U.S. patent application number 13/182774 was filed with the patent office on 2012-06-07 for method and probes for the detection of cancer.
Invention is credited to Kevin C. Halling, Larry E. Morrison, Steven A. Seelig, Irina A. Sokolova.
Application Number | 20120141987 13/182774 |
Document ID | / |
Family ID | 23030623 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141987 |
Kind Code |
A1 |
Morrison; Larry E. ; et
al. |
June 7, 2012 |
METHOD AND PROBES FOR THE DETECTION OF CANCER
Abstract
Probe sets and methods of using probes and probe sets for the
detection of cancer are described. Methods for detecting cancer
that include hybridizing a set of chromosomal probes to a
biological sample obtained from a patient, and identifying if
cancer cells are present the sample. Also included are methods of
selecting a combination of probes for the detection of cancer.
Inventors: |
Morrison; Larry E.; (Oro
Valley, AZ) ; Sokolova; Irina A.; (Villa Park,
IL) ; Seelig; Steven A.; (Elmhurst, IL) ;
Halling; Kevin C.; (Rochester, MN) |
Family ID: |
23030623 |
Appl. No.: |
13/182774 |
Filed: |
July 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12536647 |
Aug 6, 2009 |
7998678 |
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13182774 |
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11259771 |
Oct 27, 2005 |
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12536647 |
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10081393 |
Feb 20, 2002 |
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11259771 |
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60270271 |
Feb 20, 2001 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6841 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C12Q 1/68 20060101 C12Q001/68 |
Claims
1) A method of screening for lung cancer in a human subject whose
status with respect to lung cancer is unknown, the method
comprising: a) obtaining a biological sample, wherein the
biological sample comprises a lung sample, from the subject; b)
obtaining a set of chromosomal probes consisting of two probes said
two probes suitable for detecting lung cancer with greater
sensitivity and specificity together than each individual probe,
said set of two probes consisting of a 5p15 locus specific probe
and a 7p12 locus specific probe or a 7p12 locus specific probe and
a chromosome 6 enumeration probe; c) contacting the set of probes
to the biological sample under conditions sufficient to enable
hybridization of probes in the set to chromosomes in the sample, if
any; and d) detecting a hybridization pattern consisting of gains
or losses of copy number of chromosomal target regions as
determined by the set of chromosomal probes hybridized to the
chromosomes in the biological sample to determine whether the
subject has lung cancer.
2) The method of claim 1, wherein the biological sample consists of
one or more of a bronchial specimen, a lung biopsy, or a sputum
sample.
3) The method of claim 1, wherein the chromosomal probes are
fluorescently labeled.
4) The method of claim 1, further comprising performing cytological
analysis on the sample.
Description
RELATED APPLICATIONS
[0001] The present application is a Continuation of U.S. patent
application Ser. No. 11/259,771 filed on Oct. 27, 2005 (pending)
which is a Division of U.S. patent application Ser. No. 10/081,393
filed on Feb. 20, 2002 (abandoned) which is a Non-Provisional of
U.S. Patent Application Ser. No. 60/270,271 (expired) filed on Feb.
20, 2001 and is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods and probes for the
detection of cancer.
BACKGROUND OF THE INVENTION
[0003] Lung cancer is the leading cause of death due to cancer in
the United States, killing approximately 156,000 men and women each
year. There are four major bronchogenic carcinoma cell types that
account for over 95% of primary lung cancers: adenocarcinoma;
squamous cell carcinoma; large cell carcinoma; and small cell
carcinoma. These cell types occur singly or in combination. The
remaining 5% of tumors are composed of several unusual tumor
types.
[0004] When lung cancer develops, it tends to spread from the
original cancer site to the lymph nodes, and then, either at the
same time or sequentially, to other areas of the body. The most
common sites for lung cancer to spread (metastasis) are the brain,
bones, liver, adrenal glands, and any other organ with a high rate
of blood flow. It is this process of metastasis that leads to
fatality in most patients.
[0005] When a cancer is first discovered by physical examination or
by diagnostic tests (e.g., X-ray or high resolution imaging such as
spiral CT), it is usually at least 1 cm in size. A cancer that is 1
cm in size contains at least about 1 billion cells.
[0006] Changes in chromosomal DNA have been shown to accompany the
conversion of normal cells to malignant cells. Because of this,
detection of specific chromosomal alterations provides a route to
detecting and diagnosing lung cancer.
SUMMARY OF THE INVENTION
[0007] The invention is based on the discovery that specific probes
and probe sets can be used to detect lung cancer with high levels
of sensitivity. By using the probes described herein, lung cancer
can be detected with enhanced sensitivity as compared to
conventional methods. Accordingly, the probes and methods of the
invention facilitate the detection of lung cancer and/or allow for
the detection of lung cancer at early stages. The invention
includes probe sets, methods of using probes and probe sets, and
methods of selecting probe sets for the detection of cancer.
[0008] In one aspect, the invention features set of chromosomal
probes including any of the following combinations of two probes:
(a) a 5p chromosome arm probe and a probe selected from the group
consisting of a 8q24 locus specific probe, a 3q chromosome arm
probe, a 20q chromosome arm probe, a 7p12 locus specific probe, a
chromosome 16 enumeration probe, a chromosome 4 enumeration probe,
a chromosome 12 enumeration probe, a chromosome6 enumeration probe,
and a 17q21 locus specific probe; (b) a 8q24 locus specific probe
and a probe selected from the group consisting of a chromosome 17
enumeration probe, a chromosome 1 enumeration probe, and a
chromosome 6 enumeration probe; (c) a 7p12 locus specific probe and
a probe selected from the group consisting of a 3g chromosome arm
probe and a chromosome 6 enumeration probe; (d) a 3q chromosome arm
probe and a chromosome 7 enumeration probe; or (e) a chromosome 6
enumeration probe and a chromosome 7 enumeration probe.
[0009] A detection moiety can be attached to the two probes. The
detection moiety can contain a fluorescent label. The two probes
can optionally be coupled to different detection moieties. For
example, the detection moieties can contain fluorescent labels.
[0010] In another aspect, the invention features a set of
chromosomal probes including any of the following combinations of
three probes: (a) a 5p15 locus specific probe, a. 8q24 locus
specific probe, and a probe selected from the group consisting of a
9p21 locus specific probe, a chromosome 1 enumeration probe, a
chromosome 6 enumeration probe, a 7p12 locus specific probe, and a
17q21 locus specific probe; (b) a 5p15 locus specific probe, a
chromosome 12 enumeration probe, and a 9p21 locus specific probe;
(c) a 8q24 locus specific probe, a chromosome 17 enumeration probe,
and a 9p21 locus specific probe; (d) a 8g24 locus specific probe, a
chromosome 1 enumeration probe, and a 9p21 locus specific probe; or
(e) a 5p15 locus specific probe, a 3q chromosome arm probe, and a
chromosome 12 enumeration probe.
[0011] In another aspect, the invention features a set of
chromosomal probes including any of the following combinations of
four probes: (a) a 5p15 locus specific probe, a chromosome 6
enumeration probe, a 17p13 locus specific probe, and a chromosome
17 enumeration probe; (b) a 5p15 locus specific probe, a 8q24 locus
specific probe, a chromosome 1 enumeration probe, and a 7p12 locus
specific probe; (c) a 5p15 locus specific probe, a 8q24 locus
specific probe, a 3q chromosome arm probe, and a 7p12 locus
specific probe; (d) a 5p15 locus specific probe, a 8q24 locus
specific probe, a 20q chromosome arm probe, and a 7p12 locus
specific probe; (e) a 5p15 locus specific probe, a 8q24 locus
specific probe, a 7p12 locus specific probe, and a 17q21 locus
specific probe; (f) a 5p15 locus specific probe, a 8q24 locus
specific probe, a chromosome 6 enumeration probe, and a 7p12 locus
specific probe; (g) a 5p15 locus specific probe, a 8q24 locus
specific probe, a chromosome 6 enumeration probe, and a chromosome
1 enumeration probe; (h) a 5p15 locus specific probe, a 8q24 locus
specific probe, a chromosome 6 enumeration probe, and a chromosome
12 enumeration probe; (i) a 5p15 locus specific probe, a chromosome
1 enumeration probe, a chromosome 6 enumeration probe, and a
chromosome 12 enumeration probe; (j) a chromosome 7 enumeration
probe, a chromosome 1 enumeration probe, a chromosome 6 enumeration
probe, and a chromosome 12 enumeration probe; or (k) a 5p
chromosome arm probe, a chromosome 1 enumeration probe, a
chromosome 6 enumeration probe, and a chromosome 7 enumeration
probe.
[0012] In some embodiments of the probe sets described herein,
e.g., a set containing at least two, three, or four probes, a 5p
chromosome arm probe can be used in place of a 5p15 locus specific
probe. In other embodiments of the probe sets described herein, a
7p chromosome arm probe can be used in place of a 7p12 locus
specific, probe.
[0013] In another aspect, the invention features a method of
screening for lung cancer in a subject, the method including the
steps of: (a) obtaining a biological sample from the subject; (b)
obtaining a set of at least t*o different chromosomal probes, e.g.,
at least two, three, or four probes, from a set described herein;
(c) contacting the set of probes to the biological sample under
conditions sufficient to enable hybridization of probes in the set
to chromosomes in the sample, if any; and (d) detecting the
hybridization pattern of the set of chromosomal probes to the
biological sample to determine whether the subject has lung
cancer.
[0014] The probes used in the methods described herein can be
selected from the group consisting of a chromosome 1 enumeration
probe, a chromosome 3 enumeration probe, a chromosome 4 enumeration
probe, a chromosome 6 enumeration probe, a chromosome 7 enumeration
probe, a chromosome 8 enumeration probe, a chromosome 9 enumeration
probe, a chromosome 10 enumeration probe, a chromosome 11
enumeration probe, a chromosome 12 enumeration probe, a chromosome
16 enumeration probe, a chromosome 17 enumeration probe, a
chromosome 18 enumeration probe, a 3p14 locus specific probe, a
3q26 locus specific; probe, a 5p15 locus specific probe, a 5q31
locus specific probe, a 7p12 locus specific probe, a 8q24 locus
specific probe, a 9p21 locus specific probe, a 10q23 locus specific
probe, a 13q14 locus specific probe, a 17p13 locus specific probe,
a 17q21 locus specific probe, a 20q13 locus specific probe, a 21q22
locus specific probe, a 3q chromosome arm probe, a 5p chromosome
anti probe, a 7p chromosome arm probe, a 3p chromosome arm probe,
and a 20q chromosome arm probe.
[0015] The biological sample used in the methods described herein
can contain a bronchial specimen, a lung biopsy, or a sputum
sample. The chromosomal probes used in the methods described herein
can optionally be fluorescently labeled. The methods described
herein can further include performing cytological analysis on the
sample.
[0016] In another aspect, the invention features a method of
screening for king cancer in a subject, the method including the
steps of: (a) obtaining a biological sample from the subject; (b)
obtaining a chromosomal probe selected from the group consisting of
a 5p15 locus specific probe, a chromosome 1 enumeration probe, a
7p12 locus specific probe, a 8q24 locus specific probe, and a
chromosome 9 enumeration probe; (c) contacting the chromosomal
probe to the biological sample under conditions sufficient to
enable hybridization of the probe to chromosomes in the sample, if
any; and (d) detecting the hybridization pattern of the probe to
the biological sample to determine whether the subject has lung
cancer.
[0017] In another aspect, the invention features a method of
selecting a combination of probes for the detection of cancer, the
method including the steps of; (a) providing a first plurality of
chromosomal probes; (b) determining the ability of each of the
first plurality of probes to distinguish cancer specimens from
normal specimens; (c) selecting those probes within the first
plurality of probes that identify the cancer specimens as compared
to the normal specimens to yield a second plurality of probes,
wherein the second plurality of probes each identify the cancer
specimens as compared to the normal specimens at a p value of less
than 0.01 or a vector value of less than 0.500; (d) determining the
ability of a combination of probes selected from the second
plurality of probes to distinguish the cancer specimens from the
normal specimens; and (e) selecting a combination of probes that
identifies the cancer specimen as compared to the normal specimen
with a vector value of less than 0.400.
[0018] In one embodiment, the cancer specimens are lung caner
specimens. For example, the specimens can be derived from patients
diagnosed as having lung cancer. The normal specimens can be lung
tissue specimens derived from patients not diagnosed as having lung
cancer.
[0019] In one embodiment, step (c) of the method includes selecting
those probes within the first plurality of probes that identify the
cancer specimens as compared to the normal specimens to yield a
second plurality of probes, wherein the second plurality of probes
each identify the cancer specimens as compared to the normal
specimens at a p value of less than 0.005 or 0.001 and/or a vector
value of less than 0.400, 0.300, 0.200, or 0.100.
[0020] In another embodiment, step (e) of the method includes
selecting a combination of probes that identifies the cancer
specimen as compared to the normal specimen with a vector value of
less than 0.300, 0.200, or 0.100.
[0021] In another aspect, the invention features a set of
chromosomal probes including at least two different probes, wherein
the set of probes is capable of detecting lung cancer with a
sensitivity of at least about 60%, e.g., when tested on a
population containing at least 35 lung cancer patients.
[0022] In one example, the set contains at least three different
probes. In another example, the set contains at least four
different probes.
[0023] In one example, the set is capable of detecting lung cancer
with a sensitivity of at least about 60% at a cutoff value of about
10%. In another example, the set is capable of detecting lung
cancer with a sensitivity of at least about 70% when the detection
is performed on a biological sample containing a bronchial
specimen. In another example, the set is capable of detecting lung
cancer with a sensitivity of at least about 80% at a cutoff value
of about 20%.
[0024] The chromosomal probes contained in the sets described
herein, e.g., sets of at least two, three, or four different
probes, can be selected from the group consisting of a chromosome 1
enumeration probe, a chromosome 3 enumeration probe, a chromosome 4
enumeration probe, a chromosome 6 enumeration probe, a chromosome 7
enumeration probe, a chromosome 8 enumeration probe, a chromosome 9
enumeration probe, a chromosome 10 enumeration probe, a chromosome
11 enumeration probe, a chromosome 12 enumeration probe, a
chromosome 16 enumeration probe, a chromosome 17 enumeration probe,
a chromosome 18 enumeration probe, a 3p14 locus specific probe, a
3q26 locus specific probe, a 5p15 locus specific probe, a 5q31
locus specific probe, a 7p12 locus specific probe, a 8q24 locus
specific probe, a 9p21 locus specific probe, a 10q23 locus specific
probe; a 13q14 locus specific probe, a 17p13 locus specific probe,
a 17q21 locus specific probe, a 20q13 locus specific probe, a 21q22
locus specific probe, a 3q chromosome arm probe, a 5p chromosome
arm probe, a 7p chromosome aim probe, a 3p chromosome arm probe,
and a 20q chromosome arm probe.
[0025] In another aspect, the invention features a set of
chromosomal probes including at least two different probes, wherein
the set is capable of detecting lung cancer with a vector value of
less than 0.500, e.g., when tested on a population containing at
least 35 lung cancer patients and 20 normal individuals.
[0026] In one example, the set is capable of detecting lung cancer
with a vector value of less than 0.500 at a cutoff value of about
10%. In another example, the set is capable of detecting lung
cancer with a vector value of less than 0.400. In another example,
the set is capable of detecting lung cancer with a vector value of
less than 0.400 at a cutoff value of about 15%. In another example,
the set is capable of detecting lung cancer with a vector value of
less than 0.300. In another example, the set is capable of
detecting lung cancer with a vector value of less than 0.300 at a
cutoff value of about 15%. In another example, the set is capable
of detecting lung cancer with a vector valve of less than 0.200. In
another example, the set is capable of detecting lung-cancer with a
vector value of less than 0.200 at a cutoff value of about 20%.
[0027] The at least two different probes of the set can be selected
from the group consisting of a chromosome 1 enumeration probe, a
chromosome 3 enumeration probe, a chromosome 4 enumeration probe, a
chromosome 6 enumeration probe, a chromosome 7 enumeration probe, a
chromosome 8 enumeration probe, a chromosome 9 enumeration probe, a
chromosome 10 enumeration probe, a chromosome 11 enumeration probe,
a chromosome 12 enumeration probe, a chromosome 16 enumeration
probe, a chromosome 17 enumeration probe, a chromosome 18
enumeration probe, a 3p14 locus specific probe, a 3g26 locus
specific probe, a 5p15 locus specific probe, a 5q31 locus specific
probe, a 7p12 locus specific probe, a 8q24 locus specific probe, a
9p21 locus specific probe, a 10q23 locus specific probe, a 13q14
locus specific probe, a 17p13 locus specific probe, a 17q21 locus
specific probe, a 20q13 locus specific probe, a 21g22 locus
specific probe, a 3q chromosome ant probe, a 5p chromosome arm
probe, a 7p chromosome arm probe, a 3p chromosome arm probe, and a
20q chromosome arm probe.
[0028] An advantage of the invention is that it allows for the
detection of lung cancer with improved sensitivity, as compared to
conventional methods such as cytology. These probes and methods can
thus allow for the early detection of lung cancer, e.g., at a
pre-invasive stage.
[0029] Another advantage of the invention is that it allows for the
detection of cancer cells based on genetic alterations, rather than
gross morphological changes in cell structure. Genetic alterations
can be detected at an early stage, e.g., before the occurrence of
visually detectable changes in cell structure.
[0030] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
Although methods and materials similar or equivalent to those
described herein can-be used in the practice or testing of the
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of a conflict in terminology, the present specification
will control. In addition, the described materials and methods are
illustrative only and are not intended to be limiting.
[0031] Other features and advantages of the invention will be
apparent from the following detailed description and the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts a receiver operator characteristic (ROC)
curve derived from FISH analysis of specimens from cancer positive
and cancer negative patients. Sensitivity (y axis) and specificity
(x axis; 1--specificity) are depicted for cutoff values ranging
from 1 to 10 cells per specimen.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The invention includes probe sets and methods of using
probes and probe sets for the detection of lung cancer. The probes
and methods described herein allow for the rapid and sensitive
detection of lung cancer in a biological sample such as a bronchial
specimen, a lung biopsy, or a sputum sample. In addition, the
invention includes methods of selecting probe sets for the
detection of cancer.
Chromosomal Probes
[0034] Suitable probes for in situ hybridization in accordance with
the invention fall into three broad groups: chromosome enumeration
probes, which hybridize to a chromosomal region and indicate the
presence or absence of a chromosome; chromosome arm probes, which
hybridize to a chromosomal region and indicate the presence or
absence of an arm of a chromosome; and locus specific probes, which
hybridize to a specific locus on a chromosome and detect the
presence or absence of a specific locus. Chromosomal probes and
combinations thereof are chosen for sensitivity and/or specificity
when used in methods for the detection of lung cancer. Probe sets
can include any number of probes, e.g., at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, or 20 probes.
[0035] A chromosome enumeration probe can hybridize to a repetitive
sequence, located either near or removed from a centromere, or can
hybridize to a unique sequence located at any position on a
chromosome. For example, a chromosome enumeration probe can
hybridize with repetitive DNA associated with the centromere of a
chromosome. Centromeres of primate chromosomes contain a complex
family of long tandem repeats of DNA, composed of a monomer repeat
length of about 171 base pairs, that are referred to as
alpha-satellite DNA. Non-limiting examples of chromosome
enumeration probes include probes to chromosomes 1, 3,4, 6, 7, 8,
9, 10, 11, 12, 16, 17, and 18. Examples of several specific
chromosome enumeration probes and their respective target regions
are described in Table 1 of Example 1.
[0036] A chromosome arm probe can hybridize to a repetitive or
unique sequence located on an arm, either the short or long arm, of
a given chromosome. The gain or loss of the sequence to which the
chromosome arm probe hybridizes can be used to indicate the gain or
loss of the arm. Non-limiting examples of chromosome arm probes
include probes to chromosome arms 3q, 5p, 7p, 3p, and 20q. Examples
of specific chromosome arm probes and their respective target
regions are described in Table 1.
[0037] A locus specific probe hybridizes to a specific,
non-repetitive locus on a chromosome. Non-limiting examples of
locus specific probes include probes to the following loci: 3p14;
3q26; 5p15; 5q31; 7p12; 8q24; 9p21; 10q23; 13q14; 17p13; 17q21;
20q13; and 21q22. Some of these loci comprise genes, e.g.,
oncgogenes and tumor suppressor genes, that are altered in some
forms of cancer. Thus, probes that target these genes, either
exons, introns, or regulatory sequences of the genes, can be used
in the detection methods described herein. Examples of target genes
include: FHIT (3p14); EGR1 (5q31); EGFR1 (7p12); c-MYC (8q24); PTEN
(10q23); RB (13q14); P53 (17p13); and HER-2/neu (17q21).
[0038] Chromosomal probes can be of any size, but are typically
about 50 to about 5.times.10.sup.5 nucleotides in length.
Chromosomal probes can comprise repeated sequences, e.g., fragments
of about 100 to about 500 nucleotides in length. Probes that
hybridize with centromeric DNA and specific chromosomal loci are
available commercially, for example, from Vysis, Inc. (Downers
Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.), or from
Cytocell (Oxfordshire, UK). Alternatively, probes can be made
non-commercially from chromosomal or genomic DNA through standard
techniques. For example, sources of DNA that can be used include
genomic DNA, cloned DNA sequences such as a bacterial artificial
chromosome (BAC), somatic cell hybrids that contain one, or a part
of one, human chromosome along with the normal chromosome
complement of the host, and chromosomes purified by flow cytometry
or microdissection. The region of interest, e.g., a target region
indicated in Table 1, can be isolated through cloning, or by
site-specific amplification via the polymerase chain reaction
(PCR). See, for example, Nath and Johnson, Biotechnic Histochem.
1998, 73(1):6-22; Wheeless et al., Cytometry, 1994, 17:319-326; and
U.S. Pat. No. 5,491,224.
[0039] Chromosomal probes can contain a detection moiety that
facilitates the detection of the probe when hybridized to a
chromosome. Examples of detection moieties include both direct and
indirect labels, as described below.
[0040] Chromosomal probes can be directly labeled with a detectable
label. Examples of detectable labels include fluorophores, organic
molecules that fluoresce after absorbing light of lower
wavelength/higher energy, and radioactive isotopes, e.g., .sup.32P
and .sup.3H. A fluorophore can allow a probe to be visualized
without a secondary detection molecule. For example, after
covalently attaching a fluorophore to a nucleotide, the nucleotide
can be directly incorporated into the probe with standard
techniques such as nick translation, random priming, and PCR
labeling. Alternatively, deoxycytidine nucleotides within the probe
can be transaminated with a linker. The fluorophore then is
covalently attached to the transaminated deoxycytidine nucleotides.
See, U.S. Pat. No. 5,491,224.
[0041] Examples of fluorophores that can be used in the methods
described herein are as follows: 7-amino-4-methylcoumarin-3-acetic
acid (AMCA), Texas Red.TM. (Molecular Probes, Inc., Eugene, Oreg.);
5-(and-6)-carboxy-X-rhodamine, lissamine-rhodamine B,
5-(and-6)-carboxyfluorescein; fluorescein-5-isothiocyanate (FITC);
7-diethylaminocoumarin-3-carboxylic acid,
tetramethylrhodamine-5-(and-6)-isothiocyanate;
5-(and-6)-carboxytetramethylrhodamine;
7-hydroxycoumarin-3-carboxylic acid; 6-[fluorescein
5-(and-6)-carboxamido]hexanoic acid;
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic
acid; eosin-5-isothiocyanate; erythrosin-5-isothiocyanate; and
Cascade.TM. blue acetylazide (Molecular Probes, Inc., Eugene,
Oreg.).
[0042] In methods using multiple probes, fluorophores of different
colors can be chosen such that each chromosomal probe in the set
can be distinctly visualized. Alternatively, two or more probes in
a set can be labeled with the same or a similar fluorophore. Probes
can be viewed with a fluorescence microscope and an appropriate
filter for each fluorophore, or by using dual or triple band-pass
filter sets to observe multiple fluorophores. See, for example,
U.S. Pat. No. 5,776,688, Alternatively, techniques such as flow
cytometry can be used to examine the hybridization pattern of the
chromosomal probes.
[0043] Probes also can be indirectly labeled, e.g., with biotin or
digoxygenin, although secondary detection molecules or further
processing is required to visualize the labeled probes. For
example, a probe labeled with biotin can be detected by avidin
conjugated to a detectable marker, e.g., a fluorophore.
Additionally, avidin can be conjugated to an enzymatic marker such
as alkaline phosphatase or horseradish peroxidase. The enzymatic
markers can be detected in standard colorimetric reactions using a
substrate for the enzyme. Substrates for alkaline phosphatase
include 5-bromo-4-chloro-3-indolylphosphate and nitro blue
tetrazolium. Diaminobenzoate can be used as a substrate for
horseradish peroxidase.
In Situ Hybridization
[0044] The presence or absence of cells with chromosomal
aberrations is determined by in situ hybridization. Cells with
chromosomal aberrations have, for example, an abnormal number of
chromosomes and/or have chromosomal structural alterations such as
the gain or loss (e.g., hemizygous or homozygous loss) of a
specific chromosomal region, such as a locus or a chromosomal arm
as indicated in Table I. For example, a cell having one or more
chromosomal gains, e.g., three or more copies of any given
chromosome, can be considered to test positive in the methods
described herein. Cells exhibiting monosomy and nullisomy may also
be considered test positive under certain circumstances. In
general, in situ hybridization includes the steps of fixing a
biological sample, hybridizing a chromosomal probe to target DNA
contained within the fixed biological sample, washing to remove
non-specific binding, and detecting the hybridized probe.
[0045] A "biological sample" is a sample that contains cells or
cellular material, e.g., cells or cellular material derived from
pulmonary structures, including but not limited to lung parenchyme,
bronchioles, bronchial, bronchi, and trachae. Non-limiting examples
of biological samples useful for the detection of lung cancer
include bronchial specimens, lung biopsies, and sputum samples.
Examples of bronchial specimens include bronchial secretions,
washings, lavage, aspirations, and brushings. Lung biopsies can be
obtained by methods including surgery, bronchoscopy, and
transthoracic needle biopsy. In one example, touch preparations can
be made from lung biopsies.
[0046] In addition, biological samples can include effusions, e.g.,
pleural effusions, pericardial effusions, or peritoneal effusions.
In addition, biological samples can include cells or cellular
material derived from tissues to which lung cancers commonly
metastasize. These tissues include, for example, lymph nodes,
blood, brain, bones, liver, and adrenal glands. Thus, the probes
and probes sets described herein can be used to detect lung cancer
and lung cancer metastasis.
[0047] Typically, cells are harvested from-a biological sample and
prepared using techniques well known to those of skill in the art.
For example, cells can be harvested by centrifuging a biological
sample, such as a bronchial washing, and resuspending the pelleted
cells. Typically, the cells are resuspended in phosphate-buffered
saline (PBS). After centrifuging the cell suspension to obtain a
cell pellet, the cells can be fixed, for example, in acid alcohol
solutions, acid acetone solutions, or aldehydes such as
formaldehyde, para formaldehyde, and glutaraldehyde. For example, a
fixative containing methanol and glacial acetic acid in a 3:1
ratio, respectively, can be used as a fixative. A neutral buffered
formalin solution also can be used, and includes approximately 1%
to 10% of 37-40% formaldehyde in an aqueous solution of sodium
phosphate. Slides containing the cells can be prepared by removing
a majority of the fixative, leaving the concentrated cells
suspended in only a portion of the solution. The cell suspension is
applied to slides such that the cells do not overlap on the slide.
Cell density can be measured by a light or phase contrast
microscope.
[0048] Prior to in situ hybridization, chromosomal probes and
chromosomal DNA contained within the cell each are denatured. If
chromosomal probes are prepared as a single-stranded nucleic acid,
then denaturation of the probe is not be required. Denaturation
typically is performed by incubating in the presence of high pH,
heat (e.g., temperatures from about 70.degree. C. to about
95.degree. C.), organic solvents such as formamide and
tetraalkylammonium halides, or combinations thereof. For example,
chromosomal DNA can be denatured by a combination, of temperatures
above 70.degree. C. (e.g., about 73.degree. C.) and a denaturation
buffer containing 70% formamide and 2.times.SSC (0.3M sodium
chloride and 0.03 M sodium citrate). Denaturation conditions
typically are established such that cell morphology is preserved.
For example, chromosomal probes can be denatured by heat, e.g., by
heating the probes to about 73.degree. C. for about five
minutes.
[0049] After removal of denaturing chemicals or conditions, probes
are annealed to the chromosomal DNA under hybridizing conditions.
"Hybridizing conditions" are conditions that facilitate annealing
between a probe and target chromosomal DNA. Hybridization
conditions vary, depending on the concentrations, base
compositions, complexities, and lengths of the probes, as well as
salt concentrations, temperatures, and length of incubation. For
example, in situ hybridizations are typically performed in
hybridization buffer containing 1-2.times.SSC, 50-55% formamide, a
hybridization acceleratant (e.g. 10% dextran sulfate), and blocking
DNA to suppress nonspecific hybridization. In general,
hybridization conditions, as described above, include temperatures
of about 25.degree. C. to about 55.degree. C., and incubation
lengths of about 0.5 hours to about 96 hours. More particularly,
hybridization can be performed at about 32.degree. C. to about
45.degree. C. for about 2 to about 16 hours.
[0050] Non-specific binding of chromosomal probes to DNA outside of
the target region can be removed by a series of washes. Temperature
and concentration of salt in each wash depend on the desired
stringency. For example, for high stringency conditions, washes can
be carried out at about 65.degree. C. to about 80.degree. C., using
0.2.times. to about 2.times.SSC, and about 0.1% to about 1% of a
non-ionic detergent such as Nonidet P-40 (NP40). Stringency can be
lowered by decreasing the temperature of the washes or by
increasing the concentration of salt in the washes
Detection of Chromosomal Abnormalities
[0051] Gain or loss of chromosomes or chromosomal regions within, a
cell is assessed by examining the hybridization pattern of the
chromosomal probe or set of chromosomal probes (e.g., the number of
signals for each probe) in the cell, and recording the number of
signals. In a typical assay, the hybridization pattern is assessed
in a plurality of cells, e.g., about 25-5,000 cells.
[0052] Samples containing a plurality of cells, e.g., at least
about 100, of which 1 or more, at least about 5,.6, 7, 8, 9, 10,
15, or 20, cells "test positive" typically are considered cancer
positive. By "test positive" is meant possessing the gain or loss
of a chromosome, chromosomal arm, or locus as described herein.
Criteria for "test positive" can include testing positive with one,
two, three, four or more probes. In addition, "test positive" can
include performing a hybridization analysis with multiple probes,
e.g. four probes, and detecting abnormal hybridization patterns
with a subset of the probes, e.g., at least two or three
probes.
[0053] A sample containing cells, e.g. cells placed on a flat
surface such as a slide, can be evaluated by a variety of methods
and using a variety of criteria. The probes and methods described
herein are not limited to usage with a particular screening
methodology. For example, in what is known as the "scanning
method," the observer scans hundreds to thousands of cells for
cytologic abnormalities (as viewed with a DAPI filter). The number
of cells assessed depends on the cellularity of the specimen,
which, varies from patient to patient. Cytologic abnormalities
commonly but not invariably associated with neoplastic cells
include nuclear enlargement, nuclear irregularity, and abnormal
DAPI staining (frequently mottled and lighter). In the scanning
method, the observer primarily focuses the evaluation of the cells
for chromosomal abnormalities (as demonstrated by FISH) on those
cells that also exhibit cytologic abnormalities. In addition, a
proportion of the cells that do not have obvious cytologic
abnormalities can be evaluated, since chromosomal abnormalities
occur in the absence of cytologic abnormalities. The scanning
method is described in further detail in U.S. Pat. No. 6,174,681,
the content of which is incorporated by reference.
Screening, Monitoring, and Diagnosis of Patients for Lung
Cancer
[0054] The methods described herein can be used to screen
individuals for lung cancer or to monitor patients diagnosed with
lung cancer. For example, in a screening mode, individuals at risk
for lung cancer, such as individuals who smoke or have been
chronically exposed to smoke, or individuals chronically exposed to
asbestos, are screened with the goal of earlier detection of lung
cancer. In addition, the probes and methods described herein can be
used for the diagnosis of symptomatic patients. The methods
described herein can be used alone, or in conjunction with other
tests. For example, a patient having an increased risk of lung
cancer can be screened for lung cancer by performing in situ
hybridization as described herein together with other standard
tests such as imaging analysis, e.g., CT, spiral CT, and X-ray
analysis, and/or cytology. Alternatively, standard methods can be
performed first on a patient, and if the standard test gives
equivocal or negative results, then a method described herein can
be performed.
[0055] The methods described herein can also be used to select a
therapy for a patient diagnosed as having lung cancer. The methods
can thus simultaneously diagnose a lung cancer and provide useful
information as to possible treatments for the cancer. Several of
the probes described herein are directed to oncogenes and tumor
suppressor genes. If one or more of these genes is found to be
altered in the course of a determination that the patient has
cancer, then this information can be used to select a therapy,
e.g., a therapy that modulates (increases or decreases) the
presence or activity of these genes and/or their protein products.
For example, if an alteration of the 17q21 locus is discovered,
then this information could be used to design a Her-2-based therapy
(see, e.g., Cragg et al., Curr. Opin. Immunol., 1999, 11:541-547).
The loci containing specific oncogenes and tumor suppressor genes
are indicated in Table 1.
Probe Selection Methods
[0056] The selection of individual probes and probe sets can be
performed using the principles described in the examples. These
selection methods make use of discriminate and/or combinatorial
analysis to select probes and probes sets that are useful for the
detection of lung cancer with high sensitivity.
[0057] The methods described herein preferably have a combined
sensitivity and specificity that is better than that of
conventional methods, particularly for the early detection of lung
cancer. As described in the examples, 26 chromosomal probes were
hybridized to 27 different lung tumor specimens and 12 normal
adjacent tissue specimens, and the extent of gain and loss of each
target was measured. To analyze this data and select the most
useful probe sets, several rules were developed that, when
considered in combination, yield probe sets having a high
sensitivity and specificity. Each rule is not hard-and-fast but
states general preferences that are weighed against the other rules
in order to arrive at optimally performing probe sets.
[0058] (1) Each probe selected for a probe set should have an
ability on its own to discriminate between tumor and normal tissue.
Probes with high discrimination abilities are preferred. The
discrimination analysis utilizes two different approaches: (a)
comparing the means and standard deviations between the tumor
specimen set and normal adjacent tumor specimen set of the
percentage of cells with target gain and loss for each of the probe
targets, and (b) calculating the sensitivity and specificity of
each probe individually for identifying the tumor and normal
adjacent tumor specimens, for various cutoff values of the cell
percentages for targets gained and lost. Several different metrics
can be generated to evaluate approach (a), which included
calculation of D.V. (discriminate value), S.D.M. (standard
deviation at "midpoint"), and p-value. D.V. and p-value are
generally accepted methods for evaluation. The relevance of S.D.M.
is that it is the cutoff value, as a multiple of the standard
deviations from the tumor and normal means, at which the
sensitivity would equal the specificity if the means and standard.
deviations actually equaled the true values of the two populations.
For example, if the midpoint was one standard deviation, of the
tumor specimens from the mean of the tumor specimens, and one
standard deviation of the normal adjacent specimens from the mean
of the normal adjacent specimens, then the sensitivity and
specificity would each equal 84% (this also assumes normal-error
distributions for each population, which is less likely to be true
for the normal adjacent tissue distributions due to their proximity
to 0). The larger the S.D.M. the greater the sensitivity and
specificity of that probe.
[0059] (2) The primary metric for combined sensitivity, and
specificity will be the quantity called `vector` which is the
magnitude of the vector drawn between the points on a sensitivity
versus specificity plot representing the ideal
(sensitivity=specificity=1) and the measured sensitivity and
specificity. Therefore the vector value ranges from 0 for the ideal
case and 1.414 for the worst case.
[0060] (3) Each probe selected for a probe set should complement
the other selected probes, that is, it should identify additional
tumor specimens that the other probe(s) failed to identify. One
method of identifying the best complementing set of probes is to
take the probe with the lowest vector value, remove the group of
tumor specimens it identified from the full set of tumor specimens,
and then determine the probe with lowest vector value on the
remaining tumor specimens. This process can be continued as
necessary to complete the probe set. The approach selected here of
generating all possible probe combinations, and calculating the
sensitivity and specificity of each, predicts the performance of
all possible probe sets and allows selection of the minimal probe
set with the highest performance characteristics. Also, a variety
of combinations with similarly high performance characteristics is
obtained. Considering the possible errors due to the finite number
of specimens tested, several of the high ranking probe combinations
can be compared based on other practical characteristics such as
relevance to disease prognosis or difficulty in making the
probe.
[0061] (4) The ability of probes to complement one another is more
important than the discriminating ability of individual probes,
except as indicated in (5) below.
[0062] (5) Regardless of the measured ability to complement other
probes, each probe must identify a statistically different
percentage of test positive cells between the tumor and normal
adjacent tissue specimen sets. If this condition is not met then a
probe might be selected erroneously based on apparent
complementation.
[0063] (6) Data for combinations of two probes is more reliable
than data for combinations of three probes, and data for
combinations of three probes is more reliable than data for
combinations of four probes. This results from the reduced ability
to make correlations between greater numbers of probes with the
finite number of specimens tested.
[0064] (7) The dependence of probe and probe combination
performance as a function of cutoff value must be considered.
"Cutoff value" refers to the percentage of cells in a population
that must have gains or losses for the sample to be considered
positive. A sample is therefore called as positive or negative
depending upon whether the percentage of cells in the sample is
above the cutoff value or equal to or less than the cutoff
value.
[0065] In general, the combined specificity and sensitivity of
probes is better at low cutoff values. However, when the cancer
cells are distributed within a matrix containing many normal cells,
such as bronchial secretions or sputum, probes performing best at
high cutoffs are more likely to be detected. This is because good
performance at high cutoffs indicates a higher prevalence of cells
containing the abnormality. Examples of cutoff values that can be
used in the calculations include about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, and 60%.
[0066] (8) The measurement of target gain is favored over
measurement of target loss. Overlapping targets or poor
hybridization to some cells can falsely suggest monosomy.
Locus-specific or chromosomal arm probes designed to detect
deletions are generally sirialler than locus-specific or
chromosomal arm probes designed to detect gain since the deletion
probes must net extend beyond the minimally deleted region. If too
much of the "deletion probe" extends beyond the deleted,sequence,
enough signal may remain to be falsely counted. Since "deletion
probes" are usually kept small the signals are not as intense as
signals for targets typically gained. This in turn makes. it more
likely that real signals from targets being monitored for deletion
may be miscounted. Likewise, repetitive sequence probes, like some
chromosome enumeration probes used here are preferable to single
locus probes because they usually provide brighter signals and
hybridize faster than locus specific probe. On the other hand,
repetitive sequence probes are more sensitive to polymorphisms than
locus specific probes.
[0067] (9) A probe or combination of probes preferably shows an
improvement over conventional methods such as cytology. A probe or
probe combination preferably detects lung cancer with a sensitivity
of at least about 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
even 100%. A probe or probe combination preferably detects lung
cancer with a vector value of less than about 0.500, 0.450, 0.400,
0.350, 0.300, 0.250, 0.200, 0.150, or 0.100.
[0068] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Probe Selection
[0069] A collection of 26 probes was assembled as candidates for
detecting chromosomal abnormalities in lung cancer by in situ
hybridization. The probes were hybridized to a collection of lung
tumor touch preparations, and the distribution of the copy number
per cell of each probe target was determined. In order to conserve
tumor specimens, multi-color hybridizations were utilized to limit
the number of hybridization regions per specimen to 8. To achieve
this, the 26 probes were labeled with several different
fluorophores. Mixtures of 3 or 4 probes each were prepared from the
labeled probes forming the 8 probe sets. Where possible, chromosome
enumeration probes and locus specific probes that target the same
chromosome were combined in the same set to distinguish whole
chromosome aneuploidy from gains and tosses of regions within a
chromosome.
[0070] The 26 probes selected for hybridization to lung touch
preparations are described in Table 1. The probes included 13
chromosome enumeration probes (CEP.TM. probes from Vysis, Inc.;
targeting repetitive centromeric sequences) and 13 locus specific
probes (LSI.TM. from Vysis, Inc. or BAC preparations; targeting
unique sequences associated with amplified or deleted chromosomal
regions). Column 3 of Table 1 describes the target location of each
of the 26 probes. For several of the probes, oncogenes or tumor
suppressor genes that are located at the relevant locus are also
listed.
[0071] Mixtures of 3 probes, labeled with SpectrumAqua.TM.,
SpectrumGreen, and SpectrumOrange.TM., or 4 probes, labeled with
SpectrumAqua.TM., SpectrumGreen.TM., SpectrumGold.TM., and
SpectrumRed.TM., were prepared to form the 8 probe sets. The
fluorescent label used for each probe and the probe set containing
each probe are described in columns 4 and 5, respectively, of Table
1.
[0072] Tumor touch preparations, prepared from lung tumors removed
from 27 patients with a range of lung cancers, were used for
testing the 26 probes. In addition, specimens prepared from normal
lung tissue generally at some distance from the tumors (NAL=normal
adjacent lung tissue) from twelve of the same patients were also
tested in order to examine the background levels of gained and lost
targets for each probe. The characteristics of the lung tumor and
normal specimens are listed in Table 2. Touch preparations were
prepared by pressing a piece of lung tumor or normal adjacent
tissue against a glass microscope slide and fixing briefly in
ethanol. The specimens were then stored at -20.degree. C. until
ready for use.
[0073] Prior to in situ hybridization, the touch preparations were
treated to improve in situ hybridization performance by the
following protocol.
[0074] (1) Fix the specimen slide ma fresh Carnoy's solution (3:1
methanol:acetic acid) for 20 minutes at room temperature. Allow the
slide to dry in the air.
[0075] (2) Place the slide on a 45.degree. C. hot plate for 15
minutes.
[0076] (3) Incubate the slide in 2.times.SSC at 37.degree. C. for
10 minutes.
[0077] (4) Place the slide in a pepsin solution (0.05 mg pepsin per
ml 10 mM HCl) at 37.degree. C. for 13 minutes. The pepsin solution
is prepared fresh each day by diluting 25 .mu.L of a pepsin stock
solution (100 mg pepsin/mL water; use 2,500-3,000 U/mg pepsin) into
50 mL of 10 mM HCl.
[0078] (5) Place the slide in 1.times. PBS for 5 minutes at room
temperature.
[0079] (6) Fix the slide in 1% formaldehyde for 5 minutes at room
temperature. The. formaldehyde solution is prepared by mixing 1.35
mL of 37% formaldehyde with 48.15 mL of 1.times.PBS and 0.5 mL of 2
M MgCl.sub.2. Discard after each day of use.
[0080] (7) Place the slide in 1.times. PBS for 5 minutes at room
temperature.
[0081] (8) Dehydrate the specimen by placing the slide in a series
of ethanol solutions (70%, 85%, 100%), 1-5 minutes per solution.
Allow the specimen to dry in the air before denaturing.
[0082] After performing t ie above treatments, fluorescence in situ
hybridization was performed on all specimens as follows.
[0083] (1) Denature the specimen's DNA by placing the slide in a
solution of 70% formamide/2.times.SSC at 73.degree. C. for 5
minutes.
[0084] (2) Dehydrate the specimen by placing the slide in a series
of ethanol (70%, 85%, 100%), 1-5 minutes per solution. Allow the
specimen to air dry before applying denatured probe.
[0085] (3) Denature a probe solution by placing a tube containing
the probe in a 73.degree. C. water bath for 5 minutes.
[0086] (4) Apply the denatured probe solution.-to the denatured
slide, place a coverslip over the solution, and seal the coverslip
by applying rubber cement along the edges. Allow the probe to
hybridize overnight at 37.degree. C. in humidified chamber.
[0087] (5) Wash the slide in a Coplin jar in 0.4.times.SSC/0.3%
NP-40 for 3 minutes at 70.degree. C. (or 1 minute at 73.degree. C.)
Wash 4 slides simultaneously per Coplin jar.
[0088] (6) Soak the slide in 2.times.SSC/0.1% NP-40, for several
seconds to several minutes..sub.--
[0089] (7) Apply antifade/counterstain solution and cover with a
coverslip. Store the slides at -20.degree. C. until analyzed.
[0090] Hybridized specimen slides were viewed on a fluorescence
microscope using single bandpass filter sets specific for each of
the 4 fluorescent labels and the DAPI counterstain. Each touch
preparation was analyzed by counting the number of spots of each
fluorescent color in 100 consecutive non-inflammatory cells and the
copy number of each probe target recorded. Several of the specimens
did not hybridize well with all 26 probes, so the number of
specimens tested differs for each probe. In addition, probe set 8
was not tested on all specimens.
Example 2
Analysis of In Situ Hybridization Data
[0091] The target copy number data for each of the normal and tumor
specimens was analyzed for the ability of each probe to
discriminate between tumor and normal specimens (discriminate
analysis) and for the ability of probe combinations to discriminate
between tumor and normal specimens (combinatorial analysis). These
analyses were used as part of the data considered in deciding which
probes should be used individually or in concert to best identify
lung cancer cells.
Discriminate Analysis
[0092] The ability of individual probes to discriminate between the
normal specimen group and the tumor specimen group was evaluated
first by comparing the averages and standard deviations of the
percentages of abnormal cells found in each group. These data are
listed in Tables 3 (normal specimen group) and 4 (tumor specimen
group). The first 26 rows in each table lists data derived from
absolute target counts per cell, for each of the 26 probes tested.
For these calculations, individual targets present in greater than
2 copies were considered an abnormal gain in copy number, and
targets present in less than 2 copies were considered an abnormal
loss in copy number. The last 8 rows in Tables 3 and 4 list data
derived from ratios of LSI/CEP target numbers, or in the Case of
chromosome 5, the ratio of LSI 5p15/LSI 5q31 target numbers. Ratios
were only calculated when both probes were contained in the same
probe set. The ratios were calculated on a cell-by-cell basis. For
the purpose of these calculations, cells were considered to have
target gain when ratios were greater than 1, and target loss when
ratios were less than 1.
[0093] In Tables 3 and 4, the columns headed `Ave. % cells are the
averages of the percentage of cells found in each specimen with
either target copy number gain or target copy number loss, as
indicated in the heading. The columns headed `S.D. % cells . . . `
are the standard deviations of the average cell percentages for the
number of specimens (`Number of specimens . . . ` columns) in which
interpretable hybridizations for each specific probe were
obtained.
[0094] Included in Table 4 are three columns containing different
measures of the ability of each probe to discriminate between the
tumor and normal specimen groups. The discriminate value, D.V., is
calculated according to Equation 1:
DV=(M.sub.T-M.sub.N).sup.2/(SD.sub.T.sup.2+SD.sub.N.sup.2) (1)
with values being larger for greater separation between the mean of
the normal specimens, M.sub.N, and the mean of the tumor specimens,
M.sub.T, and for smaller standard deviations of the normal,
S.D..sub.N, and tumor, S.D..sub.T, specimens.
[0095] The `SD's at midpoint`, S.D.M. is calculated by Equation
2:
S.D.M.=(M.sub.T-M.sub.N)/(SD.sub.T+SD.sub.N) (2)
and is the number of standard deviations from the tumor and normal
group means which equal the separation of the means. If the means
and standard deviations were the true values for the tumor and
normal populations, then S.D.M. is the point at which the
sensitivity and specificity are equal to each other. The larger the
S.D.M., the greater the value of the sensitivity and
specificity.
[0096] The third measure of discrimination listed in Table 4 is the
probability, p, that the measured means are from the same
population. The value of p is determined from the Student's t-test.
In effect the smaller the p value, the more statistically different
the tumor population is from the normal population. A p<0.05 is
typically considered to represent a statistically significant
difference between the two groups.
[0097] The p values in Table 4 indicate that all of the 26 probes
found statistically significant (p<0.05) gains for the tumor
specimen group relative to the normal group, when using the
absolute target numbers. When viewed as ratios between LSI and
corresponding CEP or LSI target numbers, 5 of the 8 ratios showed
significant differences (last 8 rows in Table 4). By contrast, only
2 of the 26 probes found statistically significant loss of absolute
target numbers (LSI 8p24 and CEP 17), while 5 of the 8 ratios
showed significant differences.
[0098] The rows of Table 4 are sorted from highest to lowest D.V.
for gain of targets. The data derived from absolute target counts
is sorted separately from the ratio data. Examination of the D.V.,
S.D.M., and p values for target gain shows relatively good
correspondence between the three discrimination parameters. The top
5 discriminating probes selected by all three parameters are the
same, LSI 5p15, LSI 7p12, CEP 1, CEP 6, and LSI 8q24, in descending
order (all indicating gain of targets in tumor specimens).
[0099] Another approach within the overall selection method for
determining which probes provide the best discrimination between
normal and tumor specimens is to look at the number of specimens
correctly identified by each probe. This requires selecting a
cutoff number for the percentage of cells with gains or losses. A
sample is then called positive or negative for cancer depending
upon whether the percentage of cells in the sample is above the
cutoff value or equal to or less than the cutoff value,
respectively. The accuracies of identifying the positive samples
(sensitivity) and negative samples (specificity) are then used to
select the best probes.
[0100] Table 5 lists the specificity and sensitivity of gain and
loss of all 26 probe targets and the same CEP/LSI and 5p/q ratios
listed in Tables 3 and 4. The table includes the specificity and
sensitivity values at 6 different cutoff values (5%, 10%, 20%, 30%,
40%, and 50%). The table also includes two measures of the combined
specificity and sensitivity, since the overall ability to
discriminate between tumor and normal specimens depends on both
specificity and sensitivity. The first combined attribute is the
product of specificity and sensitivity. The product is largest if
both specificity and sensitivity are high, and is reduced if either
or both are low. The other combined attribute, designated as
"vector," is calculated according to Equation 3:
Vector=[(1-specificity).sup.2+(1-sensitivity).sup.2].sup.0.5
(3)
This attribute has a value of 0 when specificity and sensitivity=1,
and increases to 1.414 as both approach 0.
[0101] The rows in Table 5 are sorted by increasing vector value
for each cutoff value. The data derived from absolute target counts
is sorted separately from the ratio data. Target gains dominate the
top of the table and the same probes tend to show the lowest vector
values, although their relative order changes with cutoff value.
Probes showing consistently high discrimination ability based on
the vector value and absolute target counts include LSI 8q24, LSI
5p15, LSI 7p12, LSI 3q26, LSI 20q13, LSI 5q31, LSI 3p14, LSI 17q21,
CEP 1, CEP 4, CEP 6, CEP 7, CEP 9, and CEP 16. Each of these probes
is found in the top 10 rows for at least two of the cutoff values.
The target ratios generally showed lower vector values except for
the chromosome 5p15/5q31 ratio which had vector values comparable
to some Of the best probes based on their absolute target
counts.
Combinatorial Analysis
[0102] The ability of multiple probes used in concert to increase
assay sensitivity (complementation) was investigated using
combinatorial analysis. The analysis was initiated by generating
all possible combinations of a group of probes. The counting data
from each specimen was then examined to determine if any of the
probes in each combination identified gain or loss of their target
above a threshold number of cells. If any of the probes in a
combination were positive, then the specimen was considered
positive for cancer for that combination.
[0103] The combinations were kept to a maximum of four probes. The
entire set of 26 probes was not used to generate all combinations
due to the large number of possible combinations that would be
generated for the 26 probes and their relevant ratios, each of
which would be examined for gain and loss (866,847 possible
combinations of 1, 2, 3, and 4 probes). Instead, the set of probes
and ratios was reduced to include only those probes that identified
gains, and those probes that identified losses with p<0.01
(Table 4). This provided some assurance that probe combinations
would not be over rated as a result of randomly high target counts
of individual probes. To further reduce complexity, two different
groups of probes were examined separately. Group 1 included all of
the probes for which the absolute counts identified target gain or
loss with p<001. Group 2 replaced the members of Group 1 with
their corresponding LSI/CEP or LSI/LSI ratio, if the ratio
identified target gain or loss with p<0.01, Therefore, Group 1
consisted of all of the probes for gain listed in the first 25 rows
of Table 4 (because of its high significance, LSI 5p15/LSI 5q31 was
also included in this group) and none of the probes for losses.
Group 24 replaced LSI 7p12 and LSI 8q24 with LSI 7p12/CEP 7 and LSI
8q24/CEP 8, respectively, for gains; deleted the other LSI probes
that had corresponding LSI/CEP ratios with p>0.01; and added LSI
9p21/CEP 9 and LSI 17p13/CEP 17 for loss.
[0104] Tables 6 through 9 list the combinations of 2, 3, and 4
probes with the combined highest sensitivities and specificities,
for cutoff values of 10% (Table 6), 20% (Table 7), 30% (Table 8),
and 40% (Table 9), respectively. The measure of combined
sensitivity and specificity used to order the combinations was the
vector value. A particular combination was excluded from the tables
if a subset of probes in the combination gave an equal or lower
vector value. The probes contributing to the best combinations
changed as the cutoff value was increased. The best vector values
also increased as the cutoff was increased, as seen previously in
Table 5 for single probes. In determining the number of probes in a
combination, ratios were counted as two probes, unless one of the
probes in the ratio was also in the combination. In general, ratios
were not found in the better scoring combinations, except for the
LSI 5p15/LSI 5q31 ratio. Also, target loss rarely ranked in the top
performing probe combinations. As a result, in thither discussion
the gain of a target is implied, unless specifically denoted as a
loss.
[0105] At a percent cell cutoff value of 10% (Table 6), LSI 8q24
and LSI 5p15 were commonly found in the top performing combinations
of two probes, and complemented each other as well, LSI 8q24 was
also complemented well by LSI 17q21, LSI 5q31, LSI 9p21, CEP 1, CEP
6, CEP 7, CEP 9, CEP 11, and CEP 17. LSI. 5p15 was also
complemented well by LSI 17q21, LSI 5q31, LSI 9p21, LSI 13q14, CEP
8, CEP 12, and CEP 17.
[0106] In addition, LSI 7p12 and CEP 1 complemented one another
well. The same probes were also found in the better combinations of
three and four probes.
[0107] When the cutoff was increased to 20% (Table 7), LSI 5p15
remained in the top combinations of two, and was complemented best
by LSI 3q26, CEP 16, LSI 20q13, LSI 17q21 and CEP 4. LSI 8q24, LSI
3p14, LSI 5q31, LSI 7p12, CEP 3, CEP 6, and CEP 9 also provided
good complementation to LSI 5p15. LSI 8q24 fell lower in the list,
although still a good performer, being complemented by LSI 7p12 and
CEP 6. The better combinations of three and four probes also
included these probes as well as other probes identified above in
the better combinations at the cutoff of 10.
[0108] As the cutoff was increased to 30% (Table 8), LSI 5p15
persisted in the better combinations, and LSI 8q24 was absent from
the higher-ranking combinations. Complementation of LSI 5p15 was
provided by CEP 6, CEP 16, LSI 20q13, LSI 3q26, LSI 17q21, LSI
7p12, and LSI 3p14. Also, LSI 7p12 was complemented by CEP 6, and
CEP 6 and CEP 7 complemented one another. Detection of target loss
was only found to be important in combinations of four probes (LSI
17p13 loss relative to CEP 7).
[0109] Increasing the cutoff value to 40% (Table 9) reduced the
importance of LSI 5p15 in two probe combinations, and placed LSI
7p12 at the top of the list, which was complemented best by LSI
3q26 and CEP 6, and also by CEP 18, CEP 4, CEP 16, LSI 20q13 and
LSI 5p15. CEP 6 ranked high when complemented by either CEP 1 or
CEP 7. Other high ranking pairs of probes included LSI 3q26 with
either LSI 5p15, CEP1,or CEP 7. In combinations of three probes,
the combination of LSI 7p12 and CEP 6 with CEP 11 was at the top of
the list, just ahead of combinations of CEP 6 with either CEP 1 or
CEP 7, also complemented by CEP 11. Other probes included in the
better performing combinations of three were 17q21, LSI 3q26 CEP 4,
CEP 16, CEP 18, and LSI 20q. In combinations of four probes, CEP 6
combined with either CEP 1 or CEP 7 was at the top of the list when
complemented by 17p13/CEP 16 loss. Another loss, 9p21/CEP 9 was
next when combined with CEP 7 and LSI 3q. Other high ranking
combinations of four included LSI 7p12, LSI 10q23, CEP 10, CEP 11,
LSI 5p15, LSI 3q31, LSI 5p/LSI 5q, CEP 6, CEP 7, and CEP 9.
Example 3
Selection of Probe Sets
[0110] Table 13 lists probes and probe sets selected by analyzing
the data from the discriminate and combinatorial analyses and
applying the probe selection criteria described herein. The probe
sets of Table 13 range in size from a single probe to 4 probes.
Assays using additional probes, e.g., more than four, and
additional fluorescent labels can be performed.
[0111] The single probes listed in Table 13 are the probes that
individually showed improvement over cytology. These include LSI
5p15, LSI 7p12, LSI 8q24, CEP 1, CEP 6, and CEP 9. For each of
these probes, the vector value was less than 0.400 for two of the
cutoff values tested. Other probes described herein also gave
vector values less than 0.400 for a single cutoff. However, good
performance for two cutoff values implies that a probe is more
robust. Next, Table 13 lists 2-probe combinations. The probe pairs
placed in this group were required to have a vector value less than
0.400 and rank in the top approximately 30 probe pairs (lowest
vector values) for at least one cutoff value. The vector values are
listed in the table for each probe pair for each cutoff value in
which the probe pair was ranked in the top 30. Of special note are
the probe pairs of LSI 5p15+LSI 8q24, LSI 5p15+CEP 12, and LSI
5p15+LSI 17q21 which have vector values less than 0.400 at 3
different cutoff values.
[0112] Next, Table 13 lists 3-probe combinations. Only a few
combinations of 3 probes are listed under this heading since these
are the few sets that improved over combinations of 2 probes for
any particular cutoff value.
[0113] Next, Table 13 lists 4-probe combinations. Only one
combination of 4 probes is listed under this heading since it was
the only combination that improved over the combinations of 2 and 3
probes for any particular cutoff value.
[0114] To take advantage of the practical capability of using 3 and
4 FISH probes together, a strategy of redundancy can be introduced.
Under this strategy, a third probe could be added to a pair of
complementary probes if it also complemented one of the 2 probes.
Alternatively, it might not complement either probe well, but
instead it might be the next highest performing single probe.
Similarly, 4 probe pairs could be generated by combining pairs of
complementary probes. Some 3 and 4 probe sets generated using
redundancy of the 2-probe sets listed in Table 13 are listed in a
lower part of the same table. An alternative approach is to pick a
2-probe pair and add an additional 2 probes, one of which
complements one member of the first pair, and the other of which
complements the other member of the first pair. One benefit of
redundancy probes is that assay specificity might be improved by
requiring 2 of the targets to be gained in order to call the
specimen abnormal. Redundancy can also improve sensitivity since if
one probe hybridization should fail in an assay, the redundant
probe might still detect the target gain. Other practical issues
can be considered in probe selection. For example, the 4 probe set
of LSI 5p15+LSI 8p24+LSI 7p12+LSI 17q21 can be constructed from
probes in three of the top performing combinations of 2 probes
listed in Table 13. The significance of this probe set is that it
detects two loci of therapeutic importance, 17q21 containing the
HER-2/neu gene and 7p12 containing the epidermal growth factor
receptor gene (EGFR). The identification of abnormalities at these
loci can be used to select an appropriate treatment regimen.
Example 4
Lung Cancer Detection
[0115] Two 3-color probe sets were chosen for preliminary testing
on a series of bronchial secretion specimens. The results of this
study showed that specificity and sensitivity equivalent to or
better than conventional cytology could be obtained with Multi-cam
FISH panels.
[0116] The results of the hybridizations of 3-color probe sets to
each of 21 bronchial secretion smears are listed in Table 10,
together with specimen identification numbers, clinical diagnoses,
cytology results, and bronchoscopic biopsy results (two results
when additional biopsy was performed). Each specimen was hybridized
with two different 3-color probes sets. The first 3 color probe set
contained LSI 8q24, LSI 5p15, and CEP 1, and the second set
contained LSI 8q24, LSI 5p15, and CEP 6. Gain of the 5p15 target
was found in 13 of the 13 FISH positive specimens. Gain of the
8q24, CEP1, and CEP 6 targets were found in 11, 7, and-5 of the 13
FISH positive targets, respectively. One of the specimen slides
could not be evaluated by FISH due to poor morphology and no FISH
abnormalities were found in the remaining 7 specimens. The
performance of conventional cytology and FISH are compared to the
diagnosis in Tables 11 and 12, respectively. Clinical diagnosis was
based on the combined information available to the clinician, and
did not include the FISH result.
[0117] In the above methods, smears of bronchial secretions were
prepared by placing a specimen between two microscope slides and
sliding the slides apart from one another while applying slight
pressure. The slides were then fixed briefly with ethanol and
stored at -20.degree. C. Until ready for use.
[0118] Smears of bronchial secretions were prepared for in situ
hybridization by the following protocol.
[0119] (1) Incubate the specimen slide in 2.times.SSC at 37.degree.
C. for 10 minutes.
[0120] (2) Place the slide in a pepsin solution (0.05 mg pepsin per
mL 10 mM HCl) at 37.degree. C. for 13 minutes.
[0121] (3) Place the slide in 1.times.PBS for 5 minutes at room
temperature.
[0122] (4) Fix the Specimen by placing the slides in 1%
formaldehyde for 5 minutes at room temperature.
[0123] (5) Place the slides 1.times.PBS for 5 minutes at room
temperature,
[0124] In Situ Hybridization was performed on the specimens as
follows.
[0125] (1) Denature the specimen DNA by placing the slides in a
solution of 70% formamide/2.times.SSC at 73.degree. C. for 5
minutes.
[0126] (2) Dehydrate the specimen by placing the slide in a series
of ethanol solutions (70%, 85%, 100%), 1-5 minutes per solution.
Allow the specimen to air dry, before applying denatured probe.
[0127] (3) Denature a probe solution by placing a tube containing
the probe in a 73.degree. C. water bath for 5 minutes.
[0128] (4) Apply the denatured probe solution to the denatured
slide, place a coverslip over the solution, and seal the coverslip
by applying rubber cement along the edges.
[0129] (5) Allow the probe to hybridize overnight at 37.degree. C.
in humidified chamber.
[0130] (6) Wash the slide in a Coplin jar in 0.4.times.SSC/0.3%
NP-40 for 3 minutes at 70.degree. C. minute at 73.degree. C.). Wash
4 slides simultaneously per Coplin jar.
[0131] (7) Soak the slide in 2.times.SSC/0.1% NP-40 for several
seconds to several minutes.
[0132] (8) Apply antifade/counterstain solution and cover with a
coverslip. Store the slide at -20.degree. C. until analyzed.
[0133] Bronchial secretion smears were analyzed by scanning the
entire specimen. Each microscope field was viewed sequentially with
the 4 single bandpass filter sets (DAPI
TABLE-US-00001 TABLE 1 Probes Used for Probe Selection PROBE NAME
DNA SOURCE TARGET LOCATION LABEL PROBE SET CEP 1, sat. II/III Vysis
product 1q12 SpectrumGreen 5 CEP 3, alpha sat. Vysis product D3Z1,
3p11.1-q11.1 SpectrumAqua 6 LSI 3p14/FHIT BAC 3p14 SpectrumOrange 6
LSI 3q26/TERC BAC 3q26 SpectrumGreen 8 CEP 4, alpha sat. Vysis
product 4p11-q11 SpectrumAqua 8 LSI D5S721, D5S23 Vysis product
D5S721, D5S23, 5p15 SpectrumGreen 4 LSI EGR1 Vysis product 5q31
SpectrumOrange 4 CEP 6, alpha sat. Vysis product D6Z1, 6p11.1-q11
SpectrumGreen 6 CEP 7, alpha sat. Vysis product D7Z1, 7p11.1-q11.1
SpectrumAqua 5 LSI EGFR BAC 7p12 SpectrumOrange 5 CEP 8, alpha sat.
Vysis product D8Z2, 8p11.1-q11.1 SpectrumAqua 2 LSI c-myc Vysis
product 8q24 SpectrumOrange 2 CEP 9, alpha sat. Vysis product
9p11-q11 SpectrumGreen 3 LSI 9p21 Vysis product 9p21 SpectrumGold 3
CEP 10, alpha sat. Vysis product 10p11.1-q11.1 SpectrumGreen 7 LSI
10q23 (PTEN) BAC 10q23 SpectrumOrange 7 CEP 11, alpha sat. Vysis
product D11Z1, 11p11.1-q11 SpectrumAqua 3 CEP 12, alpha sat. Vysis
product D12Z3, 12p11.1-q11 SpectrumAqua 4 LSI 13/RB1 retinoblastoma
1 Vysis product 13q14 SpectrumGreen 2 CEP 16, sat. II Vysis product
D16Z3, 16q11.2 SpectrumGold 8 CEP 17, alpha sat. Vysis product
D17Z1, 17p11.1-q11.1 SpectrumAqua 1 LSI p53 Vysis product 17p13
SpectrumOrange 1 LSI her2/neu (ERBB2) Vysis product 17q21
SpectrumGreen 1 CEP 18, alpha sat. Vysis product D18Z1,
18p11.1-q11.1 SpectrumAqua 7 LSI 20q13 (ZNF217) Vysis product 20q13
SpectrumRed 8 LSI 21 Vysis product D21S259, D21S341, D21S342, 21q22
SpectrumRed 3
TABLE-US-00002 TABLE 2 Lung Tumor and Normal Adjacent Tissue used
for Probe Selection SPECIMEN SPECIMEN TUMOR NAM TYPE TUMOR TYPE
GRADE T1 tumor bronchial alviolar carcinoma 2 T2 tumor
adenocarcinoma 2 T3 tumor adenocarcinoma 2 T7 tumor adenocarcinoma
4 T8 tumor bronchial alviolar carcinoma 1 T9 tumor adenocarcinoma 2
T10 tumor adenocarcinoma 3 T11 tumor squamous cell carcinoma 4 T12
tumor adenocarcinoma 3 T13 tumor large cell carcinoma 4 T14 tumor
adenocarcinoma 4 T15 tumor carcinoid tumor ? T16 tumor
adenocarcinoma 3 T17 tumor adenocarcinoma 2 T18 tumor large cell
carcinoma 4 T19 tumor adenocarcinoma 4 T20 tumor squamous cell
carcinoma 4 T21 tumor squamous cell carcinoma 4 T22 tumor squamous
cell carcinoma 4 T23 tumor adenocarcinoma 3 T24 tumor
adenocarcinoma 3 T25 tumor squamous cell carcinoma 4 T26 tumor
adenocarcinoma 3 T27 tumor adenocarcinoma 2 T28 tumor ? ? T31 tumor
? ? T32 tumor ? ? N1 NAT NA NA N2 NAT NA NA N3 NAT NA NA N7 NAT NA
NA N8 NAT NA NA N12 NAT NA NA N13 NAT NA NA N14 NAT NA NA N15 NAT
NA NA N16 NAT NA NA N17 NAT NA NA N18 NAT NA NA *NAT = normal
tissue adjacent to tumor tissue, NA = not applicable, ? = status
unknown
TABLE-US-00003 TABLE 3 DISCRIMINATION ANALYSIS Ave. % S.D. % cells
cells Ave. % S.D. % Number of with with cells cells specimens gain
gain with loss with loss PROBE LSI 5p15 10 4.4000 2.8752 2.8000
1.9322 LSI 7p12 10 5.5500 2.4771 1.3000 1.9465 CEP 1 10 3.5500
0.8317 3.3000 2.5408 CEP 6 10 1.9000 2.2336 4.8000 2.5734 LSI 8q24
10 2.7500 1.9329 3.1000 1.8529 LSI 20q 10 3.9000 2.2336 4.5000
2.8771 CEP 9 10 2.1000 2.0790 7.1000 5.0211 LSI 3p14 10 4.3000
4.2439 2.9000 2.2828 CEP 16 10 2.8000 1.4757 10.1000 4.6774 CEP 4
10 2.8000 2.6162 2.6000 1.5055 LSI 3q 10 7.5000 3.2404 2.9000
3.0350 CEP 7 10 1.4000 0.9661 2.4000 2.0111 LSI 17q21 10 2.9000
2.4698 6.5000 2.8771 LSI 5q31 10 3.4000 1.6465 4.4000 2.5033 CEP 3
10 1.7000 1.4181 3.7000 2.2632 CEP 10 10 1.4000 2.0656 4.1000
2.9981 CEP 11 10 2.6500 2.3576 4.4000 1.7764 CEP 8 10 1.0000 1.0541
4.5000 2.9907 CEP 18 10 1.8000 1.9889 7.9000 3.8427 LSI 13 10
2.4500 2.2417 3.6500 2.7894 LSI 9p21 10 2.7500 2.8211 4.0000 3.0185
LSI 10q23 10 6.0000 4.5947 3.0000 2.1602 CEP 12 10 1.5000 1.2693
4.4000 2.6331 CEP 17 10 2.3000 2.6687 10.9000 4.4585 LSI 17p13 10
4.1000 3.9567 6.9000 3.2472 LSI 21 10 7.8500 5.8407 6.1500 4.9668
Ratios: 5 p/q imbal. 10 5.2234 3.4875 3.2132 1.7602 LSI 7p12/CEP 7
10 6.3000 3.6833 1.1500 2.1350 LSI 8q24/CEP 8 10 6.1561 3.3667
2.9540 1.7098 LSI 3p14/CEP 3 10 7.0000 4.9666 3.6000 2.6331 LSI
17q21/CEP 10 11.4000 5.4610 6.2000 2.1499 LSI 10q23/CEP 10 8.5000
5.9489 3.0000 1.6997 LSI 9p21/CEP 9 10 7.7041 7.4657 3.9041 3.9546
LSI 17p13/CEP 10 11.3000 5.3759 6.0000 3.0551
TABLE-US-00004 TABLE 4 DISCRIMINATION ANALYSIS Ave. S.D. Ave. S.D.
Number of % cells % cells D.V. S.D.M. p % cells % cells D.V. S.D.M.
p PROBE specimens with gain with gain gain gain gain with loss with
loss loss point + loss loss LSI 5p15 26 34.0385 25.1483 1.3710
1.0576 0.000003 1.4615 1.9022 0.2437 -0.3491 0.079773 LSI 7p12 26
30.1154 21.6505 1.2708 1.0181 0.000005 1.3462 2.2617 0.0002 0.0110
0.952130 CEP 1 26 27.7308 21.7946 1.2292 1.0687 0.000007 7.7308
19.0527 0.0531 0.2052 0.256410 CEP 6 26 27.8462 24.2976 1.1307
0.9779 0.000012 3.7692 3.2901 0.0609 -0.1758 0.332295 LSI 8q24 27
22.7407 19.3621 1.0555 0.9388 0.000013 1.5926 1.5753 0.3842 -0.4397
0.038297 LSI 20q 19 26.2632 22.4297 0.9843 0.9067 0.000395 2.6842
2.1616 0.2546 -0.3604 0.100820 CEP 9 26 19.6923 17.6176 0.9834
0.8932 0.000031 6.6154 8.2998 0.0025 -0.0364 0.832828 LSI 3p14 26
21.5385 17.1912 0.9477 0.8042 0.000043 4.2692 6.6547 0.0379 0.1532
0.365082 CEP 16 19 21.9474 20.5520 0.8635 0.8692 0.000741 8.3684
5.9368 0.0525 -0.1631 0.398174 CEP 4 19 20.7368 19.5756 0.8248
0.8083 0.000888 2.9474 1.9571 0.0198 0.1003 0.600639 LSI 3q 19
29.7895 24.3278 0.8248 0.8085 0.000889 2.6316 5.8709 0.0016 -0.0301
0.872277 CEP 7 26 23.1154 24.0704 0.8126 0.8673 0.000106 2.6154
2.8576 0.0038 0.0442 0.801649 LSI 17q21 27 22.3704 21.4658 0.8120
0.8134 0.000077 4.2593 4.5454 0.1735 -0.3019 0.087675 LSI 5q31 26
22.9231 22.2996 0.7623 0.8153 0.000154 4.2692 5.0482 0.0005 -0.0173
0.918489 CEP 3 26 21.1154 24.0671 0.6485 0.7618 0.000377 3.9231
6.5600 0.0010 0.0253 0.880460 CEP 10 25 17.1600 19.9827 0.6154
0.7148 0.000645 3.7000 2.9155 0.0091 -0.0676 0.723951 CEP 11 26
18.9231 21.5108 0.5655 0.6818 0.000770 3.4231 3.1135 0.0743 -0.1998
0.248755 CEP 8 27 17.2222 21.7155 0.5567 0.7125 0.000646 3.2593
2.9819 0.0863 -0.2077 0.278498 CEP 18 25 17.0000 21.1325 0.5128
0.6574 0.001526 5.0800 4.1122 0.2510 -0.3545 0.070839 LSI 13 27
13.4444 15.3230 0.5040 0.6259 0.001103 4.0741 3.4744 0.0091 0.0677
0.705658 LSI 9p21 26 14.9615 17.5191 0.4736 0.6004 0.001833 9.0000
15.5486 0.0997 0.2693 0.128341 LSI 10q23 25 15.8600 13.9280 0.4520
0.5323 0.003606 3.3600 4.4989 0.0052 0.0541 0.752077 CEP 12 26
19.3462 26.9250 0.4383 0.6330 0.002417 3.2308 3.4212 0.0734 -0.1931
0.286454 CEP 17 27 16.3704 21.9057 0.4065 0.5726 0.002832 6.2222
5.8001 0.4089 -0.4560 0.016682 LSI 17p13 27 14.1852 15.7774 0.3844
0.5111 0.004264 7.6296 9.9466 0.0049 0.0553 0.738974 LSI 21 26
17.7844 17.8255 0.2805 0.4198 0.016950 4.5832 3.8505 0.0622 -0.1777
0.384519 Ratios: 5 p/q imbal. 26 28.1566 22.1019 1.0505 0.8962
0.000020 5.3885 7.0458 0.0897 0.2470 0.154145 LSI 7p12/CEP 7 26
15.8237 10.9781 0.6764 0.6496 0.000446 3.8921 4.6890 0.2832 0.4018
0.022057 LSI 8q24/CEP 8 27 13.7445 8.6033 0.6747 0.6340 0.000477
5.2233 7.7125 0.0825 0.2408 0.160626 LSI 3p14/CEP 3 26 12.5385
9.8599 0.2517 0.3736 0.033580 14.6154 18.1242 0.3618 0.5307
0.005431 LSI 17q21/CEP 27 16.7089 9.9709 0.2181 0.3440 0.048699
8.4901 8.3931 0.0699 0.2172 0.200321 LSI 10q23/CEP 25 12.3232
8.1702 0.1431 0.2708 0.138690 11.8277 17.5057 0.2519 0.4596
0.019653 LSI 9p21/CEP 9 26 9.2480 9.1323 0.0171 0.0930 0.608094
13.3700 16.2368 0.3208 0.4688 0.009422 LSI 17p13/CEP 27 11.3749
9.4349 0.0000 0.0051 0.976201 17.4138 20.1651 0.3132 0.4915
0.007889
TABLE-US-00005 TABLE 5 Sensitivity and Specificity of Lung Tumor
Detection # TUMOR PROBE LOSS/GAIN SPECIFICITY SENSITIVITY SENS*SPEC
VECTOR SPECIMENS CUTOFF = 5% CELLS WITH GAINS OR LOSSES CEP 1 gain
1.000 0.923 0.923 0.077 26 8q24 gain 0.900 0.815 0.733 0.210 27 CEP
16 gain 1.000 0.737 0.737 0.263 19 CEP 6 gain 0.900 0.731 0.658
0.287 26 CEP 9 gain 0.900 0.731 0.658 0.287 26 LSI 5q31 gain 0.900
0.692 0.623 0.324 26 LSI 20q gain 0.800 0.737 0.589 0.331 19 3p14
gain 0.700 0.846 0.592 0.337 26 17q21 gain 0.800 0.704 0.563 0.357
27 CEP 4 gain 0.800 0.684 0.547 0.374 19 LSI 5p15 gain 0.600 0.923
0.554 0.407 26 CEP 8 gain 1.000 0.593 0.593 0.407 27 LSI 13 gain
0.900 0.593 0.533 0.420 27 CEP 11 gain 0.900 0.577 0.519 0.435 26
CEP 10 gain 1.000 0.560 0.560 0.440 25 CEP 17 gain 0.900 0.556
0.500 0.456 27 CEP 3 gain 1.000 0.538 0.538 0.462 26 CEP 7 gain
1.000 0.538 0.538 0.462 26 9p21 gain 0.800 0.577 0.462 0.468 26
10q23 gain 0.600 0.720 0.432 0.488 25 CEP 18 gain 0.900 0.520 0.468
0.490 25 CEP 12 gain 1.000 0.500 0.500 0.500 26 17p13 gain 0.600
0.593 0.356 0.571 27 LSI 21 gain 0.500 0.692 0.346 0.587 26 7p12
gain 0.400 0.846 0.338 0.619 26 9p21 loss 0.800 0.385 0.308 0.647
26 LSI 13 loss 0.700 0.370 0.259 0.697 27 CEP 1 loss 0.800 0.308
0.246 0.721 26 LSI 3q gain 0.300 0.789 0.237 0.731 19 10q23 loss
0.900 0.240 0.216 0.767 25 3p14 loss 0.900 0.231 0.208 0.776 26 CEP
11 loss 0.700 0.269 0.188 0.790 26 CEP 6 loss 0.700 0.269 0.188
0.790 26 CEP 12 loss 0.900 0.192 0.173 0.814 26 CEP 7 loss 0.900
0.192 0.173 0.814 26 CEP 10 loss 0.700 0.240 0.168 0.817 25 LSI 21
loss 0.500 0.346 0.173 0.823 26 17q21 loss 0.500 0.333 0.167 0.833
27 CEP 4 loss 1.000 0.158 0.158 0.842 19 CEP 16 loss 0.300 0.526
0.158 0.845 19 LSI 5q31 loss 0.600 0.231 0.138 0.867 26 CEP 8 loss
0.700 0.185 0.130 0.868 27 CEP 3 loss 0.800 0.154 0.123 0.869 26
LSI 3q loss 0.800 0.105 0.084 0.917 19 7p12 loss 0.900 0.077 0.069
0.928 26 17p13 loss 0.300 0.370 0.111 0.942 27 LSI 5p15 loss 0.900
0.038 0.035 0.967 26 8q24 loss 0.900 0.037 0.033 0.968 27 LSI 20q
loss 0.800 0.053 0.042 0.968 19 CEP 18 loss 0.200 0.400 0.080 1.000
25 CEP 9 loss 0.200 0.385 0.077 1.009 26 CEP 17 loss 0.100 0.519
0.052 1.021 27 ratios: 5 .mu./q imbal. gain 0.600 0.923 0.554 0.407
26 8q24/CEP 8 gain 0.600 0.852 0.511 0.427 27 3p14/CEP 3 loss 0.700
0.654 0.458 0.458 26 9p21/CEP 9 loss 0.800 0.577 0.462 0.468 26
10q23/CEP 1 loss 0.900 0.520 0.468 0.490 25 7p12/CEP 7 gain 0.500
0.808 0.404 0.536 26 10q23/CEP 1 gain 0.500 0.760 0.380 0.555 25
3p14/CEP 3 gain 0.400 0.808 0.323 0.630 26 8q24/CEP 8 loss 1.000
0.333 0.333 0.667 27 9p21/CEP 9 gain 0.400 0.615 0.246 0.713 26
7p12/CEP 7 loss 0.900 0.269 0.242 0.738 26 17p13/CEP 1 loss 0.300
0.667 0.200 0.775 27 5 p/q imbal. loss 0.900 0.231 0.208 0.776 26
17q21/CEP 1 loss 0.300 0.593 0.178 0.810 27 17q21/CEP 1 gain 0.100
0.889 0.089 0.907 27 17p13/CEP 1 gain 0.100 0.704 0.070 0.948 27
CUTOFF = 10% CELLS WITH GAINS OR LOSSES 8q24 gain 1.000 0.778 0.778
0.222 27 LSI 5p15 gain 1.000 0.769 0.769 0.231 26 7p12 gain 0.900
0.692 0.623 0.324 26 CEP 1 gain 1.000 0.654 0.654 0.346 26 CEP 9
gain 1.000 0.654 0.654 0.346 26 LSI 3q gain 0.900 0.632 0.568 0.382
19 CEP 6 gain 1.000 0.615 0.615 0.385 26 17q21 gain 1.000 0.593
0.593 0.407 27 CEP 16 gain 1.000 0.579 0.579 0.421 19 CEP 4 gain
1.000 0.579 0.579 0.421 19 LSI 20q gain 1.000 0.579 0.579 0.421 19
LSI 5q31 gain 1.000 0.577 0.577 0.423 26 3p14 gain 0.900 0.577
0.519 0.435 26 CEP 7 gain 1.000 0.538 0.538 0.462 26 CEP 3 gain
1.000 0.500 0.500 0.500 26 CEP 8 gain 1.000 0.481 0.481 0.519 27
9p21 gain 1.000 0.462 0.462 0.538 26 CEP 11 gain 1.000 0.462 0.462
0.538 26 10q23 gain 0.800 0.480 0.384 0.557 25 CEP 12 gain 1.000
0.423 0.423 0.577 26 LSI 21 gain 0.700 0.500 0.350 0.583 26 CEP 17
gain 1.000 0.407 0.407 0.593 27 LSI 13 gain 1.000 0.407 0.407 0.593
27 CEP 10 gain 1.000 0.400 0.400 0.600 25 CEP 18 gain 1.000 0.400
0.400 0.600 25 17p13 gain 0.900 0.407 0.367 0.601 27 17p13 loss
0.800 0.259 0.207 0.767 27 CEP 9 loss 0.800 0.192 0.154 0.832 26
LSI 5q31 loss 1.000 0.154 0.154 0.846 26 9p21 loss 0.900 0.154
0.138 0.852 26 3p14 loss 1.000 0.077 0.077 0.923 26 CEP 3 loss
1.000 0.077 0.077 0.923 26 CEP 17 loss 0.500 0.222 0.111 0.925 27
LSI 21 loss 0.900 0.077 0.069 0.928 26 17q21 loss 0.900 0.074 0.067
0.931 27 CEP 18 loss 0.800 0.080 0.064 0.941 25 LSI 3q loss 1.000
0.053 0.053 0.947 19 CEP 16 loss 0.400 0.263 0.105 0.950 19 10q23
loss 1.000 0.040 0.040 0.960 25 CEP 10 loss 1.000 0.040 0.040 0.960
25 CEP 1 loss 1.000 0.038 0.038 0.962 26 CEP 11 loss 1.000 0.038
0.038 0.962 26 CEP 6 loss 1.000 0.038 0.038 0.962 26 LSI 13 loss
1.000 0.037 0.037 0.963 27 CEP 12 loss 0.900 0.038 0.035 0.967 26
7p12 loss 1.000 0.000 0.000 1.000 26 8q24 loss 1.000 0.000 0.000
1.000 27 CEP 4 loss 1.000 0.000 0.000 1.000 19 CEP 7 loss 1.000
0.000 0.000 1.000 26 CEP 8 loss 1.000 0.000 0.000 1.000 27 LSI 5p15
loss 1.000 0.000 0.000 1.000 26 LSI 20q loss 0.900 0.000 0.000
1.005 19 5 p/q imbal. gain 0.800 0.692 0.554 0.367 26 7p12/CEP 7
gain 0.900 0.577 0.519 0.435 26 8q24/CEP 8 gain 0.800 0.593 0.474
0.454 27 3p14/CEP 3 gain 0.900 0.500 0.450 0.510 26 3p14/CEP 3 loss
1.000 0.462 0.462 0.538 26 10q23/CEP 1 gain 0.700 0.480 0.336 0.600
25 17q21/CEP 1 gain 0.500 0.667 0.333 0.601 27 17p13/CEP 1 loss
0.900 0.407 0.367 0.601 27 9p21/CEP 9 loss 0.900 0.346 0.312 0.661
26 17q21/CEP 1 loss 1.000 0.333 0.333 0.667 27 17p13/CEP 1 gain
0.600 0.407 0.244 0.715 27 10q23/CEP 1 loss 1.000 0.240 0.240 0.760
25 9p21/CEP 9 gain 0.800 0.231 0.185 0.795 26 7p12/CEP 7 loss 1.000
0.192 0.192 0.808 26 8q24/CEP 8 loss 1.000 0.111 0.111 0.999 27 5
p/q imbal. loss 1.000 0.038 0.038 0.962 26 CUTOFF = 20% CELLS WITH
GAINS OR LOSSES LSI 5p15 gain 1.000 0.654 0.654 0.346 26 7p12 gain
1.000 0.615 0.615 0.385 26 LSI 3q gain 1.000 0.579 0.579 0.421 19
CEP 1 gain 1.000 0.538 0.538 0.462 26 3p14 gain 1.000 0.500 0.500
0.500 26 CEP 6 gain 1.000 0.500 0.500 0.500 26 CEP 16 gain 1.000
0.474 0.474 0.526 19 CEP 4 gain 1.000 0.474 0.474 0.526 19 LSI 20q
gain 1.000 0.474 0.474 0.526 19 CEP 7 gain 1.000 0.462 0.462 0.538
26 17q21 gain 1.000 0.444 0.444 0.556 27 8q24 gain 1.000 0.444
0.444 0.556 27 CEP 3 gain 1.000 0.423 0.423 0.577 26 CEP 9 gain
1.000 0.423 0.423 0.577 26 LSI 5q31 gain 1.000 0.423 0.423 0.577 26
CEP 11 gain 1.000 0.385 0.385 0.615 26 CEP 10 gain 1.000 0.360
0.360 0.640 25 CEP 12 gain 1.000 0.346 0.346 0.654 26 10q23 gain
1.000 0.320 0.320 0.680 25 CEP 18 gain 1.000 0.320 0.320 0.680 25
CEP 17 gain 1.000 0.296 0.296 0.704 27 CEP 8 gain 1.000 0.296 0.296
0.704 27 LSI 21 gain 0.900 0.269 0.242 0.738 26 9p21 gain 1.000
0.231 0.231 0.769 26 17p13 gain 1.000 0.222 0.222 0.778 27 LSI 13
gain 1.000 0.148 0.148 0.852 27 9p21 loss 1.000 0.115 0.115 0.885
26 CEP 3 loss 1.000 0.077 0.077 0.923 26 17p13 loss 1.000 0.074
0.074 0.926 27 CEP 16 loss 1.000 0.053 0.053 0.947 19 LSI 3q loss
1.000 0.053 0.053 0.947 19 3p14 loss 1.000 0.038 0.038 0.962 26 CEP
1 loss 1.000 0.038 0.038 0.962 26 CEP 9 loss 1.000 0.038 0.038
0.962 26 LSI 5q31 loss 1.000 0.038 0.038 0.962 26 CEP 17 loss 1.000
0.037 0.037 0.963 27 10q23 loss 1.000 0.000 0.000 1.000 25 17q21
loss 1.000 0.000 0.000 1.000 27 7p12 loss 1.000 0.000 0.000 1.000
26 8q24 loss 1.000 0.000 0.000 1.000 27 CEP 10 loss 1.000 0.000
0.000 1.000 25 CEP 11 loss 1.000 0.000 0.000 1.000 26 CEP 12 loss
1.000 0.000 0.000 1.000 26 CEP 18 loss 1.000 0.000 0.000 1.000 25
CEP 4 loss 1.000 0.000 0.000 1.000 19 CEP 6 loss 1.000 0.000 0.000
1.000 26 CEP 7 loss 1.000 0.000 0.000 1.000 26 CEP 8 loss 1.000
0.000 0.000 1.000 27 LSI 13 loss 1.000 0.000 0.000 1.000 27 LSI 20q
loss 1.000 0.000 0.000 1.000 19 LSI 21 loss 1.000 0.000 0.000 1.000
26 LSI 5p15 loss 1.000 0.000 0.000 1.000 26 5 p/q imbal. gain 1.000
0.500 0.500 0.500 26 17q21/CEP 1 gain 1.000 0.370 0.370 0.630 27
7p12/CEP 7 gain 1.000 0.346 0.346 0.654 26 17p13/CEP 1 loss 1.000
0.259 0.259 0.741 27 3p14/CEP 3 loss 1.000 0.192 0.192 0.808 26
9p21/CEP 9 loss 1.000 0.192 0.192 0.808 26 17p13/CEP 1 gain 0.900
0.185 0.167 0.821 27 10q23/CEP 1 loss 1.000 0.160 0.160 0.840 25
3p14/CEP 3 gain 1.000 0.154 0.154 0.846 26 8q24/CEP 8 gain 1.000
0.148 0.148 0.852 27 10q23/CEP 1 gain 1.000 0.120 0.120 0.880 25
17q21/CEP 1 loss 1.000 0.111 0.111 0.889 27 9p21/CEP 9 gain 0.900
0.077 0.069 0.928 26 8q24/CEP 8 loss 1.000 0.037 0.037 0.963 27 5
p/q imbal. loss 1.000 0.000 0.000 1.000 26 7p12/CEP 7 loss 1.000
0.000 0.000 1.000 26 CUTOFF = 30% CELLS WITH GAINS OR LOSSES LSI
5p15 gain 1.000 0.577 0.577 0.423 26 7p12 gain 1.000 0.500 0.500
0.500 26 CEP 6 gain 1.000 0.500 0.500 0.500 26 LSI 20q gain 1.000
0.474 0.474 0.526 19 LSI 3q gain 1.000 0.474 0.474 0.526 19 CEP 1
gain 1.000 0.385 0.385 0.615 26 CEP 7 gain 1.000 0.385 0.385 0.615
26 3p14 gain 1.000 0.346 0.346 0.654 26 LSI 5q31 gain 1.000 0.346
0.346 0.654 26 CEP 3 gain 1.000 0.308 0.308 0.692 26 17q21 gain
1.000 0.296 0.296 0.704 27 CEP 11 gain 1.000 0.269 0.269 0.731 26
CEP 12 gain 1.000 0.269 0.269 0.731 26 CEP 16 gain 1.000 0.263
0.263 0.737 19 CEP 4 gain 1.000 0.263 0.263 0.737 19 CEP 10 gain
1.000 0.240 0.240 0.760 25 CEP 18 gain 1.000 0.240 0.240 0.760 25
8q24 gain 1.000 0.222 0.222 0.778 27 CEP 17 gain 1.000 0.222 0.222
0.778 27 CEP 9 gain 1.000 0.192 0.192 0.808 26 LSI 21 gain 1.000
0.192 0.192 0.808 26 17p13 gain 1.000 0.185 0.185 0.815 27 CEP 8
gain 1.000 0.185 0.185 0.815 27 10q23 gain 1.000 0.160 0.160 0.840
25 9p21 gain 1.000 0.154 0.154 0.846 26 9p21 loss 1.000 0.115 0.115
0.885 26 LSI 13 gain 1.000 0.111 0.111 0.889 27 CEP 1 loss 1.000
0.038 0.038 0.962 26 CEP 9 loss 1.000 0.038 0.038 0.962 26 17p13
loss 1.000 0.037 0.037 0.963 27 10q23 loss 1.000 0.000 0.000 1.000
25 17q21 loss 1.000 0.000 0.000 1.000 27 3p14 loss 1.000 0.000
0.000 1.000 26 7p12 loss 1.000 0.000 0.000 1.000 26 8q24 loss 1.000
0.000 0.000 1.000 27
CEP 10 loss 1.000 0.000 0.000 1.000 25 CEP 11 loss 1.000 0.000
0.000 1.000 26 CEP 12 loss 1.000 0.000 0.000 1.000 26 CEP 16 loss
1.000 0.000 0.000 1.000 19 CEP 17 loss 1.000 0.000 0.000 1.000 27
CEP 18 loss 1.000 0.000 0.000 1.000 25 CEP 3 loss 1.000 0.000 0.000
1.000 26 CEP 4 loss 1.000 0.000 0.000 1.000 19 CEP 6 loss 1.000
0.000 0.000 1.000 26 CEP 7 loss 1.000 0.000 0.000 1.000 26 CEP 8
loss 1.000 0.000 0.000 1.000 27 LSI 13 loss 1.000 0.000 0.000 1.000
27 LSI 20q loss 1.000 0.000 0.000 1.000 19 LSI 21 loss 1.000 0.000
0.000 1.000 26 LSI 3q loss 1.000 0.000 0.000 1.000 19 LSI 5p15 loss
1.000 0.000 0.000 1.000 26 LSI 5q31 loss 1.000 0.000 0.000 1.000 26
5 p/q imbal. gain 1.000 0.385 0.385 0.615 26 17p13/CEP 1 loss 1.000
0.185 0.185 0.815 27 10q23/CEP 1 loss 1.000 0.160 0.160 0.840 25
3p14/CEP 3 loss 1.000 0.115 0.115 0.885 26 3p14/CEP 3 gain 1.000
0.115 0.115 0.885 26 7p12/CEP 7 gain 1.000 0.115 0.115 0.885 26
9p21/CEP 9 loss 1.000 0.115 0.115 0.885 26 10q23/CEP 1 gain 1.000
0.080 0.080 0.920 25 9p21/CEP 9 gain 1.000 0.077 0.077 0.923 26
17q21/CEP 1 gain 1.000 0.074 0.074 0.926 27 17p13/CEP 1 gain 1.000
0.037 0.037 0.963 27 17q21/CEP 1 loss 1.000 0.037 0.037 0.963 27
8q24/CEP 8 loss 1.000 0.037 0.037 0.963 27 8q24/CEP 8 gain 1.000
0.037 0.037 0.963 27 5 p/q imbal. loss 1.000 0.000 0.000 1.000 26
7p12/CEP 7 loss 1.000 0.000 0.000 1.000 26 CUTOFF = 40% CELLS WITH
GAINS OR LOSSES 7p12 gain 1.000 0.385 0.385 0.615 26 CEP 1 gain
1.000 0.346 0.346 0.654 26 LSI 3q gain 1.000 0316 0.316 0.684 19
CEP 6 gain 1.000 0.308 0.308 0.692 26 CEP 7 gain 1.000 0.308 0.308
0.692 26 LSI 5p15 gain 1.000 0.308 0.308 0.692 26 LSI 20q gain
1.000 0.211 0.211 0.789 19 CEP 18 gain 1.000 0.200 0.200 0.800 25
17q21 gain 1.000 0.185 0.185 0.815 27 CEP 10 gain 1.000 0.160 0.160
0.840 25 CEP 16 gain 1.000 0.158 0.158 0.842 19 CEP 4 gain 1.000
0.158 0.158 0.842 19 CEP 11 gain 1.000 0.154 0.154 0.846 26 CEP 12
gain 1.000 0.154 0.154 0.846 26 CEP 3 gain 1.000 0.154 0.154 0.846
26 CEP 17 gain 1.000 0.148 0.148 0.852 27 3p14 gain 1.000 0.115
0.115 0.885 26 9p21 loss 1.000 0.115 0.115 0.885 26 CEP 9 gain
1.000 0.115 0.115 0.885 26 LSI 21 gain 1.000 0.115 0.115 0.885 26
LSI 5q31 gain 1.000 0.115 0.115 0.885 26 17p13 gain 1.000 0.111
0.111 0.889 27 8q24 gain 1.000 0.111 0.111 0.889 27 CEP 8 gain
1.000 0.111 0.111 0.889 27 10q23 gain 1.000 0.040 0.040 0.960 25
9p21 gain 1.000 0.038 0.038 0.962 26 CEP 1 loss 1.000 0.038 0.038
0.962 26 CEP 9 loss 1.000 0.038 0.038 0.962 26 17p13 loss 1.000
0.037 0.037 0.963 27 LSI 13 gain 1.000 0.037 0.037 0.963 27 10q23
loss 1.000 0.000 0.000 1.000 25 17q21 loss 1.000 0.000 0.000 1.000
27 3p14 loss 1.000 0.000 0.000 1.000 26 7p12 loss 1.000 0.000 0.000
1.000 26 8q24 loss 1.000 0.000 0.000 1.000 27 CEP 10 loss 1.000
0.000 0.000 1.000 25 CEP 11 loss 1.000 0.000 0.000 1.000 26 CEP 12
loss 1.000 0.000 0.000 1.000 26 CEP 16 loss 1.000 0.000 0.000 1.000
19 CEP 17 loss 1.000 0.000 0.000 1.000 27 CEP 18 loss 1.000 0.000
0.000 1.000 25 CEP 3 loss 1.000 0.000 0.000 1.000 26 CEP 4 loss
1.000 0.000 0.000 1.000 19 CEP 6 loss 1.000 0.000 0.000 1.000 26
CEP 7 loss 1.000 0.000 0.000 1.000 26 CEP 8 loss 1.000 0.000 0.000
1.000 27 LSI 13 loss 1.000 0.000 0.000 1.000 27 LSI 20q loss 1.000
0.000 0.000 1.000 19 LSI 21 loss 1.000 0.000 0.000 1.000 26 LSI 3q
loss 1.000 0.000 0.000 1.000 19 LSI 5p15 loss 1.000 0.000 0.000
1.000 26 LSI 5q31 loss 1.000 0.000 0.000 1.000 26 5 p/q imbal. gain
1.000 0.192 0.192 0.808 26 17p13/CEP 1 loss 1.000 0.185 0.185 0.815
27 10q23/CEP 1 loss 1.000 0.080 0.090 0.920 25 3p14/CEP 3 loss
1.000 0.077 0.077 0.923 26 9p21/CEP 9 loss 1.000 0.077 0.077 0.923
26 9p21/CEP 9 gain 1.000 0.038 0.038 0.962 26 5 p/q imbal. loss
1.000 0.000 0.000 1.000 26 10q23/CEP 1 gain 1.000 0.000 0.000 1.000
25 17p13/CEP 1 gain 1.000 0.000 0.000 1.000 27 17q21/CEP 1 loss
1.000 0.000 0.000 1.000 27 17q21/CEP 1 gain 1.000 0.000 0.000 1.000
27 3p14/CEP 3 gain 1.000 0.000 0.000 1.000 26 7p12/CEP 7 loss 1.000
0.000 0.000 1.000 26 7p12/CEP 7 gain 1.000 0.000 0.000 1.000 26
8q24/CEP 8 loss 1.000 0.000 0.000 1.000 27 8q24/CEP 8 gain 1.000
0.000 0.000 1.000 27 CUTOFF = 50% CELLS WITH GAINS OR LOSSES CEP 1
gain 1.000 0.231 0.231 0.769 26 LSI 20q gain 1.000 0.211 0.211
0.789 19 LSI 3q gain 1.000 0.211 0.211 0.789 19 CEP 7 gain 1.000
0.192 0.192 0.808 26 LSI 5p15 gain 1.000 0.192 0.192 0.808 26 7p12
gain 1.000 0.154 0.154 0.846 26 CEP 11 gain 1.000 0.154 0.154 0.846
26 CEP 3 gain 1.000 0.154 0.154 0.846 26 CEP 6 gain 1.000 0.154
0.154 0.846 26 CEP 12 gain 1.000 0.115 0.115 0.885 26 LSI 5q31 gain
1.000 0.115 0.115 0.885 26 17q21 gain 1.000 0.111 0.111 0.889 27
8q24 gain 1.000 0.111 0.111 0.889 27 CEP 17 gain 1.000 0.111 0.111
0.889 27 CEP 8 gain 1.000 0.111 0.111 0.889 27 CEP 16 gain 1.000
0.105 0.105 0.895 19 CEP 4 gain 1.000 0.105 0.105 0.895 19 CEP 10
gain 1.000 0.080 0.080 0.920 25 CEP 18 gain 1.000 0.080 0.080 0.920
25 3p14 gain 1.000 0.077 0.077 0.923 26 LSI 21 gain 1.000 0.077
0.077 0.923 26 17p13 gain 1.000 0.074 0.074 0.926 27 9p21 loss
1.000 0.038 0.038 0.962 26 9p21 gain 1.000 0.038 0.038 0.962 26 CEP
1 loss 1.000 0.038 0.038 0.962 26 CEP 9 gain 1.000 0.038 0.038
0.962 26 LSI 13 gain 1.000 0.037 0.037 0.963 27 10q23 loss 1.000
0.000 0.000 1.000 25 10q23 gain 1.000 0.000 0.000 1.000 25 17p13
loss 1.000 0.000 0.000 1.000 27 17q21 loss 1.000 0.000 0.000 1.000
27 3p14 loss 1.000 0.000 0.000 1.000 26 7p12 loss 1.000 0.000 0.000
1.000 26 8q24 loss 1.000 0.000 0.000 1.000 27 CEP 10 loss 1.000
0.000 0.000 1.000 25 CEP 11 loss 1.000 0.000 0.000 1.000 26 CEP 12
loss 1.000 0.000 0.000 1.000 26 CEP 16 loss 1.000 0.000 0.000 1.000
19 CEP 17 loss 1.000 0.000 0.000 1.000 27 CEP 18 loss 1.000 0.000
0.000 1.000 25 CEP 3 loss 1.000 0.000 0.000 1.000 26 CEP 4 loss
1.000 0.000 0.000 1.000 19 CEP 6 loss 1.000 0.000 0.000 1.000 26
CEP 7 loss 1.000 0.000 0.000 1.000 26 CEP 8 loss 1.000 0.000 0.000
1.000 27 CEP 9 loss 1.000 0.000 0.000 1.000 26 LSI 13 loss 1.000
0.000 0.000 1.000 27 LSI 20q loss 1.000 0.000 0.000 1.000 19 LSI 21
loss 1.000 0.000 0.000 1.000 26 LSI 3q loss 1.000 0.000 0.000 1.000
19 LSI 5p15 loss 1.000 0.000 0.000 1.000 26 LSI 5q31 loss 1.000
0.000 0.000 1.000 26 5 p/q imbal. gain 1.000 0.115 0.115 0.885 26
17p13/CEP 1 loss 1.000 0.111 0.111 0.889 27 10q23/CEP 1 loss 1.000
0.080 0.080 0.920 25 3p14/CEP 3 loss 1.000 0.077 0.077 0.923 26
9p21/CEP 9 loss 1.000 0.077 0.077 0.923 26 5 p/q imbal. loss 1.000
0.000 0.000 1.000 26 10q23/CEP 1 gain 1.000 0.000 0.000 1.000 25
17p13/CEP 1 gain 1.000 0.000 0.000 1.000 27 17q21/CEP 1 loss 1.000
0.000 0.000 1.000 27 17q21/CEP 1 gain 1.000 0.000 0.000 1.000 27
3p14/CEP 3 gain 1.000 0.000 0.000 1.000 26 7p12/CEP 7 loss 1.000
0.000 0.000 1.000 26 7p12/CEP 7 gain 1.000 0.000 0.000 1.000 26
8q24/CEP 8 loss 1.000 0.000 0.000 1.000 27 8q24/CEP 8 gain 1.000
0.000 0.000 1.000 27 9p21/CEP 9 gain 1.000 0.000 0.000 1.000 26
TABLE-US-00006 TABLE 6 Combinations of 2, 3 and 4 Probes at a
Cutoff Value of 10% SPECI- SENSI- # TUMOR PROBE 1 PROBE 2 PROBE 3
PROBE 4 FICITY TIVITY SENS*SPEC VECTOR SPECIMENS 2 probe
combinations: CEP 17 gain 8q24 gain 1.000 0.852 0.852 0.148 27 8q24
gain CEP 1 gain 1.000 0.846 0.846 0.154 26 8q24 gain LSI 5p15 gain
1.000 0.846 0.846 0.154 26 CEP 12 gain LSI 5p15 gain 1.000 0.846
0.846 0.154 26 17q21 gain 8q24 gain 1.000 0.815 0.815 0.185 27
17q21 gain CEP 1 gain 1.000 0.808 0.808 0.192 26 17q21 gain LSI
5p15 gain 1.000 0.808 0.808 0.192 26 8q24 gain CEP 6 gain 1.000
0.808 0.808 0.192 26 8q24 gain CEP 7 gain 1.000 0.808 0.808 0.192
26 8q24 gain LSI 5q31 gain 1.000 0.808 0.808 0.192 26 9p21 gain
8q24 gain 1.000 0.808 0.808 0.192 26 9p21 gain LSI 5p15 gain 1.000
0.808 0.808 0.192 26 CEP 11 gain 8q24 gain 1.000 0.808 0.808 0.192
26 CEP 17 gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 8 gain
LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 9 gain 8q24 gain 1.000
0.808 0.808 0.192 26 LSI 13 gain LSI 5p15 gain 1.000 0.808 0.808
0.192 26 LSI 5q31 gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 LSI
5p15 gain LSI 3q gain 0.875 0.842 0.737 0.201 19 17p13 gain 8q24
gain 0.900 0.815 0.733 0.210 27 8q24 gain CEP 4 gain 1.000 0.789
0.789 0.211 19 CEP 16 gain 8q24 gain 1.000 0.789 0.789 0.211 19 CEP
16 gain CEP 1 gain 1.000 0.789 0.789 0.211 19 CEP 16 gain LSI 5p15
gain 1.000 0.789 0.789 0.211 19 CEP 16 gain LSI 5q31 gain 1.000
0.789 0.789 0.211 19 CEP 17 gain CEP 16 gain 1.000 0.789 0.789
0.211 19 LSI 20q gain 8q24 gain 1.000 0.789 0.789 0.211 19 LSI 20q
gain CEP 1 gain 1.000 0.789 0.789 0.211 19 LSI 20q gain LSI 5p15
gain 1.000 0.789 0.789 0.211 19 LSI 5p15 gain CEP 4 gain 1.000
0.789 0.789 0.211 19 3 probe combinations: 9p21 gain 8q24 gain LSI
5p15 gain 1.000 0.885 0.885 0.115 26 9p21 gain 8q24 gain CEP 1 gain
1.000 0.885 0.885 0.115 26 CEP 12 gain 9p21 gain LSI 5p15 gain
1.000 0.885 0.885 0.115 26 CEP 17 gain 9p21 gain 8q24 gain 1.000
0.885 0.885 0.115 26 17q21 gain 9p21 gain 8q24 gain 1.000 0.846
0.846 0.154 26 17q21 gain 9p21 gain CEP 8 gain 1.000 0.846 0.846
0.154 26 17q21 gain 9p21 gain LSI 5p15 gain 1.000 0.846 0.846 0.154
26 17q21 gain 9p21 gain CEP 1 gain 1.000 0.846 0.846 0.154 26 17q21
gain CEP 12 gain CEP 1 gain 1.000 0.846 0.846 0.154 26 17q21 gain
CEP 8 gain LSI 5p15 gain 1.000 0.846 0.846 0.154 26 17q21 gain CEP
8 gain CEP 1 gain 1.000 0.846 0.846 0.154 26 17q21 gain LSI 13 gain
9p21 gain 1.000 0.846 0.846 0.154 26 17q21 gain LSI 13 gain LSI
5p15 gain 1.000 0.846 0.846 0.154 26 17q21 gain LSI 13 gain CEP 1
gain 1.000 0.846 0.846 0.154 26 9p21 gain 8q24 gain CEP 7 gain
1.000 0.846 0.846 0.154 26 9p21 gain 8q24 gain CEP 6 gain 1.000
0.846 0.846 0.154 26 9p21 gain 8q24 gain LSI 5q31 gain 1.000 0.846
0.846 0.154 26 9p21 gain CEP 8 gain LSI 5p15 gain 1.000 0.846 0.846
0.154 26 9p21 gain CEP 8 gain CEP 1 gain 1.000 0.846 0.846 0.154 26
9p21 gain CEP 9 gain 8q24 gain 1.000 0.846 0.846 0.154 26 9p21 gain
LSI 5q31 gain LSI 5p15 gain 1.000 0.846 0.846 0.154 26 CEP 11 gain
9p21 gain 8q24 gain 1.000 0.846 0.846 0.154 26 CEP 12 gain 9p21
gain CEP 6 gain 1.000 0.846 0.846 0.154 26 CEP 12 gain CEP 6 gain
CEP 1 gain 1.000 0.846 0.846 0.154 26 CEP 17 gain 9p21 gain LSI
5p15 gain 1.000 0.846 0.846 0.154 26 CEP 17 gain CEP 8 gain LSI
5p15 gain 1.000 0.846 0.846 0.154 26 CEP 17 gain CEP 9 gain CEP 8
gain 1.000 0.846 0.846 0.154 26 CEP 17 gain CEP 9 gain CEP 8 gain
1.000 0.846 0.846 0.154 26 CEP 17 gain LSI 13 gain CEP 9 gain 1.000
0.846 0.846 0.154 26 CEP 17 gain LSI 13 gain LSI 5p15 gain 1.000
0.846 0.846 0.154 26 CEP 8 gain LSI 5q31 gain LSI 5p15 gain 1.000
0.846 0.846 0.154 26 CEP 8 gain LSI 5q31 gain CEP 1 gain 1.000
0.846 0.846 0.154 26 LSI 13 gain 9p21 gain LSI 5p15 gain 1.000
0.846 0.846 0.154 26 LSI 13 gain 9p21 gain CEP 1 gain 1.000 0.846
0.846 0.154 26 LSI 13 gain LSI 5q31 gain LSI 5p15 gain 1.000 0.846
0.846 0.154 26 LSI 13 gain LSI 5q31 gain CEP 1 gain 1.000 0.846
0.846 0.154 26 4 probe combinations: 17q21 gain 9p21 gain CEP 8
gain LSI 5p15 gain 1.000 0.885 0.885 0.115 26 17q21 gain 9p21 gain
CEP 8 gain CEP 1 gain 1.000 0.885 0.885 0.115 26 17q21 gain CEP 12
gain 9p21 gain CEP 1 gain 1.000 0.885 0.885 0.115 26 17q21 gain CEP
17 gain LSI 13 gain 9p21 gain 1.000 0.885 0.885 0.115 26 17q21 gain
CEP 17 gain 9p21 gain CEP 8 gain 1.000 0.885 0.885 0.115 26 17q21
gain LSI 13 gain 9p21 gain LSI 5p15 gain 1.000 0.885 0.885 0.115 26
17q21 gain LSI 13 gain 9p21 gain CEP 1 gain 1.000 0.885 0.885 0.115
26 9p21 gain CEP 8 gain LSI 5q31 gain LSI 3p15 gain 1.000 0.885
0.885 0.115 26 9p21 gain CEP 8 gain LSI 5q31 gain CEP 1 gain 1.000
0.885 0.885 0.115 26 CEP 12 gain 9p21 gain CEP 8 gain CEP 1 gain
1.000 0.885 0.885 0.115 26 CEP 12 gain 9p21 gain CEP 6 gain CEP 1
gain 1.000 0.885 0.885 0.115 26 CEP 12 gain 9p21 gain CEP 3 gain
CEP 1 gain 1.000 0.885 0.855 0.115 26 CEP 17 gain 9p21 gain CEP 9
gain CEP 8 gain 1.000 0.885 0.885 0.115 26 CEP 17 gain 9p21 gain
CEP 8 gain CEP 6 gain 1.000 0.885 0.885 0.115 26 CEP 17 gain 9p21
gain CEP 8 gain LSI 5p15 gain 1.000 0.885 0.885 0.115 26 CEP 17
gain 9p21 gain CEP 8 gain CEP 1 gain 1.000 0.885 0.885 0.115 26 CEP
17 gain CEP 12 gain 9p21 gain CEP 6 gain 1.000 0.885 0.885 0.115 26
CEP 17 gain LSI 13 gain 9p21 gain CEP 9 gain 1.000 0.885 0.885
0.115 26 CEP 17 gain LSI 13 gain 9p21 gain CEP 6 gain 1.000 0.885
0.885 0.115 26 CEP 17 gain LSI 13 gain 9p21 gain LSI 5p15 gain
1.000 0.885 0.885 0.115 26 CEP 17 gain LSI 13 gain 9p21 gain CEP 1
gain 1.000 0.885 0.885 0.115 26 LSI 13 gain 9p21 gain LSI 5q31 gain
LSI 5p15 gain 1.000 0.885 0.885 0.115 26 LSI 13 gain 9p21 gain LSI
5q31 gain CEP 1 gain 1.000 0.885 0.885 0.115 26 LSI 13 gain CEP 12
gain 9p21 gain CEP 1 gain 1.000 0.885 0.885 0.115 26 CEP 17 gain
CEP 10 gain 9p21 gain CEP 8 gain 1.000 0.880 0.880 0.120 25 CEP 17
gain LSI 13 gain CEP 10 gain 9p21 gain 1.000 0.880 0.880 0.120
25
TABLE-US-00007 TABLE 7 Combinations of 2, 3 and 4 Probes at a
Cutoff Value of 20% SPECI- SENSI- # TUMOR PROBE 1 PROBE 2 PROBE 3
PROBE 4 FICITY TIVITY SENS*SPEC VECTOR SPECIMENS 2 probe
combinations LSI 5p15 gain LSI 3q gain 1.000 0.789 0.789 0.211 19
CEP 16 gain LSI 5p15 gain 1.000 0.737 0.737 0.263 19 LSI 20q gain
LSI 5p15 gain 1.000 0.737 0.737 0.263 19 LSI 5p15 gain CEP 4 gain
1.000 0.737 0.737 0.263 19 17q21 gain LSI 5p15 gain 1.000 0.731
0.731 0.269 26 8q24 gain LSI 5p15 gain 1.000 0.731 0.731 0.269 26
CEP 6 gain LSI 5p15 gain 1.000 0.731 0.731 0.269 26 CEP 9 gain LSI
5p15 gain 1.000 0.731 0.731 0.269 26 LSI 5p15 gain 3p14 gain 1.000
0.731 0.731 0.269 26 LSI 5p15 gain CEP 3 gain 1.000 0.731 0.731
0.269 26 5 p/q imbal. gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26
5 p/q imbal. gain LSI 5q31 gain 1.000 0.692 0.692 0.308 26 7p12
gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26 8q24 gain 7p12 gain
1.000 0.692 0.692 0.308 26 8q24 gain CEP 6 gain 1.000 0.692 0.692
0.308 26 CEP 12 gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26 CEP
17 gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26 CEP 7 gain LSI
5p15 gain 1.000 0.692 0.692 0.308 26 CEP 8 gain LSI 5p15 gain 1.000
0.692 0.692 0.308 26 CEP 9 gain 3p14 gain 1.000 0.692 0.692 0.308
26 LSI 13 gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26 LSI 5p15
gain CEP 1 gain 1.000 0.692 0.692 0.308 26 7p12 gain LSI 3q gain
1.000 0.684 0.684 0.316 19 CEP 12 gain LSI 3q gain 1.000 0.684
0.684 0.316 19 CEP 7 gain LSI 3q gain 1.000 0.684 0.684 0.316 19
LSI 5q31 gain LSI 3q gain 1.000 0.684 0.684 0.316 19 3 probe
combinations and 3 pr comb (1 rat + 1 abs) CEP 12 gain LSI 5p15
gain LSI 3q gain 1.000 0.842 0.842 0.158 19 5 p/q imbal. gain LSI
5q31 gain LSI 3q gain 1.000 0.789 0.789 0.211 19 8q24 gain LSI 5p15
gain CEP 4 gain 1.000 0.789 0.789 0.211 19 CEP 12 gain LSI 5p15
gain CEP 4 gain 1.000 0.789 0.789 0.211 19 CEP 16 gain 8q24 gain
LSI 5p15 gain 1.000 0.789 0.789 0.211 19 CEP 16 gain CEP 12 gain
LSI 5p15 gain 1.000 0.789 0.789 0.211 19 LSI 20q gain 8q24 gain LSI
5p15 gain 1.000 0.789 0.789 0.211 19 LSI 20q gain CEP 12 gain LSI
5p15 gain 1.000 0.789 0.789 0.211 19 17q21 gain 5 p/q imbal. gain
LSI 5q31 gain 1.000 0.769 0.769 0.231 26 17q21 gain 8q24 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 17q21 gain CEP 12 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 17q21 gain LSI 5p15 gain 3p14
gain 1.000 0.769 0.769 0.231 26 17q21 gain LSI 5p15 gain CEP 3 gain
1.000 0.769 0.769 0.231 26 5 p/q imbal. gain LSI 5p15 gain 3p14
gain 1.000 0.769 0.769 0.231 26 5 p/q imbal. gain LSI 5p15 gain CEP
3 gain 1.000 0.769 0.769 0.231 26 5 p/q imbal. gain LSI 5q31 gain
3p14 gain 1.000 0.769 0.769 0.231 26 5 p/q imbal. gain LSI 5q31
gain CEP 3 gain 1.000 0.769 0.769 0.231 26 8q24 gain 5 p/q imbal.
gain LSI 5q31 gain 1.000 0.769 0.769 0.231 26 8q24 gain 5 p/q
imbal. gain LSI 5p15 gain 1.000 0.769 0.769 0.231 26 8q24 gain CEP
6 gain LSI 5p15 gain 1.000 0.769 0.769 0.231 26 8q24 gain LSI 5p15
gain 3p14 gain 1.000 0.769 0.769 0.231 26 8q24 gain LSI 5p15 gain
CEP 3 gain 1.000 0.769 0.769 0.231 26 CEP 12 gain 8q24 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 CEP 12 gain CEP 6 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 CEP 12 gain CEP 9 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 CEP 12 gain CEP 9 gain LSI
5p15 gain 1.000 0.769 0.769 0.231 26 CEP 12 gain LSI 5p15 gain 3p14
gain 1.000 0.769 0.769 0.231 26 CEP 12 gain LSI 5p15 gain CEP 3
gain 1.000 0.769 0.769 0.231 26 CEP 6 gain 5 p/q imbal. gain LSI
5q31 gain 1.000 0.769 0.769 0.231 26 CEP 6 gain 5 p/q imbal. gain
LSI 5p15 gain 1.000 0.769 0.769 0.231 26 CEP 6 gain LSI 5p15 gain
3p14 gain 1.000 0.769 0.769 0.231 26 CEP 6 gain LSI 5p15 gain CEP 3
gain 1.000 0.769 0.769 0.231 26 CEP 9 gain 5 p/q imbal. gain LSI
5q31 gain 1.000 0.769 0.769 0.231 26 CEP 9 gain 5 p/q imbal. gain
LSI 5p15 gain 1.000 0.769 0.769 0.231 26 CEP 9 gain 8q24 gain 3p14
gain 1.000 0.769 0.769 0.231 26 CEP 9 gain LSI 5p15 gain 3p14 gain
1.000 0.769 0.769 0.231 26 CEP 9 gain LSI 5p15 gain CEP 3 gain
1.000 0.769 0.769 0.231 26 4 probe combinations and 4 pr comb (1
rat + 2 abs) CEP 12 gain 8q24 gain LSI 5p15 gain CEP 4 gain 1.000
0.842 0.842 0.158 19 CEP 12 gain 5 p/q imbal. gain LSI 5q31 gain
LSI 3q gain 1.000 0.842 0.842 0.158 19 CEP 16 gain CEP 12 gain 8q24
gain LSI 5p15 gain 1.000 0.842 0.842 0.158 19 LSI 20q gain CEP 12
gain 8q24 gain LSI 5p15 gain 1.000 0.842 0.842 0.158 19 17p13 gain
CEP 9 gain 5 p/q imbal. gain CEP 3 gain 1.000 0.808 0.808 0.192 26
17q21 gain CEP 12 gain 5 p/q imbal. gain LSI 5q31 gain 1.000 0.808
0.808 0.192 26 17q21 gain CEP 12 gain 8q24 gain LSI 5p15 gain 1.000
0.808 0.808 0.192 26 17q21 gain CEP 12 gain LSI 5p15 gain 3p14 gain
1.000 0.808 0.808 0.192 26 17q21 gain CEP 12 gain LSI 5p15 gain CEP
3 gain 1.000 0.808 0.808 0.192 26 17q21 gain 8q24 gain 5 p/q imbal.
gain LSI 5q31 gain 1.000 0.808 0.808 0.192 26 17q21 gain 8q24 gain
5 p/q imbal. gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 17q21
gain 8q24 gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808 0.192 26
17q21 gain 8q24 gain LSI 5p15 gain CEP 3 gain 1.000 0.808 0.808
0.192 26 17q21 gain 5 p/q imbal. gain LSI 5q31 gain 3p14 gain 1.000
0.808 0.808 0.192 26 17q21 gain 5 p/q imbal. gain LSI 5p15 gain
3p14 gain 1.000 0.808 0.808 0.192 26 17q21 gain 5 p/q imbal. gain
LSI 5q31 gain CEP 3 gain 1.000 0.808 0.808 0.192 26 17q21 gain 5
p/q imbal. gain LSI 5p15 gain CEP 3 gain 1.000 0.808 0.808 0.192 26
8q24 gain CEP 6 gain 5 p/q imbal. gain LSI 5q31 gain 1.000 0.808
0.808 0.192 26 8q24 gain CEP 6 gain 5 p/q imbal. gain LSI 5p15 gain
1.000 0.808 0.808 0.192 26 8q24 gain CEP 6 gain LSI 5p15 gain 3p14
gain 1.000 0.808 0.808 0.192 26 8q24 gain CEP 6 gain LSI 5p15 gain
CEP 3 gain 1.000 0.808 0.808 0.192 26 8q24 gain 5 p/q imbal. gain
LSI 5q31 gain 3p14 gain 1.000 0.808 0.808 0.192 26 8q24 gain 5 p/q
imbal. gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808 0.192 26 8q24
gain 5 p/q imbal. gain LSI 5q15 gain CEP 3 gain 1.000 0.808 0.808
0.192 26 8q24 gain 5 p/q imbal. gain LSI 5p15 gain CEP 3 gain 1.000
0.808 0.808 0.192 26 9p21 gain 8q24 gain CEP 6 gain 5 p/q imbal.
gain 1.000 0.808 0.808 0.192 26 CEP 12 gain CEP 9 gain 8q24 gain
LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain CEP 9 gain 5
p/q imbal. gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 12
gain CEP 9 gain 8q24 gain 3p14 gain 1.000 0.808 0.808 0.192 26 CEP
12 gain CEP 9 gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808 0.192
26 CEP 12 gain CEP 9 gain LSI 5p15 gain CEP 3 gain 1.000 0.808
0.808 0.192 26 CEP 12 gain CEP 9 gain 5 p/q imbal. gain LSI 8q31
gain 1.000 0.808 0.808 0.192 26 CEP 12 gain CEP 6 gain 5 p/q imbal.
gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain CEP 6
gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain
CEP 6 gain LSI 5p15 gain CEP 3 gain 1.000 0.808 0.808 0.192 26 CEP
12 gain CEP 6 gain 5 p/q imbal. gain LSI 5q31 gain 1.000 0.808
0.808 0.192 26 CEP 12 gain 8q24 gain CEP 6 gain LSI 5p15 gain 1.000
0.808 0.808 0.192 26 CEP 12 gain 8q24 gain 5 p/q imbal. gain LSI
5p15 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain 8q24 gain LSI 5p15
gain 3p14 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain 8q24 gain LSI
5p15 gain CEP 3 gain 1.000 0.808 0.808 0.192 26 CEP 12 gain 5 p/q
imbal. gain LSI 5q31 gain 3p14 gain 1.000 0.808 0.808 0.192 26 CEP
12 gain 5 p/q imbal. gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808
0.192 26 CEP 12 gain 5 p/q imbal. gain LSI 5q31 gain CEP 3 gain
1.000 0.808 0.808 0.192 26 CEP 12 gain 5 p/q imbal. gain LSI 5p15
gain CEP 3 gain 1.000 0.808 0.808 0.192 26 CEP 6 gain 5 p/q imbal.
gain LSI 5q31 gain 3p14 gain 1.000 0.808 0.808 0.192 26 CEP 6 gain
5 p/q imbal. gain LSI 5q31 gain 3p14 gain 1.000 0.808 0.808 0.192
26 CEP 6 gain 5 p/q imbal. gain LSI 5q31 gain CEP 3 gain 1.000
0.808 0.808 0.192 26 CEP 6 gain 5 p/q imbal. gain LSI 5p15 gain CEP
3 gain 1.000 0.808 0.808 0.192 26 CEP 9 gain 8q24 gain 5 p/q imbal.
gain LSI 5q31 gain 1.000 0.808 0.808 0.192 26 CEP 9 gain 8q24 gain
5 p/q imbal. gain LSI 5p15 gain 1.000 0.808 0.808 0.192 26 CEP 9
gain 8q24 gain LSI 5p15 gain 3p14 gain 1.000 0.808 0.808 0.192 26
CEP 9 gain 8q24 gain LSI 5p15 gain CEP 3 gain 1.000 0.808 0.808
0.192 26 CEP 9 gain 8q24 gain 5 p/q imbal. gain CEP 1 gain 1.000
0.808 0.808 0.192 26 CEP 9 gain 5 p/q imbal. gain 3p14 gain 1.000
0.808 0.808 0.192 26 CEP 9 gain 5 p/q imbal. gain LSI 5q31 gain CEP
3 gain 1.000 0.808 0.808 0.192 26 CEP 9 gain 5 p/q imbal. gain LSI
5p15 gain CEP 3 gain 1.000 0.808 0.808 0.192 26
TABLE-US-00008 TABLE 8 Combinations of 2, 3 and 4 Probes at a
Cutoff Value of 30% SPECI- SENSI- # TUMOR PROBE 1 PROBE 2 PROBE 3
PROBE 4 FICITY TIVITY SENS*SPEC VECTOR SPECIMENS 2 probe
combinations CEP 6 gain LSI 5p15 gain 1.000 0.692 0.692 0.308 26
CEP 16 gain LSI 5p15 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain
LSI 5p15 gain 1.000 0.684 0.684 0.316 19 LSI 5p15 gain LSI 3q gain
1.000 0.684 0.684 0.316 19 17q21 gain LSI 5p15 gain 1.000 0.654
0.654 0.346 26 7p12 gain CEP 6 gain 1.000 0.654 0.654 0.346 26 7p12
gain LSI 5p15 gain 1.000 0.654 0.654 0.346 26 CEP 7 gain CEP 6 gain
1.000 0.654 0.654 0.346 26 LSI 5p15 gain 3p14 gain 1.000 0.654
0.654 0.346 26 10q23 gain LSI 5p15 gain 1.000 0.640 0.640 0.360 25
CEP 10 gain LSI 5p15 gain 1.000 0.640 0.640 0.360 25 LSI 5p15 gain
CEP 4 gain 1.000 0.632 0.632 0.368 19 LSI 5q31 gain LSI 3q gain
1.000 0.632 0.632 0.368 19 17p13 gain LSI 5p15 gain 1.000 0.615
0.615 0.385 26 8q24 gain LSI 5p15 gain 1.000 0.615 0.615 0.385 26
CEP 17 gain LSI 5p15 gain 1.000 0.615 0.615 0.385 26 CEP 6 gain CEP
1 gain 1.000 0.615 0.615 0.385 26 CEP 6 gain LSI 5q31 gain 1.000
0.615 0.615 0.385 26 CEP 7 gain LSI 5p15 gain 1.000 0.615 0.615
0.385 26 CEP 8 gain LSI 5p15 gain 1.000 0.615 0.615 0.385 26 LSI 13
gain LSI 5p15 gain 1.000 0.615 0.615 0.385 26 LSI 5p15 gain CEP 1
gain 1.000 0.615 0.615 0.385 26 LSI 5p15 gain CEP 3 gain 1.000
0.615 0.615 0.385 26 CEP 18 gain LSI 5p15 gain 1.000 0.600 0.600
0.400 25 7p12 gain LSI 3q gain 1.000 0.579 0.579 0.421 19 CEP 16
gain 7p12 gain 1.000 0.579 0.579 0.421 19 CEP 16 gain LSI 5q31 gain
1.000 0.579 0.579 0.421 19 CEP 7 gain LSI 3q gain 1.000 0.579 0.579
0.421 19 LSI 20q gain 3p14 gain 1.000 0.579 0.579 0.421 19 LSI 20q
gain 7p12 gain 1.000 0.579 0.579 0.421 19 LSI 20q gain CEP 12 gain
1.000 0.579 0.579 0.421 19 LSI 20q gain CEP 3 gain 1.000 0.579
0.579 0.421 19 LSI 20q gain CEP 6 gain 1.000 0.579 0.579 0.421 19
LSI 20q gain LSI 5q31 gain 1.000 0.579 0.579 0.421 19 3 probe
combinations <.4 and 3 pr comb (1rat + 1 abs) 8q24 gain 7p12
gain CEP 6 gain 1.000 0.692 0.692 0.308 26 8q24 gain CEP 6 gain LSI
5q31 gain 1.000 0.692 0.692 0.308 26 8q24 gain CEP 7 gain CEP 6
gain 1.000 0.692 0.692 0.308 26 CEP 6 gain 5 p/q imbal. gain LSI
5q31 gain 1.000 0.692 0.692 0.308 26 5 p/q imbal. gain LSI 5q31
gain LSI 3q gain 1.000 0.684 0.684 0.316 19 8q24 gain LSI 5q31 gain
LSI 3q gain 1.000 0.684 0.684 0.316 19 CEP 16 gain 3 p/q imbal.
gain LSI 5q31 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain 5 p/q
imbal. gain LSI 5q31 gain 1.000 0.684 0.684 0.316 19 17p13 gain
8q24 gain LSI 5p15 gain 1.000 0.654 0.654 0.346 26 17p13 gain CEP
17 gain LSI 5p15 gain 1.000 0.654 0.654 0.346 26 17p13 gain CEP 7
gain LSI 5p15 gain 1.000 0.654 0.654 0.346 26 17p13 gain CEP 8 gain
LSI 5p15 gain 1.000 0.654 0.654 0.346 26 17p13 gain LSI 13 gain LSI
5p15 gain 1.000 0.654 0.654 0.346 26 17p13 gain LSI 5p15 gain CEP 3
gain 1.000 0.654 0.654 0.346 26 17p13 gain LSI 5p15 gain CEP 1 gain
1.000 0.654 0.654 0.346 26 17p13/CEP 17 loss LSI 5p15 gain 1.000
0.654 0.654 0.346 26 17q21 gain 5 p/q imbal. gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 17q21 gain CEP 6 gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 5 p/q imbal. gain LSI 5q31 gain 3p14
gain 1.000 0.654 0.654 0.346 26 7p12 gain 5 p/q imbal. gain LSI
5q31 gain 1.000 0.654 0.654 0.346 26 7p12/CEP 7 gain CEP 7 gain LSI
5p15 gain 1.000 0.654 0.654 0.346 26 8q24 gain CEP 6 gain CEP 1
gain 1.000 0.654 0.654 0.346 26 9p21 gain CEP 6 gain CEP 1 gain
1.000 0.654 0.654 0.346 26 CEP 12 gain CEP 6 gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 CEP 12 gain CEP 6 gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 CEP 17 gain CEP 6 gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 CEP 6 gain LSI 5q31 gain CEP 3 gain
1.000 0.654 0.654 0.346 26 CEP 6 gain LSI 5q31 gain CEP 1 gain
1.000 0.654 0.654 0.346 26 CEP 8 gain CEP 6 gain LSI 5q31 gain
1.000 0.654 0.654 0.346 26 CEP 9 gain CEP 6 gain CEP 1 gain 1.000
0.654 0.654 0.346 26 10q23 gain 5 p/q imbal. gain LSI 5q31 gain
1.000 0.640 0.640 0.360 25 CEP 10 gain 5 p/q imbal. gain LSI 5q31
gain 1.000 0.640 0.640 0.360 25 CEP 18 gain 17p13 gain LSI 5p15
gain 1.000 0.640 0.640 0.360 25 4 probe combinations <.4 and 4
pr comb (1 rat + 2 abs) 17p13/CEP 17 loss CEP 6 gain LSI 5p15 gain
1.000 0.731 0.731 0.269 26 17p13/CEP 17 loss CEP 6 gain LSI 5q31
gain 1.000 0.692 0.692 0.308 26 17p13/CEP 17 loss CEP 7 gain CEP 6
gain 1.000 0.692 0.692 0.308 26 7p12 gain CEP 6 gain 5 p/q imbal.
gain 1.000 0.692 0.692 0.308 26 9p21 gain 8q24 gain CEP 6 gain CEP
1 gain 1.000 0.692 0.692 0.308 26 CEP 7 gain CEP 6 gain 5 p/q
imbal. gain 1.000 0.692 0.692 0.308 26 CEP 9 gain 8q24 gain CEP 6
gain CEP 1 gain 1.000 0.692 0.692 0.308 26 8q24 gain 7p12 gain LSI
3q gain 3p14 gain 1.000 0.684 0.684 0.316 19 8q24 gain 7p12 gain
LSI 3q gain CEP 3 gain 1.000 0.684 0.684 0.316 19 8q24 gain CEP 7
gain LSI 3q gain 3p14 gain 1.000 0.684 0.684 0.316 19 8q24 gain CEP
7 gain LSI 3q gain CEP 3 gain 1.000 0.684 0.684 0.316 19 CEP 12
gain 8q24 gain 7p12 gain LSI 3q gain 1.000 0.684 0.684 0.316 19 CEP
12 gain 8q24 gain CEP 7 gain LSI 3q gain 1.000 0.684 0.684 0.316 19
CEP 16 gain 8q24 gain 7p12 gain LSI 5q31 gain 1.000 0.684 0.684
0.316 19 CEP 16 gain 8q24 gain 7p12 gain 3p14 gain 1.000 0.684
0.684 0.316 19 CEP 16 gain 8q24 gain CEP 7 gain 3p14 gain 1.000
0.684 0.684 0.316 19 CEP 16 gain 8q24 gain LSI 5q31 gain 3p14 gain
1.000 0.684 0.684 0.316 19 CEP 16 gain 8q24 gain 7p12 gain CEP 3
gain 1.000 0.684 0.684 0.316 19 CEP 16 gain 8q24 gain CEP 7 gain
CEP 3 gain 1.000 0.684 0.684 0.316 19 CEP 16 gain 8q24 gain LSI
5q31 gain CEP 3 gain 1.000 0.684 0.684 0.316 19 CEP 16 gain CEP 11
gain 8q24 gain LSI 5q31 gain 1.000 0.684 0.684 0.316 19 CEP 16 gain
CEP 12 gain 8q24 gain 7p12 gain 1.000 0.684 0.684 0.316 19 CEP 16
gain CEP 12 gain 8q24 gain CEP 7 gain 1.000 0.684 0.684 0.316 19
CEP 16 gain CEP 12 gain 8q24 gain LSI 5q31 gain 1.000 0.684 0.684
0.316 19 LSI 20q gain 8q24 gain 7p12 gain LSI 5q31 gain 1.000 0.684
0.684 0.316 19 LSI 20q gain 8q24 gain 7p12 gain 3p14 gain 1.000
0.684 0.684 0.316 19 LSI 20q gain 8q24 gain CEP 7 gain 3p14 gain
1.000 0.684 0.684 0.316 19 LSI 20q gain 8q24 gain LSI 5q31 gain
3p14 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain 8q24 gain 7p12
gain CEP 3 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain 8q24 gain
CEP 7 gain CEP 3 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain 8q24
gain LSI 5q31 gain CEP 3 gain 1.000 0.684 0.684 0.316 19 LSI 20q
gain 9p21 gain 8q24 gain CEP 6 gain 1.000 0.684 0.684 0.316 19 LSI
20q gain 9p21 gain 8q24 gain 3p14 gain 1.000 0.684 0.684 0.316 19
LSI 20q gain 9p21 gain 8q24 gain CEP 3 gain 1.000 0.684 0.684 0.316
19 LSI 20q gain CEP 11 gain 8q24 gain LSI 5q31 gain 1.000 0.684
0.684 0.316 19 LSI 20q gain CEP 12 gain 9p21 gain 8q24 gain 1.000
0.684 0.684 0.316 19 LSI 20q gain CEP 12 gain CEP 9 gain 8q24 gain
1.000 0.684 0.684 0.316 19 LSI 20q gain CEP 12 gain 8q24 gain 7p12
gain 1.000 0.684 0.684 0.316 19 LSI 20q gain CEP 12 gain 8q24 gain
CEP 7 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain CEP 12 gain 8q24
gain LSI 5q31 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain CEP 9
gain 8q24 gain CEP 6 gain 1.000 0.684 0.684 0.316 19 LSI 20q gain
CEP 9 gain 8q24 gain 3p14 gain 1.000 0.684 0.684 0.316 19 LSI 20q
gain CEP 9 gain 8q24 gain CEP 3 gain 1.000 0.684 0.684 0.316 19
TABLE-US-00009 TABLE 9 Combinations of 2, 3 and 4 Probes at a
Cutoff Value of 40% SPECI- SENSI- # TUMOR PROBE 1 PROBE 2 PROBE 3
PROBE 4 FICITY TIVIT SENS*SPEC VECTOR SPECIMENS 2 probe
combinations 7p12 gain LSI 3q gain 1.000 0.579 0.579 0.421 19 7p12
gain CEP 6 gain 1.000 0.538 0.538 0.462 26 LSI 3q gain CEP 1 gain
1.000 0.526 0.526 0.474 19 CEP 6 gain CEP 1 gain 1.000 0.500 0.500
0.500 26 CEP 7 gain CEP 6 gain 1.000 0.500 0.500 0.500 26 CEP 18
gain 7p12 gain 1.000 0.480 0.480 0.520 25 7p12 gain CEP 4 gain
1.000 0.474 0.474 0.526 19 CEP 16 gain 7p12 gain 1.000 0.474 0.474
0.526 19 CEP 7 gain LSI 3q gain 1.000 0.474 0.474 0.526 19 LSI 20q
gain 7p12 gain 1.000 0.474 0.474 0.526 19 LSI 5p15 gain LSI 3q gain
1.000 0.474 0.474 0.526 19 7p12 gain LSI 5p15 gain 1.000 0.462
0.462 0.538 26 CEP 10 gain 7p12 gain 1.000 0.440 0.440 0.560 25 CEP
18 gain CEP 1 gain 1.000 0.440 0.440 0.560 25 7p12 gain LSI 5q31
gain 1.000 0.423 0.423 0.577 26 CEP 11 gain 7p12 gain 1.000 0.423
0.423 0.577 26 CEP 6 gain LSI 5p15 gain 1.000 0.423 0.423 0.577 26
CEP 7 gain LSI 5p15 gain 1.000 0.423 0.423 0.577 26 LSI 5p15 gain
CEP 1 gain 1.000 0.423 0.423 0.577 26 CEP 16 gain CEP 1 gain 1.000
0.421 0.421 0.579 19 CEP 16 gain CEP 7 gain 1.000 0.421 0.421 0.579
19 CEP 4 gain CEP 1 gain 1.000 0.421 0.421 0.579 19 LSI 20q gain
CEP 1 gain 1.000 0.421 0.421 0.579 19 LSI 20q gain CEP 7 gain 1.000
0.421 0.421 0.579 19 10q23 gain 7p12 gain 1.000 0.400 0.400 0.600
25 CEP 10 gain CEP 1 gain 1.000 0.400 0.400 0.600 25 CEP 18 gain
CEP 7 gain 1.000 0.400 0.400 0.600 25 CEP 11 gain CEP 1 gain 1.000
0.385 0.385 0.615 26 CEP 11 gain CEP 7 gain 1.000 0.385 0.385 0.615
26 CEP 12 gain CEP 6 gain 1.000 0.385 0.385 0.615 26 3 probe
combinations CEP 11 gain 7p12 gain CEP 6 gain 1.000 0.577 0.577
0.423 26 CEP 11 gain CEP 6 gain CEP 1 gain 1.000 0.538 0.538 0.462
26 CEP 11 gain CEP 7 gain CEP 6 gain 1.000 0.538 0.538 0.462 26
17q21 gain CEP 7 gain LSI 3q gain 1.000 0.526 0.526 0.474 19 CEP 11
gain 7p12 gain CEP 4 gain 1.000 0.526 0.526 0.474 19 CEP 11 gain
CEP 7 gain LSI 3q gain 1.000 0.526 0.526 0.474 19 CEP 16 gain CEP
11 gain 7p12 gain 1.000 0.526 0.526 0.474 19 CEP 16 gain CEP 7 gain
LSI 3q gain 1.000 0.526 0.526 0.474 19 CEP 7 gain CEP 6 gain LSI 3q
gain 1.000 0.526 0.526 0.474 19 CEP 7 gain LSI 5p15 gain LSI 3q
gain 1.000 0.526 0.526 0.474 19 LSI 20q gain CEP 11 gain 7p12 gain
1.000 0.526 0.526 0.474 19 LSI 20q gain CEP 7 gain LSI 3q gain
1.000 0.526 0.526 0.474 19 CEP 18 gain 10q23 gain 7p12 gain 1.000
0.520 0.520 0.480 25 CEP 18 gain CEP 10 gain 7p12 gain 1.000 0.520
0.520 0.480 25 CEP 18 gain CEP 6 gain CEP 1 gain 1.000 0.520 0.520
0.480 25 CEP 18 gain CEP 7 gain CEP 6 gain 1.000 0.520 0.520 0.480
25 CEP 11 gain 7p12 gain LSI 5p15 gain 1.000 0.500 0.500 0.500 26
CEP 18 gain 7p12 gain CEP 4 gain 1.000 0.500 0.500 0.500 18 CEP 18
gain CEP 16 gain 7p12 gain 1.000 0.500 0.500 0.500 18 LSI 20q gain
CEP 18 gain 7p12 gain 1.000 0.500 0.500 0.500 18 10q23 gain 7p12
gain LSI 5p15 gain 1.000 0.480 0.480 0.520 25 CEP 10 gain 7p12 gain
LSI 5p15 gain 1.000 0.480 0.480 0.520 25 CEP 11 gain CEP 10 gain
7p12 gain 1.000 0.480 0.480 0.520 25 CEP 18 gain 10q23 gain CEP 1
gain 1.000 0.480 0.480 0.520 25 CEP 18 gain CEP 10 gain CEP 1 gain
1.000 0.480 0.480 0.520 25 CEP 11 gain CEP 4 gain CEP 1 gain 1.000
0.474 0.474 0.526 19 CEP 11 gain CEP 7 gain CEP 4 gain 1.000 0.474
0.474 0.526 19 CEP 16 gain CEP 11 gain CEP 7 gain 1.000 0.474 0.474
0.526 19 CEP 16 gain CEP 11 gain CEP 1 gain 1.000 0.474 0.474 0.526
19 LSI 20q gain CEP 11 gain CEP 7 gain 1.000 0.474 0.474 0.526 19
LSI 20q gain CEP 11 gain CEP 1 gain 1.000 0.474 0.474 0.526 19 4
probe combinations 17p13/CEP 17 loss CEP 6 gain CEP 1 gain 1.000
0.538 0.538 0.462 26 17p13/CEP 17 loss CEP 7 gain CEP 6 gain 1.000
0.538 0.538 0.462 26 9p21/CEP 9 loss CEP 7 gain LSI 3q gain 1.000
0.526 0.526 0.474 19 CEP 11 gain 10q23 gain 7p12 gain LSI 5p15 gain
1.000 0.520 0.520 0.480 25 CEP 11 gain CEP 10 gain 7p12 gain LSI
5q31 gain 1.000 0.520 0.520 0.480 25 CEP 11 gain 7p12 gain 5 p/q
imbal. gain 1.000 0.500 0.500 0.500 26 CEP 11 gain CEP 9 gain CEP 6
gain LSI 5p15 gain 1.000 0.500 0.500 0.500 26 10q23 gain 7p12 gain
5 p/q imbal. gain 1.000 0.480 0.480 0.520 25 CEP 10 gain 7p12 gain
5 p/q imbal. gain 1.000 0.480 0.480 0.520 25 CEP 11 gain 10q23 gain
7p12 gain LSI 5q31 gain 1.000 0.480 0.480 0.520 25 CEP 11 gain
10q23 gain CEP 7 gain LSI 5p15 gain 1.000 0.480 0.480 0.520 25 CEP
11 gain 10q23 gain LSI 5p15 gain CEP 1 gain 1.000 0.480 0.480 0.520
25 CEP 11 gain CEP 10 gain CEP 7 gain LSI 5q31 gain 1.000 0.480
0.480 0.520 25 CEP 11 gain CEP 10 gain LSI 5q31 gain CEP 1 gain
1.000 0.480 0.480 0.520 25 CEP 11 gain CEP 10 gain LSI 5p15 gain
CEP 1 gain 1.000 0.480 0.480 0.520 25 CEP 11 gain CEP 10 gain CEP 7
gain LSI 5q31 gain 1.000 0.480 0.480 0.520 25 CEP 11 gain CEP 10
gain CEP 7 gain LSI 5p15 gain 1.000 0.480 0.480 0.520 25 CEP 18
gain 10q23 gain CEP 7 gain LSI 5p15 gain 1.000 0.480 0.480 0.520 25
CEP 18 gain 17p13 gain 10q23 gain CEP 7 gain 1.000 0.480 0.480
0.520 25 CEP 18 gain 17p13 gain CEP 10 gain CEP 7 gain 1.000 0.480
0.480 0.520 25 CEP 18 gain 17p13/CEP 17 loss CEP 1 gain 1.000 0.480
0.480 0.520 25 CEP 18 gain 17q21 gain 10q23 gain CEP 7 gain 1.000
0.480 0.480 0.520 25 CEP 18 gain 17q21 gain CEP 10 gain CEP 7 gain
1.000 0.480 0.480 0.520 25 CEP 18 gain CEP 10 gain CEP 7 gain LSI
5p13 gain 1.000 0.480 0.480 0.520 25 CEP 18 gain CEP 11 gain 10q23
gain CEP 7 gain 1.000 0.480 0.480 0.520 25 CEP 18 gain CEP 11 gain
CEP 10 gain CEP 7 gain 1.000 0.480 0.480 0.520 25 CEP 18 gain CEP 9
gain CEP 6 gain LSI 5p15 gain 1.000 0.480 0.480 0.520 25 17p13/CEP
17 loss CEP 6 gain LSI 5p15 gain 1.000 0.462 0.462 0.538 26
17p/31CEP 17 loss CEP 7 gain LSI 5p15 gain 1.000 0.462 0.462 0.538
26 17p13/CEP 17 loss LSI 5p15 gain CEP 1 gain 1.000 0.462 0.462
0.538 26 9p21/CEP 9 loss CEP 6 gain LSI 5p15 gain 1.000 0.462 0.462
0.538 26 CEP 11 gain 5 p/q imbal. gain CEP 1 gain 1.000 0.462 0.462
0.538 26 CEP 11 gain CEP 7 gain 5 p/q imbal. gain 1.000 0.462 0.462
0.538 26 CEP 12 gain CEP 11 gain CEP 9 gain CEP 6 gain 1.000 0.462
0.462 0.538 26
TABLE-US-00010 TABLE 10 Analysis of Bronchial Secretions from 21
Patients by Cytology, Bronchus Biopsy, and FISH FISH Result
Specimen Clinical Cytology Bronchus Additional FISH I.D. Diagnosis
Result Biopsy Biopsy Probes Indicating Gain Diagnosis #3935 Small
Cell CA positive positive not done LSI 5p15 positive #3912 Squamous
Cell CA positive positive not done LSI 8q24, LSI 5p15, CEP 1 CEP 6
positive #3911 Squamous Cell CA positive positive not done LSI
8q24, LSI 5p15, CEP 1 CEP 6 positive #2870 Mesenchymal CA negative
negative positive LSI 5p15, CEP 6 positive #30582 Adenocarcinoma
positive not done not done LSI 8q24, LSI 5p15, CEP 1 CEP 6 positive
#1995 Breast CA metastasis positive positive positive LSI 8q24, LSI
5p15, CEP 1 CEP 6 positive #2786 Large Cell CA negative negative
positive none negative #2789 No malignancy negative negative not
done none negative #2545 Small Cell CA positive positive not done
LSI 8q24, LSI 5p15, CEP 1 positive #3700 Adenocarcinoma positive
positive not done LSI 8q24, LSI 5p15 positive #2363 Large Cell CA
positive positive not done LSI 8q24, LSI 5p15, CEP 1 positive #3739
Squamous Cell CA positive positive not done LSI 8q24, LSI 5p15, CEP
1 positive #30796 Small Cell CA positive positive not done LSI
8q24, LSI 5p15 positive #30671 Adenocarcinoma positive negative
positive LSI 8q24, LSI 5p15 positive #1864 Breast CA metastasis
positive negative positive LSI 8q24, LSI 5p15 positive #2546 Large
Cell CA negative not done positive -- not evaluated* #2577 No
malignancy negative negative not done none negative #2251 No
malignancy negative not done negative none negative #2603 No
malignancy negative negative not done none negative #2785 No
malignancy negative negative pos for epipharynx CA none negative
#30706 Equivocal negative Not done Equivocal none negative *cell
morphology was too poor to permit evaluation.
TABLE-US-00011 TABLE 11 Conventional Cytology Performance Compared
to Clinical Diagnosis Cytology Clinical Diagnosis Result negative
positive/equivocal negative 5 4 positive 0 12 specificity = 100%
sensitivity = 75% sensitivity = 80% excluding the slide not
evaluated by FISH
TABLE-US-00012 TABLE 12 FISH Performance Compared to Clinical
Diagnosis FISH Clinical Diagnosis Result negative
positive/equivocal negative 5 2 positive 0 13 not evaluated 0 1
specificity = 100% sensitivity = 81% including non-evaluable FISH
slide sensitivity = 87% excluding non-evaluable FISH slide
TABLE-US-00013 TABLE 13 Probe Sets Based on Discriminate and
Combinatorial Analyses VECTOR VALUE CUTOFF = CUTOFF = CUTOFF =
CUTOFF = CUTOFF = PROBE 1 PROBE 2 PROBE 3 PROBE 4 5 10 20 3 40
Single probes: LSI 5p15 0.407 0.231 0.346 0.423 0.692 CEP 1 0.077
0.346 0.462 0.615 0.654 CEP 6 0.287 0.385 0.500 0.500 0.692 LSI
7p12 0.619 0.324 0.385 0.500 0.615 LSI 8q24 0.210 0.222 0.556 0.778
0.889 CEP 9 0.287 0.346 0.577 0.808 0.885 2 Probe combinations: LSI
5p15 LSI 8q24 0.154 0.269 0.385 LSI 5p15 LSI 3q 0.211 0.316 0.526
LSI 5p15 LSI 20q 0.263 0.316 LSI 5p15 LSI 7p12 0.308 0.346 0.538
LSI 5p15 CEP 16 0.263 0.316 LSI 5p15 CEP 4 0.263 0.368 LSI 5p15 CEP
12 0.154 0.308 0.368 LSI 5p15 CEP 6 0.269 0.308 0.577 LSI 5p15 LSI
17q21 0.192 0.269 0.346 LSI 8q24 CEP 17 0.148 LSI 8q24 CEP 1 0.154
LSI 8q24 CEP 6 0.192 0.308 LSI 7p12 LSI 3q 0.316 0.421 0.421 LSI
7p12 CEP 6 0.346 0.462 LSI 3q CEP 7 0.316 0.421 0.526 CEP 6 CEP 7
0.346 0.500 3 Probe combinations: LSI 5p15 LSI 8q24 LSI 9p21 0.115
LSI 5p15 CEP 12 LSI 9p21 0.115 LSI 8q24 CEP 17 LSI 9p21 0.115 LSI
8q24 CEP 1 LSI 9p21 0.115 LSI 5p15 LSI 3q CEP 12 0.158 4 Probe
combinations: LSI 5p15 CEP 6 LSI 17p13 CEP 17 0.269 (loss) Probe
sets with redundant complementation: 3 probe combinations (sum of 2
probe pairs with 1 probe in common): LSI 5p15 LSI 8q24 LSI 3q LSI
5p15 LSI 8q24 LSI 20q LSI 5p15 LSI 8q24 LSI 7p12 LSI 5p15 LSI 8q24
CEP 16 LSI 5p15 LSI 8q24 CEP 4 LSI 5p15 LSI 8q24 CEP 12 LSI 5p15
LSI 8q24 CEP 6 LSI 5p15 LSI 8q24 LSI 17q21 LSI 5p15 LSI 8q24 CEP 17
LSI 5p15 LSI 8q24 CEP 1 LSI 5p15 LSI 3q LSI 20q LSI 5p15 LSI 3q LSI
7p12 LSI 5p15 LSI 3q CEP 16 LSI 5p15 LSI 3q CEP 4 LSI 5p15 LSI 3q
CEP 12 LSI 5p15 LSI 3q CEP 6 LSI 5p15 LSI 3q LSI 17q21 LSI 5p15 LSI
3q CEP 7 LSI 5p15 LSI 3q LSI 7p12 LSI 5p15 LSI 20q LSI 7p12 LSI
5p15 LSI 20q CEP 16 LSI 5p15 LSI 20q CEP 4 LSI 5p15 LSI 20q CEP 12
LSI 5p15 LSI 20q CEP 6 LSI 5p15 LSI 20q LSI 17q21 LSI 5p15 LSI 7p12
CEP 16 LSI 5p15 LSI 7p12 CEP 4 LSI 5p15 LSI 7p12 CEP 12 LSI 5p15
LSI 7p12 CEP 6 LSI 5p15 LSI 7p12 LSI 17q21 LSI 5p15 LSI 7p12 LSI 3q
LSI 5p15 LSI 7p12 CEP 6 LSI 5p15 CEP 16 CEP 4 LSI 5p15 CEP 16 CEP
12 LSI 5p15 CEP 16 CEP 6 LSI 5p15 CEP 16 LSI 17q21 LSI 5p15 CEP 4
CEP 12 LSI 5p15 CEP 4 CEP 6 LSI 5p15 CEP 4 LSI 17q21 LSI 5p15 CEP
12 CEP 6 LSI 5p15 CEP 12 LSI 17q21 LSI 5p15 CEP 6 LSI 17q21 LSI
5p15 CEP 6 CEP 7 LSI 8q24 CEP 17 CEP 1 LSI 8q24 CEP 17 CEP 6 LSI
8q24 CEP 1 CEP 6 LSI 8q24 LSI 7p12 CEP 6 LSI 8q24 CEP 6 CEP 7 LSI
7p12 LSI 3q CEP 6 LSI 7p12 LSI 3q CEP 7 LSI 7p12 CEP 6 CEP 7 LSI 3q
CEP 6 CEP 7 4 probe combinations--2 redundant complementary pairs:
LSI 5p15 LSI 8q24 7p12 LSI 3q LSI 5p15 LSI 8q24 7p12 CEP 6 LSI 5p15
LSI 8q24 LSI 3q CEP 7 LSI 5p15 LSI 8q24 CEP 6 CEP 7 LSI 5p15 LSI 3q
8q24 CEP 17 LSI 5p15 LSI 3q 8q24 CEP 1 LSI 5p15 LSI 3q 8q24 CEP 6
LSI 5p15 LSI 3q 7p12 CEP 6 LSI 5p15 LSI 3q CEP 6 CEP 7 LSI 5p15 LSI
20q 8q24 CEP 17 LSI 5p15 LSI 20q 8q24 CEP 1 LSI 5p15 LSI 20q 8q24
CEP 6 LSI 5p15 LSI 20q 7p12 LSI 3q LSI 5p15 LSI 20q 7p12 CEP 6 LSI
5p15 LSI 20q LSI 3q CEP 7 LSI 5p15 LSI 20q CEP 6 CEP 7 LSI 5p15
7p12 8q24 CEP 17 LSI 5p15 7p12 8q24 CEP 1 LSI 5p15 7p12 8q24 CEP 6
LSI 5p15 7p12 LSI 3q CEP 7 LSI 5p15 7p12 CEP 6 CEP 7 LSI 5p15 CEP
16 LSI 8q24 CEP 17 LSI 5p15 CEP 16 LSI 8q24 CEP 1 LSI 5p15 CEP 16
LSI 8q24 CEP 6 LSI 5p15 CEP 16 LSI 7p12 LSI 3q LSI 5p15 CEP 16 LSI
7p12 CEP 6 LSI 5p15 CEP 16 LSI 3q CEP 7 LSI 5p15 CEP 16 CEP 6 CEP 7
LSI 5p15 CEP 4 LSI 8q24 CEP 17 LSI 5p15 CEP 4 LSI 8q24 CEP 1 LSI
5p15 CEP 4 LSI 8q24 CEP 6 LSI 5p15 CEP 4 LSI 7p12 LSI 3q LSI 5p15
CEP 4 LSI 7p12 CEP 6 LSI 5p15 CEP 4 LSI 3q CEP 7 LSI 5p15 CEP 4 CEP
6 CEP 7 LSI 5p15 CEP 12 LSI 8q24 CEP 17 LSI 5p15 CEP 12 LSI 8q24
CEP 1 LSI 5p15 CEP 12 LSI 8q24 CEP 6 LSI 5p15 CEP 12 LSI 7p12 LSI
3q LSI 5p15 CEP 12 LSI 7p12 CEP 6 LSI 5p15 CEP 12 LSI 3q CEP 7 LSI
5p15 CEP 12 CEP 6 CEP 7 LSI 5p15 CEP 6 LSI 8q24 CEP 17 LSI 5p15 CEP
6 LSI 8q24 CEP 1 LSI 5p15 CEP 6 LSI 7p12 LSI 3q LSI 5p15 CEP 6 LSI
3q CEP 7 LSI 5p15 LSI 17q21 LSI 8q24 CEP 17 LSI 5p15 LSI 17q21 LSI
8q24 CEP 1 LSI 5p15 LSI 17q21 LSI 8q24 CEP 6 LSI 5p15 LSI 17q21 LSI
7p12 LSI 3q LSI 5p15 LSI 17q21 LSI 7p12 CEP 6 LSI 5p15 LSI 17q21
LSI 3q CEP 7 LSI 5p15 LSI 17q21 CEP 6 CEP 7 LSI 8q24 CEP 17 LSI
7p12 LSI 3q LSI 8q24 CEP 17 LSI 7p12 CEP 6 LSI 8q24 CEP 17 LSI 3q
CEP 7 LSI 8q24 CEP 17 CEP 6 CEP 7 LSI 8q24 CEP 1 LSI 7p12 LSI 3q
LSI 8q24 CEP 1 LSI 7p12 CEP 6 LSI 8q24 CEP 1 LSI 3q CEP 7 LSI 8q24
CEP 1 CEP 6 CEP 7 LSI 8q24 CEP 6 LSI 7p12 LSI 3q LSI 8q24 CEP 6 LSI
3q CEP 7 LSI 7p12 LSI 3q CEP 6 CEP 7 LSI 7p12 CEP 6 LSI 3q CEP 7 4
probe combinations--3 pairs with 2 common probes: examples: LSI
5p15 LSI 8q24 LSI 3q CEP 1 (probe pairs in rows 17 + 18 + 27) LSI
5p15 LSI 8q24 LSI 3q CEP 6 (probe pairs in rows 17 + 18 + 28) LSI
5p15 LSI 8q24 CEP 1 CEP 6 (probe pairs in rows 17 + 24 + 27) LSI
7p12 LSI 3q CEP 6 CEP 7 (probe pairs in rows 29 + 30 + 31)
plus 3 probe labels) looking for cells with target gains. The
number of targets for each of the 3 probes was recorded for any
cell showing gain in one or more of the 3 targets.
Example 5
Detection of Lung Cancer in Bronchial Washing Specimens
[0134] The present study used an interphase FISH assay (using a
4-probe multicolor FISH panel) to detect lung cancer in 74
bronchial washing specimens that had previously been characterized
by cytological analysis. Forty eight of the specimens were from
patients with a clinical diagnosis of positive for cancer, and 26
of the specimens were from patients with a clinical diagnosis of
negative for cancer.
[0135] Bronchial washing specimens were selected from the
cytopathology archives of the Institute of Pathology in Basel,
Switzerland. These cytology specimens were pre-stained with PAP
stain and permanently mounted under coverslips. Specimens were
archived for a period of time ranging from a few months to two
years.
[0136] The four probes used for the FISH assay included a
repetitive sequence probe centromeric to chromosome 1 (CEP 1), and
three unique-sequence probes to the loci 5p15, 8q24 (containing the
c-myc gene), and 7p12 (containing the EGFR gene), labeled
respectively with SpectrumAqua.TM., SpectrumGreen.TM.,
SpectrumGold.TM., and SpectrumRed.TM.. The probes were mixed
together and hybridized simultaneously to each bronchial wash
specimen.
[0137] The archived slides were soaked in xylene until the
coverslips fell off (approximately 4-5 days) and then washed in
fresh xylene twice, 5 minutes per wash. The slides were then placed
in 95% ethanol, 85% ethanol, and 70% ethanol, sequentially (5
minutes per solution), followed by soaking the slides in
2.times.SSC buffer for 1 minute. The slides were then incubated in
0.5 mg/ml pepsin solution in 10 mM HCl for 10 minutes at 37.degree.
C., followed by a PBS wash for 5 minutes. The slides were fixed in
a freshly prepared solution of 1% neutral buffered formalin for 5
minutes at 4.degree. C., followed by soaking in PBS for 5 minutes.
The slides were then denatured for 10 minutes in 70%
formamide/2.times.SSC at 73.degree. C., dehydrated in an ethanol
series of 70%, 85%, and 100% ethanol (5 minutes per solution), and
put on a slide warmer at 37-45.degree. C. for 1 minute to dry.
Probes in the hybridization mixture were denatured by placing the
tube containing the mixture in a 73.degree. C. water bath for 5
minutes. The denatured probe hybridization mixtures were applied to
the specimens, covered with coverslips, and sealed with rubber
cement. The slides were incubated at 37.degree. C. overnight, after
which the slides were washed in 2.times.SSC/0.3% NP40 at 73.degree.
C. for 2-5 minutes. The slides were then placed in 2.times.SSC/0.1%
NP40 for several seconds to several minutes. DAPI II was applied to
the target areas and the slides were analyzed under the
fluorescence microscope using single bandpass filter sets.
[0138] The specimen slides were evaluated under a fluorescence
microscope to first assess the technical quality of the FISH
signals and the background staining. If the quality was acceptable,
the slides were then enumerated. The overall sample appearance was
evaluated with a DAPI single bandpass filter set at 40.times.
magnification. The following sample features were important to
note: 1) the presence of thin or thick mucous fibers; 2) the degree
to which the cells were trapped within Mucous fibers; 3) the
presence of nuclear pleomorphism; and 4) the presence of disrupted
cells (no clear nuclear borders, amorphous shape). Cells or groups
of cells were selected for Signal enumeration only if they had
clearly defined nuclear borders and preferably were in the areas
free of mucous fibers.
[0139] Enumeration was carried out according to the following rules
using the DAPI single bandpass filter set and the three
probe-specific single bandpass filter sets (Vysis aqua, green,
gold, and red). All specimen evaluations were performed with the
reviewer blinded to the identity of the specimen.
[0140] (1) Select the appropriate area with cells using the DAPI
single bandpass filter set.
[0141] (2) Change to the gold or green single bandpass filter, set
and observe the field cells with signal copy gain are present,
record the copy number pattern in those cells for all 4 probes,
changing sequentially to the other three probe-specific single
bandpass filter sets (order not important). If the cells look
disomic with the gold or green filter set, change to one of the
other three probe-specific filter sets and observe the field. If
cells with signal copy gains are present, record their signal
pattern for all 4 probes. Do this until the field has been scanned
with all 4 probe-specific filter sets. Only record the pattern for
any one cell once.
[0142] (3) Move to a new area and repeat the evaluation.
[0143] (4) Stop enumeration when at least 25 cells are scored or
the end of the slide was reached.
[0144] Enumeration results of signal copy number for each probe
were analyzed us JMP 3.2 version statistical software.
[0145] The samples used in this study were selected so that
approximately half of the 48 specimens with a clinical diagnosis of
cancer were also diagnosed as positive by cytology, and
approximately half were diagnosed as negative by cytology. The
majority of the cancer positive specimens were from patients with
adenocarcinoma (23 specimens), followed by patients with squamous
cell carcinoma (11 specimens). The rest of the specimens were from
patients with large cell carcinoma (6 specimens), small cell
carcinoma (6 specimens), carcinoid tumor (1 specimen), and
leiomyosarcoma (1 specimen). All 26 specimens clinically negative
for cancer had negative cytology results. No specimens were
selected with a negative clinical diagnosis and a positive cytology
result (the cytology specificity in this study was 100% by
design).
[0146] Table 14 shows the distribution of the cytology results in
the cohort Of patients that was used in this study. The cytology
results were positive for 22 patients, negative for 48 patients and
suspicious for 4 patients. The sensitivity of cytology for the
group of 48 samples positive for cancer by clinical diagnosis was
45.8%. Thirteen specimens were rejected from FISH evaluation due to
the excessive loss of tissue (9 specimens from cancer positive
patients and 4 specimens from cancer negative patients). Excluding
the slides that were not evaluated by FISH, the cytology
sensitivity for the remaining 39 cancer positive patients was 50%.
If cytology suspicious samples were counted as positive, the
cytology sensitivity increased to 53.9%.
TABLE-US-00014 TABLE 14 Correlation Between Cytology Results and
Clinical Diagnosis Clinical Diagnosis Cytology Cancer Negative
Cancer Positive Cytology Negative 26 22 (100%) (45.83%) Cytology
Positive 0 22 (0%) (45.83%) Cytology Suspicious 0 4 (0%)
(8.33%)
[0147] The bronchial washing specimens were hybridized with the
multicolor FISH probe mixture after the coverslips were removed by
soaking in xylene. The overall appearance of each sample was
evaluated. If the specimen appeared to be extremely acellular or
the morphology of the cells was disturbed, or the hybridization
signal was too weak, then the sample was rejected for FISH
enumeration.
[0148] To evaluate the FISH results, it was necessary to develop a
cancer positivity criteria. This involved developing rules to
classify individual cells as being suspicious for malignancy
("abnormal") or not suspicious ("normal"), and setting cutoff
values for the minimum number of abnormal cells required to
classify a specimen as positive for cancer.
[0149] A cell was classified as abnormal if it showed copy number
gains for at least two probes included in the probe mix (this was
termed "Multiple DNA loci gain"). Once this rule was established,
all of the specimen data were evaluated and the number of
"abnormal" cells in each of the specimens was tabulated. To decide
what should be the "cancer positivity criteria" (a quantitative
measure to discern cancer negative from cancer positive cases), the
receiver operator characteristic (ROC) curve approach was applied
to the data analysis. Using this approach, a series of tentative
cutoff points are set and the sensitivity and specificity are
calculated at each point. For data presented here, cutoff values of
1 to 10 cells per specimen were used. For each cutoff value the
sensitivity was determined for the cohort of cancer positive
patients, and the specificity was determined for the cohort of
cancer negative patients. Then the ROC curve was plotted for
sensitivity (y axis) as a function of [1-specificity] (x axis)
(FIG. 1).
[0150] As seen in FIG. 1, there is a section on the curve, where
the sensitivity increases significantly while specificity remains
about the same. The cutoff point is often selected in the section
where the curve turns. The turning point in this assay corresponded
to a cutoff value of finding 5-6 cells that met the criteria of
cancer positivity. Consequently, the rule for classifying a
specimen as positive used in this study was as follows: if a sample
contained 6 or more abnormal cells with "multiple loci gain," it
was classified as "cancer positive." If a sample had less than 6
abnormal cells, it was classified as "cancer negative."
[0151] Table 15 shows the correlation between cytology and FISH
results for the group of "cancer positive" patients. Cytology was
positive in 22 out of 48 "cancer positive" patients, providing a
sensitivity of 45.8%. For another 4 specimens the cytology was
reevaluated by cytopathologists, and the specimens classified as
"suspicious". If "suspicious" results were interpreted as "cancer
positive", then the sensitivity of cytology became 53.8%. Several
samples were rejected from FISH evaluation due to low cellularity
and other reasons, so the number of cases evaluated by FISH was
different from the number Of cases evaluated by cytology.
Recalculating the cytology results for those cases that were also
evaluated by FISH, the sensitivity, of cytology became 46.2% (18/39
cases), if "suspicious" results are counted as positive results,
the sensitivity would be 53.8%. Thus, there was no significant
difference between the sensitivity results if FISH-rejected samples
were included or excluded from the calculations. The FISH results
for the same group of patients showed 32 positive results among the
39 "cancer positive" patients, providing a sensitivity of
82.0%.
TABLE-US-00015 TABLE 15 Cancer Positive Patients: Correlation of
FISH and Cytology Results FISH FISH FISH Negative Positive Rejected
Total Cytology Negative 3 15 4 22 Cytology Positive 3 15 4 22
Cytology Suspicious 1 2 1 4 Total 7 32 9 48
[0152] FISH was able to clarify two of the cytology suspicious
specimens (an additional specimen was rejected for FISH evaluation)
by placing them into the category of "cancer positive" specimens.
The number of abnormal cells in each of those specimens was 8 for a
small cell carcinoma specimen and 10 for a large cell carcinoma
specimen. Even more important are the results obtained for the
group of 18 cytology negative/cancer positive cases. Table 15 shows
that for these cancer patients that were missed by cytology, FISH
was positive in 15/18 cases, thus improving the diagnosis in 83.3%
of cases.
[0153] FISH and cytology results were also analyzed relative to the
type of tumor. The data showed that FISH had its lowest sensitivity
for the specimens diagnosed as squamous cell carcinoma (5/9
specimens, 55.5%). For this type of lung tumor, cytology showed
54.5% sensitivity. Adenocarcinoma, large cell carcinoma, and small
cell carcinoma demonstrated sensitivity by FISH of 86.4% (19/22
cases), 100% (5/5 cases) and 100% (3/3 cases), respectively.
Cytology sensitivity for these tumors was as follows: 60.9% for
adenocarcinoma, 50% for large cell carcinoma; and 100% for small
cell carcinoma.
[0154] The group of "cancer, negative" patients consisted of 26
patients. Cytology results were negative for all of the patients in
this selected group setting the specificity of 100%. Four specimens
were rejected from. FISH evaluation due to low cellularity, thus
only 22 specimens were evaluated. Among those 22 specimens, FISH
was clearly negative in 18. patients providing a specificity of
81.8% (Table 16). Four specimens had positive FISH results. These
four specimens contained as Many as 19, 15, 11 and 8 "abnormal"
cells per 25. evaluated suspicious cells. It is also important to
note that in two of the specimens, the magnitude of copy number
gain was as high as 7-8 copies per cell in one case and 11-12
copies per cell in another case. One of the specimens was derived
from a patient diagnosed with advanced colorectal cancer
approximately one year before the specimen was prepared(the patient
died by the time of the present study). Another patient had a
previous history of heavy smoking and had the occupational hazard
of being a miner. Thus, it is possible that these FISH positive,
but "cytology negative" specimens were derived from patients at
risk of developing lung cancer.
TABLE-US-00016 TABLE 16 Cancer Negative Patients: Correlation of
FISH and Cytology Results FISH FISH FISH Negative Positive Rejected
Total Cytology Negative 18 4 4 26 Cytology 0 0 0 0
Positive/Suspicious Total 18 4 4 26
[0155] Table 17 shows comparative data on sensitivity and
specificity for cytology and FISH for the total population of 74
patients.
TABLE-US-00017 TABLE 17 Total population of patients: Correlation
of FISH and cytology results FISH FISH FISH Negative Positive
Rejected Total Cytology Negative 21 19 8 48 Cytology Positive 3 15
4 22 Cytology Suspicious 1 2 1 4 Total 25 36 13 74
Example 6
Detection of Lung Cancer in Bronchoscopic Specimens
[0156] The present study used an interphase FISH assay (using a
4-probe multicolor FISH panel) to detect lung cancer in 191
bronchial specimens that had previously been characterized by
surgical pathology analysis. The surgical pathology results of the
specimens used in this study are summarized in Table 18. 104 of the
specimens (55%) were from patients with a clinical diagnosis of
positive for lung cancer. 84 of the specimens (44%) were from
patients with a clinical diagnosis of negative for lung cancer.
TABLE-US-00018 TABLE 18 Surgical Pathology Results of Specimens
Used in Study Number of Specimens Diagnosis (+ or - for cancer)
Percentage 104 + 55 84 - 44 3 Equivocal diagnosis 1
[0157] One of the following three sets of four probes was used for
each FISH assay: (1) a repetitive sequence probe centromeric to
chromosome 1 (CEP 1), and three unique-sequence probes to the loci
5p15, 8q24; and 7p12; (2) repetitive sequence probes centromeric to
chromosome 16 (CEP 16) and chromosome 17 (CEP 17) and two
unique-sequence probes to the loci 3q26 and 20q13; or (3) a
repetitive sequence probe centromeric to chromosome 6 (CEP 6) and
three unique-sequence probes to the loci 5p15, 8q24, and 7p12. The
probes were mixed together and hybridized simultaneously to each
bronchial specimen.
[0158] The sensitivity detected by each of FISH and cytology
analysis for the 104 cancer positive specimens is depicted in Table
19 (38 bronchial brushing samples) and Table 20 (66 bronchial
secretion samples). As shown in Table 19, FISH demonstrated a
significantly enhanced sensitivity (72%) as compared to cytology
(51%) for the bronchial brushing samples. No significant difference
between FISH and cytology was detected for . the bronchial
secretion samples (Table 20).
TABLE-US-00019 TABLE 19 Sensitivity of FISH and Cytology for
Bronchial Brushing Samples Analysis Diagnosis Number Percentage
FISH + 26/36 72 FISH - 8/36 22 FISH Equivocal diagnosis 2/36 6
Cytology + 19/37 51 Cytology - 17/37 46 Cytology Equivocal
diagnosis 1/37 3
TABLE-US-00020 TABLE 20 Sensitivity of FISH and Cytology for
Bronchial Secretion Samples Analysis Diagnosis Number Percentage
FISH + 31/65 48 FISH - 28/65 43 FISH Equivocal diagnosis 6/65 9
Cytology + 34/66 52 Cytology - 28/66 42 Cytology Equivocal
diagnosis 4/66 6
[0159] The specificity detected by FISH and cytology analysis for
the 84 specimens negative for lung cancer (as determined by
surgical pathological analysis) is depicted in Table 21 (49
bronchial brushing samples) and Table 22 (35 bronchial secretion
samples). It is expected that among those samples described in
Tables 21 and 22 that were negative by surgical pathological
analysis, but positive by FISH analysis, there may be some
specimens that contain cancerous and/or pre-cancerous cells that
were not identified by the surgical pathology methods. In such
cases, FISH can allow for an early detection of lung cancer.
TABLE-US-00021 TABLE 21 Specificity of FISH and Cytology for
Bronchial Brushing Samples Analysis Diagnosis Number Percentage
FISH + 10/49 20 FISH - 38/49 78 FISH Equivocal diagnosis 1/49 2
Cytology + 2/49 4 Cytology - 47/49 96 Cytology Equivocal diagnosis
0/49 0
TABLE-US-00022 TABLE 22 Specificity of FISH and Cytology for
Bronchial Secretion Samples Analysis Diagnosis Number Percentage
FISH + 3/35 8 FISH - 31/35 88 FISH Equivocal diagnosis 1/35 3
Cytology + 4/35 11 Cytology - 29/35 83 Cytology Equivocal diagnosis
2/35 6
Other Embodiments
[0160] It is to be understood that, while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention. Other aspects, advantages, and
modifications of the invention are within the scope of the claims
set forth below.
* * * * *