Note: Descriptions are shown in the official language in which they were submitted.
WO 99/60162 PCT/US99/10548
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A NOVEL METHOD OF DIAGNOSING,
MONITORING, AND STAGING PROSTATE CANCER
FIELD OF THE INVENTION
This invention relates, in part, to newly developed
assays for detecting, diagnosing, monitoring, staging, and
prognosticating cancers, particularly prostate cancer.
BACKGROUND OF THE INVENTION
Cancer of the prostate is the most prevalent
malignancy in adult males, excluding skin cancer, and is an
increasingly prevalent health problem in the United States.
In 1996, it was estimated that in the United States, 41,400
deaths would result from this disease, indicating that
prostate cancer is second only to lung cancer as the most
common cause of death in the same population. If diagnosed
and treated early, when the cancer is still confined to the
prostate, the chance of cure is significantly higher.
Treatment decisions for an individual are linked to
the stage of prostate cancer present in that individual. A
common classification of the spread of prostate cancer was
developed by the American Urological Association (AUA?. The
AUA classification divides prostate tumors into four stages,
A to D. Stage A, microscopic cancer within prostate, is
further subdivided into stages A1 and A2. Sub-stage A1 is a
well-differentiated cancer confined to one site within the
prostate. Treatment is generally observation, radical
prostatectomy, or radiation. Sub-stage A2 is a moderately to
poorly differentiated cancer at multiple sites within the
prostate. Treatment is radical prostatectomy or radiation.
Stage B, palpable lump within the prostate, is further
subdivided into stages B1 and B2. In sub-stage B1, the cancer
forms a small nodule in one lobe of the prostate. In sub-
stage B2, the cancer forms large or multiple nodules, or
occurs in both lobes of the prostate. Treatment for both sub-
stages B1 and B2 is either radical prostatectomy or radiation.
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Stage C is a large cancer mass involving most or all of the
prostate and is further subdivided into two stages. In sub-
stage Cl, the cancer forms a continuous mass that may have
extended beyond the prostate. In sub-stage C2, the cancer
forms a continuous mass that invades the surrounding tissue.
Treatment for both these sub-stages is radiation with or
without drugs. The fourth stage is metastatic cancer and is
also subdivided into two stages. In sub-stage D1, the cancer
appears in the lymph nodes of the pelvis. In sub-stage D2,
the cancer involves tissues beyond lymph nodes. Treatment for
both these sub-stages is systemic drugs to address the cancer
as well as pain.
However, current prostate cancer staging methods are
limited. As many as 50% of prostate cancers initially staged
as A2, B, or C are actually stage D, metastatic. Discovery
of metastasis is significant because patients with metastatic
cancers have a poorer prognosis and require significantly
different therapy than those with localized cancers. The five
year survival rates for patients with localized and metastatic
prostate cancers are 93o and 29%, respectively.
Accordingly, there is a great need for increasingly
sensitive methods for the staging of a cancer in a human to
determine whether or not such cancer has metastasized and for
monitoring the progress of a cancer in a human.
In the present invention, methods are provided for
detecting, diagnosing, monitoring, staging and prognosticating
cancers, particularly prostate cancer via seven (7) Prostate
Specific Genes (PSG). The seven PSGs 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 or 7. In the alternative, what is meant by the seven
PSGs as used herein, means the native mRNAs encoded by the
genes comprising any of the polynucleotide sequences of SEQ
ID N0: 1, 2, 3, 4, 5, 6 or 7 or levels of the genes comprising
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any of the polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4,
5, 6 or 7.
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.
SZJI~IAIEtY 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 prostate cancer in a patient which comprises
measuring levels of PSG in a sample of cells, tissue or bodily
fluid from the patient and comparing the measured levels of
PSG with levels of PSG in preferably the same cells, tissue,
or bodily fluid type of a control, wherein an increase in the
measured PSG levels in the patient versus levels of PSG in the
control is associated with prostate cancer.
Another object of the present invention is to
provide a method of diagnosing metastatic prostate cancer in
a patient which comprises measuring PSG levels in a sample of
cells, tissue, or bodily fluid from the patient and comparing
the measured PSG levels with levels of PSG in preferably the
same cells, tissue, or bodily fluid type of a control, wherein
an increase in measured PSG levels in the patient versus
levels of PSG in the control is associated with a cancer which
has metastasized.
Another object of the present invention is to provide
a method of staging prostate cancer in a patient which
comprises identifying a patient having prostate cancer,
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measuring levels of PSG in a sample of cells, tissues, or
bodily fluid obtained from the patient, and comparing the
measured PSG levels with levels of PSG in preferably the same
cells, tissue or bodily fluid type of a control. An increase
in measured PSG levels in the patient versus PSG levels in the
control can be associated with a cancer which is progressing
while a decrease or equivalent level of PSG measured in the
patient versus the control can be associated with a cancer
which is regressing or in remission. '
Another object of the present invention is to provide
a method of monitoring prostate cancer in a patient for the
onset of metastasis. The method comprises identifying a
patient having prostate cancer that is not known to have
metastasized, periodically measuring levels of PSG in a sample
of cells, tissues, or bodily fluid obtained from the patient,
and comparing the measured PSG levels with levels of PSG in
preferably the same cells, tissue, or bodily fluid type of a
control, wherein an increase in measured PSG levels versus
control PSG levels is associated with a cancer which has
metastasized.
Yet another object of the present invention is to
provide a method of monitoring the change in stage of prostate
cancer in a patient which comprises identifying a patient
having prostate cancer, periodically measuring levels of PSG
in a sample of cells, tissue, or bodily fluid obtained from
the patient, and comparing the measured PSG levels with levels
of PSG in preferably the same cells, tissues, or bodily fluid
type of a control wherein an increase in measured PSG levels
versus the control PSG levels is associated with a cancer
which is progressing and a decrease in the measured PSG levels
versus the control PSG levels is associated with a cancer
which is regressing or in remission.
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
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understood, however, that the following description and the
specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only. Various
changes and modifications within the spirit and scope of the
disclosed invention will become readily apparent to those
skilled in the art from reading the following description and
from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The present invention relates to diagnostic assays
and methods, both quantitative and qualitative for detecting,
diagnosing, monitoring, staging, and prognosticating cancers
by comparing levels of PSG measured in a patient with levels
of PSG in a control. What is meant by "levels of PSG" as used
herein, means 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 or 7. In the alternative, what is meant
by "levels of PSG" as used herein, is levels of the native
mRNA encoded by the gene comprising any of the polynucleotide
sequence of SEQ ID N0: 1, 2, 3, 4, 5, 6 or 7 or levels of the
gene comprising any of the polynucleotide sequence of SEQ ID
N0: 1, 2, 3, 4, S, 6 or 7. Such levels are preferably
measured in at least one of cells, tissues and/or bodily
fluids, and includes determination of both normal and abnormal
levels of PSGs. Thus, for instance, a diagnostic assay in
accordance with the invention for diagnosing overexpression
of PSG protein compared to control bodily fluids, cells, or
tissue samples may be used to diagnose the presence of
cancers, including prostate cancer. Any of the seven PSGs may
be measured alone in the methods of the invention, all
together or in various combinations of the seven PSGs.
By "control" it is meant a human patient without
cancer-and/or non cancerous samples from the patient, also
referred to herein as a normal human control; in the methods
for diagnosing or monitoring for metastasis, control may also
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include samples from a human patient that is determined by
reliable methods to have prostate cancer which has not
metastasized.
All the methods of the present invention may
optionally include measuring the levels of other cancer
markers as well as PSG. Other cancer markers, in addition to
PSG, useful in the present invention will depend on the cancer
being tested and are known to those of skill in the art. For
example, simultaneous testing for increases in PSA as well as
increases in PSG are also within the scope of the present
invention and believed to provide a higher level of assurance
that such cancer being tested is metastatic or the onset of
metastasis has occurred.
Diagnostic Assays
The present invention provides methods for diagnosing
the presence of prostate cancer by analyzing for changes in
levels of PSG in cells, tissues or bodily fluids compared with
levels of PSG in cells, tissues or bodily fluids of preferably
the same type from a normal human control, wherein an increase
in levels of PSG in the patient versus the normal human
control is associated with the presence of prostate cancer.
Without limiting the instant invention, typically, for a
quantitative diagnostic assay a positive result indicating the
patient being tested has cancer is one in which cells,
tissues, or bodily fluid levels of the cancer marker, such as
PSG, 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.
The present invention also provides a method of
diagnosing metastatic prostate cancer in a patient having
prostate 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 prostate cancer which
may have metastasized (but which was not previously known to
have metastasized) is identified. This is accomplished by a
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variety of means known to those of skill in the art. For
example, in the case of prostate cancer, patients are
typically diagnosed with prostate cancer following traditional
detection methods,
In the present invention, determining the presence
of PSG in cells, tissues, or bodily fluid, is particularly
useful for discriminating between prostate cancer which has
not metastasized and prostate cancer which has metastasized.
Existing techniques have difficulty discriminating
between prostate cancer which has metastasized and prostate
cancer which has not metastasized and proper treatment
selection is often dependent upon such knowledge.
In the present invention, the cancer marker levels
measured in such cells, tissue, or bodily fluid are PSGs, and
are compared with levels of PSG in preferably the same cells,
tissue, or bodily fluid type of a normal human control. That
is, if the cancer marker being observed is just PSG in serum,
this level is preferably compared with the level of PSG in
serum of a normal human patient. An increase in the PSG in
the patient versus the normal human control is associated with
prostate cancer which has metastasized.
Without limiting the instant invention, typically,
for a quantitative diagnostic assay a positive result
indicating the cancer in the patient being tested or monitored
has metastasized is one in which cells, tissues, or bodily
fluid levels of the cancer marker, such as PSG, are at least
two times higher, and most preferable are at least five times
higher, than in preferably the same cells, tissues, or bodily
fluid of a normal patient.
Staging
The invention also provides a method of staging
prostate cancer in a human patient.
The method comprises identifying a human patient
having such cancer and analyzing a sample of cells, tissues,
or bodily fluid from such patient for PSG. Then, the method
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compares PSG levels in such cells, tissues, or bodily fluid
with levels of PSG in preferably the same cells, tissues, or
bodily fluid type of a normal human control sample, wherein
an increase in PSG levels in the patient versus the normal
human control is associated with a cancer which is progressing
and a decrease in the levels of PSG is associated with a
cancer which is regressing or in remission.
Monitoring
Further provided is a method of monitoring prostate
cancer in a human having such cancer for the onset of
metastasis. The method comprises identifying a human patient
having such cancer that is not known to have metastasized;
periodically analyzing a sample of cells, tissues, or bodily
fluid from such patient for PSG; and comparing the PSG levels
in such cells, tissue, or bodily fluid with levels of PSG in
preferably the same cells, tissues, or bodily fluid type of
a normal human control sample, wherein an increase in PSG
levels 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 prostate cancer in a human
having such cancer. The method comprises identifying a human
patient having such cancer; periodically analyzing a sample
of cells, tissue, or bodily fluid from such patient for PSG;
comparing the PSG levels in such cells, tissue, or bodily
fluid with levels of PSG in preferably the same patient.
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 TecImique,s
Assay techniques that can be used to determine levels
of gene expression, such as PSG of the present invention, in
a sample derived from a host are well-known to those of skill
in the art. Such assay methods include radioimmunoassays,
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reverse transcriptase PCR (RT-PCR) assays,
immunohistochemistry assays, in situ hybridization assays,
competitive-binding assays, Western Blot analyses and ELISA
assays. Among these, ELISAs are frequently preferred to
diagnose a gene's expressed protein in biological fluids. An
ELISA assay initially comprises preparing an antibody, if not
readily available from a commercial source, specific to PSG,
preferably a monoclonal antibody. In addition a reporter
antibody generally is prepared which binds specifically to
PSG. The reporter antibody is attached to a detectable
reagent such as radioactive, fluorescent or enzymatic reagent,
for example horseradish peroxidase enzyme or alkaline
phosphatase.
To carry out the ELISA, antibody specific to PSG 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 PSG binds
to the specific antibody attached to the polystyrene dish.
Unbound sample is washed out with buffer. A reporter antibody
specifically directed to PSG and linked to horseradish
peroxidase is placed in the dish resulting in binding of the
reporter antibody to any monoclonal antibody bound to PSG.
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 PSG
antibodies, produces a colored reaction product. The amount
of color developed in a given time period is proportional to
the amount of PSG protein present in the sample. Quantitative
results typically are obtained by reference to a standard
curve.
A competition assay may be employed wherein
antibodies specific to PSG attached to a solid support and
labeled PSG and a sample derived from the host are passed over
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the solid support and the amount of label detected attached
to the solid support can be correlated to a quantity of PSG
in the sample.
Nucleic acid methods may be used to detect PSG mRNA
as a marker for prostate cancer. Polymerase chain reaction
(PCR) and other nucleic acid methods, such as ligase chain
reaction (LCR) and nucleic acid sequence based amplification
(NASABA), can be used to detect malignant cells for diagnosis
and monitoring of various malignancies. For example, reverse
transcriptase PCR (RT-PCR) is a powerful technique which can
be used to detect the presence of a specific mRNA population
in a complex mixture of thousands of other mRNA species. In
RT-PCR, an mRNA species is first reverse transcribed to
complementary DNA (cDNA) with use of the enzyme reverse
transcriptase; the cDNA is then amplified as in a standard PCR
reaction. RT-PCR can thus reveal by amplification the
presence of a single species of mRNA. Accordingly, if the
mRNA is highly specific for the cell that produces it, RT-PCR
can be used to identify the presence of a specific type of
cell.
Hybridization to clones or oligonucleotides arrayed
on a solid support (i.e., gridding) can be used to both detect
the expression of and quantitate the level of expression of
that gene. In this approach, a cDNA encoding the PSG 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 PSG
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
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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.
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.
EXAME?~ES
The present invention is further described by the
following examples. These examples are provided solely to
illustrate the invention by reference to specific embodiments.
These exemplifications, while illustrating certain specific
aspects of the invention, do not portray the limitations or
circumscribe the scope of the disclosed invention.
EXAI~I~E 1: PSGs
Searches were carried out and PSGs identified using
the following Search Tools as part of the LIFESEQ~ database
available from Incyte Pharmaceuticals, Palo Alto, CA:
1. Library Comparison (compares one library to one
other library) allows the identification of clones expressed
in tumor and absent or expressed at a lower level in normal
tissue.
2. Subsetting is similar to library comparison but
allows the identification of clones expressed in a pool of
libraries and absent or expressed at a lower level in a second
pool of libraries.
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3. Transcript Imaging lists all of the clones in
a single library or a pool of libraries based on abundance.
Individual clones can then be examined using Electronic
Northerns to determine the tissue sources of their component
ESTs.
4. Protein Function: Incyte has identified subsets
of ESTs with a potential protein function based on homologies
to known proteins. Some examples in this database include
Transcription Factors and Proteases. Some leads were
identified by searching in this database for clones whose
component ESTs showed disease specificity.
Electronic subtractions, transcript imaging and
protein function searches were used to identify clones, whose
component ESTs were exclusively or more frequently found in
libraries from specific tumors. Individual candidate clones
were examined in detail by checking where each EST originated.
Table l:
SEQ ID Clone ID # Gene ID
NO: #
1 1550426 244673 Protein Function
(Transcription Factors)
2 1255804 14878 Subletting
3 1808432 255819 Subsetting
4 3930803 none Subsetting
5 645804 235032 Subletting
6 1862352 221558 Subletting
7 1450626 236019 Subletting
EXAMPLE 2: Measurement of SEQ ID NO: l; Clone ID # 1550426;
Gene ID #244673 (pro101)
The example is carried out using standard techniques,
which are well known and routine to those of skill in the art,
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except where otherwise described in detail. Routine molecular
biology techniques of the following example are 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) .
Relative Quantitation of Gene Expression
Real-time quantitative PCR with fluorescent Taqman
probes is a quantitative detection system utilizing the 5'
3' nuclease activity of Taq DNA polymerise. 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 polymerise
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, CA, USA).
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 are used
as the basis for comparative results (calibrator).
Quantitation relative to the "calibrator" is obtained using
the standard curve method or the comparative method (User
Bulletin #2: ABI PRISM 7700 Sequence Detection System).
To evaluate the tissue distribution, and the level
of pro101 (SEQ ID N0:1) in normal and tumor tissue, total RNA
was extracted from tumor and matched normal adjacent tissues
and from unmatched tumor and normal tissues. Subsequently,
first strand cDNA was prepared with reverse transcriptase and
the polymerise chain reaction carried out using primers and
Taqman probe specific to pro101 (SEQ ID N0:1). The results
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were obtained using the ABI PRISM 7700 Sequence Detector. The
absolute numbers are relative levels of expression of pro101
(SEQ ID NO: l) compared to the calibrator.
The absolute numbers are depicted in the following
Table 2 as relative levels of expression in 12 normal tissues
of pro101 (SEQ ID N0:1) compared to kidney (calibrator).
These RNA samples were generated by pooling samples from a
particular tissue from different individuals.
Table 2: Relative levels of pro101 Expression in Pooled
Samples
Tissue NORMAL
Brain 1.2
Heart 2
Kidney 1
Liver
7.2
Lung 4g,2
Mamma r y 2 . 5
Prostate 1418.4
Spleen 1.6
Small
1.9
Testis 57.3
Thymus 1.3
Uterus J 7 , 6 '-
The relative levels of expression in Table 2 show that for the
PSG pro101 (SEQ ID NO: l) mRNA expression is more than 20 fold
higher in the pool of normal prostate compared with the other
11 normal tissue pools analyzed. These results demonstrate
that mRNA expression of the PSG is highly specific for
prostate.
The tissues shown in Table 2 correspond to pools of
samples from different individuals. The tissues shown in the
following Table 3 were obtained from individuals and are not
pooled. Hence the values for mRNA expression levels shown in
Table 2 cannot be directly compared to the values shown in
Table 3.
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The absolute numbers in Table 3 are relative levels
of expression of pro101 (SEQ ID N0:1) compared to kidney
(calibrator), in 60 pairs of matching samples. Each matching
pair contains the cancer sample for a particular tissue and
the normal adjacent sample for that same tissue from the same
individual. The results from 3 unmatched ovary tumor, 3
unmatched normal ovary, 1 unmatched mammary tumor and 1
unmatched normal mammary gland are also shown.
Table 3: Relative Levels of pro101 Expression in Individual
Samples
T SUE ANCER MATCHING U TCHED
Prostate 1 103.9 0
Prostate 2 2219 - 84.2
Prostate 3 5048.2 3623.6
Prostate 4 11052.3 2029.4
Prostate 5 229.1 41.1
Prostate 6 57.9 25.3
Prostate 7 58.5 57.069
Prostate 8 1074.6 610.8
Prostate 9 32.7 79.3
Prostate 10 15.8 2.09
Prostate 11 436.4 438
Prostate 12 49.5 59.3
_
Prostate 13 128 56
Bladder 1 0 0
Bladder 2 0 0
Bladder 3 0.7 0
Colon 1 0 0
Colon 2 0 0
Colon 3 0 0
Colon 4 3.3 1.9
Colon 5 0.1 0.8
Colon 6 0 0
Lung 1 0 0
Lung 2 0.5 1.6
Lung 3 1.4 2.1
Lung 4 0 0
Lung 5 0 0
Kidney 1 0 0
Kidne 2 0 0
Kidne 3 0 0
Kidne 4 0 0
Liver 1 1.5 5.7
Liver 2 26.9 7.9
Liver 3 0 0
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Pancreas 1 0.9 0.9
Pancreas 2 3 0
Pancreas 3 0 0
Pancreas 4 0 0
Pancreas 5 0 0
Stomach 1 0 0
Stomach 2 0 0
Stomach 3 0 0
Stomach 4 0 0
Stomach 5 0 0
Sm Int 1 0 0
Sm Int 2 0 0
Testis 1 0 0
Mammar 1 4 0
Mammar 2 5.6 0
Mamma r 3 0 . 5 0
Mamma r 4 0 . 4 0
Mamma r 5 0 . 5
Mammar 6 0
Endo 1 1.6 7.6
Endo 2 0 0
Endo 3 0 0
Endo 4 0.3 0.2
Endo 5 5.8 5
Uterus 1 0 0
Uterus 2 0 0
Uterus 3 0 0
Uterus 4 2.2 2.6
Ovar 1 1.4
Ovar 2 11.6
Ovar 3 1.5
Ovar 4 22.9
Ovar 5 0
Ovar 6 1.8
Among 128 samples in Table 3 representing 14 different
tissues, the higher levels of expression are consistently in
prostate tissues. These results confirm the tissue
specificity results obtained with normal samples shown in
Table 2. Table 2 and Table 3 represent a combined total of
90 140 samples in 18 human tissue types. Sixty-eight samples
representing 13 different tissue types excluding prostate had
no detected pro101 mRNA (Table 3). In 4 tissues (stomach
small intestine kidney and testis) no pro101 (SEQ ID N0:1)
mRNA was detected for any sample tested from individuals
(Table 3). Expression of this PSG was detected in testis in
the pooled normal sample (Table 3). The median expression in
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prostate cancer samples in Table 3 is 166.5 units. Excluding
Ovary 4 (Normal), only 1 sample in Table 3, Liver 2 (Cancer),
is greater than 10% of this value.
Comparisons of the level of mRNA expression in
prostate tumor samples and the normal adjacent tissue from the
same individuals are also shown in Table 3. The PSG pro101
(SEQ ID NO:1) is expressed at higher levels in 9 of 13 (69$)
prostate cancer tissues (Prostate 1, 2, 3, 4, 5, 6, 8, 10 and
13) compared with the corresponding normal adjacent tissue.
The level of expression of this PSG is lower in prostate tumor
compared to normal adjacent tissue in two samples (Prostate
9 and 12). Equivalent levels of expression were detected in
two matched samples (Prostate 7 and 11). Previous mRNA
expression analysis for genes coding for the diagnostic
markers PSA and PLA2 showed higher expression of the mRNA in
40o to 800 of the tumor samples compared to matching normal
adjacent tissue. Higher expression in the tumor sample
compared to the corresponding normal adjacent tissue is
observed for Bladder 3, Colon 4, Liver 2, Pancreas 2,
Endometrium 5 and. Mammary 1, 2 and 3. Higher expression in
the normal adjacent samples is observed for Colon 5, Lung 2,
Lung 3, Liver 1, Endometrium 1 and Uterus 4. However, the
levels detected are in most cases comparable amongst the
different tissues and low compared to levels found in most
prostate tissues.
The high level of tissue specificity, plus the mRNA
overexpression in 9 of 13 of the prostate tumor samples tested
compared to the normal adjacent tissues are believed to make
the PSG, pro101 (SEQ ID N0:1) a good diagnostic marker for
detection of prostate cancer using mRNA.
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SEQUENCE LISTING
<110> Ali, Shujath
Salceda, Susana
Sun, Yangming
Cafferkey, Robert
<120> A Novel Method of Diagnosing, Monitoring and Staging
Prostate Cancer
<130> DEX-0034
<140>
<141>
<150> 60/086,265
<151> 1998-05-21
<160> 7
<170> PatentIn Ver. 2.0
<210> 1
<211> 1936
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (1908)
<400> 1
aatggtatgc caacttaagt atttacaggg tggcccaaat agaacaagat gcactcgctg 60
tgattttaag acaagctgta taaacagaac tccactgcaa gagggngggc cgggccagga 120
gaatctccgc ttgtccaaga caggggccta aggagggtct ccacactgct gctaggggct 180
gttgcatttt tttattagta gaaagtggaa aggcctcttc tcaacttttt tcccttgggc 240
tggagaattt agaatcagaa gtttcctgga gttttcaggc tatcatatat actgtatcct 300
gaaaggcaac ataattcttc cttccctcct tttaaaattt tgtgttcctt tttgcagcaa 360
ttactcacta aagggcttca ttttagtcca gatttttagt ctggctgcac ctaacttatg 420
cctcgcttat ttagcccgag atctggtctt ttttntgtnt tttttttntt tccgtctccc 480
caaagcttta tctgtcttga ctttttaaaa aagtttgggg gcagattctg aattgggcta 540
aaagacatgc atttttaaaa ctaggcaact tcttatttct ttcctttaaa aatacatagc 600
attaaatccc aaatcctatt taaagacctg acagcttgag aaggtcacta ctgcatttat 660
aggaccttct ggtggttctg ctgttacgtt tgaagtctga caatccttga gaatctttgc 720
atgcagagga ggtaagaggt attggatttt cacagaggaa gaacacagcg cagaatgaag 780
ggccaggctt actgaggctg tccagtggag ggctcatggg tgggacatgg aaaagaaggc 840
agcctaggcc ctggggagcc cagtccactg agcaagcaag ggactgagtg agccttttgc 900
aggaaaaggc taagaaaaag gaaaaccatt ctaaaacaca acaagaaact gtccaaatgc 960
1
CA 02328377 2000-11-15
WO 99/60162 PCT/US99/10548
tttgggaact gtgtttattg cctataatgg gtccccaaaa tgggtaacct agacttcaga 1020
gagaatgagc agagagcaaa ggagaaatct ggctgtcctt ccattttcat tctgttatct 1080
caggtgagct ggtagagggg agacattaga aaaaaatgaa acaacaaaac aattactaat 1140
gaggtacgct gaggcctggg agtctcttga ctccactact taattccgtt tagtgagaaa 1200
cctttcaatt ttcttttatt agaagggcca gcttactgtt ggtggcaaaa ttgccaacat 1260
aagttaatag aaagttggcc aatttcaccc cattttctgt ggtttgggct ccacattgca 1320
atgttcaatg ccacgtgctg ctgacaccga ccggagtact agccagcaca aaaggcaggg 1380
tagcctgaat tgctttctgc tctttacatt tcttttaaaa taagcattta gtgctcagtc 1440
cctactgagt actctttctc tcccctcctc tgaatttaat tctttcaact tgcaatttgc 1500
aaggattaca catttcactg tgatgtatat tgtgttgcag ngaaaagaaa aaagtgtctt 1560
tgtttaaaat tacttggttt gtgaatccat cttgcttttt ccccattgga actagtcatt 1620
aacccatctc tgaactggta gaaaaacatc tgaagagcta gtctatcagc atctgacagg 1680
tgaattggat ggttctcaga accatttcac ccagacagcc tgtttctatc ctgtttaata 1740
aattagtttg ggttctctac atgcataaca aaccctgctc caatctgtca cataaaagtc 1800
tgtgacttga agtttagtca gcacccccac caaactttat ttttctatgt gttttttgca 1860
acatatgagt gttttgaaaa taaagtaccc atgtctttat taaaaaanaa aaaaaagggc 1920
ggccgccgac tagtga 1936
<210> 2
<211> 637
<212> DNA
<213> Homo Sapiens
<400> 2
gtaggggcag acttactgcc ttgaacgaaa gacgatggtc ctcgctcagc ctcactccaa 60
ttatgttcct ctaggtgggg caggtagggg gtccagcttc ctgcttgctg gtggttcagg 120
tcatgcgtcc agccttgtcc cttctgacct gggccctacc cacggggaaa tgttcccata 180
gcagaagaat cagccccaca gtgcaggggt gtgttagtgg ggaacgggct ctgggctcct 290
gtgggaacca gggaccccct atcttggtac cggtcattgg atgtatcccc agctcatgcc 300
tgtgtctgtc ttggcccgtg tggtcaccct gtgttcatct ctctcccagc catggcctct 360
caaactgggg ttttcgtctc cctatgaggg ggtcctggta tgtacgcgtt cggtgggccc 420
gcggtgcatg tctcccggtg cagtgcatgc tggggttccc tggggccctg ggcccctcgt 980
aggatagaca gagcctgtcc taaccttccg gaagtgcatg ctggggaggc cccttgcctg 540
ctgaccttct gtgctcagga cgactaatcg gccacatgac caccactctg tcccatggga 600
ttcctagaga agtctcacta agagcccagc acactca 637
<210> 3
<211> 2693
<212> DNA
<213> Homo Sapiens.
<220>
<221> unsure
<222> (2266)..{2512)
<220>
<221> unsure
<222> (586)
2
CA 02328377 2000-11-15
WO 99/60162 PCT/US99/10548
<220>
<221> unsure
<222> (1480)
<220>
<221> unsure
<222> (1532)
<220>
<221> unsure
<222> (1562)..(1566)
<220>
<221> unsure
<222> (1569)
<220>
<221> unsure
<222> (1571)
<220>
<221> unsure
<222> (1631)
<900> 3
gctcctacag ccgcatctgc gttaacatag catccctatg gccactgtct cccttgatcc 60
ccacagccat cctaggagaa aggcagaatg tcataatttg ctaaaaggga tgctgaggct 120
ctgggaggga aagggacttg cctaaagccc cagggtgaag cagcatctct ggactcccag 180
tccagtgatc ttgcccaata ctttgctgct tgcctatacc cctctaactt ggtcaacagc 240
acatcacagg gcaagcccaa tccctgcttc atttttatat atgggcgctg gtccacagcc 300,
ccactctcca gccatttgga aacaaaaaca gatgctattg ttcttcctta gagaacgtgg 360
ccagtggaga cggcacactg gaaatcagag tgaatgttct tgaaagaggg tcacgggtca 420
acaaggccca gccaaaggat gcagtagaac cattttcctt agaaatcttt gggagtgaag 480
taggcttcag ccactaccca tccctgccct tgcggctacc actaccccat tagtttagac 540
agggtcgggc ggggaggggt gtggagaaga aatgagcttg cctgtngccc ccaggctccc 600
tctgtcctag ctcaggtctg ggtgccattc tttacactcg tgtgctcgct cacgcacaca 660
tcacacacct tgctggtcac acagtcacag actcgcctct gctcctgtgg tccagtggcc 720
ggacaccccc tgggatggct caaaggagtc aggacttgga agtggggaca tcagggtagc 780
tgaaggaaat ccacacaccc agagcatctc ggagttcaga ctctcagacc tgaagtaggc 840
gcccccggga ctgggctagg agttggacgg aatggaggat ggaggacagc gagaagaaag 900
gaagagaaat gcaaagtgtg ggcagccgcc aagagtgaaa atagagggaa gtgtcatgca 960
agtgctggac agaaggcggc aggtgggacg agccccacag ccccctcctc aaaaacgacc 1020
acctccagga ctcagtgatc cctggggggc aggctctgcc agccctcggc cacacgtggc 1080
tccggcaccc atggtcccag tgccttggat ggagacggcc agttctggcg gccagatgtg 1190
gtgctctgga atccagtccc atttccttcc tggccacgcc tgttccagcg gcctctttgg 1200
ctgcattcag cccctactta cctggggacc ccggctgggg cacaagagca ccaggggggt 1260
agggcccaaa gggatcaggg gaagcctctg gcctggaggg tatggggcac gcttccccaa 1320
3
CA 02328377 2000-11-15
WO 99/60162 PCT/US99/10548
gggcggaccc ggcaggagga agcccaggag ctgggtcctg ccgcccagga gctgggccct 1380
gccacccagg ccgggctagg gacatggcag ggcctgggca tcctgacgct ggacttgggc 1440
gacctgggag gcacagggag gggagagatg ggcggccccn acccagcgca gtgccggcca 1500
caccccaagg cggttgccag agcttaaggc cnggccccag caggagaaca tcccagctcc 1560
annnnnccnc nccgcagcca gtgctccttg tcaagctccc cccgtcactc caggtgggag 1620
ccaccccggt nagggggtgt gccacttgcc cccagggcac tcctctgggc atcccgggtg 1680
ggggattttg gggccgtggg gggcagtctc tggtacctgt gtgcgtcagg gatgctctgc 1740
acctgcaacc aggtgtcgtc cacgggcggg ggcatgggca tggtgacagt ggtcctgttg 1800
atgtcaccga tgatgctgag cgcctccttc agcgcgtggt gcatgtgcag catctcgtcg 1860
tgctgctgtg cctgctctgc caactcctcc atcagtgtgt tctggttccc acatgagtac 1920
atattggcca gcggctccga gatgatgaac tccggggtct gagagtgggc aaacagggaa 1980
gaaggttggg acctggtgcc tgtgccgccc tggctgcctt gctgggccct tctgggactg 2040
tgcgctggac ttggagcccc ttggagtatg gcttttcaca cgggcttcta taccgcttcg 2100
actggaagat ccacctcccc actgcctttt ctcactcaga tggggacacc gaggtccaga 2160
ggaaaagaca cctgtcaaat gtcacagatc tgggagggga cttaagacct atcatgccaa 2220
gaggacacct gtctactcag tttttttttg gtggggcggg gggcgnnnnn nnnnnnnnnn 2280
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnggagttgg 2520
agttgatgcc tggatacagg agctctgtgg gtgggagtga gacaaaacac agggtcctga 2580
gctctgggga ccaagcaatg tcctctggtg aaaaaaatcc tggacttgct ggcagaagat 2640
ttgcctctta cttgccatgt gctctgaata catttacctg ccctctggga aaa 2693
<210>4
<211>292
<212>DNA
<213>Homo Sapiens
<220>
<221> unsure
<222> (289)
<400> 9
aagaatatga gatttgctta gaaatgaagg actggaagga gcccacagag ttatttttta 60
aactatccag taaggcttag agggtttcaa tcagaaatat gtgttagggg aaaaaatgca 120
ctttttctat attaaaaaat attattttct tcttttaaat gtaaagcatt cctattgtga 180
agaattgaga aaatacagaa aagtacaaag aaaaacatta cctacaactc caccatccgt 240
gattatcact gttcacattt gtggctcatt tttcagtatk tctnttattt as 292
<210> 5
<211> 2699
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (52)
4
CA 02328377 2000-11-15
WO 99/60162 PCTNS99/10548
<220>
<221> unsure
<222> (74)
<220>
<221> unsure
<222> (76)
<220>
<221> unsure
<222> (80)
<220>
<221> unsure
<222> (92)
<220>
<221> unsure
<222> (97)
<220>
<221> unsure
<222> (123)
<220>
<221> unsure
<222> (132)
<220>
<221> unsure
<222> (173)
<220>
<221> unsure
<222> (217)
<220>
<221> unsure
<222> (257)
<220>
<221> unsure
<222> (2539)
<400> 5
tactatattg ctcagcattt ctaagtattc tctaagtgct ctttatttat gntttaaaat 60
agctctctta cccngntgcg ncgactagaa gancttgntt taggaaacaa tgaaatatat 120
CA 02328377 2000-11-15
WO 991601b2 PCT/US99/10548
aanttgccag antcaattgg agccctctta catctaaaag atctctggtt ggntggaaat 180
caactgtcag aattacctca ggaaatagga aatctgnaga acctgctgtg tttagatgtc 240
tctgaaaaca ggttggnaag acttcctgaa gaaatcagtg gcctgacttc attaacggat 300
ttagtcattt cccagaactt attagaaacg attccggatg gcattggaaa actaaagaaa 360
ctgtcaatct tgaaggtgga tcagaataga ctcacacagt tgcctgaagc agttggggaa 420
tgtgaaagtc tcactgagtt agttcttaca gaaaatcagc tcctgaccct gcctaaaagc 480
attggaaaac taaagaagtt gagcaacttg aatgcagaca gaaataaatt agtgtcctta 540
ccaaaagaga tcggcgggtg ctgcagcctc actgtgttct gtgtacgtga caacagacta 600
actcggatac ctgcagaggt gtcacaggca acagaacttc atgtcctgga tgtggcaggg 660
aacaggttgc tgcatctacc tttatccctg actgccttga agttgaaggc tctgtggcta 720
tctgacaacc agtcccagcc cctgcttaca ttccagacag acacagacta caccacagga 780
gagaagattt taacctgtgt cttacttcct cagctgcctt ctgaacctac ttgtcaagag 840
aatctgcctc gctgtggtgc actggagaac ttggtaaatg atgtctctga tgaagcctgg 900
aacgagcgtg ctgtcaacag agtcagtgcg atccgatttg tggaggatga gaaagatgaa 960
gaagacaatg agacgagaac acttctaagg cgagccactc cacacccagg ggagttaaag 1020
cacatgaaaa agacagtgga gaatttacgg aatgacatga atgctgctaa aggactggac 1080
tcaaacaaaa acgaggtcaa tcatgccatt gaccgagtga ccacttctgt gtagagtttc 1140
acctccaagt tttacctcct gtgtcttcct ctgctgtcga gacgttcctg tctgcttccc 1200
gggagcctca cgtgctcctt gtcctaacca gcccccgcgc gccatcttcc cgtggagtgt 1260
ggggaagctg ctgtctccca ggaagtgcct tactcatccc gcaaccagtc agcgcaccag 1320
tggtctcccg gtgtgatttt tttttttttt aatttcagtt gtttgtaata agtagaatac 1380
actactgtaa acatacgacc tttgtttttg tcttatgttg gggtaaagga aagcaggaag 1440
gggaattttt atcctcctcc cttccgtaaa gtgctgggat attttgaatc ccccaagttc 1500
ccttggacct actgatgaga gatagtttta tgtatgggga aaaatggata ctttttaaac 1560
cttttttggc agctcagatg gtgtaaattt taaaattttg tataggtatt tcataacaaa 1620
aatatgtatt tcttttttgt tattttatct tgaaaacggt acatatttta gtatttgtgc 1680
agaaaaacaa gtcctaaagt atttgttttt atttgtacca tccacttgtg ccttactgta 1740
tcctgtgtca tgtccaatca gttgtaaaca atggcatctt tgaacagtgt gatgagaata 1800
ggaatgtggt gttttaaagc agtgttgcat tttaatcagt aatctacctg gtggatttgt 1860
ttttaaccaa aaagatgaat tatcaatgat ttgtaattat atcggttgat tttttttgaa 1920
aagatgaacc aaaggatttg actgctaata ttttattcct tacacttttt ttctgaataa 1980
gtctctcata atgagtgcag tgtcagactg tgcctactct gatggtatgt gccatttgta 2040
aaataaaata gagcagaaaa acacaaaaag agaacactgg ttcagacatt cagtgggcaa 2100
gtaaattatg gactgcaaaa taatgatttt tattcaagaa agctttaaaa gttttatatc 2160
cagatataca accacaataa agcaaaataa cctactatca aaatagaaat gttgctatct 2220
ttataagtgc aatttaattt gtaaatagag tttgaatcaa agtatcacaa aatactgctt 2280
caagatttaa ttttaaatct gctaatttaa gggatattgg gaaaagtttt ggtgtgtttc 2340
tgttgatttc ttttttgtat gctgtgataa aagagaaatg aaaagtgcca gtcactgtgt 2400
ggtgtctagg aaaatcatat atattttttt ctccaagaaa taaattcatc ctggacattg 2460
gccatacagc tttttaaaat tattactttg tatgttcaag tgatagcagg tagccaaatt 2520
ctttgacagt gtgctctgnt ctgttaaata tctaaattac ccgtcagttg tgagtgacct 2580
cctgtgggac ttgcattcac atggggcaga gcccagaatt gcctttgact ctggctagta 2640
attttgggtt gtggctatct ggccaattgg actccttata aacccgtctt caac 2694
<210> 6
<211> 1335
<212> DNA
<213> Homo Sapiens
6
CA 02328377 2000-11-15
WO 99/60162 PCTNS99/10548
<220>
<221> unsure
<222> (17)
<400> 6
tcatatagta ggaaganaag cacctaggtt tgaggccagg gctggctgct gtcagaacct 60
aggccctccc ctgccttgct ccacacctgg tcaggggaga gaggggagga aagccaaggg 120
aagggaccta actgaaaaca aacaagctgg gagaagcagg aatctgcgct cgggttccgc 180
agatgcagag gttgaggtgg ctgcgggact ggaagtcatc gggcagaggt ctcacagcag 240
ccaaggaacc tggggcccgc tcctcccccc tccaggccat gaggattctg cagttaatcc 300
tgcttgctct ggcaacaggg cttgtagggg gagagaccag gatcatcaag gggttcgagt 360
gcaagcctca ctcccagccc tggcaggcag ccctgttcga gaagacgcgg ctactctgtg 920
gggcgacgct catcgccccc agatggctcc tgacagcagc ccactgcctc aagccgtggc 480
cgctacatag ttcacctggg gcagcacaac ctccagaagg aggagggctg tgagcagacc 540
cggacagcca ctgagtcctt cccccacccc ggcttcaaca acagcctccc caacaaagac 600
caccgcaatg acatcatgct ggtgaagatg gcatcgccag tctccatcac ctgggctgtg 660
cgacccctca ccctctcctc acgctgtgtc actgctggca ccagctgcct catttccggc 720
tggggcagca cgtccagccc ccagttacgc ctgcctcaca ccttgcgatg cgccaacatc 780
accatcattg agcaccagaa gtgtgagaac gcctaccccg gcaacatcac agacaccatg 840
gtgtgtgcca gcgtgcagga agggggcaag gactcctgcc agggtgactc cgggggccct 900
ctggtctgta accagtctct tcaaggcatt atctcctggg gccaggatcc gtgtgcgatc 960
acccgaaagc ctggtgtcta cacgaaagtc tgcaaatatg tggactggat ccaggagacg 1020
atgaagaaca attagactgg acccacccac cacagcccat caccctccat ttccacttgg 1080
tgtttggttc ctgttcactc tgttaataag aaaccctaag ccaagaccct ctacgaacat 1140
tctttgggcc tcctggacta caggagatgc tgtcacttaa taatcaacct ggggttcgaa 1200
atcagtgaga cctggattca aattctgcct tgaaatattg tgactctggg aatgacaaca 1260
cctggtttgt tctctgttgt atccccagcc ccaaagacag ctcctgccat atatcaagtt 1320
tcaataaata tttct 1335
<210> 7
<211> 1079
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> (268)
<220>
<221> unsure
<222> (688)
<220>
<221> unsure
<222> (700)
<400> 7
7
CA 02328377 2000-11-15
WO 99/60162 PCT/US99/10548
tttttgaaga atgccctgca aggcatcaac tggaatgtgt ttattaccaa acaagacaga 60
agagaaceag ggcctgactt ggcagtggcc ccaggctgca tgggctcagg taggctcaga 120
ccggccccag gagtgggaga gcccagagaa gagggaaaaa gagtagtggc caggaggggt 180
ctggctggga catgccactc tgggccatca gcttctggat ccactcaaag tggtggctga 290
tattggtgta gacaccgggc cgattggncc gaccacagcc cactccccag ctcacgactc 300
caatctgata ccacagtcca ttcttgttac aggccaaggg tccacctgag tcaccgaagc 360
aggcatcctt cccgccttgg gcattgccag cacaaaccat gtctccaaag atgtccttgc 920
ggaaactgta cttgaggaag aggtggttgc acatagagtt gtttatgatg gcgacctgaa 480
cttcctggag ggtgtgggga gatggcagtg cctcatcctc tttgatgtac ccccagccag 540
tcacccagca gtctgtccgg ttctcaaact caaatgtgga ggcctggaga cagatgggct 600
ggatgtgttt agtgtaggtg acaggtgcag acagcttcac caaggcaatg tcatagggtg 660
aattccccag ttagcgaggg ctcagatnga tattcgatan gaagtaacgg gtgtagtagg 720
cctgcaggct ccagaaggat ggcatggaag tcagctggcc aaactggacc atccacccgg 780
agggatcact aaggtcacta taggtttcaa agcagtgcgc cgccgtgagt gcccagcggt 840
ggctgagcag gctcactccg catacgtggg aatcccacag gcgcaggctc ccctgccacg 900
gccaacgccc gagttcggcg tcctctccac ccacgatgcg cgacgtgatg acccgtcggc 960
cgcatggtcc tgataagggc gccgcctcct gcgactccgg cttcctgagt ccagcccgag 1020
ccagcagcag cgccagcagc agcgccccgc gcgcgcccat ggcctcctct cccgcggtg 1079
8
CA 02328377 2000-11-15
