U.S. patent application number 17/022345 was filed with the patent office on 2021-06-03 for assays to determine dna methylation and dna methylation markers of cancer.
The applicant listed for this patent is ZYMO RESEARCH CORPORATION. Invention is credited to Wei GUO, Xi-Yu JIA, Paolo PIATTI, Xiaojing YANG.
Application Number | 20210164031 17/022345 |
Document ID | / |
Family ID | 1000005399945 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210164031 |
Kind Code |
A1 |
GUO; Wei ; et al. |
June 3, 2021 |
ASSAYS TO DETERMINE DNA METHYLATION AND DNA METHYLATION MARKERS OF
CANCER
Abstract
Methods are provided for determining a genomic methylation
profile in a DNA sample. In certain aspects, the methods can be
used to determine if a subject has, or is at risk for developing, a
bladder cancer or other cancers of the urinary tract. Methods for
treatment of such subjects are likewise provided.
Inventors: |
GUO; Wei; (Irvine, CA)
; PIATTI; Paolo; (Irvine, CA) ; YANG;
Xiaojing; (Irvine, CA) ; JIA; Xi-Yu; (Irvine,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZYMO RESEARCH CORPORATION |
Irvine |
CA |
US |
|
|
Family ID: |
1000005399945 |
Appl. No.: |
17/022345 |
Filed: |
September 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15552825 |
Aug 23, 2017 |
10801060 |
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PCT/US2016/019310 |
Feb 24, 2016 |
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17022345 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2545/101 20130101;
C12Q 2561/113 20130101; C12Q 2521/331 20130101; C12Q 2563/107
20130101; C12Q 2537/143 20130101; C12Q 2523/125 20130101; C12Q
1/6858 20130101 |
International
Class: |
C12Q 1/6858 20180101
C12Q001/6858 |
Claims
1. A method for determining a genomic DNA methylation profile in a
sample comprising: (a) obtaining a substantially purified test
genomic DNA sample; (b) contacting a portion test genomic DNA of
the sample with: a first reaction mixture comprising: (i) at least
two methylation sensitive restriction endonucleases; (ii) a
hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv)
dNTPs; (v) DNA primer pairs for polymerase chain reaction (PCR)
amplification of at least a first, second and third different
genomic region in the DNA sample; and (vi) fluorescent probes
complementary to sequences in said first, second and third
different genomic regions for quantitative detection of amplified
sequences from the first, second and third different genomic
regions, wherein each of the probes comprises a distinct
fluorescent label, wherein: (I) the first genomic region is a
cleavage control that is known to be unmethylated; (II) the second
genomic region is a copy number control that does not include any
cut sites for the methylation sensitive restriction endonucleases
of the first reaction mixture; and (III) the third genomic region
is a test region having an unknown amount of methylation and
including at least three cut sites for the methylation sensitive
restriction endonucleases of the first reaction mixture; (c)
subjecting the first reaction mixture to digestion and thermal
cycling, while detecting fluorescent signals from the fluorescent
probes, thereby performing real time PCR on the samples in the
first and second reaction mixtures; (d) using the detected
fluorescent signals to determine the genomic DNA methylation
profile in a sample.
2. The method of claim 1, further comprising: (a) obtaining a
substantially purified test genomic DNA sample; (b) contacting a
portion test genomic DNA of the sample with: a first reaction
mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second and third different genomic region in the DNA sample; and
(vi) fluorescent probes complementary to sequences in said first,
second and third different genomic regions for quantitative
detection of amplified sequences from the first, second and third
different genomic regions, wherein each of the probes comprises a
distinct fluorescent label; and a second reaction mixture,
identical to the first reaction mixture, but lacking the at least
two methylation sensitive restriction endonucleases, wherein: (I)
the first genomic region is a cleavage control that is known to be
unmethylated; (II) the second genomic region is a copy number
control that does not include any cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture;
and (III) the third genomic region is a test region having an
unknown amount of methylation and including at least three cut
sites for the methylation sensitive restriction endonucleases of
the first reaction mixture; (c) subjecting the first and second
reaction mixtures to digestion and thermal cycling, while detecting
fluorescent signals from the fluorescent probes, thereby performing
real time PCR on the samples in the first and second reaction
mixtures; (d) using the detected fluorescent signals to determine
the genomic DNA methylation profile in a sample.
3-8. (canceled)
9. The method of claim 1, wherein the substantially purified test
genomic DNA sample is obtained from a urine, stool, saliva, blood
or tissue sample.
10. The method of claim 9, wherein the substantially purified test
genomic DNA sample is obtained from a biopsy sample.
11. (canceled)
12. The method of claim 1, wherein the first reaction mixture
comprises at least three methylation sensitive restriction
endonucleases.
13. (canceled)
14. The method of claim 1, wherein step (b) further comprises: (b)
contacting a portion test genomic DNA of the sample with: a first
reaction mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second, third and fourth different genomic region in the DNA
sample; and (vi) fluorescent probes complementary to sequences in
said first, second, third and fourth different genomic regions for
quantitative detection of amplified sequences from the first,
second, third and fourth different genomic regions, wherein each of
the probes comprises a distinct fluorescent label, wherein: (I) the
first genomic region is a cleavage control that is known to be
unmethylated; (II) the second genomic region is a copy number
control that does not include any cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture;
and (III) the third and fourth genomic regions are test regions
having an unknown amount of methylation and including at least
three cut sites for the methylation sensitive restriction
endonucleases of the first reaction mixture.
15. The method of claim 14, wherein step (b) further comprises: (b)
contacting a portion test genomic DNA of the sample with: a first
reaction mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second, third, fourth and fifth different genomic region in the DNA
sample; and (vi) fluorescent probes complementary to sequences in
said first, second, third, fourth and fifth different genomic
regions for quantitative detection of amplified sequences from the
first, second, third, fourth and fifth different genomic regions,
wherein each of the probes comprises a distinct fluorescent label,
wherein: (I) the first genomic region is a cleavage control that is
known to be unmethylated; (II) the second genomic region is a copy
number control that does not include any cut sites for the
methylation sensitive restriction endonucleases of the first
reaction mixture; and (III) the third, fourth and fifth genomic
regions are test regions having an unknown amount of methylation
and including at least three cut sites for the methylation
sensitive restriction endonucleases of the first reaction
mixture.
16. (canceled)
17. The method of claim 1, wherein at least 4, 5, 6, 7 or 8 cut
sites for the methylation sensitive restriction endonucleases of
the first reaction mixture.
18. The method of claim 1, wherein the primer pairs are
complementary to sequences no more than 300, 275, 250, 225, 200,
190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or
50 nucleotides apart.
19. The method of claim 1, wherein the first genomic region is a
genomic region of a housekeeping gene.
20. The method of claim 19, wherein the housekeeping gene is
GAPDH.
21. The method of claim 1, wherein the second genomic region is a
genomic region of the POLR2A gene.
22. The method of claim 1, wherein the third genomic region is
selected from the group provided in Table 1A of Table 2.
23. The method of claim 1, wherein the third genomic region is
selected from the group consisting of DMRTA2, EVX2, Unk21, OTX1,
SOX1, SEPT9, Unk05, Unk09, GALR1, Unk07, Unk19, TBX15, EEF1A2,
TFAP2B, DCHS2 and SOX17.
24. The method of claim 1, wherein using the detected fluorescent
signals to determine the genomic DNA methylation profile in a
sample comprises calculating the relative methylation percentages
for the sample.
25. The method of claim 1, wherein the fluorescent probes and/or
primer pairs are selected from those provided in Table 1C.
26-28. (canceled)
29. A method for determining a genomic DNA methylation profile in a
sample comprising: (a) obtaining a test genomic DNA sample, which
has been bisulfite converted; (b) contacting the test sample with a
first reaction mixture comprising: (i) a hot-start DNA polymerase;
(ii) a pH buffered salt solution; (iii) dNTPs; (iv) DNA primer
pairs for polymerase chain reaction (PCR) amplification of at least
a first, and second different genomic region in the DNA sample,
wherein the primer pairs are complementary to sequences no more
than 200 nucleotides apart; and (v) fluorescent probes
complementary to sequences in said first, and second different
genomic regions for quantitative detection of amplified sequences
from the first and second different genomic regions, wherein each
of the probes comprises a distinct fluorescent label, wherein: (I)
the first genomic region is a copy number control region that that
does not comprise CpG dinucleotides; and (II) the second genomic
region is a test region having an unknown amount of methylation and
including at least five CpG dinucleotides in sequences that are
complementary to DNA primer pairs and the probe for the second
genomic region; (c) subjecting the first reaction mixtures to
thermal cycling, while detecting fluorescent signals from the
fluorescent probes, thereby performing real time PCR on the sample
in the first reaction mixture; and (d) using the detected
fluorescent signals and fluorescent signal from a DNA methylation
standard curve to determine the genomic DNA methylation profile in
a sample.
30-58. (canceled)
59. A method of detecting the presence of, or an increased risk of,
bladder cancer or other cancers of the urinary tract in a patient
comprising determining a methylation status in one or more genomic
regions in a patient sample selected from the group provided in
Table 1A wherein an increased level of methylation in one or more
of the genomic regions of Table 1A relative to a reference level
indicates that the patient has or is at risk of developing bladder
cancer.
60. The method of claim 59, wherein the one or more genomic regions
is selected from the group consisting of Unk 09, Unk 05, DCHS2,
OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, and SOX17.
61. The method of claim 60, wherein the one or more genomic regions
is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the genomic
regions selected from the group consisting of Unk 09, Unk 05,
DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, and
SOX17.
62-66. (canceled)
67. The method of claim 59, wherein said determining comprises
determining a methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more of said genomic regions.
68. The method of claim 59, wherein said determining comprises
determining a methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more of said genomic regions, wherein the genomic regions are
selected from the group consisting of Unk 09, Unk 05, DCHS2, OTX1,
Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, SOX17, GALR1, TBX15,
EEF1A2, DMRTA2, and TFAP2B.
69. The method of claim 68, wherein said determining comprises
determining a methylation in each of the genomic regions: Unk 09,
Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21,
SOX17, GALR1, TBX15, EEF1A2, DMRTA2, and TFAP2B.
70. The method of claim 59, wherein the patient has been previously
treated for or diagnosed with bladder cancer.
71. The method of claim 70, further defined as method for detecting
bladder cancer recurrence or a risk of bladder cancer
recurrence.
72. The method of claim 59, wherein said determining comprises
analyzing DNA methylation in the sample using restriction
endonuclease digestion and qPCR.
73. The method of claim 72, wherein the digestion reaction is
completed in a first step, followed by the qPCR reaction in a
second step.
74. The method of claim 59 wherein the patient is a human.
75. The method of claim 59, wherein the sample is a urine
sample.
76. The method of claim 59, wherein the sample is a blood
sample.
77-78. (canceled)
79. The method of claim 59, wherein determining a methylation
status comprises determining the nucleotide positions in the
genomic regions that comprise methylation.
80. The method of claim 59, wherein determining a methylation
status comprises determining the proportion of methylation at
nucleotide positions in the genomic region.
81. The method of claim 59, wherein determining a methylation
status comprises determining the proportion of nucleotide positions
that are methylated in the genomic region.
82. The method of claim 59, wherein the reference level is a level
of methylation from a patient that does not have bladder
cancer.
83-85. (canceled)
86. A method of detecting the presence of, or an increased risk of,
bladder cancer in a patient comprising: (i) obtaining a patient
sample; (ii) determining a methylation status in one or more
genomic regions selected from those in Table 1; and (iii)
identifying the presence of, or an increased risk of, bladder
cancer in the patient based on an increased level of methylation in
one or more of the genomic regions relative to a reference
level.
87-94. (canceled)
95. A synthetic polynucleotide sequence comprising a sequence at
least 90% identical to one of the probe sequences selected from
those provided in Table 1B or 1C, wherein the polynucleotide is
conjugated to a reporter molecule.
96. The polynucleotide of claim 95, wherein the polynucleotide is a
fluorophore.
97. The polynucleotide of claim 95, comprising a sequence at least
95% identical to one of the probe sequences selected from those
provided in Table 1B or 1C.
98. The polynucleotide of claim 95, comprising a sequence identical
to one of the probe sequences selected from those provided in Table
1B or 1C.
99. A kit comprising at least two primer pairs and at least two
probes for amplification and detection of a gene region selected
from those provided in Table 1A.
100. The kit of claim 99, comprising at least three, four or five
two primer pairs and at least three, four or five probes for
amplification and detection of a gene region selected from those
provided in Table 1A.
101. The kit of claim 99, wherein the least two primer pairs and at
least two probes are selected from those provided in Table 1B or
1C.
102. The kit of claim 99, further comprising one or more of the
following: (i) instructions; (ii) reagents for real-time qPCR;
(iii) a methylation sensitive endonuclease; and (iv) a control DNA
sample.
Description
[0001] The present application is a continuation of co-pending U.S.
application Ser. No. 15/552,825, which was filed Aug. 23, 2017,
which is a national phase application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2016/019310, filed Feb. 24,
2016, which claims the benefit of U.S. Provisional patent
Application No. 62/120,373, filed Feb. 24, 2015, both of which are
incorporated herein by reference, in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to the fields of
molecular biology, epigenetics, and predictive medicine. More
particularly, it concerns method for determining a genomic DNA
methylation profile in a sample.
2. Description of Related Art
[0003] Cancers of the urinary tract include bladder, urethral,
kidney and prostate cancers whose cells are detectible via urine or
biopsy. Bladder cancer was one of the 10 most prevalent
malignancies in males in 2011, ranking fourth and eighth in terms
of deaths and new cases, respectively (Siegel et al., 2011; Morgan
and Clark, 2010). Nonmuscle invasive bladder cancer (NMIBC)
accounts for 80% of all the cases, and can be further classified
into mucosa only (Ta), carcinoma in situ (Tis), and lamina propria
invading, (T1) lesions (Babjuk et al., 2011, Sobin et al., 2009).
The primary treatment for NMIBC is transurethral resection of
bladder tumor (TURBT) with or without intravesical chemo or
immunotherapy; however, more than 50% of patients recur after the
TURBT procedure, with the highest rate of recurrence occurring in
patients with high-risk disease (Shelley et al., 2010;
Millan-Rodriguez et al., 2000). As a result, patients require
frequent and lifelong monitoring following TURBT, making bladder
cancer one of the most costly types of cancer to manage.
[0004] The current standard for monitoring of bladder cancer
recurrence involves the use of cystoscopy and cytology (Morgan and
Clark, 2010; Babjuk et al., 2011). Disease surveillance is
cumbersome because of the invasive nature of cystoscopic
examination and the low sensitivity of urinary cytology in the
detection of low-grade tumors (Lintula and Hotakainen, 2010). The
addition of nuclear matrix protein 22 (NMP-22), bladder tumor
antigen, or UroVysion FISH has shown to help increase the
sensitivity of cytology (Parker and Spiess, 2011). However, due to
their inconsistent performance in terms of specificity or
sensitivity, the markers proposed to date have not been widely
adopted in routine clinical practice (Reinert 2012). Therefore,
there is a need to find reliable markers to monitor bladder cancer
patients as well as to distinguish different cancers associated
with the urinary tract. Moreover, there is a need to new
methodologies for assessing genomic DNA methylation profiles in
biological samples.
SUMMARY OF THE INVENTION
[0005] In a first embodiment, the invention provides a method for
determining a genomic DNA methylation profile in a sample
comprising: (a) obtaining a substantially purified test genomic DNA
sample; (b) contacting a portion test genomic DNA of the sample
with a first reaction mixture comprising: (i) at least two
methylation sensitive restriction endonucleases; (ii) a hot-start
DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; (v)
DNA primer pairs for polymerase chain reaction (PCR) amplification
of at least a first, second and third different genomic region in
the DNA sample; and (vi) fluorescent probes complementary to
sequences in said first, second and third different genomic regions
for quantitative detection of amplified sequences from the first,
second and third different genomic regions, wherein each of the
probes comprises a distinct fluorescent label; wherein (I) the
first genomic region is a cleavage control that is known to be
unmethylated, (II) the second genomic region is a copy number
control that does not include any cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture,
and (III) the third genomic region is a test region having an
unknown amount of methylation and including at least three cut
sites for the methylation sensitive restriction endonucleases of
the first reaction mixture; (c) subjecting the first reaction
mixture to digestion and thermal cycling, while detecting
fluorescent signals from the fluorescent probes, thereby performing
real time PCR on the samples in the first and second reaction
mixtures; and (d) using the detected fluorescent signals to
determine the genomic DNA methylation profile in a sample.
[0006] In further aspects, the method additionally comprises: (a)
obtaining a substantially purified test genomic DNA sample; (b)
contacting a portion test genomic DNA of the sample with a first
reaction mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second and third different genomic region in the DNA sample; and
(vi) fluorescent probes complementary to sequences in said first,
second and third different genomic regions for quantitative
detection of amplified sequences from the first, second and third
different genomic regions, wherein each of the probes comprises a
distinct fluorescent label; and a second reaction mixture,
identical to the first reaction mixture, but lacking the at least
two methylation sensitive restriction endonucleases, wherein: (I)
the first genomic region is a cleavage control that is known to be
unmethylated; (II) the second genomic region is a copy number
control that does not include any cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture;
and (III) the third genomic region is a test region having an
unknown amount of methylation and including at least three cut
sites for the methylation sensitive restriction endonucleases of
the first reaction mixture; (c) subjecting the first and second
reaction mixtures to digestion and thermal cycling, while detecting
fluorescent signals from the fluorescent probes, thereby performing
real time PCR on the samples in the first and second reaction
mixtures; and (d) using the detected fluorescent signals to
determine the genomic DNA methylation profile in a sample.
[0007] In some aspects, the first reaction mixture further
comprises a PCR enhancer. In specific aspects, the PCR enhancer may
comprise DMSO. In certain aspects, obtaining a substantially
purified test genomic DNA sample may comprise purifying the DNA
sample. In several aspects, the substantially purified test genomic
DNA sample is of sufficient purity to provide at least 85%, 90%,
95% or 99% digestion of the DNA by said at least two methylation
sensitive restriction endonucleases in 2 hours at 30.degree. C. In
particular aspects, the substantially purified test genomic DNA
sample comprises 50 pg to 1,000 ng of DNA. In some specific
aspects, the substantially purified test genomic DNA sample
comprises less than 50 ng of DNA.
[0008] In certain aspects, the substantially purified test genomic
DNA sample may be obtained from a urine, stool, saliva, blood or
tissue sample. In some particular aspects, the substantially
purified test genomic DNA sample is obtained from a biopsy sample.
In other particular aspects, the substantially purified test
genomic DNA sample is obtained from a urine sample. In several
aspects, the first reaction mixture comprises at least three
methylation sensitive restriction endonucleases. In further
aspects, the at least three methylation sensitive restriction
endonucleases comprise AciI, HinPl1 and HpaII.
[0009] In some aspects, step (b) may further comprise contacting a
portion test genomic DNA of the sample with a first reaction
mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second, third and fourth different genomic region in the DNA
sample; and (vi) fluorescent probes complementary to sequences in
said first, second, third and fourth different genomic regions for
quantitative detection of amplified sequences from the first,
second, third and fourth different genomic regions, wherein each of
the probes comprises a distinct fluorescent label, wherein: (I) the
first genomic region is a cleavage control that is known to be
unmethylated, (II) the second genomic region is a copy number
control that does not include any cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture,
and (III) the third and fourth genomic regions are test regions
having an unknown amount of methylation and including at least
three cut sites for the methylation sensitive restriction
endonucleases of the first reaction mixture.
[0010] In further aspects, step (b) may additionally comprise
contacting a portion test genomic DNA of the sample with a first
reaction mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
second, third, fourth and fifth different genomic region in the DNA
sample; and (vi) fluorescent probes complementary to sequences in
said first, second, third, fourth and fifth different genomic
regions for quantitative detection of amplified sequences from the
first, second, third, fourth and fifth different genomic regions,
wherein each of the probes comprises a distinct fluorescent label,
and wherein (I) the first genomic region is a cleavage control that
is known to be unmethylated, (II) the second genomic region is a
copy number control that does not include any cut sites for the
methylation sensitive restriction endonucleases of the first
reaction mixture, and (III) the third, fourth and fifth genomic
regions are test regions having an unknown amount of methylation
and including at least three cut sites for the methylation
sensitive restriction endonucleases of the first reaction mixture.
In certain aspects, the third, fourth and fifth genomic regions may
be regions of Unk05, Unk09 and SOX17. In still further aspects, a
method of the embodiments further comprises determining whether the
cells comprise an aneuploidy relative to one or more gene
region.
[0011] In several aspects, at least 4, 5, 6, 7 or 8 cut sites for
the methylation sensitive restriction endonucleases of the first
reaction mixture. In some aspects, the primer pairs are
complementary to sequences no more than 300, 275, 250, 225, 200,
190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or
50 nucleotides apart. In certain aspects, the first genomic region
is a genomic region of a housekeeping gene. The housekeeping gene
may be GAPDH. In still further aspects, the second genomic region
may be a genomic region of the POLR2A gene. In some aspects, the
third genomic region is selected from the group provided in Table
1A or Table 2. In some specific aspects, the third genomic region
is selected from the group consisting of DMRTA2, EVX2, Unk21, OTX1,
SOX1, SEPT9, Unk05, Unk09, GALR1, Unk07, Unk19, TBX15, EEF1A2,
TFAP2B, DCHS2 and SOX17. In a particular aspect, using the detected
fluorescent signals to determine the genomic DNA methylation
profile in a sample comprises calculating the relative methylation
percentages for the sample. In still further aspects, a method of
the embodiments comprises the use of one or more of the probes or
primer pairs provided in Table 1C.
[0012] In a further embodiment, the invention provides a reaction
mixture comprising: (i) at least two methylation sensitive
restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a
pH buffered salt solution; (iv) dNTPs; (v) a substantially purified
genomic DNA sample; (vi) DNA primer pairs for polymerase chain
reaction (PCR) amplification of at least a first, second and third
different genomic region in the DNA sample; (vii) fluorescent
probes complementary to sequences in said first, second and third
different genomic regions for quantitative detection of amplified
sequences from the first, second and third different genomic
regions, wherein each of the probes comprises a distinct
fluorescent label wherein: (I) the first genomic region is a
cleavage control that is known to be unmethylated; (II) the second
genomic region is a copy number control that does not include any
cut sites for the methylation sensitive restriction endonucleases
of the first reaction mixture; and (III) the third genomic region
is a test region having an unknown amount of methylation and
including at least three cut sites for the methylation sensitive
restriction endonucleases of the first reaction mixture. In some
aspects, the third genomic region is selected from the group
provided in Table 1A or Table 2. In still further aspects, the
probes complementary to sequences in said first, second and third
different genomic regions are selected from the probes provided in
Table 1C.
[0013] In still a further embodiment, there is provided a method
for determining a genomic DNA methylation profile in a sample
comprising: (a) obtaining a test genomic DNA sample, which has been
bisulfite converted; (b) contacting the test sample with a first
reaction mixture comprising: (i) a hot-start DNA polymerase; (ii) a
pH buffered salt solution; (iii) dNTPs; (iv) DNA primer pairs for
polymerase chain reaction (PCR) amplification of at least a first,
and second different genomic region in the DNA sample, wherein the
primer pairs are complementary to sequences no more than 200
nucleotides apart; and (v) fluorescent probes complementary to
sequences in said first, and second different genomic regions for
quantitative detection of amplified sequences from the first and
second different genomic regions, wherein each of the probes
comprises a distinct fluorescent label, wherein: (I) the first
genomic region is a copy number control region that that does not
comprise CpG dinucleotides; and (II) the second genomic region is a
test region having an unknown amount of methylation and including
at least five CpG dinucleotides in sequences that are complementary
to DNA primer pairs and the probe for the second genomic region;
(c) subjecting the first reaction mixtures to thermal cycling,
while detecting fluorescent signals from the fluorescent probes,
thereby performing real time PCR on the sample in the first
reaction mixture; and (d) using the detected fluorescent signals
and fluorescent signal from a DNA methylation standard curve to
determine the genomic DNA methylation profile in a sample.
[0014] In yet still a further embodiment, the invention provides a
method for determining a genomic DNA methylation profile in a
sample comprising: (a) obtaining a test genomic DNA sample, which
has been bisulfite converted, and a methylation control genomic DNA
sample that has been fully methylated; (b) contacting the test
sample with a first reaction mixture comprising: (i) a hot-start
DNA polymerase; (ii) a pH buffered salt solution; (iii) dNTPs; (iv)
DNA primer pairs for polymerase chain reaction (PCR) amplification
of at least a first and second different genomic region in the DNA
sample, wherein the primer pairs are complementary to sequences no
more than 200 nucleotides apart; and (v) fluorescent probes
complementary to sequences in said first and second different
genomic regions for quantitative detection of amplified sequences
from the first and second different genomic regions, wherein each
of the probes comprises a distinct fluorescent label, wherein: (I)
the first genomic region is a copy number control region that that
does not comprise CpG dinucleotides; and (II) the second genomic
region is a test region having an unknown amount of methylation and
including at least five CpG dinucleotides in sequences that are
complementary to DNA primer pairs and the probe for the second
genomic region; (c) contacting the methylation control bisulfite
converted genomic DNA sample with a second reaction mixture having
identical components as said first reaction mixture; (d) subjecting
the first and second reaction mixtures to thermal cycling, while
detecting fluorescent signals from the fluorescent probes, thereby
performing real time PCR on the samples in the first and second
reaction mixtures; and (e) using the detected fluorescent signals
and fluorescent signal from a DNA methylation standard curve to
determine the genomic DNA methylation profile in a sample.
[0015] In some aspects of the embodiments described herein, the
methods further comprise using the detected fluorescent signals of
the first genomic region to normalize DNA quantity across all
tested samples. In certain aspects, the first reaction mixture may
further comprise a PCR enhancer. In particular aspects, the PCR
enhancer comprises DMSO. In several aspects, determining the
genomic DNA methylation profile comprises determining the copy
number of methylated DNA molecules. In still further aspects, the
method may additionally comprise using the detected fluorescent
signals to determine ratio of methylation in the test sample to the
reference methylation control. In specific aspects, the genomic DNA
sample comprises 50 pg to 10 ng of DNA. In certain aspects, the
genomic DNA sample may comprise DNA isolated from 6 to 1,500
cells.
[0016] In yet still further aspects of the embodiments described
herein, the methods may additionally comprise obtaining a genomic
DNA sample and subjecting the genomic DNA sample to bisulfate
conversion. In specific aspects, the genomic DNA is obtained from a
urine, stool, saliva, blood or tissue sample. In other aspects, the
genomic DNA is obtained from a biopsy sample. In a particular
aspect, the genomic DNA is obtained from a urine sample.
[0017] In certain aspects, step (b) further comprises contacting
the test sample with a first reaction mixture comprising: (i) a
hot-start DNA polymerase; (ii) a pH buffered salt solution; (iii)
dNTPs; (iv) DNA primer pairs for PCR amplification of at least a
first, second and third different genomic region in the DNA sample,
wherein the primer pairs are complementary to sequences no more
than 200 nucleotides apart; and (v) fluorescent probes
complementary to sequences in said first, second and third
different genomic regions for quantitative detection of amplified
sequences from the first, second and third different genomic
regions, wherein each of the probes comprises a distinct
fluorescent label, wherein: (I) the first genomic region is a copy
number control region that that does not comprise CpG
dinucleotides; and (II) the second and third genomic regions are
test regions having an unknown amount of methylation and including
at least five CpG dinucleotides in sequences that are complementary
to DNA primer pairs and the probe for the second and third genomic
regions. In a particular aspect, step (b) may still further
comprise contacting the test sample with a first reaction mixture
comprising: (i) a hot-start DNA polymerase; (ii) a pH buffered salt
solution; (iii) dNTPs; (iv) DNA primer pairs for PCR amplification
of at least a first, second, third, fourth and fifth different
genomic region in the DNA sample, wherein the primer pairs are
complementary to sequences no more than 200 nucleotides apart; and
(v) fluorescent probes complementary to sequences in said first,
second, third, fourth and fifth different genomic regions for
quantitative detection of amplified sequences from the first,
second, third, fourth and fifth different genomic regions, wherein
each of the probes comprises a distinct fluorescent label, wherein:
(I) the first genomic region is a copy number control region that
that does not comprise CpG dinucleotides; and (II) the second,
third, fourth and fifth genomic regions are test regions having an
unknown amount of methylation and including at least five CpG
dinucleotides in sequences that are complementary to DNA primer
pairs and the probe for the second, third, fourth and fifth genomic
regions
[0018] In several aspects of the embodiments described herein, the
second genomic region includes at least 6, 7, 8, 9 or 10 CpG
dinucleotides in sequences that are complementary to DNA primer
pairs and the probe for the second region. In some specific
aspects, one of said CpG dinucleotides in sequences that are
complementary to DNA primer pairs includes a C positioned in the
last five nucleotides at the 3' end of the DNA primer pairs. In a
further aspect, the C may be positioned at the 3' end of the DNA
primer pairs.
[0019] In certain aspects, the primer pairs are complementary to
sequences no more than 170, 160, 150, 140, 130, 120, 110 or 100
nucleotides apart. In some aspects, each of the probes is no more
than 40 bp in length. In some particular aspects, each of the
probes is no more than 30 bp in length. In several aspects, each of
the probes comprises a CG ratio of 30-80%. In specific aspects,
each of the primers has a Tm of 55-62.degree. C. In a further
aspect, each of the probes may have a Tm of 65-72.degree. C.
[0020] In yet still further aspects, the copy number control region
is region of the COL2A1 gene. In some aspects, the second genomic
region is selected from the group provided in Table 1A. In some
particular aspects, the second genomic region is selected from the
group consisting of DMRTA2, EVX2, Unk21, OTX1, SOX1, SEPT9, Unk05,
Unk09, GALR1, Unk07, Unk19, TBX15, EEF1A2, TFAP2B, DCHS2 and SOX17.
In certain aspects, using the detected fluorescent signals to
determine the genomic DNA methylation profile in a sample comprises
calculating the relative methylation percentages for the
sample.
[0021] In yet a further embodiment there is provided a synthetic
polynucleotide sequence comprising a sequence at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the
probe sequences selected from those provided in Table 1B or 1C,
wherein the polynucleotide is conjugated to a reporter molecule. In
some aspects, the synthetic polynucleotide comprises a sequence
identical to one of the probes of Table 1B or 1C. In certain
aspects, the reporter molecule is a fluorophore.
[0022] In still a further embodiment there is provided a primer
pair, where the primers comprise a sequence at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the primer
sequences selected from those provided in Table 1B or 1C. In some
aspects, the primer pair comprises primers having a sequence
identical to a primer pair of Table 1B or 1C.
[0023] In still further aspects, a kit is provide comprising
reagents for preforming qPCR and a recombinant polynucleotide
sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to one of the probe sequences
selected from those provided in Table 1B or 1C, wherein the
polynucleotide is conjugated to a reporter molecule.
[0024] In a further embodiment, the invention provides a method of
treating a patient comprising determining a genomic methylation
profile for the patient in accordance with any one of embodiments
and aspects described above and performing a treatment on the
patient based on the genomic methylation profile. In certain
aspects, the treatment comprises performing a biopsy of the
patient. In some aspects, the treatment comprises administering an
anti-cancer therapy to the patient. The anti-cancer therapy may be
chemotherapy, radiotherapy, gene therapy, surgery, hormonal
therapy, anti-angiogenic therapy or cytokine therapy.
[0025] In a further embodiment there is provided a method of
detecting the presence of, or an increased risk of, bladder cancer
or other cancers of the urinary tract in a patient comprising
determining a methylation status in one or more genomic regions in
a patient sample selected from the group provided in Table 1A
wherein an increased level of methylation in one or more of the
genomic regions of Table 1A relative to a reference level indicates
that the patient has or is at risk of developing bladder
cancer.
[0026] In some aspects, one or more genomic regions is selected
from the group consisting of Unk 09, Unk 05, DCHS2, OTX1, Unk 07,
EVX2, SEPT9, SOX1, Unk 19, Unk 21, and SOX17 (as indicated in Table
1A). In certain aspects, the one or more genomic regions is
selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the genomic
regions selected from the group consisting of Unk 09, Unk 05,
DCHS2, OTX1, Unk 07, EVX2, SEPT9, SOX1, Unk 19, Unk 21, and SOX17
(as indicated in Table 1A).
[0027] In other aspects, the one or more genomic regions is
selected from the group consisting of GALR1, TBX15, EEF1A2, DMRTA2,
and TFAP2B (as indicated in Table 1A). In some aspects, the one or
more genomic regions is selected from 2, 3, 4, or 5 of the genomic
regions selected from the group consisting of GALR1, TBX15, EEF1A2,
DMRTA2, and TFAP2B (as indicated in Table 1A).
[0028] In certain aspects, the one or more genomic regions is
selected from the group consisting of SCT, Unk 14, Unk 29, CERKL,
and SHH (as indicated in Table 1A). In further aspects, the one or
more genomic regions is selected from 2, 3, 4, or 5 of the genomic
regions selected from the group consisting of SCT, Unk 14, Unk 29,
CERKL, and SHH (as indicated in Table 1A).
[0029] In some aspects, said determining comprises determining a
methylation status in two, three or more of said genomic regions.
In certain aspects, said determining comprises determining a
methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of said
genomic regions.
[0030] In some aspects, said determining comprises determining a
methylation status in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of said
genomic regions, wherein the genomic regions are selected from the
group consisting of Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2,
SEPT9, SOX1, Unk 19, Unk 21, SOX17, GALR1, TBX15, EEF1A2, DMRTA2,
and TFAP2B (as indicated in Table 1A). In further aspects, said
determining comprises determining a methylation in each of the
genomic regions: Unk 09, Unk 05, DCHS2, OTX1, Unk 07, EVX2, SEPT9,
SOX1, Unk 19, Unk 21, SOX17, GALR1, TBX15, EEF1A2, DMRTA2, and
TFAP2B (as indicated in Table 1A).
[0031] In certain aspects, the patient has been previously treated
for or diagnosed with bladder cancer. In some aspects, the method
is further defined as a method for detecting bladder cancer
recurrence or a risk of bladder cancer recurrence.
[0032] In other aspects, said determining comprises analyzing DNA
methylation in the sample using restriction endonuclease digestion
and qPCR. In further aspects, the digestion reaction is completed
in a first step, followed by the qPCR reaction in a second
step.
[0033] In some aspects, the patient is a human. In certain aspects,
the sample is a urine sample. In other aspects, the sample is a
blood sample. In further aspects, the sample is obtained by drawing
blood from the patient. In other aspects, the sample is obtained
from a third party.
[0034] In certain aspects, determining a methylation status
comprises determining the nucleotide positions in the genomic
regions that comprise methylation. In some aspects, determining a
methylation status comprises determining the proportion of
methylation at nucleotide positions in the genomic region. In
further aspects, determining a methylation status comprises
determining the proportion of nucleotide positions that are
methylated in the genomic region.
[0035] In some aspects, the reference level is a level of
methylation from a patient that does not have bladder cancer. In
certain aspects, the method is further defined as a method for
determining the severity of bladder cancer.
[0036] In a further embodiment, the invention provides a method of
detecting the presence of, or an increased risk of, bladder cancer
in a patient comprising obtaining a patient sample, determining a
methylation status in one or more genomic regions selected from
those in Table 1, and identifying the presence of, or an increased
risk of, bladder cancer in the patient based on an increased level
of methylation in one or more of the genomic regions relative to a
reference level.
[0037] In yet a further embodiment, the invention provides a method
for treating a patient having bladder cancer or at risk for having
bladder cancer comprising administering a therapy to the patient,
wherein the patient was previously determined to have an increased
level of methylation in one or more of the genomic regions selected
from those provided in Table 1 relative to a reference level. In
some aspects, the therapy comprises administering an anti-cancer
therapy to the subject. In further aspects, the anti-cancer therapy
is chemotherapy, radiotherapy, gene therapy, surgery, hormonal
therapy, anti-angiogenic therapy or cytokine therapy. In certain
aspects, the anti-cancer therapy is a BCG therapy.
[0038] In still a further embodiment, the invention provides kits
for analysis of DNA methylation. In some aspects, a kit is provided
comprising a sealed container comprising primers or probes designed
to detect methylation in one, two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,
sixteen or more genomic regions of Table 1A. For example, a kit may
comprise a primer pair for amplification of an interval of sequence
in one of the genomic regions of Table 1 and a reagent for analysis
of DNA methylation (e.g., a methylation sensitive restriction
endonuclease). In a further aspect, a kit may comprise reagents for
analysis of total DNA methylation levels.
[0039] In a further aspect, kits are provided for determining one
or more methylation positions in a DNA sample. For example, a kit
can comprise, at least, an active glucosyltransferase and a DNA
endonuclease (e.g., MspI, TaqI or a methylation dependent DNA
endonuclease, such as BisI, GlaI or McrBC). Kits according to the
invention can further comprise one or more MSEs; a DNA
methyltransferase (e.g., M.SssI and/or M.CviPI methyltransferase);
an enzyme that converts 5'mC into 5'hmC (e.g., recombinant Tet1,
Tet2 and/or Tet3 proteins); one or more reference DNA samples; an
affinity purification column; a DNA ligase; a DNA polymerase; DNA
sequencing reagents; a PCR buffer; instructions; methylation
specific antibodies; and/or DNA primers (also reagents to determine
general urine parameters (pH, amount of hemoglobin, leukocytes,
e.g. Osumex 10P test kit, Uriscan (YD Diagnostic Corp.).
[0040] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0041] 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." As used herein "another" may mean at least a second or
more.
[0042] 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.
[0043] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0045] FIG. 1A-B: (A) Schematic of methylation analysis system. (B)
List of restriction enzymes with their corresponding digestion
site. The enzyme will cut the CG-containing sequence only is the C
(shown enlarged) is not methylated.
[0046] FIG. 2: Multiplex vs Singleplex qPCR reactions; 7.5 .mu.l of
PCR reaction (sub-complete amplification; 35 cycles) were loaded
for each lane; undigested Blood gDNA was used as template. The
numbers below each well correspond to the marker numbers in Table
1.
[0047] FIG. 3: Detection Limits and One-step vs. Two-step method.
0.25-1% of amplification is considered above the background level;
0.25% lends low confidence and 1% lends high confidence. LD583
bladder cancer cell line (Isabel D. C. Markl and Peter A. Jones,
Cancer Research 58, 5348-5353, Dec. 1, 1998) gDNA was mixed with
normal Urine gDNA. Reactions A, B and C were done using the
single-step method (marked with arrows) illustrating that one
step-reaction is not applicable for all the markers.
[0048] FIGS. 4-8: Relative methylation values after restriction
endonuclease digestion and Real-Time qPCR. Y axis represents the
methylation level relative to the undigested blood DNA value
(Blood-); this is the reference sample which value=1.-=undigested
template; +=digested template. Blood=Genomic DNA extracted from
blood (healthy male, 53 yo); LD583=Genomic DNA extracted from a
bladder cancer cell line; Urine=Genomic DNA extracted from urine of
non-bladder cancer human.
[0049] FIG. 9: Example of typical results after qPCR using POLR2A
primers (loading control marker) and undigested/digested gDNAs as
template (three different sample types). 15 ng of DNA was used for
each reaction. CT values are indicated.
[0050] FIG. 10: Methylation percentages for 16 different markers
(categories A and B in Table 3) in digested clinical sample.
nnU=normal Urine. Values higher than 1% are marked in red, while
values >0.25% and <1% are marked in blue. Red arrows and
circles indicate markers for those where a higher background in nnU
was observed.
[0051] FIG. 11: The samples of FIG. 10 were classified into three
categories on the basis of the % of signal in different markers:
samples that show cancer presence/development are marked in red
(values higher than 1% in multiple markers); borderline samples,
with multiple low-confidence positive values (>0.5% and <1%)
are marked in orange; samples with values <0.5% for all the
marker are considered cancer free and are marked in green.
[0052] FIG. 12: Preliminary qPCR reaction results for marker GALR1.
LD583 is a bladder cancer cell line and the marker GALR1 is
methylated in this line, but not in blood (450K data). The
undigested LD583 sample (indicated by the red arrow) is not
amplifying at all and the digested LD583 sample (green arrow) show
an amplification, but much weaker than that in the undigested blood
DNA control (first bar). The amount of template used for Blood and
LD583 is for both 10 ng and the POLR2A marker amplification is
comparable between the two samples. This problem was circumvented
by adding 5% DMSO (final concentration) to the PCR reaction.
[0053] FIG. 13: Example of analysis conducted in Example 2 showing
results with marker 19. Signal of digested samples (+) were
calculated by comparison with the signal in the corresponding
undigested samples (100%). Data were also corrected using POLR2A as
a loading control (internal control) and complete DNA digestion was
detected using GAPDH control marker.
[0054] FIG. 14A-E: Probability of bladder cancer for training
samples and hierarchical clustering using selected DNA methylation
biomarkers. (A-B) Methylation markers derived from HM450 human
methylation array data of tumor tissues and controls. (C)
Methylation markers derived from HM450 array data of urine samples
from bladder cancer patients and healthy controls. (D-E)
Methylation markers derived from Reduced Representation Bisulfite
Sequencing (RRBS) data of tumor tissues and controls.
[0055] FIG. 15: Schematic representation of CARE assay.
Unmethylated (A) and methylated (B) DNAs are analyzed using CARE
assay. DNA methylation will impede DNA cleavage in the digestion
reaction, thus robust amplification occurs during the following
qPCR step in B), but not in A). A no-digestion control reaction is
run in parallel for each experiment.
[0056] FIG. 16: Schematic depicting CARE assay general
workflow.
[0057] FIGS. 17A-17B: CARE assay analysis of a normal urine DNA
sample added with different amount of artificially methylated urine
DNA (100% methylated; X axis). A) CARE assay is able to detect the
presence of 0.5% of fully methylated urine DNA in an unmethylated
urine DNA background. B) CARE assay performances when 50% and 100%
of artificially methylated urine DNA is added to a normal urine DNA
sample. 10 ng of input DNA were used in each reaction (the DNA
amount approximately corresponding approximately to 1500 cells).
For simplicity only cancer marker belonging to set A are
represented. Experiment was performed using three technical
replicates.
[0058] FIGS. 18A-18C: Description of an exemplary CARE assay Kit
and reaction. A) Fundamental CARE assay Kit components; other
components like Multistix.RTM. 10 SG Reagent Strips, UCBTM and
urine collection container might be included in the final kit. To
note, a single D and U Reaction Mixes are represented in the figure
(enough for one marker set); more Reaction Mixes should be included
in the kit if more marker sets will be used. B) DNA sample isolated
from urine sample is diluted to the appropriate concentration using
DNA Diluent Buffer. C) Reactions are assembled: 15 .mu.l of U and D
Reaction Mixes are distributed into a 96-well plate and mixed with
5 .mu.l of E.D.C. or DNA sample/s. After digestion and qPCR steps
CARE markers methylation is calculated. In the figure, two
technical replicates for each reaction are performed.
[0059] FIGS. 19A-19F: Algorithm used to calculate the methylation
percentage of markers belonging to CARE assay set A in two
different sample (Spl 01 and 02) and in the external digestion
control (E.D.C.). Mean CT values (CTm) are collected (A) and
.DELTA.CT (B), .DELTA..DELTA.CT (C) and RQ (D) values are
calculated by the CFX96TM-Real Time System Machine. Data are then
expressed in percentage, considering the RQ value for each single
marker in each undigested sample equal to 100% (E). In the example
above the analysis can be considered valid since the methylation
level of the endogenous digestion control GAPDH (grey circles) and
of all the markers in the exogenous digestion control E.D.C. (black
circle) are below the maximum acceptable background limit (1%) in
the digested samples (+). (F) Accordingly to the data obtained Spl
01 is negative for bladder cancer, while Spl 02 is positive.
Experiment was performed using three technical replicates. Results
might also be expressed in corrected DNA copy number resistant to
digestion instead of percentage.
[0060] FIG. 20: Example of adjustment of the baseline threshold
values for marker #27 and #15.
[0061] FIG. 21A: Analysis of input amount of DNA for bisulfite
conversion in Multiplex methylation-specific qPCR assay (MMSP).
[0062] FIG. 21B: Urine DNA size distribution of four random
selected bladder patient samples.
[0063] FIG. 22: Schematic depicting MMSP assay working flow
including bisulfite conversion, multiplex real-time PCR and data
analysis all performed in one day.
[0064] FIG. 23: Exemplary depiction of 96-well assay plate set-up
for MMSP assay including methylation standard samples S1-S7,
methylation controls (MC) and no template control (NC).
[0065] FIG. 24: Standard curves and PCR efficiency for each group
A-D of each target regions for MMSP assay.
[0066] FIG. 25: Results showing linearity of MMSP assay for 16
targeted genes based on repeated measurements of relative
methylation percentage values of DNA mixtures containing 100%, 50%,
5%, 1%, 0.5% and 0% of methylated (M.SssI-treated) normal urine
DNA.
[0067] FIG. 26: Results from MMSP assay analysis of 35 normal urine
samples and 9 bladder cancer tumor tissues for 16 biomarkers
divided into four groups A, B, C and D.
[0068] FIGS. 27A-27B: Analysis of bladder cancer recurrence using
group B markers and MMSP assay.
[0069] FIG. 28: Table reflecting sensitivities and specificities of
urine tumor markers in bladder cancer.
[0070] FIG. 29: Selection of a panel of three CpG sites with the
lowest RMSE error by Random Forest for the CARE assay.
[0071] FIG. 30: Selection of one and two CpG sites with the lowest
and second lowest RMSE errors by Random Forest for the MMSP.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Embodiments of the Present Invention
[0072] The methylome of cancer cells greatly differ from that of
normal cells. In general, cancer cells show an increase in CpG
islands methylation, while other genomic elements, including
transposons, lose their normal methylated status. Thus it is
possible to detect the presence of cancer cells in a cell
population through the analysis of the methylation status of CpG
elements that are specifically methylated in cancer cells. Provided
herein are multiplex methods for determining a genomic methylation
profile in a subject. These new techniques allow for highly
quantitative and rapid methods for determine the methylation
profile from patient samples. These profiles can, in turn, be used
to determine disease risk in patients. For example, methylation
profiles can be used on their own or in conjunction with other
diagnostic tests to determine if a patient has a cancer or to
assess the aggressiveness of a cancer. Moreover, methylation
profiles can be used to select the most effective therapy for a
subject having a disease. For example, the profiles can be used to
determine whether a cancer is likely to respond to a given
chemotherapeutic agent or if surgical removal of cancer cells is
likely to provide an effective treatment and prevent
recurrence.
[0073] In some aspects, the inventors identify a panel of
methylation markers (e.g., genomic regions) that can be assessed
for methylation status to determine whether a subject has, or is at
risk for development of, a bladder cancer. Briefly, cancer specific
methylation markers were identified by analyzing tissue and or
other cancers of the urinary tract (i.e. detected from cells
present in the urine or urine sediment or alternately from normal
sample biopsy) samples from patients with bladder cancer using the
HM450 human methylation array and RRBS next-generation sequencing.
A linear regression model was utilized to select the most
significant methylation biomarkers which separated tumor and normal
samples (FIG. 14A-E). Genomic regions (and specific potential
methylation positions) that may be assessed by a method of the
embodiments are listed below in Table 1A. Example primer and probe
sets for real-time qPCR assays, such as the MMSP assay and CARE
assay are shown in Tables 1B and 1C (respectively).
TABLE-US-00001 TABLE 1A Code Genomic region Preferred methylation
site 01 OTX1 Chr2, m5C position 63281139 02 ZIC4 Chr3, m5C position
147111660 03 PCDHGA4 Chr5, m5C position 140811642 04 TFAP2B Chr6,
m5C position 50791202 05 Unknown (Unk) 05 Chr7, m5C position
149112318 06 SCT Chr11, m5C position 627152 07 Unk 07 Chr17, m5C
position 5001047 08 GALR1 Chr18, m5C position 74962133 09 Unk 09
Chr21, m5C position 38065524 10 DMRTA2 Chr1, m5C position 50886782
11 SIX3 Chr2, m5C position 45171818 12 C1ORF70 Chr1, m5C position
1475742 13 Unk 13 Chr2, m5C position 130971164 14 Unk 14 Chr1,
position 63785946 15 GALR1 Chr18, m5C position 74962369 16 SEPT9
Chr17, m5C position 75368902 17 FZD7 Chr2, m5C position 202900063
18 DCHS2 Chr4, m5C position 155411603 19 Unk 19 Chr13, m5C position
53775108 (Near PCDH8) 20 KCNA3 Chr1, m5C position 111217194 21 Unk
21 Chr4, m5C position 1400189 22 DPM2* Chr9, m5C position 130700954
(Methylated in blood) 23 TBX15 Chr1, m5C position 119522459 24 Unk
24 Chr10, m5C position 103044008 25 CCDC81 Chr11, m5C position
86085790 26 KCNA6 Chr12, m5C position 4918900 27 SOX1 Chr13, m5C
position 112721950 28 Unk 28 Chr18, m5C position 45968115 29 Unk 29
Chr2, m5C position 118593895 30 EVX2 Chr2, m5C position 176947173
31 CERKL Chr2, m5C position 182543251 32 EEF1A2 Chr20, m5C position
62119669 33 POU4F2 Chr4, m5C position 147561722 34 SHH Chr7, m5C
position 155597779 35 Unk 35 Chr7, m5C position 24323317 (Near NPY
gene) 36 SOX17 Chr8, m5C position 55370529 37 Unk 37 Chr9, m5C
position 70921347 38 HOXA9 Chr2, m5C position 176987413 39 NPY
Chr7, m5C position 24323817 40 IRAK3 Chr12, m5C position 66583004
41 TJP2 Chr9, m5C position 71736214 *indicates a non-cancer marker
that is methylated in blood.
TABLE-US-00002 TABLE 1B Example primer and probe sets for a MMSP
assay. Region Forward Reverse Probe OTX1 TCGCGGAAGTAGCGG
CACCTCCTCCCGCATAA AACGCCTCGAACACGT (#01) C (SEQ ID NO: 1) AAA (SEQ
ID NO: 2) CCAACTATAAACG (SEQ ID NO: 3) TFAP2B GGAGGGATTATTATT
CCATACCCGCGTCCAA ATACGCCGAATACAAC (#04) CGGTTCGT (SEQ ID AC (SEQ ID
NO: 5) AACACGTCCGA (SEQ ID NO: 4) NO: 6) Unk05 TCGTACGTAAGTCGC
AATCCGAACTAACCGC CGCCGCCCAAAACGCG GTATAGTATTGT CG (SEQ ID NO: 8) A
(SEQ ID NO: 9) (SEQ ID NO: 7) Unk07 AGGTAAGAGTTTCGG
CCTCAACCCTCGAACC TCGCGACAAACACGCTT CGGC (SEQ ID NO: 10) CAAC (SEQ
ID NO: 11) CCGCCTA (SEQ ID NO: 12) GALR1A TCGGCGAAGATTTGG
CGCGACCACGAAACCT TTTTATTTGCGCGGTTG (#08) AGC (SEQ ID NO: 13) AAA
(SEQ ID NO: 14) TAGTCGGC (SEQ ID NO: 15) Unk09 CGCGTAAAAGGTAGG
AACTCGAAACTCTACC AACGCCGACTCAACAA ATCGC (SEQ ID NO: CCCGA (SEQ ID
NO: 17) AAAATTATTTCGAA 16) (SEQ ID NO: 18) DMRTA2 AGCGATAGTAGCGTT
GCGAACCCCCAAATAC TCGCAACCATAACGTA (#10) TGTGGTTT (SEQ ID G (SEQ ID
NO: 20) ATATCGACCCTCA (SEQ NO: 19) ID NO: 21) SEPT9
GTTTTTGAGTTTATAG CTATCACCGCCGCCG AAATTAAACGACAACG (#16) GTCGGGATTT
(SEQ (SEQ ID NO: 23) CACGCGACTAACAA ID NO: 22) (SEQ ID NO: 24)
DCHS2 AGCGGTGACGGTAGT CTACACCCTAATACAA AACCCGTTCTTCCAATT (#18)
GGTTT (SEQ ID NO: CCGTCCG (SEQ ID NO: ACGCTACCGAA (SEQ ID 25) 26)
NO: 27) Unk19 GCGGACGTTAGTTAG CCCGAATCCCTATCCG CGCCCACTCCGACACCA
TCGGC (SEQ ID NO: AAA (SEQ ID NO: 29) ACGT (SEQ ID NO: 30) 28)
Unk21 CGCGTTTAATTGGTT TATCTATTCGTCCCGCC AAATCCGCCTCCTCGAC GCGA (SEQ
ID NO: CG (SEQ ID NO: 32) CCGAAA (SEQ ID NO: 33) 31) TBX15
GGTTGGTATATCGAG CGCGTCTACCCGCCC AAACGACGAACGAATC (#23) GTTTCGTAGTT
(SEQ (SEQ ID NO: 35) AAACGCGACT (SEQ ID ID NO: 34) NO: 36) SOX1
GGTATTTGGGATTAG CCTCAACGACCTCCAA ACTACAACTTCTAACAA (#27)
TATATGTTTAGCGT CTCG (SEQ ID NO: 38) AACGACGCGCCG (SEQ (SEQ ID NO:
37) ID NO: 39) EVX2 GATTCGCGTTAGAGT CGTCGAACTCAAACCT
CGTCGCGTTTTCGTCGT (#30) CGGAGTTA (SEQ ID CGAAA (SEQ ID NO: 41) (SEQ
ID NO: 42) NO: 40) EEF1A2 GCGTATACGGTTTTG CAAACTTCCGCGCCCA
CTCGCCACGCTCAATAC (#32) GGGTC (SEQ ID NO: AC (SEQ ID NO: 44)
CCGTTTTACC (SEQ ID 43) NO: 45) SOX17 GGACGTGGGATTCGG
TTTTCTACACAAATATA CGAACCGATCCCGCGTC (#36) ATTAC (SEQ ID NO:
ACCAATAAAACGAC GTTAA (SEQ ID NO: 48) 46) (SEQ ID NO: 47)
TABLE-US-00003 TABLE 1C Example primer and probe sets for a CARE
assay. Locus Primer Forward Primer Reverse Probe POLR2A
ACCTACCTCTCCAAG GGTGTAATTGGGACTG TACTCACCCACCAGCCC CTATTC (SEQ ID
NO: GTTGG (SEQ ID NO: 50) GAACTA (SEQ ID NO: 51) 49) GAPDH
CGTAGCTCAGGCCTC GAGGAGCAGAGAGCG CTCAGCCAGTCCCAGCC AAGAC (SEQ ID NO:
AAGC (SEQ ID NO: 53) CAAG (SEQ ID NO: 54) 52) SOX1 CCACGACTGCACCTG
GGCAGAAACACACGC TCGGCCTCTTTGGCAAG (#27) TTTGC (SEQ ID NO: 55) ACTCG
(SEQ ID NO: 56) TGGTTT (SEQ ID NO: 57) GALR1 ACCCACCCTCTCTCA
AGTTTAGGAGTCTGAG CGCAAAGACGGTGCCA (#15) GAAGG (SEQ ID NO: CTTCC
(SEQ ID NO: 59) CCAGG (SEQ ID NO: 60) 58) DMRTA2b GCAGTGCCGTAGAGC
CCCTCAAGGGCCACAA CACAGGCAGTCCTTCCA (#10) AGCT (SEQ ID NO: 61)
ACGCTA (SEQ ID NO: GCGACAG (SEQ ID NO: 62) 63) Unk05
CACTCACGTCTTCGT TTGGAGTGTGACGGTA CGTACAGCACTGCAGG (#05) AGTCAG (SEQ
ID NO: AAAGC (SEQ ID NO: 65) GTCCG (SEQ ID NO: 66) 64) Unk07
GGGAGGACCCCTCGT CTCAGCGTCCGACCCC TGGGCTCGAGGGCTGA (#07) TAGC (SEQ
ID NO: 67) ACT (SEQ ID NO: 68) GGGC (SEQ ID NO: 69) EVX2
AGGCTTCCGAGGCCT AGCGACTCTCCTTCCT CGTACCCTGGCAAACAA (#30) GAGC (SEQ
ID NO: 70) GACG (SEQ ID NO: 71) ACGACC (SEQ ID NO: 72) SOX17
TGGGCGTGGGCCTAA CCCGTGTTCTGGCCTG TGGGTACGCTGTAGACC (#36) CGAC (SEQ
ID NO: 73) TCG (SEQ ID NO: 74) AGACC (SEQ ID NO: 75) DCHS2
CGCACGCGACAGACC TGGATGAGAACGACA CGGTACTCGTCCTGCTC (#18) TCG (SEQ ID
NO: 76) ACCCG (SEQ ID NO: 77) AAAGAC (SEQ ID NO: 78) Unk09
ACCGAGGCCCCTACC GTTAGAAACGCAGGC CACTGAACGACCCCTTC (#09) TGG (SEQ ID
NO: 79) CAGGC (SEQ ID NO: 80) TCCAG (SEQ ID NO: 81) EEF1A2
GCCGACTTGGTGACC GGCCAGAGCCCTGGG CCACGTTCTTGATGACG (#32) TTGC (SEQ
ID NO: 82) GTTG (SEQ ID NO: 83) CCTACG (SEQ ID NO: 84) Unk19
GACGTGGTGTTCGCT CGCACGTGCAGCTCGT CCCGTGGATTACCAGAG (#19) TTTGGC
(SEQ ID NO: AGG (SEQ ID NO: 86) TCAGGA (SEQ ID NO: 87) 85) Unk21
ACACGCCGAACACAC CCGTCCGTGTGTCCTG CCTAATTGGCTGCGAAC (#21) GTGC (SEQ
ID NO: 88) TGC (SEQ ID NO: 89) GGTCC (SEQ ID NO: 90)
[0074] In particular, the inventors have used a unique methylation
marker panel, along with controls and methylation sensitive
restriction enzymes to generate a novel reaction design that allows
multiplex digestion and qPCR reaction in the same buffer to detect
the methylation at specific CpG dinucleotides. In certain aspects,
this method may be preferred as it has a great advantage in
allowing for the analysis of CpG methylation without threating the
DNA with sodium bisulfite at high temperature (a reaction that
strongly impact the DNA sequence and reduce the quality of DNA for
downstream experiments).
II. Reagents and Kits
[0075] The kits may comprise suitably aliquoted reagents of the
present invention, such as a glucosyltransferase (e.g., a
.beta.-glucosyltransferase) and one or more Methylation-Sensitive
DNA endonucleases (e.g., MspI, ClaI, Csp6I, HaeIII,
Taq.sup..alpha.I, MboI, or McrBC) or a methylation dependent
endonuclease such as BisI, GlaI or McrBC. Additional components
that may be included in a kit according to the invention include,
but are not limited to, MSEs (e.g., AatII, AccIII, Acil, AfaI,
Agel, AhaII, Alw26I, Alw44I, ApaLI, ApyI, Ascl, Asp718I, AvaI,
AvaII, Bme216I, BsaAI, BsaHI, BscFI, BsiMI, BsmAI, BsiEI, BsiWI,
BsoFI, Bsp105I, Bsp119I, BspDI, BspEI, BspHI, BspKT6I, BspMII,
BspRI, BspT104I, BsrFI, BssHII, BstBI, BstEIII, BstUI, BsuFI,
BsuRI, CacI, CboI, CbrI, CceI, Cfr10I, ClaI, Csp68KII, Csp45I,
CtyI, CviAI, CviSIII, DpnII, EagI, Ec1136II, Eco47I, Eco47III,
EcoRII, EcoT22I, EheI, Esp3I, Fnu4HI, FseI, FspI, Fsp4HI, GsaI,
HaeII, HaeIII, HgaI, HhaI, HinPlI, HpaII, HpyAIII, HpyCH4IV, ItaI,
KasI, Kpn2I, LlaAI, LlaKR2I, MboI, MflI, MluI, MmeII, MroI, MspI,
MstII, MthTI, NaeI, NarI, NciAI, NdeII, NgoMIV, NgoPII, NgoS II,
NlaIII, NlaIV, NotI, NruI, NspV PmeI, PmlI, Psp1406I, PvuI,
RalF40I, RsaI, RspXI, RsrII, SacII, SalI, Sau3AI, SexAI, SfoI,
SfuI, SmaI, SnaBI, SolI, SpoI, SspRFI, Sth368I, TaiI, TaqI, TflI,
TthHB8I, VpaK11BI, or XhoI), oligonucleotide primers, reference DNA
samples (e.g., methylated and non-methylated reference samples),
distilled water, probes, a PCR buffer, dyes, sample vials,
polymerase, ligase and instructions for performing methylation
assays. In certain further aspects, reagents for DNA isolation, DNA
purification and/or DNA clean-up, analysis of urine clinical
parameters, may also be included in a kit.
[0076] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there is more
than one component in the kit, the kit also will generally contain
a second, third or other additional container into which the
additional components may be separately placed. However, various
combinations of components may be comprised in a vial. The kits of
the present invention also will typically include a means for
containing reagent containers in close confinement for commercial
sale. Such containers may include cardboard containers or injection
or blow-molded plastic containers into which the desired vials are
retained.
[0077] When the components of the kit are provided in one or more
liquid solutions, the liquid solution is an aqueous solution, with
a sterile aqueous solution being preferred.
[0078] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0079] In some aspects, labeled probes may be used to detect and/or
quantify PCR amplification. Numerous reporter molecules that may be
used to label nucleic acids probes are known. Direct reporter
molecules include fluorophores, chromophores, and radiophores.
Non-limiting examples of fluorophores include, a red fluorescent
squaraine dye such as
2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dio-
-xolate, an infrared dye such as 2,4
Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,-
-3-dioxolate, or an orange fluorescent squarine dye such as
2,4-Bis[3,5-dimethyl-2-pyrrolyl]cyclobutenediylium-1,3-diololate.
Additional non-limiting examples of fluorophores include quantum
dots, Alexa Fluor.TM. dyes, AMCA, BODIPY.TM. 630/650, BODIPY.TM.
650/665, BODIPY.TM.-FL, BODIPY.TM.-R6G, BODIPY.TM.-TMR,
BODIPY.TM.-TRX, Cascade Blue, CyDye.TM., including but not limited
to Cy2.TM., Cy3.TM., and Cy5.TM., a DNA intercalating dye,
6-FAM.TM., Fluorescein, HEX.TM., 6-JOE, Oregon Green.TM. 488,
Oregon Green.TM. 500, Oregon Green.TM. 514, Pacific Blue.TM., REG,
phycobilliproteins including, but not limited to, phycoerythrin and
allophycocyanin, Rhodamine Green.TM., Rhodamine Red.TM., ROX.TM.,
TAMRA.TM., TET.TM., Tetramethylrhodamine, or Texas Red.TM.. A
signal amplification reagent, such as tyramide (PerkinElmer), may
be used to enhance the fluorescence signal. Indirect reporter
molecules include biotin, which must be bound to another molecule
such as streptavidin-phycoerythrin for detection. Pairs of labels,
such as fluorescence resonance energy transfer pairs or
dye-quencher pairs, may also be employed.
III. Definitions
[0080] As used herein, a "methylation sensitive restriction
endonuclease" (MSRE) is a restriction endonuclease that includes CG
as part of its recognition site and has altered activity when the C
is methylated as compared to when the C is not methylated (e.g.,
SmaI). Non-limiting examples of methylation sensitive restriction
endonucleases include HpaII, BssHII, BstUI, SacII, EagI and NotI.
An "isoschizomer" of a methylation sensitive restriction
endonuclease is a restriction endonuclease that recognizes the same
recognition site as a methylation sensitive restriction
endonuclease but cleaves both methylated CGs and unmethylated CGs,
such as for example, MspI is an isoschizomer of HpaII. "Restriction
endonuclease" and "restriction enzyme" are used interchangeably
herein.
[0081] As used herein the term "genomic region" refers to a region
of genomic DNA encoding and controlling expression of a particular
RNA or polypeptide (such as sequences coding for exons, intervening
introns and associated expression control sequences) and its
flanking sequence or other genomic regions of interest (e.g.
repetitive elements or repeated regions of genomic DNA such as
dispersed or interspersed retroelements, SINES; LINES; among other
such elements). Thus, in some aspects, a genomic region is defined
by the regions encoding the genomic regions listed in Table 1. It
is, however, recognized in the art that methylation in a particular
region (e.g., at a given CpG position or in an amplification
interval) is generally indicative of the methylation status at
proximal genomic sites. This is particularly true for regulatory
elements like CpG islands. Accordingly, determining a methylation
status of a particular genomic region can comprise determining a
methylation status at a site or sites within about 100, 50, or 25
kb of a named genomic region. Thus, in some aspects, assessing
methylation in genomic regions, such as those of Table 1 comprises
assessing the methylation at one or more potential sites of
methylation with-in 100, 50, or 25 kb (or preferably with 10 kb) of
a potential methylation position listed in Table 1.
[0082] As used herein the term "genomic amplification interval"
refers to a region of genomic DNA that can be amplified by PCR. As
used herein an amplification interval comprises at least one CpG
position that is a potential site of methylation. In some cases,
the amplification interval comprises 2, 3, 4 or more potential
sites of CpG methylation (e.g., wherein the CpG is in a sequence
recognized by an MSE). In general an amplification interval is less
than about 1,200 bp, such as between about 50 bp and 100, 200, 300,
400 or 500 bp. In certain aspects, the amplification interval is
130 bp or less.
[0083] As used herein "determining a methylation status" for an
indicated genomic region means determining whether one of more
position in the DNA of the genomic region is methylated. Thus, in
certain aspects, determining a methylation status for a genomic
region comprises determining the methylation status of at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more
sites of potential DNA methylation. In other aspects determining a
methylation status means determining the methylation status of one
or more methylated sites in a differential methylated region (DMR,
e.g. in a window of about 1-10 bp, 10-100 bp, 100-200 bp,
100-1,000, bp or larger window)
IV. Examples
[0084] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--Analysis of Methylation Using Restriction Endonuclease
Digestion and Real-Time PCR
[0085] The analysis system is based on the principle that some
restriction enzymes can cut sequences containing CG dinucleotides
only if the C is not methylated (FIG. 1A-B).
[0086] Marker Design--A group of 39 genomic regions (see Table 1
below) were identified to be methylated in bladder cancer and
selected for further analysis. Many of the selected markers are not
yet annotated (i.e., the gene associated to the CpG island is not
known yet; named Unknown (Unk) #). Several control regions are
included in Table 2 below. The LINE-1 controls were designed by the
inventors and are highly methylated in normal tissues and less in
cancer. The GAPDH control was designed by the inventors to serve as
a determination of digestion efficiency. Its CpG island is not
methylated in all normal and cancer tissues. The POLR2A control
marker was designed by the inventors as well and it does not
contains any restriction site, so it is a copy number control (for
the determination of the amount of template used in each reaction)
Other LINE-1 controls could be used as a copy number control (the
amplicon does not contains any restriction site). This control will
amplify about 16,500 copies of LINE-1 elements in the human genome,
circumventing the problem that a deletion affecting a single-copy
copy number control will impair the analysis. The usage of LINE-1
as a copy number marker is not yet confirmed and the inventors have
used the POLR2A control.
TABLE-US-00004 TABLE 2 Genomic regions with methylation at specific
CG sites in bladder cancer samples (tissue, urine and cell lines).
Regions 22, 42, and 43 are methylation control regions. GAPDH =
digestion control; contains several restriction sites; always
unmethylated. POLRA = copy number control; does not contains any
restriction site (not sensitive to digestion). Genomic Code region
methylation site 01 OTX1 Chr2, m5C position 63281139 02 ZIC4 Chr3,
m5C position 147111660 03 PCDHGA4 Chr5, m5C position 140811642 04
TFAP2B Chr6, m5C position 50791202 05 Unk 05 Chr7, m5C position
149112318 06 SCT Chr11, m5C position 627152 07 Unk 07 Chr17, m5C
position 5001047 08 GALR1 Chr18, m5C position 74962133 09 Unk 09
Chr21, m5C position 38065524 10 DMRTA2 Chr1, m5C position 50886782
11 SIX3 Chr2, m5C position 45171818 12 C1ORF70 Chr1, m5C position
1475742 13 Unk 13 Chr2, m5C position 130971164 14 Unk 14 Chr1,
position 63785946 15 GALR1 Chr18, m5C position 74962369 16 SEPT9
Chr17, m5C position 75368902 17 FZD7 Chr2, m5C position 202900063
18 DCHS2 Chr4, m5C position 155411603 19 Unk 19 Chr13, m5C position
53775108 (Near PCDH8) 20 KCNA3 Chr1, m5C position 111217194 21 Unk
21 Chr4, m5C position 1400189 22 DPM2* Chr9, m5C position
130700954* hypermeth in blood 23 TBX15 Chr1, m5C position 119522459
24 Unk 24 Chr10, m5C position 103044008 25 CCDC81 Chr11, m5C
position 86085790 26 KCNA6 Chr12, m5C position 4918900 27 SOX1
Chr13, m5C position 112721950 28 Unk 28 Chr18, m5C position
45968115 29 Unk 29 Chr2, m5C position 118593895 30 EVX2 Chr2, m5C
position 176947173 31 CERKL Chr2, m5C position 182543251 32 EEF1A2
Chr20, m5C position 62119669 33 POU4F2 Chr4, m5C position 147561722
34 SHH Chr7, m5C position 155597779 35 Unk 35 Chr7, m5C position
24323317 (Near NPY gene) 36 SOX17 Chr8, m5C position 55370529 37
Unk 37 Chr9, m5C position 70921347 38 HOXA9 Chr2, m5C position
176987413 39 NPY Chr7, m5C position 24323817 40 IRAK3 Chr12, m5C
position 66583004 41 TJP2 Chr9, m5C position 71736214 42 LINE-1 5'
Chr2, m5C position 66617179 (44 Kb upstr MEIS1 gene) 43 LINE-1 body
Chr2, m5C position 66618707 (44 Kb upstr MEIS1 gene) 44 GAPDH
Chr12, m5C position 6643629 45 POLR2A Within exon 29 of RNA
Polymerase II subunit A gene *Marker # 22 is methylated in blood
but not in bladder cancer; in preferred aspects the GALR1 (#15)
marker was used instead of GALR1 (#08).
[0087] MSRE qPCR design--Four different methylation sensitive
restriction endonucleases were selected (AciI, HpyCH4IV, HinP1l and
Hpall). Each restriction endonuclease is a four-base cutter that is
able to cut the restriction site only if the cytosine in the
targeted CpG dinucleotide is unmethylated. All the amplicons
(including the primer sequences) were designed in order to contain
one or more recognition sites for at least three out of four
different restriction endonucleases. This ensures a high digestion
reliability (multiple restriction sites within the same amplicon
are targeted but it is sufficient that just one site is cut to
impair the DNA amplification in the next step) and reduce the
possibility that small changes in the DNA sequence (e.g. SNPs) can
result in false positive.
[0088] Moreover, the amplicon length range is 70 to 158 bp and all
the primers were designed and verified to have efficiencies between
90 and 110% and an annealing temperature of 60.degree. C. These are
prerequisites for qPCR data reliability and for the standardization
of the qPCR condition.
[0089] Digestion Conditions--All the restriction endonucleases were
purchased from New England Biolabs and mixed together resulting in
a stock solution in which the final concentration for each enzyme
was 2.5 U/.mu.l. The digestion reaction solutions were prepared as
follows: CutSmart 1.times., Endonuclease-MIX 0.125U/.mu.l, Template
5 or 2.5 ng/.mu.l; MgCl.sub.2 4 mM (Stock solution: 20 mM
MgCl.sub.2 in 10 mM Tris-HCl pH 7.5; increasing Mg.sup.2+
concentration will increase the digestion efficiency even if the
reaction contains 0.1 mM EDTA). The final template concentration
was 5 ng/.mu.l. Digestions were incubated overnight (16 h) at
30.degree. C. in a thermocycler machine, after that enzymes were
inactivated by heating the reactions at 80.degree. for 5'. It was
previously determined by the inventors that 30.degree. C. is the
optimal working temperature for all 4 enzymes (Acil loses a
substantial percentage of its activity over time at 37.degree. C.
but not at 30.degree.). A parallel negative reaction (with no
enzymes) was also conducted.
[0090] qPCR Reaction Conditions--The Real Time reactions were
performed using the BioRad CFX machine. Preliminary trials
demonstrate that the complete denaturation of the template is a
critical step for the qPCR reactions. This is particularly true for
methylated CpG-rich templates that are slightly more difficult to
denaturate compared to the non-methylated DNA (e.g. see FIG. 12).
This problem (which magnitude is different for different markers
See FIG. 12) was resolved by adding to the qPCR reaction a final
concentration of 5% DMSO and increasing the denaturation
temperature from 95.degree. C. to 97.degree. C. (It was observed
that this is the highest temperature that will not decrease the
ZymoTaq.TM. activity).
[0091] The inventors also verified the possibility to perform a
multiplex reaction without decreasing the amplification efficiency.
FIG. 2 shows that it is possible to multiplexing up to 5 markers in
the same reaction without affecting the overall PCR efficiency
(note the intensity of the single marker bands in singleplex--left
side of the gel--compared to the band intensities when the
reactions were performed in multiplex).
[0092] The PCR Reaction solutions were prepared according to the
following formula: ZymoTaq PreMix (2.times.), 10 .mu.l; primer FW,
0.4 .mu.M (final concentration); primer RV, 0.4 .mu.M (final
concentration); template, 10 ng (2 .mu.l of digestion reaction);
DMSO, 5% (final concentration); and ddWater to 20 .mu.l. Reactions
were amplified on a CFX BioRad qPCR machine with the thermal
profile: 97.degree. C. for 2 minutes; then forty-five cycles of
95.degree. C. for 20 seconds, 60.degree. C. for 30 seconds, and a
final extension at 72.degree. C. for 1 minute (new conditions are
60.degree. C. for one minute avoiding the elongation step at
72.degree. C.). All amplifications were then subjected to melt
curve analysis with fluorescence measurements to ensure specific
amplification and identity. The melt curve was 60.degree. C. to
97.degree. C. The amplicons were then stored at 15.degree. C.
[0093] Digestion and qPCR reactions were done in separate tubes
(two-step) since the single-step reaction strongly decreased the
sensitivity of the method for some markers (FIG. 3). When the
enzyme mix was added to the qPCR mixture containing the template, a
strong reduction of the amplification efficiency was observed for
some of the markers. This was not caused by altered digestion
efficiency nor for an alteration of the polymerase activity since
the amplification of the POLR2A control marker was comparable
between the single vs the two-step reaction.
[0094] All the markers listed in Table 2 (including LINE-1 and DPM2
controls) were tested to verify their methylation status in cancer
using the MSRE qPCR approach. qPCR reactions where thus performed
using undigested and digested genomic DNA obtained from blood,
LD583 bladder cancer cell line and normal Urine (from non-cancer
individuals). All the samples were normalized against undigested
blood gDNA (reference sample; considered to be 100%) and values
were corrected for the amount of template loaded in the reaction
(this was determined using the POLR2A control marker). Digestion
efficiencies were determined using the GAPDH control marker. The
results are shown in FIGS. 4-9.
[0095] Preferred markers were markers that did not show any
methylation in blood and normal urine gDNA and were 100% methylated
in the cancer cell line DNA. From this analysis, makers that
resulted less than 2% methylated in blood or normal urine DNA were
subdivided into three categories (see Table 3 below).
TABLE-US-00005 TABLE 3 Category A - Methylation level in normal
blood and urine <1%, Methylation level in LD583 bladder cancer
cell line >70%; Category B - Methylation level in normal blood
and urine <1%, Methylation level in LD583 bladder cancer cell
line >50% < 70%; Category C - Methylation level in normal
blood and urine >1% < 2%, Methylation level in LD583 bladder
cancer cell line >70%. Category A Category B Category C 09 Unk
(Unc) 09 08/15 GALR1 .sub. 06B SCT 05 Unk05 23 TBX15 14 Unk14 18
DCHS2 32 EEF1A2 29 Unk29 01 OTX1 .sub. 10B DMRTA2 31 CERKL 07 Unk07
04 TFAP2B .sub. 34B SHH 30 EVX2 16 SEPT9 .sub. 27B SOX1 19 Unk19
.sub. 21C Unk21 36 SOX17
[0096] Blood is known to be a major urine contaminant, however also
sperm might contaminate urine sample. Because of this, the
methylation level of all the 21 above validated markers in Table 3
were also tested using sperm DNA. The results confirm that all the
21 markers are not methylated in sperm DNA (<1%).
[0097] Additionally, for the 16 markers belonging to categories A
and B in Table 3, the methylation detection limit the MSRE qPCR
system was determined and thus the minimum amplification value that
can be considered higher than the background signal (background was
determined in digested normal Urine gDNA, which is not methylated;
e.g. see FIG. 3). In general, for all the 16 markers the background
signal is lower than 0.25%. This value is therefore considered the
low-confidence background limit, so a digested sample with a
methylation signal higher than 0.25% is putatively considered
positive. To enhance the reliability of MSRE qPCR system, the
inventors increased to 1% the confidence limit and considered this
value to be the high-confidence background limit. A sample with a
value higher than 1% is considered truly positive. Samples with a
value between 0.5 and 1% are considered low-confidence positive,
while digested sample with a methylation level higher than 1% are
considered high-confidence positive for cancer.
Example 2--Human Sample Analysis with Exemplary Markers
[0098] Clinical trial urine samples from bladder cancer tumor
patients and normal (non-cancer) urine samples were analyzed using
the category A and B markers listed in Table 3. The % of the signal
detected for a specific digested sample was calculated by
comparison with the signal of the same sample but undigested (100%)
(in Example 1, all the signals were calculated in comparison with
the signal of undigested blood sample) (e.g. FIG. 13).
[0099] The methylation signals in digested clinical samples
compared with the same undigested samples are shown in FIG. 10.
Blinded patient status classification based on MSRE data is shown
in FIG. 11. The results show that 4 out of 13 patients appear to be
healthy (marked in green), while 5 are most likely developing
cancer at various stages (marked in red). Other four patients are
positive for cancer at low-confidence (borderline cases, marked in
orange).
[0100] Markers 01, 16 and 04 (see red arrows in FIG. 10) showed a
higher background in normal Urine (although <1%; red circle)
compared with the other markers. Values from these three markers
were not considered for the final clinical sample classification
(FIG. 11).
Example 3--Analysis of Methylation Using Enhanced CARE Assay
[0101] The assay of Example 1 was further developed to increase its
overall performance, including sensitivity, simplicity and
detection limit. First, the 16 cancer markers panel (Table 4) that
have methylation levels of at least 50% in the LD583 bladder cancer
cell line and generally lower than 1% in normal urine, blood and
sperm samples was further analyzed to arrive at a panel of 12
markers to validate clinical samples.
TABLE-US-00006 TABLE 4 List of the 16 biomarkers that can better
discriminate bladder cancer (LD583 bladder cancer cell line) from
normal sample (blood and urine collected from healthy individuals).
ASSOCIATED ASSOCIATED CODE GENE/EST CODE GENE/EST #09 Unknown 09
(Unc09) #15 GALR1 #05 Unk05 #23 TBX15 #18 DCHS2 #32 EEF1A2 #01 OTX1
#10 DMRTA2 #21 Unk21 #27 SOX1 #07 Unk07 #04 TFAP2B #30 EVX2 #36
SOX17 #16 SEPT9 #19 Unk19
[0102] A small cohort of normal urine DNA samples (n=9) collected
from individuals with different age, gender and ethnicity was
analyzed by the assay of Example 1. As shown in Table 5, all of the
9 samples were classified as negative for bladder cancer. Despite
the fact that the cohort was very small, no significant differences
in methylation background levels were linked to age, gender or
ethnicity.
TABLE-US-00007 TABLE 5 DNA urine samples collected from 9 healthy
individuals were analyzed with a first version of CARE assay.
Sample codes are indicated in the first column, while markers are
listed in the first row. Age (year), gender and ethnicity are
indicated for each sample. GAPDH represents the digestion control.
Methylation levels <0.25% are indicated in white; >0.25% but
<1% with YY; >1% with BB. Experiment was performed using two
technical replicates. Markers Sample GAPDH Unk09 Unk05 DHCS2 Unk07
EVX2 SOX1 Unk19 Code Age Gender Ethnicity GAPDH #09 #05 #18 #07 #30
#27 #19 Sample #09 29 F African #16 28 F Caucasian #22 29 M
Caucasian #02 48 M Caucasian #19 44 M Caucasian #20 41 M Caucasian
#03 55 M Asian #17 72 F Caucasian #24 52 F Caucasian YY Markers
Sample Unk21 SOX17 GALR1 TBX15 DMRTA2B TFAP2B OTX1 SEP19 EEF1A2
Code #21 #36 #15 #23 #10 #04 #01 #16 #32 Sample #09 BB BB YY #16 BB
YY #22 BB BB YY #02 BB BB YY #19 BB BB BB #20 #03 BB YY YY #17 BB
YY #24 YY YY YY BB YY
[0103] Moreover, the results also pointed out an increased
background level for four markers (the four rightmost markers in
Table 5). In particular marker #04 had a methylation level >1%
(up to 4%) in most of the samples, while marker #01 had an elevated
background in four out of nine samples. Marker #16 (SEPT9) and #32
(EEF1A2), that are associated with relevant malignancies including
colorectal, prostate, pancreas, breast and ovarian cancers, also
had a high background in a few, but not all, samples. It is
interesting, however, that the background level of these two
markers is very similar in terms of sample pattern and magnitude.
To note, SEPT9 methylation levels in sample #03 and #05 were
confirmed by a parallel experiment based on DNA
bisulfite-conversion instead of CARE assay. This similarity might
indicate that the increased methylation level in these samples is
not due to technical issues (e.g. incomplete digestion) but is
rather specifically elevated in some sample. This suggests that the
slightly increase in methylation of these two markers might not be
random and might perhaps be indicative, or predictive, of a
specific clinical status (i.e. risk to develop a specific clinical
condition). Thus, marker #32 was kept in the marker panel. Instead,
markers #04, #01 and #16 were not considered for further validation
experiments reducing the bladder cancer biomarker panel to 13
loci.
[0104] Next, the selected 13 bladder cancer biomarkers were tested
using a small cohort (n=10+10) of bladder cancer samples and their
correspondent adjusted normal tissues (non-cancer bladder tissue
isolated along with the tumor at the moment of tumor removal)
(Table 6). The overall methylation level of the 13 cancer
biomarkers was significantly higher in tumor samples than in the
adjusted normal one. 7 out of 10 tumor samples had all 13 markers
significantly methylated (>1%). Some markers, specifically #27,
#36 and #30 were also highly methylated in the adjusted normal
samples.
TABLE-US-00008 TABLE 6 Analysis of DNA extracted from 10 bladder
cancer tissues and their correspondent adjusted normal sample (the
last indicated in Italic) using CARE assay. Sample codes are
indicated in the first column, while markers are listed in the
first row. As in Table 5, each biomarker and sample values are
expressed as percentage of methylation present after digestion.
GAPDH represents the digestion control. Methylation levels
<0.25% are indicated in white; >0.25% but <1% with YY;
>1% with BB. Experiment was performed using two technical
replicates. Markers GAPDH Unk09 Unk05 DHCS2 Unk07 EVX2 SOX1 Unk19
Sample Code GAPDH #09 #05 #18 #07 #30 #27 #19 Sample
01-01-0036-01-D2 BB BB BB BB BB BB BB 01-02-0036-01-D2 BB BB BB BB
BB BB BB 01-01-0037-01-D2 YY YY BB 01-02-0037-01-D2 YY YY YY YY BB
YY 01-01-0038-01-D2 BB BB BB BB BB BB BB 01-02-0038-01-D2 YY YY YY
BB YY CD564833 BB BB BB BB BB BB BB CD564841 YY YY YY BB YY YY
CD564111 BB BB BB BB BB BB BB CD564102 BB BB BB BB BB BB BB
01-01-0041-01-D1 BB BB BB BB BB BB 01-02-0041-01-D2 YY YY YY BB BB
BB 01-01-0042-01-D1 BB BB BB BB BB BB BB 01-02-0042-01-D2 YY YY YY
YY YY YY YY 01-01-0043-01-D2 BB BB BB BB BB BB BB 01-02-0043-01-D2
BB BB BB BB BB BB BB 01-01-0044-01-D2 BB BB BB BB BB BB BB
01-02-0044-01-D2 BB BB BB BB BB BB YY 01-01-0045-01-D1 BB BB BB BB
BB BB 01-02-0045-01-D1 YY YY Markers Unk21 SOX17 GALR1 TBX15 EEF1A2
DMRTA2B Sample Code #21 #36 #15 #23 #32 #10 Sample 01-01-0036-01-D2
BB BB BB BB BB BB 01-02-0036-01-D2 BB BB BB BB BB BB
01-01-0037-01-D2 BB BB BB BB 01-02-0037-01-D2 YY BB BB
01-01-0038-01-D2 BB BB BB BB BB BB 01-02-0038-01-D2 YY YY YY YY
CD564833 BB BB BB BB BB BB CD564841 YY YY YY YY CD564111 BB BB BB
BB BB BB CD564102 BB BB BB BB BB 01-01-0041-01-D1 BB YY BB BB
01-02-0041-01-D2 BB BB YY 01-01-0042-01-D1 BB BB BB BB BB BB
01-02-0042-01-D2 YY YY YY BB YY 01-01-0043-01-D2 BB BB BB BB BB BB
01-02-0043-01-D2 YY BB YY YY BB BB 01-01-0044-01-D2 BB BB BB BB BB
BB 01-02-0044-01-D2 BB BB BB BB BB BB 01-01-0045-01-D1 BB BB BB BB
BB BB 01-02-0045-01-D1 YY
[0105] The data generated from this analysis were used to score
every single marker; in particular each marker was evaluated for A)
its ability to recognize a bladder malignancy; this parameter was
determined as the number of tumor samples recognized positive
(methylation level >1%) by a specific marker. Each
cancer-specific marker should be significantly methylated in all
the tumor samples; B) for the level of methylation of a specific
marker in a specific tumor sample in comparison to the average
methylation level between all the markers in the same sample (a
high methylation level in cancer samples is desirable); C) For the
number of adjusted normal sample not recognized as normal sample;
this last parameter was considered important since, contrary to
blood or normal urine, adjusted normal samples are expected to show
an increase in bladder cancer markers methylation levels since they
are tissue samples often containing cells predisposed to the
development of cancer, a phenomena named "field effect" (e.g.
Giovannucci and Ogino, 2005; Bernstein et al., 2007); it is
therefore expected that the methylome of adjusted normal sample
will be altered at some extent. It was reasoned that a good cancer
marker is supposed to detect these differences. Based on these
criteria the 13 markers were classified into three marker set
categories (A, B and C; Table 7). Set A and B contain the controls
and the markers that more likely will be included in the final
marker set, while set C include all the other markers. To note,
marker #23 showed poor performances as cancer marker (low
methylation level in many tumor samples compared to the other
markers), therefore it was not included in any set. Thus, the
marker panel contains 12 biomarkers.
TABLE-US-00009 TABLE 7 The 12-selected biomarkers (right column for
each set) were classified into three sets based on the results
obtained in the experiment presented in Table 6. Set A and B
contain the markers with the best performances and the two internal
controls POLR2A and GAPDH, while set C contains all the other
markers. SetA SetB SetC POLR2A #05 Unk05 #09 Unk09 GAPDH #07 Unk07
#32 EEF1A2 #27 SOX1 #30 EVX2 #19 Unk19 #15 GALR1 #36 SOX17 #21
Unk21 #10 DMRTA2 #18 DCHS2
[0106] All the markers included in the three sets (Table 7) of the
new panel were further validated using DNA isolated from normal
urine sample collected from a larger cohort (n=31). This experiment
confirmed the data already presented in Table 5 and methylation
background was found to be low for all the markers except for
marker #32 (that was found to be elevated in some sample).
[0107] In an additional experiment, it was tested if the selected
biomarker panel could be used to identify different type of
malignancies. To this end, DNA extracted from bladder, prostate,
endometrium and kidney tumor and their correspondent adjusted
normal sample was analyzed. Moreover, also included in the analysis
was one bladder sample isolated from a healthy individual (as a
negative control) and three multiple myeloma sample (HA, MM1.S and
CRF30).
TABLE-US-00010 TABLE 8 CARE analysis of DNA extracted from
different cancer sample (TU), their correspondent adjusted normal
samples (AN) and three different multiple myeloma samples (HA,
MM1.S and CRF30). A healthy bladder control was included in the
analysis. Samples are listed in the first column, while markers are
indicated in the first row. Values for each biomarker and sample
are expressed as percentage of methylation present after digestion.
GAPDH represents the internal digestion control. Methylation levels
<0.25% are indicated in white; >0.25% but <1% with YY;
>1% with BB. Experiment was performed using two technical
replicates. Markers GAPDH SOX1 GALR1 DMRTA2B Unk05 Unk07 EVX2 SOX17
DHCS2 Unk09 EEF1A2 Unk19 Unk21 Sample GAPDH #27 #15 #10 #05 #07 #30
#36 #18 #09 #32 #19 #21 Sample Healthy YY YY YY YY YY Bladder
Bladder TU BB BB BB BB BB BB BB BB BB BB BB BB Bladder AN BB BB BB
BB YY BB BB YY BB BB BB YY Prostate TU BB BB BB BB BB BB BB BB BB
BB BB Prostate AN YY BB YY YY BB BB Endometrium BB BB BB BB BB BB
BB BB BB BB BB BB TU Endometrium BB BB BB BB BB BB BB BB YY BB BB
AN Kidney TU YY YY YY BB YY YY YY Kidney AN YY BB BB YY HA BB BB BB
BB BB BB BB BB BB BB BB BB MM1.S BB BB BB BB BB BB BB BB BB BB BB
BB CRF30 BB BB BB BB BB YY BB BB
[0108] Despite the small cohort used, the results clearly
demonstrated that many of the biomarkers listed in sets A, B and C
are heavily methylated in different types of cancer including
bladder, prostate, endometrial and multiple myeloma tumors.
Surprisingly, no increase of methylation was detected in kidney
cancer. As expected, none of the markers was found to be
significantly methylated (methylation level >1%) in DNA isolated
from healthy bladder. Additional different types of cancer can
potentially also be detected using only urine samples.
[0109] In addition to the trials described above, a pre-clinical
study was performed in which a small cohort of 63 urine sample was
analyzed using the enhanced CARE assay. As indicated in Table 9
this cohort includes 20 samples collected from healthy individuals
("Normal sample") with different age, gender and risk behaviors
concerning bladder cancer development (the cohort in fact includes
four young smoker individuals-<55 years old-). The rest of the
cohort (n=43; "Cancer sample") is represented by samples collected
from individuals with bladder cancer and other type of tumors;
specifically, 33 samples were collected from persons affected by
bladder cancer before cancer removal ("Bladder cancer before
surgery"), 8 sample were collected from individuals who were
recently subjected to blabber tumor removal ("Bladder cancer after
surgery") and other 2 samples (collectively named "Non-bladder
cancer") were collected from individuals, #48N and #34S, affected
by stomach cancer and colon adenocarcinoma, respectively.
TABLE-US-00011 TABLE 9 CARE analysis of DNA extracted from urine
samples collected from 20 healthy individuals (normal sample;
indicated in the upper part of the table) and from 43 patients
affected by cancer (cancer sample). The first column indicates the
subgroups description (YO = years old), while sample codes are
listed in the second column. Markers are indicated in the first
row. A blood sample control was included in the analysis (second
row) while GAPDH represents the internal digestion control.
Methylation levels <0.25% are indicated in white; >0.25% but
<1% with YY; >1% with BB. Experiment was performed using two
technical replicates. Markers GAPDH SOX1 GALR1 DMRTA2B Unk05 Unk07
EVX2 SOX17 Sample GAPDH #27 #15 #10 #05 #07 #30 #36 Blood YY Female
30N <55 YO 24N YY 27N YY YY 26N YY BB YY Male 02N YY <55 YO
22N YY YY 47N YY 01N YY YY Female 39N >55 YO 17N YY 14N YY YY
28N YY YY BB YY YY Male 12N >55 YO 25N YY YY YY YY 03N YY YY YY
YY YY 38S UR9380B YY YY BB YY Smokers 49N 75N YY 74N YY 73N YY YY
YY Bladder 05S UR5753B YY Cancer 29S UR0076B YY YY before 30S
UR0077B YY YY surgery 33S UR0089B BB 31S UR0078B YY YY 25S UR0067B
BB YY 02S UR5146B YY YY 09S UR0028B YY YY YY YY YY 32S UR0084B YY
YY BB YY 27S UR0072B YY YY BB YY 18S UR0029B YY YY BB YY 41S
UR0044B YY YY BB YY YY 24S UR0057B YY YY BB YY 20S UR0031B YY YY YY
BB YY 22S UR0040B YY YY BB BB YY YY YY 08S UR0032B YY YY YY BB YY
YY YY 11S UR0030B YY YY YY BB YY YY YY 01S UR5822B YY YY BB BB YY
23S UR0053B BB YY BB BB YY BB YY 07S UR0026B BB BB YY BB YY BB YY
43S UR9088B BB BB BB BB BB BB YY 26S UR0068B BB BB BB BB BB BB 36S
UR9280B BB BB BB BB BB BB 03S UR6679B BB BB BB BB BB BB BB 16S
UR0014B BB BB BB BB BB BB BB 37S UR9349B BB BB BB BB BB BB BB 39S
UR0043B BB BB BB BB BB BB BB 35S UR9202B BB BB BB BB BB BB BB 04S
UR0008B BB BB BB BB BB BB BB 06S UR0021B BB BB BB BB BB BB BB 13S
UR0009B BB BB BB BB BB BB BB 14S UR0012B BB BB BB BB BB BB BB 28S
UR0074B BB BB BB BB BB BB BB Bladder 10S UR0028A YY Cancer 19S
UR0029A BB after 21S UR0031A YY YY BB YY surgery 15S UR0012A YY YY
BB YY YY 42S UR0044A YY BB YY BB YY YY YY 40S UR0043A BB YY YY BB
BB YY BB 12S UR0030A BB BB BB BB BB BB BB 17S UR0014A BB BB BB BB
BB BB BB Non- 48N Stomach YY bladder 34S Colon BB BB BB BB BB BB BB
Markers DHCS2 Unk09 EEF1A2 Unk19 Unk21 Sample #18 #09 #32 #19 #21
Blood YY Female 30N Normal <55 YO 24N Sample 27N YY 26N YY YY
Male 02N YY <55 YO 22N YY 47N YY YY 01N YY Female 39N >55 YO
17N YY 14N YY 28N YY Male 12N >55 YO 25N YY YY YY 03N YY YY 38S
UR9380B YY YY Smokers 49N 75N 74N YY 73N YY YY Bladder 05S UR5753B
Cancer Cancer 29S UR0076B YY Sample before 30S UR0077B YY surgery
33S UR0089B YY YY 31S UR0078B YY YY 25S UR0067B YY YY 02S UR5146B
YY YY YY 09S UR0028B YY 32S UR0084B YY YY 27S UR0072B YY YY YY 18S
UR0029B YY YY YY YY 41S UR0044B BB YY 24S UR0057B YY YY BB 20S
UR0031B YY BB YY 22S UR0040B YY YY YY YY 08S UR0032B YY YY YY BB YY
11S UR0030B YY YY YY BB YY 01S UR5822B YY YY BB YY YY 23S UR0053B
YY BB YY 07S UR0026B YY BB YY BB YY 43S UR9088B BB BB YY BB BB 26S
UR0068B BB BB BB BB BB 36S UR9280B BB BB BB BB BB 03S UR6679B BB BB
YY BB BB 16S UR0014B BB BB YY BB BB 37S UR9349B BB BB YY BB BB 39S
UR0043B BB BB YY BB BB 35S UR9202B BB BB YY BB BB 04S UR0008B BB BB
BB BB BB 06S UR0021B BB BB BB BB BB 13S UR0009B BB BB BB BB BB 14S
UR0012B BB BB BB BB BB 28S UR0074B BB BB BB BB BB Bladder 10S
UR0028A YY YY YY Cancer 19S UR0029A YY YY after 21S UR0031A YY BB
YY surgery 15S UR0012A BB BB YY 42S UR0044A YY BB YY BB 40S UR0043A
BB YY BB BB 12S UR0030A BB BB BB BB BB 17S UR0014A BB BB BB BB BB
Non- 48N Stomach bladder 34S Colon BB BB BB BB
[0110] Based on the data obtained from this analysis, a panel
marker of #05, #36 and #09 has the highest diagnostic value,
therefore they were used for a statistical cancer diagnostic model
built utilizing the generalized linear model. This model will
classify the population in positive or negative for bladder cancer
only based on the data obtained from CARE assay. 3/4 and 1/4 of the
cohort (only the 20 normal and 33 bladder cancer samples were
considered) were used as Training and Test Sets respectively and
the results are represented in Table 10. Results indicate that the
enhanced CARE assay was able to correctly classify most of the Test
Set samples (85.71% of the controls and 100% of the bladder cancer
samples). In addition, for the Test set the enhanced CARE assay has
a sensitivity of 100% and a specificity of 85.71%.
[0111] It is also interesting to note that most of the "Bladder
cancer sample after surgery" samples were diagnosed as positive by
CARE assay (Table 9). This very interesting data support the "field
effect" concept and is in agreement with previous data represented
in Table 6 in which most of the adjusted normal tissue samples were
found to be positive using CARE assay. However it is not clear at
this point if the exposition to the cancer environment is also able
to trigger epigenetic changes in normal cells present in the
vicinity of the tumor. Thus, urine from patients who were subjected
to bladder tumor removal will be collected periodically and
analyzed using CARE assay to monitor the methylation level
evolution of the bladder cancer biomarkers over time.
TABLE-US-00012 TABLE 10 Statistical analysis of a 53-individuals
cohort affected or not by bladder cancer. The analysis is based on
the methylation values detected for marker #05, #36 and #09 using
CARE assay. The population is randomly subdivided in Training (3/4)
and Test (1/4) Sets and sensitivity % {[TP/(TP + FN)] * 100},
specificity % {[TN/(TN + FP)] * 100}, positive predictive value %
{[TP/(TP + FP)] * 100} and negative predictive value % {[TN/(TN +
FN)] *100} were calculated for each set and for the whole cohort.
Whole Training Cohort Set Test Set Data Total Sample 53 39 14
Bladder Cancer 33 26 7 Control 20 13 7 True Positive (TP) 30 23 7
True Negative (TN) 17 11 6 False Positive (FP) 3 2 1 False Negative
(FN) 3 3 0 Statistics Sensitivity % 90.91 88.46 100.00 Specificity
% 85.00 84.62 85.71 Positive Predictive Value % 90.91 92.00 87.50
Negative Predictive 80.95 78.75 100.00 Value %
[0112] Another interesting result is that the colon adenocarcinoma
urine sample (male individuals) was clearly positive for CARE
assay. This might be due to the presence of metastasis in the
bladder, or again to a "field effect" phenomenon, given the
anatomical vicinity of colon and bladder. This second hypothesis is
further supported by the fact that the stomach cancer urine sample
(#48N; anatomically distant from bladder) was found to be negative
with CARE assay.
[0113] Concerning the normal urine cohort, no major differences
were observed between the different samples except in the
subpopulation of healthy males older than 55 years (M>55 YO); in
this group, three out of four individuals had a slightly elevated
methylation background (generally <1%) in at least half of the
biomarkers tested with CARE assay. To note, aging is one of the
predominant risk factor for bladder cancer development and the male
population (especially Caucasian) is way more affected by this
pathology than females. The CARE assay was able to detect slightly
increases of methylation in the older male subgroup of the normal
cohort which suggests that the CARE assay could discriminate
individuals that have more risk to develop bladder cancer from
those that do not. In this perspective, it might be also relevant
the fact that one out of four female older than 55 years (F>55
YO) also have an elevated methylation level of six biomarkers,
while the other three individuals belonging to the same subgroup
have a bladder cancer markers methylation profile similar to those
seen for younger individuals. To note, all the four smoker young
individuals were classified as negative using the enhanced CARE
assay.
[0114] In conclusion, the enhanced CARE assay was able to correctly
classify most of the normal and bladder cancer samples included in
the Test set cohort and has a sensitivity and specificity of 100%
and 85.71% respectively (Table 9 and Table 10). In addition, the
assay detected as positive a urine sample collected from one colon
adenocarcinoma affected individuals, indicating that the CARE assay
has the potential to detect non-urinary tract cancers using urine
samples. Moreover, the assay was able to detect a slight increase
of biomarker methylation in the older male (>55 years old) group
belonging to the healthy cohort (a risk population concerning
bladder cancer development); suggesting that the enhanced CARE
assay might represent a potentially interesting tool not only for
bladder cancer recurrence diagnosis, but also to better determine
the subpopulation with higher risk to develop bladder cancer for
the first time.
Example 4--Methods of Enhanced CARE Assay
[0115] The enhanced CARE assay of Example 3 has several
improvements as compared to the assay of Example 1. First, the
reaction buffer of the enhanced CARE assay is compatible with both
digestion and qPCR steps. Therefore, the only manual step that the
operator needs to do is the addition of the proper amount of
template (5-10 ng) to the reaction buffer. After mixing, the
reaction will be incubated in the qPCR machine that will
automatically perform the digestion and the qPCR steps. This new
buffer allows complete digestion in only 2 hours of incubation at
30.degree. C. The reaction mix contains all the components
necessary for both steps, including Taq polymerase, the restriction
enzymes mix, deoxyribonucleotides, DNA primers and probes,
magnesium, additives (e.g., DMSO) and salts, and the optimal
concentration of each component was determined in order to maximize
CARE assay performances in terms of accuracy, sensitivity and
specificity. The best results were obtained using ZymoTaq.TM. Taq
Polymerase, however other Hot-Start enzymes (e.g. AmpliTaq
Gold.RTM. DNA Polymerase, Phusion.RTM. High-Fidelity DNA
Polymerase) can be used to prepare the CARE assay reaction
buffer.
[0116] In addition, the enhanced CARE assay allows multiplexing and
the simultaneous analysis up to 5 biomarkers in a single reaction.
All the 12 bladder cancer biomarkers and two internal controls
present in set A, B and C (described above) were therefore grouped
in three multiplex reactions (one for each set; see Table 11). Each
amplicon was reviewed and new primers and dual-labeled TaqMan
probes were specifically designed in order to maximize the
robustness of CARE assay and the overall performances of the
system. For each marker the amplification efficiency ranges between
90 and 110% and no significant differences (in terms of
amplification efficiencies and background level) can be observed
neither when singleplex and multiplex reaction are run in parallel,
nor when digestion and qPCR steps are performed in one machine
rather than in two separate steps (digestion in a thermocycler or
incubator and qPCR in the Real-Time PCR machine). Importantly
primers and probes were designed on sequences lacking any annotated
SNPs and repeated element. Despite different methodologies and
Real-Time machines can be potentially used for the qPCR step, we
obtained the best results by combining TaqMan methodology and the
CFX96TM-Real Time System (Bio Rad). In the new CARE assay version
dual-labeled TaqMan probes are marked with FAM, HEX, Texas Red-X,
Cy5 and Quasar 705 and quenched by either BHQ1 or BHQ2 molecules.
However, other dyes like Biosearch Blue, TET, CAL Fluor Gold 540,
JOE, VIC, CAL Fluor Orange 560, Quasar 570, Cy3, NED, TAMRA, CAL
Fluor Red 590, Cy3.5, ROX, CAL Fluor Red 610, Texas Red, CAL Fluor
Red 636, Pulsar 650, Quasar 670, Cy5.5, TEX 615, TYE 563, TYE 665,
MAX, Yakima Yellow, ABI and JUN and different quenchers like TAMRA,
QSY, BHQ2, BHQ3, Iowa Black can be successfully used for the qPCR
step. Other system variants, like Molecular Bacons and Scorpions
Probes can also be successfully used for the qPCR step.
TABLE-US-00013 TABLE 11 The biomarkers present in sets A, B and C
(right column for each set) were multiplexed. Each multiplex
reaction is able to amplify up to 5 markers simultaneously. Set A
and B contain the markers with the best performances and the two
internal controls POLR2A and GAPDH, while set C contains all the
other markers. Dies and quenchers used for each marker are
indicated in the right column of each set. Q705 = Quasar 705, TR =
Texas Red, BHQ = Black Hole Quencher. SetA SetB SetC POLR2A
Q705-BHQ2 #05 TR-BHQ2 #09 Cy5-BHQ2 GAPDH FAM-BHQ1 #07 Cy5-BHQ2 #32
Q705-BHQ2 #27 TR-BHQ2 #30 Q705-BHQ2 #19 HEX-BHQ1 #15 HEX-BHQ1 #36
HEX-BHQ1 #21 TR-BHQ2 #10 Cy5-BHQ2 #18 FAM-BHQ1
[0117] A third important improvement that was made in the enhanced
CARE assay is the increase of the number of restriction sites that
are investigated for each amplicon. The newly designed amplicons
contain at least 6 different restriction sites that are targeted at
least by two out of three restriction endonucleases selected for
this assay (AciI, HinP1l and HpaII). This, combined with the
innovative chemistry of the new reaction mixture, ensures a
complete digestion in 2 hours (Table 13) and minimizes the risk to
incurring in artifacts and unspecific background signal.
[0118] In addition, all oligonucleotide (primers and probes)
sequences do not contain consensus motives for the aforementioned
restriction endonucleases; this seems to prevent the degradation
(although very slight) of the oligonucleotides during the digestion
reaction potentially caused by the temporary and probably only
partially specific interactions with other DNA molecules that might
occur at the digestion temperature (30.degree. C.).
[0119] Several other restriction enzymes, including AatII, Acc65I,
AccI, AciI, AclI, AfeI, AgeI, AhdI, AleI, ApaI, ApaLI, ApeKI, AscI,
AsiSI, AvaI, AvaII, BaeI, BanI, BbvCI, BceAI, BcgI, BcoDI, BfuAI,
BfuCI, BglI, BmgBI, BsaAI, BsaBI, BsaHI, BsaI, BseYI, BsiEI, BsiWI,
BslI, BsmAI, BsmBI, BsmFI, BspDI, BspEI, BsrBI, BsrFI, BssHII,
BssKI, BstAPI, BstBI, BstUI, BstZ17I, BtgZI, Cac8I, ClaI, DpnI,
DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoRI, EcoRV, FauI,
Fnu4HI, FokI, FseI, FspI, HaeII, HgaI, HhaI, HincII, HinfI, HinPlI,
HpaI, HpaII, Hpy166II, Hpy188III, Hpy99I, HpyAV, HpyCH4IV, KasI,
MboI, MluI, MmeI, MspA1I, MwoI, NaeI, NarI, NciI, NgoMIV, NheI,
NlaIV, NotI, NruI, Nt.BbvCI, Nt.BsmAI, Nt.CviPII, PaeR7I, PhoI,
PleI, PluTI, PmeI, PmlI, PshAI, PspOMI, PspXI, PvuI, RsaI, RsrlI,
SacII, SalI, Sau3AI, Sau96I, ScrFI, SfaNI, SfiI, SfoI, SgrAI, SmaI,
SnaBI, StyD4I, TfiI, TliI, TseI, TspMI, XhoI, XmaI, ZraI can in
principle be utilized for the CARE assay.
[0120] Controls: Another core component of the enhanced CARE assay
is represented by the controls. The actual CARE assay version
contemplates 4 different controls: A) endogenous copy number
control (POLR2A), B) endogenous digestion control (GAPDH), C)
undigested sample control and D) exogenous digestion control
(standardized blood sample). To note, the controls used for CARE
assay are not manipulated in vitro (e.g. chemically or
enzymatically modified) reducing in that way the chances to
introducing artefacts and errors.
[0121] Endogenous copy number control--POLR2A: One point for the
reliability of the assay is the normalization of the signal
obtained from each marker against the amount of DNA sample loaded
in each reaction. Tiny differences in the amount of template loaded
in different reactions are common in qPCR and they can impact the
results of the analysis if they are not monitored. Because of that
the usage of an internal copy number control that allows to
precisely quantitate the amount of template used in each reaction
and to correct the values obtained from the analysis is needed. A
portion of the coding sequence of the human POLR2A gene was chosen
as the copy number control. That choice was made because A) POLR2A
is an essential gene, so the control signal will always be
detected, B) this control marker is a single-copy gene and its
usage in multiplex qPCR will not interfere with the amplification
of the other markers (like what can happen using a multi-copy
marker like a transposable element), C) the sequence that was
selected does not contains target sites for any of the restriction
endonucleases used in the assay, so the signal of the copy number
control will always reflects the amount of DNA used independently
on the fact that the sample is digested or not. In principle, every
locus satisfying the three points listed above can be successfully
used as endogenous copy number control.
[0122] Endogenous digestion control--GAPDH: One other point for the
reliability of CARE assay is the achievement of a complete
enzymatic digestion. In principle, a single cleavage event within a
specific locus is sufficient to prevent the amplification of the
associated target region. Nevertheless, as described previously,
amplicons were designed in order to include at least 6 restriction
sites recognized by at least two out of the three restriction
endonucleases used for the assay. This, combined with the new
reaction mix composition, allows the achievement of the maximum
specific enzymatic activity and strongly abates the possibility of
incomplete digestion. However, the digestion completeness of each
reaction was monitored by including in the final CARE assay an
endogenous digestion control. This is represented by a DNA locus
that is A) essential for the cell survival, B) that contains
multiple restriction sites for all of the three enzymes used in the
CARE assay C) and that is always unmethylated in any type of cells
and tissues. The internal digestion control was designed on the CpG
island of the human GAPDH gene; this housekeeping gene have an
essential role in glycolysis and is widely used as a copy number
control for gene expression analysis in RT-qPCR reactions and
immunoblots since it is always expressed at almost constant level
in all the cell type. In all of the digested samples tested with
CARE assay (a wide variety of specimen including biological fluids,
tissues and cell types) GAPDH signal was always lower than the
maximum acceptable background level (1%) and in most of the case it
was not detectable at all (e.g. in Table 5). Also, in this case,
loci that satisfy the three points aforementioned can be used as
endogenous digestion controls; between all, possible alternative
endogenous digestion control loci can be designed on CpG islands of
genes ATP5C1, TBP, GARS, LDHA and PGK1.
[0123] Undigested sample control: This control consists of a DNA
sample processed exactly like the samples that will be subjected to
digestion, but without adding the restriction endonucleases mix.
Therefore, in this sample, the DNA will remain intact and all the
marker copies added initially to the reaction will be suitable for
amplification. The amplification signal for each marker in this
sample can therefore be considered 100%, and the percentage of
methylated (uncut) marker detected in the parallel digested sample
will be calculated accordingly. Initially, a DNA sample isolated
from a healthy blood donor was used as the undigested sample
control. However, to minimize the possible differences that might
exist between this standard sample and urine DNA samples, an
undigested reaction (run in parallel with the digested ones) was
performed for each urine sample that will be analyzed with CARE
assay. Thus, the final assay will therefore include two distinct
premixed reaction solutions, named D and U, for each marker set;
only the reaction solution D (but not the U) will contain the
restriction nucleases mix needed for the digestion step. In
general, it was concluded that running an undigested control for
each single sample represents the most reliable way to precisely
quantify the amount of methylation level for each marker in each
sample.
[0124] To further increase the precision of the assay, a second
digestion control represented by a standardized sample of blood DNA
was included. The fact that all the cancer markers included in the
current assay are not (or only weakly) methylated in blood DNA
(Table 12) allows the detection of potential problems that might
occur during the digestion step and that are listed below:
[0125] A) Different markers are cleaved by a different combination
of restriction endonucleases (some markers are cleaved by all three
enzymes, some other just by two); moreover, a different number of
CpG dinucleotides are interrogated in different markers (ranging
from 6 to 12). Therefore, although sufficient, the evaluation of
the digestion efficiency using only the GAPDH control marker will
be imprecise (GAPDH locus is targeted at 6 different sites by all 3
restriction endonucleases). In an extreme unlikely situation in
which only one out of three enzymes is fully active, GAPDH control
will be completely digested, while cancer markers that are targeted
only by the two other enzymes will be only partially digested. This
will leads to an unreal increase of some cancer marker methylation
status, generating a possible false positive situation. The
abnormal increase of methylation for some cancer markers in the
E.D.C. control will immediately inform the operator about this
situation
[0126] B) Moreover, if a general increase in methylation background
level (including for the GAPDH endogenous digestion control) is
observed for a specific sample, it will be impossible to know (in
absence of the E.D.C.) if that is caused by a decreased activity of
the restriction enzymes (in that case the reaction mix need to be
substituted with a new batch) or by a poor quality of the DNA
sample (in this second case the sample needs to be purified). Since
the quality of the exogenous digestion control is standard and all
the markers in this sample are unmethylated, an increase of the
background level for all the markers in the exogenous digestion
control will clearly indicate that the enzymatic activity of the
restriction enzymes present in the reaction mixture is impaired and
the mix has to be replaced with a new batch.
TABLE-US-00014 TABLE 12 CARE assay analysis of the exogenous
digestion control (standardized blood DNA). Markers are listed in
the first column. Values for each marker are expressed as
percentage of methylation present after digestion. GAPDH represents
the internal digestion control. Values that exceed 0.25%
(low-confidence background limit) are underlined. Experiment was
performed using three technical replicates. E.D.C. Markers GAPDH
0.05 #27 0.12 #15 0.06 #10 0.03 #05 0.71 #07 0.06 #30 0.10 #36 0.03
#18 0.04 #09 0.02 #32 0.04 #19 0.28 #21 0.03
[0127] In addition to the four controls aforementioned, information
was collected to monitor different biochemical parameters of the
urine sample before proceeding with the DNA extraction. The primary
reason was to determine the possible influences of specific
conditions (e.g. the increased presence of proteins and ketones in
the urine, the urine pH value, etc.) on the CARE assay
performances. Secondly, other parameters can perhaps support CARE
assay diagnosis.
[0128] An analysis was performed using Multistix.RTM. 10 SG Reagent
Strips (Siemens). None of the parameters monitored with this tool
have an impact on the CARE assay performances. Moreover, hematuria,
high leukocyte and protein content was detected in the majority of
the urine collected from bladder cancer patients but not in urine
collected from healthy individuals.
[0129] One of the most remarkable characteristics of CARE assay
consists in the combination between high performances (in terms of
robustness, detection limit, sensitivity and accuracy) and extreme
simplicity and versatility. Indeed, based on the data obtained so
far, the CARE assay has a sensitivity of at 100% and a specificity
of 85.71% (Table 11). In addition, CARE assay has a remarkably low
detection limit; indeed, it is able to detect with confidence less
than 7 cancer cells in a sample containing more than 1500 cells (as
shown in FIG. 17).
[0130] On the other hand, the CARE assay is very simple, rapid, and
user friendly. The whole assay (including digestion, qPCR step and
data analysis) can be performed in less than 5 hours. For each
marker set, two premixed reaction solutions (D and U) and the
exogenous digestion control sample (E.D.C.) will be provided (FIG.
18). Both reaction mixes will contain all the components necessary
to perform the qPCR step, but only the reaction solution D (but not
the U) will contain the restriction nucleases mix needed for the
digestion step. Therefore, the user only has to add 5 to 10 ng of
sample DNA (or E.D.C. sample) to 15 .mu.l of each reaction solution
and adjust the volume to 20 .mu.l using the DNA Diluent Buffer
provided with the kit. After mixing, the reactions will be directly
incubated in a CFX96TM-Real Time System Machine (Bio Rad) that will
perform the digestion and qPCR steps as well as the data analysis
in less than 4 hours (complete digestion can be achieved after only
2 hours of incubation at 30.degree. C.; see Table 13).
TABLE-US-00015 TABLE 13 CARE assay results from reactions in which
a normal urine sample was incubated for 0, 2, 4, 8 and 16 hours at
30.degree. C. in the CFX96TM-Real Time System Machine before
starting the qPCR step. Complete digestion (background <1%) is
achieved after only 2 hours of incubation. For practicality only
marker belonging to set A were analyzed. Values of each marker are
expressed in methylation percentage after digestion, with the
exception of POLR2A for which mean CT vales are indicated.
Experiment was performed using three technical replicates.
Digestion Time 0 h 2 h 4 h 8 h 16 h Markers POLR2A (CT) 24.89 24.51
24.49 24.43 24.62 GAPDH 100 n.a. 0.01 0.00 n.a. #27 100 0.27 0.16
0.21 0.17 #15 100 0.22 0.15 0.12 0.09 #10 100 n.a. 0.04 n.a.
0.04
[0131] The stability of both reaction mixes D and U were tested and
found to be stable for several months at -20.degree. C. Moreover,
10 thaw/freeze cycles did not alter the reaction performances
(Table 14).
TABLE-US-00016 TABLE 14 CARE analysis using reaction mixes D and U
thawed and frozen multiple times. No significant increase of
background (impaired digestion activity) or decrease in
amplification efficiency (impaired Taq polymerase activity;
observable by comparison CT values of the internal copy number
control POLR2A) was detected. No Tw-Fz = reaction mixes not
subjected to thaw-freeze cycles; 5 .times. Tw-Fz = reaction mixes
thaw and frozen for 5 times; 10 .times. Tw-Fz = reaction mixes thaw
and frozen for 10 times. Values of each marker are expressed in
methylation percentage after digestion, with the exception of
POLR2A for which mean CT vales are indicated. Experiment was
performed using three technical replicates. Treatment No Tw-Fz 5
.times. Tw-Fz 10 .times. Tw-Fz Markers POLR2A (CT) 27.13 27.17
27.13 GAPDH 0.13 0.03 0.03 #27 0.27 0.27 0.33 #15 0.27 0.14 0.14
#10 n.a. n.a. n.a.
[0132] To obtain the CARE assay performances described above, the
urine DNA quality (in terms of purity and integrity) should be high
enough to guarantee an efficient digestion and amplification during
the digestion and qPCR step respectively. Therefore, a preservative
agent (UCB.TM., Zymo Research) was added immediately after the
urine collection in order to prevent nucleic acids degradation
before the DNA isolation step. It was observed that the addition of
UCB.TM., while well preserving the DNA integrity in the sample over
time (Table 15), did not interfere with the CARE assay performance
when Quick-DNA.TM. Urine Kit (Zymo Research) was used to isolate
the DNA (Table 15).
TABLE-US-00017 TABLE 15 CARE analysis (marker set A) using DNA
extracted from fresh urine (No UCB) and from urine stored with UCB
for one week (UCB 7 days) or one month (UCB 30 days). The urine
necessary for this experiment derives from a single donation of a
single individual. No substantial changes in methylation
background, or amplification potential (POLR2A CT values) can be
observed between the different samples. Values of each marker are
expressed in methylation percentage after digestion, with the
exception of POLR2A for which mean CT vales are indicated.
Experiment was performed using three technical replicates.
Treatment No UCB UCB 7 days UCB 30 days Markers POLR2A (CT) 27.34
27.21 27.01 GAPDH n.a. n.a. n.a. #27 0.10 0.40 0.11 #15 0.16 0.04
n.a. #10 n.a. n.a. n.a.
[0133] As mentioned before, the DNA quality might represent a key
prerequisite for a successful CARE assay analysis. Therefore, it
was decided to test if different urine DNA isolation kits are
equally able to provide DNA with a sufficient quality to perform
CARE assay. Three kits (Quick-DNA.TM. Urine Kit-Zymo Research-,
Supplier QI and Supplier NG) were tested in parallel using a single
urine sample with or without UCB.TM. 2 hours before the extraction
(Table 16). When DNA was isolated with different kits no
substantial differences were observed neither in the total amount
of DNA recovered (DNA was quantified using Femto.TM. Human DNA
Quantification Kit, Zymo Research; data not shown), nor in the DNA
amplification potential (CT mean values for POLR2A internal control
in different CARE reactions are very similar; Table 16).
Surprisingly, however, the quality of the DNA isolated using the
kit of Supplier NG is not sufficient to obtain good CARE assay
performance. In particular for the internal digestion control GAPDH
and marker #27 the methylation background level is above the high
confidence limit (1%). Moreover, this problem is further pronounced
in the sample added with UCB.TM. (Table 16). This indicates that
while the quality of the DNA isolated with different kits is
sufficient for the qPCR step, the same is not true for the
digestion step. Therefore, it was concluded that a very high DNA
quality is a prerequisite for a successful CARE assay.
TABLE-US-00018 TABLE 16 CARE assay compatibility with upstream
sample manipulations. While all three kits are able to isolate
urine DNA with a quality suitable for qPCR (the CT values of the
internal copy number control are very similar), only Quick- DNATM
Urine Kit (Zymo Research) and supplier QI-isolated DNA is suitable
for CARE assay (digestion and qPCR steps). DNA isolated with
Supplier NG kit has an increased methylation background, especially
when UCBTM is added to the urine sample (+UCB; underlined cell
entries). Values of each marker are expressed in methylation
percentage after digestion, with the exception of POLR2A for which
mean CT vales are indicated. Experiment was performed using two
technical replicates. - UCB = No UCBTM added; + UCB = UCBTM added.
- UCB + UCB - UCB + UCB - UCB + UCB Markers POLR2A (CT) 27.66 27.14
27.21 27.09 27.35 26.91 GAPDH 0.06 0.05 0.05 n.a. 1.07 3.87 #27
0.26 0.14 0.39 0.40 1.77 2.62 #15 0.61 0.48 0.47 0.31 0.73 0.57 #10
n.a. 0.12 0.01 n.a. n.a. n.a.
[0134] Algorithm used for data analysis: As mentioned above, the
CFX96TM-Real Time System Machine will perform also the data
analysis based on the .DELTA..DELTA.CT algorithm. Briefly (an
example is represented in FIG. 19), for each sample the mean CT
values (mean Cycle Threshold; FIG. 04A) obtained for each marker
will be first normalized against the mean CT value of the copy
number control POLR2A (obtaining in that way the .DELTA.CT value;
FIG. 19B). Afterward, for each marker, the .DELTA.CT value of the
undigested sample (reference sample) will be subtracted to the
.DELTA.CT value of the digested sample, obtaining in that way the
.DELTA..DELTA.CT value (FIG. 19C). At this point relative
quantification values (RQs; in our case represents the relative
methylation level) will be easily calculated by applying the
formula "RQ=2{circumflex over ( )}-.DELTA..DELTA.CT" (FIG. 19D).
The operator will only have to adjust (when needed) the baseline
threshold for each marker (FIG. 20), exclude from the analysis the
eventual outlier technical replicates and make sure that the values
obtained from the digestion controls are between the acceptable
ranges. Methylation level in the digested sample will be
represented as percentage of the value obtained in the undigested
sample (100%; FIGS. 19E and F).
[0135] Alternatively, the degree of methylation for each marker can
be expressed in methylated DNA copies (resistant to digestion).
From this perspective, it should be considered that when 10 ng of
DNA are used (the DNA amount derived from approximately 1500 cells,
that correspond to 3000 DNA copies), the signal obtained from the
undigested sample is derived from the amplification of 3000 DNA
copies, while the signal detected in the digested sample (DNA
copies derived from cancer cells) can be calculated
accordingly.
[0136] Moreover, the signal obtained from the analysis of each
sample and marker can be corrected for the average background
signal level obtained from each single marker in a normal urine
cohort. This will further increase the precision of the analysis,
however a larger and properly selected normal urine cohort has to
be analyzed using CARE assay in order to establish the most
reliable correction value to apply for each single marker.
[0137] Thus, provided herein is a unique combination of
cancer-specific biomarkers and an optimized system design that
renders this assay a robust, accurate, sensitive, non-invasive and
very simple epigenetic-based method for the early detection of
bladder cancer from urine samples. Thanks to the newly designed
reaction mix and to the high degree of automation a complete
analysis using CARE assay can be performed in less than 5 hours
with minimal sample manipulation. Moreover, the unique combination
of endogenous and exogenous controls, the meticulous amplicon
design and the optimized reaction chemistry makes this system
incredibly robust, reproducible and accurate. CARE assay have a
sensitivity of 100%, a specificity of 85.71% and a very low
detection limit (less than 7 cancer cells can be detected in a
sample containing more than 1500 cells). In addition, the CARE
assay has the potential to detect chromosomal aneuploidies, tumor
type other than bladder cancer and perhaps to individuate persons
who have the highest risk to develop bladder cancer for the first
time.
Example 5--Analysis of Methylation Using MMSP Assay
[0138] In order to precisely detect a predisposition to, or the
incidence of bladder cancer with limited biopsy or liquid biopsy
samples from patients, a highly sensitive and specific multiplex
assay was developed to analyze the number of methylated DNA
molecular in a vast un-methylated background.
[0139] Bisulfite conversion: Genomic DNA extracted from patients'
biopsy samples, including urine, blood or tissues samples, was
bisulfite converted. As a standard methodology for DNA methylation
study, bisulfite conversion uses chemistry to specifically convert
unmethylated cytosine residues to uracil, allowing the use of
polymerase chain reaction to selectively amplify methylated genome
DNA from unmethylated DNA. The conversion efficiency of this assay
is more than 99.5% and the lowest input amount of gDNA for
bisulfite conversion is 50 pg DNA, thus the assay is feasible for
processing as low as 10 diploid cells (FIG. 21).
[0140] PCR design: The primers and probes to target the biomarker
genome regions were designed under guidelines which consider
melting temperatures (Tm) (identical Tm=58-60.degree. C. for
primers and Tm=68-70.degree. C. for probes), length of probes (less
than 30 bp) and CG ratio (30-80%) of primers and probes. In
addition, the number of CpG dinucleotides covered by primers and
probes was carefully controlled. For example, at least 7 CpG loci
should be covered by one group of primers and probes to achieve the
desired specificity of this assay. The position of CpG
dinucleotides on primer and probes wass also considered (e.g., at
least one C in the last 5 nucleotides the 3' end of primers)
[0141] To overcome the size variance of DNA fragments in biopsy
samples, the length of PCR amplicon was limited to 130 bp to150 bp
to avoid missing small fragment DNA in urine samples. (FIG. 21B;
see Yang et al., 2010). The sixteen best performing target regions
from the upstream statistical analysis were divided into 4 groups
as following.
[0142] Group A: DMRTA2, EVX2, Unk21, OTX1, CNC
[0143] Group B: SOX1, SEPT9, Unk05, Unk09, CNC
[0144] Group C: GALR1, Unk07, Unk19, TBX15, CNC
[0145] Group D: EEF1A2, TFAP2B, DCHS2, SOX17, CNC
[0146] Controls: The internal copy number control (CNC) of the
assay is collagen type II, alpha 1 gene (COL2A1), which is a single
copy gene. The entire amplicon of COL2A1 is devoid of any CpG
dinucleotide in the original genome sequence. Therefore, the amount
of input DNA can be measured for each reaction regardless of the
methylation status of template DNA (Widschwendter et al., Cancer
Research, 64: 3807-3813, 2004). Thus, for each group, the CNC
reaction was included to check for sample quantity and integrity in
the same reaction well.
[0147] M.SssI-modified gDNA (D5014-2) was used as a positive
control of 100% Methylation (MC). For each testing plate (e.g., 96
well plate), 10 ng of MC sample is tested two or three times. The
MC control allows for normalization of the intra-assay variations,
including reagent batches and PCR instruments. (FIG. 23). The
standard curve for this assay is based on 7-point serial dilutions
of known quantities of MssI-modified genomic DNA (12-3000 copy of
haploid genome). The amplification efficiency of each quantified
target in the tested group was reported for quality control. Most
importantly, for each analyte the raw copy number of each
quantified region was calculated against the primer set's own
standard curve. Therefore, the effect of amplification efficient
difference among the primer sets could be minimized using this
assay (FIGS. 23 and 24). A no template control (NTC) was also
included in each 96-well testing plate as well as a 0% methylated
control (FIG. 23). Thus, the employment of each of these controls,
significantly enhances the precision and reproducibility of the
assay.
[0148] The assay also showed good linearity for all of the 16
targeted genes based on repeated measurements of relative
methylation percentage values on DNA mixtures containing 100%, 50%,
5%, 1%, 0.5% and 0% of methylated (M.SssI-treated) normal urine DNA
(FIG. 25)
[0149] Methylation analysis: For each analyte, the raw copy number
of each targeted region and internal control was automatically
calculated according to the standard curve. If more than one PCR
reaction was performed for a sample, the mean value of duplicates
or triplicates was calculated for following analysis. The relative
methylation percentage calculation based on following equation.
RMP = ( Target mean value ) sample / ( COL 2 A 1 Mean value )
sample ( Target mean value ) P . C . / ( COL 2 A 1 Mean value ) P .
C . .times. 100 ##EQU00001##
[0150] If the standard curve was not performed, then an alternative
calculation for methylation percentage was based on delta CT as
follows:
RMP = 2 - ( Target mean CT value ) sample - ( Target mean CT value
) P . C . 2 - ( COL 2 A 1 mean CT value ) sample - ( COL 2 A 1 mean
CT value ) P . C . .times. 100 ##EQU00002##
[0151] Validation: 35 urine samples from normal healthy people were
collected and the genomic DNA was extracted using the Quick-DNA.TM.
Urine Kit (Zymo, USA). In addition, genomic DNA are extracted using
the ZR Urine DNA Isolation Kit and ZR Genomic DNA--Tissue Kits
(Zymo, USA) from 9 bladder cancer tumor tissues. The DNA
concentration was measured by Nanodrop and 250 ng of each sample
was subjected to bisulfite conversion using the EZ Direct kit
(Zymo, USA). The converted DNA was eluted into nuclease free water
and diluted into about 2 ng/ul for polymerase chain reaction.
[0152] 25 .mu.l of reaction master mix (1U AmpliGold Taq enzyme,
1.times. ampliGold Buffer, 400 uM dNTP, 5.5 mM MgCl.sub.2, 300 nM
Primers, 100 nM probes, PCR enhancer(optional)) of Group A, B, C, D
was premade separately and loaded to a 96 well plate (FIG. 23). 5
ul of sample DNA ware used per reaction. Each sample was tested in
duplicated. 5 ul of DNA standards were loaded into each designated
well. A technical duplication of standard curves was performed for
each 96-well plate. 5 ul of 100% methylated Positive Control (MC)
samples was also tested twice.
[0153] Real-time PCR was performed on the CFX96 Touch.TM. Real-Time
PCR Detection System. The raw copy number of each of the targeting
regions in each sample was automatically reported by the
instrument's program. The relative methylation percentages were
calculated as described. As shown in FIG. 26, all of 16 tested
biomarkers in the assay were highly methylated in bladder cancer
tissue samples and also showed extremely low background of DNA
methylation in healthy urine samples. Thus, these results confirmed
that these markers can be used in bladder cancer relevant
diagnostic and prognostic implications.
[0154] Validation #2: DNA was extracted from 15 bladder cancer
urine samples and bisulfite converted and stored in -20.degree. C.
for more than one year. These samples were diluted with 80 .mu.l of
H2O for this assay. 25 .mu.l of reaction master mix (1U AmpliGold
Taq enzyme, 1.times. ampliGold Buffer, 400 uM dNTP, 5.5 mM MgCl2,
300 nM Primers, 100 nM probes, 1.5 .mu.l DMSO) of Group B was
premade separately and loaded to a 96 well plate. 5 ul of sample
DNA was used per reaction. Each sample was tested in duplicate. A
technical duplication of standard curves was performed for each
96-well plate. 5 ul of 100% methylated Positive Control (MC) sample
was also tested twice.
[0155] Real-time PCR was performed on the CFX96 Touch.TM. Real-Time
PCR Detection System. The raw copy number of each targeting regions
in each sample was automatically reported by the instrument's
program. To ensure the statistical significance, only the samples
which showed at least 200 copy of COL2A1 were kept for the next
step of analysis. The relative methylation percentages were
calculated as described previously. As shown in FIG. 27A, the group
B markers clearly identified the bladder cancer samples.
[0156] In addition, 3 months after therapy, the group B markers
detected a dramatic drop in DNA methylation indicating the success
of the therapy. In the following check-up, the group B markers
confirmed that bladder cancer did not recur (FIG. 27A). For a
patient with bladder cancer recurrence, the group B markers showed
a modest increase of DNA methylation as early as 18 months after
the first clinical visit and a significant increase at 31.5 months.
This patient was diagnosed with bladder cancer recurrence at 39.5
months. Thus, these data strongly suggest that these markers can be
used to detect and predict bladder cancer recurrence (FIG.
27B).
[0157] Validation #3: A pre-clinical study was conducted to further
validate the assay in urine samples from healthy individuals and
bladder cancer patients. 33 urine samples from bladder cancer
patients were purchased from Geneticist-IIBGR (Glendale, Calif.).
The urine samples were collected before transurethral resection of
the bladder tumor (TURBT) and without any chemotherapy. 20 normal
urine samples were collected internally (consented) with diverse
age, gender and smoking history. All the urine samples were
preserved in Urine Conditioning Buffer (Zymo research, D3061-1)
upon collection. Both cellular and cell free DNA from urine samples
were extracted using Quick-DNA.TM. Urine Kit (Zymo, USA). DNA
concentration was measured by Nanodrop and 15Ong of each sample was
subjected to bisulfite conversion using EZ Direct kit (Zymo, USA).
The converted DNA was eluted into nuclease free water and diluted
into about 2 ng/ul for polymerase chain reaction.
[0158] Biomarkers in Group A, B, C, D were tested as in Assay
Validation #1, except that each sample was tested three times. The
RMP results for each sample were subjected to further statistical
analysis. The best biomarker, Unk05 in Group B, was selected based
on a special algorithm. Using Unk05 (cut-off value of 1.5%) allowed
for stratification of samples in a bladder cancer and bladder
cancer free group (Sensitivity 90.9%; Specificity 85%). Thus, the
assay has enhanced sensitivity and specificity as compared to
current urine-based tumor markers in bladder cancer (FIG. 28).
Example 6--Statistical Analysis for Preclinical Urine Cohort
[0159] Weeding trivial biomarkers using the Random Forest
algorithm: A statistical model was built to predict the probability
of the presence of bladder cancer by measuring DNA methylation of a
set of CpG sites in a urine sample. DNA methylation levels of
twelve bladder cancer specific CpG sites (Table 7) of urine samples
were obtained using the CARE assay and the multiplex
methylation-specific qPCR (MMSP) assay. Urine samples were randomly
split into a training set and a test set in the ratio of 75% and
25% respectively. A subgroup from the twelve biomarkers with the
lowest root mean squared error (RMSE) was identified by using the
recursive feature selection algorithm called random forest (Svetnik
2003; implemented in the R package caret).
[0160] Assignment of different weights to the selected biomarkers
by the Generalized Linear Model: A coefficient was assigned to each
of the selected CpG sites and an intercept was obtained using the
generalized linear regression model (Friedman, 2008; implemented in
the R package glmnet), regressing DNA methylation on cancer status
of the urine samples (absence or presence of bladder cancer). The
alpha parameter of 0.5 was used to balance having smaller variance
and less variables, and the lambda parameter which helps achieve
the smallest error was chosen using cross validation of the
training data.
[0161] Calculation of probability of presence of bladder cancer
using logistic regression: Logistic regression relates a binary
outcome variable (like presence or absence of cancer) to a group of
predictor variables (like a set of CpG sites) (Freedman 2009). The
probability of presence of bladder cancer is calculated by the
following formula: log(p/(1-p))=X0+CpG1*X1+ . . . +CpGn*Xn=X, where
p represents the probability of bladder cancer, n is the number of
selected CpG sites, X0 is the intercept, X1 is the coefficient of
the CpG site 1, CpG1 is the DNA methylation value of the CpG site
1, and so on. X is the sum of the intercept and every biomarker's
weighted DNA methylation value which is CpG*coefficient. Therefore,
p=exp(X)/(1+exp(X)).
[0162] Results of the CARE assay: DNA methylation values of the
twelve CpG sites were obtained for the 53 urine samples from 20
healthy individuals and 33 bladder cancer patients. The samples
were randomly split into a Training set (39 samples; 13 healthy and
26 cancer) and a Test set (14 samples; 7 healthy and 7 cancer). A
subgroup of three CpG sites of #05 (Unk 05), #36 (SOX17) and #09
(Unk09) was identified having the lowest RMSE error by the Random
Forest algorithm (FIG. 29). The glmnet function assigned them
coefficients of 39.0831, 104.1071, and 21.5371 respectively, along
with an intercept of -0.5092. Then the probability of bladder
cancer was calculated for all the samples. A sample was classified
as bladder cancer positive if its probability was equal or greater
than 50% and negative if less than 50%. The CARE assay achieved
sensitivity and specificity of 88.46% and 84.62% in the Training
set, and 100% and 85.71% in the Test set (Table 10).
TABLE-US-00019 TABLE 17 Statistical analysis of preclinical urine
cohort by Multilpex methylation-sensitive qPCR assay. Whole
Training Cohort Set Test Set Total Sample 53 39 14 Bladder Cancer
33 26 7 Control 20 13 7 True Positive (TP) 30 24 6 False Negative
(FN) 3 2 1 True Negative (TN) 17 11 6 False Positive (FP) 3 2 1
Sensitivity % 90.90 92.30 85.71 Specificity % 85.00 84.62 85.71
Positive Predictive Value % 90.90 92.30 85.71 Negative Predictive
Value % 85.00 84.62 85.71 Sensitivity % = [TP/(TP + FN)] * 100
Specificity % = [TN/(TN + FP)] * 100 Positive Predictive Value % =
[TP/(TP + FP)] * 100 Negative Predictive Value % = [TN/(TN + FN)] *
100
[0163] Results of the MMSP assay: DNA methylation was measured
using the MMSP assay for the same sample set as that of the CARE
assay. The Random Forest algorithm identified one single CpG site
R3N5 [See Table 12] with the lowest RMSE error (FIG. 30). The
glmnet could not assign a coefficient to a single variable.
However, a sample was classified as bladder cancer positive if its
methylation value was equal to or greater than 1.5% and negative if
less than 1.5%. The MMSP assay had its sensitivity and specificity
of 90.9% and 85.00% respectively (Table 17).
Example 7--Aneuploidy Detection
[0164] Another hallmark of cancer cells is the chromosomal
instability that frequently results in an increased chromosomal
ploidy or deletion. In particular tetraploidy of chromosomes 3, 7
and 17, and loss of the chromosomal region 9p21 are frequently
associated with urothelial carcinomas and accumulate during the
development of bladder cancer starting three years before diagnosis
(Bonberg et al., 2014). To note, markers #05, #07 and POLR2A (Set A
and B) are positioned on chromosomes 7, 17 and 17 respectively,
make them suitable for the detection of frequent chromosomal
aberrations associated with bladder cancer.
[0165] Although so far we did not deeply investigated this aspect,
an interesting aspect of CARE assay is that it can provide
information about the presence of chromosomal aneuploidy in the
sample analyzed. Indeed, the qPCR signal obtained from each marker
in the undigested sample is equal to the number of copy of DNA
added initially to the reaction (each marker, including the
endogenous controls, is a single-copy locus). Therefore, the
comparison between the signal obtained from a specific marker in an
undigested euploid sample (e.g. the undigested reaction performed
for E.D.C. control) and the signal detected for the same marker in
an undigested clinical sample (undigested sample control) might
point out the eventual presence of an extra or a missing copy of
the marker under analysis. For example, if we analyze a urine
sample collected from an individual affected by a bladder cancer in
which marker SOX1 (#27) is duplicated, we might in principle expect
that the signal detected from SOX1 (but not for the other markers)
in the undigested clinical sample is doubled compared to the signal
obtained from the undigested EDC control.
[0166] Despite this aspect might be interesting since it can
provide additional information for bladder cancer diagnostic
purposes, we have to consider that the possibility to identify
chromosomal aberration taking advantage of CARE assay is strongly
dependent from the amount of normal and cancer cells (and more
specifically aneuploid cells) present in the original sample.
Indeed, if the aberrant cancer cells represent only a small
fraction of the cell population present in the urine sample, the
detection of aneuploidy using CARE assay will not be possible. More
data regarding this aspect will be collected from future
experiments.
[0167] All of the 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 methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. 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.
REFERENCES
[0168] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0169] Babjuk et al., EAU guidelines on non-muscle-invasive
urothelial carcinoma of the bladder, the 2011 update. Eur Urol
2011; 59: 997-1008. [0170] Bernstein et al., 2007 [0171]
Giovannucci and Ogino, 2005 [0172] Lintula and Hotakainen,
Developing biomarkers for improved diagnosis and treatment outcome
monitoring of bladder cancer. Expert Opin Biol Ther 2010;
10:1169-80. [0173] Millan-Rodriguez et al., Primary superficial
bladder cancer risk groups according to progression, mortality and
recurrence. J Urol 2000; 164:680-4. [0174] Morgan and Clark,
Bladder cancer. Curr Opin Oncol 2010; 22: 242-9. [0175] Parker and
Spiess, Current and emerging bladder cancer urinary biomarkers.
Scientific World Journal 2011; 11:1103-12. [0176] Reinert T,
Methylation markers for urine-based detection of bladder cancer:
The next generation of urinary markers for diagnosis and
surveillance of bladder cancer. Adv Urol 2012; 2012:503271. [0177]
Shelley et al., Intravesical therapy for superficial bladder
cancer: A systematic review of randomised trials and meta-analyses.
Cancer Treat Rev 2010; 36:195-205. [0178] Siegel et al., Cancer
statistics, 2011: The impact of eliminating socioeconomic and
racial disparities on premature cancer deaths. CA Cancer J Clin
2011; 61: 212-36. [0179] Sobin et al., TNM classification of
malignant tumours. 7th ed. Wiley-Blackwell; 2009. [0180]
Widschwendter et al., Cancer Research, 64: 3807-3813, 2004
Sequence CWU 1
1
90116DNAArtificial sequenceSynthetic oligonucleotides 1tcgcggaagt
agcggc 16220DNAArtificial sequenceSynthetic oligonucleotides
2cacctcctcc cgcataaaaa 20329DNAArtificial sequenceSynthetic
oligonucleotides 3aacgcctcga acacgtccaa ctataaacg
29423DNAArtificial sequenceSynthetic oligonucleotides 4ggagggatta
ttattcggtt cgt 23518DNAArtificial sequenceSynthetic
oligonucleotides 5ccatacccgc gtccaaac 18627DNAArtificial
sequenceSynthetic oligonucleotides 6atacgccgaa tacaacaaca cgtccga
27727DNAArtificial sequenceSynthetic oligonucleotides 7tcgtacgtaa
gtcgcgtata gtattgt 27818DNAArtificial sequenceSynthetic
oligonucleotides 8aatccgaact aaccgccg 18917DNAArtificial
sequenceSynthetic oligonucleotides 9cgccgcccaa aacgcga
171019DNAArtificial sequenceSynthetic oligonucleotides 10aggtaagagt
ttcggcggc 191120DNAArtificial sequenceSynthetic oligonucleotides
11cctcaaccct cgaacccaac 201224DNAArtificial sequenceSynthetic
oligonucleotides 12tcgcgacaaa cacgcttccg ccta 241318DNAArtificial
sequenceSynthetic oligonucleotides 13tcggcgaaga tttggagc
181419DNAArtificial sequenceSynthetic oligonucleotides 14cgcgaccacg
aaacctaaa 191525DNAArtificial sequenceSynthetic oligonucleotides
15ttttatttgc gcggttgtag tcggc 251620DNAArtificial sequenceSynthetic
oligonucleotides 16cgcgtaaaag gtaggatcgc 201721DNAArtificial
sequenceSynthetic oligonucleotides 17aactcgaaac tctacccccg a
211830DNAArtificial sequenceSynthetic oligonucleotides 18aacgccgact
caacaaaaaa ttatttcgaa 301923DNAArtificial sequenceSynthetic
oligonucleotides 19agcgatagta gcgtttgtgg ttt 232017DNAArtificial
sequenceSynthetic oligonucleotides 20gcgaaccccc aaatacg
172129DNAArtificial sequenceSynthetic oligonucleotides 21tcgcaaccat
aacgtaatat cgaccctca 292226DNAArtificial sequenceSynthetic
oligonucleotides 22gtttttgagt ttataggtcg ggattt 262315DNAArtificial
sequenceSynthetic oligonucleotides 23ctatcaccgc cgccg
152430DNAArtificial sequenceSynthetic oligonucleotides 24aaattaaacg
acaacgcacg cgactaacaa 302520DNAArtificial sequenceSynthetic
oligonucleotides 25agcggtgacg gtagtggttt 202623DNAArtificial
sequenceSynthetic oligonucleotides 26ctacacccta atacaaccgt ccg
232728DNAArtificial sequenceSynthetic oligonucleotides 27aacccgttct
tccaattacg ctaccgaa 282820DNAArtificial sequenceSynthetic
oligonucleotides 28gcggacgtta gttagtcggc 202919DNAArtificial
sequenceSynthetic oligonucleotides 29cccgaatccc tatccgaaa
193021DNAArtificial sequenceSynthetic oligonucleotides 30cgcccactcc
gacaccaacg t 213119DNAArtificial sequenceSynthetic oligonucleotides
31cgcgtttaat tggttgcga 193219DNAArtificial sequenceSynthetic
oligonucleotides 32tatctattcg tcccgcccg 193323DNAArtificial
sequenceSynthetic oligonucleotides 33aaatccgcct cctcgacccg aaa
233426DNAArtificial sequenceSynthetic oligonucleotides 34ggttggtata
tcgaggtttc gtagtt 263515DNAArtificial sequenceSynthetic
oligonucleotides 35cgcgtctacc cgccc 153626DNAArtificial
sequenceSynthetic oligonucleotides 36aaacgacgaa cgaatcaaac gcgact
263729DNAArtificial sequenceSynthetic oligonucleotides 37ggtatttggg
attagtatat gtttagcgt 293820DNAArtificial sequenceSynthetic
oligonucleotides 38cctcaacgac ctccaactcg 203929DNAArtificial
sequenceSynthetic oligonucleotides 39actacaactt ctaacaaaac
gacgcgccg 294023DNAArtificial sequenceSynthetic oligonucleotides
40gattcgcgtt agagtcggag tta 234121DNAArtificial sequenceSynthetic
oligonucleotides 41cgtcgaactc aaacctcgaa a 214217DNAArtificial
sequenceSynthetic oligonucleotides 42cgtcgcgttt tcgtcgt
174320DNAArtificial sequenceSynthetic oligonucleotides 43gcgtatacgg
ttttggggtc 204418DNAArtificial sequenceSynthetic oligonucleotides
44caaacttccg cgcccaac 184527DNAArtificial sequenceSynthetic
oligonucleotides 45ctcgccacgc tcaatacccg ttttacc
274620DNAArtificial sequenceSynthetic oligonucleotides 46ggacgtggga
ttcggattac 204731DNAArtificial sequenceSynthetic oligonucleotides
47ttttctacac aaatataacc aataaaacga c 314822DNAArtificial
sequenceSynthetic oligonucleotides 48cgaaccgatc ccgcgtcgtt aa
224921DNAArtificial sequenceSynthetic oligonucleotides 49acctacctct
ccaagctatt c 215021DNAArtificial sequenceSynthetic oligonucleotides
50ggtgtaattg ggactggttg g 215123DNAArtificial sequenceSynthetic
oligonucleotides 51tactcaccca ccagcccgaa cta 235220DNAArtificial
sequenceSynthetic oligonucleotides 52cgtagctcag gcctcaagac
205319DNAArtificial sequenceSynthetic oligonucleotides 53gaggagcaga
gagcgaagc 195421DNAArtificial sequenceSynthetic oligonucleotides
54ctcagccagt cccagcccaa g 215520DNAArtificial sequenceSynthetic
oligonucleotides 55ccacgactgc acctgtttgc 205620DNAArtificial
sequenceSynthetic oligonucleotides 56ggcagaaaca cacgcactcg
205723DNAArtificial sequenceSynthetic oligonucleotides 57tcggcctctt
tggcaagtgg ttt 235820DNAArtificial sequenceSynthetic
oligonucleotides 58acccaccctc tctcagaagg 205921DNAArtificial
sequenceSynthetic oligonucleotides 59agtttaggag tctgagcttc c
216021DNAArtificial sequenceSynthetic oligonucleotides 60cgcaaagacg
gtgccaccag g 216119DNAArtificial sequenceSynthetic oligonucleotides
61gcagtgccgt agagcagct 196222DNAArtificial sequenceSynthetic
oligonucleotides 62ccctcaaggg ccacaaacgc ta 226324DNAArtificial
sequenceSynthetic oligonucleotides 63cacaggcagt ccttccagcg acag
246421DNAArtificial sequenceSynthetic oligonucleotides 64cactcacgtc
ttcgtagtca g 216521DNAArtificial sequenceSynthetic oligonucleotides
65ttggagtgtg acggtaaaag c 216621DNAArtificial sequenceSynthetic
oligonucleotides 66cgtacagcac tgcagggtcc g 216719DNAArtificial
sequenceSynthetic oligonucleotides 67gggaggaccc ctcgttagc
196819DNAArtificial sequenceSynthetic oligonucleotides 68ctcagcgtcc
gaccccact 196920DNAArtificial sequenceSynthetic oligonucleotides
69tgggctcgag ggctgagggc 207019DNAArtificial sequenceSynthetic
oligonucleotides 70aggcttccga ggcctgagc 197120DNAArtificial
sequenceSynthetic oligonucleotides 71agcgactctc cttcctgacg
207223DNAArtificial sequenceSynthetic oligonucleotides 72cgtaccctgg
caaacaaacg acc 237319DNAArtificial sequenceSynthetic
oligonucleotides 73tgggcgtggg cctaacgac 197419DNAArtificial
sequenceSynthetic oligonucleotides 74cccgtgttct ggcctgtcg
197522DNAArtificial sequenceSynthetic oligonucleotides 75tgggtacgct
gtagaccaga cc 227618DNAArtificial sequenceSynthetic
oligonucleotides 76cgcacgcgac agacctcg 187720DNAArtificial
sequenceSynthetic oligonucleotides 77tggatgagaa cgacaacccg
207823DNAArtificial sequenceSynthetic oligonucleotides 78cggtactcgt
cctgctcaaa gac 237918DNAArtificial sequenceSynthetic
oligonucleotides 79accgaggccc ctacctgg 188020DNAArtificial
sequenceSynthetic oligonucleotides 80gttagaaacg caggccaggc
208122DNAArtificial sequenceSynthetic oligonucleotides 81cactgaacga
ccccttctcc ag 228219DNAArtificial sequenceSynthetic
oligonucleotides 82gccgacttgg tgaccttgc 198319DNAArtificial
sequenceSynthetic oligonucleotides 83ggccagagcc ctggggttg
198423DNAArtificial sequenceSynthetic oligonucleotides 84ccacgttctt
gatgacgcct acg 238521DNAArtificial sequenceSynthetic
oligonucleotides 85gacgtggtgt tcgcttttgg c 218619DNAArtificial
sequenceSynthetic oligonucleotides 86cgcacgtgca gctcgtagg
198723DNAArtificial sequenceSynthetic oligonucleotides 87cccgtggatt
accagagtca gga 238819DNAArtificial sequenceSynthetic
oligonucleotides 88acacgccgaa cacacgtgc 198919DNAArtificial
sequenceSynthetic oligonucleotides 89ccgtccgtgt gtcctgtgc
199022DNAArtificial sequenceSynthetic oligonucleotides 90cctaattggc
tgcgaacggt cc 22
* * * * *