U.S. patent application number 14/884254 was filed with the patent office on 2016-04-21 for perinucleolar compartment as a cancer marker.
The applicant listed for this patent is Cold Spring Harbor Laboratory, Northwestern University. Invention is credited to Sui Huang, Rajesh V. Kamath, David L. Spector, Ann D. Thor, Chen Wang.
Application Number | 20160109456 14/884254 |
Document ID | / |
Family ID | 42038057 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160109456 |
Kind Code |
A1 |
Huang; Sui ; et al. |
April 21, 2016 |
PERINUCLEOLAR COMPARTMENT AS A CANCER MARKER
Abstract
The present invention relates to compositions and methods for
cancer diagnostics, prognostics and predictions, including but not
limited to, cancer markers. In particular, the present invention
provides perinucleolar compartments and their resident molecules as
cancer markers.
Inventors: |
Huang; Sui; (Chicago,
IL) ; Kamath; Rajesh V.; (Shrewsbury, MA) ;
Spector; David L.; (Cold Spring Harbor, NY) ; Thor;
Ann D.; (Greenwood Village, CO) ; Wang; Chen;
(Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northwestern University
Cold Spring Harbor Laboratory |
Evanston
Cold Spring Harbor |
IL
NY |
US
US |
|
|
Family ID: |
42038057 |
Appl. No.: |
14/884254 |
Filed: |
October 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13584921 |
Aug 14, 2012 |
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14884254 |
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12559634 |
Sep 15, 2009 |
8241862 |
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13584921 |
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11651733 |
Jan 10, 2007 |
7588904 |
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12559634 |
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10403422 |
Mar 31, 2003 |
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11651733 |
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11794346 |
Apr 8, 2008 |
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PCT/US2005/046890 |
Dec 22, 2005 |
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12559634 |
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60368822 |
Mar 29, 2002 |
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60639502 |
Dec 23, 2004 |
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Current U.S.
Class: |
424/9.2 ; 435/29;
435/6.11; 435/6.12; 435/7.1; 435/7.92 |
Current CPC
Class: |
G01N 33/57419 20130101;
G01N 33/57496 20130101; G01N 2800/56 20130101; A61K 49/0004
20130101; G01N 2500/10 20130101; C12Q 1/6886 20130101; C12Q
2600/118 20130101; C12Q 2600/112 20130101; C12Q 2600/106 20130101;
C12Q 2600/136 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 49/00 20060101 A61K049/00; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with government support under grant
numbers R01 CA77560, R21 CA84369, and CA097761 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2005 |
US |
PCT/US2005/046890 |
Claims
1. A method for predicting or prognosticating the recurrence or
metastases of cancer comprising: a) screening a sample from a
subject for the presence of perinucleolar compartments; and b)
determining if said subject is susceptible to recurrence or
metastases of cancer based on said presence of perinucleolar
compartments.
2. The method of claim 1, wherein said subject comprises a human
subject.
3. The method of claim 1, wherein said screening comprises
identifying the prevalence of said perinucleolar compartments.
4. The method of claim 1, wherein said screening comprises
detecting polypyrimidine tract binding (PTB) protein.
5. The method of claim 1, wherein said screening step comprises
immunostaining of said perinucleolar compartments.
6. The method of claim 1, further comprising the step of selecting
a therapy for said subject based on the presence of perinucleolar
compartments.
7. The method of claim 6, wherein said therapy comprises
chemotherapy.
8. The method of claim 1, wherein said subject has been
administered an anti-cancer therapy.
9. A method for predicting or prognosticating the recurrence or
metastases of cancer comprising: a) screening a sample from a
subject for the expression of PTB; and b) determining if said
subject is susceptible to recurrence or metastases of cancer based
on said amount of PTB expression.
10. The method of claim 9, wherein said subject comprises a human
subject.
11. The method of claim 9, further comprising the step of selecting
a therapy for said subject based on said amount of PTB
expression.
12. The method of claim 11, wherein said therapy comprises
chemotherapy.
13. A method for screening compounds, comprising: a) providing
cancer cells and a candidate drug; b) exposing said cancer cells to
said candidate drug; and c) detecting the presence of perinucleolar
compartments in said cancer cell.
14. The method of claim 13, wherein said candidate drug comprises a
candidate chemotherapeutic agent.
15. The method of claim 13, wherein said cancer cells are present
in a mammal.
16. The method of claim 15, wherein said mammal is a human.
17. The method of claim 13, further comprising the step of
selecting candidate drugs that affect the presence of said
perinucleolar compartments in said cancer cell.
18.-20. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/584,921, filed Aug. 14, 2012, which is a continuation of
U.S. application Ser. No. 12/559,634, filed Sep. 15, 2009, now U.S.
Pat. No. 8,241,862, which is a Continuation-In-Part of U.S. Pat.
No. 7,588,904, issued Sep. 15, 2009, which is a Divisional of U.S.
application Ser. No. 10/403,422, filed Mar. 31, 2003, now
abandoned, which claims priority to U.S. Provisional Application
Ser. No. 60/368,822, filed Mar. 29, 2002, now expired; all of which
are herein incorporated by reference in their entireties.
[0002] This application is a continuation of U.S. application Ser.
No. 12/559,634, filed Sep. 15, 2009, now U.S. Pat. No. 8,241,862,
which is also a Continuation-In-Part U.S. application Ser. No.
11/794,346, filed Apr. 8, 2008, now abandoned, which is a U.S.
national entry of International Patent Application Number
PCT/US2005/046890, now expired, filed Dec. 22, 2005, which claims
priority to U.S. Provisional Application Ser. No. 60/639,502, filed
Dec. 23, 2004, now expired; all of which are herein incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0004] The present invention relates to compositions and methods
for cancer diagnostics, prognostics and predictions, including but
not limited to, cancer markers. In particular, the present
invention provides perinucleolar compartments and their resident
molecules as cancer markers.
BACKGROUND OF THE INVENTION
[0005] The development of cancer is a complex process involving the
interplay of many genetic and epigenetic events. Cells undergo
extensive biochemical and structural alterations throughout the
course of cancer development. Tremendous efforts have been invested
to increase the survival rate of cancer patients through the
development of early detection programs and novel therapeutic
strategies. Traditional histological standards for cancer diagnosis
are well established and prognostic criteria routinely evaluated
include size, invasiveness, involvement of adjacent structures or
lymph nodes, metastases, and histological grade. More recently,
advances in cellular and molecular techniques have fostered a
better understanding of genetic and epigenetic changes during
cancer development. An increasing number of molecular tumor markers
have been identified to provide additional information for the
diagnosis and prognosis of the disease. However, only a limited
number of them have been reproducible and clinically relevant for
cancer patient management. For most types of cancer, specific and
sensitive markers that can predict the biological behavior of
cancer cells (recurrence and metastases) are still lacking What is
needed are improved methods to specifically detect, characterize,
and monitor the specific types and the progression of cancer.
Furthermore, useful tumor markers that can accurately predict the
outcome of patients suffering from cancers of various types and at
various stages are desired.
SUMMARY OF THE INVENTION
[0006] The present invention relates to compositions and methods
for cancer diagnostics, prognostics and predictions, including but
not limited to, cancer markers. In particular, the present
invention provides perinucleolar compartments and their resident
molecules as cancer markers.
[0007] For example, in some embodiments, the present invention
provides a method for predicting or prognosticating the recurrence
or metastases of cancer comprising screening a sample from a
subject (e.g., a human or other mammalian subject) for the presence
of perinucleolar compartments; and determining if the subject is
susceptible to recurrence or metastases of cancer based on the
presence of perinucleolar compartments. In some embodiments, the
screening step comprises identifying the prevalence of the
perinucleolar compartments. In some preferred embodiments, the
screening step comprises detecting polypyrimidine tract binding
(PTB) protein. While the present invention is not limited by the
method in which the perinucleolar compartments are detected, in
some preferred embodiments, the screening step comprises
immunostaining of said perinucleolar compartments.
[0008] In some embodiments, the method further comprises the step
of selecting a therapy (e.g., chemotherapy) for the subject based
on the presence of perinucleolar compartments. In some such
embodiments, the prevalence of the perinucleolar compartments
identifies the subject as being in a particular subset of subjects
that is more likely to benefit from a particular therapeutic
regimen. In some embodiments, the subject has previously been
administered an anti-cancer therapy (e.g., chemotherapy).
[0009] The present invention also provides a method for predicting
or prognosticating the recurrence or metastases of cancer
comprising: screening a sample from a subject for the expression of
PTB (e.g., protein or nucleic acid); and determining if the subject
is susceptible to recurrence or metastases of cancer based on the
amount of PTB expressed.
[0010] The present invention further provides methods for screening
compounds (e.g., drugs), comprising: providing cancer cells (e.g.,
in vitro, ex vivo, in vivo) and a candidate drug; exposing the
cancer cells to the candidate drug; and detecting the presence of
perinucleolar compartments in the cancer cell. In some embodiments,
a number of drugs are screened (e.g., using large compound
libraries). In some embodiments, cells from multiple tissues are
analyzed. In some preferred embodiments, the candidate drug
comprises a candidate chemotherapeutic agent. In some embodiments,
the method further comprises the step of selecting candidate drugs
that affect the presence of perinucleolar compartments in the
cancer cell.
[0011] The present invention also provides a kit (e.g., an in vitro
diagnostic kit) comprising: a label that labels perinucleolar
compartments (e.g., a molecule that finds use in direct or indirect
detection of perinucleolar compartments); and a written component
comprising instruction for performing a screen of a sample with the
label such that a degree of malignancy of cells in the sample is
determined (e.g., written instruction as mandated by the FDA
regulations on in vitro diagnostic products). In some preferred
embodiments, the label comprises an antibody (e.g., an antibody
directed against PTB).
[0012] In some embodiments, the present invention provides methods
for predicting or prognosticating the recurrence or metastases of
cancer comprising: (a) screening a sample from a subject for the
presence of perinucleolar compartments; and (b) determining if the
subject is susceptible to recurrence or metastases of cancer based
on the presence of perinucleolar compartments, wherein the cancer
is selected from the group consisting of ovarian cancer, prostate
cancer, and colon cancer.
[0013] In certain embodiments, the present invention provides
methods for predicting metastases of cancer (e.g., colon cancer,
ovarian cancer, breast cancer, or prostate cancer) comprising: a)
screening a cancer sample from a subject for the prevalence of
perinucleolar compartments, wherein the sample comprises cancer
cells; and b) determining if the subject is susceptible to
metastases of the cancer (e.g., colon cancer, ovarian cancer,
breast cancer, or prostate cancer) based on the prevalence of the
perinucleolar compartments, wherein the subject is susceptible to
metastases if the prevalence of the perinucleolar compartments is
over about 80% of the cancer cells.
[0014] In certain embodiments, the present invention provides
methods of identifying the stage of cancer (e.g., colon cancer,
ovarian cancer, breast cancer, or prostate cancer) progression
comprising: a) providing cancer tissue (e.g., colon, ovarian,
breast, or prostate cancer tissue) from a subject, wherein the
cancer tissue comprises cells; b) identifying the prevalence of
perinucleolar compartments in the cancer tissue; and c) correlating
the prevalence of perinucleolar compartments in the cancer tissue
with the stage of cancer progression in the subject (e.g., Stage I,
II, III, or IV).
[0015] In particular embodiments, the prevalence of perinucleolar
compartments is at or above 20% of the cells, and the stage of
colon cancer is at least Stage I. In other embodiments, the
prevalence of perinucleolar compartments is at or above 65% of the
cells, and the stage of colon cancer is at least Stage II. In
further embodiments, the prevalence of perinucleolar compartments
is at or above 75% of the cells, and the stage of colon cancer is
at least Stage III. In other embodiments, the prevalence of
perinucleolar compartments is at or above 85% of the cells, and the
stage of colon cancer is Stage IV.
[0016] In certain embodiments, the present invention provides
methods for predicting or prognosticating the recurrence or
metastases of cancer comprising: (a) screening a sample from a
subject for the expression of PTB; and (b) determining if the
subject is susceptible to recurrence or metastases of cancer based
on the amount of PTB expression. wherein the cancer is selected
from the group consisting of ovarian cancer, prostate cancer, and
colon cancer.
[0017] In certain embodiments, the subject comprises a human
subject. In other embodiments, the screening comprises identifying
the prevalence of the perinucleolar compartments. In further
embodiments, the screening comprises detecting polypyrimidine tract
binding (PTB) protein. In particular embodiments, the screening
step comprises immunostaining of the perinucleolar compartments. In
some embodiments, the method further comprises the step of
selecting a therapy for the subject based on the presence of
perinucleolar compartments. In other embodiments, the therapy
comprises chemotherapy. In additional embodiments, the subject has
been administered an anti-cancer therapy.
[0018] In particular embodiments, the present invention provides
methods for screening compounds, comprising: (a) providing cancer
cells and a candidate drug; (b) exposing the cancer cells to the
candidate drug; and (c) detecting the presence of perinucleolar
compartments in the cancer cell, wherein the cancer cells are
derived from the group consisting of ovarian cancer, prostate
cancer, and colon cancer.
[0019] In some embodiments, the candidate drug comprises a
candidate chemotherapeutic agent. In other embodiments, the cancer
cells are present in a mammal. In further embodiments, the mammal
is a human. In additional embodiments, the methods further comprise
the step of selecting candidate drugs that affect the presence of
the perinucleolar compartments in the cancer cell.
[0020] In additional embodiments, the present invention provides
kits comprising: (a) a label that labels perinucleolar
compartments; and (b) a written component comprising instruction
for performing a screen of a sample with the label such that a
degree of malignancy of cells in the sample is determined. In some
embodiments, the label comprises an antibody. In other embodiments,
the antibody binds to PTB.
DESCRIPTION OF THE FIGURES
[0021] FIG. 1 provides a histogram showing that the percentage of
cells that contain one or more PNCs (PNC prevalence) correlates
with human cancer. The histogram indicates the statistical
evaluation of PNC prevalence among human cancer cells and diploid
cells.
[0022] FIG. 2 shows quantitation of immunohistochemical staining of
breast tissue samples at different stages of breast cancer
progression.
[0023] FIG. 3 shows Kaplan-Meier survival curves for breast cancer
patients.
[0024] FIG. 4 shows immunohistochemical staining of PTB for the
presence of PNC in normal and malignant ovarian tissue samples.
[0025] FIG. 5 shows quantization of immunohistochemical staining of
PTB for the presence of PNC in malignant ovarian tissue
samples.
[0026] FIG. 6 shows Kaplan-Meier survival curves for ovarian cancer
patients using a PNC value of 28.35 as the cut off value.
[0027] FIG. 7 shows Kaplan-Meier survival curves for ovarian cancer
patients using a PNC value of 37.74 as the cut off value.
[0028] FIG. 8 shows Kaplan-Meier survival curves for ovarian cancer
patients using a PNC value of 49.87 as the cut off value.
[0029] FIG. 9 shows PTB immunohistochemical staining of normal
(left) and malignant (right) prostate tissues.
[0030] FIG. 10 shows a comparisons of PNC prevalence for various
diagnostic subgroups of colorectal cancer cells as described in
Example 12.
[0031] FIG. 11 shows a Kaplan-Meier disease-specific survival
analysis for colorectal carcinoma using PNC prevalence data as
described in Example 12. Median patient follow-up time was 120
months.
DEFINITIONS
[0032] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0033] The term "epitope" as used herein refers to that portion of
an antigen that makes contact with a particular antibody.
[0034] When a protein or fragment of a protein is used to immunize
a host animal, numerous regions of the protein may induce the
production of antibodies which bind specifically to a given region
or three-dimensional structure on the protein; these regions or
structures are referred to as "antigenic determinants." An
antigenic determinant may compete with the intact antigen (i.e.,
the "immunogen" used to elicit the immune response) for binding to
an antibody.
[0035] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of an antibody and a protein
or peptide means that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A," the presence of a protein containing epitope A (or
free, unlabelled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0036] As used herein, the terms "non-specific binding" and
"background binding" when used in reference to the interaction of
an antibody and a protein or peptide refer to an interaction that
is not dependent on the presence of a particular structure (i.e.,
the antibody is binding to proteins in general rather that a
particular structure such as an epitope).
[0037] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0038] As used herein, the term "subject suspected of having
cancer" refers to a subject that presents one or more symptoms
indicative of a cancer (e.g., a noticeable lump or mass) or is
being screened for a cancer (e.g., during a routine physical). A
subject suspected of having cancer may also have one or more risk
factors. A subject suspected of having cancer has generally not
been tested for cancer. However, a "subject suspected of having
cancer" encompasses an individual who has received an initial
diagnosis but for whom the stage of cancer is not known. The term
further includes people who once had cancer (e.g., an individual in
remission).
[0039] As used herein, the term "subject at risk for cancer" refers
to a subject with one or more risk factors for developing a
specific cancer. Risk factors include, but are not limited to,
gender, age, genetic predisposition, environmental exposure,
previous incidents of cancer, preexisting non-cancer diseases, and
lifestyle.
[0040] As used herein, the term "characterizing cancer in a
subject" refers to the identification of one or more properties of
a cancer sample in a subject, including but not limited to, the
presence of benign, pre-cancerous or cancerous tissue and the stage
of the cancer. Cancers may be characterized by the identification
of the expression of or presence of one or more cancer markers,
including but not limited to, the cancer markers disclosed
herein.
[0041] As used herein, the term "cancer marker genes" refers to a
gene whose expression level, alone or in combination with other
genes, is correlated with cancer or prognosis of cancer. The
correlation may relate to either an increased or decreased
expression of the gene. For example, the expression of the gene may
be indicative of cancer, or lack of expression of the gene may be
correlated with poor prognosis in a cancer patient.
[0042] As used herein, the term "detecting a decreased or increased
expression relative to non-cancerous control" refers to measuring
the level of expression of a gene (e.g., the level of mRNA or
protein) relative to the level in a non-cancerous control
sample.
[0043] As used herein, the term "instructions for using said kit
for characterizing cancer in a subject" includes instructions
comprising the statement of intended use required by the U.S. Food
and Drug Administration (FDA) in labeling in vitro diagnostic
products. The FDA classifies in vitro diagnostics as medical
devices and required that they be approved through the 510(k)
procedure. Information required in an application under 510(k)
includes: 1) The in vitro diagnostic product name, including the
trade or proprietary name, the common or usual name, and the
classification name of the device; 2) The intended use of the
product; 3) The establishment registration number, if applicable,
of the owner or operator submitting the 510(k) submission; the
class in which the in vitro diagnostic product was placed under
section 513 of the FD&C Act, if known, its appropriate panel,
or, if the owner or operator determines that the device has not
been classified under such section, a statement of that
determination and the basis for the determination that the in vitro
diagnostic product is not so classified; 4) Proposed labels,
labeling and advertisements sufficient to describe the in vitro
diagnostic product, its intended use, and directions for use. Where
applicable, photographs or engineering drawings should be supplied;
5) A statement indicating that the device is similar to and/or
different from other in vitro diagnostic products of comparable
type in commercial distribution in the U.S., accompanied by data to
support the statement; 6) A 510(k) summary of the safety and
effectiveness data upon which the substantial equivalence
determination is based; or a statement that the 510(k) safety and
effectiveness information supporting the FDA finding of substantial
equivalence will be made available to any person within 30 days of
a written request; 7) A statement that the submitter believes, to
the best of their knowledge, that all data and information
submitted in the premarket notification are truthful and accurate
and that no material fact has been omitted; 8) Any additional
information regarding the in vitro diagnostic product requested
that is necessary for the FDA to make a substantial equivalency
determination. Additional information is available at the Internet
web page of the U.S. FDA.
[0044] As used herein, the term "stage of cancer" refers to a
qualitative or quantitative assessment of the level of advancement
of a cancer. Criteria used to determine the stage of a cancer
include, but are not limited to, the size of the tumor, whether the
tumor has spread to other parts of the body and where the cancer
has spread (e.g., within the same organ or region of the body or to
another organ).
[0045] As used herein, the term "providing a prognosis" refers to
providing information regarding the impact of the presence of
cancer (e.g., as determined by the diagnostic methods of the
present invention) on a subject's future health (e.g., expected
morbidity or mortality, the likelihood of getting cancer, and the
risk of metastasis).
[0046] As used herein, the term "post surgical tumor tissue" refers
to cancerous tissue that has been removed from a subject (e.g.,
during surgery).
[0047] As used herein, the term "subject diagnosed with a cancer"
refers to a subject who has been tested and found to have cancerous
cells. The cancer may be diagnosed using any suitable method,
including but not limited to, biopsy, x-ray, blood test, and the
diagnostic methods of the present invention. As used herein, the
term "initial diagnosis" refers to results of initial cancer
diagnosis (e.g. the presence or absence of cancerous cells).
[0048] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, aves,
etc.
[0049] As used herein, the term "purified" or "to purify" refers to
the removal of components (e.g., contaminants) from a sample. For
example, antibodies are purified by removal of contaminating
non-immunoglobulin proteins; they are also purified by the removal
of immunoglobulin that does not bind to the target molecule. The
removal of non-immunoglobulin proteins and/or the removal of
immunoglobulins that do not bind to the target molecule results in
an increase in the percent of target-reactive immunoglobulins in
the sample. In another example, recombinant polypeptides are
expressed in bacterial host cells and the polypeptides are purified
by the removal of host cell proteins; the percent of recombinant
polypeptides is thereby increased in the sample.
[0050] As used herein the term "portion" when in reference to a
protein (as in "a portion of a given protein") refers to fragments
of that protein. The fragments may range in size from four amino
acid residues to the entire amino acid sequence minus one amino
acid.
[0051] The terms "test compound" and "candidate compound" refer to
any chemical entity, pharmaceutical, drug, and the like that is a
candidate for use to treat or prevent a disease, illness, sickness,
or disorder of bodily function (e.g., cancer). Test compounds
comprise both known and potential therapeutic compounds. A test
compound can be determined to be therapeutic by screening using the
screening methods of the present invention.
[0052] As used herein, the term "sample" is used in its broadest
sense. In one sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from animals (including
humans) and encompass fluids, solids, tissues, and gases.
Biological samples include blood products, such as plasma, serum
and the like. Environmental samples include environmental material
such as surface matter, soil, water, crystals and industrial
samples. Such examples are not however to be construed as limiting
the sample types applicable to the present invention.
GENERAL DESCRIPTION OF THE INVENTION
[0053] The present invention relates to compositions and methods
for cancer diagnostics, including but not limited to, cancer
markers. In particular, the present invention provides
perinucleolar compartments as cancers markers.
[0054] The perinucleolar compartment (PNC) is an ribonucleoprotein
(RNP) enriched subnuclear structure whose prevalence, as shown by
experiments conducted during the development of the present
invention, correlates with the degree of malignancy. In some
embodiments of the present invention, the PNC is detected by an
antibody (e.g., an monoclonal antibody) that specifically
recognizes an RNA binding protein, polypyrimidine tract binding
protein (PTB), highly enriched in the PNC. Any molecule associated
with the PNC may be used and targeted to detect the PNC (e.g.,
antibodies directed against RNA binding proteins present in the PNC
including, but not limited to, PTB, Raver1, nucleolin, Rpp40,
CUG-BP, KSRP, and ROD1). Preferred RNA binding proteins for
detection are those that stain strongly without strong labeling of
other cellular components. Nucleic acid molecules (e.g., small RNAs
transcribed in the PNC such as RnaseP, MRP RNAs, hY RNA, and SRP
RNA) may also be used to identify the PNC. It is contemplated that
PNC prevalence provides useful information regarding the degrees of
malignant progression and chances of recurrences. As described
below, the PNC is sensitive to a subset of anti-tumor drugs. Thus,
the present invention also provides methods for monitoring
therapeutic effects of particular chemotherapy drugs. Moreover, PTB
changes its expression level and its nuclear distribution during
malignant transformation. These alterations are indicative of the
degree of cancer progression and also find use as markers for
diagnosis and prognosis.
[0055] The perinucleolar compartment (PNC) is a dynamic,
irregularly shaped, and electron dense nuclear structure that is
physically associated with the nucleolus. The presence of the PNC
is found predominantly in transformed cells in culture (Huang et
al., J. Cell Biol., 137:965 [1997]) and in breast cancer tissue
samples examined. Studies have shown that the PNC is involved in
transcription and RNA metabolism (Huang et al., J. Cell Biol.,
143:35 [1998]).
[0056] During the development of the present invention, a
substantial amount of tissue samples from breast cancer, colon
cancer, ovarian cancer and prostate cancer at various stages were
examined. It was found that PNC prevalence is statistically
different among samples derived from cancers at various stages of
malignancy. It is particularly interesting that PNC prevalence is
significantly different between node negative breast cancer
patients who later develop distal metastasis and node negative
breast cancer patients who later do not develop distal metastasis.
PNC prevalence is also significantly higher in patients with lower
survivability rates. It was also found that PNCs disappear when
cultured cells were treated with some of the routinely used
chemotherapeutic drugs, particularly the ones involved in
regulating transcription and RNA metabolism. Furthermore,
quantitative Western blot analyses and immunohistochemical
detection show that PTB, the heterogeneous nuclear RNP (hnRNP)
protein marker for the PNC, is significantly elevated in malignant
cells.
[0057] The present invention is not limited to the analysis of
breast cancer. Experiments conducted during the course of
development of the present invention further demonstrated that PNC
prevalence is associated with colon cancer, ovarian cancer and
prostate cancer. Accordingly, in some embodiments, PNC prevalence
is used to provide diagnostic, prognostic, and predictive
information for colon cancers. The present invention contemplates
the use of PNC prevalence for characterization of any type of
cancer. The correlation of PNC prevalence with cancer, progression
of cancer and survivability in tissues including breast, ovaries,
prostate and colon demonstrates that PNC prevalence may serve as a
marker for cancer in other tissues. In further support of the
general applicability of PNC prevalence as a marker and
prognosticator for cancer from all tissues, high rates of PNC
prevalence have been found in all tested cell lines derived from
malignant tissues including cancers of breast, ovary, prostate,
colon, skin, lung, and bone, whereas all nontransformed cells
tested have significantly lower rates of PNC prevalence. See, e.g.,
Example 1. PNC prevalence may, therefore, be used as a marker and
prognosticator for a variety of cancers including, but not limited
to, the following: carcinoma including that of the bladder
(including accelerated and metastatic bladder cancer), breast,
colon (including colorectal cancer), kidney, liver, lung (including
small and non-small cell lung cancer and lung adenocarcinoma),
ovary, prostate, testes, genitourinary tract, lymphatic system,
rectum, larynx, pancreas (including exocrine pancreatic carcinoma),
esophagus, stomach, gall bladder, cervix, thyroid, and skin
(including squamous cell carcinoma); hematopoietic tumors of
lymphoid lineage including leukemia, acute lymphocytic leukemia,
acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma,
Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma,
histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors
of myeloid lineage including acute and chronic myelogenous
leukemias, myelodysplastic syndrome, myeloid leukemia, and
promyelocyte leukemia; tumors of the central and peripheral nervous
system including astrocytoma, neuroblastoma, glioma, and
schwannomas; tumors of mesenchymal origin including fibrosarcoma,
rhabdomyoscarcoma, and osteosarcoma; and other tumors including
melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma,
thyroid follicular cancer, teratocarcinoma, and cancers of
gastrointestinal tract.
[0058] Thus, in some embodiments, the present invention provides
compositions, kits, systems, and methods for detection of PNC
and/or factor(s) expressed in PNCs as a means for detecting the
presence of, or status of, one or more cancers. The present
invention also provides screening methods for analyzing the
effectiveness of compounds (e.g., drugs) at stimulating or
inhibiting cell division, including an analysis of the effect of
compounds on the management, treatment, or prevention of cancers.
Since most patients treated with currently available
chemotherapeutic agents suffer tremendously undesirable side
effects, there is an urgent need to search for inhibitors of cancer
expansion with minimal cytotoxicity. Accordingly, in some
embodiments, the present invention provides methods of using the
PNC as a marker to search for chemicals that eliminate the
structure without killing the treated cells. In other embodiments,
the present invention provides methods to search for chemicals that
target cells with elevated PNCs (See e.g., Example 9).
[0059] PNC prevalence is indicative of the nature of the malignant
cells and is used to predict the chance of recurrence in patients
who are at the early stages of cancer development, thus providing
guidelines for future therapeutic strategy. The finding of the PNC
in benign lesions forecasts the possibility of these tumors
becoming malignant. Experiments showed that the level of PTB
correlates with the degree of malignancy and is used to evaluate
the progression the disease. PTB can be detected using non-invasive
protocols, when cancers originate in organs such as bladder,
kidney, or uterus, by collecting bodily fluids.
[0060] PTB is a 57-kDa hnRNP protein that specifically binds
pyrimidine rich RNA sequences (Ghetti et al., Nucl. Acids Res.
20:3671 [1992]). PTB has been shown to be involved in multiple
cellular functions including pre-mRNA splicing (Patton et al.,
Genes & Dev. 7:393 [1993]; Gonzani et al., EMBO J. 13:3356
[1994]; Singh et al., Science 268:1173 [1995]; and Ashiya and
Grabowski, RNA 3:996 [1997]), splice site selection in alternative
pre-mRNA splicing (Lin and Patton, RNA 1:234 [1995]; Perez et al.,
RNA 3:764 [1997]; and Grossman et al., RNA 4:613 [1998]), RNA
polyadenylation (Lou et al., Genes Dev. 10:208 [1996]; Moreira et
al., Genes & Dev. 12:2522 [1998]), and translational regulation
of certain viral RNA transcripts (Hellen et al., J. Virol. 68:941
[1994]; Kaminski et al., RNA 1:924[1995]; Witherell et al.,
Virology 214:660 [1995]; and Kaminski and Jackson, RNA 4:626
[1998]). PTB apparently participates in these functions through the
binding of pyrimidine rich RNA sequences. Thus, PTB may serve as a
bridge between the pyrimidine tract containing RNAs and a variety
of cellular factors that fulfill different functions.
[0061] The advantages of detection of the PNC over existing tumor
markers include, but are not limited to: 1) PNCs are easily
identifiable, distinct structures; 2) PNC prevalence provides
prognostic value for cancers that are detected at early stages; and
3) at least two distinct techniques can be used to detect and
quantify resident molecules of the PNC.
[0062] The advantages of the present invention over existing
methods are illustrated in the analysis of breast cancer. Breast
cancer is a highly prevalent and morbid disease. Each year in the
United States, about 110 new cases per 100,000 women are diagnosed,
and 45,000 breast cancer patients die from the disease. Breast
cancer is traditionally subclassified using the American Joint
Committee on cancer staging guidelines (Lester and Cotran, Robbins
Pathologic basis of disease, Cotran et al., eds., W.B. Saunders
Company, 1093-1120, 1999). Stage I tumors are small and localized
whereas stage IV patients have distant metastases. In general,
survival rates decrease with disease progression. For lymph node
positive patients, prediction of outcome is based predominantly on
tumor size and histological grade. Approximately 10-20% of
node-negative patients develop recurrence and distant metastases.
Specific markers that can accurately forecast recurrence or
metastases are not currently available. In addition to the classic
histological criteria, a growing number of molecular genetic and
immunocytochemical markers are being used or on trial to provide
additional information for breast cancer diagnosis. For instance,
80-90% of breast cancer patients with hereditary link to family
history have mutations in one of the two breast cancer genes BRCA1
and BRCA2 (See e.g., Duncan et al., J. Clin. Path. Clin. Mol. Path.
Ed. 51:237 [1998]). The detection of mutations in BRCA1 and BRCA2
genes is now being used to monitor high-risk woman with genetic
predisposition and prepare them for early diagnosis and effective
treatment. In addition, estrogen and progesterone receptors have
been shown to be associated with 50-85% of the breast cancers that
are most commonly found in post-menopausal patients (See e.g.,
Ferguson et al., Cancer Treat. & Res. 94:255 [1998]). Another
molecular marker erb-B2, a growth hormone receptor, is also found
to be correlative with the aggressive behavior of breast cancer
(See e.g., Mack et al., Human Path. 28:974 [1997]). While these
markers provide important information in breast cancer diagnosis,
and contribute to the prevention and treatment of the disease, they
fall short in accurately predicting the future behavior of each
breast cancer in forms of recurrence or metastases. Particularly
for node negative patients, the methods of the present invention
provide risk evaluation that is independent from other existing
parameters of cancer prognosis. The present invention provides such
a method, wherein the PNC represents collective changes of
malignant transformation in cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
[0063] Certain non-limiting, illustrative and preferred embodiments
of the present invention are provided below.
PNC Detection
[0064] PNCs are detected using any suitable method. In some
embodiments, PNCs are detected using immunohistolabeling methods
using a monoclonal antibody specifically recognizing PTB (e.g.,
SH54; Huang et al., 1997, supra). In some embodiments, antigen
retrieval-immunohistochemical technique is used to prepare
paraffin-embedded tissue sections for optimal antibody-antigen
interactions. The basic protocol involves deparaffinization and
microwave retrieval for 2 minutes in 10 mM citric buffer pH 6.0,
prior to conventional immunolabeling protocol (including incubation
in primary antibody and subsequently in avidin conjugated secondary
antibody). Immunolableling protocols are provided in Kamath et.
al., 2002.
[0065] In some embodiments, flow cytometry is employed, as well as
Enzyme-linked Immunosorbent Assay (ELISA) technique for
quantification of the PTB protein. Hence, the present invention
provides a marker that is detected by at least two different
techniques, each technique being independent of the other.
[0066] 1. Detection of RNA
[0067] In some preferred embodiments, detection of markers (e.g.,
PTB) is carried out by measuring the expression of corresponding
mRNA in a tissue sample. mRNA expression may be measured by any
suitable method, including but not limited to, those disclosed
below.
[0068] In some embodiments, RNA is detection by Northern blot
analysis. Northern blot analysis involves the separation of RNA and
hybridization of a complementary labeled probe. In other
embodiments, RNA (or corresponding cDNA) is detected by
hybridization to a oligonucleotide probe. A variety of
hybridization assays using a variety of technologies for
hybridization and detection are available. For example, in some
embodiments, the TaqMan assay (PE Biosystems, Foster City, Calif.;
See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is
herein incorporated by reference) is utilized. The assay is
performed during a PCR reaction. The TaqMan assay exploits the
5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A
probe consisting of an oligonucleotide with a 5'-reporter dye
(e.g., a fluorescent dye) and a 3'-quencher dye is included in the
PCR reaction. During PCR, if the probe is bound to its target, the
5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves
the probe between the reporter and the quencher dye. The separation
of the reporter dye from the quencher dye results in an increase of
fluorescence. The signal accumulates with each cycle of PCR and can
be monitored with a fluorimeter.
[0069] In yet other embodiments, reverse-transcriptase PCR (RT-PCR)
is used to detect the expression of RNA. In RT-PCR, RNA is
enzymatically converted to complementary DNA or "cDNA" using a
reverse transcriptase enzyme. The cDNA is then used as a template
for a PCR reaction. PCR products can be detected by any suitable
method, including but not limited to, gel electrophoresis and
staining with a DNA specific stain or hybridization to a labeled
probe. In some embodiments, the quantitative reverse transcriptase
PCR with standardized mixtures of competitive templates method
described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978
(each of which is herein incorporated by reference) is
utilized.
[0070] 2. Detection of Protein
[0071] In other embodiments, gene expression of cancer markers
(e.g., PTB) is detected by measuring the expression of the
corresponding protein or polypeptide. Protein expression may be
detected by any suitable method. In some embodiments, proteins are
detected by their binding to an antibody raised against the
protein. The generation of antibodies is described below.
[0072] Antibody binding is detected by techniques known in the art
(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays (e.g., using colloidal gold, enzyme or radioisotope
labels, for example), Western blots, precipitation reactions,
agglutination assays (e.g., gel agglutination assays,
hemagglutination assays, etc.), complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0073] In one embodiment, antibody binding is detected by detecting
a label on the primary antibody. In another embodiment, the primary
antibody is detected by detecting binding of a secondary antibody
or reagent to the primary antibody. In a further embodiment, the
secondary antibody is labeled. Many methods are known in the art
for detecting binding in an immunoassay and are within the scope of
the present invention.
[0074] In some embodiments, an automated detection assay is
utilized. Methods for the automation of immunoassays include those
described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and
5,358,691, each of which is herein incorporated by reference. In
some embodiments, the analysis and presentation of results is also
automated. For example, in some embodiments, software that
generates a prognosis based on the presence or absence of a series
of proteins corresponding to cancer markers is utilized.
[0075] In other embodiments, the immunoassay is described in U.S.
Pat. Nos. 5,599,677 and 5,672,480; each of which is herein
incorporated by reference. The immunoassay described therein can be
utilized to detect expression of resident molecules of the PNC
[0076] 3. Kits
[0077] In yet other embodiments, the present invention provides
kits for the detection and characterization of cancer. In some
embodiments, the kits contain antibodies specific for a cancer
marker, in addition to detection reagents and buffers. In other
embodiments, the kits contain reagents specific for the detection
of mRNA or cDNA (e.g., oligonucleotide probes or primers). In
preferred embodiments, the kits contain all of the components
necessary to perform a detection assay, including all controls,
directions for performing assays, and any necessary software for
analysis and presentation of results.
Antibodies
[0078] The present invention provides isolated antibodies. In
preferred embodiments, the present invention provides monoclonal
antibodies that specifically bind to an isolated polypeptide
comprised of at least five amino acid residues of the cancer
markers described herein. These antibodies find use in the
diagnostic methods described herein.
[0079] An antibody against a protein of the present invention may
be any monoclonal or polyclonal antibody, as long as it can
recognize the protein. Antibodies can be produced by using a
protein of the present invention as the antigen according to a
conventional antibody or antiserum preparation process.
[0080] The present invention contemplates the use of both
monoclonal and polyclonal antibodies. Any suitable method may be
used to generate the antibodies used in the methods and
compositions of the present invention, including but not limited
to, those disclosed herein. For example, for preparation of a
monoclonal antibody, protein, as such, or together with a suitable
carrier or diluent is administered to an animal (e.g., a mammal)
under conditions that permit the production of antibodies. For
enhancing the antibody production capability, complete or
incomplete Freund's adjuvant may be administered. Normally, the
protein is administered once every 2 weeks to 6 weeks, in total,
about 2 times to about 10 times. Animals suitable for use in such
methods include, but are not limited to, primates, rabbits, dogs,
guinea pigs, mice, rats, sheep, goats, etc.
[0081] For preparing monoclonal antibody-producing cells, an
individual animal whose antibody titer has been confirmed (e.g., a
mouse) is selected, and 2 days to 5 days after the final
immunization, its spleen or lymph node is harvested and
antibody-producing cells contained therein are fused with myeloma
cells to prepare the desired monoclonal antibody producer
hybridoma. Measurement of the antibody titer in antiserum can be
carried out, for example, by reacting the labeled protein, as
described hereinafter with the antiserum and then measuring the
activity of the labeling agent bound to the antibody. The cell
fusion can be carried out according to known methods, for example,
the method described by Koehler and Milstein (Nature 256:495
[1975]). As a fusion promoter, for example, Sendai virus (HVJ) or,
preferably, polyethylene glycol (PEG), is used.
[0082] Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1
and the like. The proportion of the number of antibody producer
cells (spleen cells) and the number of myeloma cells to be used is
preferably about 1:1 to about 20:1. PEG (preferably PEG 1000-PEG
6000) is preferably added in concentration of about 10% to about
80%. Cell fusion can be carried out efficiently by incubating a
mixture of both cells at about 20.degree. C. to about 40.degree.
C., preferably about 30.degree. C. to about 37.degree. C. for about
1 minute to 10 minutes.
[0083] Various methods may be used for screening for a hybridoma
producing the antibody (e.g., against a tumor antigen or
autoantibody of the present invention). For example, where a
supernatant of the hybridoma is added to a solid phase (e.g.,
microplate) to which antibody is adsorbed directly or together with
a carrier and then an anti-immunoglobulin antibody (if mouse cells
are used in cell fusion, anti-mouse immunoglobulin antibody is
used) or Protein A labeled with a radioactive substance or an
enzyme is added to detect the monoclonal antibody against the
protein bound to the solid phase. Alternately, a supernatant of the
hybridoma is added to a solid phase to which an anti-immunoglobulin
antibody or Protein A is adsorbed and then the protein labeled with
a radioactive substance or an enzyme is added to detect the
monoclonal antibody against the protein bound to the solid
phase.
[0084] Selection of the monoclonal antibody can be carried out
according to any known method or its modification. Normally, a
medium for animal cells to which HAT (hypoxanthine, aminopterin,
thymidine) are added is employed. Any selection and growth medium
can be employed as long as the hybridoma can grow. For example,
RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal
bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a
serum free medium for cultivation of a hybridoma (SFM-101, Nissui
Seiyaku) and the like can be used. Normally, the cultivation is
carried out at 20.degree. C. to 40.degree. C., preferably
37.degree. C. for about 5 days to 3 weeks, preferably 1 week to 2
weeks under about 5% CO.sub.2 gas. The antibody titer of the
supernatant of a hybridoma culture can be measured according to the
same manner as described above with respect to the antibody titer
of the anti-protein in the antiserum.
[0085] Separation and purification of a monoclonal antibody (e.g.,
against a cancer marker of the present invention) can be carried
out according to the same manner as those of conventional
polyclonal antibodies such as separation and purification of
immunoglobulins, for example, salting-out, alcoholic precipitation,
isoelectric point precipitation, electrophoresis, adsorption and
desorption with ion exchangers (e.g., DEAE), ultracentrifugation,
gel filtration, or a specific purification method wherein only an
antibody is collected with an active adsorbent such as an
antigen-binding solid phase, Protein A or Protein G and
dissociating the binding to obtain the antibody.
[0086] Polyclonal antibodies may be prepared by any known method or
modifications of these methods including obtaining antibodies from
patients. For example, a complex of an immunogen (an antigen
against the protein) and a carrier protein is prepared and an
animal is immunized by the complex according to the same manner as
that described with respect to the above monoclonal antibody
preparation. A material containing the antibody against is
recovered from the immunized animal and the antibody is separated
and purified.
[0087] As to the complex of the immunogen and the carrier protein
to be used for immunization of an animal, any carrier protein and
any mixing proportion of the carrier and a hapten can be employed
as long as an antibody against the hapten, which is crosslinked on
the carrier and used for immunization, is produced efficiently. For
example, bovine serum albumin, bovine cycloglobulin, keyhole limpet
hemocyanin, etc. may be coupled to an hapten in a weight ratio of
about 0.1 part to about 20 parts, preferably, about 1 part to about
5 parts per 1 part of the hapten.
[0088] In addition, various condensing agents can be used for
coupling of a hapten and a carrier. For example, glutaraldehyde,
carbodiimide, maleimide activated ester, activated ester reagents
containing thiol group or dithiopyridyl group, and the like find
use with the present invention. The condensation product as such or
together with a suitable carrier or diluent is administered to a
site of an animal that permits the antibody production. For
enhancing the antibody production capability, complete or
incomplete Freund's adjuvant may be administered. Normally, the
protein is administered once every 2 weeks to 6 weeks, in total,
about 3 times to about 10 times.
[0089] The polyclonal antibody is recovered from blood, ascites and
the like, of an animal immunized by the above method. The antibody
titer in the antiserum can be measured according to the same manner
as that described above with respect to the supernatant of the
hybridoma culture. Separation and purification of the antibody can
be carried out according to the same separation and purification
method of immunoglobulin as that described with respect to the
above monoclonal antibody.
[0090] The protein used herein as the immunogen is not limited to
any particular type of immunogen. For example, a cancer marker of
the present invention (further including a gene having a nucleotide
sequence partly altered) can be used as the immunogen. Further,
fragments of the protein may be used. Fragments may be obtained by
any methods including, but not limited to expressing a fragment of
the gene, enzymatic processing of the protein, chemical synthesis,
and the like.
Experimental
[0091] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
EXAMPLE 1
[0092] The Presence of PNC is Correlated with the Transformed
Phenotype
[0093] A large number of human cancer cell lines and normal human
diploid cells were examined for the presence of PNC. The results
showed that the PNC was predominantly present in cancer cells and
was rarely found in normal, primary human cells. The result was
evaluated statistically and summarized in the histogram in FIG. 1.
PNC prevalence, the percentage of cells that contain a PNC, shows a
large diversity among cancer cell lines examined. Some of the cell
lines, such as HeLa (cervical epithelial carcinoma) and T84 (colon
carcinoma) cells, show PNC prevalence of over 80%. In contrast, the
PNC prevalence of other cell lines, including SW620 and MG63 cells,
ranges from 25% to 50%. When a primary human diploid fibroblast,
WI38, is transformed by SV40 large T antigen (WI38-VA13), the PNC
prevalence rises to over 60% compared to 2% in its non-transformed
parental cells.
EXAMPLE 2
Evaluation of the PNC Prevalence in Breast Cancer Cells
[0094] To examine the presence of PNCs in breast cancer cells, a
group of cancerous and normal breast cell lines were compared by
PNC prevalence following immunolabeling with monoclonal antibody
SH54. The results are shown in Table 1, below.
TABLE-US-00001 TABLE 1 Tumor PNC in nude prevalence % Karyotype
Passage number mice MDA-MB-157 41.8 +/- 3.2 52-69 39 + MDA-MB-468
52.3 +/- 4.6 60-67 332 + MDA-134-VI 18.6 +/- 2.2 80-89 33 -
MDA-MB-453 18.8 +/- 3.1 87-91 345 - Hs578T 11.4 +/- 4.3 21-57 Na -
HNME <1 46 18 na Note: MDA cells and H578T are clonal cell lines
derived from breast adenocarcinoma or carcinoma. HNME is human
normal mammary epithelium. na = not available.
[0095] This study demonstrates that there is no direct correlation
between PNC prevalence and the number of chromosomes or the number
of passages of cells in culture, showing that the formation of the
PNC does not directly result from chromosome instability or tissue
culture alterations. The data shows a positive correlation between
PNC prevalence in the cell lines examined and the ability of these
cells lines to be tumorogenic in nude mice.
EXAMPLE 3
[0096] PNC Prevalence is Correlated with Degree of Malignancy
[0097] Antigen retrieval-Immunohistochemical technique was
performed on paraffin-embedded normal and cancerous breast tissue
samples. SHH17, a monoclonal antibody that specifically recognizes
PTB, was used to immunolabel the tissue samples. The immunostaining
patterns of PTB show remarkable differences between normal and
cancerous breast tissue samples. Five hundred or more cell nuclei
were counted for PNC prevalence (defined as the percentage cells
that contain one or more PNCs) in each tissue sample. The
quantitative data are summarized in Table 2 and FIG. 2.
TABLE-US-00002 TABLE 2 Comparison of PNC prevalence in normal and
cancerous breast tissues In situ Invasive Invasive Invasive
Invasive Normal Benign carcinoma carcinoma carcinoma carcinoma
carcinoma Metastic Lymph node - - - - - Less More involved than 4
than 4 Recurrence - - - - + + + as distal metastasis Metastasis - -
- - - - - + No. of 5 10 18 15 5 13 12 22 samples analyzed Average 0
4.5% 17.1% 18.2% 25.8% 29.5% 35.5% 95% percentage of cells positive
for PNC marker per sample analyzed
[0098] These experimental data show: 1) PNC prevalence correlates
with the degree of malignancy; 2) In cancer patients with negative
lymph nodes at primary diagnosis, there is a correlation between
PNC prevalence and recurrence of the cancer. 3) The number of PNCs
increases dramatically along with the progression of cancer from
1-2 per nucleus in carcinoma in situ to 10-20 per nucleus in
metastatic cancers; 4) Multiple PNCs are present in nearly all
cells of metastasized cancer.
EXAMPLE 4
PTB Expression is Significantly Increased During Malignant
Transformation
[0099] Quantitative Western blot analyses on various normal and
cancerous cell lines in culture system demonstrated that PTB
expresses at least ten fold higher in tumor cells lines than normal
primary human fibroblasts. For example, in one experiment, cell
lysates containing equal amounts of protein measured by a protein
quantitation kit (BCA protein assay, Pierce) were loaded on the
same gel (further confirmed by Ponceau S staining of the filter)
and blotted with monoclonal antibody SH54, showing the 10-fold
increase.
[0100] These observations are further supported by the
immunolabeling of various normal and cancerous breast tissues with
SHH17. The level of PTB is similar between the nucleus and the
cytoplasm in normal breast tissues. With the progression of the
malignancy, PTB becomes highly enriched in the nucleus and forming
multiple PNCs.
EXAMPLE 5
[0101] PNCs Disappear Upon Treatment with Therapeutic Agents
[0102] Cultured HeLa cells, whose PNC prevalence is above 95%, were
treated with various chemotherapeutic drugs. The observations are
summarized in Table 3.
TABLE-US-00003 TABLE 3 Drug Category Dose Effect on PNC Cisplatin
Pol I inhibitor 0.5 .mu.g/ml, 5 hours 95% cell negative for PNC
Actinomycin D Pol I inhibitor 0.04 .mu.g/ml, 2 hours 100% cell
negative for PNC Campothecin Anti-Topoisomerase I 1.25 to 2.5
.mu.g/ml, 1 hour 100% cell negative for PNC Methotrexate RNA
Anti-metabolite 0.5 .mu.g/ml, 16 hours 30% cells negative for PNC
1.0 .mu.g/ml, 16 hours 60% cells negative for PNC 2.5 .mu.g/ml, 16
hours 80% cells negative for PNC 5'-fluorouracil RNA
Anti-metabolite 1 to 5 .mu.g/ml, 16 hours No effect on PNC
structure Paclitaxel Anti-mitotic 5 to 40 .mu.M, 16 hours No effect
on PNC structure Vincristine Anti-mitotic 5 to 40 .mu.M, 16 hours
No effect on PNC structure Hydroxyurea DNA anti-metabolite 0.5 to
1.5 .mu.M, 16 hours No effect on PNC structure Cytosine Arabinoside
DNA anti-metabolite 10 to 40 .mu.g/ml, 16 hours No effect on PNC
structure AMSA Anti-Topoisomerase II 5 to 40 .mu.M, 16 hours No
effect on PNC structure Adriamycin Anti-Topoisomerase II 5 to 40
.mu.M, 16 hours No effect on PNC structure
[0103] These data demonstrate that PNC prevalence is reduced in
cells treated with some chemotherapeutic agents. For methotrexate,
the response is dose-dependent.
EXAMPLE 6
PNC Prevalence Contains Independent Prognostic Information for
Early Stage Breast Carcinoma Patients
[0104] To analyze the relationship between PNC prevalence in
primary tumors and the outcome of those patients, univariate
analyses were performed. PNC prevalence, patient age, histological
tumor grade, tumor size, ER status or PR status were each shown to
be significantly associated with disease free survival (DFS) (Table
4A and B). However, only PNC prevalence, histological tumor grade,
and tumor size were significantly associated with overall survival
(OS) (Table 4A). A separate univariate analysis showed that PNC
prevalence was inversely correlated with DFS in patients with
negative nodal status (p<0.0001), with 1-3 LN positive
(p<0.0128) or with .gtoreq.4 LN positive (p<0.0304). These
results suggest that PNC prevalence contains prognostic
information.
TABLE-US-00004 TABLE 4 A. Univariate Analysis for Factors
associated with Survival DFS OS odd odd Factors n ratios 95% CI
p-value ratios 95% CI p-value Age <50 52 1 1 Age .gtoreq.50 77
0.378 (0.21, 0.67) 0.0009 1.006 (0.61, 1.65) 0.98 Size .ltoreq.2 cm
56 1 1 Size >2 cm 68 2.336 (1.22, 4.48) 0.0105 2.038 (1.19,
3.48) 0.0092 LN neg 59 1 1 LN pos 70 1.34 (0.76, 2.36) 0.31 1.237
(0.76, 2.01) 0.39 LN = 0 59 1 1 LN 1-3 26 1.462 (0.70, 3.04) 0.56
1.271 (0.67, 2.41) 0.69 LN 4+ 44 1.272 (0.67, 2.41) 1.217 (0.70,
2.11) ER neg 43 1 1 ER pos 78 0.46 (0.26, 0.83) 0.0094 0.637 (0.38,
1.06) 0.08 PgR neg 59 1 1 PgR pos 54 0.482 (0.26, 0.88) 0.0193
0.681 (0.41, 1.15) 0.15 PNC <18.6 46 1 1 PNC .gtoreq.18.6 83
4.018 (1.80, 8.97) 0.0007 2.048 (1.16, 3.61) 0.013 PNC <23.6 64
1 1 PNC .gtoreq.23.6 65 1.907 (1.16, 3.14) 0.0113 1.907 (1.16,
3.14) 0.0113 Grade I 42 1 1 Grade II 38 2.568 (1.07, 6.15) 0.0011
2.273 (1.14, 4.53) 0.0054 Grade III 45 3.944 (1.78, 8.77) 2.855
(1.50, 5.42) B. Cox Proportional Hazards Model Multivariate
Survival for Lymph Node Negative Patients n n chi .DELTA. chi
Factors (patients) events square square.sup.a p value DFS Grade 56
20 7.205 Grade + PNC 56 20 15.14 11.742 0.0048 OS Size + Grade 56
27 8.811 Size + Grade + 56 27 13.614 4.803 0.0284 PNC .sup.a.DELTA.
Chi square, change in the chi square from the base model using the
log-rank statistic n = number of patients; age = patient age at the
time of diagnosis; ER status, negative (<10 fmol/mg protein or
<20% immunopositivity) or positive (10 fmol/mg protein or 20%
immunopositivity); LN, lymph node. All values are calculated as
continuous variables except ER and PR, which were calculated as a
negative or positive value. PNC prevalence 18.6, median for all
cases tested, and 23.6, median for all primary invasive cases with
follow up.
[0105] Further studies using multivariate analyses demonstrated
that adding PNC prevalence to tumor grade and size significantly
improve the survival prediction model for both DFS and OS in node
negative patients (Table 4B). Kaplan-Meier analyses (See FIG. 3)
graphically demonstrate these survival associations using the
median PNC prevalence for invasive carcinoma (23%) as the cut off
value. Patients with node negative or with less than 4 node
positive tumors, but with high PNC prevalence 23%) had a
significantly shorter disease free survival, as compared to
similarly diagnosed patients with low PNC prevalence (<23%)
(p=0.0013). Altogether, the analyses indicate that PNC prevalence
has independent prognostic value for early stage invasive carcinoma
patients.
EXAMPLE 7
Standardized Protocol for Scoring PNC Prevalence in Paraffin
Embedded Sample Sections
[0106] This Example describes a standard protocol to score PNC
prevalence in paraffin embedded tissue samples. The PNC are
detected by immunocytochemical labeling using an antigen retrieval
protocol with monoclonal antibodies (SH54 or SHH17) that
specifically recognize one of the PNC-associated proteins, PTB. The
basic protocol involves deparaffinization and microwave antigen
retrieval for 2-3 minutes in 10 mM citric buffer (pH 6.0), prior to
the conventional immunolabeling protocol (including incubation with
primary antibody and subsequently with avidin conjugated secondary
antibody). Signals are detected using horseradish peroxidase (HRP)
conjugated biotin that binds to avidin. The enzyme converts
3,3'-Diaminobenzidine (DAB) into dark precipitates (Spector, 1997
Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
2100 pp). Signals are visualized using light microscopy and images
are captured through a 60.times. objective using a SenSys CCD
camera (Princeton Instrument) that is controlled by the Metamorph
image acquisition system (Universal Imaging). Nuclear PTB labeling
aggregates that are at least 2-fold higher in intensity than the
diffuse nucleoplasmic labeling are scored PNC-positive. The
labeling intensity is determined using the densitometry software
contained in the Metamorph image acquisition system. At least 500
epithelium cells in contiguous at the diseased area (the most
aggressive areas, i.e., histologically high grade areas) are
evaluated and scored for PNC prevalence. The scoring is performed
in a blind manner such that examiners are unaware of the patient
information (e.g., tumor size, nodal status, and patient outcome).
Paraffin embedded HeLa cells, whose average PNC prevalence is 97%,
are used as a positive control. Normal breast tissues (NCI shared
tissue network) are used as a negative control for each round of
labeling and scoring.
[0107] Tissue samples were generally fixed in buffered formalin
from 2 hours to overnight. HeLa cells that were fixed either for 2
hours or overnight did not show significant difference in their PNC
prevalence, demonstrating that the antigen-retrieval labeling
method is not obviously affected by the length of fixation. Most
samples were put in fixatives from less than one hour to several
hours after the stop of blood supply. The ones that were not
immediately fixed were temporally stored at 4.degree. C. Samples
with deteriorated cellular morphology were not selected for
database or for studies.
[0108] In some embodiments, an automated scoring method is utilized
to minimize the potential human errors. Threshold is used to
distinguish the differences in labeling intensity. Each nucleus is
evaluated individually due to slight differences in overall nuclear
labeling intensity from cell to cell. However, the ratio of PNC
labeling vs. the diffused nuclear labeling should remain
similar.
EXAMPLE 8
Drug Screening Assay
[0109] The following example provides an assay that finds use for
identifying compounds that affect PNC prevalence.
[0110] The PNC as a marker can be detected easily by
immunofluorescence using the specific monoclonal antibody, SH54,
that recognizes PTB. The fluorescence intensity of PTB labeling is
at least ten fold stronger in the PNC over the diffuse nuclear
labeling. The step-wise screening protocol is following:
[0111] 1) Cells with PNC prevalence over 95% are cultured in 96
well plates. Chemical libraries are added to the corresponding
wells in concentrations initially at an .mu.M range. Cells are
cultivated in the presence of drugs for 1-2 days. The initial
screening uses two different concentration and two different
treatment durations and allows evaluation of 100 chemicals each
day. A chemical library containing 800 chemicals can be screened
within 2 weeks using these methods.
[0112] 2) Cells are fixed in paraformaldehyde at 24 or 48 hours
after drug additions, and are immunolabeled with SH54. The labeling
signals are detected by incubation with a FITC conjugated secondary
antibody. The cells are also counter stained with Dapi stain (a DNA
specific dye to evaluate cell death).
[0113] 3) Labeled cells are visualized in the 96 well plates on an
inverted Zeiss Axiovert 135 fluorescence microscope and are scanned
manually for surviving cells and the presence or absence of PNC in
these cells.
[0114] The end point of the screening measures two parameters: a)
cellular survival; and b) a reduction of PNC prevalence from 95% to
below at least 50% or a reduction of the size of PNC to pin-points
from normally irregular structures.
[0115] 4) Once initial candidate chemicals are identified, more
elaborate cellular characterizations is carried out including
global status of chromatin structure, transcription, translation
and cytoskeleton organization etc. Simultaneously, animal cancer
models are used to test the effectiveness of the chemical in
inhibition of tumor growth.
EXAMPLE 9
PNC Prevalence is Correlated with the Progression of Ovarian
Cancer
[0116] Over 90 cases of ovarian cancer were analyzed for PNC
prevalence in primary tumors after surgery removal. Five different
types of ovarian cancer were investigated, including: R,Serous
Cystadenocarcinoma, R&L,Serous Cystadenocarcinoma, L,Clear cell
carcinoma, R&L,Endometrioid adenocarcinoma, L,Endometrioid
adenocarcinoma, and L,Mixed carcinoma. Antigen
retrieval-Immunohistochemical technique was performed on
paraffin-embedded cancerous ovarian tissue samples. SHH1 7, a
monoclonal antibody that specifically recognized PTB, was used to
immunolabel the tissue samples.
[0117] The immunostaining patterns of PTB in the early grade
(Gleason grading) cancerous ovarian samples was much lower than
that in later grade samples, with a mean percent PNC of 29.17% for
grade 1 samples compared to 37.38% and 51.18% for grades 2 and 3,
respectively (Table 5: FIGS. 4 and 5). The significant increasing
of PNC prevalence in parallel with increases of grading scores
demonstrates a strong positive correlation between PNC prevalence
and grading of all tumor types.
TABLE-US-00005 TABLE 5 Grade N Mean % PNC Median % PNC Std. Dev %
PNC 1 13 29.17 28.35 3.92 2 34 37.39 37.74 3.88 3 47 51.18 49.87
6.10
EXAMPLE 10
PNC Prevalence Contains Independent Prognostic Information for
Ovarian Cancer
[0118] To analyze the relationship between PNC prevalence in
primary ovarian tumors and the outcome of those patients, survival
of the patients from Example 9 was followed up to 90 months.
Invariant risk factor modeling demonstrated that patients with
primary tumors of higher PNC prevalence have significant reduced
survival as compared with those with tumors of lower PNC prevalence
(FIGS. 6 and 7).
[0119] Using the a PNC value of the median grade 1 value (28.35) to
construct Kaplan Meier survival curves shows that patients with
28.35 or fewer PNC per sample had a high survival rate (mean follow
up period 2.5 years), while patients with PNC prevalence greater
than 28.35 mean percent PNC had a 50% fatality rate within 3 years
after surgery (FIG. 6). Patients with grade 2 tumors and PNC
prevalence below the median (37.74) had a higher survival rate than
patients with tumors of higher PNC prevalence. Similarly, patients
with grade 3 tumors and PNC prevalence below the median (49.87) had
a higher survival rate than patients with tumors of higher PNC
prevalence. Moreover, patents with a PNC prevalence rate above
37.74 had a lower overall survival rate than patients with a PNC
prevalence rate of 28.35 (FIGS. 6 and 7). These findings together
show that the formation of the PNC correlates with the progression
of the malignancy and high PNC prevalence in primary tumor is
indicative of poor survival of patients.
[0120] These findings are consistent with the observations in
breast cancer studies that higher PNC prevalence in primary tumor
associates with advance stage of the malignancy.
EXAMPLE 11
PNC Prevalence is Correlated with the Progression of Prostate and
Colon Cancer
[0121] PNC prevalence is also found to be positively correlated
with disease progression in prostate and colon cancers. Comparing
the staining pattern of PTB in normal prostate samples and in
malignant prostate samples FIG. 9 shows significantly higher
prevalence of PNC in the malignant samples compared to the normal
samples. Similarly observations were made for colon cancer as well
(data not shown).
EXAMPLE 12
Colorectal Cancer Outcome is Directly Correlated with PNC
Prevalence
[0122] To examine the relationship between PNC prevalence and
disease progression, colorectal cancer was employed as one of the
best-defined models for cancer progression. To evaluate the
diagnostic value of PNC prevalence, a large number of benign,
localized and invasive carcinomas were evaluated and the
correlation between PNC prevalence and staging of colorectal cancer
was investigated. Furthermore, to analyze PNC as a prognostic and
predictive tumor marker, PNC prevalence was correlated with various
clinical and prognostic criteria including tumor stage,
histological grade and lymph node involvement with reference to
patient outcome.
[0123] Results, shown in FIGS. 10 and 11, demonstrate that PNC
prevalence correlates with progression of colorectal carcinoma in a
gradual fashion from a median of 51.4% in primary tumors to the
82.8% in most advanced tumors. In addition further statistical
analyses show correlations between PNC prevalence, disease
recurrences and patient outcome.
[0124] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the relevant fields
are intended to be within the scope of the following claims.
* * * * *