U.S. patent application number 12/898571 was filed with the patent office on 2011-04-21 for methylation of estrogen receptor alpha and uses thereof.
Invention is credited to Dave S.B. Hoon.
Application Number | 20110091970 12/898571 |
Document ID | / |
Family ID | 38625489 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091970 |
Kind Code |
A1 |
Hoon; Dave S.B. |
April 21, 2011 |
METHYLATION OF ESTROGEN RECEPTOR ALPHA AND USES THEREOF
Abstract
Methods for diagnosis, prognosis, and treatment of cancer based
on the methylation status of the ER-.alpha. gene promoter are
disclosed. Methylation of the ER-.alpha. gene promoter is
indicative of cancer and unfavorable prognosis. The cancer can be
treated with a demethylation agent.
Inventors: |
Hoon; Dave S.B.; (Los
Angeles, CA) |
Family ID: |
38625489 |
Appl. No.: |
12/898571 |
Filed: |
October 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11693673 |
Mar 29, 2007 |
7829283 |
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12898571 |
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60787719 |
Mar 29, 2006 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
A61P 1/18 20180101; C12Q
2600/118 20130101; C12Q 2600/154 20130101; C12Q 2600/112 20130101;
A61P 1/16 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61P
17/00 20180101; A61P 1/04 20180101; C12Q 1/6886 20130101; C12Q
2600/106 20130101; C12Q 2600/156 20130101; A61P 35/04 20180101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/09 20100101
C12N005/09 |
Goverment Interests
FUNDING
[0002] This invention was made with support in part by grants from
NIH (NCI Project II P0 CA029605, CA012582, and R33-CA100314).
Therefore, the U.S. government has certain rights.
Claims
1. A method of reducing DNA methylation in a cell, comprising
contacting a melanoma, pancreatic cancer, hepatacellular cancer,
esophageal cancer, sarcoma, or gastric cancer cell with a
demethylation agent, thereby reducing methylation of the ER-.alpha.
gene promoter in the cell.
2. The method of claim 1, wherein the demethylation agent is
5-aza-2-deoxycytidine.
3. The method of claim 1, further comprising contacting the cell
with an HDAC (histone deacetylase) inhibitor.
4. The method of claim 3, wherein the HDAC inhibitor is
trichostatin A.
5. The method of claim 1, wherein the melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer is primary or metastatic melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/693,673, which claims priority to U.S. Provisional
Application Ser. No. 60/787,719, filed on Mar. 29, 2006. Both of
these applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates in general to the ER-.alpha.
(estrogen receptor alpha) gene. More specifically, the invention
relates to methylation of the ER-.alpha. gene promoter and its
utility in cancer diagnosis, prognosis, and treatment.
BACKGROUND OF THE INVENTION
[0004] Because it is difficult to predict which primary tumors will
progress to regional or distant metastases, cutaneous melanoma
remains a challenging disease to manage (1). New strategies for the
identification of epigenetic biomarkers may improve the clinical
management of melanoma by facilitating earlier disease diagnosis
and providing more accurate prognostic information. No major study
has examined the epigenetic alterations of hormone receptors in the
progression from primary to metastatic melanoma in a large series
of patients.
[0005] Hypermethylation of gene promoter CpG islands plays a
significant role in the development and progression of various
cancers, including melanoma (2-6). The identification of
hypermethylated genes in tumors has become an accepted approach to
assess tumor-related gene inactivation (6-9). Tumor-related gene
hypermethylation in primary and metastatic melanomas was previously
reported (10). Thereafter, the hypermethylation of multiple
tumor-related and tumor suppressor genes during progression from
primary to metastatic lesions was demonstrated (11). Several genes
methylated in primary and metastatic melanomas were also detected
in serum as methylated circulating DNA (11). The observation that
tumor-related DNA could be detected in circulating serum provided a
method of disease surveillance independent of the availability of
gross tumor tissue (12-17).
[0006] ER-.alpha. belongs to a superfamily of transcription
activators (18, 19) involved in many physiological processes,
including tumor progression (20-22). Loss of ER-.alpha. expression
has been associated with aberrant CpG island hypermethylation in
breast cancer cell lines and tumors (23-27), and shown to modulate
breast cancer progression (5). Several studies have reported the
presence of estrogen receptor in melanoma cell lines, but analysis
of human melanomas have shown variable ER-.alpha. expression
(28-31). Several in vitro experiments established that tamoxifen is
an effective growth inhibitor of melanoma cells (32, 33). Based on
the variable presence of ER-.alpha. in melanoma cells, as well as
anecdotal reports of clinical responses to anti-estrogen therapy,
several studies of hormonal and chemohormonal treatments were
coordinated. Initial trials were encouraging, with improved
response rates and median overall survival in patients, receiving
tamoxifen, particularly women (34, 35). Subsequent trials, however,
failed to show significant differences in response rates or overall
survival when tamoxifen was used alone or in combination with
systemic therapies (36-42). Reasons for the discrepancies in
response to anti-estrogen therapy between these trials are
unknown.
SUMMARY OF THE INVENTION
[0007] This invention relates to the utility of methylation of the
ER-.alpha. gene promoter in diagnosis, prognosis, and treatment of
cancer.
[0008] In one aspect, the invention provides methods for diagnosis
and prognosis of cancer based on methylation of the ER-.alpha. gene
promoter in acellular DNA in a body fluid of a subject.
[0009] More specifically, the invention features a method of
determining whether a subject is suffering from cancer. The method
comprises (1) providing a body fluid sample from a subject, wherein
the sample contains DNA that exists as acellular DNA in the body
fluid; and (2) determining the methylation level of the ER-.alpha.
gene promoter in the DNA. The methylation level of the ER-.alpha.
gene promoter in the DNA, if higher than a control methylation
level, indicates that the subject is likely to be suffering from
cancer.
[0010] The invention also features a method of determining the
outcome of cancer. The method comprises (1) providing a body fluid
sample from a subject suffering from cancer, wherein the sample
contains DNA that exists as acellular DNA in the body fluid; and
(2) determining the methylation level of the ER-.alpha. gene
promoter in the DNA. The methylation level of the ER-.alpha. gene
promoter in the DNA, if higher than a control methylation level,
indicates that the subject is likely to have an unfavorable outcome
of the cancer. In one embodiment, the higher methylation level of
the ER-.alpha. gene promoter in the DNA is indicative of a
decreased response to a cancer therapy, progression-free survival,
or overall survival.
[0011] In the methods described above, the cancer may be melanoma,
colorectal cancer, pancreatic cancer, hepatacellular cancer,
esophageal cancer, sarcoma, lung cancer, breast cancer, or gastric
cancer; the cancer may be a primary or metastatic cancer; the
sample may be a serum, plasma, peritoneal/pleural, or cerebral
spinal sample.
[0012] In another aspect, the invention provides a method for
prognosis of cancer based on methylation of the ER-.alpha. gene
promoter in cellular DNA contained in a PE (paraffin-embedded)
cancer tissue sample of a subject. More specifically, the invention
features a method of determining the outcome of cancer. The method
comprises (1) providing a PE cancer tissue sample of a subject,
wherein the sample contains cellular DNA; and (2) determining the
methylation level of the ER-.alpha. gene promoter in the DNA. The
methylation level of the ER-.alpha. gene promoter in the DNA, if
higher than a control methylation level, indicates that the subject
is likely to have an unfavorable outcome of the cancer. In this
method, the cancer may be melanoma, colorectal cancer, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, lung
cancer, breast cancer, or gastric cancer; the cancer may be a
primary or metastatic cancer.
[0013] In addition, the invention provides a method for prognosis
of cancer based on methylation of the ER-.alpha. gene promoter in
cellular DNA contained in a cancer tissue or cancer cells in a body
fluid from a subject. More specifically, the invention features a
method of determining the outcome of cancer. The method comprises
(1) providing a cancer tissue sample or a body fluid sample from a
subject, wherein the sample contains cellular DNA, the body fluid
contains cancer cells, and the subject is suffering from melanoma,
pancreatic cancer, hepatacellular cancer, esophageal cancer,
sarcoma, or gastric cancer; and (2) determining the methylation
level of the ER-.alpha. gene promoter in the DNA. The methylation
level of the ER-.alpha. gene promoter in the DNA, if higher than a
control methylation level, indicates that the subject is likely to
have an unfavorable outcome of the melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer. In this method, the melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer may be primary or metastatic melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer.
[0014] Also within the invention is a method of treating cancer by
reducing methylation of the ER-.alpha. gene promoter in a cell.
More specifically, the invention features a method of reducing DNA
methylation in a cell, comprising contacting a melanoma, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, or
gastric cancer cell with a demethylation agent (e.g.,
5-aza-2-deoxycytidine), thereby reducing methylation of the
ER-.alpha. gene promoter in the cell. The method may further
comprise contacting the cell with a histone deacetylase inhibitor
such as trichostatin A. In this method, the melanoma, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, or
gastric cancer may be primary or metastatic melanoma, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, or
gastric cancer.
[0015] The above-mentioned and other features of this invention and
the manner of obtaining and using them will become more apparent,
and will be best understood, by reference to the following
description, taken in conjunction with the accompanying drawings.
These drawings depict only typical embodiments of the invention and
do not therefore limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows representative expression and re-expression of
ER-.alpha. in three melanoma lines (MCA, MCB, MCC) treated with
5-Aza (5-aza-2-deoxycytidine) and TSA (trichostatin A). mRNA
expression was analyzed by RT-PCR (reverse-transcription polymerase
chain reaction). The housekeeping gene GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) was included as a RT-PCR
control. NT: cell line not treated with 5-Aza and TSA. T: cell line
treated with 5-Aza and TSA.
[0017] FIG. 2 depicts representative MSP (methylation-specific
polymerase chain reaction) results of melanoma cell line (MCA) with
and without 5-Aza plus TSA treatment. M: methylated-specific
product. U: unmethylated-specific product. Only a methylated peak
was initially observed (untreated). An unmethylated peak appeared
after treatment with 5-Aza plus TSA (treated).
[0018] FIG. 3. A. The frequency of methylated ER-.alpha. DNA in
melanoma tumors according to AJCC (American Joint Committee on
Cancer) stage. Prim: Primary melanoma tumor. Met: Metastatic
melanoma tumor. B. The frequency of methylated ER-.alpha. DNA in
melanoma patients' sera according to AJCC stage. Norm <50:
Normal healthy volunteers younger than 50 years. Norm .gtoreq.60:
Normal healthy volunteers aged 60 years or older.
[0019] FIG. 4 illustrates representative MSP results of sera and
tissue specimens. No methylation peak appeared in serum of healthy
donor (a). A methylation peak appeared in normal liver tissue (b).
A single methylation peak was detected in sera and PE specimens
from stage IV melanoma patients (c-h). Figures e and f are paired
specimens from the same patient.
[0020] FIG. 5. A. Kaplan-Meier curves showing the correlation of
pre-BC (biochemotherapy) serum ER-.alpha. methylation status with
progression-free survival (Cox proportional hazard, p=0.004).
Methylated: Patients with serum methylated ER-.alpha. DNA; No
methylation: Patients with no detectable serum methylated
ER-.alpha.. B. Kaplan-Meier curves showing the correlation of
pre-BC serum ER-.alpha. methylation status with overall survival
(Cox proportional hazard, p=0.003). Methylated: Patients with serum
methylated ER-.alpha.; No methylation: Patients with no detectable
serum methylated ER-.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0021] The role of ER-.alpha. in melanoma is unknown. Mechanisms
regulating the expression of ER-.alpha. in melanoma are poorly
defined; to date, no mutation or other gross structural alteration
of the ER-.alpha. gene has been reported in melanoma.
[0022] The invention is based at least in part upon the unexpected
discovery that ER-.alpha. gene silencing via gene promoter
hypermethylation in primary and metastatic melanoma plays an
important role in melanoma progression, and can be used as a
prognostic molecular biomarker. More specifically, ER-.alpha.
hypermethylation in primary and metastatic melanomas and sera as a
potential tumor progression marker was assessed. ER-.alpha.
methylation status in tumor (n=107) and sera (n=109) from AJCC
stage I-IV melanoma patients was examined by MSP. The clinical
significance of serum methylated ER-.alpha. was assessed among AJCC
stage IV melanoma patients receiving BC with tamoxifen. Rates of
ER-.alpha. methylation in AJCC stage I, II, and III primary
melanomas were 36% (4 of 11), 26% (5 of 19), and 35% (8 of 23),
respectively. Methylated ER-.alpha. was detected in 42% (8 of 19)
of stage III and 86% (30 of 35) of stage IV metastatic melanomas.
ER-.alpha. was methylated more frequently in metastatic than
primary melanomas (p=0.0003). Of 109 melanoma patients' sera in
AJCC stage I, II, III, and IV, methylated ER-.alpha. was detected
in 10% (2 of 20), 15% (3 of 20), 26% (5 of 19), and 32% (16 of 50),
respectively. Serum methylated ER-.alpha. was detected more
frequently in advanced than localized melanomas (p=0.03) and was
the only factor predicting progression-free (RR 2.64, 95%
confidence interval (CI) 1.36-5.13, p=0.004) and overall survival
(RR 2.31, 95% CI 1.41-5.58, p=0.003) in BC patients.
Hypermethylated ER-.alpha. is a significant factor in melanoma
progression. Serum methylated ER-.alpha. is an unfavorable
prognostic factor.
[0023] Accordingly, the invention provides various methods for
cancer diagnosis, prognosis, and treatment. A diagnostic method of
the invention generally involves analyzing the methylation level of
the ER-.alpha. gene promoter in a biological sample from a subject.
If the methylation level of the ER-.alpha. gene promoter in the
sample is higher than a control value, the subject is likely to be
suffering from cancer.
[0024] One diagnostic method of the invention involves a body fluid
sample from a subject. The sample contains DNA that exists as
acellular DNA in the body fluid. The methylation level of the
ER-.alpha. gene promoter in the DNA is determined. If the
methylation level of the ER-.alpha. gene promoter in the DNA is
higher than a control methylation level, the subject is likely to
be suffering from cancer.
[0025] As used herein, a "subject" refers to a human or animal,
including all mammals such as primates (particularly higher
primates), sheep, dog, rodents (e.g., mouse or rat), guinea pig,
goat, pig, cat, rabbit, and cow. In a preferred embodiment, the
subject is a human. In another embodiment, the subject is an
experimental animal or animal suitable as a disease model.
[0026] The term "body fluid" refers to any body fluid in which
acellular DNA or cells (e.g., cancer cells) may be present,
including, without limitation, blood, serum, plasma, bone marrow,
cerebral spinal fluid, peritoneal/pleural fluid, lymph fluid,
ascite, serous fluid, sputum, lacrimal fluid, stool, and urine.
"Acellular DNA" refers to DNA that exists outside a cell in a body
fluid of a subject or the isolated form of such DNA, while
"cellular DNA" refers to DNA that exists within a cell or is
isolated from a cell.
[0027] Body fluid samples can be obtained from a subject using any
of the methods well known in the art. Methods for extracting
acellular DNA from these samples are also well known in the art.
Commonly, acellular DNA in a body fluid sample is separated from
cells, precipitated in alcohol, and dissolved in an aqueous
solution.
[0028] A "promoter" is a region of DNA extending 150-300 bp
upstream from the transcription start site that contains binding
sites for RNA polymerase and a number of proteins that regulate the
rate of transcription of the adjacent gene. The promoter region of
the ER-.alpha. gene is well known in the art. Methylation of the
ER-.alpha. gene promoter can be assessed by any method commonly
used in the art, for example, MSP, bisulfite sequencing, or
pyrosequencing.
[0029] MSP is a technique whereby DNA is amplified by PCR dependent
upon the methylation state of the DNA. See, e.g., U.S. Pat. No.
6,017,704. Determination of the methylation state of a nucleic acid
includes amplifying the nucleic acid by means of oligonucleotide
primers that distinguish between methylated and unmethylated
nucleic acids. MSP can rapidly assess the methylation status of
virtually any group of CpG sites within a CpG island, independent
of the use of methylation-sensitive restriction enzymes. This assay
entails initial modification of DNA by sodium bisulfite, converting
all unmethylated, but not methylated, cytosines to uracils, and
subsequent amplification with primers specific for methylated
versus unmethylated DNA. MSP requires only small quantities of DNA,
is sensitive to 0.1% methylated alleles of a given CpG island
locus, and can be performed on DNA extracted from body fluid,
tissue, and PE samples. MSP eliminates the false positive results
inherent to previous PCR-based approaches which relied on
differential restriction enzyme cleavage to distinguish methylated
from unmethylated DNA. This method is very simple and can be used
on small amounts of tissue or few cells and fresh, frozen, or PE
sections. MSP product can be detected by gel electrophoresis, CAE
(capillary array electrophoresis), or real-time quantitative
PCR.
[0030] Bisulfite sequencing is widely used to detect 5-MeC
(5-methylcytosine) in DNA, and provides a reliable way of detecting
any methylated cytosine at single-molecule resolution in any
sequence context. The process of bisulfite treatment exploits the
different sensitivity of cytosine and 5-MeC to deamination by
bisulfite under acidic conditions, in which cytosine undergoes
conversion to uracil while 5-MeC remains unreactive.
[0031] To determine whether a subject (i.e., a test subject) is
suffering from cancer, the methylation level of the ER-.alpha. gene
promoter in the acellular DNA of the test subject is compared with
a control value. A suitable control value may be, e.g., the
methylation level of the ER-.alpha. gene promoter in acellular DNA
from a body fluid of a normal subject. If the methylation level of
the ER-.alpha. gene promoter in the acellular DNA from the test
subject is higher than the control value, the test subject is
likely to be suffering from cancer.
[0032] As used herein, "cancer" refers to a disease or disorder
characterized by uncontrolled division of cells and the ability of
these cells to spread, either by direct growth into adjacent tissue
through invasion, or by implantation into distant sites by
metastasis. Exemplary cancers include, but are not limited to,
primary cancer, metastatic cancer, AJCC stage I, II, III, or IV
cancer, carcinoma, lymphoma, leukemia, sarcoma, mesothelioma,
glioma, germinoma, choriocarcinoma, prostate cancer, lung cancer,
breast cancer, colorectal cancer, gastrointestinal cancer, bladder
cancer, pancreatic cancer, endometrial cancer, ovarian cancer,
melanoma, brain cancer, testicular cancer, kidney cancer, skin
cancer, thyroid cancer, head and neck cancer, hepatacellular
cancer, esophageal cancer, gastric cancer, intestinal cancer, colon
cancer, rectal cancer, myeloma, neuroblastoma, and retinoblastoma.
Preferably, the cancer is a cancer associated with the biological
function of the ER-.alpha. gene, such as melanoma, colorectal
cancer, pancreatic cancer, hepatacellular cancer, esophageal
cancer, sarcoma, lung cancer, breast cancer, and gastric
cancer.
[0033] A prognostic method of the invention generally involves
analyzing the methylation level of the ER-.alpha. gene promoter in
a biological sample from a subject suffering from cancer. If the
methylation level of the ER-.alpha. gene promoter in the sample is
higher than a control value, the subject is likely to have an
unfavorable outcome of the cancer. For instance, the subject may
have a decreased response to a cancer therapy such as BC,
progression-free survival, or overall survival.
[0034] One prognostic method of the invention involves a body fluid
sample from a subject suffering from cancer. The sample contains
DNA that exists as acellular DNA in the body fluid. Another
prognostic method of the invention involves a PE cancer tissue
sample of a subject. The sample contains cellular DNA. In both
methods, the methylation level of the ER-.alpha. gene promoter in
the DNA is determined. If the methylation level of the ER-.alpha.
gene promoter in the DNA is higher than a control methylation
level, the subject is likely to have an unfavorable outcome of the
cancer.
[0035] A third prognostic method of the invention involves a cancer
tissue sample or a body fluid sample from a subject. The sample
contains cellular DNA. The body fluid contains cancer cells. The
subject is suffering from melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer. The methylation level of the ER-.alpha. gene promoter in
the DNA is determined. If the methylation level of the ER-.alpha.
gene promoter in the DNA is higher than a control methylation
level, the subject is likely to have an unfavorable outcome of the
melanoma, pancreatic cancer, hepatacellular cancer, esophageal
cancer, sarcoma, or gastric cancer.
[0036] A tissue sample from a subject may be a biopsy specimen
sample, a normal or benign tissue sample, a cancer or tumor tissue
sample, a freshly prepared tissue sample, a frozen tissue sample, a
PE tissue sample, a primary cancer or tumor sample, or a metastasis
sample. Exemplary tissues include, but are not limited to,
epithelial, connective, muscle, nervous, heart, lung, brain, eye,
stomach, spleen, bone, pancreatic, kidney, gastrointestinal, skin,
uterus, thymus, lymph node, colon, breast, prostate, ovarian,
esophageal, head, neck, rectal, testis, throat, thyroid,
intestinal, melanocytic, colorectal, hepatacellular, gastric, and
bladder tissues.
[0037] To practice the prognostic methods of the invention,
acellular DNA can be obtained using the methods described above.
Tissue samples can be obtained from a subject using any of the
methods well known in the art. Methods for extracting cellular DNA
from tissue and body fluid samples are also well known in the art.
Typically, cells are lysed with detergents. After cell lysis,
proteins are removed from DNA using various proteases. DNA is then
extracted with phenol, precipitated in alcohol, and dissolved in an
aqueous solution.
[0038] The methylation level of the ER-.alpha. gene promoter in
acellular and cellular DNA can be determined using the methods
described above and compared with corresponding control values. As
mentioned above, a control value for the methylation level of the
ER-.alpha. gene promoter in the acellular DNA of a test subject may
be, e.g., the methylation level of the ER-.alpha. gene promoter in
acellular DNA from a body fluid of a normal subject. A control
value for the methylation level of the ER-.alpha. gene promoter in
the cellular DNA of a test subject may be, e.g., the methylation
level of the ER-.alpha. gene promoter in cellular DNA from a cell
line, a tissue, or cells in a body fluid where methylation of the
ER-.alpha. gene promoter is non-detectable. Preferably, the control
cell line is a cancer cell line and the control tissue is a cancer
tissue, where the control cell line, the control tissue, and the
cancer tissue from the test subject are of the same cancer type.
The methylation level of the ER-.alpha. gene promoter in the DNA
from the test subject, if higher than the control value, is
indicative of an unfavorable outcome of cancer.
[0039] The discovery that the ER-.alpha. gene promoter is
methylated in melanoma, pancreatic cancer, hepatacellular cancer,
esophageal cancer, sarcoma, and gastric cancer cells is useful for
identifying compounds for treating melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, and gastric
cancer. For example, a melanoma, pancreatic cancer, hepatacellular
cancer, esophageal cancer, sarcoma, or gastric cancer cell may be
contacted with a test compound. The methylation levels of the
ER-.alpha. gene promoter in the cell prior to and after the
contacting step are compared. If the methylation level of the
ER-.alpha. gene promoter in the cell decreases after the contacting
step, the test compound is identified as a candidate compound for
treating melanoma, pancreatic cancer, hepatacellular cancer,
esophageal cancer, sarcoma, or gastric cancer.
[0040] Similarly, a subject suffering from melanoma, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, or
gastric cancer may be contacted with a test compound. Samples of
cancer tissues or body fluids containing cancer cells or acellular
DNA are obtained from the subject. The methylation level of the
ER-.alpha. gene promoter in cellular or acellular DNA in a sample
obtained from the subject prior to the contacting step is compared
with the methylation level of the ER-.alpha. gene promoter in
cellular or acellular DNA in a sample obtained from the subject
after the contacting step. If the methylation level of the
ER-.alpha. gene promoter in cellular or acellular DNA decreases
after the contacting step, the test compound is identified as a
candidate compound for treating melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer.
[0041] The test compounds of the present invention can be obtained
using any of the numerous approaches (e.g., combinatorial library
methods) known in the art. See, e.g., U.S. Pat. No. 6,462,187. Such
libraries include, without limitation, peptide libraries, peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone that is resistant
to enzymatic degradation), spatially addressable parallel solid
phase or solution phase libraries, synthetic libraries obtained by
deconvolution or affinity chromatography selection, and the
"one-bead one-compound" libraries. Compounds in the last three
libraries can be peptides, non-peptide oligomers, or small
molecules. Examples of methods for synthesizing molecular libraries
can be found in the art. Libraries of compounds may be presented in
solution, or on beads, chips, bacteria, spores, plasmids, or
phages.
[0042] The candidate compounds so identified, as well as compounds
known to demethylate DNA (i.e., demethylation agents such as 5-Aza)
in a cell or subject, can be used to demethylate the ER-.alpha.
gene promoter in melanoma, pancreatic cancer, hepatacellular
cancer, esophageal cancer, sarcoma, and gastric cancer cells in
vitro and in vivo. In one embodiment, the method involves
contacting a melanoma, pancreatic cancer, hepatacellular cancer,
esophageal cancer, sarcoma, or gastric cancer cell with a
demethylation agent, thereby reducing methylation of the ER-.alpha.
gene promoter in the cell. To treat a subject suffering from
melanoma, pancreatic cancer, hepatacellular cancer, esophageal
cancer, sarcoma, or gastric cancer, an effective amount of a
demethylation agent is administered to the subject to reduce the
methylation level of the ER-.alpha. gene promoter in the subject. A
subject to be treated may be identified in the judgment of the
subject or a health care professional, and can be subjective (e.g.,
opinion) or objective (e.g., measurable by a test or diagnostic
method such as those described above).
[0043] A "treatment" is defined as administration of a substance to
a subject with the purpose to cure, alleviate, relieve, remedy,
prevent, or ameliorate a disorder, symptoms of the disorder, a
disease state secondary to the disorder, or predisposition toward
the disorder.
[0044] An "effective amount" is an amount of a compound that is
capable of producing a medically desirable result in a treated
subject. The medically desirable result may be objective (i.e.,
measurable by some test or marker) or subjective (i.e., subject
gives an indication of or feels an effect).
[0045] In some embodiments, a melanoma, pancreatic cancer,
hepatacellular cancer, esophageal cancer, sarcoma, or gastric
cancer cell or a subject suffering from melanoma, pancreatic
cancer, hepatacellular cancer, esophageal cancer, sarcoma, or
gastric cancer is further treated with other compounds or
radiotherapy. For example, one type of other compounds are HDAC
(histone deacetylase) inhibitors such as TSA (trichostatin A) which
can modify histones in chromatin regions and activate genes
silenced by methylation of CpG islands in promoter regions.
[0046] For treatment of cancer, a compound is preferably delivered
directly to tumor cells, e.g., to a tumor or a tumor bed following
surgical excision of the tumor, in order to treat any remaining
tumor cells. For prevention of cancer invasion and metastases, the
compound can be administered to, for example, a subject that has
not yet developed detectable invasion and metastases but is found
to have increased methylation level of the ER-.alpha. gene
promoter.
[0047] The compounds of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the compounds and pharmaceutically acceptable carriers.
"Pharmaceutically acceptable carriers" include solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration.
[0048] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. See, e.g., U.S. Pat. No.
6,756,196. Examples of routes of administration include parenteral,
e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates; and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0049] In one embodiment, the compounds are prepared with carriers
that will protect the compounds against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0050] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form," as used herein, refers to
physically discrete units suited as unitary dosages for the subject
to be treated, each unit containing a predetermined quantity of an
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0051] The dosage required for treating a subject depends on the
choice of the route of administration, the nature of the
formulation, the nature of the subject's illness, the subject's
size, weight, surface area, age, and sex, other drugs being
administered, and the judgment of the attending physician. Suitable
dosages are in the range of 0.01-100.0 mg/kg. Wide variations in
the needed dosage are to be expected in view of the variety of
compounds available and the different efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization as is
well understood in the art. Encapsulation of the compound in a
suitable delivery vehicle (e.g., polymeric microparticles or
implantable devices) may increase the efficiency of delivery,
particularly for oral delivery.
[0052] The following examples are intended to illustrate, but not
to limit, the scope of the invention. While such examples are
typical of those that might be used, other procedures known to
those skilled in the art may alternatively be utilized. Indeed,
those of ordinary skill in the art can readily envision and produce
further embodiments, based on the teachings herein, without undue
experimentation.
EXAMPLE I
ER-A Methylation Predicts Melanoma Progression
Materials and Methods
Melanoma Cell Line and Tumor DNA Isolation
[0053] DNA was extracted from 11 melanoma cell lines established
from metastatic tumors at John Wayne Cancer Institute (JWCI) and
one breast cancer cell line (MCF-7) from American Type Culture
Collection (ATCC) (Manassas, Va.) as described previously (14).
Institutional Review Board approval for the use of human tissues
was obtained from Saint John's Health Center and JWCI prior to
beginning the study. Patients who underwent surgery for AJCC stage
I, II, III, and IV melanoma (11 stage I primary tumors; 19 stage II
primary tumors; 23 stage III primary tumors; 19 stage III
metastatic tumors; and 35 stage IV metastatic tumors) were selected
consecutively by the database coordinator from the institutional
melanoma patient and specimen database (Table 1A). PE tumor
specimens from these patients were obtained from the Division of
Surgical Pathology at Saint John's Health Center.
TABLE-US-00001 TABLE 1A Clinical characteristics of melanoma
patients Patient Characteristics n (%) Total patients (tissue) 107
Sex male 58 (54%) female 49 (46%) Age (median) <50 25 (23%)
.gtoreq.60 50 (47%) Stage I 11 (10%) II 19 (18%) III (primary) 23
(21%) III (metastasis) 19 (18%) IV (metastasis) 35 (33%) Total
patients (serum) 109 Sex male 73 (67%) female 34 (31%) unknown 2
(2%) Age (median 45) <50 43 (41%) .gtoreq.60 51 (48%) Stage I 20
(19%) II 20 (19%) III 19 (18%) IV 50 (48%)
[0054] Several 8-.mu.m sections were cut from formalin-fixed, PE
blocks as described previously (43). One section from each tumor
block was deparaffinized, mounted on a glass slide, and stained
with hematoxylin and eosin for microscopic analysis. Light
microscopy was used to confirm tumor location and assess tissue
homogeneity. Additional sections from the tumor block were mounted
on glass slides and microdissected under light microscopy.
Dissected tissues were digested with 50 .mu.l of proteinase
K-containing lysis buffer at 50.degree. C. for 12 hr, followed by
heat deactivation of proteinase K at 95.degree. C. for 10 min (5).
DNA was extracted as previously described (10).
Serum DNA Isolation
[0055] AJCC stage I (n=20), stage II (n=20), stage III (n=19), and
stage IV patients (n=50) diagnosed with melanoma were assessed for
this study (Table 1A). Stage I, II, and III patients received no
additional adjuvant therapy, but stage IV patients received a
systemic concurrent BC regimen of dacarbazine (DTIC) or
temazolamide, cisplatin, vinblastine, interferon .alpha.-2b,
interleukin-2 (IL-2), and tamoxifen in the setting of one of
several phase II trials, as previously reported (40-42).
[0056] AJCC stage IV patients (Table 1B) were selected and coded by
the clinical study coordinator and assessed in laboratory and
statistical analyses in a blinded fashion. The selection of stage
IV patients was based on patient response or non-response to BC,
availability of clinical follow-up data, completion of the BC
trial, and specimen availability. Patients were categorized as
responders or non-responders to BC based on clinical response
criteria (42). Those showing a complete response (CR, n=13) or
partial response (PR, n=10) were included in the responder group
(n=23), whereas patients demonstrating progressive disease (PD,
n=24) were deemed non-responders. Patients exhibiting stable
disease (SD, n=3) were considered neither responders nor
non-responders. One patient in the responder group was lost to
follow-up and excluded from the survival analysis. Serum drawn from
healthy donors (n=40) served as normal controls.
TABLE-US-00002 TABLE 1B Clinical demographics of stage IV melanoma
patients receiving biochemotherapy Patient Characteristics (serum
donors) n (%) Total patients 50 Sex male 38 (76%) female 12 (24%)
Age (median 45) <50 34 (68%) .gtoreq.60 16 (32%) ECOG 0 14 (28%)
1 12 (24%) 2 24 (48%) BC response Responder CR 13 (26%) PR 10 (20%)
Non-responder PD 24 (48%)
[0057] Stage IV patients' blood was drawn for serum prior to
administration of BC. Ten ml of blood was collected in serum
separator tubes, centrifuged, run through a 13-mm serum filter
(Fisher Scientific, Pittsburgh, Pa.), aliquoted, and cryopreserved
at -30.sup.2C. DNA was extracted and processed from serum as
previously described (6). DNA quantification was performed on all
serum specimens using the PicoGreen quantification assay (Molecular
Probes, Eugene, Oreg.) (44).
Cell Line and Tissue DNA Sodium Bisulfite Modification
[0058] Extracted DNA from cell lines and PE melanoma tumors was
subjected to sodium bisulfite modification (11). Briefly, 2 .mu.g
DNA was denatured in 0.3 M NaOH for 3 min at 95.degree. C. and then
550 .mu.l of a 2.5 M sodium bisulfite/125 mM hydroquinone solution
was added. Samples were incubated under mineral oil in the dark for
3 hr at 60.degree. C. Salts were removed using the Wizard DNA
Clean-Up System (Promega, Madison, Wis.) and desulfonated in 0.3 M
NaOH at 37.degree. C. for 15 min. Modified DNA was precipitated
with ethanol using Pellet Paint NF (Novagen, Madison, Wis.) as a
carrier and re-suspended in molecular grade H.sub.2O. DNA samples
were cryopreserved at -30.degree. C. until MSP was performed.
Serum DNA Sodium Bisulfite Modification
[0059] Extracted DNA from serum was subjected to sodium bisulfite
modification (44). Briefly, DNA from 500 .mu.l of serum was
supplemented with 1 .mu.g salmon sperm DNA (Sigma, St. Louis, Mo.)
and denatured in 0.3 M NaOH for 3 min at 95.degree. C. Overall, 550
.mu.l of a 2.5 M sodium bisulfite/125 mM hydroquinone solution was
added. Samples were incubated under mineral oil in the dark for 3
hr at 60.degree. C. Salts were removed using the Wizard DNA
Clean-Up System (Promega, Madison, Wis.) and desulfonated in 0.3 M
NaOH at 37.degree. C. for 15 min. Modified serum DNA was prepared
and stored identically to tissue samples.
Detection of Methylated ER-.alpha.
[0060] ER-.alpha. methylation status was assessed using two sets of
fluorescent labeled primers specifically designed to amplify
methylated or unmethylated DNA sequences of the ER-.alpha. promoter
region. Primer sequences are provided as methylated sense and
antisense followed by unmethylated sense and antisense sequences,
with annealing temperatures and PCR product size: ER-.alpha.
methylated-specific forward, 5'-TAAATAGAGATATATCGGAGTTTGGTACG-3'
and reverse, 5'-AACTTAAAATAAACGCGAAAAACGA-3' (61.degree. C., 96
bp); unmethylated-specific forward,
5'-TAAATAGAGATATATTGGAGTTTGGTATGG-3' and reverse,
5'-AACTTAAAATAAACACAAAAAACAAA-3' (58.degree. C., 96 bp).
Bisulfite-modified DNA was subjected to PCR amplification in a
final reaction volume of 20 .mu.l containing PCR buffer, 2.5 mM
MgCl.sub.2, dNTPs, 0.3 .mu.M primers, and 0.5 U of AmpliTaq Gold
DNA polymerase (Applied Biosystems, Foster, Calif.). PCR was
performed with an initial incubation at 95.degree. C. for 10 min,
followed by 40 cycles of denaturation at 95.degree. C. for 30 sec,
annealing for 30 sec, extension at 72.degree. C. for 30 sec, and
final hold at 72.degree. C. for 7 min. DNA from the ER-.alpha.
positive breast cancer cell line MCF-7 was used as a control to
verify the presence of ER-.alpha.. DNA from the ER-.alpha. negative
melanoma cell line MCA was used as a control to verify the absence
of ER-.alpha.. A universal unmethylated control was synthesized
from normal DNA by phi-29 DNA polymerase and served as a positive
unmethylated control (45). Unmodified lymphocyte DNA was used as a
negative control for methylated and unmethylated reactions. Sssl
methylase-(New England Bio Labs, Beverly, Mass.) treated lymphocyte
DNA was used as a positive methylated control. PCR products were
visualized using CAE (CEQ 8000XL; Beckman Coulter, Inc., Fullerton,
Calif.) in a 96-well microplate format (6). Methylated and
unmethylated PCR products from each sample were assessed
simultaneously using forward primers labeled with Beckman Coulter
WellRED dye-labeled phosphoramidites (Genset oligos, Boulder,
Colo.). Forward methylated-specific primers were labeled with D4pa
dye, and forward unmethylated-specific primers were labeled with
D2a dye. One .mu.l methylated PCR product and 1 .mu.l unmethyated
PCR product were mixed with 40 .mu.l loading buffer and a 0.5 .mu.l
dye-labeled size standard (Beckman Coulter, Inc., Fullerton,
Calif.). Each marker was optimized with methylated and unmethylated
controls. Samples demonstrating a peak at the base pair size marker
for unmethylated DNA were considered unmethylated, while those
demonstrating a peak at the base pair size marker for methylated
DNA were considered methylated.
5-Aza and TSA Treatment of Melanoma Cell Lines
[0061] To confirm down-regulation of ER-.alpha. expression by
hypermethylation of the ER-.alpha. promoter region, cell lines were
treated with the DNA-demethylating agent, 5-Aza, and the HDAC
inhibitor, TSA. In combination with 5-Aza treatment, TSA can
up-regulate the mRNA expression of genes silenced due to
hypermethylation (26, 27). The MCF-7 cell line was used as an
ER-.alpha. positive control and the MCA cell line was used as an
ER-.alpha. negative control. Cell lines were maintained in RPMI
1640 medium supplemented with heat-inactivated 10% fetal bovine
serum (FBS), penicillin G, and streptomycin (100 U/ml). Cells were
treated with 1000 nM TSA for 24 hr (Wako Biochemicals, Osaka,
Japan) and 1000 nM 5-Aza for five days (Sigma Chemical Co., St
Louis, Mo.). After treatment with 5-Aza and TSA, melanoma cells
were washed with phosphate buffered saline (PBS) and harvested with
0.25% trypsin-0.53 mM EDTA (Gibco, Auckland, N.J.). The mRNA
expression level of ER-.alpha. was assessed by RT-PCR before and
after 5-Aza and TSA treatment.
mRNA Analysis
[0062] Total cellular RNA from melanoma cell lines was extracted
using Tri-Reagent (Molecular Research Center, Inc., Cincinnati,
Ohio) as previously described (6). The RNA was quantified and
assessed for purity using ultraviolet spectrophotometry and the
RIBOGreen detection assay (Molecular Probes, Eugene, Oreg.). The
expression of mRNA for GAPDH, an internal reference housekeeping
gene, was assessed by RT-PCR on all RNA samples to verify the
integrity of RNA and to indicate equal loading of PCR products for
gel electrophoresis.
[0063] All RT reactions were performed using Moloney murine
leukemia virus reverse-transcriptase (Promega, Madison, Wis.) with
oligo-dT (GeneLink, Hawthorne, N.Y.) priming as previously
described (6). cDNA from 250 ng of total RNA was used for each
reaction (46). The RT-PCR reaction mixture consisted of 1 .mu.M of
each primer, 1 U AmpliTaq Gold polymerase (Applied Biosystems,
Foster City, Calif.), 200 .mu.M of each dNTP, 4.5 mM MgCl.sub.2 and
AmpliTaq buffer to a final volume of 25 .mu.l. The primer sequences
used were as follows: ER-.alpha.: 5'-AGACATGAGAGCTGCCAACC-3'
(forward); 5'-GCCAGGCACATTCTAGAAGG-3' (reverse). GAPDH:
5'-GGGTGTGAACCATGAGAAGT-3' (forward); 5'-GACTGTGGTCATGAGTCCT-3'
(reverse). Samples were amplified with 40 cycles of denaturation at
95.degree. C. for 30 sec, annealing at 58.degree. C. for 30 sec,
and extension at 72.degree. C. for 30 sec for ER-.alpha. and GAPDH,
respectively.
[0064] ER-.alpha. positive (MCF-7 cell line) and negative (MCA
melanoma cell line) controls and reagent controls for RT-PCR assays
were included as previously described (46). All PCR products were
separated on 1.5% Tris-borate EDTA agarose gels for ER-.alpha. and
2% Tris-borate EDTA agarose gels for GAPDH and stained with SYBR
Gold (Invitrogen Detection Techonologies, Eugene, Oreg.). Each
assay was repeated in triplicate.
Biostatistical Analysis
[0065] The correlation between ER-.alpha. methylation status of
primary and metastatic melanomas with AJCC stage was assessed using
the Chi square method. Similarly, the correlation between
ER-.alpha. methylation status of circulating serum DNA with known
clinical prognostic factors and BC response was assessed by the Chi
square method. Additionally, a multivariate logistic regression
model was developed to correlate clinical prognostic factors and
serum circulating ER-.alpha. methylation status with response to
BC.
[0066] Survival length was determined from the first day of BC
treatment to death or the date of last clinical follow-up. Survival
curves were derived using the Kaplan-Meier method and the
differences between curves were analyzed using the log-rank test.
Cox's proportional hazards regression model was used for
multivariate analyses. Age, gender, ECOG (Eastern Cooperative
Oncology Group) status, lactate dehydrogenase (LDH) level, number
of metastasis sites, and ER-.alpha. methylation status were
included in the multivariate model using a stepwise method for
variable selection.
Results
Detection of Methylated ER-.alpha. DNA in Cell Lines
[0067] Initially, ER-.alpha. in established metastatic melanoma
cell lines was assessed. The frequency of hypermethylated
ER-.alpha. in metastatic melanoma cell lines was 91% (10 of 11).
Among these lines, six had only a methylated-specific peak while
four cell lines demonstrated both methylated- and
unmethylated-specific peaks. These experiments optimized the MSP
assay for ER-.alpha. and demonstrated the high frequency of
hypermethylated ER-.alpha. in metastatic melanoma cells cultured in
vitro.
ER-.alpha. Re-Expression with 5-Aza and TSA Treatment
[0068] To determine if cells with hypermethylated ER-.alpha. can be
induced to re-express ER-.alpha. mRNA, cell lines were treated with
5-Aza and TSA. In untreated cell lines, ER-.alpha. mRNA was
detected in MCB, and MCC, but not MCA (FIG. 1). ER-.alpha. mRNA
expression was restored to a detectable level in MCA after 5-Aza
and TSA treatment (FIG. 1). After treatment with 5-Aza for five
days followed by treatment with TSA for 24 hr, the MCA showed an
unmethylated-specific DNA peak when assessed by MSP (FIG. 2). To
further verify hypermethylation of the ER-.alpha. gene promoter
region in melanoma, purified PCR products after sodium bisulfite
modification were directly sequenced using a CEQ DYE Terminator
Cycle Sequencing Kit (Beckman Coulter, Inc.). Promoter region CpG
islands were fully methylated in the MCA cell line, which does not
express ER-.alpha., whereas MCC, a cell line that expresses
ER-.alpha., showed no evidence of promoter region CpG island
hypermethylation. With an optimized assay for the detection of
methylated ER-.alpha., and demonstration that reversal of
methylation leads to re-expression of ER-.alpha. mRNA, the
detection of methylated ER-.alpha. was approached in PE melanoma
specimens.
Detection of Methylated ER-.alpha. in Melanomas
[0069] 53 PE primary melanomas (stage I, n=11; stage II, n=19;
stage III, n=23) were evaluated using MSP. Overall, the frequency
of methylation ER-.alpha. in primary melanomas was 32% (17 of 53).
Similar rates of methylated ER-.alpha. were detected in primary
tumors among the patients assessed, regardless of stage. The
frequency of ER-.alpha. methylation in AJCC stage I, II, and III
primary melanoma tumors was 36% (4 of 11), 26% (5 of 19), and 35%
(8 of 23), respectively (FIG. 3A).
[0070] Additionally, 54 PE metastatic melanomas were assessed,
including stage III lymph node metastases (n=19) and stage IV
distant metastases (n=35; 14 subcutaneous, 9 lymph nodes, 6 lung, 5
colorectal, and 1 liver). Methylated ER-.alpha. was detected in 42%
(8 of 19) of stage III and 86% (30 of 35) of IV metastatic
melanomas (FIG. 3A). The frequency of methylated ER-.alpha.
detected in stage IV metastatic tumors was significantly higher
than in stage III metastatic tumors (p=0.0003). Overall, ER-.alpha.
was methylated in 70% (38 of 54) of metastatic tumors, a more than
two-fold increase in frequency compared to primary melanomas.
[0071] ER-.alpha. methylation status was also determined for 10 PE
normal tissues from various organ sites (pancreas, n=2; liver, n=2;
thymus, n=2; lung, n=2; and skin, n=2). Methylated ER-.alpha. was
detected in 90% (9 of 10) of normal tissues, indicating that
ER-.alpha. is usually methylated and silenced in normal tissue.
[0072] Because methylated ER-.alpha. in PE primary and metastatic
melanomas was frequently detected, the detection of methylated
ER-.alpha. in the serum of AJCC stage I-IV melanoma patients was
assessed to evaluate its role as a blood marker for disease
detection.
Detection of Circulating Methylated ER-.alpha. DNA in Serum
[0073] Previously, it was showed that circulating methylated DNA
markers can be valuable surrogates of tumor progression (11, 44).
Hence, an optimized assay was developed to detect the presence of
free circulating methylated ER-.alpha. DNA in serum. The frequency
with which methylated ER-.alpha. was detected in serum increased
with tumor progression and according to AJCC stage. In the analysis
of 109 melanoma patients' sera, the frequency of circulating
methylated ER-.alpha. in AJCC stage I, II, III, and IV sera was 10%
(2 of 20), 15% (3 of 20), and 26% (5 of 19), and 32% (16 of 50),
respectively (FIG. 3B). The frequency of serum methylated
ER-.alpha. was increased in patients with more advanced disease;
methylated ER-.alpha. was detected in stage III/IV more frequently
than in stage I/II (p=0.034). Methylated ER-.alpha. was detected in
the sera of only 1 of 40 healthy normal donors, an 82 year old
female. Representative methylation peaks from normal donor sera,
normal liver tissue, melanoma patient sera, and melanoma tumors are
provided in FIG. 4. Healthy normal donors ranged in age from 20 to
84 (mean, 56); the gender distribution of normal volunteers was
comparable to that of melanoma patients assessed. Having
established that methylated ER-.alpha. can be reliably detected in
the sera of melanoma patients but not in normal volunteers, and is
a marker of disease progression, attention was focused on assessing
the clinical utility of methylated ER-.alpha. as a predictor of
disease outcome.
Clinical Utility of Circulating Methylated ER-.alpha.
[0074] Prior to receiving systemic concurrent BC, blood from AJCC
stage IV melanoma patients was obtained and retrospectively assayed
for the detection of circulating methylated ER-.alpha. DNA. Serum
ER-.alpha. methylation from stage IV patients was assessed to
predict the patients most likely to respond to BC. The median time
of clinical follow-up after the initial blood draw was 12.5 months.
The frequency of circulating methylated ER-.alpha. for responders
(4 of 23, 17%) was significantly lower (p=0.018) than
non-responders (12 of 24, 50%). In a multivariate logistic
regression model that included known clinical prognostic factors
for melanoma, the presence of circulating serum methylated
ER-.alpha. DNA was the only factor that significantly correlated
with response to BC (OR=0.21, 95% CI=0.06 to 0.81; p=0.023).
Patients categorized as BC responders had significantly better
overall survival compared to patients deemed BC non-responders (Log
Rank, p<0.0001).
[0075] Regardless of response to BC, patients with serum methylated
ER-.alpha. had significantly worse progression-free survival
compared to patients in whom methylated ER-.alpha. was not detected
(Log Rank, p=0.002). Serum methylated ER-.alpha., LDH>190 IU/L
and age <50 were significantly correlated with progression-free
survival in a univariate analysis (Log Rank; methylated ER-.alpha.,
p=0.002; LDH>190 IU/L, p=0.013; age <50, p=0.028).
[0076] Similarly, patients with circulating methylated ER-.alpha.
had significantly worse overall survival compared to patients in
whom methylated ER-.alpha. was not detected (Log Rank, p=0.002).
Circulating methylated ER-.alpha. and serum LDH>190 IU/L
significantly correlated with overall survival (Log Rank;
methylated ER-.alpha., p=0.002; LDH>190 IU/L, p=0.015). Other
prognostic factors (gender, age, ECOG, and the number of metastatic
sites) were not significant.
[0077] A multivariate Cox's proportional hazard regression model
was developed to correlate clinical factors and ER-.alpha.
methylation status with progression-free and overall survival. Age,
gender, ECOG status, LDH level, number of metastasis sites, and
ER-.alpha. methylation status were included in the model using a
stepwise method for variable selection. Serum methylated ER-.alpha.
was the only independent factor predicting progression-free (FIG.
5A; RR 2.64, 95% CI 1.36-5.13, p=0.004) and overall survival (FIG.
5B; RR 2.31, 95% CI 1.41-5.58, p=0.003).
Methylated ER-.alpha.: Gender and Age
[0078] Because ER-.alpha. hypermethylation is influenced by both
age and gender in other cancers, the relation of these factors to
methylated ER-.alpha. status in primary and metastatic melanomas
and serum was assessed. There was no significant difference in the
frequency of methylated ER-.alpha. in PE tumors or sera between
male and female patients, nor was there any significant difference
in the frequency of methylated ER-.alpha. in tumors between
patients .gtoreq.60 years old and patients <50 years old.
Discussion
[0079] Methylated ER-.alpha. has been detected in neoplasia of the
colorectum, lung, and breast (21, 22, 24, 26, 27). The reported
expression level of ER-.alpha. in melanoma has been variable, with
several studies failing to demonstrate the presence of ER-.alpha.
using monoclonal antibodies (28-31). Tamoxifen has been used in
chemotherapy and BC regimens for over a decade (36-39). Although
improved response rates have been reported with its use, tamoxifen
has not been shown to significantly improve overall survival in
advanced melanoma (40, 41). This is the first study reporting a
potential mechanism for the failure of tamoxifen in the treatment
of melanoma. It has been shown that the variable down-regulation of
ER in melanoma is due to epigenetic control of its expression via
gene promoter region hypermethylation.
[0080] These studies demonstrate that methylated ER-.alpha. can be
detected in melanoma cell lines and ER-.alpha. mRNA expression can
be re-established after demethylation with 5-Aza and TSA.
Additionally, methylated ER-.alpha. can be detected in PE primary
and metastatic melanoma tumors, demonstrating its value as a
biomarker of tumor progression. Methylated ER-.alpha. DNA was
detected in the serum of melanoma patients with AJCC stage I-IV
disease and was a biomarker of disease progression. Furthermore,
serum circulating hypermethylated ER-.alpha. in AJCC stage IV
melanoma patients predicted response to BC, progression-free
survival, and overall survival.
[0081] The in vitro experiments demonstrated that all but one of
the 11 metastatic melanoma cell lines assayed had methylated
ER-.alpha.. This suggests that in vitro culturing may promote the
epigenetic silencing of ER-.alpha. or select for a subpopulation of
cells with methylated ER-.alpha.. 5-Aza alone did not significantly
increase ER-.alpha. mRNA expression (data not shown); the histone
deacetylase inhibitor TSA was necessary to significantly increase
expression above pretreatment levels. A HDAC inhibitor, such as
TSA, modulates chromatin histones and, together with 5-Aza can
effectively activate gene expression. That TSA treatment was a
necessary step for ER-.alpha. mRNA re-expression suggests histone
acetylation also plays an important regulatory role in ER-.alpha.
expression (26, 27). Similar epigenetic regulation in breast
cancer, ovarian cancer, prostate cancer, and hepatocellular cancer
has been reported (3, 23).
[0082] The frequency of ER-.alpha. methylation served as a marker
of progression from primary to metastatic disease and from regional
nodal metastasis to distant visceral metastasis. As with breast
cancer, the expression of ER-.alpha. mRNA as regulated by
ER-.alpha. methylation is directly or indirectly related to the
development of metastasis.
[0083] Because methylated ER-.alpha. in primary and metastatic
melanomas was able to be detected, whether or not methylated
ER-.alpha. could function as a blood-based biomarker for diagnosis
and disease surveillance was assessed. In the current study,
methylated ER-.alpha. was detected in the serum of AJCC stage I-IV
melanoma patients in a pattern related to disease progression. In a
subset of matched melanoma tumor and serum sample pairs, all
patients with methylated DNA detected in serum had primary or
metastatic tumors with methylated ER-.alpha. as well (data not
shown).
[0084] Knowing that methylated ER-.alpha. in serum could be
detected, the predictive utility of this marker in a selected
population of stage IV melanoma patients enrolled in a concurrent
BC trial was assessed. Prediction of the response to therapy based
on the methylation status of circulating ER-.alpha. was attempted.
Response rates for systemic therapies in advanced metastatic
melanoma are alarmingly low. BC, the use of chemotherapy in
conjunction with immune modulators, has produced better response
rates (40-42), but outcomes differ greatly between responders and
non-responders. It has been difficult to predict tumor response
before or in the early phases of BC. Identifying molecular
predictors of therapeutic response may permit physicians to treat
those patients most likely to respond to therapy while sparing
non-responsive patients unnecessary treatment and its associated
morbidity. Methylated ER-.alpha. was more commonly detected in the
serum of patients who failed to respond to BC and was the only
factor predictive of response to BC. Serum methylated ER-.alpha.
was the only independent predictor of progression-free and overall
survival in a multivariate analysis, surpassing even known clinical
prognostic factors.
[0085] There are several possible explanations for these findings.
First, tamoxifen, a member of the selective estrogen receptor
modulator family, was used in the BC regimen of 44 out of 50
patients. Patients without serum methylated ER-.alpha., who
therefore express ER-.alpha., may be more likely to respond to the
anti-tumor effects of tamoxifen. Conversely, the failure of
patients to respond to BC may be partially explained by the
inability of tamoxifen to exert its anti-tumor effects when
ER-.alpha. expression is silenced due to promoter region
hypermethylation. This is akin to the clinical situation seen in
breast cancer, where tumors not expressing ER-.alpha. do not
respond to hormone therapy and carry a poorer prognosis (23).
ER-.alpha. methylation could also reflect a pathophysiological
event that includes a more global hypermethylation of tumor-related
genes, thereby providing tumor cells with a growth advantage
(8).
[0086] Methylated ER-.alpha. is present in normal cells of
different histology (47, 48). In the serum analysis, however,
ER-.alpha. was not detected in the serum from normal healthy
donors. Normal cells containing methylated ER-.alpha. would be
expected to release this DNA into the bloodstream. Why, then, was
methylated ER-.alpha. not detected in normal healthy donors? It is
believed that methylated ER-.alpha. from tumors is cleared less
efficiently than methylated ER-.alpha. from normal cells. The
destruction of normal cells is primarily through apoptosis-related
events, resulting in the release of small, characteristic
enzyme-degraded fragments of DNA. As a result, the DNA released
from normal cells is cleared rapidly and not readily detected in
blood. On the contrary, tumor cells disrupted by physical trauma or
cell necrosis release intact, large fragments of DNA (49). Melanoma
patients release both free DNA and tumor cells into the blood
stream. Circulating tumor cells may release large fragments of DNA
due to non-apoptotic death mechanisms (unpublished results). The
detection of methylated ER-.alpha. in melanoma patients strongly
suggests that the circulating DNA is tumor-related.
[0087] Age-dependent methylation of ER-.alpha. has been previously
implicated in other studies (50). In this study, age differences in
ER-.alpha. methylation was not found. Among 40 healthy volunteers,
methylated ER-.alpha. was detected only in one 82 year-old donor,
which may be due to factors unrelated to aging, including
subclinical cancer. Further detailed studies will validate the
presence and significance of ER-.alpha. methylation in healthy
elderly volunteers.
[0088] This is the first study demonstrating the detection of
methylated ER-.alpha. in both melanoma patients' tumor tissues and
sera. The detection of methylated ER-.alpha. in tumors or sera
correlates with tumor progression, and is therefore prognostically
important. These findings indicate that detection of methylated
ER-.alpha. in serum may identify a population of patients with poor
melanoma outcomes and poor response to systemic therapy in whom
alternative treatment management should be considered. Furthermore,
these data support the initiation of a prospective BC trial for
stage IV melanoma based on serum ER-.alpha. methylation status.
Such a trial would provide valuable information regarding the
clinical value of tamoxifen in the treatment of melanoma and
further test the ability of the ER-.alpha. methylation assay to
predict response to BC.
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EXAMPLE II
ER-A Methylation in Breast, Pancreatic, and Colon Cancer
[0139] Methylation of the ER-.alpha. gene promoter in circulating
acellular DNA has been detected in breast cancer (about 30%),
pancreatic cancer (39 of 50; 78%), and colon cancer (15 of 63; 24%)
patients, respectively.
[0140] The contents of all references cited herein are incorporated
by reference in their entirety.
Sequence CWU 1
1
8129DNAArtificialSynthetic oligonucleotide 1taaatagaga tatatcggag
tttggtacg 29225DNAArtificialSynthetic oligonucleotide 2aacttaaaat
aaacgcgaaa aacga 25330DNAArtificialSynthetic oligonucleotide
3taaatagaga tatattggag tttggtatgg 30426DNAArtificialSynthetic
oligonucleotide 4aacttaaaat aaacacaaaa aacaaa
26520DNAArtificialSynthetic oligonucleotide 5agacatgaga gctgccaacc
20620DNAArtificialSynthetic oligonucleotide 6gccaggcaca ttctagaagg
20720DNAArtificialSynthetic oligonucleotide 7gggtgtgaac catgagaagt
20819DNAArtificialSynthetic oligonucleotide 8gactgtggtc atgagtcct
19
* * * * *