Note: Descriptions are shown in the official language in which they were submitted.
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IDENTIFICATION OF METASTASIS-SPECIFIC MIRNA AND
HYPOMETHYLATION SIGNATURES IN HUMAN COLORECTAL CANCER
TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of cancer detection and
diagnosis, and more
particularly, to novel metastasis-specific miRNA and hypomethylation
signatures in metastatic
human colorectal cancer.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in
connection with
colorectal cancer detection.
United States Patent Application Publication No. 20110183859, filed by Harris;
et al., entitled,
Inflammatory Genes and Microrna-21 as Biomarkers for Colon Cancer Prognosis,
discloses
methods for detecting a more aggressive form of a colon adenocarcinoma in a
subject, thereby
predicting the prognosis of the subject. The methods taught include
determining an
inflammatory gene expression signature in the colon adenocarcinoma and/or the
adjacent non-
cancerous tissue. In some embodiments, the inflammatory genes include, but are
not limited to,
PRG1, ANXA1, IL-17a, IL-23a FOXP3, HLA-DRA, IL-10, CD68 and IL-12a. In some
embodiments, the method further includes detecting expression of microRNA-21
(miR-21) in
the colon adenocarcinoma. Altered expression of one or more of the
inflammatory genes or
miR-21 indicates the prognosis of the subject. Also provided were arrays
consisting essentially
of probes specific for PRG1, ANXAL IL-17a, IL-23a, FOXP3, HLA-DRA, IL-10,
CD68, IL-
12a and miR-21.
International Patent Publication No. WO 2011128900A2, filed by Aviram, et al.,
entitled,
Plasma Based Micro-RNA Biomarkers and Methods for Early Detection of
Colorectal Cancer,
discloses compositions, methods and kits for diagnosing cancer, specifically
the diagnosis of
colorectal cancer (CRC). More specifically, the invention provides simple
assays, with high
sensitivity and specificity for CRC, wherein a panel of microRNA (miRNA) are
used as
biomarkers. A method is taught for diagnosing metastasis of CRC in a subject
is taught in which
the expression level of miRs selected from the group consisting of miR566,
miR96, miR183,
miR194, miR200a, miR200b, miR200c, miR203 and miR429, or combinations thereof,
in a
biological sample obtained from the subject, wherein a significant elevation
in the expression
levels of the miRNAs in the biological sample compared to control values
indicates that said
subject is afflicted with metastasis of CRC. Other biomarkers included hsa-miR-
16-2*, hsa-
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miR-25, hsa-miR-7, hsa-miR-93, hsa-miR-345, hsa-miR-409-3p, hsa-miR-671-3p and
hsa-miR-
331-3p.
Slaby, et al., Oncology 2007; 72: 397-402, "Altered Expression of miR-21, miR-
31, miR-143
and miR-145 is Related to Clinicopathologic Features of Colorectal Cancer,"
analyzes the
expression levels of miR-21, miR-31, miR-143 and miR-145 in 29 primary
colorectal
carcinomas and 6 non-tumor adjacent tissue specimens were examined by real-
time polymerase
chain reaction.
Huang, et al., Journal of Digestive Diseases, Volume 10, Issue 3, pages 188-
194, August 2009,
"MicroRNA expression profile in non-cancerous colonic tissue associated with
lymph node
metastasis of colon cancer", analyzes MicroRNAs isolated from six frozen non-
cancerous
surrounding colonic tissues derived from stage II¨III colon cancer patients
with (n = 3) and
without (n = 3) lymph node metastasis. They compared microRNA expression
profiles for six
non-cancerous colonic tissues from two colon cancer patient groups; those with
confirmed
lymph node metastasis, termed the lymph node positive group, and those without
detectable
lymph node metastasis, termed the lymph node negative group. MicroRNA
expression was
analyzed with Agilent microarrays containing 723 human microRNA probes and
they validated
the expression level of differentially expressed microRNA using quantitative
real-time PCR
analysis, such as, hsa-miR-129*, hsa-miR-137, miR-15b, miR-181b, miR-19 and
miR-200c.
SUMMARY OF THE INVENTION
In one embodiment the present invention includes a method for diagnosing or
detecting
colorectal cancer metastasis in a human subject comprising the steps of:
obtaining one or more
biological samples from the human subject; determining an Alu repeat
methylation level for the
one or more biological samples; and comparing the Alu repeat methylation level
in the
biological sample to an Alu repeat methylation control level from a normal non-
cancerous
sample, wherein a decrease in the Alu repeat methylation level is indicative
of at least one of
colorectal cancer disease progression or colorectal cancer metastasis. In one
aspect, the
biological samples are selected from the group consisting of a tissue sample,
a plasma sample, a
fecal sample, a cell homogenate, a blood sample, one or more biological
fluids, or any
combinations thereof In another aspect, the Alu repeat methylation level is
determined by
quantitative bisulfite pyrosequencing, thin layer chromatography (TLC), high
performance
liquid chromatography (HPLC), mass spectrometry (MS), nanopore amperometry,
nanopore
sequencing, single-molecule, real-time (SM-RT) sequencing, endonuclease
digestion,
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microarrays, matrix-assisted laser desorption ionization time-of-flight (MALDI-
TOF) mass
spectrometry, and next-generation sequencing. In another aspect, the Alu
repeat methylation
level is determined by measuring the expression status of miR-520c and miR-373
in the
biological sample when compared to a normal non-cancerous sample from the
human subject
obtained from a human subject that does not have cancer. In another aspect,
the non-cancerous
sample is from the same patient. In another aspect, the metastatic cancer is a
liver metastasis of
a colorectal cancer. In another aspect, the method further comprises the steps
of: determining an
expression level for the one or more biological samples for at least one first
marker selected
from let-7i, miR-10b, miR-200b, miR-320a, and miR-518d and at least one second
marker
selected from miR-141, miR-200c, and miR-203; and comparing the expression
levels of the
first and second markers to the biological sample, wherein a decrease in the
level of expression
of the at least one first marker and an increase in the level of expression of
the second marker
when compared to the level of expression in a normal tissue sample is
indicative of colorectal
cancer metastasis. In another aspect, the method further comprises the steps
of treating the
colorectal cancer with a therapeutic agent, obtaining one or more patient
samples and
determining if there has been a change in the expression of the one or more
microRNAs,
wherein a change in expression is indicative of colorectal cancer metastasis.
Another embodiment of the present invention includes a kit for determining a
detection,
prediction, or prognosis for colorectal cancer in a human subject comprising:
a biomarker
detecting reagent for measuring at least one first marker selected from let-
7i, miR-10b, miR-
320a, and miR-221 in a sample obtained from the human subject; and
instructions for the use of
the biomarker detecting reagent in the prognosis of colorectal cancer
metastasis, wherein the
instructions comprise providing step-by-step directions to compare the level
of expression of let-
7i, miR-10b, miR-320a, and miR-221 in the sample with the level of expression
of let-7i, miR-
10b, miR-320a, and miR-221 from normal colorectal tissue, wherein high
expression of at least
on of let-7i or miR-320a is indicative of a good prognosis for the colorectal
cancer, while the
low expression of at least one of miR-10b or miR-221 is indicative of a good
prognosis for the
colorectal cancer or colorectal cancer metastasis. In one aspect, the
biological samples are
selected from the group consisting of a tissue sample, a plasma sample, a
fecal sample, a cell
homogenate, a blood sample, one or more biological fluids, or any combinations
thereof In
another aspect, the level of expression of let-7i, miR-10b and miR-320a in
primary colorectal
cancer is prognostic of distant metastasis. In another aspect, the level of
expression of let-7i,
miR-10b correlated with the TNM stage. In another aspect, a high level of
expression of let-7i
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indicates a good prognosis for colorectal cancer survival. In another aspect,
a low level of
expression of let-7i indicates a poor prognosis for colorectal cancer
survival. In another aspect,
a high level of expression of at least one of let-7i or miR-320a is predictive
of less cancer
metastasis. In another aspect, a low level of expression of at least one of
miR-10b or miR-221 is
predictive of less cancer metastasis. In another aspect, the metastatic cancer
is a liver metastasis
of a colorectal cancer. In another aspect, the level of expression is
determined by digital color-
coded barcode technology analysis, microarray expression profiling, PCR,
reverse transcriptase
PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-
point PCR, multiplex
end-point PCR, cold PCR, ice-cold PCR, mass spectrometry, or nucleic acid
sequencing. In
another aspect, the method further comprises the detection of miR-122, wherein
an increase in
miR-122 is correlated with liver metastasis. In another aspect, the method
further comprises the
detection of one or more microRNAs selected from miR-199b-5p, miR-484, miR-490-
3p, miR-
520e, miR-337-5p, miR-485-39, miR-145, miR-144, miR-216a, miR-92b, miR-365,
miR-708 or
miR-143, wherein a decrease in expression is correlated with liver metastasis
from a primary
colorectal tumor. In another aspect, the method further comprises the steps of
treating the
colorectal cancer with a therapeutic agent, obtaining one or more patient
samples and
determining if there has been a change in the expression of the one or more
microRNAs,
wherein a change in expression is indicative of colorectal cancer metastasis.
Another embodiment of the present invention includes a biomarker for detecting
colorectal
cancer metastasis in a human subject comprising: a biomarker to determine a
methylation level
of an Alu repeat, wherein a lower methylation level of the Alu repeat is
indicative of colorectal
cancer and colorectal cancer metastasis in the human subject. In one aspect,
the biomarker
further comprises at least one first marker selected from let-7i, miR-10b, miR-
200b, miR-320a,
and miR-518d and at least one second marker selected from miR-141, miR-200c,
and miR-203.
Yet another embodiment of the present invention includes a kit for determining
colorectal cancer
metastasis in a human subject comprising: a biomarker detecting reagent for
measuring an Alu
repeat methylation level in a sample obtained from the human subject; and
instructions for the
use of the biomarker detecting reagent in diagnosing the presence of
colorectal cancer
metastasis, wherein the instructions comprise providing step-by-step
directions to compare the
Alu repeat methylation level in the sample with an Alu repeat methylation
level from normal
colorectal tissue, wherein a decrease in Alu repeat methylation is indicative
of colorectal cancer
and colorectal cancer metastasis. In one aspect, the sample is selected from
the group consisting
of a tissue sample, a fecal sample, a plasma sample, a cell homogenate, a
blood sample, one or
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more biological fluids, or any combinations thereof In another aspect, the Alu
repeat
methylation level is determined by measuring the expression status of miR-520c
and miR-373 in
the biological sample when compared to a normal non-cancerous sample from the
human
subject. In another aspect, the metastatic cancer is a liver metastasis of a
colorectal cancer. In
Yet another embodiment of the present invention includes a method for
selecting a cancer
therapy for a suspect diagnosed with colorectal cancer metastasis, the method
comprising the
evaluate a candidate drug believed to be useful in treating colorectal cancer
metastasis, the
method comprising: (a) determining the presence of colorectal cancer
metastasis by a method
comprising the steps of: determining an overall Alu repeat methylation level
in one or more cells
obtained from a biological sample of the subject, wherein a lower overall Alu
repeat methylation
In yet another embodiment the present invention includes a method of
performing a clinical trial
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of the first marker and a higher expression level in the second marker, when
compared to a
reference control, is indicative of colorectal cancer metastasis; (b)
administering a candidate
drug to a first subset of the patients, and a placebo to a second subset of
the patients; a
comparable drug to a second subset of the patients; or a drug combination of
the candidate drug
and another active agent to a second subset of patients; (c) repeating step
(a) after the
administration of the candidate drug or the placebo, the comparable drug or
the drug
combination; and (d) monitoring a change in the overall expression level of
the at least one of
let-7i, miR-10b, miR-320a, or miR-221as compared to any change in the second
subset of
patients, wherein a statistically significant decrease in the first marker and
an increase in the
second marker indicates that the candidate drug is useful in treating the
disease state.
Yet another embodiment of the present invention includes a method for
detecting colorectal
cancer metastasis in a human subject comprising the steps of: identifying the
human subject
suspected of suffering from a colorectal cancer metastasis; obtaining one or
more biological
samples suspected of being metastatic from the human subject; determining an
Alu repeat
methylation level for the one or more biological samples; and comparing the
Alu repeat
methylation level from the human subject to an Alu repeat methylation level
from normal
colorectal tissue, wherein a lower degree of methylation in the Alu repeat
level from the human
subject is indicative of colorectal cancer and colorectal cancer metastasis.
In one aspect, the
biological samples are selected from the group consisting of a tissue sample,
a plasma sample, a
fecal sample, a cell homogenate, a blood sample, one or more biological
fluids, or any
combinations thereof In another aspect, the Alu repeat methylation level is
determined by
quantitative bisulfite pyrosequencing, thin layer chromatography (TLC), high
performance
liquid chromatography (HPLC), mass spectrometry (MS), nanopore amperometry,
nanopore
sequencing, single-molecule, real-time (SM-RT) sequencing, endonuclease
digestion,
microarrays, matrix-assisted laser desorption ionization time-of-flight (MALDI-
TOF) mass
spectrometry, and next-generation sequencing. In another aspect, the Alu
repeat methylation
level is determined by measuring the expression status of miR-520c and miR-373
in the
biological sample when compared to a normal non-cancerous sample from the
human subject.
In another aspect, the metastatic cancer is a liver metastasis of a colorectal
cancer. In another
aspect, the method further comprises the steps of: determining an expression
level for the one or
more biological samples for at least one first marker selected from let-7i,
miR-10b, miR-200b,
miR-320a, and miR-518d and at least one second marker selected from miR-141,
miR-200c, and
miR-203; and comparing the expression levels of the markers to a colorectal
sample that is
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normal control level, wherein a decrease in the level of expression of the at
least one first marker
and an increase in the level of expression of the second marker is also
indicative of colorectal
cancer metastasis.
Another embodiment of the present invention includes a method for diagnosis,
prediction, or
prognosis of colorectal cancer in a human subject comprising the steps of:
obtaining one or more
biological samples from the human subject; determining a level of expression
of let-7i, miR-10b,
miR-320a, and miR-221 in the sample from the one or more biological samples;
and comparing
the level of expression of let-7i, miR-10b, miR-320a, and miR-221 in the
sample with the level
of expression of let-7i, miR-10b, miR-320a, and miR-221 from normal colorectal
tissue, wherein
high expression of at least on of let-7i or miR-320a is indicative of a good
prognosis for the
colorectal cancer, while the low expression of at least one of miR-10b or miR-
221 is indicative
of a good prognosis for the colorectal cancer or colorectal cancer metastasis.
In one aspect, the
biological samples are selected from the group consisting of a tissue sample,
a plasma sample, a
fecal sample, a cell homogenate, a blood sample, one or more biological
fluids, or any
combinations thereof In another aspect, the level of expression of let-7i, miR-
10b and miR-
320a in primary colorectal cancer is prognostic of distant metastasis. In
another aspect, the level
of expression of let-7i, miR-10b correlated with the TNM colorectal cancer
stage. In another
aspect, a high level of expression of let-7i indicates a good prognosis for
colorectal cancer
survival. In another aspect, a low level of expression of let-7i indicates a
poor prognosis for
colorectal cancer survival. In another aspect, a high level of expression of
at least one of let-7i
or miR-320a is predictive of less cancer metastasis. In another aspect, a low
level of expression
of at least one of miR-10b or miR-221 is predictive of less cancer metastasis.
In another aspect,
the metastatic cancer is a liver metastasis of a colorectal cancer. In another
aspect, the level of
expression is determined by digital color-coded barcode technology analysis,
microarray
expression profiling, PCR, reverse transcriptase PCR, reverse transcriptase
real-time PCR,
quantitative real-time PCR, end-point PCR, multiplex end-point PCR, cold PCR,
ice-cold PCR,
mass spectrometry, or nucleic acid sequencing. In another aspect, the method
further comprises
the detection of miR-122, wherein an increase in miR-122 is correlated with
liver metastasis. In
another aspect, the method further comprises the detection of one or more
microRNAs selected
from miR-199b-5p, miR-484, miR-490-3p, miR-520e, miR-337-5p, miR-485-39, miR-
145,
miR-144, miR-216a, miR-92b, miR-365, miR-708 or miR-143, wherein a decrease in
expression
is correlated with liver metastasis from a primary colorectal tumor.
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Yet another embodiment of the present invention includes a method for
diagnosing or detecting
colorectal cancer metastasis in a human subject comprising the steps of:
obtaining one or more
biological samples from the human subject suspected of comprising a metastatic
cancer;
determining an expression level for the one or more biological samples for at
least one first
marker selected from let-7i, miR-10b, miR-200b, miR-320a, and miR-518d and at
least one
second marker selected from miR-141, miR-200c, and miR-203; and comparing the
expression
levels of the markers to a colorectal sample that is normal control level,
wherein a decrease in
the level of expression of the at least one first marker and an increase in
the level of expression
of the second marker is indicative of colorectal cancer metastasis. In one
aspect, the biological
samples are selected from the group consisting of a tissue sample, a plasma
sample, a fecal
sample, a cell homogenate, a blood sample, one or more biological fluids, or
any combinations
thereof In another aspect, the expression level of 2, 3, 4, or 5 of the first
marker microRNAs are
downregulated. In another aspect, the expression level of 2 or 3 of the second
marker
microRNAs are upregulated. In another aspect, the expression level of the
first and second
markers is measured by microarray expression profiling, PCR, reverse
transcriptase PCR,
reverse transcriptase real-time PCR, quantitative real-time PCR, end-point
PCR, multiplex end-
point PCR, cold PCR, ice-cold PCR, mass spectrometry, or nucleic acid
sequencing. In another
aspect, the metastatic cancer is a liver metastasis of a colorectal cancer. In
another aspect, the
method further comprises the step of determining an Alu repeat methylation
level for the one or
more biological samples; and comparing the Alu repeat methylation level to an
Alu repeat
methylation level from normal colorectal tissue, wherein a decrease in the Alu
repeat
methylation level is also indicative of colorectal cancer and colorectal
cancer metastasis.
Yet another embodiment of the present invention also includes a biomarker for
colorectal
metastasis that comprises at least one first marker selected from microRNAs:
let-7i, miR-10b,
miR-200b, miR-320a, and miR-518d; and at least one second marker selected from
microRNAs:
miR-141, miR-200c, and miR-203, wherein a change in the overall expression of
the one or
more first and the second marker a sample obtained from a patient is
indicative of colorectal
metastasis when compared to the overall expression of the first and second
marker expression in
normal colorectal neoplasia cells or colorectal neoplasia cells obtained at an
earlier timepoint
from the same patient. In another aspect, the first markers are underexpressed
in metastatic
cancer and are selected from 2, 3, 4, or 5 of the microRNAs. In another
aspect, 2 or 3 of the
second marker microRNAs are overexpressed in metastatic cancer. In another
aspect, the
biomarker further comprises an Alu repeat methylation level of the sample and
an Alu repeat
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methylation control level, wherein a lower degree of the Alu repeat
methylation level is also
indicative of colorectal cancer and colorectal cancer metastasis.
Yet another embodiment of the present invention includes a kit for determining
colorectal cancer
metastasis in a human subject comprising: a biomarker detecting reagent for
measuring at least
one first marker selected from microRNAs: let-7i, miR-10b, miR-200b, miR-320a,
and miR-
518d; and at least one second marker selected from microRNAs: miR-141, miR-
200c, and miR-
203 level in a sample; and instructions for the use of the biomarker detecting
reagent in
diagnosing the presence of colorectal cancer metastasis, wherein the
instructions comprise
providing step-by-step directions to compare the expression level for the
first and second
markers in the sample with a normal colorectal tissue which comprises a
control level. In
another aspect, the sample is selected from the group consisting of a tissue
sample, a fecal
sample, a plasma sample, a cell homogenate, a blood sample, one or more
biological fluids, or
any combinations thereof In another aspect, the normal colorectal tissue
control level is
obtained from a biological sample obtained from a healthy subject, wherein the
healthy subject
is a human subject not suffering from cancer metastasis or non-cancerous
tissue from the same
patient. In another aspect, the kit further comprises an Alu repeat
methylation detecting reagent
to determine the Alu repeat methylation level of the sample and an Alu repeat
methylation
control level, wherein a lower degree of the Alu repeat methylation level is
also indicative of
colorectal cancer and colorectal cancer metastasis. In another aspect, the
metastatic cancer is a
liver metastasis of a colorectal cancer.
Another embodiment of the present invention includes a method of performing a
clinical trial to
evaluate a candidate drug believed to be useful in treating colorectal cancer
metastasis, the
method comprising: (a) determining the presence of colorectal cancer
metastasis by a method
comprising the steps of: determining an overall level of expression of at
least one first marker
selected from microRNAs: let-7i, miR-10b, miR-200b, miR-320a, and miR-518d;
and at least
one second marker selected from microRNAs: miR-141, miR-200c, and miR-203 in
one or more
cells obtained from a biological sample of the subject, wherein a lower
overall expression level
of the first marker and a higher expression level in the second marker, when
compared to a
reference control, is indicative of colorectal cancer metastasis; (b)
administering a candidate
drug to a first subset of the patients, and a placebo to a second subset of
the patients; a
comparable drug to a second subset of the patients; or a drug combination of
the candidate drug
and another active agent to a second subset of patients; (c) repeating step
(a) after the
administration of the candidate drug or the placebo, the comparable drug or
the drug
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combination; and (d) monitoring a change in the overall expression level of
the first and second
markers as compared to any change in the second subset of patients, wherein a
statistically
significant decrease in the first marker and an increase in the second marker
indicates that the
candidate drug is useful in treating the disease state.
5 Yet another embodiment of the present invention includes a method for
detecting colorectal
cancer metastasis in a human subject comprising the steps of: identifying the
human subject
suspected of suffering from a colorectal cancer; obtaining one or more
biological samples from
the human subject; determining the level of expression of at least one first
marker selected from
microRNAs: let-7i, miR-10b, miR-200b, miR-320a, and miR-518d; and at least one
second
10 marker selected from microRNAs: miR-141, miR-200c, and miR-203 for the
one or more
biological samples; and comparing the expression level of the first and second
markers to the
expression level of the first and second markers in normal tissues, wherein a
decrease in the
expression level of the first marker and an increase in the expression level
of the second marker
as compared to normal tissue is indicative of colorectal cancer metastasis. In
one aspect, the
biological samples are selected from the group consisting of a tissue sample,
a plasma sample, a
fecal sample, a cell homogenate, a blood sample, one or more biological
fluids, or any
combinations thereof In another aspect, the expression level of 2, 3, 4, or 5
of the first marker
microRNAs are downregulated. In another aspect, the expression level of 2 or 3
of the second
markers microRNAs are upregulated. In another aspect, the expression level of
the first and
second markers is measured by microarray expression profiling, PCR, reverse
transcriptase
PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-
point PCR, multiplex
end-point PCR, cold PCR, ice-cold PCR, mass spectrometry, or nucleic acid
sequencing. In
another aspect, the metastatic cancer is a liver metastasis of a colorectal
cancer. In another
aspect, the method further comprises the step of determining an Alu repeat
methylation level for
the one or more biological samples; and comparing the Alu repeat methylation
level to an Alu
repeat methylation control level, wherein a lower degree of the Alu repeat
methylation level is
also indicative of colorectal cancer and colorectal cancer metastasis. Yet
another embodiment of
the present invetion includes a method of diagnosing or providing a prognosis
for colorectal
cancer metastasis, the method comprising the steps of: obtaining a biological
sample from a
human subject suspected of or having colorectal cancer metastasis; detecting
in the biological
sample altered expression for an Alu repeat methylation level for the one or
more biological
samples, wherein a decrease in the Alu repeat methylation level is indicative
of at least one of
colorectal cancer disease progression or colorectal cancer metastasis; and
providing a diagnosis
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or prognosis for colorectal cancer metastasis. Another embodiment includes a
method of
diagnosing or providing a prognosis for colorectal cancer metastasis, the
method comprising the
steps of: obtaining a biological sample from a human subject suspected of or
having colorectal
cancer metastasis; detecting in the biological sample altered expression (over
or under
expression of at least 50% compared to a subject without colorectal cancer
metastasis) of let-7i,
miR-10b, miR-320a, and miR-221, wherein high expression of at least one of let-
7i or miR-320a
is indicative of a good prognosis for the colorectal cancer, while the low
expression of at least
one of miR-10b or miR-221 is indicative of a good prognosis for the colorectal
cancer or
colorectal cancer metastasis; and providing a diagnosis or prognosis for
colorectal cancer
metastasis.
BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the features and advantages of the
present invention,
reference is now made to the detailed description of the invention along with
the accompanying
figures and in which:
Figure 1 shows the expression analysis of metastasis predictive microRNAs
expression
comparing Primary Colorectal (PC) cancer microRNA expression compared to
colorectal cancer
(CRC) liver metastasis (LM) microRNA expression.
Figure 2 is an analysis of the expression of the miR-200 family (-200b, -200c,
-141 and -429),
and miR-203 in serum samples from CRC patients with metastasis (Stage IV) and
without
metastasis (Stage I) by qRT-PCR. The expression of mir-200c and miR-203 were
significantly
elevated in serum samples from CRC patients with metastasis (Stage IV)
compared to patients
without metastasis (Stage I).
Figure 3 shows the role of Alu methylation as a surrogate marker for global
methylation, CRC
cell lines were treated with the demethylating agent (5-azacytidine), which
were used to confirm
methylation status of global Alu repetitive element by quantitative
pyrosequencing for each of
the listed CRC cell lines.
Figure 4 is an analysis of the global Alu methylation status in matched
corresponding primary
CRC (PC) and liver metastasized CRC (LM) human tissues by pyrosequencing.
Figure 5A are maps that show the Alu hypomethylation regulated the expression
of down-
stream miR-373, but not miR-520c.
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Figure 5B shows that Alu methylation inversely correlated with miR-373
expression in CRC cell
lines.
Figure 5C shows that Alu methylation did not correlate with miR-520c
expression in CRC cell
lines.
Figure 6A is an analysis of the expression of miR-520c. All cell lines showed
low level of miR-
520c expression, except Lovo cell.
Figure 6B shows that the expression of miR-520c was induced by a demethylating
agent. This
data indicates that miR-520c expression is regulated by Alu methylation in
promoter region.
Figure 7 is an analysis of the expression status all 4 miRNAs that are located
downstream of Alu
repetitive sequences in matched corresponding primary CRC (PC) and liver
metastasized CRC
(LM) human tissues by qRT-PCR. LM showed significantly lower expression of miR-
30b, miR-
518d, and miR-520c compared to PC.
Figure 8A shows that Alu hypomethylation positively correlated with miR-373
expression in
LM.
Figure 8B shows that Alu hypomethylation did not correlate with miR-520c
expression in LM.
Figure 9 shows that an RNA pol II inhibitor did not suppress activation of miR-
373, but
inhibited the expression of miR-520c.
Figure 10 shows the results of the qRT-PCR validation for selected miRNAs in
58 PCs and LMs.
Figure 11 shows the results from the microarray validation for selected miRNAs
in 84 PCs.
Figure 12 shows the results of qRT-PCR validation for miR-7i (left graph) and
miR-10b (right
graph) in 175 PCs.
Figure 13 shows the ISH validation for the expression of miR-7i and miR-10b in
CRC tissues
and liver metastasis.
DETAILED DESCRIPTION OF THE INVENTION
While the making and using of various embodiments of the present invention are
discussed in
detail below, it should be appreciated that the present invention provides
many applicable
inventive concepts that can be embodied in a wide variety of specific
contexts. The specific
embodiments discussed herein are merely illustrative of specific ways to make
and use the
invention and do not delimit the scope of the invention.
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13
To facilitate the understanding of this invention, a number of terms are
defined below. Terms
defined herein have meanings as commonly understood by a person of ordinary
skill in the areas
relevant to the present invention. Terms such as "a", "an" and "the" are not
intended to refer to
only a singular entity, but include the general class of which a specific
example may be used for
illustration. The terminology herein is used to describe specific embodiments
of the invention,
but their usage does not delimit the invention, except as outlined in the
claims.
The present invention includes biomarkers and methods for detecting Colorectal
Cancer (CRC)
metastasis and exploring curative target of metastasized CRC, including but
not limited to
cancer research, cancer screening, diagnosis of metastasis, planning of cancer
treatment and
molecular target of anti-cancer drug.
As used herein, the term "colorectal cancer" includes the well-accepted
medical definition that
defines colorectal cancer as a medical condition characterized by cancer of
cells of the intestinal
tract below the small intestine (i.e., the large intestine (colon), including
the cecum, ascending
colon, transverse colon, descending colon, sigmoid colon, and rectum).
Additionally, as used
herein, the term "colorectal cancer" also further includes medical conditions,
which are
characterized by cancer of cells of the duodenum and small intestine (jejunum
and ileum).
As used herein, the term "tissue sample" (the term "tissue" is used
interchangeably with the term
"tissue sample") includes any material composed of one or more cells, either
individual or in
complex with any matrix obtained from a patient. The definition includes any
biological or
organic material and any cellular subportion, product or by-product thereof
The definition of
"tissue sample" should be understood to include without limitation colorectal
tissue samples,
tissues suspected of including colorectal cancer cells, blood components, and
even fecal matter
or fluids that includes colorectal cells. Also included within the definition
of "tissue" for
purposes of this invention are certain defined acellular structures such as
dermal layers of
epithelium that have a cellular origin but are no longer characterized as
cellular. The term
"stool" or "feces" as used herein is a clinical term that refers to feces
obtained from a mammal
such as a human.
As used herein, the term "biological fluid" refers to a fluid containing cells
and compounds of
biological origin, and may include blood, stool or feces, lymph, urine, serum,
pus, saliva,
seminal fluid, tears, urine, bladder washings, colon washings, sputum or
fluids from the
respiratory, alimentary, circulatory, or other body systems. For the purposes
of the present
invention the "biological fluids", the nucleic acids containing the biomarkers
may be present in a
circulating cell or may be present in cell-free circulating DNA or RNA.
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As used herein, the term "gene" refers to a functional protein, polypeptide or
peptide-encoding
unit. As will be understood by those in the art, this functional term includes
both genomic
sequences, cDNA sequences, or fragments or combinations thereof, as well as
gene products,
including those that may have been altered by the hand of man. Purified genes,
nucleic acids,
protein and the like are used to refer to these entities when identified and
separated from at least
one contaminating nucleic acid or protein with which it is ordinarily
associated. The term
"allele" or "allelic form" refers to an alternative version of a gene encoding
the same functional
protein but containing differences in nucleotide sequence relative to another
version of the same
gene.
As used herein, "nucleic acid" or "nucleic acid molecule" refers to
polynucleotides, such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides,
fragments generated
by the polymerase chain reaction (PCR), and fragments generated by any of
ligation, scission,
endonuclease action, and exonuclease action. Nucleic acid molecules can be
composed of
monomers that are naturally-occurring nucleotides (such as DNA and RNA), or
analogs of
naturally-occurring nucleotides (e.g., a-enantiomeric forms of naturally-
occurring nucleotides),
or a combination of both. Modified nucleotides can have alterations in sugar
moieties and/or in
pyrimidine or purine base moieties. Sugar modifications include, for example,
replacement of
one or more hydroxyl groups with halogens, alkyl groups, amines, and azido
groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire sugar moiety
can be replaced with
sterically and electronically similar structures, such as aza-sugars and
carbocyclic sugar analogs.
Examples of modifications in a base moiety include alkylated purines and
pyrimidines, acylated
purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic
acid monomers
can be linked by phosphodiester bonds or analogs of such linkages. Analogs of
phosphodiester
linkages include phosphorothioate,
phosphorodithioate, phosphoros elenoate,
phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,
phosphoramidate, and the like.
The term "nucleic acid molecule" also includes so-called "peptide nucleic
acids," which
comprise naturally-occurring or modified nucleic acid bases attached to a
polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
As used herein, a "biomarker" refers to a molecular indicator that is
associated with a particular
pathological or physiological state. The "biomarker" as used herein is a
molecular indicator for
cancer, more specifically an indicator for primary CRCs and distant metastasis
of primary
CRCs. Examples of "biomarkers" include let-7i, miR-10b, miR-30b, miR-34a, miR-
141, miR-
200b, miR-200c, miR-203, miR-221, miR-320a, miR-373, miR-429, miR-518d, and
miR-520c.
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As used herein, the term "statistically significant" refers to differences
between the groups
studied, relates to condition when using the appropriate statistical analysis
(e.g. Chi-square test,
t-test) the probability of the groups being the same is less than 5%, e.g.
p<0.05. In other words,
the probability of obtaining the same results on a completely random basis is
less than 5 out of
5 100 attempts. The skilled artisan will recognize that there will be
variability in certain
measurements, for example, the level of mir-148a expression was determined by
normalizing the
expression to, e.g., miR-16, thus, the number 0.069-fold is not a definitive
number. As a general
matter, when the terms "higher" or "lower" are used to indicate the level of
expression of a MiR,
this indicates a statistically "higher" or "lower" level of expression for
that same marker (e.g.,
10 miR-148a) in a CRC sample versus normal mucosa. As demonstrated in the
figures disclosed
herein (where expression is generally shown as a range), the skilled artisan
can determine the
statistical significance of the measured biomarker in relation to that
expressed in normal
colorectal tissue from the same patient. Thus, the cut-off value can be
determined in the context
of the same patient, thus yielding a statistically significant measurement for
an increase or
15 decrease in expression. It is also possible to measure invariant markers
from CRC, e.g., miR-16,
that can also be used to normalize levels of expression.
As used herein, the term "kit" or "testing kit" denotes combinations of
reagents and adjuvants
required for an analysis. Although a test kit consists in most cases of
several units, one-piece
analysis elements are also available, which must likewise be regarded as
testing kits.
As used herein, the term "TNM" refers to the internationally recognized TNM
classification of
malignant tumors developed and maintained by the International Union Against
Cancer, which
has been adopted by the American Joint Committee on Cancer (AJCC) and the
International
Federation of Gynecology and Obstetrics (FIGO). T refers to the size or direct
extent of the
primary tumor; N to the degree of spread to regional lymph nodes, and M to the
presence of
metastasis.
The present invention includes the identification and use of miRNA biomarkers
(let-7i, miR-10b,
miR-30b, miR-34a, miR-141, miR-200b, miR-200c, miR-203, miR-221, miR-320a, miR-
373,
miR-429, miR-518d, and miR-520c) that have been found to be very specific for
detecting liver
metastasized CRC. Most of the existing cancer metastasis biomarkers are
developed through
comparison between primary tissues with metastasis and without metastasis. In
contrast, the
present invention is based on a detailed analysis and discovery that certain
miRNA biomarkers
were identified by direct comparison between primary CRC and matching liver
metastasis
tissues, rather than a comparison to primary tissues. These biomarkers were
validated using
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16
tissue sample miRNAs expression, but also using serum samples of CRC patients
with distant
metastasis. Furthermore, it was found that Alu repetitive element was
hypomethylated in distant
metastasized tissues compared to primary CRC, which can regulate the
expression of some of
the miRNA biomarkers (miR-30b, miR-373, miR-518d, and miR-520c) of the present
invention.
DNA modification is biologically and chemically stable than RNA transcription.
Thus, the
miRNA biomarkers of the present invention are more accurate and specific
compared to
biomarkers developed using just primary cancer tissues.
The present invention has several advantages when compared to existing miRNA
biomarkers.
First, the miRNAs biomarkers of the present invention are metastasis specific
biomarkers
because they are derived from the direct comparison between primary CRC and
matching liver
metastasis tissues. Second, the miRNAs biomarkers of the present invention are
more specific
for the detection of CRC metastasis, as validated using miRNAs expression of
serum samples
from CRC patients with and without distant metastasis. Third, the miRNAs
biomarkers of the
present invention can be applied to detect any diseases which related with
global
hypomethylation, as it was also found that hypomethylation (decrease of
methylation) of Alu
repetitive sequence in distant metastasis tissue compared to primary CRC
tissue, which can
regulate Alu sequence downstream located miRNAs expression (miR-30b, miR-373,
miR-518d,
and miR-520c).
Materials and Methods. A commercially available kit for miRNA extraction from
cell lines,
Formalin-Fixed, Paraffin-Embedded (FFPE) tissue, and human serum samples with
some
modifications. To compare miRNAs expression status between primary CRC and
distant
metastasized CRC, we have analyzed expression of fourteen metastasis-related
miRNAs (let-7i,
miR-10b, miR-30b, miR-34a, miR-141, miR-200b, miR-200c, miR-203, miR-221, miR-
320a,
miR-373, miR-429, miR-518d, and miR-520c) in matched primary colorectal cancer
and
corresponding liver metastasis tissues from 59 patients. MicroRNA expression
levels were
determined by quantitative real-time PCR (qRT-PCR) and the data were
normalized relative to
miR-16 expression. Second, we have also investigated global Alu methylation
and local Alu
methylation status by quantitative pyrosequencing analysis in matched primary
colorectal cancer
and corresponding liver metastasis tissues. The skilled artisan will
recognize, however, that
many different methods for determining methylation can be used with the
present invention,
e.g., thin layer chromatography (TLC), high performance liquid chromatography
(HPLC), mass
spectrometry (MS), nanopore amperometry, nanopore sequencing, single-molecule,
real-time
(SM-RT) sequencing, endonuclease digestion, microarrays, matrix-assisted laser
desorption
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ionization time-of-flight (MALDI-TOF) mass spectrometry, and next-generation
sequencing
(Laird, Peter W., "Principles and Challenges of Genomewide DNA Methylation
Analysis,"
Nature Review Genetics, Vol. 11, March 2010, pgs 191-203, relevant portions
incorporated
herein by reference).
The present invention may include the use of digital color-coded barcode
technology analysis
(e.g., NANOSTRINGO technology (such as the nCounter Analysis System,
NanoString
Technologies, Inc., Seattle, Washington). The NANOSTRING0protocol includes the
following
steps: (1) Hybridization: two ¨50 base probes per mRNA that hybridize in
solution, a reporter
probe that carries the signal, while a capture probe allows the complex to be
immobilized for
data collection. (2) Purification and Immobilization: following hybridization,
excess probes are
removed and the probe/target complexes are aligned and immobilized in, e.g.,
an nCounter
Cartridge; and (3) Data is collected from the sample cartridges, which can be
placed in the
digital analyzer instrument for data acquisition using color codes on the
surface of the cartridge
that are counted and tabulated for each target molecule.
Cell lines and 5-aza-2-deoxy-cytidine treatment. Seven CRC cell lines, HCT116,
RKO, 5W48,
Caco-2, HT29, 5W480, and 5W620 were obtained from the American Type Culture
Collection
(ATCC, Rockville, MD). Cell lines were treated with 2.5 uM 5-Aza-2"-
deoxycytidine (5-aza-dC;
Sigma-Aldrich) for 72 hours, and fresh medium containing 5-aza-dC was replaced
every 24
hours.
Tissue specimens. A total of 59 formalin-fixed, paraffin-embedded (FFPE)
matched
corresponding normal cololectal mucosa (NM), primary CRC tissues (PC), and
liver metastasis
tissues (LM) were enrolled in this study. Written informed consent was
obtained from all
patients and the study was approved by the institutional review boards of all
participating
institutions. Careful microdissection was performed in order to enrich for
tumor cells.
Isolation of RNA and DNA. Total RNA (including miRNAs) from CRC cell lines was
extracted
using miRNeasy0 Mini Kits (Qiagen). For RNA extraction from FFPE specimens, a
Total
Nucleic Acid Isolation Kit for FFPE tissues (Ambion, Austin, TX, USA) was used
according to
the manufacturer's instructions. DNA was extracted from CRC cell lines using a
QIAamp0
DNA Mini Kit (Qiagen) and from FFPE specimens using a QIAamp0 DNA FFPE Tissue
Kit
(Qiagen).
miRNA expression analysis. Expression of fourteen metastasis-related miRNAs
(let-7i, miR-
10b, miR-30b, miR-34a, miR-141, miR-200b, miR-200c, miR-203, miR-221, miR-
320a, miR-
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373, miR-429, miR-518d, and miR-520c) was analyzed using TaqMan miRNA assays
(Applied
Biosystems Inc., Foster City, CA). Expression of RNU6B (Applied Biosystems
Inc., Foster City,
CA) and miR-16 were used as endogenous controls for cell lines and FFPE
tissues, respectively.
DNA methylation analysis. Methylation levels of repetitive sequences (global
Alu and local
Alu) were analyzed by quantitative bisulfite pyrosequencing using the PSQ HS
96A
pyrosequencing system (Qiagen) following bisulfite modification of genomic DNA
using EZ
DNA methylation Gold Kits (Zymo Research), as described previously.
Statistical analysis. Data were analyzed with GraphPad Prism 5.0 software. To
evaluate
significant differences between two matched pair groups of samples, paired t-
tests was used,
whereas the difference between two independent groups of samples was analyzed
using the
Mann-Whitney U test.
Additional materials and methods. CRC Cell line: CACO2, HCT116, RKO, 5W48,
5W480, and
5W620. Tissue specimens: A total of 59 formalin-fixed, paraffin-embedded
(FFPE) primary
CRC tissues and corresponding liver metastasis tissues were analyzed. 5-Aza-2"-
deoxycytidine
(5-aza-dC) treatment: CRC cell lines were treated with 2.5 uM 5-aza-dC for 72
hours. a-
amanitin treatment: CRC cell lines were treated with 50 ug/m1 a-amanitin, a
RNA pol II
inhibitor, for 7 hours. Methylation analysis: Methylation levels were
analyzed by bisulfite
pyrosequencing for quantitative methylation analysis using PSQ HS 96A
pyrosequencing system
(Qiagen) on bisulfite modified genomic DNA template. microRNAs expression
analysis:
Expression of miR-373 and miR-520c was analyzed using TaqMan miRNA assays.
Figure 1 shows a metastasis predictive microRNAs expression colorectal cancer
(CRC). Briefly,
all 10 metastasis predictive candidate miRNAs expression status in matched
corresponding
primary CRC (PC) and liver metastasized CRC (LM) human tissues by qRT-PCR.
Five miRNAs
(let7i, miR-10b, miR-200b, miR-221, and miR-320) were significantly down-
regulated in LM
compared to PC. On the contrary, three miRNAs (miR-141, miR-200c, and miR-203)
were
significantly up-regulated in LM compared to PC.
Figure 2 is an analysis of the expression of the miR-200 family (-200b, -200c,
-141 and -429),
and miR-203 in serum samples from CRC patients with metastasis (Stage IV) and
without
metastasis (Stage I) by qRT-PCR. The expression of mir-200c and miR-203 were
significantly
elevated in serum samples from CRC patients with metastasis (Stage IV)
compared to patients
without metastasis (Stage I). These data strongly indicate that our invented
miRNAs are useful
biomarker for CRC metastasis prediction.
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Figure 3 shows the percent methylation the role of Alu methylation as a
surrogate marker for
global methylation, CRC cell lines were treated with the demethylating agent
(5-azacytidine),
which were used to confirm methylation status of global Alu repetitive
element. Briefly,
quantitative pyrosequencing was performed in CRC cell lines. All MSI and MSS
CRC cell lines
showed hypomethylation of Alu element. The Alu element was markedly
demethylated by 5-
aza treatment indicating Alu methylation can be used as a global DNA
methylation indicator.
Figure 4 is an analysis of the global Alu methylation status in matched
corresponding primary
CRC (PC) and liver metastasized CRC (LM) human tissues by pyrosequencing. LM
showed
significantly lower Alu methylation compared to PC. This data suggests that
Alu
hypomethylation is involved in CRC metastasis, which indicates usefulness of
Alu methylation
as a marker of CRC metastasis. The present inventors analyzed the location of
the various
miRNAs using the UCSC Genome Browser. Briefly, the miRNA sequences were found
in the
context of the genome in which they are located and it was found that miR-30b,
miR-373, miR-
518d, and miR-520c are located downstream of Alu elements (data not shown).
Figure 5A are maps that show the Alu hypomethylation regulated the expression
of down-
stream miR-373, but not miR-520c.
Figure 5B shows that Alu methylation inversely correlated with miR-373
expression in CRC cell
lines. An analysis of the expression of miR-373 and methylation of Alu (which
is located in
miR-373 promoter region) in CRC cell lines. It was found that the expression
of miR-373 was
inversely correlated with Alu methylation. These data demonstrate that miR-373
expression is
regulated by promoter region Alu methylation.
Figure 5C shows that Alu methylation did not correlate with miR-520c
expression in CRC cell
lines. The expression of miR-373 was induced by demethylating agent, and Alu
element in
miR-373 promoter region was demethylated by 5Aza treatment. These data
indicate that miR-
373 expression is regulated by Alu methylation.
Figure 6A is an analysis of the expression of miR-520c, which is located in
Alu element
downstream. All cell lines showed low level of miR-520c expression, except
Lovo cell.
Figure 6B shows that the expression of miR-520c was induced by a demethylating
agent. This
data indicates that miR-520c expression is regulated by Alu methylation in
promoter region.
Figure 7 is an analysis of the expression status all 4 miRNAs that are located
downstream of Alu
repetitive sequences in matched corresponding primary CRC (PC) and liver
metastasized CRC
(LM) human tissues by qRT-PCR. LM showed significantly lower expression of miR-
30b, miR-
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518d, and miR-520c compared to PC. On the other hand, miR-373 was
significantly up-
regulated in LM compared to PC. This data shows that the expression of these
four miRNAs
distinguished between PC and LM. Moreover, the expression of these four miRNAs
expression
was regulated by Alu elements methylation, which are located in promoter
region.
5 Figure 8A shows that Alu hypomethylation positively correlated with miR-
373 expression in
LM.
Figure 8B shows that Alu hypomethylation did not correlate with miR-520c
expression in LM.
Figure 9 shows that an RNA pol II inhibitor did not suppress activation of miR-
373, but
inhibited the expression of miR-520c.
10 These results show that methylation of Alu repeat elements was not
different between MSS and
MSI CRC cell lines. Alu methylation in liver metastasized CRC was
significantly lower
compared to matched primary CRC (80% and 83%, respectively; P<0.001). It was
also found
that Alu sequences in the promoter region of miR-373 and miR-520c were
significantly
hypomethylated in liver metastasized CRC compared to matched primary CRC
(P<0.05 and
15 P<0.01, respectively). Thus, hypomethylation of Alu elements correlated
with the activation and
subsequent expression of miR-373, which is transcribed in same direction as
Alu elements in
liver metastasis tissues compared to matched primary CRC(P<0.01). However,
this correlation
was not observed for miR-520c that is transcribed in the opposite direction.
Finally, treatment
of RNA polymerase II inhibitor did not suppress miR-373 transcription, but
inhibited the
20 expression of miR-520c.
These figures show that a miRNA signature can be used to distinguish between
primary CRC
and liver metastasis. It was found that a subset of miRNAs, including: let-7i,
miR-10b, miR-30b,
miR-200b, miR-320a, and miR-518d were significantly downregulated in liver
metastasis tissues
compared to primary CRC. In contrast, miRNAs such as miR-141, miR-200c, and
miR-203
were significantly over-expressed in liver metastasis tissues. In a further
evaluation step using
serum samples from CRC patients, it was found that the serum expression levels
of miR-200c
and miR-203 were upregulated in CRC patients with distant metastasis compared
to CRC
patients without metastasis.
The methylation status of Alu elements was determined by quantitative
bisulfite pyrosequencing,
and the expression of miRNAs (miR-30b, miR-373 and miR-520c) was measured by
quantitative real-time PCR. Global hypomethylation of Alu sequences was
observed by
treatment of the DNA demethylating agent, 5-azacytidine, in the CRC cell
lines. When the
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21
global Alu methylation levels were analyzed in clinical specimens, it was
found that the levels of
Alu methylation in LM were significantly lower compared to the matched PC
(mean
methylation value; PC = 83% vs. LM = 80%; P<0.001). Furthermore, methylation
analysis of
surrounding normal liver tissues revealed higher levels of Alu methylation
(mean methylation
value; 85%) when compared to both PC and LM. The expression of miRNAs (miR-
30b, miR-
373 and miR-520c) located downstream from Alu regions. For example, it was
found that miR-
373 expression was significantly increased in liver metastasis compared to
matching primary
CRC clinical samples. In contrast, expression of miR-30b and miR-520c was
significantly
decreased in liver metastasis when compared to matching primary CRC tissues.
These data
demonstrate that Alu hypomethylation is involved in CRC distant metastasis,
which regulates
metastasis-related miRNAs (miR-30b, miR-373 and miR-520c).
Thus, these results show that increased Alu hypomethylation occurs in liver
metastasis tissues
from patients with CRC. As such, hypomethylation of Alu repeat elements permit
activation of
metastasis-related miRNAs, which in turn may facilitate a more aggressive
malignant phenotype
in these advanced stage cancers.
Next, additional studies were conducted to identify the specific subsets of
miRNAs that may
serve as diagnostic and therapeutic biomarkers for patients with metastatic
CRC. A recent and
accurate technology to identify novel metastasis related miRNA biomarkers
(NANOSTRINGO),
plus additional studies were conducted to validate screened miRNA biomarkers
using two
different assay techniques in a large number of CRC tissues.
The screening step included the following materials: 9 pairs of primary CRC
(PC) and matched
liver metastasis (LM), Frozen tissue, Not-microdissected, method used:
NANOSTRINGO.
The validation step in matched PCs and LMs included the following materials:
58 pairs of PC
and matched LM, formalin-fixed, paraffin-embedded (FFPE) tissue,
Microdissected. The
method for analysis was TaqMan miRNA assays, miR-16 was used as endogenous
control.
A microarray validation step included the following materials: 84 pairs of PC
and corresponding
normal mucosa (NM), frozen tissue, not-microdissected. The method used was
MicroRNA
microarray (quadruplicates of 389 human miRNAs) as published in JAMA. 2008 Jan
30;299(4):425-36.
A qRT-PCR Validation step included the following materials: 175 PCs, FFPE
tissue,
microdissected. The method for analysis was TaqMan miRNA assays, with miR-16
used as
endogenous control.
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22
Table 1 is a summary of the clinicopathology characteristics of the colorectal
cancer patients.
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using the
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WO- m.1.R...tin M 217'
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0.39 3.E-01
hut-itilk-92b 44:38 3231 .. 0.73 039
3õE-02
...... .......
....=
hsn-n.P.,365 56.55 2Ø25i 0,52 039
.3.,E-02ji
Im-IiiiP.,-708 269 8S 155,4 0.58 039
...=
... ...
Ina-milt-143 2652.8.8, 1$36.82i 0.5 ....... 0.39
Figure 10 shows the Results - qRT-PCR validation for selected miRNAs in 58 PCs
and LMs.
Figure 11 shows the results from the microarray validation for selected miRNAs
in 84 PCs
CA 02859865 2014-06-18
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PCT/US2012/071455
23
(Kaplan-Meier survival curves), in which high expression of has-let-7i and has-
miR-320a
indicated a good prognosis, which a low expression of has-miR-10b and has-miR-
221 indicated
a good prognosis.
Table 3 shows the results from the microarray validation for 4 miRNAs in 84
PCs, briefly, it was
found that The expression of let-7i, miR-10b and miR-320a in PC was
significantly associated
with the distant metastasis, while the expression of let-7i and miR-10b was
significantly
associated with the TNM stage.
itq.koi ka.tala 1)u WR =2z.1 k4-ta -÷,fiZ.
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Table 4 shows the results of microarray validation for 4 miRNAs in 84 PCs,
using a Cox
proportional hazards model. It was found that low expression of let-7i was an
independent
prognostic factor.
CA 02859865 2014-06-18
WO 2013/096888
PCT/US2012/071455
24
Utlit aim
Vetio14et
RR CI
Aeo 5:R11 0 75)3 0,1N1 to
470t ON
Sec Nolo n. Reale) 1.342:5 0.5%5-
W3.2147 1:$474
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to$ 6750 U):006.
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la. Low) 1,900 0,,n1
3.8300 0.0$99
Itu.-ta-M. w.
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RR, lama ratio; Ci. ordiere iaterml
Table 5 shows the results of microarray validation for 4 miRNAs in 84 PCs,
using a logistic
regression model. It was found that all 4 miRNAs (let-7i, miR-320a, miR-10b
and miR-221)
expression in PCs was significantly associated with the distant metastasis. It
was also found that
low expression of let-7i and high expression of miR-10b in PCs were an
independent metastasis
prediction marker, respectively.
CA 02859865 2014-06-18
WO 2013/096888
PCT/US2012/071455
Uttiviniat
Age
Variat43
OR .0,÷fe
(`.,Rt vs. &50) .;,941 0 .0e65
7.37N 0,3423
Sez (Male w. Female) 1.1034 0.213) 571g1
osoo
T (TIM vs.. Tin) 2.45E+07
omoo to 0 0{100 0.0673
N stage (N1,s2a vs. le 9 1,06,50 to
74.6570 0.010
bsa-let-A (Low IT. 110) 191) 4.1
32. 10 89 a% 10,00011
hgainit-342 (Low 1,1. ITO) 55K1 13N8
213i20 t2:014,11
Uttimtim
Vadat& s
OR 05% CI
55% O.741 0.0SfiS to 7
3723 08423
Sa (Ma k tl= Fmk) 1.1034 0.2129 to
5.7192 0,906
'I siam (T3.4 T1t2) 2,05E47 0,M0 00a) co73
N m.z4F CiI17/3 VI; NO) 91 039.) la
74.070 0,01
hilaitaaviCt(H.Ovs. Low) 7,625 1,8645 to
31.1334 004.41
(1411 m Low) 9 . 1
4150 to 71.570 t 0101 t
OR, oold.t ratio; CI, toddeace iatetval
Figure 12 shows the results of qRT-PCR validation for miR-7i and miR-10b in
175 PCs.
Survival analysis of 2 microarray validated miRNAs is shown.
Table 6 shows the results of qRT-PCR validation for miR-7i and miR-10b in 175
PCs. It was
5 found that the expression of let-7i, miR-10b and miR-320a in PC was
significantly associated
with the distant metastasis (using the Kreskal-Wallis test). The expression of
let-7i and miR-10b
was significantly associated with the TNM stage.
CA 02859865 2014-06-18
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PCT/US2012/071455
26
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IV 24 a 6133 o. I
Table 7 shows the results from qRT-PCR validation for miR-7i and miR-10b in
175 PCs using
the Cox proportional hazards model. It was found that Low expression of let-7i
was
significantly associated with CRC patient's prognosis, which was an
independent prognostic
factor.
CA 02859865 2014-06-18
WO 2013/096888
PCT/US2012/071455
27
\ \\
tAtiv:wiak=
Vatioblt
P ,
A P. t >:',Weefal0 is)=4 04370;0 I.47?"2:
Sex (1,õtok 'Kt. Ennio) 11504 0.6133 =1i16"
0.662
T stap i;T:34 vt; 7.9376 1.9310 to 6160
0.-001
41qt mit; =Outwit fftto n. No) MEV 5,.t117 to 4,1 0A5,6
0,0001
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0.0001
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0 2:V5
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:Lu&
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1-3R 05% CI P
(Att=&tt rt. ale:-}Oriµt 0 ;>34 0.4370 to 4772
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0.662
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0.0001
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0=170 A C00011
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hoo,11-.ra,=M (Br vs. Low) I. MO 03203 to 4.3747 ,
,
HP, Itaurd nth; CL. etstildeate
Table 8 shows the results from qRT-PCR validation for miR-7i and miR-10b in
175 PCs using a
logistic regression model. It was found that expression of let-7i and miR-10b
in PCs was
significantly associated with the distant metastasis. Low expression of let-7i
and high
expression of miR-10b in PCs were an independent metastasis prediction marker,
respectively.
CA 02859865 2014-06-18
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PCT/US2012/071455
28
Univariot
OR
Vatiatite s
95% Ci
Agc 3:::-Mcdna -:=<.,Mediat* 0. 2a6u.)
wri
=sex (mak Fnttille) 0,5W4 to
2.1662
T taw t:TY4 TI72) 6.07E+00
1.7714 to 20. 7K3 0,0095
N ntvc (N112V3 rs. NO)
2$,6744 7.479 $O4 < 0.0 001
in441-7i (Low vs HO) 59853 [3679 to
2.!2
OR 05% Cl
A. (Me. 0 5152 0 248016
0671 0 0711
Sex CMak. Fennle) 1,0505 0.3i,V4 to
2,1 %I ams
=
T Mgt cr514-1.n. TI72)
6.07E410 1.7714 20 323 (1.0095
N (Ni25n, NO) 25,6744 7,470
0324 <0,0001
0411 vs, Low) 0,91.76 to lo.A..)17
3,41,21,AL
OR, ockin CI, confidence inntreal.
Figure 13 shows the ISH validation for the expression of miR-7i and miR-10b in
CRC tissues
and liver metastasis.
As such, it was found that 19 metastasis specific miRNAs were identified
through screening step
using NANOSTRINGOanalysis. Among 19 screened miRNAs, 4 miRNAs were validated
in a
large number of matched PC and LM tissues (58 pairs). High expression of let-
7i was
significantly associated with better survival, which was an independent
prognostic marker in
CRC patients. Low expression of let-7i and high expression of miR-10b were
independent
metastasis prediction markers in PCs, respectively. Finally, it was found that
let-7i and miR-10b
expression was successfully validated through ISH analysis in CRC tissues.
It is contemplated that any embodiment discussed in this specification can be
implemented with
respect to any method, kit, reagent, or composition of the invention, and vice
versa.
Furthermore, compositions of the invention can be used to achieve methods of
the invention.
It will be understood that particular embodiments described herein are shown
by way of
illustration and not as limitations of the invention. The principal features
of this invention can
be employed in various embodiments without departing from the scope of the
invention. Those
skilled in the art will recognize, or be able to ascertain using no more than
routine
experimentation, numerous equivalents to the specific procedures described
herein. Such
CA 02859865 2014-06-18
WO 2013/096888 PCT/US2012/071455
29
equivalents are considered to be within the scope of this invention and are
covered by the
claims.
All publications and patent applications mentioned in the specification are
indicative of the level
of skill of those skilled in the art to which this invention pertains. All
publications and patent
applications are herein incorporated by reference to the same extent as if
each individual
publication or patent application was specifically and individually indicated
to be incorporated
by reference.
The use of the word "a" or "an" when used in conjunction with the term
"comprising" in the
claims and/or the specification may mean "one," but it is also consistent with
the meaning of
"one or more," "at least one," and "one or more than one." The use of the term
"or" in the
claims is used to mean "and/or" unless explicitly indicated to refer to
alternatives only or the
alternatives are mutually exclusive, although the disclosure supports a
definition that refers to
only alternatives and "and/or." Throughout this application, the term "about"
is used to indicate
that a value includes the inherent variation of error for the device, the
method being employed to
determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words "comprising" (and any
form of comprising,
such as "comprise" and "comprises"), "having" (and any form of having, such as
"have" and
"has"), "including" (and any form of including, such as "includes" and
"include") or
"containing" (and any form of containing, such as "contains" and "contain")
are inclusive or
open-ended and do not exclude additional, unrecited elements or method steps.
As used herein,
the phrase "consisting essentially of' limits the scope of a claim to the
specified materials or
steps and those that do not materially affect the basic and novel
characteristic(s) of the claimed
invention. As used herein, the phrase "consisting of" excludes any element,
step, or ingredient
not specified in the claim except for, e.g., impurities ordinarily associated
with the element or
limitation.
The term "or combinations thereof' as used herein refers to all permutations
and combinations
of the listed items preceding the term. For example, "A, B, C, or combinations
thereof' is
intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order
is important in a
particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing
with this
example, expressly included are combinations that contain repeats of one or
more item or term,
such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled
artisan will understand that typically there is no limit on the number of
items or terms in any
combination, unless otherwise apparent from the context.
CA 02859865 2014-06-18
WO 2013/096888 PCT/US2012/071455
As used herein, words of approximation such as, without limitation, "about",
"substantial" or
"substantially" refers to a condition that when so modified is understood to
not necessarily be
absolute or perfect but would be considered close enough to those of ordinary
skill in the art to
warrant designating the condition as being present. The extent to which the
description may vary
5 will depend on how great a change can be instituted and still have one of
ordinary skilled in the
art recognize the modified feature as still having the required
characteristics and capabilities of
the unmodified feature. In general, but subject to the preceding discussion, a
numerical value
herein that is modified by a word of approximation such as "about" may vary
from the stated
value by at least 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
10 All of the compositions and/or methods disclosed and claimed herein can
be made and executed
without undue experimentation in light of the present disclosure. While the
compositions and
methods of this invention have been described in terms of preferred
embodiments, it will be
apparent to those of skill in the art that variations may be applied to the
compositions and/or
methods and in the steps or in the sequence of steps of the method described
herein without
15 departing from the concept, spirit and scope of the invention. All such
similar substitutes and
modifications apparent to those skilled in the art are deemed to be within the
spirit, scope and
concept of the invention as defined by the appended claims.
