U.S. patent application number 13/211767 was filed with the patent office on 2012-01-19 for novel method of diagnosing, monitoring, staging, imaging and treating various cancers.
This patent application is currently assigned to diaDexus, Inc.. Invention is credited to Robert Cafferkey, Herve Recipon, Susana Salceda, Yongming Sun.
Application Number | 20120014872 13/211767 |
Document ID | / |
Family ID | 37848825 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120014872 |
Kind Code |
A1 |
Salceda; Susana ; et
al. |
January 19, 2012 |
Novel Method of Diagnosing, Monitoring, Staging, Imaging and
Treating Various Cancers
Abstract
The present invention provides a new method for detecting,
diagnosing, monitoring, staging, prognosticating, imaging and
treating selected cancers including gynecologic cancers such as
breast, ovarian, uterine and endometrial cancer and lung
cancer.
Inventors: |
Salceda; Susana; (San Jose,
CA) ; Sun; Yongming; (San Jose, CA) ; Recipon;
Herve; (San Francisco, CA) ; Cafferkey; Robert;
(San Jose, CA) |
Assignee: |
diaDexus, Inc.
South San Francisco
CA
|
Family ID: |
37848825 |
Appl. No.: |
13/211767 |
Filed: |
August 17, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12787675 |
May 26, 2010 |
8029787 |
|
|
13211767 |
|
|
|
|
09763978 |
Apr 25, 2001 |
7737255 |
|
|
PCT/US99/19655 |
Sep 1, 1999 |
|
|
|
12787675 |
|
|
|
|
60098880 |
Sep 2, 1998 |
|
|
|
Current U.S.
Class: |
424/1.49 ;
424/138.1; 424/178.1; 424/9.1; 435/6.12; 435/7.92; 530/387.1;
536/23.1; 536/24.5 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; G01N 33/57411 20130101; Y10T 29/49108
20150115; G01N 33/57449 20130101; A61P 43/00 20180101; G01N
33/57442 20130101; C12Q 2600/16 20130101; G01N 33/57415 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
424/1.49 ;
424/9.1; 424/138.1; 424/178.1; 536/23.1; 536/24.5; 530/387.1;
435/7.92; 435/6.12 |
International
Class: |
A61K 51/10 20060101
A61K051/10; A61K 39/395 20060101 A61K039/395; C07H 21/04 20060101
C07H021/04; C12Q 1/68 20060101 C12Q001/68; C07K 16/00 20060101
C07K016/00; A61P 43/00 20060101 A61P043/00; A61P 35/00 20060101
A61P035/00; G01N 33/574 20060101 G01N033/574; A61K 49/16 20060101
A61K049/16; C07H 21/02 20060101 C07H021/02 |
Claims
1. A method for detecting the presence of cancer in a patient
comprising: (a) measuring the level of the native protein expressed
by the gene comprising the polynucleotide sequence of any of SEQ ID
NOs: 1, 10, 11, 12 and 13 in a sample from a patient; and (b)
comparing the measured level of said protein in said patient with
the level of said protein in a control, wherein an increase in the
measured level of said protein in said patient versus said control
is associated with the presence of a cancer.
2. A method of detecting metastases of a cancer in a patient
comprising: (a) identifying a patient having a cancer that is not
known to have metastasized; (b) measuring the level of the native
protein expressed by the gene comprising the polynucleotide
sequence of any of SEQ JD NOs: 1, 10, 11, 12 and 13 in a sample
from a patient; and (c) comparing the measured level of said
protein with the level of said protein in a control, wherein an
increase in measured levels of said protein in the patient versus
the control is associated with a cancer which has metastasized.
3. A method of staging cancer in a patient having cancer
comprising: (a) identifying a patient having cancer; (b) measuring
the level of the native protein expressed by the gene comprising
the polynucleotide sequence of any of SEQ ID NOs: 1, 10, 11, 12 and
13 in a sample from a patient; and (c) comparing the measured level
of said protein with level of said protein in a control, wherein an
increase in the measured level of said protein in said patient
versus the control is associated with a cancer which is progressing
and a decrease in the measured level of said protein in said
patient versus the control is associated with a cancer which is
regressing or in remission.
4. A method of monitoring cancer in a patient for the onset of
metastasis comprising: (a) identifying a patient having cancer that
is not known to have metastasized; (b) periodically measuring the
level of the native protein expressed by the gene comprising the
polynucleotide sequence of any of SEQ ID NOs: 1, 10, 11, 12 and 13
in a sample from a patient; and (c) comparing the periodically
measured levels of said protein with level of said protein in a
control, wherein an increase in any one of the periodically
measured levels of said protein in the patient versus the control
is associated with cancer which has metastasized.
5. A method of monitoring the change in stage of cancer in a
patient comprising: (a) identifying a patient having cancer; (b)
periodically measuring the level of the native protein expressed by
the gene comprising the polynucleotide sequence of any of SEQ ID
NOs: 1, 10, 11, 12 and 13 in a sample from a patient; and (c)
comparing the periodically measured of said protein with level of
said protein a control, wherein an increase in any one of the
periodically measured levels of said protein in the patient versus
the control is associated with a cancer which is progressing in
stage and a decrease is associated with a cancer which is
regressing in stage or in remission.
6. A method of imaging cancer in a patient comprising administering
to the patient an antibody which binds to the native protein
expressed by the gene comprising the polynucleotide sequence of any
of SEQ ID NOs: 1, 10, 11, 12 and 13.
7. The method of claim 6 wherein said antibody is labeled with
paramagnetic ions or a radioisotope.
8. A method of treating cancer in a patient comprising
administering to the patient an antibody which binds to the native
protein expressed by the gene comprising the polynucleotide
sequence of any of SEQ ID NOs: 1, 10, 11, 12 and 13.
9. The method of claim 8 wherein the antibody is conjugated to a
cytotoxic agent.
10. An isolated nucleic acid molecule selected from the group
consisting of: (a) a nucleic acid molecule comprising a nucleic
acid sequence of SEQ ID NOs: 1, 10, 11, 12 or 13; (b) a nucleic
acid sequence of SEQ ID NOs: 1, 10, 11, 12 or 13; (c) the native
mRNA encoded by the gene comprising any of the polynucleotide
sequences of SEQ ID NOs: 1, 10, 11, 12 or 13; or (d) the gene
comprising any of the polynucleotide sequences of SEQ ID NOs: 1,
10, 11, 12 or 13.
11. The nucleic acid molecule according to claim 10, wherein the
nucleic acid molecule is a cDNA.
12. The nucleic acid molecule according to claim 10, wherein the
nucleic acid molecule is a RNA.
13. The nucleic acid molecule according to claim 1, wherein the
nucleic acid molecule is a human nucleic acid molecule.
14. A native protein expressed by the nucleic acid molecule
according to claim 10.
15. An isolated polypeptide selected from the group consisting of:
(a) the native protein expressed by the gene comprising the
polynucleotide sequences of SEQ ID NOs: 1, 10, 11, 12 or 13; or (b)
the protein encoded by polynucleotide sequence of SEQ ID NOs: 1,
10, 11, 12 or 13.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 12/787,675 filed May 26, 2010, which is a continuation of U.S.
application Ser. No. 09/763,978 filed Apr. 25, 2001, now issued as
7,737,255, which is the U.S. National Phase of PCT/US1999/019655
filed Sep. 1, 1999, which claims the benefit of priority from U.S.
Provisional Application Ser. No. 60/098,880 filed Sep. 2, 1998,
each of which are herein incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] This invention relates, in part, to newly developed assays
for detecting, diagnosing, monitoring, staging, prognosticating,
imaging and treating various cancers, particularly gynecologic
cancer including ovarian, uterine endometrial and breast cancer,
and lung cancer.
BACKGROUND OF THE INVENTION
[0003] The American Cancer Society has estimated that over 560,000
Americans will die this year from cancer. Cancer is the second
leading cause of death in the United States, exceeded only by heart
disease. It has been estimated that over one million new cancer
cases will be diagnosed in 1999 alone.
[0004] In women, gynecologic cancers account for more than
one-fourth of the malignancies.
[0005] Of the gynecologic cancers, breast cancer is the most
common. According to the Women's Cancer Network, 1 out of every 8
women in the United States is as risk of developing breast cancer,
and 1 out of every 28 women are at risk of dying from breast
cancer. Approximately 77% of women diagnosed with breast cancer are
over the age of 50. However, breast cancer is the leading cause of
death in women between the ages of 40 and 55.
[0006] Carcinoma of the ovary is another very common gynecologic
cancer. Approximately one in 70 women will develop ovarian cancer
during her lifetime. An estimated 14,500 deaths in 1995 resulted
from ovarian cancer. It causes more deaths than any other cancer of
the female reproductive system. Ovarian cancer often does not cause
any noticeable symptoms. Some possible warning signals, however,
are an enlarged abdomen due to an accumulation of fluid or vague
digestive disturbances (discomfort, gas or distention) in women
over 40; rarely there will be abnormal vaginal bleeding. Periodic,
complete pelvic examinations are important; a Pap test does not
detect ovarian cancer. Annual pelvic exams are recommended for
women over 40.
[0007] Also common in women is endometrial cancer or carcinoma of
the lining of the uterus. According to the Women's Cancer Center
endometrial cancer accounts for approximately 13% of all
malignancies in women. There are about 34,000 cases of endometrial
cancer diagnosed in the United States each year.
[0008] Uterine sarcoma is another type of uterine malignancy much
more rare as compared to other gynecologic cancers. In uterine
sarcoma, malignant cells start growing in the muscles or other
supporting tissues of the uterus. Sarcoma of the uterus is
different from cancer of the endometrium, a disease in which cancer
cells start growing in the lining of the uterus. This uterine
cancer usually begins after menopause. Women who have received
therapy with high-dose X-rays (external beam radiation therapy) to
their pelvis are at a higher risk to develop sarcoma of the uterus.
These X-rays are sometimes given to women to stop bleeding from the
uterus. Lung cancer is the second most prevalent type of cancer for
both men and women in the United States and is the most common
cause of cancer death in both sexes. Lung cancer can result from a
primary tumor originating in the lung or a secondary tumor which
has spread from another organ such as the bowel or breast. Primary
lung cancer is divided into three main types; small cell lung
cancer; non-small cell lung cancer; and mesothelioma. Small cell
lung cancer is also called "Oat Cell" lung cancer because the
cancer cells are a distinctive oat shape. There are three types of
non-small cell lung cancer. These are grouped together because they
behave in a similar way and respond to treatment differently to
small cell lung cancer. The three types are squamous cell
carcinoma, adenocarcinoma, and large cell carcinoma. Squamous cell
cancer is the most common type of lung cancer. It develops from the
cells that line the airways. Adenocarcinoma also develops from the
cells that line the airways. However, adenocarcinoma develops from
a particular type of cell that produces mucus (phlegm). Large cell
lung cancer has been thus named because the cells look large and
rounded when they are viewed under a microscope. Mesothelioma is a
rare type of cancer which affects the covering of the lung called
the pleura. Mesothelioma is often caused by exposure to
asbestos.
[0009] Procedures used for detecting, diagnosing, monitoring,
staging, and prognosticating each of these types of cancer are of
critical importance to the outcome of the patient. In all cases,
patients diagnosed early in development of the cancer generally
have a much greater five-year survival rate as compared to the
survival rate for patients diagnosed with a cancer which has
metastasized. New diagnostic methods which are more sensitive and
specific for early detection of various types of cancer are clearly
needed.
[0010] In the present invention methods are provided for detecting,
diagnosing, monitoring, staging, prognosticating, in vivo imaging
and treating selected cancers including, but not limited to,
gynecologic cancers such as ovarian, breast endometrial and/or
uterine cancer, and lung cancer via detection of a Cancer Specific
Genes (CSGs). Nine CGSs have been identified and refer, among other
things, to native proteins expressed by the genes comprising the
polynucleotide sequences of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,
8 or 9. In the alternative, what is meant by the nine CSGs as used
herein, means the native mRNAs encoded by the genes comprising any
of the polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,
8 or 9 or it can refer to the actual genes comprising any of the
polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9.
Fragments of the CSGs such as those depicted in SEQ ID NO:10, 11,
12, 13 or 14 can also be detected.
[0011] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in the art
from the following description. It should be understood, however,
that the following description and the specific examples, while
indicating preferred embodiments of the invention are given by way
of illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
SUMMARY OF THE INVENTION
[0012] Toward these ends, and others, it is an object of the
present invention to provide a method for diagnosing the presence
of selected cancers by analyzing for changes in levels of CSG in
cells, tissues or bodily fluids compared with levels of CSG in
preferably the same cells, tissues, or bodily fluid type of a
normal human control, wherein a change in levels of CSG in the
patient versus the normal human control is associated with the
selected cancer. For the purposes of this invention, by "selected
cancer" it is meant to include gynecologic cancers such as ovarian,
breast, endometrial and uterine cancer, and lung cancer.
[0013] Further provided is a method of diagnosing metastatic cancer
in a patient having a selected cancer which is not known to have
metastasized by identifying a human patient suspected of having a
selected cancer that has metastasized; analyzing a sample of cells,
tissues, or bodily fluid from such patient for CSG; comparing the
CSG levels in such cells, tissues, or bodily fluid with levels of
CSG in preferably the same cells, tissues, or bodily fluid type of
a normal human control, wherein an increase in CSG levels in the
patient versus the normal human control is associated with a cancer
which has metastasized.
[0014] Also provided by the invention is a method of staging
selected cancers in a human patient by identifying a human patient
having such cancer; analyzing a sample of cells, tissues, or bodily
fluid from such patient for CSG; comparing CSG levels in such
cells, tissues, or bodily fluid with levels of CSG in preferably
the same cells, tissues, or bodily fluid type of a normal human
control sample, wherein an increase in CSG levels in the patient
versus the normal human control is associated with a cancer which
is progressing and a decrease in the levels of CSG is associated
with a cancer which is regressing or in remission.
[0015] Further provided is a method of monitoring selected cancers
in patients for the onset of metastasis. The method comprises
identifying a human patient having a selected cancer that is not
known to have metastasized; periodically analyzing a sample of
cells, tissues, or bodily fluid from such patient for CSG;
comparing the CSG levels in such cells, tissues, or bodily fluid
with levels of CSG in preferably the same cells, tissues, or bodily
fluid type of a normal human control sample, wherein an increase in
CSG levels in the patient versus the normal human control is
associated with a cancer which has metastasized.
[0016] Further provided is a method of monitoring the change in
stage of selected cancers in humans having such cancer by looking
at levels of CSG. The method comprises identifying a human patient
having a selected cancer; periodically analyzing a sample of cells,
tissues, or bodily fluid from such patient for CSG; comparing the
CSG levels in such cells, tissue, or bodily fluid with levels of
CSG in preferably the same cells, tissues, or bodily fluid type of
a normal human control sample, wherein an increase in CSG levels in
the patient versus the normal human control is associated with a
cancer which is progressing and a decrease in the levels of CSG is
associated with a cancer which is regressing or in remission.
[0017] Further provided are antibodies against CSG or fragments of
such antibodies which can be used to detect or image localization
of CSG in a patient for the purpose of detecting or diagnosing
selected cancers. 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 CSG. 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.
[0018] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in the art
from the following description. It should be understood, however,
that the following description and the specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention relates to diagnostic assays and
methods, both quantitative and qualitative for detecting,
diagnosing, monitoring, staging and prognosticating selected
cancers by comparing levels of CSG with those of CSG in a normal
human control. What is meant by levels of CSG as used herein is
levels of the native protein expressed by the gene comprising the
polynucleotide sequence of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8
or 9. In the alternative, what is meant by levels of CSG as used
herein is levels of the native mRNA encoded by the gene comprising
any of the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6,
7, 8 or 9 or levels of the gene comprising any of the
polynucleotide sequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9.
Fragments of CSGs such as those depicted in SEQ ID NO: 10, 11, 12,
13 and 14 can also be detected. 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 diagnosing
over-expression of CSG protein compared to normal control bodily
fluids, cells, or tissue samples may be used to diagnose the
presence of selected cancers. What is meant by "selected cancers"
as used herein is a gynecologic cancer such as ovarian, breast,
endometrial or uterine cancer, or lung case.
[0020] Any of the 9 CSGs can be measured alone in the methods of
the invention, or all together or any combination thereof. However,
for methods relating to gynecologic cancers including ovarian,
breast, endometrial and uterine cancer, it is preferred that levels
of CSG comprising SEQ ID NO:1 or a fragment thereof be determined.
Exemplary fragments of this CSG which can be detected are depicted
in SEQ ID NO: 10, 11, 12, and 13. For methods relating to lung
cancer and gynecologic cancers including ovarian, endometrial and
uterine, it is preferred that levels of CSG comprising SEQ ID NO:2
or 9 be determined. Fragments of this CSG such as that depicted in
SEQ ID NO:14 can also be detected. For methods relating to ovarian
cancer, determination of levels of CSG comprising SEQ ID NO:3 is
also preferred.
[0021] All the methods of the present invention may optionally
include measuring the levels of other cancer markers as well as
CSG. Other cancer markers, in addition to CSG, useful in the
present invention will depend on the cancer being tested and are
known to those of skill in the art.
Diagnostic Assays
[0022] The present invention provides methods for diagnosing the
presence of selected cancers by analyzing for changes in levels of
CSG in cells, tissues or bodily fluids compared with levels of CSG
in cells, tissues or bodily fluids of preferably the same type from
a normal human control, wherein a change in levels of CSG in the
patient versus the normal human control is associated with the
presence of a selected cancer.
[0023] Without limiting the instant invention, typically, for a
quantitative diagnostic assay a positive result indicating the
patient being tested has cancer is one in which cells, tissues or
bodily fluid levels of the cancer marker, such as CSG, are at least
two times higher, and most preferably are at least five times
higher, than in preferably the same cells, tissues or bodily fluid
of a normal human control.
[0024] The present invention also provides a method of diagnosing
metastases of selected cancers in a patient having a selected
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 a selected 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 ovarian
cancer, patients are typically diagnosed with ovarian cancer
following surgical staging and monitoring of CAl25 levels.
Traditional detection methods are also available and well known for
other selected cancers which can be diagnosed by determination of
CSG levels in a patient.
[0025] In the present invention, determining the presence of CSG
levels in cells, tissues or bodily fluid, is particularly useful
for discriminating between a selected cancer which has not
metastasized and a selected cancer which has metastasized. Existing
techniques have difficulty discriminating between cancers which
have metastasized and cancers which have not metastasized and
proper treatment selection is often dependent upon such
knowledge.
[0026] In the present invention, the cancer marker levels measured
in such cells, tissues or bodily fluid is CSG, and are compared
with levels of CSG in preferably the same cells, tissue or bodily
fluid type of a normal human control. That is, if the cancer marker
being observed is CSG in serum, this level is preferably compared
with the level of CSG in serum of a normal human patient. An
increase in the CSG in the patient versus the normal human control
is associated with a cancer which has metastasized.
[0027] Without limiting the instant invention, typically, for a
quantitative diagnostic assay a positive result indicating the
cancer in the patient being tested or monitored has metastasized is
one in which cells, tissues or bodily fluid levels of the cancer
marker, such as CSG, are at least two times higher, and most
preferably are at least five times higher, than in preferably the
same cells, tissues or bodily fluid of a normal patient.
[0028] Normal human control as used herein includes a human patient
without cancer and/or non cancerous samples from the patient; in
the methods for diagnosing or monitoring for metastasis, normal
human control may also include samples from a human patient that is
determined by reliable methods to have a selected cancer which has
not metastasized.
Staging
[0029] The invention also provides a method of staging selected
cancers in human patients. The method comprises identifying a human
patient having a selected cancer and analyzing a sample of cells,
tissues or bodily fluid from such human patient for CSG. Then, the
method compares CSG levels in such cells, tissues or bodily fluid
with levels of CSG in preferably the same cells, tissues or bodily
fluid type of a normal human control sample, wherein an increase in
CSG levels in the human patient versus the normal human control is
associated with a cancer which is progressing and a decrease in the
levels of CSG is associated with a cancer which is regressing or in
remission.
Monitoring
[0030] Further provided is a method of monitoring selected cancers
in humans for the onset of metastasis. The method comprises
identifying a human patient having a selected cancer that is not
known to have metastasized; periodically analyzing a sample of
cells, tissues or bodily fluid from such human patient for CSG;
comparing the CSG levels in such cells, tissues or bodily fluid
with levels of CSG in preferably the same cells, tissues or bodily
fluid type of a normal human control sample, wherein an increase in
CSG levels in the human patient versus the normal human control is
associated with a cancer which has metastasized.
[0031] Further provided by this invention is a method of monitoring
the change in stage of selected cancers in humans having such
cancers. The method comprises identifying a human patient having a
selected cancer; periodically analyzing a sample of cells, tissues
or bodily fluid from such human patient for CSG; comparing the CSG
levels in such cells, tissues or bodily fluid with levels of CSG in
preferably the same cells, tissues or bodily fluid type of a normal
human control sample, wherein an increase in CSG levels in the
human patient versus the normal human control is associated with a
cancer which is progressing in stage and a decrease in the levels
of CSG is associated with a cancer which is regressing in stage or
in remission.
[0032] 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
[0033] Assay techniques that can be used to determine levels of
gene expression, such as CSG of the present invention, in a sample
derived from a patient 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.
[0034] An ELISA assay initially comprises preparing an antibody, if
not readily available from a commercial source, specific to CSG,
preferably a monoclonal antibody. In addition a reporter antibody
generally is prepared which binds specifically to CSG. The reporter
antibody is attached to a detectable reagent such as radioactive,
fluorescent or enzymatic reagent, for example horseradish
peroxidase enzyme or alkaline phosphatase.
[0035] To carry out the ELISA, antibody specific to CSG 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 CSG binds to the specific antibody attached
to the polystyrene dish. Unbound sample is washed out with buffer.
A reporter antibody specifically directed to CSG and linked to
horseradish peroxidase is placed in the dish resulting in binding
of the reporter antibody to any monoclonal antibody bound to CSG.
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 CSG
antibodies, produces a colored reaction product. The amount of
color developed in a given time period is proportional to the
amount of CSG protein present in the sample. Quantitative results
typically are obtained by reference to a standard curve.
[0036] A competition assay may be employed wherein antibodies
specific to CSG attached to a solid support and labeled CSG 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 CSG in the sample.
[0037] Nucleic acid methods may be used to detect CSG mRNA as a
marker for selected cancers. 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 the various
selected 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.
[0038] 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 CSG 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 CSG 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.
[0039] 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.
[0040] 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
[0041] Antibodies against CSG can also be used in vivo in patients
suspected of suffering from a selected cancer including lung cancer
or gynecologic cancers such as ovarian, breast, endometrial or
uterine cancer. Specifically, antibodies against a CSG can be
injected into a patient suspected of having a selected cancer 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
CSGs can be used in a similar manner. Labeled antibodies against a
CSG can be injected into patients suspected of having a selected
cancer for the purpose of diagnosing or staging of the disease
status of the patient. The label used will be selected in
accordance with the imaging modality to be used. For example,
radioactive labels such as Indium-111, Technetium-99m or Iodine-131
can be used for planar scans or single photon emission computed
tomography (SPECT). Positron emitting labels such as Fluorine-19
can be used in positron emission tomography. Paramagnetic ions such
as Gadlinium (III) or Manganese (II) can used in magnetic resonance
imaging (MRI). Localization of the label permits determination of
the spread of the cancer. The amount of label within an organ or
tissue also allows determination of the presence or absence of
cancer in that organ or tissue.
[0042] For patients diagnosed with a selected cancer, injection of
an antibody against a CSG 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-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 CSGs.
[0043] 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.
[0044] The present invention is further described by the following
examples. These examples are provided solely to illustrate the
invention by reference to specific embodiments. The
exemplifications, while illustrating certain aspects of the
invention, do not portray the limitations or circumscribe the scope
of the disclosed invention.
EXAMPLES
Example 1
[0045] Identification of CSGs 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.
[0046] 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. The CLASP allows the
identification of highly expressed organ and cancer specific genes.
A final manual in depth evaluation is then performed to finalize
the CSGs selection.
TABLE-US-00001 TABLE 1 CSG Sequences SEQ ID NO: Clone ID Gene ID 1
16656542 234617 2 1283171 332459 3 1649377 481154 4 236044H1 none
assigned 5 none assigned 255687 6 none assigned 251313 7 none
assigned 12029 8 none assigned 251804
[0047] The following examples are 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
[0048] Real-Time quantitative PCR with fluorescent Taqman probes is
a quantitation 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).
[0049] Amplification of an endogenous control is 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) is 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).
[0050] The tissue distribution and the level of the target gene for
every example in normal and cancer tissue were evaluated. Total RNA
was extracted from normal tissues, cancer tissues, and from cancers
and the corresponding matched adjacent tissues. Subsequently, first
strand cDNA was prepared with reverse transcriptase and the
polymerase chain reaction was done using primers and Taqman probe
specific to each target gene. The results are analyzed using the
ABI PRISM 7700 Sequence Detector. The absolute numbers are relative
levels of expression of the target gene in a particular tissue
compared to the calibrator tissue.
Measurement of Ovr110; Clone ID16656542; Gene ID 234617 (SEQ ID
NO:1, 10, 11, 12 or 13)
[0051] The absolute numbers depicted in Table 2 are relative levels
of expression of Ovr110 (SEQ ID NO:1 or a fragment thereof as
depicted in SEQ ID NO:10, 11, 12, or 13) in 12 normal different
tissues. All the values are compared to normal stomach
(calibrator). These RNA samples are commercially available pools,
originated by pooling samples of a particular tissue from different
individuals.
TABLE-US-00002 TABLE 2 Relative Levels of Ovr110 Expression in
Pooled Samples Tissue NORMAL colon 0.00 endometrium 8.82 kidney
7.19 liver 0.36 ovary 1.19 pancreas 21.41 prostate 2.79 small
intestine 0.03 spleen 0.00 00000000000000stomach 1.00 testis 8.72
uterus 0.93
[0052] The relative levels of expression in Table 2 show that
Ovr110 is expressed at comparable levels in most of the normal
tissues analyzed. Pancreas, with a relative expression level of
21.41, endometrium (8.82), testis (8.72), and kidney (7.19) are the
only tissues expressing high levels of Ovr110 mRNA.
[0053] The absolute numbers in Table 2 were obtained analyzing
pools of samples of a particular tissue from different individuals.
They can not be compared to the absolute numbers originated from
RNA obtained from tissue samples of a single individual in Table
3.
[0054] The absolute numbers depicted in Table 3 are relative levels
of expression of Ovr110 in 73 pairs of matching samples. All the
values are compared to normal stomach (calibrator). A matching pair
is formed by mRNA from the cancer sample for a particular tissue
and mRNA from the normal adjacent sample for that same tissue from
the same individual. In addition, 15 unmatched cancer samples (from
ovary and mammary gland) and 14 unmatched normal samples (from
ovary and mammary gland) were also tested.
TABLE-US-00003 TABLE 3 Relative Levels of Ovr110 Expression in
Individual Samples Matching Normal Sample ID Tissue Cancer Adjacent
Normal Ovr103X Ovary 1 86.22 0.53 Ovr1040O Ovary 2 168.31 Ovr1157
Ovary 3 528.22 Ovr63A Ovary 4 1.71 Ovr773O Ovary 5 464.65 Ovr1005O
Ovary 6 18.32 Ovr1028 Ovary 7 7.78 Ovr1118 Ovary 8 0.00 Ovr130X
Ovary 9 149.09 Ovr638A Ovary 10 3.14 OvrA1B Ovary 11 21.26 OvrA1C
Ovary 12 1.83 OvrC360 Ovary 13 0.52 Ovr18GA Ovary 14 1.07 Ovr20GA
Ovary 15 1.88 Ovr25GA Ovary 16 2.52 Ovr206I Ovary 17 2.51 Ovr32RA
Ovary 18 3.01 Ovr35GA Ovary 19 5.17 Ovr40G Ovary 20 0.45 Ovr50GB
Ovary 21 2.69 OvrC087 Ovary 22 0.47 OvrC179 Ovary 23 1.46 OvrC004
Ovary 24 4.99 OvrC007 Ovary 25 13.36 OvrC109 Ovary 26 6.61 MamS516
Mammary 16.39 13.74 Gland 1 MamS621 Mammary 826.70 4.60 Gland 2
MamS854 Mammary 34.60 18.30 Gland 3 Mam59X Mammary 721.57 27.00
Gland 4 MamS079 Mammary 80.73 5.10 Gland 5 MamS967 Mammary 6746.90
72.80 Gland 6 MamS127 Mammary 7.00 20.00 Gland 7 MamB011X Mammary
1042.00 29.00 Gland 8 Mam12B Mammary 1342.00 Gland 9 Mam82XI
Mammary 507.00 Gland 10 MamS123 Mammary 24.85 4.24 Gland 11 MamS699
Mammary 84.74 5.54 Gland 12 MamS997 Mammary 482.71 11.84 Gland 13
Mam162X Mammary 15.73 10.59 Gland 14 MamA06X Mammary 1418.35 8.20
Gland 15 Mam603X Mammary 294.00 Gland 16 Mam699F Mammary 567.40
86.60 Gland 17 Mam12X Mammary 425.00 31.00 Gland 18 MamA04 Mammary
2.00 Gland 19 Mam42DN Mammary 46.05 31.02 Gland 20 Utr23XU Uterus 1
600.49 27.95 Utr85XU Uterus 2 73.52 18.83 Utr135XO Uterus 3 178.00
274.00 Utr141XO Uterus 4 289.00 26.00 CvxNKS54 Cervix 1 2.47 0.61
CvxKS83 Cervix 2 1.00 2.00 CvxNKS18 Cervix 3 1.00 0.00 CvxNK23
Cervix 4 5.84 14.47 CvxNK24 Cervix 5 20.32 33.13 End68X Endometrium
1 167.73 544.96 End8963 Endometrium 2 340.14 20.89 End8XA
Endometrium 3 1.68 224.41 End65RA Endometrium 4 303.00 5.00 End8911
Endometrium 5 1038.00 74.00 End3AX Endometrium 6 6.59 1.69 End4XA
Endometrium 7 0.43 15.45 End5XA Endometrium 8 17.81 388.02 End10479
Endometrium 9 1251.60 31.10 End12XA Endometrium 312.80 33.80 10
Kid107XD Kidney 1 2.68 29.65 Kid109XD Kidney 2 81.01 228.33 Kid10XD
Kidney 3 0.00 15.30 Kid6XD Kidney 4 18.32 9.06 Kid11XD Kidney 5
1.38 20.75 Kid5XD Kidney 6 30.27 0.19 Liv15XA Liver 1 0.00 0.45
Liv42X Liver 2 0.81 0.40 Liv94XA Liver 3 12.00 2.16 Lng LC71 Lung 1
5.45 3.31 LngAC39 Lung 2 1.11 0.00 LngBR94 Lung 3 4.50 0.00 LngSQ45
Lung 4 15.03 0.76 LngC20X Lung 5 0.00 1.65 LngSQ56 Lung 6 91.77
8.03 ClnAS89 Colon 1 0.79 7.65 ClnC9XR Colon 2 0.03 0.00 ClnRC67
Colon 3 0.00 0.00 ClnSG36 Colon 4 0.81 0.35 ClnTX89 Colon 5 0.00
0.00 ClnSG45 Colon 6 0.00 0.06 ClnTX01 Colon 7 0.00 0.00 Pan77X
Pancreas 1 0.89 2.62 Pan71XL Pancreas 2 3.99 0.12 Pan82XP Pancreas
3 59.92 28.44 Pan92X Pancreas 4 17.21 0.00 StoAC93 Stomach 1 7.54
6.43 StoAC99 Stomach 2 19.49 3.19 StoAC44 Stomach 3 3.62 0.37
SmI21XA Small 0.00 0.00 Intestine 1 SmIH89 Small 0.00 0.00
Intestine 2 Bld32XK Bladder 1 0.00 0.21 Bld46XK Bladder 2 0.36 0.32
BldTR17 Bladder 3 0.28 0.00 Tst39X Testis 11.24 2.24 Pro84XB
Prostate 1 2.60 24.30 Pro90XB Prostate 2 1.40 2.00 0.00 =
Negative
[0055] Table 2 and Table 3 represent a combined total of 187
samples in 16 different tissue types. In the analysis of matching
samples, the higher levels of expression were in mammary gland,
uterus, endometrium and ovary, showing a high degree of tissue
specificity for the gynecologic tissues. Of all the samples
different than those mentioned before analyzed, only a few samples
(Kid109XD, LngSQ56, and Pan82XP) showed high levels of expression
of Ovr110.
[0056] Furthermore, the level of mRNA expression was compared in
cancer samples and the isogenic normal adjacent tissue from the
same individual. This comparison provides an indication of
specificity for the cancer stage (e.g. higher levels of mRNA
expression in the cancer sample compared to the normal adjacent).
Table 3 shows overexpression of Ovr110 in 15 of 16 mammary gland
cancer tissues compared with their respective normal adjacent
(mammary gland samples MamS516, MamS621, MamS854, Mam59X, MamS079,
MamS967, MamB011X, MamS123, MamS699, MamS997, Mam162X, MamA06X,
Mam699F, Mam12X, and Mam42DN). There was overexpression in the
cancer tissue for 94% of the mammary gland matching samples
tested.
[0057] For uterus, Ovr110 is overexpressed in 3 of 4 matching
samples (uterus samples Utr23XU, Utr85XU, and Utr141XO). There was
overexpression in the cancer tissue for 75% of the uterus matching
samples analyzed.
[0058] For endometrium, Ovr110 is overexpressed in 6 of 10 matching
samples (endometrium samples End8963, End65RA, End8911, End3AX,
End10479, and End12XA). There was overexpression in the cancer
tissue for 60% of the endometrium matching samples.
[0059] For ovary, Ovr110 shows overexpression in 1 of 1 matching
sample. For the unmatched ovarian samples, 8 of 12 cancer samples
show expression values of Ovr110 higher than the median (2.52) for
the normal unmatched ovarian samples. There was overexpression in
the cancer tissue for 67% of the unmatched ovarian samples.
[0060] Altogether, the level of tissue specificity, plus the mRNA
overexpression in most of the matching samples tested are
indicative of Ovr110 (including SEQ ID NO:1, 10, 11, 12 or 13)
being a diagnostic marker for gynecologic cancers, specifically,
mammary gland or breast, uterine, ovarian and endometrial
cancer.
Measurement of Ovr114; Clone ID1649377; Gene ID 481154 (SEQ ID
NO:3)
[0061] The numbers depicted in Table 4 are relative levels of
expression in 12 normal tissues of Ovr114 compared to pancreas
(calibrator). These RNA samples were obtained commercially and were
generated by pooling samples from a particular tissue from
different individuals.
TABLE-US-00004 TABLE 4 Relative Levels of Ovr114 Expression in
Pooled Samples Tissue Normal Colon 2.3 Endometrium 7.6 Kidney 0.5
Liver 0.6 Ovary 5.2 Pancreas 1.0 Prostate 2.1 Small Intestine 1.3
Spleen 2.4 Stomach 1.5 Testis 15.8 Uterus 8.8
[0062] The relative levels of expression in Table 4 show that
Ovr114 mRNA expression is detected in all the pools of normal
tissues analyzed.
[0063] 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.
[0064] The numbers depicted in Table 5 are relative levels of
expression of Ovr114 compared to pancreas (calibrator), in 46 pairs
of matching samples and 27 unmatched tissue samples. Each matching
pair contains the cancer sample for a particular tissue and the
normal adjacent tissue sample for that same tissue from the same
individual. In cancers (for example, ovary) where it was not
possible to obtain normal adjacent samples from the same
individual, samples from a different normal individual were
analyzed.
TABLE-US-00005 TABLE 5 Relative Levels of Ovr114 Expression in
Individual Samples Normal & Matching Borderline Normal Tissue
Sample ID Cancer Type Cancer Malignant Adjacent Ovary 1
Ovr1037O/1038O Papillary serous 17.04 3.93 adenocarcinoma, G3 Ovary
2 OvrG021SPI/SN2 Papillary serous 1.62 4.34 adenocarcinoma Ovary 3
OvrG010SP/SN Papillary serous 0.50 1.12 adenocarcinoma Ovary 4
OvrA081F/A082D Mucinous tumor, low 0.84 0.96 malignant potential
Ovary 5 OvrA084/A086 Mucinous tumor, grade 5.24 6.00 G-B,
borderline Ovary 6 Ovr14604A1C Serous 5.33 cystadenofibroma, low
malignancy Ovary 7 Ovr14638A1C Follicular cysts, low 8.11 malignant
potential Ovary 8 Ovr1040O Papillary serous 13.27 adenocarcinoma,
G2 Ovary 9 Ovr1157O Papillary serous 106.08 adenocarcinoma Ovary 10
Ovr1005O Papillary serous 77.04 endometricarcinoma Ovary 11
Ovr1028O Ovarian carcinoma 14.78 Ovary 12 Ovr14603A1D
Adenocarcinoma 22.23 Ovary 13 Ovr9410C360 Endometrioid 4.74
adenocarcinoma Ovary 14 Ovr1305X Papillary serous 96.49
adenocarcinoma Ovary 15 Ovr773O Papillary serous 8.40
adenocarcinoma Ovary 16 Ovr988Z Papillary serous 6.40
adenocarcinoma Ovary 17 Ovr9702C018GA Normal Cystic 12.06 Ovary 18
Ovr2061 Normal left atrophic, 10.11 small cystic Ovary 19
Ovr9702C020GA Normal-multiple 12.70 ovarian cysts Ovary 20
Ovr9702C025GA Normal-hemorrhage CL 22.09 cysts Ovary 21
Ovr9701C050GB Normal-multiple 9.01 ovarian cysts Ovary 22
Ovr9701C087RA Normal-small follicle 1.86 cysts Ovary 23
Ovr9702C032RA 7.81 Ovary 24 Ovr9701C109RA Normal 1.50 Ovary 25
Ovr9411C057R Benign large 5.22 endometriotic cyst Ovary 26
Ovr9701C179a Normal 3.09 Ovary 27 Ovr1461O Serous 3.53
cystadenofibroma, no malignancy Ovary 28 Ovr9701C035GA Normal 6.32
Ovary 29 Ovr9702C007RA Normal 0 Ovary 30 Ovr9701C087RA Normal-small
follicle 1.97 cysts Ovary 31 Ovr9411C109 Normal 9.49 Ovary 32
Ovr9701C177a Normal-cystic 3.85 follicles Endometrium 1
End14863A1A/A2A Moderately differ. 1.30 0.70 Endome. carcinoma/NAT
Endometrium 2 End9709C056A/55A Endometrial 1.83 11.90
adenocarcinoma/NAT Endometrium 3 End9704C281A/2A Endometrial 13.32
7.76 adenocarcinoma/NAT Endometrium 4 End9705A125A/6A Endometrial
3.62 3.34 adenocarcinoma/NAT Mammary Mam00042D01/N01 3.13 0.76
Gland 1 Mammary MamS99-522A/B 4.45 0.45 Gland 2 Mammary
Mam1620F/1621F 0.74 1.91 Gland 3 Mammary Mam4003259a/g 3.48 2.00
Gland 4 Uterus 1 Utr850U/851U Stage 1 endometrial 46.96 11.96
cancer/NAT Uterus 2 Utr233U96/234U96 Adenocarcinoma/NAT 20.02 5.90
Uterus 3 Utr1359O/1358O Tumor/NAT 10.23 7.74 Uterus 4
Utr1417O/1418O Malignant tumor/NAT 7.52 4.92 Cervix 1
CvxVNM00083/83 Keratinizing squamous 5.47 14.31 cell carcinoma
Cervix 2 CvxIND00023D/N Large cell 4.99 3.99 nonkeratinizing
carcinoma Cervix 3 CvxIND00024D/N Large cell 10.14 14.22
nonkeratinizing carcinoma Bladder 1 Bld665T/664T 1.43 4.03 Bladder
2 Bld327K/328K Papillary transitional 1.15 0.99 cell carcinoma/NAT
Kidney 1 Kid4003710C/F 0.03 0.35 Kidney 2 Kid1242D/1243D 1.61 0.14
Lung 1 Lng750C/751C Metastatic osteogenic 2.44 5.73 sarcoma/NAT
Lung 2 Lng8890A/8890B Cancer/NAT 1.11 5.19 Lung 3 Lng9502C109R/10R
1.99 0.80 Liver 1 Liv1747/1743 Hepatocellular 0.67 1.07
carcinoma/NAT Liver 2 LivVNM00175/175 Cancer/NAT 15.46 2.85 Skin 1
Skn2S9821248A/B Secondary malignant 2.83 0.70 melanoma Skin 2
Skn4005287A1/B2 0.91 4.02 Small Int. 1 SmI9802H008/009 0.87 0.82
Stomach 1 Sto4004864A4/B4 Adenocarcinoma/NAT 0.81 1.22 Stomach 2
StoS9822539A/B Adenocarcinoma/NAT 1.22 1.39 Stomach 3 StoS99728A/C
Malignant 0.47 0.35 gastrointestinal stromal tumor Prostate 1
Pro1012B/1013B Adenocarcinoma/NAT 2.39 2.61 Prostate 2
Pro1094B/1095B 0.10 0.38 Pancreas 1 Pan776p/777p Tumor/NAT 2.39
0.52 Pancreas 2 Pan824p/825p Cystic adenoma 1.66 1.22 Testis 1
Tst239X/240X Tumor/NAT 1.24 1.72 Colon 1 Cln9706c068ra/
Adenocarcinoma/NAT 0.38 0.65 69ra Colon 2 Cln4004732A7/B6
Adenocarcinoma/NAT 0.44 1.26 Colon 3 Cln4004695A9/B8 1.94 1.53
Colon 4 Cln9612B006/005 Asc. Colon, Cecum, 3.38 1.10 adenocarcinoma
Colon 5 Cln9704C024R/25R Adenocarcinoma/NAT 1.66 2.77
[0065] Table 4 and Table 5 represent a combined total of 129
samples in 17 human tissue types. Among 117 samples in Table 5
representing 16 different tissues high levels of expression are
seen only in ovarian cancer samples. The median expression of
Ovr114 is 14.03 (range: 0.5-106.08) in ovarian cancer and 4.34
(range: 0-22.09) in normal ovaries. In other words, the median
expression levels of Ovr114 in cancer samples is increased 3.5 fold
as compared with that of the normal ovarian samples. Five of 12
ovarian cancers (42%) showed increased expression relative to
normal ovary (with 95% specificity). The median expression of
Ovr114 in other gynecologic cancers is 4.99, and 2 out of 15
samples showed expression levels comparable with that in ovarian
cancer. The median of the expression levels of Ovr114 in the rest
of the cancer samples is 1.24, which is more than 11 fold less than
that detected in ovarian cancer samples. No individual showed an
expression level comparable to that of ovarian cancer samples
(except Liver 2; LivVNM00175/175).
[0066] The 3.5 fold increase in expression in 42% of the individual
ovarian cancer samples and no compatible expression in other
non-gynecologic cancers is indicative of Ovr114 being a diagnostic
marker for detection of ovarian cancer cells. It is believed that
the Ovr114 marker may also be useful in detection of additional
gynecologic cancers.
Measurement of Ovr115; Clone ID1283171; Gene ID 332459 (SEQ ID NO:2
or 14)
[0067] The numbers depicted in Table 6 are relative levels of
expression Ovr115 compared to their respective calibrators. The
numbers are relative levels of expression in 12 normal tissues of
ovaries compared to Testis (calibrator). These RNA samples were
obtained commercially and were generated by pooling samples from a
particular tissue from different individuals.
TABLE-US-00006 TABLE 6 Relative Levels of Ovr115 Expression in
Pooled Samples Tissue Normal Colon 858.10 Endometrium 12.34 Kidney
3.76 Liver 0.00 Ovary 0.43 Pancreas 0.00 Prostate 8.91 Small
Intestine 62.25 Spleen 0.00 Stomach 37.53 Testis 1.00 Uterus
47.67
[0068] The relative levels of expression in Table 6 show that
Ovr115 mRNA expression is detected in all the 12 normal tissue
pools analyzed.
[0069] The tissues shown in Table 6 are pooled samples from
different individuals. The tissues shown in Table 7 were obtained
from individuals and are not pooled. Hence the values for mRNA
expression levels shown in Table 6 cannot be directly compared to
the values shown in Table 7.
[0070] The numbers depicted in Table 7 are relative levels of
expression of Ovr115 compared to testis (calibrator), in 46 pairs
of matching samples and 27 unmatched tissue samples. Each matching
pair contains the cancer sample for a particular tissue and the
normal adjacent tissue sample for that same tissue from the same
individual. In cancers (for example, ovary) where it was not
possible to obtain normal adjacent samples from the same
individual, samples from a different normal individual were
analyzed.
TABLE-US-00007 TABLE 7 Relative Levels of Ovr115 Expression in
Individual Samples Normal & Matching Borderline Normal Tissue
Sample ID Cancer Type Cancer Malignant Adjacent Ovary 1
Ovr1037O/1038O Papillary serous 193.34 0.24 adenocarcinoma, G3
Ovary 3 OvrG021SPI/SN2 Papillary serous 0.38 0.31 adenocarcinoma
Ovary 4 OvrG010SP/SN Papillary serous 231.25 0.45 adenocarcinoma
Ovary 2 OvrA084/A086 Mucinous tumor, 143.34 16.65 grade G-B,
borderline Ovary 5 OvrA081F/A082D Mucinous tumor, low 314.13 0
malignant potential Ovary 19 Ovr14604A1C Serous 299.87
cystadenofibroma, low malignancy Ovary 26 Ovr14638A1C Follicular
cysts, 1278.32 low malignant potential Ovary 6 Ovr1040O Papillary
serous 144.25 adenocarcinoma, G2 Ovary 22 Ovr9410C360 Endometrioid
0.29 adenocarcinoma Ovary 23 Ovr1305X Papillary serous 157.41
adenocarcinoma Ovary 27 Ovr773O Papillary serous 340.04
adenocarcinoma Ovary 28 Ovr988Z Papillary serous 464.75
adenocarcinoma Ovary 7 Ovr1157O Papillary serous 432.07
adenocarcinoma Ovary 8 Ovr1005O Papillary serous 74.23
endometricarcinoma Ovary 9 Ovr1028O Ovarian carcinoma 1408.79 Ovary
10 Ovr14603A1D Adenocarcinoma 0.00 Ovary 11 Ovr9702C018GA Normal
Cystic 0.16 Ovary 12 Ovr2061 Normal left 0.00 atrophic, small
cystic Ovary 13 Ovr9702C020GA Normal-multiple 0.00 ovarian cysts
Ovary 14 Ovr9702C025GA Normal-hemorrhage CL 0.00 cysts Ovary 15
Ovr9701C050GB Normal-multiple 0.91 ovarian cysts Ovary 16
Ovr9701C087RA Normal-small 0.00 follicle cysts Ovary 17
Ovr9702C032RA 0.28 Ovary 18 Ovr9701C109RA Normal 0.00 Ovary 20
Ovr9411C057R Benign large 38.87 endometriotic cyst Ovary 21
Ovr9701C179a Normal 0.08 Ovary 24 Ovr1461O Serous 0.00
cystadenofibroma, no malignancy Ovary 25 Ovr9701C035GA Normal 0.00
Ovary 29 Ovr9702C007RA Normal 0.00 Ovary 30 Ovr9701C087RA
Normal-small 0.00 follicle cysts Ovary 31 Ovr9411C109 Normal 0.00
Ovary 32 Ovr9701C177a Normal-cystic 0.00 follicles Uterus 1
Utr850U/851U Stage 1 endometrial 39.95 13.60 cancer/NAT Uterus 2
Utr233U96/234U96 Adenocarcinoma/NAT 140.37 22.67 Uterus 3
Utr1359O/1358) Tumor/NAT 16.45 32.50 Uterus 4 Utr1417O/1418O
Malignant tumor/NAT 288.52 5.29 Endometrium 1 End14863A1A/A2A
Moderately differ. 2.61 6.24 Endome. carcinoma/NAT Endometrium 2
End9709C056A/55A Endometrial 2.10 49.40 adenocarcinoma/NAT
Endometrium 3 End9704C281A/2A Endometrial 480.77 19.22
adenocarcinoma/NAT Endometrium 4 End9705A125A/6A Endometrial 322.07
31.08 adenocarcinoma/NAT Lung 1 Lng750C/751C Metastatic 38.81 7.36
osteogenic sarcoma/NAT Lung 2 Lng8890A/8890B Cancer/NAT 690.12
14.71 Lung 3 Lng9502C109R/10R 1756.90 2.86 Skin 1 Skn2S9821248A/B
Secondary malignant 10.56 0.00 melanoma Skin 2 Skn4005287A1/B2
331.30 47.23 Prostate 1 Pro1012B/1013B Adenocarcinoma/NAT 14.64
4.39 Prostate 2 Pro1094B/1095B 0.09 2.54 Bladder 1 Bld665T/664T
404.56 90.20 Bladder 2 Bld327K/328K Papillary 77.35 177.37
transitional cell carcinoma/NAT Kidney 1 Kid4003710C/F 0.17 12.72
Kidney 2 Kid1242D/1243D 0.00 13.74 Mammary Mam1620F/1621F 0.27 0.12
Gland 1 Mammary Mam4003259a/g 5.71 0.00 Gland 2 Liver 1
Liv1747/1743 Hepatocellular 0.14 0.69 carcinoma/NAT Liver 2
LivVNM00175/175 Cancer/NAT 0.00 0.00 Small Int. 1 SmI9802H008/009
128.44 151.38 Stomach 1 Sto4004864A4/B4 Adenocarcinoma/NAT 303.01
116.72 Stomach 2 StoS9822539A/B Adenocarcinoma/NAT 24.12 17.76
Stomach 3 StoS99728A/C Malignant 0.00 9.10 gastrointestinal stromal
tumor Pancreas 1 Pan776p/777p Tumor/NAT 0.00 0.43 Pancreas 2
Pan824p/825p Cystic adenoma 0.00 3.17 Testis 1 Tst239X/240X
Tumor/NAT 24.05 1.37 Colon 1 Cln9706c068ra/69ra Adenocarcinoma/NAT
605.60 169.77 Colon 2 Cln4004732A7/B6 Adenocarcinoma/NAT 367.20
281.32 Colon 3 Cln4004695A9/B8 316.15 295.77 Colon 4
Cln9612B006/005 Asc. Colon. Cecum, 820.89 543.52 adenocarcinoma
Colon 5 Cln9704C024R/25R Adenocarcinoma/NAT 161.18 150.07 Cervix 1
CvxVNM00083/83 Keratinizing 738.17 1195.88 squamous cell carcinoma
Cervix 2 CvxIND00023D/N Large cell 1473.04 1229.80 nonkeratinizing
carcinoma Cervix 3 CvxIND00024D/N Large cell 2877.48 1275.02
nonkeratinizing carcinoma
Table 6 and Table 7 represent a combined total of 129 samples in 17
human tissue types. Comparisons of the level of mRNA expression in
ovarian cancer samples and the normal adjacent tissue from the same
individuals or normal tissues from other individuals are shown in
Table 7. Ovr115 was expressed at higher levels in 9 of 12 cancer
tissues (75%), relative to the maximum level detected in all 21
normal or normal adjacent ovarian samples. All 4 of 4 (100%)
ovarian tumors with borderline malignancy had elevated Ovr115
expression. The median expression in ovarian cancers (including the
ones with borderline malignancy) was 212.30 while the median
expression in normal ovaries was 0. When compared with their own
normal adjacent tissue samples, expression levels of Ovr115 were
also elevated in 3 of 3 (100%) lung cancers, 3 of 4 (75%) uterus
cancers and 2 of 4 (50%) endometrial cancers.
[0071] The relatively high expression levels of Ovr115 in ovarian
and other selected cancer samples is indicative of Ovr115 being a
diagnostic marker for detection of ovarian, lung, uterine and
endometrial cancer.
[0072] A homolog of Ovr115 has also been identified in public data
base; g2597613 as gi|2507612|gb|U75329.1|HSU75329 Human serine
protease mRNA, complete CDS. This homolog is depicted herein as SEQ
ID NO:9. It is believed that SEQ ID NO:9 or the protein encoded
thereby (SEQ ID NO:15) may also be useful as a diagnostic marker
for detection of ovarian, lung, uterine and endometrial cancer in
human patients.
Sequence CWU 1
1
1612587DNAHomo sapien 1ggaaggcagc gggcagctcc actcagccag tacccagata
cgctgggaac cttccccagc 60catggcttcc ctggggcaga tcctcttctg gagcataatt
agcatcatca ttattctggc 120tggagcaatt gcactcatca ttggctttgg
tatttcaggg agacactcca tcacagtcac 180tactgtcgcc tcagctggga
acattgggga ggatggaatc ctgagctgca cttttgaacc 240tgacatcaaa
ctttctgata tcgtgataca atggctgaag gaaggtgttt taggcttggt
300ccatgagttc aaagaaggca aagatgagct gtcggagcag gatgaaatgt
tcagaggccg 360gacagcagtg tttgctgatc aagtgatagt tggcaatgcc
tctttgcggc tgaaaaacgt 420gcaactcaca gatgctggca cctacaaatg
ttatatcatc acttctaaag gcaaggggaa 480tgctaacctt gagtataaaa
ctggagcctt cagcatgccg gaagtgaatg tggactataa 540tgccagctca
gagaccttgc ggtgtgaggc tccccgatgg ttcccccagc ccacagtggt
600ctgggcatcc caagttgacc agggagccaa cttctcggaa gtctccaata
ccagctttga 660gctgaactct gagaatgtga ccatgaaggt tgtgtctgtg
ctctacaatg ttacgatcaa 720caacacatac tcctgtatga ttgaaaatga
cattgccaaa gcaacagggg atatcaaagt 780gacagaatcg gagatcaaaa
ggcggagtca cctacagctg ctaaactcaa aggcttctct 840gtgtgtctct
tctttctttg ccatcagctg ggcacttctg cctctcagcc cttacctgat
900gctaaaataa tgtgccttgg ccacaaaaaa gcatgcaaag tcattgttac
aacagggatc 960tacagaacta tttcaccacc agatatgacc tagttttata
tttctgggag gaaatgaatt 1020catatctaga agtctggagt gagcaaacaa
gagcaagaaa caaaaagaag ccaaaagcag 1080aaggctccaa tatgaacaag
ataaatctat cttcaaagac atattagaag ttgggaaaat 1140aattcatgtg
aactagacaa gtgtgttaag agtgataagt aaaatgcacg tggagacaag
1200tgcatcccca gatctcaggg acctccccct gcctgtcacc tggggagtga
gaggacagga 1260tagtgcatgt tctttgtctc tgaattttta gttatatgtg
ctgtaatgtt gctctgagga 1320agcccctgga aagtctatcc caacatatcc
acatcttata ttccacaaat taagctgtag 1380tatgtaccct aagacgctgc
taattgactg ccacttcgca actcaggggc ggctgcattt 1440tagtaatggg
tcaaatgatt cactttttat gatgcttcca aaggtgcctt ggcttctctt
1500cccaactgac aaatgccaaa gttgagaaaa atgatcataa ttttagcata
aacagagcag 1560tcggcgacac cgattttata aataaactga gcaccttctt
tttaaacaaa caaatgcggg 1620tttatttctc agatgatgtt catccgtgaa
tggtccaggg aaggaccttt caccttgact 1680atatggcatt atgtcatcac
aagctctgag gcttctcctt tccatcctgc gtggacagct 1740aagacctcag
ttttcaatag catctagagc agtgggactc agctggggtg atttcgcccc
1800ccatctccgg gggaatgtct gaagacaatt ttggttacct caatgaggga
gtggaggagg 1860atacagtgct actaccaact agtggataaa ggccagggat
gctgctcaac ctcctaccat 1920gtacaggacg tctccccatt acaactaccc
aatccgaagt gtcaactgtg tcaggactaa 1980gaaaccctgg ttttgagtag
aaaagggcct ggaaagaggg gagccaacaa atctgtctgc 2040ttctcacatt
agtcattggc aaataagcat tctgtctctt tggctgctgc ctcagcacag
2100agagccagaa ctctatcggg caccaggata acatctctca gtgaacagag
ttgacaaggc 2160ctatgggaaa tgcctgatgg gattatcttc agcttgttga
gcttctaagt ttctttccct 2220tcattctacc ctgcaagcca agttctgtaa
gagaaatgcc tgagttctag ctcaggtttt 2280cttactctga atttagatct
ccagaccctt cctggccaca attcaaatta aggcaacaaa 2340catatacctt
ccatgaagca cacacagact tttgaaagca aggacaatga ctgcttgaat
2400tgaggccttg aggaatgaag ctttgaagga aaagaatact ttgtttccag
cccccttccc 2460acactcttca tgtgttaacc actgccttcc tggaccttgg
agccacggtg actgtattac 2520atgttgttat agaaaactga ttttagagtt
ctgatcgttc aagagaatga ttaaatatac 2580atttcct 258722070DNAHomo
sapien 2cacagagaga ggcagcagct tgctcagcgg acaaggatgc tgggcgtgag
ggaccaaggc 60ctgccctgca ctcgggcctc ctccagccag tgctgaccag ggacttctga
cctgctggcc 120agccaggacc tgtgtgggga ggccctcctg ctgccttggg
gtgacaatct cagctccagg 180ctacagggag accgggagga tcacagagcc
agcatgttac aggatcctga cagtgatcaa 240cctctgaaca gcctcgatgt
caaacccctg cgcaaacccc gtatccccat ggagaccttc 300agaaaggtgg
ggatccccat catcatagca ctactgagcc tggcgagtat catcattgtg
360gttgtcctca tcaaggtgat tctggataaa tactacttcc tctgcgggca
gcctctccac 420ttcatcccga ggaagcagct gtgtgacgga gagctggact
gtcccttggg ggaggacgag 480gagcactgtg tcaagagctt ccccgaaggg
cctgcagtgg cagtccgcct ctccaaggac 540cgatccacac tgcaggtgct
ggactcggcc acagggaact ggttctctgc ctgtttcgac 600aacttcacag
aagctctcgc tgagacagcc tgtaggcaga tgggctacag cagcaaaccc
660actttcagag ctgtggagat tggcccagac caggatctgg atgttgttga
aatcacagaa 720aacagccagg agcttcgcat gcggaactca agtgggccct
gtctctcagg ctccctggtc 780tccctgcact gtcttgcctg tgggaagagc
ctgaagaccc cccgtgtggt gggtggggag 840gaggcctctg tggattcttg
gccttggcag gtcagcatcc agtacgacaa acagcacgtc 900tgtggaggga
gcatcctgga cccccactgg gtcctcacgg gcagcccact gcttcaggaa
960acataccgat gtgttcaact ggaaggtgcg ggcaggctca gacaaactgg
gcagcttccc 1020atccctggct gtggccaaga tcatcatcat tgaattcaac
cccatgtacc ccaaagacaa 1080tgacatcgcc ctcatgaagc tgcagttccc
actcactttc tcaggcacag tcaggcccat 1140ctgtctgccc ttctttgatg
aggagctcac tccagccacc ccactctgga tcattggatg 1200gggctttacg
aagcagaatg gagggaagat gtctgacata ctgctgcagg cgtcagtcca
1260ggtcattgac agcacacggt gcaatgcaga cgatgcgtac cagggggaag
tcaccgagaa 1320gatgatgtgt gcaggcatcc cggaaggggg tgtggacacc
tgccagggtg acagtggtgg 1380gcccctgatg taccaatctg accagtggca
tgtggtgggc atcgttagct ggggctatgg 1440ctgcgggggc ccgagcaccc
caggagtata caccaaggtc tcagcctatc tcaactggat 1500ctacaatgtc
tggaaggctg agctgtaatg ctgctgcccc tttgcagtgc tgggagccgc
1560ttccttcctg ccctgcccac ctggggatcc cccaaagtca gacacagagc
aagagtcccc 1620ttgggtacac ccctctgccc acagcctcag catttcttgg
agcagcaaag ggcctcaatt 1680cctataagag accctcgcag cccagaggcg
cccagaggaa gtcagcagcc ctagctcggc 1740cacacttggt gctcccagca
tcccagggag agacacagcc cactgaacaa ggtctcaggg 1800gtattgctaa
gccaagaagg aactttccca cactactgaa tggaagcagg ctgtcttgta
1860aaagcccaga tcactgtggg ctggagagga gaaggaaagg gtctgcgcca
gccctgtccg 1920tcttcaccca tccccaagcc tactagagca agaaaccagt
tgtaatataa aatgcactgc 1980cctactgttg gtatgactac cgttacctac
tgttgcattg ttattacagc tatggccact 2040attattaaag agctgtgtaa
catctctggc 207031709DNAHomo sapien 3agcagactca caccagaact
acattccctg gccccctgcc tgtgtgcttc tggccaggcc 60ttggttggca agtctgaccc
gagaaaagga tctgcagaaa atcagactat gggatcactt 120tgtttgtgca
ttgggaatga cattctttcc caccccagga aaacctttgg gactttcaga
180gacattgtgg ctagccaacc acatggtcag cctcaaagtt gagaggctca
gtaaccctcc 240tatccctaga gaattccaaa gtgtggatgt aatttaacta
gaaagccatt ggtgactatc 300tgtgatcctc tggaagtatg ctatgttgtg
tatatcttgc atccaaagcc agagggaacc 360acaatgacta gtaaaacggt
ggtctcaatg cccacttagc ctctgcctct gaatttgacc 420atagtggcgt
tcagctgata gagcgggaag aagaaatatg cattttttat gaaaaaataa
480atatccaaga gaagatgaaa ctaaatggag aaattgaaat acatctactg
gaagaaaaga 540tccaattcct gaaaatgaag attgctgaga agcaaagaca
aatttgtgtg acccagaaat 600tactgccagc caagaggtcc ctggatgccg
acctagctgt gctccaaatt cagttttcac 660agtgtacaga cagaattaaa
gacctggaga aacagttcgt aaagcctgat ggtgagaata 720gagctcgctt
ccttccaggg aaagatctga ccgaaaaaga aatgatccaa aaattagaca
780agctggaact acaactggcc aagaaggagg agaagctgct ggagaaggat
ttcatctatg 840agcaggtctc caggctcaca gacaggctct gcagcaaaac
tcagggctgc aagcaggaca 900cactgctctt agccaagaag atgaatggct
atcaaagaag gatcaaaaat gcaactgaga 960aaatgatggc tcttgttgct
gagctgtcca tgaaacaagc cctaaccatt gaactccaaa 1020aggaagtcag
ggagaaagaa gacttcatct tcacttgcaa ttccaggata gaaaaaggtc
1080tgccactcaa taaggaaatt gagaaagaat ggttgaaagt ccttcgagat
gaagaaatgc 1140acgccttggc catcgctgaa aagtctcagg agttcttgga
agcagataat cgccagctgc 1200ccaatggtgt ttacacaact gcagagcagc
gtccgaatgc ctacatccca gaagcagatg 1260ccactcttcc tttgccaaaa
ccttatggtg ctttggctcc ttttaaaccc agtgaacctg 1320gagccaatat
gaggcacata aggaaacctg ttataaagcc agttgaaatc tgaatatgtg
1380aacaaatcca ggcctctcaa ggaaaagact tcaaccaggc ttccttgtac
ccacaggtga 1440aaaatgtgag cataatactt ctaatattat tgataagtaa
ggtaaccaca attagtcagc 1500aacagagtac aacagggttt ctatttaccc
accaactact atacctttca tgacgttgaa 1560tgggacatag aactgtccta
catttatgtc aaagtatata tttgaatcgc ttatattttc 1620tttttcactc
tttatattga gtacattcca gaaatttgta gtaggcaagg tgctataaaa
1680atgcactaaa aataaatctg ttctcaatg 17094257DNAHomo sapien
4ttaatgggta agtatttttt atatgcttta gctatagcta aagaaaactg atacttaaca
60aagttgaata gtattattca ctggtgctcc taaaatattg tttttcagtg taaaatatgc
120atatcttcta tatttaatat gaaagtcttg aaatgtatca gacagaaggg
gatttcagtt 180tgcaaataat gagcaatgta gcaattttaa cacatttcat
aaatatatat tttgtcattg 240gtggagagca ccatttg 2575359DNAHomo sapien
5gcctgagagc acttagcgtt catgagtgtc cccaccatgg cctggatgat gcttctcctc
60ggactccttg cttatggatc aggtcaggga gtggattctc agactgtggt gacccaagag
120ccatcgttat cagtgtcccc tggagggaca gtcacactca cttgtggctt
ggcctctgac 180tcagtctcta ctaatttctt ccccacctgg taccagcaga
ccccaggcca ggctccacgc 240acgctcatct acagcacaag cactcgctct
tctggggtcc ctgatcgttt ctctggctcc 300atccttggga acaaagctgc
cctcaccatt acgggggccc aggcagatga tgaatctga 35961372DNAHomo
sapienmisc_feature(6)..(6)n = a, c, g or t 6ccttanagnc ttggttgcca
aacagaatgc ccatatccgt cttacttgtg aggaagcttg 60ccttgggcgc cctctgctgg
ccctcctgaa gctaacaggg gcgagtgctc ggtggtttac 120aaattgcctc
catgcagact atgaaactgt tcagcctgct atagttagat ctctggcact
180ggcccaggag gtcttgcaga tttgcagatc aaggagaacc caggagtttc
aaagaagcgg 240ctagtaaagg tctctgagat ccttgcacta gctacatcct
cagggtagga ggaagatggc 300ttccagaagc atgcggctgc tcctattgct
gagctgcctg gccaaaacag gagtcctggg 360tgatatcatc atgagaccca
gctgtgctcc tgggatggtt ttaccacaag tccaattgct 420atggttactt
caggaagctg aggaactggt ctgatgccga gctcgagtgt cagtcttacg
480gaaacggagc ccacctggca tctatcctga gtttaaagga agccagcacc
atagcagagt 540acataagtgg ctatcagaga agccagccga tatggattgg
cctgcacgac ccacagaaga 600ggcagcagtg gcagtggatt gatggggcca
tgtatctgta cagatcctgg tctggcaagt 660ccatgggtgg gaacaagcac
tgtgctgaga tgagctccaa taacaacttt ttaacttgga 720gcagcaacga
atgcaacaag cgccaacact tcctgtgcaa gtaccgacca tagagcaaga
780atcaagattc tgctaactcc tgcacagccc cgtcctcttc ctttctgcta
gcctggctaa 840atctgctcat tatttcagag gggaaaccta gcaaactaag
agtgataagg gccctactac 900actggctttt ttaggcttag agacagaaac
tttagcattg gcccagtagt ggcttctagc 960tctaaatgtt tgccccgcca
tccctttcca cagtatcctt cttccctcct cccctgtctc 1020tggctgtctc
gagcagtcta gaagagtgca tctccagcct atgaaacagc tgggtctttg
1080gccataagaa gtaaagattt gaagacagaa ggaagaaact caggagtaag
cttctagccc 1140ccttcagctt ctacaccctt ctgccctctc tccattgcct
gcaccccacc ccagccactc 1200aactcctgct tgtttttcct ttggccatgg
gaaggtttac cagtagaatc cttgctaggt 1260tgatgtgggc catacattcc
tttaataaac cattgtgtac ataagaggtt gctgtgttcc 1320agttcagtaa
atggtgaatg tggaaaagtg aaataagacc aagaaataca aa 13727291DNAHomo
sapienmisc_feature(277)..(277)n= a, c, g, or t 7agaatggtag
tagtaagaag aagaaaaata gaggatctga atgtattttg aaggtagagt 60ccactggact
tagagatgga ttgaatgtgg aagattaagg aaagggagaa atgaaagata
120gtcttaggtt tcatcttcag atgactgggt gaacagcagt gttctttgct
aagatgggga 180agactaggga aaagagccag ttctgtattg agcatattat
atttaagaca atcccatctg 240ggtccaaaga caatgttgat tttttttctt
agatacntgc cctttagacc t 29181275DNAHomo
sapienmisc_feature(410)..(410)n= a, c, g, or t 8attctagaac
atatgtataa gctaaaaaca gtattttact cagatcagta gttatcgtgt 60ctatcagcta
taaaaaaaat caactgccag ccaagaactt taaaacttta agctgtgtat
120tatagaaccg ttttgtgtag cattggaata ttgtccattt tgtaagtcat
tgtgaatgtt 180cttaattatc agcttgaagg tatttttgta ttaaaagttg
acattgaaga acctaagtgg 240atgatgggat ttggggccag tagtgaaagt
atgtttcctc taaaatattt ccctaaacag 300tggtatacat ggttatttta
ttatgagatt tgtatatgtt ctgtgtttct ctgtgaacaa 360tgtttcagtc
tctctgtcac catatgtaag gggaagtcca caaatatagn actacattgc
420acaaaactaa aattgttaat tacaagaaaa tataggtgct taccttttga
aggtttatta 480atacatatgg ttgtcacaat acgtatatat gataaatggt
gtacatatac agatgtttat 540ggtgtataaa tttttctata cccaattaga
attatcttcc tgattcttta ttcaataaca 600tgctaattcc tcttctatgt
tctatagtga cagaatgcta acttttctta taccctggca 660gaggacagag
gagtctggtc taggatgggg aactgaattt ttgaacgaaa aggaaagaga
720aaggatgnnn nnnnnnnnnn nnnnnnnnnn nnnnnntaat gtttcttagt
cattttgatt 780ggccatttga acagtctaca agtttaacgt tatttccagt
gaagtaggat ggctgaccta 840gcaatacatg tttcttcaaa agggtaaaca
tgctttagtg acctaaagct aaattttgta 900catttgacat caggggtgtt
ataagtactg cacttaatac aaagctattt ctcaatngtg 960ttatttttga
gacaaatttt tcttcaccat taacttcttg ttggtagctt tttgttttgt
1020aaaaattgag agatggcaat gcttatctca accagattat ccatctgcag
aattaaggta 1080tgcaactggt aaataaaaga caaatgctcc agtttgtctt
tctcaacctt tgagttctta 1140acctttgagt taaaacctag tctaaatagt
gggaatgtct tggtttacag taaggttttc 1200ttgggaagga tcttggtttt
gtgatctatt tgtgaattaa ggagtagatg ttaaccatta 1260ttttatagat aagtg
127592479DNAHomo sapien 9gtcatattga acattccaga tacctatcat
tactcgatgc tgttgataac agcaagatgg 60ctttgaactc agggtcacca ccagctattg
gaccttacta tgaaaaccat ggataccaac 120cggaaaaccc ctatcccgca
cagcccactg tggtccccac tgtctacgag gtgcatccgg 180ctcagtacta
cccgtccccc gtgccccagt acgccccgag ggtcctgacg caggcttcca
240accccgtcgt ctgcacgcag cccaaatccc catccgggac agtgtgcacc
tcaaagacta 300agaaagcact gtgcatcacc ttgaccctgg ggaccttcct
cgtgggagct gcgctggccg 360ctggcctact ctggaagttc atgggcagca
agtgctccaa ctctgggata gagtgcgact 420cctcaggtac ctgcatcaac
ccctctaact ggtgtgatgg cgtgtcacac tgccccggcg 480gggaggacga
gaatcggtgt gttcgcctct acggaccaaa cttcatcctt cagatgtact
540catctcagag gaagtcctgg caccctgtgt gccaagacga ctggaacgag
aactacgggc 600gggcggcctg cagggacatg ggctataaga ataattttta
ctctagccaa ggaatagtgg 660atgacagcgg atccaccagc tttatgaaac
tgaacacaag tgccggcaat gtcgatatct 720ataaaaaact gtaccacagt
gatgcctgtt cttcaaaagc agtggtttct ttacgctgtt 780tagcctgcgg
ggtcaacttg aactcaagcc gccagagcag gatcgtgggc ggtgagagcg
840cgctcccggg ggcctggccc tggcaggtca gcctgcacgt ccagaacgtc
cacgtgtgcg 900gaggctccat catcaccccc gagtggatcg tgacagccgc
ccactgcgtg gaaaaacctc 960ttaacaatcc atggcattgg acggcatttg
cggggatttt gagacaatct ttcatgttct 1020atggagccgg ataccaagta
caaaaagtga tttctcatcc aaattatgac tccaagacca 1080agaacaatga
cattgcgctg atgaagctgc agaagcctct gactttcaac gacctagtga
1140aaccagtgtg tctgcccaac ccaggcatga tgctgcagcc agaacagctc
tgctggattt 1200ccgggtgggg ggccaccgag gagaaaggga agacctcaga
agtgctgaac gctgccaagg 1260tgcttctcat tgagacacag agatgcaaca
gcagatatgt ctatgacaac ctgatcacac 1320cagccatgat ctgtgccggc
ttcctgcagg ggaacgtcga ttcttgccag ggtgacagtg 1380gagggcctct
ggtcacttcg aacaacaata tctggtggct gataggggat acaagctggg
1440gttctggctg tgccaaagct tacagaccag gagtgtacgg gaatgtgatg
gtattcacgg 1500actggattta tcgacaaatg aaggcaaacg gctaatccac
atggtcttcg tccttgacgt 1560cgttttacaa gaaaacaatg gggctggttt
tgcttccccg tgcatgattt actcttagag 1620atgattcaga ggtcacttca
tttttattaa acagtgaact tgtctggctt tggcactctc 1680tgccatactg
tgcaggctgc agtggctccc ctgcccagcc tgctctccct aaccccttgt
1740ccgcaagggg tgatggccgg ctggttgtgg gcactggcgg tcaattgtgg
aaggaagagg 1800gttggaggct gcccccattg agatcttcct gctgagtcct
ttccaggggc caattttgga 1860tgagcatgga gctgtcactt ctcagctgct
ggatgacttg agatgaaaaa ggagagacat 1920ggaaagggag acagccaggt
ggcacctgca gcggctgccc tctggggcca cttggtagtg 1980tccccagcct
acttcacaag gggattttgc tgatgggttc ttagagcctt agcagccctg
2040gatggtggcc agaaataaag ggaccagccc ttcatgggtg gtgacgtggt
agtcacttgt 2100aaggggaaca gaaacatttt tgttcttatg gggtgagaat
atagacagtg cccttggtgc 2160gagggaagca attgaaaagg aacttgccct
gagcactcct ggtgcaggtc tccacctgca 2220cattgggtgg ggctcctggg
agggagactc agccttcctc ctcatcctcc ctgaccctgc 2280tcctagcacc
ctggagagtg aatgcccctt ggtccctggc agggcgccaa gtttggcacc
2340atgtcggcct cttcaggcct gatagtcatt ggaaattgag gtccatgggg
gaaatcaagg 2400atgctcagtt taaggtacac tgtttccatg ttatgtttct
acacattgat ggtggtgacc 2460ctgagttcaa agccatctt 247910576DNAHomo
sapien 10ttcaaagaca tattagaagt tgggaaaata attcatgtga actagacaag
tgtgttaaga 60gtgataagta aaatgcacgt ggagacaagt gcatccccag atctcaggga
cctccccctg 120cctgtcacct ggggagtgag aggacaggat agtgcatgtt
ctttgtctct gaatttttag 180ttatatgtgc tgtaatgttg ctctgaggaa
gcccctggaa agtctatccc aacatatcca 240catcttatat tccacaaatt
aagctgtagt atgtacccta agacgctgct aattgactgc 300cacttcgcaa
ctcaggggcg gctgcatttt agtaatgggt caaatgattc actttttatg
360atgcttccaa aggtgccttg gcttctcttc ccaactgaca aatgccaaag
ttgagaaaaa 420tgatcataat tttagcataa acagagcagt cggcgacacc
gattttataa ataaactgag 480caccttcttt ttaaacaaac aaatgcgggt
ttatttctca gatgatgttc atccgtgaat 540ggtccaggga aggacctttc
accttgacta tatggc 57611890DNAHomo sapien 11caagctctga ggcttctcct
ttccatcctg cgtggacagc taagacctca gttttcaata 60gcatctagag cagtgggact
cagctggggt gatttcgccc cccatctccg ggggaatgtc 120tgaagacaat
tttggttacc tcaatgaggg agtggaggag gatacagtgc tactaccaac
180tagtggataa aggccaggga tgctgctcaa cctcctacca tgtacaggga
cgtctcccca 240ttacaactac ccaatccgaa gtgtcaactg tgtcaggact
aagaaaccct ggttttgagt 300agaaaagggc ctggaaagag gggagccaac
aaatctgtct gcttcctcac attagtcatt 360ggcaaataag cattctgtct
ctttggctgc tgcctcagca cagagagcca gaactctatc 420gggcaccagg
ataacatctc tcagtgaaca gagttgacaa ggcctatggg aaatgcctga
480tgggattatc ttcagcttgt tgagcttcta agtttctttc ccttcattct
accctgcaag 540ccaagttctg taagagaaat gcctgagttc tagctcaggt
tttcttactc tgaatttaga 600tctccagacc cttcctggcc acaattcaaa
ttaaggcaac aaacatatac cttccatgaa 660gcacacacag acttttgaaa
gcaaggacaa tgactgcttg aattgaggcc ttgaggaatg 720aagctttgaa
ggaaaagaat actttgtttc cagccccctt cccacactct tcatgtgtta
780accactgcct tcctggacct tggagccacg gtgactgtat tacatgttgt
tatagaaaac 840tgattttaga gttctgatcg ttcaagagaa tgattaaata
tacatttcct 89012406DNAHomo sapienmisc_feature(30)..(30)n= a, c, g,
or t 12gtgaatgtgg actataatgc cagctcagan accttgcggt gtgaggctcc
ccgatggttc 60ccccagccca cagtggtctg ggcatcccaa gttgaccagg gagccaactt
ctcggaagtc 120tccaatacca gctttgagct gaactctgag aatgtgacca
tgaaggttgt gtctgtgctc 180tacaatgtta cgatcaacaa cacatactcc
tgtatgattg aaaatgacat tgccaaagca 240acaggggnta tcaaagtgac
agaatcggag atcaaaaggc ggagtcacct acagctgcta 300aactcaaagg
cttctctgtg tgtctcttct ttctttgcca tcagctgggc acttctgcct
360ctcagccctt acctgatgct aanataatgt gccttggcca caaaaa
40613462DNAHomo sapien 13ggaaggcagc ggcagctcca ctcagccagt
acccagatac gctgggaacc ttccccagcc
60atggcttccc tggggcagat cctcttctgg agcataatta gcatcatcat tattctggct
120ggagcaattg cactcatcat tggctttggt atttcaggga gacactccat
cacagtcact 180actgtcgcct cagctgggaa cattggggag gatggaatcc
tgagctgcac ttttgaacct 240gacatcaaac tttctgatat cgtgatacaa
tggctgaagg aaggtgtttt aggcttggtc 300catgagttca aagaaggcaa
agatgagctg tcggagcagg atgaaatgtt cagaggccgg 360acagcagtgt
ttgctgatca agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg
420caactcacag atgctggcac ctacaaatgt tatatcatca ct 46214272DNAHomo
sapien 14gcagcttgct cagcggacaa ggatgctggg cgtgagggac caaggcctgc
cctgcactcg 60ggcctcctcc agccagtgct gaccagggac ttctgacctg ctggccagcc
aggacctgtg 120tggggaggcc ctcctgctgc cttggggtga caatctcagc
tccaggctac agggagaccg 180ggaggatcac agagccagca tggatcctga
cagtgatcaa cctctgaaca gcctcgtcaa 240ggtgattctg gataaatact
acttcctctg cg 27215492PRTHomo sapien 15Met Ala Leu Asn Ser Gly Ser
Pro Pro Ala Ile Gly Pro Tyr Tyr Glu1 5 10 15Asn His Gly Tyr Gln Pro
Glu Asn Pro Tyr Pro Ala Gln Pro Thr Val 20 25 30Val Pro Thr Val Tyr
Glu Val His Pro Ala Gln Tyr Tyr Pro Ser Pro 35 40 45Val Pro Gln Tyr
Ala Pro Arg Val Leu Thr Gln Ala Ser Asn Pro Val 50 55 60Val Cys Thr
Gln Pro Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Lys65 70 75 80Thr
Lys Lys Ala Leu Cys Ile Thr Leu Thr Leu Gly Thr Phe Leu Val 85 90
95Gly Ala Ala Leu Ala Ala Gly Leu Leu Trp Lys Phe Met Gly Ser Lys
100 105 110Cys Ser Asn Ser Gly Ile Glu Cys Asp Ser Ser Gly Thr Cys
Ile Asn 115 120 125Pro Ser Asn Trp Cys Asp Gly Val Ser His Cys Pro
Gly Gly Glu Asp 130 135 140Glu Asn Arg Cys Val Arg Leu Tyr Gly Pro
Asn Phe Ile Leu Gln Met145 150 155 160Tyr Ser Ser Gln Arg Lys Ser
Trp His Pro Val Cys Gln Asp Asp Trp 165 170 175Asn Glu Asn Tyr Gly
Arg Ala Ala Cys Arg Asp Met Gly Tyr Lys Asn 180 185 190Asn Phe Tyr
Ser Ser Gln Gly Ile Val Asp Asp Ser Gly Ser Thr Ser 195 200 205Phe
Met Lys Leu Asn Thr Ser Ala Gly Asn Val Asp Ile Tyr Lys Lys 210 215
220Leu Tyr His Ser Asp Ala Cys Ser Ser Lys Ala Val Val Ser Leu
Arg225 230 235 240Cys Leu Ala Cys Gly Val Asn Leu Asn Ser Ser Arg
Gln Ser Arg Ile 245 250 255Val Gly Gly Glu Ser Ala Leu Pro Gly Ala
Trp Pro Trp Gln Val Ser 260 265 270Leu His Val Gln Asn Val His Val
Cys Gly Gly Ser Ile Ile Thr Pro 275 280 285Glu Trp Ile Val Thr Ala
Ala His Cys Val Glu Lys Pro Leu Asn Asn 290 295 300Pro Trp His Trp
Thr Ala Phe Ala Gly Ile Leu Arg Gln Ser Phe Met305 310 315 320Phe
Tyr Gly Ala Gly Tyr Gln Val Gln Lys Val Ile Ser His Pro Asn 325 330
335Tyr Asp Ser Lys Thr Lys Asn Asn Asp Ile Ala Leu Met Lys Leu Gln
340 345 350Lys Pro Leu Thr Phe Asn Asp Leu Val Lys Pro Val Cys Leu
Pro Asn 355 360 365Pro Gly Met Met Leu Gln Pro Glu Gln Leu Cys Trp
Ile Ser Gly Trp 370 375 380Gly Ala Thr Glu Glu Lys Gly Lys Thr Ser
Glu Val Leu Asn Ala Ala385 390 395 400Lys Val Leu Leu Ile Glu Thr
Gln Arg Cys Asn Ser Arg Tyr Val Tyr 405 410 415Asp Asn Leu Ile Thr
Pro Ala Met Ile Cys Ala Gly Phe Leu Gln Gly 420 425 430Asn Val Asp
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Thr Ser 435 440 445Asn
Asn Asn Ile Trp Trp Leu Ile Gly Asp Thr Ser Trp Gly Ser Gly 450 455
460Cys Ala Lys Ala Tyr Arg Pro Gly Val Tyr Gly Asn Val Met Val
Phe465 470 475 480Thr Asp Trp Ile Tyr Arg Gln Met Lys Ala Asn Gly
485 49016890DNAHomo sapienmisc_feature(168)..(237)n=a, c, g or t
16caagctctga ggcttctcct ttccatcctg cgtggacagc taagacctca gttttcaata
60gcatctagag cagtgggact cagctggggt gatttcgccc cccatctccg ggggaatgtc
120tgaagacaat tttggttacc tcaatgaggg agtggaggag gatacagnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnncat 240tacaactacc caatccgaag tgtcaactgt
gtcaggacta agaaaccctg gttttgagta 300gaaaagggcc tgggaaagag
gggagccaac aaatctgtct gcttcctcac attagtcatt 360ggcaaataag
cattctgtct ctttggctgc tgcctcagca cagagagcca gaactctatc
420gggcaccagg ataacatctc tcagtgaaca gagttgacaa ggcctatggg
aaatgcctga 480tgggattatc ttcagcttgt tgagcttcta agtttctttc
ccttcattct accctgcaag 540ccaagttctg taagagaaat gcctgagttc
tagctcaggt tttcttactc tgaatttaga 600tctccagacc ctgcctggcc
acaattcaaa ttaaggcaac aaacatatac cttccatgaa 660gcacacacag
acttttgaaa gcaaggacaa tgactgcttg aattgaggcc ttgaggaatg
720aagctttgaa ggaaaagaat actttgtttc cagccccctt cccacactct
tcatgtgtta 780accactgcct tcctggacct tggagccacg gtgactgtat
tacatgttgt tatagaaaac 840tgattttaga gttctgatcg ttcaagagaa
tgattaaata tacatttcct 890
* * * * *