U.S. patent application number 12/404964 was filed with the patent office on 2009-07-16 for method of detection of prostate cancer.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to David Sidransky.
Application Number | 20090181400 12/404964 |
Document ID | / |
Family ID | 23302188 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181400 |
Kind Code |
A1 |
Sidransky; David |
July 16, 2009 |
Method of Detection of Prostate Cancer
Abstract
The present invention provides methods and kits useful for
detecting neplasia by measuring the methylation level of
biomarkers, especially the promoter region of GSTP1 for the
detection of prostate adenocarcinoma.
Inventors: |
Sidransky; David;
(Baltimore, MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.;DLA PIPER LLP (US)
Suite 1100, 4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
23302188 |
Appl. No.: |
12/404964 |
Filed: |
March 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11825479 |
Jul 6, 2007 |
7524633 |
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12404964 |
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10295483 |
Nov 15, 2002 |
7252935 |
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11825479 |
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60333296 |
Nov 16, 2001 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/154 20130101;
C12Q 1/6851 20130101; C12Q 2523/125 20130101; C12Q 1/6827 20130101;
C12Q 1/6886 20130101; C12Q 1/6827 20130101; C12Q 2523/125
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made in part with government support
under Grant No. 1U01CA84986 awarded by the National Institutes of
Health. The United States government has certain rights in this
invention.
Claims
1. A kit useful for detecting prostate adenocarcinoma comprising: a
first container containing a pair of primers for amplification of a
promoter region of glutathione-S-transferase (GSTP1), a second
container containing a pair of primers for amplification of a
region in a reference gene, and a third container containing a
first and second oligonucleotide probe, wherein the first
oligonucleotide probe is specific to the amplification of the
promoter region of GSTP1 and the second oligonucleotide probe is
specific to the amplification of the region in the reference gene,
wherein at least one of the primers for the amplification of the
promoter region of GSTP1 or one of the oligonucleotide probes is
capable of distinguishing between methylated and unmethylated
nucleic acid.
2. The kit of claim 1 further comprising a fourth container
containing a modifying agent that modifies unmethylated cytosine to
produce a converted nucleic acid.
3. The kit of claim 1 further comprising an instruction disclosing
a methylation ratio indicative of prostate adenocarcinoma.
4. The kit of claim 1 further comprising an instruction disclosing
that a methylation ratio greater than 5 is indicative of prostate
adenocarcinoma.
5. The kit of claim 1 further comprising an instruction disclosing
that a methylation ratio greater than 10 is indicative of prostate
adenocarcinoma.
6. The kit of claim 1 further comprising an instruction disclosing
that a methylation ratio greater than 50 is indicative of prostate
adenocarcinoma.
7. The kit of claim 1, wherein the reference gene is MYOD or
ACTB.
8. The kit of claim 1, wherein the region of the reference gene is
devoid of CpG dinucleotides.
9. The kit of claim 1, wherein the region of the reference gene
contains a first and second primer binding site and an
oligonucleotide binding site and wherein the first and second
primer binding site and the olionucleotide binding site are devoid
of CpG dinucleotides.
10. The kit of claim 1, wherein the primers for amplification of
the promoter region of GSTP1 are capable of distinguishing between
methylated and unmethylated nucleic acid.
11. The kit of claim 1, wherein the first oligonucleotide probe is
capable of distinguishing between methylated and unmethylated
nucleic acid.
12. The kit of claim 1 further comprising an instruction disclosing
that the kit is useful for detecting prostate adenocarcinoma in a
prostate tissue sample from a subject.
13. The kit of claim 1 further comprising a probe for PSA.
14. A kit useful for detecting prostate adenocarcinoma comprising:
a first container containing a pair of primers for amplification of
a promoter region of glutathione-S-transferase (GSTP1), wherein the
primers are capable of distinguishing between methylated and
unmethylated nucleic acid, and an instruction disclosing that the
kit is useful for detecting prostate adenocarcinoma in a bodily
fluid sample from a subject and that a methylation level of the
promoter region of GSTP1 as determined by conventional polymerase
chain reaction using the primers in the first container that is
higher than the methylation level of the promoter region of GSTP1
in a normal subject is indicative of prostate adenocarcinoma in the
subject with a sensitivity no less than 40%.
15. The kit of claim 14, wherein the sensitivity is no less than
50%.
16. The kit of claim 14, wherein the bodily fluid is serum, plasma,
ejaculate, or urine.
17. The kit of claim 14, further comprising a second container
containing a pair of primers for amplification of a region in a
reference gene, and a third container containing a first and second
oligonucleotide probe, wherein the first oligonucleotide probe is
specific to the amplification of the promoter region of GSTP1 and
the second oligonucleotide probe is specific to the amplification
of the region in the reference gene, wherein at least one of the
primers for the amplification of the promoter region of GSTP1 and
one of the oligonucleotide probes is capable of distinguishing
between methylated and unmethylated nucleic acid.
18. The kit of claim 17, wherein the reference gene is MYOD or
ACTB.
19. The kit of claim 17, wherein the first oligonucleotide probe is
capable of distinguishing between methylated and unmethylated
nucleic acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/825,479 filed Jul. 6, 2007; which is a
continuation application of U.S. application Ser. No. 10/295,483
filed Nov. 15, 2002, now issued as U.S. Pat. No. 7,252,935; which
claims the benefit under 35 USC .sctn. 119(e) to U.S. Application
Ser. No. 60/333,296 filed Nov. 16, 2001, now abandoned. The
disclosure of each of the prior applications is considered part of
and is incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates generally to the field of methylation
status of genes and regulatory elements and more specifically to
detection of prostate cancer by conventional and quantitative PCR
methods.
[0005] 2. Background Information
[0006] Prostatic adenocarcinoma is the most commonly diagnosed
non-cutaneous cancer for men in the United States. The incidence is
likely to continue to increase as people survive longer and more
middle-aged men undergo routine screening for the disease. Men
diagnosed with early stage small volume disease have the best
outcome following curative treatment. Therefore the aim of early
detection programs is to diagnose cancer at an early curable
stage.
[0007] The gold standard algorithm for diagnosis currently entails
digital rectal exam and measurement of serum prostate-specific
antigen (PSA) and if either is suspicious it is followed by
trans-rectal prostatic needle biopsy. However, serum PSA can be
elevated in benign conditions and needle biopsy may fail to
identify even significant amounts of cancer due to sampling error.
Therefore, the introduction of additional diagnostic tests is
needed to improve the sensitivity of prostate cancer diagnosis.
[0008] Although several specific genetic alterations have been
described in prostate adenocarcinoma, such as TP53 and PTEN
inactivation, the single most common and earlier of these is
methylation of the 5'-regulatory region of the GSTP1 gene. The
detection of this epigenetic alteration in bodily fluids has been
successfully accomplished using DNA-based techniques. However,
these earlier studies either included only a relatively small
number of patients or focused mainly on cases of advanced
disease.
[0009] Recently, a specific real-time quantitative methyl specific
PCR (RTQ-MSP) method, allowing the performance of non-isotopic,
rapid, and highly accurate quantitative amplification analysis via
the continuous optical monitoring of a fluorogenic PCR assay was
developed. The application of this method to evaluate the
methylation status of the p16 gene in bone marrow aspirates from
patients with multiple myeloma, revealed complete concordance with
conventional MSP (C-MSP) analysis. In this same study, it was shown
that RTQ-MSP was sensitive enough to detect down to 10 genome
equivalents of methylated p16 sequence.
[0010] However, there is a need in the art to develop sensitive and
accurate early stage diagnostic assays for detecting prostate
adenocarcinoma.
SUMMARY OF THE INVENTION
[0011] The present invention is based, in part, on the discovery
that quantitative measurement of the methylation level of
biomarkers, e.g., promoter region of glutathione-S-transferase
(GSTP1), can be used to detect neoplasia. Accordingly, the present
invention provides methods and kits useful for detecting neoplasia,
especially prostate adenocarcinoma. In one embodiment, the present
invention provides a method for detecting prostate neoplasia. The
method includes determining a methylation ratio of a sample, e.g.,
a tissue sample from a subject. The methylation ratio is a ratio
between the level of methylation of a promoter region, e.g., of
glutathione-S-transferase (GSTP1), relative to the level of
methylation of a region of a reference gene. If the methylation
ratio is higher in the sample, e.g., tissue, from the test subject
than the methylation ratio in a sample, or tissue from normal
subjects or from a tissue or sample from a subject with
hyperplasia, it is indicative of prostate neoplasia in the test
subject.
[0012] In another embodiment, the present invention provides a
method for detecting prostate neoplasia by determining the
methylation level of a promoter region of glutathione-S-transferase
(GSTP1) in a sample of bodily fluid, such as urine or serum, from a
subject. The methylation level is determined using a conventional
polymerase chain reaction (PCR), or the real-time/quantitative PCR
method. If the methylation level is higher in the test subject than
the methylation level in a normal subject it is indicative of
prostate neoplasia in the subject.
[0013] In yet another embodiment, the present invention provides a
kit for detecting prostate hyperplasia. The kit includes a first
container containing a pair of primers for amplification of a
promoter region of glutathione-S-transferase (GSTP1), a second
container containing a pair of primers for amplification of a
region of a reference gene, and a third container containing a
first and second oligonucleotide probe, with the first
oligonucleotide probe specific for the amplification of the
promoter region of GSTP1 and the second oligonucleotide probe
specific for the amplification of the region in the reference gene.
In one aspect, at least one of the primers for the amplification of
the promoter region of GSTP1 or one of the oligonucleotide probes
is capable of distinguishing between methylated and unmethylated
nucleic acid.
[0014] In another aspect, the kit contains a first container
containing a pair of primers for amplification of a promoter region
of glutathione-S-transferase (GSTP1), with the primers being
capable of distinguishing between methylated and unmethylated
nucleic acid, and an instruction disclosing that the kit is useful
for detecting prostate adenocarcinoma in a bodily fluid sample from
a subject and that a methylation level of the promoter region of
GSTP1 as determined by conventional polymerase chain reaction using
the primers in the first container that is higher than the
methylation level of the promoter region of GSTP1 in a normal
subject is indicative of prostate adenocarcinoma in the subject
with a sensitivity no less than 40%.
[0015] In another embodiment, the present invention provides a
method for detecting prostate neoplasia by amplifying a promoter
region of glutathione-S-transferase (GSTP1) in a biological sample
from a subject by means of oligonucleotide primers in the presence
of at least one specific oligonucleotide probes, with the promoter
region being modified by an agent that modifies unmethylated
cytosine to produce a converted nucleic acid and at least one
oligonucleotide primer or specific oligonucleotide probe being
capable of distinguishing between unmethylated and methylated
nucleic acid, and determining the methylation level of the promoter
region by determining the amplification level of the promoter
region based on amplification-mediated displacement of the specific
oligonucleotide probe. If the methylation level is higher in the
test subject than the methylation level in a normal subject, it is
indicative of prostate neoplasia in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the amplification curves for a case positive
for carcinoma by GSTP1 QMSP on 4/6 biopsies (LB=left base, LM=left
mid, RA=right apex, RB=right base). Each biopsy was run in
quadruple and compared to standard dilutions of positive control
(S1-5=100 ng, 10 ng, 1 ng, 100 pg, 10 pg standard DNA
respectively). This case was also positive for carcinoma by
histology on 2/6 biopsies (Left Base 2 mm, Left Mid 1 mm). The
extent of tumor seen histologically on biopsy corresponded to the
levels of GSTP1 methylation measured.
[0017] FIG. 2 shows the ROC curves for histology (blue), GSTP1 QMSP
(green) and combined tests (red).
[0018] FIGS. 3A and 3B show the distribution of GSTP1 methylation
levels in tissue and bodily fluids. (a) GSTP1 methylation was
detected by RTQ-MSP in 29% of patients with BPH and 91.3% of
patients with clinically localized prostate adenocarcinoma (TRP).
Solid bars indicate the median within a group of patients.
Asterisks indicate the samples with 0-values which cannot be
plotted on a log scale (BPH: n=31; TRP: n=69).(b) GSTP1 methylation
levels (RTQ-MSP) in positive paired urine (n=13) and plasma (n=9)
samples. Solid bars indicate the median within a group of patients.
Asterisks indicate the samples with 0-values which cannot be
plotted on a log scale (urine: n=56, plasma: n=60).
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention is based, in part, on the discovery
that quantitative measurement of methylation level of biomarkers,
e.g., promoter region of glutathione-S-transferase (GSTP1), can be
used to detect neoplasia, e.g., prostate adenocarcinoma.
Accordingly, the present invention provides methods and kits useful
for detecting neoplasia, especially prostate adenocarcinoma by
determining the methylation level or ratio of biomarkers.
[0020] According to one aspect of the present invention, neoplasia
in a cell or tissue can be detected by quantitatively measuring the
methylation level of one or more biomarkers. The biomarkers of the
present invention can be any marker whose methylation level is
characteristically associated with the abnormal growth or
proliferation of a cell or tissue. For example, the methylation
levels of various tumor suppressor genes are associated with
neoplasia or tumor growth and can be used as biomarkers of the
present invention. In one embodiment, the biomarker of the present
invention is glutathione-S-transferase (GSTP1).
[0021] In another embodiment, the biomarker is a promoter region of
GSTP1. The promoter region of GSTP1 can be any region within the
promoter of GSTP1, e.g., any region containing one or more sites
associated with methylation such as CpG dinucleotides and suitable
for quantitative measurement such as amplification by polymerase
chain reaction (PCR). The promoter of GSTP1 usually includes the
regulatory region located upstream or 5' to GSTP1. Sequence
analysis of the promoter region of GSTP1 shows that nearly 72% of
the nucleotides are CG and about 10% are CpG dinucleotides.
[0022] According to the present invention, neoplasia in a cell or
tissue can be detected by quantitatively measuring the methylation
level of one or more biomarkers using, e.g., real-time polymerase
chain reaction (PCR) with at least one oligonucleotide primer or
oligonucleotide specific probe being capable of distinguishing
between methylated and unmethylated nucleic acid. In general, a
quantitatively measured methylation level in a biological sample
from a subject, e.g., human that is higher than the quantitatively
measured methylation level in a biological sample from a normal
subject is indicative of neoplasia in the subject.
[0023] A normal subject as used in the present invention can be any
subject that does not have detectable neoplasia by means other than
the methods provided by the present invention. For example, a
normal subject for prostate adenocarcinoma can be any human that
does not have any clinical symptom of prostate adenocarcinoma, a
normal PSA level, and a histological diagnosis free of prostate
adenocarcinoma. In general, a level associated with a normal
subject as used in the present invention includes statistically
obtained value associated with a population of normal subjects. For
example, the methylation level in a normal subject as used in the
present invention includes the mean or average methylation level
and a range for a statistically significant population of normal
subjects.
[0024] The biological sample of the present invention can be any
sample suitable for the methods provided by the present invention.
In one embodiment, the biological sample of the present invention
is a tissue sample, e.g., a biopsy specimen such as samples from
needle biopsy. In another embodiment, the biological sample of the
present invention is a sample of bodily fluid, e.g., serum, plasma,
urine, and ejaculate.
[0025] According to one embodiment of the present invention,
prostate adenocarcinoma in a cell or tissue can be detected by
quantitatively measuring the methylation level of the promoter
region of GSTP1, e.g., using real-time methylation specific PCR.
For example, the methylation level of the promoter region of GSTP1
can be determined by determining the amplification level of the
promoter region of GSTP1 based on amplification-mediated
displacement of one or more probes whose binding sites are located
within the amplicon.
[0026] In general, real-time quantitative methylation specific PCR
is based on the continuous monitoring of a progressive fluorogenic
PCR by an optical system. Such PCR systems usually use two
amplification primers and an additional amplicon-specific,
fluorogenic hybridization probe that specifically binds to a site
within the amplicon. The probe can include one or more fluorescence
label moieties. For example, the probe can be labeled with two
fluorescent dyes: 1) a 6-carboxy-fluorescein (FAM), located at the
5'-end, which serves as reporter, and 2) a
6-carboxy-tetramethyl-rhodamine (TAMRA), located at the 3'-end,
which serves as a quencher. When amplification occurs, the 5'-3'
exonuclease activity of the Taq DNA polymerase cleaves the reporter
from the probe during the extension phase, thus releasing it from
the quencher. The resulting increase in fluorescence emission of
the reporter dye is monitored during the PCR process and represents
the number of DNA fragments generated.
[0027] In one embodiment, oligonucleotide primers are designed to
specifically bind methylated primer binding sites, e.g.,
bisulfite-converted DNA within the 3'-end of the promoter region of
the GSTP1 gene, and a probe is designed to anneal specifically
within the amplicon during extension. In another embodiment,
oligonucleotide primers are designed to bind either methylated or
unmethylated primer binding sites and the probe is designed to
anneal specifically to methylated probe binding site, e.g.,
bisulfite-converted binding site. In yet another embodiment,
oligonucleotide primers and probes are designed to specifically
bind methylated binding sites, e.g., bisulfite-converted binding
sites.
[0028] According to another aspect of the present invention,
neoplasia of a biological sample is indicated when a methylation
ratio of a biomarker is higher than the methylation ratio in a
normal subject. The methylation ratio of the present invention
includes the ratio of the methylation level of a biomarker and the
level of a region in a reference gene determined by the same means
used for the determination of the methylation level of the
biomarker. Usually, the methylation ratio of the present invention
is represented by the ratio of the methylation level of a biomarker
and the level of a region in a reference gene determined by the
same means used for the determination of the methylation level of
the biomarker.
[0029] In one embodiment, the methylation ratio of the present
invention is the ratio of the methylation level of a biomarker and
the level of a region of a reference gene, both of which are
quantitatively measured using real-time polymerase chain reaction
(PCR). For example, the methylation level of a biomarker from a
sample of a subject can be quantitatively measured using a pair of
primers and an oligonucleotide probe, where one primer, both
primers, the oligonucleotide probe, or both primers and the
oligonucleotide probe are capable of distinguishing between
methylated and unmethylated nucleic acid, e.g., after the nucleic
acid being modified by a modifying agent, e.g., bisulfite
converting unmethylated cytosine to a converted nucleic acid.
[0030] In another embodiment, the methylation ratio of the present
invention is the ratio of the methylation level of a promoter
region of GSTP1 and the level of a region of a reference gene, both
of which are quantitatively measured using real-time PCR. In yet
another embodiment, the methylation ratio of the present invention
is the ratio of the methylation level of a promoter region of GSTP1
measured by methylation specific real-time PCR and the level of a
region of a reference gene measured by real-time PCR.
[0031] The region of a reference gene of the present invention can
be any region of a gene having one or more sites or regions that
are devoid of methylation sites, e.g., devoid of CpG dinucleotides.
For example, the region of a reference gene can be a region that
having two primer binding sites for amplification such as PCR that
are devoid of CpG dinucleotdies or a region having at least one
specific oligonucleotide probe binding site for real-time PCR that
is devoid of CpG dinucleotides. In one aspect, the region of a
reference gene of the present invention is a region of MYOD gene.
In another aspect, the region of a reference gene of the present
invention is a region of ACTB gene. In yet another embodiment, the
region of a reference gene of the present invention is a region
that is not frequently subject to copy number alterations, such as
gene amplification or deletion.
[0032] In general, according to the present invention the level of
a region of a reference gene is quantitatively measured using
real-time PCR with primers and specific probes that specifically
bind to sites after bisulfite conversion but without discriminating
directly or indirectly the methylation status of the sites.
[0033] According to one embodiment of the present invention,
prostate adenocarcinoma is indicated in a subject when a
methylation ratio of a promoter region of GSTP1 in a biological
sample from the subject is greater than 5, or 10, as the
methylation ratio is represented by the ratio of the methylation
level of a promoter region of GSTP1 and the level of a region of a
reference gene, e.g., MYOD or ACTB times 1000 fold. For example,
when a methylation ratio times 1000 is greater than 3, or 5 or even
10, it is indicative of prostate adenocarcinoma in the subject.
[0034] According to another aspect of the present invention, the
methylation ratio of the present invention can be used for
diagnosing neoplasia either independently or in combination with
other diagnostic methods, e.g., diagnostic methods based on genomic
information, proteomic assessment, or histological analysis of
tissue samples. In one embodiment, the methylation ratio of the
present invention is used independently or in combination with
histological analysis in detecting neoplasia.
[0035] In another embodiment, the methylation ratio of a promoter
region of GSTP1 is used independently of histological analysis for
the detection of prostate adenocarcinoma. In general, according to
the present invention the methods of using methylation ratio for
detection of prostate adenocarcinoma as provided by the present
invention is more sensitive than the histological analysis in its
currently available form. A subject having a methylation ratio of a
promoter region of GSTP1 higher than the methylation ratio in a
normal subject, e.g., greater than 5 or 10, is indicative of
prostate adenocarcinoma in light of the fact that the subject is
determined free of prostate adenocarcinoma by histological
analysis.
[0036] Alternatively, the methylation ratio of a promoter region of
GSTP1 can be used in combination with histological analysis for the
detection of prostate adenocarcinoma. For example, according to the
present invention detection of prostate adenocarcinoma in a subject
can include determination of the methylation ratio and a
histological analysis of prostate tissue samples, e.g., needle
biopsy from the subject and a higher than normal methylation ratio,
e.g., greater than 5 or 10, either alone or in combination with a
histological diagnosis of prostate adenocarcinoma is indicative of
prostate adenocarcinoma.
[0037] In yet another embodiment, the methylation ratio of the
present invention can be used independently or in combination with
diagnostics based on proteomic assessment. For example, according
to the present invention the methylation ratio of a promoter region
of GSTP1 in a subject can be used either independently or in
association with the determination of PSA level in the subject in
detecting prostate adenocarcinoma.
[0038] According to the present invention, the methylation ratio of
a promoter region of GSTP1 generally does not directly correlate
with the PSA level, thus the methylation ratio can be used as an
assessment independent of the PSA level for detecting prostate
adenocarcinoma, e.g. a higher than normal methylation ratio of a
promoter region of GSTP1 in a subject is indicative of prostate
adenocarcinoma in the subject with a normal PSA level. Likewise,
the methylation ratio can rule out cancer in a subject with a
falsely high PSA value. Alternatively, detection of prostate
adenocarcinoma can include determining the methylation ratio of a
promoter region of GSTP1 and the PSA level; a higher than normal
methylation ratio either alone or in association with an abnormal
PSA level is indicative of prostate adenocarcinoma.
[0039] According to another aspect of the present invention,
different ways for determination of the methylation level of
biomarkers in association with different types of biological
samples from a subject provide different levels of sensitivity with
respect to neoplasia detection. For example, according to the
present invention for bodily fluid samples determination of the
methylation level of biomarkers using conventional PCR generally
provides better sensitivity than the sensitivity obtained by using
real-time PCR.
[0040] In one embodiment, the methylation level of a promoter
region of GSTP1 in a bodily fluid sample, e.g., not limited to
serum, plasma, urine, or ejaculate as determined by conventional or
non-real-time PCR provides better sensitivity for prostate
adenocarcinoma detection than the sensitivity obtained by using
real-time PCR. Therefore, according to the present invention the
methylation level of a promoter region of GSTP1 in a sample from
bodily fluid from a subject can be determined by using conventional
non-real-time PCR, or real-time PCR and a methylation level higher
than the methylation level in a normal subject is indicative of
prostate adenocarcinoma, e.g., in light of the fact that the
subject has a normal methylation level of the promoter region of
GSTP1 as determined by real-time PCR.
[0041] According to another aspect of the present invention, it
provides kits useful for detecting neoplasia in a cell or tissue,
e.g., using the methods provided by the present invention for the
detection of neoplasia. In one embodiment, the present invention
provides a kit, e.g., a compartmentalized carrier including a first
container containing a pair of primers for amplification of a
biomarker, a second container containing a pair of primers for
amplification of a region in a reference gene, and a third
container containing a first and second oligonucleotide probe
specific for the amplification of the biomarker and the region of
the reference gene, respectively.
[0042] In another embodiment, the kit provided by the present
invention further includes a fourth container containing a
modifying agent that modifies unmethylated cytosine to produce a
converted nucleic acid, e.g., uracil. Any suitable modifying agent
can be included in the kit provided by the present invention. For
example, the modifying agent can be sodium bisulfite.
[0043] In yet another embodiment, the kit provided by the present
invention further includes a probe for PSA determination. In still
another embodiment, the kit provided by the present invention
further includes an instruction insert disclosing normal and/or
abnormal methylation ratio ranges for the detection of neoplasia,
describing the types of samples suitable or unsuitable for the
application of the kit, and/or the specificity or sensitivity
provided by the assays utilizing the kit of the present
invention.
[0044] According to one embodiment of the present invention, the
kit provided by the present invention includes a first container
containing at least one pair of primers for amplification of a
promoter region of GSTP1, a second container containing at least
one pair of primers for amplification of a region of a reference
gene, and a third container containing a first and second
oligonucleotide probe specific to the amplification of the promoter
region of GSTP1 and the region of the reference gene, respectively,
provided that one or both primers for amplification of the promoter
region of GSTP1 or one or more first oligonucleotide probes
specific to the amplicon of the promoter region of GSTP1 are
capable of distinguishing between methylated and unmethylated
nucleic acid, either directly or indirectly, e.g., after bisulfite
modification. Optionally the kit provided by this embodiment of the
present invention can further include an instruction insert, e.g.,
disclosing that a methylation ratio times 1000 of a promoter region
of GSTP1 of the present invention that is greater than 3, 5, or 10,
is indicative of prostate adenocarcinoma or that the kit can be
used with a prostate tissue sample, e.g., most suitable to be used
with a prostate tissue sample.
[0045] The present invention also provides a kit useful for
detecting prostate adenocarcinoma, especially in bodily fluid
samples. The kit includes a first container containing at least one
pair of primers capable of distinguishing between methylated and
unmethylated nucleic acid for amplification of a promoter region of
GSTP1 and an instruction insert disclosing, among other things,
that the kit is useful for detecting prostate adenocarcinoma in a
bodily fluid sample of a subject and that a methylation level of
the promoter region of GSTP1 as determined by conventional or
non-real-time PCR using the primers provided that is higher than
the methylation level of the promoter region of GSTP1 in a normal
subject is indicative of prostate adenocarcinoma in the subject
with a sensitivity no less than 40%.
EXAMPLES
[0046] The following examples are intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
Example 1
Quantitative GSTP1 Methylation Improves the Detection of Prostate
Adenocarcinoma Sextant Biopsies
[0047] In this study, a direct comparison of GSTP1 QMSP was done
with standard histological review of needle biopsies to see if it
could improve the sensitivity of cancer diagnosis. Analysis of
sextant biopsies allowed direct comparison of the two approaches on
tissue with subsequent final pathological diagnosis. The
combination of histology with GSTP1 could significantly improve the
diagnosis of prostate cancer without affecting specificity.
Materials and Methods
Patients and Sample Collection
[0048] 56 patients undergoing prostatectomy for prostate
adenocarcinoma (PSA range 0.5-25.8, median 5.0) and 16 patients
undergoing cystoprostatectomy for bladder carcinoma at The Johns
Hopkins Hospital between November 2001 and May 2002 were included
in this study. Immediately after resection, sextant biopsies (from
left and right apex, mid and base) were taken from all 72 resected
prostate specimens using a thin (18 gauge) needle and promptly
frozen at -80.degree. C.
[0049] All the needle biopsies were cut into 10 .mu.m sections and
placed in a mixture of 1% sodium dodecyl sulfate and proteinase K
(0.5 mg/ml) at 48.degree. C. overnight to digest. DNA was then
extracted using phenol/chloroform and ethanol precipitation as
previously described (10). In addition, 5 .mu.m frozen sections
were taken every 10 slices and stained with hematoxylin-eosin for
blinded examination by light. All the resected prostates were then
serially sectioned and totally submitted for histological
examination, which was considered the gold standard for determining
the presence of prostatic carcinoma.
Bisulfite Treatment
[0050] Sodium bisulfite conversion of 2 .mu.g DNA from each biopsy
sample was performed as described previously. Briefly, DNA samples
were denatured with sodium hydroxide, incubated for 2 hours at
70.degree. C. with sodium bisulfite and hydroquinone, purified
using the Wizard purification kit (Promega, Madison, Wis.), further
denatured and deaminated and finally ethanol precipitated. Samples
were resuspended in 60 .mu.l 10 mM Tris (pH8).
Real-Time Quantitative MSP
[0051] The modified DNA samples were analyzed for methylation of
GSTP1 using fluorogenic real-time QMSP. In addition samples were
analyzed for amplification of 2 internal reference genes, ACTB and
MYOD. Primers and probes for GSTP1(8), MYOD(8) and ACTB (9) have
been described previously. Primers were obtained from Invitrogen
(Gaithersburg, Md.) and probes from Applied Biosystems (Foster
City, Calif.). Reactions were performed on 384 well plates using an
Applied Biosystems 7900 Sequence Detector (Foster City, Calif.).
The final (20 .mu.l) reaction mix consisted of 600 nM each primer,
200 nM probe, 200 .mu.M of each dATP, dCTP, dGTP, dTTP, 16.6 mM
ammonium sulfate, 67 mM Trizma, 6.7 mM magnesium chloride, 10 mM
mercaptoethanol, 0.1% dimethylsulfoxide and 3 .mu.l (100 ng)
modified DNA. Amplification conditions were 95.degree. C.
initiation for 2 mins, followed by 50 cycles at 95.degree. C. for
15 secs and 60.degree. C. for 1 min.
[0052] All samples were run in quadruple and were considered
positive for GSTP1 if 3/4 of the replicates showed amplification.
Each PCR plate also included serial dilutions of 2 positive
controls for construction of standard curves, a negative control
and multiple water blanks. Leucocyte DNA from a healthy individual
was used as the negative control. The same leucocyte DNA was
methylated in vitro with excess Sss I Methyltransferase (New
England Biolabs, Beverly, Mass.) to generate fully methylated DNA
and used as one positive control. In addition, DNA from a human
prostate cancer cell line, LNCaP (ATCC, Manassas, Va.), known to be
methylated at the GSTP1 locus, was also run as a second positive
control.
[0053] With this assay, detection of methylated GSTP1 DNA down to 4
genome equivalents was possible, determined by serial dilutions of
the positive controls using a conversion factor of 6.6 pg DNA per
diploid cell. The relative level of methylated GSTP1 in a
particular sample was calculated using the ratio of the averaged
GSTP1 value to the corresponding internal reference gene value.
This ratio was then multiplied by 1000 for easier tabulation.
[0054] In this study we used 2 different reference genes, MYOD and
ACTB. This was because we have converted from using MYOD as a
reference gene to a more robust ACTB primer/probe set and wished to
directly compare the previously reported GSTP1/MYOD (G/M) values to
GSTP1/ACTB (G/A) values. All statistics and figures are based on
the G/M values but G/A values were virtually identical and the same
threshold values apply using this alternative reference gene.
Statistical Analysis
[0055] Exact binomial 95% confidence intervals are reported for all
proportions. All analyses were done using the case as the unit of
analysis. The statistical significance and precision of increases
in sensitivity were calculated using a McNemar test, taking into
account the pairing of readings within cases. The various
sensitivity-specificity combinations produced by using different
thresholds for GSTP1 are displayed in the form of receiver operator
characteristic (ROC) curves. All calculations were performed with
the statistical package Stata 7.0 (Stata Corp., College Station,
Tex.).
Results
[0056] Seventy-two sets of biopsies, using both histology and GSTP1
methylation (56 known positives and 16 presumed negative) were
analyzed in a blinded manner for the presence of prostate
adenocarcinoma in the resection specimen. Final surgical pathology
review of the cystoprostatectomy specimens detected clinically
undiagnosed prostate adenocarcinoma in 5/16 (31%) cases, which
increased the number of true positives to 61, leaving 11 true
negative cases. This was taken to be the gold standard to which the
blinded biopsy analysis by histology and GSTP1 methylation were
compared. The pathological stages and grades of the 61 cases were:
19 T2a (Gleason 4-7), 29 T2b (Gleason 6-7), 11T3a (Gleason 6-7) and
2 T3b (one Gleason 7 and one ductal carcinoma).
[0057] Only 1 biopsy out of the 6 from each case needed to be
called positive using either test for the case to be called
positive by that test. Based on this, the sensitivity and
specificity of histology and GSTP1 methylation as diagnostic tests
were calculated separately and in combination (Table 1).
TABLE-US-00001 TABLE 1 Sensitivity and Specificity for Histology
and GSTP1 QMSP Using Different Thresholds Sensitivity Histology
Sensitivity and combined Sensitivity increment for combined Test
with GSTP1 GSTP1 alone tests (95% Cl) Histology 64% (39/61) GSTP1
> 10 75% (46/61) 70% (43/61) 11% (5-22) GSTP1 > 5 79% (48/61)
75% (46/61) 15% (7-26) GSTP1 > 2 85% (52/61) 82% (50/61) 21%
(12-34) GSTP1 > 1 89% (54/61) 89% (54/61) 25% (15-37)
[0058] Blinded histological assessment of the biopsies detected
prostate adenocarcinoma in 39/61 cases, a sensitivity of 64% (CI
51-76%). The extent of carcinoma detected on biopsies for each case
ranged from 0-20 mm, median 1 mm. All 11 true negative cases were
found to be negative, a specificity of 100%. As the histologic
criteria applied to the biopsy specimens was the same as that used
for the total prostate examination, a specificity of 100% for
biopsy would be expected unless the biopsy removed all abnormal
cells, so the precision for this 100% estimate is higher than a
statistical confidence interval would indicate, which assumes
independence of a test and gold standard test.
[0059] For GSTP1 alone, using the previously established threshold
value of 10 in pilot studies, cancer was detected in 43/61 cases
(range 0-791.4, median 41.3), a sensitivity of 70% (an example is
shown in FIG. 1). All 11 true negative cases were found to be
negative (range 0-2.5, median 0), a specificity of 100% (CI:
72%-100%). Using GSTP1 in combination with histology (defining
positivity in either as a positive test), 46/61 cases were
correctly diagnosed as positive, increasing sensitivity to 75%, an
11% (CI 5-22%) improvement over histology alone. A specificity of
100% was still maintained using a lower GSTP1 threshold of 5. For
this threshold, GSTP1 alone had a sensitivity of 75% (46/61)
{p=0.06 for being more sensitive than histology}. In combination
with histology, 48/61 cases were correctly diagnosed, a sensitivity
of 79% and a 15% (CI 7-26%) improvement over histology alone. ROC
curves were used to display and contrast the performance of biopsy
alone, GSTP1 alone and GSTP1 in combination with histology (FIG.
2)).
[0060] At the optimum threshold, GSTP1 QMSP analysis correctly
diagnosed 9 cases of prostate cancer missed by histology and
histology detected 2 cases missed by GSTP1 QMSP, which shows the
importance of using both criteria. Pure primary tumor DNA samples
from the 2 cases missed by QMSP were subsequently obtained by
microdissection of paraffin block sections and analyzed for GSTP1
methylation (data not shown). One tumor was GSTP1 negative (0.1 mm
carcinoma detected in one biopsy by histology); the other tumor was
weakly GSTP1 positive and histology detected 1 mm and 0.1 mm
carcinoma on 2/6 biopsies, yet GSTP1 values did not reach the
positive threshold. For the 9 cases detected by GSTP1 QMSP but not
histology, GSTP1 values ranged from 6.2 to 64.2 (median 13.0) and
were positive in 2 biopsies for 3/9 and 1 biopsy for 6/9 cases. One
case was an atypical extensive ductal carcinoma, 3 were small T2a
and 5 were moderate to extensive T2a-b on formal surgical pathology
review.
[0061] The 5 cystoprostatectomy cases found to incidentally contain
prostate cancer included 2/5 containing a moderate extent of cancer
(Gleason 7) present in both lobes, both of which were detected by
GSTP1 QMSP and one of which was detected by histology on biopsy
analysis; the remaining 3/5 cases each contained only a minute
focus of carcinoma (Gleason grade 6 or lower) and were not detected
by either test. The natural progression of these minute incidental
lesions and appropriate therapy (if any) remains to be fully
investigated.
[0062] In order to investigate whether GSTP1 could improve the
sensitivity of routine diagnosis of prostate adenocarcinoma on
needle biopsy, sextant biopsies taken from patients undergoing
prostatectomy for previously diagnosed prostate adenocarcinoma and
from patients undergoing cystoprostatectomy for bladder carcinoma,
with no known diagnosis of prostate cancer were analyzed. These
sextant biopsies were analyzed in a blinded manner for prostate
adenocarcinoma in the resection specimen by histology and by GSTP1
QMSP.
[0063] At an optimal threshold of 5, the addition of GSTP1 QMSP
analysis improved the sensitivity of diagnosis by 15% (from 64 to
79%) while maintaining 100% specificity. The marked improvement in
the diagnosis of a cancer by the addition of a simple molecular
test offers strong support for the use of GSTP1 methylation
analysis prospectively as an adjunct to histology on diagnostic
trans-rectal needle biopsies.
[0064] Histopathology is considered the gold standard by which the
diagnosis of prostate cancer is established. However, on biopsies,
sampling error and various technical artifacts are recognized as
limitations of the approach. This study shows that a significant
number of small cancers (22/61, 36%) can be missed by frozen
section histological analysis of sextant biopsies. This included 18
out of 56 cases that had already been pre-selected with a previous
histological diagnosis of prostate cancer based on a directed or
random needle biopsy prior to prostatectomy. In a population of men
undergoing diagnostic sextant biopsies, this number is likely to be
similar or even higher.
[0065] Histology review on frozen sections is technically more
difficult than from paraffin sections and this may explain why some
tumors may have been missed on histology. A comparison of formalin
fixed paraffin embedded sections to GSTP1 analysis on the same
needle biopsy specimens may have resulted in more favorable results
for histology. Also frozen sections make the diagnosis of high
grade PIN particularly difficult. It is possible that GSTP1
analysis detected high grade PIN in some of the 9 cases missed by
histology (a known histologic marker of cancer).
[0066] However all pure tumor DNA tested from these cases was
strongly positive for GSTP1 and 4/4 other cases, which were
subsequently reported to contain high grade PIN on formal surgical
pathology review, were not found positive by GSTP1 analysis. It is
therefore more likely that GSTP1 QMSP improves sensitivity by
overcoming sampling error. This may be due to pure sensitivity
(able to detect down to 4 cancer cells) or to the recognized field
effect in many cancers, where surrounding cells to the neoplasm
harbor some but not all of the genetic alterations in the primary
tumor and thus do not always display neoplastic morphologic
characterizations.
[0067] For the investigation and diagnosis of prostate
adenocarcinoma, multiple trans-rectal needle biopsies are taken
although only one needs to be found positive to establish the
diagnosis. In this study, as a secondary analysis, we found that
while the same number of biopsies were found positive for both
tests in 37/72 cases, more biopsies from each patient with cancer
were positive by GSTP1 QMSP than by histology in 31/72 cases and
4/72 cases had more biopsies positive on histology than by GSTP1
QMSP. The commonest (modal) number of positive biopsies per case
was 3/6 for GSTP1 QMSP and 1/6 for histology. This supports the
benefit of molecular analysis for every biopsy taken and could be
especially useful for the diagnosis of very small cancers where
evidence suggests that an increased number of biopsies can increase
diagnostic accuracy.
[0068] In each specific case, GSTP1 levels were highest in biopsies
from that case which also had the greatest extent of tumor seen on
histology (as in FIG. 1). However, absolute GSTP1 values did not
directly correlate with a specific amount of tumor seen on biopsy
across all cases due to the wide range of GSTP1 methylation levels
between different primary tumors. GSTP1 QMSP levels did not
correlate with PSA values as shown previously.
[0069] At various threshold ratios from 3-10 for GSTP1 QMSP, all 11
true negative cases were negative although the confidence intervals
are still wide due to the small numbers analyzed. High specificity
is very important for any diagnostic test particularly when
treatment options for diagnosed disease include major surgery. Even
using a very conservative threshold ratio of 10 for GSTP1 QMSP, the
sensitivity of diagnosis was improved substantially (11%),
providing a safe way to improve diagnostic sensitivity for prostate
cancer on needle biopsy.
[0070] At the very least, patients found to have negative histology
but elevated GSTP1 on needle biopsy could be prioritized as high
risk for early repeat biopsy to improve the chance of earliest
possible diagnosis of cancer. Additional imaging techniques could
also be employed to identify suspicious areas for more directed
biopsies. (REF.) The addition of GSTP1 QMSP to routine (paraffin)
histology is likely to improve the sensitivity of diagnostic needle
biopsies by parallel amounts to that shown in this study but
requires further study.
[0071] GSTP1 QMSP is a robust, reproducible and highly specific
diagnostic test for prostate adenocarcinoma that could dramatically
improve the sensitivity of prostate cancer diagnosis when used in
combination with routine histology. Multiple pilot studies and this
current prospective trial continue to support the use of this
molecular approach to improve the accuracy of routine diagnostic
biopsies for prostate cancer.
Example 2
Quantitative GSTP1 Hypermethylation in Bodily Fluids of Prostate
Cancer Patients
[0072] We investigated the potential of GSTP1 hypermethylation
detection in voided urine and plasma DNA as a prostate cancer
specific marker in two groups of patients, one of them harboring
clinically localized prostate cancer, and a control group
consisting of patients with benign prostatic hyperplasia (BPH).
RTQ-MSP was used to quantify the GSTP1 methylation level. The
results were compared to C-MSP. The rationale for the former
approach is that RTQ-MSP allows for rapid analysis of a larger
number of samples in a highly reproducible assay using small
amounts of template DNA. Moreover, quantification may allow
discrimination between benign and neoplastic disease, and could be
useful in monitoring this disease.
Material and Methods
Patients and Sample Collection
[0073] Sixty-nine patients with clinically localized prostate
adenocarcinoma, consecutively diagnosed and primarily treated with
radical prostatectomy at the Portuguese Oncology Institute--Porto,
were selected for this study. All cases were identified by raised
serum prostate specific antigen (PSA) in routine analysis and
confirmed by sextant prostate biopsy (stage T1c). Additionally, 31
patients with BPH, submitted to transurethral resection of the
prostate (TURP), were included for control purposes.
[0074] All histological slides were reviewed and each tumor was
staged (TNM staging system) and graded (Gleason grading system).
Snap-frozen tissue stored at -80.degree. C., or paraffin-embedded
prostatic tissue was collected from each surgical specimen.
Sections were cut for the identification of adenocarcinoma (radical
prostatectomy specimens), and BPH (TURP tissue). For DNA
extraction, these areas were micro-dissected from an average of
fifty 12-.mu.m thick sections for enrichment (>70%) of
adenocarcinoma and hyperplastic tissue. Paraffin-embedded tissue
was similarly micro-dissected, but was placed in xylene for 3 hours
at 48.degree. C. to remove the paraffin. DNA was also extracted
from plasma and voided urine collected from each patient, as
previously described. Briefly, DNA was digested overnight at
48.degree. C. in 1% SDS/Proteinase K (0.5 mg/ml), extracted by
phenol-chloroform, and ethanol precipitated.
Bisulfite Treatment
[0075] To perform the sodium bisulfite conversion of genomic DNA, a
modification of a previously described method was used. Details of
this method are given elsewhere.
Real-Time Quantitative MSP
[0076] Methylation levels of GSTP1 gene promoter and copy number of
MYOD1 gene (used as a control for the amplification quality of the
template DNA) were determined by fluorescence based RTQ-MSP, as
previously described..sup.20 Briefly, primers and probes were
designed to specifically amplify either bisulfite-converted
promoter DNA for the gene of interest, GSTP1. For tissue samples,
the relative level of methylated GSTP1 DNA was expressed as the
ratio between the values of GSTP1 versus MYOD1 obtained by the
RTQ-MSP analysis, in each particular sample, and then multiplied by
1000.
[0077] All plasma and urine samples were also subjected to RTQ-MSP
analysis, both for GSTP1 methylation and MYOD1 gene. The GSTP1
methylation level in bodily fluids was expressed as copies of
methylated GSTP1 (genome equivalents--GE) per 50 ml for urine
samples, and per 1 ml for plasma samples..sup.21 The specificity of
the reaction for the methylated DNA was confirmed separately using
a positive control (LNCaP cell line,) and a negative control (Du145
cell line). Multiple water blanks were included on each plate. The
primer and probe sequences used, were described in a previous
article of ours..sup.15
[0078] The lowest number of genome equivalents detected by RTQ-MSP
was 3.16 GE, determined by serial dilutions of the positive control
(LNCaP DNA). This figure was calculated based on a conversion
factor of 6.6 pg of DNA per cell..sup.22
Conventional MSP
[0079] Primer sequences for either methylated or modified
unmethylated GSTP1 have been described previously. C-MSP was
carried out using the appropriate negative and positive controls as
described above. Forty cycles of PCR were performed using an
annealing temperature of 62.degree. C. The PCR products were
directly loaded onto a non-denaturing 6% polyacrylamide gel,
stained with ethidium bromide, and visualized under UV
illumination.
Statistical Analysis
[0080] Mann-Whitney U tests were carried out to compare the age
distribution and serum PSA levels between the patients with BPH and
those with adenocarcinoma. Correlations between the tumor
methylation ratios and PSA level, Gleason score, and pathological
stage were determined by calculating Spearman's correlation
coefficient. Associations between urine or plasma GSTP1 methylation
status, and pathological stage and Gleason score, were examined
using the chi-square test, and Fisher's exact test. Statistical
analyses were performed with Statistica for Windows, version 6.0
(StatSoft, Tulsa, Okla.), and Epi Info, version 6 (CDC, Atlanta,
Ga.). Statistical significance was reached at P<0.05.
Results
[0081] We prospectively studied 69 patients with clinically
localized prostate adenocarcinoma with a median age of 63 years
(range: 52-74). As a control group, 31 patients with BPH were
included (median age=64 years, range: 53-82). No statistically
significant difference was found between the age distributions of
these two groups (p=0.33). The median value of the preoperative
serum PSA was 10.3 ng/mL (range: 1.69-48.3), and 3.43 ng/mL (range:
0.67-31), for cancer and BPH patients, respectively
(p<1E-5).
[0082] We determined the promotor methylation status of the GSTP1
gene in the tissue samples, both for prostate cancer patients and
for controls, by C-MSP and RTQ-MSP (FIG. 3a). Sixty-three of 69
(91.3%) adenocarcinomas were found to be positive for GSTP1
methylation. No correlation was found between the methylation ratio
in the tumor samples and PSA levels (r=0.04, p=0.74), Gleason score
(r=0.13, p=0.36), or pathological stage (r=0.23, p=0.57). In the
BPH group, 9 of 31 (29%) tissue samples also showed GSTP1
hypermethylation. No discordance was found between the two MSP
methods.
[0083] After screening for methylation changes in tissue, we
analyzed the paired urine and plasma DNA samples, using both MSP
methods in blinded fashion. In every case we were able to amplify
DNA from all samples, i.e., tissue, urine, and plasma. GSTP1
hypermethylation was found in 13 of 69 (18.8%) urine sediments, and
9 of 69 (13.0%) plasma DNA samples from prostate cancer patients,
using RTQ-MSP (FIG. 3b). The median and interquartile ranges (IQR)
of GE of methylated GSTP1 were 3.039 GE/ml (IQR: 0.857-3.529), and
140.533 GE/ml (IQR: 54.6-552,267), for urine and plasma samples,
respectively. C-MSP was able to detect GSTP1 methylation in 21/69
(30.4%) urines, and in 25/69 (36.2%) plasmas from the same
samples.
[0084] Moreover, all cases positive for GSTP1 hypermethylation by
RTQ-MSP (plasma and/or urine) where also positive by C-MSP.
Importantly, there was no case in which urine sediment or plasma
DNA harbored methylation when the corresponding tumor was negative.
No association was found between plasma GSTP1 methylation status
and pathological stage or Gleason score (p=0.84 and p=0.26,
respectively). Likewise, we found no correlation between GSTP1
methylation statuses in urine samples and the pathological stage or
Gleason score (p=0.09 and p=0.83, respectively).
[0085] In BPH patients, GSTP1 hypermethylation was detected in 1/31
(3.2%) urine samples, and both MSP methods were concordant (5.549
GE/ml). The matched BPH tissue did not harbor GSTP1
hypermethylation (representing a potential false positive or
laboratory labeling error: see discussion below). All plasma
samples from BPH patients were negative (using both methods) for
GSTP1 hypermethylation.
[0086] GSTP1 promoter methylation was found in more than 90% of
tumor tissue samples and to a lower degree in paired serum and
urine as previously reported. These findings confirm the high
frequency of this genetic alteration, and continue to support its
application in DNA-based prostate cancer detection approaches. The
median levels of GSTP1 hypermethylation in serum were significantly
higher than urine DNA levels, by quantitative analysis (FIG.
3b).
[0087] This study clearly shows that higher amounts of DNA are
present in plasma than in urine, especially when considering the
much larger total volume that is sampled. This finding could be
related to the extraction of DNA from urine sediments, i.e.,
predominantly from tumor cells shed in urine. Thus, it is suggested
that free tumor DNA is preferentially released into the circulation
rather than the prostate ductal system. These results are also
consistent with the propensity of prostate cancer to disseminate
early throughout the body.
[0088] Among the prostate cancer patients who had GSTP1
hypermethylation in the primary tumor, 37 (53.6%) also displayed
this alteration in urine or plasma DNA using C-MSP. The number of
positive cases in plasma slightly outnumbered those found in urine
(36.2% vs. 30.4%). The same trend was reported in a previous study,
in which 72% of patients were positive in plasma or serum, and only
36% in urine.
[0089] However, there are some major differences between Goessl et
al. and our study, preventing direct comparisons between them.
Goessl et al. included a large number (45%) of stage IV patients
(not amenable to curable surgical resection) in which the
likelihood of circulating tumor cells is rather high, perhaps
resulting in a higher detection rate. Indeed, all advanced stage
patients were positive for GSTP1 methylation in serum in their
study. The rate of detection in urine was also superior to ours,
but in their cases prostatic massage was performed previous to
sample collection, increasing the shedding of prostate cells in
urine.
[0090] The rate of detection in urine found in this study,
reinforces the results of our previous preliminary work. Thus,
several strategies can be considered to improve the detection rate
of GSTP1 hypermethylation in bodily fluids. One approach would be
to increased the number and/or volume of urine and plasma samples,
enabling a larger sampling of tumor DNA.
[0091] Moreover, prostatic massage might increase cell shedding in
urine, but this procedure could limit the acceptability of the
test. Although a higher rate of GSTP1 hypermethylation was detected
in ejaculates (approaching 50%), the nature of the sampling
procedure, especially in older men, may preclude its widespread
use. Eventually, further technical refinements of the PCR method
could contribute to an increase in sensitivity, although these
procedures have been substantially optimized.
[0092] The specificity of GSTP1 hypermethylation remains high since
it was rarely detected in the urine and plasma DNA from patients in
whom this marker was not altered in the tumor tissue. Moreover,
GSTP1 methylation has not been generally detected in other
genitourinary malignancies, including bladder carcinomas.
[0093] Thirty-one BPH patients, with no evidence of harboring
prostate adenocarcinoma were used as controls. Although GSTP1
hypermethylation was reported to be rare in normal tissue, 9 of
these patients (29%) displayed this alteration in prostatic tissue.
Our findings could be explained by age-related GSTP1
hypermethylation, since recent evidence suggests that promoter
methylation of certain genes in normal-appearing tissues is
associated with aging. However, we saw no age-related patterns in
our sample set (both BPH and cancer). Moreover, we cannot disregard
the possibility that small foci of adenocarcinoma with GSTP1
hypermethylation could have been resected during the TURP
procedure, along with hyperplastic glands.
[0094] In one BPH patient, GSTP1 hypermethylation was detected in
urine but not in matched tissue, by both MSP methods. This result
could be interpreted as a false positive, diminishing the
specificity of this method. In our patients with prostate cancer no
hypermethylation was detected in urine or plasma DNA of paired
unmethylated tumors. Thus, it is tempting to suggest that this BPH
patient could harbor occult prostate adenocarcinoma, localized in
the peripheral region of the organ, not sampled by TURP. Careful
follow-up may clarify this interesting observation.
[0095] In previous studies, promoter hypermethylation of several
genes has been successfully used to detect tumor DNA in bodily
fluids from several types of cancer, namely bronchial lavage fluid,
sputum, and serum from lung cancer patients, and serum from head
and neck cancer patients. In these studies, C-MSP method was found
to have a high sensitivity (1:1000). However, this method does not
permit quantification of the extent of gene methylation status.
[0096] In this study, a larger number of urine and plasma samples
were positive for GSTP1 hypermethylation using C-MSP, comparing to
RTQ-MSP (53.6% vs. 31.9%). This finding suggests that the former
method is significantly more sensitive than the latter, perhaps due
to the greater specificity of the internal probe designed for
quantitative analysis and the high background level of fluorescence
intrinsic to the RTQ-MSP analysis.
[0097] Notwithstanding, the lower limit of RTQ-MSP detection
determined in the present study (3.16 GE) was more sensitive than
the level reported by Lo et al. (10 GE) in myeloma. However, the
amount of DNA from prostate cancer cells present in urine and
plasma may be very low, impairing its detection by RTQ-MSP. Indeed,
Lo and co-workers were able to detect hypermethylation in
reasonable amounts of cells obtained from bone marrow aspirates of
their patients.
[0098] These results suggest that RTQ-MSP could be particularly
useful in the identification of neoplastic disease in cell-rich
clinical material, such as needle biopsies. In this regard, RTQ-MSP
has the advantage of enabling the quantification of the number of
GSTP1 methylated copies, which may allow the discrimination between
methylated normal tissue and carcinoma.
[0099] GSTP1 hypermethylation may be detected in urine and plasma
in a large proportion of early stage prostate cancer patients
harboring DNA methylation in the tissue as shown herein. Because so
many patients die of prostate cancer each year, these results could
have significant implications for the development of molecular
approaches as adjuncts to cancer detection. Furthermore, such
assays may be useful in patient monitoring and detection of minimal
residual disease, once the GSTP1 methylation status of the primary
tumor is established.
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[0138] U.S. Pat. Nos. 6,265,171; 6,200,756; 6,017,704; 5,786,146;
5,552,277 (all herein incorporated by reference).
[0139] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
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