtgccaa 1440 gataaccctt taacgggcaa cactttctta
aatgaagact atttctttca tgaaaaaatt 1500 cacttttatg actttcttgt
taaaataaaa agtctgcttt taaaaaaaaa aaaaaaaaaa 1560 5 1227 DNA Homo
sapiens unsure (327) a, c, g or t 5 attttgtagt tcagcaaatc
ctccaaatac acagcatgtt acaaggcact ggtggcacag 60 ggcacaacag
gaaatgatat ttatttagca aattcattta acaaatatta ttgggcacct 120
gttatgtgag acactgtcct aggcactgtg ggataacaac agcaaacact tcacacaaca
180 gcctggcctt cctgtgtttt acaacagctc ctaaagatag ctgatatcaa
gacatttgag 240 ggacacagtt catgtagaat caaaatatta gtatttcaga
ataaggattt tttttctgaa 300 aagcatacag agaggaaaca gcttaanaat
aggtcaagac ctaaaaacag antataatca 360 cggaataanc tggataaccc
agacagtccc cacagaattt ctttcaggtc acagatttct 420 taaaactcac
ccccaaaatg tgcctgcttg gttgtttgaa tcttgcataa ttaatgtcac 480
aggcgcaagc cgctgaactt agttgagatg cagaaaacaa acaaatgcaa tgacatatct
540 gagaagcatt tatgtaactc cggttaagtg gtgaggaggg gtgtgtgaag
acagtgtgca 600 tgcatgagtg tgtattcata tatatgtgta tacatatgaa
tttcactgtt attttccagg 660 gtctatggac aatgtggcag taagagtcta
tgatgttctg aaacttttca cagtaaatcc 720 aaagattaca gaccttacaa
ggtgcttgca ttctgttgct tttccatctg tcacttctca 780 ggttatttga
ctgtgttcaa accttctttt ctttttcatt gagtttcatt ttttaagctt 840
gttaaatgcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tgtcattttt cacattatcc
900 tctcttctct gcaacaagga tagtaagatg tagatgaatg caaaaataat
aacaacaata 960 aggaaatata ttaaagcttt aaaatatgca catatgtagt
tctaaagagc aataacggta 1020 gtatctattt cgaacatgca ttaggcaaaa
aagaaatcaa aactgaaatt ttcgtgtatt 1080 tttccccttg taagatgttc
aaatgctaac ttcattttct cctttcctct atgtggcact 1140 ttctcaaaat
atctatgaaa tacttttaga caaagattga gctggagaaa gagatacaaa 1200
tttccatccc cccagacagn gagacat 1227 6 253 DNA Homo sapiens unsure
(181) a, c, g or t 6 gaacagcctc acttgtgttg ctgtcagtgc cagtagggca
ggcaggaatg cagcagagag 60 gactcgccat cgtggccttg gctgtctgtg
cggccctaca tgcctcagaa gccatacttc 120 ccattgcctc cagctgttgc
acggaggttt cacatcatat ttccagaagg ctcctggaaa 180 nagtgaatat
gtgtcgcatc naganagctg atggggattg tgacttggct gctgtcancc 240
nncatgtcan gcg 253 7 943 DNA Homo sapiens unsure (128) a, c, g or t
7 gggggcctgg ccccggcccc tgtgaggacc ccgcgggtgc tggggtaaga ggctctagac
60 ccttcacctg tcagtcacct gagggaggct gaggccaagc cccatccctc
agaatcaagg 120 cttgcaancn cccctcacct gcccagtctc tgtccacacc
cctcgggctg aagacggccc 180 tgaccaggcc ctgggcctca gcgaccaccc
ctccccctcc tgcctggacc cagggagcag 240 gtgcaggggg ctccgagccc
ctggtgactg tcaccgtgca gtgcgccttc acagtggccc 300 tgagggcacg
aagaggagcc gacctgtcca gcctgcgggc actgctgggc caagccctcc 360
ctcaccaggc ccagcttggg caactcaggt gggccagaaa gcccccggtg gctgcggtgg
420 agctgggcac cgccccgact gaggcagctg ctggaagagg gggtggcaga
ggtcactgcc 480 ctccctgcag gccccaccca ggaggccccc tctgaggaat
ctctttgcag ttacctagcc 540 ccaggtgagg acgggcactg ggtccccatc
cccgaggagg agtcgctgca gagggcctgg 600 caggacgcag ctgcctgccc
cagggggctg cagctgcagt gcaggggagc cgggggtcgg 660 ccggtcctct
accaggtggt ggcccagcac agctactccg cccaggggcc agaggacctg 720
ggcttccgac agggggacac ggtggacgtc ctgtgtgaag tggaccaggc atggctggag
780 ggccactgtg acggccgcat cggcatcttc cccaagtgct tcgtggtccc
cgccggccct 840 cggatgtcag gagcccccgg ccgcctgccc cgatcccagc
agggagatca gccctaatga 900 tgctgtgtcc atgatgcttt taatnaaaaa
aacccccact gca 943 8 249 DNA Homo sapiens unsure (110) a, c, g or t
8 atcacattaa gtcattgcta attttataaa caaaaacaat ggttttantt tgcatctccc
60 tgattggtat tgctgtagaa catatttgga gaagtttgtt tgtctttggn
gtttatttca 120 tgaatagatt gtgtgcccat tttctcttgg ggtattcagt
tttttattac tgatgtgagc 180 atgtgtatgg gngattattt gatgnttatc
agttttgntt agtagactgg caatatttag 240 tcttgctgt 249 9 690 DNA Homo
sapiens 9 gacgcccagt gacctgccga ggtcggcagc acagagctct ggagatgaag
accctgttcc 60 tgggtgtcac gctcggcctg gccgctgccc tgtccttcac
cctggaggag gaggatatca 120 cagggacctg gtacgtgaag gccatggtgg
tcgataagga ctttccggag gacaggaggc 180 ccaggaaggt gtccccagtg
aaggtgacag ccctgggcgg tgggaagttg gaagccacgt 240 tcaccttcat
gagggaggat cggtgcatcc agaagaaaat cctgatgcgg aagacggagg 300
agcctggcaa atacagcgcc tatgggggca ggaagctcat gtacctgcag gagctgccca
360 ggagggacca ctacatcttt tactgcaaag accagcacca tgggggcctg
ctccacatgg 420 gaaagcttgt gggtaggaat tctgatacca accgggaggc
cctggaagaa tttaagaaat 480 tggtgcagcg caagggactc tcggaggagg
acattttcac gcccctgcag acgggaagct 540 gcgttcccga acactaggca
gcccccgggt ctgcacctcc agagcccacc ctaccaccag 600 acacagagcc
cggaccacct ggacctaccc tccagccatg acccttccct gctcccaccc 660
acctgactcc aaataaagtc cttctccccc 690 10 294 PRT Homo sapiens 10 Met
Leu Val Leu His Asn Ser Gln Lys Leu Gln Ile Leu Tyr Lys Ser 1 5 10
15 Leu Glu Lys Ser Ile Pro Glu Ser Ile Lys Val Tyr Gly Ala Ile Phe
20 25 30 Asn Ile Lys Asp Lys Asn Pro Phe Asn Met Glu Val Leu Val
Asp Ala 35 40 45 Trp Pro Asp Tyr Gln Ile Val Ile Thr Arg Pro Gln
Lys Gln Glu Met 50 55 60 Lys Asp Asp Gln Asp His Tyr Thr Asn Thr
Tyr His Ile Phe Thr Lys 65 70 75 80 Ala Pro Asp Lys Leu Glu Glu Val
Leu Ser Tyr Ser Asn Val Ile Ser 85 90 95 Trp Glu Gln Thr Leu Gln
Ile Gln Gly Cys Gln Glu Gly Leu Asp Glu 100 105 110 Ala Ile Arg Lys
Val Ala Thr Ser Lys Ser Val Gln Val Asp Tyr Met 115 120 125 Lys Thr
Ile Leu Phe Ile Pro Glu Leu Pro Lys Lys His Lys Thr Ser 130 135 140
Ser Asn Asp Lys Met Glu Leu Phe Glu Val Asp Asp Asp Asn Lys Glu 145
150 155 160 Gly Asn Phe Ser Asn Met Phe Leu Asp Ala Ser His Ala Gly
Leu Val 165 170 175 Asn Glu His Trp Ala Phe Gly Lys Asn Glu Arg Ser
Leu Lys Tyr Ile 180 185 190 Glu Arg Cys Leu Gln Asp Phe Leu Gly Phe
Gly Val Leu Gly Pro Glu 195 200 205 Gly Gln Leu Val Ser Trp Ile Val
Met Glu Gln Ser Cys Glu Leu Arg 210 215 220 Met Gly Tyr Thr Val Pro
Lys Tyr Arg His Gln Gly Asn Met Leu Gln 225 230 235 240 Ile Gly Tyr
His Leu Glu Lys Tyr Leu Ser Gln Lys Glu Ile Pro Phe 245 250 255 Tyr
Phe His Val Ala Asp Asn Asn Glu Lys Ser Leu Gln Ala Leu Asn 260 265
270 Asn Leu Gly Phe Lys Ile Cys Pro Cys Gly Trp His Gln Trp Lys Cys
275 280 285 Thr Pro Lys Lys Tyr Cys 290
* * * * *