U.S. patent application number 17/169293 was filed with the patent office on 2021-08-26 for method of screening for colorectal cancer.
The applicant listed for this patent is Clinical Genomics Pty Ltd. Invention is credited to Rohan Kartin BAKER, Snigdha GAUR, Lawrence Charles LAPOINTE, Susanne K. PEDERSEN, Melissa THOMAS.
Application Number | 20210262038 17/169293 |
Document ID | / |
Family ID | 1000005553424 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210262038 |
Kind Code |
A1 |
LAPOINTE; Lawrence Charles ;
et al. |
August 26, 2021 |
METHOD OF SCREENING FOR COLORECTAL CANCER
Abstract
The present invention relates generally to a method of
determining one or more probabilities of respective classifications
of a neoplasm into one or more neoplastic categories. More
particularly, the present invention relates to a method of
determining the probability of classification of a large intestine
neoplasm into one or more categories selected from adenoma, stage
I, stage II, stage III or stage IV by screening for changes to the
methylation levels of a panel of gene markers, including BCAT1,
IKZF1, IRF4, GRASP and/or CAHM. The method of the present invention
is useful in a range of applications including, but not limited to,
those relating to the diagnosis and/or monitoring of colorectal
neoplasms, such as colorectal adenocarcinosis.
Inventors: |
LAPOINTE; Lawrence Charles;
(Bridgewater, NJ) ; PEDERSEN; Susanne K.; (New
South Wales, AU) ; BAKER; Rohan Kartin; (New South
Wales, AU) ; GAUR; Snigdha; (New South Wales, AU)
; THOMAS; Melissa; (New South Wales, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clinical Genomics Pty Ltd |
Elsternwick |
|
AU |
|
|
Family ID: |
1000005553424 |
Appl. No.: |
17/169293 |
Filed: |
February 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14401157 |
Nov 14, 2014 |
10941449 |
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PCT/AU2013/000519 |
May 17, 2013 |
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17169293 |
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61648821 |
May 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/112 20130101;
C12Q 2600/154 20130101; C12Q 1/6886 20130101; G16H 50/30
20180101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574 |
Claims
1. (canceled)
2. A method comprising measuring a methylation level of a DNA
region of a sample from an individual having or suspected of having
a large intestine neoplasm, wherein said measuring of a methylation
level of a DNA region of a sample from said individual comprises:
selecting an individual from a patient population known or
suspected to have a large intestine neoplasm staged as adenoma,
stage I, stage II, stage III, or stage IV; obtaining or having
obtained a blood-derived sample from said individual, the
blood-derived sample comprising circulating cell-free DNA;
extracting circulating cell-free DNA from the sample; bisulfite
converting the circulating cell-free DNA; and detecting a level of
methylation of a DNA region of the bisulfite converted DNA, wherein
said detecting comprises hybridizing methylation-specific
oligonucleotide primers to the DNA region of the bisulfite
converted DNA, wherein the DNA region comprises: (i) the region,
including 2 kb upstream of the transcription start site, defined by
chr7:50344378..50472798 and at least one of Hg19 coordinates: (1)
chr12:24962958..25102393; (2) chr6:391739..411443; (3)
chr12:52400748..52409671; or (4) chr6:163834097..163834982; or (ii)
the gene region, including 2 kb upstream of IKZF1 and at least one
of: (1) BCAT1; (2) IRF4; (3) GRASP; or (4) CAHM; determining a
probability whether said individual has a premalignant neoplasm, an
early stage malignant neoplasm or a late stage malignant neoplasm
from said measurement of said methylation level of said DNA region
of said sample from said individual.
3. The method of claim 2, wherein said methylation level is
measured in (1) IKZF1 subregions: chr7:50343867-50343961 (SEQ ID
NO: 3 or corresponding minus strand) and chr7:50343804-5033895 (SEQ
ID NO: 4 or corresponding minus strand), and one or more
chromosomal subregions selected from: (2) BCAT1 subregions
chr12:25101992-25102093 (SEQ ID NO: 1 or corresponding minus
strand) and chr12:25101909-25101995 (SEQ ID NO: 2 or corresponding
minus strand) (3) IRF4 subregions chr6:392036-392145 (SEQ ID NO: 5
or corresponding minus strand) (4) GRASP subregions:
chr12:52399672-52399922, chr12:52400821-52401051 (SEQ ID NO: 6 or
corresponding minus strand), chr12:52401407-52401664 (SEQ ID NO: 7
or corresponding minus strand) chr12:52400866-52400973 and
Chr12:52401107-52401664, or (5) CAHM subregions:
chr6:163834295-163834500 (SEQ ID NO: 8 or corresponding minus
strand), chr6:163834621-163834906, chr6:163834393-163834455 and
chr6: 163834393-163834519.
4. The method of claim 3, wherein said subregion is selected from
SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or
corresponding minus strands.
5. The method of claim 3, said method comprising measuring the
methylation of one or more cytosine residues selected from:
TABLE-US-00017 (IKZF1) chr7: 50343869 chr7: 50343872 chr7: 50343883
chr7: 50343889 chr7: 50343890 chr7: 50343897 chr7: 50343907 chr7:
50343909 chr7: 50343914 chr7: 50343934 chr7: 50343939 chr7:
50343950 chr7: 50343959 chr7: 50343805 chr7: 50343822 chr7:
50343824 chr7: 50343826 chr7: 50343829 chr7: 50343831 chr7:
50343833 chr7: 50343838 chr7: 50343847 chr7: 50343850 chr7:
50343858 chr7: 50343864 chr7: 50343869 chr7: 50343872 chr7:
50343890 (BCAT1) chr12: 25101998 chr12: 25102003 chr12: 25102006
chr12: 25102009 chr12: 25102017 chr12: 25102022 chr12: 25102039
chr12: 25102048 chr12: 25102050 chr12: 25102053 chr12: 25102061
chr12: 25102063 chr12: 25102071 chrl12: 25101921 chr12: 25101934
chr12: 25101943 chr12: 25101951 chr12: 25101962 chr12: 25101964
chr12: 25101970 (GRASP) chr12: 52399713 chr12: 52399731 chr12:
52399749 chr12: 52399783 chr12: 52399796 chr12: 52399808 chr12:
52399823 chr12: 52399835 chr12: 52399891 chr12: 52400847 chr12:
52400850 chr12: 52400859 chr12: 52400866 chr12: 52400869 chr12:
52400873 chr12: 52400881 chr12: 52400886 chr12: 52400893 chr12:
52400895 chr12: 52400899 chr12: 52400902 chr12: 52400907 chr12:
52400913 chr12: 52400919 chr12: 52400932 chr12: 52400938 chr12:
52400958 chr12: 52400962 chr12: 52400971 chr12: 52400973 chr12:
52400976 chr12: 52400998 chr12: 52401008 chr12: 52401010 chr12:
52401012 chr12: 52401016 chr12: 52401019 chr12: 52401025 chr12:
52401041 chr12: 52401044 chr12: 52401053 chr12: 52401060 chr12:
52401064 chr12: 52401092 chr12: 52401118 chr12: 52401438 chr12:
52401448 chr12: 52401460 chr12: 52401465 chr12: 52401474 chr12:
52401477 chr12: 52401479 chr12: 52401483 chr12: 52401504 chr12:
52401514 chr12: 52401523 chr12: 52401540 chr12: 52401553 chr12:
52401576 chr12: 52401588 chr12: 52401595 chr12: 52401599 chr12:
52401604 chr12: 52401606 chr12: 52401634 chr12: 52401640 chr12:
52401644 chr12: 52401659 chr12: 52401160 chr12: 52401165 chr12:
52401174 chr12: 52401177 chr12: 52401179 chr12: 52401183 chr12:
52401204 chr12: 52401215 chr12: 52401223 chr12: 52401240 chr12:
52401253 chr12: 52401288 chr12: 52401295 chr12: 52401299 chr12:
52401304 chr12: 52401334 chr12: 52401340 chr12: 52401344 chr12:
52401359 (CAHM) chr6: 163834330 chr6: 163834332 chr6: 163834357
chr6: 163834373 chr6: 163834384 chr6: 163834390 chr6: 163834392
chr6: 163834406 chr6: 163834412 chr6: 163834419 chr6: 163834443
chr6: 163834448 chr6: 163834452 chr6: 163834464 chr6: 163834483
chr6: 163834653 chr6: 163834660 chr6: 163834672 chr6: 163834675
chr6: 163834678 chr6: 163834681 chr6: 163834815 chr6: 163834824
chr6: 163834835 chr6: 163834840 chr6: 163834853 chr6: 163834855
chr6: 163834858 chr6: 163834863 chr6: 163834869 chr6: 163834872
(IRF4) chr6: 392036 chr6: 392047 chr6: 392049 chr6: 392057 chr6:
392060 chr6: 392066 chr6: 392080 chr6: 392094 chr6: 392102 chr6:
392131
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
6. The method of claim 2, wherein any one of said DNA regions
exhibit a higher level of methylation relative to a control
sample.
7. The method of claim 2, wherein any two or more of said DNA
regions exhibits a higher level of methylation relative to a
control sample.
8. The method of claim 2, wherein said neoplasm is an adenoma or an
adenocarcinoma.
9. The method of claim 2, wherein said neoplasm is a colorectal
neoplasm.
10. The method of claim 2, wherein said level of methylation is
used to determine one or more probabilities of respective
classifications of said large intestine neoplasm of said individual
into one or more neoplastic categories selected from adenoma, stage
I, stage II, stage III, or stage IV categories.
11. The method of claim 10, wherein said level of methylation is
used to determine one or more probabilities of respective
classifications of said large intestine neoplasm of said individual
into one or more aggregates of fewer than five of said neoplastic
categories.
12. The method of claim 2, wherein said level of methylation is
used to determine a probability that said large intestine of said
individual would be classified as non-neoplastic, based on
comparison of said level of methylation relative to said
corresponding measured levels of methylation and to corresponding
measured levels of methylation from a population of individuals
whose large intestines were classified as non-neoplastic.
13. The method of claim 11, wherein said aggregates include one or
more of: (i) one or more aggregates of fewer than five of said
neoplastic categories and an aggregate of the non-neoplastic
category with at least the adenoma category; (ii) a pre-malignant
neoplasm category consisting of an aggregate of the non-neoplastic
and adenoma categories; (iii) an early stage malignant neoplasm
category consisting of an aggregate of the stage I and stage II
categories; (iv) a late stage malignant neoplasm category
consisting of an aggregate of the stage III and stage IV
categories; or (v) the pre-malignant neoplasm category, the early
stage malignant neoplasm category, and the late stage malignant
neoplasm category.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/401,157, filed Nov. 14, 2014, which is a U.S. National
Phase Application of PCT International Application Number
PCT/AU2013/000519, filed on May 17, 2013, designating the United
States of America and published in the English language, which is
an International Application of and claims the benefit of priority
to U.S. Provisional Application No. 61/648,821, filed May 18, 2012,
the disclosures of which are hereby expressly incorporated by
reference in their entireties.
FIELD
[0002] The present invention relates generally to a method of
classifying a large intestine neoplasm and, in particular,
classifying a colorectal neoplasm in a mammal. The present
invention more specifically provides a method for assessing the
probability that a large intestine neoplasm is a premalignant
neoplasm, an early stage malignant neoplasm or a late stage
malignant neoplasm. The method of the present invention is based on
screening for modulation in the DNA methylation levels of one or
more gene markers in blood samples from said mammal.
BACKGROUND
[0003] Colorectal cancer includes cancerous growths in the colon,
rectum and appendix. With 655,000 deaths worldwide per year, it is
the fourth most common form of cancer in the United States, and the
third leading cause of cancer-related deaths in the Western world.
Colorectal cancers arise from adenomatous polyps in the colon.
These mushroom-shaped growths are usually benign, but some develop
into cancer over time. Localized colon cancer is usually diagnosed
through colonoscopy.
[0004] Invasive cancers that are confined within the wall of the
colon (Stage I and II) are curable with surgery. If untreated, they
spread to regional lymph nodes (stage III), where up to 73% are
curable by surgery and chemotherapy. Cancer that metastasizes to
distant sites (stage IV) is usually not curable, although
chemotherapy can extend survival, and in rare cases, surgery and
chemotherapy together have seen patients through to a cure
(Markowitz and Bertagnolli, 2009, N. Engl. J. Med. 361(25):
2449-60). Radiation is used with rectal cancer.
[0005] Colorectal cancer is preceded by adenomas. Adenomas are
benign tumours, or neoplasms, of epithelial origin which are
derived from glandular tissue or exhibit clearly defined glandular
structures. Some adenomas show recognisable tissue elements, such
as fibrous tissue (fibroadenomas) and epithelial structure, while
others, such as bronchial adenomas, produce active compounds that
might give rise to clinical syndromes.
[0006] Adenomas may progress to become an invasive neoplasm, and
are then termed adenocarcinomas. Accordingly, adenocarcinomas are
defined as malignant epithelial tumours arising from glandular
structures, which are constituent parts of many organs of the body.
The term adenocarcinoma is also applied to tumours showing a
glandular growth pattern. These tumours may be sub-classified
according to the substances that they produce, for example mucus
secreting and serous adenocarcinomas, or to the microscopic
arrangement of their cells into patterns, for example papillary and
follicular adenocarcinomas. These carcinomas may be solid or cystic
(cystadenocarcinomas). Each organ may produce tumours showing a
variety of histological types, for example the ovary may produce
both mucinous and cystadenocarcinoma.
[0007] Adenomas in different organs behave differently. In general,
the overall chance of carcinoma being present within an adenoma
(i.e., a focus of cancer having developed within a benign lesion)
is approximately 5%. However, this is related to size of an
adenoma. For instance, in the large bowel (colon and rectum
specifically), occurrence of a cancer within an adenoma is rare in
adenomas of less than 1 centimetre. Such a development is estimated
at 40 to 50% in adenomas which are greater than 4 centimetres and
show certain histopathological change such as villous change, or
high grade dysplasia. Adenomas with higher degrees of dysplasia
have a higher incidence of carcinoma. In any given colorectal
adenoma, the predictors of the presence of cancer now or the future
occurrence of cancer in the organ include size (especially greater
than 9 mm), degree of change from tubular to villous morphology,
presence of high grade dysplasia and the morphological change
described as "serrated adenoma". In any given individual, the
additional features of increasing age, familial occurrence of
colorectal adenoma or cancer, male gender or multiplicity of
adenomas, predict a future increased risk for cancer in the
organ--so-called risk factors for cancer. Except for the presence
of adenomas and its size, none of these is objectively defined, and
all those other than number and size are subject to observer error
and to confusion as to precise definition of the feature in
question. Because such factors can be difficult to assess and
define, their value as predictors of current or future risk for
cancer is imprecise.
[0008] Once a sporadic adenoma has developed, the chance of a new
adenoma occurring is approximately 30% within 26 months.
[0009] The symptoms of colorectal cancer depend on the location of
tumour in the bowel, and whether it has metastasised.
Unfortunately, many of the symptoms may occur in other diseases as
well, and hence symptoms may not be conclusively diagnostic of
colorectal cancer.
[0010] Local symptoms are more likely if the tumour is located
closer to the anus. There may be a change in bowel habit (new-onset
constipation or diarrhea in the absence of another cause), a
feeling of incomplete defecation, and reduction in diameter of
stools. Tenesmus and change in stool shape are both characteristic
of rectal cancer. Lower gastrointestinal bleeding, including the
passage of bright red blood in the stool, may indicate colorectal
cancer, as may the increased presence of mucus. Melena, black stool
with a tarry appearance, normally occurs in upper gastrointestinal
bleeding (such as from a duodenal ulcer), but is sometimes
encountered in colorectal cancer when the disease is located in the
beginning of the large bowel.
[0011] A tumour that is large enough to fill the entire lumen of
the bowel may cause bowel obstruction. This situation is
characterized by constipation, abdominal pain, abdominal distension
and vomiting. This occasionally leads to the obstructed and
distended bowel perforating and causing peritonitis.
[0012] Certain local effects of colorectal cancer occur when the
disease has become more advanced. A large tumour is more likely to
be noticed on feeling the abdomen, and it may be noticed by a
doctor on physical examination. The disease may invade other
organs, and may cause blood or air in the urine or vaginal
discharge.
[0013] If a tumour has caused chronic occult bleeding, iron
deficiency anaemia may occur. This may be experienced as fatigue,
palpitations and noticed as pallor. Colorectal cancer may also lead
to weight loss, generally due to a decreased appetite.
[0014] More unusual constitutional symptoms are an unexplained
fever and one of several paraneoplastic syndromes. The most common
paraneoplastic syndrome is thrombosis, usually deep vein
thrombosis.
[0015] Colorectal cancer most commonly spreads to the liver. This
may go unnoticed, but large deposits in the liver may cause
jaundice and abdominal pain (due to stretching of the capsule). If
the tumour deposit obstructs the bile duct, the jaundice may be
accompanied by other features of biliary obstruction, such as pale
stools.
[0016] Colorectal cancer can take many years to develop and early
detection of colorectal cancer greatly improves the prognosis. Even
modest efforts to implement colorectal cancer screening methods can
result in a drop in cancer deaths. Despite this, colorectal cancer
screening rates remain low.
[0017] In addition to screening for the onset of a colorectal
neoplasm, determining the stage or grade of a neoplasm is also
extremely valuable since this provides a patient with the
possibility of better tailored treatment regimen and potentially a
significantly better prognosis. Currently, staging of large
intestine neoplasms is an invasive procedure since it requires the
harvesting of a tissue specimen which is histologically
analysed.
[0018] The most commonly used staging system for colorectal cancer
is that of the American Joint Committee on Cancer (AJCC), sometimes
also known as the TNM system. The TNM system describes 3 key pieces
of information:
[0019] T describes how far the main (primary) tumour has grown
through the layers of the intestine and whether it has grown into
nearby areas. These layers, from the inner to the outer,
include:
[0020] The inner lining (mucosa)
[0021] A thin muscle layer (muscularis mucosa)
[0022] The fibrous tissue beneath this muscle layer (submucosa)
[0023] A thick muscle layer (muscularis propria) that contracts to
force the contents of the intestines along
[0024] The thin, outermost layers of connective tissue (subserosa
and serosa) that cover most of the colon but not the rectum
[0025] N describes the extent of spread to nearby (regional) lymph
nodes, and, if so, how many lymph nodes are involved.
[0026] Nx: No description of lymph node involvement is possible
because of incomplete information.
[0027] N0: No cancer in nearby lymph nodes.
[0028] N1a: Cancer cells are found in 1 nearby lymph node.
[0029] N1b: Cancer cells are found in 2 to 3 nearby lymph
nodes.
[0030] NU: Small deposits of cancer cells are found in areas of fat
near lymph nodes, but not in the lymph nodes themselves.
[0031] N2a: Cancer cells are found in 4 to 6 nearby lymph
nodes.
[0032] N2b: Cancer cells are found in 7 or more nearby lymph
nodes.
[0033] M indicates whether the cancer has metastasized.
[0034] M0: No distant spread is seen.
[0035] M1a: The cancer has spread to 1 distant organ or set of
distant lymph nodes.
[0036] M1b: The cancer has spread to more than 1 distant organ or
set of distant lymph nodes, or it has spread to distant parts of
the peritoneum (the lining of the abdominal cavity).
[0037] Numbers or letters appear after T, N, and M to provide more
details about each of these factors. The numbers 0 through 4
indicate increasing severity. The letter X means "cannot be
assessed because the information is not available."
T Categories for Colorectal Cancer
[0038] T categories of colorectal cancer describe the extent of
spread through the layers that form the wall of the colon and
rectum.
Stage Grouping
[0039] Once a person's T, N, and M categories have been determined,
usually after surgery, this information is combined in a process
called stage grouping. The stage is expressed in Roman numerals
from stage I (the least advanced) to stage IV (the most advanced).
Some stages are subdivided with letters.
Stage 0
[0040] Tis, N0, M0: The cancer is in the earliest stage. It has not
grown beyond the inner layer (mucosa) of the colon or rectum. This
stage is also known as carcinoma in situ or intramucosal
carcinoma.
Stage I
[0041] T1-T2, N0, M0: The cancer has grown through the muscularis
mucosa into the submucosa (T1) or it may also have grown into the
muscularis propria (T2). It has not spread to nearby lymph nodes or
distant sites.
Stage IIA
[0042] T3, N0, M0: The cancer has grown into the outermost layers
of the colon or rectum but has not gone through them. It has not
reached nearby organs. It has not yet spread to the nearby lymph
nodes or distant sites.
Stage IIB
[0043] T4a, N0, M0: The cancer has grown through the wall of the
colon or rectum but has not grown into other nearby tissues or
organs. It has not yet spread to the nearby lymph nodes or distant
sites.
Stage IIC
[0044] T4b, N0, M0: The cancer has grown through the wall of the
colon or rectum and is attached to or has grown into other nearby
tissues or organs. It has not yet spread to the nearby lymph nodes
or distant sites.
Stage IIIA
[0045] One of the following applies.
[0046] T1-T2, N1, M0: The cancer has grown through the mucosa into
the submucosa (T1) or it may also have grown into the muscularis
propria (T2). It has spread to 1 to 3 nearby lymph nodes (N1a/N1b)
or into areas of fat near the lymph nodes but not the nodes
themselves (N1c). It has not spread to distant sites.
[0047] T1, N2a, M0: The cancer has grown through the mucosa into
the submucosa.
[0048] It has spread to 4 to 6 nearby lymph nodes. It has not
spread to distant sites.
Stage IIIB
[0049] One of the following applies.
[0050] T3-T4a, N1, M0: The cancer has grown into the outermost
layers of the colon or rectum (T3) or through the visceral
peritoneum (T4a) but has not reached nearby organs. It has spread
to 1 to 3 nearby lymph nodes (N1a/N1b) or into areas of fat near
the lymph nodes but not the nodes themselves (N1c). It has not
spread to distant sites.
[0051] T2-T3, N2a, M0: The cancer has grown into the muscularis
propria (T2) or into the outermost layers of the colon or rectum
(T3). It has spread to 4 to 6 nearby lymph nodes. It has not spread
to distant sites.
[0052] T1-T2, N2b, M0: The cancer has grown through the mucosa into
the submucosa (T1) or it may also have grown into the muscularis
propria (T2). It has spread to 7 or more nearby lymph nodes. It has
not spread to distant sites.
Stage IIIC
[0053] One of the following applies.
[0054] T4a, N2a, M0: The cancer has grown through the wall of the
colon or rectum (including the visceral peritoneum) but has not
reached nearby organs. It has spread to 4 to 6 nearby lymph nodes.
It has not spread to distant sites.
[0055] T3-T4a, N2b, M0: The cancer has grown into the outermost
layers of the colon or rectum (T3) or through the visceral
peritoneum (T4a) but has not reached nearby organs. It has spread
to 7 or more nearby lymph nodes. It has not spread to distant
sites.
[0056] T4b, N1-N2, M0: The cancer has grown through the wall of the
colon or rectum and is attached to or has grown into other nearby
tissues or organs. It has spread to 1 or more nearby lymph nodes or
into areas of fat near the lymph nodes. It has not spread to
distant sites.
Stage IVA
[0057] Any T, Any N, M1a: The cancer may or may not have grown
through the wall of the colon or rectum, and it may or may not have
spread to nearby lymph nodes. It has spread to 1 distant organ
(such as the liver or lung) or set of lymph nodes.
Stage IVB
[0058] Any T, Any N, M1b: The cancer may or may not have grown
through the wall of the colon or rectum, and it may or may not have
spread to nearby lymph nodes. It has spread to more than 1 distant
organ (such as the liver or lung) or set of lymph nodes, or it has
spread to distant parts of the peritoneum (the lining of the
abdominal cavity).
[0059] Another factor that can affect the outlook for survival is
the grade of the cancer. Grade is a description of how closely the
cancer resembles normal colorectal tissue when looked at under a
microscope.
[0060] The scale used for grading colorectal cancers goes from G1
(where the cancer looks much like normal colorectal tissue) to G4
(where the cancer looks very abnormal). The grades G2 and G3 fall
somewhere in between. The grade is often simplified as either
"low-grade" (G1 or G2) or "high-grade" (G3 or G4). Low-grade
cancers tend to grow and spread more slowly than high-grade
cancers.
[0061] In the context of large intestine neoplasms, the
histological analysis of tissue specimens is both relatively slow
and highly invasive. Due to its invasiveness, it is also not a
procedure which one would want to perform repeatedly. The
development of a means to reliably and routinely assess a patient
to determine whether an identified neoplasm is premalignant (e.g.,
adenoma), early stage or late stage (e.g., metastatic) is highly
desirable if it can be performed quickly and repeatedly, since this
would enable decisions in relation to treatment regimes to be made
and implemented more accurately. It would also enable ongoing
monitoring to be performed during a treatment regime, such as in
the context of treating an adenoma or early stage cancer, to assess
transition to a more advanced stage without the need to perform
invasive biopsies. This would also enable more flexibility in terms
of adapting treatment regimes to reflect changes to the stage of a
neoplasm.
[0062] In work leading up to the present invention, it has been
determined that a panel of gene markers which are known to be
diagnostic of large intestine neoplasms can, in fact, also provide
valuable information in relation to the classification of a
neoplasm. Specifically, whereas the level of increase in the
methylation of the DNA of these gene markers is similar in most
biological samples, irrespective of how advanced the neoplasm is,
when assessed in a blood-derived sample, such as plasma, there is
found an increase in the level of methylation as the stage of the
neoplasm becomes more advanced.
[0063] This finding has therefore now provided a means to assess
the probability that a given neoplasm is premalignant, early stage
malignant, or late stage malignant. This information in relation to
the classification of the neoplasm can then inform the development
of the therapeutic treatment and ongoing monitoring which is
appropriate for the patient. Importantly, particularly in the
context of premalignant or early stage malignant neoplasms, it
provides a means for non-invasive ongoing monitoring. The method of
the present invention can be performed either after initial
diagnosis, or may itself form part of the screening of patients
presenting for initial diagnosis but where, in addition to the
diagnostic result, there is also provided classification
information.
SUMMARY
[0064] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0065] As used herein, the term "derived from" shall be taken to
indicate that a particular integer or group of integers has
originated from the species specified, but has not necessarily been
obtained directly from the specified source. Further, as used
herein the singular forms of "a", "and" and "the" include plural
referents unless the context clearly dictates otherwise.
[0066] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0067] The subject specification contains nucleotide sequence
information prepared using the programme PatentIn Version 3.5,
presented herein after the bibliography. Each nucleotide sequence
is identified in the sequence listing by the numeric indicator
<210> followed by the sequence identifier (eg. <210>1,
<210>2, etc). The length, type of sequence (DNA, etc) and
source organism for each sequence is indicated by information
provided in the numeric indicator fields <211>, <212>
and <213>, respectively. Nucleotide sequences referred to in
the specification are identified by the indicator SEQ ID NO:
followed by the sequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2,
etc.). The sequence identifier referred to in the specification
correlates to the information provided in numeric indicator field
<400> in the sequence listing, which is followed by the
sequence identifier (eg. <400>1, <400>2, etc). That is
SEQ ID NO:1 as detailed in the specification correlates to the
sequence indicated as <400>1 in the sequence listing.
[0068] A method of determining one or more probabilities of
respective classifications of a large intestine neoplasm in an
individual, said method comprising assessing the methylation status
of a DNA region selected from: [0069] (i) the region, including 2
kb upstream of the transcription start site, defined by at least
one of Hg19 coordinates: [0070] (1) chr12:24962958..25102393 [0071]
(2) chr7:50344378 . . . 50472798 [0072] (3) chr6:391739..411443;
[0073] (4) chr12:52400748..52409671; and [0074] (5)
chr6:163834097..163834982; or [0075] (ii) the gene region,
including 2 kb upstream of at least one of:
TABLE-US-00001 [0075] (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and
(5) CAHM
[0076] in a blood-derived sample from said individual, wherein a
level of methylation of at least one of the DNA regions of group
(i) and/or (ii) relative to corresponding measured levels of
methylation from a population of individuals with known neoplastic
categories of corresponding large intestine neoplasms is used to
determine one or more probabilities of respective classifications
of said large intestine neoplasm of said individual into one or
more neoplastic categories selected from adenoma, stage I, stage
II, stage III, and stage IV categories or into one or more
aggregates of fewer than five of said neoplastic categories.
[0077] In one embodiment, the method is directed to determining one
or more probabilities of respective classifications of said large
intestine neoplasms of said individual into one or more aggregates
of fewer than five of said neoplastic categories.
[0078] The method may further include using said level of
methylation for said individual to determine a probability that
said large intestine of said individual would be classified as
non-neoplastic, based on comparison of said level of methylation
relative to said corresponding measured levels of methylation and
to further corresponding measured levels of methylation from a
further population of individuals whose large intestines were
classified as non-neoplastic.
[0079] The subregions which have been determined to exhibit
particular utility are listed below with reference to the gene and
chromosomal region within which they are found: [0080] (1) BCAT1
subregions chr12:25101992-25102093 (SEQ ID NO:1 or the
corresponding minus strand) and chr12:25101909-25101995 (SEQ ID
NO:2 or the corresponding minus strand) [0081] (2) IKZF1
subregions: chr7:50343867-50343961 (SEQ ID NO:3 or the
corresponding minus strand) and chr7:50343804-5033895 (SEQ ID NO:4
or the corresponding minus strand) [0082] (3) IRF4 subregions
chr6:392036-392145 (SEQ ID NO:5 or the corresponding minus strand)
[0083] (4) GRASP subregions: chr12:52399672-52399922,
chr12:52400821-52401051 (SEQ ID NO:6 or the corresponding minus
strand), chr12:52401407-52401664 (SEQ ID NO:7 or the corresponding
minus strand) chr12:52400866-52400973 and Chr12:52401107-52401664.
[0084] (5) CAHM subregions: chr6:163834295-163834500 (SEQ ID NO:8
or the corresponding minus strand), chr6:163834621-163834906
chr6:163834393-163834455 and chr6: 163834393-163834519.
[0085] Without limiting the present invention to any one theory or
mode of action, the skilled person may screen one or more
subregions for each gene marker.
[0086] To the extent that the method of the present invention
includes analysing the methylation of BCAT1, the subject
residues:
TABLE-US-00002 chr12: 25101998 chr12: 25102003 chr12: 25102006
chr12: 25102009 chr12: 25102017 chr12: 25102022 chr12: 25102039
chr12: 25102048 chr12: 25102050 chr12: 25102053 chr12: 25102061
chr12: 25102063 chr12: 25102071 chrl12: 25101921 chr12: 25101934
chr12: 25101943 chr12: 25101951 chr12: 25101962 chr12: 25101964
chr12: 25101970
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0087] To the extent that the method of the present invention
includes analysing the methylation of GRASP, the subject residues
are:
TABLE-US-00003 chr12: 52399713 chr12: 52399731 chr12: 52399749
chr12: 52399783 chr12: 52399796 chr12: 52399808 chr12: 52399823
chr12: 52399835 chr12: 52399891 chr12: 52400847 chr12: 52400850
chr12: 52400859 chr12: 52400866 chr12: 52400869 chr12: 52400873
chr12: 52400881 chr12: 52400886 chr12: 52400893 chr12: 52400895
chr12: 52400899 chr12: 52400902 chr12: 52400907 chr12: 52400913
chr12: 52400919 chr12: 52400932 chr12: 52400938 chr12: 52400958
chr12: 52400962 chr12: 52400971 chr12: 52400973 chr12: 52400976
chr12: 52400998 chr12: 52401008 chr12: 52401010 chr12: 52401012
chr12: 52401016 chr12: 52401019 chr12: 52401025 chr12: 52401041
chr12: 52401044 chr12: 52401053 chr12: 52401060 chr12: 52401064
chr12: 52401092 chr12: 52401118 chr12: 52401438 chr12: 52401448
chr12: 52401460 chr12: 52401465 chr12: 52401474 chr12: 52401477
chr12: 52401479 chr12: 52401483 chr12: 52401504 chr12: 52401514
chr12: 52401523 chr12: 52401540 chr12: 52401553 chr12: 52401576
chr12: 52401588 chr12: 52401595 chr12: 52401599 chr12: 52401604
chr12: 52401606 chr12: 52401634 chr12: 52401640 chr12: 52401644
chr12: 52401659 chr12: 52401160 chr12: 52401165 chr12: 52401174
chr12: 52401177 chr12: 52401179 chr12: 52401183 chr12: 52401204
chr12: 52401215 chr12: 52401223 chr12: 52401240 chr12: 52401253
chr12: 52401288 chr12: 52401295 chr12: 52401299 chr12: 52401304
chr12: 52401334 chr12: 52401340 chr12: 52401344 chr12: 52401359
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0088] To the extent that the method of the present invention
includes analysing the methylation of CAHM, the subject residues
are:
TABLE-US-00004 chr6: 163834330 chr6: 163834332 chr6: 163834357
chr6: 163834373 chr6: 163834384 chr6: 163834390 chr6: 163834392
chr6: 163834406 chr6: 163834412 chr6: 163834419 chr6: 163834443
chr6: 163834448 chr6: 163834452 chr6: 163834464 chr6: 163834483
chr6: 163834653 chr6: 163834660 chr6: 163834672 chr6: 163834675
chr6: 163834678 chr6: 163834681 chr6: 163834815 chr6: 163834824
chr6: 163834835 chr6: 163834840 chr6: 163834853 chr6: 163834855
chr6: 163834858 chr6: 163834863 chr6: 163834869 chr6: 163834872
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0089] To the extent that the method of the present invention
includes analysing the methylation of IKZF1, the subject residues
are:
TABLE-US-00005 chr7: 50343869 chr7: 50343872 chr7: 50343883 chr7:
50343889 chr7: 50343890 chr7: 50343897 chr7: 50343907 chr7:
50343909 chr7: 50343914 chr7: 50343934 chr7: 50343939 chr7:
50343950 chr7: 50343959 chr7: 50343805 chr7: 50343822 chr7:
50343824 chr7: 50343826 chr7: 50343829 chr7: 50343831 chr7:
50343833 chr7: 50343838 chr7: 50343847 chr7: 50343850 chr7:
50343858 chr7: 50343864 chr7: 50343869 chr7: 50343872 chr7:
50343890
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0090] To the extent that the method of the present invention
includes analysing the methylation of IRF4, the subject residues
are:
TABLE-US-00006 chr6: 392036 chr6: 392047 chr6: 392049 chr6: 392057
chr6: 392060 chr6: 392066 chr6: 392080 chr6: 392094 chr6: 392102
chr6: 392131
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0091] FIG. 1. Measurement of CAHM methylation levels in tissue and
blood plasma specimens. A: The levels of methylated CAHM in
colorectal tissue specimens including normals (n=26, solid
circles), adenomas (n=17, solid squares), Stage 1 (n=20, open
squares), Stage II (n=21, open circles), Stage III (n=30, open
diamonds) and Stage IV (n=8, crosses) were measured using the
methylation specific CAHM assay described in M&M on 15 ng of
bisulfite converted DNA extracted from 122 tissue specimens (Left
figure). The signal output was converted to a mass/well using a
calibration curve. Data is given as the % methylated CAHM measured
in 15 ng of total bisulfite converted tissue DNA per reaction. Data
are mean values of triplicate. B: The levels of methylated CAHM was
measured in the equivalent of triplicate analysis of 0.5 mL plasma
from colonoscopy-confirmed patients including normals (n=74, solid
circles), adenomas (n=73, solid squares), Stage 1 (n=12, open
squares), Stage II (n=21, open circles), Stage III (n=23, open
diamonds) and Stage IV (n=12, black crosses). The signal output was
converted to a mass using a calibration curve. Data is given as pg
methylated CAHM per mL plasma. Data are mean values of triplicate.
Increased masses of methylated CAHM was observed in blood plasma
from patients as a function of disease progression (i.e. pg/mL CAHM
was calculated to be higher in Stage III-IV compared Stage I-II).
In contrast, high and similar levels of pg methylated CAHM was
measured in colorectal tissue from the earliest onset of disease
(precancerous) to late stage cancer. C: The levels of methylated
CAHM was measured and plotted in a scatter graph of pg methylated
CAHM/ml plasma (log 10) vs. wt DNA ng/mL plasma from
colonoscopy-confirmed patients including normal (n=74, solid
circles), adenomas (n=73, solid squares), Stage I (n=12, open
squares), Stage II (n=21, open circles), Stage III (n=23, open
diamonds) and Stage IV (n=12, black crosses).
[0092] FIG. 2. Increased levels of methylated colorectal cancer
biomarkers in blood plasma as a function of disease progression.
Measurement of CAHM methylation levels in tissue and blood plasma
specimens. The levels of methylated IRF4 (A), GRASP (B), BCAT1 (C)
and IKZF1 (D) were measured in the equivalent of triplicate
analysis of 0.5 mL plasma from colonoscopy-confirmed patients
including normals (solid circles), adenomas (solid squares), Stage
1 (open squares), Stage II (open circles), Stage III (open
diamonds) and Stage IV (crosses). The signal output was converted
to a mass/well using a calibration curve. Data is given as pg
methylated biomarker per mL plasma. Data are mean values of
triplicate. As exemplified in FIG. 1 for CAHM, FIG. 2 demonstrates
another four biomarkers, namely, GRASP, IRF4, BCAT1 and IKZF1,
where measurement of blood plasma methylation levels may be
indicative of colorectal cancer progression.
[0093] FIGS. 3A and 3B. Density distributions of methylated CAHM
grouped by phenotype classification estimated from assay determined
methylation levels. FIG. 3A) Empirical probability density curves
for CAHM based on methylation levels previously determined by
methylated CAHM assays (cf FIG. 1B). Density is estimated using
only positive assay values for non-neoplastic plasma specimens
(black); premalignant adenomas (blue); early stage cancers
including plasma from patients diagnosed with Stage 1 or Stage 2
(red); and late stage cancers including plasma from patients
diagnosed with Stage 3 or Stage 4 (purple). FIG. 3B) Estimated
normal (Gaussian) distribution using mean and standard deviation
estimates determined from positive methylation levels measured in
plasma drawn from patients with premalignant neoplastic lesions
(blue); early stage cancers (red) and late stage cancers
(purple).
DETAILED DESCRIPTION
[0094] The present invention is predicated, in part, on the
determination that several genes which are known to exhibit
increased levels of methylation in individuals exhibiting large
intestine neoplasms are also an indicator of the classification of
the neoplasm. Specifically, it has been surprisingly determined
that although the relative increase in DNA methylation levels of a
given gene marker is relatively consistent in a tissue biopsy
irrespective of the stage or grade of the neoplasm in issue, the
same is not true if a blood-derived sample, such as plasma, is
analysed. In particular, whereas a blood-derived sample will also
show increased levels of DNA methylation in the context of the
presence of a large intestine malignancy, these increased
methylation levels become progressively more increased as the
malignancy becomes more advanced. This is not observed in tissue
specimens. Accordingly, by assessing DNA methylation levels in
accordance with the method of the present invention, one can
determine not only whether or not malignant transformation has
occurred, but further the likely probability that the malignancy
would be classified as early stage or late stage.
[0095] Accordingly, one aspect of the present invention is directed
to a method of determining one or more probabilities of respective
classifications of a large intestine neoplasm in an individual,
said method comprising assessing the methylation status of a DNA
region selected from: [0096] (i) the region, including 2 kb
upstream of the transcription start site, defined by at least one
of Hg19 coordinates: [0097] (1) chr12:24962958..25102393 [0098] (2)
chr7:50344378 . . . 50472798 [0099] (3) chr6:391739..411443; [0100]
(4) chr12:52400748..52409671; and [0101] (5)
chr6:163834097..163834982; or [0102] (ii) the gene region,
including 2 kb upstream of at least one of:
TABLE-US-00007 [0102] (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and
(5) CAHM
[0103] in a blood-derived sample from said individual, wherein a
level of methylation of at least one of the DNA regions of group
(i) and/or (ii) relative to corresponding measured levels of
methylation from a population of individuals with known neoplastic
categories of corresponding large intestine neoplasms is used to
determine one or more probabilities of respective classifications
of said large intestine neoplasm of said individual into one or
more neoplastic categories selected from adenoma, stage I, stage
II, stage III, and stage IV categories or into one or more
aggregates of fewer than five of said neoplastic categories.
[0104] In one embodiment, the method is directed to determining one
or more probabilities of respective classifications of said large
intestine neoplasms of said individual into one or more aggregates
of fewer than five of said neoplastic categories.
[0105] The method may further include using said level of
methylation for said individual to determine a probability that
said large intestine of said individual would be classified as
non-neoplastic, based on comparison of said level of methylation
relative to said corresponding measured levels of methylation and
to further corresponding measured levels of methylation from a
further population of individuals whose large intestines were
classified as non-neoplastic.
[0106] The aggregate categories may include one or more aggregates
of fewer than five of said neoplastic categories and an aggregate
of the non-neoplastic category with at least the adenoma
category.
[0107] The aggregate categories may include a pre-malignant
neoplasm category consisting of an aggregate of the non-neoplastic
and adenoma categories.
[0108] The aggregate categories may include an early stage
malignant neoplasm category consisting of an aggregate of the stage
I and stage II categories.
[0109] The aggregate categories may include a late stage malignant
neoplasm category consisting of an aggregate of the stage III and
stage IV categories.
[0110] The aggregate categories may include the pre-malignant
neoplasm category, early stage malignant neoplasm category, and
late stage malignant neoplasm categories.
[0111] Given measured levels of methylation in a population of
individuals with known classifications of large intestine neoplasms
into neoplastic categories selected from adenoma, stage I, stage
II, stage III, and stage IV categories or aggregates of fewer than
five of said neoplastic categories, and further measured levels of
methylation in a further population of individuals whose large
intestines were classified as non-neoplastic, the prior statistical
relationships between the measured methylation levels and the
corresponding known categories for the populations of individuals
can be used to determine one or more probabilities that a further
measured methylation level of an individual corresponds to
respective ones of the same categories of the large intestine
neoplasm or non-neoplastic large intestine of said individual. As
will be apparent to those skilled in the art, the determination can
be made using any one of a variety of standard statistical methods,
including Bayesian statistics and machine learning.
[0112] In some embodiments, the statistical method includes
generating histograms wherein the measured levels of methylation
corresponding to known categories are allocated into bins of
respective ranges of methylation levels and using these histograms
to estimate at least one probability that a further measured
methylation level corresponds to at least one of the known
categories. As will be apparent to those skilled in the art, there
are many methods to estimate classification probabilities. For
example, by adding the total sizes of the bins corresponding to
methylation levels of equal or lesser levels to the methylation
level in question and dividing this sum by the total across all
bins, one can estimate the proportion of methylation levels at or
below a given level which are of a known category and thus the
probability that a measured methylation level is of said
category.
[0113] In some embodiments, the statistical distributions of said
measured levels of methylation are modelled by standard statistical
distributions such as a Gaussian distribution, for example, and a
standard fitting or regression procedure such as maximum likelihood
is used to determine the parameters of the distributions, which can
then be applied to a further measured level of methylation from an
individual to determine one or more probabilities that the large
intestine of that individual would be classified as respective ones
of the neoplastic categories and the non-neoplastic category.
[0114] In some embodiments, the method may include using the
determined probabilities to automatically classify the large
intestine neoplasm of the individual into one of the prior known
classifications, whether neoplastic or non-neoplastic. However,
given the overlap between measured levels of methylation for the
different categories, the probabilities are generally more useful
for the selection of possible further medical treatment
options.
[0115] In other embodiments, the statistical associations between
the measured methylation levels on the one hand and the
corresponding categories observed by colonoscopy or surgery on the
other are used as a training set for supervised learning. The
resulting weights are then applied to at least one further measured
methylation level for at least one further individual in order to
determine corresponding probabilities for membership of those
categories for that at least one individual. As will be apparent to
those skilled in the art, any one of a number of standard
supervised training methods can be used.
[0116] The categories used for supervised learning may include all
five neoplastic categories and the non-neoplastic category, or
aggregates of those six categories, such as the aggregate
categories described above. The selection of which categories to
use for supervised training may be based on diagnostic/treatment
requirements and/or the available number and/or classification
confidence of the observed categories (e.g., in order to improve
sensitivity and specificity of classification). In any case, the
weights generated by supervised learning can also be provided as
inputs to a standard classifier in order to classify the large
intestine of the individual into a neoplastic or non-neoplastic
class or category based on the corresponding measured methylation
level.
[0117] Reference to "neoplasm" should be understood as a reference
to a lesion, tumour or other encapsulated or unencapsulated mass or
other form of growth which comprises neoplastic cells. A
`neoplastic cell" should be understood as a reference to a cell
exhibiting abnormal growth. The term "growth" should be understood
in its broadest sense and includes reference to proliferation. In
this regard, an example of abnormal cell growth is the uncontrolled
proliferation of a cell. Another example is failed apoptosis in a
cell, thus prolonging its usual life span. The neoplastic cell may
be a benign cell or a malignant cell. In a preferred embodiment,
the subject neoplasm is an adenoma or an adenocarcinoma. Without
limiting the present invention to any one theory or mode of action,
an adenoma is generally a benign tumour of epithelial origin which
is either derived from epithelial tissue or exhibits clearly
defined epithelial structures. These structures may take on a
glandular appearance. It can comprise a malignant cell population
within the adenoma, such as occurs with the progression of a benign
adenoma or benign neoplastic legion to a malignant
adenocarcinoma.
[0118] Reference to "large intestine" should be understood as a
reference to a cell derived from one of the eight anatomical
regions of the large intestine, which regions commence after the
terminal region of the ileum, these being:
[0119] (i) the cecum;
[0120] (ii) the ascending colon;
[0121] (iii) the transverse colon;
[0122] (iv) the descending colon;
[0123] (v) the sigmoid colon;
[0124] (vi) the rectum;
[0125] (vii) the splenic flexure; and
[0126] (viii) the hepatic flexure.
[0127] Preferably, said neoplastic cell is an adenoma or
adenocarcinoma and even more preferably a colorectal adenoma or
adenocarcinoma.
[0128] Reference to "DNA region" should be understood as a
reference to a specific section of genomic DNA. These DNA regions
are specified either by reference to a gene name or a set of
chromosomal coordinates. Both the gene names and the chromosomal
coordinates would be well known to, and understood by, the person
of skill in the art. As detailed hereinbefore, the chromosomal
coordinates correspond to the Hg19 version of the genome. In
general, a gene can be routinely identified by reference to its
name, via which both its sequences and chromosomal location can be
routinely obtained, or by reference to its chromosomal coordinates,
via which both the gene name and its sequence can also be routinely
obtained.
[0129] Reference to each of the genes/DNA regions detailed above
should be understood as a reference to all forms of these molecules
and to fragments or variants thereof. As would be appreciated by
the person of skill in the art, some genes are known to exhibit
allelic variation between individuals or single nucleotide
polymorphisms. SNPs encompass insertions and deletions of varying
size and simple sequence repeats, such as dinucleotide and
trinucleotide repeats. Variants include nucleic acid sequences from
the same region sharing at least 90%, 95%, 98%, 99% sequence
identity i.e. having one or more deletions, additions,
substitutions, inverted sequences etc. relative to the DNA regions
described herein. Accordingly, the present invention should be
understood to extend to such variants which, in terms of the
present diagnostic applications, achieve the same outcome despite
the fact that minor genetic variations between the actual nucleic
acid sequences may exist between individuals. The present invention
should therefore be understood to extend to all forms of DNA which
arise from any other mutation, polymorphic or allelic
variation.
[0130] It should be understood that the "individual" who is the
subject of testing may be any human or non-human mammal. Examples
of non-human mammals includes primates, livestock animals (e.g.
horses, cattle, sheep, pigs, donkeys), laboratory test animals
(e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g.
dogs, cats) and captive wild animals (e.g. deer, foxes).
[0131] Preferably, said mammal is a human.
[0132] As detailed hereinbefore, the method of the present
invention enables one to assess the probability that a neoplasm is
a premalignant neoplasm, early stage malignancy or late stage
malignancy. Although the staging and grading systems which are
commonly used by pathologists in the context of large intestine
neoplasms (such as colorectal cancers) can purportedly stage such
cancers quite precisely, the fact is that they require a biopsy
specimen to be harvested, this being a procedure which is invasive.
Since the assessment required to be performed is the preparation
and histological analysis of a tissue section, it can also take
some time to obtain results. In terms of accurately assessing
histological specimens, the interpretation of the sections is
subjective and can be extremely difficult and unreliable,
particularly in the context of moderate grade cancers, as opposed
to very early stage cancers or metastatic cancers. Accordingly, the
identification of a molecular basis upon which to classify a
neoplasm is a significant advance due to the fact that such
analyses are not subjective. Still further, since the DNA
methylation levels of the genes at issue are assessed in a blood
sample, this is the first available non-invasive means of
classifying a large intestine neoplasm.
[0133] Without limiting the present invention to any one theory or
mode of action, the most commonly used staging system for
colorectal cancer is that of the American Joint Committee on Cancer
(AJCC), sometimes also known as the TNM system. The TNM system
describes 3 key pieces of information:
[0134] T describes how far the main (primary) tumour has grown into
the wall of the intestine and whether it has grown into nearby
areas.
[0135] N describes the extent of spread to nearby (regional) lymph
nodes.
[0136] M indicates whether the cancer has metastasized.
[0137] Numbers or letters appear after T, N, and M to provide more
details about each of these factors. The numbers 0 through 4
indicate increasing severity.
[0138] A detailed description in relation to how the TNM system is
applied has been detailed earlier.
[0139] Once a person's T, N, and M categories have been determined,
usually after surgery, this information is combined in a process
called stage grouping. The stage is expressed in Roman numerals
from stage I (the least advanced) to stage IV (the most advanced).
Nevertheless, despite the apparent theoretical precision with which
staging parameters are classified, the reality in terms of
assessing these parameters is far less precise. The present method
has enabled a simpler and more reliable staging system to be made
available. Specifically, a patient is assessed to determine the
probability that a neoplasm is a "premalignant neoplasm", "early
stage malignant neoplasm" or "late stage malignant neoplasm". Based
on these results, one may elect to also have a biopsy or some other
diagnostic procedure performed. Alternatively one may use these
results to inform what therapeutic or palliative care regime should
be designed and implemented. Of particular advantage is the fact
that this method enables ongoing testing to be performed. This may
be particularly relevant where a premalignant neoplasm has been
identified and a decision has been made not to surgically remove
the neoplasm in the first instance but to attempt to treat it.
[0140] In the context of the present invention, reference to
"premalignant neoplasm" should be understood as a reference to a
neoplasm which is not malignant. An example of a non-malignant
neoplasm is an adenoma. Without limiting the present invention in
any way, the histological and functional characteristics of a
premalignant large intestine neoplasm are evidence of new, abnormal
tissue growth without evidence of invasion.
[0141] Reference to an "early stage malignant neoplasm" is a
reference to a large intestine neoplasm which has become malignant
but which is unlikely to extend beyond the bowel wall.
[0142] Reference to a "late stage malignant neoplasm" should be
understood as a reference to a large intestine neoplasm which is
malignant and which has spread to lymph nodes or distant organs.
Reference to late stage malignant neoplasms includes, for example,
neoplasms which have become metastatic.
[0143] In terms of screening for the methylation of these gene
regions, it should be understood that the assays can be designed to
screen either the specific regions listed herein (which correspond
to the "plus" strand of the gene) or the complementary "minus"
strand. It is well within the skill of the person in the art to
choose which strand to analyse and to target that strand based on
the chromosomal coordinates provided herein. In some circumstances,
assays may be established to screen both strands.
[0144] Without limiting the present invention to any one theory or
mode of action, although measuring the methylation levels across
these DNA regions is diagnostic of the classification of a large
intestine neoplastic condition, it has been determined that
discrete subregions are particularly useful in this regard since
these subregions contain a high density of CpG dinucleotides which
are frequently hypermethylated in large intestine neoplasias, such
as colorectal cancers. This finding renders these subregions a
particularly useful target for analysis since it both simplifies
the screening process due to a shorter more clearly defined region
of DNA requiring analysis and, further, the fact that the results
from these regions will provide a significantly more definitive
result in relation to the presence, or not, of hypermethylation
than would be obtained if analysis was performed across the DNA
region as a whole. This finding therefore both simplifies the
screening process and increases the sensitivity of large intestine
neoplasia diagnosis.
[0145] The subregions which have been determined to exhibit
particular utility are listed below with reference to the gene and
chromosomal region within which they are found:
[0146] (1) BCAT1 subregions chr12:25101992-25102093 (SEQ ID NO:1 or
the corresponding minus strand) and chr12:25101909-25101995 (SEQ ID
NO:2 or the corresponding minus strand)
[0147] (2) IKZF1 subregions: chr7:50343867-50343961 (SEQ ID NO:3 or
the corresponding minus strand) and chr7:50343804-5033895 (SEQ ID
NO:4 or the corresponding minus strand)
[0148] (3) IRF4 subregions chr6:392036-392145 (SEQ ID NO:5 or the
corresponding minus strand)
[0149] (4) GRASP subregions: chr12:52399672-52399922,
chr12:52400821-52401051 (SEQ ID NO:6 or the corresponding minus
strand), chr12:52401407-52401664 (SEQ ID NO:7 or the corresponding
minus strand) chr12:52400866-52400973 and
Chr12:52401107-52401664.
[0150] (5) CAHM subregions: chr6:163834295-163834500 (SEQ ID NO:8),
chr6:163834621-163834906, chr6:163834393-163834455 and
chr6:163834393-163834519.
[0151] Without limiting the present invention to any one theory or
mode of action, the skilled person may screen one or more
subregions for each gene marker.
[0152] Without limiting the present invention to any one theory or
mode of action, DNA methylation is universal in bacteria, plants,
and animals. DNA methylation is a type of chemical modification of
DNA that is stable over rounds of cell division but does not
involve changes in the underlying DNA sequence of the organism.
Chromatin and DNA modifications are two important features of
epigenetics and play a role in the process of cellular
differentiation, allowing cells to stably maintain different
characteristics despite containing the same genomic material. In
eukaryotic organisms DNA methylation occurs only at the number 5
carbon of the cytosine pyrimidine ring. In mammals, DNA methylation
occurs mostly at the number 5 carbon of the cytosine of a CpG
dinucleotide. CpG dinucleotides comprise approximately 1% human
genome.
[0153] 70-80% of all CpGs are methylated. CpGs may be grouped in
clusters called "CpG islands" that are present in the 5' regulatory
regions of many genes and are frequently unmethylated. In many
disease processes such as cancer, gene promoters and/or CpG islands
acquire abnormal hypermethylation, which is associated with
heritable transcriptional silencing. DNA methylation may impact the
transcription of genes in two ways. First, the methylation of DNA
may itself physically impede the binding of transcriptional
proteins to the gene, thus blocking transcription. Second,
methylated DNA may be bound by proteins known as Methyl-CpG-binding
domain proteins (MBDs). MBD proteins then recruit additional
proteins to the locus, such as histone deacetylases and other
chromatin remodelling proteins that can modify histones, thereby
forming compact, inactive chromatin termed silent chromatin. This
link between DNA methylation and chromatin structure is very
important. In particular, loss of Methyl-CpG-binding Protein 2
(MeCP2) has been implicated in Rett syndrome and Methyl-CpG binding
domain protein 2 (MBD2) mediates the transcriptional silencing of
hypermethylated genes in cancer.
[0154] In humans, the process of DNA methylation is carried out by
three enzymes, DNA methyltransferase 1, 3a and 3b (DNMT1, DNMT3a,
DNMT3b). It is thought that DNMT3a and DNMT3b are the de novo
methyltransferases that set up DNA methylation patterns early in
development. DNMT1 is the proposed maintenance methyltransferase
that is responsible for copying DNA methylation patterns to the
daughter strands during DNA replication. DNMT3L is a protein that
is homologous to the other DNMT3s but has no catalytic activity.
Instead, DNMT3L assists the de novo methyltransferases by
increasing their ability to bind to DNA and stimulating their
activity. Finally, DNMT2 has been identified as an "enigmatic" DNA
methylstransferase homolog, containing all 10 sequence motifs
common to all DNA methyltransferases; however, DNMT2 may not
methylate DNA but instead has been shown to methylate a small
RNA.
[0155] "Methylation status" should therefore be understood as a
reference to the presence, absence and/or quantity of methylation
at a particular nucleotide, or nucleotides, within a DNA region.
The methylation status of a particular DNA sequence (e.g. DNA
region as described herein) can indicate the methylation state of
every base in the sequence or can indicate the methylation state of
a subset of the base pairs (e.g., of cytosines or the methylation
state of one or more specific restriction enzyme recognition
sequences) within the sequence, or can indicate information
regarding regional methylation density within the sequence without
providing precise information of where in the sequence the
methylation occurs. The methylation status can optionally be
represented or indicated by a "methylation value." A methylation
value can be generated, for example, by quantifying the amount of
intact DNA present following restriction digestion with a
methylation dependent restriction enzyme. In this example, if a
particular sequence in the DNA is quantified using quantitative
PCR, an amount of template DNA approximately equal to a mock
treated control indicates the sequence is not highly methylated
whereas an amount of template substantially less than occurs in the
mock treated sample indicates the presence of methylated DNA at the
sequence. Accordingly, a value, i.e., a methylation value, for
example from the above described example, represents the
methylation status and can thus be used as a quantitative indicator
of the methylation status. This is of particular use when it is
desirable to compare the methylation status of a sequence in a
sample to a threshold value.
[0156] The term "methylation" shall be taken to mean the presence
of a methyl group added by the action of a DNA methyl transferase
enzyme to a cytosine base or bases in a region of nucleic acid,
e.g. genomic DNA. As described herein, there are several methods
known to those skilled in the art for determining the level or
degree of methylation of nucleic acid.
[0157] By "higher level" is meant that there are a higher number of
methylated CpG dinucleotides in the subject diagnosed than in a
control sample, that is, either the proportion of DNA molecules
methylated at a particular CpG site is higher or there are a higher
number of separate CpG sites methylated in the subject. It should
be understood that the terms "enhanced" and "increased" are used
interchangeably with the term "higher".
[0158] The present invention is not to be limited by a precise
number of methylated residues that are considered to be diagnostic
of neoplasia in a subject, because some variation between patient
samples will occur. The present invention is also not limited by
positioning of the methylated residue.
[0159] Nevertheless, a number of specific cytosine residues which
undergo hypermethylation within these subregions have also been
identified. In another embodiment, therefore, a screening method
can be employed which is specifically directed to assessing the
methylation status of one or more of either these residues or the
corresponding cytosine at position n+1 on the opposite DNA
strand.
[0160] To this end, detailed in Table 2 are the cytosine residues
which have been identified in this regard. It should be appreciated
by the person of skill in the art that these individual residues
are numbered by reference to Hg19, which also corresponds to the
numbering of the specific subregions listed hereinbefore and which
can be further identified when the coordinate numbering for each
subregion is applied to the corresponding subregion sequences which
are provided in the sequence listing. It should be understood that
these residues have been identified in the context of the subregion
DNA. However, there are other residues which are hypermethylated
outside the subregions themselves but within the larger DNA region
from which the subregions derive. Accordingly, these specified
residues represent a particularly useful subset of individual
cytosine residues which undergo hypermethylation within the context
of the DNA regions and subregions herein disclosed. These
individual residues are grouped below according to the DNA region
within which they occur. These DNA regions are identified by
reference to both the Hg19 chromosomal coordinates and the gene
region name.
[0161] To the extent that the method of the present invention
includes analysing the methylation of BCAT1, the subject
residues:
TABLE-US-00008 chr12: 25101998 chr12: 25102003 chr12: 25102006
chr12: 25102009 chr12: 25102017 chr12: 25102022 chr12: 25102039
chr12: 25102048 chr12: 25102050 chr12: 25102053 chr12: 25102061
chr12: 25102063 chr12: 25102071 chrl12: 25101921 chr12: 25101934
chr12: 25101943 chr12: 25101951 chr12: 25101962 chr12: 25101964
chr12: 25101970
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0162] To the extent that the method of the present invention
includes analysing the methylation of GRASP, the subject residues
are:
TABLE-US-00009 chr12: 52399713 chr12: 52399731 chr12: 52399749
chr12: 52399783 chr12: 52399796 chr12: 52399808 chr12: 52399823
chr12: 52399835 chr12: 52399891 chr12: 52400847 chr12: 52400850
chr12: 52400859 chr12: 52400866 chr12: 52400869 chr12: 52400873
chr12: 52400881 chr12: 52400886 chr12: 52400893 chr12: 52400895
chr12: 52400899 chr12: 52400902 chr12: 52400907 chr12: 52400913
chr12: 52400919 chr12: 52400932 chr12: 52400938 chr12: 52400958
chr12: 52400962 chr12: 52400971 chr12: 52400973 chr12: 52400976
chr12: 52400998 chr12: 52401008 chr12: 52401010 chr12: 52401012
chr12: 52401016 chr12: 52401019 chr12: 52401025 chr12: 52401041
chr12: 52401044 chr12: 52401053 chr12: 52401060 chr12: 52401064
chr12: 52401092 chr12: 52401118 chr12: 52401438 chr12: 52401448
chr12: 52401460 chr12: 52401465 chr12: 52401474 chr12: 52401477
chr12: 52401479 chr12: 52401483 chr12: 52401504 chr12: 52401514
chr12: 52401523 chr12: 52401540 chr12: 52401553 chr12: 52401576
chr12: 52401588 chr12: 52401595 chr12: 52401599 chr12: 52401604
chr12: 52401606 chr12: 52401634 chr12: 52401640 chr12: 52401644
chr12: 52401659 chr12: 52401160 chr12: 52401165 chr12: 52401174
chr12: 52401177 chr12: 52401179 chr12: 52401183 chr12: 52401204
chr12: 52401215 chr12: 52401223 chr12: 52401240 chr12: 52401253
chr12: 52401288 chr12: 52401295 chr12: 52401299 chr12: 52401304
chr12: 52401334 chr12: 52401340 chr12: 52401344 chr12: 52401359
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0163] To the extent that the method of the present invention
includes analysing the methylation of CAHM, the subject residues
are:
TABLE-US-00010 chr6: 163834330 chr6: 163834332 chr6: 163834357
chr6: 163834373 chr6: 163834384 chr6: 163834390 chr6: 163834392
chr6: 163834406 chr6: 163834412 chr6: 163834419 chr6: 163834443
chr6: 163834448 chr6: 163834452 chr6: 163834464 chr6: 163834483
chr6: 163834653 chr6: 163834660 chr6: 163834672 chr6: 163834675
chr6: 163834678 chr6: 163834681 chr6: 163834815 chr6: 163834824
chr6: 163834835 chr6: 163834840 chr6: 163834853 chr6: 163834855
chr6: 163834858 chr6: 163834863 chr6: 163834869 chr6: 163834872
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0164] To the extent that the method of the present invention
includes analysing the methylation of IKZF1, the subject residues
are:
TABLE-US-00011 chr7: 50343869 chr7: 50343872 chr7: 50343883 chr7:
50343889 chr7: 50343890 chr7: 50343897 chr7: 50343907 chr7:
50343909 chr7: 50343914 chr7: 50343934 chr7: 50343939 chr7:
50343950 chr7: 50343959 chr7: 50343805 chr7: 50343822 chr7:
50343824 chr7: 50343826 chr7: 50343829 chr7: 50343831 chr7:
50343833 chr7: 50343838 chr7: 50343847 chr7: 50343850 chr7:
50343858 chr7: 50343864 chr7: 50343869 chr7: 50343872 chr7:
50343890
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0165] To the extent that the method of the present invention
includes analysing the methylation of IRF4, the subject residues
are:
TABLE-US-00012 chr6: 392036 chr6: 392047 chr6: 392049 chr6: 392057
chr6: 392060 chr6: 392066 chr6: 392080 chr6: 392094 chr6: 392102
chr6: 392131
or a corresponding cytosine at position n+1 on the opposite DNA
strand.
[0166] The detection method of the present invention can be
performed on any suitable blood sample. To this end, reference to a
"blood sample" should be understood as a reference to any sample
deriving from blood such as, but not limited to, whole blood, serum
or plasma. The blood sample which is tested according to the method
of the present invention may be tested directly or may require some
form of treatment prior to testing. For example, it may require
permeabilisation prior to testing. In one embodiment, the blood
sample is a plasma sample.
[0167] To the extent that the DNA region of interest is present in
a biological sample, the biological sample may be directly tested
or else all or some of the nucleic acid present in the biological
sample may be isolated prior to testing. In yet another example,
the sample may be partially purified or otherwise enriched prior to
analysis. For example, to the extent that a biological sample
comprises a very diverse cell population, it may be desirable to
enrich for a sub-population of particular interest. It is within
the scope of the present invention for the target cell population
or molecules derived therefrom to be treated prior to testing, for
example, inactivation of live virus. It should also be understood
that the biological sample may be freshly harvested or it may have
been stored (for example by freezing) prior to testing or otherwise
treated prior to testing (such as by undergoing culturing).
[0168] The choice of what type of sample is most suitable for
testing in accordance with the method disclosed herein will be
dependent on the nature of the situation.
[0169] Although the present method is directed to classifying a
large intestine neoplasm, the method of the invention is also
useful as a means to monitor disease progression. This can be
important in situations such as where a decision has been made not
to excise an early stage tumour or, even where surgery has been
performed on the primary tumour, to monitor for the development of
metastases which may not have been visually detectable at the time
that the primary tumor was identified. One may also seek to monitor
a patient during a treatment regime for a premalignant or early
stage malignancy in order to detect likely transition to a higher
stage malignancy.
[0170] The method of the invention can be used to evaluate
individuals known or suspected to have a neoplasia or as a routine
clinical test, i.e., in an individual not necessarily suspected to
have a neoplasia.
[0171] Further, the present methods may be used to assess the
efficacy of a course of treatment. For example, the efficacy of an
anti-cancer treatment can be assessed by monitoring DNA methylation
of the sequences described herein over time in a mammal having
cancer.
[0172] The method of the present invention is therefore useful as a
one-time test or as an on-going monitor of those individuals
thought to be at risk of neoplasia development or as a monitor of
the effectiveness of therapeutic or prophylactic treatment regimes
directed to inhibiting or otherwise slowing neoplasia development.
In these situations, mapping the modulation of methylation levels
in any one or more classes of biological samples is a valuable
indicator of the status of an individual or the effectiveness of a
therapeutic or prophylactic regime which is currently in use.
[0173] Any method for detecting DNA methylation can be used in the
methods of the present invention. A number of methods are available
for detection of differentially methylated DNA at specific loci in
either primary tissue samples or in patient samples such as blood,
urine, stool or saliva (reviewed in Kristensen and Hansen Clin
Chem. 55:1471-83, 2009; Ammerpohl et al. Biochim Biophys Acta.
1790:847-62, 2009; Shames et al. Cancer Lett. 251:187-98, 2007;
Clark et al. Nat Protoc. 1:2353-64, 2006). For analysis of the
proportion or extent of DNA methylation in a target gene, DNA is
normally treated with sodium bisulfite and regions of interest
amplified using primers and PCR conditions that will amplify
independently of the methylation status of the DNA. The methylation
of the overall amplicon or individual CpG sites can then be
assessed by sequencing, including pyrosequencing, restriction
enzyme digestion (COBRA) or by melting curve analysis.
Alternatively ligation-based methods for analysis of methylation at
specific CpG sites may be used. Detection of aberrantly methylated
DNA released from tumours and into bodily fluids is being developed
as a means of cancer diagnosis. Here, in the case of
hypermethylated sequences, it is necessary to use sensitive methods
that allow the selective amplification of the methylated DNA
sequence from a background of normal cellular DNA that is
unmethylated. Such methods based on bisulfite-treated DNA, for
example; include methylation selective PCR (MSP), Heavymethyl PCR,
Headloop PCR and Helper-dependent chain reaction
(PCT/AU2008/001475).
[0174] Briefly, in some embodiments, methods for detecting
methylation include randomly shearing or randomly fragmenting the
genomic DNA, cutting the DNA with a methylation-dependent or
methylation-sensitive restriction enzyme and subsequently
selectively identifying and/or analyzing the cut or uncut DNA.
Selective identification can include, for example, separating cut
and uncut DNA (e.g., by size) and quantifying a sequence of
interest that was cut or, alternatively, that was not cut. See,
e.g., U.S. Pat. No. 7,186,512. Alternatively, the method can
encompass amplifying intact DNA after restriction enzyme digestion,
thereby only amplifying DNA that was not cleaved by the restriction
enzyme in the area amplified. See, e.g., U.S. patent application
Ser. Nos. 10/971,986; 11/071,013; and Ser. No. 10/971,339. In some
embodiments, amplification can be performed using primers that are
gene specific. Alternatively, adaptors can be added to the ends of
the randomly fragmented DNA, the DNA can be digested with a
methylation-dependent or methylation-sensitive restriction enzyme,
intact DNA can be amplified using primers that hybridize to the
adaptor sequences. In this case, a second step can be performed to
determine the presence, absence or quantity of a particular gene in
an amplified pool of DNA. In some embodiments, the DNA is amplified
using real-time, quantitative PCR.
[0175] In some embodiments, the methods comprise quantifying the
average methylation density in a target sequence within a
population of genomic DNA. In some embodiments, the method
comprises contacting genomic DNA with a methylation-dependent
restriction enzyme or methylation-sensitive restriction enzyme
under conditions that allow for at least some copies of potential
restriction enzyme cleavage sites in the locus to remain uncleaved;
quantifying intact copies of the locus; and comparing the quantity
of amplified product to a control value representing the quantity
of methylation of control DNA, thereby quantifying the average
methylation density in the locus compared to the methylation
density of the control DNA.
[0176] The quantity of methylation of a locus of DNA can be
determined by providing a sample of genomic DNA comprising the
locus, cleaving the DNA with a restriction enzyme that is either
methylation-sensitive or methylation-dependent, and then
quantifying the amount of intact DNA or quantifying the amount of
cut DNA at the DNA locus of interest. The amount of intact or cut
DNA will depend on the initial amount of genomic DNA containing the
locus, the amount of methylation in the locus, and the number
(i.e., the fraction) of nucleotides in the locus that are
methylated in the genomic DNA. The amount of methylation in a DNA
locus can be determined by comparing the quantity of intact DNA or
cut DNA to a control value representing the quantity of intact DNA
or cut DNA in a similarly-treated DNA sample. The control value can
represent a known or predicted number of methylated nucleotides.
Alternatively, the control value can represent the quantity of
intact or cut DNA from the same locus in another (e.g., normal,
non-diseased) cell or a second locus.
[0177] By using at least one methylation-sensitive or
methylation-dependent restriction enzyme under conditions that
allow for at least some copies of potential restriction enzyme
cleavage sites in the locus to remain uncleaved and subsequently
quantifying the remaining intact copies and comparing the quantity
to a control, average methylation density of a locus can be
determined. A methylation-sensitive enzyme is one which cuts DNA if
its recognition sequence is unmethylated while a
methylation-dependent enzyme cuts DNA if its recognition sequence
is methylated. If the methylation-sensitive restriction enzyme is
contacted to copies of a DNA locus under conditions that allow for
at least some copies of potential restriction enzyme cleavage sites
in the locus to remain uncleaved, then the remaining intact DNA
will be directly proportional to the methylation density, and thus
may be compared to a control to determine the relative methylation
density of the locus in the sample. Similarly, if a
methylation-dependent restriction enzyme is contacted to copies of
a DNA locus under conditions that allow for at least some copies of
potential restriction enzyme cleavage sites in the locus to remain
uncleaved, then the remaining intact DNA will be inversely
proportional to the methylation density, and thus may be compared
to a control to determine the relative methylation density of the
locus in the sample. Such assays are disclosed in, e.g., U.S.
patent application Ser. No. 10/971,986.
[0178] Kits for the above methods can include, e.g., one or more of
methylation-dependent restriction enzymes, methylation-sensitive
restriction enzymes, amplification (e.g., PCR) reagents, probes
and/or primers.
[0179] Quantitative amplification methods (e.g., quantitative PCR
or quantitative linear amplification) can be used to quantify the
amount of intact DNA within a locus flanked by amplification
primers following restriction digestion. Methods of quantitative
amplification are disclosed in, e.g., U.S. Pat. Nos. 6,180,349;
6,033,854; and 5,972,602, as well as in, e.g., Gibson et al.,
Genome Research 6:995-1001 (1996); DeGraves, et al., Biotechniques
34(1):106-10, 112-5 (2003); Deiman B, et al., Mol. Biotechnol.
20(2):163-79 (2002). Amplifications may be monitored in "real
time."
[0180] Additional methods for detecting DNA methylation can involve
genomic sequencing before and after treatment of the DNA with
bisulfite. See, e.g., Frommer et al., Proc. Natl. Acad. Sci. USA
89:1827-1831 (1992). When sodium bisulfite is contacted to DNA,
unmethylated cytosine is converted to uracil, while methylated
cytosine is not modified.
[0181] In some embodiments, restriction enzyme digestion of PCR
products amplified from bisulfite-converted DNA is used to detect
DNA methylation. See, e.g., Sadri & Hornsby, Nucl. Acids Res.
24:5058-5059 (1996); Xiong & Laird, Nucleic Acids Res.
25:2532-2534 (1997).
[0182] In some embodiments, a methylation-specific PCR ("MSP")
reaction is used alone or in combination with other methods to
detect DNA methylation. An MSP assay entails initial modification
of DNA by sodium bisulfite, converting all unmethylated, but not
methylated, cytosines to uracil, and subsequent amplification with
primers specific for methylated verses unmethylated DNA. See,
Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826 (1996); U.S.
Pat. No. 5,786,146.
[0183] In some embodiments, a MethyLight assay is used alone or in
combination with other methods to detect DNA methylation (see, Eads
et al., Cancer Res. 59:2302-2306 (1999)). Briefly, in the
MethyLight process genomic DNA is converted in a sodium bisulfite
reaction (the bisulfite process converts unmethylated cytosine
residues to uracil). Amplification of a DNA sequence of interest is
then performed using PCR primers that hybridize to CpG
dinucleotides. By using primers that hybridize only to sequences
resulting from bisulfite conversion of methylated DNA, (or
alternatively to unmethylated sequences) amplification can indicate
methylation status of sequences where the primers hybridize.
Furthermore, the amplification product can be detected with a probe
that specifically binds to a sequence resulting from bisulfite
treatment of an unmethylated DNA. If desired, both primers and
probes can be used to detect methylation status. Thus, kits for use
with MethyLight can include sodium bisulfite as well as primers or
detectably-labelled probes (including but not limited to Taqman or
molecular beacon probes) that distinguish between methylated and
unmethylated DNA that have been treated with bisulfite. Other kit
components can include, e.g., reagents necessary for amplification
of DNA including but not limited to, PCR buffers, deoxynucleotides;
and a thermostable polymerase.
[0184] In some embodiments, a Ms-SNuPE (Methylation-sensitive
Single Nucleotide Primer Extension) reaction is used alone or in
combination with other methods to detect DNA methylation (see,
Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531 (1997)). The
Ms-SNuPE technique is a quantitative method for assessing
methylation differences at specific CpG sites based on bisulfite
treatment of DNA, followed by single-nucleotide primer extension
(Gonzalgo & Jones, supra). Briefly, genomic DNA is reacted with
sodium bisulfite to convert unmethylated cytosine to uracil while
leaving 5-methylcytosine unchanged. Amplification of the desired
target sequence is then performed using PCR primers specific for
bisulfite-converted DNA, and the resulting product is isolated and
used as a template for methylation analysis at the CpG site(s) of
interest.
[0185] Typical reagents (e.g., as might be found in a typical
Ms-SNuPE-based kit) for Ms-SNuPE analysis can include, but are not
limited to: PCR primers for specific gene (or methylation-altered
DNA sequence or CpG island); optimized PCR buffers and
deoxynucleotides; gel extraction kit; positive control primers;
Ms-SNuPE primers for a specific gene; reaction buffer (for the
Ms-SNuPE reaction); and detectably-labelled nucleotides.
Additionally, bisulfite conversion reagents may include: DNA
denaturation buffer; sulfonation buffer; DNA recovery regents or
kit (e.g., precipitation, ultrafiltration, affinity column);
desulfonation buffer; and DNA recovery components.
[0186] Additional methylation detection methods include, but are
not limited to, methylated CpG island amplification (see, Toyota et
al., Cancer Res. 59:2307-12 (1999)) and those described in, e.g.,
U.S. Patent Publication 2005/0069879; Rein, et al. Nucleic Acids
Res. 26 (10): 2255-64 (1998); Olek, et al. Nat. Genet. 17(3): 275-6
(1997); and PCT Publication No. WO 00/70090.
[0187] More detailed information in relation to several of these
generally described methods is provided below:
(a) Probe or Primer Design and/or Production
[0188] Several methods described herein for the diagnosis of a
neoplasia use one or more probes and/or primers. Methods for
designing probes and/or primers for use in, for example, PCR or
hybridization are known in the art and described, for example, in
Dieffenbach and Dveksler (Eds) (In: PCR Primer: A Laboratory
Manual, Cold Spring Harbor Laboratories, N Y, 1995). Furthermore,
several software packages are publicly available that design
optimal probes and/or primers for a variety of assays, e.g. Primer
3 available from the Center for Genome Research, Cambridge, Mass.,
USA.
[0189] Clearly, the potential use of the probe or primer should be
considered during its design. For example, should the probe or
primer be produced for use in a methylation specific PCR or ligase
chain reaction (LCR) assay the nucleotide at the 3' end (or 5' end
in the case of LCR) should preferably correspond to a methylated
nucleotide in a nucleic acid.
[0190] Probes and/or primers useful for detection of a sequence
associated with a neoplasia are assessed, for example, to determine
those that do not form hairpins, self-prime or form primer dimers
(e.g. with another probe or primer used in a detection assay).
Furthermore, a probe or primer (or the sequence thereof) is often
assessed to determine the temperature at which it denatures from a
target nucleic acid (i.e. the melting temperature of the probe or
primer, or Tm). Methods for estimating Tm are known in the art and
described, for example, in Santa Lucia, Proc. Natl. Acad. Sci. USA,
95: 1460-1465, 1995 or Bresslauer et al., Proc. Natl. Acad. Sci.
USA, 83: 3746-3750, 1986.
[0191] Methods for producing/synthesizing a probe or primer of the
present invention are known in the art. For example,
oligonucleotide synthesis is described, in Gait (Ed) (In:
Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford,
1984). For example, a probe or primer may be obtained by biological
synthesis (e.g. by digestion of a nucleic acid with a restriction
endonuclease) or by chemical synthesis. For short sequences (up to
about 100 nucleotides) chemical synthesis is preferable.
[0192] For longer sequences standard replication methods employed
in molecular biology are useful, such as, for example, the use of
M13 for single stranded DNA as described by Messing, Methods
Enzymol, 101, 20-78, 1983. Other methods for oligonucleotide
synthesis include, for example, phosphotriester and phosphodiester
methods (Narang, et al. Meth. Enzymol 68: 90, 1979) and synthesis
on a support (Beaucage, et al. Tetrahedron Letters 22:1859-1862,
1981) as well as phosphoramidate technique, Caruthers, M. H., et
al., Methods in Enzymology, Vol. 154, pp. 287-314 (1988), and
others described in "Synthesis and Applications of DNA and RNA," S.
A. Narang, editor, Academic Press, New York, 1987, and the
references cited therein. Probes comprising locked nucleic acid
(LNA) are synthesized as described, for example, in Nielsen et al.
J. Chem. Soc. Perkin Trans., 1:3423, 1997; Singh and Wengel, Chem.
Commun. 1247, 1998. While, probes comprising peptide-nucleic acid
(PNA) are synthesized as described, for example, in Egholm et al.,
Am. Chem. Soc., 114:1895, 1992; Egholm et al., Nature, 365:566,
1993; and Orum et al., Nucl. Acids Res., 21:5332, 1993.
(b) Methylation-Sensitive Endonuclease Digestion of DNA
[0193] In one example, the increased methylation in a sample is
determined using a process comprising treating the nucleic acid
with an amount of a methylation-sensitive restriction endonuclease
enzyme under conditions sufficient for nucleic acid to be digested
and then detecting the fragments produced. Exemplary
methylation-sensitive endonucleases include, for example, HhaI or
HpaII. Preferably, assays include internal controls that are
digested with a methylation-insensitive enzyme having the same
specificity as the methylation-sensitive enzyme employed. For
example, the methylation-insensitive enzyme MspI is an isoschizomer
of the methylation-sensitive enzyme HpaII.
Hybridization Assay Formats
[0194] In one example, the digestion of nucleic acid is detected by
selective hybridization of a probe or primer to the undigested
nucleic acid. Alternatively, the probe selectively hybridizes to
both digested and undigested nucleic acid but facilitates
differentiation between both forms, e.g., by electrophoresis.
Suitable detection methods for achieving selective hybridization to
a hybridization probe include, for example, Southern or other
nucleic acid hybridization (Kawai et al., Mol. Cell. Biol.
14:7421-7427, 1994; Gonzalgo et al., Cancer Res. 57:594-599,
1997).
[0195] Suitable hybridization conditions are determined based on
the melting temperature (Tm) of a nucleic acid duplex comprising
the probe. The skilled artisan will be aware that optimum
hybridization reaction conditions should be determined empirically
for each probe, although some generalities can be applied.
Preferably, hybridizations employing short oligonucleotide probes
are performed at low to medium stringency. In the case of a GC rich
probe or primer or a longer probe or primer a high stringency
hybridization and/or wash is preferred. A high stringency is
defined herein as being a hybridization and/or wash carried out in
about 0.1.times.SSC buffer and/or about 0.1% (w/v) SDS, or lower
salt concentration, and/or at a temperature of at least 65.degree.
C., or equivalent conditions. Reference herein to a particular
level of stringency encompasses equivalent conditions using
wash/hybridization solutions other than SSC known to those skilled
in the art.
[0196] In accordance with the present example, a difference in the
fragments produced for the test sample and a negative control
sample is indicative of the subject having a neoplasia. Similarly,
in cases where the control sample comprises data from a tumor,
cancer tissue or a cancerous cell or pre-cancerous cell,
similarity, albeit not necessarily absolute identity, between the
test sample and the control sample is indicative of a positive
diagnosis (i.e. cancer).
Amplification Assay Formats
[0197] In an alternative example, the fragments produced by the
restriction enzyme are detected using an amplification system, such
as, for example, polymerase chain reaction (PCR), rolling circle
amplification (RCA), inverse polymerase chain reaction (iPCR), in
situ PCR (Singer-Sam et al., Nucl. Acids Res. 18:687, 1990), strand
displacement amplification (SDA) or cycling probe technology.
[0198] Methods of PCR are known in the art and described, for
example, by McPherson et al., PCR: A Practical Approach. (series
eds, D. Rickwood and B. D. Hames), IRL Press Limited, Oxford. pp
1-253, 1991 and by Dieffenbach (ed) and Dveksler (ed) (In: PCR
Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, N Y,
1995), the contents of which are each incorporated in their
entirety by way of reference. Generally, for PCR two
non-complementary nucleic acid primer molecules comprising at least
about 18 nucleotides in length, and more preferably at least 20-30
nucleotides in length are hybridized to different strands of a
nucleic acid template molecule at their respective annealing sites,
and specific nucleic acid molecule copies of the template that
intervene the annealing sites are amplified enzymatically.
Amplification products may be detected, for example, using
electrophoresis and detection with a detectable marker that binds
nucleic acids. Alternatively, one or more of the oligonucleotides
are labelled with a detectable marker (e.g. a fluorophore) and the
amplification product detected using, for example, a lightcycler
(Perkin Elmer, Wellesley, Mass., USA, Roche Applied Science,
Indianapolis, Ind., USA).
[0199] Strand displacement amplification (SDA) utilizes
oligonucleotide primers, a DNA polymerase and a restriction
endonuclease to amplify a target sequence. The oligonucleotides are
hybridized to a target nucleic acid and the polymerase is used to
produce a copy of the region intervening the primer annealing
sites. The duplexes of copied nucleic acid and target nucleic acid
are then nicked with an endonuclease that specifically recognizes a
sequence at the beginning of the copied nucleic acid. The DNA
polymerase recognizes the nicked DNA and produces another copy of
the target region at the same time displacing the previously
generated nucleic acid. The advantage of SDA is that it occurs in
an isothermal format, thereby facilitating high-throughput
automated analysis.
[0200] Cycling Probe Technology uses a chimeric synthetic primer
that comprises DNA-RNA-DNA that is capable of hybridizing to a
target sequence. Upon hybridization to a target sequence the
RNA-DNA duplex formed is a target for RNaseH thereby cleaving the
primer. The cleaved primer is then detected, for example, using
mass spectrometry or electrophoresis.
[0201] For primers that flank or are adjacent to a
methylation-sensitive endonuclease recognition site, it is
preferred that such primers flank only those sites that are
hypermethylated in neoplasia to ensure that a diagnostic
amplification product is produced. In this regard, an amplification
product will only be produced when the restriction site is not
cleaved, i.e., when it is methylated. Accordingly, detection of an
amplification product indicates that the CpG dinucleotide/s of
interest is/are methylated.
[0202] As will be known to the skilled artisan, the precise length
of the amplified product will vary depending upon the distance
between the primers. Clearly this form of analysis may be used to
determine the methylation status of a plurality of CpG
dinucleotides provided that each dinucleotide is within a
methylation sensitive restriction endonuclease site. In these
methods, one or more of the primers may be labelled with a
detectable marker to facilitate rapid detection of amplified
nucleic acid, for example, a fluorescent label (e.g. Cy5 or Cy3) or
a radioisotope (e.g. .sup.32P).
[0203] The amplified nucleic acids are generally analyzed using,
for example, non-denaturing agarose gel electrophoresis,
non-denaturing polyacrylamide gel electrophoresis, mass
spectrometry, liquid chromatography (e.g. HPLC or dHPLC), or
capillary electrophoresis. (e.g. MALDI-TOF). High throughput
detection methods, such as, for example, matrix-assisted laser
desorption/ionization time of flight (MALDI-TOF), electrospray
ionization (ESI), mass spectrometry (including tandem mass
spectrometry, e.g. LC MS/MS), biosensor technology, evanescent
fiber-optics technology or DNA chip technology (e.g., WO98/49557;
WO 96/17958; Fodor et al., Science 767-773, 1991; U.S. Pat. Nos.
5,143,854; and 5,837,832, the contents of which are all
incorporated herein by reference), are especially preferred for all
assay formats described herein. Alternatively, amplification of a
nucleic acid may be continuously monitored using a melting curve
analysis method as described herein and/or in, for example, U.S.
Pat. No. 6,174,670, which is incorporated herein by reference.
(c) Other Assay Formats
[0204] In an alternative example, the increased methylation in a
sample is determined by performing a process comprising treating
chromatin containing the nucleic acid with an amount of DNaseI
under conditions sufficient for nucleic acid to be digested and
then detecting the fragments produced. This assay format is
predicated on the understanding that chromatin containing
methylated DNA, e.g., hyper methylated DNA, has a more
tightly-closed conformation than non-hyper methylated DNA and, as a
consequence, is less susceptible to endonuclease digestion by DNase
I.
[0205] In accordance with this method, DNA fragments of different
lengths are produced by DNase I digestion of methylated compared to
non-methylated DNA. Such different DNA fragments are detected, for
example, using an assay described earlier. Alternatively, the DNA
fragments are detected using PCR-SSCP essentially as described, for
example, in Gregory and Feil, Nucleic Acids Res., 27, e32i-e32iv,
1999. In adapting PCR-SSCP to the present invention, amplification
primers flanking or comprising one or more CpG dinucleotides in a
nucleic acid that are resistant to DNase I digestion in a neoplasia
sample but not resistant to DNase I digestion in a healthy/normal
control or healthy/normal test sample are used to amplify the DNase
I-generated fragments. In this case, the production of a specific
nucleic acid fragment using DNase I is diagnostic of neoplasia,
because the DNA is not efficiently degraded. In contrast, template
DNA from a healthy/normal subject sample is degraded by the action
of DNase I and, as a consequence, amplification fails to produce a
discrete amplification product. Alternative methods to PCR-SSCP,
such as for example, PCR-dHPLC are also known in the art and
contemplated by the present invention.
(d) Selective Mutagenesis of Non-Methylated DNA
[0206] In an alternative method the increased methylation in a
sample is determined using a process comprising treating the
nucleic acid with an amount of a compound that selectively mutates
a non-methylated cytosine residue within a CpG dinucleotide under
conditions sufficient to induce mutagenesis.
[0207] Preferred compounds mutate cytosine to uracil or thymidine,
such as, for example, a salt of bisulfite, e.g., sodium bisulfite
or potassium bisulfite (Frommer et al., 1992, supra). Bisulfite
treatment of DNA is known to distinguish methylated from
non-methylated cytosine residues, by mutating cytosine residues
that are not protected by methylation, including cytosine residues
that are not within a CpG dinucleotide or that are positioned
within a CpG dinucleotide that is not subject to methylation.
Sequence Based Detection
[0208] In one example, the presence of one or more mutated
nucleotides or the number of mutated sequences is determined by
sequencing mutated DNA. One form of analysis comprises amplifying
mutated nucleic acid using an amplification reaction described
herein, for example, PCR. The amplified product is then directly
sequenced or cloned and the cloned product sequenced. Methods for
sequencing DNA are known in the art and include for example, the
dideoxy chain termination method or the Maxam-Gilbert method (see
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed.,
CSHP, New York 1989) or Zyskind et al., Recombinant DNA Laboratory
Manual, (Acad. Press, 1988)).
[0209] As the treatment of nucleic acid with a compound, such as,
for example, bisulfite results in non-methylated cytosines being
mutated to uracil (and hence thymidine after an amplification
process), analysis of the sequence determines the presence or
absence of a methylated nucleotide. For example, by comparing the
sequence obtained using a control sample or a sample that has not
been treated with bisulfite, or the known nucleotide sequence of
the region of interest with a treated sample facilitates the
detection of differences in the nucleotide sequence. Any thymine
residue detected at the site of a cytosine in the treated sample
compared to a control or untreated sample may be considered to be
caused by mutation as a result of bisulfite treatment. Suitable
methods for the detection of methylation using sequencing of
bisulfite treated nucleic acid are described, for example, in
Frommer et al., 1992, supra or Clark et al., Nucl. Acids Res.
22:2990-2997, 1994.
[0210] In another method, the presence of a mutated or non-mutated
nucleotide in a bisulfite treated sample is detected using
pyrosequencing, such as, for example, as described in Uhlmann et
al., Electrophoresis, 23: 4072-4079, 2002. Essentially this method
is a form of real-time sequencing that uses a primer that
hybridizes to a site adjacent or close to the site of a cytosine
that is methylated. Following hybridization of the primer and
template in the presence of a DNA polymerase each of four modified
deoxynucleotide triphosphates are added separately according to a
predetermined dispensation order. Only an added nucleotide that is
complementary to the bisulfite treated sample is incorporated and
inorganic pyrophosphate (PPi) is liberated. The PPi then drives a
reaction resulting in production of detectable levels of light.
Such a method allows determination of the identity of a specific
nucleotide adjacent to the site of hybridization of the primer.
[0211] Methods of solid phase pyrosequencing are known in the art
and reviewed in, for example, Landegren et al., Genome Res., 8(8):
769-776, 1998. Such methods enable the high-throughput detection of
methylation of a number of CpG dinucleotides.
[0212] A related method for determining the sequence of a bisulfite
treated nucleotide is methylation-sensitive single nucleotide
primer extension (Me-SnuPE) or SNaPmeth. Suitable methods are
described, for example, in Gonzalgo and Jones, 1997, supra, or
Uhlmann et al., Electrophoresis, 23:4072-4079, 2002. An
oligonucleotide is used that hybridizes to the region of a nucleic
acid adjacent to the site of a cytosine that is methylated. This
oligonucleotide is then used in a primer extension protocol with a
polymerase and a free nucleotide diphosphate or dideoxynucleotide
triphosphate that corresponds to either or any of the possible
bases that occur at this site following bisulfite treatment (i.e.,
thymine or cytosine). Preferably, the nucleotide-diphosphate is
labelled with a detectable marker (e.g. a fluorophore). Following
primer extension, unbound labelled nucleotide diphosphates are
removed, e.g. using size exclusion chromatography or
electrophoresis, or hydrolyzed, using for example, alkaline
phosphatase, and the incorporation of the labelled nucleotide to
the oligonucleotide is detected, indicating the base that is
present at the site.
[0213] Clearly other high throughput sequencing methods are
encompassed by the present invention. Such methods include, for
example, solid phase minisequencing (as described, for example, in
Southern et al., Genomics, 13:1008-1017, 1992), or minisequencing
with FRET (as described, for example, in Chen and Kwok, Nucleic
Acids Res. 25:347-353, 1997).
Restriction Endonuclease-Based Assay Format
[0214] In one method, the presence of a non-mutated sequence is
detected using combined bisulfite restriction analysis (COBRA)
essentially as described in Xiong and Laird, 2001, supra. This
method exploits the differences in restriction enzyme recognition
sites between methylated and unmethylated nucleic acid after
treatment with a compound that selectively mutates a non-methylated
cytosine residue, e.g., bisulfite.
[0215] Following bisulfite treatment a region of interest
comprising one or more CpG dinucleotides that are methylated and
are included in a restriction endonuclease recognition sequence is
amplified using an amplification reaction described herein, e.g.,
PCR. The amplified product is then contacted with the restriction
enzyme that cleaves at the site of the CpG dinucleotide for a time
and under conditions sufficient for cleavage to occur. A
restriction site may be selected to indicate the presence or
absence of methylation. For example, the restriction endonuclease
TaqI cleaves the sequence TCGA, following bisulfite treatment of a
non-methylated nucleic acid the sequence will be TTGA and, as a
consequence, will not be cleaved. The digested and/or non-digested
nucleic acid is then detected using a detection means known in the
art, such as, for example, electrophoresis and/or mass
spectrometry. The cleavage or non-cleavage of the nucleic acid is
indicative of cancer in a subject. Clearly, this method may be
employed in either a positive read-out or negative read-out system
for the diagnosis of a cancer.
Positive Read-Out Assay Format
[0216] In one embodiment, the assay format of the invention
comprises a positive read-out system in which DNA from a sample
that has been treated, for example, with bisulfite is detected as a
positive signal. Preferably, the non-hypermethylated DNA from a
healthy or normal control subject is not detected or only weakly
detected.
[0217] In a preferred embodiment, the increased methylation in a
subject sample is determined using a process comprising:
[0218] (i) treating the nucleic acid with an amount of a compound
that selectively mutates a non-methylated cytosine residue under
conditions sufficient to induce mutagenesis thereby producing a
mutated nucleic acid;
[0219] (ii) hybridizing a nucleic acid to a probe or primer
comprising a nucleotide sequence that is complementary to a
sequence comprising a methylated cytosine residue under conditions
such that selective hybridization to the non-mutated nucleic acid
occurs; and
[0220] (iii) detecting the selective hybridization.
[0221] In this context, the term "selective hybridization" means
that hybridization of a probe or primer to the non-mutated nucleic
acid occurs at a higher frequency or rate, or has a higher maximum
reaction velocity, than hybridization of the same probe or primer
to the corresponding mutated sequence. Preferably, the probe or
primer does not hybridize to the non-methylated sequence carrying
the mutation(s) under the reaction conditions used.
Hybridization-Based Assay Format
[0222] In one embodiment, the hybridization is detected using
Southern, dot blot, slot blot or other nucleic acid hybridization
means (Kawai et al., 1994, supra; Gonzalgo et al., 1997, supra).
Subject to appropriate probe selection, such assay formats are
generally described herein above and apply mutatis mutandis to the
presently described selective mutagenesis approach.
[0223] Preferably, a ligase chain reaction format is employed to
distinguish between a mutated and non-mutated nucleic acid. Ligase
chain reaction (described in EP 320,308 and U.S. Pat. No.
4,883,750) uses at least two oligonucleotide probes that anneal to
a target nucleic acid in such a way that they are juxtaposed on the
target nucleic acid. In a ligase chain reaction assay, the target
nucleic acid is hybridized to a first probe that is complementary
to a diagnostic portion of the target sequence (the diagnostic
probe) e.g., a nucleic acid comprising one or more methylated CpG
dinucleotide(s), and with a second probe that is complementary to a
nucleotide sequence contiguous with the diagnostic portion (the
contiguous probe), under conditions wherein the diagnostic probe
remains bound substantially only to the target nucleic acid. The
diagnostic and contiguous probes can be of different lengths and/or
have different melting temperatures such that the stringency of the
hybridization can be adjusted to permit their selective
hybridization to the target, wherein the probe having the higher
melting temperature is hybridized at higher stringency and,
following washing to remove unbound and/or non-selectively bound
probe, the other probe having the lower melting temperature is
hybridized at lower stringency. The diagnostic probe and contiguous
probe are then covalently ligated such as, for example, using T4
DNA ligase, to thereby produce a larger target probe that is
complementary to the target sequence, and the probes that are not
ligated are removed by modifying the hybridization stringency. In
this respect, probes that have not been ligated will selectively
hybridize under lower stringency hybridization conditions than
probes that have been ligated. Accordingly, the stringency of the
hybridization can be increased to a stringency that is at least as
high as the stringency used to hybridize the longer probe, and
preferably at a higher stringency due to the increased length
contributed by the shorter probe following ligation.
[0224] In another example, one or both of the probes is labelled
such that the presence or absence of the target sequence can be
tested by melting the target-probe duplex, eluting the dissociated
probe, and testing for the label(s). Where both probes are
labelled, different ligands are used to permit distinction between
the ligated and unligated probes, in which case the presence of
both labels in the same eluate fraction confirms the ligation
event. If the target nucleic acid is bound to a solid matrix e.g.,
in a Southern hybridization, slot blot, dot blot, or microchip
assay format, the presence of both the diagnostic and contiguous
probes can be determined directly.
[0225] Methylation specific microarrays (MSO) are also useful for
differentiating between a mutated and non-mutated sequence. A
suitable method is described, for example, in Adorjan et al. Nucl.
Acids Res., 30: e21, 2002. MSO uses nucleic acid that has been
treated with a compound that selectively mutates a non-methylated
cytosine residue (e.g., bisulfite) as template for an amplification
reaction that amplifies both mutant and non-mutated nucleic acid.
The amplification is performed with at least one primer that
comprises a detectable label, such as, for example, a fluorophore,
e.g., Cy3 or Cy5.
[0226] To produce a microarray for detection of mutated nucleic
acid oligonucleotides are spotted onto, for example, a glass slide,
preferably, with a degree of redundancy (for example, as described
in Golub et al., Science, 286:531-537, 1999). Preferably, for each
CpG dinucleotide analyzed two different oligonucleotides are used.
Each oligonucleotide comprises a sequence N.sub.2-16CGN.sub.2-16 or
N.sub.2-16TGN.sub.2-16 (wherein N is a number of nucleotides
adjacent or juxtaposed to the CpG dinucleotide of interest)
reflecting the methylated or non-methylated status of the CpG
dinucleotides.
[0227] The labelled amplification products are then hybridized to
the oligonucleotides on the microarray under conditions that enable
detection of single nucleotide differences. Following washing to
remove unbound amplification product, hybridization is detected
using, for example, a microarray scanner. Not only does this method
allow for determination of the methylation status of a large number
of CpG dinucleotides, it is also semi-quantitative, enabling
determination of the degree of methylation at each CpG dinucleotide
analyzed. As there may be some degree of heterogeneity of
methylation in a single sample, such quantification may assist in
the diagnosis of cancer.
Amplification-Based Assay Format
[0228] In an alternative example, the hybridization is detected
using an amplification system. In methylation-specific PCR formats
(MSP; Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1992),
the hybridization is detected using a process comprising amplifying
the bisulfite-treated DNA. Accordingly, by using one or more probe
or primer that anneals specifically to the unmutated sequence under
moderate and/or high stringency conditions an amplification product
is only produced using a sample comprising a methylated nucleotide.
Alternate assays that provide for selective amplification of either
the methylated or the unmethylated component from a mixture of
bisulfite-treated DNA are provided by Cottrell et al., Nucl. Acids
Res. 32: e10, 2003 (HeavyMethyl PCR), Rand et al. Nucl. Acids Res.
33:e127, 2005 (Headloop PCR), Rand et al. Epigenetics 1:94-100,
2006 (Bisulfite Differential Denaturation PCR) and PCT/AU07/000389
(End-specific PCR).
[0229] Any amplification assay format described herein can be used,
such as, for example, polymerase chain reaction (PCR), rolling
circle amplification (RCA), inverse polymerase chain reaction
(iPCR), in situ PCR (Singer-Sam et al., 1990, supra), strand
displacement amplification, or cycling probe technology. PCR
techniques have been developed for detection of gene mutations
(Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991)
and quantitation of allelic-specific expression (Szabo and Mann,
Genes Dev. 9: 3097-3108, 1995; and Singer-Sam et al., PCR Methods
Appl. 1: 160-163, 1992). Such techniques use internal primers,
which anneal to a PCR-generated template and terminate immediately
5' of the single nucleotide to be assayed. Such as format is
readily combined with ligase chain reaction as described herein
above. The use of a real-time quantitative assay format is also
useful. Subject to the selection of appropriate primers, such assay
formats are generally described herein above and apply mutatis
mutandis to the presently described selective mutagenesis
approach.
[0230] Methylation-specific melting-curve analysis (essentially as
described in Worm et al., Clin. Chem., 47:1183-1189, 2001) is also
contemplated by the present invention. This process exploits the
difference in melting temperature in amplification products
produced using bisulfite treated methylated or unmethylated nucleic
acid. In essence, non-discriminatory amplification of a bisulfite
treated sample is performed in the presence of a fluorescent dye
that specifically binds to double stranded DNA (e.g., SYBR Green
I). By increasing the temperature of the amplification product
while monitoring fluorescence the melting properties and thus the
sequence of the amplification product is determined. A decrease in
the fluorescence reflects melting of at least a domain in the
amplification product. The temperature at which the fluorescence
decreases is indicative of the nucleotide sequence of the amplified
nucleic acid, thereby permitting the nucleotide at the site of one
or more CpG dinucleotides to be determined. As the sequence of the
nucleic acids amplified using the present invention.
[0231] The present invention also encompasses the use of real-time
quantitative forms of PCR, such as, for example, TaqMan (Holland et
al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991; Lee et al.,
Nucleic Acid Res. 21:3761-3766, 1993) to perform this embodiment.
For example, the MethylLight method of Eads et al., Nucl. Acids
Res. 28: E32, 2000 uses a modified TaqMan assay to detect
methylation of a CpG dinucleotide. Essentially, this method
comprises treating a nucleic acid sample with bisulfite and
amplifying nucleic acid comprising one or more CpG dinucleotides
that are methylated in a neoplastic cell and not in a control
sample using an amplification reaction, e.g., PCR. The
amplification reaction is performed in the presence of three
oligonucleotides, a forward and reverse primer that flank the
region of interest and a probe that hybridizes between the two
primers to the site of the one or more methylated CpG
dinucleotides. The probe is dual labelled with a 5' fluorescent
reporter and a 3' quencher (or vice versa). When the probe is
intact, the quencher dye absorbs the fluorescence of the reporter
due to their proximity. Following annealing of to the PCR product
the probe is cleaved by 5' to 3' exonuclease activity of, for
example, Taq DNA polymerase. This cleavage releases the reporter
from the quencher thereby resulting in an increased fluorescence
signal that can be used to estimate the initial template
methylation level. By using a probe or primer that selectively
hybridizes to unmutated nucleic acid (i.e. methylated nucleic acid)
the level of methylation is determined, e.g., using a standard
curve.
[0232] Alternatively, rather than using a labelled probe that
requires cleavage, a probe, such as, for example, a Molecular
Beacon is used (see, for example, Mhlanga and Malmberg, Methods
25:463-471, 2001). Molecular beacons are single stranded nucleic
acid molecules with a stem-and-loop structure. The loop structure
is complementary to the region surrounding the one or more CpG
dinucleotides that are methylated in a neoplastic sample and not in
a control sample. The stem structure is formed by annealing two
"arms" complementary to each other, which are on either side of the
probe (loop). A fluorescent moiety is bound to one arm and a
quenching moiety that suppresses any detectable fluorescence when
the molecular beacon is not bound to a target sequence is bound to
the other arm. Upon binding of the loop region to its target
nucleic acid the arms are separated and fluorescence is detectable.
However, even a single base mismatch significantly alters the level
of fluorescence detected in a sample. Accordingly, the presence or
absence of a particular base is determined by the level of
fluorescence detected. Such an assay facilitates detection of one
or more unmutated sites (i.e. methylated nucleotides) in a nucleic
acid.
[0233] Fluorescently labelled locked nucleic acid (LNA) molecules
or fluorescently labelled protein-nucleic acid (PNA) molecules are
useful for the detection of nucleotide differences (e.g., as
described in Simeonov and Nikiforov, Nucleic Acids Research,
30(17):1-5, 2002). LNA and PNA molecules bind, with high affinity,
to nucleic acid, in particular, DNA. Fluorophores (in particular,
rhodomine or hexachlorofluorescein) conjugated to the LNA or PNA
probe fluoresce at a significantly greater level upon hybridization
of the probe to target nucleic acid. However, the level of increase
of fluorescence is not enhanced to the same level when even a
single nucleotide mismatch occurs. Accordingly, the degree of
fluorescence detected in a sample is indicative of the presence of
a mismatch between the LNA or PNA probe and the target nucleic
acid, such as, in the presence of a mutated cytosine in a
methylated CpG dinucleotide. Preferably, fluorescently labelled LNA
or PNA technology is used to detect at least a single base change
in a nucleic acid that has been previously amplified using, for
example, an amplification method known in the art and/or described
herein.
[0234] As will be apparent to the skilled artisan, LNA or PNA
detection technology is amenable to a high-throughput detection of
one or more markers by immobilizing an LNA or PNA probe to a solid
support, as described in Orum et al., Clin. Chem. 45:1898-1905,
1999.
[0235] Alternatively, a real-time assay, such as, for example, the
so-called HeavyMethyl assay (Cottrell et al., 2003, supra) is used
to determine the presence or level of methylation of nucleic acid
in a test sample. Essentially, this method uses one or more
non-extendible nucleic acid (e.g., oligonucleotide) blockers that
bind to bisulfite-treated nucleic acid in a methylation specific
manner (i.e., the blocker/s bind specifically to unmutated DNA
under moderate to high stringency conditions). An amplification
reaction is performed using one or more primers that may optionally
be methylation specific but that flank the one or more blockers. In
the presence of unmethylated nucleic acid (i.e., non-mutated DNA)
the blocker/s bind and no PCR product is produced. Using a TaqMan
assay essentially as described supra the level of methylation of
nucleic acid in a sample is determined.
[0236] Other amplification based methods for detecting methylated
nucleic acid following treatment with a compound that selectively
mutates a non-methylated cytosine residue include, for example,
methylation-specific single stranded conformation analysis
(MS-SSCA) (Bianco et al., Hum. Mutat., 14:289-293, 1999),
methylation-specific denaturing gradient gel electrophoresis
(MS-DGGE) (Abrams and Stanton, Methods Enzymol., 212:71-74, 1992)
and methylation-specific denaturing high-performance liquid
chromatography (MS-DHPLC) (Deng et al. Chin. J. Cancer Res.,
12:171-191, 2000). Each of these methods use different techniques
for detecting nucleic acid differences in an amplification product
based on differences in nucleotide sequence and/or secondary
structure. Such methods are clearly contemplated by the present
invention.
[0237] As with other amplification-based assay formats, the
amplification product is analyzed using a range of procedures,
including gel electrophoresis, gel filtration, mass spectrometry,
and in the case of labelled primers, by identifying the label in
the amplification product. In an alternative embodiment,
restriction enzyme digestion of PCR products amplified from
bisulfite-converted DNA is performed essentially as described by
Sadri and Hornsby, Nucl. Acids Res. 24:5058-5059, 1996; and Xiong
and Laird, Nucl. Acids Res. 25:2532-2534, 1997), to analyze the
product formed.
[0238] High throughput detection methods, such as, for example,
matrix-assisted laser desorption/ionization time of flight
(MALDI-TOF), electrospray ionization (ESI), Mass spectrometry
(including tandem mass spectrometry, e.g. LC MS/MS), biosensor
technology, evanescent fiber-optics technology or DNA chip
technology, can also be employed.
[0239] As with the other assay formats described herein that
utilize hybridization and/or amplification detection systems,
combinations of such processes as described herein above are
particularly contemplated by the selective mutagenesis-based assay
formats of the present invention. In one example, the increased
methylation is detected by performing a process comprising:
[0240] (i) treating the nucleic acid with an amount of a compound
that selectively mutates a non-methylated cytosine residue within a
CpG dinucleotide under conditions sufficient to induce mutagenesis
thereby producing a mutated nucleic acid;
[0241] (ii) hybridizing the nucleic acid to two non-overlapping and
non-complementary primers each of which comprises a nucleotide
sequence that is complementary to a sequence in the DNA comprising
a methylated cytosine residue under conditions such that
hybridization to the non-mutated nucleic acid occurs;
[0242] (iii) amplifying nucleic acid intervening the hybridized
primers thereby producing a DNA fragment consisting of a sequence
that comprises a primer sequence;
[0243] (iv) hybridizing the amplified DNA fragment to a probe
comprising a nucleotide sequence that corresponds or is
complementary to a sequence comprising a methylated cytosine
residue under conditions such that hybridization to the non-mutated
nucleic acid occurs; and detecting the hybridization.
Negative Read-Out Assays
[0244] In another example, the assay format comprises a negative
read-out system in which reduced methylation of DNA from a
healthy/normal control sample is detected as a positive signal and
preferably, methylated DNA from a neoplastic sample is not detected
or is only weakly detected.
[0245] In a preferred embodiment, the reduced methylation is
determined using a process comprising:
[0246] (i) treating the nucleic acid with an amount of a compound
that selectively mutates a non-methylated cytosine residue within a
CpG island under conditions sufficient to induce mutagenesis
thereby producing a mutated nucleic acid;
[0247] (ii) hybridizing the nucleic acid to a probe or primer
comprising a nucleotide sequence that is complementary to a
sequence comprising the mutated cytosine residue under conditions
such that selective hybridization to the mutated nucleic acid
occurs; and
[0248] (iii) detecting the selective hybridization.
[0249] In this context, the term "selective hybridization" means
that hybridization of a probe or primer to the mutated nucleic acid
occurs at a higher frequency or rate, or has a higher maximum
reaction velocity, than hybridization of the same probe or primer
to the corresponding non-mutated sequence. Preferably, the probe or
primer does not hybridize to the methylated sequence (or
non-mutated sequence) under the reaction conditions used.
Hybridization-Based Assay Format
[0250] In one embodiment the hybridization is detected using
Southern, dot blot, slot blot or other nucleic acid hybridization
means (Kawai et al., 1994, supra; Gonzalgo et al., 1997, supra).
Subject to appropriate probe selection, such assay formats are
generally described herein above and apply mutatis mutandis to the
presently described selective mutagenesis approach. Preferably, a
ligase chain reaction format is employed to distinguish between a
non-mutated and mutated nucleic acid. In this respect, the assay
requirements and conditions are as described herein above for
positive read-out assays and apply mutatis mutandis to the present
format. However the selection of probes will differ. For negative
read-out assays, one or more probes are selected that selectively
hybridize to the mutated sequence rather than the non-mutated
sequence.
[0251] Preferably, the ligase chain reaction probe(s) have
3'-terminal and/or 5'-terminal sequences that comprise a CpG
dinucleotide that is not methylated in a healthy control sample,
but is hypermethylated in cancer, such that the diagnostic probe
and contiguous probe are capable of being ligated only when the
cytosine of the CpG dinucleotide is mutated to thymidine e.g., in
the case of a non-methylated cytosine residue.
[0252] As will be apparent to the skilled artisan the MSO method
described supra is amenable to either or both positive and/or
negative readout assays. This is because the assay described
detects both mutated and non-mutated sequences thereby facilitating
determining the level of methylation. However, an assay detecting
only methylated or non-methylated sequences is contemplated by the
invention.
Amplification-Based Assay Format
[0253] In an alternative example, the hybridization is detected
using an amplification system using any amplification assay format
as described herein above for positive read-out assay albeit using
primers (and probes where applicable) selectively hybridize to a
mutated nucleic acid.
[0254] In adapting the HeavyMethyl assay described supra to a
negative read-out format, the blockers that bind to
bisulfite-treated nucleic acid in a methylation specific manner
bind specifically to mutated DNA under moderate to high stringency
conditions. An amplification reaction is performed using one or
more primers that may optionally be methylation specific (i.e. only
bind to mutated nucleic acid) but that flank the one or more
blockers. In the presence of methylated nucleic acid (i.e., mutated
DNA) the blocker/s bind and no PCR product is produced.
[0255] In one example, the reduced methylation in the
normal/healthy control subject is detected by performing a process
comprising:
[0256] (i) treating the nucleic acid with an amount of a compound
that selectively mutates non-methylated cytosine residues under
conditions sufficient to induce mutagenesis thereby producing a
mutated nucleic acid;
[0257] (ii) hybridizing the nucleic acid to two non-overlapping and
non-complementary primers each of which comprises a nucleotide
sequence that is complementary to a sequence in the DNA comprising
a mutated cytosine residue under conditions such that hybridization
to the mutated nucleic acid occurs;
[0258] (iii) amplifying nucleic acid intervening the hybridized
primers thereby producing a DNA fragment consisting of a sequence
that comprises a primer sequence;
[0259] (iv) hybridizing the amplified DNA fragment to a probe
comprising a nucleotide sequence that corresponds or is
complementary to a sequence comprising a mutated cytosine residue
under conditions such that hybridization to the mutated nucleic
acid occurs; and
[0260] (v) detecting the hybridization.
[0261] As will be apparent to the skilled artisan, a negative
read-out assay preferably includes a suitable control sample to
ensure that the negative result is caused by methylated nucleic
acid rather than a reaction failing.
[0262] This invention also provides kits for the detection and/or
quantification of the diagnostic sequences of the invention, or
expression or methylation thereof using the methods described
herein.
[0263] For kits for detection of methylation, the kits of the
invention can comprise at least one polynucleotide that hybridizes
to at least one of the diagnostic sequences of the invention and at
least one reagent for detection of gene methylation. Reagents for
detection of methylation include, e.g., sodium bisulfite,
polynucleotides designed to hybridize to sequence that is the
product of a biomarker sequence of the invention if the biomarker
sequence is not methylated (e.g., containing at least one
C.fwdarw.U conversion), and/or a methylation-sensitive or
methylation-dependent restriction enzyme. The kits may also include
control natural or synthetic DNA sequences representing methylated
or unmethylated forms of the sequence. The kits can provide solid
supports in the form of an assay apparatus that is adapted to use
in the assay. The kits may further comprise detectable labels,
optionally linked to a polynucleotide, e.g., a probe, in the kit.
Other materials useful in the performance of the assays can also be
included in the kits, including test tubes, transfer pipettes, and
the like. The kits can also include written instructions for the
use of one or more of these reagents in any of the assays described
herein.
[0264] The present invention is further described by reference to
the following non-limiting examples.
Examples
Materials and Methods
Specimen Collection
[0265] Tissue DNA samples were acquired through a commercial
specimen bank (BioServe, @, US) and a tertiary referral hospital
tissue bank in Adelaide, Australia. Blood plasma specimens were
acquired from a commercial specimen bank (Proteogenex, Culver City,
Calif.) and a tertiary referral hospital in Adelaide, Australia.
Blood specimens were classified as normal, adenoma or cancer based
on colonoscopy results verified (where appropriate) by
histopathology. This also identified the stage of the cancer.
Peripheral blood was drawn into K.sub.3EDTA VACUETTE blood tubes
(Greiner-One, Monroe, N.C.) and transported to the processing
laboratory on wet ice. Whole blood was centrifuged at 1,500 g
(4.degree. C.) for 10 minutes within 4 hours of blood draw and
plasma was collected. The plasma was centrifuged for a second time
at 1,500 g (4.degree. C.) for 10 minutes, where after the plasma
was collected and stored at -80.degree. C. until further use.
Tissue DNA Extraction & Bisulfite Conversion
[0266] Tissue specimens were homogenised using a bead homogeniser
and genomic DNA extracted using a Wizard.RTM. Genomic DNA
Purification Kit (Promega, Sydney, Australia).
[0267] Commercially acquired DNA was extracted by BioServe (MD,
USA). DNA concentration was determined by Nanodrop ND 1000 20
spectrophometor (Nanodrop Technologies, Wilmington, Del.). The EZ
DNA Methylation-Gold Kit (Zymo Research Corporation, Orange, Calif.
USA) was used for bisulfite conversion of 1 .mu.g of
tissue-extracted DNA in accordance with the manufacturers
instructions with the following modification to the bisulfite
reaction cycling conditions: 99.degree. C. for 5 minutes,
60.degree. C. for 25 minutes, 99.degree. C. for 5 minutes,
60.degree. C. for 85 minutes, 99.degree. C. for 5 minutes and
60.degree. C. for 175 minutes. The concentration of purified
bisulfite converted DNA was determined by qPCR using bisulfite
conversion specific primers to beta-Actin [ACTB1] as described in
Table 1 and previously by Devos et al. (Clin. Chem., 2009;
55(7):1337-1346). The bisulfite converted DNA samples were stored
at -80.degree. C. until further use.
Plasma DNA Extraction & Bisulfite Conversion:
[0268] DNA was prepared from 4 mL of plasma using the QIAamp
Circulating Nucleic Acid Kit (QIAGEN, Dusseldorf, Germany)
according to the manufacturer's specifications with the following
modifications: The column was washed twice with 750 ul of ACW2 and
twice with 750 ul of absolute ethanol (200 proof). The resulting
DNA was eluted in 35 ul of buffer AVE and this eluate was then
reapplied to the column and eluted again to increase the
concentration of the DNA. The optimised protocol resulted in a 20%
improvement in DNA yield compared to the recommended manufacturer
protocol (data not shown). The final volume of plasma DNA was
.about.324 per 4 mL of patient plasma. Real-time PCR was used to
measure the total DNA recovery: 2.times.14 aliquots of the
resulting plasma DNA was used in a previously described CFF1 assay
(Devos et al. 2009. Clin Chem) (Table 1).
[0269] All PCRs were performed on the LightCycler 480 Real-Time PCR
System, model II (Roche). A 4-fold serial dilution of sonicated
genomic blood DNA (Roche, Mannheim, Germany) was used as a standard
to determine the amount (ng) of DNA extracted per mL of plasma 304
of DNA extracted from 4 mL plasma was stripped of DNA-binding
proteins by incubation at 37.degree. C. for 1 hour after adding 3
uL of a Lysis Buffer consisting of 1 mg/mL tRNA, 2 mg/mL Proteinase
K and 10% SDS. The samples were subsequently bisulphite converted
using either the EZ DNA Methylation-Gold Kit.TM. as recommended by
the manufacturer (Zymo Research Corp. Orange, Calif. USA), with the
same modification to thermal cycling conditions described above, or
the Epitect Plus DNA Bisulfite Kit using Epitect Fast bisulfite
reagent (QIAGEN, Dusseldorf, Germany) using conditions recommended
by the manufacturer. In both cases, the purified DNA was eluted
with 40 uL nuclease-free water. The resulting bisulphite DNA
concentration was calculated by analysing 24 in the ACTB PCR assay
as described above. Triplicate 5 uL aliquots of the resulting 36 uL
bisulphite converted DNA extracted from 4 mL plasma (the equivalent
of 5554 plasma per aliquot) were analysed in methylation-specific
qPCR assays as described below.
Measurement of Methylation
[0270] Methylation specific oligonucleotide primers and probes were
designed to interrogate the methylation status of sites within
CAHM, GRASP, IRF4, BCAT1 and IKZF1. PCR was performed in triplicate
on bisulfite-converted tissue DNA (5 ng) or 54 plasma DNA in a
total volume of 154 (see Table 1 for primer/probe sequences and
reaction conditions). Melt peaks of 78.4.degree.+/-0.9.degree. C.
(CAHM) and 82.9.4.degree.+/-0.3.degree. C. (IKZF1) were
characteristic for the amplicons which ran without a probe.
Methylation levels were quantified against an in house made
standard curve consisting of a serial dilution of
bisulfite-converted sheared methylated DNA (CpGenome Universal
Methylated DNA, Chemicon, Temecula, Calif., USA) in a background of
sheared bisulfite-converted white blood cell DNA (Roche, Mannheim,
Germany). The standard curve contained the following dilution
points: 5000-, 1250-, 312.5-, 78.125, 19.53-, 4.88-, 1.22- and 0 pg
mCpG in a background of WBC DNA (5 ng total DNA per reaction).
Estimation of Class Probabilities
[0271] The R open source programming language and environment for
statistical computing and graphics (http://www.r-project.org) was
installed on a standard Intel IA-32 personal computer system and
used to access and process input data representing the measured
methylation levels and corresponding observed non-neoplastic and
neoplastic categories, as described below.
[0272] Specifically, the observed methylated CAHM mass by phenotype
classification (FIG. 1B) was used to determine the empirical
probability density plots for phenotype classes (premalignant,
early stage cancer, late stage cancer) (FIG. 3A), and the estimated
means and standard deviation values were then used to generate the
modelled density plots of FIG. 3B assuming the observed methylation
levels are drawn from a normal distribution. The density
distributions were then used to estimate the probability that an
assayed CAHM methylation level for a patient specimen is drawn from
one of these classifications.
[0273] Using only positive (i.e., greater than zero)_methylation
levels determined for plasma specimens of known phenotype (and
shown in FIG. 1B), the corresponding estimated distribution
profiles are shown in FIG. 3B. These density functions were used to
calculate the relative probability that a plasma specimen with a
methylated CAHM level of 2.8 pg, 148 pg or 22,000 pg is likely to
be drawn from a patient diagnosed with colorectal lesions
classified as pre-malignant (i.e., adenoma), early-stage cancer
(Stage 1 or Stage 2), or late stage cancer (Stage 3 or Stage
4).
TABLE-US-00013 RELATIVE Probability (normalised to premalignant)
Early-Stage Late-Stage Log(methyl CAHM pg) Premalignant Cancer
Cancer 1.0 (2.8 pg) 1.0 1.0 0.175 5.0 (148 pg) 1.0 3.2 4.95 10.0
(22,000 pg) 1.0 8.3 627
[0274] These data were also used to estimate the probability that a
plasma specimen from a known classification would yield an observed
methylation level equal to or greater than the hypothetical CAHM
methylation levels. Using the probability density functions shown
in FIG. 3B and determined from the raw CAHM methylation levels
shown in FIG. 1B, it was determined that only 3.0% of premalignant
plasma specimens are found to contain at least 148 pg methylated
CAHM, while 66% late stage cancers show 148 pg methylation or more
(relative value of 23:1). Further, a plasma specimen yielding 22 ng
of methylated CAHM is approximately 1600 times more likely to be
drawn from a patient with late cancer than a patient with a
premalignant neoplasm.
TABLE-US-00014 Cumulative probability methylated CAHM is greater
than or equal to (ratio to premalignant) Log(methyl Early-Stage
Late-Stage CAHM pg) Premalignant Cancer Cancer 1.0 36% (1.0) 68%
(1.8) 96% (2.6) 5.0 3% (1.0) 12% (4.1) 66% (23.2) 10.0 0.0058%
(1.0) 0.052% (8) 9% (1600)
[0275] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
TABLE-US-00015 TABLE 1 Oligonucleotide sequences and reaction
conditions for PCR, Methylation Specific PCR and Methylight assays.
PCR Primer/Probe Sequence Mastermix Cycling Conditions CAHM
Forward: GAAGGAAGTAT Each 15 .mu.L reaction contained: Activation -
95.degree. C. 2 min. TTCGAGTACGATTGAC 0.1 .mu.L 5 U/.mu.L Platinum
Taq DNA 3 Cycles: 92.degree. C. 15 sec Reverse: CCCGAACGCAA
polymerase (Invitrogen), 1.5 .mu.L (4.4.degree. C./sec), 62.degree.
C. 15 sec CGACTTAA 10X Platinum Buffer (Invitrogen), (2.2.degree.
C./sec), 72.degree. C. 20 sec 0.9 .mu.L 50 mM MgCl.sub.2 (f 3 mM
Final, 50 Cycles: 82.degree. C. 15 sec Invitrogen), 0.3 .mu.L 10 mM
dNTPs (4.4.degree. C./sec), 63.degree. C. 15 sec (200 uM Final,
Promega), 0.1 .mu.L of (2.2.degree. C./sec), 72.degree. C. 20 sec
the forward and reverse primers Cooling - 40.degree. C. 5 sec (50
uM/200 nM Final) and 0.125 .mu.L (2.2.degree. C./sec) followed by a
1/1000 SYBR Green and Nuclease Melt analysis to confirm Free Water
correct product GRASP Forward: Each 15 .mu.L reaction contained:
Activation - 95.degree. C. 2 min. CGGAAGTCGCGTTCGT 0.1 .mu.L 5
U/.mu.L Platinum Taq DNA 3 Cycles: 92.degree. C. 15 sec C
polymerase (Invitrogen), 1.5 .mu.L (4.4.degree. C./sec), 64.degree.
C. 15 sec Reverse: 10X Platinum Buffer (Invitrogen), (2.2.degree.
C./sec), 72.degree. C. 20 sec GCGTACAACTCGTCCG 1.2 .mu.L 50 mM
MgCl.sub.2 (4 mM Final, (4.4.degree. C./sec) CTAA Invitrogen), 0.3
.mu.L 10 mM dNTPs 47 Cycles: 85.degree. C. 15 sec Probe: [HEX] (0.2
mM Final, Promega), 0.06 .mu.L (4.4.degree. C./sec), 64.degree. C.
15 sec TTCGATTTCGGGATTTT of the forward and reverse primers
(2.2.degree. C./sec), 72.degree. C. 20 sec TTGTCGTAGTC[BHQ1] (50
uM/200 nM Final) and 0.03 .mu.L of (4.4.degree. C./sec) probe (50
uM stock/100 nM Final) cooling - 40.degree. C. 10 sec (2.2.degree.
C./sec). IRF4 Forward: Each 15 .mu.L reaction contained: Activation
- 95.degree. C. 2 min. TGGGTGTTTTGGACGGT 0.1 .mu.L 5 U/.mu.L
Platinum Taq DNA 3 Cycles: 95.degree. C. 15 sec TTC polymerase
(Invitrogen), 1.5 .mu.L (4.4.degree. C./sec), 64.degree. C. 30 sec
Reverse: 10X Platinum Buffer (Invitrogen), (2.2.degree. C./sec),
72.degree. C. 30 sec CGCCTACCCTCCGCG 1.2 .mu.L 50 mM MgCl.sub.2 (4
mM Final, (4.4.degree. C./sec) Probe: [HEX] Invitrogen), 0.3 .mu.L
10 mM dNTPs 50 Cycles: 86.degree. C. 15 sec TCGTTTAGTTTGTGGCG (0.2
mM Final, Promega), 0.12 .mu.L (4.4.degree. C./sec), 62.degree. C.
30 sec ATTTCGTCG {BHQ1] of the forward and reverse primers
(2.2.degree. C./sec), 72.degree. C. 30 sec (50 uM/400 nM Final) and
0.3 ul of (4.4.degree. C./sec) probe (10 uM stock/200 nM Final)
cooling - 40.degree. C. 10 sec (2.2.degree. C./sec). BCAT1 Forward:
Each 15 .mu.L reaction contained: Activation - 95.degree. C. 2 min.
GTTTTTTTGTTGATGTA 0.1 .mu.L 5 U/.mu.L Platinum Taq DNA 50 Cycles:
95.degree. C. 15 sec ATTCGTTAGGTC polymerase (Invitrogen), 1.5
.mu.L (4.4.degree. C./sec), 62.degree. C. 30 sec Reverse: 10X
Platinum Buffer (Invitrogen), (2.2.degree. C./sec), 72.degree. C.
30 sec CAATACCCGAAACGAC 1.2 .mu.L 50 mM MgCl.sub.2 (4 mM Final,
(4.4.degree. C./sec) GACG Invitrogen), 0.3 .mu.L 10 mM dNTPs
cooling - 40.degree. C. 5 sec Probe: HEX-5' (0.2 mM Final,
Promega), 0.06 .mu.L (2.2.degree. C./sec). TTCGTCGCGAGAGGGT of the
forward and reverse primers CGGTT -BHQ (50 uM/200 nM Final) and
0.15 uL of probe (10 uM stock/100 nM Final) IKZF1 Forward: Each 15
.mu.L reaction contained: Activation - 95.degree. C. 2 min.
GACGACGTATTTTTTTC 7.5 ul of 2x GoTaq Hot Start buffer 50 Cycles:
95.degree. C. 15 sec GTGTTTC w/MgCL2, 0.3 .mu.L 50 mM MgCl.sub.2
(4.4.degree. C./sec), 62.degree. C. 30 sec Reverse: (4 mM Final,
Invitrogen), 0.06 .mu.L (2.2.degree. C./sec), 72.degree. C. 30 sec
GCGCACCTCTCGACCG of the forward and reverse primers (4.4.degree.
C./sec) (50 uM/200 nM Final) and 0.15 ul of cooling - 40.degree. C.
5 sec SYBR (1:1000 stock/1:100,000 (2.2.degree. C./sec). Final)
CFF1 Forward: TAAGAGTAATA Each 15 .mu.L reaction contained:
Activation - 95.degree. C. 2 min. ATGGATGGATGATG 0.15 .mu.L 5
U/.mu.L Platinum Taq DNA 50 Cycles: 95.degree. C. 10 sec Reverse:
polymerase (Invitrogen), 1.5 .mu.L (4.4.degree. C./sec), 58.degree.
C. 60 sec CCTCCCATCTCCCTTCC 10X Platinum Buffer (Invitrogen),
(2.2.degree. C./sec) Probe: 6FAM- 0.9 .mu.L 50 mM MgCl.sub.2 (3 mM
Final, cooling - 40.degree. C. 5 sec ATGGATGAAGAAAGAA Invitrogen),
0.3 .mu.L 10 mM dNTPs (2.2.degree. C./sec). AGGATGAGT-BHQ-1 (0.2 mM
Final, Promega), 0.189 .mu.L of the forward and reverse primers (50
uM/200 nM Final) and 0.3 ul of probe (10 uM stock/200 nM Final)
.beta.-actin Forward: GTGATGGAGGA Each 15 .mu.L reaction contained:
Activation - 95.degree. C. 2 min. GGTTTAGTAAGTT 0.15 .mu.L 5
U/.mu.L Platinum Taq DNA 60 Cycles: 95.degree. C. 10 sec Reverse:
CCAATAAAACC polymerase (Invitrogen), 1.5 .mu.L (4.4.degree.
C./sec), 57.degree. C. 40 sec TACTCCTCCCTTAA 10X Platinum Buffer
(Invitrogen), (2.2.degree. C./sec), 72.degree. C. l0 sec Probe:
FAM- 0.6 .mu.L 50 mM MgCl.sub.2 (2 mM Final, (4.4.degree. C./sec)
ACCACCACCCAACACA Invitrogen), 0.3 .mu.L 10 mM dNTPs cooling -
40.degree. C. 5 sec CAATAACAAACACA- (0.2 mM Final, Promega), 0.27
.mu.L (2.2.degree. C./sec). BHQ1 of the forward and reverse primers
(50 uM/900 nM Final) and 0.15 ul of probe (10 uM stock/100 nM
Final)
TABLE-US-00016 TABLE 2 Coordinates Oligonucleotide sequences
Genomic Current sub-region(s) of interest (genomic of sub-
Resulting bisulphite converted sequence for measurement of Gene
Strand sequence) region(s) (strands no longer complementary)
methylation levels BCAT1 top 5'-cagtgccCGaggCGgCGgCGagtacaCGtggC
25,101,992- 5'-tagtgttCGaggCGgCGgCGagtataCGtggCGggttgga strand
GggctggattgcagacCGgccctctCGCGgCGgagactCGC 25,102,093
ttgtagatCGgttttttCGCGgCGgagattCGCGatttagCGgatt
GacctagCGgattgcatcagcaggaagac (SEQ ID No: 1) gtattagtaggaagat minus
3'-gtcacggGCtccGCcGCcGCtcatgtGCaccGCc
3'-gttatggGCtttGCtGCtGCttatgtGCattGCttgatttaatg
5'-gtttttttgttgatgtaattcgtt strand
cgacctaacgtctgGCcgggagaGCGCcGCctctgaGCGCt
tttgGCtgggagaGCGCtGCttttgaGCGCtggattGCttaatgt aggtc
ggatcGCctaacgtagtcgtccttctg agttgtttttttg 5'-caatacccgaaacgacgacg
5'-ttcgtcgcgagagggtcggtt 5'-tttttgttgatgtaattcgttagg tc
5'-attacaaaccgaccctctcg top
5'-agatcccaagggtCGtagcccctggcCGtgtggacCGg 25,101,909- This sequence
is for measuring CpG methylation 5'-agatcccaagggtcgtagc strand
gtctgCGgctgcagagCGCGgtccCGgctgcagcaagacctgg 25,101,995 levels using
methylation sensitive restriction 5'-actgccccaggtcttgct ggcagt
((SEQ ID No: 2) enzymes (e.g. HbaII, HhaI (underlined) minus
3'-tctagggttcccaGCatcggggaccgGCacacctgGCc strand
cagacGCcgacgtctcGCGCcaggGCcgacgtcgttctggacc ccgtca IKZF1 top
5'-gaCGaCGcaccctctcCGtgtccCGctctgCGccctt 50,343,867-
5'-gaCGaCGtatttttttCGtgtttCGttttgCGtttttttgCGCG
5'-gacgacgtatttttttcgtgtttc strand
ctgCGCGcccCGctccctgtacCGgagcagCGatcCGggag 50,343,961
tttCGttttttgtatCGgagtagCGattCGggaggCGgtCGagagg 5'-gcgcacctctcgaccg
gCGgcCGagaggtgCGc (SEQ ID No: 3) tgCGt 5'-tttgtatcggagtagcgattcggg
ag minus 3'-ctGCtGCgtgggagagGCacaggGCgagacGCgg
3'-ttGCtGCgtgggagagGCataggGCgagatGCgggaa strand
gaagacGCGCgggGCgagggacatgGCctcgtcGCtagGC
gatGCGCgggGCgagggatatgGCtttgttGCtagGCtttttGCt cctccGCcgGCtctccacGCg
gGCtttttatGCg top 5'-cCGgagttgCGgctgagaCGCGCGcCGCGCG 50,343,804-
This sequence is for measuring CpG methylation 5'-ggagttgcggctgagac
strand agcCGggggactCGgCGaCGgggCGgggaCGggaCGa 50,343,895 levels
using methylation sensitive restriction 5'-agagcgggacacggaga
CGcaccctctcCGtgtccCGctct (SEQ ID No: 4) enzymes (e.g. HbaII, HhaI
(underlined) minus 3'-gGCctcaacGCcgactctGCGCGCgGCGCGC strand
tcgGCcccctgaGCcGCtGCcccGCccctGCcctGCtGC gtgggagagGCacaggGCgaga IRF4
top 5'-CGcctgccctcCGCGctcctgCGaCGgggtCGcc 392,036-
5'-CGtttgtttttCGCGtttttgCGaCGgggtCGttataagttg 5'- gtttttgcgacggggtc
strand acaagctggaCGggatgagctaacCGgactgtCGgggccccag 392,145
gaCGggatgagttaatCGgattgtCGgggttttaggagtggttgagg
5'-taaaaccccgacaatccg gagtggctgaggCGgggcCGtccaaggcaccca (SEQ ID
CGgggtCGtttaaggtattta No: 5) minus
3'-GCggacgggagGCGCgaggacGCtGCcccaGC
3'-GCggatgggagGCGCgaggatGCtGCtttaGCggtgtttg 5'-tgggtgttttggacggtttc
strand ggtgttcgacctGCcctactcgattgGCctgacaGCcccggggtc
atttGCtttatttgattgGCttgataGCtttggggtttttattgat
5'-tagttatttttggggtttcgatagt ctcaccgactccGCcccgGCaggttccgtgggt
tttGCtttgGCaggttttgtgggt tc 5'-cgcctaccctccgcg
5'-tcgtttagtttgtggcgatttcgt cg GRASP top
5'-caggaagctgcagcagaaggaggaggCGgCGgcca 52,400,821-
5'-taggaagttgtagtagaaggaggaggCGgCGgttatttCGga 5'-cggaagtcgcgttcgtc
strand cccCGgacccCGcCGccCGgactccCGactCGgaagtCG 52,401,051
tttCGtCGttCGgattttCGattCGgaagtCGCGttCGtCGttt
5'-gcgtacaactcgtccgctaa CGccCGcCGctcCGgtccCGacccCGggaccccctgcCG
CGgtttCGatttCGggattttttgtCGtagtCGttatttttgggtt
5'-ttcgatttcgggattttttgtcgt
cagcCGccacccctgggcccccagCGgaCGagctgtaCGCG
tttagCGgaCGagttgtaCGCGgCGttggaggattattattttgtCG agtc
gCGctggaggactatcaccctgcCGagctgtacCGCGCGctC
agttgtatCGCGCGttCGtCGtgttCGggggtattttgtttCGtCGa
5'-cggattttcgattcggaagt GcCGtgtcCGggggcaccctgcccCGcCGaaaggtgCGtcc
aaggtgCGtttttCGttCGtttttaggatttgtttagttttttttCG
ccCGccCGccttcaggatctgctcagcccctctcCGactccctaca
attttttatagggtttgttgatttCG gggcctgctgactcCG (SEQ ID No: 6) minus
3'-gtccttcgacgtcgtcttcctcctccGCcGCcggtgggG
3'-gttttttgatgttgttttttttttttGCtGCtggtgggGCttggg
5'-ggtagggtgttttcggatac strand
CctgggGCgGCggGCctgaggGCtgaGCctcaGCGCgg
GCgGCggGCttgaggGCtgaGCtttaGCGCggGCgGCgagG 5'-aacgaacgaactatacgcgac
GCgGCgagGCcaggGCtgggGCcctgggggacgGCgtcg
CtaggGCtgggGCtttgggggatgGCgttgGCggtggggatttgg
GCggtggggacccgggggtcGCctGCtcgacatGCGCcGC
gggttGCttGCttgatatGCGCtGCgattttttgatagtgggatgGC
gacctcctgatagtgggacgGCtcgacatgGCGCGCgaGCg
ttgatatgGCGCGCgaGCgGCatagGCttttgtgggatgggGC
GCacagGCccccgtgggacgggGCgGCtttccacGCagggg
gGCtttttatGCaggggGCggGCggaagttttagatgagttgggga
GCggGCggaagtcctagacgagtcggggagagGCtgagggatg
gagGCtgagggatgttttggatgattgagGC tcccggacgactgagGC top
5'-gacagagacagccccaggcaagttgaaggtcCGagagc 52,401,407-
5'-gatagagatagttttaggtaagttgaaggttCGagagttttCGgt strand
cccCGgtgggagaagCGggcCGgtggctgCGcCGCGtgC 52,401,664
gggagaagCGggtCGgtggttgCGtCGCGtgCGtttttattttgagg
GttctcactctgaggaagtgCGtggggagcCGctgactcCGgata
aagtgCGtggggagtCGttgatttCGgatagtatattttttCGagggg
gcacacccttcCGaggggactcccCGattcctgggctgggggcct
atttttCGatttttgggttgggggtttgtCGtttggttttaCGtttga
gcCGcctggccccaCGtctgaCGtaCGgggCGCGagggcc
CGtaCGgggCGCGagggttattgttttttggatttttgtCGgaatCGg
actgctccctggacttctgtCGgaacCGgaCGcagtgggaggggt
aCGtagtgggaggggtCGtagg CGcagg (SEQ ID No: 7) minus
3'-ctgtctctgtcggggtccgttcaacttccagGCtctcgggg
3'-ttgtttttgttggggtttgtttaatttttagGCttttggggGCtat
5'-cggagttagcggttttttacg strand
GCcaccctcttcGCccgGCcaccgacGCgGCGCacGCaa
tttttttGCttgGCtattgatGCgGCGCatGCaagagtgagattttttt
5'-cgataaaaaaaacgaaccga
gagtgagactccttcacGCacccctcgGCgactgagGCctatcgt
atGCattttttgGCgattgagGCttattgtgtgggaagGCttttttgag
5'-agagtgagaacgtacgcggc
gtgggaagGCtcccctgagggGCtaaggacccgacccccggacg
ggGCtaaggatttgatttttggatgGCggattggggtGCagattGCatG
GCggaccggggtGCagactGCatGCcccGCGCtcccggtg
CtttGCGCttttggtgatgagggatttgaagataGCtttgGCttGCgtt
acgagggacctgaagacaGCcttgGCctGCgtcaccctccccaG attttttttaGCgttt Cgtcc
top 5'-gacagagacagccccaggcaagttgaaggtcCGagag This sequence is for
measuring CpG methylation 5'-caagttgaaggtccgagagc strand
ccccCGgtgggagaagCGggcCGgtggctgCGcCGCGtg levels using methylation
sensitive restriction 5'-cgcacttcctcagagtgaga
CGttctcactctgaggaagtgCGtggggagcCGctgactcCGga enzymes (e.g. HbaII,
HhaI (underlined) tagcacacccttcCGaggggactcccCGattcctgggctgggggcc
tgcCGcctggccccaCGtctgaCGtaCGgggCGCGagggc
cactgctccctggacttctgtCGgaacCGgaCGcagtgggaggg gtCGcagg minus
3'-ctgtctctgtcggggtccgttcaacttccagGCtctcgggg strand
GCcaccctcttcGCccgGCcaccgacGCgGCGCacGCaa
gagtgagactccttcacGCacccctcgGCgactgagGCctatcgt
gtgggaagGCtcccctgagggGCtaaggacccgacccccggacg
GCggaccggggtGCagactGCatGCcccGCGCtcccggtg
acgagggacctgaagacaGCcttgGCctGCgtcaccctccccaG Cgtcc CAHM top
5'-atctgtaaaaatgttgacttctgcttttcagactaCGCGcac 163,834,295
5'-atttgtaaaaatgttgatttttgttttttagattaCGCGtatagtt
5'-gaaggaagtatttcgagtacgatt strand
agcctctttatttcctactgCGgcttcattccctcaCGgaacactg 163,834,500
tttttattttttattgCGgttttatttttttaCGgaatattgaCGttat gacc
aCGccatCGCGaaggaagcatttCGagcaCGactgaCGctcccc
CGCGaaggaagtatttCGagtaCGattgaCGttttttttattatttgtt
5'-cccgaacgcaacgacttaa
ttattatttgctaagcCGctgCGctCGggtctggctaCGatttgct
aagtCGttgCGttCGggtttggttaCGatttgtttttagaataaCGgga
5'-gcctctaaaaaaacgatcttatta ttcagaataaCGggaaggtgcaacaaga (SEQ ID
No: 8) aggtgtaataaga cacc minus
3'-tagacatttttacaactgaagacgaaaagtctgatGCGCg
3'-tagatatttttataattgaagatgaaaagtttgatGCGCgtgttgg
5'-gaaacactaacgccatcg strand
tgtcggagaaataaaggatgacGCcgaagtaagggagtGCcttgtg
agaaataaaggatgatGCtgaagtaagggagtGCtttgtgattGCggta
5'-cgtagttagattcgagcgtag
actGCggtaGCGCttccttcgtaaaGCtcgtGCtgactGCgag
GCGCtttttttgtaaaGCttgtGCtgattGCgaggggaataataaatg
5'-aggggagcgttagtcgtgttcg
gggaataataaacgattcgGCgacGCgaGCccagaccgatGCt
atttgGCgatGCgaGCttagattgatGCtaaatgaaagttttattGCtt aaa
aaacgaaagtcttattGCccttccacgttgact ttttatgttgtttt minus
3'-cgGCacgacgaaaggtcggagagtcgtttagtGCttgt 163,834,621
tgGCatgatgaaaggttggagagttgtttagtGCttgtgGCtttttttg
5'-gtttttttcggcgataaagc strand
gGCtttcttcggtGCcGCcGCtGCcctccccGCaGCGCG 163,834,906
gtGCtGCtGCtGCtttttttGCaGCGCGCatgaagggaGCtGC 5'-cgcctctacgaaactctacg
CacgaagggaGCcGCtgtttcGCcctcgGCccGCGCgGC
tgttttGCttttgGCttGCGCgGCtgGCttttGCggGCtGCgttt 5'-cgtcggtcgagggcgttc
cgGCtcccGCggGCcGCgtctcaggGCgtctccGCctGCg
taggGCgtttttGCttGCgGCGCtgtGCGCggaGCtttttggag
GCGCcgtGCGCggaGCttttcggagtttgagaataggaGCcg
tttgagaataggaGCtgagaggGCggggtggagGCgggGCgttg
agaggGCggggtggagGCgggGCgtcggttctggGCGCgG
gttttggGCGCgGCattGCttggGCtGCtggttttttttgggtggttg
CaccGCccggGCtGCcggttcctttcgggtggtcgggagGC ggagGC tgGCat tgGCac
BIBLIOGRAPHY
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DNA Laboratory Manual, (Acad. Press, 1988)
Sequence CWU 1
1
891102DNAArtificial sequenceSynthetic construct 1cagtgcccga
ggcggcggcg agtacacgtg gcgggctgga ttgcagaccg gccctctcgc 60ggcggagact
cgcgacctag cggattgcat cagcaggaag ac 102287DNAArtificial
sequenceSynthetic construct 2agatcccaag ggtcgtagcc cctggccgtg
tggaccgggt ctgcggctgc agagcgcggt 60cccggctgca gcaagacctg gggcagt
87395DNAArtificial sequenceSynthetic construct 3gacgacgcac
cctctccgtg tcccgctctg cgcccttctg cgcgccccgc tccctgtacc 60ggagcagcga
tccgggaggc ggccgagagg tgcgc 95492DNAArtificial sequenceSynthetic
construct 4ccggagttgc ggctgagacg cgcgccgcgc gagccggggg actcggcgac
ggggcgggga 60cgggacgacg caccctctcc gtgtcccgct ct
925110DNAArtificial sequenceSynthetic construct 5cgcctgccct
ccgcgctcct gcgacggggt cgccacaagc tggacgggat gagctaaccg 60gactgtcggg
gccccaggag tggctgaggc ggggccgtcc aaggcaccca 1106299DNAArtificial
sequenceSynthetic construct 6caggaagctg cagcagaagg aggaggcggc
ggccaccccg gaccccgccg cccggactcc 60cgactcggaa gtcgcgcccg ccgctccggt
cccgaccccg ggaccccctg ccgcagccgc 120cacccctggg cccccagcgg
acgagctgta cgcggcgctg gaggactatc accctgccga 180gctgtaccgc
gcgctcgccg tgtccggggg caccctgccc cgccgaaagg tgcgtccccc
240gcccgccttc aggatctgct cagcccctct ccgactccct acagggcctg ctgactccg
2997258DNAArtificial sequenceSynthetic construct 7gacagagaca
gccccaggca agttgaaggt ccgagagccc ccggtgggag aagcgggccg 60gtggctgcgc
cgcgtgcgtt ctcactctga ggaagtgcgt ggggagccgc tgactccgga
120tagcacaccc ttccgagggg actccccgat tcctgggctg ggggcctgcc
gcctggcccc 180acgtctgacg tacggggcgc gagggccact gctccctgga
cttctgtcgg aaccggacgc 240agtgggaggg gtcgcagg 2588206DNAArtificial
sequenceSynthetic construct 8atctgtaaaa atgttgactt ctgcttttca
gactacgcgc acagcctctt tatttcctac 60tgcggcttca ttccctcacg gaacactgac
gccatcgcga aggaagcatt tcgagcacga 120ctgacgctcc ccttattatt
tgctaagccg ctgcgctcgg gtctggctac gatttgcttt 180cagaataacg
ggaaggtgca acaaga 206927DNAArtificial sequenceSynthetic construct
9gaaggaagta tttcgagtac gattgac 271019DNAArtificial
sequenceSynthetic construct 10cccgaacgca acgacttaa
191117DNAArtificial sequenceSynthetic construct 11cggaagtcgc
gttcgtc 171220DNAArtificial sequenceSynthetic construct
12gcgtacaact cgtccgctaa 201328DNAArtificial sequenceSynthetic
construct 13ttcgatttcg ggattttttg tcgtagtc 281420DNAArtificial
sequenceSynthetic construct 14tgggtgtttt ggacggtttc
201515DNAArtificial sequenceSynthetic construct 15cgcctaccct ccgcg
151626DNAArtificial sequenceSynthetic construct 16tcgtttagtt
tgtggcgatt tcgtcg 261729DNAArtificial sequenceSynthetic construct
17gtttttttgt tgatgtaatt cgttaggtc 291820DNAArtificial
sequenceSynthetic construct 18caatacccga aacgacgacg
201921DNAArtificial sequenceSynthetic construct 19ttcgtcgcga
gagggtcggt t 212024DNAArtificial sequenceSynthetic construct
20gacgacgtat ttttttcgtg tttc 242116DNAArtificial sequenceSynthetic
construct 21gcgcacctct cgaccg 162221DNAArtificial sequenceSynthetic
construct 22taagagtaat aatggatgga t 212317DNAArtificial
sequenceSynthetic construct 23cctcccatct cccttcc
172425DNAArtificial sequenceSynthetic construct 24atggatgaag
aaagaaagga tgagt 252524DNAArtificial sequenceSynthetic construct
25gtgatggagg aggtttagta agtt 242625DNAArtificial sequenceSynthetic
construct 26ccaataaaac ctactcctcc cttaa 252730DNAArtificial
sequenceSynthetic construct 27accaccaccc aacacacaat aacaaacaca
3028102DNAArtificial sequenceSynthetic construct 28tagtgttcga
ggcggcggcg agtatacgtg gcgggttgga ttgtagatcg gttttttcgc 60ggcggagatt
cgcgatttag cggattgtat tagtaggaag at 10229102DNAArtificial
sequenceSynthetic construct 29gtcacgggct ccgccgccgc tcatgtgcac
cgcccgacct aacgtctggc cgggagagcg 60ccgcctctga gcgctggatc gcctaacgta
gtcgtccttc tg 10230102DNAArtificial sequenceSynthetic construct
30gttatgggct ttgctgctgc ttatgtgcat tgcttgattt aatgtttggc tgggagagcg
60ctgcttttga gcgctggatt gcttaatgta gttgtttttt tg
1023129DNAArtificial sequenceSynthetic construct 31gtttttttgt
tgatgtaatt cgttaggtc 293220DNAArtificial sequenceSynthetic
construct 32caatacccga aacgacgacg 203321DNAArtificial
sequenceSynthetic construct 33ttcgtcgcga gagggtcggt t
213426DNAArtificial sequenceSynthetic construct 34tttttgttga
tgtaattcgt taggtc 263520DNAArtificial sequenceSynthetic construct
35attacaaacc gaccctctcg 203619DNAArtificial sequenceSynthetic
construct 36agatcccaag ggtcgtagc 193718DNAArtificial
sequenceSynthetic construct 37actgccccag gtcttgct
183887DNAArtificial sequenceSynthetic construct 38tctagggttc
ccagcatcgg ggaccggcac acctggccca gacgccgacg tctcgcgcca 60gggccgacgt
cgttctggac cccgtca 873995DNAArtificial sequenceSynthetic construct
39gacgacgtat ttttttcgtg tttcgttttg cgtttttttg cgcgtttcgt tttttgtatc
60ggagtagcga ttcgggaggc ggtcgagagg tgcgt 954024DNAArtificial
sequenceSynthetic construct 40gacgacgtat ttttttcgtg tttc
244115DNAArtificial sequenceSynthetic construct 41gcgcacctct cgacc
154226DNAArtificial sequenceSynthetic construct 42tttgtatcgg
agtagcgatt cgggag 264395DNAArtificial sequenceSynthetic construct
43ctgctgcgtg ggagaggcac agggcgagac gcgggaagac gcgcggggcg agggacatgg
60cctcgtcgct aggccctccg ccggctctcc acgcg 954495DNAArtificial
sequenceSynthetic construct 44ttgctgcgtg ggagaggcat agggcgagat
gcgggaagat gcgcggggcg agggatatgg 60ctttgttgct aggctttttg ctggcttttt
atgcg 954517DNAArtificial sequenceSynthetic construct 45ggagttgcgg
ctgagac 174617DNAArtificial sequenceSynthetic construct
46agagcgggac acggaga 174792DNAArtificial sequenceSynthetic
construct 47ggcctcaacg ccgactctgc gcgcggcgcg ctcggccccc tgagccgctg
ccccgcccct 60gccctgctgc gtgggagagg cacagggcga ga
9248110DNAArtificial sequenceSynthetic construct 48cgtttgtttt
tcgcgttttt gcgacggggt cgttataagt tggacgggat gagttaatcg 60gattgtcggg
gttttaggag tggttgaggc ggggtcgttt aaggtattta 1104917DNAArtificial
sequenceSynthetic construct 49gtttttgcga cggggtc
175018DNAArtificial sequenceSynthetic construct 50taaaaccccg
acaatccg 1851110DNAArtificial sequenceSynthetic construct
51gcggacggga ggcgcgagga cgctgcccca gcggtgttcg acctgcccta ctcgattggc
60ctgacagccc cggggtcctc accgactccg ccccggcagg ttccgtgggt
11052110DNAArtificial sequenceSynthetic construct 52gcggatggga
ggcgcgagga tgctgcttta gcggtgtttg atttgcttta tttgattggc 60ttgatagctt
tggggttttt attgattttg ctttggcagg ttttgtgggt 1105320DNAArtificial
sequenceSynthetic construct 53tgggtgtttt ggacggtttc
205427DNAArtificial sequenceSynthetic construct 54tagttatttt
tggggtttcg atagttc 275515DNAArtificial sequenceSynthetic construct
55cgcctaccct ccgcg 155626DNAArtificial sequenceSynthetic construct
56tcgtttagtt tgtggcgatt tcgtcg 2657299DNAArtificial
sequenceSynthetic construct 57taggaagttg tagtagaagg aggaggcggc
ggttatttcg gatttcgtcg ttcggatttt 60cgattcggaa gtcgcgttcg tcgtttcggt
ttcgatttcg ggattttttg tcgtagtcgt 120tatttttggg tttttagcgg
acgagttgta cgcggcgttg gaggattatt attttgtcga 180gttgtatcgc
gcgttcgtcg tgttcggggg tattttgttt cgtcgaaagg tgcgtttttc
240gttcgttttt aggatttgtt tagttttttt tcgatttttt atagggtttg ttgatttcg
2995817DNAArtificial sequenceSynthetic construct 58cggaagtcgc
gttcgtc 175920DNAArtificial sequenceSynthetic construct
59gcgtacaact cgtccgctaa 206028DNAArtificial sequenceSynthetic
construct 60ttcgatttcg ggattttttg tcgtagtc 286120DNAArtificial
sequenceSynthetic construct 61cggattttcg attcggaagt
2062298DNAArtificial sequenceSynthetic construct 62gtccttcgac
gtcgtcttcc tcctccgccg ccggtggggc ctggggcggc gggcctgagg 60gctgagcctc
agcgcgggcg gcgaggccag ggctggggcc ctgggggacg gcgtcggcgg
120tggggacccg ggggtcgcct gctcgacatg cgccgcgacc tcctgatagt
gggacggctc 180gacatggcgc gcgagcggca caggcccccg tgggacgggg
cggctttcca cgcagggggc 240gggcggaagt cctagacgag tcggggagag
gctgagggat gtcccggacg actgaggc 29863298DNAArtificial
sequenceSynthetic construct 63gttttttgat gttgtttttt ttttttgctg
ctggtggggc ttggggcggc gggcttgagg 60gctgagcttt agcgcgggcg gcgaggctag
ggctggggct ttgggggatg gcgttggcgg 120tggggatttg ggggttgctt
gcttgatatg cgctgcgatt ttttgatagt gggatggctt 180gatatggcgc
gcgagcggca taggcttttg tgggatgggg cggcttttta tgcagggggc
240gggcggaagt tttagatgag ttggggagag gctgagggat gttttggatg attgaggc
2986420DNAArtificial sequenceSynthetic construct 64ggtagggtgt
tttcggatac 206521DNAArtificial sequenceSynthetic construct
65aacgaacgaa ctatacgcga c 2166258DNAArtificial sequenceSynthetic
construct 66gatagagata gttttaggta agttgaaggt tcgagagttt tcggtgggag
aagcgggtcg 60gtggttgcgt cgcgtgcgtt tttattttga ggaagtgcgt ggggagtcgt
tgatttcgga 120tagtatattt tttcgagggg atttttcgat ttttgggttg
ggggtttgtc gtttggtttt 180acgtttgacg tacggggcgc gagggttatt
gttttttgga tttttgtcgg aatcggacgt 240agtgggaggg gtcgtagg
25867258DNAArtificial sequenceSynthetic construct 67ctgtctctgt
cggggtccgt tcaacttcca ggctctcggg ggccaccctc ttcgcccggc 60caccgacgcg
gcgcacgcaa gagtgagact ccttcacgca cccctcggcg actgaggcct
120atcgtgtggg aaggctcccc tgaggggcta aggacccgac ccccggacgg
cggaccgggg 180tgcagactgc atgccccgcg ctcccggtga cgagggacct
gaagacagcc ttggcctgcg 240tcaccctccc cagcgtcc 25868258DNAArtificial
sequenceSynthetic construct 68ttgtttttgt tggggtttgt ttaattttta
ggcttttggg ggctattttt tttgcttggc 60tattgatgcg gcgcatgcaa gagtgagatt
tttttatgca ttttttggcg attgaggctt 120attgtgtggg aaggcttttt
tgaggggcta aggatttgat ttttggatgg cggattgggg 180tgcagattgc
atgctttgcg cttttggtga tgagggattt gaagatagct ttggcttgcg
240ttattttttt tagcgttt 2586921DNAArtificial sequenceSynthetic
construct 69cggagttagc ggttttttac g 217020DNAArtificial
sequenceSynthetic construct 70cgataaaaaa aacgaaccga
207120DNAArtificial sequenceSynthetic construct 71agagtgagaa
cgtacgcggc 2072258DNAArtificial sequenceSynthetic construct
72gacagagaca gccccaggca agttgaaggt ccgagagccc ccggtgggag aagcgggccg
60gtggctgcgc cgcgtgcgtt ctcactctga ggaagtgcgt ggggagccgc tgactccgga
120tagcacaccc ttccgagggg actccccgat tcctgggctg ggggcctgcc
gcctggcccc 180acgtctgacg tacggggcgc gagggccact gctccctgga
cttctgtcgg aaccggacgc 240agtgggaggg gtcgcagg 2587319DNAArtificial
sequenceSynthetic construct 73caagttgaag gtccgagag
197420DNAArtificial sequenceSynthetic construct 74cgcacttcct
cagagtgaga 2075258DNAArtificial sequenceSynthetic construct
75ctgtctctgt cggggtccgt tcaacttcca ggctctcggg ggccaccctc ttcgcccggc
60caccgacgcg gcgcacgcaa gagtgagact ccttcacgca cccctcggcg actgaggcct
120atcgtgtggg aaggctcccc tgaggggcta aggacccgac ccccggacgg
cggaccgggg 180tgcagactgc atgccccgcg ctcccggtga cgagggacct
gaagacagcc ttggcctgcg 240tcaccctccc cagcgtcc 25876206DNAArtificial
sequenceSynthetic construct 76atttgtaaaa atgttgattt ttgtttttta
gattacgcgt atagtttttt tattttttat 60tgcggtttta tttttttacg gaatattgac
gttatcgcga aggaagtatt tcgagtacga 120ttgacgtttt ttttattatt
tgttaagtcg ttgcgttcgg gtttggttac gatttgtttt 180tagaataacg
ggaaggtgta ataaga 2067728DNAArtificial sequenceSynthetic construct
77gaaggaagta tttcgagtac gattgacc 287819DNAArtificial
sequenceSynthetic construct 78cccgaacgca acgacttaa
197928DNAArtificial sequenceSynthetic construct 79gcctctaaaa
aaacgatctt attacacc 2880206DNAArtificial sequenceSynthetic
construct 80tagacatttt tacaactgaa gacgaaaagt ctgatgcgcg tgtcggagaa
ataaaggatg 60acgccgaagt aagggagtgc cttgtgactg cggtagcgct tccttcgtaa
agctcgtgct 120gactgcgagg ggaataataa acgattcggc gacgcgagcc
cagaccgatg ctaaacgaaa 180gtcttattgc ccttccacgt tgttct
20681206DNAArtificial sequenceSynthetic construct 81tagatatttt
tataattgaa gatgaaaagt ttgatgcgcg tgttggagaa ataaaggatg 60atgctgaagt
aagggagtgc tttgtgattg cggtagcgct ttttttgtaa agcttgtgct
120gattgcgagg ggaataataa atgatttggc gatgcgagct tagattgatg
ctaaatgaaa 180gttttattgc ttttttatgt tgtttt 2068218DNAArtificial
sequenceSynthetic construct 82gaaacactaa cgccatcg
188321DNAArtificial sequenceSynthetic construct 83cgtagttaga
ttcgagcgta g 218425DNAArtificial sequenceSynthetic construct
84aggggagcgt tagtcgtgtt cgaaa 2585285DNAArtificial
sequenceSynthetic construct 85cggcacgacg aaaggtcgga gagtcgttta
gtgcttgtgg ctttcttcgg tgccgccgct 60gccctccccg cagcgcgcac gaagggagcc
gctgtttcgc cctcggcccg cgcggccggc 120tcccgcgggc cgcgtctcag
ggcgtctccg cctgcggcgc cgtgcgcgga gcttttcgga 180gtttgagaat
aggagccgag agggcggggt ggaggcgggg cgtcggttct gggcgcggca
240ccgcccgggc tgccggttcc tttcgggtgg tcgggaggct ggcac
28586285DNAArtificial sequenceSynthetic construct 86tggcatgatg
aaaggttgga gagttgttta gtgcttgtgg ctttttttgg tgctgctgct 60gctttttttg
cagcgcgcat gaagggagct gctgttttgc ttttggcttg cgcggctggc
120ttttgcgggc tgcgttttag ggcgtttttg cttgcggcgc tgtgcgcgga
gctttttgga 180gtttgagaat aggagctgag agggcggggt ggaggcgggg
cgttggtttt gggcgcggca 240ttgcttgggc tgctggtttt ttttgggtgg
ttgggaggct ggcat 2858720DNAArtificial sequenceSynthetic construct
87gtttttttcg gcgataaagc 208820DNAArtificial sequenceSynthetic
construct 88cgcctctacg aaactctacg 208918DNAArtificial
sequenceSynthetic construct 89cgtcggtcga gggcgttc 18
* * * * *
References