U.S. patent application number 11/236310 was filed with the patent office on 2006-02-16 for differential diagnosis of cancer and other conditions based on expression of p63.
This patent application is currently assigned to Mount Sinai School of Medicine of New York University. Invention is credited to David E. Burstein.
Application Number | 20060035292 11/236310 |
Document ID | / |
Family ID | 26720494 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035292 |
Kind Code |
A1 |
Burstein; David E. |
February 16, 2006 |
Differential diagnosis of cancer and other conditions based on
expression of p63
Abstract
This invention relates to methods of distinguishing among
various types of differentiated and undifferentiated epithelial
carcinomas, and non-epithelial carcinomas, by detecting the
presence of p63 nucleic acid or protein expression. The invention
also provides methods for detecting p63 nucleic acids and proteins,
as well as methods for diagnosing and treating certain tumors based
on whether the tumors express p63.
Inventors: |
Burstein; David E.; (New
York, NY) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
Mount Sinai School of Medicine of
New York University
New York
NY
|
Family ID: |
26720494 |
Appl. No.: |
11/236310 |
Filed: |
September 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10043502 |
Jan 9, 2002 |
6972181 |
|
|
11236310 |
Sep 26, 2005 |
|
|
|
60261603 |
Jan 12, 2001 |
|
|
|
Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/57484 20130101;
G01N 33/5748 20130101; G01N 33/57423 20130101; G01N 2800/046
20130101; G01N 2800/7095 20130101; G01N 33/564 20130101 |
Class at
Publication: |
435/007.23 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Goverment Interests
[0002] The research leading to the present invention was supported,
in part, by the National Cancer Institute Grant No. R21-CA81362.
Accordingly, the U.S. Government may have certain rights in the
invention.
Claims
1. A method for distinguishing a differentiated lung cancer from an
undifferentiated lung cancer, which method comprises detecting p63
expression in cells from a lung cancer, wherein p63 expression
indicates that the lung cancer is a differentiated lung cancer and
the absence of p63 expression indicates that the lung cancer is an
undifferentiated lung cancer.
2. The method according to claim 1 wherein detecting p63 expression
comprises detecting expression of p63 protein.
3. The method according to claim 2 wherein detecting p63 protein
expression comprises detecting the p63 protein with an
immunoassay.
4. The method according to claim 3 wherein the immunoassay is an
immunohistochemical assay.
5. The method according to claim 1 wherein the differentiated lung
cancer is selected from the group consisting of a poorly
differentiated squamous cell carcinoma, a moderately differentiated
squamous cell carcinoma, a well differentiated squamous cell
carcinoma, an adenosquamous carcinoma, and an adenocarcinoma.
6. The method according to claim 1 wherein the differentiated lung
cancer is a poorly differentiated squamous cell carcinoma.
7. The method according to claim 1 wherein the undifferentiated
lung cancer is a small cell undifferentiated carcinoma.
8. A method of treatment of lung cancer in a patient, which method
comprises administering a chemotherapeutic agent to a patient
diagnosed with a small cell undifferentiated carcinoma lung cancer,
wherein the small cell undifferentiated carcinoma is distinguished
from a squamous cell carcinoma by detecting an absence of p63
expression in cells from the lung cancer.
9. A method according to claim 8 wherein detecting p63 expression
comprises detecting expression of p63 protein.
10. The method according to claim 9 wherein detecting p63 protein
expression comprises detecting the p63 protein with an
immunoassay.
11. The method according to claim 10 wherein the immunoassay is an
immunohistochemical assay.
12. A method of treatment of lung cancer in a patient, which method
comprises surgically resecting a squamous cell carcinoma from a
lung of a patient diagnosed with squamous cell carcinoma lung
cancer, wherein the squamous cell carcinoma is distinguished from a
small cell carcinoma by detecting p63 expression in cells from the
lung cancer.
13. A method according to claim 12 wherein detecting p63 expression
comprises detecting expression of p63 protein.
14. The method according to claim 13 wherein detecting p63 protein
expression comprises detecting the p63 protein with an
immunoassay.
15. The method according to claim 12 wherein the immunoassay is an
immunohistochemical assay.
16. A method for distinguishing a carcinoma of epithelial cells
with squamous cell potential from a non-epithelial cell carcinoma,
which method comprises detecting p63 expression in cells from a
carcinoma, wherein p63 expression indicates that the carcinoma is a
carcinomal of epithelial cells with squamous cell potential and the
absence of p63 expression indicates that the carcinoma is a
non-epithelial carcinoma or a carcinoma without squamous
differentiation potential.
17. The method according to claim 16, wherein the carcinoma without
squamous differentiation potential is a glandular carcinoma.
18. The method according to claim 17, wherein the glandular
carcinoma is a renal carcinoma.
19. The method according to claim 16, wherein the epithelial cells
with squamous cell potential are selected from the group consisting
of squamous epithelia, transitional cells, and glandular
epithelia.
20. A method for distinguishing a thyroid papillary carcinoma from
another thyroid neoplasm, nodule, or enlargement, which method
comprises detecting p63 expression in cells from a thyroid
neoplasm, nodule, or enlargement, wherein p63 expression indicates
that the neoplasm, nodule, or enlargement is a papillary carcinoma
and the absence of p63 expression indicates that the neoplasm,
nodule, or enlargement is not a papillary carcinoma.
21. The method according to claim 20, wherein the neoplasm that is
not a papillary carcinoma is a follicular adenoma, a medullary
carcinoma, an anaplastic carcinoma, or a Hurthle cell
carcinoma.
22. A method for distinguishing a Hashimoto's thyroiditis from
another thyroid inflammatory condition, which method comprises
detecting p63 expression in cells from a thyroid inflammatory
condition, wherein p63 expression indicates that the pathology is
Hashimoto's thyroiditis.
23. The method according to claim 22, wherein the inflammatory
condition is not Hashimoto's thyroiditis.
24. The method according to claim 23, wherein the inflammatory
condition is Grave's disease.
Description
[0001] This Application is a Division of application Ser. No.
10/043,502 filed on Jan. 9, 2002. Ser. No. 10/043,502 is a
Non-Provisional of Provisional (35 USC 119(e)) application
60/261,603 filed on Jan. 12, 2001.
FIELD OF THE INVENTION
[0003] The present invention relates to a gene product, referred to
herein as p63, and to nucleic acids, including the gene, mRNAs, and
cDNAs, encoding it, in particular, to methods of using the gene and
gene product to diagnose and/or treat certain diseases and
disorders such as cancer. The invention also relates to kits which
may be used in such diagnostic and treatment methods.
BACKGROUND OF THE INVENTION
[0004] The nuclear protein p63, located on chromosome 3q, has been
shown to possess homology with p53, a tumor-suppressor gene
encoding a multi-functional DNA-binding protein important in cell
cycle and cell death regulation. p63 exists in three isoforms, each
of which can encode two categories of transcripts under the control
of two alternative promoters. The first encodes full length
proteins with an N-terminal transactivation domain that, like p53,
can activate transcription and induce apoptosis. The second encodes
truncated proteins lacking the N-terminal transactivation domain
(ANp63), and potentially acting in a dominant-negative manner to
suppress transactivation by p53 and/or full length p63.
[0005] Little is known about the function of p63. Evidence suggests
that p63 is necessary for normal development (Levrero et al., J.
Cell Sci. 2000, 113:1661-70). Immunohistochemical studies using an
anti-p63 polyclonal antiserum show that the protein is widely
detectable, with predominant association in the proliferative
compartments in epithelia but also in non-proliferative populations
(Hall et al., Carcinogenesis 2000, 21:153-60). Normal human
epidermis, hair follicles, and stratified epidermal cultures show
p63 expression primarily in cells with proliferative potential,
particularly basal cells, and not in cells undergoing terminal
differentiation (Parsa et al., J. Invest. Dermatol. 1999,
113:1099-1105). The p63 RNA present in the basal cells was mainly
truncated, potentially dominant-negative isotypes (Parsa et al.,
supra).
[0006] Investigators have studied p63 expression in neoplasms. In
keratinocyte squamous cell carcinomas, the number of cells
containing p63 and their distribution depends on the degree of
anaplasia (Parsa et al., supra). However, p63 does not yet have a
defined role in neoplasia and other physiological and pathological
situations (Hall et al., supra). Thus, there is a need in the art
to determine what, if any role p63 plays in neoplasms and
cancer.
SUMMARY OF THE INVENTION
[0007] The present invention are methods for detecting p63 nucleic
acid and protein expression in a cell or tissue that can be used
for diagnostic and prognostic purposes. Such methods include
nucleic acid hybridization, PCR and RT-PCR, and various protein
assays employing antibodies that bind to p63 polypeptides. In a
preferred embodiment, the protein assay immunohistochemical
staining using the anti-p63 4A4 monoclonal antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.). The present invention
also provides a kit comprising the reagents required for detection
of p63 nucleic acids or polypeptides.
[0008] The present invention also provides methods for
distinguishing differentiated lung cancers from undifferentiated
lung cancers by evaluating lung tissue for the expression of p63
nucleic acids or proteins. In particular, the expression of p63
indicates that the lung cancer is a differentiated carcinoma and
absence of p63 expression is indicative of a undifferentiated
carcinoma.
[0009] In a preferred embodiment, the undifferentiated lung cancer
is a small cell undifferentiated carcinoma and the differentiated
lung cancer is a poorly differentiated squamous cell carcinoma, a
moderately differentiated squamous cell carcinoma, a
well-differentiated squamous cell carcinoma, an adenosquamous
carcinoma or an adenocarcinoma.
[0010] The present invention also provides methods of
distinguishing an epithelial squamous cell carcinoma from a
non-epithelial cell carcinoma by detecting p63 nucleic acid or
protein expression in cells derived from a carcinoma. In
particular, p63 expression indicates that the carcinoma is an
epithelial squamous cell carcinoma and the absence of p63
expression indicates that the carcinoma is a non-epithelial cell
carcinoma or a carcinoma without squamous differentiation
potential.
[0011] In a preferred embodiment, the p63 positive cells are
squamous epithelial cells, transitional cells or glandular
epithelial cells and the p63 negative cells are glandular carcinoma
cells, or most preferably, renal carcinoma cells.
[0012] Also provided by the present invention is a method for
distinguishing a thyroid papillary carcinoma from another thyroid
disorder in thyroid cells derived from a neoplasm, nodule, or
enlargement by detecting p63 nucleic acid or protein expression.
p63 expression is indicative of a papillary carcinoma and absence
of p63 expression is indicative of a follicular adenomata, a
medullary carcinoma, an anaplastic carcinoma or a Hurthle cell
carcinoma.
[0013] The present invention also provides a method of
distinguishing Hashimoto's thyroiditis from another inflammatory
thyroid condition by detecting p63 nucleic acid or protein
expression in inflammatory thyroid cell. The presence p63 nucleic
acid or protein expression indicates that the pathology is
Hashimoto's thyroiditis and the absence of p63 expression indicates
that the inflammatory pathology is Grave's disease.
[0014] The present invention additionally provides a method for
treating lung cancer in a patient by diagnosing the lung cancer as
a squamous cell carcinoma by detecting p63 expression, followed by
surgical resection of the p63-expressing tumor.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention provides a method for differentially
distinguishing various types of cancer and other pathological
conditions. For example, the invention permits distinguishing a
specific type of lung cancer, such as a poorly differentiated
squamous cell carcinoma, a moderately differentiated squamous
carcinoma, a well differentiated squamous cell carcinoma, an
adenosquamous carcinoma, and an adenocarcinoma, from an
undifferentiated lung cancer, such as a small cell undifferentiated
carcinoma (oat cell carcinoma). Alternatively, the invention
provides for distinguishing papillary thyroid carcinoma from other
thyroid neoplasms. In yet another embodiment, the invention
provides for distinguishing transitional cell carcinomas (from the
kidney and ureter) from renal cell carcinomas. P63 detection may be
employed in any differential diagnosis between a tumor of squamous
origin, differentiation or squamous potential, and a tumor without
squamous origin, differentiation or potential.
[0016] In another aspect, the invention permits detection or
diagnosis of precancerous dysplasia, intra-epithelial neoplasms, or
cancer (malignancy) in epithelial cells by detecting p63 expression
in surface (not basal stem) epithelial cells, which ordinarily lack
p63 expression. For example, cervical cells obtained by
non-traumatic brushing or lavage that express p63 are likely in a
state of pre-malignancy or have become malignant. Other cell types
amenable to this diagnostic approach include, but are not limited
to, squamous epithelia (e.g., mouth, esophagus, trachea, anus);
transitional cells (e.g., bladder, ureter, urethra, epithelia
lining the respiratory tract); glandular epithelial cells (e.g.,
thyroid, endometrium, endocervix); and other cells that are or have
the capacity to become squamous. This aspect of the invention
yields better results when the basal stem cells, that continuously
divide to reform the epithelial tissue, are not assayed, since
these cells express p63 endogenously.
[0017] In each case, the method comprises detecting p63 expression
in cells from the cancer or other condition. The invention is
based, in part, on the observation that tumors or conditions
arising from non-squamous epithelia having the capacity to undergo
squamous differentiation are capable of expressing p63. In
contrast, tumors that never undergo squamous differentiation do not
express p63.
[0018] In the case of cancer, p63 expression indicates that the
lung cancer is a differentiated lung cancer. The absence of p63
expression indicates that the lung cancer is likely an
undifferentiated lung cancer. The invention specifically provides
for distinguishing poorly differentiated squamous carcinoma (p63
expression) from small cell undifferentiated carcinoma (no p63
expression). In another embodiment, the invention provides for
distinguishing bronchioloalveolar carcinoma (no p63 expression)
from adenocarcinoma with alveolar spread pattern (p63 expression).
The invention further provides for diagnosing a new subtype of
adenosquamous carcinoma marked by basal p63 staining.
[0019] In the case of distinguishing between transitional cell and
renal cell cancer, p63 expression indicates that the renal cancer
is a transitional cell carcinoma, and the absence of p63 expression
indicates that the renal cancer is a renal cell carcinoma. For
example, the invention provides for distinguishing upper uretal or
renal pelvic transitional carcinoma (urothelial carcinoma), in
which cells express p63, from renal cell carcinoma or adrenal
carcinoma, in which the cells are negative for p63 expression.
[0020] In the case of thyroid cancer, p63 expression indicates that
the thyroid cancer is a papillary thyroid carcinoma, and the
absence of p63 expression indicates that the thyroid cancer is a
different thyroid neoplasm. In particular, the invention provides
for distinguishing papillary carcinoma, in which cells are positive
for p63 expression, from follicular carcinoma, medullary carcinoma,
follicular adenoma, Hurthle cell adenoma, Hurthle cell carcinoma,
and nodular goiter in which cells are negative for p63
expression.
[0021] In addition, the invention advantageously provides for
differentiation of non-cancerous thyroid conditions. In particular,
it provides for diagnosing antibody-positive and antibody-negative
Hashimoto's thyroiditis. This is particularly advantageous in the
case of antibody-negative Hashimoto's. It further permits
distinguishing Hashimoto's thyroiditis (p63 positive) from other
inflammatory thyroid conditions, such as Grave's disease (which is
p63 negative).
[0022] Various methods are available to detect p63 expression,
including biochemical assays, immunoassays, Northern and
reverse-transcriptase polymerase chain reaction (RT-PCR) assays,
and the like. In a specific embodiment, detecting p63 expression
comprises detecting expression of p63 protein. More particularly,
detecting p63 protein expression comprises detecting the p63
protein with an immunoassay. In specific embodiments exemplified
infra, the immunoassay is an immunohistochemical assay or an
immunocytopathology assay.
[0023] The assays of the invention can be performed on various cell
samples. For example, tumor cell tissue obtained by biopsy,
resection, or other technique can be tested. Alternatively,
cytological samples can be tested.
[0024] The invention further provides a method of treatment of lung
cancer in a patient. The method comprises administering a
chemotherapeutic agent to a patient diagnosed with a small cell
undifferentiated carcinoma lung cancer, wherein the small cell
undifferentiated carcinoma is distinguished from a squamous cell
carcinoma by detecting an absence of p63 expression in cells from
the lung cancer. In another embodiment, the method comprises
surgically resecting a squamous cell carcinoma from a lung of a
patient diagnosed with squamous cell carcinoma lung cancer, wherein
the squamous cell carcinoma is distinguished from a small cell
carcinoma by detecting p63 expression in cells from the lung
cancer.
[0025] As used herein, the term "p63" refers to a protein
homologous to the tumor suppressor protein p53, which contains a
multi-functional DNA-binding protein important in cell cycle and
cell death regulation. p63 exists in three isoforms, each of which
can encode two categories of transcripts under the control of two
alternative promoters. The first encodes full length proteins with
an acidic N-terminal transactivation domain that, like p53, can
activate transcription and induce apoptosis. The second encodes
truncated proteins lacking the N-terminal transactivation domain
(.DELTA.Np63), and potentially acting in a dominant-negative manner
to suppress transactivation by p53 and full length p63. Unless
otherwise specified, p63 means any of the three isoforms. The three
p63 splice forms, .alpha., .beta., and .gamma. may be
differentially expressed in tumors.
[0026] A tumor is "p63 positive" or has detectable p63 expression
when any cells in the tumor express p63. A tumor is "p63 negative"
or has no detectable p63 expression when no cells express p63 at
detectable levels.
[0027] As used herein, the term "cancer" refers to any malignant
tumor, particularly arising in the lung, kidney, or thyroid. The
cancer manifests itself as a "tumor" or tissue comprising malignant
cells of the cancer. Examples of tumors that can be detected,
diagnosed and treated according to the invention include sarcomas
and carcinomas such as, but not limited to: fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma. As noted above, the invention specifically permits
differential diagnosis of lung, kidney, and thyroid tumors.
[0028] In particular, the invention permits differential diagnosis
of various lung cancers. These include poorly differentiated,
moderately differentiated, and high differentiated squamous cell
carcinomas; bronchioloalveolar carcinoma, carcinoid, and small cell
undifferentiated carcinoma. Bronchioloalveolar carcinoma is a
well-differentiated adenocarcinoma probably derived from lung
alveolar epithelium. Carcinoid is a generally much less aggressive
form of tumor than small cell undifferentiated. Small cell
undifferentiated carcinoma is a highly aggressive cancer.
[0029] The present invention also advantageously provides for
assessing the tumorigenic potential of thyroid nodules. At present,
detection of thyroid nodules leads to fine needle aspiration to
obtain cells from the nodule. Cytologic analysis of these cells can
provide an indication of whether or not they are neoplastic or
malignant. For example, follicular adenomas and papillary carcinoma
may present as thyroid nodules. Fine needle aspiration can fail to
yield cells amenable to definitive pathological analysis. Detecting
p63 expression in nodule cells deemed neoplastic permits a
confident diagnosis of papillary carcinoma; the absence of p63
expression leads to a diagnosis of a different pathology, such as
follicular adenoma.
[0030] Accuracy in the pre-surgical determination of malignancy in
thyroid nodules is essential, in order to avoid unnecessary surgery
for non-malignant thyroid conditions presenting as a nodule or
enlargement. Although pre-surgical testing including
ultrasonographic scans and fine needle aspiration cytologic
evaluation is useful for definitively identifying some of these
lesions pre-surgically, in many cases, the definitive decision to
perform surgery or to choose the appropriate surgery cannot be
made.
[0031] For example, papillary thyroid carcinoma may have cytologic
features on fine needle aspiration which are considered abnormal or
suspicious, but non-definitive, or may have features which are
indistinguishable from benign entities such as follicular adenomas.
In these and other circumstances, detection and evaluation of p63
at the gene, mRNA, or protein level will allow for definitive
diagnosis, reducing either unnecessary surgery for benign entities,
inappropriate delays of months to years for surgical removal of
malignancy, and reduce the need for second surgical procedures,
such as when a papillary carcinoma is misdiagnosed as a benign
adenoma. p63 may be detected alone, or in a multiplex panel with
non-p63 probes, e.g., other antibodies, on a DNA or proteomics
chip.
[0032] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. In the case of nucleic acid
molecules, an isolated nucleic acid includes a PCR product, an
isolated mRNA, a cDNA, or a restriction fragment. In another
embodiment, an isolated nucleic acid is preferably excised from the
chromosome in which it may be found, and more preferably is no
longerjoined to non-regulatory, non-coding regions, or to other
genes, located upstream or downstream of the gene contained by the
isolated nucleic acid molecule when found in the chromosome. In yet
another embodiment, the isolated nucleic acid lacks one or more
introns. Isolated nucleic acid molecules include sequences inserted
into plasmids, cosmids, artificial chromosomes, and the like. Thus,
in a specific embodiment, a recombinant nucleic acid is an isolated
nucleic acid. An isolated protein may be associated with other
proteins or nucleic acids, or both, with which it associates in the
cell, or with cellular membranes if it is a membrane-associated
protein. An isolated organelle, cell, or tissue is removed from the
anatomical site in which it is found in an organism. An isolated
material may be, but need not be, purified.
[0033] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate the
presence of unrelated materials, i.e., contaminants, including
native materials from which the material is obtained. For example,
a purified protein is preferably substantially free of other
proteins or nucleic acids with which it is associated in a cell; a
purified nucleic acid molecule is preferably substantially free of
proteins or other unrelated nucleic acid molecules with which it
can be found within a cell. As used herein, the term "substantially
free" is used operationally, in the context of analytical testing
of the material. Preferably, purified material substantially free
of contaminants is at least 50% pure; more preferably, at least 90%
pure, and more preferably still at least 99% pure. Purity can be
evaluated by chromatography, gel electrophoresis, immunoassay,
composition analysis, biological assay, and other methods known in
the art.
[0034] Methods for purification are well-known in the art. For
example, nucleic acids can be purified by precipitation,
chromatography (including preparative solid phase chromatography,
oligonucleotide hybridization, and triple helix chromatography),
ultracentrifugation, and other means. Polypeptides and proteins can
be purified by various methods including, without limitation,
preparative disc-gel electrophoresis, isoelectric focusing, HPLC,
reversed-phase HPLC, gel filtration, ion exchange and partition
chromatography, precipitation and salting-out chromatography,
extraction, and countercurrent distribution. For some purposes, it
is preferable to produce the polypeptide in a recombinant system in
which the protein contains an additional sequence tag that
facilitates purification, such as, but not limited to, a
polyhistidine sequence, or a sequence that specifically binds to an
antibody, such as FLAG and GST. The polypeptide can then be
purified from a crude lysate of the host cell by chromatography on
an appropriate solid-phase matrix. Alternatively, antibodies
produced against the protein or against peptides derived therefrom
can be used as purification reagents. Cells can be purified by
various techniques, including centrifugation, matrix separation
(e.g., nylon wool separation), panning and other immunoselection
techniques, depletion (e.g., complement depletion of contaminating
cells), and cell sorting (e.g., fluorescence activated cell sorting
[FACS]). Other purification methods are possible. A purified
material may contain less than about 50%, preferably less than
about 75%, and most preferably less than about 90%, of the cellular
components with which it was originally associated. The
"substantially pure" indicates the highest degree of purity which
can be achieved using conventional purification techniques known in
the art.
[0035] A "sample" as used herein refers to a biological material
which can be tested, e.g., for the presence of p63 polypeptides or
p63 nucleic acids, e.g., to identify cells that specifically
express the p63 gene and its gene product. Such samples can be
obtained from any source, including tissue biopsies, blood and
blood cells, pleural effusions, pericardial effussions,
cerebrospinal fluid (CSF), urine, ascites fluid, cyst fluids,
bronchial aspiration, bronchoscopic washes and ravages, peritoneal
washes, brushes and brush specimens, fine needle aspiration, pap
smear, and cell culture. In preferred embodiments samples are
obtained, e.g., in a biopsy, from cancerous tissue (e.g., a tumor)
or from tissue that is suspected of being cancerous or of
containing cancer cells. In one particularly preferred embodiment
samples are obtained from lung tissue. In another preferred
embodiment for testing epithelial cells for dysplasia,
intra-epithelial pre-malignant changes, or malignancy, the cells
are obtained from the surface by gentle brushing, washing, or
lavage to avoid obtaining p63-positive basal cells.
[0036] Non-human animals include, without limitation, laboratory
animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.;
domestic animals such as dogs and cats; and, farm animals such as
sheep, goats, pigs, horses, and cows.
[0037] In preferred embodiments, the terms "about" and
"approximately" shall generally mean an acceptable degree of error
for the quantity measured given the nature or precision of the
measurements. Typical, exemplary degrees of error are within 20
percent (%), preferably within 10%, and more preferably within 5%
of a given value or range of values. Alternatively, and
particularly in biological systems, the terms "about" and
"approximately" may mean values that are within an order of
magnitude, preferably within 5-fold and more preferably within
2-fold of a given value. Numerical quantities given herein are
approximate unless stated otherwise, meaning that the term "about"
or "approximately" can be inferred when not expressly stated.
[0038] The invention provides for administration of a
therapeutically effective treatment in conjunction with the
diagnostic and prognostic methods. The term "therapeutically
effective" refers to that amount of a treatment regimen that is
sufficient to result in a desired activity. Thus, as used to
describe a cancer therapy, a therapeutically effective treatment
refers to the amount of a chemotherapeutic compound or
compositions, radiation, resection, or gene therapy, for example,
that is sufficient to produce an effective outcome, such as tumor
regression, increase time of survival, increased time of remission,
and the like.
[0039] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction (for
example, gastric upset, dizziness and the like) when administered
to an individual. Preferably, and particularly where a vaccine is
used in humans, the term "pharmaceutically acceptable" may mean
approved by a regulatory agency (for example, the U.S. Food and
Drug Agency) or listed in a generally recognized pharmacopeia for
use in animals (for example, the U.S. Pharmacopeia).
[0040] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which a compound is administered. Sterile water or
aqueous saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. Exemplary suitable pharmaceutical carriers
are described in "Reminington's Pharmaceutical Sciences" by E. W.
Martin.
Detection of p63 Expression
[0041] A variety of methods can be employed for diagnostic and
prognostic methods using reagents. For example, using the methods
described here it is possible to detect expression of a p63 nucleic
acid or protein in cells or tissues from an individual, such as in
cells or tissues in a sample (e.g., from a biopsy) obtained or
derived from an individual subject or patient. As explained above,
p63 nucleic acids and polypeptides are expressed at elevated levels
in certain cancerous cells and tissues including, for example, in
the various types of cancer and tumor cells and tissues identified
above, and not expressed in other tumor types.
[0042] Thus, using the methods described here (as well as other
methods known in the art) a skilled artisan may detect expression
of p63 in a sample of cells or tissue from an individual, and may
thereby detect and/or identify cells or tissue in that sample as
being cancer cells or tissue of a particular pathological type. For
example, in preferred embodiments a skilled artisan may use such
methods to identify cells or tissue in a sample as being a
particular type of cancer cell or tissue which is known to express
elevated levels of a p63 nucleic acid or polypeptide. Such cancer
cells and tissues may be, for example, any of the particular cancer
and tumor cell/tissue types described supra. In certain preferred
embodiments the particular type of cancer cell or tissue identified
in such methods are lung cancer cells or tissue, such as cells or
tissues of bronchogenic carcinoma, lung carcinoma or small cell
lung carcinoma. By using such methods to detect cancer cells or
tissue in an individual, a skilled user may thereby diagnose the
presence of the cancer in that individual. Similarly, the absence
of p63 expression provides information about the nature of the
cancer.
[0043] In preferred embodiments the methods described herein are
performed using pre-packaged diagnostic kits. Such kits may
comprise at least one specific p63 nucleic acid or a p63 specific
antibody reagent. The kit and any reagent(s) contained therein may
be used, for example, in a clinical setting, to diagnose patients
exhibiting or suspected of having a disorder such as a type of
cancer.
[0044] A sample comprising a nucleated cell (of any cell type) from
an individual may be used in such diagnostic and prognostic methods
as a starting source for genomic nucleic acid and to detect
mutations of a p63 gene. A sample comprising a cell of any cell
type or tissue of any tissue type in which a p63 gene is expressed
may also be used in such diagnostic methods, e.g., for detection of
p63 gene expression or of p63 gene products (such as p63 proteins),
as well as for identifying cells, particularly cancer and tumor
cells, that express or do not express p63.
Immunoassays
[0045] The diagnostic and prognostic methods of the invention
include ones that comprise detecting levels of a p63 protein or
other p63 polypeptides and including functionally conserved
variants and fragments thereof. For example, antibodies directed
against unimpaired, wild-type or mutant p63 gene products or
against functionally conserved variants or peptide fragments of a
p63 gene product may be used as diagnostic and prognostic reagents,
e.g., to detect various types of cancer cells and tumors. Such
reagents may be used, for example, to detect abnormalities in the
level of p63 gene product synthesis or expression. Antibodies and
immunoassay methods such as those described hereinbelow also have
important in vitro applications for assessing the efficacy of
treatments, e.g., for cancer. Compounds that may have beneficial
effects on a disorder associated with abnormal p63 expression
(e.g., any of the types of cancer identified supra) can be
identified and a therapeutically effective dose for such compounds
may be determined using such assays.
[0046] In vitro immunoassays can also be used to assess the
efficacy of cell-based gene therapy for a cancer or other disorder
associated with abnormal p63 expression. For example, antibodies
directed against p63 polypeptides may be used in vitro to determine
the level of p63 gene or polypeptide expression achieved in tumor
cells. Such methods may be used to detect intracellular p63 gene
products, preferably using whole cells, but also including cell
lysates or extracts, to detect expression of p63 gene products.
[0047] The isolated cells may be cells derived from cell culture or
from an individual (e.g., a biopsy sample from a patient suspected
of having a type of cancer or other disorder associated with
abnormal levels of p63 expression, or suspected of having a
propensity for such a cancer or other disorder).
[0048] As one example, antibodies or fragments of antibodies may be
used to detect the presence of a p63 gene product, a variant of a
p63 gene product or fragments thereof, for example, by
immunofluorescence techniques employing a fluorescently labeled
antibody coupled with light microscopic, flow cytometric or
fluorimetric detection methods.
[0049] In particularly preferred embodiments, antibodies or
fragments thereof may also be employed histologically, for example
in immunostaining, immunofluorescence or immunoelectron microscopy
techniques, for in situ detection of a p63 gene product. In situ
detection may be accomplished by removing a histological specimen
(e.g., a tissue sample) from a patient and applying thereto a
labeled antibody of the present invention or a fragment of such an
antibody. The antibody or antibody fragment is preferably applied
by overlaying the labeled antibody or antibody fragment onto a
biological sample. Through the use of such a procedure, it is
possible to detect, not only the presence of a p63 gene product,
but also the gene product's distribution in the examined tissue. A
wide variety of histological methods that are well known in the art
(for example, staining procedures) can be readily modified by those
skilled in the art without undue experimentation to achieve such in
situ detection. Immunoassays for p63 gene products will typically
comprise incubating a biological sample (for example, a biological
fluid, a tissue extract, freshly harvested cells or cell lysates)
in the presence of a detectably labeled antibody that is capable of
specifically binding a p63 gene product (including, for example, a
functionally conserved variant or a peptide fragment thereof). The
bound antibody may then be detected by any of a number of
techniques well known in the art.
[0050] According to the invention, p63 polypeptide produced
recombinantly or by chemical synthesis, and fragments or other
derivatives or analogs thereof, including fusion proteins, may be
used as an immunogen to generate antibodies that recognize the p63
polypeptide. Such antibodies include but are not limited to
polyclonal, monoclonal, chimeric, single chain, Fab fragments, and
an Fab expression library. The anti-p63 antibodies of the invention
may be cross reactive, e.g., they may recognize p63 from different
species. Polyclonal antibodies have greater likelihood of cross
reactivity. Alternatively, an antibody of the invention may be
specific for a single form of p63, such as murine p63. Preferably,
such an antibody is specific for human p63. Antibodies for use in
the invention can also be specific for a specific isoform of p63,
such as any of the three p63 splice forms, .alpha., .beta., and
.gamma.. Since p63 forms have different cellular functions, it will
be useful to develop a panel consisting of an N-terminal-sequence
specific antibody (that would detect only the full length p63
forms), and an antibody such as the one used in this study, that
recognizes both full length and truncated A-N forms. Additional
clinically useful differences in expression patterns are
expected.
[0051] Various procedures known in the art may be used for the
production of polyclonal antibodies to p63 polypeptides or
derivatives or analogs thereof. For the production of antibody,
various host animals can be immunized by injection with the p63
polypeptide, or a derivative (e.g., fragment or fusion protein)
thereof, including but not limited to rabbits, mice, rats, sheep,
goats, etc. In one embodiment, the p63 polypeptide or fragment
thereof can be conjugated to an immunogenic carrier, e.g., bovine
serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various
adjuvants may be used to increase the immunological response,
depending on the host species, including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette--Guerin) and Corynebacterium
parvum.
[0052] For preparation of monoclonal antibodies directed toward the
p63 polypeptide, or fragment, analog, or derivative thereof, any
technique that provides for the production of antibody molecules by
continuous cell lines in culture may be used. These include but are
not limited to the hybridoma technique originally developed by
Kohler and Milstein (Nature 256:495-497, 1975), as well as the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al., Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad.
Sci. U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,
1985). In an additional embodiment of the invention, monoclonal
antibodies can be produced in germ-free animals (International
Patent Publication No. WO 89/12690, published 28 Dec. 1989). In
fact, according to the invention, techniques developed for the
production of "chimeric antibodies" (Morrison et al., J. Bacteriol.
159:870, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda
et al., Nature 314:452-454, 1985) by splicing the genes from a
mouse antibody molecule specific for an p63 polypeptide together
with genes from a human antibody molecule of appropriate biological
activity can be used; such antibodies are within the scope of this
invention. Such human or humanized chimeric antibodies are
preferred for use in therapy of human diseases or disorders
(described infra), since the human or humanized antibodies are much
less likely than xenogenic antibodies to induce an immune response,
in particular an allergic response, themselves.
[0053] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to
produce p63 polypeptide-specific single chain antibodies. Indeed,
these genes can be delivered for expression in vivo. An additional
embodiment of the invention utilizes the techniques described for
the construction of Fab expression libraries (Huse et al., Science
246:1275-1281, 1989) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for an p63
polypeptide, or its derivatives, or analogs.
[0054] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0055] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme--linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. 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 means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of an p63 polypeptide, one may assay generated hybridomas
for a product which binds to an p63 polypeptide fragment containing
such epitope. For selection of an antibody specific to an p63
polypeptide from a particular species of animal, one can select on
the basis of positive binding with p63 polypeptide expressed by or
isolated from cells of that species of animal.
[0056] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of the p63
polypeptide, e.g., for Western blotting, imaging p63 polypeptide in
situ, measuring levels thereof in appropriate physiological
samples, etc. using any of the detection techniques mentioned above
or known in the art.
[0057] In a specific embodiment, antibodies that agonize or
antagonize the activity of p63 polypeptide can be generated. Such
antibodies can be tested using the assays described infra for
identifying ligands.
Molecular Biological Assays
[0058] The diagnostic and prognostic methods of the invention
include methods for assaying the level of p63 gene expression. A
variety of methods known in the art may be used to detect assay
levels of p63 nucleic acid sequences in a sample. For example, RNA
from a cell type or tissue, such a tumor cell or tissue type, that
is known or suspected to express the p63 gene may be isolated and
tested utilizing hybridization or PCR techniques known in the art.
The isolated cells may be, for example, cells derived from a cell
culture or from an individual. The analysis of cells taken from a
cell culture may be useful, e.g., to test the effect of compounds
on the expression of a p63 gene, or alternatively, to verify that
the cells are ones of a particular cell type that expresses a p63
gene.
[0059] As an example, and not by way of limitation, diagnostic
methods for the detection of p63 nucleic acids can involve
contacting and incubating nucleic acids (including recombinant DNA
molecules, cloned genes or degenerate variants thereof) obtained
from a sample with one or more labeled nucleic acid reagents, such
as recombinant p63 DNA molecules, cloned genes or degenerate
variants thereof, under conditions favorable for specifically
annealing or hybridizing these reagents to their complementary
sequences in the sample nucleic acids. Preferably the lengths of
these nucleic acid reagents are at least 15 to 30 nucleotides.
After incubation, all non-annealed or non-hybridized nucleic acids
are removed. The presence of nucleic acids that have hybridized, if
any such molecules exist, is then detected and the level of p63
nucleic acid sequences to which the nucleic acid reagents have
annealed may be compared to the annealing pattern or level expected
from a control sample (e.g., from a sample of normal, non-cancerous
cells or tissues) to determine whether p63 nucleic acid is
expressed at an elevated level.
[0060] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m (melting temperature) of
55.degree. C., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS).
Moderate stringency hybridization conditions correspond to a higher
T.sub.m, e.g., 40% formamide, with 5.times. or 6.times.SCC. High
stringency hybridization conditions correspond to the highest
T.sub.m, e.g., 50% formamide, 5.times. or 6.times.SCC. SCC is a
0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two
nucleic acids contain complementary sequences, although depending
on the stringency of the hybridization, mismatches between bases
are possible. The appropriate stringency for hybridizing nucleic
acids depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-9.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al., supra, 11.7-11.8). A minimum length for a hybridizable nucleic
acid is at least about 10 nucleotides; preferably at least about 15
nucleotides; and more preferably the length is at least about 20
nucleotides.
[0061] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C. In a specific embodiment, "high
stringency" refers to hybridization and/or washing conditions at
68.degree. C. in 0.2.times.SSC, at 42.degree. C. in 50% formamide,
4.times.SSC, or under conditions that afford levels of
hybridization equivalent to those observed under either of these
two conditions.
[0062] In a preferred embodiment of such a detection scheme, the
nucleic acid from the cell type or tissue of interest may be
immobilized, for example, to a solid support such as a membrane or
a plastic surface (for example, on a nylon membrane, a microtiter
plate or on polystyrene beads). After incubation, non-annealed,
labeled p63 nucleic acid reagents may be easily removed and
detection of the remaining, annealed, labeled p63 nucleic acid
reagents may be accomplished using standard techniques that are
well-known in the art.
[0063] Alternative diagnostic methods for the detection of p63
nucleic acids in patient samples or in other cell or tissue sources
may involve their amplification, e.g., by PCR (see, for example,
the experimental embodiment taught in U.S. Pat. No. 4,683,202)
followed by detection of the amplified molecules using techniques
that are well known to those of skilled in the art. The resulting
level of amplified p63 nucleic acid may be compared to those levels
that would be expected if the sample being amplified contained only
normal levels of p63 nucleic acid, as normal cells or tissues, to
determine whether elevated levels of a p63 nucleic acid are
expressed.
[0064] In one preferred embodiment of such a detection scheme, a
cDNA molecule is synthesized from an RNA molecule of interest
(e.g., by reverse transcription). A sequence within the cDNA may
then be used as a template for a nucleic acid amplification
reaction such as PCR. Nucleic acid reagents used as synthesis
intitation reagents (e.g., primers) in the reverse transcription
and amplification steps of such an assay are preferably chosen from
the p63 nucleic acid sequences described herein or are fragments
thereof. Preferably, the nucleic acid reagents are at least about 9
to 30 nucleotides in length. The amplification may be performed
using, e.g., radioactively labeled or fluorescently labeled
nucleotides, for detection. Alternatively, enough amplified product
may be made such that the product can be visualized by standard
ethidium bromide or other staining methods.
[0065] p63 gene expression assays of the invention may also be
performed in situ (i.e., directly upon tissue sections of patient
tissue, which may be fixed and/or frozen), thereby eliminating the
need of nucleic acid purification. p63 nucleic acid reagents may be
used as probes or as primers for such in situ procedures (see, for
example, Nuovo, PCR In Situ Hybridization: Protocols And
Application, 1992, Raven Press, New York). Alternatively, if a
sufficient quantity of the appropriate cells can be obtained,
standard Northern analysis can be performed to determine the level
of p63 gene express by detecting levels of p63 mRNA.
EXAMPLES
[0066] The present invention may be better understood by reference
to the following examples, which are provided by way of
illustration and are not limiting.
Example 1
Immunohistochemical Study of Expression of p53-Homolog p63 in
Pulmonary Neoplasms
[0067] p63 is a nuclear protein recently discovered in a screen for
genes with homology to the tumor suppressor p53 gene. This Example
describes p63 expression in benign lung tissue and neoplasms of
pulmonary origin.
Materials and Methods
[0068] Tissues. Archival, routinely processed, formalin-fixed,
paraffin-embedded surgical pathology specimens from bronchoscopic
biopsy and pneumonectomy and/or lobectomy specimens were
examined.
[0069] Antibody. The antibody used in this study was anti-p63
monoclonal antibody 4A4 (Santa Cruz Biotechnology, Inc., Santa Cruz
Calif.). This antibody is reactive against a region common to both
the full-length and truncated forms of p63.
[0070] Immunohistochemical staining. Five-micron-thick sections
were heated and deparaffinized and were treated with 0.3% hydrogen
peroxide to block endogenous peroxidase activity and pretreated
with citric acid (pH 6.0) for antigen retrieval. Slides were then
incubated overnight at room temperature with an anti-p63 antibody
and stained using a streptavidin biotin-based immunoperoxidase
staining kit (BioGenex, San Ramon, Calif.), according to the
manufacturer's instructions. The kit employs diaminobenzidine as
chromagen, and then counterstain with hematoxylin and/or eosin.
[0071] p63 expression was considered positive only if distinct
nuclear staining was present. Intense positive staining of
bronchial reserve cell nuclei was noted as an internal positive
control for p63 staining of histologic sections.
Results
[0072] Normal epithelium. In normal lung, p63 intensely stained
nuclei of bronchial reserve cells but did not stain ciliated cells,
alveolar epithelial cells or non-epithelial cells. The lower strata
of squamous metaplastic bronchial epithelium stained positively for
p63.
[0073] Squamous cell carcinoma. Twenty-six out of twenty-seven
cases (96%) of squamous cell carcinoma stained positively for p63.
In well-differentiated carcinomas staining was generally inversely
proportional to the degree of differentiation or keratinization as
assessed by counterstaining slides with eosin. Staining, noted in
basilar peristromal areas, diminished as cells became keratinized.
In many keratinizing carcinomas, p63 expression was lost with onset
of keratinization. However, in some cases, we noted keratinization
with persisting p63 positivity, suggesting relative loss of cell
regulatory suppression of proliferation concommitant with
differentiation. Poorly differentiated carcinomas showed very high
proportions (80-100%) of p63-positive nuclei.
[0074] Bronchioloalveolar carcinoma. Eight out of eight cases
(100%) showed no detectable staining of tumor cell nuclei.
[0075] Adenocarcinoma. Staining of adenocarcinomas was variable: 9
out of 19 tumors showed no detectable staining. One out of 4
adenocarcinomas with alveolar spread showed positive staining.
[0076] Adenosquanous carcinomas. Two out of 3 adenosquamos
carcinomas revealed a unique basalar staining pattern that
approximated the pattern in normal ciliated bronchial
epithelium.
[0077] Small cell undifferentiated carcinoma. Ten out often cases
(100%) showed no detectable staining of tumor cell nuclei.
[0078] Carcinoid. Six out of six cases were almost entirely
negative. Rare positive cells were of indeterminate, probably
non-neoplastic origin and appeared morphologically distinct from
the tumor cells.
[0079] Large cell carcinoma. Eight out of 12 tumors stained
positively for p63.
Discussion
[0080] p63 is a p53 homolog postulated to play a role in stem cell
commitment in squamous epithelia. The activation of cell pathways
that suppress cell division is essential for the commitment of
undifferentiated dividing basalar cells to undergo maturation.
Consistent with the known role of p53 as an activator of such
suppressive pathways in cells responding to DNA damage, p63 might
serve in a homologous physiologic role in normal stem cell
commitment.
[0081] Accumulated data from several laboratories demonstrates
expression of p63 in normal human cervical epidermis, hair
follicles, and in stratified epidermal cultures, p63 protein is
primarily restricted to cells with high proliferative potential and
is absent from the cells that are undergoing terminal
differentiation. In squamous cell carcinomas of the epidermis, the
number of cells containing p63 and their distribution was found to
depend on the degree of anaplasia. (Parsa et al, J. Invest.
Dermatol. 1999, 113:1099-1105). In highly differentiated tumors,
p63 was confined to a ring of basal-like cells surrounding, but at
a distance from, centers of terminal differentiation. In less
differentiated tumors, most cells contained p63 and their
distribution was chaotic with respect to centers of terminal
differentiation (Yamaguchi et al, Int. J. Cancer 2000, 86:684-9).
FISH analysis shows amplification of the locus containing the p63
gene in primary head and neck carcinoma and in cervical
carcinoma.
[0082] The results of the current study indicate that p53 homolog
p63 stains poorly differentiated squamous cell carcinomas in a
consistent and uniform manner with very high percentages
(approaching 100%) of tumor cells showing nuclear staining.
Staining in well differentiated squamous cell carcinomas is also
observed to be strong but is apparent in basalar, para-stromal
areas lacking keratinization. Staining of other lung tumor types
revealed no staining in undifferentiated small-cell carcinomas and
bronchioloalveolar carcinomas, and essentially negligible staining
in carcinoids.
[0083] Adenocarcinomas showed varied staining patterns. When
positive, the patterns differed from the staining pattern seen in
squamous carcinomas.
[0084] One highly unusual pattern was that seen in two cases of
adenosquamous carcinoma. The similarity of the unique basalar
staining pattern to the staining of reserve cells in normal
bronchial epithelium raises the possibility that this might be a
form of adenocarcinoma in situ.
[0085] p63 expression appears to differ between bronchioloalveolar
carcinoma and adenocarcinoma with alveolar spread: no cases of the
former stained positively, whereas 1 out of 4 cases of the latter
were p63-positive.
[0086] We have noted in other studies p63 staining in myoepithelial
cells of breast; expression in thyroid follicular epithelial cells
in Hashimoto's thyroiditis, and expression in a spectrum of other
neoplasms. These include papillary thyroid carcinoma, urothelial
carcinoma, endometrial adenocarcinoma and endocervical
adenocarcinoma (see Examples 2 and 3, infra). Of potential
significance, all of these tumors arising from non-squamous
epithelia have the capacity to undergo squamous differentiation,
and all are capable of expressing p63. In contrast, renal cell
carcinomas, which never undergo squamous differentiation, do not
express p63.
[0087] Ascertaining the specific tumor type in primary lung cancer
has important implications, both for treatment and prognosis. Small
cell undifferentiated carcinoma differs from the other histologic
types in that it is a more aggressive tumor with a greater capacity
for dissemination, therefore having a worse prognosis. For this
reason, and because of greater chemosensitivity, the treatment of
patients with small cell undifferentiated carcinoma is primarily
chemotherapy. Surgery, on the other hand, is the therapy of choice
for the remaining non-small cell lung carcinomas.
[0088] Distinction of poorly differentiated squamous carcinoma from
small cell undifferentiated carcinoma can be difficult,
particularly in small tumor samples found in cytopathologic
specimens and in bronchoscopic biopsies. Previous studies have
reported that the accuracy of diagnosis on bronchial brush
specimens was 62 to 97.5% and specifically 53.8% for small cell
undifferentiated carcinoma. Hence, using immunohistochemistry to
determine the presence of p63 in cytologic specimens, which will
distinguish a poorly-differentiated squamous cell carcinoma from a
small cell undifferentiated carcinoma has evident clinical
utility.
Example 2
Expression of p63 Protein in Subtypes of Transitional Cell and
Renal Cell Carcinomas
[0089] The p53 family of tumor suppressor genes includes p63, which
is highly expressed in the basal layers of epithelial tissues. P63
may either promote apoptosis or antagonize p53, depending upon the
expressed protein isoform. Expression of p63 has been found in
transitional epithelium. Expression of p63 in transitional cell
carcinomas was compared with expression in renal cell carcinomas.
Non-cancerous tissues associated with the urinary tract were also
examined.
Methods
[0090] Formalin-fixed, paraffin-embedded archival tissue from 81
patients was immunostained with p63 monoclonal antibody 4A4 (Santa
Cruz) reactive against all subtypes of p63 protein as described
above for Example 1. Immunostaining patterns and routine H&E
staining patterns were interpreted by four observers. Nuclear
staining was considered positive.
Results
[0091] Normal tissue. Benign adrenal cortex (1), prostate (2), and
other benign kidney specimens were negative for p63 staining.
[0092] Transitional cell carcinomas. 96% of transitional cell
carcinomas stained diffusely for p63, (low grade bladder 9/9, high
grade bladder 10/22, ureter 2/2, renal pelvis 8/8). Bladder
carcinoma-in-situ cases all stained (n=18) positively to varying
degrees, including basal (about 1/2 of cases), diffuse, and rare.
Cystitis cystica with focal dysplasia showed strong basal staining
(1/1) for p63.
[0093] Renal cell carcinomas. In contrast all renal cell carcinoma
specimens (21/21), regardless of type and grade, (including clear
cell, papillary, chromophobe, and collecting duct) were negative as
were xanthogranulomatous pyelonephritis (1).
CONCLUSION
[0094] Staining of p63 is strongly positive in transitional cell
carcinomas and uniformly negative in renal cell carcinomas. This
sharp distinction may be a useful diagnostic tool in preoperative
evaluation and therapeutic planning in patients with upper urinary
tract lesions.
Example 3
Expression of p63 in Papillary Thyroid Carcinoma and in Hashimoto's
Thyroiditis
[0095] Nuclear proteins were discovered as a consequence of a
screen for p53-homologous genes. Thus far, p63 expression has been
demonstrated in basal cells of squamous epithelium and in
urothelium. Immunohistochemical detection of p63 expression was
evaluated in normal and abnormal thyroid tissue.
Materials and Methods
[0096] Five micron sections from routinely fixed and processed
archival thyroid resection specimens were pretreated with citric
acid pH 6.0 for antigen retrieval at 100.degree. C. for 5 minutes,
then incubated overnight with anti-p63 monoclonal antibody 4A4
(Santa Cruz). Slides were stained using a streptavidinbiotin kit
(BioGenex), followed by reaction with diaminobenzidine, and then
counterstaining with hematoxylin.
Results
[0097] Conditions negative for p63. No p63 expression was seen in
normal thyroid tissue, twelve follicular adenomata, eleven Hurthle
cell adenomata, six medullary carcinomas, two anaplastic
carcinomas, and six Hurthle cell carcinomas. There was rare
staining in one of seven follicular carcinomas. In addition, there
was no p63 staining in ten nodular goiter samples, and one of 8
Grave's disease samples stained.
[0098] Hashimoto's thyroiditis. In contrast, seventy five percent
(12/16) of Hashimoto's thyroiditis cases and seventy six percent
(22/29) of papillary thyroid carcinomas showed epithelial cells
with nuclear positivity for p63. Staining of tumor cells ranged
from strong and/or frequent to weak and/or uncommon. In Hashimoto's
cases, staining was characteristically intense but limited to
several follicles. Of note, on parallel sections, CK19 positivity
was found in p63-positive carcinomas but also in numerous benign
follicles within areas of p63-positive thyroiditis.
Conclusions
[0099] p63 is expressed in papillary thyroid carcinoma and in
Hashimoto's thyroiditis. Considering the debated association of
papillary thyroid carcinoma with Hashimoto's thyroiditis, it is
possible that p63 may be a potential pathobiologic link between the
two disorders.
[0100] p63 may also be of use in distinguishing papillary carcinoma
from other thyroid neoplasms, particularly on fine needle
aspirates. This observation has particular relevance for avoiding
unnecessary surgery because of a misdiagnosis or the inability to
diagnose nodular goiter instead of papillary carcinoma.
Example 4
Detecting p63 in Non-Traumatically Obtained Samples
[0101] In this example, we describe results of examination of
pre-cancerous squamous intra-epithelial lesions of the uterine
cervix compared with normal cervical epithelium.
Methods
[0102] Cervical and vaginal epithelial cells were obtained from
gentle swabbing of the uppermost layers and subjected to
immunostaining for p63 as described infra.
Results
[0103] In contrast to normal epithelium, in which p63 is expressed
in the lower cell layers of the epithelium, in accordance with
findings by others, p63 expression is found in the uppermost layers
of high grade squamous intraepithelial lesions.
Discussion
[0104] Since the papanicolaou "pap" smear of the cervical and
vaginal epithelium involves sampling of the uppermost cell layers
of these epithelia via gentle, non-traumatic swabbing, brushing or
other non-traumatic sampling means, detection of p63 alone, or in a
multiplex panel with other markers, in cells from cervix or vaginal
squamous epithelium can be used to signify the presence of a
clinically advanced squamous intraepithelial lesion. This can be
adapted for automated screening for aberrant expression of p63 in
cells from the uppermost layers of cervical-vaginal squamous
epithelium in cervical-vaginal smears, as a substitute for manual
screening presently performed on millions of pap smears to detect
such lesions.
[0105] Similarly, detection of cancer or pre-cancerous changes by
detecting p63 (at the gene, mRNA, or protein level) in a cell or
fluid sample following non-traumatic sampling (such as lavage,
gentle brush, wash) of other epithelia in which abnormal
p63-positive epithelia replace normal epithelia (the uppermost
layer of cells of which are normally p63-negative) constitutes
another claim of this patent. This applies not only to cervical
vaginal sampling, but to sampling of oral, upper respiratory tract,
lower respiratory tract, esophageal, cyst content samples or cyst
lining, urinary tract, endocervical, endometrial, and other
surfaces or mucosae.
[0106] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0107] It is further to be understood that all values are
approximate, and are provided for description.
[0108] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
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