Abstrict
The present invention provides a new method of detecting, diagnosing,
monitoring, staging, prognosticating, imaging and treating breast
cancer.
Claims
What is claimed is:
1. An isolated antibody that specifically binds a protein encoded
by Mam004 (SEQ ID NO:4).
2. A method of imaging breast cancer in a patient comprising administering
to the patient an antibody of claim 1 and detecting the antibody
in the patient wherein detection of the antibody within the breast
allows determination of the presence or absence of cancer in the
breast.
3. The method of claim 2 wherein said antibody is labeled with
paramagnetic ions or a radioisotope.
Description FIELD OF THE INVENTION
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
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.
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.
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.
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.
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.
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.
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.
In the present invention methods are provided for detecting, diagnosing,
monitoring, staging, prognosticating, imaging and treating breast
cancer via 9 Breast Specific Genes (BSGs). The 9 BSGs refer, among
other things, to native proteins expressed by the genes comprising
the polynucleotide sequences of any of SEQ ID NO: 1-9. In the alternative,
what is meant by the 9 BSGs as used herein, means the native mRNAs
encoded by the genes comprising any of the polynucleotide sequences
of SEQ ID NO: 1-9 or it can refer to the actual genes comprising
any of the polynucleotide sequences of SEQ ID NO: 1-9.
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
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.
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.
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.
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.
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.
Further provided are antibodies against the BSGs or fragments of
such antibodies which can be used to detect or image localization
of the BSGs in a patient for the purpose of detecting or diagnosing
a disease or condition. Such antibodies can be polyclonal or monoclonal,
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 labels including, but
not limited to, radioisotopes and paramagnetic metals. These antibodies
or fragments thereof can also be used as therapeutic agents in the
treatment of diseases characterized by expression of a BSG. In therapeutic
applications, the antibody can be used without or with derivatization
to a cytotoxic agent such as a radioisotope, enzyme, toxin, drug
or a prodrug.
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
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. What is meant
by levels of BSG as used herein, means levels of the native protein
expressed by the genes comprising the polynucleotide sequence of
any of SEQ ID NO: 1-9. In the alternative, what is meant by levels
of BSG as used herein, means levels of the native mRNA encoded by
any of the genes comprising any of the polynucleotide sequences
of SEQ ID NO: 1-9 or levels of the gene comprising any of the polynucleotide
sequence of SEQ ID NO: 1-9. 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) 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.
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.
Diagnostic Assays
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) 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.
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.
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.
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.
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) 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.
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.
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,
such as earlier samples of the same patient.
Staging
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) 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) 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 or in remission.
Monitoring
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) 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.
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) 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) 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.
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.
Assay Techniques
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 radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays,
immunohistochemistry assays, in situ hybridization assays, competitive-binding
assays, Western Blot analyses, ELISA assays and proteomic approaches.
Among these, ELISAs are frequently preferred to diagnose a gene's
expressed protein in biological fluids.
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.
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.
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.
Nucleic acid methods may be used to detect 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 (NASABA), can be used to detect malignant
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 of a specific type of cell.
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, a cDNA encoding the BSG gene 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 gene 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.
Of the proteomic approaches, 2D electrophoresis is a technique
well known to those 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.
The above tests can be carried out on samples derived from a variety
of patients' cells, bodily fluids and/or tissue extracts (homogenates
or solubilized tissue) such as 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. Blood
can include whole blood, plasma, serum, or any derivative of blood.
In Vivo Antibody Use
Antibodies against BSGs can also be used in vivo in patients with
disease of the breast. Specifically, antibodies against a BSG can
be injected into a patient suspected of having a disease of the
breast for diagnostic and/or therapeutic purposes. The use of antibodies
for in vivo diagnosis 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
BSGs can be used in a similar manner. Labeled antibodies against
a BSG can be injected into patients suspected of having a disease
of the breast such as 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 bemused 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 used in magnetic resonance
imaging (MRI). Localization of the label within the breast or external
to the breast permits determination of the spread of the disease.
The amount of label within the breast also allows determination
of the presence or absence of cancer in the breast.
For patients diagnosed with breast cancer, injection of an antibody
against a BSG can also have a therapeutic benefit. The antibody
may exert its therapeutic effect alone. Alternatively, the antibody
is conjugated to a cytotoxic agent such as a drug, toxin or radionuclide
to enhance its therapeutic effect. Drug monoclonal antibodies have
been described in the art for example by Garnett and Baldwin, Cancer
Research 1986 46:2407-2412. The use of toxins conjugated to monoclonal
antibodies for the therapy of various cancers has also been described
by Pastan et al. Cell 1986 47:641-648). Yttrium-90 labeled monoclonal
antibodies have been described for maximization of dose delivered
to the tumor while limiting toxicity to normal tissues (Goodwin
and Meares Cancer Supplement 1997 80:2675-2680). Other cytotoxic
radionuclides including, but not limited to Copper-67, Iodine-131
and Rhenium-186 can also be used for labeling of antibodies against
BSGs.
Antibodies which can be used in these in vivo methods include both
polyclonal and monoclonal 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.
EXAMPLES
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
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.
The CLASP performs the following steps:
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.
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.
CLASP allows the identification of highly expressed organ and cancer
specific genes useful in the diagnosis of breast cancer.
TABLE 1 BSQs 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
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
Relative Quantitation of Gene Expression
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).
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) 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) 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) and Mam005 (SEQ ID NO:3) compared to their respective
calibrators.
Measurement of SEQ ID NO:2; Clone ID;1730886 Gene ID: 238469 (Mam001)
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.
TABLE 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
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
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.
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.
TABLE 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 5997 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 Hnd6 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
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 represents 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).
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.
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.
Measurement of SEQ ID NO:1; Clone ID: 2740238; Gene ID 242151 (Mam002)
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.
TABLE 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
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.
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.
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. |