U.S. patent application number 16/469946 was filed with the patent office on 2019-10-24 for epigenetic markers and related methods and means for the detection and management of ovarian cancer.
The applicant listed for this patent is EUROFINS GENOMICS EUROPE SEQUENCING GMBH, GENEDATA AG, UCL BUSINESS PLC. Invention is credited to Johannes EICHNER, Iona EVANS, Allison JONES, Harri LEMPPIAINEN, Tobias PAPROTKA, Tamas RUJAN, Benjamin WAHL, Martin WIDSCHWENDTER, Timo WITTENBERGER.
Application Number | 20190323090 16/469946 |
Document ID | / |
Family ID | 57609702 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190323090 |
Kind Code |
A1 |
WIDSCHWENDTER; Martin ; et
al. |
October 24, 2019 |
EPIGENETIC MARKERS AND RELATED METHODS AND MEANS FOR THE DETECTION
AND MANAGEMENT OF OVARIAN CANCER
Abstract
The present invention relates to methods of determining the
presence or absence of an ovarian cancer in a woman, as well as to
related methods to determine the response to therapy against
ovarian cancer in a woman. Such methods are based on the
detection--from cell-free DNA of said woman--of one or more
methylated (or un-methylated) CpGs being associated with
differentially methylated regions (DMRs) of the present
invention;such as methylation (or un-methylation) at one or more or
all of certain CpGs being associated with such DMRs. Accordingly,
such methods have diagnostic, prognostic and/or predictive utility
for detectingor managing ovarian cancer in women. The present
invention further relates to nucleic acids comprising certain
sequences that may be detected during the method, or nucleic acids
(such as probes and/or primers) that are usefulto detect such
sequences, as wells as compositions, kits, computer program
products and other aspects that are useful for or related to the
practice or application of such methods.
Inventors: |
WIDSCHWENDTER; Martin;
(Wildermieming, AT) ; JONES; Allison; (London,
GB) ; EVANS; Iona; (London, GB) ; LEMPPIAINEN;
Harri; (Binningen, CH) ; EICHNER; Johannes;
(Lorrach, DE) ; RUJAN; Tamas; (Bottmingen, CH)
; WITTENBERGER; Timo; (Konstanz, DE) ; PAPROTKA;
Tobias; (Konstanz, DE) ; WAHL; Benjamin;
(Laupheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EUROFINS GENOMICS EUROPE SEQUENCING GMBH
GENEDATA AG
UCL BUSINESS PLC |
Konstanz
Basel
London |
|
DE
CH
GB |
|
|
Family ID: |
57609702 |
Appl. No.: |
16/469946 |
Filed: |
December 15, 2017 |
PCT Filed: |
December 15, 2017 |
PCT NO: |
PCT/EP2017/083159 |
371 Date: |
June 14, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/118 20130101; G16H 20/00 20180101; C12Q 2600/154
20130101; G16H 50/20 20180101; G16B 40/10 20190201; G16B 20/00
20190201; C12Q 2600/112 20130101; C12Q 2600/106 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G16B 20/00 20060101 G16B020/00; G16H 20/00 20060101
G16H020/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2016 |
EP |
16204822.7 |
Claims
1. A method of determining the presence or absence of, or response
to therapy against, an ovarian cancer in a woman, said method
comprising the steps: providing a biological sample from said
woman, said sample comprising cell-free DNA of said woman; and
determining, in at least one molecule of said cell-free DNA, the
methylation status at one or more CpGs located within one or more
of the nucleotide sequences independently selected from the group
consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 and 31, or a nucleotide sequence present within about 2,000 bp
5' or 3' thereof, or an allelic variant and/or complementary
sequence of said nucleotide sequence(s), wherein, the presence in
at least one of said cell-free DNA molecules of one or more: (i)
methylated CpGs associated with one or more of said nucleotide
sequences independently selected from: SEQ ID NOs: 1, 2, 3, 4, 10,
12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29 and 30;
and/or (ii) un-methylated CpGs associated with one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 5, 6,
7, 8, 9, 11, 13, 16, 17, 22 and 31, indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman.
2. The method of claim 1, wherein said biological sample is liquid
biological sample selected from the group consisting of: a blood
sample, a plasma sample and a serum sample.
3. The method of claim 1 or 2, wherein said CpGs for a given
nucleotide sequence are identifiable by a genome position for the
cytosine (C) thereof, independently selected from the list of
genome positions corresponding to said nucleotide sequence set
forth in TABLE 1C.
4. The method of any one or claims 1 to 3, wherein the methylation
status is determined at a number being two, three, four, five, six,
seven, eight, nine, ten, about 12, about 15, about 20, about 25 or
more of said CpGs located within said nucleotide sequence; wherein,
the presence in at least one of said cell-free DNA molecules of at
least one, such as up to said number, of: (i) methylated CpGs
associated with one or more of said nucleotide sequences
independently selected from: SEQ ID NOs: 1, 2, 3, 4, 10, 12, 14,
15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29 and 30; and/or (ii)
un-methylated CpGs associated with one or more of said nucleotide
sequences independently selected from: SEQ ID NOs: 5, 6, 7, 8, 9,
11, 13, 16, 17, 22 and 31, indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
5. The method of any one of claims 1 to 4, wherein the presence in
at least one of said cell-free DNA molecules of one or more pattern
of methylation and/or un-methylation as set forth in TABLE 2B for
the respective nucleotide sequence(s), indicates the presence of,
or a reduced response to therapy against, an ovarian cancer in said
woman.
6. The method of any one of claims 1 to 5, wherein the methylation
status at one or more CpGs located within a number of two, three,
four or more of said nucleotide sequences is determined; wherein,
the presence in at least one of said cell-free DNA molecules of one
or more: (i) methylated CpGs associated with one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 1, 2,
3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29
and 30; and/or (ii) un-methylated CpGs associated with one or more
of said nucleotide sequences independently selected from: SEQ ID
NOs: 5, 6, 7, 8, 9, 11, 13, 16, 17, 22 and 31, indicates the
presence of, or a reduced response to therapy, against an ovarian
cancer in said woman.
7. The method of any one of claims 1 to 6, wherein said nucleotide
sequence(s) is/are associated with DMR(s) #141 and/or #204 and/or
#228 (eg, SEQ ID NOs: 1, 2 and/or 3), or an allelic variant and/or
complementary sequence of said nucleotide sequence(s).
8. The method of claim 7, wherein the methylation status is
determined at one or more of said CpGs located within each of said
three nucleotide sequences; wherein, the presence in at least one
of said cell-free DNA molecules of one or more methylated CpGs,
and/or of one or more pattern of methylation and/or un-methylation
as set forth in TABLE 2B for the respective nucleotide sequence(s),
located within any one of said nucleotide sequences indicates the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman.
9. The method of claim 7 or 8, wherein the methylation status is
determined at a number of between about 5 and about 18 of said CpGs
located within said nucleotide sequence(s); wherein, the presence
in at least one of said cell-free DNA molecules of at least said
number of methylated CpGs, and/or of one or more pattern of
methylation and/or un-methylation as set forth in TABLE 2B for the
respective nucleotide sequence(s), located within any one of said
nucleotide sequences indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
10. The method of any one of claims 7 to 9, wherein the methylation
status is determined at about 7 CpGs located within nucleotide
sequence SEQ ID NO 1 and/or at about 16 to 18 CpGs located within
nucleotide sequence SEQ ID NO 2 and/or at about 7 to 9 CpGs located
within nucleotide sequence SEQ ID NO 3.
11. The method of any one of claims 7 to 10, wherein the
methylation status is determined at about 7 CpGs located within
nucleotide sequence SEQ ID NO 1 and at about 16 to 18 CpGs located
within nucleotide sequence SEQ ID NO 2 and about 7 to 9 CpGs
located within nucleotide sequence SEQ ID NO 3; wherein, the
presence in at least one of said cell-free DNA molecules of at
least said number of methylated said CpGs, and/or of one or more
pattern of methylation and/or un-methylation as set forth in TABLE
2B for the respective nucleotide sequence(s), located within any
one of said nucleotide sequences indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman.
12. The method of any one of claims 1 to 11, comprising the step of
isolating said cell-free DNA from said biological sample.
13. The method of any one of claims 1 to 12, comprising the step of
treating said cell-free DNA with an agent that differentially
modifies said cell-free DNA based on the methylation status of one
or more CpGs located within; preferably a methylation sensitive
restriction enzyme and/or bisulphite.
14. The method of claim 13, wherein said agent is bisulphite and
said determining step comprises the detection of at least one
bisulphite-converted un-methylated cytosine within one or more of
the nucleotide sequences independently selected from those set
forth in TABLE 2A (eg, independently selected the group consisting
of: SEQ ID NOs 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61
and 62), wherein one or more of the bases identified by "Y" therein
is a U or T and, preferably, where one or more of the bases
identified by "Y" in a CpG therein is a C, or an allelic variant
and/or complementary sequence of said nucleotide sequence(s).
15. The method of claim 13 or 14, wherein said agent is bisulphite
and said determining step comprises the detection of at least one
bisulphite-converted un-methylated cytosine within a nucleotide
sequence having a length of at least 50 bp comprised in SEQ ID NO
32 and/or SEQ ID NO 33 and/or SEQ ID NO 34, wherein one or more of
the bases identified by "Y" therein is a U or T and, preferably,
where one or more of the bases identified by "Y" in a CpG therein
is a C, or an allelic variant and/or complementary sequence of said
nucleotide sequence(s).
16. The method of any one of claims 1 to 15, comprising the step of
amplifying one or more regions of said cell-free DNA to produce DNA
prior to or as part of said determining step, and; preferably after
said treating step.
17. The method of claim 16, wherein said amplified region(s)
comprises at least one of said nucleotide sequences.
18. The method of claim 17, wherein said amplification comprises
PCR using the primer-pair(s) for the respective nucleotide
sequence(s) as independently selected from the group of
primer-pairs set forth in each row of TABLE 3.
19. The method of any one of claims 1 to 18, wherein the
methylation status of said CpGs is determined by a technology
selected from the group consisting of: methylation specific
PCR/MethylLight, Epityper, nucleic acid chip-hybridisation, nucleic
acid mass-spectrometry, Methylated DNA immunoprecipitation (MeDIP),
Raindance and nucleic acid sequencing, preferably, (single) strand
sequencing, nanopore sequencing, bisulphite sequencing, such as
targeted bisulphite sequencing; preferably wherein said
determination step is conducted as a pool when in respect of two,
three, four or more of said nucleotide sequences.
20. The method of any one of claims 1 to 19, wherein the
methylation status of said CpG(s) is determined in multiple
molecules of said cell-free DNA and/or amplified DNA representing
each of said nucleotide sequences.
21. The method of claim 20, wherein the presence in at least a
plurality of said cell-free DNA molecules of one or more methylated
and/or un-methylated CpGs (as applicable), and/or the presence in
at least a plurality of said cell-free DNA molecules of one or more
pattern of methylation and/or un-methylation as set forth in TABLE
2B for the respective nucleotide sequence(s), located within one or
more of said nucleotide sequences, indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman.
22. The method of claim 20 or 21, wherein said plurality of
cell-free DNA molecules with one or more of said methylated and/or
un-methylated CpGs (as applicable), and/or the presence in at least
a plurality of said cell-free DNA molecules of one or more pattern
of methylation and/or un-methylation as set forth in TABLE 2B for
the respective nucleotide sequence(s), located is at least 2, 3, 4,
5, 6, 7, 18, 9 or 10, or at least about 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 70, 80, 90, 100, 125, 150, 175 or 200, or a greater
number such as greater than about 500, 1,000, 5,000, 7,500, 1,000,
2,500, 5,000 or greater than 5,000 molecules.
23. The method of any one of claims 20 to 22, wherein the
methylation status of said CpG(s) is determined in a number of
molecules of said cell-free DNA and/or amplified DNA representing
each of said nucleotide sequences selected from the group
consisting of at least about: 1,000, 5,000, 10,000, 50,000,
100,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000,
2,500,000, 3,000,000, 3,500,000, 4,000,000 and 5,000,000 molecules,
or more than 5,000,000 molecules.
24. The method of any one of claims 20 to 23, wherein a fraction or
ratio of, or an absolute number of, cell-free DNA molecules in said
sample having said methylated and/or un-methylated CpG(s) (as
applicable) located within said nucleotide sequence(s), and/or
having said pattern of methylation and/or un-methylation as set
forth in TABLE 2B for the respective nucleotide sequence(s), is
estimated.
25. The method of claim 24, further comprising a step of comparing
said fraction or ratio with a standard or cut-off value; wherein a
fraction or ratio greater than the standard or cut-off value
indicates the presence of or a reduced response to therapy against,
an ovarian cancer in said woman.
26. The method of claim 25, wherein said standard or cut-off value
is about 0.0008 for said methylated/un-methylated CpG or said
pattern of methylation and/or un-methylation associated with
nucleotide sequence SEQ ID NO 1 and/or about 0.00003 for said
methylated/un-methylated CpG or said pattern of methylation and/or
un-methylation associated with nucleotide sequence SEQ ID NO 2
and/or about 0.00001 for said methylated/un-methylated CpG or said
pattern of methylation and/or un-methylation associated with
nucleotide sequence SEQ ID NO 3.
27. The method of claim 25 or 26, wherein a fraction or ratio of
cell-free DNA molecules with said methylated and/or un-methylated
CpG(s) (as applicable) located within each of said nucleotide
sequence(s), and/or with said pattern of methylation and/or
un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), is estimated and compared to a respective
standard or cut-off value; wherein any one of such fraction or
ratios being greater than its respective standard or cut-off value
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman.
28. The method of any one of claims 25 to 27, wherein said standard
or cut-off value(s) is/are modified for a given sample based on:
the amount or concentration of total cell-free DNA present in said
sample; and/or a baseline value of said fraction or ratio
previously determined for said woman; and/or a value of said
fraction or ratio determined from multiple samples from a
population of women representative of said woman; and/or the
specificity and/or sensitivity desired for said method of
determination.
29. The method of claim 28, wherein said standard or cut-off value
is/are reduced for a given sample that has an amount or
concentration of total cell-free DNA present in said sample that is
greater than a standard or cut-off value.
30. The method of any one of claims 1 to 29, which is practiced on
multiple samples; wherein each sample is collected from the same
woman at different time points.
31. The method of claim 30, wherein said multiple samples are
collected from said woman with an interval between them selected
from the group consisting of about: 2 days, 3 days, 4 days, 5 days,
7 days, 10, days, 14 days, 21 days, 24 days, 3weeks, 4 weeks, 5
weeks, 6 weeks, 6, weeks, 8 weeks, 3 months, 4 months, 5 months, 6
months, 8 months, 12 months, 18 months, 2 years, 3 years and 5
years.
32. The method of claim 30 or 31, wherein in comparison to a
previous sample, the presence of, or an increase in the absolute
number of, or an increase in the fraction or ratio of, cell-free
DNA molecules in said sample having said methylated and/or
un-methylated CpG(s) (as applicable) located within said nucleotide
sequence(s), and/or having said pattern of methylation and/or
un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
33. The method of any one of claims 1 to 32, comprising the step of
determining (in-vitro), from a blood sample from said woman, the
amount present therein of one or more proteins independently
selected from the group consisting of: CA-125, HE4, transthyretin,
apolipoprotein Al, beta-2-microglobin and transferrin; wherein,
either or both of: the presence in at least one of said cell-free
DNA molecules of one or more methylated and/or un-methylated CpGs
(as applicable) located within one or more of said nucleotide
sequences, and/or the presence in at least one of said cell-free
DNA molecules of one or more pattern of methylation and/or
un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s); an amount of said protein(s) present in
said blood sample is greater than a standard or cut-off value for
such amount or protein, indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
34. The method of claim 33, wherein said protein is determined by a
ROCA, a ROMA and/or an OVA1 diagnostic test.
35. The method of any one of claims 1 to 34, wherein said ovarian
cancer is an invasive ovarian cancer, such as an invasive
epithelial ovarian cancer; in particular one selected from the
group consisting of: high grade serious (HGS), endometroid,
cell-cell and mucinous ovarian cancers; and/or is a peritoneal
cancer or a Fallopian tube cancer.
36. The method of any one of claims 1 to 35, for distinguishing the
presence of ovarian cancer from the presence of a benign pelvic
mass.
37. The method of any one of claims 1 to 36, for determining the
response of a woman suffering from ovarian cancer to a therapy
comprising chemotherapeutic agent(s) against said ovarian
cancer.
38. The method of claim 37, wherein the risk of death of said woman
is predicted.
39. The method of claim 37 or 38, practiced on said woman after
one, two, three, four and/or five cycles of said
(chemo)therapy.
40. The method of any one of claims 37 to 39, wherein said sample
is obtained from said woman within a period after completion of
said cycle or (chemo)therapy that is selected from the group
consisting of about: 2 hours, 4 hours, 6 hours, 12 hours, 18 hours,
24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6, days, 7
days, 8 days, 10 days, 12 days, 14 days, 16, days, 18 days, 21
days, 24 days, 4 weeks, 5 weeks, 6 weeks, and 8 weeks.
41. The method of any one of claims 37 to 40, wherein said
(chemo)therapy includes one or more chemotherapeutic agent(s)
independently selected from the group consisting of: a
platinum-based antineoplastic and a taxane.
42. The method of claim 41, wherein at least one of said
chemotherapeutic agents is carboplatin, cisplatin, paclitaxel or
docetaxel
43. The method of any one of claims 37 to 42, wherein said therapy
is a neoadjuvant (chemo)therapy.
44. The method of any one of claims 37 to 43, wherein if said woman
is determined to respond to said (chemo)therapy, then said woman is
designated as being eligible for tumour de-baulking surgery.
45. The method of any one of claims 37 to 43, wherein if said woman
is determined to not respond to said (chemo)therapy, then said
woman is designated as eligible for therapy with one or more
second-line chemotherapeutic agent(s) against said ovarian
cancer.
46. The method of claim 45, wherein said second-line (chemo)therapy
includes one or more chemotherapeutic agent(s) independently
selected from the list consisting of: paclitaxel, carboplatin,
cisplatin, liposomal doxorubicin, gemcitabine, trabectedin,
etoposide, cyclophosphamide, an angiogenesis inhibitor and a PARP
inhibitor.
47. A chemotherapeutic agent, such as one selected from the list
consisting of: carboplatin, paclitaxel, docetaxel, cisplatin,
liposomal doxorubicin, gemcitabine, trabectedin, etoposide,
cyclophosphamide an angiogenesis inhibitor and a PARP inhibitor,
for use in a method of therapy of ovarian cancer in a woman,
wherein said chemotherapeutic agent is administered to a woman
within about 3 months of said woman having been determined, using a
method of any one of claims 37 to 43, to not respond to a therapy
against ovarian cancer.
48. A nucleic acid comprising at least 10 (preferable at least
about 15, such as at least 50, for any SEQ ID other than SEQ ID
NO:58) contiguous bases comprised in a sequence selected from the
group consisting of: SEQ ID NOs 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61 and 62, wherein said nucleic acid sequence includes
one or more of the bases identified by "Y" therein is a U or T and,
preferably, where one or more of the bases identified by "Y"
therein is a C, or an allelic variant and/or complementary sequence
of said nucleotide sequence.
49. The nucleic acid sequence of claim 48, comprising at least 50
contiguous bases comprised in a sequence of SEQ ID NO 32, SEQ ID NO
33 or SEQ ID NO 34, wherein said nucleic acid sequence includes one
or more of the bases identified by "Y" therein is a U or T and,
preferably, where one or more of the bases identified by "Y"
therein is a C, or an allelic variant and/or complementary sequence
of said nucleotide sequence.
50. The nucleic acid sequence of claim 48 or 49, which is comprised
in a sequence as set forth in TABLE 2B (eg, a sequence selected
from the group consisting of: SEQ ID NOs 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92 and 93.
51. A nucleic acid probe complementary to a nucleic acid sequence
recited in any one of claims 48 to 50, preferably for detection of
said nucleic acid.
52. The nucleic acid probe of claim 51, that differentially binds
to said nucleic acid sequence depending on the methylation status
of one or more CpGs within said nucleic acid sequence.
53. The nucleic acid probe of claim 51 or 52, comprising a label,
preferably a label being a detectable fluorescent moiety.
54. A nucleic acid primer pair for amplifying a nucleic acid
sequence consisting of at least 10 (preferable at least about 15,
such as at least 50, for any SEQ ID other than SEQ ID NO:89)
contiguous bases comprised in a sequence) selected from the group
consisting of: SEQ ID NOs: SEQ ID NOs 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92 and 93, or a nucleotide sequence present
within about 2,000 bp 5' or 3' thereof, or an allelic variant
and/or complementary sequence said nucleotide sequence(s),
preferably wherein at least one primer of said pair includes a
sequence corresponding to at least one bisulphite-converted CpG
present in said nucleotide sequence(s).
55. The primer pair of claim 54, selected from the group of
primer-pairs set forth in each row of TABLE 3.
56. A plurality of nucleic acids comprising at least two or three
nucleic acid sequences of any one of claims 48 to 50 and/or at
least two or three nucleic acid probes of any one of claims 51 to
53 and/or at least two or three primer pairs of claim 54 or 55.
57. The plurality of nucleic acids of claim 56, as an admixture or
array of said nucleic acid sequences and/or nucleic acid probes
and/or primer pairs.
58. A kit, preferably for determining the presence or absence of,
or response to therapy against, an ovarian cancer in a woman, said
kit comprising: one or more nucleic acid sequences of any one of
claims 48 to 50 and/or nucleic acid probes of any one of claims 51
to 53 and/or primer pairs of claim 54 or 55 and/or the plurality of
nucleic acids of claim 56 or 57; and optionally, said kit further
comprising: (i) a printed manual or computer readable memory
comprising instructions to use said nucleic acid sequence(s),
nucleic acid probe(s), primer pair(s) and/or plurality of nucleic
acids to practice a method of any one of claims 1 to 46 and/or to
produce or detect the nucleic acid sequence(s) of any one of any
one of claims 48 to 50; and/or (ii) one or more other claim,
component or reagent useful for the practice of a method of any one
of claims 1 to 46 and and/or the production or detection of the
nucleic acid sequence(s) of any one of claims 48 to 50, including
any such item, component or reagent disclosed herein useful for
such practice, production or detection.
59. The kit of claim 58, further comprising one or more of the
following components. means to collect and/or store a biological
sample, such as blood, to be taken from said woman, preferably
wherein said means is a blood collection tube; and/or means to
extract DNA, preferably cell-free DNA, from the sample to be taken
from said woman, preferably wherein said means is a cell-free DNA
extraction kit; and/or an agent to differentially modify DNA based
on the methylation status of one or more CpGs located within said
DNA, preferably wherein said agent is bisulphite; and/or one or
more reagents to detect a nucleic acid sequence, preferably for
detecting the sequence of a bisulphite-converted nucleotide
sequence; and/or a printed manual or computer readable memory
comprising instructions to identify, obtain and/or use one or more
of said means, agent or reagent(s) in the context of a method of
any one of claims 1 to 46.
60. A computer program product comprising: a computer readable
medium encoded with a plurality of instructions for controlling a
computing system to perform and/or manage an operation for
determining the presence or absence of, or response to therapy
against, an ovarian cancer in a woman, from a biological sample
from said woman, said sample comprising cell-free DNA of said
woman, and determining, in at least one molecule of said cell-free
DNA, the methylation status at one or more CpGs located within one
or more nucleotide sequences in accordance with a method as set
forth in any one of claims 1 to 46; said operation comprising the
steps of: receiving a first signal representing the number of
molecules of said cell-free DNA comprising one or more methylated
and/or un-methylated CpGs (as applicable), and/or comprising one or
more pattern of methylation and/or un-methylation as set forth in
TABLE 2B for the respective nucleotide sequence(s), located within
one or more of the nucleotide sequences independently selected from
the group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30 and 31, or a nucleotide sequence present within about
2,000 bp 5' or 3' thereof, or an allelic variant and/or
complementary sequence of said nucleotide sequence(s); and
determining a classification of the presence or absence of, or
response to therapy against, an ovarian cancer in said woman based
on their being at least one molecules of said cell-free DNA
comprising one or more said methylated and/or un-methylated CpGs
(as applicable), and/or comprising one or more said pattern of
methylation and/or un-methylation, located within one or more of
said nucleotide sequences.
61. The computer program product of claim 60, wherein said
operation further comprising the steps of: receiving a second
signal representing the number of molecules of said cell-free DNA
comprising said nucleotide sequence(s); and estimating a fraction
or ratio of molecules of said cell-free DNA comprising one or more
said methylated and/or un-methylated CpGs (as applicable), and/or
comprising one or more said pattern of methylation or
un-methylation, located within one or more of the nucleotide
sequences within all of said nucleotide sequences.
62. The computer program product of claim 61, wherein said
classification is determined by comparing said a fraction or ratio
to a standard or cut-off value.
63. The computer program product of claim 62, wherein said
operation further comprising the steps of: receiving a third signal
representing: (i) the amount or concentration of total cell-free
DNA present in said sample; and/or (ii) a baseline value of said
fraction or ratio previously determined for said woman; and
modifying said standard or cut-off value for a given sample based
on said third signal.
64. The computer program product of any one of claims 60 to 63,
wherein said first signal, and optional second signal, is
determined from nucleotide sequence and/or methylation status
information of a plurality of said molecules of said cell-free DNA
and/or amplified DNA representing each of said nucleotide
sequences, preferably wherein said plurality is a number selected
from the group consisting of at least about: 1,000, 5,000, 10,000,
50,000, 100,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000,
2,500,000, 3,000,000, 3,500,000, 4,000,000 and 5,000,000 molecules,
or more than 5,000,000 molecules.
65. The computer program product of claim 64, wherein said
operation further comprises the steps of: for each of said
molecule's sequence and/or methylation status information,
determining if said molecule comprises none, one or more methylated
and/or un-methylated CpGs (as applicable), and/or comprises none,
one or more pattern of methylation or un-methylation as set forth
in TABLE 2B for the respective nucleotide sequence(s), located
within one or more of the nucleotide sequences independently
selected from the group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 and 31, or a nucleotide sequence present
within about 2,000 bp 5' or 3' thereof, or an allelic variant
and/or complementary sequence of said nucleotide sequence(s); and
calculating said first signal, and optional second signal, based on
said determination for all or a portion of said plurality of
molecules.
66. The computer program product of any one of claims 60 to 65,
wherein said operation further comprises the steps of: receiving a
signal representing the amount present, in a sample of blood taken
from said women, of one or more proteins independently selected
from the group consisting of: CA-125, HE4, transthyretin,
apolipoprotein A1, beta-2-microglobin and transferrin; and
comparing said a fraction or ratio to a standard or cut-off value
for said protein; and determining a classification of the presence
or absence of, or response to therapy against, an ovarian cancer in
said woman based on their being either or both of: (i) at least one
molecules of said cell-free DNA comprising one or more said
methylated and/or un-methylated CpGs (as applicable), and/or
comprising one or more said pattern of methylation or
un-methylation, located within one or more of said nucleotide
sequences; and/or (ii) an amount of said protein(s) present in said
blood sample is greater than said standard or cut-off value for
such amount or protein.
67. A use of a nucleic acid sequences of any one of claims 48 to 50
and/or a nucleic acid probes of any one of claims 51 to 53 and/or a
primer pair of claim 54 or 55 and/or a plurality of nucleic acids
of claim 56 or 57 and/or a kit of claim 58 or 59 and/or a computer
program product of any one of claims 60 to 66, in each case for
determining the presence or absence of, or response to therapy
against, an ovarian cancer in a woman.
Description
[0001] The present invention relates to methods of determining the
presence or absence of an ovarian cancer in a woman, as well as to
related methods to determine the response to therapy against
ovarian cancer in a woman. Such methods are based on the
detection--from cell-free DNA of said woman--of one or more
methylated (or un-methylated) CpGs being associated with
differentially methylated regions (DMRs) of the present invention;
such as methylation (or un-methylation) at one or more or all of
certain CpGs being associated with such DMRs. Accordingly, such
methods have diagnostic, prognostic and/or predictive utility for
detecting or managing ovarian cancer in women. The present
invention further relates to nucleic acids comprising certain
sequences that may be detected during the method, or nucleic acids
(such as probes and/or primers) that are useful to detect such
sequences, as wells as compositions, kits, computer program
products and other aspects that are useful for or related to the
practice or application of such methods.
[0002] Three quarters of ovarian cancers are diagnosed once the
tumour has spread into the abdomen and long-term survival rates of
these women are low (10-30%) (Ref. 1). High-grade serous (HGS)
ovarian cancer (OC) accounts for 70-80% of OC deaths and the
survival figures have not changed significantly over the past few
decades (Ref. 2). Early diagnosis and personalised treatment still
remain the biggest unmet needs in combatting this devastating
disease (Ref. 2).
[0003] A number of ovarian cancer biomarkers have been studied in
the past. Amongst those, CA125, which was discovered more than 30
years ago (Ref. 3), is still the `gold standard` despite the
relatively low sensitivity for early epithelial ovarian cancer and
the modest positive predictive value (Ref. 4). The 35 most
promising ovarian cancer biomarkers were evaluated in samples taken
up to 6 months prior to OC diagnosis from 118 women and 951
age-matched controls from the Prostate, Lung, Colorectal, and
Ovarian (PLCO) Cancer Screening Trial. At a fixed specificity of
95%, CA125 had the best sensitivity (Ref. 5). The performance of
CA125 dropped dramatically when samples taken >6 months prior to
diagnosis were evaluated (Ref. 5). Recently it was demonstrated
that the performance of the Risk of Ovarian Cancer Algorithm (ROCA)
demonstrates superior performance characteristics during screening,
but this requires serial blood samples that are not available in
patients presenting clinically (Refs. 6, 7). In addition, the
dynamics of CA125 in women undergoing neoadjuvant chemotherapy
(NACT) are of limited use in predicting disease response and
outcome (Ref. 8).
[0004] The vast majority of protein-based tumour markers are
produced not only by cancerous but also non-neoplastic normal
cells; CA125 is produced by mesothelial cells (i.e. peritoneum and
pleura) and hence benign or inflammatory processes can result in
aberrant elevations of serum CA125.
[0005] Recently, markers based on DNA shed from tumour cells, have
shown great promise in monitoring treatment response and predicting
prognosis (Refs. 9-13). But efforts to characterise the cancer
genome have shown that only a few genes are frequently mutated in
most cancers with the gene mutation site differing across
individuals for similar tumours. Hence, the detection of somatic
mutations is limited to patients that harbour a predefined set of
mutations. The necessity of prior knowledge regarding specific
genomic composition of tumour tissues is one of the limiting
factors when using these `liquid biopsy` approaches for early
detection or differential diagnosis of a pelvic mass. Current
technology allows for the detection of a mutant allele fraction of
0.1% (which is one mutant molecule in a background of 1000
wild-type molecules) (Refs. 9, 14).
[0006] The development of cell-free DNA based early cancer
detection tests poses two major challenges: (1) a very low
abundance of cancer-DNA in the blood and (2) an extremely high
level of "background DNA" (shed from white blood cells (Ref. 15))
in all population based cohorts which allow for the validation of
potential screening markers years in advance of current
diagnosis.
[0007] Alteration of DNA methylation (DNAme) is (i) an early event
in cancer development, (ii) more frequently observed than somatic
mutations and (iii) centred around specific regions, i.e. CpG
islands (Ref. 17). Together with its chemical and biological
stability, the detection of aberrant DNA methylation patterns in
serum or plasma provide a novel strategy for cancer diagnosis as
evidenced by several proof of principle studies in the past (Refs.
9, 10, 13, 15, 18-20). The fact that technologies to detect DNA
methylation allow for the detection of specific methylation
patterns (for example, full methylation or un-methylation) of all
of (for example, between 7 and 16) certain linked CpGs in a region
of 120-150 base-pairs as opposed to single point mutations (e.g. in
the TP53 gene) is likely to improve both the performance
characteristics of the test and the detection limit of the assay.
Plasma SEPT9 methylation analyses--currently the only cell-free DNA
which is available for cancer screening in the clinical
setting--demonstrates a specificity of 79% and a sensitivity of 68%
for detection of colon cancers (Ref. 21). Maternal plasma cell-free
DNA testing for foetal trisomy has already become clinical practice
as it has a higher sensitivity and a lower false positive rate
compared to imaging-based techniques (Ref. 22).
[0008] The inventors have employed two different epigenome-wide
approaches to identify the most promising DNAme-based markers,
developed serum tests and validated their performance benchmarking
against serum ovarian cancer marker CA125.
[0009] It has been suggested that DNA methylation markers have
promise (and challenges) for early detection of women's cancers
such as ovarian cancer (Ref. 20). Indeed, there are a number of
publications that disclose various epigenetic biomarkers and their
association with various cancers, including women's cancers such as
ovarian cancer, and the use of such biomarkers (including in
certain combinations) for the detection and/or management of one or
more of such cancers: WO2002/018631A2; WO2002/018632A2;
WO2007/019670A1; EP1862555A1; WO2009/153667A2; WO2012/104642A1;
WO2012/138609A2; WO2012/143481A2; US2013/0041047A1;
WO2013/09661A1). In particular, single-CpG-resolution methylation
analysis (including patterns/signatures) in certain specific
markers or genes (such as DNA hypermethylation of the CpG sites on
the FAM150A, GRM6, ZNF540, ZFP42, EOMES HOXA9, POU4F2, TWIST1, VIM,
ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, C2CD4D, PRAC, WNT3A,
TRH, FAM78A, ZNF671, SLC13A5, NKX6-2, GP5 and HOTAIR genes) has
identified cancers, or aggressive types thereof, such as renal cell
carcinomas (Arai et al, 2012; Carcinogenesis 33:1487), bladder
cancer (Reinert et al, 2012; PLos ONE 10:e46297), other cancers
including breast cancer (Refs. 30, 31. Legendre et al, 2015;
Clinical Epigenomics 7:100), ovarian cancer (Teschendorff et al,
2009; PLos ONE 4: e8274) and/or association with chemotherapeutic
response in ovarian cancer (Ref. 25).
[0010] Hence, there is still a need, from one or more of the above
or perspectives, for improved methods to determine the presence or
absence of ovarian cancer in a woman, preferably in a non-invasive
manner, such as by the use of cell-free DNA of said woman (eg
isolated from a sample of a circulatory fluid). Preferably, such
methods will have improved ability to discriminate ovarian cancer
from benign pelvic mass, and/or high-grade serious (HGS) ovarian
cancer from less severe or aggressive forms of ovarian cancer, such
as by having improved specificity and/or sensitivity for the
phenotype/disease to be detected. Such methods would provide a
significant shift in the clinical paradigm for early-detection,
diagnosis (eg by an in-vitro method) and/or management of ovarian
cancer, in particular of HGS ovarian cancer and or
chemotherapy-responsive ovarian cancer, and in particular providing
the potential for individualisation of treatment for women
suffering from ovarian cancer.
[0011] Accordingly, it is an object of the present invention to
provide alternative, improved, simpler, cheaper and/or integrated
methods, means, compounds, compositions, kits and other aspects
that address one or more of these or other problems (such as those
set forth elsewhere herein). Such an object underlying the present
invention is solved by the subject matter as disclosed or defined
anywhere herein, for example by the subject matter of itemised
embodiments or the claimed embodiments.
[0012] Generally, and by way of brief description, the main aspects
of the present invention can be described as follows:
[0013] In a first aspect, and as may be further described, defined,
claimed or otherwise disclosed herein, the invention relates to a
method of determining the presence or absence of, or response to
therapy against, an ovarian cancer in a woman, said method
comprising the steps: [0014] Providing a biological sample from
said woman, said sample comprising cell-free DNA of said woman; and
[0015] Determining, in at least one molecule of said cell-free DNA,
the methylation status at one or more CpGs located within one or
more of the nucleotide sequences comprised in one or more of the
respective DMRs of the present invention independently selected
from the group consisting of DMR#: #141, #204, #228, #144, #123,
#129, #137, #148, #150, #154, #158, #164, #176, #178, #180, #186,
#188, #190, #192, #200, #202, #208,#210, #213, #214, #219, #222,
#223, #224, #225 and #226, or a nucleotide sequence present within
about 2,000 bp (such as within about 200 bp) 5' or 3' thereof, or
an allelic variant and/or complementary sequence of any of said
nucleotide sequences, wherein, the presence in at least one of said
cell-free DNA molecules of one or more: (i) methylated CpGs
associated with one or more of the hyper-methylated DMRs of the
present invention; and/or (ii) un-methylated CpGs associated with
one or more of the hypo-methylated DMRs of the present invention,
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman.
[0016] In a second aspect, and as may be further described,
defined, claimed or otherwise disclosed herein, the invention
relates to a chemotherapeutic agent, such as one selected from the
group consisting of: carboplatin, paclitaxel, docetaxel, cisplatin,
liposomal doxorubicin, gemcitabine, trabectedin, etoposide,
cyclophosphamide, an angiogenesis inhibitor (such as bevacizumab)
and a PARP inhibitor (such as olaparib), for use in a method of
therapy of ovarian cancer in a woman, wherein said chemotherapeutic
agent is administered to a woman within about 3 months of said
woman having been predicted and/or determined, using a method of
the first aspect, to not respond to a therapy against ovarian
cancer.
[0017] In a third aspect, and as may be further described, defined,
claimed or otherwise disclosed herein, the invention relates to a
nucleic acid comprising a nucleic acid sequence consisting of at
least about 10 contiguous bases (preferably at least about 15
contiguous bases for any DMR other than DMR #222) comprised in a
sequence producible by bisulphite conversion of a sequence
comprised within a DMR selected from the group consisting of DMR#:
#141, #204, #228, #144, #123, #129, #137, #148, #150, #154, #158,
#164, #176, #178, #180, #186, #188, #190, #192, #200, #202,
#208,#210, #213, #214, #219, #222, #223, #224, #225 and #226, or an
allelic variant and/or complementary sequence of any of said
nucleotide sequences.
[0018] In other aspects, the invention also relates to a nucleic
acid probe, a nucleic acid PCR primer pair, a population of nucleic
acids of the invention, a kit and a computer program product, in
each case as may be described, defined, claimed or otherwise
disclosed herein, and in each case related to use within or in
connection with a method of the invention and/or to detect one or
more a nucleic acid of the invention.
[0019] The figures show:
[0020] FIG. 1 depicts the study design. Using two different
epigenome-wide technologies, 711 human tissue samples have been
analysed to identify a total of 31 regions whose methylation status
has been analysed in two sets consisting of 151 serum samples.
Three markers have been validated in two independent settings: (1)
Serum Set 3 which consisted of 250 serum samples from women with
various benign and malignant conditions of the female genital
tract; and (2) NACT (NeoAdjuvant ChemoTherapy) Set consisting of
serial samples from women with advanced stage ovarian cancer before
and during chemotherapy. Samples obtainable from the UKCTOCS
(United Kingdom Collaborative Trial of Ovarian Cancer Screening)
sample collection may be included in a third validation set to
include serum samples from those women in the control arm who
developed ovarian cancer within 2 years; for each case, a number of
(such as three) control women who did not develop ovarian cancer
within 5 years of sample donation can been matched to those women
who did so develop ovarian cancer.
[0021] FIG. 2 depicts the principles of methylation pattern
discovery in tissue and analyses in serum. Reduced Representation
Bisulfite Sequencing (RRBS) was used in tissue samples in order to
identify those CpG regions for which methylation patterns
discriminate ovarian cancer from other tissues, in particular blood
cells which were deemed to be the most abundant source of cell-free
DNA. An example of region #141 is provided which is a 136 base-pair
long region containing 7 linked CpGs. A cancer pattern may consist
of reads in which all linked CpGs are methylated, indicated by
"1111111" (Panel A). The tissue RRBS data have been processed
through a bioinformatic pipeline in order to identify the most
promising markers (Panel B). The principles of the serum DNA
methylation assay are demonstrated in Panel C.
[0022] FIG. 3 depicts serum DNA methylation analysis in women with
benign and malignant conditions of the female genital tract.
Pattern frequencies for the different regions and CA125 levels
analysed in Serum Set 3 samples are shown and horizontal bars
denote the mean (Panels A-C; ns not significant; *p<0.05,
**p<0.01, ***p<0.001; Mann-Whitney U test compared to HGS; H,
Healthy; BPM, benign pelvic mass; BOT, borderline tumours; NET,
non-epithelial tumours; OCM other cancerous malignancies; NHGS,
non-high grade serous ovarian cancers; HGS, high grade serous; OC
ovarian cancers). Based on Set 1&2 analyses cut-off thresholds
of 0.0008, 0.0001 and 0.0001 for regions #141, #204 and #228,
respectively, to discriminate HGS OC from H or BPM women were
chosen and validated in Set 3; combining Sets 1-3 the cut-off
thresholds have been refined for regions #204 and #228 so that the
final cut-offs were 0.0008, 0.00003 and 0.00001, respectively; the
sample was called positive if at least one of the three regions
showed a pattern frequency above the cut-off; sensitivities and
specificities to discriminate HGS from H&BPM are shown in Panel
E. The overlap between CA125 positive samples (cut-off >35
IU/mL) and the three DNA methylation (DNAme) marker panel in cases
and controls is shown in Panel F.
[0023] FIG. 4 depicts the dynamics of serum DNA methylation markers
and CA125 as a function of exposure to carboplatin-based
chemotherapy. The changes in pattern frequency of the three markers
as well as CA125 is shown before compared to after 2 cycles of
chemotherapy (Panels A-D). The changes of markers during
chemotherapy and whether this can predict response (as described in
Supplementary Information) to chemotherapy in all patients and in
those who had no macroscopic residual disease after
interval-debulking surgery (R0/1) is shown (Panel E). Definitions
of CA125 and DNA methylation positivity are provided in FIG. 3.
[0024] FIG. 5 depicts the algorithm which first determines sets of
consecutive CpG sites of maximum size, from which multiple
potentially overlapping subsets are derived, which still meet the
selection criteria.
[0025] FIG. 6 depicts cancer-specific differentially methylated
region (DMR) discovery with Illumina 450K methylation arrays. (A)
Schematic illustration of DMRs that are discovered by the single
CpG and range approaches. Each horizontal line of lollipops
indicates neighbouring CpGs in a single DNA molecule extracted from
the indicated tissue. Filled lollipop indicates a methylated CpG,
and an unfilled lollipop indicates an unmethylated CpG. 450K
methylation arrays measure the ratio (% of methylation) of
methylated and unmethylated molecules at a given single CpG
location. See Supplementary Information for details on the DMR
discovery methods. "up-arrow" (.uparw.) Single CpG DMRs (high
scoring); "left/right-arrow" (.rarw..fwdarw.) Range of DMR (high
scoring); asterisk (*) Not identified as DMRs because of the
methylation in WBCs, "hash" (#) Not identified as high scoring DMRs
with single CpG approach because the methylation difference between
OC and other control tissues (=colon, lung, liver, rectum,
endometrium, fimbriae and benign ovarian tissue) is not large
enough. Identified as DMRs with range approach because the pooling
of neighbouring CpG information increases statistical robustness.
(B) Example of the methylation data for a high scoring DMR. The
#228 targeted BS reaction was designed for this DMR.
[0026] FIG. 7 depicts a procedure for the isolation of cell-free
DNA from a plasma or serum biological sample.
[0027] FIG. 8 depicts pattern frequencies for the different regions
analysed in Serum Set 1 samples. H, Healthy; BPM, benign pelvic
mass; NHGS, non-high grade serous ovarian cancers; HGS, high grade
serous ovarian cancers. Horizontal bar denotes mean. ns not
significant; *p<0.05, Mann-Whitney U test compared to HGS.
[0028] FIG. 9 depicts pattern frequencies for the different regions
analysed in Serum Set 2 samples. H, Healthy; BPM, benign pelvic
mass; BOT, borderline tumour; NET, non-epithelial tumours; OCM,
other cancerous malignancies; NHGS, non-high grade serous ovarian
cancers; HGS, high grade serous ovarian cancers. Horizontal bar
denotes mean. ns not significant; * p<0.05; ** p<0.01; ***
p<0.001, Mann-Whitney U test compared to HGS.
[0029] FIG. 10 depicts coverage (number of reads) for the three
different regions analysed in Serum Set 3 samples. H, Healthy; BPM,
benign pelvic mass; BOT, borderline tumour; NET, non-epithelial
tumours; OCM, other cancerous malignancies; NHGS, non-high grade
serous ovarian cancers; HGS, high grade serous ovarian cancers.
Horizontal bar denotes mean. ns not significant; * p<0.05;
Mann-Whitney U test compared to HGS.
[0030] FIG. 11 depicts CA125 levels measured in the NACT (the
neoadjuvant chemotherapy) Serum Set samples. Samples taken before
chemotherapy, after the first cycle of chemotherapy, and after the
second cycle of chemotherapy. ns not significant; **p<0.01;
Mann-Whitney U test compared to before chemotherapy.
[0031] FIG. 12 depicts pattern frequencies for the top 3 reactions
measured in NACT Serum Set samples. Samples taken before
chemotherapy, after the first cycle of chemotherapy, and after the
second cycle of chemotherapy. * p<0.05; ** p<0.01; **
p<0.01; Mann-Whitney U test compared to before chemotherapy.
[0032] FIG. 13 depicts coverage (number of reads) for the top 3
reactions measured in NACT Serum Set samples. Samples taken before
chemotherapy, after the first cycle of chemotherapy, and after the
second cycle of chemotherapy. ns not significant; Mann-Whitney U
test compared to before chemotherapy.
[0033] The present invention, and particular non-limiting aspects
and/or embodiments thereof, can be described in more detail as
follows:
[0034] In a first aspect, the invention relates to a method of
determining the presence or absence of, or response to therapy
against, an ovarian cancer in a woman, said method comprising the
steps: [0035] Providing a biological sample from said woman, said
sample comprising cell-free DNA of said woman; and [0036]
Determining, in at least one molecule of said cell-free DNA, the
methylation status at one or more CpGs located within one or more
of the nucleotide sequences independently selected from the group
consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 and 31 (for example, within one or more of the nucleotide
sequences comprised in one or more of the respective DMRs of the
present invention independently selected from the group consisting
of DMR#: #141, #204, #228, #144, #123, #129, #137, #148, #150,
#154, #158, #164, #176, #178, #180, #186, #188, #190, #192, #200,
#202, #208,#210, #213, #214, #219, #222, #223, #224, #225 and
#226), or a nucleotide sequence present within about 2,000 bp (such
as within about 200 bp) 5' or 3' thereof, or an allelic variant
and/or complementary sequence of any of said nucleotide sequences,
wherein, the presence in at least one of said cell-free DNA
molecules of one or more: (i) methylated CpGs associated with (such
as located within) one or more of the hyper-methylated DMRs of the
present invention (eg, as identified in TABLE 1A), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 1, 2,
3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29
and 30; and/or (ii) un-methylated CpGs associated with (such as
located within) one or more of the hypo-methylated DMRs of the
present invention (eg, as identified in TABLE 1B), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 5, 6,
7, 8, 9, 11, 13, 16, 17, 22 and 31, indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman.
[0037] The genomic sequence and genome coordinates (hg19) of one
class of the regions of the genome used in the present invention as
a source of epigenetic markers, ie those where the presence of
methylation at one or more CpGs therein (or associated therewith,
such as within about 2,000 bp--such as within about 200 bp--5' or
3' thereof), and represent the hyper-methylated cancer-specific
differentially methylated regions (DMRs) of the present invention,
are set out in TABLE 1A. Any of such CpGs (including those of an
allelic variant and/or complementary sequence of any of the
respective said nucleotide sequence/s) is one considered
"associated with" the respective hyper-methylated cancer-specific
DMR of the present invention.
TABLE-US-00001 TABLE 1A Identity, source, genome-coordinates and
genomic sequences of the hyper-methylated DMRs of the present
invention Amplicon DMR Data coordinates Amplicon genomic sequence
SEQ ID # basis (hg19) (relevant CpGs are underlined) Class NO. 141
RRBS chr5:178004395- CATCCGGAGGCCCAGGGGTGAGGACTTCGCCACGGGAAGG Hyper
1 178004530 AGGCACACGATTCAGCCCATGACACCGCCACCTCGGCGTG
GTGCTGTAGGGGGAAGCTCAGGCACTCACCGAGGACAGGA CCCGGGGAATCCGCTG 204 RRBS
chr1:151810784- GATATTCGGTGGAGAGCCGCAGCTGCCCGCCGCGGGGCCC Hyper 2
151810937 CAGGCGCAGCACGCTCTCGCGCGTGGGCCGCAGCTGGCAG
CACAGGAAGTCCAGGTGGAAGAGCGGCGGCGTGGGCGGCC
CGGCGCGGCGCGGCGAGTGCGGGCTGGTATCGGC 228 450K chr2:219736276-
GTTCTATGGGCGAGCTGCTGCAGTGCGGCTGCCAGGCGCC Hyper 3 219736386
CCGCGGGCGGGCCCCTCCCCGGCCCTCCGGCCTGCCCGGC
ACCCCCGGACCCCCTGGCCCCGCGGGCTCCC 144 RRBS chr19:58220413-
CCCAGGCCTGACGTGGGTCCCCCAGGGCGGCGTCGCCAAG Hyper 4 58220552
GCTTAGACGCTTTCGTGCAGGAGGGACGACGACTCCCCTC
ACGCCTTCGTGGCCCCAACTCGGCGCTCTGCTATCTCTGA TCCGGTGAACACACCTCAGA 154
RRBS chr17:70112132- TCCCCGGAGTCCGGAGCTCAGGCCAGTGGCAGTCGACCCA Hyper
10 70112268 GCCCCCGAGACTCCCTCACGCCGCTCCAAAACCAAAACGG
AGCCCAACACGAAGCTGGGTGAAGCCGTAGCTTGCAGGAG CCAGGGAGATGCGCTCT 164 RRBS
chr4:174427917- GACTCGCGAGGTTTTCCAGCAGCTCATTCCGGGACGGCGG Hyper 12
174428054 TGTCTAGTCCAGTCCAGGGTAACTGGGCTCTCTGAGAGTC
CGACCTCCATCGGTCTGGGAGCGAGTGGTTCGAGTTCAGA TGCTGGGAACCGTCGCTT 178
RRBS chr19:13215409- GGCAGGAGCGCCCCACTATGCGCAAGCCCGTGGCCTGGAG Hyper
14 13215550 AGCGCTGAAGGTGGGAGGGGGAAGAGGGGcAGAAcccccG
CGGGAGCGAGCGCACAGCTGCCGCCCCGTGGCCGCTTCGG GAATCGCTGGCTCCGGCTCTGG 180
RRBS chr3:192125846- CTGCAGAAGCGCACTTTGCTGAACACCCCGAGGACGTGCC Hyper
15 192125980 TCTCGCACAGGGAGCGCCCGTCTTTGCTGGGGCTGGAGCG
GCGCTTGGAGGCCGACACTCGGTCGCTGTTGGACTCCCTC GCCTGCCGCTTCTGC 190 RRBS
chr9:79629064- GTCAGACGAGAGCCTGGGGTCAATGTCGAGGTGGAGCGAC Hyper 18
79629172 GCTGGCACGGCAACCCTGAGCCTGCGCGGCCCGGCGCTAT
CCCCTGGCTCTCCGCTGCTGGCTGGACCC 192 RRBS chr12:75601294-
CGGTAGGTCATCCAGCAGCAGGGCTCCACGTCGGTCTCGT Hyper 19 75601437
CGATGCCCCAGAAGGCCAGCTCCTCCTCGAAGAGCGGCCC
GCACACGTCTGCGGGGCAGTGCAGCTTGCCGGTGCGGTAG TAATTGAGCACATAGGCGAAGACG
200 RRBS chr9: 138999180- AATCAGCCCAGCAACCGGCGACCCCAAGCGCGGCGACCGC
Hyper 20 138999294 AAAGGGAGTGCTTGCCCATCCGCGTTTGAAAGCAGACTTT
TTCTCGGCAGGAACACAGGACTCACCTGCCAGTGG 202 RRBS chr1:2987508-
GTGCGAACAAGACCGGGCGTTTCGCCGCCGACGCGAAGGG Hyper 21 2987655
GCTGTCTGTGCGCGGCGTTGCGGGCCCTCCGCGCGTGGGG
TGTGCGTGTGCGTGTTCGGGTTCGGTTCTGTGTGTGCACC
GCGGGCCTGCTCAGAGTCGGGACCACCG 210 450K chr12:123713499-
TGCATACAGATTACTGTAGGACCATTTCCTGTGCCTTTTA Hyper 23 123713590
AAATTTCCTTTTCTCGTTTTATTTCACATATTCCTTTGTT TTTTACAACTCC 213 450K
chr2:106776938- CCGCTCGGGAATGGGAATATAGCTACATATGGGAAAACGC Hyper 24
106777040 GGTGCAGGGAGAAAACCAATTCAGTGAGGAGCGGAGGCGC
AGGACTGTGGAGTGTGCATCCGG 214 450K chr3:141516260-
CTGCTTAAAGGCGCAGAGGAGCAGCTGGGAACGAGAACAA Hyper 25 141516353
AGCGGCCAGGCCCCCCTCGGAGGAAGGAAGGAGAGAGCCC CAGGAAACAGCTGA 219 450K
chr16:30484157- GGATGAAGGATTCCTGCATCACTGTGATGGCCATGGCGCT Hyper 26
30484257 GCTGTCTGGGTTCTTTTTCTTCGGTAGGCAAGGGAGGAGG
CAGGGGAAGGGACATGTGTCT 222 450K chr3:111809437-
TAGGCTACAGGAAGAGGCATTTCCTATAGATGACGGCTGT Hyper 27 111809506
AAAATTTTAAGCTGAGTTCCTCCAGGAAGT 223 450K chr10:120489250-
AAGAGAGAGTGGTTGATAATCAGTAGAGAGAGGTTTCTAA Hyper 28 120489333
CTCACGGAAGTGTTTGCAATACAACCTCTTTGTACATCAG CTGT 224 450K
chr11:1874037- GGTCCCCCTCCCCGAGCCATGAAGAGCTGCCTGCGGCCAT Hyper 29
1874133 CTTGGCCCTCGCACCCCGTCTCTGTCACCCCAGGCCCCTG TAACTTGCTTAACGCTT
225 450K chr7:142422193- GAAGCTTGACACTCCTGGCCCCAAACACTGCCTGGCTACA
Hyper 30 142422278 ACACGATATCCAGGGACAGATACCTTCCATGTACAGCAAG
CTGTGG
[0038] The genomic sequence and genome coordinates (hg19) of the
other class of the regions of the genome used in the present
invention as a source of epigenetic markers, ie those where the
absence of methylation at one or more CpGs therein, (or associated
therewith, such as within about 2,000 bp--such as within about 200
bp--5' or 3' thereof), and represent the hypo-methylated
cancer-specific DMRs of the present invention, are set out in TABLE
1B. Any of such CpGs (including those of an allelic variant and/or
complementary sequence of the respective said nucleotide
sequence/s) is one considered "associated with" the respective
hypo-methylated cancer-specific DMR of the present invention.
TABLE-US-00002 TABLE 1B Identity, source, genome-coordinates and
genomic sequences of the hypo-methylated DMRs of the present
invention Amplicon DMR Data coordinates Amplicon genomic sequence
SEQ ID # basis (hg19) (relevant CpGs are underlined) Class NO. 123
RRBS chr16:1271152- GCGAAGCAGGAGTAGCTGCCGGGCCCCACGAGCCTCCGTC Hypo 5
1271271 CGTTCTGGTTCGGGTTTCTCCGAGTTTTGCTACCAGCCGA
GGCTGTGCGGGCAACTGGGTCAGCCTCCCGTCAGGAGAGA 129 RRBS chr11:69054638-
AACTCTGCTGAGTGAGCTCACAAACAGGGCATAACCGAGA Hypo 6 69054757
CGCGGGAATGCCTGGGTCGCCGCGCAGTCACCGGGCAGGG
CCGCCCTCCCCTGTGGGTCAGCAAAAACGGTGTCAAGTGA 137 RRBS chr12:132896275-
ACTCCGCCACACACACAGCTGTACCCGGCACAACACGCGG Hypo 7 132896404
CCACAGGTCACCTCAGGTCGCCTCGGGTGCTCCTCCCGCA
GCCCCACGTAGACAGAAGACATTCCTCGGGCCTGGGTGCC CAGCCTCCCG 148 RRBS
chr2:72359599- GAGGTAATGGAAGCGGCCATCCTTGTCCTCGCTCCGCGCC Hypo 8
72359718 TGGCTGAAGCGATCGGGGTCGAACACGTTCACGTCTTTGA
ACACGGGCGCTGTGTCATGGGTGTCCCGGATGCTATACAT 150 RRBS chr7: 156735029-
GCGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCG Hypo 9 156735165
GGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGCGAG
CCGAGATCGCGCCACCGCACTCCAGCCTGGGCGACAGAGC GAGACTCCGTCTAAAAA 158 RRBS
chr16:74441696- GACTCTGTCTCAAAAAAGAAAAAAATAGGGCCGGGCGCGG Hypo 11
74441831 TGGCTCACGCCTGTCATCCCAGCACTTTGGGAGGCCGAGG
CGGGTGGATCACGAGGTCGGGAGATCGATACCATCCTGGG TAACACGGTGAAACCC 176 RRBS
chr6:119107203- TAACCCATTTCTTTATTAAATTGCATGAAGAAGGCCGGGC Hypo 13
119107340 GCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCC
GAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCACGG TGAAACCCCGTCTCTACT 186
RRBS chr22:21483239- CGTGTTAGCCAGGATGGTCTCGACCTCCTGACCTCGTGAT Hypo
16 21483384 CAGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTAAAGGCG
TGAGCCACCGCGCCGGGCCGAGACTCTGTCTTAAAAAAAA AAGGCCTGGGCTGTGGCACTTTGGGA
188 RRBS chr19:18497131- AGAGTTGCACTCCGAAGACTCCAGATTCCGAGAGTTGCGG
Hypo 17 18497271 AAACGCTACGAGGACCTGCTAACCAGGCTGCGGGCCAACC
AGAGCTGGGAAGATTCGAACACCGACCTCGTCCCGGCCCC TGCAGTCCGGATACTCACGCC 208
RRBS chr8:55467518- ATATTAATCTTGTCCGGGCACGGTGGCTCACGCCTGTAAT Hypo
22 55467638 CCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGT
CAGGAGATCGAGACCATCCTGGCGAACATGGTGAAACCTC G 226 450K chr1:3086452-
GGGGGGACTGTCGTTAATTCACTGCCTAATGACCGCGGCC Hypo 31 3086542
CGCGCGCTCCGAGTAATCGGGTGATGTATGTGGACTGTGC ACACCTCGTGG
[0039] In particular embodiments, the present invention may not
include (or not include the use of) one or more of the specific
epigenetic markers (eg the presence of absence of methylation at
one or more CpGs therein, that are within (or associated therewith,
such as within about 2,000 bp--such as within about 200 bp--5' or
3' of) one or more of the DMRs set forth in TABLE 1A or TABLE 1B),
for example, the present invention may not encompass one, two,
three, four, five, six, seven, eight, nine, ten, or about 12, 15,
20, 25 28, 29, or 30 of the DMRs independently selected from the
group consisting of DMR#: #141, #204, #228, #144, #123, #129, #137,
#148, #150, #154, #158, #164, #176, #178, #180, #186, #188, #190,
#192, #200, #202, #208,#210, #213, #214, #219, #222, #223, #224,
#225 and #226. In certain of such embodiments, the present
invention may not encompass DMR #144 (SEQ ID NO. 4).
[0040] As set out herein, the epigenetic marker (eg the
presence/absence of methylation at a CpG) may be within the
nucleotide sequence of a DMR of the present invention, or may be
present within about 2,000 bp (such as within about 200 bp) 5' or
3' of such DMR sequence; ie is upstream or downstream of the DMR
sequence disclosed herein. This is because (CpG) "islands" are
present throughout the genome and the cancer-specific pattern of
methylation/un-methylation described herein may equally be
detectable elsewhere in such "island" such at one or more CpGs
located within about 2,000 bp (eg about 200 bp) 5' or 3' of the DMR
sequence disclosed herein. Following the disclosure herein, the
person of ordinary skill will readily recognise that inspection of
the human genome sequence can identify other CpGs potentially
useful epigenomic markers within any of such "islands" such as
around or associated with any of the DMRs of the present invention,
and hence such other epigenomic markers (eg other CpGs within about
2,000 bp--such as within about 200 bp--5' or 3' of such DMR
sequence) are specifically envisioned as being within the scope of
the present invention. In certain embodiments, one or more of said
CpGs is located within about 1,750, 1,500, 1,250, 1,000, 750, 500,
250, 200, 150, 125, 100, 75, 60, 50, 40, 30, 25, 20, 15, 10 or 5
base pairs 5' of a DMR described herein; or within about 1,750,
1,500, 1,250, 1,000, 750, 500, 250, 200, 150, 125, 100, 75, 60, 50,
40, 30, 25, 20, 15, 10 or 5 base pairs 3' of a DMR described
herein.
[0041] In further embodiment, the DMR of the present invention may
be a variant of the respective sequence given herein. For example,
by the deletion, addition or substitution of one or more (such as
2, 3, 4, 5 or more than 5) base pairs compared to the respective
sequences. As will be understood by the person of ordinary skill,
such variants can exist in any population of women, such as by
being an allelic variant or a SNP.
[0042] As will also be appreciated by the person of ordinary skill,
any of such sequences may be represented by (or analysed as) a
complementary sequence to any of the sequences set out herein.
[0043] As used herein, "determining" may be understood in the
broadest sense as any recognition, including detection,
localisation, diagnosis, classifying, staging or quantification of
ovarian cancer. Determining may be performed as set out herein. In
the context of the present invention, determining is, preferably,
performed in respect of the woman in-vitro, for example as an in
vitro method of diagnosis. That is, the biological sample
comprising cell-free DNA is obtained from the woman, and the method
of determining (or, eg diagnosis) is conducted on such sample that
is isolated and separated from said woman. For example, the
biological sample is processed in a laboratory and/or in plastic or
glass receptacles to analyses that the cell-free DNA of said woman
by a method of the present invention.
[0044] As will be appreciated, a method that determines the
response to therapy against ovarian cancer in a woman can be
understood as a method of monitoring the increase (or reduction) of
ovarian cancer in a woman previously diagnosed (eg by other methods
or tests) with ovarian cancer; in particular providing a method of
monitoring--in an individual-specific manner--the success of
(chemo)therapy administered to said woman in reducing or otherwise
treating the ovarian cancer, or other symptoms thereof.
[0045] It will be understood by the person of ordinary skill that
"determining" the presence or absence of (or response to therapy
against) ovarian cancer may not be, in every and all circumstance,
100% accurate. Such determination (or diagnoses) may be reported as
a likelihood of the present or absence of the ovarian cancer,
and/or interpreted in the context of the false-positive and/or
false-negative rates of such a method or test. As is conventional
in diagnostic tests, such rates can also be represented by the
sensitivity and/or specificity of the test.
[0046] Accordingly, in particular embodiments of the present
invention, the tests or methods hereof provide a test for the
determination of ovarian cancer that has a sensitivity and/or
specificity that is superior to that provided by a CA125 test,
and/or has non-overlapping false-positives and/or false-negatives
with a CA125 test. Also envisioned are embodiments of the present
invention wherein a methods or test may be provided having: (i) a
sensitivity (ie true-positive rate) of greater than about 30%, 40%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 94%, 95%,
96%, 97%, 98%, 99% or 99.5%, in particular greater than about 90%,
95% or 98%; and/or (ii) a specificity (ie, true-negative rate) of
greater than about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, in
particular greater than about 55%, 60%, 70% or 80%. For example, in
one embodiment, a test or method of the present invention may have
a specificity of greater than about 95% (such as about 98%) and a
sensitivity of greater than about 60% (such as about 63%). In
another embodiment, a test or method of the present invention may
have a specificity of greater than about 85% (such as about 90%)
and a sensitivity of greater than about 55% (such as about 58%). In
yet another embodiment, a test or method of the present invention
may have a specificity of greater than about 98% (such as greater
than about 99.5%) and a sensitivity of greater than about 70% (such
as greater than about 75%), for example if applied to a general
population. As will be appreciated, such test parameters can depend
on the population of women being screened, and in particular the
prevalence of OC in such population. In this regard are presented
two examples: (1) in a population which has a dramatically
increased prevalence of OC (eg BRCA mutation carriers who have an
OC lifetime risk of up to 60%), a lower specificity may be
applicable as the rate of false positives will be substantially
lower and a false-positive result would have substantial less
impact in such women--who may eventually opt for risk reducing
surgery anyway; and (2) in a general population setting, the
prevalence of OC in those women actually tested using the present
invention may be "artificially increased" by conducting a
pre-screen for OC (eg, by using ROCA or other diagnostic
tests/methods as described elsewhere herein) and then conducting a
test of the present invention only in the sub-population of women
who have an intermediate or elevated OC risk as determined by such
pre-screen (which may be about 8% of general female population),
and in this sub-population of women, a lower specificity may also
be applicable.
[0047] The term "ovarian cancer" is art recognised, and encompasses
any cancer that forms in tissue associated with the ovary; in
particular those that result in abnormal cells that have the
ability to invade or spread to other parts of the body. The most
common type of ovarian cancer, comprising more than 95% of cases,
is ovarian carcinoma. There are five main subtypes of ovarian
carcinoma, of which high-grade serous (HGS) is most common. The
other main subtypes include: low-grade serous, endometrioid, clear
cell and mucinous carcinomas. These tumours are believed to start
in the cells covering the ovaries, though some may form at the
Fallopian tubes. Other types of ovarian cancer include germ cell
tumours and sex cord stromal tumours. For the purpose of the
present invention, the term "ovarian cancer" can also include
peritoneal cancer and Fallopian tube cancer, in each case in women,
as both are high-grade serous, have analogous biology to ovarian
cancer and have the same treatment modalities as ovarian
cancer.
[0048] The various aspects and embodiments of the present
invention, may apply to any one (or more) of such specific
types/subtypes of ovarian cancer, and in particular the
discrimination of one types/subtypes of ovarian cancer from others
or from other disorders (such as gynaecological disorders). For
example, in certain embodiments, the present invention is used to
discriminate ovarian cancer from benign pelvic mass, and/or
high-grade serious (HGS) ovarian cancer from less severe or
aggressive forms of ovarian cancer, and/or or
chemotherapy-resistant from chemotherapy-responsive ovarian
cancer.
[0049] In other aspects or embodiments of the present invention,
the test may determine (or diagnose) the presence or absence of a
cancer in said woman other than ovarian cancer (instead of, or as
well as, determining the presence or absence of, or the response to
therapy against, ovarian cancer). Such other cancer may be another
gynaecological cancer (such as uterine cancer, vaginal cancer,
cervical cancer, and vulvar cancer) or a cancer of the colon or
breast. Such aspects or embodiments are particular envisioned for
the present invention which makes use of one or more epigenetic
markers (eg, one or more methylated CpGs, in particular the related
CpGs thereof) located within (or within about 2,000 bp--such as
within about 200 bp--5' or 3' of) a nucleic acid sequence comprised
in DMR #204 [SEQ ID No. 2], optionally where the present invention
makes use of one or more further epigenetic markers within one or
more other DMRs of the present invention (such as #141, #228 and/or
#144 [SEQ ID NOs: 1, 3, and/or 4, respectively]; in particular
#141, and #228).
[0050] The biological sample to be provided in this aspect of the
present invention may be obtained from the woman (eg, a woman
having, suspected of having or being investigated for having,
ovarian cancer) by any procedure, process or step that the person
of ordinary skill will recognise. For example, a biological sample
may be obtained by surgery, biopsy, swab, collection of biological
fluids etc. The biological sample may be a sample of tissue and/or
fluid of the woman. Examples of a biological fluid include whole
blood or a blood fraction (eg, such as plasma or serum). In
alternative examples, the sample may be a biological fluid selected
from the group consisting of: urine, saliva, sweat, tears, phlegm,
beast milk, breast aspirate, vaginal secretion, vaginal wash and
colonic wash.
[0051] In one embodiment, the biological sample may be a liquid
biological sample selected from the group consisting of: a blood
sample, a plasma sample and a serum sample. In more particular
embodiments, the sample is a plasma or serum sample from the woman,
or is urine from the woman female. In certain embodiments, the
sample is substantially (or essentially) free from cells, and/or is
not a whole blood sample.
[0052] Methods of collecting such biological samples will be known
to the person of ordinary skill, in particular the collection of
whole blood (eg by needle-puncture of a suitable vein of the
woman), and the subsequent preparation of plasma or serum from the
whole blood (such as described in the examples hereof). In
particular embodiments, the blood may be collected, stored and/or
transported in a cell-free DNA blood collection tube, such as one
with a formaldehyde-free preservative that stabilises nucleated
blood cells. Such stabilisation would be expected to prevent, or
reduce, the release of genomic DNA (eg from nucleated blood cells),
enhancing the isolation of high-quality cell-free DNA which can be
further used in the method or other aspects of the present
invention. The use of such tubes in the present invention can, in
certain embodiments, reduce the need for immediate plasma
preparation. For example, cell-Free DNA is stable for up to 14
days, at room temperature, allowing convenient sample collection,
transport and storage over such period. Suitable blood collection
tubes include the "Cell-Free DNA BCT.RTM." of Streck Inc, such as
their research grade or CE-marked versions of this product.
[0053] Accordingly, in certain embodiments, the whole blood
collected, for example collected in such a free DNA blood
collection tube, may be processed within about 14 days of
collection, such as within about 10 days, 7 days, 5 days, 4 days, 3
days or 2 days, or between about 30 mins and 24 hours (such as
within about 12 or 8 hours) of collection. Between collection and
processing (for example during storage and/or transport) the sample
may be kept at ambient (such as room) temperature, or may be
maintained at a reduced temperature by refrigeration of use of
cooling materials. Suitable reduced temperatures include about
10.degree. C., 4.degree. C. or lower, such as about 0.degree. C.,
-18.degree. C. or -70.degree. C., or lower such as about
-200.degree. C. (as may be provided by storage in liquid
nitrogen).
[0054] Steps of subsequent processing can include, centrifugation
or other methods to separate intact cells (such as red and
nucleated blood cells) from the biological sample, preparation of
plasma or serum from a blood sample and/or extraction of cell-free
DNA from the biological. Suitable methods for extraction of cell
free DNA, in particular from plasma or serum are described in the
examples herein. For example, the QIAamp Circulating Nucleic Acid
and/or DNeasy Blood and Tissue extraction product series of Qiagen,
as well as automated systems for DNA extraction such as the
QiaSymphony (Qiagen), Chemagen 360 (PerkinElmer). The same,
analogous or modified procedures may be used to subsequently
process other biological fluids, such as urine, tears, breast
aspirate or vaginal swabs, to isolate cell-free DNA therefrom.
[0055] The biological sample from the woman comprises cell-free DNA
of said woman. The term "cell-free DNA" (or "cfDNA") is art
recognised, and includes the meaning of DNA that is found outside
of a cell, such as in a biological fluid (eg blood, or a blood
fraction) of an individual. In particular embodiments, the
cell-free DNA may be circulating.
[0056] "Circulating" is also an art-recognised term, and includes
the meaning that an entity or substance (eg cfDNA) is present in,
detected or identified in, or isolated from, a circulatory system
of the individual, such as the blood system or the lymphatic
system. In particular, when cfDNA is "circulating" it is not
located in a cell, and hence may be present in the plasma or serum
of blood, or it may be present in the lymph of lymphatic fluid.
[0057] The cell-free DNA present in the biological sample may arise
from different sources (ie, tissues or cells) present in or of the
woman. For example, cfDNA may derive from nucleated (such as white)
blood cells and/or other "normal" cells of the body such as
dead/dying (or apoptotic/necrotic) epithelial or other cells. Such
cfDNA can be deemed "somatic" cfDNA as it is derived from cells
that are assumed to comprise a normal genomic complement, genetic
and epigenetic make up of the woman. In addition to such somatic
cfDNA, the biological sample may contain cfDNA derived from other
sources, and hence the total cfDNA present in, or extracted from,
the biological sample (such as plasma or serum) may be an admixture
of cfDNA derived from two or more different sources, each source
providing cfDNA which may have a different genomic complement
and/or genetic or epigenetic make up. In the present invention, the
determination of the presence or absence (or response to therapy
against) of ovarian cancer in a woman is based on a differential
epigenetic make up--as described for the first time herein for the
DMRs of the present invention--of cfDNA derived from cells of the
ovarian cancer compared to that of the somatic cfDNA present in the
biological sample. cfDNA derived from a tumour (such as an ovarian
cancer cell) can be described as circulating tumour DNA ("ctDNA").
In the present invention, the determination of the presence or
absence (or response to therapy against) of ovarian cancer is based
on the (eg single-molecule) analysis of certain epigenetic markers
present on the cfDNA derived from ovarian cancer. However, the
cfDNA present in the biological sample may contain cfDNA from
sources other than, or in additional to, cfDNA derived from ovarian
cancer. For example, the cfDNA may comprise an admixture of somatic
cfDNA of the woman, and cfDNA derived from one or more other
cancers (or tumorous tissues/cells) that may be present in the
woman (such as breast cancer). Furthermore, if the woman is
pregnant, the cfDNA may comprise cfDNA derived from the foetus
and/or the placenta of such foetus (Lo et al 1997, Lancet 350:485),
or if the woman has received a tissue, cell or blood
transplant/transfusion donated by another individual, the cfDNA of
the woman may comprise DNA derived from the cells of such other
individual.
[0058] The amount of total cfDNA isolated from a biological sample,
in particular from a blood or blood-fraction sample, may differ
from woman to woman and sample to sample (such as, dependent on the
storage, transport, temperature and other environmental conditions
the samples is subjected to, as described in the example). For
example, between about 0 ng (ie, absence, or essentially absent)
and 5000 ng cfDNA per mL of plasma/serum, such as about between
about 2 ng/mL or 10 ng/mL and about 2000 or 1000 ng/mL, in
particular between about 2 ng/mL or 10 ng/mL and about 500 ng/mL or
between about 15 ng/mL or 20 ng/mL or 30 ng/ml or 40 ng/ml or 50
ng/mL and about 500 ng/ ml, 400 ng/mL or 300 mg/mL or 250 mg/mL or
200 mg/mL, such as (eg in cases when blood is collected in a free
DNA blood collection tube) between about 2 ng/mL or 20 ng/mL and
about 500 ng/mL. In any of such embodiments (in particular, when
the total cfDNA is at an amount of between about 2 ng/mL or 20
ng/mL and about 500 ng/mL), the cfDNA (such as that derived from
the ovarian cancer) comprises at (or more than) about 0.001%,
0.0025%, 0.005%, 0.0075%, 0.01%, 0.025%, 0.05%, 0.075%, 0.1%,
0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 7.5% or 10% of the total
cfDNA, such as between about 0.001% and about 10%, or between about
0.1% and about 10%, or between about 0.5% and about 10%, or between
about 0.5% and about 5%, or between about 1% and about 5%, and/or
the frequency of an epigenetic marker of the present invention
(such as one associated with DMR #141, #204, or #228) is at (or
more than) about one molecule of the epigenetic marker to about 3,
5, 10, 15, 20, 25, 50, 60, 75, 100, 150, 200, 250, 500, 750, 1000,
1500, 2000, 3000, 4000, 5000, 10000 or more than 10000, such as
about 100000 molecules of total cfDNA (or fragments) presenting in
or isolated from such biological sample.
[0059] Cell-free DNA present in, or isolated from, the biological
sample (such as plasma or serum) is, typically, fragmented. In
certain embodiments, the average fragment size of such cfDNA may be
between about 50 bp and 5000 bp, for example between about 50 bp
and 3000 bp, between about 50 bp and 3000 bp, between about 50 bp
and 2000 bp, 50 bp and 2000 bp, such as between about 75 bp and
1000 bp or between about 75 bp and 750 bp, or between about 100 bp
and 300 bp, such as between about 150 bp and 200 bp. The average
fragment size (and amount/concentration) of cfDNA may be determined
by any suitable methodology, as will be apparent to the person of
ordinary skill, including by capillary electrophoresis analysis
and/or size fractional analysis, such as the Fragment Analyzer and
the High Sensitivity Large Fragment Analysis Kit (MTI, USA). Such
characterisation can occur prior to the determination step, as its
outcome may be used to influence the number of molecules to be
analysed and/or the number of those molecules exhibiting the
cancer-specific DNAme marker (as described elsewhere herein).
[0060] The inventors have identified that, for certain of the DMRs
of the present invention, one or more of the CpGs therein have
particular relevance for the association of such CpG's/s'
methylation status to the presence or absence of, or response to
therapy against, an ovarian cancer in a woman. The identify of
those CpGs (each, a "relevant CpG") is underlined in TABLE 1A and
TABLE 1B, as applicable, and the genomic position (hg19) of the
cytosine (C) of each such relevant CpGs for such DMRs of the
present invention is set forth in TABLE 1C.
TABLE-US-00003 TABLE 1C Genome-coordinates of the Cs for each
relevant CpGs of the DMRs of the present invention DMR# Genome
coordinates (hg19) of the Cs for relevant CpGs Class 141 chr5:
178004422-178004427-178004442-178004460-178004468-178004471- Hyper
178004504 204 chr1:
151810811-151810814-151810816-151810828-151810835-151810841- Hyper
151810843-151810845-151810852-151810887-151810890-151810893-151810899-
151810904-151810907-151810909 228 chr2:
219736312-219736317-219736319-219736335-219736343-219736352- Hyper
219736361 144 chr19:
58220440-58220443-58220446-58220460-58220466-58220479-58220482-
Hyper 58220494-58220500-58220513-58220516 123 chr16:
1271180-1271188-1271192-1271202-1271212-1271229-1271239 Hypo 129
chr11: 69054678-69054680-69054700-69054709 Hypo 137 chr12:
132896310-132896312-132896333-132896338-132896351-132896361- Hypo
132896381 148 chr2: 72359633-72359635-72359648-72359652-72359682
Hypo 150 chr7:
156735054-156735067-156735078-156735086-156735093-156735105- Hypo
156735110-156735116-156735118-156735124-156735140 154 chr17:
70112177-70112190-70112193-70112209-70112221-70112237 Hyper 158
chr16:
74441733-74441743-74441771-74441776-74441787-74441793-74441801 Hypo
164 chr4: 174427997-174428007-174428018-174428027 Hyper 176 chr6:
119107242-119107244-119107282-119107287 Hypo 178 chr19:
13215489-13215495-13215499-13215510-13215521 Hyper 180 chr3:
192125900-192125924-192125927-192125938-192125945-192125949 Hyper
186 chr22: 21483289-21483317-21483327-21483329-21483332-21483337
Hypo 188 chr19: 18497159-18497226-18497233-18497239 Hypo 190 chr9:
79629090-79629100-79629103-79629111-79629128-79629130-79629135-
Hyper 79629138 192 chr12:
75601322-75601325-75601331-75601334-75601361-75601368-75601373-
Hyper 75601379-75601385-75601403-75601408 200 chr9:
138999208-138999210-138999213-138999217-138999240-138999242- Hyper
138999264 202 chr1:
2987558-2987560-2987577-2987579-2987581-2987592-2987598-2987604-
Hyper 2987610-2987627-2987629 208 chr8:
55467548-55467576-55467581-55467585-55467592-55467606 Hypo 210
chr12: 123713553 Hyper 213 chr2:
106776975-106776977-106777009-106777015 Hyper 214 chr3:
141516291-141516302-141516317 Hyper 219 chr16: 30484193-30484218
Hyper 222 chr3: 111809470 Hyper 223 chr10: 120489294 Hyper 224
chr11: 1874070-1874086-1874093 Hyper 225 chr7: 142422236 Hyper 226
chr1: 3086485-3086487-3086492-3086494-3086496-3086501-3086509
Hypo
[0061] An epigenetic marker of, or for use in, the present
invention may comprise the presence/absence, as applicable, of
methylation at a CpG associated with (such as located within) any
of the DMRs of the present invention (or within about 2,000
bp--such as within about 200 bp--5' or 3' thereof), and in
particular the presence/absence, as applicable, of methylation at
one of the relevant CpGs associated with a given DMR of the present
invention as set forth in TABLE 1C. However, as set out herein, the
determination of the presence or absence of (or response to therapy
against) an ovarian cancer in a woman may be enhanced if, in
respect of one or more of such DMRs, the methylation status at a
plurality of CpGs for such DMR is determined.
[0062] Accordingly, the present invention specifically includes
embodiments where the methylation status is determined at a number
being two, three, four, five, six, seven, eight, nine, ten, about
12, about 15, about 20, about 25 or more of said CpGs located
within said nucleotide sequence and in particular at such number
of--or up to the maximum number of--any CpGs (or the relevant CpGs
as set forth in TABLE 1C) associated with a given DMR of the
present invention. In such embodiments, the presence in at least
one of said cell-free DNA molecules of at least one, up to the
respective said number (such as a number between about three and
about fifteen) of methylated CpGs or un-methylated CpGs (as
applicable) located within one or more of said nucleotide sequences
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman.
[0063] For example, the present invention includes embodiments
where the methylation status is determined at two, three, four,
five, six, seven, eight, nine, ten, 11, 12, 13, 14 or 15 CpGs (such
as between about four and about ten) located within a
hyper-methylated DMR (eg, those set forth in TABLE 1A) of the
present invention (or associated therewith, such as within about
2,000 bp--such as within about 200 bp--5' or 3' thereof), and
wherein the presence of at least one methylated such CpG associated
with such a hyper-methylated DMR of the invention, such as all such
CpGs or at least all of the applicable relevant CpGs in such DMR as
set forth in TABLE 1C (such as at all two, three, four, five, six,
seven, eight, nine, ten, 11, 12, 13, 14 or 15, up to the maximum
number of such CpGs associated with said DMR), indicates the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman. Preferably, in such embodiments, the presence
of methylation at all of the relevant CpGs (see TABLE 1C) for a
given hyper-methylated DMR (eg, those set forth in TABLE 1A)
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman.
[0064] As another example, the present invention also includes
embodiments where the methylation status is determined at two,
three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14 or
15 CpGs (such as between about four and about ten) located within a
hypo-methylated DMR (eg, those set forth in TABLE 1B) of the
present invention (or associated therewith, such as within about
2,000 bp--such as within about 200 bp--5' or 3' thereof), and
wherein the absence of at least one methylated such CpG associated
with such a hypo-methylated DMR of the invention, such as all such
CpGs or at least all of the applicable relevant CpGs in such DMR as
set forth in TABLE 1C (such as at all two, three, four, five, six,
seven, eight, nine, ten, 11, 12, 13, 14 or 15, up to the maximum
number of such CpGs associated with said DMR), indicates the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman. Preferably, in such embodiments, the absence
of methylation at all of the relevant CpGs (see TABLE 1C) for a
given hypo-methylated DMR (eg, those set forth in TABLE 1B)
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman.
[0065] As will be apparent to the person of ordinary skill, the
maximum number of CpGs for which such determination of methylation
status may be made will be the number of CpGs located within the
sequence that is analysed. For example, for DMR #141 (SEQ ID No.
1), the wild-type sequence shows that 7 CpGs are located therein
(of which all 7 are underlined in TABLE 1A, are further identified
in TABLE 1C and are the relevant CpGs for such DMR), and hence such
number of CpGs would represent a maximum number of CpGs in respect
of such sequence for which the methylation status may be determined
and hence used to investigate the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
[0066] The status of methylation at any of such CpGs may be
determined to be absent (un-methylated) or to be present
(methylated), such as in the form of methylcytosine and/or
hydroxymethylcytosine and/or formylcytosine, in particular
5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) or
5-formylcytosine (5fC) (Li & Liu, 2011; Journal of Nucleic
Acids, article ID 870726. Ito et al, 2011; Science 333:1300). In
one embodiment, the present invention relates to the determination
of the 5-methylation and/or 5-hydroxymethylcytosine status at one
or more Cs in said CpGs, wherein the presence of one or more
5-methylcytosine and/or 5-hydroxymethylcytosine in the CpGs (in
particular, in the relevant CpGs for a DMR, as set out in TABLE 1C)
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman. Any of those CpGs
investigated which are determined not to comprise 5-methylcytosine,
are typically un-methylated cytosine, but in certain embodiments
one or more may comprise modifications other than 5-methylcytosine,
such as 5hmC or 5fC. Investigation of the respective modification
comprised in CpGs is art know, including methylation-sensitive
restriction enzyme or bisulphite conversion/analysis (eg for 5mC
and/or 5hmC) and reduced bisulphite sequencing (redBS-Seq) (eg, for
5fC), as may be conducted by or products obtained from Cambridge
Epigenetix Ltd (UK). Alternatively, single-molecule DNA
sequencing/analysis techniques (such as those utilised in the
PacBio or Nanopore instruments described elsewhere herein) may be
used to determine the status and form of the methylation present at
the CpGs that are interrogated as part of the present
invention.
[0067] As well as the present invention including embodiments were
the methylation status at one or more CpGs (in particular, at one
or more relevant CpGs) is determined located within a single DMR of
the present invention (or associated therewith, such as within
about 2,000 bp--such as 200 bp--5' or 3' thereof), for example a
DMR selected from the group consisting of #141, #204 and #228 (SEQ
ID NOs.: 1, 2 and 3, respectively), it specifically envisions other
embodiments wherein such CpGs are located within a plurality of
DMRs of the present invention (or associated therewith, such as
within about 2,000 bp--such as within 200 bp--5' or 3' thereof).
Also as described herein, the determination of the presence or
absence of (or response to therapy against) an ovarian cancer in a
woman may be enhanced if, in respect of such plurality DMRs, the
methylation status of at least one (or more) CpGs of such DMRs is
determined, especially those embodiments where in respect of each
of such plurality of DMRs, the methylation status of a plurality of
CpGs (such as two, three, four, five, six, seven, eight, nine, ten
or more than ten, such as 11, 12, 13, 14 or 15, or between about
four and about ten), and in particularly of the applicable relevant
CpGs for a DMR, is determined.
[0068] Accordingly, in certain embodiments of the present
invention, the methylation status at one or more CpGs (in
particular, of the applicable relevant CpGs) located within a
number of two, three, four, five, six, seven, eight, nine, ten or
more than ten (such as 11, 12, 13, 14 or 15, or between about four
and about ten) of said nucleotide sequences (in particular, within
at least two, three or four nucleotide sequences) is determined;
wherein, the presence in at least one of said cell-free DNA
molecules of one or more (such as between about four and about
ten): (i) methylated such CpGs (eg the relevant CpGs set out in
TABLE 1C) located within one or more of said nucleotide sequences
of the hyper-methylated DMRs (eg, those of TABLE 1A); and/or (ii)
un-methylated such CpGs (eg the relevant CpGs set out in TABLE 1C)
located within one or more of said nucleotide sequences of the
hypo-methylated DMRs (eg, those of TABLE 1B), indicates the
presence of, or a reduced response to therapy, against an ovarian
cancer in said woman. In certain of such embodiments, one or more
other CpGs within the nucleotide sequence(s) may be either
methylated or un-methylated (or their methylation status
undetermined), wherein said pattern of methylation can also
indicate the presence of (or reduced response to therapy against)
ovarian cancer in said woman. For example, one pattern of
methylation that indicates the presence of (or reduced response to
therapy against) ovarian cancer in said woman may be for a
hyper-methylated DMRs (eg, those of TABLE 1A) that all (or all but
one, two or three) of the relevant CpGs set out in TABLE 1C for
such DMR are methylated, and that all other CpGs are either
methylated or un-methylated (or their methylation status is
undetermined); ie that for DMR #228, a pattern of methylation of
linked CpGs therein of "X111X111" indicates the presence of (or
reduced response to therapy against) ovarian cancer in said woman
(where "1" represents the presence of a methylated CpG and "X"
represents the presence of either a methylated or an un-methylated
CpG; and the relative position of the non-relevant CpG for #228).
In another example, one pattern of methylation that indicates the
presence of (or reduced response to therapy against) ovarian cancer
in said woman may be for a hypo-methylated DMRs (eg, those of TABLE
1A) that all (or all but one, two or three) of the relevant CpGs
set out in TABLE 1C for such DMR are un-methylated, and that all
other CpGs are either methylated or un-methylated (or their
methylation status is undetermined). In certain embodiments of the
present invention, the pattern of methylation/un-methylation for a
given DMR that is associated with the presence of (or reduced
response to therapy against) ovarian cancer in said woman is shown
in Table 2B.
[0069] The present invention additionally provides for particularly
advantageous nucleotide sequences associated with one or more CpGs
(in particularly at least one of the applicable relevant CpGs), the
methylation status of which is associated with the presence or
absence of, or response to therapy against, an ovarian cancer in a
woman. As described above, such nucleotide sequences include those
selected from the group consisting of DMR#: #141, #204, #228, #144,
#123, #129, #137, #148, #150, #154, #158, #164, #176, #178, #180,
#186, #188, #190, #192, #200, #202, #208,#210, #213, #214, #219,
#222, #223, #224, #225 and #226; in particular #141 and/or #204
and/or #228 and/or #144 and/or #154 and/or #158 and/or #186 and/or
#188 and/or #202 , such as preferably #141 and/or #204 and/or #228
(SEQ ID NOs: 1, 2 and 3, respectively), or in certain other
embodiments a nucleotide sequence present within about 2,000 bp
(such as within about 200 bp) 5' or 3' thereof, and alternatively,
in each case, an allelic variant and/or a complementary sequence of
any of said nucleotide sequences.
[0070] Accordingly, in a particular embodiment, the method of the
present invention said nucleotide sequence(s) is/are #141 and/or
#204 and/or #228 (SEQ ID NOs: 1, 2 and/or 3); for example, at least
one of said nucleotide sequences is #141; or an allelic variant
and/or complementary sequence of any of said nucleotide sequences.
In an alternative embodiment, the method of the present invention
said nucleotide sequence is #144 (SEQ ID NO 4), or an allelic
variant and/or complementary sequence of any of said nucleotide
sequences; which embodiment may also be include the determination
of methylation status at CpGs (in particularly at the applicable
relevant CpGs) located in one or more of the nucleotide sequence
#141 and/or #204 and/or #228 (SEQ ID NOs: 1, 2 and/or 3). As set
out elsewhere, also envisioned are embodiments where the CpGs are
located in a nucleotide sequence present within about 2,000 bp
(such as within about 200 bp) 5' or 3' of each or any of the
respective DMRs. In particular of such embodiments, said nucleotide
sequences and the relevant CpGs thereof are, respectively, those
set out in TABLE 1D, or an allelic variant and/or complementary
sequence of any of said nucleotide sequences.
TABLE-US-00004 TABLE 1D Genomic sequences of the non-primer portion
of particular DMRs of the present invention Marker Genome
coordinates SEQ coordinates Marker genomic sequence (hg19) of the
Cs for ID DMR# (hg19) (relevant CpGs are underlined)] relevant CpGs
NO. 141 chr5:178004422- CGCCACGGGAAGGAGGCACACGATTCAGCCCA
chr5:178004422- 156 178004505 TGACACCGCCACCTCGGCGTGGTGCTGTAGGG
178004427-178004442- GGAAGCTCAGGCACTCACCG 178004460-178004468-
178004471-178004504 204 chr1:151810811-
CGCCGCGGGGCCCCAGGCGCAGCACGCTCTCG chr1:151810811- 157 151810917
CGCGTGGGCCGCAGCTGGCAGCACAGGAAGTC 151810814-151810816-
CAGGTGGAAGAGCGGCGGCGTGGGCGGCCCGG 151810828-151810835- CGCGGCGCGGC
151810841-151810843- 151810845-151810852- 151810887-151810890-
151810893-151810899- 151810904-151810907- 151810909 228
chr2:219736301- CGGCTGCCAGGCGCCCCGCGGGCGGGCCCCTC chr2:219736312-
158 219736362 CCCGGCCCTCCGGCCTGCCCGGCACCCCCG 219736317-219736319-
219736335-219736343- 219736352-219736361 144 chr19:58220438-
GGCGGCGTCGCCAAGGCTTAGACGCTTTCGTG chr19:58220440-58220443- 159
58220517 CAGGAGGGACGACGACTCCCCTCACGCCTTCG 58220446-58220460-
TGGCCCCAACTCGGCG 58220466-58220479- 58220482-58220494-
58220500-58220513- 58220516
[0071] As shown in the examples, a particularly useful test is
provided by those embodiments of the present invention in which the
methylation status may be determined at one or more CpGs associated
with each of a plurality of the DMRs described herein. For example,
more than one (such as two, three, four, five, six, seven, eight,
nine or ten) of such nucleotides sequence may be investigated for
the presence or absence of methylated CpGs located therein. In
particular, where at least one methylated CpG (such as a plurality
of methylated GpCs)--in particular of the respective relevant
CpGs--is determined in any one of the plurality of such nucleotide
sequences associated with a hyper-methylated DMR analysed (such as
DMR #141, #204, #228 and/or #144), then a determination may be made
that ovarian cancer is present in said woman, or that ovarian
cancer in a woman is not responding to (chemo)therapy.
Alternatively, where at least one un-methylated CpG (such as a
plurality of un-methylated GpCs)--in particular of the respective
relevant CpGs--is determined in any one of the plurality of such
nucleotide sequences associated with a hypo-methylated DMR analysed
(such as those set forth in TABLE 1B), then a determination may be
made that ovarian cancer is present in said woman, or that ovarian
cancer in a woman is not responding to (chemo)therapy. As will also
be understood, the plurality of nucleotide sequences analysed for
the presence or absence of methylated CpGs located therein may
include at least one nucleotide sequence associated with a
hyper-methylated DMR (such as those set forth in TABLE 1A, and in
particular DMR #141, #204, #228 and/or #144 as set forth in TABLE
1D) and at least one nucleotide sequence associated with a
hypo-methylated DMR (such as those set forth in TABLE 1B), wherein
the determination of at least one methylated CpG (such as a
plurality of methylated GpCs)--in particular of the respective
relevant CpGs--located in said hyper-methylated DMR and/or the
determination of at least one un-methylated CpG (such as a
plurality of un-methylated GpCs)--in particular of the respective
relevant CpGs--located in said hypo-methylated DMR may be used to
determine that ovarian cancer is present in said woman, or that
ovarian cancer in a woman is not responding to (chemo)therapy.
[0072] Accordingly, in a particular embodiment of the method of the
present invention the methylation status may be determined at one
or more of said CpGs located within each of the nucleotide
sequences so analysed; wherein, the presence in at least one of
said cell-free DNA molecules of one or more: (i) methylated CpGs
(in particular, the applicable relevant CpGs set forth in TABLE 1C)
located within any one of said nucleotide sequences associated with
the hyper-methylated DMRs (eg as set forth in TABLE 1A); and/or
(ii) un-methylated CpGs (in particular, the applicable relevant
CpGs set forth in TABLE 1C) located within one or more of said
nucleotide sequences associated with the hypo-methylated DMRs (eg
as set forth in TABLE 1B), indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman. For
example, the method includes embodiments where the methylation
status of one or more CpGs (in particular, of the applicable
relevant CpGs set forth in TABLE 1C) located in each of the
nucleotide sequence #141 and #204 and #228 (SEQ ID NOs: 1, 2 and
3)--such as at least one (such as all) of such CpG in each of said
sequences--is determined for at least one molecule of said
cell-free DNA, and wherein the presence in at least one of said
cell-free DNA molecules of one or more methylated CpGs located
within any one of said nucleotide sequences (such as methylation at
all GpCs, or all relevant CpGs, therein, or methylation at all but
one, two or three of such GpCs) indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman. In particular of such embodiments, one or more (such as all)
of said nucleotide sequences (and the applicable relevant CpGs)
are, respectively, those set out in TABLE 1D, or an allelic variant
and/or complementary sequence of any of said nucleotide
sequences.
[0073] One particular feature described in the examples is the
presence of patterns of methylation/un-methylation at CpGs
associated with the same DMR, in particular at the relevant CpGs
for such DMR. Such examples support the assertion that the
investigation, analysis and/or determination of such patterns of
methylation/un-methylation are particularly useful or advantageous
tools for determining presence or absence of, or response to
therapy against, an ovarian cancer in a woman. In particular, to
provide tests that have a performance that enables them to be used
in diagnostic settings; such as having a sensitivity and/or
specificity as set out herein. The number of CpGs associated with a
given DMR for which the methylation status is determined will
depend on the length of nucleotide sequence analysed and the number
of CpGs present therein. For a given nucleotide sequence associated
with (such as within, or within about 2,000 bp--such as within
about 200 bp--5' and/or 3' of) a DMR of the present invention, the
number of CpGs for which the methylation status is determined can
range from two or more (such as three, four and five, up to the
maximum number of CpGs within such nucleotide sequence, and/or up
to about nine, ten, 11, 12, 13, 14, 15, 18, about 20, about 25 or
about 30. In particular embodiments, the methylation status is
determined at a number of between about 5 and about 15 of said CpGs
located within the nucleotide sequence(s), and in particular at
such number of the relevant CpGs for the nucleotide sequence(s)
(eg, as set forth in TABLE 1C). In certain embodiments of the
present invention, the number of (eg relevant) CpGs for which the
methylation status is determined the pattern of
methylation/un-methylation for a given DMR that is associated with
the presence of (or reduced response to therapy against) ovarian
cancer in said woman is show in Table 2B.
[0074] Accordingly, in certain methods of the present invention,
the methylation status may be determined at a number of between
about 2 and about 15 (for example, between about four and about
ten, such as five, six, seven, eight or nine) of said CpGs (in
particular of the relevant CpGs set forth in TABLE 1C) located
within said nucleotide sequence(s)--in particular within the
nucleotide sequence(s) selected from: #141, #204 and/or #228 (SEQ
ID NOs: 1, 2 and/or 3), or an allelic variant and/or complementary
sequence thereof; wherein the presence in at least one of said
cell-free DNA molecules of at least said number of methylated CpGs
(in particular of methylated relevant CpGs set forth in TABLE 1C)
located within any one of said nucleotide sequences indicates the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman.
[0075] In specific of such embodiments, the methylation status may
be determined at about 7 CpGs (in particular, the 7 relevant CpGs)
located within nucleotide sequence #141 (SEQ ID NO 1) and/or at
about 16 CpGs (in particular, the 16 relevant CpGs) located within
nucleotide sequence #204 (SEQ ID NO 2) and/or at about 7 CpGs (in
particular, the 7 relevant CpGs) located within nucleotide sequence
#228 (SEQ ID NO 3) and/or at about 11 CpGs (in particular, the 11
relevant CpGs) located within nucleotide sequence #144 (SEQ ID NO
4)--for example those CpGs (in particular, the applicable relevant
CpGs) located within SEQ ID NOs: 156, 157, 158 and/or 159,
respectively--or in each case an allelic variant and/or
complementary sequence of any of said nucleotide sequences. In
particular embodiments, the methylation status of all CpGs, or at
least all of the relevant CpGs, in the given nucleotide sequence
may be determined. In one embodiment, the presence of methylation
at all of said CpGs in a given nucleotide sequence for the
hyper-methylated DMRs #141, #204, #228 and/or #144 can indicate the
presence of (or reduced response to therapy against) ovarian cancer
in said woman. In other embodiments, the presence of methylation at
all but one, two or three of said CpGs in one or more of said
nucleotide sequence can indicate the presence of (or reduced
response to therapy against) ovarian cancer in said woman. For
example, all of the relevant CpGs for DMR #141, #204, #228 and/or
#144 may be determined to be methylated, and one or more other CpGs
therein they may be either methylated or un-methylated (or their
methylation status undetermined), wherein said pattern of
methylation can also indicate the presence of (or reduced response
to therapy against) ovarian cancer in said woman. In certain
embodiments of the present invention, the number of (eg relevant)
CpGs for which the methylation status is determined the pattern of
methylation/un-methylation for DMR #141, #204, #228 and/or #144
that is associated with the presence of (or reduced response to
therapy against) ovarian cancer in said woman is show in Table
2B.
[0076] As will now be apparent to the person of ordinary skill, and
as shown in the examples within a clinical setting to be superior
and/or complementary to a CA125 test (FIGS. 3E and 3F), one
particular embodiment of the present invention is based on the
analysis and determination of the methylation pattern of three sets
of CpGs (in particular, of the applicable relevant CpGs), each set
associated with the respective one or three DMRs: #141, #204 and
#208; wherein the presence of a (marker) methylation pattern in any
one of said DMRs determines the presence or absence of, or response
to therapy against, an ovarian cancer in a woman. Therefore, one
specific embodiment of the method of the present invention includes
where the methylation status is determined at about 7 CpGs (in
particular, the 7 relevant CpGs) located within nucleotide sequence
#141 (SEQ ID NO 1) and at about 16 CpGs (in particular, the 16
relevant CpGs) located within nucleotide sequence #204 (SEQ ID NO
2) and at about 7 CpGs (in particular, the 7 relevant CpGs)located
within nucleotide sequence #228 (SEQ ID NO 3)--for example located
within SEQ ID NOs: 156, 157 and/or 158, respectively--or in each
case an allelic variant and/or complementary sequence of any of
said nucleotide sequences; wherein, the presence in at least one of
said cell-free DNA molecules of at least said number of methylated
said CpGs (in particular, said relevant CpGs) located within any
one of said nucleotide sequences indicates the presence of, or a
reduced response to therapy against, an ovarian cancer in said
woman. As described above, in certain of such embodiments other
CpGs within such nucleotide sequence may (or may not) be analysed
for their methylation status; and if their methylation status is
determined then they such CpGs may be determined to be either
methylated or un-methylated, and the presence of, or a reduced
response to therapy against, an ovarian cancer may be determined in
said woman.
[0077] For one example of such embodiment, the determination of the
presence of methylation (in at least one of said cell-free DNA
molecules, such as more than 10, 20, 50, 100, 500 or 1000, or
another number as set out below) at all 7 of the relevant CpGs
located within nucleotide sequence #141 (SEQ ID NO 1) indicates the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman, regardless of the methylation status of any
other CpG therein and/or regardless of the methylation status of
CpGs located within nucleotide sequence #204 (SEQ ID NO 2) or
nucleotide sequence #228 (SEQ ID NO 3). As a first alternative
example of such embodiment, the determination of the presence of
methylation (in at least one of said cell-free DNA molecules, such
as more than 10, 20, 50, 100, 500 or 1000, or another number as set
out below) at all 16 of the relevant CpGs located within nucleotide
sequence #204 (SEQ ID NO 2) indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman,
regardless of the methylation status of any other CpG therein
and/or regardless of the methylation status of CpGs located within
nucleotide sequence #141 (SEQ ID NO 1) or nucleotide sequence #228
(SEQ ID NO 3). As a second alternative example of such embodiment,
the determination of the presence of methylation (in at least one
of said cell-free DNA molecules, such as more than 10, 20, 50, 100,
500 or 1000, or another number as set out below) at all 7 of the
relevant CpGs located within nucleotide sequence #228 (SEQ ID NO 3)
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman, regardless of the
methylation status of any other CpG therein and/or regardless of
the methylation status of CpGs located within nucleotide sequence
#141 (SEQ ID NO 1) or nucleotide sequence #204 (SEQ ID NO 2). As
will be now be appreciated, in an alternative embodiment, the
presence of, or a reduced response to therapy against, an ovarian
cancer in said woman may be also indicated when the presence of
methylation is determined at all of the relevant CpGs located
within each of nucleotide sequences 141 (SEQ ID NO 1), #204 (SEQ ID
NO 2) and #228 (SEQ ID NO 3). In such an alternative embodiment
(and as described elsewhere herein), the number of cell-free DNA
molecules in which any of such methylation patterns is determined
may be less than if only one of such DMRs is found to have such a
methylation pattern. In certain embodiments of the present
invention, the number of (eg relevant) CpGs for which the
methylation status is determined the pattern of
methylation/un-methylation for DMR #141, #204, #228 and/or #144
that is associated with the presence of (or reduced response to
therapy against) ovarian cancer in said woman is show in Table
2B.
[0078] In any of the embodiments of the present invention, the
biological sample can be further processed, such as comprising a
step of isolating cell-free DNA therefrom. Such isolation can
include particular steps of centrifugation (such as density
gradient ultracentrifugation, Jonathan et al (2015), J Cancer Prey
Curr Res 3:00064), treatment with ionic solutions and/or organic
solvents to selectively solubilise/precipitate nucleic acids (such
as cell-free DNA), addition of (cell-free DNA) selective binding
and separation moieties (such as magnetic beads) and/or filtration
or chromatographic steps; and in particular, steps of lysis of
sample, absorption to a silica membrane (or column or beads),
removal of residual contaminates and/or election of pure nucleic
acids, such as cell-free DNA. Other methods of cfDNA isolation can
include rapid electrokinetic isolation directly from blood
(Sonnenberg et al (2014), Clin Chem 60:500).
[0079] In certain of such embodiments, the biological sample (such
as plasma or serum) may be processed to isolate the cell-free DNA
generally according to a process as set out in FIG. 5. For
example:
[0080] Lysing samples: Free-circulating nucleic acids in biological
fluids are usually bound to proteins or enveloped in vesicles,
which may utilise an efficient lysis step in order to release
nucleic acids for selective binding to the column (or to Solid
Phase Reversible Immobilisation--SPRI--[paramagnetic] beads).
Hence, samples may be lysed under highly denaturing conditions at
elevated temperatures in the presence of proteinase K and
appropriate buffer, such as Buffer ACL from Qiagen (cat. no.
19076), which together provide for inactivation of DNases and
RNases and complete release of nucleic acids from bound proteins,
lipids, and vesicles.
[0081] Adsorption to a silica membrane: Binding conditions can be
adjusted by adding an appropriate buffer, such as Buffer ACB
(Qiagen) to allow binding of the circulating nucleic acids to the
silica membrane. Lysates may then then be transferred onto a
separation column (such as the QIAamp Mini column, Qiagen), and
circulating nucleic acids adsorbed from a large volume onto the
small silica membrane as the lysate is drawn through by vacuum
pressure. Appropriate salt and pH conditions can ensure that
proteins and other contaminants, which can inhibit PCR and other
downstream enzymatic reactions, are not retained on the separation
column. A vacuum manifold (e.g., the QIAvac 24 Plus with the QIAvac
Connecting System) and a vacuum pump capable of producing a vacuum
of -800 to -900 mbar (e.g., QIAGEN Vacuum Pump) may be used for the
protocol. A vacuum regulator can be used for easy monitoring of
vacuum pressures and convenient vacuum release.
[0082] Removal of residual contaminants: Nucleic acids remain bound
to the membrane, while contaminants can be efficiently washed away
during a plurality of wash steps, such as 2 or 3 wash steps. In a
single step, highly pure circulating nucleic acids can be eluted in
an appropriate buffer (such as in Buffer AVE, Qiagen), equilibrated
to room temperature.
[0083] Elution of pure nucleic acids: Elution can be performed
using Buffer AVE. The elution volume may be 50 ul (or greater, such
as 100 ul or 150 ul). If higher nucleic acid concentrations are
required, the elution volume can be reduced, such as by using 20 ul
(or 50 ul). Low elution volume leads to highly concentrated nucleic
acid eluates. For downstream applications that use small starting
volumes (e.g., some PCR and RT-PCR assays), a more concentrated
eluate may increase assay sensitivity. For downstream applications
that use a larger starting volume, the elution volume can be
increased up to 150 ul. However, an increase in elution volume can
decrease the concentration of nucleic acids in the eluate. Eluted
nucleic acids can collected in 1.5 ml microcentrifuge tubes or in
microtitre plates. If the purified circulating nucleic acids are to
be stored for up to 24 hours, they can be stored at 2-8.degree. C.;
and/or for storage longer than 24 hours at -15 to -30.degree.
C.
[0084] In certain embodiments of the present invention, the
cell-free DNA may be subjected to an agent that differentially
modifies said DNA based on the methylation status of one or more of
the CpGs. Such a modification can facilitate the detection of
differences in the methylation status. However, as described
elsewhere herein, methods are available to detect differences in
the methylation status of CpGs without use of such a modifying
agent.
[0085] Accordingly, the present invention also includes those
embodiments of the method that include a step of treating said DNA
with an agent that differentially modifies said DNA based on the
methylation status of one or more CpGs located within. Such agents
will be known to the person of ordinary skill and include the use
of one or more methylation sensitive restriction enzyme and/or of a
bisulphite-based reaction. The use of bisulphite or
methylation-sensitive restriction enzymes to study differential
methylation will be well known to the person of ordinary skill, who
may apply teachings of standard texts or adaptation of published
methods such as Poon et al (2002), Nygren et al (2010) or
Yegnasubramanian et al (2006, Nuc Acid Res 34:e19).
[0086] A methylation sensitive is a restriction enzyme that is
sensitive to the DNA methylation states. Cleavage of such a
restriction enzyme's recognition sequence may be blocked, or
impaired, when a particular base in the enzyme's recognition site
is modified, eg methylated. In particular embodiments of all
aspects of the invention, the agent comprises a
methylation-sensitive restriction enzyme, such as a
methylation-sensitive restriction enzyme disclosed herein;
including such embodiments that comprise two, three, four, five or
more of such methylation-sensitive restriction enzymes. In
particular embodiments, the reagent agent comprises: at least one
methylation sensitive enzyme; at least one methylation sensitive
restriction enzyme; and/or an agent selected from the group
consisting of: AatII, AciI, AcII, AfeI, AgeI, AgeI-HF, AscI, AsiSI,
AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI. BsiWI, BsmBI, BspDI,
BsrFI, BssHII, BstBI, BstUI, ClaI, EagI, FauI, FseI, FspI, HaeII,
Hgal, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, Kasl, MluI, NaeI,
NarI, NgoMIV, NolI, NotI-HF, NruI, Nt.BsmAI, Nt.CviPII, PaeR7I,
PIuTI, PmII, PvuI, PvuI-HF, RsrII, SacII, Sall, SaII-HF, Sfol,
SgrAI, Smal, SnaBI, TspMI and Zral. In particular embodiments, said
reagent is one selected from the group consisting of: BstUI, HhaI
and HpaII.
[0087] Treatment of DNA with an agent comprising bisulphite
(bisulfite) converts un-methylated cytosine residues to uracil, but
leaves 5-methylcytosine residues unaffected. Thus, bisulphite
treatment introduces specific changes in the DNA sequence that
depend on the methylation status of individual cytosine residues,
yielding single nucleotide resolution information about the
methylation status of a segment of DNA. Various analyses can be
performed on the altered sequence to retrieve this information,
including the use of PCR primers and/or probes and/or sequencing
that can distinguish between such singe-nucleotide changes. As
described above, other agents that may uses in methods to determine
methylation at CpGs include oxidative bisulphite (eg for analysis
of 5hmC).
[0088] Bisulphite modification may be conducted using eg the EZ-96
DNA methylation kit (Zymo Research), and/or may include the steps
of adding an effective amount of a bisulphite reagent to each
sample, and incubating (eg in the dark) at about 50.degree. C. for
12-16 hours (e.g., using a thermal cycler). After such incubation,
prior to analysis, a step of incubating the sample at 0-4.degree.
C. (e.g., on ice or using a thermal cycler) for 10 minutes may be
included.
[0089] In particular embodiments of the present invention, said
agent may be bisulphite and said determining step may comprise the
detection of at least one bisulphite-converted cytosine (such as
one in a CpG) within one or more of the nucleotide sequences
selected from the group consisting of a sequence produced or
producible following bisulphite conversion of a sequence comprised
within a DMR selected from the group consisting of DMR#: #141,
#204, #228, #144, #123, #129, #137, #148, #150, #154, #158, #164,
#176, #178, #180, #186, #188, #190, #192, #200, #202, #208,#210,
#213, #214, #219, #222, #223, #224, #225 and #226; such as a
sequence consisting of at least about 10 contiguous bases
(preferably at least about 15 contiguous bases for any SEQ ID other
than SEQ ID NO: 58) comprised in a sequence selected from the group
consisting of SEQ ID NOs (see TABLE 2A): 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61 and 62 (such as SEQ ID NO: 32, 33, 34
and/or 35, or in particular of SEQ ID NOs: 32 and 33 and 34,
optionally also 35), or an allelic variant and/or complementary
sequence of any of said nucleotide sequences. In particular
embodiments, said number of contiguous bases is at least about 16,
18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 110, 120, 10, 140, 150 or 160 bases, such as
between about 80 and about 160, such as between about 100 and about
160, in each case, independently, up to the maximum number of bases
within the sequence selected from said group; wherein one or more
of the bases identified by "Y" therein is a U or T (in particular,
of a C within a CpG, such as of a relevant CpG of a hypo-methylated
DMR) and, preferably, where one or more of another of the bases
identified by "Y" therein is a C (in particular, of a C within a
CpG, such as of a relevant CpG of a hyper-methylated DMR), or an
allelic variant and/or complementary sequence of any of said
nucleotide sequences. In particular embodiments, two, three, four,
five, six, seven, eight, nine, ten or more than ten of the bases
identified by "Y" therein is a U or T (preferably T), such as all
of the bases identified by "Y" therein is a U or T (preferably T),
or all but one, two, three, four, five, six, seven, eight, nine,
ten or more than ten of the bases identified by "Y" therein is a U
or T (preferably T), in particular where such other one, two,
three, four, five, six, seven, eight, nine, ten or more than ten of
the bases identified by another "Y" is a C within a CpG, such as of
a relevant CpG of a hyper-methylated DMR).
[0090] In particular of such embodiments, the method of the present
invention may also include those wherein said agent is bisulphite
and said determining step comprises the detection of at least one
(non-natural) bisulphite-converted cytosine within a nucleotide
sequence having a length of at least about 15 bp comprised in a
bisulphite conversion of a sequence comprised within DMR #141
and/or #204 and/or #228 (SEQ ID NOs: 32, 33 and/or 34,
respectively), wherein one or more of the bases identified by "Y"
therein is a U or T and, preferably, where one or more of the bases
identified by "Y" (in particular, those within a CpG, such as one
or more (or all) of a relevant CpG) therein is a C, or an allelic
variant and/or complementary sequence of any of said nucleotide
sequences. Such a nucleotide sequence may be at least about 16 bp,
18 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60
bp or 70 bp, for example up to about 90 bp, 100 bp or 150 bp, such
as between about 100 bp and about 160 bp.
[0091] In particular embodiments, the sequence detected may be one
comprised in a bisulphite conversion of a sequence comprised within
DMR #141 and/or #204 and/or #228 and/or #144 (SEQ ID NOs: 32, 33,
34 and/or 35, respectively), such as SEQ ID NOs: 32 or 33 or 34,
wherein all (or all but one, two, three, four or five) of the
cytosines of CpGs located (in particular of the relevant CpG) in
the analysed sequence are detected as a C (ie, such cytosines are
determined to be methylated) and all other cytosines located in the
analysed sequence (in particular, the cytosines not of a CpG
therein) are detected a U or T (ie, such other cytosines/CpGs are
determined to be un-methylated).
[0092] As will now be apparent, such a detected BS converted
sequence will be a non-natural sequence, as any un-methylated
cytosine (such as one outside a CpG) will have been converted to U
by the bisulphite treatment, and detected as either a U or a T.
[0093] In other certain embodiments of the present invention, the
methylation status of the one or more the CpGs present in the
cell-free DNA may be determined without use of an agent that
differentially modifies said DNA based on such methylation. For
example, single molecule sequencing/analysis of DNA may be used to
determine such methylation status. Examples of such technologies
include those utilised in the: (1) PacBio instruments (Pacific
Biosciences) that use Single Molecule, Real-Time (SMRT) sequencing,
based on zero-mode waveguides (ZMWs) and phospholinked nucleotides.
ZMWs allow light to illuminate only the bottom of a well in which a
DNA polymerase/template complex is immobilized. Phospholinked
nucleotides allow observation of the immobilized complex as the DNA
polymerase produces a completely natural DNA strand. Such
instruments can be used for the direct detection of epigenetic
modifications (Mol Genetics & Genomics, 2016; 291:1491); and
(2) Nanopore instruments (Oxford Nanopore) that use nanopored
membranes to detect variations in current flow, characteristic to
the base/modified-base, as single strands of DNA pass through such
nanopore (Nature 467:190). Accordingly, in certain embodiments of
the present invention the methylation status at said one or more
CpGs is determined using single molecule DNA sequencing or
analysis, such as by SMRT or nanopore sequencing.
[0094] Analysis of the cell-free DNA present or isolated from the
biological sample may, in some embodiments, be subjected to an
amplification process, for example prior to or as part of the step
of determining the methylation status of the one or more CpGs
associated with the DMR.
[0095] Accordingly, certain embodiments of the present invention
may include a method that also comprises a step of amplifying one
or more regions of said cell-free DNA to produce DNA prior to or as
part of said determining step, and preferably after any optional
step of treating with said agent. If more than one region of
cell-free DNA is to be amplified, this may occur as a multiplex or
pool (eg, conducted in a single mixed reaction), or each region may
be amplified separately and/or individually, with the possibility
that such independent amplified regions are subsequently mixed or
pooled so enable pooled and/or multiplex analysis thereon. As will
be apparent, any regions of DNA so amplified, in particular those
amplified in in-vitro processes, will be synthetic (of in-vitro
produced) DNA molecules.
[0096] Amplification of regions of cell-free DNA may occur by any
suitable method, including polymerase chain reaction (PCR) and
rolling circle amplification. Those embodiments of the present
invention that comprise PCR amplification can further comprises
specific steps that are related to the practice of PCR, such as any
of those described herein, or in particular the steps of: (A)
providing a reaction mixture comprising a double-stranded target
DNA, a pair of primers (for example, a pair of primers disclosed
herein) designed to amplify a region of such DNA (such as a DMR as
described herein) wherein the first primer is complementary to a
sequence on the first strand of the target DNA and the second
primer is complementary to a sequence on the second strand of the
target DNA, Taq polymerase, and a plurality of free nucleotides
comprising adenine, thymine, cytosine and guanine; (B) heating the
reaction mixture to a first predetermined temperature for a first
predetermined time to separate the strands of the target DNA from
each other; (C) cooling the reaction mixture to a second
predetermined temperature for a second predetermined time under
conditions to allow the first and second primers to hybridise with
their complementary sequences on the first and second strands of
the target DNA, and to allow the Taq polymerase to extend the
primers; and (D) repeating steps (B) and (C) at least 20 times. The
person of ordinary skill will readily be able to design such PCR
primers for use in the method of the invention, for example by use
of primer design algorithms and programs such as Clone Manager
Professional 9 (Sci-Ed Software), Vector NTI (Life Technologies),
or web-based tools such as those found from
www.ncbi.nlm.nih.gov/tools/primer-blast/ or
molbiol-tools.ca/PCR.htm.
[0097] In embodiments utilising amplification of regions of
cell-free DNA, include those wherein said amplified region(s)
comprises at least one of the nucleotide sequences to be analysed
for the methylation status of one or more CpGs; such as the
methylation status at a plurality of CpGs (in particular of the
relevant CpGs) associated in any of DMRs of the present invention;
in particular DMR #141, #204 and/or #228.
[0098] Any amplified region may also comprise other sequences, such
as (non-natural) synthetic sequences that are used to identify the
source (eg sample/woman) and/or the reaction. Such "molecular
barcoding" is art-known.
[0099] In particular embodiments, said amplification may comprise
the use of the primer-pair(s) for the respective nucleotide
sequence(s) as independently selected from the group of
primer-pairs set forth in each row of TABLE 3. For example, for
that embodiment of the present invention utilising the DMRs #141,
#204 and #228, the applicable regions of cell-free DNA may be
amplified with the primers comprising (eg, consisting of) the
sequences set forth in SEQ ID NOs 94 and 125; 95 and 126; and 96
and 127, respectively. In particular embodiments, for any of said
primers comprising a Y, such primer may be a mixture of
(degenerate) primers wherein the base at each Y is either a C or a
T; and for those of said primers comprising a R, such primer may be
a mixture of (degenerate) primers wherein the base at each R is
either a G or an A.
[0100] Analysis of the methylation status of the CpGs within the
nucleotide sequence of interests (such as associated with or
located within a DMR of the present invention) can be conducted by
any suitable methodology. For example, the present invention
includes those method wherein the methylation status of said CpGs
is determined by a technology selected from the group consisting
of: methylation specific PCR/MethylLight (eg, via use of real-time
quantitative PCR), Epityper, nucleic acid chip-hybridisation,
nucleic acid mass-spectrometry, xMAP (Luminex) Methylated DNA
immunoprecipitation (MeDIP, in which methylated DNA fragments are
isolated/enriched via an antibody raised against 5-methylcytosine
(5mC)), Raindance (and other droplet digital PCR
methodology--ddPCR) and nucleic acid sequencing, preferably,
(single) strand sequencing, nanopore sequencing, bisulphite
sequencing, such as targeted bisulphite sequencing. Sequencing
(such targeted bisulphite sequencing) may be conducted to enable
ultra-high coverage. Also envisioned are embodiments wherein said
determination step may be conducted as a pool and/or essentially
simultaneously when in respect of two, three, four or more of said
nucleotide sequences. As described above, the present in invention
include embodiments where the methylation status at said one or
more CpGs may be determined using single molecule DNA sequencing or
analysis, such as by SMRT or nanopore sequencing.
[0101] It will be apparent to the person of ordinary skill that
bisulphite-modified DNA methylation sites may be detected using eg
methylation-specific PCR (such as using primers and/or probes that
selectively bind to the bisulphite-modified sequences) and/or by
the subsequent use of restriction enzymes the recognition site of
which is created upon such bisulphite-modification.
Methylation-specific PCR ("MSP") is described by Herman et al (U.S.
Pat. No. 5,6200,756, EP0954608 and related family members); and a
further development of MSP using probe-based PCR (known as
"MethylLight") is described by Laird et al (U.S. Pat. No.
56,331,393, EP1185695 and related family members).
[0102] Alternative methods of detecting differences in sequences
that have been converted by bisulphite-modification include
mass-spectrometry methodologies (eg MASS-Array of Sequenom) or
bead-chip technologies such as the Infinium MethylationEPIC Array
or Infinium HumanMethylation450 BeadChip technologies of
Illumina.
[0103] In certain of said embodiments, the methylation status of
said CpGs may be determined by bisulphite sequencing, such as by
single-read and/or high coverage bisulphite sequencing, such as
described in the examples.
[0104] As described in the example, one advantage of the present
invention is that the analysis of cancer-specific DNAme patterns
from ctDNA is that a greater dynamic range can be achieved than
with alternative tests, such as CA125. The dynamic range desired
for such a DNAme pattern-based test for ovarian cancer is related
to the number of molecules of said cell-free DNA (and/or amplified
DNA) that are analysed in the method. For example, the more such
molecules are investigated for the presence (or absence) of the
cancer-specific epigenetic markers, the greater the dynamic range
can be achieved; such as the detection of cancer-specific markers
in very rarely found ctDNA molecules present in the total cfDNA of
the woman.
[0105] Accordingly, in certain embodiments, the method of the
present invention includes those when the methylation status of
said CpG(s) is determined in multiple molecules of said cell-free
DNA and/or amplified DNA representing each of said nucleotide
sequences.
[0106] As will be appreciated, the detection of more than one
molecule carrying the cancer-specific DNAme marker would increase
the confidence in the determination of the presence of (or reduced
response to therapy against) ovarian cancer that has been returned
by the test. Accordingly, in particular of such embodiments, the
method can include where the presence in at least a plurality of
said cell-free DNA molecules of one or more methylated or
methylated (as applicable) CpGs located (in particular, the
relevant CpGs) within one or more of said nucleotide sequences
indicates the presence of, or a reduced response to therapy
against, an ovarian cancer in said woman. In such embodiments, the
plurality of cell-free DNA molecules with one or more of said
methylated or methylated (as applicable) CpGs located may be a
number that is at least 2, 3, 4, 5, 6, 7, 18, 9 or 10, or at least
about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125,
150, 175 or 200, or a greater number such as greater than about
500, 1,000, 5,000, 7,500, 1,000, 2,500, 5,000 or greater than 5,000
molecules.
[0107] To achieve the desired performance characteristics of the
test (such as in terms of sensitivity, specificity and/or dynamic
range), a greater number of total cell-free DNA molecules may need
to be analysed than the number of those exhibiting the
cancer-specific DNAme marker (ie, one or more methylated or
un-methylated--as applicable--CpGs). For example, in particular
embodiments of the present invention, the methylation status of
said CpG(s) is determined in a number of molecules of said
cell-free DNA and/or amplified DNA representing each of said
nucleotide sequences selected from the group consisting of at least
about: 1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 500,000,
1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000,
4,000,000 and 5,000,000 molecules, or more than 5,000,000
molecules.
[0108] The total number of DNA molecules to be analysed and/or that
number of which, when exhibiting the cancer-specific DNAme marker,
determines the presence of (or reduced response to therapy against)
ovarian cancer, can differ from test to test, sample to sample,
woman to woman or population to population. For example, particular
such numbers may apply when the woman carries a typical amount of
or proportion of ctDNA and total cfDNA, and another when she, or
the sample obtained from her, is atypical in response of
total/ctDNA quality or amount/ratio.
[0109] Accordingly, the method of the present invention includes
those embodiments wherein a fraction or ratio of, or an absolute
number of, cell-free DNA molecules in said sample having said
methylated or un-methylated (as applicable) CpG(s) located within
said nucleotide sequence(s) is estimated. To aid the process of
determining the presence or absence of, or response to therapy
against, an ovarian cancer in a woman, the present invention also
includes certain embodiments comprising a step of comparing said
fraction or ratio with a standard or cut-off value. For example, if
the measured fraction or ratio is greater than such standard or
cut-off value, then the test is interpreted to indicate the
presence of an ovarian cancer in said woman; or it is interpreted
to indicate a reduced response to a therapy against an ovarian
cancer in said woman, such as persistence of the OC or no response
of such OC to such therapy. Exemplary standard or cut-off values
for such fraction or ratio of cancer-specific patterns/markers for
certain of the DMRs of the present invention include about 0.0008
for DMR #141 and/or about 0.00003 for DMR #204 and/or about 0.00001
for DMR #228.
[0110] As described above, improved performance of the test of the
present invention is achieved by various means; including by the
analysis of the methylation status of multiple CpGs (eg a
particular DNAme marker pattern) associated with multiple DMRs
and/or the analysis of multiple cell-free DNA molecules for such
multiple CpGs at such multiple DMRs. Hence, an excess over a
standard or cut-off value for the number of DNAme marker patterns
found at any of the multiple DMRs can be a particularly
advantageous embodiment for the test of the present invention.
Accordingly, the test of the present invention includes those
particular embodiments wherein a fraction or ratio of cell-free DNA
molecules with said methylated or un-methylated (as applicable)
CpG(s) present in each of said nucleotide sequence(s) is estimated
and compared to a respective standard or cut-off value; wherein any
one of such fraction or ratios being greater than its respective
standard or cut-off value indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman. For
example, if the methylation status of CpGs located in each of DMRs
#141 and #208 and #228 is determined in a test of the present
invention, and the fraction or ratios of a SEQ ID No 63 at DMR #141
is greater than about 0.0008 or the fraction or ratios of a SEQ ID
No 64 at DMR #204 is greater than about 0.00003 or the fraction or
ratios of a SEQ ID No 65 at DMR #228 is greater than about 0.00001,
then the presence of (or reduced response to therapy again) ovarian
cancer is determined for the woman. Alternatively, the use of
multiple DMRs would enable a decision rule to be applied that takes
into account the multiple DMRs by using a different cut-off for
some markers depending on how many markers are positive for
existing cut-off values in a single sample. Generally, for example,
lower individual cut-offs can be applied the more DMRs that are
found to give a positive result. Another example could be to define
a "hyperplane" of cut-off values in N-dimensional space (N =number
of markers measured), and for any combination of marker values, it
can be determined if they fall "below" (=positive) or "above"
(=negative) the hyperplane. Finally, a logistic regression model
could be applied that determines, for any combination of marker
values, the likelihood of the outcome to be positive. Accordingly,
the invention also includes embodiments that may incorporate such
analysis approaches when two or more of the DMRs of the invention
are utilised.
[0111] As described herein, the present invention also provides
advantages in that the standard or cut-off value used in respect of
a particular DNAme marker can be adapted, for example in response
to desired characteristics of the test or in response to the
characteristics of the cfDNA isolated of the woman.
[0112] Accordingly, certain embodiments of the test of the present
invention include those where said standard or cut-off value(s)
is/are modified for a given sample based on one or more of the
following factors: (i) the amount or concentration of total
cell-free DNA present in said sample; and/or (ii) a baseline value
of said fraction or ratio previously determined for said woman;
and/or; (Hi) a value of said fraction or ratio determined from
multiple samples from a population of women representative of said
woman; and/or (iv) the specificity and/or sensitivity and/or
dynamic range desired for said method of determination. In
particular embodiments, said standard or cut-off value(s) may be
increased when cfDNA blood collection tubes such as Streck Tubes,
are used. For example, in such embodiments, the applicable said
standard or cut-off value(s) may be increased by a factor of about
2, 5, 10, 20, 50, 100, 200, 500 or 1000, or by a factor that is
greater than 1000.
[0113] In particular of such embodiments, the standard or cut-off
value may be reduced for a given sample that has an amount and/or
concentration and/or quality of total cell-free DNA present in said
sample that is greater than a standard or cut-off value. Suitable
methods for the inspection, estimation or determination of amount
and/or concentration and/or quality of total cell-free DNA include
those described elsewhere herein. For example, if the quality of
the total cfDNA of the woman is lower than expected (such as a
higher average fragment size than as described herein), and/or if
the total cfDNA amount is higher (in each case indicating that
somatic DNA may have been released from eg WBCs during sample
collection, transport, storage and/or processing), then the
standard or cut-off value used for the respective fraction or
ration for the DNAme marker can be reduced. This allows for more
possibility to adapt the test to the individual situation or woman
taking the test.
[0114] As a further embodiment of the test, it may be practiced
multiple times on a given woman, such as 2, 3, 4, 5, 6, 7, 8, 9,
10, about 15, about 20 or more than about 20 times (such as about
50 times). Such a repeated test can enhance the (early) detection
of ovarian cancer in the woman and/or the long-term response to
therapy against (or monitoring of) ovarian cancer. Accordingly, the
test may include those embodiments when it is practiced on multiple
samples; wherein each sample is collected from the same woman at
different time points. For example, said multiple samples are
collected from said woman with an interval between them selected
from the group consisting of about: 2 days, 3 days, 4 days, 5 days,
7 days, 10, days, 14 days, 21 days, 24 days, 3weeks, 4 weeks, 5
weeks, 6 weeks, 6, weeks, 8 weeks, 3 months, 4 months, 5 months, 6
months, 8 months, 12 months, 18 months, 2 years, 3 years and 5
years.
[0115] In certain of such embodiments, it may be that the test of
the present invention is conducted as part of a routine screen of
one or more women, such as part of an annual screen for the
presence or absence of ovarian cancer. For certain women (or groups
thereof), the period of repeat testing may be shorter. For example,
women in high risk groups (such as those with BRAC 1/2 positive/
and/or a family history of ovarian cancer and/or belonging to
certain sub-populations eg Ashkenazi women) may be tested more
frequently, for example every 6, 3, 2, or 1 month. Furthermore,
those women for which ovarian cancer has already been diagnosed
(and perhaps already treated with chemotherapy) may be
repeat-tested using the present invention at a frequency of about
every 6, 3, 2, 1 month, or even more frequently such as once about
every two weeks or about every week.
[0116] As has been shown for the CA125-based ROCA test, a deviation
or change from an earlier patient-specific standard or cut-off
value can provide a further enhanced test for ovarian cancer.
Accordingly, for those embodiments of the present invention where a
woman is tested multiple times, the presence of, or a reduced
response to therapy against, an ovarian cancer in a woman is
indicated by--in comparison to a previous sample of said woman--the
presence of, or an increase in the absolute number of, or an
increase in the fraction or ratio of, cell-free DNA molecules in
said sample having said methylated or un-methylated (as applicable)
CpG(s) located within said nucleotide sequence(s).
[0117] As shown by the examples herein, the test of the present
invention can be used in combination with other tests for ovarian
cancer, in particular with those which reduced (or no) overlap of
false positives and/or false negatives to the test of the present
invention. Exemplary such tests, including those based on CA-125,
HE4, transthyretin, apolipoprotein Al, beta-2-microglobin and
transferrin (such as ROCA, ROMA and OVA1) are described in more
detail elsewhere herein.
[0118] Accordingly, in certain embodiments of the present
invention, one or more additional steps may be conducted in respect
of such other tests for ovarian cancer. In particular, one optional
an additional step may comprise that of determining (eg by an
in-vitro procedure), from a blood sample from said woman, the
amount present therein of one or more proteins independently
selected from the group consisting of: CA-125, HE4, transthyretin,
apolipoprotein Al, beta-2-microglobin and transferrin; wherein,
either or both of: (i) the presence in at least one of said
cell-free DNA molecules of one or more methylated or un-methylated
(as applicable) CpGs located within one or more of said nucleotide
sequences (such as by an excess of any one of the DNAme marker
patterns of the present invention being in excess of a standard or
cut-off value); or (ii) an amount of said protein(s) present in
said blood sample is greater than a standard or cut-off value for
such amount or protein; indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman. As
will be appreciated, such additional step related to the other test
for ovarian cancer may be conducted either before or after
conducting the DNAme-based aspects of the test. Indeed, the conduct
of one (or other) of the tests may be dependent on the outcome of
the first, for example to provide additional sensitivity and/or
sensitivity to the other all determination, test or diagnosis.
[0119] In such embodiments, the protein may be determined by a
ROCA, a ROMA and/or an OVA1 diagnostic test.
[0120] As described elsewhere herein, the test of the present
invention may be applied to different types of ovarian cancer. For
example, in certain embodiments the present invention the ovarian
cancer may be an invasive ovarian cancer, such as an invasive
epithelial ovarian cancer; in particular one selected from the
group consisting of: high grade serious (HGS), endometroid,
cell-cell and mucinous ovarian cancers. In alternative embodiments,
the cancer may be peritoneal cancer or Fallopian tube cancer.
[0121] In particular, the test of the present invention may be used
(or useful) for distinguishing the presence of ovarian cancer (such
as one described elsewhere herein) from the presence of a benign
pelvic mass in the woman.
[0122] In other embodiments, the test of the present invention may
be used (or useful) for determining the response of a woman
suffering from ovarian cancer to a therapy comprising
chemotherapeutic agent(s) against said ovarian cancer (such as one
described elsewhere herein). For example, such a test may be
conducted to predict the risk of death of said woman, in particular
from risk of death by ovarian cancer that is not responding to
(chemo)therapy.
[0123] In certain of such embodiments, the test of the present
invention may be practiced on said woman after one, two, three,
four and/or five cycles of said (chemo)therapy.
[0124] And in further such embodiments, said sample may be obtained
from said woman within a period after completion of said cycle or
(chemo)therapy that is selected from the group consisting of about:
2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours,
48 hours, 3 days, 4 days, 5 days, 6, days, 7 days, 8 days, 10 days,
12 days, 14 days, 16, days, 18 days, 21 days, 24 days, 4 weeks, 5
weeks, 6 weeks, and 8 weeks.
[0125] In particular such embodiments, it may be that a short-lived
increase in the ratio or fraction of the DNAme marker is observed
shortly after said (chemo)therapy. Without being bound by theory,
this may arise due to death/lysis of cancer cells in response to
the (chemo)therapy and their shedding of ctDNA into the blood
stream of the woman. Accordingly, the test of the present invention
may include the monitoring of such short lived (eg for 1, 2, 3, 5,
7, or 14 days) increase in DNAme marker(s) as an indication of
(initial) response/success of the (chemo)therapy. The test may then
be repeated to monitor the reduction of the DNAme marker(s) in
cfDNA after such increases, and whether the ratio or fraction of
the DNAme marker increases at a later time (indicating a reduced
response to therapy against, and/or the recurrence of, the ovarian
cancer.
[0126] In such embodiments of the present invention, said therapy
includes one or more chemotherapeutic agent(s), such as those one
that is used and/or is approved (eg, by the European Medicines
Agency in Europe and/or by the Food and Drug Administration in the
US) for the treatment of a cancer, in particular for the treatment
of ovarian cancer. In alternative such embodiments, the
chemotherapeutic agent may be one in development on the date
hereof, or in the future. Examples of such chemotherapeutic agent
include one or more independently selected from the group
consisting of: a platinum-based antineoplastic (such as
carboplatin, cisplatin, oxaliplatin, nedplatin, picoplatin or
satraplatin) and a taxane (such paclitaxel or docetaxel). In
particular, the chemotherapeutic agent may be one selected from the
group consisting of: carboplatin, paclitaxel, docetaxel, cisplatin,
liposomal doxorubicin, gemcitabine, trabectedin, etoposide,
cyclophosphamide an angiogenesis inhibitor (such as bevacizumab)
and a PARP inhibitor (such as olaparib). Other PARP inhibitors in
development include: veliparib (ABT-888) from Abbot, MK4827 from
Merck, AG-014699 of Pfizer and Iniparib (BSI-201) of
Sanofi-Aventis. Other angiogenesis inhibitors in development
include aflibercept, AMG386, cediranib, sorafenib, sunitinib and
pazopanib. Antibody-drug conjugates in development include T-DM1,
IMGN388, lorvotuzumab mertansine, AN-152, S1(dsFv)-PE38, BIIB015,
SAR566658, VB6-845, Thio Hu3A5-VC-MMAE, CDX-014, MEDI-547, SGN-75
and MDX-1203. In particular embodiments, the chemotherapeutic
agents may be carboplatin, cisplatin, paclitaxel or docetaxel, or
may be combination therapies thereof.
[0127] A particular form of (chemo)therapy that may be used in the
treatment of ovarian cancer, and one following which the test may
be conducted, is neoadjuvant (chemo)therapy ("NACT").
[0128] One particular advantage of the test of the present
invention is that it can provide individual-specific therapeutic
options. For example, in certain embodiments of the test of the
present invention, if said woman is determined to respond to (for
example, has responded to) said (chemo)therapy, then said woman may
be designated as being eligible for tumour de-baulking surgery. As
will be recognised, if a woman is determined to respond to such
chemotherapy then tumour de-baulking surgery is an intervention
that could prove life-saving. In contrast, if a woman is determined
to not respond to said (chemo)therapy, then said woman may not be
suitable for such invasive tumour de-baulking surgery. However, in
such embodiments, she may be designated as eligible for therapy
with one or more second-line chemotherapeutic agent(s) against said
ovarian cancer. The response to therapy with such second-line
chemotherapeutic agent(s) may also be determined using a test of
the present invention, and if such second-line chemotherapy leads
to a response (such as determined sooner by a test of the present
invention), then such woman may then be designated as being
eligible for tumour de-baulking surgery.
[0129] Said second-line (chemo)therapy includes one or more
chemotherapeutic agent(s) independently selected from the group
consisting of: carboplatin, paclitaxel (such as alone, and as a
weekly treatment), docetaxel, cisplatin, liposomal doxorubicin,
gemcitabine, trabectedin, etoposide, cyclophosphamide, an
angiogenesis inhibitor (such as bevacizumab) and a PARP inhibitor
(such as olaparib), or any of those chemotherapeutic agent(s)
described above. The second line chemotherapeutic agent may, in
same embodiments, be the same as that used in said first therapy;
but in such alternative embodiment said (same) subsequent
chemotherapeutic agent is used at a different dosage, different
administration route, different treatment regimen and/or in
combination therapy together with other treatment modalities. For
example, carboplatin in combination with paclitaxel is commonly
used as first line therapy, and carboplatin (alone) may be used as
the second line therapy, such as if the patient is relapse-free for
12 months.
[0130] In this way, the test of the present invention provides a
faster and more accurate test for determining their response to
(chemo)therapy against ovarian cancer, such that the most
appropriate, or additional, therapeutic interventions can be made;
ultimately increasing the success of treatment for ovarian cancer,
an increase in progression free survival, overall survival and/or
quality of life (such as may be measured by pain suffered or
reported and/or pain-killer use).
[0131] In a first related aspect, the invention relates to a method
of determining the methylation status at one or more CpGs of
cell-free DNA comprising the steps: [0132] Providing a biological
sample, said sample comprising cell-free DNA; and [0133]
Determining, in at least one molecule of said cell-free DNA, the
methylation status at one or more CpGs (such as one or more
relevant CpGs) located within one or more of nucleotide sequences,
wherein, said one or more of the nucleotide sequences are
independently selected from the group consisting of: SEQ ID NOs: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31 (for example, within
one or more of the nucleotide sequences comprised in one or more of
the respective DMRs of the present invention independently selected
from the group consisting of DMR#: #141, #204, #228, #144, #123,
#129, #137, #148, #150, #154, #158, #164, #176, #178, #180, #186,
#188, #190, #192, #200, #202, #208,#210, #213, #214, #219, #222,
#223, #224, #225 and #226), or a nucleotide sequence present within
about 2,000 bp (such as within about 200 bp) 5' or 3' thereof, or
an allelic variant and/or complementary sequence of any of said
nucleotide sequences.
[0134] In a second related aspect, the invention relates to a
method of detecting one or more: (i) methylated CpGs associated
with (such as located within) one or more of the hyper-methylated
DMRs of the present invention;
[0135] and/or (ii) un-methylated CpGs associated with (such as
located within) one or more of the hypo-methylated DMRs of the
present invention, in each case comprised in at least one molecule
of cell-free DNA of a woman comprising the steps: [0136] Providing
a biological sample, said sample comprising cell-free DNA of said
woman; and [0137] Determining, in at least one molecule of said
cell-free DNA, the methylation status at one or more CpGs (such as
one or more relevant CpGs) located within one or more of the
nucleotide sequences independently selected from the group
consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 and 31 (for example, within one or more of the nucleotide
sequences comprised in one or more of the respective DMRs of the
present invention independently selected from the group consisting
of DMR#: #141, #204, #228, #144, #123, #129, #137, #148, #150,
#154, #158, #164, #176, #178, #180, #186, #188, #190, #192, #200,
#202, #208,#210, #213, #214, #219, #222, #223, #224, #225 and
#226), or a nucleotide sequence present within about 2,000 bp (such
as within about 200 bp 5' or 3') thereof, or an allelic variant
and/or complementary sequence of any of said nucleotide sequences,
wherein, the presence in at least one of said cell-free DNA
molecules of one or more: (i) methylated CpGs associated with (such
as located within) one or more of the hyper-methylated DMRs of the
present invention (eg, as identified in TABLE 1A), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 1, 2,
3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29
and 30; thereby detects said methylated CpG(s) in the cell-free DNA
of said woman; and/or (ii) un-methylated CpGs associated with (such
as located within) one or more of the hypo-methylated DMRs of the
present invention (eg, as identified in TABLE 1B), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 5, 6,
7, 8, 9, 11, 13, 16, 17, 22 and 31; thereby detects said
un-methylated CpG(s) in the cell-free DNA of said woman.
[0138] In a third related aspect, the invention relates to a method
of diagnosing and treating a woman having an ovarian cancer
comprising the steps: [0139] Providing a biological sample from
said woman, said sample comprising cell-free DNA of said woman;
[0140] Detecting, in at least one molecule of said cell-free DNA,
whether one or more methylated or un-methylated CpGs (such as one
or more relevant CpGs) is located within one or more of the
nucleotide sequences independently selected from the group
consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 and 31 (for example, within one or more of the nucleotide
sequences comprised in one or more of the respective DMRs of the
present invention independently selected from the group consisting
of DMR#: #141, #204, #228, #144, #123, #129, #137, #148, #150,
#154, #158, #164, #176, #178, #180, #186, #188, #190, #192, #200,
#202, #208,#210, #213, #214, #219, #222, #223, #224, #225 and
#226), or a nucleotide sequence present within about 2,000 bp (such
as within about 200 bp) 5' or 3' thereof, or an allelic variant
and/or complementary sequence of any of said nucleotide sequences;
[0141] Diagnosing the woman with an ovarian cancer, such as an
ovarian cancer that does not respond to a first therapy, when one
or more of: (i) said methylated CpGs associated with (such as
located within) one or more of the hyper-methylated DMRs of the
present invention (eg, as identified in TABLE 1A), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 1, 2,
3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29
and 30 is detected; and/or (ii) un-methylated CpGs associated with
(such as located within) one or more of the hypo-methylated DMRs of
the present invention (eg, as identified in TABLE 1B), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 5, 6,
7, 8, 9, 11, 13, 16, 17, 22 and 31 is detected; and [0142] Treating
said diagnosed woman by: [0143] Administering, or recommending
administration of, an effective amount of a chemotherapeutic agent
to a woman diagnosed as having an ovarian cancer, such as one that
does not respond to the first therapy; or [0144] Conducting, or
recommending the conduct of, tumour debaulking surgery to a woman
diagnosed as having an ovarian cancer that does respond to the
first therapy.
[0145] In such related aspect, the chemotherapeutic agent and/or
the first therapy may be one as described elsewhere herein.
[0146] In certain embodiments of such related aspect(s), the
methylation status of said CpG(s) may be determined according to
one or more of the corresponding embodiments of the other aspects
of the present invention; including for example, that said
cell-free DNA of the woman may be subjected to an agent that
differentially modifies said DNA based on the methylation status of
one or more of the CpGs, and/or that the methylation status of the
one or more the CpGs is determined without use of such an agent
(such as, by single molecule sequencing/analysis of DNA). In
certain embodiments of such related aspect(s), the methylation
status of said CpG(s) may be determined by the detection of a
nucleic acid of the third aspect, such as the detection of a (eg
non-natural) nucleic acid comprising at least about 15 contiguous
bases comprised in SEQ ID No. 32, 33, 34 and/or 35.
[0147] In further certain embodiments of such related aspect(s),
the/a biological sample may be collected and/or further processed
according to one or more of the corresponding embodiments of the
other aspects of the present invention. For example, the biological
sample may be obtained from a woman having or suspected of having
ovarian cancer, or suspected to not have responded to therapy
against ovarian cancer. In any such embodiments, the biological
sample may be, or may be obtained by, any of the corresponding
embodiments of the other aspects of the present invention.
[0148] In further of such embodiments, the method of detecting said
methylated or un-methylated (as applicable) CpG(s) (in particular,
one or more relevant CpGs) may comprise the step of determining the
presence or absence of, or response to therapy against, an ovarian
cancer in said woman, wherein the detection in at least one of said
cell-free DNA molecules of one or more methylated un-methylated (as
applicable) said CpGs located within one or more of said nucleotide
sequences indicates the presence of, or a reduced response to
therapy against, an ovarian cancer in said woman.
[0149] In a second aspect, the invention relates to a
chemotherapeutic agent for use in a method of therapy of ovarian
cancer in a woman, wherein said chemotherapeutic agent is
administered to a woman within about three months of said woman
having been predicted and/or determined, using a method of the
first aspect, to not respond to a therapy against ovarian
cancer.
[0150] In a related second aspect, the invention also relates to a
method of treating ovarian cancer, comprising administering an
effective amount of a chemotherapeutic agent is administered to a
woman within about three months of said woman having been predicted
and/or determined, using a method of the first aspect, to not
respond to a therapy against ovarian cancer.
[0151] In certain embodiments of such second aspect, the
chemotherapeutic agent is one that is used and/or is approved (eg,
by the European Medicines Agency in Europe and/or by the Food and
Drug Administration in the US) for the treatment of a cancer, in
particular for the treatment of ovarian cancer. Examples of such
chemotherapeutic agent include one or more independently selected
from the group consisting of: carboplatin, paclitaxel, docetaxel,
cisplatin, liposomal doxorubicin, gemcitabine, trabectedin,
etoposide, cyclophosphamide an angiogenesis inhibitor (such as
bevacizumab) and a PARP inhibitor (such as olaparib). In other
embodiments, the chemotherapeutic agent may be any one of those
described elsewhere herein.
[0152] In any embodiments of such second aspect, the
chemotherapeutic agent is administered to said woman within about 3
months, or about 70, 56, 53, 49, 46, 42, 39, 35, 32, 28, 25, 21,
18, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days or less,
such as within about 24, 20, 18, 15, 12, 10, 8, 6, 5, 4, 3 or 2
hours, or within about 1 hour, of said prediction and/or
determination. Also envisioned by the present invention, is that
such chemotherapeutic agents can be used in combination
formulations and/or combination treatment regimens with another
chemotherapeutic agent, other pharmaceutical agents and /or medical
intervention such as radiation treatment or surgery.
[0153] A chemotherapeutic agent may be administered to said woman
by the applicable or conventional mode of administration, such as
intravenously, intramuscularly, intradermally, orally.
[0154] In such second aspect, said prediction and/or determination
is made using a method of the first aspect. That is, an embodiment
of such method of the invention may be practiced, such as to
determine or to predict (eg by an increased likelihood) that said
woman has not responded (or will not respond) to a first therapy
against the ovarian cancer she is suffering from; or that she has
not responded (or will not respond) completely, sufficiently and/or
within a predetermined period of time (such as about 12 months, 6
months, 4 months, 3 months, 2 months, or about 4 weeks or 2 weeks,
or about 1 week).
[0155] A first therapy against the ovarian cancer may be surgery,
such as tumour de-baulking surgery, or it may be treatment with a
chemotherapeutic agent such as one described above. Whilst in one
embodiment, the subsequent chemotherapeutic agent used after said
prediction and/or determination is different to a chemotherapeutic
agent used for said first therapy, in another aspect said
subsequent chemotherapeutic agent is the same as that used in said
first therapy; but in such alternative embodiment said (same)
subsequent chemotherapeutic agent is used at a different dosage,
different administration route, different treatment regimen and/or
in combination therapy together with other treatment
modalities.
[0156] In the context of the invention, an effective amount of a
chemotherapeutic agent can be any one that will elicit the
biological, physiological, pharmacological, therapeutic or medical
response of the woman that is being sought by the pharmacologist,
pharmacist, medical doctor, or other clinician, eg, lessening of
the effects/symptoms of the ovarian cancer.
[0157] In a third aspect, the invention relates to a nucleic acid
comprising a nucleic acid sequence consisting of at least about 10
contiguous bases (preferably at least about 15 contiguous bases for
any DMR other than DMR #222) comprised in a particular (eg
non-natural) sequence derived from one selected from the group
consisting of one set out in TABLE 1A, TABLE 1B or TABLE 2A (for
example, a sequence consisting of at least about 10 contiguous
bases (preferably at least about 15 contiguous bases for any DMR
other than DMR #222) comprised in a sequence producible by (such as
produced following) bisulphite conversion of a sequence comprised
within a DMR selected from the group consisting of DMR#: #141,
#204, #228, #144, #123, #129, #137, #148, #150, #154, #158, #164,
#176, #178, #180, #186, #188, #190, #192, #200, #202, #208,#210,
#213, #214, #219, #222, #223, #224, #225 and #226; such as a
sequence consisting of at least about 10 contiguous bases
(preferably at least about 15 contiguous bases for any SEQ ID other
than SEQ ID: NO:58) comprised in a sequence selected from the group
consisting of: SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61 and 62 (such as SEQ ID NO: 32, 33, 34 and/or 35), or an
allelic variant and/or complementary sequence of any of said
nucleotide sequences. In particular embodiments, said number of
contiguous bases is at least about 16, 18, 20, 22, 24, 26, 28, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,
10, 140, 150 or 160 bases, such as between about 80 and about 160,
such as between about 100 and about 160, in each case,
independently, up to the maximum number of bases within the
sequence selected from said group.
TABLE-US-00005 TABLE 2A DMR-correspondence and possible sequences
of the bisulphite-converted DMRs of the present invention SEQ ID
DMR# Bisulphite-converted amplicon sequence (all CpGs are
underlined) NO. 141
TATTYGGAGGTTTAGGGGTGAGGATTTYGTTAYGGGAAGGAGGTATAYGATTTAGTTTATGATATYG
32
TTATTTYGGYGTGGTGTTGTAGGGGGAAGTTTAGGTATTTATYGAGGATAGGATTYGGGGAATTYGT
TG 204
GATATTYGGTGGAGAGTYGTAGTTGTTYGTYGYGGGGTTTTAGGYGTAGTAYGTTTTYGYGYGTGGG
33
TYGTAGTTGGTAGTATAGGAAGTTTAGGTGGAAGAGYGGYGGYGTGGGYGGTTYGGYGYGGYGYGGY
GAGTGYGGGTTGGTATYGGT 228
GTTTTATGGGYGAGTTGTTGTAGTGYGGTTGTTAGGYGTTTYGYGGGYGGGTTTTTTTTYGGTTTTT
34 YGGTTTGTTYGGTATTTTYGGATTTTTTGGTTTYGYGGGTTTTT 144
TTTAGGTTTGAYGTGGGTTTTTTAGGGYGGYGTYGTTAAGGTTTAGAYGTTTTYGTGTAGGAGGGAY
35
GAYGATTTTTTTTAYGTTTTYGTGGTTTTAATTYGGYGTTTTGTTATTTTTGATTYGGTGAATATAT
TTTAGA 123
GYGAAGTAGGAGTAGTTGTYGGGTTTTAYGAGTTTTYGTTYGTTTTGGTTYGGGTTTTTTYGAGTTT
36 TGTTATTAGTYGAGGTTGTGYGGGTAATTGGGTTAGTTTTTYGTTAGGAGAGA 129
AATTTTGTTGAGTGAGTTTATAAATAGGGTATAATYGAGAYGYGGGAATGTTTGGGTYGTYGYGTAG
37 TTATYGGGTAGGGTYGTTTTTTTTTGTGGGTTAGTAAAAAYGGTGTTAAGTGA 137
ATTTYGTTATATATATAGTTGTATTYGGTATAATAYGYGGTTATAGGTTATTTTAGGTYGTTTYGGG
38 TGTTTTTTTYGTAGTTTTAYGTAGATAGAAGATATTTTTYGGGTTTGGGTGTTTAGTTTTTYG
148
GAGGTAATGGAAGYGGTTATTTTTGTTTTYGTTTYGYGTTTGGTTGAAGYGATYGGGGTYGAATAYG
39 TTTAYGTTTTTGAATAYGGGYGTTGTGTTATGGGTGTTTYGGATGTTATATAT 150
GYGGGTATTTGTAGTTTTAGTTATTYGGGAGGTTGAGGYGGGAGAATGGYGTGAATTYGGGAGGYGG
40
AGTTTGTAGYGAGTYGAGATYGYGTTATYGTATTTTAGTTTGGGYGATAGAGYGAGATTTYGTTTAA
AAA 154
TTTTYGGAGTTYGGAGTTTAGGTTAGTGGTAGTYGATTTAGTTTTYGAGATTTTTTTAYGTYGTTTT
41
AAAATTAAAAYGGAGTTTAATAYGAAGTTGGGTGAAGTYGTAGTTTGTAGGAGTTAGGGAGATGYGT
TTT 158
GATTTTGTTTTAAAAAAGAAAAAAATAGGGTYGGGYGYGGTGGTTTAYGTTTGTTATTTTAGTATTT
42
TGGGAGGTYGAGGYGGGTGGATTAYGAGGTYGGGAGATYGATATTATTTTGGGTAATAYGGTGAAAT
TT 164
GATTYGYGAGGTTTTTTAGTAGTTTATTTYGGGAYGGYGGTGTTTAGTTTAGTTTAGGGTAATTGGG
43
TTTTTTGAGAGTTYGATTTTTATYGGTTTGGGAGYGAGTGGTTYGAGTTTAGATGTTGGGAATYGTY
GTTT 176
TAATTTATTTTTTTATTAAATTGTATGAAGAAGGTYGGGYGYGGTGGTTTAYGTTTGTAATTTTAGT
44
ATTTTGGGAGGTYGAGGYGGGYGGATTAYGAGGTTAGGAGATYGAGATTAYGGTGAAATTTYGTTTT
TATT 178
GGTAGGAGYGTTTTATTATGYGTAAGTTYGTGGTTTGGAGAGYGTTGAAGGTGGGAGGGGGAAGAGG
45
GGTAGAATTTTYGYGGGAGYGAGYGTATAGTTGTYGTTTYGTGGTYGTTTYGGGAATYGTTGGTTTY
GGTTTTGG 180
TTGTAGAAGYGTATTTTGTTGAATATTTYGAGGAYGTGTTTTTYGTATAGGGAGYGTTYGTTTTTGT
46
TGGGGTTGGAGYGGYGTTTGGAGGTYGATATTYGGTYGTTGTTGGATTTTTTYGTTTGTYGTTTTTG
T 186
YGTGTTAGTTAGGATGGTTTYGATTTTTTGATTTYGTGATTAGTTYGTTTYGGTTTTTTAAAGTGTT
47
GGGATTAAAGGYGTGAGTTATYGYGTYGGGTYGAGATTTTGTTTTAAAAAAAAAAGGTTTGGGTTGT
GGTATTTTGGGA 188
AGAGTTGTATTTYGAAGATTTTAGATTTYGAGAGTTGYGGAAAYGTTAYGAGGATTTGTTAATTAGG
48
TTGYGGGTTAATTAGAGTTGGGAAGATTYGAATATYGATTTYGTTTYGGTTTTTGTAGTTYGGATAT
TTAYGTT 190
GTTAGAYGAGAGTTTGGGGTTAATGTYGAGGTGGAGYGAYGTTGGTAYGGTAATTTTGAGTTTGYGY
49 GGTTYGGYGTTATTTTTTGGTTTTTYGTTGTTGGTTGGATTT 192
YGGTAGGTTATTTAGTAGTAGGGTTTTAYGTYGGTTTYGTYGATGTTTTAGAAGGTTAGTTTTTTTT
50
YGAAGAGYGGTTYGTATAYGTTTGYGGGGTAGTGTAGTTTGTYGGTGYGGTAGTAATTGAGTATATA
GGYGAAGAYG 200
AATTAGTTTAGTAATYGGYGATTTTAAGYGYGGYGATYGTAAAGGGAGTGTTTGTTTATTYGYGTTT
51 GAAAGTAGATTTTTTTTYGGTAGGAATATAGGATTTATTTGTTAGTGG 202
GTGYGAATAAGATYGGGYGTTTYGTYGTYGAYGYGAAGGGGTTGTTTGTGYGYGGYGTTGYGGGTTT
52
TTYGYGYGTGGGGTGTGYGTGTGYGTGTTYGGGTTYGGTTTTGTGTGTGTATYGYGGGTTTGTTTAG
AGTYGGGATTATYG 208
ATATTAATTTTGTTYGGGTAYGGTGGTTTAYGTTTGTAATTTTAGTATTTTGGGAGGTYGAGGYGGG
53 YGGATTAYGAGGTTAGGAGATYGAGATTATTTTGGYGAATATGGTGAAATTTYG 210
TGTATATAGATTATTGTAGGATTATTTTTTGTGTTTTTTAAAATTTTTTTTTTTYGTTTTATTTTAT
54 ATATTTTTTTGTTTTTTATAATTTT 213
TYGTTYGGGAATGGGAATATAGTTATATATGGGAAAAYGYGGTGTAGGGAGAAAATTAATTTAGTGA
55 GGAGYGGAGGYGTAGGATTGTGGAGTGTGTATTYGG 214
TTGTTTAAAGGYGTAGAGGAGTAGTTGGGAAYGAGAATAAAGYGGTTAGGTTTTTTTYGGAGGAAGG
56 AAGGAGAGAGTTTTAGGAAATAGTTGA 219
GGATGAAGGATTTTTGTATTATTGTGATGGTTATGGYGTTGTTGTTTGGGTTTTTTTTTTTYGGTAG
57 GTAAGGGAGGAGGTAGGGGAAGGGATATGTGTTT 222
TAGGTTATAGGAAGAGGTATTTTTTATAGATGAYGGTTGTAAAATTTTAAGTTGAGTTTTTTTAGGA
58 AGT 223
AAGAGAGAGTGGTTGATAATTAGTAGAGAGAGGTTTTTAATTTAYGGAAGTGTTTGTAATATAATTT
59 TTTTGTATATTAGTTGT 224
GGTTTTTTTTTTYGAGTTATGAAGAGTTGTTTGYGGTTATTTTGGTTTTYGTATTTYGTTTTTGTTA
60 TTTTAGGTTTTTGTAATTTGTTTAAYGTTT 225
GAAGTTTGATATTTTTGGTTTTAAATATTGTTTGGTTATAATAYGATATTTAGGGATAGATATTTTT
61 TATGTATAGTAAGTTGTGG 226
GGGGGGATTGTYGTTAATTTATTGTTTAATGATYGYGGTTYGYGYGTTTYGAGTAATYGGGTGATGT
62 ATGTGGATTGTGTATATTTYGTGG
Each Y, Independently Either C or T/U
[0158] In certain embodiments of such aspect, one or more of the
bases identified by "Y" in such sequences of contiguous bases is a
U or T (preferably, a T). As will now be apparent to the person of
ordinary skill, such sequences (ie, those comprising a U and/or a T
at one or more bases identified by "Y") are non-natural sequence as
the sequence is produced following conversion of a cytosine in a
natural sequence by bisulphite. In particular of such embodiments,
such a nucleic acid of the present invention comprising two, three,
four, five, six, seven, eight, nine or ten (up to the maximum
number of "Ys" present in such sequence of contiguous bases) Us
and/or Ts; and in more particular of such embodiments, such a
nucleic acids may comprise at least one, two, three, four, five,
six, seven, eight, nine, ten or more than ten (up to the maximum
number of CpGs present in such sequence of contiguous bases) Cs at
any of the bases identified by "Y" therein, such as those "Ys"
within CpGs (such as relevant CpGs) located in such sequence of
contiguous bases. In particular embodiments, two, three, four,
five, six, seven, eight, nine, ten or more than ten of the bases
identified by "Y" therein may be a U or T (preferably, a T), such
as all of the bases identified by "Y" therein may be a U or T
(preferably, a T), or all but one, two, three, four or five of the
bases identified by "Y" therein is a U or T (preferably, a T) and
preferably all other Y's therein (in particular, those in CpGs) may
be Cs.
[0159] In other embodiments, such a nucleic acid of the invention
comprises a nucleic acid sequence consisting of said number of
contiguous bases--for example, at least about 10 contiguous bases
(preferably at least about 15 contiguous bases for any SEQ ID other
than SEQ ID: NO:89)--comprised in a particular (non-natural)
sequence selected from the group consisting of those set forth in
TABLE 2B: SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92 and 93 (such as SEQ ID NO: 63, 64, 65 and/or 66, or SEQ ID
NOs: 63 and 64 and 65), and in particular of such embodiments, said
nucleic acid comprises a sequence selected from said group, or in
each case an allelic variant and/or complementary sequence of any
of said nucleotide sequences. As will be apparent, typically a
bisulphite converted (un-methylated) cytosine would be detected as
a T, particularly after an amplification (such as a PCR) step.
However, certain detection technologies (for example, those able to
detect single molecules) may be able to directly detect a
bisulphite converted (un-methylated) cytosine as a U. Accordingly,
the sequences of TABLE 2B are also envisioned to encompass the
analogous sequences where, instead of a T representing a bisulphite
converted (un-methylated) cytosine, such T is instead a U. The
location of such Ts (eg, C in the genome sequence outside of CpGs)
will readily be identifiable by the person of ordinary skill by
comparison of the sequences presented in this TABLE 2B to the
corresponding genomic sequence presented in TABLE 1A or TABLE 1B,
as applicable.
TABLE-US-00006 TABLE 2B Particular patterns of epigenetic markers
associated with the presence of ovarian cancer detected by
particular (non-natural) sequences of the DMRs of the present
invention Marker Detected sequence for Total No. of SEQ DMR
coordinates marker (relevant CpGs No. of relevant OC specific
pattern ID # (hg19) are underlined) CpGs CpGs of methylation NO.
141 chr5:178004422- CGTTACGGGAAGGAGGTATAC 7 7 1111111 63 178004505
GATTTAGTTTATGATATCGTT ATTTCGGCGTGGTGTTGTAGG GGGAAGTTTAGGTATTTATCG
204 chr1:151810811- CGTCGCGGGGTTTTAGGCGTA 18 16 1111111111111111XX
64 151810917 GTACGTTTTCGCGCGTGGGTC GTAGTTGGTAGTATAGGAAGT
TTAGGTGGAAGAGCGGCGGCG TGGGCGGTTCGGCGCGGYGYG GT 228 chr2:219736301-
YGGTTGTTAGGCGTTTCGCGG 9 7 X111X1111 65 219736362
GYGGGTTTTTTTTCGGTTTTT CGGTTTGTTCGGTATTTTCG 144 chr19:58220438-
GGCGGCGTCGTTAAGGTTTAG 11 11 11111111111 66 58220517
ACGTTTTCGTGTAGGAGGGAC GACGATTTTTTTTACGTTTTC GTGGTTTTAATTCGGCG 123
chr16:1271180- TGAGTTTTTGTTTGTTTTGGT 7 7 0000000 67 1271240
TTGGGTTTTTTTGAGTTTTGT TATTAGTTGAGGTTGTGTG 129 chr11:69054672-
TYGAGATGTGGGAATGTTTGG 8 4 X00XX00X 68 69054732
GTYGTYGTGTAGTTATTGGGT AGGGTYGTTTTTTTTTGTG 137 chr12:132896310-
TGTGGTTATAGGTTATTTTAG 7 7 0000000 69 132896382
GTTGTTTTGGGTGTTTTTTTT GTAGTTTTATGTAGATAGAAG ATATTTTTTG 148
chr2:72359624- TTTTYGTTTTGTGTTTGGTTG 10 5 X0000XXX0X 70 72359687
AAGTGATTGGGGTYGAATAYG TTTAYGTTTTTGAATATGGGY G 150 chr7:156735054-
TGGGAGGTTGAGGTGGGAGAA 11 11 00000000000 71 156735141
TGGTGTGAATTTGGGAGGTGG AGTTTGTAGTGAGTTGAGATT GTGTTATTGTATTTTAGTTTG
GGTG 154 chr17:70112163- GTYGATTTAGTTTTCGAGATT 7 6 X111111 72
70112238 TTTTTACGTCGTTTTAAAATT AAAACGGAGTTTAATACGAAG TTGGGTGAAGTCG
158 chr16:74441727- YGGGYGTGGTGGTTTATGTTT 9 7 XX0000000 73 74441802
GTTATTTTAGTATTTTGGGAG GTTGAGGTGGGTGGATTATGA GGTTGGGAGATTG 164
chr4:174427946- YGGGAYGGYGGTGTTTAGTTT 7 4 XXX1111 74 174428028
AGTTTAGGGTAATTGGGTTTT TTGAGAGTTCGATTTTTATCG GTTTGGGAGCGAGTGGTTCG
176 chr6:119107238- YGGGTGTGGTGGTTTAYGTTT 9 4 X00X00XXX 75
119107313 GTAATTTTAGTATTTTGGGAG GTTGAGGTGGGYGGATTAYGA GGTTAGGAGATYG
178 chr19:13215437- YGTGGTTTGGAGAGYGTTGAA 10 5 XXX1111X1X 76
13215527 GGTGGGAGGGGGAAGAGGGGT AGAATTTTYGCGGGAGCGAGC
GTATAGTTGTCGTTTYGTGGT CGTTTYG 180 chr3:192125880-
YGTGTTTTTYGTATAGGGAGC 9 6 XX1X11111 77 192125950
GTTYGTTTTTGTTGGGGTTGG AGCGGCGTTTGGAGGTCGATA TTCGGTCG 186
chr22:21483273- YGTGATTAGTTYGTTTTGGTT 8 6 XX000000 78 21483360
TTTTAAAGTGTTGGGATTAAA GGTGTGAGTTATTGTGTTGGG TTGAGATTTTGTTTTAAAAAA
AAAA 188 chr19:18497159- TGAGAGTTGYGGAAAYGTTAY 9 4 0XXXX000X 79
18497245 GAGGATTTGTTAATTAGGTTG YGGGTTAATTAGAGTTGGGAA
GATTTGAATATTGATTTTGTT TYG 190 chr9:79629090- CGAGGTGGAGCGACGTTGGTA
8 8 11111111 80 79629145 CGGTAATTTTGAGTTTGCGCG GTTCGGCGTTATTT 192
chr12:75601322- CGTCGGTTTCGTCGATGTTTT 11 11 11111111111 81 75601409
AGAAGGTTAGTTTTTTTTCGA AGAGCGGTTCGTATACGTTTG CGGGGTAGTGTAGTTTGTCGG
TGCG 200 chr9:138999208- CGCGGCGATCGTAAAGGGAGT 7 7 1111111 82
138999265 GTTTGTTTATTCGCGTTTGAA AGTAGATTTTTTTTCG 202 chr1:2987530-
YGTYGTYGAYGYGAAGGGGTT 18 11 XXXXX11XX111111111 83 2987630
GTTTGTGCGCGGYGTTGYGGG TTTTTCGCGCGTGGGGTGTGC GTGTGCGTGTTCGGGTTCGGT
TTTGTGTGTGTATCGCG 208 chr8:55467547- ATGTTTGTAATTTTAGTATTT 6 6
000000 84 55467607 TGGGAGGTTGAGGTGGGTGGA TTATGAGGTTAGGAGATTG 210
chr12:123713533- TTTTTAAAATTTTTTTTTTTC 1 1 1 85 123713562 GTTTTATTT
213 chr2:106776970- GAAAACGCGGTGTAGGGAGAA 4 4 1111 86 106777018
AATTAATTTAGTGAGGAGCGG AGGCGTA 214 chr3:141516288-
GAACGAGAATAAAGCGGTTAG 3 3 111 87 141516321 GTTTTTTTCGGAG 219
chr16:30484192- GCGTTGTTGTTTGGGTTTTTT 2 2 11 88 30484232
TTTTTCGGTAGGTAAGGGAG 222 chr3:111809469- NNACGGTTNN 1 1 1 89
111809474 223 chr10:120489276- AGAGAGGTTTTTAATTTACGG 1 1 1 90
120489298 AA 224 chr11:1874066- TTTGCGGTTATTTTGGTTTTC 3 3 111 91
1874099 GTATTTCGTTTTT 225 chr7:142422227- GTTATAATACGATATTTAG 1 1 1
92 142422245 226 chr1:3086483- ATTGTGGTTTGTGTGTTTTGA 7 7 0000000 93
3086511 GTAATTGG
In sequences, each Y, independently, C or T/U
In methylation pattern, each independently: 1=methylation,
0=un-methylation and X=either methylation or un-methylation
[0160] In a fourth aspect, the invention relates to a nucleic acid
probe that is complementary to the particular (eg non-natural)
sequence of a nucleic acid of the third aspect, preferably wherein
said nucleic acid probe is labelled.
[0161] In certain of such embodiments, the label is a detectable
moiety such as detectable fluorescent moiety. Examples of suitable
fluorescent moieties include, but are not limited to,
6-carboxyfluorescein (6-FAM), VIC, HEX, Cy3, Cy5, TAMRA, JOE, TET,
ROX, R-phycoerythrin, fluorescein, rhodamin, Alexa, or Texas Red.
Said nucleic acid probe may further comprise enhancers and/or
quencher molecules Iowa Black FQ, ZEN, Iowa Black RQ TAMRA,
Eclipse, BHQ-1.
[0162] Such a nucleic acid probe has utility to detect the nucleic
acid of the invention to which it is complementary, such as in
the/a method of the first aspect of the invention, such as by
hybridisation (and detection thereof) between said probe and its
complementary nucleic acid of the invention.
[0163] In particular embodiments of said aspect, a nucleic acid
probe of the present invention differential may bind to its
complementary nucleic acid of the invention depending on the
methylation status of one or more CpCs within said nucleic acid
sequence. For example, a nucleic acid probe may be designed to bind
only (or preferably) to: (i) a nucleic acid used in the method of
the present invention when an un-methylated cytosine in a CpG has
been converted to U or T (following bisulphite treatment of an
un-methylated such CpG); compared to (ii) a nucleic acid used in
the method of the present invention when said cytosine has not been
converted following bisulphite treatment (ie, when such CpG
comprises 5-methylcytosine). As will now be apparent to the person
of ordinary skill, such specifically (or preferentially) for
binding/hybridisation nucleic acid probes may be designed using
routine methodology following the disclosure of the complementary
sequences herein; and will readily be usable in array based and/or
PCR-based detection technology, such as MethylLight, MSP, or
BeadChip (eg Illumina Epic arrays).
[0164] In a fifth aspect, the invention relates to a PCR primer
pair for amplifying a nucleic acid sequence consisting of at least
10 contiguous bases (such as the number of contiguous bases
described elsewhere herein, and preferably at least 15 contiguous
bases for any SEQ ID other than SEQ ID NO:58) comprised in a
sequence selected from the group consisting of: SEQ ID NOs: 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 and 62 (such as SEQ ID
NO: 32, 33, 34 and/or 35), or a nucleotide sequence present within
about 2,000 bp (such as within about 200 bp) 5' or 3' thereof, or
an allelic variant and/or complementary sequence of any of said
nucleotide sequences.
[0165] In certain embodiments of such aspect, at least one primer
of said pair may include a sequence corresponding to at least one
(eg non-natural) bisulphite-converted CpG; such a primer pair
having particular utility in amplifying (eg by PCR amplification)
bisulphite converted sequences. Indeed, in other embodiments, the
primer pair may be a degenerate primer pair such that two version
of at least one of said primer pair are present such that both the
bisulphite-converted CpG (ie the CpG is un-methylated and converted
to U/T) and the non-converted CpG (ie the CpG comprises
5-methylcytosine and remains as C) are amplified/detected.
[0166] In particular embodiments, a primer pair of the present
invention is one selected from the group primer-pairs set forth in
each row of TABLE 3.
TABLE-US-00007 TABLE 3 Particular primer pairs and predicted
amplicon size SEQ ID SEQ ID Amplicon DMR# Primer sequence 1 NO.
Primer sequence 2 NO. size 141 TATTYGGAGGTTTAGGGGTGAGGATT 94
CAACRAATTCCCCRAATCCTAT 125 136 bp T CCT 204
GATATTYGGTGGAGAGTYGTAGTTGT 95 ACCRATACCAACCCRCACTC 126 154 bp T 228
GTTTTATGGGYGAGTTGTTGTAGTG 96 AAAAACCCRCRAAACCAAAAAA 127 111 bp TC
144 TTTAGGTTTGAYGTGGGTTTTTTAG 97 TCTAAAATATATTCACCRAATC 128 140 bp
AAAAATAACAAAA 123 GYGAAGTAGGAGTAGTTGTYGGGTTT 98
TCTCTCCTAACRAAAAACTAAC 129 120 bp TA CCAATTACC 129
AATTTTGTTGAGTGAGTTTATAAATA 99 TCACTTAACACCRTTTTTACTA 130 120 bp
GGGTATAA ACC 137 ATTTYGTTATATATATAGTTGTATTY 100
CRAAAAACTAAACACCCAAACC 131 130 bp GGTATAATA 148
GAGGTAATGGAAGYGGTTATTTTTG 101 ATATATAACATCCRAAACACCC 132 120 bp
ATAACACAA 150 GYGGGTATTTGTAGTTTTAGTTATT 102 TTTTTAAACRAAATCTCRCTCT
133 137 bp AT 154 TTTTYGGAGTTYGGAGTTTAGGTTAG 103
AAAACRCATCTCCCTAACTCCT 134 137 bp TGGTA ACAAACTA 158
GATTTTGTTTTAAAAAAGAAAAAAAT 104 AAATTTCACCRTATTACCCAAA 135 136 bp
AGGGT ATAATAT 164 GATTYGYGAGGTTTTTTAGTAGTTTA 105
AAACRACRATTCCCAACATCTA 136 138 bp TTT AACT 176
TAATTTATTTTTTTATTAAATTGTAT 106 AATAAAAACRAAATTTCACCRT 137 138 bp
GAAGAAGGT AATCT 178 GGTAGGAGYGTTTTATTATGYGTAAG 107
CCAAAACCRAAACCAACRATTC 138 142 bp TT C 180
TTGTAGAAGYGTATTTTGTTGAATAT 108 ACAAAAACRACAAACRAAAAAA 139 135 bp
TTYGAGGA TCCAACAA 186 YGTGTTAGTTAGGATGGTTTYGATTT 109
TCCCAAAATACCACAACCCAAA 140 146 bp TTTGATTT CC 188
AGAGTTGTATTTYGAAGATTTTAGAT 110 AACRTAAATATCCRAACTACAA 141 141 bp TT
AAAC 190 GTTAGAYGAGAGTTTGGGGTTAATGT 111 AAATCCAACCAACAACRAAAAA 142
109 bp CCAAA 192 YGGTAGGTTATTTAGTAGTAGGGTTT 112
CRTCTTCRCCTATATACTCAAT 143 144 bp TA TACTAC 200
AATTAGTTTAGTAATYGGYGATTTTA 113 CCACTAACAAATAAATCCTATA 144 115 bp AG
TTCCTAC 202 GTGYGAATAAGATYGGGYGTTT 114 CRATAATCCCRACTCTAAACAA 145
148 bp ACC 208 ATATTAATTTTGTTYGGGTAYGGTGG 115
CRAAATTTCACCATATTCRCCA 146 121 bp TTT AAATAATCT 210
GGAGTTGTAAAAAATAAAGGAATATG 116 TACATACAAATTACTATAAAAC 147 92 bp TG
CATTTCCTATAC 213 TYGTTYGGGAATGGGAATATAGTTAT 117
CCRAATACACACTCCACAATCC 148 103 bp ATATGG 214
TTGTTTAAAGGYGTAGAGGAGTAGTT 118 TCAACTATTTCCTAAAACTCTC 149 94 bp GG
TCCTTCCTTC 219 GGATGAAGGATTTTTGTATTATTGTG 119
AAACACATATCCCTTCCCCTAC 150 101 bp ATGGTTATG CTC 222
TAGGTTATAGGAAGAGGTATTTTTTA 120 ACTTCCTAAAAAAACTCAACTT 151 70 bp
TAGATG AAAATTTTAC 223 AAGAGAGAGTGGTTGATAATTAGTAG 121
ACAACTAATATACAAAAAAATT 152 84 bp ATATTACAAACAC 224
GGTTTTTTTTTTYGAGTTATGAAGAG 122 AAACRTTAAACAAATTACAAAA 153 97 bp TTG
ACCTAAAATAAC 225 GAAGTTTGATATTTTTGGTTTTAAAT 123
CCACAACTTACTATACATAAAA 154 86 bp ATTGTTTG AATATCTATCC 226
GGGGGGATTGTYGTTAATTTATTGTT 124 CCACRAAATATACACAATCCAC 155 91 bp
TAATG ATACATCAC
Each Y, independently, C or T; each R, independently, G or A
[0167] In a sixth aspect, the invention relates to a plurality of
nucleic acids comprising least two, three, four, five, six, seven,
eight, nine, ten or more (such as about 12, 15, 20, 25 or 30)
nucleic acid sequences of the third aspect and/or of the nucleic
acid probes of the fourth aspect and/or of the PCR primer pairs of
the fifth aspect (for example, such PCR primers for amplifying at
least 15 contiguous bases of SEQ ID NO: 32, 33, 34 and/or 35, in
particular at least 3 pairs of primers for amplifying at least 15
contiguous bases of at least SEQ ID NO: 32, 33 and 34).
[0168] In certain embodiments of such aspect, said plurality of
nucleic acids may be in any form that is applicable to the practice
of the invention (or its storage or preparation), such as in the
form of an admixture or an array. For example, such arrays may
comprise a microtitire plate or a hybridisation chip.
[0169] As will now be apparent to the person of ordinary skill, any
of such nucleic acids (including the probes and/or PCR primer
pairs) of the present invention may be synthetic (ie, synthesised
by chemical and/or enzymatic means/methods practiced in-vitro),
and/or may be isolated and/or are not natural occurring or are used
or present in a non-natural composition or mixture. Furthermore,
any of the methods of the present invention may produce (and hence
a composition or any nucleic acid of the present invention may
comprise) an in-vitro-produced nucleic acid molecule, such as a DNA
product of a PCR reaction (eg a "PCR product"). One or more of such
in-vitro-produced nucleic acid molecules may be non-natural because
they comprise a nucleotide primer and/or probe that includes at
least one non-natural substituted base, detectable label or bases,
such a nucleic acid molecule having been generated by polymerase
extension (or partial nuclease digestion) or bisulphite or
enzymatic conversion of such nucleic acid (eg a labelled primer
and/or probe), and hence providing at least a fraction of such
nucleic acid molecules that include a non-natural base or
detectable label, such that even though the nucleic acid sequence
of the nucleic acid molecules may otherwise comprise a naturally
occurring sequence (or fragment thereof), such an in-vitro-produced
nucleic acid molecule is non-natural by virtue of (at least) the
non-natural base and/or detectable label that it includes.
[0170] In a seventh aspect, the invention relates to a kit (for
example, one for determining the presence or absence of, or
response to therapy against, an ovarian cancer in a woman), such as
a kit of parts comprising two or more separate compartments,
holders, vessels or containers (eg each holding a different
component of the kit), wherein said kit comprises: [0171] two,
three, four, five, six, seven, eight, nine, ten or more (such as
about 12, 15, 20, 25 or 30) nucleic acid sequences of the third
aspect and/or of the nucleic acid probes of the fourth aspect
and/or of the PCR primer pairs of the fifth aspect (for example,
such PCR primers for amplifying at least 15 contiguous bases of SEQ
ID NOs: 32, 33, 34 and/or 35, in particular at least 3 pairs of
primers for amplifying at least 15 contiguous bases of at least SEQ
ID NO: 32, 33 and 34) and/or the population of nucleic acids of the
sixth aspect; and [0172] optionally, said kit further comprising:
[0173] (i) a printed manual or computer readable memory comprising
instructions to use said synthetic nucleic acid sequence(s),
labelled nucleic acid probe(s) and/or population of nucleic acids
to practice a method of the first aspect and/or to produce or
detect the nucleic acid sequence(s) of the third aspect; and/or
[0174] (ii) one or more other item, component or reagent useful for
the practice of a method of the first aspect and and/or the
production or detection of the nucleic acid sequence(s) of third
aspect, including any such item, component or reagent disclosed
herein and/or useful for such practice, production or
detection.
[0175] In certain embodiments said kit further comprises one or
more additional components. For example, such a kit may comprise
one or more (such as two, three, four, all) of the following:
[0176] means to collect and/or store a biological sample, such as
blood, to be taken from said woman, preferably wherein said means
is a blood collection tube; and/or [0177] means to extract DNA,
preferably cell-free DNA, from the sample to be taken from said
woman, preferably wherein said means is a cell-free DNA extraction
kit; and/or [0178] an agent to differentially modify DNA based on
the methylation status of one or more CpGs located within said DNA,
preferably wherein said agent is bisulphite; and/or [0179] one or
more reagents to detect a nucleic acid sequence, preferably for
detecting the sequence of a bisulphite-converted nucleotide
sequence; and/or [0180] a printed manual or computer readable
memory comprising instructions to identify, obtain and/or use one
or more of said means, agent or reagent(s) in the context of a
method of the first aspect.
[0181] In some embodiments, any method of the invention may be a
computer-implemented method, or one that is assisted or supported
by a computer. In some embodiments, information reflecting the
determination, detection, presence or absence of one or more
methylated or un-methylated (as applicable) CpGs located within one
or more of said nucleotide sequences comprised in at least one
molecule of cell-free DNA of a woman in a sample is obtained by at
least one processor, and/or information reflecting the
determination, detection, presence or absence of one or more
methylated CpGs located within one or more of said nucleotide
sequences comprised in at least one molecule of cell-free DNA of a
woman in a sample is provided in user readable format by another
processor. The one or more processors may be coupled to random
access memory operating under control of or in conjunction with a
computer operating system. The processors may be included in one or
more servers, clusters, or other computers or hardware resources,
or may be implemented using cloud-based resources. The operating
system may be, for example, a distribution of the LinuxTM operating
system, the UnixTM operating system, or other open-source or
proprietary operating system or platform. Processors may
communicate with data storage devices, such as a database stored on
a hard drive or drive array, to access or store program
instructions other data. Processors may further communicate via a
network interface, which in turn may communicate via the one or
more networks, such as the Internet or other public or private
networks, such that a query or other request may be received from a
client, or other device or service. In some embodiments, the
computer-implemented method (or the one that is assisted or
supported by a computer) of detecting the determination, detection,
presence or absence of one or more methylated CpGs located within
one or more of said nucleotide sequences comprised in at least one
molecule of cell-free DNA of a woman in a sample may be provided as
a kit.
[0182] In an eighth aspect, the invention relates to a to a
computer program product comprising: a computer readable medium
encoded with a plurality of instructions for controlling a
computing system to perform and/or manage an operation for
determining the presence or absence of, or response to therapy
against, an ovarian cancer in a woman, from a biological sample
from said woman, said sample comprising cell-free DNA of said
woman, and determining, in at least one molecule of said cell-free
DNA, the methylation status at one or more CpGs located within one
or more nucleotide sequences in accordance with a method of the
first aspect; said operation comprising the steps of: [0183]
receiving a first signal representing the number of molecules of
said cell-free DNA comprising one or more methylated or
un-methylated CpGs (such as relevant CpGs) located within one or
more of the nucleotide sequences independently selected from the
group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 and 31 (for example, within one or more of the nucleotide
sequences comprised in one or more of the respective DMRs of the
present invention independently selected from the group consisting
of DMR#: #141, #204, #228, #144, #123, #129, #137, #148, #150,
#154, #158, #164, #176, #178, #180, #186, #188, #190, #192, #200,
#202, #208,#210, #213, #214, #219, #222, #223, #224, #225 and
#226), or a nucleotide sequence present within 200 bp 5' or 3'
thereof, or an allelic variant and/or complementary sequence of any
of said nucleotide sequences; and [0184] determining a
classification of the presence or absence of, or response to
therapy against, an ovarian cancer in said woman based on their
being at least one molecules of said cell-free DNA comprising one
or more: (i) said methylated CpGs associated with (such as located
within) one or more of the hyper-methylated DMRs of the present
invention (eg, as identified in TABLE 1A), for example associated
with (such as located within) one or more of said nucleotide
sequences independently selected from: SEQ ID NOs: 1, 2, 3, 4, 10,
12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29 and 30;
and/or (ii) said un-methylated CpGs associated with (such as
located within) one or more of the hypo-methylated DMRs of the
present invention (eg, as identified in TABLE 1B), for example
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 5, 6,
7, 8, 9, 11, 13, 16, 17, 22 and 31.
[0185] In certain embodiments of a computer program product of the
present invention the operation further comprises steps of:
receiving a second signal representing the number of molecules of
said cell-free DNA comprising said nucleotide sequence(s); and
estimating a fraction or ratio of molecules of said cell-free DNA
comprising one or more methylated or un-methylated (as applicable)
CpGs located within one or more of the nucleotide sequences within
all of said nucleotide sequence(s).
[0186] In particular embodiments of the computer program product of
the present invention, said classification is determined by
comparing said a fraction or ratio to a standard or cut-off value;
such as a standard or cut-off value described elsewhere herein.
Such an embodiment has particular utility where different
populations of women (such as patient stratification; or even
individualised therapy) is desired. The establishment of the
applicable standard or cut-off value for each population of women
will be apparent to the person of ordinary skill, such as by the
collection and analysis of data from a statistically meaningful
number of women within the desired population, and/or
stratification of sub-populations from large population studies. In
particular of such embodiments, the computer program may comprise,
or the cut-off values may be calculated by, a machine-learning
algorithm that is trained on the DMR-based data generated from
samples from women with OC vs. control samples, for example, the
(sub)populations of women described herein and/or from samples from
other (sub)populations such as UKCTOCS, by using any number and
combination of the methylation patterns/DMRs as described herein.
In other embodiments, the standard or cut-off value may be modified
based on the amount of total DNA present in a sample (for example,
if increased such as by contamination from genomic DNA leaking from
WBCs) and/or if the average sample size of the extracted DNA is
increased (for example, a fragment size of greater than about 1000
bp), which can also indicate contamination from genomic DNA. By way
of none limiting examples, such a standard or cut-off value may be
reduced by factor, such as by a factor of 2, 3, 4, 5, 6, 8 or 10
(in particular, by a factor of 3) if the DNA extracted from one or
more samples used in a study or test exhibits characteristics (such
as those described herein) of contamination from genomic DNA.
[0187] In other particular embodiments, the computer program
product of the present invention may be for an operation that
further comprises the steps of: receiving a third signal
representing: (i) the amount or concentration of total cell-free
DNA present in said sample; and/or (ii) a baseline value of said
fraction or ratio previously determined for said woman; and
modifying said standard or cut-off value for a given sample based
on said third signal. As will now be apparent to the person of
ordinary skill, such patient-specific modification of the standard
or cut-off value can provide increased personalisation of detection
(such as by an increase in specificity and/or sensitivity for
individual women), analogously to that provided by the ROCA
test.
[0188] The computer program product of the present invention may
include embodiments wherein said first signal, and optional second
signal, is determined from nucleotide sequence and/or methylation
status information of a plurality of said molecules of said
cell-free DNA and/or amplified DNA representing each of said
nucleotide sequences, preferably wherein said plurality is a number
selected from the group consisting of at least about: 50, 100,
1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 500,000, 1,000,000,
1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000
and 5,000,000 molecules, or more than 5,000,000 molecules. In
particular of such embodiments, said operation may further comprise
the steps of: for each of said molecule's sequence and/or
methylation status information, determining if said molecule
comprises none, one or more: (i) methylated CpGs associated with
(such as located within) one or more of the hyper-methylated DMRs
of the present invention (eg, as identified in TABLE 1A), for
example associated with (such as located within) one or more of
said nucleotide sequences independently selected from: SEQ ID NOs:
1, 2, 3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28,
29 and 30;, or a nucleotide sequence present within about 2,000 bp
(such as 200 bp) 5' or 3' thereof, or an allelic variant and/or
complementary sequence of any of said nucleotide sequences; and/or
(ii) un-methylated CpGs associated with (such as located within)
one or more of the hypo-methylated DMRs of the present invention
(eg, as identified in TABLE 1B), for example associated with (such
as located within) one or more of said nucleotide sequences
independently selected from: SEQ ID NOs: 5, 6, 7, 8, 9, 11, 13, 16,
17, 22 and 31, or a nucleotide sequence present within about 2,000
bp (such as 200 bp) 5' or 3' thereof, or an allelic variant and/or
complementary sequence of any of said nucleotide sequences; and
calculating said first signal, and optional second signal, based on
said determination for all or a portion of said plurality of
molecules. In certain embodiments, the number of said cell-free DNA
and/or amplified DNA molecules to be analysed may be predetermined.
For example, depending on the expected stage of ovarian cancer, age
or general (or specific) health of the woman or the total number of
cell-free DNA found in the biological sample of the woman, the said
number may be of the higher (eg, greater than about 100,000,
500,000, 1,000,000, 1,500,000 or 2,000,000) or lower (eg less than
about 100,000, 500,000, 1,000,000, 1,500,000 or 2,000,000) regions
of said range. The number of DNA molecules analysed can be
modified, predetermined and/or selected for reasons such as to
achieve a particular sensitivity and/or specificity of the test; or
may be increased for a second or subsequent test conducted for a
woman who has had a borderline result for a previous test or where
said woman may desire increased sensitivity and/or specificity (ie
confidence of the test result) before making a decision on further
therapy.
[0189] The ability to increase the number of said DNA molecules
analysed is one particular advantage of the test of the present
invention, as it enables the dynamic range of the test to be
increased (eg, to that desired), including to a dynamic range that
is greater than alternative (eg protein-based) tests for OC such as
those based on the CA125.
[0190] In further embodiments of the computer program product of
the present invention, said operation may further comprise the
steps of: [0191] receiving a signal representing the amount
present, in a sample of blood taken from said woman, of one or more
proteins independently selected from the group consisting of:
CA-125, HE4, transthyretin, apolipoprotein A1, beta-2-microglobin
and transferrin; and [0192] comparing said a fraction or ratio to a
standard or cut-off value for said protein; and [0193] determining
a classification of the presence or absence of, or response to
therapy against, an ovarian cancer in said woman based on their
being either of (i) at least one molecules of said cell-free DNA
comprising one or more of said methylated or un-methylated (as
applicable) CpGs located within one or more of said nucleotide
sequences; and/or (ii) an amount of said protein(s) present in said
blood sample is greater than said standard or cut-off value for
such amount or protein.
[0194] As will be known to gynecological oncologists, each (or
certain combinations) of said proteins are associated with and used
for the diagnosis of ovarian cancer. Accordingly, the present
invention envisions that it is used in combination with such
protein-based diagnostic tests. In particular, and as set out
elsewhere herein, the combination of the test of the present
invention shows independent sensitivity and/or specificity to CA125
(and/or other protein-based tests). Importantly, in the data
presented below, there was no overlap between the DNAme-false
positives and the CA125-false positives. Therefore, the application
of a test of the present invention with one or more of such
(independent) protein-based diagnostic tests would be particularly
advantageous to women in that such a combination would provide
greater confidence in the result of such a combined test; for
example that their (combination) result was not a false positive or
false negative; ie that they have been correctly diagnosed for: (i)
the presence of ovarian cancer (such as HGS ovarian cancer; or one
that is not responding, or has not responded, to chemotherapy
treatment), or (ii) the absence of ovarian cancer (such as having a
benign pelvic mass), or the absence of HGS ovarian cancer (or
having an ovarian cancer that is responding, or has responded, to
chemotherapy treatment).
[0195] A number of such protein-based tests for ovarian cancer are
used, including those which have been validated in clinical trials
and are commercially available. In particular, suitable
protein-based tests that may be used in combination with the test
of the present invention include tests for CA125 (in particular
when conducted in a routine manner for each woman, such as in the
ROCA.RTM. test of Abcodia Ltd. Refs. 6, 7) and/or HE4 (such as the
ELEXSYS.RTM./COBAS.RTM. versions thereof of Roche Diagnostics), as
well as when CA125 and HE4 are used in a combined test (such as the
ROMA--Risk of Ovarian Malignancy Algorithm--test. Moore et al,
2009; Gynecological Oncology 112:49. Malkasian et al, 1988; Am J
Obstet Gynecol 159:341) and/or when CA125 is used in a test that
also involves other proteins including transthyretin,
apolipoprotein Al, beta-2-microglobin and/or transferrin (such as
in the OVA1 test of Aspira Labs. Ueland et al, 2009: Obstet Gynecol
117:1289. Ware Miller et al, 2011; Obstet Gynecol 117:1298).
[0196] In other embodiments, the test of the present invention is
used in combination with other DNA-based diagnostic tests for
ovarian cancer. For example, determination of the woman's germline
mutational status of BRCA1 and/or BRCA2 gene or any other high risk
genes including but not limited to RAD51, PALB2, ATM, BRIP1, CHEK2,
PTEN, CDH1. Also envisioned for such other DNA-based diagnostic
tests for ovarian cancer, are those tests which may include the
analysis of one or more single-nucleotide polymorphisms (SNPs) that
are associated with OC. In other embodiments, the test of the
present invention is used in combination with epidemiological-based
models for ovarian cancer, such as those which use various
combinations of family history, number of lifetime ovulatory cycles
(eg a function of period taking oral contraceptive pill, number of
pregnancies and time of breastfeeding) as well as body mass index
and hormone replacement therapy use.
[0197] Any of such other diagnostic tests for ovarian cancer may be
used to identify a sub-population of women who are at a
higher/highest risk of developing ovarian cancer, and as described
above, can be used to "artificially increase" the prevalence of OC
(ie, only in that identified group at highest risk). In such
sub-populations, then the specificity of the inventive test can be
lower without a major impact on the rate of false positives.
[0198] In another aspect, the present invention also relates to a
use of a nucleic acid sequences of the third aspect of the present
invention and/or a labeled nucleic acid probes of the fourth aspect
of the present invention and/or a PCR primer pair of the fifth
aspect of the present invention (for example, such PCR primers for
amplifying at least 15 contiguous bases of SEQ ID NO: 32, 33, 34
and/or 35, in particular at least 3 pairs of primers for amplifying
at least 15 contiguous bases of at least SEQ ID NO: 32, 33 and 34)
and/or a plurality of nucleic acids of the sixth aspect of the
present invention and/or a kit of the seventh aspect of the present
invention and/or a computer program product of the eighth aspect of
the present invention, in each case for determining (such in-vitro,
including in an in-vitro diagnostic test) the presence or absence
of, or response to therapy against, an ovarian cancer in a woman.
It being envisioned that all embodiments set forth herein for other
aspects are also encompassed within this use of the present
invention.
[0199] It is to be understood that application of the teachings of
the present invention to a specific problem or environment, and the
inclusion of variations of the present invention or additional
features thereto (such as further aspects and embodiments), will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein.
[0200] Terms as set forth herein are generally to be understood by
their common meaning unless indicated otherwise. Where the term
"comprising" or "comprising of" is used herein, it does not exclude
other elements. For the purposes of the present invention, the term
"consisting of" is considered to be a particular embodiment of the
term "comprising of". If hereinafter a group is defined to comprise
at least a certain number of embodiments, this is also to be
understood to disclose a group that consists of all and/or only of
these embodiments. Where used herein, "and/or" is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. For example, "A and/or B" is
to be taken as specific disclosure of each of (i) A, (ii) B and
(iii) A and B, just as if each is set out individually herein.
Analogously, the terms "in particular", "particular" or "certain"
(and the like), when used in the context of any embodiment of the
present invention, in each case is to be interpreted as a non
limiting example of just one (or more) possible such embodiment(s)
amongst others, and not that such "particular" or "certain"
embodiment is the only one envisioned by the present invention. In
the context of the present invention, the terms "about" and
"approximately" denote an interval of accuracy that the person
skilled in the art will understand to still ensure the technical
effect of the feature in question. The term typically indicates
deviation from the indicated numerical value by .+-.20%, .+-.15%,
.+-.10%, and for example .+-.5%. As will be appreciated by the
person of ordinary skill, the specific such deviation for a
numerical value for a given technical effect will depend on the
nature of the technical effect. For example, a natural or
biological technical effect may generally have a larger such
deviation than one for a man-made or engineering technical effect.
Where an indefinite or definite article is used when referring to a
singular noun, e.g. "a", "an" or "the", this includes a plural of
that noun unless something else is specifically stated. The terms
"of the [present] invention", "in accordance with the [present]
invention", or "according to the [present] invention" as used
herein are intended to refer to all aspects and embodiments of the
invention described and/or claimed herein.
[0201] Unless context dictates otherwise, the descriptions and
definitions of the features set out above are not limited to any
particular aspect or embodiment of the invention and apply equally
to all aspects and embodiments which are described.
[0202] All references, patents, and publications cited herein are
hereby incorporated by reference in their entirety; provided that
In case of conflict, the present specification, including
definitions, will control.
[0203] In view of the above, it will be appreciated that the
present invention also relates to the following items: [0204] Item
1. A method of determining the presence or absence of, or response
to therapy against, an ovarian cancer in a woman, said method
comprising the steps: [0205] providing a biological sample from
said woman, said sample comprising cell-free DNA of said woman; and
[0206] determining, in at least one molecule of said cell-free DNA,
the methylation status at one or more CpGs located within one or
more of the nucleotide sequences independently selected from the
group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 and 31, or a nucleotide sequence present within about 2,000
bp (such as within about 200 bp) 5' or 3' thereof, or an allelic
variant and/or complementary sequence of said nucleotide seq
uence(s), [0207] wherein, the presence in at least one of said
cell-free DNA molecules of one or more: (i) methylated CpGs
associated with (such as located within) one or more of said
nucleotide sequences independently selected from: SEQ ID NOs: 1, 2,
3, 4, 10, 12, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29
and 30; and/or (ii) un-methylated CpGs associated with (such as
located within) one or more of said nucleotide sequences
independently selected from: SEQ ID NOs: 5, 6, 7, 8, 9, 11, 13, 16,
17, 22 and 31, indicates the presence of, or a reduced response to
therapy against, an ovarian cancer in said woman; preferably
wherein said biological sample is liquid biological sample selected
from the group consisting of: a blood sample, a plasma sample and a
serum sample. [0208] Item 2. The method of item 1, wherein said
CpGs for a given nucleotide sequence are identifiable by a genome
position for the cysteine thereof, independently selected from the
list of genome positions corresponding to said nucleotide sequence
set forth in TABLE 1C. [0209] Item 3. The method of item 1 or 2,
wherein the presence in at least one of said cell-free DNA
molecules of one or more pattern of methylation and/or
un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
[0210] Item 4. The method of any one of items 1 to 3, wherein said
nucleotide sequence(s) is/are associated with DMR(s) #141 and/or
#204 and/or #228 (eg, SEQ ID NOs: 1, 2, 3 and/or 4), or an allelic
variant and/or complementary sequence of said nucleotide
sequence(s). [0211] Item 5. The method of item 4, wherein the
methylation status is determined at one or more of said CpGs
located within each of said three nucleotide sequences; wherein,
the presence in at least one of said cell-free DNA molecules of one
or more methylated CpGs, and/or of one or more pattern of
methylation and/or un-methylation as set forth in TABLE 2B for the
respective nucleotide sequence(s), located within any one of said
nucleotide sequences indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman.
[0212] Item 6. The method of item 5, wherein the methylation status
is determined at about 7 CpGs located within nucleotide sequence
SEQ ID NO 1 and at about 16 to 18 CpGs located within nucleotide
sequence SEQ ID NO 2 and about 7 to 9 CpGs located within
nucleotide sequence SEQ ID NO 3; wherein, the presence in at least
one of said cell-free DNA molecules of at least said number of
methylated said CpGs, and/or of one or more pattern of methylation
and/or un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), located within any one of said nucleotide
sequences indicates the presence of, or a reduced response to
therapy against, an ovarian cancer in said woman [0213] Item 7. The
method of any one of items 1 to 6, comprising the step of treating
said cell-free DNA with an agent that differentially modifies said
cell-free DNA based on the methylation status of one or more CpGs
located within;
[0214] preferably a methylation sensitive restriction enzyme and/or
bisulphite; preferably wherein said agent is bisulphite and said
determining step comprises the detection of at least one
bisulphite-converted un-methylated cytosine within one or more of
the nucleotide sequences independently selected from those set
forth in TABLE 2A (eg, independently selected the group consisting
of: SEQ ID NOs 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61
and 62), wherein one or more of the bases identified by "Y" therein
is a U or T and, preferably, where one or more of the bases
identified by "Y" in a CpG therein is a C, or an allelic variant
and/or complementary sequence of said nucleotide sequence(s).
[0215] Item 8. The method of item 7, wherein said agent is
bisulphite and said determining step comprises the detection of at
least one bisulphite-converted un-methylated cytosine within a
nucleotide sequence having a length of at least 15 bp (such as at
least 50 bp) comprised in SEQ ID NO 32 and/or SEQ ID NO 33 and/or
SEQ ID NO 34, wherein one or more of the bases identified by "Y"
therein is a U or T and, preferably, where one or more of the bases
identified by "Y" in a CpG therein is a C, or an allelic variant
and/or complementary sequence of said nucleotide sequence(s).
[0216] Item 9. The method of any one of items 1 to 8, wherein the
methylation status of said CpG(s) is determined in multiple
molecules of said cell-free DNA and/or amplified DNA representing
each of said nucleotide sequences; preferably wherein: [0217] (a)
the presence in at least a plurality of said cell-free DNA
molecules of one or more methylated and/or un-methylated CpGs (as
applicable), and/or the presence in at least a plurality of said
cell-free DNA molecules of one or more pattern of methylation
and/or un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), located within one or more of said
nucleotide sequences, indicates the presence of, or a reduced
response to therapy against, an ovarian cancer in said woman;
and/or [0218] (b) said plurality of cell-free DNA molecules with
one or more of said methylated and/or un-methylated CpGs (as
applicable), and/or the presence in at least a plurality of said
cell-free DNA molecules of one or more pattern of methylation
and/or un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), located is at least 2, 3, 4, 5, 6, 7, 18, 9
or 10, or at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
70, 80, 90, 100, 125, 150, 175 or 200, or a greater number such as
greater than about 500, 1,000, 5,000, 7,500, 1,000, 2,500, 5,000 or
greater than 5,000 molecules; and/or (c) the methylation status of
said CpG(s) is determined in a number of molecules of said
cell-free DNA and/or amplified DNA representing each of said
nucleotide sequences selected from the group consisting of at least
about: 1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 500,000,
1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000,
4,000,000 and 5,000,000 molecules, or more than 5,000,000
molecules. [0219] Item 10. The method of item 9, wherein a fraction
or ratio of, or an absolute number of, cell-free DNA molecules in
said sample having said methylated and/or un-methylated CpG(s) (as
applicable) located within said nucleotide sequence(s), and/or
having said pattern of methylation and/or un-methylation as set
forth in TABLE 2B for the respective nucleotide sequence(s), is
estimated. [0220] Item 11. The method of item 10, further
comprising a step of comparing said fraction or ratio with a
standard or cut-off value; wherein a fraction or ratio greater than
the standard or cut-off value indicates the presence of or a
reduced response to therapy against, an ovarian cancer in said
woman; preferably wherein said standard or cut-off value(s) is/are
modified for a given sample based on: [0221] the amount or
concentration of total cell-free DNA present in said sample; and/or
[0222] a baseline value of said fraction or ratio previously
determined for said woman; and/or [0223] a value of said fraction
or ratio determined from multiple samples from a population of
women representative of said woman; and/or [0224] the specificity
and/or sensitivity desired for said method of determination; [0225]
more preferably wherein said standard or cut-off value is/are
reduced for a given sample that has an amount or concentration of
total cell-free DNA present in said sample that is greater than a
standard or cut-off value. [0226] Item 12.A nucleic acid comprising
at least 10 (preferable at least about 15, such as at least 50, for
any SEQ ID other than SEQ ID NO:58) contiguous bases comprised in a
sequence selected from the group consisting of:
[0227] SEQ ID NOs 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61 and 62, wherein said nucleic acid sequence includes one or more
of the bases identified by "Y" therein is a U or T and, preferably,
where one or more of the bases identified by "Y" therein is a C, or
an allelic variant and/or complementary sequence of said nucleotide
sequence; [0228] preferably wherein said nucleic acid sequence
comprises at least 15, (such as at least 50) contiguous bases
comprised in a sequence of SEQ ID NOs 32, SEQ ID NOs 33 or SEQ ID
NOs 34, wherein said nucleic acid sequence includes one or more of
the bases identified by "Y" therein is a U or T and, preferably,
where one or more of the bases identified by "Y" therein is a C, or
an allelic variant and/or complementary sequence of said nucleotide
sequence; and [0229] more preferably wherein said nucleic acid
sequence is comprised in a sequence as set forth in TABLE 2B (eg, a
sequence selected from the group consisting of: SEQ ID NOs 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93. [0230] Item 13.A
nucleic acid primer pair for amplifying a nucleic acid sequence
consisting of at least 10 (preferable at least about 15, such as at
least 50, for any SEQ ID other than SEQ ID NO: 89) contiguous bases
comprised in a sequence selected from the group consisting of: SEQ
ID NOs: SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92 and 93, or a nucleotide sequence present within about 2,000 bp
(such as within about 200 bp) 5' or 3' thereof, or an allelic
variant and/or complementary sequence said nucleotide sequence(s);
[0231] preferably wherein: [0232] (a) at least one primer of said
pair includes a sequence corresponding to at least one
bisulphite-converted CpG present in said nucleotide sequence(s);
and/or [0233] (b) said primer pair is selected from the group of
primer-pairs set forth in each row of TABLE 3. [0234] Item 14. A
kit, preferably for determining the presence or absence of, or
response to therapy against, an ovarian cancer in a woman, said kit
comprising: [0235] one or more nucleic acid sequences of item 12
and/or primer pairs of item 13; and [0236] optionally, said kit
further comprising: [0237] (i) a printed manual or computer
readable memory comprising instructions to use said nucleic acid
sequence(s) and/or primer pair(s) to practice a method of any one
of items 1 to 11 and/or to produce or detect the nucleic acid
sequence(s) of item 12; and/or [0238] (ii) one or more other item,
component or reagent useful for the practice of a method of any one
of items 1 to 11 and and/or the production or detection of the
nucleic acid sequence(s) of item 12, including any such item,
component or reagent disclosed herein useful for such practice,
production or detection. [0239] Item 15.A computer program product
comprising: a computer readable medium encoded with a plurality of
instructions for controlling a computing system to perform and/or
manage an operation for determining the presence or absence of, or
response to therapy against, an ovarian cancer in a woman, from a
biological sample from said woman, said sample comprising cell-free
DNA of said woman, and determining, in at least one molecule of
said cell-free DNA, the methylation status at one or more CpGs
located within one or more nucleotide sequences in accordance with
a method as set forth in any one of items 1 to 11; said operation
comprising the steps of: [0240] receiving a first signal
representing the number of molecules of said cell-free DNA
comprising one or more methylated and/or un-methylated CpGs (as
applicable), and/or comprising one or more pattern of methylation
and/or un-methylation as set forth in TABLE 2B for the respective
nucleotide sequence(s), located within one or more of the
nucleotide sequences independently selected from the group
consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 and 31, or a nucleotide sequence present within about 2,000 bp
(such as within about 200 bp) 5' or 3' thereof, or an allelic
variant and/or complementary sequence of said nucleotide
sequence(s); and [0241] determining a classification of the
presence or absence of, or response to therapy against, an ovarian
cancer in said woman based on their being at least one molecules of
said cell-free DNA comprising one or more said methylated and/or
un-methylated CpGs (as applicable), and/or comprising one or more
said pattern of methylation and/or un-methylation, located within
one or more of said nucleotide sequences; [0242] preferably wherein
said operation further comprises the steps of: [0243] receiving a
second signal representing the number of molecules of said
cell-free DNA comprising said nucleotide sequence(s); and [0244]
estimating a fraction or ratio of molecules of said cell-free DNA
comprising one or more said methylated and/or un-methylated CpGs
(as applicable), and/or comprising one or more said pattern of
methylation or un-methylation, located within one or more of the
nucleotide sequences within all of said nucleotide sequences; and
[0245] more preferably wherein said classification is determined by
comparing said a fraction or ratio to a standard or cut-off
value.
[0246] Certain aspects and embodiments of the invention will now be
illustrated by way of examples and with reference to the
description, figures and tables set out herein. Such examples of
the Methods, Results, Supplementary Information, Discussion,
conclusions and other uses or aspects of the present invention are
representative only, and should not be taken to limit the scope of
the present invention to only such representative examples.
Furthermore, any embodiments, text or other descriptions found in
such examples are also to encompassed and considered as part of the
description of the present invention.
[0247] Methods:
[0248] Patients and Sample Collection:
[0249] We have used a total of 703 tissue and 251 serum samples in
seven sets (FIG. 1). For Serum Sets 1-3 and the NACT Serum Set,
women attending the hospitals in London and Prague were invited,
consented and 20-40 mL blood obtained (VACUETTE.RTM. Z Serum Sep
Clot Activator tubes, Cat 455071, Greiner Bio One International
GmbH), centrifuged at 3,000 rpm for 10 minutes with serum stored at
-80.degree. C. Serum CA125 was analysed for the validation sets
using the CA125 Cobas immunoassay platform (Roche Diagnostics,
Burgess Hill, UK). For detailed information see the Supplementary
Information.
[0250] Additionally, a seventh validation set may be provided from
the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS)
(Refs. 6, 7, 22). For example, all women (among those in the
control arm) who donated serum samples at recruitment and developed
invasive epithelial OC within two years of recruitment and the
corresponding age and centre matched women who did not develop
ovarian cancer within five years of recruitment can be analysed.
Blood samples from all UKCTOCS volunteers can be spun down for
serum separation after being couriered at room temperature to a
central laboratory, and aliquoted and stored in liquid nitrogen
vapour phase until thawing and analysed such as described herein;
even if only 1 mL of serum per UKCTOCS volunteer may be
available.
[0251] Isolation and bisulphite modification of DNA:
[0252] DNA was isolated from tissue and serum samples at GATC
Biotech (Constance, Germany). Tissue DNA was quantified using
NanoDrop and Qubit (both Thermo Fisher Scientific, USA); the size
was assessed by agarose gel electrophoresis. Serum DNA was
quantified using the Fragment Analyzer and the High Sensitivity
Large Fragment Analysis Kit (AATI, USA). DNA was bisulfite
converted at GATC Biotech.
[0253] DNAme Analysis in Tissue:
[0254] Genome wide methylation analysis was performed either by the
Illumina Infinium Human Methylation 450K beadchip array (Illumina
Inc USA, WG-314-1003) as previously described (Refs. 24, 25) or
using Reduced
[0255] Representation Bisulfite Sequencing (RRBS) at GATC Biotech.
For the 450k methylation data we developed a pipeline in order to
select the most promising cancer-specific differentially methylated
regions (DMRs) that are most likely to fulfil the strict
specificity criteria of a serum based test (see Supplementary
Information).
[0256] For RRBS, DNA was digested by the restriction endonuclease
Mspl that is specific for the CpG containing motif CCGG; a size
selection of the library provides an enhanced coverage for the
CpG-rich regions including CpG islands, promoters and enhancer
elements (Refs. 26, 27). The digested DNA was adapter ligated,
bisulfite modified and PCR amplified. The libraries were sequenced
on Illumina's HiSeq 2500 with 50 bp or 100 bp paired-end mode.
Using Genedata Expressionist.RTM. for Genomic Profiling v9.1, we
have established a bioinformatics pipeline for the detection of
cancer specific DMRs. The most promising DMRs have been taken
forward for the development and validation of serum based clinical
assays (see Supplementary Information).
[0257] Targeted ultra-high coverage bisulphite sequencing of serum
DNA:
[0258] Targeted bisulfite sequencing libraries were prepared at
GATC Biotech. In brief, bisulfite modification was performed with 1
mL serum equivalent. Modified DNA was used to test up to three
different markers using a two-step PCR approach. Ultra-high
coverage sequencing was performed on Illumina's MiSeq or HiSeq 2500
with 75 bp or 125 bp paired-end mode (see Supplementary
Information).
[0259] Statistical/Data Analyses:
[0260] For DMR discovery the data analysis pipelines are described
within the respective sections in the Supplementary Information. In
brief, Genedata Expressionist.RTM. for Genomic Profiling was used
to map reads to human genome version hg19, identify regions with
tumor specific methylation patterns, quantify the occurrence of
those patterns, and calculate relative pattern frequencies per
sample. Pattern frequencies were calculated as number of reads
containing the pattern divided by total reads covering the pattern
region. The 95% CI intervals for sensitivity and specificity have
been calculated according to the efficient-score method (Ref. 28).
Differences in pattern frequencies or coverage have been analysed
using Mann Whitney U test.
[0261] Results:
[0262] Study Design:
[0263] The samples, techniques and purpose of the three phases of
the study--marker discovery, assay development and test
validation--are summarized in FIG. 1.
[0264] DNA methylation marker discovery in tissue:
[0265] We have used two independent epigenome-wide approaches in
order to discover DMRs which have the potential for diagnosing
ovarian cancer with high sensitivity and specificity. (1) Illumina
Infinium Human Methylation450 BeadChip Array (450K) technology was
used to interrogate the methylation status of .about.485,000
genomic sites in 218 ovarian cancer (Ref. 28) and 438 control
samples (FIG. 1; see also Supplementary Information). A set of 19
high scoring and ranking DMRs were selected for targeted-BS based
serum assay development. FIG. 6 shows an example of a selected top
DMR (reaction #228). (2) Using RRBS, we first determined the
methylation pattern frequencies in relevant genomic regions in
different tissues. The algorithm that we have developed scans the
whole genome and identifies regions that contain at least 10
aligned paired-end reads. These read bundles are split into smaller
regions of interest which contain at least 4 CpGs in a stretch of,
at most, 100 base pairs (bp). For each region and tissue/sample the
absolute frequency (number of supporting reads) for all observed
methylation patterns was determined (FIG. 2A). This led to tens of
millions of patterns per tissue/sample. The patterns were filtered
in a multi-step procedure to identify the methylation patterns
which specifically occur in tumour samples. In order to increase
sensitivity and specificity, respectively, of our pattern discovery
procedure, we pooled reads from different tumour or white blood
cell (WBC) samples, respectively, and scored patterns based on
over-representation within tumour tissue. The results were
summarized in the specificity score Sp, which reflects the cancer
specificity of the patterns. After applying a cut-off of
Sp.gtoreq.10, 2.6 million patterns for OC remained and were further
filtered according to the various criteria demonstrated in FIG. 2B
(and Supplementary Information).
[0266] For the filtered unique cancer specific patterns for OC
identified in the Array (n=19) and RRBS (n=45) approach,
respectively, bisulfite sequencing primers have been designed and
technically validated, eventually leading to 31 candidate markers,
The genome coordinates (hg19) and genomic sequence of each DMR is
shown in TABLE 1A and TABLE 1B, and the possible
bisulphite-converted sequences thereof (where "Y" symbolises either
a C or a U/T, preferably a C or a T) are shown in TABLE 2A (with
CpGs--sites of possible 5-methylcytosine--shown underlined).
[0267] Serum DNAme Assay Establishment:
[0268] We used ultra-deep BS sequencing (FIG. 2C) to develop serum
assays for the 31 candidate regions in 59 serum samples from Set 1
(FIG. 1 and Supplementary Information and FIG. 8). Based on
sensitivity and specificity, nine markers have been selected for
further validation in Set 2 (n=92). In Sets 1&2 combined, the
specificity/sensitivity of the top four candidate markers (FIG. 9)
referred to Regions #141, #144, #204 and #228 (#228 was only
analysed in Set 2) to discriminate HGS OC from healthy women or
those with a benign pelvic mass at pattern frequency thresholds of
0.0008, 0.0001, 0.0001 and 0.0001 was 95.7%/42.4%, 93.5%/48.5%,
100%/25.0% and 100%/36.8%, respectively. Interestingly region #144
has already been defined as a promising cell-free DNA marker for
cancer, in particular ovarian cancer (Refs. 30, 31). The
combination of Regions #141, #204 and #228 (at least one of these
regions with a pattern frequency above the aforementioned
threshold) resulted in a 98.1% specificity and a 63.2% sensitivity.
These regions are linked to genes COL23A1, C2CD4D and WNT6,
respectively.
[0269] Clinical Validation of the Serum DNAme Assay:
[0270] We validated the combination of the three markers in Set 3
(FIG. 3A-C) alongside the CA125 serum marker (FIG. 3D). The average
coverage (i.e. DNA strands read by the sequencer for each sample
and region) is >500,000 (FIG. 10). Applying the above indicated
cut-off thresholds for the three DNAme markers and 35 IU/mL for
serum CA125 led to specificities of 90.7% and 87.1% and
sensitivities of 41.4% and 82.8%, respectively (FIG. 3E). Due to
the fact that Reaction #228 was only analysed in Set 2, we combined
Set 2 and Set 3 in order to redefine the thresholds. Whereas for
#141 the threshold of 0.0008 remained unchanged, for #204 and #228
we further lowered the pattern frequency threshold to 0.00003 and
0.00001, respectively, leading to a specificity and sensitivity now
of 91.8% and 58.3%, respectively (FIG. 3E). Importantly, there was
no overlap between the DNAme- and CA125-false positive controls
(FIG. 3F).
[0271] Serum DNAme to Predict Response to Platinum-Based
Neoadjuvant Chemotherapy:
[0272] We recruited 25 ovarian cancer patients who received
carboplatin-based neoadjuvant chemotherapy. Compared with the
pre-treatment sample, all three DNAme markers dropped substantially
and more dramatically compared to CA125 after one and two cycles
(FIG. 4A-D and FIGS. 11, 12, 13). Whereas CA125 dynamics was not
able to discriminate chemotherapy-responders from non-responders
(FIG. 4E and Supplementary Information), serum DNAme dynamics (i.e.
serum DNAme as defined in Set 2&3, before as compared to after
two cycles) correctly identified 78% and 86% of responders and
non-responders (Fisher's exact test p=0.04) overall and 78% and
100% amongst those women who were left without residual disease
after interval debulking surgery (Fisher's exact test p=0.007)
(FIG. 4E).
[0273] Serum DNAme for Early Diagnosis of Ovarian Cancer:
[0274] In order to judge whether our marker panel is able to
diagnose ovarian cancer early, samples predating OC diagnosis by up
to 2 years (cases) and matched controls can be used from the
control (no screening)--arm of the UKCTOCS cohort. As at the time
of their collection, UKCTOCS samples were not obtained, treated or
stored with the analysis cell-free DNA in mind. Hence, it is to be
expected that upon DNA extraction it will be found that either or
both the amount of DNA/mL serum as well as the average DNA fragment
size may be higher (such as dramatically higher) in UKCTOCS samples
compared with the other samples used in this study. As has been
previously observed and proposed (Anjum et al, 2014, Genome Med
6:47), without being bound by theory, such effect may be due to DNA
from WBCs leaking into the serum during the 24-48 hour blood sample
transport time--in particular in the warm season). This
"contaminating" high quality [genomic rather than tumour] DNA would
not only dilute the cancer signal but also skew the target sequence
amplification towards WBC DNA. In order to adjust for these
factors, an a priori decision may be made to reduce the threshold
for the three regions by a factor, such as by a factor of 3, and/or
to split the analyses in samples above (high) and below (low) the
median amount of DNA.
[0275] Such adjustments can enable the three DNAme-marker panel to
yet further confirm its validation above and its utility for the
early detection of ovarian cancer. Indeed, it can then be used to
identify cases with a specificity of over 70%, 80%, or 90% (such as
between about 70% and 80% or between about 80% and 90%) and a
sensitivity of over 45% 50%, 55% or 60% (such as between about 50%
and 55% or between about 55% and 60%) [indeed, the sensitivity may
be even higher in CA125 negative (<35U/mL) samples] in samples
with a lower than median amount of DNA and may remain literally
unchanged within two years between sample collection and cancer
diagnosis. Given the greater dynamic range of DNAme panel test and
the results above, it can have higher sensitivity but lower
specificity compared to that of CA125 using a cut-off of 35 U/ml in
the early detection of ovarian cancer, and/or to have no overlap of
false positives. The DNAme panel has higher sensitivity but lower
specificity compared to that of CA125 using a cut-off of 35 U/ml in
the early detection of ovarian cancer.
[0276] Supplementary Information:
[0277] Subjects and Sample Collection:
[0278] We analysed a total of 6 sets as detailed in FIG. 1:
[0279] 1. Array-Set: Ovarian cancer samples (Refs. S1, S2), WBC
samples (Ref. S3) and Fallopian Tube samples (Ref. S4) have been
described before. Ten benign pelvic tumours (2x
endometriosis-ovarian cyst, 1.times. fibroma, 2.times. papillary
serous cystadenoma, lx mucinous cystadenoma, 2.times. serous
cystadenoma, 1.times. mucinous cystadeonoma with Brenner tumour and
1 dermoid cyst), 96 endometrial samples (Ref. 51) (Haukeland
University Hospital, Bergen, 52 patients with primary and
metastatic samples equalling 87, 8.times. benign endometrial (all
hyperplasia) & 1 cell line) and 170 samples (38 colon (COAD
controls), 50 liver (LIHC controls), 75 lung (LUSC and LUAD
controls), 7 rectum (READ controls) from the publically available
The Cancer Genome Atlas (TCGA) repository were analysed.
[0280] 2. RRBS Set: 11 prospectively collected invasive epithelial
ovarian cancer samples (high grade serous n=8, low grade serous
n=1, endometrioid n=1, mean age=54.7 years), one benign tumour
(papillary serous cystadenoma, age=86 years), 18 normal tissue
samples (normal breast n=7 and normal ovarian n=11, mean age=60.2
years), two normal endometrial tissues mean age=68, and twenty
three white blood cell samples (breast cancer patients n=10 &
ovarian cancer patients n=13 [11 of which match corresponding OC
tissue samples, 1 matches corresponding normal endometrial sample
and 1 matches normal ovarian sample], mean age=57.8) were assessed
by RRBS.
[0281] All samples of the RRBS Set were collected prospectively at
the University College London Hospital in London (University
College London Hospital, 235 Euston Rd, Fitzrovia, London NW1 2BU)
and at the Charles University Hospital in Prague (Gynecological
Oncology Center, Department of Obstetrics and Gynecology, Charles
University in Prague, First Faculty of Medicine and General
University Hospital, Prague, Apolinarska 18128 00 Prague 2, Czech
Republic.). The study was approved by the local research ethics
committees: UCL/UCLH Biobank for Studying Health & Disease
NC09.13) and the ethics committee of the General University
Hospital, Prague approval No.: 22/13 GRANT--7. RP--EPI-FEM-CARE
respectively. All patients provided written informed consent.
[0282] 3. Serum Set 1: Serum samples from the following volunteers
have been collected (at the time of diagnosis, prior to treatment):
[0283] Healthy volunteers (n=19, mean age 41.1 years). [0284] Women
with benign pelvic masses (n=22, mean age 41.3 years) with the
following histologies: endometriosis (n=6), fibroids (n=5),
hydrosalpinx (n=1), serous cystadenoma (n=5) and mucinous
cystadenoma (n=5). [0285] Patients with ovarian cancers (n=18, mean
age 62.2 years): endometrioid (n=2) and clear cell (n=1) and high
grade serous (n=15) ovarian cancers; 10 and 8 women had a stage
I/II and stage III/IV ovarian cancer, respectively.
[0286] All samples of Serum Set 1 were collected prospectively at
the University College London Hospital in London and at the Charles
University Hospital in Prague. The study was approved by the local
research ethics committees: UCL/UCLH Biobank for Studying Health
& Disease (NC09.13) and the ethics committee of the General
University Hospital, Prague approval No.: 22/13 GRANT--7.
RP--EPI-FEM-CARE respectively. All patients provided written
informed consent.
[0287] 4. Serum Set 2: Serum samples from the following volunteers
have been collected (at the time of diagnosis, prior to treatment):
[0288] Healthy volunteers (n=20, mean age 42.8 years). [0289] Women
with benign pelvic masses (n=34, mean age 40.0 years) with the
following histologies: endometriosis (n=7), fibroids (n=8), pelvic
inflammatory disease or pelvic abscess (n=9), serous cystadenoma
(n=5) and mucinous cystadenoma (n=5). [0290] Patients with
borderline ovarian tumors (n=11, mean age 47.3 years): mucinous
(n=6) and serous (n=5) borderline tumor. [0291] Patients with
ovarian cancers (n=27, mean age 62.9 years): endometrioid (n=3) and
clear cell (n=3), mucinous (n=2) and high grade serous (n=19)
ovarian cancers; 10 and 17 women had a stage I/II and stage III/IV
ovarian cancer, respectively.
[0292] All samples of Serum Set 2 were collected prospectively at
the University College London Hospital in London and at the Charles
University Hospital in Prague. The study was approved by the local
research ethics committees:
[0293] UCL/UCLH Biobank for Studying Health & Disease NC09.13)
and the ethics committee of the General University Hospital, Prague
approval No.: 22/13 GRANT--7. RP--EPI-FEM-CARE respectively. All
patients provided written informed consent.
[0294] 5. Serum Set 3: Serum samples from the following volunteers
have been collected (at the time of diagnosis, prior to treatment):
[0295] Healthy volunteers (n=21, mean age 50.8 years). [0296] Women
with benign pelvic masses (n=119, mean age 41.4 years) with the
following histologies: endometriosis (n=21), fibroids (n=21),
pelvic inflammatory disease or pelvic abscess (n=7), serous
cystadenoma (n=20), mucinous cystadenoma (n=20) and dermoid cysts
(n=29). [0297] Patients with borderline ovarian tumors (n=27, mean
age 57.1 years): mucinous (n=7) and serous (n=20) borderline tumor.
[0298] Patients with non-epithelial tumors (n=5, mean age 55.8
years): granulosa cell tumors. [0299] Patients with non-ovarian
cancers (n=37, mean age 58.3 years): cervical (n=10), endometrial
(n=20) and colorectal (n=7) cancers. [0300] Patients with ovarian
cancers (n=41, mean age 59.6 years): endometrioid (n=3) and clear
cell (n=5), mucinous (n=4) and high grade serous (n=29) ovarian
cancers; 16 and 25 women had a stage I/II and stage III/IV ovarian
cancer, respectively.
[0301] All samples of Serum Set 3 were collected prospectively at
the University College London Hospital in London and at the Charles
University Hospital in Prague; CA125 analysis was performed using
the CA125 Cobas immunoassay and platform (Roche Diagnostics,
Burgess Hill, UK). The study was approved by the local research
ethics committees. The study was approved by the local research
ethics committees: UCL/UCLH Biobank for Studying Health &
Disease (NC09.13) and the ethics committee of the General
University Hospital, Prague approval No.: 22/13 GRANT--7.
RP--EPI-FEM-CARE respectively. All patients provided written
informed consent.
[0302] Of note: For the Serum Sets 1-3 which have been
prospectively collected within EpiFemCare there is a substantial
age difference between women who presented with benign pelvic
masses and women who presented with ovarian cancer. We were
completely aware of this as the main purpose was to benchmark DNAme
markers against CA125 and to assess whether CA125 false positive
controls are also DNAme-false positive. The main source of false
positivity are endometriosis, pelvic inflammatory disease and
fibroids--all conditions which are substantially more prevalent (or
occur exclusively) in premenopausal (i.e. younger women) whereas
ovarian cancer is far more prevalent in older women.
[0303] 6. NACT ("Neoadjuvant Chemotherapy") Set: Patients (n=25) at
the Gynaecological Oncology Centre in Prague deemed not to be
suitable for up-front surgery have been recruited. The average age
was 62.8 years. High grade serous ovarian cancers were the most
prevalent histology (n=23) and the remaining two patients had clear
cell ovarian cancers. Twenty-four patients received
Carboplatin-Paclitaxel combination chemotherapy and one patient
received Carboplatin only. All but two patients had interval
debulking surgery. Among the 23 patients, 14 had no residual
disease, 5 had macroscopic residual disease and 4 had microscopic
residual disease (i.e. tumor reaches the edge of at least one of
the resected specimens--according to TNM classification). Twelve
patients were deemed to be platinum-sensitive (no recurrence within
6 months after successful completion of neoadjuvant and adjuvant
chemotherapy and interval debulking surgery) and eight patients
were deemed to be platinum-refractory (n=2, no response to
chemotherapy or progression on chemotherapy) or platinum-resistant
(n=6, recurrence within 6 months after successful completion of
neoadjuvant and adjuvant chemotherapy and interval debulking
surgery). For 5 patients no data were available on
platinum-sensitivity.
[0304] All serum samples of the NACT Set were collected
prospectively at the Charles University Hospital in Prague. Each
patient provided three samples at the following time-points: [0305]
At the time of histological diagnosis, prior to chemotherapy.
[0306] Three weeks after the first cycle of chemotherapy
(immediately prior to the second cycle). [0307] Three weeks after
the second cycle of chemotherapy (immediately prior to the third
cycle).
[0308] CA125 analysis on the NACT Set was performed using the CA125
Cobas immunoassay and platform (Roche Diagnostics, Burgess Hill,
UK). The study was approved by the local research ethics
committees: UCL/UCLH Biobank for Studying Health & Disease
(NC09.13) and the ethics committee of the General University
Hospital, Prague approval No.: 22/13 GRANT--7. RP--EPI-FEM-CARE
respectively. All patients provided written informed consent.
Eighteen and seven patients presented with a stage IIIC and IV
ovarian cancer respectively.
[0309] In addition to these six sets, a seventh set could be used
to provide further confirmation on the validation of the present
assay provided by the other sets. Such a seventh set could comprise
samples from the UKCTOCS collection. For example, among those women
who were randomised into the control arm of UKCTOCS between 2001
and 2005, the subset of such women who developed an invasive
epithelial ovarian cancer within 2 years of serum sample donation
and had at least 4mL of non-haemolysed serum available, and such
number of women stratified into those women which developed a high
grade serous, mucinous, endometrioid, clear cell, carcinosarcoma or
a carcinoma not otherwise specified, respectively. The average age
at sample donation can be calculated in years; as can the number of
such women, the number of women who were diagnosed within one year
and those who were diagnosed between 1-2 years after sample
donation, as well as the respective number of women who were
diagnosed with a stage I/II and stage III/IV cancer, respectively.
For each of the such cases, three women who did not develop any
cancer within the first five years after recruitment can be matched
with regards to age at recruitment, centre and month of recruitment
(controls).
[0310] DNA Methylation Analyses in Tissue Samples:
[0311] DNA isolation: DNA was isolated from tissue samples using
the Qiagen DNeasy Blood and Tissue Kit (Qiagen Ltd, UK, 69506) and
600ng was bisulfite converted using the Zymo methylation Kits (Zymo
Research Inc, USA, D5004/8).
[0312] Illumina Infinium Human Methylation450 BeadChip Array data
analysis: Genome wide methylation analysis was performed using the
Illumina Infinium Methylation 450K beadchip (Illumina Inc USA,
WG-314-1003). The raw data processing and quality control was
performed in R/Bioconductor (versions 2.15.0/2.11) (Ref. S5) using
minfi (Ref. S6) and BMIQ (Ref. S7) packages. Identification of
differentially methylated regions (DMRs) was carried out using
Genedata Expressionist.RTM. for Genomic Profiling as described
below.
[0313] To correct for the individual probe to probe variation in
the affinity/sensitivity to unnmethylated vs. methylated DNA we
used fully methylated (Sssl treated) and unmethylated (whole genome
amplified; WGA) genomic DNA from WBC samples of different
individuals. These technical controls were used for both filtering
out probes that do not show sufficient specificity (i.e. cannot
discriminate between methylated vs. unmethylated state) and to
perform array-wide recalibration of the biological sample data to
normalize for the probe to probe variation in background and
dynamic range, respectively. The removal of the non-specific probes
was achieved by doing a T-test with Sssl vs WGA samples (using
M-values) and removing the probe sets that have p-value <0.01
and effect size below <5 i.e. cannot discriminate between fully
methylated and unmethylated DNA. The normalization was done for
each sample individually for each probe set with the formula
M.sub.true=(M.sub.measured-M.sub.WGA)/(M.sub.SssI-M.sub.WGA);
M.sub.SssI and M.sub.WGA values used were average (arithmetic mean)
values of the respective sample groups. For the downstream analyses
the individual sample Sssl and WGA data were also normalized with
the same formula. This leads to efficient removal of background
noise from the probe to probe variation and increases the power to
detect homogenously methylated or unmethylated genomic regions.
T-test and normalization were performed in Genedata
Expressionist.RTM. for Genomic Profiling software.
[0314] The control sample set was selected to identify DMRs that
are cancer specific and would also be specific in a serum based
clinical assay. Therefore, in addition to the ovarian (including
Fallopian Tube and endometrium) control tissues we used a large
panel of tissues that are likely to shed DNA into the serum [i.e.
white blood cells (WBCs), lung, liver, rectum and colon], with WBCs
being the most abundant source of normal germline DNA in serum
samples. Two statistical approaches were used to identify DMRs from
the 450K data: (1) a statistical test to identify single probes
showing differential methylation between ovarian cancer and WBC
samples, and (2) a sliding window ANOVA approach that scans the
whole genome and identifies sets of neighboring probes (Ranges)
showing correlated methylation differences between ovarian cancer
and WBC samples. Only the DMRs showing no methylation in WBCs were
considered for downstream analysis steps. The identified DMRs where
ranked and scored based on the following criteria: (1) Differences
in methylation levels between ovarian cancer and the control
tissues (with WBC difference being emphasized). (2) Feasibility of
designing a clinical assay (number of CpGs in the region to allow
designing an assay with sufficient sensitivity/specificity). (3)
For ranges only: Reliability of the DMRs (number of probes within
the Range).
[0315] In the sliding window approach, the algorithm performs a
pooling of all features in a given sliding window (120 bp) before
it calculates an ANOVA p-value between sample groups. The pooling
increases statistical robustness and also results in smoother ANOVA
p-Values. The smoothed ANOVA p-Values are then used to detect
regions containing one or more p-values exceeding the given Maximum
P-Value threshold (le-5). If a gap of more than 1000 bp is detected
between similar methylation differences, two different regions are
reported. Note, that the algorithm also reports single probes
showing significant methylation difference (if no neighbouring
similar methylation difference is present), but groups of probes
with similar profile do get lower p-values and are therefore
preferentially reported. The sliding window approach was used for
OC vs WBC samples to detect cancer DMRs (using normalized M-values)
and arithmetic mean M-values of the probes per detected DMR (here
after referred as "Range") were reported for all the relevant
samples for downstream analysis. The Range discovery was performed
in Genedata Expressionist.RTM. for Genomic Profiling v8.0 software.
The Ranges varied in size between 1 and 45432 bp, with average
(arithmetic mean) size being 368 bp. The Ranges showing methylation
in WBC were removed by a T-test with WBC vs. WGA samples
(p-value<1e-6 and/or directed effect>0.15; i.e.
M(WBC)>M(WGA)+0.15). Next, a T-test for OC vs WBC was used and
Ranges showing significant difference (p-value <le-6) and
difference (directed effect) of WBC upper quartile vs OC lower
quartile >0.15 were selected as differentially methylated
regions. For different control tissue samples the methylation
values were calculated for the same OC vs WBC Ranges (arithmetic
mean M-values of the probes per detected DMR).
[0316] For ranking of the DMRs the effect sizes of methylation of
cancer samples versus different relevant controls tissues were
calculated. In addition to "direct" control tissues
(fimbrial/endometrial/benign ovarian tissues) also large tissues
with high turnover (liver, lung, rectum and colon) were included;
data were download from TCGA data portal
(https://tcga-data.nci.nih.gov/tcga/) as level 3 data as detP
filtered beta-values; data normalization was carried out as
described above. The effect sizes were always calculated with
cancer lower quartile vs control tissue upper quartile values
(based on Sssl/WGA normalized M-values).
[0317] Two different scoring methods were used for the effect
sizes. In Method 1 the OC vs WBC effect size gets the weight
6.times., and all the control tissue 1/4.times.. In Method 2 the OC
vs WBC effect size gets the weight 6.times., and all the control
tissue 1.times.. The Method 2 takes more into account the data from
all the control tissues whereas Method 1 maximises the effect of
the difference between WBC and cancers samples. However, for both
methods only DMRs prefiltered for low methylation in WBCs were used
(as described above). The final scores are the sum of the tissue
scores and the feasibility and confidence scores (see next
paragraph). If data were not available for a certain probe for a
certain tissue (i.e. was filtered out due to high detection
p-value), the score for the tissue was 0.
[0318] For further ranking of the DMRs feasibility (for designing a
functional clinical assay) and confidence (for ranges) scores were
calculated. The feasibility score is based on number of CpG
dinucleotides within (or close by; +/-60 bp) a probe/range. If the
number of CpGs is <5, the score is -0.5, if the number of CpGs
is between 5 and 9 the score is 0 and if the number of CpGs is
>=10 the score is 0.5. The number of CpGs per range was
calculated using EMBOSS cpgreport tool in Galaxy (Refs. S8-S10)
using the range genomic coordinates as input. The confidence score
for ranges is 0.5 if 2 or more probes are within the range, if only
one probe the score is 0.
[0319] Reduced Representation Bisulfite Sequencing (RRBS):
[0320] RRBS: RRBS libraries were prepared by GATC Biotech AG using
INVIEW RRBS-Seq according to proprietary SOPs. In brief, DNA was
digested with the restriction endonuclease Mspl that is specific
for the CpG containing motif CCGG; a subsequent size selection
provides enhanced coverage for the CpG-rich regions including CpG
islands, promoters and enhancer elements (Refs. S11, S12). The
digested DNA is then adapter ligated, bisulfite modified and
PCR-amplified. The libraries were sequenced on Illumina's HiSeq
2500 with 50 bp or 100 bp paired-end mode.
[0321] After sequencing raw data was trimmed using Trimmomatic
(0.32) to remove adapter sequences and low quality bases at the
beginning and end of reads. Subsequently, reads were trimmed with
TrimGalore (0.3.3) to remove cytosines derived from library
preparation which must not be included in the methylation analysis.
Read pairs were mapped to the human genome (hg19) in Genedata
Expressionist.RTM. for Genomic Profiling 8.0 applying Bisulfite
Mapper based on BOWTIE v2.1.0 (Ref. S13) with the
settings--no-discordant--reorder-p 8--end-to-end--no-mixed-D 50 -k
2--fr--norc-X 400-I 0--phred33. Further analysis was done using
Genedata Expressionist.RTM. for Genomic Profiling v9.1.
[0322] Computation of RRBS-determined methylation pattern
frequencies: In order to allow the sensitive detection of
methylation patterns with low abundance, the read data available
for each sample type (e.g. breast cancer, ovarian cancer and white
blood cells) were pooled across patients and sequencing runs.
Candidate genomic regions for methylation pattern analysis were
defined based on bundles of at least 10 paired-end reads covering
at least 4 consecutive CpG sites which are located within a genomic
range of at most 150 bp. As illustrated in FIG. 5, our algorithm
first determines sets of consecutive CpG sites of maximum size,
from which multiple potentially overlapping subsets are derived,
which still meet the selection criteria. CpG sites located in the
gap between the mate reads are ignored. For each derived set of CpG
sites, the absolute and relative frequencies of all methylation
patterns observed in the corresponding reads are determined. The
methylation patterns are represented in terms of binary strings in
which the methylation state of each CpG site is denoted by 1 if
methylated or 0 if unmethylated. The algorithm for selecting
candidate regions and calculating methylation pattern frequencies
was implemented in our software platform Genedata
Expressionist.RTM. for Genomic Profiling.
[0323] Procedure for the selection of tumour-specific
RRBS-determined methylation patterns: In order to ensure that the
pattern exclusively occurs in tumour samples, all patterns present
in white blood cells were excluded. A score for assessing the
relevance of each pattern was determined by integrating multiple
subordinate scores which quantitatively capture desired properties
of candidate biomarker patterns. First, for each pattern a Tumour
Specificity Score Sp=DL-TP-TE-AF was calculated, which consists of
the four components Dilution Factor DL, Tumour Prevalence TP,
Tumour Enrichment Factor TE and Avoiding Factor AF. The formal
definitions of the score components are given in the following:
DL WBC = # total reads # reads with pattern 1 10 3 ##EQU00001## TP
tumor = # reads with pattern in tumor # total reads in tumor 10
##EQU00001.2## TE tumor = # observed reads with pattern in tumor #
expected reads with pattern in tumor ##EQU00001.3## AF WBC = #
expected reads with pattern in WBC # observed reads with pattern in
WBC ##EQU00001.4##
[0324] The Dilution Factor DL and Tumour Prevalence TP favour
patterns which are supported by a high proportion of reads in
tumour and low proportion of reads in WBC, respectively. A pattern
observed in 1 out of 10 reads in tumour and in 1 out of 1000 reads
in WBC scores 1 for both factors. The Tumour Enrichment Factor TE
and Avoiding Factor AF were included to assess the
overrepresentation of the pattern in tumour samples and its
underrepresentation in WBC samples, respectively, relative to an
expected number of pattern reads which is based on the observed
overall methylation level in those tissues. In order to estimate
the number of expected reads supporting the pattern, the
methylation frequencies are calculated for each CpG site
individually. Next, the number of expected reads with a specific
pattern is calculated as the product of the relative frequencies of
the tumour specific methylation states observed for each CpG site
in the pattern times the number of reads stretching across the
pattern. A TE >1 indicates that a pattern is more frequent in
tumour than expected when randomly distributing the observed
methylation levels across reads. Besides favouring tumour
specificity our scoring procedure was also designed to make
patterns with high variance of the highest priority (i.e., patterns
for which a high number of transitions in the methylation state is
observed between consecutive CpG sites). Such patterns may be a
product of the epigenetic reprogramming of tumour cells and in
order to account for the potentially increased biological relevance
of these patterns another score component was introduced. The
normalized variance V.sub.p of a pattern p is defined as the
pattern variance divided by the maximum variance, i.e., the pattern
length minus 1. The scores for the tumour specificity S.sub.p and
pattern variance V.sub.p were combined in the tumor-specific
variance score SV.sub.p=V.sub.p log(S.sub.p). In order to
facilitate the ranking of each candidate genomic region r based on
the relevance of patterns p.sub.1, . . . , p.sub.N observed in the
region the aggregation score AS.sub.r was calculated based on the
following formula:
AS r = i = 1 n 1 i SV Pi ##EQU00002##
[0325] The aggregation score AS.sub.r corresponds to a weighted sum
of the tumour-specific variance scores of the observed patterns.
The weighting was included since an ordinary sum would introduce a
bias towards regions, in which a high number of patterns have been
observed due to a high read coverage and/or high CpG site density.
All of the presented statistics for assessing the relevance of
methylation patterns and genomic regions were implemented in
Genedata Expressionist.RTM. for Genomic Profiling and R,
respectively.
[0326] DNA Methylation Analyses in Serum Samples:
[0327] Serum separation: For Serum Sets 1-3 and the NACT Serum Set,
women attending the hospitals in London and Prague have been
invited, consented and 20-40 mL blood has been obtained
(VACUETTE.RTM. Z Serum Sep Clot Activator tubes, Cat 455071,
Greiner Bio One International GmbH), centrifuged at 3,000 rpm for
10 minutes and serum collected and stored at -80.degree. C. We have
applied non-stringent measures (i.e. allowed for up to 12 hours
between blood draw and centrifugation) purposely in order to mimic
the situation of UKCTOCS samples which could be used to compare the
results presented herein, which samples had been sent from the
recruiting centre to UCL within 24-48 hours before
centrifugation.
[0328] Serum DNA isolation and bisulfite modification: DNA was
isolated using the DNeasy Blood and Tissue Kit (Qiagen Ltd, UK,
69506) at GATC Biotech (Constance, Germany). Serum DNA was
quantified using the Fragment Analyzer and the High Sensitivity
Large Fragment Analysis Kit (AATI, USA). DNA was bisulfite
converted using the EZ-96 DNA Methylation Kit (Zymo Research Inc,
USA, D5004/8) at GATC Biotech.
[0329] Targeted ultra-high coverage bisulfite sequencing: Targeted
bisulfite sequencing was performed at GATC. To this end, a two-step
PCR approach was used similar to the recently published BisPCR2
(Ref. S14). Bisulfite modification was performed with 1 mL serum
equivalent. For each batch of samples, positive and non-template
controls were processed in parallel. Bisulfite converted DNA was
used to test up to three different markers using automated
workflows. After bisulfite modification the target regions were
amplified using primers carrying the target specific sequence and a
linker sequence. Amplicons were purified and quantified. All
amplicons of the same sample were pooled equimolarly. In a second
PCR, primers specific to the linker region were used to add
sequences necessary for the sequencing and multiplexing of samples.
Libraries were purified and quality controlled. Sequencing was
performed on Illumina's MiSeq or HiSeq 2500 with 75 bp or 125 bp
paired-end mode. Trimming of adapter sequences and low quality
bases was performed with Trimmomatic as described for the RRBS
data.
[0330] Assessment of RRBS-determined methylation pattern frequency
in serum DNA: After sequencing, raw data were trimmed using
Trimmomatic (0.32) to remove adapter sequences and low quality
bases at the beginning and end of reads. Subsequently, reads were
trimmed with TrimGalore (0.3.3) to remove cytosines derived from
library preparation which must not be included in the methylation
analysis. Further analysis was done using Genedata
Expressionist.RTM. for Genomic Profiling 9.1. Read pairs were
mapped to the human genome (hg19) applying Bisulfite Mapper based
on BOWTIE v2.2.5 (13) with the settings--no-discordant-p
8--norc--reorder-D 50--fr--end-to-end-X 500-I 0--phred33-k
2--no-mixed. Coverage was calculated per sample and target region
using Numeric Data Feature Quantification activity by calculating
the arithmetic mean of the coverage in each region. As part of the
data quality control, efficiency of the bisulfite conversion was
estimated in each sample by quantifying the methylation levels of
CpHpG and CpHpH sites (where H is Any Nucleotide Except G), with
minimum coverage of 10, within the target regions. The median
bisulfite conversion efficiency was 99.4%, with efficiency for no
sample being lower than 97.7%. Methylation pattern frequencies in
serum samples for target regions were determined as described
above. Relative pattern frequencies were calculated by dividing the
number of reads containing the pattern by the total number of reads
covering the pattern region.
[0331] Discussion:
[0332] Circulating tumour DNA analysis--using cancer-specific DNAme
markers and/or patterns of the present invention--shows independent
sensitivity/specificity to that of CA125 and has a greater dynamic
range correlating with changes in tumour burden and response to
treatment.
[0333] Consistent with published data (Ref. 8), CA125 change after
2 cycles of chemotherapy was not able to indicate responsiveness to
chemotherapy (in this case carboplatin alone or in combination with
paclitaxel). The fact that serum DNAme-dynamics--as analysed using
a method of the present invention--correctly identified 7/9 and 6/7
neoadjuvant chemotherapy responders and non-responders,
respectively, provides a proof of principle and a basis for
prospective clinical trials to individualise pre-operative systemic
treatment in advanced ovarian cancer.
[0334] In healthy individuals, cell-free DNA is present at
concentrations between 0 and 100 ng/mL and an average of 30 ng/mL
(Ref. 32). DNA derived from tumour cells is shorter than that from
non-malignant cells in the plasma of cancer patients (Ref. 33). One
problem to be solved is the development of DNAme based markers (and
an assay) for ovarian cancer detection, in particular early
detection of ovarian cancer. Samples available in order to carry
out this task are from large population based screening studies.
For example, the largest of such studies being UKCTOCS. Serum
samples from 100,000 women need to be collected in order to secure
sufficient numbers (i.e. between 40-50) of women who develop
ovarian cancer within 2 years of sample donation. Within the
UKCTOCS setting whole blood samples were couriered to the central
laboratory with median time to spin of 22 hours. Prospectively
collected blood samples were spun down between 2-12 hours after
collection in order to mimic the collection-setting typically used
for large studies like UKCTOCS. It is expected for such an analysis
of UKCTOCS samples that, and as has been already seen for other
prospectively collected sets including UKCTOCS (Anjum et al, 2014),
samples from such prospectively collected sets contain higher than
average amounts of cell-free DNA and fragments being longer on
average. Both factors are likely to reflect the leakage of WBC DNA
into serum. Despite these complicating factors the three-DNAme
marker panel can outperform CA125 in detecting ovarian cancer, also
for detecting ovarian cancer early.
[0335] False CA125-positivity can usually be explained by a CA125
producing benign condition (Ref. 34). The fact that in Serum Set 3
there was no overlap at all between false CA125 and false DNAme
positive samples indicates that the DNAme-false positivity is
largely triggered by technical artefacts as a result of extremely
low thresholds down to a pattern frequency of 0.000003 (i.e. 3
cancer patterns in the background of 1.000.000 DNA fragments with a
non-cancer pattern).
[0336] Based on the UKCTOCS prevalence screen (Ref. 23), the Risk
of Ovarian Cancer Algorithm (ROCA) identified 0.65% of women at an
elevated risk of which 13% (42/327) have eventually been diagnosed
after having been assessed by ultrasound, additional imaging and
clinical assessment. Applying the three-marker DNAme test of the
present invention with a conservative estimate of specificity and
sensitivity of 90% and 60%, respectively, in ROCA-elevated risk
women would immediately enable diagnosis and treatment of the 0.05%
of women within a population with an ovarian cancer with a positive
predictive value of 44% (i.e. only 2.3 operations are necessary to
diagnose/treat 1 ovarian cancer patient).
[0337] At the UKCTOCS prevalence screen (Ref. 22), the Risk of
Ovarian Cancer Algorithm (ROCA) identified elevated risk in 0.65%
of women of whom 13% (42/327) were diagnosed after repeat CA125
testing, ultrasound, additional imaging and clinical assessment.
Applying the three-marker DNAme test of the present invention with
a conservative estimate (i.e. excessive background DNA will not be
an issue in prospective samples) of specificity and sensitivity of
90% and 60%, as a second line test to ROCA-elevated risk women
could substantially decrease time to diagnosis in at least half the
women with ovarian cancer.
[0338] Overall and for the first time, the present invention
provides serum DNAme markers and assays (and other means and
methods) that can diagnose ovarian cancers (or are useful for such
diagnosis), and it is likely that they are able to detect/diagnose
OC up to two years in advance of conventional methods of diagnosis,
and are able to individualise ovarian cancer treatment. The recent
advance of purpose-made blood collection tubes (such as those from
Streck as described above) that stabilise circulating DNA and
prevent leakage of DNA from blood cells (Ref. 35) will facilitate
clinical implementation of DNAme pattern detection in cell free DNA
as a clinical tool in cancer medicine.
[0339] Note: The work leading to this invention has received
funding from the European Union Seventh Framework Programme
(FP7/2007-2013) under Grant Agreement Number 305428 (Project
EpiFemCare).
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Sequence CWU 1
1
1591136DNAHomo sapiens 1catccggagg cccaggggtg aggacttcgc cacgggaagg
aggcacacga ttcagcccat 60gacaccgcca cctcggcgtg gtgctgtagg gggaagctca
ggcactcacc gaggacagga 120cccggggaat ccgctg 1362154DNAHomo sapiens
2gatattcggt ggagagccgc agctgcccgc cgcggggccc caggcgcagc acgctctcgc
60gcgtgggccg cagctggcag cacaggaagt ccaggtggaa gagcggcggc gtgggcggcc
120cggcgcggcg cggcgagtgc gggctggtat cggc 1543111DNAHomo sapiens
3gttctatggg cgagctgctg cagtgcggct gccaggcgcc ccgcgggcgg gcccctcccc
60ggccctccgg cctgcccggc acccccggac cccctggccc cgcgggctcc c
1114140DNAHomo sapiens 4cccaggcctg acgtgggtcc cccagggcgg cgtcgccaag
gcttagacgc tttcgtgcag 60gagggacgac gactcccctc acgccttcgt ggccccaact
cggcgctctg ctatctctga 120tccggtgaac acacctcaga 1405120DNAHomo
sapiens 5gcgaagcagg agtagctgcc gggccccacg agcctccgtc cgttctggtt
cgggtttctc 60cgagttttgc taccagccga ggctgtgcgg gcaactgggt cagcctcccg
tcaggagaga 1206120DNAHomo sapiens 6aactctgctg agtgagctca caaacagggc
ataaccgaga cgcgggaatg cctgggtcgc 60cgcgcagtca ccgggcaggg ccgccctccc
ctgtgggtca gcaaaaacgg tgtcaagtga 1207130DNAHomo sapiens 7actccgccac
acacacagct gtacccggca caacacgcgg ccacaggtca cctcaggtcg 60cctcgggtgc
tcctcccgca gccccacgta gacagaagac attcctcggg cctgggtgcc
120cagcctcccg 1308120DNAHomo sapiens 8gaggtaatgg aagcggccat
ccttgtcctc gctccgcgcc tggctgaagc gatcggggtc 60gaacacgttc acgtctttga
acacgggcgc tgtgtcatgg gtgtcccgga tgctatacat 1209137DNAHomo sapiens
9gcgggcacct gtagtcccag ctactcggga ggctgaggcg ggagaatggc gtgaacccgg
60gaggcggagc ttgcagcgag ccgagatcgc gccaccgcac tccagcctgg gcgacagagc
120gagactccgt ctaaaaa 13710137DNAHomo sapiens 10tccccggagt
ccggagctca ggccagtggc agtcgaccca gcccccgaga ctccctcacg 60ccgctccaaa
accaaaacgg agcccaacac gaagctgggt gaagccgtag cttgcaggag
120ccagggagat gcgctct 13711136DNAHomo sapiens 11gactctgtct
caaaaaagaa aaaaataggg ccgggcgcgg tggctcacgc ctgtcatccc 60agcactttgg
gaggccgagg cgggtggatc acgaggtcgg gagatcgata ccatcctggg
120taacacggtg aaaccc 13612138DNAHomo sapiens 12gactcgcgag
gttttccagc agctcattcc gggacggcgg tgtctagtcc agtccagggt 60aactgggctc
tctgagagtc cgacctccat cggtctggga gcgagtggtt cgagttcaga
120tgctgggaac cgtcgctt 13813138DNAHomo sapiens 13taacccattt
ctttattaaa ttgcatgaag aaggccgggc gcggtggctc acgcctgtaa 60tcccagcact
ttgggaggcc gaggcgggcg gatcacgagg tcaggagatc gagaccacgg
120tgaaaccccg tctctact 13814142DNAHomo sapiens 14ggcaggagcg
ccccactatg cgcaagcccg tggcctggag agcgctgaag gtgggagggg 60gaagaggggc
agaacccccg cgggagcgag cgcacagctg ccgccccgtg gccgcttcgg
120gaatcgctgg ctccggctct gg 14215135DNAHomo sapiens 15ctgcagaagc
gcactttgct gaacaccccg aggacgtgcc tctcgcacag ggagcgcccg 60tctttgctgg
ggctggagcg gcgcttggag gccgacactc ggtcgctgtt ggactccctc
120gcctgccgct tctgc 13516146DNAHomo sapiens 16cgtgttagcc aggatggtct
cgacctcctg acctcgtgat cagcccgcct cggcctccca 60aagtgctggg attaaaggcg
tgagccaccg cgccgggccg agactctgtc ttaaaaaaaa 120aaggcctggg
ctgtggcact ttggga 14617141DNAHomo sapiens 17agagttgcac tccgaagact
ccagattccg agagttgcgg aaacgctacg aggacctgct 60aaccaggctg cgggccaacc
agagctggga agattcgaac accgacctcg tcccggcccc 120tgcagtccgg
atactcacgc c 14118109DNAHomo sapiens 18gtcagacgag agcctggggt
caatgtcgag gtggagcgac gctggcacgg caaccctgag 60cctgcgcggc ccggcgctat
cccctggctc tccgctgctg gctggaccc 10919144DNAHomo sapiens
19cggtaggtca tccagcagca gggctccacg tcggtctcgt cgatgcccca gaaggccagc
60tcctcctcga agagcggccc gcacacgtct gcggggcagt gcagcttgcc ggtgcggtag
120taattgagca cataggcgaa gacg 14420115DNAHomo sapiens 20aatcagccca
gcaaccggcg accccaagcg cggcgaccgc aaagggagtg cttgcccatc 60cgcgtttgaa
agcagacttt ttctcggcag gaacacagga ctcacctgcc agtgg 11521148DNAHomo
sapiens 21gtgcgaacaa gaccgggcgt ttcgccgccg acgcgaaggg gctgtctgtg
cgcggcgttg 60cgggccctcc gcgcgtgggg tgtgcgtgtg cgtgttcggg ttcggttctg
tgtgtgcacc 120gcgggcctgc tcagagtcgg gaccaccg 14822121DNAHomo
sapiens 22atattaatct tgtccgggca cggtggctca cgcctgtaat cccagcactt
tgggaggccg 60aggcgggcgg atcacgaggt caggagatcg agaccatcct ggcgaacatg
gtgaaacctc 120g 1212392DNAHomo sapiens 23tgcatacaga ttactgtagg
accatttcct gtgcctttta aaatttcctt ttctcgtttt 60atttcacata ttcctttgtt
ttttacaact cc 9224103DNAHomo sapiens 24ccgctcggga atgggaatat
agctacatat gggaaaacgc ggtgcaggga gaaaaccaat 60tcagtgagga gcggaggcgc
aggactgtgg agtgtgcatc cgg 1032594DNAHomo sapiens 25ctgcttaaag
gcgcagagga gcagctggga acgagaacaa agcggccagg cccccctcgg 60aggaaggaag
gagagagccc caggaaacag ctga 9426101DNAHomo sapiens 26ggatgaagga
ttcctgcatc actgtgatgg ccatggcgct gctgtctggg ttctttttct 60tcggtaggca
agggaggagg caggggaagg gacatgtgtc t 1012770DNAHomo sapiens
27taggctacag gaagaggcat ttcctataga tgacggctgt aaaattttaa gctgagttcc
60tccaggaagt 702884DNAHomo sapiens 28aagagagagt ggttgataat
cagtagagag aggtttctaa ctcacggaag tgtttgcaat 60acaacctctt tgtacatcag
ctgt 842997DNAHomo sapiens 29ggtccccctc cccgagccat gaagagctgc
ctgcggccat cttggccctc gcaccccgtc 60tctgtcaccc caggcccctg taacttgctt
aacgctt 973086DNAHomo sapiens 30gaagcttgac actcctggcc ccaaacactg
cctggctaca acacgatatc cagggacaga 60taccttccat gtacagcaag ctgtgg
863191DNAHomo sapiens 31ggggggactg tcgttaattc actgcctaat gaccgcggcc
cgcgcgctcc gagtaatcgg 60gtgatgtatg tggactgtgc acacctcgtg g
9132136DNAArtificial SequenceDMR 141 32tattyggagg tttaggggtg
aggatttygt taygggaagg aggtatayga tttagtttat 60gatatygtta tttyggygtg
gtgttgtagg gggaagttta ggtatttaty gaggatagga 120ttyggggaat tygttg
13633154DNAArtificial SequenceDMR 204 33gatattyggt ggagagtygt
agttgttygt ygyggggttt taggygtagt aygttttygy 60gygtgggtyg tagttggtag
tataggaagt ttaggtggaa gagyggyggy gtgggyggtt 120yggygyggyg
yggygagtgy gggttggtat yggt 15434111DNAArtificial SequenceDMR 228
34gttttatggg ygagttgttg tagtgyggtt gttaggygtt tygygggygg gtttttttty
60ggtttttygg tttgttyggt attttyggat tttttggttt ygygggtttt t
11135140DNAArtificial SequenceDMR 144 35tttaggtttg aygtgggttt
tttagggygg ygtygttaag gtttagaygt tttygtgtag 60gagggaygay gatttttttt
aygttttygt ggttttaatt yggygttttg ttatttttga 120ttyggtgaat
atattttaga 14036120DNAArtificial SequenceDMR 123 36gygaagtagg
agtagttgty gggttttayg agttttygtt ygttttggtt ygggtttttt 60ygagttttgt
tattagtyga ggttgtgygg gtaattgggt tagtttttyg ttaggagaga
12037120DNAArtificial SequenceDMR 129 37aattttgttg agtgagttta
taaatagggt ataatygaga ygygggaatg tttgggtygt 60ygygtagtta tygggtaggg
tygttttttt ttgtgggtta gtaaaaaygg tgttaagtga 12038130DNAArtificial
SequenceDMR 137 38atttygttat atatatagtt gtattyggta taataygygg
ttataggtta ttttaggtyg 60tttygggtgt ttttttygta gttttaygta gatagaagat
atttttyggg tttgggtgtt 120tagtttttyg 13039120DNAArtificial
SequenceDMR 148 39gaggtaatgg aagyggttat ttttgtttty gtttygygtt
tggttgaagy gatyggggty 60gaataygttt aygtttttga ataygggygt tgtgttatgg
gtgtttygga tgttatatat 12040137DNAArtificial SequenceDMR 150
40gygggtattt gtagttttag ttattyggga ggttgaggyg ggagaatggy gtgaattygg
60gaggyggagt ttgtagygag tygagatygy gttatygtat tttagtttgg gygatagagy
120gagatttygt ttaaaaa 13741137DNAArtificial SequenceDMR 154
41ttttyggagt tyggagttta ggttagtggt agtygattta gttttygaga tttttttayg
60tygttttaaa attaaaaygg agtttaatay gaagttgggt gaagtygtag tttgtaggag
120ttagggagat gygtttt 13742136DNAArtificial SequenceDMR 158
42gattttgttt taaaaaagaa aaaaataggg tygggygygg tggtttaygt ttgttatttt
60agtattttgg gaggtygagg ygggtggatt aygaggtygg gagatygata ttattttggg
120taatayggtg aaattt 13643138DNAArtificial SequenceDMR 164
43gattygygag gttttttagt agtttattty gggayggygg tgtttagttt agtttagggt
60aattgggttt tttgagagtt ygatttttat yggtttggga gygagtggtt ygagtttaga
120tgttgggaat ygtygttt 13844138DNAArtificial SequenceDMR 176
44taatttattt ttttattaaa ttgtatgaag aaggtygggy gyggtggttt aygtttgtaa
60ttttagtatt ttgggaggty gaggygggyg gattaygagg ttaggagaty gagattaygg
120tgaaatttyg tttttatt 13845142DNAArtificial SequenceDMR 178
45ggtaggagyg ttttattatg ygtaagttyg tggtttggag agygttgaag gtgggagggg
60gaagaggggt agaattttyg ygggagygag ygtatagttg tygtttygtg gtygtttygg
120gaatygttgg tttyggtttt gg 14246135DNAArtificial SequenceDMR 180
46ttgtagaagy gtattttgtt gaatatttyg aggaygtgtt tttygtatag ggagygttyg
60tttttgttgg ggttggagyg gygtttggag gtygatatty ggtygttgtt ggatttttty
120gtttgtygtt tttgt 13547146DNAArtificial SequenceDMR 186
47ygtgttagtt aggatggttt ygattttttg atttygtgat tagttygttt yggtttttta
60aagtgttggg attaaaggyg tgagttatyg ygtygggtyg agattttgtt ttaaaaaaaa
120aaggtttggg ttgtggtatt ttggga 14648141DNAArtificial SequenceDMR
188 48agagttgtat ttygaagatt ttagatttyg agagttgygg aaaygttayg
aggatttgtt 60aattaggttg ygggttaatt agagttggga agattygaat atygatttyg
tttyggtttt 120tgtagttygg atatttaygt t 14149109DNAArtificial
SequenceDMR 190 49gttagaygag agtttggggt taatgtygag gtggagygay
gttggtaygg taattttgag 60tttgygyggt tyggygttat tttttggttt ttygttgttg
gttggattt 10950144DNAArtificial SequenceDMR 192 50yggtaggtta
tttagtagta gggttttayg tyggtttygt ygatgtttta gaaggttagt 60tttttttyga
agagyggtty gtataygttt gyggggtagt gtagtttgty ggtgyggtag
120taattgagta tataggygaa gayg 14451115DNAArtificial SequenceDMR 200
51aattagttta gtaatyggyg attttaagyg yggygatygt aaagggagtg tttgtttatt
60ygygtttgaa agtagatttt ttttyggtag gaatatagga tttatttgtt agtgg
11552148DNAArtificial SequenceDMR 202 52gtgygaataa gatygggygt
ttygtygtyg aygygaaggg gttgtttgtg ygyggygttg 60ygggttttty gygygtgggg
tgtgygtgtg ygtgttyggg ttyggttttg tgtgtgtaty 120gygggtttgt
ttagagtygg gattatyg 14853121DNAArtificial SequenceDMR 208
53atattaattt tgttygggta yggtggttta ygtttgtaat tttagtattt tgggaggtyg
60aggygggygg attaygaggt taggagatyg agattatttt ggygaatatg gtgaaattty
120g 1215492DNAArtificial SequenceDMR 210 54tgtatataga ttattgtagg
attatttttt gtgtttttta aaattttttt ttttygtttt 60attttatata tttttttgtt
ttttataatt tt 9255103DNAArtificial SequenceDMR 213 55tygttyggga
atgggaatat agttatatat gggaaaaygy ggtgtaggga gaaaattaat 60ttagtgagga
gyggaggygt aggattgtgg agtgtgtatt ygg 1035694DNAArtificial
SequenceDMR 214 56ttgtttaaag gygtagagga gtagttggga aygagaataa
agyggttagg tttttttygg 60aggaaggaag gagagagttt taggaaatag ttga
9457101DNAArtificial SequenceDMR 219 57ggatgaagga tttttgtatt
attgtgatgg ttatggygtt gttgtttggg tttttttttt 60tyggtaggta agggaggagg
taggggaagg gatatgtgtt t 1015870DNAArtificial SequenceDMR 222
58taggttatag gaagaggtat tttttataga tgayggttgt aaaattttaa gttgagtttt
60tttaggaagt 705984DNAArtificial SequenceDMR 223 59aagagagagt
ggttgataat tagtagagag aggtttttaa tttayggaag tgtttgtaat 60ataatttttt
tgtatattag ttgt 846097DNAArtificial SequenceDMR 224 60ggtttttttt
ttygagttat gaagagttgt ttgyggttat tttggtttty gtatttygtt 60tttgttattt
taggtttttg taatttgttt aaygttt 976186DNAArtificial SequenceDMR 225
61gaagtttgat atttttggtt ttaaatattg tttggttata ataygatatt tagggataga
60tattttttat gtatagtaag ttgtgg 866291DNAArtificial SequenceDMR 226
62ggggggattg tygttaattt attgtttaat gatygyggtt ygygygttty gagtaatygg
60gtgatgtatg tggattgtgt atatttygtg g 916384DNAArtificial
SequenceDMR 141 63cgttacggga aggaggtata cgatttagtt tatgatatcg
ttatttcggc gtggtgttgt 60agggggaagt ttaggtattt atcg
8464107DNAArtificial SequenceDMR 204 64cgtcgcgggg ttttaggcgt
agtacgtttt cgcgcgtggg tcgtagttgg tagtatagga 60agtttaggtg gaagagcggc
ggcgtgggcg gttcggcgcg gygyggt 1076562DNAArtificial SequenceDMR 228
65yggttgttag gcgtttcgcg ggygggtttt ttttcggttt ttcggtttgt tcggtatttt
60cg 626680DNAArtificial SequenceDMR 144 66ggcggcgtcg ttaaggttta
gacgttttcg tgtaggaggg acgacgattt tttttacgtt 60ttcgtggttt taattcggcg
806761DNAArtificial SequenceDMR 123 67tgagtttttg tttgttttgg
tttgggtttt tttgagtttt gttattagtt gaggttgtgt 60g 616861DNAArtificial
SequenceDMR 129 68tygagatgtg ggaatgtttg ggtygtygtg tagttattgg
gtagggtygt ttttttttgt 60g 616973DNAArtificial SequenceDMR 137
69tgtggttata ggttatttta ggttgttttg ggtgtttttt ttgtagtttt atgtagatag
60aagatatttt ttg 737064DNAArtificial SequenceDMR 148 70ttttygtttt
gtgtttggtt gaagtgattg gggtygaata ygtttaygtt tttgaatatg 60ggyg
647188DNAArtificial SequenceDMR 150 71tgggaggttg aggtgggaga
atggtgtgaa tttgggaggt ggagtttgta gtgagttgag 60attgtgttat tgtattttag
tttgggtg 887276DNAArtificial SequenceDMR 154 72gtygatttag
ttttcgagat ttttttacgt cgttttaaaa ttaaaacgga gtttaatacg 60aagttgggtg
aagtcg 767376DNAArtificial SequenceDMR 158 73ygggygtggt ggtttatgtt
tgttatttta gtattttggg aggttgaggt gggtggatta 60tgaggttggg agattg
767483DNAArtificial SequenceDMR 164 74ygggayggyg gtgtttagtt
tagtttaggg taattgggtt ttttgagagt tcgattttta 60tcggtttggg agcgagtggt
tcg 837576DNAArtificial SequenceDMR 176 75ygggtgtggt ggtttaygtt
tgtaatttta gtattttggg aggttgaggt gggyggatta 60ygaggttagg agatyg
767691DNAArtificial SequenceDMR 178 76ygtggtttgg agagygttga
aggtgggagg gggaagaggg gtagaatttt ygcgggagcg 60agcgtatagt tgtcgtttyg
tggtcgttty g 917771DNAArtificial SequenceDMR 180 77ygtgttttty
gtatagggag cgttygtttt tgttggggtt ggagcggcgt ttggaggtcg 60atattcggtc
g 717888DNAArtificial SequenceDMR 186 78ygtgattagt tygttttggt
tttttaaagt gttgggatta aaggtgtgag ttattgtgtt 60gggttgagat tttgttttaa
aaaaaaaa 887987DNAArtificial SequenceDMR 188 79tgagagttgy
ggaaaygtta ygaggatttg ttaattaggt tgygggttaa ttagagttgg 60gaagatttga
atattgattt tgtttyg 878056DNAArtificial SequenceDMR 190 80cgaggtggag
cgacgttggt acggtaattt tgagtttgcg cggttcggcg ttattt
568188DNAArtificial SequenceDMR 192 81cgtcggtttc gtcgatgttt
tagaaggtta gttttttttc gaagagcggt tcgtatacgt 60ttgcggggta gtgtagtttg
tcggtgcg 888258DNAArtificial SequenceDMR 200 82cgcggcgatc
gtaaagggag tgtttgttta ttcgcgtttg aaagtagatt ttttttcg
5883101DNAArtificial SequenceDMR 202 83ygtygtygay gygaaggggt
tgtttgtgcg cggygttgyg ggtttttcgc gcgtggggtg 60tgcgtgtgcg tgttcgggtt
cggttttgtg tgtgtatcgc g 1018461DNAArtificial SequenceDMR 208
84atgtttgtaa ttttagtatt ttgggaggtt gaggtgggtg gattatgagg ttaggagatt
60g 618530DNAArtificial SequenceDMR 210 85tttttaaaat tttttttttt
cgttttattt 308649DNAArtificial SequenceDMR 213 86gaaaacgcgg
tgtagggaga aaattaattt agtgaggagc ggaggcgta 498734DNAArtificial
SequenceDMR 214 87gaacgagaat aaagcggtta ggtttttttc ggag
348841DNAArtificial SequenceDMR 219 88gcgttgttgt ttgggttttt
ttttttcggt aggtaaggga g 418910DNAArtificial SequenceDMR
222misc_feature1..2/note="n can be any
nucleotide"misc_feature9..10/note="n can be any nucleotide"
89nnacggttnn 109023DNAArtificial SequenceDMR 223 90agagaggttt
ttaatttacg gaa 239134DNAArtificial SequenceDMR 224 91tttgcggtta
ttttggtttt cgtatttcgt tttt 349219DNAArtificial SequenceDMR 225
92gttataatac gatatttag 199329DNAArtificial SequenceDMR 226
93attgtggttt gtgtgttttg agtaattgg 299427DNAArtificial SequenceDMR
141 for 94tattyggagg tttaggggtg aggattt
279527DNAArtificial SequenceDMR 204 for 95gatattyggt ggagagtygt
agttgtt 279625DNAArtificial SequenceDMR 228 for 96gttttatggg
ygagttgttg tagtg 259725DNAArtificial SequenceDMR 144 for
97tttaggtttg aygtgggttt tttag 259828DNAArtificial SequenceDMR 123
for 98gygaagtagg agtagttgty gggtttta 289934DNAArtificial
SequenceDMR 129 for 99aattttgttg agtgagttta taaatagggt ataa
3410035DNAArtificial SequenceDMR 137 for 100atttygttat atatatagtt
gtattyggta taata 3510125DNAArtificial SequenceDMR 148 for
101gaggtaatgg aagyggttat ttttg 2510225DNAArtificial SequenceDMR 150
for 102gygggtattt gtagttttag ttatt 2510331DNAArtificial SequenceDMR
154 for 103ttttyggagt tyggagttta ggttagtggt a 3110431DNAArtificial
SequenceDMR 158 for 104gattttgttt taaaaaagaa aaaaataggg t
3110529DNAArtificial SequenceDMR 164 for 105gattygygag gttttttagt
agtttattt 2910635DNAArtificial SequenceDMR 176 for 106taatttattt
ttttattaaa ttgtatgaag aaggt 3510728DNAArtificial SequenceDMR 178
for 107ggtaggagyg ttttattatg ygtaagtt 2810834DNAArtificial
SequenceDMR 180 for 108ttgtagaagy gtattttgtt gaatatttyg agga
3410934DNAArtificial SequenceDMR 186 for 109ygtgttagtt aggatggttt
ygattttttg attt 3411028DNAArtificial SequenceDMR 188 for
110agagttgtat ttygaagatt ttagattt 2811126DNAArtificial SequenceDMR
190 for 111gttagaygag agtttggggt taatgt 2611228DNAArtificial
SequenceDMR 192 for 112yggtaggtta tttagtagta gggtttta
2811328DNAArtificial SequenceDMR 200 for 113aattagttta gtaatyggyg
attttaag 2811422DNAArtificial SequenceDMR 202 for 114gtgygaataa
gatygggygt tt 2211529DNAArtificial SequenceDMR 208 for
115atattaattt tgttygggta yggtggttt 2911628DNAArtificial SequenceDMR
210 for 116ggagttgtaa aaaataaagg aatatgtg 2811732DNAArtificial
SequenceDMR 213 for 117tygttyggga atgggaatat agttatatat gg
3211828DNAArtificial SequenceDMR 214 for 118ttgtttaaag gygtagagga
gtagttgg 2811935DNAArtificial SequenceDMR 219 for 119ggatgaagga
tttttgtatt attgtgatgg ttatg 3512032DNAArtificial SequenceDMR 222
for 120taggttatag gaagaggtat tttttataga tg 3212126DNAArtificial
SequenceDMR 223 for 121aagagagagt ggttgataat tagtag
2612229DNAArtificial SequenceDMR 224 for 122ggtttttttt ttygagttat
gaagagttg 2912334DNAArtificial SequenceDMR 225 for 123gaagtttgat
atttttggtt ttaaatattg tttg 3412431DNAArtificial SequenceDMR 226 for
124ggggggattg tygttaattt attgtttaat g 3112525DNAArtificial
SequenceDMR 141 rev 125caacraattc cccraatcct atcct
2512620DNAArtificial SequenceDMR 204 rev 126accratacca acccrcactc
2012724DNAArtificial SequenceDMR 228 rev 127aaaaacccrc raaaccaaaa
aatc 2412835DNAArtificial SequenceDMR 144 rev 128tctaaaatat
attcaccraa tcaaaaataa caaaa 3512931DNAArtificial SequenceDMR 123
rev 129tctctcctaa craaaaacta acccaattac c 3113025DNAArtificial
SequenceDMR 129 rev 130tcacttaaca ccrtttttac taacc
2513122DNAArtificial SequenceDMR 137 rev 131craaaaacta aacacccaaa
cc 2213231DNAArtificial SequenceDMR 148 rev 132atatataaca
tccraaacac ccataacaca a 3113324DNAArtificial SequenceDMR 150 rev
133tttttaaacr aaatctcrct ctat 2413430DNAArtificial SequenceDMR 154
rev 134aaaacrcatc tccctaactc ctacaaacta 3013529DNAArtificial
SequenceDMR 158 rev 135aaatttcacc rtattaccca aaataatat
2913626DNAArtificial SequenceDMR 164 rev 136aaacracrat tcccaacatc
taaact 2613727DNAArtificial SequenceDMR 176 rev 137aataaaaacr
aaatttcacc rtaatct 2713823DNAArtificial SequenceDMR 178 rev
138ccaaaaccra aaccaacrat tcc 2313930DNAArtificial SequenceDMR 180
rev 139acaaaaacra caaacraaaa aatccaacaa 3014024DNAArtificial
SequenceDMR 186 rev 140tcccaaaata ccacaaccca aacc
2414126DNAArtificial SequenceDMR 188 rev 141aacrtaaata tccraactac
aaaaac 2614227DNAArtificial SequenceDMR 190 rev 142aaatccaacc
aacaacraaa aaccaaa 2714328DNAArtificial SequenceDMR 192 rev
143crtcttcrcc tatatactca attactac 2814429DNAArtificial SequenceDMR
200 rev 144ccactaacaa ataaatccta tattcctac 2914525DNAArtificial
SequenceDMR 202 rev 145crataatccc ractctaaac aaacc
2514631DNAArtificial SequenceDMR 208 rev 146craaatttca ccatattcrc
caaaataatc t 3114734DNAArtificial SequenceDMR 210 rev 147tacatacaaa
ttactataaa accatttcct atac 3414822DNAArtificial SequenceDMR 213 rev
148ccraatacac actccacaat cc 2214932DNAArtificial SequenceDMR 214
rev 149tcaactattt cctaaaactc tctccttcct tc 3215025DNAArtificial
SequenceDMR 219 rev 150aaacacatat cccttcccct acctc
2515132DNAArtificial SequenceDMR 222 rev 151acttcctaaa aaaactcaac
ttaaaatttt ac 3215235DNAArtificial SequenceDMR 223 rev
152acaactaata tacaaaaaaa ttatattaca aacac 3515334DNAArtificial
SequenceDMR 224 rev 153aaacrttaaa caaattacaa aaacctaaaa taac
3415433DNAArtificial SequenceDMR 225 rev 154ccacaactta ctatacataa
aaaatatcta tcc 3315531DNAArtificial SequenceDMR 226 rev
155ccacraaata tacacaatcc acatacatca c 3115684DNAHomo sapiens
156cgccacggga aggaggcaca cgattcagcc catgacaccg ccacctcggc
gtggtgctgt 60agggggaagc tcaggcactc accg 84157107DNAHomo sapiens
157cgccgcgggg ccccaggcgc agcacgctct cgcgcgtggg ccgcagctgg
cagcacagga 60agtccaggtg gaagagcggc ggcgtgggcg gcccggcgcg gcgcggc
10715862DNAHomo sapiens 158cggctgccag gcgccccgcg ggcgggcccc
tccccggccc tccggcctgc ccggcacccc 60cg 6215980DNAHomo sapiens
159ggcggcgtcg ccaaggctta gacgctttcg tgcaggaggg acgacgactc
ccctcacgcc 60ttcgtggccc caactcggcg 80
* * * * *
References