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A NOVEL METHOD OF DIAGNOSTNG,
MONITORJ:NG, STAGING, IMAGING AND TREATING BREAST CANCER
FIELD OF T:HE 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 bz'east cancer and approximately 46,000 will
die of the disease.
Every woman i.s 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 n« known risk factors. Less than 10 percent
of breast cancer case; are thought to be related to the BRCA1
gene disco~rered in LS~94. Researchers are now investigating
the role other factor's 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.
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Mammograms, special x:-rays of the breast, can detect more than
90 percent: of all lareast cancer's. If breast cancer is found
early, tl-..e chance of cure i:> greater than 90 percent.
Treatment options include surgery, chemotherapy, and radiation
therapy depending on tree stage of the cancer.
Procedures used for detect:ing, diagnosing, monitoring,
staging, :~rognostica.t:ing and imaging breast cancer are of
critical importance r_o 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 ~~atients diagnosed with distant metastasized breast
cancer. New di_agno:~t.ic methods which are more sensit=ive and
specific for detect:i.ng early breast cancer are clearly needed.
Bre~~st cancer ~at.ients az~e closely monitored following
initial therapy arid during ac~juvant therapy to determine
response too therapy and to detect persistent or recurrent
disease of metastasis. There is clearly a need for a breast
cancer marker whir.~h is more sensitive and specific in
detecting breast cancer and its recurrence and progression.
Another impoi:t<~nt 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 lea:~t 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 b~~ detecting new markers in cells, tissues, or bodily
fluids which could c:ii.f_ferentiate between different stages of
invasion.
In the present invention methods are provided for
detecting, diagnosing, moni~oring, staging, prognosticating,
imaging anc3 treating breast cancer via 9 Breast Specific Genes
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(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 BSCxs as used herein, means the native mRNAs
encoded b:~ the genes comprising any of the polynucleotide
sequences of SEQ ID NO: 1--9 or it can refer to the actual
genes comprising arty of the polynucleotide sequences of SEQ
ID NO: 1-9.
Other object~~, 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 t:he Following description and the
specific examples, while indicating preferred embodiments of
the invention, are rliven by way cf illustration only. Various
changes and modificat:.ions within the spirit and scope of the
disclosed inventiorv will. become readily apparent to those
skilled in the art t:rom 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 ~~referably the same cells, tissues, or bodily fluid
type of a normal human control, wherein a change in levels of
BSG in tree patient versus the normal human control is
associated with brea~ct: 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 havin_~ breast. cancer that has metastasized;
analyzing a sample of cells, tissues, or. bodily fluid from
such patient for BSC~; comparing the BSG levels in such cells,
tissues, or bodily fluid with levels of BSG in preferably the
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same cell:, tissue;, or bodily fauid type of a normal human
control, wherein a ch<~nge i:n 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, ti:~sues, or bodily fluid from such patient zor BSG;
comparing BSG levels in such cells, tissues, or bodily fluid
with levels of BSG in preferably the same cells, tissues, or
bodily fluid t:ype of a normal human control sample, wherein
a change in BSG levels in the p,:~~ient: versus the normal human
control i~~ associat:.ed with a c<~ncer which is progressing or
regressing or in remission.
Further provided is a method of monitoring breast cancer
in a human having sur_h 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; compai_-ing 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, whewein 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 c>f 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 r~wc:h patient for BSG; comparing the BSG
levels in .such cell;, tissue, or bodily fluid with levels of
BSG in preferably t:he 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
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associated with a cancer which is progressing or regressing
or in remission.
Furvher provided are antibodies against the BSGs or
fragments of such ant:ibodies which can be used to detect or
image loc<~lization of the BSGs in a patient for the purpose
of detecting or diagnosing a disease or condition. Such
antibodie:~ can be polyclonal c>r monoclonal, or prepared by
molecular biology t:ec:hniques . 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 SEL~EX and we_L1 known tc those skilled in the art.
Antibodies can be labeled with <~ variety of detectable labels
including, but not l.~.mited 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
derivatiza.tion 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 iol.lowing description. It should be
understood., however, that t:he following description and the
specific examples, Wh=Lle 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 IPdVENTION
The present invention relates to diagnostic assays and
methods, both quant~_tative and qualitative for detecting,
diagnosing, monitoring, staging, prognosticating and imaging
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cancers by comparing levels of BSG with those of BSG in a
normal human contrcil. What is meant by levels of BSCi as used
herein, means level: of the native protein expressed by the
genes comprising the pc~lynuclectide sequence of any of SEQ ID
NO: 1-9. In the alternative, what is meant by levels of BSG
as used herein, means level: 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 fo:r measuring changes in levels of any one
of the BSCi proteins compared to normal control bodily fluids,
cells, or tissue samples may be used to diagnose the presence
of cancer~~, including breast cancer. By "change" it is meant
either an increase or decrease in levels of the BSG. For
example, :Eor BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ
ID N0:4) ;end Mam00~:~ (SEQ ID N0:3), an increase in levels as
compared 1~o normal human controls is associated with breast
cancer, metastasis and progression of the cancer, while a
decrease in levels is as;~ociation with regression and/or
remission. For thE: BSG Mam002 (SEQ ID NO:1), a decrease in
levels as compared t:o 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 a:ny 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
BRCAl are known to t.'.~ose of skill in the art.
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Diagnostic' Assays
The present invention provides methods for diagnosing
the presence of bre<~st 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 norma7_ human control. As demonstrated
herein an increase i:n level.s of= BSGs such as Mam001 (SEQ ID
N0:2), Mam004 (SEQ ID N0:4) or Mam005 (SEQ ID N0:3) in the
patient versus the normal human control is associated with the
presence of breast c=ancer, while a decrease in levels of BSGs
such as Mam002 (SEQ TD NO:1) in the patient versus the normal
human cont=rol is a~;sociatc=_d with the presence of breast
cancer.
Without limiting the instant invention, typically, for
a quantitative diagnostic a:~say a positive result indicating
the patient being trested has cancer is one in which cells,
tissues, o:r 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 contro:L.
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 o:f 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 discriminat::ing between breast cancer which has not
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metastasi~:ed and breast cancer which has metastasized.
Existing technique.:; 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 cel:Ls, tissues, or bodily fluid is BSG, and
are compared with levels of BSG in preferably the same cells,
tissue, or bodily f:LL;id type of a normal human control. That
is, if the cancer m,~rke:r being observed is just BSG in serum,
this level- is preferably compared with the level of BSG in
serum of a normal han:an pat-gent. An increase in BSGs such as
Mam001 (SEQ ID N0:2), Mam004 (SEQ ID N0:4) or Mam00S (SEQ ID
N0:3) in the patient: versus the normal human control is
associated with brea:~t cancer which has metastasized while a
decrease in BSGs such as Mam002 (SEQ ID NO: l) 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 ~,~atient being tested or monitored has
metastasized is one in which cells, tissues, or bodily fluid
levels of the cancer marker, sucr: as BSC:~, are at least two
times higher or lower, and most preferably are at least five
times higher or lowe r, than in preferably the same cells,
tissues, or bodily f7_uid of a normal patient.
Normal human control. as used herein includes a human
patient without cancer and/or non cancerous samples from the
patient; ~~n the methods for diagnosing or monitoring for
metastasis, normal human contro~_ preferably comprises samples
from a human patient that i.s determined by reliable methods
to have breast cancer- which has not metastasized, such as
earlier samples of the same patient.
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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, tissue~~, or bodily fluid with levels
of BSG in preferably t:he same cells, tissues, or bodily fluid
type of a normal human control sample, wherein an increase in
levels of BSGs such Gm~ Mam001 (SEQ ID N0:2), Mam004 (SEQ ID
N0:4) or M~.m005 (SEQ ..D N0:3) or a decrease in levels of BSGs
such as Mam002 (SEQ TD 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 N0:2),
Mam004 (SEQ ID N0:4) c>r Mam005 (SEQ ID N0:3) or an increase
in levels of BSGs such as Mam002 (SEQ ID NO: l) 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 h:nown to have metastasized; periodically
analyzing ~i sample ~:~f cells, tissues, or bodily fluid from
such patient fo:r 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 N0:2), Mam004 (SEQ ID N0:4) or Mam005 (SEQ ID
N0:3) or a decrease in levels of BSGs such as Mam002 (SEQ ID
NO:l} 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
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patient having such cancer; periodically analyzing a sample
of cells, tissues, oz- bodily fluid from such patient for BSG;
comparing the BSG 1.=vets 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) , MamC)04 (SEQ TD N0:4) or Mam005 (SEQ ID N0:3)
or a decrease in leve_Ls of BSGs such as Mam002 (SEQ :ID NO:1)
in the patient versu:~ 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 N0:2), Mam004 (SEQ
ID N0:4) or Mam005 (SEQ ID N0:3) or an increase in levels of
BSGs such as Mam002 {SEQ ID NO:1) is associated with a cancer
which is regressing -_.n stage or in remission.
Monitoring such patient for onset of metastasis is
periodic and preferab-.~y 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 ex:oression, such as BSG of the present invention, in
a sample df:rived frc:>m a host are well-known to those of skill
in the art:. Such a:~say methods include radioimmunoassays,
reverse transcri.ptase PCR (RT-PCR) assays,
immunohist~achemistry 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 biologi~~al fluids.
An ELISA assay initially comprises preparing an
antibody, _Lf not readily available from a commercial source,
specific t:o BSG, preferably a monoclonal antibody. In
addition a reporter antibody generally is prepared which binds
specifical=Ly to BSG. The reporter antibody is attached to a
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detectable reagent:: such as radioactive, fluorescent or
enzymatic reagent, for example horseradish peroxidase enzyme
or alkalize 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 f ree 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 wa~,hed outs with buffer. A reporter antibody
specifica7_ly directed to BSG and linked to horseradish
peroxidase is placed in the dish resulting in binding of the
reporter antibody tc> any monoclonal antibody bound to BSG.
Unattached reporter antibod~~ is then washed out. Reagents for
peroxidase activity, including a colorimetric substrate are
then added to the dash. Immobilized peroxidase, linked to BSG
antibodie~~, produces a colored reaction product. The amount
of color c.eveloped in a given time period is proportional to
the amount of BSG protein present. in the sample. Quantitative
results tarpically ar_e obtained by reference to a standard
curve.
A competitiozx essay may be employed wherein antibodies
specific t:o 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.
Nuc7.eic acid methods may be used to detect BSG mRNA as
a marker for breast cancer. Polymerase chain reaction (PCR)
and other nucleic ac~id methods, such as ligase chain :reaction
(LCR) and nucleic acid sequence based amplification (NASABA),
can be u~~ed to detect malignant cells for diagnosis and
monitoring' of various malignancies. For example, reverse-
transcriptase PCR (R7:--PCR) is a powerful technique which can
be used to detect the presence of a specific mRNA population
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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 c:DNA 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 spec:ifi.c for the cell that produces it, RT-PCR
can be usE~d to identify the presence of a specific type of
cell.
Hybridization to clones or oligonucleotides arrayed on
a solid support (i.e., griddinc~) can be used to both detect
the expre~;sion of and quant:itat:.e the level of expression of
that gene. In th~.s approach, a cDNA encoding the BSG gene
is fixed to a subst~:-ate. 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 subs>trate 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 detE~cted and qi~antitated by several means including but
not limited to radi«ac:tive :Labeling or fluorescence labeling
of the analyte or a secondary molecule designed to detect the
hybrid. c2uantitation 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, quant:itating the yield, and then using that
material to generate a standard curve.
Of the proteomic approaches, 2D electrophoresis is a
technique well known t.o 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
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current. The current acts uniformly on all proteins, so
smaller proteins move farther on the gel than larger proteins.
The second dimensi<:7n 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 ~.Nhich 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 out=opsy material. Bodily fluids useful in
the present inventi.en 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 ac.~ai.nst 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 :Lndium-11_L have been described for use
in the radioimmunoscintographic imaging of carcinoembryonic
antigen expressing tumors 1 Sumerdon et al . Nucl . Med. Biol .
1990 17:2~E7-254) . ~_n 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). Anti_bod~es with paramagnetic ions as
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labels fox- use in magnetic resonance imaging have also been
described (Lauffer, R.B. Magnetic Resonance in Medicine 1991
22:339-342). Antibod.i.es 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 brea:~t 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 examp:l.e, radioactive labels such as Indium-111,
Technetium-99m or Iodine-131 can be used for planar scans or
single photon emission computed tomography (SPELT). Positron
emitting labels such as Fluorine-19 can be used in positron
emission t:omography. Paramagnetic ions such as Gadlinium
(III) or Manganese (I:II can used in magnetic resonance imaging
(MRI). hocalization of the 1_abel 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 antibod~r against a BSG can also have a therapeutic benefit.
The antibody may exert its therapeutic effect alone.
Alternatively, the ant::ibody is conjugated to a cytotoxic agent
such as a drug, t~~.xin or radionuclide to enhance its
therapeutic effect. Drug monoclonal antibodies have been
described in the arty 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-
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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 exemplificati.o:ns, while illustrating certain specific
aspects of: the invf:ant:ion, do not portray the limitations or
circumscribe the sco~.~e 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, Pa:Lo Alto, CA, using the data mining Cancer
Leads Automatic Search Package (CLASP) developed by diaDexus
LLC, Santa Clara, CA.
The CLASP performs the fo7_lowing steps:
Selection of highly expressed organ specific genes based
on the abundance level of the corresponding EST in the
targeted organ versu;~ 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.
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CLAS P allows the identification of highly expressed
organ and cancer spE~cific genes useful in the diagnosis of
breast cancer.
Table 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
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 c~irried out as described in standard laboratory
manuals, :such as .~ambrook 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 quantitat=.ive detection system utilizing the 5'-
3' nuclease activity of Taq DNA polymerase. The method uses
an interna7_ fluorescent oligonuc~Leotide probe (Taqman) labeled
with a 5' reporter dye and a downstream, 3' quencher dye.
During PCR,, the 5'-~' nuclease activity of Taq DNA polymerase
releases tree reporter, whose fluorescence can then be detected
by the laser detector of the Model 7700 Sequence Detection
System (PE Applied Biosysterns, Foster City, CA, USA).
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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 t:he basis f:or comparative results (calibrator).
Quantitation relative to the "calibrator" can be obtained
using the standard c:llrVe method or the comparative method
(User Bull.°tin #2: ABI PRISM 7700 Sequence Detection System).
To evaluate the tissLe distribution, and the level of. breast
specific markers (BSM) Mam001 (SEQ ID N0:2), Mam002 (SEQ ID
NO: l), Mam004 (SEQ ID N0:4) and Mam005 (SEQ ID N0:3) in normal
and cancer tissue, total RNA was extracted from cancer and
matched normal adjacent tissues (NAT) and from unmatched
cancer and normal. ti;~sues. Subsequently, first strand cDNA
was prepared with reverse transcriptase and the pol.ymerase
chain reaction carried out using primers and Taqman probes
specific t.o each of Mam001 (SEQ ID N0:2), Mam002 (SEQ ID
N0 : 1 ) , MamO 04 ( SEQ ID N0 : 4 ) and MamO 0 5 ( SEQ II) N0 : 3 )
respectively. The results are obtained using the ABI PRISM
7700 Sequence Detector. The numbers are relative levels of
expression of Mam001 (SEQ ID DJ0:2), Mam002 (SEQ ID NO: l),
Mam004 (SEQ ID N0:4) and Mam005 (SEQ ID N0:3) compared to
their respective ca:l.ibrator:~.
Measurement: of SEQ ID N0:2; Clone ID:1730886; Gene ID: 238469
(Mam001)
The numbers deb>i.cted :in Table 2 are relative levels of
expression in 12 normal tissues of Mam001 (SEQ ID N0:2)
compared t.o testis (~~alibrator). These RNA samples were
obtained commercially and were generated by pooling samples
from a parvicular tissue from different individuals.
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Table 2: Relative levels of Mam001 (SEQ ID N0:2) Expression
in Pooled Samples
Tissue NORMAL
Brai.n_ 0
Heart 0
Kidney 0
Liver 0
Lunc:f _ 0
Mammary 6
Prostate 0
Muscl~_ 0
Small Intestine 0
Test.i;s 1
Thymus 0
Utazu~ 0
The relative levels of expression in Table 2 show that
Mam001 (S~~Q ID N0:2) mRNA expression is detected in the pool
of normal mammary and in testis but not in the other 1.0 normal
tissue pools analyzed. These results demonstrate that Mam001
(SBQ ID N0:2) mRNA expression is highly specific for mammary
tissue and is als~_:~ 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 individua~.s. The tissues shown in Table 3 were
obtained from individuals and are not pooled. Hence the
values fo=r mRNA expression leve:Ls 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 N0:2) compared tc> testis
(calibrator), in 24 lairs of matching samples. Each matching
pair contains the cancer sample for a particular tissue and
the norma:L adjacent tissue (NAT) sample for that same tissue
from the ~~ame individual.
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Table 3: Relative levels of Mam001 (SEQ ID N0:2) Expression
in Individual Sample;
Sample ID Tissue Cancer Matching
Normal
Mam 47XP Mammary Gland 0 0
Mam A06X Mammary Gland 23 1
Mam BOllX Mammary Gland 0 5
Mam 603X/C034 Mammary Gland 0 2.10
Mam 16a?X Mammary Gland 1.96 0.15
Mam 42DN Mammary Gland 0.38 1.27
Mam 5079 Mammary Gland 0.34 0.36
Mam Sla?3 Mammary Gland 0.03 0.87
Mam S5u6 Mammary Gland 0.43 0.53
Mam 5639 Mammary Gland 0.40 0.66
Mam S9~)7 Mammary Gland 0.41 0.51
Sto AC~64 ~>tomach 0 0
TST 39X Testis 0 ()
Cln SG~65 Colon 0 0
Cln TX01 Colon 0 0
Cvx NK.?3 Cervix 0 0
Cvx NKa?4 Cervix 0 0
Endo 3~~X 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
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
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normal sarlples 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 N0:2) rciRNA {Tabl.e 2 and 3) . Other than breast tissue,
Mam001 (SEC) ID N0:2) i:~ detected only in one other tissue type
(Testis) and then or~l.y in the pooled tissue sample (Table 2)
but not in the matched testis cancer samples (Table 3).
Comparisons of t=he level of mRNA expression in breast
cancer samples and the normal adjacent tissue from the same
individual: are shown in Table 3. Mam001 (SEQ ID N0:2) is
expressed ~~t higher levels in 2 of 11 breast cancer tissues
(Mam A06X a.nd Mam 16:.'X) compared with the corresponding normal
adjacent tissue. The level of Mam001 (SEQ ID N0:2) expression
is lower in breast cancer compared to normal adjacent tissue
in four matched samples (Mam BO11X, Mam 603X/C034, 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 5699 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 Mam00=L (SEQ Ii:) 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 (calibratorj. These RNA samples were
obtained commercially and were generated by pooling samples
from a particular ti.s;~ue from different individuals.
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Table 4: Relative levels of Mam002 (SEQ ID NO:1) Expression
in Pooled Samples
Tissue NORMAL
Brain 0.03
Heart O.Ol
Kidney 0
Liver 0
~
Lung 0.06
Mammary 289.01
Muscle 0
Prostate 0.31
Smal l :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 ti.:>sues shown in Table 5 were
obtained i_rom individuals and are not pooled. Hence the
values for mRNA expression levels shown in Table 4 cannot be
directly compared to t=he values shown in Table 5.
The numbers depicted in Table 5 are relative levels of
expression of Mam002 (SEQ ID NO: l) 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 ;game indiv=_dual. In addition 2 unmatched mammary
samples from normal tissues and one unmatched ovarian cancer
and one normal (non--cancerous) ovary were also tested.
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Table 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 42DrJ Mammary 1051 2075
Glanci
Mam 59X Mammary 7.0 15.5
Gland
Mam A06?F Mamm;:~z~y 1655 1781
G 1 axlCi
Mam B011X Mamm<~ry 32.1 2311
Gland
Mam S12',~ Mamm~x~y 1 . 7 3 0
Glanc;
Mam S51F> Mammary 9.72 69.95
Gland
Mam S 6 9 Mamm,::~ 8 3 . 4 6 7 5 . 6 5
~3 zwy
Gland
Mam S85~6 Mammary 133.23 836.56
Gland
Mam S967 Mammary 59.77 188.28
Glarncl
Mam S99'' Mammary 94.14 73.64
GlancL
Mam 162; Mammary 674.0 31.1
Gland
Mam Cola; Mammaz~y N/A N/A 11379.3
Gland
Mam C039: Mammary N/A N/A 3502.6
Gland
Mam 507~~ Mammary 11772.5 903.5
GlancL
Mam S 12 Mamma~~y 3 . 4 17 0 . 5
_;
Gland
Ovr 103; Ovarv 0 0
Ovr 1118 Ovary 0.13 ~N/A
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Ovr 35GA Ovary N/A N/A 0.13
Utr 23XU Uteru:~ 5.6 0
Utr 135X0 Uteru:> 0 0
Cvx NK24 Cervix: 0.9 1.4
End 4XA Endometz~iu 32.2 0
m
Cln AS43 Colon 2.3 0
Cln AS45 Colon 0 0
Cln RCO1 Colc:>n 0.2 0
Lng AC90 Lung 0 2.0
Lng LC10 Lun:~ 0 0 . 6
~~
Lng SC32 Lung 0.8 0
Sto AC93 Stomach 0 0
Tst 39X Testi~> 1.97 0
Among 58 sam~:~les in Table 5 representing 9 different
tissues, the highest expression is seen in mammary tissues.
Amongst the non-breasr_ 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 7C) samples in 17 human tissue
types. Twenty-two samples representing 11 different. tissue
types excluding brea:~t had no detected Mam002 (SEQ ID NO:1)
mRNA (Table 4 and Table 5).
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 ?'. of 13 matched breast cancer
tissues (Samples Mam 5127, Mam 162X and Mam 5079) compared
with the correspondin.c~ normal adjacent tissue. The level of
Mam002 (SE;Q ID NO:'1) expression is lower in breast cancer
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compared to normal adjacent tissue in eight individuals (Mam
12X, Mam ~~2DN, Mam 59X, Mam BOllX, Mam S516, Mam 5854, Mam
S967, and Mam 5123). Equivalent levels or very similar levels
of expres:~ion were detected in three other matched samples
(Samples Nfam A06X, M<~m 5699 and Mam 5997).
The high level of tissue specificity is demonstrative
of Mam002 (SEQ ID NO:l) being a diagnostic marker for
detection of mammary cancer cells using mRNA. Breast tissue
is the on7_y signif a.cant source of this gene' s expression so
far detected. Eight:. of 13 matched samples have lower levels
of express>ion in cancer than normal adjacent tissue. Thus,
decreased expression of this gene appears to be diagnostic of
cancer presence.
Measurement of SEQ ID N0:4; Clone ID: 2613064; Gene ID: 27052
(Mam004)
The numbers depicted in Table 6 are relative levels of
expression in 12 :uc%rmal tissues of Mam004 (SEQ ID N0:4)
compared t:o mammary (calibrator). These RNA samples were
obtained commerciall~~ and were generated by pooling samples
from a particular ti;~sue from different individuals.
Table 6: Relative levels of Mam004 (SEQ ID N0:4) 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
The relative levels of expression in Table 6 show that Mam004
(SEQ ID N0:4) mRNA expression is detected in the pool of
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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 N0:4) mRNA expression is not specific for
mammary gl<~nd.
The tissues shown. in Table 6 are pooled samples from
different individual:. 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.
The :numbers depicted in Table 7 are relative levels of
expression of Mam004 (SEQ ID N0:4) 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 s<~me indiv:i.dual.
Table 7: Relative levels of Mam004 (SEQ ID N0:4) Expression
in Individual Samples
Sample :ID T:Lssue Cancer Matching
Mam 1213 Mammary Gland 0 0
Mam 12:~ Mammary Gland 13.454 0
Mam 603X Mammary Gland 30.484 0
Mam 59:~ 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 5997 Mammary Gland 10.43 1.29
Sto AC44 '.:>tomach 0.61 0
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Cln SG45 Colon 0.04 0
Cln TXO1 Colon 0 0
Cvx NK23 Cervix 0 0
Cvx NK24 Cervix 0 0
Endo 3F.X Enc3ometriurn 0 0
Endo 4xA En<~ometrium 0 0
Endo 5xA Enciometrium 0 2.73
Kid 11X:D K_~dney 0 0
Kid 5X1 Kidney 0 2.63
Lng C20X Dung 0 0
Lng SQ56 Lung 10.37 0
Among 46 samples in Table 7 representing 7 different
tissues expression i.s highest in breast tissues particularly
cancers. Expression comparable to that seen in breast samples
is also seen in 1 of 4 lung camp-les (Sample 23), 1 of 4 kidney
samples (Sample 21) <~:nd 1 of 6 endometrial samples (Sample
19). Table E~ and Table 7 represent a combined total of 58
samples in 16 human ti.~~sue types. Twenty samples representing
7 different tissue types excluding breast had no detected
Mam004 (SE(~ ID N0:4 j mRNA (''able 6 and Table 7) .
Comparisons o:f the level of mRNA expression in breast
cancer samples and the normal adjacent tissue from the same
individual; are shown in Table 7. Mam004 (SEQ ID N0:4) is
expressed ~~t higher 1_evels in 8 of 11 breast cancer tissues
(Mam 12X, N(am 603X, Mam 59X, Mam 162X, Mam 5079, Mam S123, Mam
S516 and hlam 5997) compared with the corresponding normal
adjacent tissue. The level of Mam004 (SEQ ID N0:4) expression
is lower in breast <:ancer compared to normal adjacent tissue
in two matched samples (Mam 42DN and Mam 5699). No expression
was detected in one matched sample (Mam 12B).
Elevated expre:~sion in the majority of matched cancer
samples compared to normal adjacent tissue is indicative of
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Mam004 (SEQ ID N0:4) being a diagnostic marker for detection
of mammary cancer cells using mRNA.
Measurement. of SEQ ID N0:3; Clone ID:y155b03; Gene ID: 348845
(Mam005)
The numbers depicted in Table 8 are relative levels of
expression in 12 normal tissues of Mam005 (SEQ ID N0:3)
compared to testis (calibrator). These RNA samples were
obtained commercially and were generated by pooling samples
from a part:icul.ar ta.ssue from d~.fferent individuals.
Table 8: Relative levels of Mam005 (SEQ ID N0:3) 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 )intestine0
Testis 1
~
Thymus 0
Uterus 0
The relati~le levels o:E expression in Table 8 show that Mam005
(SEQ ID NO:3) mRNA E=xpression is detected in the pool of
normal mammary and in testis but is not present at significant
levels in the other 7..0 normal tissue pools analyzed. These
results demonstrate that Mam005 (SEQ ID N0:3) 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.
The tissues shown in Table 8 are pooled samples from
different individua:l:~. The tissues shown in Table 9 were
obtained from individuals and are not pooled. Hence the
values for mRNA expression leve:Ls shown in Table 8 cannot be
directly c~smpared to t:he va:Lues shown in Table 9.
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The numbers depicted in Table 9 are relative levels of
expression of Mam00!~ (SEQ ID N0:3) compared to testis
(calibrator), in 46 pairs of matching samples. Each matching
pair contains the canee:r sample for a particular tissue and
the normal adjacent tissue sample for that same tissue from
the same individual. In additioru 2 unmatched mammary samples
from norma7_ tissues and one unmatched ovarian cancer and one
normal (non-cancerous) ovary were also tested.
Table 9: Relative levels of Mam005 (SEQ ID N0:3) Expression
in Individual Samples
Sample ID Tissue Cancer Matching Normal
Mam 12X Mamma:r~r 0 . 3 0 . 71
Gland
Mam 42DN Mammar~r 0.22 0.63
Gland
Mam 59X Mammary 0.03 0.23
Gland
Mam A06X Mammary 70.77 0.56
Gland
Mam BO11X Mammary 0.0:~ 1.52
Gland
Mam 162X Mammary 0.43 0.09
Glanca
Mam C012 Mammary N/A N/A 1..6
Gland
Mam C034 Mammary N/A N/A 2.9
Gland
2 Mam S 0 7 Mamma z~y 0 . 2 2 0 . 13
0 9 )
Gland
Mam 5123 Mammary 0.01. 0.23
Gland
Mam 5127 Mammary 0 0.28
Gland
Mam 5516 Mammary 0.15 0.05
Gland
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Mam S699 Mammary 0.21 0.42
Gland
Mam S854 Mammary 1.12 0.54
Gland
Mam 5967 Mammary 30.61 0.54
Gland
Mam 5997 Mammary 0.40 0.22
Gland
Mam 14DN Mammary 0.07 0
Gland
Mam 6 9 9 Mamma.r~T 0 . 01 0 . 0 9
F Gland
Mam 5621 Mammary 1.82 0
Gl.an;~
Mam 5918 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 RCO1 Colon 0.0012 0.0003
Lng AC90 Lung 0 0
Lng LC109 Lung 0 0
Lng SQ3 2 Lunch 0 0
Lng SQ43 Lung 0 0
Ovr 103X Ovary 0 0
Ovr 1118 Ovary 0 N/A
Ovr A084 Ovary 0 0
Ovr 6021 Ovary 0 0
Ovr 35GA Ovary N/A N/A 0
Cvx NK23 Cervix 0 0
Cvx NK24 Cervix 0 0
Endo 3AX Endomet::riu0 0
m
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Endo 4XA Endomet:.riu0 0
m
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 135X0 Uterus 0 0
Utr 23XU Uterus 0 0
Kid 124D Kidney 0 0
Lvr 15XA Liver 0 0
Pan C044 Pancreas 0 0
Skn 4485 Skirl. 0 0
SmInt 21X~~ Small 0 0
Intestines
Among 96 samples in 'rar>le 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 ~. combined total of 108 samples in 18 human tissue
types. Sixay-seven s<~mples representing 16 different tissue
types excluding breast and t=estis had either no or very low
levels of detected M<~m005 (SEQ ID N0:3) mRNA {Table 8 and
Table 9 ) .
Comp<zrisons of. the level of mRNA expression in breast
cancer samples and r~he normal adjacent tissue from the same
individuals are shown in Table 9. Mam005 (SEQ ID NO:3) is
expressed ;~t higher levels in 7.0 of 18 cancer and normal
adjacent tissue samples (Mam A06X, Mam 162X, Mam 5079, Mam
5516, Mam 3854, Mam 5967, Mam S997, Mam 14DN, Mam 5621, and
Mam 5918) compared with the corresponding normal adjacent
tissue. The level oi: Mam005 (SEQ ID N0:3) expression is lower
in breast cancer compared to normal adjacent tissue in eight
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cancer and normal adjacent tissue samples (Mam 12X, Mam 42DN,
Mam 59X, Mam BO11X, Mam 51.23, Mam 5127, Mam S699 and Mam
699F). No expression was detected in two matching samples.
The :high leve7_ of tissue specificity and overexpression
in 10 of lf3 matched cancer and normal adjacent tissue samples
is indicative of Marn005 (SEQ ID N0:3) being a diagnostic
marker for detection of mammary cancer cells using mRNA.
CA 02347906 2001-02-02
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SEQUENCE LISTING
<1.10> Sun, Yongming
Recipon, Herv~>_
Cafferkey, Robert
DIADEXUS LLC
<1.20> A NOVEL METHOD OF DIAGNOSING, MONITORII'IG, STAGING ,
IMAGING AND T:ftEATING BREAST CANCER
<7.30> DEX-0040
<7.40>
<7.41>
<~_50> 60/095,232
<:_51> 1998-08-04
<=_60> 9
<:_70> PatentIn Ver. 2.0
W?10>1
W?11>544
<;?12>DNA
<;?13>Homo sapiens
<:?20>
<;?21> unsure
<:?22> (505) . . (506)
<220>
<221> unsure
<222> (510)
< 220>
<221> unsure
<222> (521?
<220>
<221> unsure
<:022> (527)..(528)
<220>
<221> unsure
<222> (531>
<220>
1
CA 02347906 2001-02-02
WO 00108210 PCT/US99/16811
<221> unsure
<222> (534)..(535)
<220>
<221> unsure
<222> (540)..(541)
<400> 1
ctagtctcga gtctagagcg ccttgcctt:.c tcttaggctt t.gaagcattt ttgtctgtgc 60
tccctgatct tcatgtcacc accatgaacxt.tcttagcagt c:ctggtactc ttgggagttt 120
ccatctttct ggtctctgcc cagaatcc::xa. caacagctgc t:ccagctgac acgtatccag 180
ctactggtcc tgctgatgat gaagcccct:.g ,atgctgaaac cactgctgct gc aaccactg 240
cgaccactgc tgctcctacc actgcaacaa ccg ctgcttc taccactgct cgtaaagaca 300
ttccagtttt acccaaatc~g gttgggga;-c tcccgaatgg t.agagtgtgt ccctgagatg 360
gaatcagctt gagtcttci:g caattggt::::a caactattca tccttcc~gt gatttcatcc 420
aactacttac cttgcctacg atatcccc;:t tatctctaat c:agttta-tt tctttcaaat 980
aaaaaataac tatgagcaac taaannaa~:~n aaaaaaaaaa naaaaannaa n<3annaaaan 540
naga 544
<210> 2
<211> 1066
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (729)..(813)
<400> 2
gttgaccagt ggtcatgcca ctgcctgtt:g atttgttgaa aatattgttt acacgtatgt 60
tcttgttact gattgtcac~a aagctggt?::t tgagactgca gcttggacta aattcagtca 120
tctggctgtc tggggaagc:a tgctgaccag tctggtgttc tttggca~ct actcagccat 180
ctggtccacc attctcatt:g ccccaaatat gagaggacag aagaatggta ccggtactgc 240
caatggagat ggaggaagc~a gacagaaac~a <3acagagccc agaccctagg gaccaccagc 300
atttgcagaa tggataaaca gccttctt:::c taacaaagga agcacagcaa ctgtgatcct 360
gagctgtgca cacttctgc~t tgggattat::t tctggtttct acttcctgtt tgaagatgtg 420
gcatggagag tgaacaagca gct=gcccacc <3cctggcatc acagccccag aactcagcta 480
tttccatggg accacagcat ctcatctct~g g gctgagcca gaaagacccc tactgaagtc 590
cagaggcact tttctgaaag gctctgctt~t gacctgaagt attttatcta tc ctcagtct 600
caggacactg ttgatggaat taaggccaag cacatctgca aaaaagacat tgctggagga 660
ggtgcaaaga gctggaaac:c aagtctcc<_ig tcctgggaaa agcagtggta tggaaaagca 720
atggaaagnn nnnnnnnnnn nnnnnnnnvn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780
nnnnnnnnnn nnnnnnnnnn nnnnnnnn:un nnncatagca ccaatgacct gaagagcctt 890
gttgaaggaa gactccatct gatgactc_ig agcaagtatt ttttagtgtg ttattgttat 900
tagcagaaag agggccataa aatacatg.~g c~caagctgaa tatatcttag gcaaaagaag 960
aaaatattca aattcttat:g ttatttt-_at::c taattatttt atctcttttt gtgtgtgact 1020
tataatgtgt gtattgtat:t aataaaagt:a tataaacatg tagttt 1066
CA 02347906 2001-02-02
WO 00/08210 PCT/US99/16811
<:?10>3
<:?11>649
< DNA
212>
<213>Homo Sapiens
<<~00> 3
gcaatgttta atatctcata agctatacac acctcgaagc catcaatgac aaccttttct 60
tc~ctgaatag aacagtgatt gatgtcatga agacaat:ttt <stctcctttt gccttccata 120
aiatgtacca ggttatataa tagtataaca ctgccaagga g,~ggattatc tcatcttcat 180
cca gtaattc cagtgtttgt cacgtggttg ttgaataaat gaataaagaa tgagaaaacc 240
ac~aagctctg atacataatc ataatgataa ttatttc:aat :3cacaactac gggtggtgct 300
gaactagaat ctatattttc tgaaactggc_ tcctctagga ~c~tactaatg atttaaatct 360
a<~aagatgaa gttagtaaag catcagaaaa aaaaggt.aaa ,~aaattgctc ctgtggagat 920
g<3ttggcatc acatggtgtt ttgagctgat ac:accc<taca ~ttgagctca ctgcaacagt 480
ac~cagatttt caccgctatg cctcctttca ctctggc;agt .~rt:cagagg tcttgcactc 540
gc~gagagcat gctcaggttt ccccagctct acaaaat:cac ~c~agaatgcc aaagacttca 600
ac~acaagggt aaataaggtt gatct:,agaa ttgtcac:ctc aaaaaggcc 649
<2I0>9
<211>388
<212>DNA
<213>Homo Sapiens
<220>
<:?21> unsure
<222> (378)
< 220>
<221> unsure
<222> (385)
<~00> 4
agctgctcaa tacggaacat attctcagtc: ctcctctggt ctacaaagcc tgtgatttct 60
t~~tctatgga cagaacgtct ggtttaat~~t acaggaaccc ataacttcct gaagctttat 120
g~~ttaacagt gacaacgtga gtcagttgaa ttttatt~gtg r_-tcagtccg tagagtatta 180
g~~taacagaa acctttccat tgccatac-tc~ agaaactggc .ag caggcagt gtgcctacag 240
gt ctacaaag aaacttcaga tcatcttctt gagggaaaga agctgaagtg ctacataaga 300
t~~cttgtgct tcataactct cagaagctg c agattct:gta r_aaatcctta gaaaagagca 360
t~~ccctgaat ccataaangt at,stngcc; 388
<.210> 5
<211> 1227
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> (327)
3
CA 02347906 2001-02-02
WO 00/08210 PCT/US99/16811
<220>
<221> unsure
<222> (352)
<220>
<221> unsure
<222> (369)
<2 20>
<221> unsure
<222> (850)..(880)
<<:20>
<<:21> unsure
<<:22> (122C)
<900> 5
at.tttgtagt tcagcaaa~c ct~~caaatac acagcatgtt acaaggcact ggtggcacag 60
gc~cacaacag gaaatgat,~t ttatttag:::a aattcattta acaaatatta ttgggcacct 120
gt:tatgtgag acactgtc~~t aggcactgtg ggataacaac agcaaacact t~~acacaaca 180
gc:ctggcctt cctgt.gttvt acaacagcl::c ctaaagatag ctgatat ~aa gacatttgag 240
gc~acacagtt catgtaga~3t caaaatatt:a gtatttcaga at.aaggattt tttttctgaa 300
aagcatacag agaggaaaca gcttaanaat aggtcaagac caaaaaa~,ag antataatca 360
cctgaataanc tggataac~~c: agacagtc:::c: r-acagaattt cattcaggtc acagatttct 420
t~.aaactcac ccccaaaarg tg~~ctgctt~g gttgtttgaa t.cttgcataa tt aatgtcac 980
actgcgcaagc cgctgaacvt agttgagal:g cagaaaacaa acaaatgcaa tgacatatct 540
ga.gaagcatt tatgtaacvc cggttaagt:g gtgaggaggg <xtgtgtgaag acagtgtgca 600
tcfcatgagtg tgtattca~-a tatatgtgta tacatatgaa tttcactgtt attttccagg 660
gtctatggac aatgtggc<3g taagagtcta tgatgttctg aaacttttca ca gtaaatcc 720
aa.agattaca gaccttacaa ggtgcttg~:a ttctgttgct tttccatctg tc acttctca 780
gcatatttga ctgtgttcaa accttcttt~t ctttttcatt gagtttcatt ttttaagctt 840
gttaaatgcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn t.gtcatt-_rt cacattatcc 900
tcacttctct gcaacaagc~a tagtaagat:g tagatgaatg c:aaaaataat aacaacaata 960
acgaaatata ttaaagcttt aaaatatg-,:a catatgtagt t:.ctaaagagc aataacggta 1020
gtatctattt cgaacatgca ttaggcaaaa aagaaatcaa aactgaaatt ttcgtgtatt 1080
tttccccttg taagatgttc aaatgcta,ac ttcattttct cctttcctct atgtggcact 1140
ttctcaaaat atctatgaaa tactttta~Ta c:aaagattga c;ctggagaaa gagatacaaa 1200
tttccatccc cccagacagn gagacat 1227
<210>6
<<11>253
<<12>DNA
<2I3>Homo sa~iens
<<:20>
<<:21> unsure
<<:22> (181)
n
CA 02347906 2001-02-02
WO 00/08210 PCT/US99/16811
< 220>
<:?21> unsure
<222> (201)
<2 20>
<221> unsure
<2 22> (205)
< 220>
<;?21> unsure
<<'?22> (238)
<:?20>
<;?21> unsure
<;?22> (241) .. (242)
<;?20>
<<'?21> unsure
<;?22> (250)
«!00> 6
gaacagcctc acttgtgttg ctgtcagtgc cagtagggca <~gcaggaatg cagcagagag 60
gactcgccat cgtggccttg gctgtctgt=g cggwcctaca t_gcctcagaa gccatacttc 120
ccattgcctc cagctgtt~~c acc3gaggtt=t cacatcatat t: t:ccagaagg ctcctggaaa 180
nagtgaatat gtgtcgca~c naganagct:g atggggattg t_qacttggct gctgtcancc 240
nncatgtcan gcg 253
<:?10> 7
<<?11> 943
< a? 12 > DNA
<i?13> Homo Sapiens
W?20>
W?21> unsure
W?22> ( 128 )
<i?20>
<i?21> unsure
<i?22> ( 130 )
W?20>
W?21> unsure
W?22> (925)
<400> 7
gclgggcctgg ccccggcc~~c tgtgaggacc ccgcgggtgc tggggtaaga ggctctagac 60
ccttcacctg tcagtcac~~t gagggagg<~t gaggccaagc cccatccctc agaatcaagg 120
:p
CA 02347906 2001-02-02
WO 00/08210 PCT/1JS99/16811
cttgcaancn cccctcac:ct gcccagtctc tgtccacacc cctcgggctg aagacggccc 180
tgaccaggcc ctgggcct:ca gcgaccaccc ctccccctcc tgcctggacc cagggagcag 240
gtgcaggggg ctccgagc:cc ctggtgactg tcaccgtgca gtgcgccttc acagtggccc 300
tgagggcacg aagaggac;cc gacctgtc:-ca gcctgcgggc actgctgggc c aagccctcc 360
ctcaccaggc ccagcttc;gg caactca=:;gt gggccagaaa gcccccggtg gctgcggtgg 420
agctgggcac cgccccgact gaggcagc:a~~ ctggaagagg gggtggcaga ggtcactgcc 480
ctccctgcag gccccacc:ca ggaggcc;.:c~ t~~tgaggaat ctctttgcag ttacctagcc 540
ccaggtgagg acgggcaca g ggtcccc<atc c~~cgaggagg agtcgctgca gagggcctgg 600
caggacgcag ctgcctgc:cc cagggggc:a~3 cagctg~agt_ gcaggggagc cgggggtcgg 660
ccggtcctct accaggtc;gt ggcccagc:a~~ agctactccg cc~aggggcc agaggacctg 720
ggcttccgac agggggac:ac ggtggac~;tc ctgtgtgaag tggaccaggc atggctggag 780
ggccactgtg acggccgc:at cggcatct:t~: c~~caagtgct tcgtggtccc cgccggccct 840
cggatgtcag gagccccc:gg ccgccr_g:-:c.. cgatcc_agc agggagatca gccctaatga 900
tgctgtgtcc atgatgct:tt taatnaa<aaa aacccc~~act gca 943
<210> 8
<211> 249
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (48)
<220>
<221> unsure
<222> (110)
<220>
<221> unsure
<222> (192)
<220>
<221> unsure
<222> (205)
<220>
<221> unsure
<222> (218)
<400> 8
atcacattaa gtcattgcaa attttataaa caaaaacaat ggttttantt tgcatctccc 60
tgattggtat tgctgtacaa catatttc;ga gaagtttgtt tgtctttggn gtttatttca 120
tgaatagatt gtgtgcccat tttctctt.gc; ggtattc~agt ttr_ttattac tgatgtgagc 180
atgtgtatgg gngattattt gatgnttast<: agttttgntt agtagactgg c:aatatttag 240
tcttgctgt 249
<210> 9
6
CA 02347906 2001-02-02
WO 00/08210 PCT/US99/16811
<2L1> 690
<2L2> DNA
<2:L3> Homo sapiens
<400> 9
gac~gcccagt gacctgccga ggtcggcagc acagagct:ct gg<sgatgaag accctgttcc 60
tgc3gtgtcac gctcggcctg gccgctgcc:c tgtccttcac c~~rggaggag gaggatatca 120
cagggacctg gtacgtgaag gccatggtc:g tcgataagga cttt:ccggag gacaggaggc 180
cc<iggaaggt gtccccagtg aaggtgacag ccctgggc:gg tgggaagttg gaagccacgt 240
tc<3ccttcat gagggaggat cggtgcatc:c a.gaagaaaat c~tgatgcgg aagacggagg 300
ago ctggcaa atacagcgcc tatgggggc:a ggaagctc:at g=acctgcag gagctgccca 360
gga gggacca ctacatcttt tactgcaaag accagca<:ca tc)ggggcr_tg ctccacatgg 420
gaa agcttgt gggtaggaat tctgatacc:a acc:gggaqgc c;tgqaagaa tttaagaaat 480
tgc)tgcagcg caagggactc tcggaggagg acattttcac g;~ccctgcag acgggaagct 540
gcc~ttcccga acactaggca gccc~ccggc~~ ctgcacctcc agagcccacc: ctaccaccag 600
ac<icagagcc cggaccacct ggacctaccc tccagccatg a--ccatc:cct gctcccaccc 660
acc~tgactcc aaataaagtc cttctcccc~= 690
7
