U.S. patent application number 10/071521 was filed with the patent office on 2003-04-17 for pin1 as a marker for prostate cancer.
This patent application is currently assigned to Pintex Pharmaceuticals, Inc.. Invention is credited to Bao, Lere, Wang, Da Gong.
Application Number | 20030073096 10/071521 |
Document ID | / |
Family ID | 26952505 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030073096 |
Kind Code |
A1 |
Bao, Lere ; et al. |
April 17, 2003 |
Pin1 as a marker for prostate cancer
Abstract
This invention provides methods for diagnosing prostate cancer,
methods for measuring the aggressiveness of prostate cancer, and
methods for identifying prostate cancer likely to metastasize. The
diagnostic and prognostic assays of this invention include methods
involving the antibody-based detection of Pin 1 and the
amplification of Pin 1 RNA. The diagnostic and prognostic assays of
this invention may be used in combination with other methods of
prostate cancer diagnosis including the PSA test, digital rectal
exam, and Gleason prostate tumor grading system.
Inventors: |
Bao, Lere; (Newton, MA)
; Wang, Da Gong; (Chestnut Hill, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Pintex Pharmaceuticals,
Inc.
313 Pleasant Street
Watertown
MA
02472
|
Family ID: |
26952505 |
Appl. No.: |
10/071521 |
Filed: |
February 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60267552 |
Feb 9, 2001 |
|
|
|
60347546 |
Jan 10, 2002 |
|
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Current U.S.
Class: |
435/6.15 ;
435/7.23 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/158 20130101; G01N 33/57434 20130101 |
Class at
Publication: |
435/6 ;
435/7.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What is claimed is:
1. A method for facilitating the diagnosis of prostate cancer in a
subject, comprising: assessing the level of Pin1 in a biological
sample from the subject, wherein an elevation in the level of Pin1
is indicative of prostate cancer; and evaluating a TDPCA on the
subject such that the diagnosis of prostate cancer is
facilitated.
2. A method for facilitating the diagnosis of prostate cancer in a
subject, comprising: assessing the level of Pin1 in a biological
sample from the subject, wherein an elevation in the level of Pin1
is indicative of prostate cancer, and wherein the subject was
previously categorized by a TDPCA as being likely to have prostate
cancer.
3. A method for measuring the aggressiveness of prostate cancer in
a subject, comprising assessing the level of Pin1 in a biological
sample from the subject, wherein an elevation in the level of Pin1
is indicative of the aggressiveness of the prostate cancer.
4. A method for identifying metastatic prostate cancer in a
subject, comprising assessing the level of Pin1 in a biological
sample from the subject, wherein an elevation in the level of Pin1
is indicative of metastatic prostate cancer.
5. The method of claim 1, 2, 3, or 4, wherein assessing the level
of Pin1 in a biological sample from the subject comprises
contacting the biological sample with an antibody to Pin1 or a
fragment thereof, determining the amount of binding of the antibody
to the biological sample; and comparing the amount of antibody
bound to the biological sample to a predetermined base level.
6. The method of claim 1 or 2, wherein the subject is receiving, or
has received, therapy for a state associated with prostate cancer
and the level of Pin1 is indicative of the subject's response to
the therapy.
7. The method of claim 1, 2, 3, or 4, wherein the biological sample
comprises a body fluid.
8. The method of claim 7, wherein the body fluid is selected from
the group consisting of blood, serum, semen, prostate fluid,
seminal fluid, and urine.
9. The method of claim 5, wherein the antibody is a polyclonal
antibody.
10. The method of claim 1, 2, 3, or 4, wherein the biological
sample comprises prostate tissue.
11. The method of claim 5, wherein the antibody is a monoclonal
antibody.
12. The method of claim 5, wherein the antibody is a labeled
antibody.
13. The method of claim 12, wherein the amount of binding of the
antibody to the biological sample is determined by the intensity of
the signal emitted by the labeled antibody.
14. The method of claim 12, wherein the amount of binding of the
antibody to the biological sample is determined by the number cells
in the biological sample bound to the labeled antibody.
15. The method of claim 5, wherein the amount of binding of the
antibody to the biological sample is determined by a
radioimmunoassay.
16. The method of claim 5, wherein the amount of binding of the
antibody to the biological sample is determined by an enzyme
immunoassay.
17. A method of diagnosing prostate cancer in a subject,
comprising: detecting a level of Pin1 nucleic acid in a biological
sample; and comparing the level of Pin1 in the biological sample
with a level of Pin1 in a control sample, wherein an elevation in
the level of Pin1 in the biological sample compared to the control
sample is indicative of prostate cancer.
18. A method of diagnosing prostate cancer metastasis in a subject,
comprising the steps of: detecting a level of Pin1 nucleic acid in
a biological sample; and comparing the level of Pin1 in the
biological sample with a level of Pin1 in a control sample, wherein
an elevation in the level of Pin1 in the biological sample compared
to the control sample is indicative of prostate cancer
metastasis.
19. The method of claim 17 or 18, wherein the detecting a level of
Pin1 nucleic acid in a biological sample comprises amplifying Pin1
RNA.
20. The method of claim 19, wherein the biological sample is
selected from the group consisting of: saliva, sputum, mucus, bone
marrow, serum, blood, urine, lymph, tears, semen, seminal fluid,
prostate fluid, and prostate tissue.
21. The method of claim 20, wherein the biological sample is a
blood sample.
22. The method of claim 20, wherein the biological sample is a
prostate tissue sample.
23. The method of claim 19, wherein the amplifying comprises
performing a polymerase chain reaction.
24. The method of claim 1 or 2, wherein the TDPCA is a digital
rectal exam showing the subject as having a prostate
abnormality.
25. The method of claim 1 or 2, wherein the TDPCA is a test for the
detection of a prostate cancer marker is selected from the group
consisting of: prostatic acid phosphatase, prostate secreted
protein, prostate specific membrane antigen, human kallekrein 2,
prostate specific transglutaminase, and interleukin 8.
26. The method of claim 1 or 2, wherein the TDPCA is a test for the
detection of prostate-specific antigen.
27. The method of claim 1 or 2, wherein the TDPCA is a test for the
detection of prostate-specific antigen in the blood serum of the
subject.
28. The method of claim 27, wherein the subject has a blood serum
concentration of prostate-specific antigen of between about 2 and
about 10 ng/ml.
29. The method of claim 27, wherein the subject has a blood serum
concentration of the prostate-specific antigen of between about 4
and about 8 ng/ml.
30. The method of claim 27, wherein the subject has a blood serum
concentration of the prostate-specific antigen of between about 3
and about 7 ng/ml and the subject is between about 40 and about 60
years old.
31. The method of claim 27, wherein the subject has a blood serum
concentration of the prostate-specific antigen of between about 5
and about 9 ng/ml and the subject is between about 60 and about 80
years old.
32. The method of claim 27, wherein the subject has a blood serum
concentration of the prostate-specific antigen of less than about 4
ng/ml and a PSA velocity of greater than about 0.7 ng/ml per
year.
33. The method of claim 27, wherein the subject has a blood serum
concentration of the prostate-specific antigen of between about 4
and about 8 ng/ml and a percent-free prostate-specific antigen of
between about 15 and about 25%.
34. The method of claim 4, wherein the prostate cancer sample has a
Gleason sum of 4-9.
35. The method of claim 34, wherein the Gleason sum is 6 or 7.
36. A method for determining whether a subject having prostate
cancer is likely to respond to treatment comprising a Pin1
inhibitor compound, the method comprising: assessing the level of
Pin1 in a test sample from the subject; and comparing the level of
Pin1 in the test sample to the level of Pin1 in normal tissue,
whereby an increased level of Pin1 in the test sample is indicative
that the subject is likely to respond to treatment comprising a
Pin1 inhibitor compound.
37. A method of determining if a subject is at risk of developing
metastatic prostate cancer comprising: assessing the level of Pin1
in a test sample from the subject; and determining if the level of
Pin1 in the test sample is indicative of a cancer that will become
metastatic.
38. The method of claim 37 wherein the test sample is collected by
needle biopsy.
39. The method of claim 37 where the test sample is collected from
human serum.
40. A method of determining if a subject is at risk of developing
PSA failure comprising: assessing the level of Pin1 in a test
sample from the subject; and determining if the level of Pin1 in
the test sample is indicative of developing PSA failure.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/267,552, filed Feb. 9, 2001 and U.S.
Provisional Application Serial No. 60/XXX,XXX, filed on Jan. 10,
2002, the entire contents of which are incorporated herein by this
reference.
BACKGROUND OF THE INVENTION
[0002] The increased number of cancer cases reported in the United
States, and, indeed, around the world, is a major concern.
Currently there are only a handful of treatments available for
specific types of cancer, and these treatments require not only an
early detection of the malignancy, but also a reliable assessment
of the severity of the malignancy. Carcinoma of the prostate (PCA)
is the most frequently diagnosed cancer in men in the United
States, and is the second leading cause of male cancer deaths (Karp
et al., 1996, Cancer Res. 56:5547-5556). Over 40,000 Americans are
estimated to have died of PCA in 1995, and about 244,000 new cases
of prostate cancer were detected (Cancer Facts and Figures-1995,
American Cancer Society, Inc., 1995) and these numbers have
increased annually at an alarming rate. Further, the rate of
appearance of prostate cancer in African-American men is 37% higher
than for their white counterparts (Jaroff, L. (Apr. 1, 1996),
Time).
[0003] An unusual challenge presented by prostate cancer is that
most prostate tumors do not represent life threatening conditions.
Projections from autopsy surveys indicate that as many as 11
million American men have prostate cancer (Dhom, 1983, J. Cancer
Res. Clin. Oncol., 106:210-218). Cancer cells are generally found
in the prostates of men who live into their seventies or eighties.
However not all of these men develop prostate cancer (PCA), and
autopsies show microscopic clusters of prostate cancer cells in
one-third of men who die of other causes (Thayer, W., (Mar. 19,
1996), quoted in Nutr. Act. Newsletter, 23(2):12). Death rates from
prostate cancer rise after age 55, and new cases of prostate
cancer, are increasing even faster than the death rate. These
figures are consistent with clinical observations of prostate
carcinomas, which normally exhibit a slow and lingering course of
progression. Such disease progression results in relatively few
prostate tumors developing into cases of clinical concern during
the lifetime of the patient. If, upon detection with available
methods, the cancer appears well-differentiated, organ-confined and
focal, treatment normally cannot extend the life expectancy of
older patients.
[0004] Unfortunately, prostate carcinomas that are progressive in
nature frequently have already metastasized by the time of clinical
detection with available methods. Survival rates for individuals
with metastatic prostate cancer are quite low. Between these two
extremes are patients with prostate tumors that will metastasize
during their lifetimes, but have not yet done so. For these
patients, surgical removal of the prostate is curative and extends
life expectancy. Therefore, accurate determination of which group a
newly diagnosed patient falls into is critical in determining
optimal treatment and patient survival.
[0005] The current primary diagnostic tool for disorders of the
prostate is measurement of the level of prostate-specific antigen
(PSA) in blood, which in normal men ranges from 0 to 4
nanograms/milliliters. The presence of Prostate Specific Antigen
(PSA) can be measured with relative ease from blood samples using
standard antibody-based detection kits. Prostate enlargement, a
condition known as benign prostatic hyperplasia (BPH), is found in
about half of men over age 45. With BPH, PSA levels rise in
proportion to prostate size, possibly obscuring diagnosis of PCA.
In addition, a significant proportion of men with PCA have normal
PSA levels. The PSA test is somewhat non-specific for
distinguishing PCA and DPH, and produces a degree of false negative
results (Garnick, M., (1993), Am. Inst. Med., 118:804-818). In the
majority of cases, PSA elevation is due to BPH or prostatitis
rather than carcinoma. The PSA test, a major advance over previous
procedures, thus leaves much to be desired.
[0006] Although clinical and pathologic stage and histological
grading systems (e.g., Gleason's) have been used to indicate
prognosis for groups of patients based on the degree of tumor
differentiation or the type of glandular pattern (Carter and
Coffey, In: J. P. Karr and H. Yamanak (eds.), Prostate Cancer: The
Second Tokyo Symposium, pp. 19 -27, New York: Elsevier, 1989.;
Diamond et al., J. Urol., 128: 729-734, 1982), these systems do not
adequately predict the progression rate of the cancer. While the
use of computer-system image analysis of histologic sections of
primary lesions for "nuclear roundness" has been suggested as an
aide in the management of individual patients (Diamond et al.,
1982, J. Urol., 128:729-734), this method is of limited use in
studying the progression of the disease.
[0007] There currently is a need for new methods in the fight
against prostate cancer and it would therefore be beneficial to
provide specific methods and reagents for the diagnosis, staging,
prognosis, and monitoring of prostate cancer.
SUMMARY OF THE INVENTION
[0008] This invention provides a method for facilitating the
diagnosis of prostate cancer in a subject, comprising: assessing
the level of Pin1 in a biological sample from the subject, wherein
an elevation in the level of Pin1 is indicative of prostate cancer;
and evaluating a TDPCA on the subject such that the diagnosis of
prostate cancer is facilitated.
[0009] This invention also provides a method for facilitating the
diagnosis of prostate cancer in a subject, comprising: assessing
the level of Pin1 in a biological sample from the subject, wherein
an elevation in the level of Pin1 is indicative of prostate cancer,
and wherein the subject was previously categorized by a TDPCA as
being likely but not certain to have prostate cancer.
[0010] In another aspect, this invention includes a method for
measuring the aggressiveness of prostate cancer in a subject,
comprising assessing the level of Pin1 in a biological sample from
the subject, wherein an elevation in the level of Pin1 is
indicative of the aggressiveness of the prostate cancer.
[0011] Also provided by this invention is a method for identifying
metastatic prostate cancer in a subject, comprising assessing the
level of Pin1 in a biological sample from the subject, wherein an
elevation in the level of Pin1 is indicative of metastatic prostate
cancer.
[0012] The invention further provides a method for identifying a
subject at risk for developing metastatic prostate cancer
comprising assessing the level of Pin1 in a biological sample from
the subject, wherein an elevation in the level of Pin1 is
indicative of that the subject is at risk for developing metastatic
prostate cancer.
[0013] Further, provided by this invention are the above methods,
where assessing the level of Pin1 in a biological sample from the
subject includes contacting the biological sample with an antibody
to Pin1 or a fragment thereof, determining the amount of binding of
the antibody to the biological sample; and comparing the amount of
antibody bound to the biological sample to a predetermined base
level. The amount of binding of the antibody to the biological
sample can be determined by the intensity of the signal emitted by
the labeled antibody and/or by the number cells in the biological
sample bound to the labeled antibody.
[0014] Also encompassed by this invention are the above methods
wherein the level of Pin1 is assessed by detecting a level of Pin1
nucleic acid in a biological sample; and comparing the level of
Pin1 in the biological sample with a level of Pin1 in a control
sample. For example, in certain embodiments Pin1 nucleic acid is
detected using hybridization probes and/or nucleic acid
amplification methods.
[0015] The diagnostic and prognostic assays of this invention can
be used in combination with other methods of prostate cancer
diagnosis. Examples of prostate diagnostic methods that can be used
in combination with the assays of the invention include, but are
not limited to, current diagnostic methods for PCA known to medical
practitioners skilled in the art such as the PSA test, rectal
examination, transrectal ultrasonography or magnetic resonance
imaging (MRI), bone scanning, X-rays, skeletal survey, intravenous
pyelography, CAT-scan, and biopsy. In an embodiment, the subject in
the above diagnostic and prognostic assays of this invention was
identified by a digital rectal exam as having a prostate
abnormality. In another embodiment, the subject was identified as
having elevated PSA levels. In other embodiments, markers for
prostate cancer which can be used in combination with the methods
of the invention include prostatic acid phosphatase, prostate
secreted protein, prostate specific membrane antigen, human
kallekrein 2, prostate specific transglutaminase, keratin-19, and
interleukin 8.
[0016] The diagnostic and prognostic assays of this invention are
particularly useful when the PSA level falls within a gray zone
where it is not clear whether a prostate biopsy should be
performed. For example, the diagnostic and prognostic assays of
this invention may be used when the subject has a blood serum
concentration of the prostate-specific antigen of between about 4
and about 8 ng/ml. The prognostic assays of this invention are also
particularly useful when the Gleason sum falls within a gray zone
where the prognosis is usually unclear, for example a Gleason score
between 4 and 8, or a Gleason score of 6 to 7.
[0017] The diagnostic and prognostic assays of this invention can
also be used in combination with other methods of prostate cancer
staging. The assays of this invention are particularly useful when
conventional staging methods leave unclear the prognosis of the
prostate cancer. For example, the aggressiveness of a T2 or T3
stage prostate cancer can be better assessed using the assays of
this invention.
[0018] The present invention also includes methods of determining
whether a subject is likely to respond to a treatment regimen
comprising agents, or modulators which have a stimulatory or
inhibitory effect on Pin1 activity (e.g., Pin1 gene expression or
enzyme activity). For example, Pin1 inhibitors can be administered
to individuals, such as those identified using the diagnostic and
prognostic methods of the invention as having elevated levels of
Pin1, to treat (prophylactically or therapeutically) disorders
(e.g, proliferative disorders such as cancer) associated with
aberrant Pin1 activity. In conjunction with such treatment,
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug.
[0019] Thus, a physician or clinician may consider applying
knowledge obtained in relevant pharmacogenomics studies in
determining whether use of a Pin1 inhibitor would be efficatious.
Information generated from pharmacogenomics approaches can be used
to determine appropriate dosage and treatment regimens for
prophylactic or therapeutic treatment an individual. This
knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a Pin1 molecule or Pin1 modulator, such as a modulator identified
by one of the exemplary screening assays described herein. Thus,
the methods described herein can be used to determine which Pin1
modulator or inhibitor to administer and whether to administer a
Pin1 molecule or Pin1 modulator as well as tailoring the dosage
and/or therapeutic regimen of treatment with a Pin1 molecule or
Pin1 modulator.
[0020] The diagnostic and prognostic assays of this invention are
also useful in assessing the recovery of a subject who is
receiving, or has received, therapy for a state associated with
prostate cancer. For example, the assays of this invention can be
used alone or in combination with PSA failure to assess recovery
after prostate treatment (i.e. prostate removal surgery).
[0021] The invention also encompasses kits for detecting the
presence of a Pin1 polypeptide or nucleic acid in a biological
sample according to the methods described herein. Such kits can be
used to determine if a subject is suffering from or is at increased
risk of developing prostate cancer, and for identifying subjects
who have, or are at risk of developing metastatic prostate cancer.
For example, the kit can comprise a labeled compound or agent
capable of detecting Pin1 or an mRNA encoding a Pin1 in a
biological sample and means for determining the amount of the Pin1
or Pin1 mRNA in the sample (e.g., an antibody which binds the
polypeptide or an oligonucleotide probe which binds to DNA or mRNA
encoding Pin1). Kits can also include immunomagnetic beads that can
be used to facilitate serum assays. Kits can also include
instructions for carrying out the methods of the invention and/or
for interpreting the results obtained from using the kit.
[0022] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide corresponding to a marker of the invention;
and, optionally, (2) a second, different antibody which binds to
either the polypeptide or the first antibody and is conjugated to a
detectable label.
[0023] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a polypeptide corresponding to a marker of the invention
or (2) a pair of primers useful for amplifying a nucleic acid
molecule corresponding to a marker of the invention. The kit can
also comprise, e.g., a buffering agent, a preservative, or a
protein stabilizing agent. The kit can further comprise components
necessary for detecting the detectable label (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the
biological sample. Each component of the kit can be enclosed within
an individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0024] In certain embodiments kit for diagnosing prostate cancer
includes: at least one reagent for assaying levels of Pin1 in a
sample from a subject, and instructions for using the at least one
reagent to diagnose prostate cancer based on assay levels of Pin1
in a sample from a subject. An example of a kit for assessing the
aggressiveness of prostate cancer in a subject includes: at least
one reagent for assaying levels of Pin1 in a sample from a subject,
and instructions for using the at least one reagent to assess the
aggressiveness prostate cancer based on assay levels of Pin1 in a
sample from a subject.
DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows the correlation between Pin 1 expression and
Gleason sum based on 42 specimens of human prostate carcinomas with
Gleason scores of 4-10. Each symbol represents a specimen from a
different individual.
[0026] FIG. 2 shows the recurrence free survival as a function of
time using Pin 1 High (P1H) intensity value of 100 as a cutoff
between the two groups.
[0027] FIG. 3 shows the recurrence free survival as a function of
time using Pin 1 Average (P1A) intensity value of 100 as a cutoff
between the two groups.
[0028] FIG. 4 shows the recurrence free survival as a function of
time using Pin % High (P%H) intensity of value of 70 as a cutoff
between the two groups.
[0029] FIG. 5 shows the recurrence free survival as a function of
time using Pin % Average (P%A) intensity of value of 60 as a cutoff
between the two groups.
[0030] FIG. 6 depicts a multivariate analysis of Pin 1 as a marker
of biochemical recurrence in Gleason 6 and 7 patients using a P1H
score of 100 as a cutoff between the two groups.
[0031] FIG. 7 depicts analysis of recurrence free survival as a
function of time using visual Pin1 Intensity High (PMIH) values of
0 and >0 as a cutoff between the two groups.
DETAILED DESCRIPTION OF THE INVENTION
[0032] This invention provides methods for diagnosing prostate
cancer, methods for measuring the aggressiveness of prostate
cancer, and methods for identifying prostate cancer likely to
metastasize. The diagnostic and prognostic assays of this invention
include methods involving the antibody-based detection of Pin1 and
the amplification of Pin1 RNA. The diagnostic and prognostic assays
of this invention may be used in combination with other methods of
prostate cancer diagnosis including the PSA test, digital rectal
exam, and Gleason prostate tumor grading system. The methods are an
improvement over currently available methods due to the precise
nature of the measurement and the ability to use these methods
without invasive surgery.
[0033] Definitions
[0034] "Pin1" is a highly conserved protein that catalyzes the
isomerization of only phosphorylated Ser/Thr-Pro bonds (Accession
Nos. AAC50492 and U49070; Lu et al., 1996 Nature 380:544-547;
Rananathan, R. et al. 1997 Cell 89:875-86; Yaffe, et al. 1997,
Science 278:1957-1960; Shen, et al. 1998,Genes Dev. 12:706-720; Lu,
et al. 1999 Science 283:1325-1328; Crenshaw, et al. 1998, Embo J.
17:1315-1327; Lu, et al. 1999, Nature 399:784-788; Zhou, et al.
1999, Cell Mol. Life Sci. 56:788-806). In addition, Pin1 contains
an N-terminal WW domain, which functions as a phosphorylated
Ser/Thre-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol.
Biol. 65:113-32). This phosphorylation-depende- nt interaction
targets Pin1 to a subset of phosphorylated substrates, including
Cdc25, Wee 1, Myt1, Tau-Rad4, and the C-terminal domain of RNA
polymerase II large domain (Crenshaw, D. G., et al. (1998) Embo. J.
17:1315-27; Shen, M. (1998) Genes Dev. 12:706-20; Wells, N. J.
(1999) J. Cell. Sci. 112: 3861-71)
[0035] "Cancer" includes a malignant neoplasm characterized by
deregulated or uncontrolled cell growth. The term "cancer" includes
primary malignant tumors (e.g., those whose cells have not migrated
to sites in the subject's body other than the site of the original
tumor) and secondary malignant tumors (e.g., those arising from
metastasis, the migration of tumor cells to secondary sites that
are different from the site of the original tumor).
[0036] "Anaplasia" refers to the histological features of cancer.
These features include derangement of the normal tissue
architecture, the crowding of cells, lack of cellular orientation
termed dyspolarity, cellular heterogeneity in size and shape termed
"pleomorphism." The cytologic features of anaplasia include an
increased nuclear-cytoplasmic ratio (the nuclear-cytoplasmic ratio
can be over 50% for malignant cells), nuclear pleomorphism,
clumping of the nuclear chromatin along the nuclear membrane,
increased staining of the nuclear chromatin, simplified endoplasmic
reticulum, increased free ribosomes, pleomorphism of mitochondria,
decrease in size and number of organelles, enlarged and increased
numbers of nucleoli, and sometimes the presence of intermediate
filaments.
[0037] "Neoplasia" or "neoplastic transformation" is the pathologic
process that results in the formation and growth of a neoplasm,
tissue mass, or tumor. Such process includes uncontrolled cell
growth, including either benign or malignant tumors. Neoplasms
include abnormal masses of tissue, the growth of which exceeds and
is uncoordinated with that of the normal tissues and persists in
the same excessive manner after cessation of the stimuli which
evoked the change. Neoplasms may show a partial or complete lack of
structural organization and functional coordination with the normal
tissue, and usually form a distinct mass of tissue.
[0038] Neoplasms tend to morphologically and functionally resemble
the tissue from which they originated. For example, neoplasms
arising within the islet tissue of the pancreas resemble the islet
tissue, contain secretory granules, and secrete insulin. Clinical
features of a neoplasm may result from the function of the tissue
from which it originated.
[0039] By assessing the histologic and other features of a
neoplasm, it can be determined whether the neoplasm is benign or
malignant. Invasion and metastasis (the spread of the neoplasm to
distant sites) are definitive attributes of malignancy. Despite the
fact that benign neoplasms may attain enormous size, they remain
discrete and distinct from the adjacent non-neoplastic tissue.
Benign tumors are generally well circumscribed and round, have a
capsule, and have a grey or white color, and a uniform texture. By
contrast, malignant tumor generally have fingerlike projections,
irregular margins, are not circumscribed, and have a variable color
and texture. Benign tumors grow by pushing on adjacent tissue as
they grow. As the benign tumor enlarges it compresses adjacent
tissue, sometimes causing atrophy. The junction between a benign
tumor and surrounding tissue may be converted to a fibrous
connective tissue capsule allowing for easy surgical remove of
benign tumors. By contrast, malignant tumors are locally invasive
and grow into the adjacent tissues usually giving rise to irregular
margins that are not encapsulated making it necessary to remove a
wide margin of normal tissue for the surgical removal of malignant
tumors. Benign neoplasms tends to grow more slowly than malignant
tumors. Benign neoplasms also tend to be less autonomous than
malignant tumors. Benign neoplasms tend to closely histologically
resemble the tissue from which they originated. More high
differentiated cancers, cancers that resemble the tissue from which
they originated, tend to have a better prognosis than poorly
differentiated cancers. Malignant tumors are more likely than
benign tumors to have an aberrant function (i e. the secretion of
abnormal or excessive quantities of hormones).
[0040] "Prostate cancer" is an adenocarcinoma of the prostate
gland. The prostate gland is a walnut sized organ that helps the
body form semen. The prostate gland is located where the urethra
joins the neck of the urinary bladder. The term "prostate cancer"
(PCA) as used herein refers to both the appearance of a palpable
tumor of the prostate, and also to microscopically detectable
neoplastic or transformed cells in the prostate gland. In the
latter case, the said cytologically-detectable prostate cancer may
be asymptomatic, in that neither the patient nor the medical
practitioner detects the presence of the cancer cells. Prostate
cancer usually progresses gradually and usually expands to adjacent
tissue and lymph glands before detection. In the event that
prostate cancer metastasizes to additional sites distal to the
prostate, the condition is described as metastatic cancer (MC), or
prostate cancer, metastasized, to distinguish this condition from
organ-confined prostate cancer. PCA fatality results from
metastatic dissemination of prostatic adenocarcinoma cells to
distant sites, usually in the axial skeleton. As the disease
progresses, other organs including the bones, lungs, and liver can
become cancerous.
[0041] "Benign prostatic hypertrophy," comprises an age-related
non-cancerous enlargement of the prostate, and affects more than
50% of men over age 45. Benign prostatic hypertrophy (BPH) may be
asymptomatic, that is, have no negative consequences for the
individual, and is not intended here to imply the necessary
development of prostate cancer. BPH is accompanied by an increase
in production of the protein prostate specific antigen,
proportional to the extent of growth of the prostate gland. For
this reason, the diagnosis of PCA in a BPH patient may be difficult
to distinguish from further asymptomatic growth by sole use of the
PSA test.
[0042] BPH may appear as or may progress to "problematic" prostatic
hyperplasia, with symptoms that include urinary urgency, frequency,
and hesitancy, and penile erectile difficulties. Since these same
symptoms are associated with PCA (M. Garnick, (1993), Annals Int.
Med., 118(10):804-818), the clinician distinguishes PCA and
problematic prostatic hyperplasia by the suddenness in onset of
symptoms, and by additional diagnostic tests. BPH and problematic
prostatic hypertrophy may also progress to PCA, however these terms
are meant neither to exclude nor to imply disease progression, as
the full range of diagnostic possibilities is found for the BPH
patient population as for the normal subject.
[0043] "Invasive" or "aggressive" as used herein with respect to
cancer refers to the proclivity of a tumor for expanding beyond its
boundaries into adjacent tissue, or to the characteristic of the
tumor with respect to metastasis (Darnell, J. (1990), Molecular
Cell Biology, Third Ed., W. H. Freeman, NY). Invasive cancer can be
contrasted with organ-confined cancer. For example, a basal cell
carcinoma of the skin is a non-invasive or minimally invasive
tumor, confined to the site of the primary tumor and expanding in
size, but not metastasizing. In contrast, the cancer melanoma is
highly invasive of adjacent and distal tissues. The invasive
property of a tumor is often accompanied by the elaboration of
proteolytic enzymes, such as collagenases, that degrade matrix
material and basement membrane material to enable the tumor to
expand beyond the confines of the capsule, and beyond confines of
the particular tissue in which that tumor is located.
[0044] "Organ-confined" as used herein with respect to prostate
cancer refers to prostate cancer that has not metastasized beyond
the boundaries of the prostate gland, i.e., has not been found by
techniques familiar to those skilled in the art to occur in any
organs or tissues beyond the prostate gland. It can not be ruled
out, however, that some number of cells have metastasized, however
they are not detected by ordinary techniques used by those with
skill in the art.
[0045] The term "metastasis" as used herein refers to the condition
of spread of cancer from the organ of origin to additional distal
sites in the patient. The process of tumor metastasis is a
multistage event involving local invasion and destruction of
intercellular matrix, intravasation into blood vessels, lymphatics
or other channels of transport, survival in the circulation,
extravasation out of the vessels in the secondary site and growth
in the new location (Fidler, et al., Adv. Cancer Res. 28, 149-250
(1978), Liotta, et al., Cancer Treatment Res. 40, 223-238 (1988),
Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and Zetter, N.
Eng. J. Med. 322, 605-612 (1990)). Increased malignant cell
motility has been associated with enhanced metastatic potential in
animal as well as human tumors (Hosaka, et al., Gann 69, 273-276
(1978) and Haemmerlin, et al., Int. J. Cancer 27, 603-610
(1981)).
[0046] "Metastatic prostate cancer" includes prostate cancers which
have spread to regional lymph nodes or to distant sites. The most
common site for prostate cancer metastasis is bone. Prostate cancer
bone metastases are, on balance, characteristically osteoblastic
rather than osteolytic (i.e., resulting in net bone formation).
Bone metastases are found most frequently in the spine, followed by
the femur, pelvis, rib cage, skull and humerus. Other common sites
for metastasis include lymph nodes, lung, liver and brain.
Metastatic prostate cancer is typically diagnosed by open or
laparoscopic pelvic lymphadenectomy, whole body radionuclide scans,
skeletal radiography, and/or bone lesion biopsy.
[0047] "Subject" includes living organisms, e.g., prokaryotes and
eukaryotes. Examples of subjects include mammals, e.g., humans,
dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice,
rabbits, rats, and transgenic non-human animals. Most preferably
the subject is a human.
[0048] "TDPCA" is a test done to facilitate diagnosis of prostate
cancer. For example, TDPCA includes a test for the detection of a
prostate cancer marker is selected from the group consisting of:
prostatic acid phosphatase, prostate secreted protein, prostate
specific membrane antigen, human kallekrein 2, prostate specific
transglutaminase, and interleukin 8. Examples of TDPCA include the
PSA test, rectal examination, transrectal ultrasonography or
magnetic resonance imaging (MRI), bone scanning, X-rays, skeletal
survey, intravenous pyelography, CAT-scan, and biopsy. In an
embodiment, TDPCA is a digital rectal exam. In a preferred
embodiment, TDPCA is a test for the detection of prostate-specific
antigen in the blood serum of the subject. TDPCA does not include a
test to detect Pin1.
[0049] "Biological samples" include solid and body fluid samples.
The biological samples of the present invention may include cells,
protein or membrane extracts of cells, blood or biological fluids
such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
Examples of solid biological samples include samples taken from
feces, the rectum, central nervous system, bone, breast tissue,
renal tissue, the uterine cervix, the endometrium, the head/neck,
the gallbladder, parotid tissue, the prostate, the brain, the
pituitary gland, kidney tissue, muscle, the esophagus, the stomach,
the small intestine, the colon, the liver, the spleen, the
pancreas, thyroid tissue, heart tissue, lung tissue, the bladder,
adipose tissue, lymph node tissue, the uterus, ovarian tissue,
adrenal tissue, testis tissue, the tonsils, and the thymus.
Examples of "body fluid samples" include samples taken from the
blood, serum, semen, prostate fluid, seminal fluid, urine, saliva,
sputum, mucus, bone marrow, lymph, and tears. For amplifying Pin1
RNA, the preferred samples include peripheral venous blood samples
and prostate tissue samples. Samples for use in the assays of the
invention can be obtained by standard methods including venous
puncture and surgical biopsy. In one embodiment, the biological
sample is a prostate tissue sample obtained by needle biopsy.
[0050] "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment according to that individual's drug response
genotype.
[0051] Diagnostic and Prognostic Methods
[0052] As described in more detail below, the detection methods of
the invention can be used to detect mRNA, protein, cDNA, or genomic
DNA, for example, in a biological sample in vitro as well as in
vivo. For example, in vitro techniques for detection of mRNA
include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of a polypeptide corresponding to a
marker of the invention include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations, immunohistochemistry
and immunofluorescence. In vitro techniques for detection of
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of a polypeptide corresponding to a marker
of the invention include introducing into a subject a labeled
antibody directed against the polypeptide. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques. Nucleic acid probes as well as antibodies to Pin1 for
use in these methods can readily be designed since the nucleic and
amino acid sequence of Pin1 is known (Hunter et al., WO 97/17986
(1997); Hunter et al., U.S. Pat. Nos. 5,952,467 and 5,972,697).
[0053] I. Antibody-Based Assays
[0054] In embodiments of the above methods, assessing the level of
Pin1 in a biological sample from the subject includes contacting
the biological sample with an antibody to Pin1 or a fragment
thereof; determining the amount of binding of the antibody to the
biological sample; and comparing the amount of antibody bound to
the biological sample to a predetermined base level.
[0055] The level of Pin-1 in normal (i.e. non-cancerous) biological
samples can be assessed in a variety of ways. In one embodiment,
this normal level of expression is determined by assessing the
level of Pin-1 in a portion of prostate cells which appears to be
non-cancerous and by comparing this normal level with the level of
Pin-1 in a portion of the prostate cells which is suspected of
being cancerous. Alternatively, the `normal` level of expression of
a marker may be determined by assessing the level of Pin-1 in a
sample or samples obtained from a non-cancer-afflicted
individuals.
[0056] "Antibody" includes immunoglobulin molecules and
immunologically active determinants of immunoglobulin molecules, i
e., molecules that contain an antigen binding site which
specifically binds (immunoreacts with) an antigen. Structurally,
the simplest naturally occurring antibody (e.g., IgG) comprises
four polypeptide chains, two copies of a heavy (H) chain and two of
a light (L) chain, all covalently linked by disulfide bonds.
Specificity of binding in the large and diverse set of antibodies
is found in the variable (V) determinant of the H and L chains;
regions of the molecules that are primarily structural are constant
(C) in this set. Antibody includes polyclonal antibodies,
monoclonal antibodies, whole immunoglobulins, and antigen binding
fragments of the immunoglobulins.
[0057] The binding sites of the proteins that comprise an antibody,
i.e., the antigen-binding functions of the antibody, are localized
by analysis of fragments of a naturally-occuring antibody. Thus,
antigen-binding fragments are also intended to be designated by the
term "antibody." Examples of binding fragments encompassed within
the term antibody include: a Fab fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; an F.sub.d fragment
consisting of the V.sub.H and C.sub.H1 domains; an F.sub.v,
fragment consisting of the V.sub.L and V.sub.H domains of a single
arm of an antibody; a dAb fragment (Ward et al., 1989 Nature
341:544-546) consisting of a V.sub.H domain; an isolated
complementarity determining region (CDR); and an F(ab').sub.2
fragment, a bivalent fragment comprising two Fab'fragments linked
by a disulfide bridge at the hinge region. These antibody fragments
are obtained using conventional techniques well-known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies. The term "antibody" is
further intended to include bispecific and chimeric molecules
having at least one antigen binding determinant derived from an
antibody molecule.
[0058] In the diagnostic and prognostic assays of the invention,
the antibody can be a polyclonal antibody or a monoclonal antibody
and in a preferred embodiment is a labeled antibody.
[0059] Polyclonal antibodies are produced by immunizing animals,
usually a mammal, by multiple subcutaneous or intraperitoneal
injections of an immunogen (antigen) and an adjuvant as
appropriate. As an illustrative embodiment, animals are typically
immunized against a protein, peptide or derivative by combining
about 1 .mu.g to 1 mg of protein capable of eliciting an immune
response, along with an enhancing carrier preparation, such as
Freund's complete adjuvant, or an aggregating agent such as alum,
and injecting the composition intradermally at multiple sites.
Animals are later boosted with at least one subsequent
administration of a lower amount, as 1/5 to {fraction (1/10)} the
original amount of immunogen in Freund's complete adjuvant (or
other suitable adjuvant) by subcutaneous injection at multiple
sites. Animals are subsequently bled, serum assayed to determine
the specific antibody titer, and the animals are again boosted and
assayed until the titer of antibody no longer increases (i.e.,
plateaus).
[0060] Such populations of antibody molecules are referred to as
"polyclonal" because the population comprises a large set of
antibodies each of which is specific for one of the many differing
epitopes found in the immunogen, and each of which is characterized
by a specific affinity for that epitope. An epitope is the smallest
determinant of antigenicity, which for a protein, comprises a
peptide of six to eight residues in length (Berzofsky, J. and I.
Berkower, (1993) in Paul, W., Ed., Fundamental Immunology, Raven
Press, N.Y., p.246). Affinities range from low, e.g. 10.sup.-6 M,
to high, e.g., 10.sup.-11 M. The polyclonal antibody fraction
collected from mammalian serum is isolated by well known
techniques, e.g. by chromatography with an affinity matrix that
selectively binds immunoglobulin molecules such as protein A, to
obtain the IgG fraction. To enhance the purity and specificity of
the antibody, the specific antibodies may be further purified by
immunoaffinity chromatography using solid phase-affixed immunogen.
The antibody is contacted with the solid phase-affixed immunogen
for a period of time sufficient for the immunogen to immunoreact
with the antibody molecules to form a solid phase-affixed
immunocomplex. Bound antibodies are eluted from the solid phase by
standard techniques, such as by use of buffers of decreasing pH or
increasing ionic strength, the eluted fractions are assayed, and
those containing the specific antibodies are combined.
[0061] "Monoclonal antibody" or "monoclonal antibody composition"
as used herein refers to a preparation of antibody molecules of
single molecular composition. A monoclonal antibody composition
displays a single binding specificity and affinity for a particular
epitope. Monoclonal antibodies can be prepared using a technique
which provides for the production of antibody molecules by
continuous growth of cells in culture. These include but are not
limited to the hybridoma technique originally described by Kohler
and Milstein (1975, Nature 256:495-497; see also Brown et al. 1981
J. Immunol 127:539-46; Brown et al., 1980, J. Biol Chem
255:4980-83; Yeh et al., 1976, PNAS 76:2927-31; and Yeh et al.,
1982, Int. J. Cancer 29:269-75) and the more recent human B cell
hybridoma technique (Kozbor et al., 1983, Immunol Today 4:72),
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma
techniques. The technology for producing hybridomas is well known
(see generally Current Protocols in Immunology, Coligan et al. ed.,
John Wiley & Sons, New York, 1994). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the
hybridoma culture supernatants for antibodies that bind the
polypeptide of interest, e.g., using a standard ELISA assay.
[0062] A monoclonal antibody can be produced by the following
steps. In all procedures, an animal is immunized with an antigen
such as a protein (or peptide thereof) as described above for
preparation of a polyclonal antibody. The immunization is typically
accomplished by administering the immunogen to an immunologically
competent mammal in an immunologically effective amount, i.e., an
amount sufficient to produce an immune response. Preferably, the
mammal is a rodent such as a rabbit, rat or mouse. The mammal is
then maintained on a booster schedule for a time period sufficient
for the mammal to generate high affinity antibody molecules as
described. A suspension of antibody-producing cells is removed from
each immunized mammal secreting the desired antibody. After a
sufficient time to generate high affinity antibodies, the animal
(e.g., mouse) is sacrificed and antibody-producing lymphocytes are
obtained from one or more of the lymph nodes, spleens and
peripheral blood. Spleen cells are preferred, and can be
mechanically separated into individual cells in a physiological
medium using methods well known to one of skill in the art. The
antibody-producing cells are immortalized by fusion to cells of a
mouse myeloma line. Mouse lymphocytes give a high percentage of
stable fusions with mouse homologous myelomas, however rat, rabbit
and frog somatic cells can also be used. Spleen cells of the
desired antibody-producing animals are immortalized by fusing with
myeloma cells, generally in the presence of a fusing agent such as
polyethylene glycol. Any of a number of myeloma cell lines suitable
as a fusion partner are used with to standard techniques, for
example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma
lines, available from the American Type Culture Collection (ATCC),
Rockville, Md.
[0063] The fusion-product cells, which include the desired
hybridomas, are cultured in selective medium such as HAT medium,
designed to eliminate unfused parental myeloma or lymphocyte or
spleen cells. Hybridoma cells are selected and are grown under
limiting dilution conditions to obtain isolated clones. The
supernatants of each clonal hybridoma is screened for production of
antibody of desired specificity and affinity, e.g., by immunoassay
techniques to determine the desired antigen such as that used for
immunization. Monoclonal antibody is isolated from cultures of
producing cells by conventional methods, such as ammonium sulfate
precipitation, ion exchange chromatography, and affinity
chromatography (Zola et al, Monoclonal Hybridoma Antibodies:
Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press,
1982). Hybridomas produced according to these methods can be
propagated in culture in vitro or in vivo (in ascites fluid) using
techniques well known to those with skill in the art.
[0064] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
an antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J.
12:725-734.
[0065] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0066] "Labeled antibody" as used herein includes antibodies that
are labeled by a detectable means and includes enzymatically,
radioactively, fluorescently, chemiluminescently, and/or
bioluminescently labeled antibodies.
[0067] One of the ways in which an antibody can be detectably
labeled is by linking the same to an enzyme. This enzyme, in turn,
when later exposed to its substrate, will react with the substrate
in such a manner as to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or by
visual means. Enzymes which can be used to detectably label the
Pin1-specific antibody include, but are not limited to, malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase,
triose phosphate isomerase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,
glucoamylase and acetylcholinesterase.
[0068] Detection may be accomplished using any of a variety of
immunoassays. For example, by radioactively labeling an antibody,
it is possible to detect the antibody through the use of
radioimmune assays. A description of a radioimmune assay (RIA) may
be found in Laboratory Techniques and Biochemistry in Molecular
Biology, by Work, T. S., et al., North Holland Publishing Company,
NY (1978), with particular reference to the chapter entitled "An
Introduction to Radioimmune Assay and Related Techniques" by Chard,
T.
[0069] The radioactive isotope can be detected by such means as the
use of a gamma counter or a scintillation counter or by
audioradiography. Isotopes which are particularly useful for the
purpose of the present invention are: .sup.3H, .sup.131I, .sup.35S,
.sup.14C, and preferably .sup.125I.
[0070] It is also possible to label an antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0071] An antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentaacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0072] An antibody also can be detectably labeled by coupling it to
a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, luciferin, isoluminol, theromatic
acridinium ester, imidazole, acridinium salt and oxalate ester.
[0073] Likewise, a bioluminescent compound may be used to label an
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0074] In the diagnostic and prognostic assays of the invention,
the amount of binding of the antibody to the biological sample can
be determined by the intensity of the signal emitted by the labeled
antibody and/or by the number cells in the biological sample bound
to the labeled antibody.
[0075] Serum Assays
[0076] A serum assay for detecting a cancer marker is a non-evasive
method, which is more acceptable to patients and also provides a
tool for screening large number of samples. Additional advantages
include that the antibody recognizes an antigen that is related to
the early events rather than the later stages of progression to the
metastatic phenotype. Serum assays can be used in conjunction with
other assays presented herein to diagnose cancer.
[0077] Antibodies directed toward a protein of interest can be
connected to magnetic beads and used to enrich a population.
Immunomagnetic selection has been used previously for this purpose
and examples of this method can be found, for example, at U.S.
patent Ser. No.: 5,646,001; Ree et al. (2002) Int. J. Cancer
97:28-33; Molnar et al. (2001) Clin. Cancer Research 7:4080-4085;
and Kasimir-Bauer et al. (2001) Breast Cancer Res. Treat.
69:123-32. An antibody, either polyclonal or monoclonal, that is
specific for a cell surface protein on a cell of interest is
attacthed to a magnetic substrate thereby allowing selection of
only those cells that express the surface protein of interest. Once
a population of cells is selected, the following assays can be
performed to test for the presence of Pin1.
[0078] Immunoassays
[0079] The amount of an antigen (i.e. Pin1) in a biological sample
may be determined by a radioimmunoassay, an immunoradiometric
assay, and/or an enzyme immunoassay.
[0080] "Radioimmunoassay" is a technique for detecting and
measuring the concentration of an antigen using a labeled (i.e.
radioactively labeled) form of the antigen. Examples of radioactive
labels for antigens include .sup.3H, .sup.14C, and .sup.125I. The
concentration of antigen (i.e. Pin1) in a sample (i.e. biological
sample) is measured by having the antigen in the sample compete
with a labeled (i.e. radioactively) antigen for binding to an
antibody to the antigen. To ensure competitive binding between the
labeled antigen and the unlabeled antigen, the labeled antigen is
present in a concentration sufficient to saturate the binding sites
of the antibody. The higher the concentration of antigen in the
sample, the lower the concentration of labeled antigen that will
bind to the antibody.
[0081] In a radioimmunoassay, to determine the concentration of
labeled antigen bound to antibody, the antigen-antibody complex
must be separated from the free antigen. One method for separating
the antigen-antibody complex from the free antigen is by
precipitating the antigen-antibody complex with an anti-isotype
antiserum. Another method for separating the antigen-antibody
complex from the free antigen is by precipitating the
antigen-antibody complex with formalin-killed S. aureus. Yet
another method for separating the antigen-antibody complex from the
free antigen is by performing a "solid-phase radioimmunoassay"
where the antibody is linked (i.e. covalently) to Sepharose beads,
polystyrene wells, polyvinylchloride wells, or microtiter wells. By
comparing the concentration of labeled antigen bound to antibody to
a standard curve based on samples having a known concentration of
antigen, the concentration of antigen in the biological sample can
be determined.
[0082] A "Immunoradiometric assay" (IRMA) is an immunoassay in
which the antibody reagent is radioactively labeled. An IRMA
requires the production of a multivalent antigen conjugate, by
techniques such as conjugation to a protein e.g., rabbit serum
albumin (RSA). The multivalent antigen conjugate must have at least
2 antigen residues per molecule and the antigen residues must be of
sufficient distance apart to allow binding by at least two
antibodies to the antigen. For example, in an IRMA the multivalent
antigen conjugate can be attached to a solid surface such as a
plastic sphere. Unlabeled "sample" antigen and antibody to antigen
which is radioactively labeled are added to a test tube containing
the multivalent antigen conjugate coated sphere. The antigen in the
sample competes with the multivalent antigen conjugate for antigen
antibody binding sites. After an appropriate incubation period, the
unbound reactants are removed by washing and the amount of
radioactivity on the solid phase is determined. The amount of bound
radioactive antibody is inversely proportional to the concentration
of antigen in the sample.
[0083] The most common enzyme immunoassay is the "Enzyme-Linked
Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent
Assay (ELISA)" is a technique for detecting and measuring the
concentration of an antigen using a labeled (i.e. enzyme linked)
form of the antibody.
[0084] In a "sandwich ELISA" , an antibody (i.e. to Pin1) is linked
to a solid phase (i.e. a microtiter plate) and exposed to a
biological sample containing antigen (i.e. Pin1). The solid phase
is then washed to remove unbound antigen. A labeled (i.e. enzyme
linked) is then bound to the bound-antigen (if present) forming an
antibody-antigen-antibody sandwich. Examples of enzymes that can be
linked to the antibody are alkaline phosphatase, horseradish
peroxidase, luciferase, urease, and .beta.-galactosidase. The
enzyme linked antibody reacts with a substrate to generate a
colored reaction product that can be assayed for.
[0085] In a "competitive ELISA" , antibody is incubated with a
sample containing antigen (i.e. Pin1). The antigen-antibody mixture
is then contacted with an antigen-coated solid phase (i. e. a
microtiter plate). The more antigen present in the sample, the less
free antibody that will be available to bind to the solid phase. A
labeled (i.e. enzyme linked) secondary antibody is then added to
the solid phase to determine the amount of primary antibody bound
to the solid phase.
[0086] In a "immunohistochemistry assay" a section of tissue for is
tested for specific proteins by exposing the tissue to antibodies
that are specific for the protein that is being assayed. The
antibodies are then visualized by any of a number of methods to
determine the presence and amount of the protein present. Examples
of methods used to visualize antibodies are, for example, through
enzymes linked to the antibodies (e.g, luciferase, alkaline
phosphatase, horseradish peroxidase, or .beta.-galactosidase), or
chemical methods (e.g., DAB/Substrate chromagen).
[0087] II. Pin1 Nucleic Acid-Based Diagnostic and Prognostic
Methods
[0088] Also encompassed by this invention is a method of diagnosing
prostate cancer in a subject, comprising: detecting a level of Pin1
nucleic acid in a biological sample; and comparing the level of
Pin1 in the biological sample with a level of Pin1 in a control
sample, wherein an elevation in the level of Pin1 in the biological
sample compared to the control sample is indicative of prostate
cancer.
[0089] In addition, this invention pertains to a method of
diagnosing prostate cancer metastasis in a subject, comprising the
steps of: detecting a level of Pin1 nucleic acid in a biological
sample; and comparing the level of Pin1 in the biological sample
with a level of Pin1 in a control sample, wherein an elevation in
the level of Pin1 in the biological sample compared to the control
sample is indicative of prostate cancer metastasis.
[0090] In an embodiment of the above methods, the detecting a level
of Pin1 nucleic acid in a biological sample includes amplifying
Pin1 RNA. In another embodiment of the above methods, the detecting
a level of Pin1 nucleic acid in a biological sample includes
hybridizing the Pin1 RNA with a probe.
[0091] As an alternative to making determinations based on the
absolute expression level of the Pin1 marker, determinations may be
based on the normalized expression level of the marker. Expression
levels are normalized by correcting the absolute expression level
of a marker by comparing its expression to the expression of a gene
that is not a marker, e.g., a housekeeping gene that is
constitutively expressed. Suitable genes for normalization include
housekeeping genes such as the actin gene, or epithelial
cell-specific genes. This normalization allows the comparison of
the expression level in one sample, e.g., a patient sample, to
another sample, e.g., a non-prostate cancer sample, or between
samples from different sources.
[0092] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a marker, the level of expression of the marker is determined
for 10 or more samples of normal versus cancer cell isolates,
preferably 50 or more samples, prior to the determination of the
expression level for the sample in question. The mean expression
level of each of the genes assayed in the larger number of samples
is determined and this is used as a baseline expression level for
the marker. The expression level of the marker determined for the
biological sample (absolute level of expression) is then divided by
the mean expression value obtained for that marker. This provides a
relative expression level.
[0093] One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. Probes based on the sequence of a nucleic acid
molecule of the invention can be used to detect transcripts
corresponding to Pin-1. The nucleic acid probe can be, for example,
a full-length cDNA, or a portion thereof, such as an
oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to a mRNA or genomic DNA encoding a
marker of the present invention. Hybridization of an mRNA with the
probe indicates that the marker in question is being expressed. In
an embodiment, the probe includes a label group attached thereto,
e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor.
[0094] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the markers of the present invention.
[0095] "Amplifying" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal. As used herein, the term template-dependent
process is intended to refer to a process that involves the
template-dependent extension of a primer molecule. The term
template dependent process refers to nucleic acid synthesis of an
RNA or a DNA molecule wherein the sequence of the newly synthesized
strand of nucleic acid is dictated by the well-known rules of
complementary base pairing (see, for example, Watson, J. D. et al.,
In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc.,
Menlo Park, Calif. (1987). Typically, vector mediated methodologies
involve the introduction of the nucleic acid fragment into a DNA or
RNA vector, the clonal amplification of the vector, and the
recovery of the amplified nucleic acid fragment. Examples of such
methodologies are provided by Cohen et al. (U.S. Pat. No.
4,237,224), Maniatis, T. et al., Molecular Cloning (A Laboratory
Manual), Cold Spring Harbor Laboratory, 1982.
[0096] A number of template dependent processes are available to
amplify the target sequences of interest present in a sample. One
of the best known amplification methods is the polymerase chain
reaction (PCR) which is described in detail in Mullis, et al., U.S.
Pat. No. 4,683,195, Mullis, et al., U.S. Pat. No. 4,683,202, and
Mullis, et al., U.S. Pat. No. 4,800,159, and in Innis et al., PCR
Protocols, Academic Press, Inc., San Diego Calif., 1990. Briefly,
in PCR, two primer sequences are prepared which are complementary
to regions on opposite complementary strands of the target
sequence. An excess of deoxynucleoside triphosphates are added to a
reaction mixture along with a DNA polymerase (e.g., Taq
polymerase). If the target sequence is present in a sample, the
primers will bind to the target and the polymerase will cause the
primers to be extended along the target sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
target to form reaction products, excess primers will bind to the
target and to the reaction products and the process is repeated.
Preferably a reverse transcriptase PCR amplification procedure may
be performed in order to quantify the amount of mRNA amplified.
Polymerase chain reaction methodologies are well known in the
art.
[0097] Another method for amplification is the ligase chain
reaction (LCR), disclosed in European Patent No. 320,308B1. In LCR,
two complementary probe pairs are prepared, and in the presence of
the target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR, bound ligated units dissociate from
the target and then serve as "target sequences"for ligation of
excess probe pairs. Whiteley, et al., U.S. Pat. No. 4,883,750
describes an alternative method of amplification similar to LCR for
binding probe pairs to a target sequence.
[0098] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880 may also be used as still another amplification
method in the present invention. In this method, a replicative
sequence of RNA which has a region complementary to that of a
target is added to a sample in the presence of an RNA polymerase.
The polymerase will copy the replicative sequence which can then be
detected.
[0099] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis, i.e.
nick translation. A similar method, called Repair Chain Reaction
(RCR) is another method of amplification which may be useful in the
present invention and is involves annealing several probes
throughout a region targeted for amplification, followed by a
repair reaction in which only two of the four bases are present.
The other two bases can be added as biotinylated derivatives for
easy detection. A similar approach is used in SDA.
[0100] Prostate specific sequences can also be detected using a
cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5'
sequences of non-prostate specific DNA and middle sequence of
prostate specific RNA is hybridized to DNA which is present in a
sample. Upon hybridization, the reaction is treated with RNaseH,
and the products of the probe identified as distinctive products
generating a signal which are released after digestion. The
original template is annealed to another cycling probe and the
reaction is repeated. Thus, CPR involves amplifying a signal
generated by hybridization of a probe to a prostate cancer specific
expressed nucleic acid.
[0101] Still other amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025 may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR like, template and enzyme dependent synthesis. The primers
may be modified by labeling with a capture moiety (e.g., biotin)
and/or a detector moiety (e.g., enzyme). In the latter application,
an excess of labeled probes are added to a sample. In the presence
of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0102] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (Kwoh D., et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:1173, Gingeras T. R., et
al., PCT Application WO 88/1D315), including nucleic acid sequence
based amplification (NASBA) and 3SR. In NASBA, the nucleic acids
can be prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has prostate specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second prostate specific primer, followed
by polymerization. The double stranded DNA molecules are then
multiply transcribed by a polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNAs are reverse transcribed into
double stranded DNA, and transcribed once against with a polymerase
such as T7 or SP6. The resulting products, whether truncated or
complete, indicate prostate cancer specific sequences.
[0103] Davey, C., et al., European Patent No. 329,822B1 disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention. The ssRNA is a first template for a first
primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from
resulting DNA:RNA duplex by the action of ribonuclease H (RNase H,
an RNase specific for RNA in a duplex with either DNA or RNA). The
resultant ssDNA is a second template for a second primer, which
also includes the sequences of an RNA polymerase promoter
(exemplified by T7 RNA polymerase) 5' to its homology to its
template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase I), resulting as a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0104] Miller, H. I., et al., PCT Application WO 89/06700 discloses
a nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic; i.e. new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "race" disclosed by Frohman, M.
A., In: PCR Protocols: A Guide to Methods and Applications 1990,
Academic Press, New York) and "one-sided PCR" (Ohara, O., et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:5673-5677).
[0105] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide (Wu, D. Y. et al., Genomics 1989, 4:560), may
also be used in the amplification step of the present
invention.
[0106] Following amplification, the presence or absence of the
amplification product may be detected. The amplified product may be
sequenced by any method known in the art, including and not limited
to the Maxam and Gilbert method. The sequenced amplified product is
then compared to a sequence known to be in a prostate cancer
specific sequence. Alternatively, the nucleic acids may be
fragmented into varying sizes of discrete fragments. For example,
DNA fragments may be separated according to molecular weight by
methods such as and not limited to electrophoresis through an
agarose gel matrix. The gels are then analyzed by Southern
hybridization. Briefly, DNA in the gel is transferred to a
hybridization substrate or matrix such as and not limited to a
nitrocellulose sheet and a nylon membrane. A labeled probe is
applied to the matrix under selected hybridization conditions so as
to hybridize with complementary DNA localized on the matrix. The
probe may be of a length capable of forming a stable duplex. The
probe may have a size range of about 200 to about 10,000
nucleotides in length, preferably about 200 nucleotides in length.
Various labels for visualization or detection are known to those of
skill in the art, such as and not limited to fluorescent staining,
ethidium bromide staining for example, avidin/biotin, radioactive
labeling such as .sup.32P labeling, and the like. Preferably, the
product, such as the PCR product, may be run on an agarose gel and
visualized using a stain such as ethidium bromide. The matrix may
then be analyzed by autoradiography to locate particular fragments
which hybridize to the probe.
[0107] III. Assays for Use in Combination with Pin1 Detection
[0108] A. PSA Assays
[0109] The level of "prostate-specific antigen" (PSA) in the blood
is currently the primary diagnostic tool for disorders of the
prostate. The presence of Prostate Specific Antigen (PSA) can be
measured with relative ease from blood samples using standard
antibody-based detection kits. Prostate enlargement, a condition
known as benign prostatic hyperplasia (BPH), is found in about half
of men over age 45. With BPH, PSA levels rise in proportion to
prostate size, possibly obscuring diagnosis of PCA. In addition, a
significant proportion of men with PCA have normal PSA levels. The
PSA test is somewhat non-specific for distinguishing PCA and BPH,
and produces a degree of false negative results (Garnick, M.,
(1993), Am. Inst. Med., 118:804-818). In the majority of cases, PSA
elevation is due to BPH or prostatitis rather than carcinoma. The
normal level of prostate-specific antigen (PSA) in blood increases
with age and ranges from 0-2.5 ng/ml for men 40 to 49 years old to
0-6.5 ng/ml for men 70-79 years old. (See Table 1, which follows,
for variation of normal PSA levels with age. Prostate cancer will
be detected in approximately one-third of men with PSA levels
between 4.1 and 10 ng/ml. Approximately two-thirds of men with PSA
levels above 10 ng/ml have prostate cancer. Approximately 20% of
men with prostate cancer have PSA levels below 4 ng/ml.
1TABLE 1 Age-Specific Prostate-Specific Antigen (PSA) Values Normal
PSA values (ng/ml)* Mayo Clinic Walter Reed Age range (yr) White
White Black 40-49 0-2.5 0-2.5 0.2.0 50-59 0-3.5 0-3.5 0-4.0 60-69
0-4.5 0-3.5 0-4.5 70-79 0-6.5 0-3.5 0-5.5
[0110] In addition to the serum PSA concentration, the percent-free
PSA and PSA velocity can also be used to help diagnose prostate
cancer.
[0111] PSA exists in 2 forms in the blood: protein bound and free.
Examples of bound PSA are PSA bound with .alpha.1-antichymotrypsin
(PSA-ACT) or .alpha.2-macroglobulin. In BPH, there is generally a
higher percentage free (as opposed to bound) PSA than in prostate
cancer. For example, a percent-free PSA between 0 and 14% with a
PSA level between about 4.1 and 10 ng/ml is associated with a 64%
chance of a biopsy positive for prostate cancer. In another
example, a percent-free PSA between 15 and 24% with a PSA level
between about 4.1 and 10 ng/ml is associated with a 37% chance of a
biopsy positive for prostate cancer. See Table 2, which follows,
shows the probability of a positive prostate biopsy based on the
percent-free PSA.
2TABLE 2 Probability of Positive Prostate Biopsy Based on
Percent-Free Prostate- Specific Antigen (PSA) Percent-Free PSA
Estimated probability of positive biopsy Patients with PSA in
4.1-10 ng/mL range: 0%-14% 64% 15-24% 37% >24% 10% Patients with
PSA in 2.6-4 ng/mL range: 0%-14% 25% 15%-24% 23% >24% 18%
[0112] "PSA velocity" is the rate of change in PSA concentration.
Because prostate cancer may raise the PSA level more quickly than
BPH, a higher PSA velocity correlates with an increased likelihood
of prostate cancer. For example, a PSA velocity of greater than
0.75 ng/ml per year may indicate prostate cancer even with a PSA of
less than 4 ng/ml.
[0113] The diagnostic and prognostic assays of this invention are
particularly useful when the PSA level falls within a gray zone
where it is not clear whether a prostate biopsy should be
performed. For example, the diagnostic and prognostic assays of
this invention may be used to detect prostate cancer and assess the
subject's prospects when: (a) the subject has a blood serum
concentration of the prostate-specific antigen of between about 2
and about 10 ng/ml; (b) the subject has a blood serum concentration
of the prostate-specific antigen of between about 4 and about 8
ng/ml; (c) the subject has a blood serum concentration of the
prostate-specific antigen of between about 3 and about 7 ng/ml and
is between about 40 and about 60 years old; (d) the subject has a
blood serum concentration of the prostate-specific antigen of
between about 5 and about 9 ng/ml and is between about 60 and about
80 years old; (e) the subject has a blood serum concentration of
the prostate-specific antigen of less than about 4 ng/ml and a PSA
velocity of greater than about 0.7 ng/ml per year; and (f) the
subject has a blood serum concentration of the prostate-specific
antigen of between about 4 and about 8 ng/ml and a percent-free
prostate-specific antigen of between about 15 and about 25%.
[0114] B. Methods of Staging Prostate Cancer
[0115] Beyond detection of elevated PSA and other prostate cancer
marker levels such as those disclosed herein, other current methods
for diagnosing and staging PCA are known to medical practitioners
skilled in the art and include rectal examination, transrectal
ultrasonography (TRUS) or magnetic resonance imaging (MRI), bone
scanning, X-rays, skeletal survey, intravenous pyelography,
CAT-scan, and biopsy (reviewed in Garnick, M. (1993), Annals of
Internal Medicine, 118:803-818; and Garnick, M. (1994), Scientific
American, 270:72-81).
[0116] Digital Rectal Exam
[0117] In a "digital rectal exam" (DRE), a clinician uses a gloved
finger to assess the posterior aspect of the prostate through the
rectal wall. The clinician will check the rear surface of the
prostate for abnormalities such as hardness, a lump, or an enlarged
prostate. In an embodiment, the subject in the above diagnostic and
prognostic assays of this invention was identified by a digital
rectal exam as having a prostate abnormality.
[0118] ABCD Scale and TNM Staging System
[0119] Prostate cancer (PCA) stages are commonly evaluated
according to a scale divided as A, B, C and D. Tumors in stage A
are microscopic; stage A.sub.1 designates tumors confined to a
relatively small area and composed of well-differentiated tissue;
stage A.sub.2 tumors are more diffuse and less well differentiated;
stage B tumors are large enough to be felt during a rectal
examination; and stage C prostate cancers have spread throughout
the gland and typically have pushed past the borders of the
prostate into surrounding structures. Stage D tumors have
metastasized, e.g., to lymph nodes, bone, or other organs.
Alternatively, tumors are also staged by the TNM staging system, in
which tumors are ranked on a scale of progressively worsening
disease from T1a to T4b (e.g., T1c tumors are non-palpable and
non-visible that were detected by elevated blood levels of prostate
specific antigen). The TNM system for staging prostate cancer is
summarized in Table 3, which follows. Of tumors characterized as
being in stages A2, B, or C, 25% to 50% turn out, on further
testing, to be metastatic. Methods involving procedures for removal
or destruction of prostatic tumor tissue usually are employed with
non-metastasized cancers. For example, radical prostatectomy
preferably is used with stage A, B and some stage C tumors (i. e.,
where the tumor growth has not extended considerably beyond the
borders of the prostate gland) as well as stage T1c tumors. X-ray
therapy (e.g., external or interstitial) preferably is used with
stage A, B or C tumors as well as T1c tumors. Additional diagnostic
tools might aid in distinguishing cases suitable for various
treatments.
3TABLE 3 The TMN System of Prostate Tumor Classification
Classification Description Incidental prostate cancer TX Primary
tumour cannot be assessed T0 No evidence of primary tumour T1
Clinically unapparent tumour, not palpable nor visible by imaging
T1a Tumour an incidental histological finding in 5% or less of
tissue resected T1b Tumour an incidental histological finding in
more than 5% of tissue resected T1c Tumour identified by needle
biopsy (e.g because of elevated serum PSA level) Palpable or
visible carcinoma confined to the prostrate T2* Tumour confined
within the prostate T2a Tumour involves half of a lobe or less T2b
Tumour involves more than half a lobe but not both lobes T2c Tumour
involves both lobes Locally extensive prostate cancer T3** Tumour
extends through the prostate capsule T3a Unilateral extracapsular
extension T3b Bilateral extracapsular extension T3c Tumour invades
seminal vesicle(s) Locally extensive tumours with fixation or
invasion into neighbouring organs T4 Tumour is fixed or invades
adjacent structures other than seminal vesicles T4a Tumour invades
bladder neck and/or external sphincter and/or rectum T4b Tumour
invades levator muscles and/or is fixed to pelvic wall *Tumor found
in one or both lobes by needle biopsy, not palpable or visible by
imaging, is classified as T1c. **Invasion into the prostatic apex
or into (but not beyond) the prostatic capsule is not classified as
T3 but as T2.
[0120] Gleason Scoring
[0121] Another commonly used system for determining the prognosis
of a patient with prostate cancer is the Gleason scoring system.
The "Gleason score" or "Gleason grade" is a value from 1 (well
differentiated) to 5 (poorly differentiated) based on the
examination of slices of prostate cancer tissue under a microscope.
The lower the Gleason score the more the prostate cancer tissue
resembles the structure of normal prostate tissue and the less
aggressive the cancer is likely to be.
[0122] The "combined Gleason score" or "Gleason sum" is a value
from 2 (least anaplastic) to 10 (most anaplastic) based on the
Gleason scores of the 2 most common 10 histological patterns in the
prostate cancer tissue. The lower the Gleason sum, the better the
prognosis for the patient. The most common Gleason sums are 6 and
7, which often represent a gray zone for cancer prognosis. Tables
4A-4D (the Partin Coefficient Tables), which follow, show the
probability of organ-confined prostate cancer based on the PSA
levels, Gleason score, and stage of the prostate cancer.
4TABLE 4A Prediction of Probability of Organ-Confined Disease (for
PSA = 0.0-4.0 ng/ml) Gleason Stage Stage Stage Stage Stage Stage
Stage score T1a T1b T1c T2a T2b T2c T3a 2-4 90 80 89 81 72 77 . . .
5 82 66 81 68 57 62 40 6 78 61 78 64 52 57 35 7 . . . 43 63 47 34
38 19 8-10 . . . 31 52 36 24 27 . . . All numbers represent percent
predictive probabilities (95% confidence interval); ellipses
indicate lack of sufficient data to calculate probability
[0123]
5TABLE 4B Prediction of Probability of Organ-Confined Disease (for
PSA = 4.1-10.0 ng/ml) Gleason Stage Stage Stage Stage Stage Stage
Stage score T1a T1b T1c T2a T2b T2c T3a 2-4 84 70 83 71 61 66 43 5
72 53 71 55 43 49 27 6 67 47 67 51 38 43 23 7 49 29 49 33 22 25 11
8-10 35 18 37 23 14 15 6 All numbers represent percent predictive
probabilities (95% confidence interval); ellipses indicate lack of
sufficient data to calculate probability
[0124]
6TABLE 4C Prediction of Probability of Organ-Confined Disease (for
PSA = 10.1-20.0 ng/ml) Gleason Stage Stage Stage Stage Stage Stage
Stage score T1a T1b T1c T2a T2b T2c T3a 2-4 76 58 75 60 48 53 . . .
5 61 40 60 43 32 36 18 6 . . . 33 55 38 26 31 14 7 33 17 35 22 13
15 6 8-10 . . . 9 23 14 7 8 3 All numbers represent percent
predictive probabilities (95% confidence interval); ellipses
indicate lack of sufficient data to calculate probability
[0125]
7TABLE 4D Prediction of Probability of Organ-Confined Disease (for
PSA => 20.0 ng/ml) Gleason Stage Stage Stage Stage Stage Stage
Stage score T1a T1b T1c T2a T2b T2c T3a 2-4 . . . 38 58 41 29 . . .
. . . 5 . . . 23 40 26 17 19 8 6 . . . 17 35 22 13 15 6 7 . . . . .
. 18 10 5 6 2 8-10 . . . 3 10 5 3 3 1 All numbers represent percent
predictive probabilities (95% confidence interval); ellipses
indicate lack of sufficient data to calculate probability
[0126] The development of the prostate specific antigen (PSA) test
as a diagnostic tool for prostate cancer has allowed the earlier
and more accurate detection of clinical prostate carcinoma. Before
the development of this test, most tumors were discovered by manual
examination and these tumors were necessarily large and often high
grade. A significant portion of these tumors were already
metastatic. The advent of PSA test has enabled the detection of
smaller tumors, the majority of which are intermediate grade
(Gleason score 6-7).
[0127] Despite our increased ability to detect early prostate
tumors, it is difficult, especially in patients with mid-grade
tumors, to determine which patients will have an indolent disease
course and which patients will go on to develop metastatic disease.
The therapeutic decision of which patients should have aggressive
treatment and which should have less aggressive treatment followed
by PSA monitoring is particularly difficult to make for these
mid-grade patients. The ability to distinguish the potential
outcome of a particular tumor would potentially have a great effect
on the selection of a treatment course for that patient. Therefore,
the prognostic assays of this invention are also particularly
useful when the Gleason sum falls within a gray zone where the
prognosis is usually unclear (i.e. a Gleason sum of 6 or 7).
[0128] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference
EXAMPLES
Example 1
Primary Screen for Pin1 Expression in Human Tissues
[0129] Materials and Methods
[0130] Human Biological Samples
[0131] Formalin-fixed, paraffin-embedded sections of normal human
organs were obtained from Novagen (Madison, Wis.). Organs examined
included: prostate, brain, pituitary gland, kidney, muscle,
esophagus, stomach, small intestines, colon, liver, spleen,
pancreas, thyroid, heart, lung, bladder, adipose, lymph node,
uterus, ovary, adrenal, testis, tonsil and thymus.
[0132] Formalin-fixed, paraffin-embedded sections of 19 different
human tumor tissues were obtained from both Novagen and Imgenix
(San Diego, Calif.). Cancers examined included: prostate, stomach,
breast, pancrease, lung, liver, renal, ovary, thyroid, bladder,
uterine cervix, colon, esophagus, lymphoma, endometrium, head/neck,
gallbladder, melanoma, parotid.
[0133] Antibody
[0134] A commercial polyclonal antibody (Ab-1) (Oncogene Research
Products, MA) was employed in this study, which was generated by
immunizing rabbits with recombinant human Pin1. The specificity of
the antibody was tested and confirmed by Western blotting and
affinity purification.
[0135] Immunohistochemistry
[0136] Immunohistochemistry was performed on formalin-fixed tissues
embedded in paraffin and sectioned at 4 to 6 um for both normal and
tumor tissues. The sections were deparaffinized in xylene,
rehydrated in graded ethanols (100, 95 and 75%), followed by
immersed in 3% H202/methanol for 15 minutes. For antigen retrieval,
sections were microwaved in citrate buffer (pH 6.0) (BioGenex) for
15 minutes. Sections were then blocked in 10% normal goat serum in
TBS, followed by incubation with primary antibody 1:800 overnight
at 4.degree. C. Incubation with biotinylated goat anti-rabbit
antibody (Vector Laboratories, Burlingame, Calif.) for 30 minutes
at room temperature was followed by the standard
avidin-biotin-complex (ABC) process (Vectastain Elite ABC kit,
Vector). Diaminobenzidine (DAB) was used as a chromogen, followed
by counterstaining with hematoxylin. For negative controls, the
primary antibody was omitted and prior immunostaining with
preabsorbed antibody did not reveal any specific reactivity.
[0137] Results
[0138] Normal Tissues
[0139] Normal human biological samples from 25 organs were studies.
In normal tissues, the Pin1 level was low except for normal kidney,
brain, pancreatic islet cells and testis tissues where higher
levels of Pin1 were detected.
[0140] Tumor Tissues
[0141] 260 tumor samples from 19 different types of common human
cancers were studied. All 19 different types of cancers have shown
Pin1 over-expression. The incidence of Pin1 protein over-expression
varies in different types of cancers (Table 5).
8TABLE 5 Pin1 Expression in Human Tumors Tumor Type Total number %
Positive Prostate 49 92% Stomach 18 28% Breast 17 100% Pancreatic
16 33% Lung 14 50% Liver 13 31% Renal 13 23% Ovary 12 58% Thyroid
12 58% Bladder 11 81% Uterine Cervix 11 73% Colon 11 55% Esophagus
11 55% Malignant lymphoma 10 90% Endometrium 10 90% Head/Neck 10
60% Gallbladder 10 45% Malignant melanoma 9 100% Parotid 3 33%
Example 2
Use of Pin1 as a Prognostic Marker in Human Prostate Cancer
[0142] Materials and Methods
[0143] A total of 42 patients with prostatic adenocarcinoma
underwent radical prostatectomy between 1988 and 1996. The clinical
stage of the prostate tumor was assessed retrospectively by a
review of the medical records. The grade of each neoplasm was
determined using the Gleason scoring system.
[0144] Antibody
[0145] A commercially available human polyclonal Pin1 antibody
(Oncogene Research Products, MA) was used in this study. The
antibody was affinity-purified using CNBr-activated Sepharose 4B
column (Amersham Pharmacia Biotech). The purified antibody was
tested on a Western blot which contained recombinant human Pin1
protein.
[0146] Immunohistochemical Staining
[0147] Human prostate cancer sections were stained for Pin1 using
an avidin-biotin-peroxidase complex (ABC) method (Vector,
Burlingame, Calif.). Formalin-fixed, paraffin-embedded 5 .mu.m
tissue sections were deparaffinized in xylenes, rehydrated in
graded alcohols, and blocked for endogenous peroxidase activity by
3% hydrogen peroxide (Sigma) in methanol for 15 min. For antigen
retrieval, sections were microwaved in citrate buffer, pH 6.0
(BioGenex) for 15 min. The sections were then treated with 10%
normal serum same specie as secondary antibody for 40 min to
prevent nonspecific binding before incubating with an anti Pin1
antibody overnight at 4.degree. C. at 1:800 (v/v) dilution. The
sections were washed 4 times (5 min each) with TBS followed by
incubation with a biotinylated goat anti-rabbit IgG antibody for 40
min. After incubation with a preformed avidin-biotin complex for 40
min, specifically bound antibodies were visualized by using
peroxidase substrate, 3,3'-diaminobenzidine tetrahydrochloride
(DAB). Sections were counterstained with Gill's hematoxylin.
Negative controls included sections without primary antibody or
with normal serum instead of Pin1 antibody.
[0148] Discussion
[0149] Using affinity purified polyclonal Pin1 antibody, an
immunohistochemical study on paraffin sections of 42 human prostate
carcinoma cases was conducted. Positive immunostaining was observed
in the cytoplasm as well as the nucleus of epithelial cells in
neoplastic prostates but not in or very little in normal prostates.
The stromal cells surrounding tumors showed no or very little Pin1
expression. Among the specimens investigated, well differentiated
carcinomas with Gleason scores 4-5 generally showed no Pin1
staining or very low levels of staining. In some cases, high-grade
prostatic interstitial neoplasia (PIN) showed Pin1 immunostaining,
but usually to a lesser extent than the malignant lesions.
Moderately differentiated prostate carcinomas with Gleason 6-7
showed partially positive immunostaining, in which not all cancer
cells expressed Pin1, nor all cancerous lesions in a same specimen.
Poorly differentiated prostate carcinomas with Gleason scores 8-10
displayed the most extensive and intense Pin1 immunoreaction.
[0150] Pin1 staining levels for all prostatic carcinoma specimens
are summarized in FIG. 1. The results showed a general correlation
between Pin1 expression and the Gleason scores, with high grade
tumors (Gleason scores of 8-10) showing a higher percentage of
positive staining than low grade (Gleason scores of 4-5) tumors.
Interestingly, moderately differentiated prostate carcinomas with
Gleason scores of 6-7 could be divided into three groups according
to the levels of Pin1 expression. Group I: less than 30% of cancer
cells in a whole section stained for Pin1; group II: 30-50% of
cancer cells stained for Pin 1; group III: more than 50% stained
for Pin1. 8 out of 42 cases (19%) were classified as group I; 15
(36%) cases were classified as group II; and 19 (45%) were
classified as group III.
[0151] Gleason grading system is the most common clinical practice
for prostate cancer, with high Gleason scores showing high rate of
recurrence and metastasis, and low Gleason scores showing low rate
of mortality. Patients in intermediate grade (Gleason score 6-7)
have various outcomes. Most people diagnosed as having prostate
cancer belong to this group and present the biggest challenge to
the diagnosing clinician. Patients in group I appear to represent
an indolent disease course and have a low risk of developing
metastatic disease; patients in group III are likely to go on to
develop metastatic disease. Therefore, Pin1 staining of prostate
cancer is a useful tool to measure the degree of biological
aggressiveness of prostate cancer.
[0152] Clinical follow-up for three years or more on patients is
summarized in Table 6. Prostate specific antigen (PSA) is commonly
used for early diagnosis of prostate cancer and monitoring the
effectiveness of treatment. After surgery, PSA level is
undetectable. In the follow-up, if PSA level becomes detectable, it
is called PSA failure that indicates either primary tumor
recurrence or development of metastasis. Based on Pin1 expression
and PSA follow-up, it was found that there is a tendency that at
the time of surgery, patients whose tumor showed high levels of
Pin1 expression were likely to experience PSA failure. Table 7
shows the correlation between Pin1 expression and PSA failure.
Patients with low levels of Pin1 expression (0-30% of tumor cells
positive) showed low rate of PSA failure (12.5%), followed by
medium levels of Pin1 expression (30% -50%) showing higher rate of
failure (64%). Patients with high levels of Pin1 expression
(50-100%) exhibited the highest rate of PSA failure (78%).
9TABLE 6 Pin1 Expression and Clinical Outcome in Prostate Cancer %
Cells Pin1 Express Gleason PSA PSA failure- Intensity ing Sum
Surgery date Failure Date 1 + 50% 7 9/6/1991 1 8/24/1992 2 + 50% 4
1/24/1992 0 3/30/1995 3 + 80% 7 8/17/1992 0 9/16/1992 4 + 90% 10
10/20/1992 1 7/1/1993 5 + 40% 7 11/11/1992 n/a 6 ++ 70% 7 12/7/1992
1 7/1/1993 7 + 30% 7 3/1/1993 0 11/7/1996 8 + 50% 7 5/14/1993 0
8/8/1996 9 + 60% 7 9/28/1993 1 1/12/1995 10 + 70% 8 11/7/1994 1
5/16/1995 11 + 40% 8 12/5/1994 1 9/3/1996 12 ++ 70% 7 1/4/1995 1
9/13/1996 13 + 80% 7 2/15/1995 1 7/31/1995 14 + 80% 5 2/24/1995 0
11/22/1996 15 + 50% 7 4/13/1995 1 6/16/1995 16 + 80% 7 3/8/1995 0
5/21/1997 17 + 30% 7 5/12/1995 0 12/13/1995 18 + 80% 7 5/9/1995 0
10/24/1996 19 + 40% 7 9/22/1995 0 4/11/1997 20 + 80% 8 1/29/1996
n/a 21 ++ 70% 7 09/13/89 1 08/01/91 22 + 40% 7 01/23/90 1 07/10/92
23 ++ 30% 7 12/20/89 1 07/26/91 24 + 40% 6 02/07/90 0 0 25 + 40% 7
5/6/1988 0 2/11/1993 26 ++ 70% 9 11/17/1988 1 9/10/1990 27 + 40% 7
2/13/1990 1 5/27/1993 28 ++ 60% 8 4/24/1991 1 10/7/1999 29 + 60% 7
9/18/1991 1 5/12/1995 30 + 50% 7 10/21/1991 0 10/25/1999 31 + 50% 6
10/25/1991 1 7/1611992 32 + 30% 5 5/18/1999 0 3/21/1995 33 + 50% 7
2/22/1989 1 3/13/1989 34 + 60% 7 2/22/1989 1 3/13/1989 35 + 10% 7
5/24/1989 0 6/14/1999 36 + 70% 7 1/2/1990 0 1/17/1992 37 + 50% 7
2/16/1990 1 11/15/1990 38 + 70% 7 11/8/1990 1 9/11/1991 39 + 60% 7
12/14/1990 1 9/27/1995 40 + 50% 7 2/27/1991 1 6/11/1992 41 + 10% 6
5/9/1991 0 7/11/1997 42 + 10% 7 7/8/1994 0 7/1/1995
[0153]
10TABLE 7 Pin1 Expression and PSA Failure in Prostate Cancer % Pin1
positive PSA Failure cells + - % 0-30 1 7 12.5 30-50 9 5 64 50-100
14 4 78
[0154] As the above data show, patients with the most extensive
Pin1 staining are at greater risk to develop recurrent disease than
those with low Pin1 staining, and Pin1 can be used as a biomarker
that functions as an indicator of metastatic progression and
disease outcome in human prostate cancer patients.
Example 3
Pin 1 as a Prognostic Marker for Biochemical Failure
[0155] Materials and Methods
[0156] Study Subjects
[0157] Over 3400 of patients with Benign Prostatic Hyperplasia
(BPH) or cancer underwent radical prostatectomies at one of the
Baylor College of Medicine affiliated institutions (The Methodist
Hospital, Ben Taub Hospital, Saint Luke's Hospital and the Houston
Veterans Affairs Medical Center), and provided tissues for the
Baylor Prostate SPORE Tissue Bank in the Histology Core. Radical
prostatectomy specimens from these patients were processed using
whole mount slides according to procedures previously described by
for example, Sakr, W. A., et al. (1996) Cancer 15; 78:366-778 or
Ohori, M. et al. (1999) J. Urol. 161:500-4. Of these patients 1291
were operated by a single surgeon between 1983 and 1998 without any
previous form of adjuvant therapy such as radiation or hormonal
therapy. These are 87.9% Caucasians, 7.4% Hispanic, 3.5%
African-American and 1.2% Asian or Middle Eastern. Complete
demographic, clinical and follow-up data was available in the
Medical Informatics Core and includes patient's age, blood PSA
levels, clinical staging, and biopsy pathologic information. Entry
criteria for the retrospective cohort study to create a radical
prostatectomy 1) No preoperative treatment 2) Operated by a single
surgeon between 1983 and 1998 3) Radical prostatectomy specimen in
the tissue bank 4) Prostate cancer present in the surgical specimen
and large enough to be cored for tissue microarrays. A total of 622
patients fulfilled the above mentioned criteria and were cored to
produce a large outcomes array.
[0158] Radical Prostatectomy Specimens
[0159] After surgery, the prostate specimens were sliced into 5
mm-thick tissue whole mount according to a procedure previously
described by for example, Sakr, W. A., et al. (1996) Cancer 15;
78:366-778 or Ohori, M. et al. (1999) J. Urol. 161:500-4. The
tissue slices were then fixed in 10% neutral buffered formalin and
embedded in paraffin according to a routine procedure. A single
pathologist following a standard protocol evaluated H & E
stained whole mount sections from each specimen.
[0160] Cohort Enrollment and Follow-up
[0161] Data about the population of patients mentioned above have
been accrued to using SPORE protocols and gathered in the Medical
Informatics Core using the SPORE in Prostate Cancer Information
System (SPIS). A single pathologist performed the pathologic
analysis that includes staging, pathologic stage, margins, capsular
penetration, seminal vesicle invasion, biopsy and prostatectomy
primary and secondary Gleason grades, lymph node status, tumor
volume and geographic location. The clinical and pathologic data of
patients who meet the entry criteria is available for analysis with
patient identifiers removed. Institutional Review Board approved
consent forms were used to obtain assent from all patients.
[0162] Array Construction and Tissue Sections
[0163] The index tumor, defined as the largest and/or highest
Gleason cancer focus, was identified and mapped on the whole mount
sections for each specimen. The tissue microarrays were built using
a visual tissue arrayer (Beecher Instruments, Silver Spring, Md.).
Previous studies indicate that triplicate 0.6 mm. punches reliably
reproduce immunohistochemical marker study results of full sections
of prostate, even for low expression markers such as Ki67. We
therefore punched triplicate 0.6-millimeter cores of the areas with
highest Gleason grade within the mapped index tumor and transferred
them to a recipient block. Areas of normal peripheral zone away
from the tumor were also circled as well as areas of BPH.
Transition zone without BPH was selected in the absence of BPH. The
circled areas were then transferred on to the blocks and triplicate
0.6 mm. cores were also obtained from the circled areas of normal
peripheral zone and BPH. Internal controls were placed at a
pre-established pattern throughout each one of the blocks in order
to assess adequacy of the stain throughout the sections. Sausage
internal controls, which included up to 10 different types of
tissues within each 0.6-mm.-control core, were also placed with the
standard controls.
[0164] The final tissue array set consisted of 15 blocks with 9
cores for every one of the 622 patients for a grand total of
approximately 5500 cores. A database was built for every block
produced, including the coordinates of each core and the area and
case of origin. Five-.mu.m sections from the array blocks and
tissues were cut without use of the transfer tapes for
immunostaining.
[0165] Immunohistochemistry
[0166] Sections were deparaffinized and rehydrated. They were then
heated in Antigen Retrieval Citra solution (pH6.0) (BioGenex, San
Ramon, Calif., Cat. #HK086-9K), with in an 1300 w microwave oven
(Panasonic, Inverter, the Genius 1300W) for 2 minutes at full power
level to bring the solution to boiling temperature. Once it starts
boiling, immediately reduce the power level of microwave to 10% and
continue heating the slides for 15 min. Endogenous peroxidase in
sections was inactivated in 3% H2O2 for 10 min. The sections were
then blocked in 3% normal horse serum in 0.2 M
phosphate-buffered-saline (PBS) pH 7.4 and followed by incubation
in a Pin1 polyclonal antibody (Oncogene Research Products,
Cambridge, Mass., Cat. #PC270, 4 mg/ml) diluted at 1:10,000 in TBS
overnight at 4.degree. C. They were then processed following a
standard avidin biotin complex (ABC) immunostaining procedure with
an ABC kit (Vector Lab, Calif.). Immuno-reaction products were
visualized in a 3,3'-diaminobenzidine/H2O2 solution. To verify the
specificity of the immunoreactions, some sections were incubated
either in PBS or in normal rabbit serum replacing for the Pin 1
antibody.
[0167] Digitizing
[0168] Each slide was digitized twice, using different imaging
systems. The BLISS automated imaging system was used to digitize
the micorarray slides initially. It produces a high-resolution
image of every micorarray dot, with information as to the dot
coordinates on the microarray slide. These images were used at
Baylor College of Medicine for visual semiquantitation and
subsequent correlation with the clinical database.
[0169] The ChromaVision System (ChromVision Medical Systems, Inc.,
San JuanCapistrano, Calif.) was used to digitize the microarray
slides and perform automated image analysis.
[0170] Quantitation of Immunohistochemstry
[0171] Pin 1 immunostaining in normal prostate epithelium, BPH and
prostate cancer were evaluated microscopically and by image
analysis and recorded as percentage of PIN 1 positive cells and
intensity of Pin 1 staining using automated image analysis and
visual semiquantitation. Because of the triplicate nature of the
arrays, three values were obtained for every measurement. The
highest value and the average of the three values were considered
for analysis, giving rise to 4 interpretative values: Intensity
high (PIH), Intensity Average (PIA), Percentage High (P%H) and
Percentage Average (P%A).
[0172] Automated image analysis: Automated image analysis: Each
micro-histoarry section was scanned and images were captured using
automated cellular imaging system (ACIS) (ChromaVision Medical
Systems, Inc., San Juan Capistrano, Calif.) which combines
automated microscopy and computerized image processing in analysis
of multiple tissues on a single slide. In this study, ACIS was used
to analyze micro tissue array sections on glass slides stained
using a diaminodenzidine chromagen (DAB) and hematoxylin
counterstain. Positive staining (brown color) as viewed by light
microscope indicates the presence of the protein, and color
intensity correlates directly with protein quantity (expression).
The ACIS is able to recognize 255 levels of immnohistochemical
staining intensity (0-255) and convert these to fractional scores
for the selected individual areas. However, because the system is
very sensitive the base limit on the threshold for the Generic DAB
is pre-set at 30 by the manufacturer. Therefore, any intensity
below 30, which would have to be an extremely light brown, was
treated as 0 in this study. Entire immunostained tissue sections
were scanned using the 4X objective and images were captured using
the 10X objective. In this study, we used the intensity scoring and
percent positive scoring (the percentage of brown divided by blue
plus brown area) for entire individual tissue dot selected. The
immunohistochemical staining was quantitated without knowledge of
the pathologist's score.
[0173] Visual Semiquantitation: The percentage of positive cells
(labeling rate %) was defined as follows: 0 when no cells stained;
1, 2 and 3 when up to 33%, 66% and 100% of the cells were
immunoreactive. The labeling intensity(I) was semiquantified where
0 defined as lack of staining, 1 weak but distinct staining, 2
moderate staining seen at medium power and 3 intense staining seen
at low power.
[0174] Statistical Analysis
[0175] Associations between clinical/pathological parameters and
Pin 1 expressions were evaluated using Spearman correlation
coefficient testing. For survival analysis, the end point used was
defined as cancer biochemical recurrence, defined as serum PSA
level higher than 0.4 ng/ml on two successive measurements
(Hybritech, Inc., San Diego, Calif.). Time to recurrence was
defined as the time interval between the date of surgery and the
date of identification of biochemical recurrence. The predictive
value of Pin 1 for recurrence-free survival were determined using
the Kaplan-Meier actuarial analysis and the log rank test.
Kaplan-Meier survival curves were constructed for PIN 1-positive
and Pin 1-negative patients. The differences between the survival
curves between groups were tested for statistical significance by
the log-rank test. The Cox univariate and multivariate proportional
hazard regression model was used to evaluate the HR (relative risk
of recurrence). In the multivariate analysis, the models were
adjusted for LN, Surgical Margins, SVI, Gleason grade, ECE, UICC,
and Preoperative PSA levels. All analyses were performed with a
statistical software (Power and Precision software by Biostat (PC
Version, Englewood, N.J.).).
[0176] Results
[0177] Valid information was obtained from tumor specimens of 580
patients. Patient ages ranged from 37 to 80 years, with a median of
63 years. Follow-up information (median follow-up period, 61
months) was available from 578 patients, among whom 111 (19.2%) had
recurred. Other clinical characteristics are seen in Table 8.
11TABLE 8 Clinical/ Intensity Intensity % High % Average
Pathological High p Average P mean p mean p Factors N(%) mean(sd)
value mean(sd) value (sd) value (sd) value TNM stage T1 182 70.7
0.158 66.1 0.172 24.7 0.191 17.6 0.229 (31) (15.4) (13.5) (25.3)
(19.5) T2 356 71.7 66.6 25.3 18.0 (61) (16.7) (16.3) (25.0) (19.8)
T3 42(7) 74.6 70.4 31.0 23.3 (16.0) (14.9) (25.2) (21.0) Gleason
score 4-6 232 71.5 0.779 67.5 0.949 24.1 0.499 17.3 0.611 (40)
(13.8) (12.5) (23.6) (18.8) 7 300 71.6 66.0 26.3 18.6 (52) (16.8)
(15.9) (26.0) (20.3) 8-10 47(8) 72.6 68.0 27.8 21.0 (23.3) (23.4)
(26.8) (21.9) Extraprostatic extension Positive 258 72.0 0.328 67.0
0.396 25.9 0.437 18.6 0.429 (45) (16.4) (16.2) (24.7) (19.4)
Negative 322 71.2 66.5 25 2 18.0 (55) (16.1) (14.7) (25.5) (20.1)
Seminal vesicle invasion Positive 72(12) 73.8 0.175 68.7 0.124 27.8
0.269 20.6 0.240 (15.3) (13.8) (25.5) (21.1) Negative 508 71.3 66.4
25.2 17.9 (88) (16.4) (15.6) (25.1) (19.6) Lymph node metastasis
Positive 37(3) 73.3 0.114 70.2 0.031 32.7 0.026 25.9 0.011 (19.1)
(17.6) (r.sup.2 = (24.3) (r.sup.2 = (22.0) (r.sup.2 = Negative 543
71.5 66.5 0.09) 25.0 0.09) 17.7 0.11) (97) (16.5) (15.2) (25.1)
(19.6) Surgical margin Positive 86(15) 70.3 0.581 63.9 0.353 24.2
0.730 16.6 0.504 (19.2) (19.2) (23.8) (17.8) Negative 494 71.8 67.2
25.7 18.6 (85) (15.7) (14.6) (25.4 (20.1) UICC Staging Age 580
0.330 0.316 0.456 0.401 Preop PSA 564 0.724 0.985 0.149 0.173
[0178] Normal and BPH
[0179] Pin 1 staining was nuclear and seen in normal prostate,
prostate cancer and BPH in varying intensities and percentages.
However, a trend towards Pin 1 over-expression was noted in
prostate cancer as compared to normal prostatic epithelium and BPH
(see Table 9).
12 TABLE 9 Tumor % Average 18.27 10.34 Normal Intensity High 66.6
65 Normal % Average 8.1 3.3 BPH Intensity High 61.5 60 BPH %
Average 6.7 1.2
[0180] Univariate analysis demonstrates that only the Pin 1 average
intensity value in BPH tissues was statistically significant, but
was lost on multivariate analysis (Table 10). The data indicates
that Pin 1 expression in normal or hyperplastic prostatic tissues
cannot be used to predict biochemical failure in PCa.
13 TABLE 10 Descriptives Cox Univarate N Mean (SD) Median (Range)
p-value Intensity High Normal 569 66.6 (13.8) 65 (0-127) 0.094
Intensity Average Normal 570 59.9 (15.4) 61 (0-109.3) 0.731 % High
Normal 567 12.3 (15.5) 6.03 (0-85.8) 0.437 % Average Normal 566 8.1
(11.4) 3.3 (0-63.5) 0.349 Intensity High BPH 573 61.5 (14.7) 60
(0-110) 0.055 Intensity Average BPH 572 55.8 (16) 57.7 (0-99.5)
0.039 (HR = 0.99) % High BPH 574 10.4 (15.8) 2.2 (0-74.9) 0.903 %
Average BPH 575 6.7 (10.9) 1.2 (0-56.9) 0.837
[0181] Prostate Cancer
[0182] Pin 1 expression in prostate cancer is nuclear and seen
between 0 and 91% of the cells, with a median of 18% and a mean of
10% of the cells.
[0183] A) Image Analysis
[0184] 1) Correlation
[0185] Correlation with clinical and pathologic factors showed that
Pin 1 expression was associated with lymph node metastasis and
clinical staging. (see Table 8).
[0186] 2) Survival Analysis
[0187] Continuous measures "Pin 1 Intensity High" (PIH), "Pin 1%
High" (P%H), "PIN 1% Average" (P%A) were significant predictors for
time to PSA recurrence, while "PIN 1 Intensity Average" (PIA) was
not significant, univariately (Table 11). This finding is not
surprising, for intensity of staining is usually best associated
with the areas of greatest intensity or hot spots. In the same
vein, the average of percentage of immunoreactive cells is a better
measure of labeling rate.
14TABLE 11 Univariate and multivariate Analysis of PIN 1 as a
Predictor of Biochemical Recurrence Models HR (95% Cl) p-value PIH
Univariate PIH (continuous) 1.015 (1.003, 1.026) 0.0120 PIH (split
at median) 1.512 (1.034, 2.213) 0.0331 PIH (split at 100) 2.642
(1.554, 4.493) 0.0003 Mulivariate PIH (split at 100) 3.902 (2.215,
6.873) <0.0001 LN 3.385 (2.087, 5.489) <0.0001 Margins 3.201
(2.117, 4.839) 0.0460 Seminal Vesicel 2.883 (1.827, 4.549)
<0.0001 Invasion (SVI) Gleason 2.161 (1.629 2.867) 0.0090 Extra
Prostatic 2.119 (1.202, 3.733) <0.0001 Extension (ECE) Clinical
Stage 1.166 (1.003, 1.356) <0.0001 (IUCC) PreOpPSA 1.019 (1.009,
1.03) <0.0001 PIA Univariate PIA(continuous) 1.011 (0.999,
1.024) 0.0763 PIA (split at median) 1.446 (0.991, 2.110) 0.0557 PIA
(split at 100) 3.566 (1.655, 7.682) 0.0012 Mulivariate PIA (split
at 100) 5.318 (2.335, 12.116) 0.0001 LN 3.265 (2.026, 5.263)
<0.0001 Margins 3.278 (2.151, 4.198) <0.0001 SVI 2.671
(1.699, 3.660) <0.0001 Gleason 2.121 (1.598, 2.816) <0.0001
ECE 2.074 (1.175, 3.660) 0.0119 UIOC 1.137 (0.976, 1.324) 0.0982
PreOpPSA 1.020 (1.010, 1.031) 0.0002 P % H Univariate P % H
(continuous) 1.008 (1.001, 1.015) 0.0221 P % H (split at median)
1.554 (1.062, 2.273) 0.0233 P % H (split at 70) 2.013 (1.167, 3.47)
0.0118 Mulivariate P % H (split at 70) 3.357 (1.905, 5.916)
<0.0001 PreOpPSA 1.020 (1.01, 1.031) <0.0001 UICC 1.152
(0.992, 1.338) 0.0631 LN 3.091 (1.916, 4.986) <0.0001 ECE 2.173
(1.233, 3.828) 0.0072 SVI 2.732 (1.736, 4.301) <0.0001 Margins
3.258 (2.145, 4.948) <0.0001 Gleason 2.269 (1.707 3.016)
<0.0001 P % A Univariate P % A (continuous) 1.011 (1.003, 1.02)
0.0088 P % A (split at median) 1.587 (1.085, 2.322) 0.0174 P % A
(split at 60) 2.896 (1.662, 5.172) 0.0003 Mulivariate P % A (split
at 60) 2.708 (1.416, 5.179) 0.0026 LN 2.557 (1.568, 4.169) 0.0002
Margins 3.457 (2.249, 5.315) <0.0001 SVI 2.617 (1.662, 4.121)
<0.0001 Gleason 2.112 (1.595 2.795) <0.0001 ECE 2.122 (1.205,
3.739) 0.0092 UIOC 1.147 (0.987, 1.332) 0.0744 PreOpPSA 1.021
(1.01, 1.031) 0.0001
[0188] Since all measures are not normally distributed, first the
medians were tested as possible cutoffs. Conclusions remained the
same as for continuous measures. Then the optimal cutoffs were
identified for all four measures of Pin 1 and tested for ability to
predict time to recurrence.
[0189] PIH.+-. were defined as "Pin 1 Intensity High" <100 and
"Pin 1 Intensity High" .gtoreq.100. The hazard ratio for PIH.+-.
was 2.6 (1.6,4.5) with p-value=0.0003 (FIG. 2). The cutoff of 100
was also used for PIA.+-., and the hazard ratio was 3.6 (1.7,7.7)
with p-value=0.0012 (FIG. 3).
[0190] For "Pin 1% High" and "Pin 1% Average", cutoffs of 70 and 60
were used respectively. Both P%H.+-. [HR=2.0 (1.2, 3.5)] (FIG. 3)
and P%A.+-. [2.9 (1.7, 5.2)] (FIG. 4) were significant predictors
for time to recurrence (p=0.0118 and p=0.0003).
[0191] Kaplan-Meier survival curves (Table 8) also demonstrate that
patients with PIN 1-positive tumors have substantially shorter time
to recurrence compared with those who are Pin 1 -negative.
[0192] In multivariate models these relationships were adjusted for
LN, Surgical Margins, SVI, Gleason grade, ECE, UICC, and
Preoperative PSA levels. All four discrete measures were shown to
be significant independent predictors. Hazard ratios (Table 8) show
that Pin 1 is as good, and possible much better, predictor for
recurrence as these commonly used markers.
[0193] 3) Gleason 6 and 7 Patients
[0194] PIH.+-. was also tested in the subgroup of patients with
Gleason grade 6-7. While the majority of PCA cancer patients falls
into this category, this group of patients represents the most
difficult predictive category in PCA. Currently there are no
markers that will predict biochemical recurrence, metastasis or
death. Univariately, PIN 1 remain a strong predictor of biochemical
recurrence in this category of patients, with a p value of 0.0356
and a hazard ratio of 2.6 (1.6,4.5). The later increases on
multivariate analysis to 3.4 (1.7,6.9), among the highest (FIG. 6).
This data strongly suggests that Pin 1 status remains a strong
significant and independent predictor for time to recurrence in
this difficult group of patients with prostate cancer.
[0195] B) Visual Semiquantitation
[0196] Pin 1 expression was identified in 57.5% of the patients
examined by visual semiquantitation, with the vast majority of
these being low expressers (256 of 322). The human eye was not able
to discriminate intensity of expression as well as the image
analysis, for the results indicate significance only as an on/off
phenomenon. The presence of stain, regardless of intensity, was a
significant predictor of biochemical recurrence. While only 37 of
238 patients with no expression recurred, 72 of 307 patients with
expression recurred, for a hazard ration of 1.6 on univariate and
1.9 (1.3-3.0) on multivariate analysis (FIG. 7).
[0197] When Pin 1 is expressed in PCA, the majority of the cancer
cells were immunoreactive, with over 50% of the cases having
immunoreactivity in 100% of the cells. Percent Positive Tumor was a
significant continuous predictor (HR=1.004 p=0.0343) of time to
recurrence. As the result of the optimal cutoff chosen at 10%, the
division of patients 100% agreed with the Intensity Method
described above. This cutoff is substantially lower than the one
obtained by image analysis. It is important to stress however, that
this technique measures the percent of cancer cells expressing PIN
1 while image analysis measures the percentage of the core that is
immunoreactive.
[0198] Percent Positive in Core (<90% Vs 90-100) appeared to be
a significant predictor of time to recurrence on the univariate
analysis (p<0.0001), but when adjusted for know clinical and
pathological predictors in a multivariate model, it was no longer
significant (p=0.7242)
[0199] C) Correlation Between Visual and Image Analysis Results
[0200] There was a highly significant correlation between visual
and machine measures. Although correlation coefficients were
relatively low (Table 12), we believe that is due to the much more
discrete nature of visual measurements.
15TABLE 12 Correlation Between Visual and Image Analysis Visual
Intensity Visual % in Tumor Positive in Tumor Machine rho p-value
rho p-value Intensity High in Tumor 0.4382 <0.0001 0.4247
<0.0001 Intensity Average in Tumor 0.4294 <0.0001 0.4068
<0.0001 % High in Tumor 0.3265 <0.0001 0.3192 <0.0001 %
Average in Tumor 0.3347 <0.0001 0.3220 <0.0001
CONCLUSIONS
[0201] The data here presented establishes Pin 1 as an excellent
prognostic marker for biochemical failure in prostate cancer
patients treated with radical prostatectomy. The association of
elevated Pin 1 expression with cancer's lymph node metastasis and
clinical staging as well as poor clinical outcome indicates its
involvement in disease progression.
[0202] The visual studies tell us that any intensity of expression
of Pin 1 by a minority of Pca cells (>10%) is a very strong
predictor of biochemical recurrence in patients with PCA. Although
the results of visual and automated image analysis are
significantly correlated, the higher hazard ratios obtained with
the latter demonstrate a greater discriminatory power. It is
apparent that image analysis is able to discriminate different
intensities of expression and that a cutoff value can be used to
determine groups with different survival. The patients with
intensities over 100 have a higher rate of biochemical
recurrence.
[0203] These results clearly indicate that Pin 1 can be used on
radical prostatectomy specimens. We suggest that visual observation
of labeling ratio, coupled with the enhanced discriminatory power
of measuring intensity of expression by image analysis can be used
to discriminate biochemical recurrence in patients that have
undergone radical prostatectomies. Furthermore, because tissue
microarray cores have similar amounts of tissue than biopsies, we
are very hopeful that Pin 1 will have applications in pre therapy
biopsies. If so, Pin 1 could be used to triage patients into
watchful waiting with grater certainty.
EQUIVALENTS
[0204] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
* * * * *