U.S. patent application number 09/977876 was filed with the patent office on 2002-08-29 for methods for disease diagnosis from stool samples.
Invention is credited to Lapidus, Stanley N., Shuber, Anthony P..
Application Number | 20020119472 09/977876 |
Document ID | / |
Family ID | 22024768 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119472 |
Kind Code |
A1 |
Lapidus, Stanley N. ; et
al. |
August 29, 2002 |
Methods for disease diagnosis from stool samples
Abstract
The present invention provides methods for preparing a stool
sample in order to screen for the presence of indicators of a
disease, for example a subpopulation of cancerous or precancerous
cells. The methods take advantage of the recognition that cellular
debris from cancerous and precancerous cells is deposited onto only
a longitudinal stripe of stool as the stool is forming in the
colon. Accordingly, methods of the invention comprise obtaining a
representative sample, such as a circumferential or cross-sectional
sample of stool in order to ensure that any disease indicator, such
as cellular debris that is shed by colonic cells, is obtained in
the sample.
Inventors: |
Lapidus, Stanley N.;
(Bedford, NH) ; Shuber, Anthony P.; (Milford,
MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
22024768 |
Appl. No.: |
09/977876 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09977876 |
Oct 15, 2001 |
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09243311 |
Feb 2, 1999 |
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6303304 |
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09243311 |
Feb 2, 1999 |
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09059713 |
Apr 13, 1998 |
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5952178 |
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09059713 |
Apr 13, 1998 |
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08699678 |
Aug 14, 1996 |
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5741650 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2521/537 20130101;
C12Q 1/6806 20130101; C12Q 2600/156 20130101; C12Q 1/6827 20130101;
C12Q 1/6806 20130101; G01N 33/57419 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of screening for the presence of a disease in a
patient, the method comprising the steps of: a) obtaining a sample
comprising a circumferential surface of a stool voided by a
patient; and b) performing an assay to detect in the sample
characteristics indicative of the presence of said disease.
2. A method for screening for the presence of a colorectal cancer
or precancerous lesion in a patient, the method comprising the
steps of: a) obtaining a stool voided by a patient; b) removing a
circumferential surface from said stool; and c) performing an assay
on said surface to detect characteristics indicative of the
presence of cells or cellular debris shed onto said stool.
3. The method of claim 2, wherein said assay detects debris from a
clonal population of transformed cells comprising said lesion.
4. The method of claim 3, wherein said assay detects a protein
expressed by the transformed cells indicative of the presence of
said cells.
5. The method of claim 3, wherein said assay detects a DNA
characteristic indicative of the presence of said cells.
6. The method of claim 1, further comprising the step of
homogenizing said portion in a physiologically compatible buffer
prior to step (b).
7. The method of claim 6, wherein said physiologically compatible
buffer comprises a detergent and a proteinase.
8. The method of claim 1, wherein said assay detects the presence
of carcinoembryonic antigen shed from said lesion.
9. The method of claim 1, wherein said assay comprises the step of
exposing said sample to an antibody which specifically binds a
molecule characteristic of the presence of said debris.
10. The method of claim 5, wherein said DNA characteristic is a
mutation.
11. The method of claim 10, wherein said mutation is selected from
the group consisting of loss of heterozygosity and microsatellite
instability.
12. The method of claim 10, wherein said characteristics comprise a
deletion in a tumor suppressor allele.
13. The method of claim 1, wherein said assay comprises the step of
determining whether a difference exists in said sample between a
number X of a first allele known or suspected to be mutated in a
subpopulation of cells in the sample and a number Y of a second
allele that is known or suspected not to be mutated in a
subpopulation of cells in the sample, the presence of a
statistically-significant difference being indicative of a mutation
in a subpopulation of cells in the sample and the potential
presence of a cancerous or precancerous lesion.
14. The method of claim 1, wherein said assay comprises the step of
determining whether a difference exists between a number of a
target tumor suppressor allele in the sample and a number of a
non-cancer-associated reference allele in the sample, the presence
of a statistically-significant difference being indicative of a
deletion of the target tumor suppressor allele in a subpopulation
of cells in the sample and the potential presence of a cancerous or
precancerous lesion.
15. The method of claim 5, wherein said characteristics comprise a
loss of heterozygosity encompassing a polymorphic locus.
16. The method of claim 13, wherein said assay comprises the steps
of: a) detecting an amount of a maternal allele at a polymorphic
locus in the biological sample; b) detecting an amount of a
paternal allele at the polymorphic locus in the biological sample;
and c) determining whether a difference exists between the amounts
of maternal and paternal allele, the presence of a
statistically-significant difference being indicative of a deletion
at the polymorphic locus in a subpopulation of cells in the
biological sample and the potential presence of a lesion.
17. The method of claim 1, comprising the additional step of
performing a visual examination of the colon of a patient
presenting positive assay results.
18. The method of claim 1, wherein said at least cross-sectional
portion comprises an entire stool voided by a patient.
19. A method for reducing morbidity from colorectal cancer in a
population, the method comprising the steps of: a) obtaining a
sample comprising at least a representative portion of a stool
voided by a patient; b) performing an assay to detect in the sample
the presence of cellular debris shed from the lesion into the
voided stool; and c) performing a visual examination of the colon
of a patient presenting positive results in said assay to detect
the presence of a lesion.
20. The method of claim 19, wherein said assay detects a loss of
DNA in a portion of debris shed from a clonal population of
transformed epithelial cells comprising said lesion.
Description
[0001] This patent application is a continuation-in-part of U.S.
Ser. No. 08/699,678, filed Aug. 14, 1996, the disclosure of which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods for disease diagnosis,
including the early detection of colon cancer in patients, and more
particularly to methods for preparing stool samples for disease
diagnosis, including the detection of colon cancer,-so as to assure
or increase the likelihood that the sample will contain the
diagnostically relevant information if the patient has a disease,
for example a cancerous or precancerous lesion, and to methods for
stool sample analysis.
BACKGROUND OF THE INVENTION
[0003] Stool samples frequently must be prepared for medical
diagnostic analysis. Stool samples may be analyzed to help diagnose
medical conditions ranging from parasitic, bacterial or viral
infections to inflammatory bowel disease and colorectal cancer.
[0004] Colorectal cancer is a leading cause of death in Western
society. However, if diagnosed early, it may be treated effectively
by surgical removal of the cancerous tissue. Colorectal cancers
originate in the colorectal epithelium and typically are not
extensively vascularized (and therefore not invasive) during the
early stages of development. Colorectal cancer is thought to result
from the clonal expansion of a single mutant cell in the epithelial
lining of the colon or rectum. The transition to a highly
vascularized, invasive and ultimately metastatic cancer which
spreads throughout the body commonly takes ten years or longer. If
the cancer is detected prior to invasion, surgical removal of the
cancerous tissue is an effective cure. However, colorectal cancer
is often detected only upon manifestation of clinical symptoms,
such as pain and black tarry stool. Generally, such symptoms are
present only when the disease is well established, often after
metastasis has occurred, and the prognosis for the patient is poor,
even after surgical resection of the cancerous tissue. Early
detection of colorectal cancer therefore is important in that
detection may significantly reduce its morbidity.
[0005] Invasive diagnostic methods such as endoscopic examination
allow for direct visual identification, removal, and biopsy of
potentially cancerous growths such as polyps. Endoscopy is
expensive, uncomfortable, inherently risky, and therefore not a
practical tool for screening populations to identify those with
colorectal cancer. Non-invasive analysis of stool samples for
characteristics indicative of the presence of colorectal cancer or
precancer is a preferred alternative for early diagnosis, but no
known diagnostic method is available which reliably achieves this
goal.
[0006] Current non-invasive diagnostic methods involve assaying
stool samples for the presence of fecal occult blood or for
elevated levels of carcinoembryonic antigen, both of which are
suggestive of the presence of colorectal cancer. Additionally,
recent developments in molecular biology provide methods of great
potential for detecting the presence of a range of DNA mutations or
alterations associated with and indicative of the presence of
colorectal cancer. The presence of such mutations theoretically can
be detected in DNA found in stool samples during the early stages
of colorectal cancer. However, stool comprises cells and cellular
debris from the patient, from microorganisms, and from food,
resulting in a heterogeneous population of cells. This makes
detection of a small, specific subpopulation impossible to detect
reliably.
[0007] Stool diagnostic assays for colorectal cancer described in
the art typically are performed on samples prepared from randomly
sampled portions of voided stool. However, samples prepared
according to such methods do not reproducibly yield characteristics
indicative of the presence of colorectal cancer or precancer, even
when prepared from stool voided by a patient with colorectal cancer
or precancer. There is therefore a need in the art for methods for
early diagnosis of colorectal cancer or precancer that will
reproducibly detect characteristics indicative of the presence of
cancerous or precancerous material in samples prepared from stool
voided by a patient with colorectal cancer or precancer. Such
methods are provided herein.
SUMMARY OF THE INVENTION
[0008] It has now been appreciated that cells and cellular debris
are shed from colonic epithelial cells onto forming stool in a
longitudinal "stripe" of material along the length of the stool.
The shed material is confined to this longitudinal stripe, as shown
in FIG. 1 (designated "C"). Based upon this recognition, Applicants
teach that stool sample preparation for diagnostic testing must
include taking a representative sample in order to ensure that the
sample will contain any cells or cellular debris that was shed into
the stool as it passed through the colon. Accordingly, methods of
the invention comprise obtaining at least a representative (e.g. a
cross-section or circumferential surface) portion of stool voided
by a patient, and performing an assay to detect in the sample the
presence of cells or cellular debris shed from epithelial cells
lining the colon that may be indicative of cancer or precancer.
Most often, such cells will be derived from a polyp or a cancerous
or precancerous lesion at a discrete location along the colon. For
purposes of the present invention, a precancerous lesion comprises
precancerous cells, and precancerous cells are cells that have a
mutation that is associated with cancer and which renders such
cells susceptible to becoming cancerous. As shown in FIG. 1, a
cross-sectional sample is a sample that contains at least a
circumferential surface of the stool (or portion of a stool
comprising an entire cross-sectional portion), as, for example, in
a coronal section or a sagittal section. A sample comprising the
surface layer of a stool (or of a cross-section of a stool) also
contains at least a circumferential surface of the stool. Both
cross-sections and circumferential surfaces comprise longitudinal
stripes of sloughed colonic epithelium, and are therefore
representative samples.
[0009] In a preferred embodiment, methods of the invention comprise
the steps of obtaining at least a circumferential surface or
cross-sectional portion of a stool voided by a patient, and
performing an assay to detect debris indicative of disease. For
example, such debris may comprise a clonal subpopulation of cells
having one or more mutations (for purposes of the present
application, a mutation is a deletion, substitution, addition,
modification, intercalation or rearrangement of DNA). Preferred
methods of the invention comprise detection of characteristics of
such transformed cells, including, for example, mutations, proteins
expressed uniquely or in altered amounts in transformed cells, and
blood. Particularly preferred methods of the invention comprise
obtaining at least a circumferential surface or cross-sectional
portion of a stool sample, and performing an assay to detect DNA
characteristics indicative of the presence of a clonal
subpopulation of cells in the sample. The clonal subpopulation may
be, for example, a subpopulation of cancerous or precancerous
cells, having a mutation in, for example, a p53 tumor suppressor
gene. Clonal subpopulations of cells detected by methods according
to the invention are often characterized by a massive loss of DNA,
resulting in a loss of heterozygosity that renders ineffective the
gene or genes encompassed by the deletion. Alternative methods of
the invention comprise performing an assay to detect the presence
of an infectious disease, for example debris from a
microorganism.
[0010] Methods of the invention also comprise obtaining a
representative (i.e., cross-sectional or circumferential) sample of
stool and homogenizing the stool in a buffer, such as a buffer
comprising a detergent and a proteinase and optionally a DNase
inhibitor.
[0011] In methods according to the invention, an assay performed on
at least a circumferential surface or cross-sectional portion of
stool may be an assay to detect a disease when a diagnostic
indicator of the disease is incorporated into stool. Methods of the
invention are useful to detect cellular debris shed from the
epithelial lining of the colon, and from other tissue sources that
shed cellular material into the gastrointestinal tract and
preferably into the colon. Shed material may be from colonic
epithelial cells, pancreatic cells, or may be indicative of
infection (e.g. bacterial or viral nucleic acids or proteins). In
methods according to the invention, an assay performed on at least
a circumferential surface or cross-sectional portion of stool may
be an assay to detect the presence of elevated levels of
carcinoembryonic antigen shed from cells lining the colon. Such an
assay may also comprise detecting the presence of occult blood.
However, methods of the invention preferably comprise an assay
wherein the sample is exposed to an antibody that specifically
binds to a molecule characteristic of cellular debris shed from
cells comprising a subpopulation of cells having a mutation that is
potentially associated with cancer.
[0012] Methods of the invention are especially and most preferably
useful for detecting DNA characteristics indicative of a
subpopulation of transformed cells in a representative stool
sample. The DNA characteristics may be, for example, mutations,
including loss of heterozygosity, microsatellite instability, and
others. An assay for DNA characteristics in a method of the
invention may comprise the step of determining whether a difference
exists in a number X of a first allele known or suspected to be
mutated in a subpopulation of cells in a representative stool
sample, and a number Y of an allele known or suspected not to be
mutated in the sample, a statistically-significant difference being
indicative of a mutation and the possible presence of cancer in a
subpopulation of cells in the sample. In an embodiment of the
invention, the difference between a number of a tumor suppressor
gene and a number of a non-cancer-associated gene are compared, a
statistically-significant difference in the numbers being
indicative of a mutation in the tumor suppressor gene.
[0013] Assays useful in the practice of methods according to the
invention also include an assay to detect the presence of a
deletion or other mutation in a region encompassing a polymorphic
nucleotide. In such an assay, a number of a polymorphic nucleotide
present at maternal and paternal alleles, wherein the patient is
heterozygous for the polymorphic nucleotide, is determined. A
statistically significant difference between a number of a
polymorphic nucleotide in a maternal allele and a paternal allele
is indicative of the presence of a deletion in one of the two
alleles. Another useful assay is one to detect an infectious
disease.
[0014] Methods of the invention typically include, following sample
preparation and an assay for characteristics of cells or cellular
debris, a visual examination of the colon in order to determine if
a polyp or other lesion is, in fact, present. Finally, surgical
resection of abnormal tissue may be done in order to prevent the
spread of cancerous or precancerous tissue.
[0015] Accordingly, methods of the invention provide means for
screening for the presence of a cancerous or precancerous
subpopulation of cells in a heterogeneous sample, such as a stool
sample. Methods of the invention reduce morbidity and mortality
associated with lesions of the colonic epithelium. Moreover,
methods of the invention comprise more accurate screening methods
than are currently available in the art, because current methods
take advantage of the observation that cancerous or precancerous
cells shed debris only onto or into part of the surface of the
forming stool. The present methods reliably assay over the entire
circumference of the stool, thereby increasing the likelihood of
detecting an abnormality if one exists. Further aspects and
advantages of the invention are contained in the following detailed
description thereof.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a diagram of a cylinder which represents a formed
stool and shows various cross-sections which will contain material
from the entire circumference of a stool. The Section labeled "A"
is a typical coronal section and the section labeled "B" is a
typical sagittal section. The strip labeled "C" represents material
shed from cancerous tissue which is deposited in a longitudinal
stripe.
[0017] FIG. 2 is a schematic diagram of a receptacle for containing
a stool sample.
[0018] FIG. 3 is a schematic diagram of a multi-orifice impedance
counter; wherein reference numeral 1 indicates the direction of
flow through the column; reference numeral 2 indicates a plunger
means for forcing material downward in the column; reference
numerals 3 and 4 are different-sized hybridization beads; reference
numeral 5 is an optional filter for extracting unwanted particles;
reference numeral 6 indicates an array of orifices for measuring
differential impedance; and reference numeral 7 is a collection
chamber.
[0019] FIG. 4 is a diagram showing primers useful for the detection
of single base polymorphisms.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Methods according to the present invention are useful for
the preparation of stool samples that will reproducibly contain
cells or cellular debris shed from a clonal population of cancerous
or precancerous cells, if such a population is present at any site
along the colon of a patient. These samples are then used to
perform assays to detect characteristics indicative of cancer in a
highly-reproducible and accurate way. In preferred methods of the
invention, a sample representative of the surface of a stool is
obtained. Such methods provide an improvement over the art inasmuch
as they teach removing the portion of stool onto which the colonic
epithelium sheds debris. Two preferred methods for obtaining such a
portion are cross-sectioning and extracting the surface of the
stool. In a more preferred method, the surface of a cross-section
is obtained. Without the recognition that at least a
cross-sectional sample must be obtained, there is no means for
reproducibly obtaining a sample that will contain a cancerous or
precancerous subpopulation of cells, if one exists.
[0021] Methods described in the art do not recognize that, unlike
infection by parasites, bacteria and viruses, characteristics
indicative of the presence of colon cancer, especially early stage
colon cancer, are found only in a specific portion of voided stool.
If the sampled portion of stool does not include the portion that
happens to contain cells and cellular debris shed from early-stage
cancer tissue, the diagnostic assay necessarily will fail to detect
the characteristics indicative of the presence of colorectal cancer
in a reliable manner even if homogenized, i.e., will produce a
false-negative result. Methods of the invention are also useful to
detect the presence of bacteria, virus or parasites, as they will
also be present on the surface of formed stool.
[0022] Sloughed cells from, for example, a polyp forming in the
epithelial lining of the colon, or on early stage cancerous
lesions, are sloughed onto only the portion of the forming stool
that comes into contact with the polyp or lesion. Accordingly, in
early stage disease, only a small portion of the surface layer of
the forming stool will contain sloughed cells, and if that portion
happens not to be taken as part of the sample, an assay for indicia
of colon cancer necessarily will produce a false-negative result. A
brief review of the anatomy and physiology of the colon will aid in
an understanding of this phenomenon.
[0023] A typical adult colon is approximately six feet in length,
with a diameter of about two to three inches. Numerous bends and
folds are present throughout its length. The colon removes water
from liquid or semi-liquid waste material that enters the colon,
and relatively solid stool begins to form in the proximal third of
the colon. Epithelial cells line the lumen of the colon, and the
lumenal surface is organized into microscopic crypts. Colorectal
epithelial cells are replaced every four to five days. The
epithelial cells divide rapidly at the base of the crypts and
migrate to the apeces, where cells appear to undergo apoptosis
(programmed cell death), and cellular debris is shed into the
lumen. The lining of the colorectal lumen is elastic and the
diameter of the lumen is determined by the volume of stool that is
passing through the colon at any given time. As a result, the
surface of the forming stool passing through the colon is in direct
contact with the epithelial lining of the lumen. Shed epithelial
cells (which may or may not have undergone apoptosis) and cellular
debris therefore are incorporated onto the surface of stool as it
passes through the colon.
[0024] Cells and cellular debris from colorectal epithelial cancers
therefore also are shed onto forming stool. Most colorectal cancers
develop in regions of the colon where stool is relatively solid,
indeed approximately one third of such cancers develop in the
rectum. Markers indicative of the presence of cancer, including
cells, cellular debris, DNA, blood, and carcinoembryonic antigen,
are shed onto the portion of the forming stool that contacts the
cancerous tissue as the stool passes through the colon. Since the
stool is relatively solid, these markers will remain on or near the
surface of the stool where they were deposited and will not be
homogeneously dispersed throughout the stool. As stool passes over
a cancerous or precancerous growth, material from the growth will
be deposited along the stool, but only on the part of the stool
circumference that comes into direct contact with the cancerous or
precancerous tissue comprising the lesion. Stool voided by a
patient with colorectal cancer or precancer is therefore
characterized by a longitudinal "stripe" of diagnostically relevant
material derived from the cancerous or precancerous tissue.
[0025] A sample that does not include material from the entire
circumference of a stool voided by a patient with colorectal cancer
or precancer will not reproducibly contain material derived from
the cancerous or precancerous tissue. Currently, random,
non-cross-sectional samples ("smears") of voided stool are analyzed
in clinical settings. In these, sloughed cancerous or precancerous
cells and cellular debris have no possibility of detection unless
the sample happens by chance to contain the portion of stool which
made contact with the region of the colon from which cells were
sloughed.
[0026] Furthermore, cancers typically develop by clonal expansion
of a single mutant cell, and in the early stages of the disease,
i.e., when surgical removal is an effective cure, the cancerous
lesion will be very small and may lie on a small arc of the
circumference of the colon. Material derived from such an early
stage cancer therefore will be shed onto or into stool in a very
narrow stripe (labeled C in FIG. 1). Consequently, a sample that
does not contain the entire circumference of a stool voided by a
patient with early stage colorectal cancer or precancer only by
chance will contain material indicative of the presence of the
early stage cancerous or precancerous condition. However, early
detection of colorectal cancer is very important for effective
surgical intervention. The present invention provides Methods for
reproducible early detection of characteristics indicative of the
presence of cancer or precancer in a patient.
[0027] Similarly, the present invention provides methods for
reproducibly detecting an indicator of the presence of an
infectious agent, such as a virus, bacterium, parasite or other
microorganism, in stool from an infected patient. Methods of the
invention are particularly useful for reproducibly detecting an
infectious agent, or an indicator characteristic thereof, that is
deposited onto narrow area of stool as it forms in the colon.
[0028] Analysis of at least a circumferential surface or
cross-sectional sample of stool ensures that a disease indicator,
incorporated on a stool as it moves through the colon, will be
present in a diagnostic assay sample. For example, analysis of at
least a circumferential surface or cross-sectional sample of stool
(including a whole stool), as shown in FIG. 1, ensures that at
least a portion of cells and cellular debris shed from any existing
cancerous or pre-cancerous cells (even if shed from small early
stage cancerous or pre-cancerous tissue, e.g., small polyps) will
be present in the portion of the stool sample to be analyzed.
Indeed taking at least a circumferential surface or cross-section
of the stool sample avoids the possibility of analyzing stool
portions that will not contain sloughed cancerous or precancerous
cells even when the patient has colorectal cancer or precancer.
[0029] Once a circumferential surface or cross-sectional sample of
stool is obtained, it may be homogenized by known methods to
distribute cells and cellular debris throughout the sample. An
assay then is performed on the homogenate, or an extract of the
homogenate, to detect the presence of cells and/or cellular debris
in the sample.
[0030] The assay may be any one or a combination of histological
cellular assays, antibody based immunoassays (or other formats)
designed to detect the presence of a molecule characteristic of
transformation such as a protein, or DNA-based assays for detecting
mutations or genetic characteristics indicative of colorectal
cancer. Known assay protocols, those disclosed herein or in U.S.
Pat. No. 5,670,325, or assays hereafter developed may be used in
the practice of the invention. Non-limiting examples of useful
known assay protocols include those disclosed in U.S. Pat. No.
5,137,806 (detection of sequences in selected DNA molecules), U.S.
Pat. No. 5,348,855 (assay for nucleic acid sequences), U.S. Pat.
No. 5,512,441 (detection of mutant alleles), U.S. Pat. No.
5,272,057 and U.S. Pat. No. 5,380,645 (RFLP analysis), U.S. Pat.
No. 5,527,676 (detection of p53 gene sequences), U.S. Pat. No.
5,330,892 (detection of MCC gene sequences), U.S. Pat. No.
5,352,775 (detection of APC gene sequences), U.S. Pat. No.
5,532,108 (detection of DCC gene sequences), and in WO96/08514
(monoclonal antibodies against human colon carcinoma-associated
antigens), the disclosures of each of which are incorporated by
reference herein. Alternatively, or additionally, an assay for
fecal occult blood may be performed as reported in U.S. Pat. Nos.
4,333,734 and 5,196,167, incorporated by reference herein. Assays
useful in the context of the present invention also include an
assay for carcinoembryonic antigen as reported in U.S. Pat. No.
5,380,647, incorporated by reference herein. In addition, assays
described in U.S. Ser. Nos. 08/808,763; 08/815,576; 08/876,857;
08/876,638; 08/877,333; and No. 60/063,219; incorporated by
reference herein, are also useful in the context of the present
invention. Finally, the sample may be prepared, as reported in U.S.
Pat. No. 4,857,300, incorporated by reference herein, for
histological examination to detect characteristics indicative of
the presence of cancerous or precancerous cells.
[0031] The purpose of any assay protocol used in connection with
obtaining at least a cross-sectional sample is to identify
candidates for subsequent invasive diagnostic procedure such as
colonoscopy or sigmoidoscopy. The assay accordingly need not
definitively detect the presence of a cancerous or precancerous
lesion, although false negatives obviously are to be avoided. The
goal of the test protocol is not to determine whether in the vast
quantities of cell debris in the sample there are a few cells which
bear a mutation commonly associated with early stage
transformation, but rather whether the sample contains debris
indicative of clonal expansion of a mutant cellular subpopulation.
Maximum benefit will come from an assay designed to detect the
likely presence of a clonally expanded cell population, i.e., of
transformed colonic epithelial cells which comprise the cancerous
or precancerous lesions. Assays having the ability to detect cancer
or precancer in early stages are preferred in methods of the
invention. Assays using polymerase chain reaction (PCR),
restriction fragment length polymorphism (RFLP) or other methods
for nucleic acid analysis may be used to detect known DNA
characteristics indicative of the presence of colorectal cancer or
precancer. More precise methods for quantitative detection of
cellular debris, such as DNA fragments or segments, may be used to
analyze cross-sectional samples according to methods described
herein.
[0032] A preferred assay interrogates the sample for DNA
characteristics indicative of the development of cancer or
precancer. However, assays for use with methods of the invention
may detect any abnormal cellular debris shed from
clinically-relevant transformed tissue. Thus, in accordance with a
preferred aspect of the invention, an assay is used to detect the
presence of characteristics of cells which have experienced loss of
heterozygosity, microsatellite instability or other mutation.
[0033] The following examples provide details of methods according
to the invention. However, numerous additional aspects of the
invention, especially in terms of assays to be performed, will
become apparent upon consideration of the following detailed
description thereof.
EXAMPLE 1
[0034] Preparation of a Stool Sample
[0035] A sample is prepared such that it contains at least a
circumferential surface or cross-sectional portion of a stool
voided by a patient.
[0036] Stool is voided into a receptacle that is preferably small
enough to be transported to a testing facility. The receptacle may
be fitted to a conventional toilet such that the receptacle accepts
stool voided in a conventional manner. The receptacle may comprise
a mesh or screen of sufficient size and placement such that stool
is retained while urine is allowed to pass through the mesh or
screen and into the toilet. The receptacle additionally may
comprise means for removing a circumferential surface or
cross-sectional portion from the stool. Moreover, the receptacle
may comprise means for introducing homogenization buffer or one or
more preservatives, such as alcohol, a solution of high salt
concentration, antibiotics, and chaotropic salts in order to
neutralize bacteria present in the stool sample. The homogenization
buffer may be a physiologically compatible buffer such as phosphate
buffered saline, and may comprise salt such as 20-100 mM NaCl or
KCl. The homogenization buffer may also comprise a detergent, such
as 1-1 0% SDS or triton, and/or a proteinase, such as proteinase K.
The buffer may also contain inhibitors of DNA and RNA degrading
enzymes.
[0037] The receptacle, whether adapted to fit a toilet or simply
adapted for receiving the voided stool sample, should include
sealing means sufficient to contain the voided stool sample and any
solution added thereto and to prevent the emanation of odors. An
exemplary receptacle is shown in FIG. 2. A shown in that figure,
the receptacle has a support frame I which is placed directly over
the toilet bowl 2. The support frame 1 has attached thereto an
articulating cover 3 which may be placed in a raised position, as
shown in FIG. 2, for depositing of sample or a closed position (not
shown) for sealing voided stool within the receptacle. The support
frame 1 additionally has a central opening 4 traversing from a top
surface 5 through to a bottom surface 6 of the support frame 1. The
bottom surface 6 directly communicates with a top surface 7 of the
toilet 2. Extending from the bottom surface 6 of the support frame
1 is a means 8 for capturing voided stool. Means 8 may be fixedly
attached to the support frame 1 or may be removably attached for
removal subsequent to deposition of stool. Means 8 may comprise a
further means for removing at least a circumferential surface or
cross-sectional portion from the voided stool. A preferable sample
size is at least 5-10 g or at least 5-10 ml. A means to assess the
presence of a minimal sample size may comprise a physical diagram
indicating the minimal sample size. Alternatively a means to assess
the presence of a minimal sample size may comprise the displacement
of a liquid or of a mechanical device to a minimal level upon
deposit of the stool sample.
[0038] In a preferred embodiment, a cross-sectional portion is
removed from the voided stool by making one or more sagittal or
coronal section through the stool, as shown in FIG. 1. The removed
portion comprises material from an entire circumferential surface
of the stool. Alternatively, a circumferential surface of a stool
is prepared by taking the entire surface layer of the stool, or the
entire surface layer of a cross-sectional portion of the stool. In
a preferred embodiment, the surface layer is approximately 0.001 to
0.1 mm thick. In another preferred embodiment, the surface layer is
approximately 0.1 to 20 mm thick. In a more preferred embodiment,
the surface layer is approximately 5 mm thick. In a most preferred
embodiment, the surface layer is representative of material
deposited on the forming stool, and reproducibly contains markers
or indicators, such as shed cellular material, of a disease that is
present in the patient. In one embodiment, a stool surface layer is
obtained by cutting or scraping off the surface of the stool or
cross-sectional portion of the stool. In a preferred embodiment, a
stool surface layer is prepared using a cylindrical cutting device
(for example a rigid loop) that has a diameter that is smaller than
the diameter of the stool or cross-sectional portion of the stool.
The surface layer is obtained when the stool sample is forced
through the cutting device. In a preferred embodiment, the diameter
of the cutting device is adjustable. Alternatively, a
circumferential surface of a stool is obtained by contacting the
surface of a stool or cross-sectional portion of a stool with a
sampling device. In one embodiment, the sampling device is first
wrapped around the stool sample and subsequently removed or peeled
off. The layer of stool that is peeled off with the sampling device
is then processed as described herein. Alternatively, the stool
sample is rolled along the surface of the sampling device, and the
layer of stool that adheres to the surface is then processed as
described herein. Alternatively, a diagnostic assay is performed
directly on the layer of material on the sampling device. It is
important that the entire circumference of the stool is sampled. In
a preferred embodiment, the sampling device has a surface that
stool adheres to. In a more preferred embodiment, the sampling
device surface preferentially binds diagnostic markers or
indicators of a disease (for example nucleic acid or protein
molecules), and thereby removes them from the surface of the stool
sample. In one embodiment, the sampling device is an adherent paper
or membrane. In a preferred embodiment, a stool is frozen prior to
processing. In one embodiment the stool is frozen to between -5 and
-30.degree. C. In a preferred embodiment the cross-sectional
portion or circumference of the stool is obtained from the frozen
or partially thawed stool. In an alternative embodiment, a whole
stool may be used for diagnostic assays. The stool or portion of
stool that is obtained contains sufficient material to allow
subsequent diagnostic assays to be performed.
[0039] Once obtained, the entire circumference or cross-sectional
sample of stool is homogenized in an appropriate buffer, such as
phosphate buffered saline. Homogenization means and materials for
homogenization are generally known in the art. Thus, particular
homogenization methods may be selected by the skilled artisan and
may depend upon the assay to be employed. The buffer may contain
detergent, salt, proteinase, inhibitors of DNA and RNA degrading
enzymes. The composition of the buffer will depend on the type of
assay to be performed. If a fecal occult blood assay is to be
performed, the buffer may contain chemical compounds which react
with blood to produce a color, the intensity of which can be
measured. Buffers useful for detecting the presence of fecal occult
blood are known in the art. If a test is to be performed for the
presence of a particular protein, the buffer should not contain a
proteinase capable of degrading such tumor marking antigens.
[0040] DNA or RNA may be isolated from the homogenate using methods
known in the art. Subsequent tests may be performed on the isolated
DNA and RNA.
EXAMPLE 2
[0041] Exemplary Enumerative Methods for Detection of Colorectal
Cancer or Precancer in Stool Samples
[0042] DNA characteristics associated with the presence of
colorectal cancer or a precancerous lesion may be detected in stool
samples prepared according to the invention, using, for example,
the methods described in the following sections. A careful
endoscopic examination preferably is performed on positive
Individuals, followed by early surgical excision of any diseased
tissue.
[0043] A. Reference-Target
[0044] Methods of the invention are used to prepare a stool sample
followed by detection of a deletion or other mutation in the p53
tumor suppressor gene. The p53 gene is a good choice because a loss
of heterozygosity in p53 is often associated with colorectal
cancer. An mRNA sequence corresponding to the DNA coding region for
p53 is reported as GenBank Accession No. M92424. At least a
cross-section of a voided stool sample is obtained and prepared
according to methods of the invention as described immediately
above. The sample need not be further processed for analysis.
However, DNA or RNA may optionally be isolated from the sample
according to methods known in the art. See, Smith-Ravin, et al.,
Gut, 36: 81-86 (1995), incorporated by reference herein.
[0045] Nucleic acids may be sheared or cut into small fragments by,
for example, restriction digestion. The size of nucleic acid
fragments produced is not critical, subject to the limitations
described below. A target allele that is suspected of being mutated
(p53 in this example) and a reference allele are chosen. A
reference allele may be any allele known normally not to be mutated
in colon cancer.
[0046] Either portions of a coding strand or its complement may be
detected. For exemplification, detection of the coding strand of
p53 and reference allele are described herein. Complement to both
p53 and reference allele are removed by hybridization to
anti-complement oligonucleotide probes (isolation probes) and
subsequent removal of duplex formed thereby. Methods for removal of
complement strands from a mixture of single-stranded
oligonucleotides are known and include techniques such as affinity
chromatography. Upon converting double-stranded DNA to
single-stranded DNA [See, e.g., Sambrook, et al., Molecular
Cloning, A Laboratory Manual (1989) incorporated by reference
herein], sample is passed through an affinity column packed with
bound isolation probe that is complementary to the sequence to be
isolated away from the sample. Conventional column chromatography
is appropriate for isolation of complement. An affinity column
packed with sepharose or other appropriate materials with attached
complementary nucleotides may be used to isolate complement DNA in
the column, while allowing DNA to be analyzed to pass through the
column. See Sambrook, Supra. As an alternative, isolation beads may
be used to exclude complement as discussed in detail below.
[0047] After removal of complement strands, first oligonucleotide
probes which hybridize to at least a portion of the p53 allele and
second oligonucleotide probes that hybridize to at least a portion
of the reference allele are obtained. The probes are labeled with a
detectable label, such as fluorescein or with detectable particles.
Distinct labels for the probes are preferred. However, identical
labels may be used if, for example, sample is assayed in two
separate aliquots. Probes may be labeled with identical or with
distinct labels. However, distinct labels are preferred.
[0048] Labeled probes then are exposed to sample under
hybridization conditions. Such conditions are well-known in the
art. See, e.g., Wallace, et al., Nucleic Acids Res., 6:3543-3557
(1979), incorporated by reference herein. First and Second
oligonucleotide probes that are distinctly labeled (i.e. with
different radioactive isotopes, fluorescent means, or with beads of
different size, See infra) are applied to a single aliquot of
sample. After exposure of the probes to sample under hybridization
conditions, sample is washed to remove any unhybridized probe.
Thereafter, hybridized probes are detected separately for p53
hybrids and reference allele hybrids. Standards may be used to
establish background and to equilibrate results. Also, if
differential fluorescent labels are used, the number of probes may
be determined by counting differential fluorescent events in a
sample that has been diluted sufficiently to enable detection of
single fluorescent events in the sample. Duplicate samples may be
analyzed in order to confirm the accuracy of results obtained.
[0049] If there is a statistically-significant difference between
the amount of p53 detected and the amount of the reference allele
detected, it may be assumed that a mutation has occurred in p53 so
as to alter its sequence and prevent hybridization of the probe, or
that at least a portion of the region of the genome containing p53
has been lost in a subpopulation of cells shed from the colon. The
patient therefore may be at risk for developing or may have
developed colon cancer. Statistical significance may be determined
by any known method. See, e.g., Steel, et al., Principles and
Procedures of Statistics: A Biometrical Approach (McGraw, Hill,
1980). Statistical methods are also outlined in co-owned U.S. Pat.
No. 5,670,325.
[0050] The determination of a p53 mutation allows a clinician to
recommend further treatment, such as endoscopy procedures, in order
to further diagnose and, if necessary, treat the patient's
condition. The following examples illustrate methods that allow
direct quantification of hybridization events.
[0051] 1. Method for Quantitation of Target and Reference
Polynucleotides
[0052] Enhanced quantification of binding events between
hybridization probes and target or reference is accomplished by
coupling hybridization probes to particles, such as beads
(hybridization beads). In order to obtain a precise quantitative
measure of the amount of a polynucleotide in a sample,
hybridization beads are constructed such that each bead has
attached thereto a single oligonucleotide probe.
[0053] a. Method for Preparation of Probe-Bead Combinations
[0054] A single probe is attached to a bead by incubating a large
excess of hybridization beads with oligonucleotide probes of a
given type (i.e., either first or second oligonucleotide probes).
Coupling of probe to bead is accomplished using an affinity-binding
pair. For example, beads may be coated with avidin or streptavidin
and probes may be labeled with biotin to effect attachment of the
probe to the bead. The mixture of beads and probes is agitated such
that 100% of the probes are bound to a bead. The mixture is then
exposed to a matrix, such as an affinity column or a membrane
coated with oligonucleotides that are complementary to the probe.
Only beads that have an attached probe will adhere to the matrix,
the rest being washed away. Beads with coupled probe are then
released from the matrix by melting hybridizations between probe
and complement. Multiple exposures to the matrix and pre-washing of
the column reduces non-specific binding. Moreover, naked beads
(i.e., without attached probe) may be exposed to the matrix to
determine a background number of beads that can be expected to
attach to the matrix in the absence of probe.
[0055] By using a vast excess of beads relative to probe as
described above, the vast majority of recovered beads will have
only one attached probe. For example, if a mixture has a ratio of 1
probe to 1000 beads, it is expected that only about 1 bead in a
million will have two attached probes and even less than one bead
in a million will have more than two attached probes. Accordingly,
hybridization beads are provided in an effective 1:1 ratio with
probe which allows for precise quantification of target and
reference polynucleotide as described below.
[0056] For each assay described below, two distinct hybridization
beads are used. A first hybridization bead has attached thereto a
single first oligonucleotide probe that is complementary to at
least a portion of a target polynucleotide (e.g., a p53 allele). A
second hybridization bead, of a size distinct from the first
hybridization bead, has attached thereto a single second
oligonucleotide probe that is complementary to at least a portion
of a reference polynucleotide (i.e., one that is known or suspected
not to be mutated in the sample).
[0057] b. Use of Beads to Quantify Target and Reference
Polynucleotides
[0058] DNA is melted (denatured to form single-stranded DNA) by
well-known methods See, e.g., Gyllensten, et al., in Recombinant
DNA Methodology II, 565-578 (Wu, ed., 1995), incorporated by
reference herein. One may detect either a coding strand or its
complement in order to quantify target and/or reference
polynucleotide. For purposes of illustration, the present example
assumes detection of the coding strand.
[0059] 2. Removal of Complement
[0060] Single-stranded complement of the target polynucleotide
(e.g., p53) and reference polynucleotide are removed from the
sample by binding to oligonucleotide probes that are complementary
to target or reference complement. Such probes, referred to herein
as isolation probes, are attached to isolation beads prior to their
introduction into the sample. The beads may be magnetized. Thus,
when magnetized isolation beads [with attached isolation probe(s)]
are introduced into the sample, the attached isolation probes
hybridize to complement of target or reference (or vice versa).
Isolation beads preferably are introduced in vast excess in order
to saturate complement binding. Once hybridization is complete, a
magnetic field is applied to the sample to draw the magnetized
isolation beads (both with and without hybridized complement) out
of the sample. Assuming that a sufficient quantity of isolation
beads are introduced into the sample, removal of the isolation
beads effectively removes all target and reference complement from
the sample.
[0061] In an alternative method for complement removal, an excess
of oligonucleotide probe labeled with biotin is exposed to the
melted or dehybridized (single stranded) sample under hybridization
conditions. Once hybridization is complete, the sample is exposed
to a column containing immobilized avidin. The biotin-labeled
probe, whether free or hybridized to complement, is bound by avidin
on the column. The remainder of the DNA, including target and
reference coding strands to be detected, pass through the column.
In contrast to the description of hybridization beads above, beads
for removal of complement may each comprise multiple complementary
oligonucleotide probes.
[0062] 3Quantitation of Target and Reference
[0063] Two sets of hybridization beads are prepared as described
above. Each member of a first set of hybridization beads (all of
which are identical to each other) has attached thereto a single
oligonucleotide probe that is complementary to at least a portion
of the target polynucleotide, i.e., the portion of the genome which
is altered in the cells of a cancerous lesion. Each member of a
second set of identical hybridization beads (all of which are
identical to each other but not to the first set) has attached
thereto a single oligonucleotide probe that is complementary to at
least a portion of the reference polynucleotide, i.e., a portion of
the genome which is not likely to be altered in malignant cells.
Members of the second set of hybridization beads are of a size or
color distinct from that of members of the first set of
hybridization beads. First and second hybridization beads may also
be distinguished on the basis of other characteristics. For
example, beads may have fluorescent markers that are distinguished
by their fluorescence wavelength. Beads with distinct
electrochemical charges also may be used. The precise modality used
for distinguishing beads is not essential as long as it is possible
to distinguish between first and second probe on the basis of
distinctions between attached first and second beads.
[0064] Both sets of hybridization beads are exposed to the sample
under hybridization conditions thereby allowing hybridization to
reference and target. The sample then is washed to remove
unhybridized bead/probe combinations. Unhybridized bead/probe
combinations are removed by, for example, passing the sample
through a column lined of immobilized DNA complementary to the
probe sequence. Thus, any unhybridized bead/probe combinations are
retained on the column while duplex passes through. Subsequently,
the sample is exposed to means for differentially counting
hybridization beads in order to quantify first and second
hybridization probes which have formed duplexes. The numbers
obtained provide a precise estimate of the number of copies of the
reference and target polynucleotide in the population because
differential counting means count individual beads. One bead is
equal to one probe which, in turn, signifies one copy of the
nucleic acid being measured.
[0065] An example of a differential counting means is an impedance
measuring device, such as a Coulter counter (Coulter Electronics,
Inc., Miami, Fla). Sample is passed through the device which
differentially detects the two types of hybridization beads by
measuring their differential impedance of an electric current.
Alternatively, the device may measure fluorescence, color, or other
parameters. In order to increase the speed of the assay, a
multi-orifice device may be used. A multi-orifice impedance counter
is shown schematically in FIG. 2. A multi-orifice array is placed
at one end of a column filled with an electrically-conductive
fluid, such as saline. Hybridization beads with either hybridized
target or reference segments are inserted at an opposite end of the
column. Each orifice is large enough to accommodate only one
hybridization bead at a time and sufficiently wide to allow
reliable impedance measurements. A voltage is set across each
orifice. Each hybridization bead (which is non-conducting), as it
passes through one of the orifices, displaces a volume of saline,
thus creating an impedance that is proportional to its size. This,
in turn, creates a measurable decrease in current that is directly
correlated with the size of the bead. By compiling the number of
each of the two distinct impedance events, a precise estimate of
the number of hybridization beads and, therefore, the number of
probes of each type in the population may be obtained.
[0066] Upon quantitative measurement of first and second
hybridization beads, the data may be analyzed to determine whether
any difference between the amounts of first and second
hybridization beads is statistically significant. A reduction in
the amount of target relative to the reference is indicative of a
mutation in or deletion of the target allele in a subpopulation of
cells in the sample. Where the p53 gene is the target allele, such
a mutation is indicative of a cancerous or precancerous condition.
A clinician may use such results as a basis for prescribing
additional treatment, such as endoscopy and polypectomy
procedures.
[0067] B. Detection of Mutations in Single-Base Polymorphisms
[0068] The basic method described above may also be applied to
detect a loss of heterozygosity or other mutation at a single base
polymorphic site between maternal and paternal alleles. Such
detection is typically an indication of a larger deletion or other
mutation. However, a mutation at a single polymorphic nucleotide
may be all that is necessary to inhibit gene function in one of the
two alleles. A mutation in a single-base polymorphic region may be
difficult to detect due to a recently-discovered phenomenon called
complementary reduplication. In complementary reduplication, the
loss of one of two alleles at a particular locus results in
"reduplication" of the surviving allele. Reduplication usually
takes place on the chromosome containing the surviving allele and
involves the production of one or more copies of the surviving
allele in close proximity on the chromosome to the position of the
surviving allele. In the case of a locus that displays one or more
single-base allelic polymorphisms (i.e., heterozygosity at the
locus is determined by virtue of one or more single-base
differences in one or more regions of the locus), complementary
reduplication results in the insertion on the chromosome containing
the surviving allele of a duplicate of the sequence corresponding
to that which was deleted. Even under the most stringent
hybridization conditions, some of a probe directed against the
deleted sequence will bind to the reduplicated sequence at a locus
of a single-base polymorphism. Accordingly, in such circumstances,
the deletion may not be detected because any true difference in the
number of probes binding to the polymorphic site (i.e., the allelic
region encompassing the single-base polymorphism) may be obscured
by an increase resulting from the other allele's reduplicated
region.
[0069] The problems associated with complementary reduplication,
and with non-specific probe binding generally, are alleviated by
the practice of the methods described herein. Such methods allow
detection of a deletion in one of two alleles present at a specific
locus in a subpopulation of cells contained in a biological sample.
Numerous alleles, including tumor suppressor alleles, contain
single polymorphic nucleotides in the context of a constant nucleic
acid region. Individuals normally may be either homozygous or
heterozygous for the polymorphic nucleotide. Since numerous
single-base polymorphic nucleotide sites exist in most alleles, the
probability that a given individual is heterozygous at least one of
the single-base polymorphism sites is high. A
statistically-significant reduction in one of the two nucleotides
at a single-base polymorphic site (at which the individual is
heterozygous) may be used as a marker for a deletion in the allele
encompassing that site.
[0070] Genomic regions containing known single-base polymorphisms
may be identified by reference to a nucleotide database, such as
GenBank, EMBL, or any other appropriate database. The existence of
polymorphisms may be determined by methods taught herein, gel
electrophoresis or by other standard methods. For purposes of the
invention, a single-base polymorphism is intended to be a single
polymorphic nucleotide adjacent to a non-polymorphic region of the
allele regardless of whether the single polymorphic nucleotide
forms part of a larger polymorphic site (i.e. the single-base
polymorphism may be the terminal nucleotide of a larger,
polynucleotide polymorphism). For cancer detection, the regions
considered are regions in which loss of heterozygosity is
prevalent, such as regions containing tumor suppressor genes. A
given individual may be homozygous or heterozygous for the
polymorphic nucleotide in any identified single-base polymorphic
region. Accordingly, if a number of single-base polymorphic regions
are identified, the probability increases that at least one
heterozygous single-base polymorphic region is found in a
sample.
[0071] Once single-base polymorphic sites are identified, a DNA
sample is obtained from a patient, e.g., from blood cells, to
determine which of those sites is heterozygous in normal (i.e.,
non-cancerous or non pre-cancerous) cells for that individual.
Then, a stool sample is prepared as described above. Double
stranded DNA in the sample is converted to single-stranded DNA.
Then, either the coding strand or the anti-coding strand for both
alleles is removed from the sample. As will be evident from the
following discussion, methods disclosed herein are indifferent as
to whether coding strand or anti-coding strand is tested.
[0072] An oligonucleotide probe is constructed that is
complementary to a portion of the region of single-base
polymorphism, said portion ending at the nucleotide that is
immediately 3' to the polymorphic nucleotide, regardless of whether
the 5'-3' (coding) strand or the 3'-5' (anticoding) strand is used
as a template. FIG. 3 shows four possible probes that are
immediately 3' to the polymorphic nucleotide for each of four
possible template strands as described above (the Sequences in FIG.
3 are hypothetical and are not intended to represent any actual
sequence). The sequence labeled M1 is SEQ ID NO:1; the sequence
labeled M2 is SEQ ID NO:2; the sequence labeled M3 is SEQ ID NO: 3;
the sequence labeled M4 is SEQ ID NO:4; the sequence labeled F1 is
SEQ ID NO: 5; the sequence labeled F2 is SEQ ID NO: 6; the sequence
labeled F3 is SEQ ID NO: 7; and the sequence labeled F4 is SEQ ID
NO: 8. While either strand may be used as a template for probe
binding to determine heterozygosity and/or the loss thereof, the
sequence of the probe that is hybridized to the template will be
different depending upon the strand used. Probes may be of any
length that allows efficient and specific hybridization. FIG. 3
merely illustrates four hypothetical probes that are useful for
hybridization to the hypothetical sequence shown. The length of
probe sequences may be determined as appropriate for each genomic
region that is analyzed. A preferable length is between about 10
and about 100 nucleotides. The size of the probe will also depend
upon the size of the region surrounding the single-base
polymorphism (i.e., the region 5' or 3' to the next adjacent
polymorphism, if any). Details concerning the construction and
hybridization of oligonucleotide probes are known in the art.
[0073] Unique probes for each polymorphic region will hybridize to
regions of both maternal and paternal alleles up to, but not
including, the polymorphic nucleotide, which, in a heterozygote,
will be different in the maternal and paternal alleles. FIG. 3
shows only a small portion of the region surrounding the
polymorphic nucleotide. The alleles shown in FIG. 3 are
heterozygous at the polymorphic site.
[0074] Probe is hybridized to its specific template DNA by standard
methods. The sample may optionally be washed to remove unhybridized
probe. To determine whether each target region bound by a probe is
heterozygous or homozygous at the polymorphic nucleotide, a
modification of the dideoxy chain termination method as reported in
Sanger, Proc. Nat'l Acad. Sci. (USA), 74: 5463-5467 (1977),
incorporated by reference herein, is used. The method involves
using at least two of the four common 2', 3'-dideoxy nucleoside
triphosphates (ddATP, ddCTP, ddGTP, and ddTTP). A different
detectable label is attached to each dideoxy nucleoside
triphosphate (ddNTP) according to methods known in the art.
Differentially-labeled ddNTPs are available commercially, for
example, from Perkin Elmer Corporation (Cat. No. 401456). At least
two labeled ddNTPs then are exposed to each sample having probe
hybridized to maternal and paternal alleles as described above. The
choice of which two ddNTPs are used will depend upon the
nucleotides at the heterozygous polymorphic site. Any 3' modified
nucleoside triphosphate may be used in the method as long as the 3'
modification prevents binding of an additional 3' nucleotide (i.e.
probe extension) and does not inhibit binding of the modified
nucleotide to the 3' end of the probe. A DNA polymerase, such as
Sequenase.TM. (Perkin-Elmer), is added to the sample mixture. Using
the allelic strands as primer, the polymerase will add one ddNTP to
the 3' end of the probe, the incorporated ddNTP will be
complementary to the nucleotide that exists at the single-base
polymorphic site. Because the ddNTPs have no 3' hydroxyl, further
elongation of the hybridized probe will not occur. After
completion, the sample is washed to remove excess ddNTPs. Label is
then counted in each sample. The presence of two
differentially-labeled ddNTPs in a sample is indicative of
heterozygosity at the polymorphic site.
[0075] It is not necessary to determine the amount of each label
present in the sample in order to establish heterozygosity or
homozygosity. For Example, differentially-labeled deoxynucleoside
triphosphates may be used for a determination of heterozygosity or
homozygosity. The mere fact that two different labeled dideoxy
nucleotides are incorporated into the probe means that the
single-base polymorphic site being analyzed is heterozygous.
However, determination of sites at which a patient is polymorphic
is useful in order to establish a baseline of polymorphisms which
may be used in future tests to detect changes in polymorphic sites
which may be indicative of cancer. The existence of polymorphisms
may be determined by methods taught herein, by gel electrophoresis
or by other standard methods.
[0076] In the case in which heterozygosity exists at the
polymorphic site, counting the amount of each of the two
differentially-labeled ddNTPs allows a determination of whether
there is a loses of heterozygosity (i.e., a deletion) in a
subpopulation of cells in the sample. In a normal (ie.,
non-cancerous) sample containing cells that are heterozygous at the
single-base polymorphic site, it is expected that the detected
amount of each of the two ddNTPs added to the probe will be
identical (within chosen limits of statistical significance).
However, if a deletion has occurred in one of the two alleles in a
subpopulation of cells in the sample, there will be a
statistically-significant difference between the amounts of each of
the two alleles detected via the incorporated (labeled) ddNTPs. The
detection of such a difference is indicative of genomic instability
within the sample. Such genomic instability indicates the
possibility of cancerous or pre-cancerous cells in the sample.
[0077] In order to improve the ability to count alleles to which
ddNTPs have attached accurately, ddNTPs are labeled with
hybridization-type beads of different sizes as described above.
Alleles with bound probe comprising a labeled ddNTP are counted as
described above using a counting device, such as a Coulter counter.
Also as described above, differential fluorescent labels or other
counting means may be used to separately detect incorporated
ddNTPs.
[0078] The detection of heterozygosity at single-base polymorphic
sites and the detection of the loss of heterozygosity may be
determined in separate steps. For example, probes may be hybridized
immediately adjacent to but not including the nucleotide determined
to be polymorphic as described above. The four ddNTPs may then be
added to the sample, washed, and the presence or absence of each
label may be detected. Detection of only one label indicates that
the individual from whom the sample was obtained is homozygous at
the site of the potential polymorphic nucleotide. Detection of two
labels means that the individual is heterozygous. The heterozygous
loci are recorded. As noted above, baseline determinations of
heterozygosity may be done using standard methods. Once a baseline
is established, future tests on that individual are performed
exploiting the heterozygous loci in order to detect a loss of
heterozygosity. For the detection of cancer, the heterozygous loci
are typically chromosomal areas containing tumor suppressor genes,
including p53, dcc, apc, and others. Using methods described
herein, a "fingerprint" of heterozygous tumor suppressor loci may
be constructed. Future deviation from the fingerprint (i.e.,
deletions) provides valuable information as to the development of
cancer.
[0079] A preferred use of the foregoing methods is in the detection
of colon cancer. A representative stool sample is prepared as
described above. Double-stranded DNA is converted to
single-stranded DNA and complement of the strand to be detected is
removed from the sample. The remaining single-stranded DNA is
exposed to multiple copies of a probe designed on the basis of
known single-base polymorphisms in a cancer-associated allele such
that the probe hybridizes with a desired number of nucleotides
immediately adjacent the polymorphic nucleotide as described above.
After hybridization is complete, the sample is washed and exposed
to differentially-labeled ddNTPs and a DNA polymerase. The sample
then is washed to remove unincorporated ddNTPs. The presence of any
labeled ddNTPs is determined. If two labels are detected, the
individual from whom the sample is obtained is heterozygous at the
polymorphic nucleotide. The heterozygosity of the allele and the
probe sequence matching the site immediately adjacent to the
polymorphic allele are noted for reference in future testing for
the loss of heterozygosity. Alternatively, once the patient is
determined to be heterozygous at a locus, an assay may be performed
immediately in the manner described above in order to determine an
existing loss of heterozygosity in a subpopulation of cells in the
sample.
[0080] C. Analysis of Microsatellite Instability
[0081] Microsatellites are di- or trinucleotide repeats found
throughout the genome. A particular array of microsatellite repeats
is often associated with a particular genomic sequence and is
stably inherited under normal conditions. Expansions of
microsatellite copy number typically, called "microsatellite
instability," are associated with defects in mismatch repair.
Accordingly, changes in a microsatellite region indicate that the
patient is at risk for a mutation in other genomic regions.
[0082] In order to detect microsatellite instability as an
indicator of a mutation in a cancer-associated gene, one must first
identify a microsatellite region associated with the gene of
interest. Such regions are typically identified on a database, such
as GenBank, EMBL, and others. Once a wild-type microsatellite
region associated with, for example, the p53 tumor suppressor gene,
is identified, an oligonucleotide probe is constructed that spans
the microsatellite region and the regions immediately 5' and
immediately 3' to the microsatellite region. The precise length of
probes may be determined by the experimenter. Probes are
constructed that hybridize to the microsatellite region, including
portions extending 5' and 3', on both the maternal and paternal
alleles with which the microsatellite is associated (e.g.,
p53).
[0083] An appropriate sample of body tissue or fluid is obtained
and processed as described herein. Double stranded DNA is denatured
and an excess of maternal and paternal probes, as described above,
are introduced into the sample under hybridization conditions. The
probes are detectably labeled as described above. Complement of the
strands to be detected may optionally be removed by methods
described above. The sample is then washed to remove unhybridized
probe and the amount of hybridized probe in quantitatively
detected.
[0084] Quantitative detection may be accomplished by any means
described herein. For example, probes may be attached to
hybridization beads such that probes that bind to maternal allele
are attached to beads of one size and probes that bind to paternal
allele are attached to beads of a second size that is
distinguishable from beads of the first size. Beads with attached
probe may be counted as described above.
[0085] The detection of a statistically-significant difference
between the amount of probe binding to the maternal allele and the
amount of probe binding to the paternal allele is indicative of
microsatellite instability. As previously mentioned, microsatellite
instability can be indicative of a mutation at the locus in which
the microsatellite resides. If the microsatellite region is
associated with a tumor suppressor gene or an oncogene, the
detection of microsatellite instability in an allele in a
subpopulation of cells in a biological sample is indicative of the
potential for cancer or that cancer or precancer may have already
developed. Further testing as described herein (either by invasive
or noninvasive means) may then be conducted.
[0086] In an alternative embodiment, a "fingerprint" of
microsatellites is taken from regions associated with
cancer-causing genes in a sample obtained from a patient. Such a
fingerprint may be obtained by standard methods. The fingerprint
comprises the sequence of wild-type microsatellites associated with
the cancer-causing gene or genes. Once obtained, the fingerprint is
stored and is used in future tests of samples from the same patient
in order to monitor changes in microsatellite regions (i.e.
microsatellite instability) that may be associated with the
development of cancer. Changes in microsatellite length and/or
sequence over time may be used to prescribe additional testing
and/or treatment in order to detect and remove cancerous tissue at
an early stage in its etiology.
[0087] Additional embodiments of the invention are apparent upon
consideration of the following claims.
Sequence CWU 1
1
8 1 9 DNA Artificial Sequence Description of Artificial Sequence
Hypothetical M1 1 ggcatcgca 9 2 19 DNA Artificial Sequence
Description of Artificial Sequence Hypothetical M2 2 atcggcttac
tgcgatgcc 19 3 19 DNA Artificial Sequence Description of Artificial
Sequence Hypothetical M3 3 ggcatcgcag taagccgat 19 4 9 DNA
Artificial Sequence Description of Artificial Sequence Hypothetical
M4 4 atcggctta 9 5 9 DNA Artificial Sequence Description of
Artificial Sequence Hypothetical F1 5 ggcatcgca 9 6 19 DNA
Artificial Sequence Description of Artificial Sequence Hypothetical
F2 6 atcggcttat tgcgatgcc 19 7 19 DNA Artificial Sequence
Description of Artificial Sequence Hypothetical F3 7 ggcatcgcaa
taagccgat 19 8 9 DNA Artificial Sequence Description of Artificial
Sequence Hypothetical F4 8 atcggctta 9
* * * * *