Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF COPY NUMBER CHANGES IN COLON CANCER
FIELD OF THE INVENTION
This invention pertains to the field of cancer genetics and cytogenetics. In
particular, this invention pertains to the identification of an association
between
amplification of regions on chromosome 20 and colorectal cancer.
BACKGROUND OF THE INVENTION
Chromosome abnormalities are often associated with genetic disorders,
degenerative diseases, and cancer. The deletion or multiplication of copies of
whole
chromosomes and the deletion or amplifications of chromosomal segments or
specific
regions are common occurrences in cancer (Smith (1991) Breast Cancers Res.
Treat. 18:
Suppl. 1:5-14; van de Vijer (1991) Biochim. Bio~ahys. Acta. 1072:33-50). In
fact,
amplifications and deletions of DNA sequences can be the cause of a cancer.
For
example, proto-oncogenes and tumor-suppressor genes, respectively, are
frequently
characteristic of tumorigenesis (Dutrillaux (1990) Cancers Genet. Cytogenet.
49: 203-
217). Clearly, the identification and cloning of specific genomic regions
associated with
cancer is crucial both to the study of tumorigenesis and in developing better
means of
diagnosis and prognosis.
One of the amplified regions found in studies of breast cancer cells is on
chromosome 20, specifically, 20q13.2 (see, e.g. W098/02539). Amplification of
20q13.2
was subsequently found to occur in a variety of tumor types and to be
associated with
aggressive tumor behavior. Increased 20q13.2 copy number has been found in 40%
of
breast cancer cell lines and 18% of primary breast tumors (Kalliioniemi (1994)
Ps°oc.
Natl. Acad. Sci. USA 91: 2156-2160). Copy number gains at 20q13.2 have also
been
reported in greater than 25% of cancers of the ovary (Iwabuchi (1995) Cancer
Res.
55:6172-6180), colon (Schlegel (1995) Cancer Res. 55: 6002-6005), head-and-
neck
(Bockmuhl (1996) Laryngor. 75: 408-414), brain (Mohapatra (1995) Genes
Chromosomes Cancer 13: 86-93), and pancreas (Solinas-Toldo (1996) Genes
Chromosomes Cancer 20:399-407).
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A number of studies have elucidated genetic alterations that occur during
the development of colorectal tumors. For instance, deletions of p53 genes on
chromosome 17p are often late events associated with the transition from the
benign
(adenoma) to the malignant (carcinoma) state. See Vogelstein et al., New
England
Journal ofMedicine, 319:525 (1988), Fearon and Vogelstein, Cell, 61:759-767
(1990)
and Baker et al. Cancef~ Res. 50:7717-22 (1990). More recently, comparative
genomic
hybridization has shown that specific patterns of chromosomal gains and losses
take place
during colorectal carcinogenesis (see, e.g. Schlegel, et al. Cancer Research.
55, 6002-
6005 (1995); Ried, et al. Genes, Ch~ornosomes & Cancer 15, 234-245 (1996); and
Nakao et al., Jpn. J. Sufg. 28, 567-569 (1998). These changes included
overrepresentation (amplification) of large portion of chromosome 20 material.
Because carcinomas are often lethal, while the precursor adenomas are
uniformly curable, the early detection of chromosomal changes associated with
this
transition are of considerable importance. .The identification of regions of
chromosomal
abnormalities in other cancers is obviously great use in diagnosis, prognosis
and
treatment of these diseases. The present invention addresses these and other
needs.
SUMMARY OF THE INVENTION
The present invention provides methods of screening for colon carcinoma
cells in a sample. The methods comprise providing a nucleic acid sample from a
premalignant lesion in colorectal tissue from a human patient and contacting
the sample
with a nucleic acid probe that selectively hybridizes to a chromosomal region
on 20q.
The formation of a hybridization complex is then detected and the presence or
absence of
increased copy number at 20q (usually 20q13.2) is determined.
The sample used can be, for instance, one suitable for in situ hybridization
techniques, such as a metaphase spread or an interphase nucleus. Usually, the
probe is
labeled, e.g. with fluorescent label, which can be attached directly or
indirectly to the
probe. In some embodiments, a second probe that selectively hybridizes to a
second
chromosomal region is used as a reference. In these embodiments, the second
probe is
labeled with a fluorescent label distinguishable from the label on the probe
that
selectively hybridizes to a chromosomal region on 20q.
The sample can be derived from any premalignant colorectal tissue
suspected of containing cancer cells. Often, the premalignant tissue is an
ademomatous
polyp or tissue showing high grade dysplasia.
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In some embodiments the probe may comprise repetitive sequences. In
this the methods may further comprise the step of blocking the hybridization
capacity of
repetitive sequences in the probe. This can be done by, for example, including
unlabeled
blocking nucleic acids with the labeled probe. The unlabeled blocking nucleic
acids can be,f
for example, Cot-1 DNA.
A number of hybridization formats can be used. In some formats, the
probe is bound to a solid substrate as a member of an array of probes. In
these
embodiments, the probe is not labeled and the sample nucleic acids are
labeled.
Definitions
To facilitate understanding of the invention, a number of terms are defined
below.
The term "amplicon" as used herein refers to a region of genomic nucleic
acid which, when present in altered copy number, is associated with cancer.
For example,
the invention provides nucleic acid sequences which, when present in aberrant
copy
number, are associated with colon cancer.
The term "20q13.2 amplicon" refers to a region on the q arm of human
chromosome 20 at about band 13.2 that has been identified in cancer cells.
This amplicon
has been extensively analyzed (see, e.g., WO 95/02539) in breast cancer cells.
A 1.5
megabase (Mb) wide amplified region within 20q13.2 was identified (Stokke
(1995)
Genomics 26: 134-137); Tanner (1994) Cancer Res. 54:4257- 4260). Interphase
FISH
revealed low-level (>1.SX) and high level (>3X) 20q13.2 sequence amplification
in 29%
and 7% of breast cancers, respectively (Tanner (1995) Clin. Cancer Res. 1:
1455-1461).
High level amplification was associated with an aggressive tumor phenotype
(Tanner
(1995) supra; Courjal (1996) Br. J. Cancer 74: 1954). Another study, using
FISH to
analyze 14 loci along chromosome 20q in 146 uncultured breast carcinomas,
identified
three independently amplified regions, including RMC20C001 region at 20q13.2
(highly
amplified in 9.6% of the cases), PTPNl region 3 Mb proximal (6.2%), and AIB3
region
at 20q11 (6.2%) (Tanner (1996) Cancer Res. 56:3441-3445).
An "animal" refers to a member of the kingdom Animalia, characterized
by multicellularity, the possession of a nervous system, voluntary movement,
internal
digestion, etc. An "animal" can be a human or other mammal. Preferred animals
include
humans, non-human primates, and other mammals. Thus, it will be recognized
that the
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methods of this invention contemplate veterinary applications as well as
medical
applications directed to humans.
A "cancer" in an animal refers to the presence of cells possessing
characteristics typical of cancer-causing cells, such as uncontrolled
proliferation,
immortality, metastatic potential, rapid growth and proliferation rate, and
certain
characteristic morphological features. Often, cancer cells will be in the form
of a tumor,
but such cells may exist alone within an animal, or may be a non-tumorigenic
cancer cell,
such as a leukemia cell. Cancers include, but are not limited to breast
cancer, lung
cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreas cancer,
stomach
cancer, ovarian cancer, urinary bladder cancer, brain or central nervous
system cancer,
peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine
or
endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney
cancer,
testis cancer, biliary tract cancer, small bowel or appendix cancer, salivary
gland cancer,
thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and
the like.
The phrase "detecting a cancer" refers to the ascertainment of the presence
or absence of cancer in an animal, in this case, colorectal premalignant
tissue. "Detecting
a cancer" can also refer to obtaining indirect evidence regarding the
likelihood of the
presence of cancerous cells in the animal or to the likelihood or predilection
to
development of a cancer. Detecting a cancer can be accomplished using the
methods of
this invention alone, or in combination with other methods or in light of
other information
regarding the state of health of the animal.
The terms "hybridizing specifically to" and "specific hybridization" and
"selectively hybridize to," as used herein refer to the binding, duplexing, or
hybridizing of
a nucleic acid molecule preferentially to a particular nucleotide sequence
under stringent
conditions. The term "stringent conditions" refers to conditions under which a
probe will
hybridize preferentially to its target subsequence, and to a lesser extent to,
or not at all to,
other sequences. A "stringent hybridization" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization (e.g., as in array,
Southern or
Northern hybridizations) are sequence dependent, and are different under
different
environmental parameters. An extensive guide to the hybridization of nucleic
acids is
found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and
Molecula~~
Biology--Hybridization with Nucleic Acid Probes past I, chapt 2, "Overview of
principles
of hybridization and the strategy of raucleic acid probe assays," Elsevier, NY
("Tij ssen").
Generally, highly stringent hybridization and wash conditions are selected to
be about
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5°C lower than the thermal melting point (Tm) for the specific sequence
at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at
which 50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent conditions are selected to be equal to the Tm for a particular
probe. An example
of stringent hybridization conditions for hybridization of complementary
nucleic acids
which have more than 100 complementary residues on an array or on a filter in
a
Southern or northern blot is 42°C using standard hybridization
solutions (see, e. g.,
Sambrook (1989) Molecular Clonirag: A Laboratory Manual (end ed.) Irol. 1-3,
Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed
discussion,
below), with the hybridization being carried out overnight. An example of
highly
stringent wash conditions is 0.15 M NaCI at 72°C for about 15 minutes.
An example of
stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes
(see, e.g., Sambrook
supra.) for a description of SSC buffer). Often, a high stringency wash is
preceded by a
low stringency wash to remove background probe signal. An example medium
stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at
45°C for 15
minutes. An example of a low stringency wash for a duplex of, e.g., more than
100
nucleotides, is 4x to 6x SSC at 40°C for 15 minutes.
The term "labeled with a detectable composition", as used herein, refers to
a nucleic acid attached to a detectable composition, i.e., a label. The
detection can be by,
e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or
chemical
means. For example, useful labels include 32P, 3sS, 3H, laC, lash isih
fluorescent dyes
(e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense
reagents (e.g.
gold), enzymes, e.g., as corninonly used in an ELISA (e.g., horseradish
peroxidase, beta-
galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g.
colloidal gold),
magnetic labels (e.g. DynabeadsTM ), biotin, dioxigenin, or haptens and
proteins for which
antisera or monoclonal antibodies are available. The label can be directly
incorporated
into the nucleic acid, peptide or other target compound to be detected, or it
can be
attached to a probe or antibody that hybridizes or binds to the target. A
peptide can be
made detectable by incorporating predetermined polypeptide epitopes recognized
by a
secondary reporter (e.g., leucine zipper pair sequences, binding sites for
secondary
antibodies, transcriptional activator polypeptide, metal binding domains,
epitope tags).
Label can be attached by spacer arms of various lengths to reduce potential
steric
hindrance or impact on other useful or desired properties. See, e.g.,
Mansfield (1995)
Mol Cell Probes 9: 145-156.
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The term "nucleic acid" as used herein refers to a deoxyribonucleotide or
ribonucleotide in either single- or double-stranded form. The term encompasses
nucleic
acids, i.e., oligonucleotides, containing known analogues of natural
nucleotides which
have similar or improved binding properties, for the purposes desired, as the
reference
nucleic acid. The term also includes nucleic acids which are metabolized in a
manner
similar to naturally occurnng nucleotides or at rates that are improved for
the purposes
desired. The term also encompasses nucleic-acid-like structures with synthetic
backbones. DNA backbone analogues provided by the invention include
phosphodiester,
phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate,
alkyl
phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-
carbamate,
morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides
and
Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford
University
Press (1991); Antisense Strategies, Annals of the New York Academy of
Sciences,
Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med.
Chem.
36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs
contain
non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate
linkages
are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.
Pharmacol.
144:189-197. Other synthetic backbones encompasses by the term include methyl-
phosphonate linkages or alternating methylphosphonate and phosphodiester
linkages
(Strauss-Soukup (1997) Biochemistf~y 36: 8692-8698), and benzylphosphonate
linkages
(Samstag (1996) Antiser~se Nucleic Acid Drug Dev 6: 153-156). The term nucleic
acid is
used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and
amplification product.
The term a "nucleic acid array" as used herein is a plurality of target
elements, each target element comprising one or more nucleic acid molecules
(probes)
immobilized on one or more solid surfaces to which sample nucleic acids can be
hybridized. The nucleic acids of a target element can contain sequences) from
specific
genes or clones, e.g. from the regions identified here. Other target elements
will contain,
for instance, reference sequences. Target elements of various dimensions can
be used in
the arrays of the invention. Generally, smaller, target elements are
preferred. Typically,
a target element will be less than about 1 cm in diameter. Generally element
sizes are
from 1 ~m to about 3 mm, preferably between about S ~,m and about 1 mm. The
target
elements of the arrays may be arranged on the solid surface at different
densities. The
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target element densities will depend upon a number of factors, such as the
nature of the
label, the solid support, and the like. One of skill will recognize that each
target element
may comprise a mixture of nucleic acids of different lengths and sequences.
Thus, for
example, a target element may contain more than one copy of a cloned piece of
DNA, and
each copy may be broken into fragments of different lengths. The length and
complexity
of the nucleic acid fixed onto the target element is not critical to the
invention. One of
skill can adjust these factors to provide optimum hybridization and signal
production for a
given hybridization procedure, and to provide the required resolution among
different
genes or genomic locations. In various embodiments, target element sequences
will have
a complexity between about 1 kb and about 1 Mb, between about 10 kb to about
500 kb,
between about 200 to about 500 kb, and from about 50 kb to about 150 kb.
The terms "nucleic acid sample" or "sample of human nucleic acid" as
used herein refers to a sample comprising human DNA or RNA in a form suitable
for
detection by hybridization or amplification. Typically, it will be prepared
from a
premalignant tissue sample from a patient who has or is suspected of having
colorectal
cancer. The sample will most usually be prepared from polyp tissue.
In many instances, the nucleic acid sample will be a tissue or cell sample
prepared for standard iu situ hybridization methods described below. The
sample is
prepared such that individual chromosomes remain substantially intact and
typically
comprises metaphase spreads or interphase nuclei prepared according to
standard
techniques. Alternatively, the nucleic acid may be isolated, cloned or
amplified. It may
be, e.g., genomic DNA, mRNA, or cDNA from a particular chromosome, or selected
sequences (e.g. particular promoters, genes, amplification or restriction
fragments, cDNA,
ete.) within particular amplicons or deletions disclosed here.
The nucleic acid sample is extracted from colon adenoma cells or tissues.
Methods of isolating cell and tissue samples are well known to those of skill
in the art and
include, but are not limited to, aspirations, tissue sections, needle
biopsies, and the like.
Frequently the sample will be a "clinical sample" which is a sample derived
from a
patient, including sections of tissues such as frozen sections or paraffin
sections taken for
histological purposes. The sample can also be derived from supernatants (of
cells) or the
cells themselves from cell cultures, cells from tissue culture and other media
in which it
may be desirable to detect chromosomal abnormalities or determine amplicon
copy
number. In some cases, the nucleic acids may be amplified using standard
techniques
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such as PCR, prior to the hybridization. The sample may be isolated nucleic
acids
immobilized on a solid.
A "premalignant lesion", as used herein refers to benign adenomatous
colon tissue (e.g. a polyp) that has the potential of malignant
transformation. Polyps are
exceedingly common. Cytologically adenomas show varying degrees of dysplasia
ranging form mild to severe. There is virtually no distinction between severe
dysplasia
and carcinoma in situ, except that, dysplastic lesions, by definition, show no
evidence of
visible invasion. Polyps are generally removed by colonoscopic polypectomy.
The term "probe" or a "nucleic acid probe", as used herein, is defined to be
a collection of one or more nucleic acid fragments whose hybridization to a
sample can
be detected. The probe may be unlabeled or labeled as described below so that
its
binding to the target or sample can be detected. The probe is produced from a
source of
nucleic acids from one or more particular (preselected) portions of the
genome, e.g., one
or more clones, an isolated whole chromosome or chromosome fragment, or a
collection
of polyrnerase chain reaction (PCR) amplification products. The probes of the
present
invention are produced from nucleic acids found in the regions described
herein. The
probe or genomic nucleic acid sample may be processed in some manner, e.g., by
blocking or removal of repetitive nucleic acids or enrichment with unique
nucleic acids.
The word "sample" may be used herein to refer not only to detected nucleic
acids, but to
the detectable nucleic acids in the form in which they are applied to the
target, e.g., with
the blocking nucleic acids, etc. The blocking nucleic acid may also be
referred to
separately. What "probe" refers to specifically is clear from the context in
which the
word is used. The probe may also be isolated nucleic acids immobilized on a
solid
surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an
array. In some
embodiments, the probe may be a member of an array of nucleic acids as
described, for
instance, in WO 96/17958. Techniques capable of producing high density arrays
can also
be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston
(1998) Curr.
Biol. 8: 8171-8174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997)
Biotechniques 23: 120-124; U.S. Patent No. 5,143,854). One of skill will
recognize that
the precise sequence of the particular probes described herein can be modified
to a certain
degree to produce probes that are "substantially identical" to the disclosed
probes, but
retain the ability to specifically bind to (i.e., hybridize specifically to)
the same targets or
samples as the probe from which they were derived (see discussion above). Such
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modifications are specifically covered by reference to the individual probes
described
herein.
"Providing a nucleic acid sample" means to obtain a biological sample for
use in the methods described in this invention. Most often, this will be done
by removing
a sample of cells from an animal, but can also be accomplished by using
previously
isolated cells (e.g. isolated by another person), or by performing the methods
of the
invention in vivo.
"Tissue biopsy" refers to the removal of a biological sample for diagnostic
analysis. In a patient with cancer, tissue may be removed from a tumor,
allowing the
analysis of cells within the tumor.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention is based, at least in part, on the identification of
particular chromosomal abnormalities associated with the transition from an
adenoma to
carcinoma in colorectal cancer. Colorectal cancer is the second leading cause
of cancer
related deaths in the United States and other Western countries. Unlike lung
cancer, for
example, in which smoking has been identified as the prime etiologic factor
responsible
for the disease, the principle mechanisms underlying colorectal cancer are
complex and
incompletely understood.
The development of colon adenocarcinoma, like many other cancers, is a
mufti-step progression to malignancy. The concept of mufti-step carcinogenesis
means
that cancers evolve slowly over time during which the surrounding tissue
becomes
increasingly abnormal. The features of the early, intermediate and advanced
stages of
mufti-step malignant progression have been described using microscopy. The
first stage
of neoplastic progression is an increased number of relatively normal
appearing cells, the
hyperplastic stage. There are several stages of hyperplasia in which the cells
progressively accumulate and begin to develop an abnormal appearance, which is
the
emergence of the dysplastic phase. Dysplastic cells resemble immature
epithelial cells,
and during this phase of neoplastic progression, an increasing percentage of
the
epithelium is composed of these immature cells. Eventually, invasive cancers
develop in
tissue severely affected by dysplasia.
The critical issue in treatment of benign polyps is to decide whether or not
to proceed with colectomy in the case in which the removed polyp contains
malignant
foci. Generally, if the carcinomatous tissue is restricted superficially and
does not
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penetrate the muscolaris mucosa the carcinoma (in situ carcinoma or
intramucosal
carcinoma) no further surgical treatment is performed, while if the malignant
foci has
penetrated into the muscolaris mucosa the lesion is considered invasive and
therefore
colonic resection may be carried out. However the distinction between
intramucosal
carcinomas/high dysplastic lesion and invasive carcinoma may sometime
difficult to
achieve even by an experienced pathologist especially in the case of sessile
or friable
lesions or when the lesion have been morphologically altered in resection.
The present invention provides alternative criteria by which this transition
can be determined. As demonstrated below, amplification of the human
chromosomal
region at 20q (particularly at 20q13.2), is a frequent event in colon
adenocarcinomas,
occurring in approximately 80% of the cases. This molecular alteration is,
however, a
vary rare event in premalignant lesions, i.e. adenomas (polyps). In addition,
intermediary
diagnostic stages in the colon cancer natural history , i.e. high grade
dysplasia and
intramucosal carcinoma, display increased copy number of this chromosomal
region in
approximately 50% of the cases. These intermediary stages are thought to be
the
precursors of adenocarcinoma. Thus, detection of the 20q13.2 amplicon or other
regions
an 20q in colon tissue polyps can be used to the diagnose the stage and
therefore with the
gravity of the lesion.
The assays of the invention can also be used for prognosis of disease. For
instance, the presence of the amplicon in high grade dysplasia/ intramucosal
carcinoma
lesions can be used to predict whether the lesions will display
hystopathological and
cytopathological features similar to the invasive colon carcinomas. Thus, the
assays of
the invention can be an import tool in decision malting for both surgery and
therapy of
those patients that have high grade preadenomatous lesions. Similarly, the
presence of
20q13.2 amplicon in colon adenocarcinomas can be correlated with poor clinical
outcome.
DETECTION OF COPY NUMBER
In the preferred embodiment, the presence of colon carcinoma cells is
evaluated by a determination of copy number of regions on 20q identified here.
Methods
of evaluating the copy number of a particular gene or chromosomal region are
well
known to those of skill in the art.
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Hybridization-based assays
Preferred hybridization-based assays include, but are not limited to,
traditional "direct probe" methods such as Southern Blots or Ira Situ
Hybridization (e.g.,
FISH), and "comparative probe" methods such as Comparative Genomic
Hybridization
(CGH). The methods can be used in a wide vaxiety of formats including, but not
limited
to substrate- (e.g. membrane or glass) bound methods or array-based approaches
as
described below.
In situ hybridization assays are well known (e.g., Angerer (1987) Meth.
Erazymol 152: 649). Generally, ifz situ hybridization comprises the following
major steps:
(1) fixation of tissue or biological structure to analyzed; (2)
prehybridization treatment of
the biological structure to increase accessibility of target DNA, and to
reduce nonspecific
binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid
in the
biological structure or tissue; (4) post-hybridization washes to remove
nucleic acid
fragments not bound in the hybridization and (5) detection of the hybridized
nucleic acid
fragments. The reagent used in each of these steps and the conditions for use
vary
depending on the particular application.
In a typical ih situ hybridization assay, cells are fixed to a solid support,
typically a glass slide. If a nucleic acid is to be probed, the cells are
typically denatured
with heat or alkali. The cells are then contacted with a hybridization
solution at a
moderate temperature to permit annealing of labeled probes specific to the
nucleic acid
sequence encoding the protein. The targets (e.g., cells) are then typically
washed at a
predetermined stringency or at an increasing stringency until an appropriate
signal to
noise ratio is obtained.
The probes are typically labeled, e.g., with radioisotopes or fluorescent
reporters. The preferred size range is from about 200 by to about 1000 bases,
more
preferably between about 400 to about 800 by for double stranded, nick
translated nucleic
acids.
In some applications it is necessary to block the hybridization capacity of
repetitive sequences. Thus, in some embodiments, human genomic DNA or Cot-1
DNA
is used to block non- specific hybridization.
In Comparative Genomic Hybridization methods a first collection of
(sample) nucleic acids (e.g. from a possible tumor) is labeled with a first
label, while a
second collection of (control) nucleic acids (e.g. from a healthy cell/tissue)
is labeled with
a second label. The ratio of hybridization of the nucleic acids is determined
by the ratio of
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the two (first and second) labels binding to each fiber in the array. Where
there are
chromosomal deletions or multiplications, differences in the ratio of the
signals from the
two labels will be detected and the ratio will provide a measure of the copy
number.
Hybridization protocols suitable for use with the methods of the invention
are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988)
Proc.
Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular
Biology, hol. 33: In Situ Hyb~idizatiorz Protocols, Choo, ed., Humana Press,
Totowa, NJ
(1994), etc. In one particularly preferred embodiment, the hybridization
protocol of
Pinkel et al. (1998) Nature Genetics 20: 207-211 or of Kallioniemi (1992)
P~oc. Natl
Acad Sci USA 89:5321-5325 (1992) is used.
The methods of this invention are particularly well suited to array-based
hybridization formats. For a description of one preferred array-based
hybridization
system see Pinkel et al. (1998) Nature Genetics, 20: 207-211.
Arrays are a multiplicity of different "probe" or "target" nucleic acids (or
other compounds) attached to one or more surfaces (e.g., solid, membrane, or
gel). In a
preferred embodiment, the multiplicity of nucleic acids (or other moieties) is
attached to a
single contiguous surface or to a multiplicity of surfaces juxtaposed to each
other.
In an array format a large number of different hybridization reactions can
be run essentially "in parallel." This provides rapid, essentially
simultaneous, evaluation
of a number of hybridizations in a single "experiment". Methods of performing
hybridization reactions in array based formats are well known to those of
skill in the art
(see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature
Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958.
Arrays, particularly nucleic acid arrays can be produced according to a
wide variety of methods well known to those of skill in the art. For example,
in a simple
embodiment, "low density" arrays can simply be produced by spotting (e.g. by
hand using
a pipette) different nucleic acids at different locations on a solid support
(e.g. a glass
surface, a membrane, etc.).
This simple spotting, approach has been automated to produce high
density spotted arrays (see, e.g., U.S. Patent No: 5,807,522). This patent
describes the
use of an automated systems that taps a microcapillary against a surface to
deposit a small
volume of a biological sample. The process is repeated to generate high
density arrays.
Arrays can also be produced using oligonucleotide synthesis technology. Thus,
for
example, U.S. Patent No. 5,143,854 and PCT patent publication Nos. WO 90/15070
and
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92/10092 teach the use of light-directed combinatorial synthesis of high
density
oligonucleotide arrays.
In another embodiment the array, particularly a spotted array, can include
genomic DNA, e.g. overlapping clones that provide a high resolution scan of
the
amplicon corresponding to the region of interest. Amplicon nucleic acid can be
obtained
from, e.g., MACs, YACs, BACs, PACs, Pls, cosmids, plasmids, inter-Alu PCR
products
of genomic clones, restriction digests of genomic clone, cDNA clones,
amplification
(e.g., PCR) products, and the like.
In various embodiments, the array nucleic acids are derived from
previously mapped libraries of clones spanning or including the taxget
sequences of the
invention, as well as clones from other areas of the genome, as described
below. The
arrays can be hybridized with a single population of sample nucleic acid or
can be used
with two differentially labeled collections (as with an test sample and a
reference sample).
Many methods for immobilizing nucleic acids on a variety of solid
surfaces are known in the art. A wide variety of organic and inorganic
polymers, as well
as other materials, both natural and synthetic, can be employed as the
material for the
solid surface. Illustrative solid surfaces include, e.g., nitrocellulose,
nylon, glass, quartz,
diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose,
and
cellulose acetate. In addition, plastics such as polyethylene, polypropylene,
polystyrene,
and the like can be used. Other materials which may be employed include paper,
ceramics, metals, metalloids, semiconductive materials, cermets or the like.
In addition,
substances that form gels can be used. Such materials include, e.g., proteins
(e.g.,
gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Where
the solid
surface is porous, various pore sizes may be employed depending upon the
nature of the
system.
In preparing the surface, a plurality of different materials maybe
employed, particularly as laminates, to obtain various properties. For
example, proteins
(e.g., bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt's
solution)
can be employed to avoid non-specific binding, simplify covalent conjugation,
enhance
signal detection or the like. If covalent bonding between a compound and the
surface is
desired, the surface will usually be polyfunctional or be capable of being
polyfunctionalized. Functional groups wluch may be present on the surface and
used for
linking can include carboxylic acids, aldehydes, amino groups, cyano groups,
ethylenic
groups, hydroxyl groups, mercapto groups and the like. The manner of linking a
wide
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variety of compounds to various surfaces is well known and is amply
illustrated in the
literature.
For example, methods for immobilizing nucleic acids by introduction of
various functional groups to the molecules is known (see, e.g., Bischoff
(1987) Anal.
Biochem., 164: 336-344; Kremsky (1987) Nucl. Acids Res. 15: 2891-2910).
Modified
nucleotides can be placed on the target using PCR primers containing the
modified
nucleotide, or by enzymatic end labeling with modified nucleotides. Use of
glass or
membrane supports (e.g., nitrocellulose, nylon, polypropylene) for the nucleic
acid arrays
of the invention is advantageous because of well developed technology
employing
manual and robotic methods of arraying targets at relatively high element
densities. Such
membranes are generally available and protocols and equipment for
hybridization to
membranes is well known.
Target elements of various sizes, ranging from 1 mm diameter down to 1
~,m can be used. Smaller target elements containing low amounts of
concentrated, fixed
probe DNA are used for high complexity comparative hybridizations since the
total
amount of sample available for binding to each target element will be limited.
Thus it is
advantageous to have small array target elements that contain a small amount
of
concentrated probe DNA so that the signal that is obtained is highly localized
and bright.
Such small array target elements are typically used in arrays with densities
greater than
104/cm~. Relatively simple approaches capable of quantitative fluorescent
imaging of 1
cm2 areas have been described that permit acquisition of data from a large
number of
target elements in a single image (see, e.g., Wittrup (1994) Cytometry 16:206-
213).
Arrays on solid surface substrates with much lower fluorescence than
membranes, such as glass, quartz, or small beads, can achieve much better
sensitivity.
Substrates such as glass or fused silica are advantageous in that they provide
a very low
fluorescence substrate, and a highly efficient hybridization environment.
Covalent
attachment of the target nucleic acids to glass or synthetic fused silica can
be
accomplished according to a number of known techniques (described above).
Nucleic
acids can be conveniently coupled to glass using commercially available
reagents. For
instance, materials for preparation of silanized glass with a number of
functional groups
are commercially available or can be prepared using standard techniques (see,
e.g., Gait
(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Wash.,
D.C.).
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Quartz cover slips, which have at least 10-fold lower autofluorescence than
glass, can
also be silanized.
Alternatively, probes can also be immobilized on commercially available
coated beads or other surfaces. For instance, biotin end-labeled nucleic acids
can be
bound to commercially available avidin-coated beads. Streptavidin or anti-
digoxigenin
antibody can also be attached to silanized glass slides by protein-mediated
coupling using
e.g., protein A following standard protocols (see, e.g., Smith (1992) Sciefzce
258: 1122-
1126). Biotin or digoxigenin end-labeled nucleic acids can be prepared
according to
standard techniques. Hybridization to nucleic acids attached to beads is
accomplished by
suspending them in the hybridization mix, and then depositing them on the
glass substrate
for analysis after washing. Alternatively, paramagnetic particles, such as
ferric oxide
particles, with or without avidin coating, can be used.
In one particularly preferred embodiment, probe nucleic acid is spotted
onto a surface (e.g., a glass or quartz surface). The nucleic acid is
dissolved in a mixture
of dimethylsulfoxide (DMSO) and nitrocellulose and spotted onto amino-silane
coated
glass slides. Small capillaries tubes can be used to "spot" the probe mixture.
A variety of other nucleic acid hybridization formats are known to those
skilled in the art. For example, common formats include sandwich assays and
competition or displacement assays. Hybridization techniques are generally
described in
Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL
Press;
Gall and Pardue (1969) Proc. Natl. Aead. Sci. USA 63: 378-383; and John et al.
(1969)
Nature 223: 582-587.
Sandwich assays are commercially useful hybridization assays for
detecting or isolating nucleic acid sequences. Such assays utilize a "capture"
nucleic acid
covalently immobilized to a solid support and a labeled "signal" nucleic acid
in solution.
The sample will provide the target nucleic acid. The "capture" nucleic acid
and "signal"
nucleic acid probe hybridize with the target nucleic acid to form a "sandwich"
hybridization complex. To be most effective, the signal nucleic acid should
not hybridize
with the capture nucleic acid.
Detection of a hybridization complex may require the binding of a signal
generating complex to a duplex of target and probe polynucleotides or nucleic
acids.
Typically, such binding occurs through ligand and anti-ligand interactions as
between a
ligand-conjugated probe and an anti-ligand conjugated with a signal.
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The sensitivity of the hybridization assays may be enhanced through use of
a nucleic acid amplification system that multiplies the target nucleic acid
being detected.
Examples of such systems include the polymerase chain reaction (PCR) system
and the
ligase chain reaction (LCR) system. Other methods recently described in the
art are the
nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,
Ontario)
and Q Beta Replicase systems.
Nucleic acid hybridization simply involves providing a denatured probe
and target nucleic acid under conditions where the probe and its complementary
target
can form stable hybrid duplexes through complementary base pairing. The
nucleic acids
that do not form hybrid duplexes are then washed away leaving the hybridized
nucleic
acids to be detected, typically through detection of an attached detectable
label. It is
generally recognized that nucleic acids are denatured by increasing the
temperature or
decreasing the salt concentration of the buffer containing the nucleic acids,
or in the
addition of chemical agents, or the raising of the pH. Under low stringency
conditions
(e.g., low temperature and/or high salt and/or high target concentration)
hybrid duplexes
(e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed
sequences are not perfectly complementary. Thus specificity of hybridization
is reduced
at lower stringency. Conversely, at higher stringency (e.g., higher
temperature or lower
salt) successful hybridization requires fewer mismatches.
One of skill in the art will appreciate that hybridization conditions may be
selected to provide any degree of stringency. In a preferred embodiment,
hybridization is
performed at low stringency to ensure hybridization and then subsequent washes
are
performed at higher stringency to eliminate mismatched hybrid duplexes.
Successive
washes may be performed at increasingly higher stringency (e.g., down to as
low as 0.25
X SSPE-T at 37°C to 70°C) until a desired level of hybridization
specificity is obtained.
Stringency can also be increased by addition of agents such as formamide.
Hybridization
specificity may be evaluated by comparison of hybridization to the test probes
with
hybridization to the various controls that can be present.
In general, there is a tradeoff between hybridization specificity
(stringency) and signal intensity. Thus, in a preferred embodiment, the wash
is performed
at the highest stringency that produces consistent results and that provides a
signal
intensity greater than approximately 10% of the background intensity. Thus, in
a
preferred embodiment, the hybridized array may be washed at successively
higher
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stringency solutions and read between each wash. Analysis of the data sets
thus produced
will reveal a wash stringency above which the hybridization pattern is not
appreciably
altered and which provides adequate signal for the particular probes of
interest.
In a preferred embodiment, background signal is reduced by the use of a
detergent (e.g., C-TAB) or a blocking reagent (e.g., sperm DNA, cot-1 DNA,
etc.) during
the hybridization to reduce non-specific binding. In a particularly preferred
embodiment,
the hybridization is performed in the presence of about 0.1 to about 0.5 mg/ml
DNA (e.g.,
cot-1 DNA). The use of blocking agents in hybridization is well known to those
of skill
in the art (see, e.g., Chapter ~ in P. Tijssen, supra.)
Methods of optimizing hybridization conditions are well known to those of
skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques in
Biochemistry afZd
Molecular Biology, hol. 24: Hybridization With Nucleic Acid Probes, Elsevier,
N.Y.).
Optimal conditions are also a function of the sensitivity of label (e.g.,
fluorescence) detection for different combinations of substrate type,
fluorochrome,
excitation and emission bands, spot size and the like. Low fluorescence
background
membranes can be used (see, e.g., Chu (1992) Electroplzo~esis 13:105-114). The
sensitivity for detection of spots ("target elements") of various diameters on
the candidate
membranes can be readily determined by, e.g., spotting a dilution series of
fluorescently
end labeled DNA fragments. These spots are then imaged using conventional
fluorescence microscopy. The sensitivity, linearity, and dynamic range
achievable from
the various combinations of fluorochrome and solid surfaces (e.g., membranes,
glass,
fused silica) can thus be determined. Serial dilutions of pairs of
fluorochrome in known
relative proportions can also be analyzed. This determines the accuracy with
which
fluorescence ratio measurements reflect actual fluorochrome ratios over the
dynamic
range permitted by the detectors and fluorescence of the substrate upon which
the probe
has been fixed.
Labeling and detection of nucleic acids.
In a preferred embodiment, the hybridized nucleic acids are detected by
detecting one or more labels attached to the sample or probe nucleic acids.
The labels
may be incorporated by any of a number of means well known to those of skill
in the art.
Means of attaching labels to nucleic acids include, for example nick
translation or end-
labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and
subsequent
attachment (ligation) of a nucleic acid linker joining the sample nucleic acid
to a label
(e.g., a fluorophore). A wide variety of linkers for the attachment of labels
to nucleic
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acids are also known. In addition, intercalating dyes and fluorescent
nucleotides can also
be used.
Detectable labels suitable for use in the present invention include any
composition detectable by spectroscopic, photochemical, biochemical,
immunochemical,
electrical, optical or chemical means. Useful labels in the present invention
include biotin
fox staining with labeled streptavidin conjugate, magnetic beads (e.g.,
DynabeadsTM),
fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent
protein, and
the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels
(e.g., 3H, lash
3s5, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others
commonly used in an ELISA), and colorimetric labels such as colloidal gold
(e.g., gold
particles in the 40 -80 nm diameter size range scatter green light with high
efficiency) or
colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents
teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752;
3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241.
A fluorescent label is preferred because it provides a very strong signal
with low background. It is also optically detectable at high resolution and
sensitivity
through a quick scanning procedure. The nucleic acid samples can all be
labeled with a
single label, e.g., a single fluorescent label. Alternatively, in another
embodiment,
different nucleic acid samples can be simultaneously hybridized where each
nucleic acid
sample has a different label. For instance, one target could have a green
fluorescent label
and a second target could have a red fluorescent label. The scanning step will
distinguish
cites of binding of the red label from those binding the green fluorescent
label. Each
nucleic acid sample (target nucleic acid) can be analyzed independently from
one another.
Suitable chromogens which can be employed include those molecules and
compounds which absorb light in a distinctive range of wavelengths so that a
color can be
observed or, alternatively, which emit light when irradiated with radiation of
a particular
wave length or wave length range, e.g., fluorescers.
Desirably, fluorescers should absorb light above about 300 nm, preferably
about 350 nm, and more preferably above about 400 nm, usually emitting at
wavelengths
greater than about 10 nm higher than the wavelength of the light absorbed. It
should be
noted that the absorption and emission characteristics of the bound dye can
differ from
the unbound dye. Therefore, when referring to the various wavelength ranges
and
characteristics of the dyes, it is intended to indicate the dyes as employed
and not the dye
which is unconjugated and characterized in an arbitrary solvent.
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Fluorescers are generally preferred because by irradiating a fluorescer with
light, one can obtain a plurality of emissions. Thus, a single label can
provide for a
plurality of measurable events.
Detectable signal can also be provided by chemiluminescent and
bioluminescent sources. Chemiluminescent sources include a compound which
becomes
electronically excited by a chemical reaction and can then emit light which
serves as the
detectable signal or donates energy to a fluorescent acceptor. Alternatively,
luciferins can
be used in conjunction with luciferase or lucigenins to provide
bioluminescence.
Spin labels are provided by reporter molecules with an unpaired electron spin
which can
be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin
labels
include organic free radicals, transitional metal complexes, particularly
vanadium,
copper, iron, and manganese, and the like. Exemplary spin labels include
nitroxide free
radicals.
The label may be added to the target (sample) nucleic acids) prior to, or
after the hybridization. So called "direct labels" are detectable labels that
are directly
attached to or incorporated into the target (sample) nucleic acid prior to
hybridization. In
contrast, so called "indirect labels" are joined to the hybrid duplex after
hybridization.
Often, the indirect label is attached to a binding moiety that has been
attached to the
target nucleic acid prior to the hybridization. Thus, for example, the target
nucleic acid
may be biotinylated before the hybridization. After hybridization, an avidin-
conjugated
fluorophore will bind the biotin bearing hybrid duplexes providing a label
that is easily
detected. For a detailed review of methods of labeling nucleic acids and
detecting labeled
hybridized nucleic acids see Labo~atoYy Techniques in Biochemistry ahd
Molecular
Biology, Irol. 24: HybridizatioyZ With Nucleic Acid Probes, P. Tijssen, ed.
Elsevier, N.Y.,
(1993)).
Fluorescent labels are easily added during an in vitro transcription
reaction. Thus, for example, fluorescein labeled UTP and CTP can be
incorporated into
the RNA produced in an ira vitro transcription.
The labels can be attached directly or through a linker moiety. In general,
the site of label or linker-label attachment is not limited to any specific
position. For
example, a label may be attached to a nucleoside, nucleotide, or analogue
thereof at any
position that does not interfere with detection or hybridization as desired.
For example,
certain Label-ON Reagents from Clontech (Palo Alto, CA) provide for labeling
interspersed throughout the phosphate backbone of an oligonucleotide and for
terminal
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labeling at the 3' and 5' ends. As shown for example herein, labels can be
attached at
positions on the ribose ring or the ribose can be modified and even eliminated
as desired.
The base moieties of useful labeling reagents can include those that are
naturally
occurring or modified in a manner that does not interfere with the purpose to
which they
are put. Modified bases include but are not limited to 7-deaza A and G, 7-
deaza-8-aza A
and G, and other heterocyclic moieties.
It will be recognized that fluorescent labels are not to be limited to single
species organic molecules, but include inorganic molecules, mufti-molecular
mixtures of
organic and/or inorganic molecules, crystals, heteropolymers, and the like.
Thus, for
example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be
easily
derivatized for coupling to a biological molecule (Bruchez et al. (1998)
Science, 281:
2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped
cadmium
selenide) have been covalently coupled to biomolecules for use in
ultrasensitive
biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
Amplification-based assays.
In another embodiment, amplification-based assays can be used to measure
copy number. In such amplification-based assays, the nucleic acid sequences
act as a
template in an amplification reaction (e.g. Polymerase Chain Reaction (PCR).
In a
quantitative amplification, the amount of amplification product will be
proportional to the
amount of template in the original sample. Comparison to appropriate (e.g.
healthy
tissue) controls provides a measure of the copy number of the desired target
nucleic acid
sequence. Methods of "quantitative" amplification are well known to those of
skill in the
art. For example, quantitative PCR involves simultaneously co-amplifying a
known
quantity of a control sequence using the same primers. This provides an
internal standard
that may be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR
are provided in Innis et al. (1990) PCR Protoc~ls, A Cruide to Methods and
Applications,
Academic Press, Inc. N.Y.).
Other suitable amplification methods include, but are not limited to ligase
chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et
al.
(1988) Science 241: 1077, and Barringer et al. (1990) Gene 89: 117,
transcription
amplification (Kwoh et al. (1989) P~oc. Natl. Acad. Sci. USA 86: 1173), and
self
sustained sequence replication (Guatelli et al. (1990) P~oc. Nat. Acad. Sci.
USA 87:
1874).
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DETECTION OF GENE E~I'RESSION
As indicated below, a number of oncogenes are found in the regions of
amplification disclosed here. Thus, oncogene activity can be detected by, for
instance,
measuring levels of the gene transcript (e.g. mRNA), or by measuring the
quantity of
translated protein.
Detection of gene transcripts.
Methods of detecting and/or quantifying t gene transcripts using nucleic
acid hybridization techniques are known to those of skill in the art (see
Sambrook et al.
supra). For example , a Northern transfer may be used for the detection of the
desired
mRNA directly. In brief, the mRNA is isolated from a given cell sample using,
for
example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is
then
electrophoresed to separate the mRNA species and the mRNA is transferred from
the gel
to a nitrocellulose membrane. As with the Southern blots, labeled probes are
used to
identify and/or quantify the target mRNA.
In another preferred embodiment, the gene transcript can be measured
using amplification (e.g. PCR) based methods as described above for directly
assessing
copy number of the target sequences.
Detection of expressed protein
The "activity" of the target onocgene can also be detected and/or
quantified by detecting or quantifying the expressed polypeptide. The
polypeptide can be
detected and quantified by any of a number of means well known to those of
skill in the
art. These may include analytic biochemical methods such as electrophoresis,
capillary
electrophoresis, high performance liquid chromatography (HPLC), thin layer
chromatography (TLC), hyperdiffusion chromatography, and the like, or various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single
or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and
the
like.
KITS FOR USE IN DIAGNOSTIC AND/OR PROGNOSTIC APPLICATIONS.
For use in diagnostic, research ,and therapeutic applications suggested
above, kits are also provided by the invention. In the diagnostic and research
applications
such kits may include any or all of the following: assay reagents, buffers,
nucleic acids
for detecting the target sequesences and other hybridization probes and/or
primers. A
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therapeutic product may include sterile saline or another pharmaceutically
acceptable
emulsion and suspension base.
In addition, the kits may include instructional materials containing
directions (i.e., protocols) for the practice of the methods of this
invention. While the
instructional materials typically comprise written or printed materials they
are not limited
to such. Any medium capable of storing such instructions and communicating
them to an
end user is contemplated by this invention. Such media include, but are not
limited to
electronic storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media
(e.g., CD ROM), and the like. Such media may include addresses to Internet
sites that
provide such instructional materials.
EXAMPLES
This examples provides a detailed protocol using FISH analysis to detect
the 20q13.2 amplicon in colon tissue sections. The method was tested in 140
archival
tissue specimens.
Materials and Methods
Tissue specimens and slide preparation
Four micron sections were cut from archival tissue which had been fixed
in buffered formalin and embedded in paraffin and placed on aminoalkylsilane-
treated
slides. Silanization of the slides was for 5 min in a 2% 3-
aminopropyltriethoxysilane
solution (Sigma Chemical Co., St. Louis, MO) in acetone, followed by
successive washes
in acetone and distilled water. Slide mounted tissue sections were then baked
overnight
at 65°C. and deparaffmized in xylenes for 10 min. x 3, followed by
immersion in ethanol.
Air dried sections were subsequently treated in 1N. Sodium thiocyanate for
20min. at
80°C., washed 3X in 2X SSC and then treated in as solution of
proteinase I~ in 2xSSC
(250~,g.lml.) for 20 min at 37C. Tissue section were then washed in 2X SSC as
above
and dehydrated in 70% EtOH and then in acetone for 2 min each.
Probe
A probe spanning approximately 200kb of the chromosomal region
containing the ZNF217 gene was utilized to detect the presence or absence of
20q13.2
amplification. The probe spans from about D20S854 to about D2020S876 (see,
Collins et
al. Proc. Natl. Acad. Sci. U.S.A. 95:8703-8708 (1998) for a description of the
20q13.2
amplicon and ZNF217 gene). The probe was directly labeled with the fluorophore
SpectrumOrangeT"" (Vysis, Inc., Downers Grove, IL).
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In situ hybridization
Slide mounted tissue sections were denatured in 70% formamide/2X SSC,
pH7.0 at ~5°C for 10 min and then immersed in 100% EtOH al -
20°C. Before applying
the hybridization mixture onto tissue sections, slides were air-dried and
warmed to 45°C.
The hybridization mixture contained 50% formamide/10% dextran sulfate/2X SSC.
Concentration of probe was l Ong/ml and hybridization of repetitive DNA
sequences was
suppressed by inclusion of O.S~g of human Cot-1 DNA (Gibco BRL, Grand Island,
NY).
The hybridization mixture was denatured for 10 min at 100°C and allowed
to reanneal for
20-30 min at 37°C. Hybridization was carned out overnight at
42°C (direct-labeled
probe) under a coverslip in a moist chamber. Washes were performed
respectively at
45°Cas follows: twice in 50% formamide/2X SSC for 15 min each, twice in
0.1% Triton
X-100/2X SSC for 10 min each. Tissue sections were counterstained using 0.15
mM 4,6
diamidino-2-phenylindole (DAPI) (Sigma Chemical Co., St. Louis, MO) in 2X SSC
for 5
min, destained in 2X SSC for 5 min, dehydrated in 100% EtOH, air dried and
mounted in
antifade solution (Vector Laboratories, Inc., Burlingame, CA).
Fluorescence Microscoby
A Zeiss epifluorescence microscope equipped with a 100 watt mercury-arc
lamp and high numerical aperture Neofluor objectives was used with the
following
single-band pass fitter combinations: DAPI 02 (Carl Zeiss, Inc., Thornwood,
NY), High
Q FITC (Chroma Technology Corp., Brattleboro, VT), FITC 09 (Carl Zeiss, Inc.,
Thornwood, NY) and a specially designed fitter combination optimized for
SpectrumOrange detection (Vysis, Inc., Downers Grove, IL).
Results
Using the techniques described above, it was found that more than 2/3 of
colon cancers contain amplification at 20q13.2. The finding is especially
useful in
diagnostics. The only methods presently available for the diagnosis of colon
lesions is by
direct examination by a pathologist. The 20q13.2 amplicon can easily be tested
for using
this approach and provides clear-cut confirmation of diagnosis. Second, the
diagnosis of
intramucosal carcinomas (IMCa) is particularly difficult and somewhat
subjective
depending heavily on the pathologist. Determination of a diagnosis of IMCa
could malce
the difference between more or less aggressive therapies.
23
SUBSTITUTE SHEET (RULE 26)
