U.S. patent application number 10/288609 was filed with the patent office on 2004-03-18 for markers for prostate cancer.
This patent application is currently assigned to Sloan-Kettering Institute for Cancer Research. Invention is credited to Cordon-Cardo, Carlos, Koff, Andrew, Scher, Howard I..
Application Number | 20040053247 10/288609 |
Document ID | / |
Family ID | 31996461 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040053247 |
Kind Code |
A1 |
Cordon-Cardo, Carlos ; et
al. |
March 18, 2004 |
Markers for prostate cancer
Abstract
This invention provides a method for determining the
aggressiveness of a prostate carcinoma comprising: (a) obtaining a
sample of the prostate carcinoma; and (b) detecting the presence of
p27 protein in the prostate carcinoma, the absence of p27
indicating that the prostate carcinoma is aggressive. This
invention also provides a method for diagnosing a beign prostate
hyperplasia comprising: (a) obtaining an appropriate sample of the
hyperplasia; and (b) detecting the presence of the p27 RNA, a
decrease of the p27 RNA indicating that the hyperplasia is beign.
This invention provides various uses of p27 in prostate cancer.
Finally, this invention also provides different marker for prostate
cancer.
Inventors: |
Cordon-Cardo, Carlos; (New
York, NY) ; Scher, Howard I.; (Tenafly, NJ) ;
Koff, Andrew; (Westbury, NY) |
Correspondence
Address: |
John P. White, Esq.
Cooper & Dunham, LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Sloan-Kettering Institute for
Cancer Research
|
Family ID: |
31996461 |
Appl. No.: |
10/288609 |
Filed: |
November 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10288609 |
Nov 5, 2002 |
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09329917 |
Jun 10, 1999 |
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09329917 |
Jun 10, 1999 |
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PCT/US98/25483 |
Dec 1, 1998 |
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60067190 |
Dec 1, 1997 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 33/57434 20130101; C07K 14/4738 20130101; A61K 38/1709
20130101; C12Q 2600/118 20130101; C12Q 2600/112 20130101; A61K
48/00 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Goverment Interests
[0002] This invention was made in part with support under United
States Government NIH Grant CA-DK-47650. Accordingly, the United
States Government has certain rights in the invention.
Claims
What is claimed is:
1. A method for determining the aggressiveness of a prostate
carcinoma comprising: (a) obtaining a sample of the prostate
carcinoma; and (b) detecting the presence of p27 protein in the
prostate carcinoma, the absence of p27 indicating that the prostate
carcinoma is aggressive.
2. A method for diagnosing a beign prostate hyperplasia comprising:
(a) obtaining an appropriate sample of the hyperplasia; and (b)
detecting the presence of the p27 RNA, a decrease of the p27 RNA
indicating that the hyperplasia is beign.
3. A method of claim 2, further comprising detecting the protein
expression of p27 wherein this additional step may be performed
before or after the detection of the presence of the p27 RNA.
4. A method for predicting the life-span of patient with prostate
carcinoma comprising: (a) obtaining a sample of the prostate
carcinoma; and (b) detecting the presence of p27 protein in the
prostate carcinoma, the presence of the p27 protein indicating that
the patient can live longer than the patient who are undetectable
p27 protein.
5. A method for increasing the life-span of patient with prostate
carcinoma comprising inducing the expression of p27 protein in the
prostate carcinoma.
6. A method for prolong life-span of patient with prostate
carcinoma which comprises introducing a nucleic acid molecule
having sequence encoding a p27 protein into the carcinoma cell
under conditions permitting expression of said gene so as to
prolong the life-span of the patient with said prostate
carcinoma.
7. The method of claim 6, wherein the nucleic acid molecule
comprises a vector.
8. The method of claim 7, wherein the vector is an adenovirus
vector, adenoassociated virus vector, Epstein-Barr virus vector,
retrovirus vector or vaccinia virus vector.
9. A method for prolong life-span of patient with prostate
carcinoma which comprises introducing an effective amount of p27
protein into the carcinoma cell so as to thereby prolong the
life-span of the patient with said prostate carcinoma.
10. A method for prolong life-span of patient with prostate
carcinoma which comprises introducing an effective amount of a
substance capable of stabilizing the p27 protein into the carcinoma
cell so as to thereby prolong the life-span of the patient with
said prostate carcinoma.
11. A composition for prolong life-span of patient with prostate
carcinoma which comprises an effective amount of a nucleic acid
molecule having sequence encoding a p27 protein and a suitable
carrier.
12. A composition for prolong life-span of patient with prostate
carcinoma which comprises an effective amount of the p27 protein
and a suitable carrier.
13. A composition for prolong life-span of patient with prostate
carcinoma which comprises an effective amount a substance capable
of stabilizing the p27 protein and a suitable carrier.
14. A method for determining the rate of proliferation of a
prostate cancer comprising: (a) obtaining a sample of the prostate
cancer; and (b) detecting the presence of p21 protein in the
prostate cancer, the presence of p21 indicating that the prostate
cancer will have a high proliferation rate.
15. A method for determining the rate of proliferation of a
prostate cancer comprising: (a) obtaining a sample of the prostate
cancer; and (b) detecting the mdm2 expression in the prostate
cancer, the overexpression of mdm2 indicating that the prostate
cancer will have high proliferation rate.
16. A method for determining whether a prostate cancer would be
metastatic comprising: (a) obtaining a sample of the prostate
cancer; and (b) detecting the level of cyclin D1 expression in the
prostate cancer, the overexpression of cyclin D1 indicating that
the prostate cancer will be metastatic.
17. The method of claim 16, wherein the prostate cancer is
metastatic to bone.
18. A method for determining the tumor recurrence in prostate
cancer comprising: (a) obtaining a sample of the prostate cancer;
and (b) detecting the expression of the cyclin-dependent kinase
inhibitor p16 in the prostate cancer, the overexpression of p16
indicating that the prostate cancer will have high tumor
recurrence.
Description
[0001] This application is a Continuation-In-Part application of
International Application No. PCT/US98/25483, filed Dec. 1, 1998,
which claims the benefit of U.S. Provisional Application No.
60/067,190, filed Dec. 1, 1997, the content of which are
incorporated into this application by reference.
[0003] Throughout this application, various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end the specification, preceding the
claims.
BACKGROUND OF THE INVENTION
[0004] It has been postulated that the loss of function of a new
family of negative cell cycle regulators, which act as
cyclin-dependent kinase inhibitors and have beeen termed CKI, might
lead to tumor development. CKIs fall into two families, Kip and
Ink, on the basis of sequence homology. p27.sup.Kip1 is implicated
in G1 phase arrest by associating with multiple G1 cyclin-dependent
kinases, abrogating their activity. However, no tumor-specific
p27.sup.KiP1 genomic mutations have been found in a large group of
primary human cancers studied. More recently, it has been reported
that proteasome-mediated degradation of p27 protein occurs during
the cell cycle and that this degradation is increased in a subset
of breast and colon carcinomas of poor prognosis. Purpose: The
present study was undertaken in order to assess for potential
alterations of p27 expression in benign prostatic hyperplasia (BPH)
and in a well characterized cohort of patients with prostatic
cancer.
[0005] Inactivation of the p53 and RB tumor suppressor genes has
been implicated in the development and progression of a number of
different cancers (1,2). It has also been postulated that the loss
of function of a new family of negative cell cycle regulators,
which act as cyclin-dependent kinase inhibitors and have been
termed CKI, might also lead to tumor development (3). CKIs fall
into two families, Kip and Ink, on the basis of sequence homology
(4). Kip family members include p21 (also known as WAF1, Cip1, or
Sdi1) (5-7), p27.sup.KiP1 (8-10) and p57.sup.Kip2 (11,12). The Ink
group includes four members: p16.sup.INK4A/MTS1/CDKN2 (13),
p15.sup.INK4B/MTS2 (14) p18.sup.INK4C (15), and p19.sup.INK4D (16).
p27 is a negative regulator implicated in G1 phase arrest by
TGF.beta., cell-cell contact, agents that elevate cyclic AMP, and
the growth inhibitory drug rapamycin (17-21). p27 associates with
multiple G1 cyclin-dependent kinases in non-proliferating cells,
abrogating their activity (4, 8-10).
[0006] To assess its role as a potential tumor suppressor, the
p27.sup.Kip1 gene was mapped to 12p12-12p13.1 and no tumor-specific
genomic mutations in a large group of primary human cancers were
observed (22-24). More recently, it has been reported that
proteasome-mediated degradation of p27 occurs during the cell cycle
and that this degradation is increased in a subset of breast and
colon carcinomas of poor prognosis (25-28). The present study was
undertaken in order to assess for potential alterations of p27
expression in BPH and in a well characterized cohort of patients
with primary and metastatic prostatic cancer.
[0007] 74 prostate carcinomas from primary and metastatic sites,
representing different hormone sensitivities were analyzed. Normal
prostatic tissues and cases of benign prostatic hyperplasia were
also studied. In order to evaluate prostatic tissue of p27 null
mice, eight 7 month old and six greater than 12 month old
littermate pairs of wild-type and p27 knockout animals were used.
Levels of expression and microanatomical localization of p27
protein and RNA transcripts were determined by immunohistochemistry
and in situ hybridization with specific antibodies and probes,
respectively. Comparative analyses between immunohistochemistry,
immunoblotting and immunodepletion assays were also conducted in a
subset of cases. Association between alterations in p27 expression
and clinicopathological variables were evaluated using the
two-tailed Fisher's exact test. Disease relapse-free survivals were
evaluated using the Kaplan-Meier method and the Logrank test.
Distinct anomalies in the expression of p27 in benign and malignant
human prostate tissues are reported. The normal human prostate
shows abundant amounts of p27 and high levels of p27 messenger in
both epithelial and stroma cells. However, p27 protein and
transcripts are almost undetectable in epithelial and stroma cells
of BPH lesions. It is also reported that p27-null mice develop
hypercellular prostatic glands which histologically resemble human
BPH. Based on these findings we postulate that the loss of p27
expression in human prostate may be causally linked to BPH.
Prostatic carcinomas can be categorized into two groups: those that
contain detectable p27 protein and those that do not. In contrast
to BPH, however, both groups of prostatic carcinomas contain
abundant p27 transcripts. Moreover, primary prostatic carcinomas
displaying the p27-negative phenotype appear to be biologically
more aggressive, based on their association with time to prostate
specific antigen (PSA) failure following radical prostatectomy.
These results support the postulate that BPH is not a premalignant
lesion in the pathway of prostate cancer development. Data also
suggest that prostatic carcinoma develops along two different
pathways, one involving the loss of p27 and the other using other
processes that circumvent the growth suppressive effects of
p27.
SUMMARY OF THE INVENTION
[0008] This invention provides a method for determining the
aggressiveness of a prostate carcinoma comprising: (a) obtaining a
sample of the prostate carcinoma; and (b) detecting the presence of
p27 protein in the prostate carcinoma, the absence of p27
indicating that the prostate carcinoma is aggressive.
[0009] This invention also provides a method for diagnosing a beign
prostate hyperplasia comprising: (a) obtaining an appropriate
sample of the hyperplasia; and (b) detecting the presence of the
p27 RNA, a decrease of the p27 RNA indicating that the hyperplasia
is beign. In an embodiment, the above method further detects the
protein expression of p27 wherein this additional step may be
performed before or after the detection of the presence of the p27
RNA.
[0010] This invention provides a method for predicting the
life-span of patient with prostate carcinoma comprising: (a)
obtaining a sample of the prostate carcinoma; and (b) detecting the
presence of p27 protein in the prostate carcinoma, the presence of
the p27 protein indicating that the patient can live longer than
the patient who are undetectable p27 protein.
[0011] This invention also provides a method for increasing the
life-span of patient with prostate carcinoma comprising inducing
the expression of p27 protein in the prostate carcinoma.
[0012] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing a
nucleic acid molecule having sequence encoding a p27 protein into
the carcinoma cell under conditions permitting expression of said
gene so as to prolong the life-span of the patient with said
prostate carcinoma. In an embodiment, the nucleic acid molecule
comprises a vector. The vector includes, but is not limited to, an
adenovirus vector, adeno-associated virus vector, Epstein-Barr
virus vector, Herpes virus vector, attenuated HIV virus, retrovirus
vector and vaccinia virus vector.
[0013] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing an
effective amount of p27 protein into the carcinoma cell so as to
thereby prolong the life-span of the patient with said prostate
carcinoma.
[0014] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing an
effective amount of a substance capable of stabilizing the p27
protein into the carcinoma cell so as to thereby prolong the
life-span of the patient with said prostate carcinoma.
[0015] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount of a nucleic acid molecule having sequence encoding a p27
protein and a suitable carrier.
[0016] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount of the p27 protein and a suitable carrier.
[0017] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount a substance capable of stabilizing the p27 protein and a
suitable carrier.
BRIEF DESCRIPTION OF THE FIGURES
Figures for the First Series of Experiments
[0018] FIG. 1
[0019] Histological analysis, immunohistochemistry, and in situ
hybridization of human primary and metastatic prostatic
carcinomas.
[0020] (A-C) Photomicrographs of primary prostatic carcinomas
processed as follows: (A) Immunohistochemical staining against p27
of a prostatic intra-epithelial neoplastic (PIN) lesion; note the
intense positive immunoreactivities observed in the nuclei of the
tumor cells growing into the lumen. (B) Immunohistochemical
staining against p27 of another PIN lesion showing dysplastic
changes; note the intense positive immunostaining in the nuclei of
normal epithelial cell and the low-to-undetectable staining of the
tumor cells dissecting the gland and growing into the lumen. (C)
Undetectable levels of p27 protein in an invasive primary prostatic
carcinoma; note the staining of a normal gland trapped into the
tumor.
[0021] (D-F) Photomicrographs of metastatic prostate carcinomas
processed as follows: (D) Immunohistochemical staining against p27
of a metastatic prostate carcinoma to lymph node; note the intense
nuclear staining of both tumor cells and lymphocytes (cells in the
germinal center display low p27 levels). (E) Immunohistochemical
staining against p27 of another metastatic prostate carcinoma to
lymph node; note the intense positive immunostaining in the nuclei
of lymphocytes and the undetectable levels of p27 staining on the
tumor cells. (F) Immunohistochemical staining against p27 of a
metastatic prostate carcinoma to bone; note the positive
immunoreactivities in the nuclei of osteoblasts and the lack of
staining of tumor cells.
[0022] (F and G) Photomicrographs of a primary invasive prostatic
carcinoma processed as follows: (F) Low-to undetectable
immunohistochemical staining against p27 in the tumor cells; note
the staining of a normal gland trapped into the tumor. (G) In situ
hybridization on a consecutive section from the case illustrated in
panel (F) showing high mRNA levels of p27.sup.KiP1 even in
p27-negative tumor cells utilizing the anti-sense probe to
p27.sup.KiP1. Original magnification (A) trough (F) 400.times..
[0023] FIG. 2
[0024] In certain prostatic carcinomas p27 protein is a functional
cyclin-dependent kinase inhibitor. (A) Immunohistochemical staining
correlates with the presence of p27 by immunoblotting. Tumors #1
and #2 were negative and tumor #3 positive for p27 protein
expression, paralleling their IHC patterns. (B) Immunodepletion of
p27 extracts. Extracts obtained from tumors #2 and #3 were
subjected to sequential depletion with antibodies specific to p27
or a non-specific rabbit anti-mouse (RaM). Following depletion, the
proteins in the supernatants were resolved and the presence of p27
determined by immunoblotting. (C) Depletion of p27 depletes heat
stable cyclin-dependent kinase inhibitory activity. The supernatant
shown in panel B was boiled and following clarification the soluble
fraction was incubated with different amounts of recombinant cyclin
E/CDK2 kinase and the degree of inhibition of cylin E/CDK2 activity
on histone H1 substrate was measured. The amount of each kinase
used is shown in the panel and the bars are representative
activities on an arbitrary scale. Depletion with either RaM or p27
specific antibodies did not affect the inhibitory activity of the
p27 negative tumor; however, depletion of p27 from the positive
tumor extract completely ablated the heat stable inhibitor
activity.
[0025] FIG. 3
[0026] Recurrence-free proportion analysis of patients with primary
prostate carcinoma (n=42) as assessed by time to detectable PSA.
Patients who had PSA relapse were classified as failures, and
patients with PSA relapse, or those who were still alive or died
from other disease or lost to follow-up during the study period,
were coded as censored. Time to relapse was defined as the time
from date of surgery to the endpoint (relapse or censoring).
Disease relapse-free survivals were evaluated using the
Kaplan-Meier method and the Logrank test. A trend was observed
between a p27 negative phenotype and early relapse (p=0.08).
[0027] FIG. 4
[0028] Histological analysis, immunohistochemistry, and in situ
hybridization of human normal prostate and benign prostatic
hyperplasia.
[0029] (A-C) Photomicrographs of consecutive sections of normal
prostate tissue processed as follows: (A) Immunohistochemical
staining against p27; intense positive immunoreactivities are
observed in the nuclei of epithelial cells in the luminal side of
the acinus, with decreased reactivities in the nuclei of basal and
stroma cells. (B) In situ hybridization showing high mRNA levels of
p27.sup.KiP1 in both epithelial and stroma cells utilizing the
anti-sense probe. (C) In situ hybridization utilizing the sense
probe to p27.sup.KiP1 showing lack of signals in both epithelial
and stroma cells.
[0030] (D-F) Photomicrographs of consecutive tissue sections of a
benign prostatic hyperplastic nodule processed as follows: (D)
Immunohistochemical staining against p27; note the lack or almost
undetectable levels of immunoreactivity observed in the nuclei of
both epithelial and stroma cells in the luminal side of the acinus,
with decreased reactivities in the nuclei of basal and stroma
cells. (E) In situ hybridization showing low-to-undetectable
p27.sup.KiP1 transcripts also in both epithelial and stroma cells
utilizing the anti-sense probe; note the strong signal of the
cellular inflammatory infiltrates that serve as an internal
positive control. (F) In situ hybridization utilizing the sense
probe to p27.sup.KiP1 showing lack of signals in epithelial and
stroma cells, as well as cellular inflammatory elements. Original
magnifications: (A), (B) and (C) 1000.times.; (D), (E) and (F)
400.times..
[0031] FIG. 5
[0032] Histopathological analysis of the prostatic tissues of 12
month old p27+/+ (A) and p27-/- (B-D) mice. Photomicrographs of
tissue sections of normal prostate samples processed as follows:
(A) Hematoxylin and eosin staining of a prostate gland of a p27+/+
mouse showing well defined acini of epithelial cells surrounded by
a stroma containing few fibroblasts and poor in supportive
connective tissue components. (B) Hematoxylin and eosin staining of
a prostate gland of a p27-/- mouse showing multiple and complex
glands and hypercellular acini of epithelial cells surrounded by
fibromuscular stroma cells in a connective tissue displaying
abundant supportive components. (C and D) Hematoxylin and eosin
stainings of a prostate gland of a p27-/- mouse, high power
details, illustrating the complexity of the glands and abundant
fibromuscular stroma elements (C), as well as the hypercellularity
of the acini (D). Original magnifications: (A) and (B) 200.times.;
(C) and (D) 400.times..
Figures for the Second Series of Experiments
[0033] FIG. 6. Photomicrographs of selected primary prostate
carcinoma cases analyzed by immunohistochemistry utilizing mouse
monoclonal antibodies PAB1801 (anti-p53, A), 2A10 (anti-mdm2, B),
Ab-1 (anti-p21, C), and MIBI (anti-Ki67, D). A, p53 nuclear
overexpression in tumor cells. Note the positive nuclear staining
of tumor cells at a perineural invasion site (arrow). B, mdm2
nuclear overexpression in tumor cells. C,p21 nuclear overexpression
in tumor cells. D, high Ki67 proliferative index. Note the intense
Ki67 nuclear staining of tumor cells at a perineural invasion site
(arrow).
[0034] FIG. 7. Progression-free and survival curves for patients
with primary prostate cancer. The Kaplan-Meier method was used to
estimate overall disease free survival. The log-rank analysis was
used to compare the different curves. A, progression was
significantly reduced in patients with tumors displaying a
p53-postive phenotype (P<0.01). B, progression was not related
to mdm2 status. C, progression was significantly reduced in
patients with tumors displaying a p21 positive phenotype
(P=0.0165).
[0035] FIG. 8. Diagrammatic representation of the p53-pathway (A),
and alterations that may develop during tumor progression in
prostate cancer (B). (A) p53 regulates the expression of several
genes involved in cell cycle arrest (ie, p21) and apoptosis (ie,
bax). p21 binds to heterodimeric protein kinases formed by cyclins
and cyclin-dependent kinases (Cdk's), blocking phosphorylation of
pRB/E2F1 complexes and abrogating S-phase entry. p53 also produces
an autoregulatory feed back loop by transactivating mdm2. (B)
Overexpression of mdm2 has been observed to occur in several tumor
types, and it is considered an oncogenic event. Upon binding to
mdm2, p53 products are trasncriptionally inactivated and triggered
for degradation. This will release the G1 arrest imposed, in part,
by p21 and abolish the apoptotic signals of the pathway. Thus,
inactivation of p53 will favor proliferative activity, immortality,
and development/accumulation of further DNA damage or mutations.
The increased p21 expression observed in our study could be
produced via growth factor signaling, which would also impact on
cyclin D1 expression. The increment of p21 does not appear to be
able to control the proliferative activity of tumor cells, as
attested by the association of p21 positive phenotype and high Ki67
proliferative index. Taken together, mdm2 overexpression will
inactivate the p53-pathway, while increased mitogenic activity will
offset the RB-pathway. The mechanistic basis for this dual
requirement stems, in part, from the deactivation of a
p53-dependent cell suicide program that would normally be brought
about as a response to uncheked cellular proliferation resulting
from RB-deficiency.
Figures for the Third Series of Experiments
[0036] FIG. 9A., FIG. 9B., FIG. 9C., FIG. 9D., FIG. 9E., and FIG.
9F.
Figures for the Fourth Series of Experiments
[0037] FIGS. 10A-B. Immunohistochemistry and in situ hybridization
of human benign prostatic hyperplasia (BPH). Consecutive sections
of benign hyperplastic prostate tissue were processed as follows:
A) Immunohistochemical staining of p16 is shown. Protein expression
levels are undetectable in both epithelial and stromal components.
B) In situ hybridization shows undetectable mRNA levels of p16 in
both epithelial and stromal components when the antisense probe is
used.
[0038] FIGS. 11A-D. Immunohistochemistry and in situ hybridization
of human primary prostatic carcinomas. Consecutive sections of
primary human prostate cancer tissue were processed as follows: A)
Immunohistochemical staining of p16 is shown. Lack of
immunoreaction noted in the nuclei and cytoplasm of both epithelial
and stromal components. B) In situ hybridization reveals
undetectable mRNA levels of p16 in both epithelial and stromal
components when the antisense probe is used. (C-D) Histologic
analysis, immunohistochemistry, and in situ hybridization of human
primary prostatic carcinoma showing p16 overexpression. Consecutive
sections of primary human prostate cancer tissue were processed as
follows: C) Immunohistochemical staining of p16 is shown. Note
strong brown immunoreaction observed in the nuclei of cells. Faint
cytoplasmic staining is noted as well. D) In situ hybridization
shows high mRNA levels of p16 in epithelial cells when the
antisense probe is used. A normal gland (see pointer) serves as an
internal negative control in both the immunohistochemical analysis
in FIG. 11C and also the in situ hybridization analysis in FIG.
11D.
[0039] FIG. 12. Kaplan-Meier curves, using the log rank test,
stratified by p16 groups (group A or group B) of patients with
primary prostate carcinoma (n=88) as assessed by time to detectable
PSA level post prostatectomy. Time to relapse was defined as the
time from the date of surgery to the time of PSA elevation after
surgery. The median time to relapse for group A has not been
reached. The median time to relapse for group B was 46.25 months.
Patients who had PSA relapse were classified as having treatment
failures and tumor recurrence.
DETAILED DESCRIPTION OF THE INVENTION
[0040] This invention provides a method for determining the
aggressiveness of a prostate carcinoma comprising: (a) obtaining a
sample of the prostate carcinoma; and (b) detecting the presence of
p27 protein in the prostate carcinoma, the absence of p27
indicating that the prostate carcinoma is aggressive.
[0041] This invention also provides a method for diagnosing a beign
prostate hyperplasia comprising: (a) obtaining an appropriate
sample of the hyperplasia; and (b) detecting the presence of the
p27 RNA, a decrease of the p27 RNA indicating that the hyperplasia
is beign. In an embodiment, the above method further detects the
protein expression of p27 wherein this additional step may be
performed before or after the detection of the presence of the p27
RNA.
[0042] This invention provides a method for predicting the
life-span of patient with prostate carcinoma comprising: (a)
obtaining a sample of the prostate carcinoma; and (b) detecting the
presence of p27 protein in the prostate carcinoma, the presence of
the p27 protein indicating that the patient can live longer than
the patient who are undetectable p27 protein.
[0043] This invention also provides a method for increasing the
life-span of patient with prostate carcinoma comprising inducing
the expression of p27 protein in the prostate carcinoma.
[0044] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing a
nucleic acid molecule having sequence encoding a p27 protein into
the carcinoma cell under conditions permitting expression of said
gene so as to prolong the life-span of the patient with said
prostate carcinoma. In an embodiment, the nucleic acid molecule
comprises a vector. The vector includes, but is not limited to, an
adenovirus vector, adeno-associated virus vector, Epstein-Barr
virus vector, Herpes virus vector, attenuated HIV virus, retrovirus
vector and vaccinia virus vector.
[0045] Methods to introduce a nucleic acid molecule into cells have
been well known in the art. Naked nucleic acid molecule may be
introduced into the cell by direct transformation. Alternatively,
the nucleic acid molecule may be embedded in liposomes.
Accordingly, this invention provides the above methods wherein the
nucleic acid is introduced into the cells by naked DNA technology,
adenovirus vector, adeno-associated virus vector, Epstein-Barr
virus vector, Herpes virus vector, attenuated HIV vector,
retroviral vectors, vaccinia virus vector, liposomes,
antibody-coated liposomes, mechanical or electrical means. The
above recited methods are merely served as examples for feasible
means of introduction of the nucleic acid into cells. Other methods
known may be also be used in this invention.
[0046] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing an
effective amount of p27 protein into the carcinoma cell so as to
thereby prolong the life-span of the patient with said prostate
carcinoma.
[0047] This invention provides a method for prolong life-span of
patient with prostate carcinoma which comprises introducing an
effective amount of a substance capable of stabilizing the p27
protein into the carcinoma cell so as to thereby prolong the
life-span of the patient with said prostate carcinoma. Such
substance may be either inhibiting the protease which degrade the
p27 protein or it may interact with p27 in such a way that the
protein will be resistant to degradation. By administering such
substance into the cell, the effective amount of p27 protein will
be increased.
[0048] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount of a nucleic acid molecule having sequence encoding a p27
protein and a suitable carrier.
[0049] As used herein, the term "suitable carrier" encompasses any
of the standard carriers. The composition may be constituted into
any form suitable for the mode of administration selected.
Compositions suitable for oral administration include solid forms,
such as pills, capsules, granules, tablets, and powders, and liquid
forms, such as solutions, syrups, elixirs, and suspensions. Forms
useful for parenteral administration include sterile solutions,
emulsions, and suspensions.
[0050] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount of the p27 protein and a suitable carrier.
[0051] This invention provides a composition for prolong life-span
of patient with prostate carcinoma which comprises an effective
amount a substance capable of stabilizing the p27 protein and a
suitable carrier.
[0052] This invention provides a method for determining the rate of
proliferation of a prostate cancer comprising: (a) obtaining a
sample of the prostate cancer; and (b) detecting the presence of
p21 protein in the prostate cancer, the presence of p21 indicating
that the prostate cancer will have a high proliferation rate.
[0053] This invention also provides a method for determining the
rate of proliferation of a prostate cancer comprising: (a)
obtaining a sample of the prostate cancer; and (b) detecting the
mdm2 expression in the prostate cancer, the overexpression of mdm2
indicating that the prostate cancer will have high proliferation
rate.
[0054] This invention provides a method for determining whether a
prostate cancer would be metastatic comprising: (a) obtaining a
sample of the prostate cancer; and (b) detecting the level of
cyclin D1 expression in the prostate cancer, the overexpression of
cyclin D1 indicating that the prostate cancer will be metastatic.
In an embodiment, the prostate cancer is metastatic to bone.
[0055] This invention provides a method for determining the tumor
recurrence in prostate cancer comprising: (a) obtaining a sample of
the prostate cancer; and (b) detecting the expression of the
cyclin-dependent kinase inhibitor p16 in the prostate cancer, the
overexpression of p16 indicating that the prostate cancer will have
high tumor recurrence.
[0056] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Experimental Details for First Series of Experiments
MATERIALS AND METHODS
[0057] Patient Characteristics and Tissues. A cohort of 74 patients
with prostatic carcinoma were evaluated. Tissues were obtained from
the Department of Pathology, Memorial Sloan-Kettering Cancer
Center, New York. Samples were formalin-fixed, paraffin-embedded
tissue specimens. Fourty-two primary prostate adenocarcinoma
specimens were evaluated, as well as 9 metastases to lymph node and
23 metastases to bone. Normal prostatic tissue and/or areas of
benign prostatic hyperplasia adjacent to tumor were observed in the
majority of the primary cases studied. These tissues were also
analyzed as part of the study. In addition, 10 pairs of frozen
normal and tumor prostate tissues were utilized for antibody
titration, as well as comparative analyses between
immunohistochemistry, immunoblotting and immunodepletion assays
(see below). Representative hematoxylin-eosin stained sections were
examined to evaluate the histopathological characteristics of the
lesions to be analyzed, including the ratio of normal-to-tumor
content for microdissection techniques.
[0058] In order to evaluate prostatic tissue of p27 null mice,
eight 7 month old and six greater than 12 month old littermate
pairs of wild-type and p27 knockout animals were used. Tissues were
dissected, weighted and processed for histology by formalin
fixation and paraffin embedding. Tissue sections were cutted and
stained with hematoxylin-eosin for histologic analysis. All
sections were utilized to count the number of acini per gland, a
process that was conducted utilizing magnifications of
200.times..
[0059] Antibodies and Immunohistochemistry. The following well
characterized antibodies and corresponding final working dilutions
were used for the present study: monoclonal antibody p27/Kip1
(Ab-2, Oncogene Science, Boston, Mass.--0.1 ug/ml final
concentration) and anti-p27 affinity purified rabbit antiserum
(1:500 dilution). A non-immune rabbit serum and mouse monoclonal
antibody MIgS-KpI were used as negative controls at similar working
dilutions. Deparaffinized sections were treated with 3%
H.sub.2O.sub.2 in order to block endogenous peroxidase activity.
Sections were subsequently immersed in boiling 0.01% citric acid
(pH 6.0) in a microwave oven for 15 minutes to enhance antigen
retrieval, allowed to cool, and incubated with 10% normal horse or
normal goat sera to block non-specific tissue immunoreactivities.
Primary antibodies were then incubated overnight at 4.degree. C.
Biotinylated horse anti-mouse IgG antibodies (Vector Laboratories,
Burlingame, Calif.--1:500 dilution) or goat anti-rabbit antibodies
(Vector Laboratories--1:800 dilution) were applied for 1 hour,
followed by avidin-biotin peroxidase complexes for 30 minutes
(Vector Laboratories--1:25 dilution). Diaminobenzidine was used as
the final chromogen and hematoxylin was used as the nuclear
counterstain. Nuclear immunoreactivities were classified as a
continuum data (undetectable levels or 0% to homogeneous staining
or 100%). Tumors were grouped into two categories defined as
follows: negative (0% or undetectable staining to <20% nuclear
immunoreactivity in tumor cells), and positive (neoplasms with
.gtoreq.20% tumor cells with nuclear staining) (see statistical
section).
[0060] Probes and In Situ Hybridization. Digoxigenin-labeled probes
were used for in situ hybridization and 1 ug of recombinant plasmid
pCR.TM.II (Invitrogen, San Diego, Calif.), containing the full
length human p27 gene (gift of Dr. M. Pagano, New York University
School of Medicine, NY) was linearized by BamHI and XbaI to
generate antisense and sense transcripts. Riboprobes were generated
with T7 and SP6 polymerase for 2 hours at 37.degree. C. in 1.times.
transcription buffer (Boehringer Mannheim, Indianapolis, Ind.), 20
U of RNAse inhibitor, 1 mmol/L each of ATP, GTP, CTP, 6.5 mmol/L
UTP and 3.35 mmol/L digoxigenin-UTP. Deparaffinized tissue sections
were rinsed in water and PBS for 10 minutes. The slides were
digested with Proteinase K (50 ug/ml) for 18 minutes at 37.degree.
C. in PBS, and post-fixed at .degree.4 C. in a freshly prepared
solution of 4% paraformaldehyde in PBS for 5 minutes.
Prehybridization was done for 30 minutes at 45.degree. C. in 50%
formamide and 2.times.SSC. The hybridization buffer consisted of
50% deionized formamide (v/v), 10% dextran sulphate (50% stock
solution), 2.times.SSC (20.times. stock solution), 1% SDS (10%
stock solution), and 0.25 mg/ml of herring sperm DNA (10 mg/ml).
Hybridization was peformed overnight at 45.degree. C. applying 10
pmol/L digoxigenin-labeled riboprobe in 50 ul of hybridization
buffer per section under a coverslip. The coverslips were removed
and the slides were washed in pre-warmed 2.times.SSC for 20 minutes
at 60.degree. C. twice, followed by washes in pre-warmed
0.5.times.SSC and 0.01.times.SSC at 60.degree. C. for 20 minutes,
respectively. After these washes the slides were incubated in
normal sheep serum diluted in buffer pH 7.5 and successively in the
same buffer with antibody anti-digoxigenin-AP (Boehringer Mannheim,
Indianapolis, Ind.) at dilution of 1:1500 for 1 hour at room
temperature. The visualization was accomplished by nitro-blue
tetrazolium 5-bromo-4-chloro-3-indoylphosphate. The slides were
counterstained with methyl green and mounted.
[0061] Immunoblotting and Immunodepletion Assays. Proteins were
extracted from three OCT-embedded prostatic carcinomas and resolved
on polyacrylamide gels for immunoblotting with p27-specific
antibodies. Extracts obtained from p27 positive and negative tumors
were subjected to sequential depletion with antibodies specific to
p27 or a non-specific rabbit anti-mouse (RaM). Following depletion,
the proteins in the supernatants were resolved and the presence of
p27 determined by immunoblotting. Aliquots of these supernatants
were briefly boiled and following clarification the soluble
fraction was incubated with different amounts of recombinant cyclin
E/CDK2 kinase and the degree of inhibition of cylin E/CDK2 activity
on histone H1 substrate was measured.
[0062] Statistical Methods. The statistical analyses were conducted
as follows. For alterations of the p27, we divided patients into
two groups: p27 negative (0% or no immunohistochemical staining to
<20% tumor cells displaying nuclear reactivities) or p27
positive (.gtoreq.20% tumor cells with nuclear immunostaining with
IHC). The data analyses were conducted to explore the relationship
between p27 alterations and clinicopathological variables such as
presentation (primary, lymph node metastases, and bone metastases),
clinical stage (B, C, D), total Gleason score (6 or less versus 7
or more), and hormonal status (nave versus androgen-independent) in
a total 74 patients. For 42 patients with primary prostate cancer
who underwent radical prostatectomy, further analysis was conducted
to evaluate the relationship between p27 alterations and clinical
variables, including those described above and PSA relapse (yes and
no). Two-tail Fisher's exact test was utilized to assess these
associations and two tailed p-values were employed as a significant
level (29). The FREQ procedure in SAS was used in this study (30).
In the analysis of disease relapse-free survival, patients who had
PSA relapse were classified as lost failures, and patients with PSA
relapse, or those who were still alive or died from other disease
or to follow-up during the study period, were coded as censored.
Disease relapse-free survivals were evaluated using the
Kaplan-Meier method (31) and the Logrank test (32). The LIFETEST
procedure in SAS was used (30). Proportional hazards analysis was
used to obtain maximum likelihood estimates of relative risks and
their 95% confidence intervals (33,34).
EXPERIMENTAL RESULTS AND DISCUSSION
Experimental Results and Discussion for the First Series of
Experiments
[0063] To determine whether loss of p27 expression was a common
feature in prostate cancer, we analyzed 74 prostate carcinomas from
primary and metastatic sites, representing different hormone
sensitivities. Included were 42 hormone-nave primary tumors, some
with associated prostatic intraepithelial neoplastic (PIN) lesions,
and 32 metastatic carcinomas from lymph node tumors (n=9) and bone
metastases (n=23). Thirteen of these metastatic lesions were from
hormone-nave cases, while the remaining 19 metastases were obtained
after hormonal treatment. PIN lesions displaying a cribiform or
pseudopapillary pattern expressed high levels of p27 protein (FIG.
1A) and were associated with p27-positive invasive prostatic
carcinomas. In contrast, PIN lesions displaying a flat growth
pattern had low to undetectable p27 levels (FIG. 1B) and were
associated with p27-negative invasive tumors. Of the invasive
primary prostatic carcinomas studied, 12 of 42 (28.5%) cases had an
intense nuclear immunoreactive p27 pattern in the malignant cells
(data not shown). The remaining 30 (71.5%) primary neoplasms
displayed altered patterns of expression: 12 cases had undetectable
p27 levels (FIG. 1C), while 18 cases had a heterogeneous pattern of
expression (data not shown). In metastatic lesions, 7 of 32 (21.9%)
showed intense p27 nuclear immunostaining in most tumor cells (FIG.
1D). The remaining 25 (78.1) metastatic lesions had either
heterogeneous (data not shown) or undetectable nuclear expression
of p27 (FIGS. 1E and 1F). Interestingly, all but one of the nine
patients with hormone-independent bone lesions displayed altered
p27 expression. Four of these 9 cases had undetectable p27 protein
expression (FIG. 1F), 4 cases had heterogeneous patterns of p27
expression ranging from 30% to 40% tumor cells with weak positive
staining, and one case displayed 80% positive tumor cells. However,
high levels of p27.sup.KiP1 mRNA, as determined by in situ
hybridization to a p27 cDNA probe, were found in all tumors even
when the lesions displayed undetectable levels of p27 protein
(FIGS. 1G and 1H).
[0064] In the group of tumors that expressed p27, we next
determined if the p27 protein was inactivated. To accomplish this
we extracted protein from fresh frozen samples and measured the
heat stable Cdk inhibitory activity, using cyclin E/CDK2 as a
substrate, remaining in extracts following depletion with
p27-specific antibodies as described previously (17) (FIG. 2).
Depletion of p27 protein was confirmed by immunoblotting. As
expected, the depletion of extracts derived from p27 negative
tumors did not affect the heat stable inhibitory activity, nor did
depletion of p27 positive tumor extract with a non-specific
rabbit-anti-mouse immunoglobulin. However, depletion of extracts
derived from p27 positive tumors with the p27-specific antibody
completely removed the inhibitory activity, indicating that p27 was
functional as a Cdk inhibitor in these samples.
[0065] Taken together, these data suggest that prostatic carcinomas
develop along two different pathways, one involving the loss of p27
and the other using alternative processes that may circumvent the
growth suppressive effects of p27. In order to determine if these
distinct pathways of prostate tumorigenesis correlate with clinical
parameters, as reported for other tumor types (25-28), associations
between p27 immunostaining, stage, total Gleason score, and
hormonal status of the tumor were assessed. No associations between
detectable versus undetectable p27 protein, Gleason score (6 or
less versus 7 or more), or hormonal status (nave versus
androgen-independent) were observed. To assess disease
aggressiveness, we evaluated the time to PSA failure, the most
sensitive indicator of success or failure following radical
prostatectomy, in patients treated for localized disease. Only
patients who had an undetectable PSA level after surgery, an
indication that the resection was complete, were considered. A
trend toward an association was observed between a p27 negative
phenotype and early relapse (p=0.08) (FIG. 3). This difference did
not reach statistical significance due to the limited sample size
of the cohort analyzed. Supporting this concept is the fact that in
a multivariate proportional hazards analysis, after controlling for
stage and Gleason score, p27 status still was the strongest factor
in predicting PSA relapse (p=0.07).
[0066] These data suggest extending the characterization of p27
expression to normal prostate and benign prostatic hyperplasia. In
the normal human prostate, abundant amounts of p27 protein were
detected in the ductal and acinar cells, mainly luminal elements,
as well as stroma cells using immunohistochemistry. Epithelial
cells displayed a strong nuclear immunostaining signal (FIG. 4A).
Likewise, both epithelial and stroma cells expressed abundant p27
transcripts (FIGS. 4B and 4C), as detected by in situ
hybridization. Strikingly, in 12 cases of BPH p27 expression was
low to undetectable in epithelial and stroma cells in the
hyperplastic nodules. Immunohistochemical staining revealed low to
undetectable immunoreactivities in both epithelial and
fibromuscular cells in the hyperplastic nodules (FIG. 4D). This
contrasts with the strong p27 nuclear immunostaining phenotype
observed in the normal prostate. Likewise, p27 mRNA transcript
levels were low to undetectable on consecutive sections of BPH by
in situ hybridization (FIGS. 4E and 4F). In some of these BPH
tissue samples we found areas of basal cell hyperplasia. These
cellular elements also had low to undetectable amounts of p27
protein and transcripts (data not shown). Nevertheless, in the
non-hyperplastic regions of these same BPH samples, normal ductal
and acinar epithelial cells, as well as stroma elements, showed
high levels of p27 expression. These results indicate that in the
development of BPH, p27 transcription may be down-regulated. This
finding was quite unexpected as this gene product is generally
regulated at post-transcriptional levels (35-37), although members
of the nuclear hormone receptor superfamily are suggested to
regulate p27.sup.KIP1 mRNA levels (38).
[0067] The targeted deletion of the p27 locus in a murine model was
recently reported (39-41). p27 deficient mice are viable and
display organomegaly, increased body size and female infertility.
These anomalies could not be attributed to a defect of the growth
hormone/IGF-1 axis, rather, they resulted from excess proliferation
prior to withdrawal of cells into a terminally differentiated state
(39). No increased incidence of spontaneous tumors was observed;
however, many p27-null mice developed a pituitary hyperplasia
reminiscent of adenoma in the intermediate lobe. These data suggest
that p27 deficiency leads to hyperplasia in many tissues and
organs. The high frequency of benign prostatic hyperplasia (BPH) in
men and the alterations on p27 expression in that condition
suggested a parallel to p27 deficiency. Previous reports of
histopathological analyses of p27 null mice did not include the
prostate (39-41). We next set up to determine the morphologic
characteristics of the prostate gland in p27 deficient animals.
Comparing the total mean prostate weights of 7 month old
age-matched p27+/+ (n=8) and p27-/- (n=8) mice, the differences
were not significant [mean +/- SD: 80.6 mg (+/-8.6 mg) and 90.1 mg
(+/-13.3 mg), respectively (p=0.1)]. However, the mean acini counts
of the total gland in these groups were significantly different
[mean +/- SD: 50.4 (+/-8.5) and 74.9 (+/-8.9), respectively
(p<0.01)]. A similar relationship was observed in the mean total
prostate weights of the old (greater than 12 months) p27+/+ (n=6)
and p27-/- (n=6) mice [mean +/- SD: 114.0 mg (+/-18.5 mg) and 119.0
mg (+/-26.8 mg), respectively (p=0.7)], and the mean acini counts
[mean +/- SD: 54.7 (+/-6.5) and 73.8 (+/-5.3), respectively
(p<0.01)]. The significant increase in the number of acini in
both young and old p27 deficient mice was associated with
histopathological differences that became more accentuated in the
elderly group. The hyperplastic prostate of the older p27-/- mice
showed enlarged glands, development of hypercellular acini of
epithelial cells, and an increase in fibromuscular stroma cells
(FIG. 5). These histological changes are reminiscent of BPH in
humans and support the hypothesis that the loss of p27 expression
in human prostate may be causally linked to BPH.
[0068] It has been suggested that BPH and malignant prostate growth
share a common pathway because they commonly coexist and
demonstrate androgen dependency (42-44). However, this relationship
remains unclear since BPH tends to develop in the transition zone,
while the majority of carcinomas develop in the peripheral zone
(45-48). Results from the present study reveal that, unlike in the
BPH lesions, prostatic carcinoma cells regulate p27 expression at
the post-transcriptional level. Taken together these data support
the postulate that BPH is not a premalignant lesion in prostate
cancer development.
[0069] Coordinate inactivation of the pathways involving the p53
and RB genes appears to be an essential requirement for the genesis
of most human cancers. However, both p53 mutations and RB
alterations are reported to be late and uncommon events in prostate
tumor progression (49-52). Contrary to these results, data from
this study indicate that inactivation of p27 is a frequent and
early event in some prostate cancers. It is thus our working
hypothesis that p27 represents another pathway of tumor suppression
in certain human tumors, prostate cancer being a paradigm in which
this concept could be further tested.
[0070] In summary, data from this study suggest that p27.sup.KiP1
gene ablation in the mouse causes a pronounced prostatic
hyperplasia, and that the loss of p27 expression in human prostate
may be causally linked to BPH. In addition, data from this study
suggest that prostatic carcinoma develops along two different
pathways, one involving the loss of p27 and the other using
alternative processes that circumvent the growth suppressive
effects of p27. These phenotypes can be identified as early as in
the PIN stage. Moreover, primary prostatic carcinomas displaying
the p27-negative phenotype appear to be biologically more
aggressive, based on their association with time to PSA failure
following radical prostatectomy while controlling for other
variables. The consistent alteration of p27 expression observed in
all androgen-independent metastatic lesions suggests an association
with tumor progression, which may be the result of the metastatic
process itself. Alternatively, it may be postulated that p27
positive tumors are more sensitive to androgen ablation, the
primary treatment of metastatic disease. Finally, two dissimilar
mechanisms appear to be involved in the loss of p27 expression in
BPH versus a subset of prostatic carcinomas. p27.sup.KiP1 mRNA
levels are extensively reduced in BPH, whereas p27 proteins are
diminished to undetectable levels in some prostatic carcinomas
despite detectable p27 mRNA as the result of a post-transcriptional
event. These results support the postulate that BPH is not a
premalignant lesion in the pathway of prostate cancer
development.
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A., Hammond, E. H., Pilepich M V, Forman J D, Mesic J, Fu K K,
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SECOND SERIES OF EXPERIMENTS
[0123] To determine the potential role of p53 inactivation in
prostate cancer, we studied a well characterized cohort of 86
patients treated with radical prostatectomy. We analyzed patterns
of p53, mdm2, and p21/WAF1 expression by immunohistochemistry.
Results were then correlated with clinicopathological parameters of
poor outcome, including time to PSA relapse. In addition, data were
also correlated with proliferative index, as assessed by Ki67
antigen detection. p53 positive phenotype, defined as
identification of nuclear immunoreactivity in >20% tumor cells,
was observed in 6 of 86 cases (7%). An association was observed
between p53 positive phenotype and decreased time to PSA relapse
(P<0.01). mdm2 positive phenotype, defined as .gtoreq.20% tumor
cells displaying nuclear immunoreactivity, was observed in 28 of 86
cases (32.5%). mdm2 positive phenotype was found to be associated
with advanced stage (P=0.009). p21 positive phenotype, defined as
>5% tumor cells with nuclear immunoreactivity, was observed in
28 of 86 cases (32.5%). An association was observed between p21
positive phenotype and high Ki67 proliferative index (P=0.002).
Patients with p21 positive phenotype had a significant association
with decreased time to PSA relapse (P=0.0165). In addition, a
significant association was found between p21 positive phenotype
and co-expression of mdm2 (P<0.01). Fourty-three of 86 cases
(50%) were found to have one or more alterations, and patients with
any alteration were found to have a higher rate of PSA relapse
(P<0.01). It is our hypothesis that a pathway of prostate cancer
progression involves p53 inactivation caused by mdm2
overexpression, and that p21 transactivation in this setting is due
to an alternative signaling system rather than through a
p53-dependent mechanism.
[0124] p53 responds to different forms of cellular stress by
targeting and activating genes involved in growth arrest and cell
death. A target of p53-induce transcription is the p21/WAF1 gene,
which encodes a cyclin-dependent kinase inhibitor (1). In addition,
levels of p53 are tightly regulated by mdm2, which binds to p53
repressing its activity and triggering its degradation. The MDM2
gene is itself under the transcriptional control of p53, creating
an autoregulatory feedback loop (2).
[0125] Alterations in the TP53 gene appear to be uncommon in
prostate cancer, and their clinical significance has not been fully
investigated. A recognized limitation of most studies is that they
are confined to the analysis of p53 alterations, without analyzing
other critical components that regulate its functions. The MDM2
gene is amplified in a variety of tumors, and mdm2 overexpression
without amplification appears to be a common mechanism of p53
inactivation in certain cancers (3,4). Lack of data regarding the
functional status of the p53 products encountered in the tumors
analyzed represents another drawback. It has been reported that
p21/WAF1 gene expression may serve as an indicator of p53 activity,
since p21/WAF1 is under the transcriptional control of p53.
However, serum or individual growth factors, such as epidermal
growth factor (EGF), and fibroblast growth factor (FGF), were shown
to induce p21 expression in p53-deficient cells (5,6). Thus, there
are at least two separate pathways accounting for the induction of
p21, one linked to DNA-damage recognition, and the other produced
by signaling mechanisms caused by certain cellular mitogens.
[0126] In the present study, we have analyzed the patterns of p53
expression and those of critical components of its pathway, namely
mdm2 and p21, in 86 patients with prostate cancer.
[0127] The association between these markers and
clinicopathological parameters of poor outcome, including time to
PSA relapse and proliferative index, were also examined.
EXPERIMENTAL DETAILS
Experimental Details for Second Series of Experiments
MATERIALS AND METHODS
[0128] Patients. A total number of 86 patients who underwent
radical prostatectomy at Memorial Sloan-Kettering Cancer Center in
the period between 1990 through 1991 were studied. Patient
selection was based on the availability of both adequate clinical
follow up and representative archival pathological materials for
immunohistochemical analysis. The median age at the time of surgery
was 65 years (range 46-74). Their median follow up was 64.5 months
(range 10-94 months). Formalin-fixed, paraffin embedded prostate
tissues were obtained from our archival tumor bank. Representative
hematoxylin-eosin stained sections were examined to evaluate the
histopathological characteristics of each case.
[0129] Clinicopathological parameters examined include
pre-treatment PSA, pathologic stage and Gleason score, both
determined based on the radical prostatectomy specimen. Time to PSA
relapse was calculated from the day of surgery to the first
detectable PSA. PSA relapse was defined as three consecutive rise
in PSA at least one week apart. Only patients who had undetectable
PSA level after surgery were included in this analysis.
[0130] Tumors were staged pT2 (n=51) and pT3 (n=35). Twenty-nine
patients were Gleason score <7, while 18 patients were Gleason
.gtoreq.7. In six cases, due to scarcity of tumor representation in
the specimen, grade was considered to be not interpretable.
Thirty-three patients (38.3%) received neoadjuvant hormone
treatment preoperatively, and were defined as hormone-treated.
These patients had non-evaluable Gleason scores. Patients who did
not receive neoadjuvant hormone treatment were defined as
hormone-naive.
[0131] Monoclonal Antibodies and Immunohistochemistry. The
following well characterized mouse monoclonal antibodies and
corresponding final working dilutions were used for the present
study: anti-p53 monoclonal antibody PAB1801 (Ab-2 clone;
CalBiochem/Oncogene Science, Boston, Mass.; 1:500 dilution);
anti-mdm2 monoclonal antibody 2A10 (a gift from Dr. Arnold Levine,
Rockefeller University, New York, N.Y.; 1:500 dilution); and an
anti-p21 monoclonal antibody (Ab-1 clone; CalBiochem/Oncogene
Science; 1:20 dilution). An anti-Ki67 mouse monoclonal antibody
(clone MIB1; Immunotech SA, France; 1:50 dilution) was used to
assess proliferative index. MIgS-Kp1, a mouse monoclonal antibody
of the same subclass as the primary antibodies listed above was
used as negative control.
[0132] An avidin-biotin immunoperoxidase method was utilized.
Briefly, sections were subsequently immersed in boiling 0.0% citric
acid (pH 6.0) for 15 minutes to enhance antigen retrieval and
incubated with primary antibodies overnight at 4.degree. C.
Biotinylated horse anti-mouse IgG antibodies were applied for 1 h
(Vector Laboratories, Burlingame, Calif.; 1:500 dilution), followed
by avidin-biotin peroxidase complexes for 30 minutes (Vector
Laboratories; 1:25 dilution). Diaminobenzidine was used as the
final chromogen and hematoxylin was used as the nuclear
counterstain. Nuclear immunoreactivity were classified on a
continuous scale with values that ranged from undetectable levels
or 0% to homogeneous staining or 100%.
[0133] Statistical Analysis. The three markers were analyzed both
as percentage of tumor cells and as discrete variables based on a
priori cut-points. The cut-point for p53 of >20% was based on
our previous analysis of p53 alterations in bladder cancer that
revealed a strong association between p53 point mutation and p53
nuclear accumulation in >20% of tumor cells (7,8). For mdm2, the
cut-point was based on what have been published correlating mdm2
overexpression in .gtoreq.20% of tumor cells with worse
clinicopathological parameters (9,10). The same principle applied
to the Ki67 cut-point determination (11,12). For p21 the cut-point
of >5% was based on our finding that normal prostate glands lack
p21 expression, and the observation of p21 nuclear staining and
presence of mitotic figures indicating high proliferative activity
of the tumors.
[0134] The association of percentage of tumor cells expressing the
markers with time to PSA relapse, while adjusting for other
variables with known prognostic significance, was assessed using
the Cox proportional hazards model (13). In addition, Kaplan-Meier
estimation (14) was performed and the log rank test (15) employed
to assess the univariate relationship between the individual
markers using cut points and time to PSA relapse.
[0135] The associations between Gleason group and the three
biomarkers were assessed using Fisher's exact test (16). Also,
associations between the three markers and variables such as Ki67
proliferative index, stage, and hormone status were also assessed
using the above test.
EXPERIMENTAL RESULTS
Experimental Results for the Second Series of Experiments
[0136] Table 1 summarizes the data in relation to
clinicopathological parameters, including pre-treatment PSA, tumor
stage, Gleason tumor grade, hormone status, proliferative index,
and immunophenotype profile. FIG. 7 illustrates the univariate
relationships of the three markers with time to PSA relapse with
Kaplan-Meier curves estimated.
1TABLE 1 Summary Of Data In Relation To Immunophenotype Profile P53
p21 mdm-2 (#) (%) (#) (%) (#) (%) PSA <4 0/18 0 5/18 27 3/18 16
4-10 3/28 10 7/28 25 7/28 25 >10 3/40 7.5 16/40 40 18/40 45 p
value .374 .382 .060 Stage T < 3 3/51 5.8 14/51 27 11/51 21 T =
3 3/35 8.5 14/35 40 17/35 48 p value .631 .222 .009 Gleason Score
<7 0/29 0 7/29 24 7/29 24 =7 2/18 11 10/18 22 9/18 50 NE 4/33 2
10/33 30 11/33 33 p value .157 .074 .190 Hormone Status Naive 2/53
3 18/53 33 17/53 32 Treated 4/33 12 10/33 30 11/33 33 p value .139
.725 .904 Proliferation Index Ki67 Low 5/75 6 20/75 26 22/75 29
High 1/11 6 8/11 72 6/11 54 p value .768 .002 .096
[0137] p53 nuclear overexpression of >20% was observed in 6 of
86 cases. The distribution of p53% expression was primarily
patients expressing less than 5% p53 (n=76). The other 10 patients
had varying levels of p53% expression, indicating a very low
frequency of p53 alteration in this group of patients. There is no
correlation between p53 positive phenotype and pretreatment PSA,
tumor stage, tumor grade, hormone status, or high proliferative
index. Also, there is no association between p53 overexpression and
p21 or mdm2 overexpression. A significant association was observed
between p53 status determined by the cut-point and time to PSA
relapse. This association is illustrated in FIG. 7. Using the log
rank test to examine the overall differences between p53 negative
phenotype and p53 positive phenotype revealed a statistical
significant difference P<0.01. This indicates an obvious PSA
relapse time advantage for patients who do not overexpress p53.
However, the magnitude of this difference may not be reliably
estimated due to the small number of patients and events in the p53
positive phenotype group.
[0138] mdm2 nuclear overexpression of .gtoreq.20% tumor cells was
observed in 28 of 86 cases (32.5%). mdm2 positive phenotype was
associated with advanced stage (P=0.009). In addition, mdm2
overexpression was observed not to be significant with respect to a
decreased time to PSA relapse (FIG. 7). A trend was observed
between mdm2 overexpression and higher pretreatment PSA
(P=0.06).
[0139] p21 nuclear overexpression of >5% tumor cells was
observed in 28 of 86 patients (32.5%). Patients with p21 positive
phenotype were observed to have a significant association with high
Ki67 proliferative index (P=0.002). High Ki67 porileferative index
was identified in 11 of 86 patients (12.7%). Patients with p21
positive phenotype had a significant association with decreased
time to PSA relapse, as illustrated in FIG. 7. Also, p21
overexpression was associated with mdm2 overexpression (P<0.0l).
However, no association was observed between identification of p21
and/or mdm2 positive phenotype and p53 overexpression.
[0140] Forty-three of the total 86 patients had one or more altered
markers. Patients with any alteration (p53 or mdm2 or p21) were
observed to have a higher rate of PSA relapse (P<0.01).
[0141] The multivariate relationship between the markers and time
to PSA relapse was assessed using Cox proportional hazards model.
It was of interest to examine the effect of the markers while
adjusting for variables with know prognostic significance. Both,
p53 and p21 positive phenotypes were significant while adjusting
for pre-treatment PSA and Gleason group (P<0.01 for both
markers). Examination of the overexpression of at least one marker
(p53, mdm2 or p21) with respect to time to PSA relapse showed that
this variable was also significant (P<0.01) while adjusted for
pre-treatment PSA and Gleason group. Tumor stage (<3 vs.
.gtoreq.3) was not significant in either the univariate or
multivariate analyses, and was thus excluded from the model. The
model that seemed to account for the most information included p53
and p21, along with pre-treatment PSA.
EXPERIMENTAL DISCUSSION
Experimental Discussion for the Second Series of Experiments
[0142] Reports dealing with the frequency of TP53 mutations and p53
overexpression in prostate cancer have yielded conflicting results,
alterations ranging from 2% to 65% of cases studied (17-21). This
discrepancy might be explained by the relatively small number of
cases and different disease stages analyzed in some reports, the
distinct methodologies employed, and the cutoff points used for
evaluation of IHC results. However, a general finding was the
association between p53 alterations and clinicopathological
parameters of poor clinical outcome, such as high grade and late
stage (18,19,22). In this study, we observed a relatively low
frequency of p53 nuclear overexpression in patients with localized
prostate cancer, as previously reported (23,24). To determine the
potential clinical relevance of identifying a p53 positive
phenotype, we correlated phenotypic characteristics of the tumors
with the time to PSA relapse. This is considered the most sensitive
indicator of success or failure following radical prostatectomy in
patients treated for localized disease. Analysis of data revealed
that p53 overexpression was significantly associated with PSA
relapse (P<0.01) and independent of pretreatment PSA and Gleason
group. However, the magnitude of this difference may not be
reliably estimated due to the small number of patients and events
in the positive phenotype. We also observed that all patients who
received neoadjuvant hormone treatment prior to surgery and had
tumors that overexpressed p53 relapsed. This finding could be due
to the advanced stage at which patients presented and were selected
for treatment using this modality. Mechanistically, an altered p53
status in this setting could have conferred resistance to
castration-induced apoptosis, ultimately leading to disease
relapse. The association between p53 overexpression and hormone
refractory prostate cancer has been reported in locally advanced
and metastatic disease (25). However, to our knowledge, this is the
first report to suggest that this association might be an early
event in the evolution of hormone refractory disease in clinically
localized prostate cancer. In the present study we also analyzed
alterations affecting other regulators of the p53 pathway in
primary prostate cancer, including mdm2 and p21. The MDM2 gene maps
to 12q13 and is found overexpressed in certain tumors, due to its
amplification as a component of an amplicon that includes other
relevant genes, such as CDK4. The MDM2 is under transcriptional
regulation by p53, and encodes a 90-kDa zinc finger protein (mdm2)
which contains a p53-binding site (26). It has been shown that mdm2
binds to p53, and acts as a negative regulator by inhibiting p53
transcriptional activity and targeting its degradation, thus
creating an autoregulatory feedback loop (27). In this study,
nuclear mdm2 overexpression was found in 32.5% of cases. We
observed that mdm2 positive phenotype was significantly associated
with advanced stage. It has been previously reported that MDM2 is
not amplified on primary prostate cancer, based on a study of 29
tumors analyzed by Southern blot hybridization (28). The
discrepancy between the rate of MDM2 gene amplification and protein
overexpression has been described in Burkitt's lymphoma and breast
cancer (29,30). Furthermore, it was observed in soft tissue
sarcomas that mdm2 overexpression, rather than its amplification,
was associated with worse clinical outcome (10). Based on data from
this study, we can postulate that mdm2 overexpression is a frequent
mechanism of p53 inactivation in prostate cancer, and in this
context the MDM2 gene can be classified as an oncogene in this
setting.
[0143] The p21/WAF1 gene encodes a nuclear protein nember of the
cyclin-dependent kinase inhibitory KIP family involved in
senescence and cell quiescence (31). The p21/WAF1 gene is also
transcriptionally regulated by p53. However, p21 induction could
also be accomplished by a p53-independent pathway. Serum or
individual growth factors, such as EGF and FGF, were shown to
induce p21 in p53-deficient cells (32). Based on these data, it has
been postulated that p21 induction could be activated through two
separate pathways. The rate of p21/WAF1 mutations in human cancer
is very low (33). However, there is an association between altered
patterns of p21 expression and clinical outcome in certain tumors,
such as bladder, colon, and heptocellular carcinomas (34-36). Lack
of p21 expression in these studies was correlated with poor
clinical outcome, an expected finding if one postulates that p21
deficiency reflects p53 inactivation. As a corollary to this
hypothesis, the p21 negative phenotype observed in the above
referred studies was usually associated with p53 alterations.
However, in our study we found that p21 positive phenotype was
significantly associated with high proliferative index and mdm2
overexpression, but not with p53 status. Moreover, patients with
p21 positive phenotype had a significant association with decreased
time to PSA relapse. p21 overexpression has been reported to be
associated with worse prognosis in other tumor types, including,
breast, esophageal carcinoma, and squamous cell carcinomas of head
and neck (37-39). Moreover, p21 overexpression was found to be
associated with resistance to chemotherapy in acute myeloid
leukemia and glioblastoma (40,41).
[0144] These data could be interpreted as follows (see FIG. 8). A
positive p21 phenotype could signify activation of p53 in response
to DNA damage or cellular stress. This effect would result in G1
arrest of the prostate tumor cells expressing p21. We observed, on
the contrary, an association between p21 positive phenotype and
increased proliferative activity. Thus, it is more plausible to
postulate that the p21 overexpression observed is caused by a
p53-independent transactivation mechanism. In the setting of
prostate cancer, the alternative mechanism could be due to
mitogenic stimuli via growth factor signaling. There is abundant
evidence regarding the upregulation of growth factor
receptor/ligand activity in prostate tumors (42-46). An additional
aberration causing p53 inactivation would be required in this model
to explain the lack of cell death and association with
proliferative activity. It is our hypothesis that the increased
mdm2 expression discussed above provides this requirement, further
supporting the oncogenic role of mdm2 in prostate cancer.
[0145] Finally, the association between p21 and hiah proliferative
index might also reflect deregulated cyclinD1/CDK4 activity. In
fact, we observed a strong association between p21 positive
phenotype and cyclin D1 overexpression in this cohort of patients
(Drobnjak et al, personal communication). Taken together, these
data supports the concept that p21 overexpression denotes an
inefficient pRB control on S-phase entry.
[0146] Growth control in mammalian cells is accomplished largely by
the action of the RB protein, regulating exit from the G1 phase,
and the p53 protein, triggering growth arrest or apoptotic
processes. In this group of patients, there is enough evidence to
suggest that both mechanisms are defective in prostate cancer. The
high proliferative index reflects the inefficient pRB control. We
postulate that this phenomenon is produced by deregulated
cyclinD1/CDK4 activity, which is associated with a p21 positive
phenotype. The deactivation of a p53-dependent apoptosis could be
explained by the degradation of p53 induced by mdm2
overexpression.
[0147] In sum, alterations affecting the p53 pathway are frequent
events in prostate cancer. It is our hypothesis that a pathway of
prostate cancer progression involves p53 inactivation caused by
mdm2 overexpression, and that p21 transactivation in this setting
is due to an alternative signaling system rather than through a
p53-dependent mechanism.
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suppressor gene in normal and malignant ovarian epithelial cells.
Clin Cancer Res, 2:1571-1575,1996.
[0181] 34. Stein, J. P., Ginsberg, D. A., Grossfeld, G. D.,
Chatterjee, S. J., Esrig, D., Dickinson, M. G., Groshen, S.,
Taylor, C. R., Jones, P. A., Skinner, D. G., and Cote, R. J. Effect
of p21WAF1/CIP1 expression on tumor progression in bladder cancer.
J Natl. Cancer Inst., 90:1072-1079, 1998.
[0182] 35. Valassiadou, K. E., Stefanaki, K., Tzardi, M., Datseris,
G., Georgoulias, V., Melissas, J., Tsiftsis, D. D., Delides, G.,
Kanavaros, P. Immunohistochemical expression of p53, bcl-2, mdm2
and waf1/p21 proteins in colorectal adenocarcinomas. Anticancer
Res, 17:2571-2576,1997.
[0183] 36. Qin, L. F., Ng, I. O., Fan, S. T., and Ng, M. p21/WAF1,
p53 and PCNA expression and p53 mutation status in hepatocellular
carcinoma. Int J Cancer, 79:424-428, 1998.
[0184] 37. Caffo, O., Doglioni, C., Veronese, S., Bonzanini, M.,
Marchetti, A., Buttitta, F., Fina, P., Leek, R., Morelli, L.,
Palma, P. D., Harris, A. L., and Barbareschi, M. Prognostic Value
of p21(WAF1) and p53 Expression in Breast Carcinoma: An
Immunohistochemical Study in 261 Patients with long-term Follow-Up.
Clin Cancer Res, 2:1591-1599,1996.
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Wang, L., Fink, U., Heep, H., Dutkowski, P., Willers, R., Muller,
W., Seeber, S., and Gabbert, H. Expression of p21 predicts outcome
of esophageal cancer patients treated by surgery alone or by
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Conradt, C., Bartek, J., and Bosch, F. X. Aberrant p21(CIP1/WAF1)
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Claxton, D., and Deisseroth, A. B. High levels of constitutive
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M., Levin, V., and Zhang, W. Overexpressed WAF1/Cip1 renders
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K., Kamiakito, T., Kobayashi, Y., Yoshida, H., Koike, M., and
Fukayama M. High frequency of fibroblast growth factor (FGF) 8
expression in clinical prostate cancers and breast tissues,
immunohistochemically demonstrated by a newly established
neutralizing monoclonal antibody against FGF 8. Cancer
Res,58:2053-2056,1998.
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PDGF modify TGFbeta-induction of smooth muscle cell phenotype in
human prostatic stromal cells. Prostate,35:125-134,1998.
[0191] 44. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C.,
Hittmair, A., Zhang, J., Thurnher, M., Bartsch, G., and Klocker, H.
Regulation of prostatic growth and function by peptide growth
factors. Prostate, 28:392-405,1996.
[0192] 45. Scher, H. I., Sarkis, A., Reuter, V., Cohen, D., Netto,
G., Petrylak, D., Lianes, P., Fuks, Z., Mendelson, J., and
Cordon-Cardo, C. Changing pattern of expression of the epidermal
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Scher, H. I., and Cordon-Cardo, C. Expression of transforming
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THIRD SERIES OF EXPERIMENTS
[0194] Cyclin D1 is a key regulator of the G1 phase progression of
the cell division cycle. There is an increasing evidence that
deregulated cyclin D1 expression is implicated in tumorigenesis and
tumor progression in certain neoplasms. The present study was
conducted in order to analyze the alterations affecting cyclin D1
in prostate cancer, as well as to assess its potential clinical
significance. We studied 116 cases of primary (n=86) and metastatic
(n=30) prostate carcinomas using immunohistochemistry and a well
characterized monoclonal antibody to cyclin D1. The results were
correlated with proliferative index, as assessed by Ki67 antigen
expression and with clinicopathologic variables of poor prognosis.
Cyclin D1 positive phenotype, defined as identification of
immunoreactivity in the nuclei of .gtoreq.20% tumor cells, was
found in 26 of 116 (22%) cases. A significant association was
observed between cyclin D1 positive phenotype and clinicopathologic
parameters, such as advanced tumor stage (T.gtoreq.3) (P=0.045),
evidence of bone metastases (P=0.001) and with elevated
preoperative prostate specific antigen measurements (PSA>10
ng/ml) (P=0.01) Ki67 proliferative index was considered high when
.gtoreq.20% tumor cells displayed positive nuclear staining, a
phenotype that was observed in 20 of 107 (19%) evaluable cases.
Moreover, high Ki67 proliferative index was associated with cyclin
D1 overexpression (P=0.01) These data support the hypothesis that
alterations of cyclin D1 may represent an oncogenic event in human
prostate cancer. Furthermore, it appears that cyclin D1
overexpression contributes to tumor progression in a subset of
particularly aggressive prostate carcinomas, especially those
developing osseous metastases.
[0195] Prostate cancer has been reported to be a neoplastic disease
of a slow growth rate. Nevertheless, it still represents the second
leading cause of cancer deaths in men in the United States. There
is an obvious discrepancy between the clinical impression of a
slowly growing neoplasm and tendency to produce an aggressive
metastatic disease in individual patients. The prognostic
indicators of histologic grade, pathologic stage, DNA ploidy, and
tumor cell proliferative index proved to be of limited value in
determining the biologic behavior of prostate cancer (1-3). Tumor
suppressor genes, particularly p53 and RB, implicated in the
molecular genetics of many human malignancies, were reported to be
altered in a rather low frequency in prostate cancer (4-8).
[0196] Cell cycle transitions are controlled by functional
heterodimers composed of a cyclin, acting as a regulatory subunit,
and cyclin-dependent kinase (Cdk), which acts as the catalytic
component (9). Multiple cyclins have been isolated and
characterized, and a temporal map of their expression has been
delineated. It is postulated that the complexes formed by cyclin D1
and Cdk4 govern G1 progression, while cyclin E-Cdk2 controls entry
into S-phase and cyclin A-Cdk2 affects the regulation through
S-phase (10). Cyclin D1-Cdk4 complexes exert their function through
the phosphorylation of the product encoded by the retinoblastoma
gene, pRb, in order to overcome the cell cycle block imposed by
hypophosphorylated pRb (10). Several studies suggest that gene
amplification and overexpression of cyclin D1 and Cdk4 are
oncogenic events in certain tumors, including breast cancer (11),
head and neck tumors (12, 13), esophageal (14, 15) and colorectal
carcinoma (16). We undertook this study in order to analyze
patterns of cyclin D1 expression in prostate cancer. The
alterations identified were correlated with Ki67 proliferative
index, as well as relevant clinicopathologic parameters, in an
attempt to define their potential biologic significance in prostate
cancer.
EXPERIMENTAL DETAILS
Experimental Details for Third Series of Experiments
MATERIALS AND METHODS
[0197] Patients Characteristics and Tissues. A cohort of 116
patients with prostate carcinoma were evaluated, consisting of 86
primary and 30 metastatic cases (eight metastases to lymph node and
22 metastases to bone). All primary tumors (n=86) represented
consecutive cases of patients who underwent radical prostatectomy
at Memorial Sloan-Kettering Cancer Center, in the period of 1990
and 1991. All metastatic cases (n=30) were selected on the basis of
the availability of tissue in the tumor bank. Samples were
formalin-fixed, paraffin embedded tissue specimens, obtained from
the Department of Pathologic at Memorial Sloan-Kettering Cancer
Center. Representative hematoxylin-eosin stained sections of each
paraffin block were examined microscopically to confirm the
presence of tumor, as well as to evaluate the pathologic grade and
stage of the tumors analyzed. Thirty-three of 86 patients with
primary carcinoma received preoperatively neoadjuvant hormone
therapy (hormone treated), while the remaining 53 patients were not
treated with such protocols and were considered hormone-naive.
Hormone-naive primary tumors with sufficient tumor representation
on tissue sections were assigned histologic grade (n=47).
Histologic grade was categorized into two groups: low grade
(Gleason score <7), and high grade (Gleason score .gtoreq.7).
According to pathologic stage, cases were grouped into early (organ
confined tumors, T.sub.2), or advanced tumors (extending beyond
prostatic capsule, .gtoreq.T.sub.3) The response variable time to
prostate-specific antigen (PSA) relapse was defined as the time
from radical prostatectomy to the time of the first detectable (non
zero) PSA measurement. Three consecutive increases of PSA were
required to confirm PSA relapse. Only patients who had a
nonmeasurable PSA after radical prostatectomy were included in the
analysis.
[0198] Monoclonal Antibodies and Immunohistochemistry. The
following well characterized antibodies and corresponding final
working concentrations were used for the present study: anti-cyclin
D1 mouse monoclonal antibody (Ab-3, clone DCS-6, IgG1, Oncogene,
Calbiochem, Cambridge, Mass.; 1 .mu.g/ml); anti-Ki67 mouse
monoclonal antibody (clone MIB-1, IgG1, Immunotech, Marseille,
France; 4 .mu.g/ml). A nonspecific mouse IgG1 kappa monoclonal
antibody was used as a negative control at similar working
concentrations. Immunohistochemistry was performed on 5 .mu.m
tissue sections using avidin-biotin-peroxidase method and antigen
retrieval. Briefly, sections were immersed in boiling 0.01 M citric
acid (pH 6.0) and heated in microwave oven for 15 minutes, to
enhance epitope exposure. After cooling to room temperature, slides
were incubated with 10% normal horse serum for 30 minutes.
Subsequently, appropriately diluted primary antibodies were applied
for overnight incubation at 4.degree. C. Biotinylated horse
anti-mouse IgG antibodies were used as secondary reagents, applied
for an incubation period of 30 minutes (Vector Laboratories,
Burlingame, Calif.; 1:500 dilution), followed by
avidin-biotin-peroxidase complexes incubated for 30 minutes (Vector
Laboratories--1:25 dilution). Diaminobenzidine was used as the
final chromogen and hematoxylin as the nuclear counterstain.
[0199] Immunohistochemistry Evaluation. Nuclear immunoreactivities
for both cyclin D1 and Ki67 antigens, were classified into two
categories defined as follows: negative (<20% tumor cells
displaying nuclear immunostaining), and positive (.gtoreq.20% tumor
cells with nuclear immunostaining). The appropriateness of this
cutoff point was validated graphically by using predicted survival
time and looking at specific immunoreactivities as a continuum data
in this group of patients. Ultimately, results were interpreted as
defined above.
[0200] Statistical Methods. The baseline variables examined were
PSA (ng/ml) at time of diagnosis (divided into three categories:
<4, 4-10 and >10), Tumor grade (Gleason score) (divided into
two mutually exclusive categories: <7 or .gtoreq.7), Pathologic
stage T (2 or .gtoreq.3), and percent cyclin D1 and Ki67
expression. Statistical analyses were conducted to assess: 1) the
correlation between immunophenotypic variables and
clinicopathologic parameters such as: presentation status, tumor
grade, pathologic stage, preoperative PSA, and hormonal status; 2)
the correlation among immunophenotypic variables; 3) association
between immunophenotypes and PSA relapse free survival. The
Mantel-Haenszel chi-square test was used to assess the associations
among the different variables and results were considered
significant if the P value was <0.05. The FREQ procedure in SAS
was used for this study (17). The associations between time to PSA
relapse and the immunophenotypes were evaluated using the Log Rank
test and Kaplan Meier estimates (18).
EXPERIMENTAL RESULTS
Experimental Results for the Third Series of Experiments
[0201] Table 2 summarizes immunohistochemical data in relation to
clinicopathologic parameters. FIG. 9 illustrates the
immunohistochemical staining patterns of cyclin D1 in
representative cases of primary tumors and bone metastases.
2TABLE 2 Clinicopathologic Parameters in Relation to Cyclin D1
Immunoreactivity cyclin cylcin D1 - (<20%) D1 + (.gtoreq.20%)
Parameter N (%) N (&) Total P-value Total Patients 90 (77.6) 26
(22.4) 116 Presentation Primary tms 76 (88.4) 10 (11.6) 86 LN
metastases 7 (87.5) 1 (12.5) 8 NS Primary tms 76 (88.4) 10 (11.6)
86 Bone metastases 7 (31.8) 15 (68.2) 22 0.001 Ki67 proliferative
index Low (<20%) 71 (81.6) 16 (18.4) 87 High (.gtoreq.20%) 11
(55.0) 9 (45.0) 20 0.01 Primary tumors 76 (88.4) 10 (11.6) 86 Tm.
Grade (Gleason)* Low (<7) 27 (93.1) 2 (6.9) 29 High (.gtoreq.7)
17 (94.4) 1 (5.6) 18 NS Path. Stage Early (T.sub.2) 48 (94.1) 3
(5.9) 51 Advanced (.gtoreq.T.sub.3) 28 (80.0) 7 (20.0) 35 0.045
Hormonal status H. naive 49 (92.5) 4 (7.5) 53 H. treated 27 (81.8)
6 (18.2) 33 NS Pretreatment PSA <4 ng/ml 17 (94.4) 1 (5.6) 18
4-10 ng/ml 28 (100.0) 0 (0.0) 28 >10 ng/ml 31 (77.5) 9 (22.5) 40
0.01 *Patients who had received neoadjuvant hormonal therapy (n =
33) and tissue sections with inadequate tumor representation (n =
6) were not assigned Gleason grade and were excluded from this
analysis.
[0202] Cyclin D1 was expressed in .gtoreq.20% tumor cells in 26 of
116 (22%) evaluable cases, corresponding to 10 of 86 (12%) primary
lesions and 16 of 30 (53%) metastases. There was a statistically
significant association between cyclin D1 overexpression and the
presence of bone metastases. We observed that 15 of 22 (68%) bone
metastases lesions overexpressed cyclin D1, while only 10 of 86
(12%) primary tumors presented with this positive phenotype
(p=0.001). Cyclin D1 overexpression was also associated with
advanced pathologic stage in primary tumors. We found that 7 of 35
(20%) tumors of advanced stage (extending beyond the prostatic
capsule, .gtoreq.T.sub.3) displayed cyclin D1 nuclear
overexpression, compared to only 3 of 51 (6%) organ confined tumors
(T.sub.2) (P=0.045). Cyclin D1 was also detected at increased
percentage of tumor cells in patients with high initial
pretreatment PSA values. Nine of 68 (13%) patients with PSA
.gtoreq.10 ng/ml were cyclin D1 positive compared to only 1 of 18
(5%) patients with PSA <4 ng/ml (P=0.01). There was no
association between cyclin D1 overexpression and tumor grade
(Gleason score) or hormonal status (hormone naive vs. hormone
treated). In order to assess disease progression we evaluated the
time to PSA failure after radical prostatectomy. There was no
association between cyclin D1 nuclear overexpression and early
relapse as defined by increased PSA measurements after radical
prostatectomy in these group of patients (p=0.2).
[0203] Cyclin D1 overexpression correlated well with high Ki67
proliferative index, which was scored as being high in 20 of 107
(19%) evaluable tumors. Nine of 20 (45%) tumors displaying high
Ki67 proliferative index also possessed cyclin D1 nuclear
overexpression, while only 16 of 87 (18%) cases with low Ki67
proliferative index overexpressed cyclin D1 (P=0.01). Nevertheless,
Ki67 proliferative index alone was not associated with
clinicopathologic parameters of poor outcome in this cohort of
patients.
EXPERIMENTAL DISCUSSION
Experimental Discussion for the Third Series of Experiments
[0204] Autopsy records show that by the age of 80, approximately
60-70% of men around the world, have histologic evidence of
prostatic carcinoma (19). Although this fact indicates generally
slow growing nature of this malignancy, there are vast differences
in the progression rate and development of clinically evident or
metastatic disease in these patients during their lifetime.
Prostate cancer progression tends to follow periprostatic and
perivascular penetration, invasion along perineural spaces, pelvic
lymph node metastases and particularly bone metastases (20). Almost
one fourth of newly diagnosed cases presents with lymph node and/or
osseous metastases and only one fourth of those survive five years
(21). We were interested in analyzing the molecular events that
might be responsible for the progression of prostate cancer from
indolent to a life threatening, metastatic disease.
[0205] Some earlier reports on determining prostate tumor
proliferation, measured by flow cytometric S-phase fraction showed
a positive predictive value of this variable and prostate cancer
progression (2). Tumors that demonstrate a higher proliferation
rate are more likely to grow to and beyond prostatic capsule and to
produce distant metastases. Recently, the cell division cycle
regulatory mechanisms and their oncogenic role have become a major
focus of cancer research. There is ever growing literature on
cyclins and their associated kinases and their role in
tumorigenesis (22). Particularly D-family cyclins were implicated
in specific human tumors. Bartkova et al. (23) report on a large
group of various human malignancies, including carcinoma of the
breast, uterus, colon, melanomas and soft tissue sarcomas, high
proportion of which exhibit immunoreactivity for cyclin D1. By far
the most frequent chromosomal abnormality that affects cyclin D1 in
majority of tumors is DNA amplification that results in increased
expression of the RNA transcripts and protein levels (24). In some
tumor types, however, immunohistochemistry proved to be the most
accurate technique in determining deregulated expression of cyclin
D1 (11, 23). In this study we used immunohistochemistry to
determine cyclin D1 expression in patients with prostate carcinoma.
To our knowledge this first expression study on cyclin D1 in both
primary tumors and bone metastases specimens. Only recently
Kallakury et al (25) evaluated the expression of p34.sup.cdc2 and
cyclin D1 in patients with radical prostatectomy. Results were
correlated with conventional markers of poor prognosis. The authors
showed no association between cyclin D1 immunoreactivity and
clinicopathologic parameters, such as tumor grade, pathologic
stage, lymph node metastases and with disease free survival. Our
data in this study, on the other hand, suggest the involvement of
cyclin D1 in the progression of human prostate cancer. There was a
remarkably significant difference in the levels of cyclin D1
expression between bone metastases and primary tumors. There was an
association between cyclin D1 immunoreactivity and tumors with
advanced pathologic stage. Cyclin D1 further correlated well with
high Ki67 proliferative index. Taken together, these results
support the theory that increased levels of cyclin D1 expression
contribute to cell cycle imbalance with extremely shortened G1
phase and possibly with reduced cell requirements for growth
factors to proliferate (26, 27). Subclones of tumor cells with
elevated cyclins expression may acquire uncontrolled growth
advantage and contribute to tumor progression.
[0206] In this study we conclude that cyclin D1 may play an
oncogenic role in prostate cancer. Our data indicate that cyclin D1
is involved in tumor progression, particularly in a development of
bone metastases. However, in order to determine the timeframe and
prognostic value of this marker in prostate cancer, from the early
onset to the evolution of metastatic disease, we intend to evaluate
cyclin D1 immunophenotypes in paired samples of primary tumors and
metastatic sites from the same patients.
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FOURTH SERIES OF EXPERIMENTS
[0234] The INK4A gene maps to the region 9p21, and was initially
described as encoding a 148 amino acid protein termed p16. The p16
protein associates exclusively with Cdk4 and Cdk6, inhibiting their
complexation with D-type cyclins, and the consequent
phosphorylation of pRB. This contributes to cell cycle arrest. The
purpose of the present study was to evaluate patterns of p16
expression in a well characterized cohort of prostatic
adenocarcinomas, while exploring potential associations between
alterations of p16 and clinicopathological variables.
[0235] Normal and malignant tissues from 88 patients with prostate
carcinoma were examined. In situ hybridization and
immunohistochemistry assays were used to determine the status of
the INK4A exon 1.alpha. transcripts and levels of p16 protein,
respectively. Associations between altered patterns of expression
and clinicopathological variables, including pre-treatment
prostate-specific intigen (PSA) level, Gleason grade, pathologic
stage, and hormonal status, were evaluated using the
Mantel-Haenszel chi-square test. Biochemical (PSA) relapse after
surgery was evaluated using the Kaplan-Meier method and the Log
rank test. The levels of p16 expression and INK4A exon 1.alpha.
transcripts in normal prostate and benign hyperplastic tissues were
undetectable. However, p16 nuclear overexpression was observed in
38 (43%) prostate carcinomas, while the remaining 50 (57%) cases
showed undetectable p16 levels. Overexpression of p16 protein was
found to correlate with increased INK4A exon 1.alpha. transcripts.
Moreover, p16 overexpression was associated with a higher
pre-treatment PSA level (P=0.018), the use of neoadjuvant androgen
ablation (P=0.001), and a sooner time to PSA relapse after radical
prostatectomy (P=0.002). These data suggest that p16 overexpression
is associated with tumor recurrence and a poor clinical course in
patients with prostate cancer.
[0236] The INK4A gene maps to the short arm of chromosome 9 (9p21),
and was initially described as encoding a protein of Mr 15,845,
termed p16. (1,2). The p16 protein forms binary complexes
exclusively with Cdk4 and Cdk6, inhibiting their kinase activity
and subsequent pRb phosphorylation during the G.sub.1 phase of the
cell cycle. (1,3). Additional complexity results from the presence
of a second INK4A product termed p19.sup.ARF. (4-6) The p19.sup.ARF
protein nas recently been shown to interact with mdm2 and to block
mdm2-induced p53 degradation and transactivational silencing(7,8).
The two products, p16 and p19.sup.ARF, share exons 2 and 3 of the
INK4A gene, but have distinct promoters and exon 1 units, exon
1.alpha. (p16) and exon 1.beta. (p19.sup.ARF). The INK4A gene is
mutated in a wide variety of tumor cell lines and certain primary
tumors (2, 9-14). In addition, methylation of the 5' CpG island of
the exon 1.alpha. promoter region is a frequent mechanism of p16
inactivation in primary tumors (15, 16).
[0237] In prostate cancer the role of INK4A has not been well
elucidated, though analyses utilizing microsatellite markers in the
vicinity of the INK4A gene have revealed loss of heterozygosity in
a subset of primary and metastatic prostate tumors (17). Unlike
reports of other primary tumors, INK4A inactivation, either through
deletions, mutations, or through promoter methylation, appears to
be an infrequent event in prostate cancer (18-23). The present
study utilizes immunohistochemical and in situ hybridization assays
to examine patterns of p16 expression in a well characterized
cohort of prostate cancer patients treated with radical retropubic
prostatectomy. Associations between altered p16 phenotypes and
clinicopathological variables were also studied to further define
their potential implications in prostate cancer.
EXPERIMENTAL DETAILS
Experimental Details for Fourth Series of Experiments
MATERIALS AND METHODS
[0238] Patient Characteristics and Tissues. A cohort of patients
with prostatic adenocarcinoma undergoing radical prostatectomy at
the Memorial Sloan-Kettering Cancer Center from 1990-1991 was
retrospectively evaluated. A total of 88 patients had adequate
clinical follow-up and available pathological materials. The median
age at the time of surgery was 65 years (range 46-74 years) The
median follow-up time was 64.5 months (range 10-94 months)
Formalin-fixed, paraffin-embedded prostate tissues were obtained
from the Department of Pathology. Representative hematoxylin-eosin
stained sections were examined to evaluate the histopathological
characteristics of each tissue section.
[0239] Clinicopathologic parameters examined included pre-treatment
PSA, pathologic stage (24) and Gleason grade (25), both determined
based on the radical prostatectomy specimen. Hormonal status of the
patients was also evaluated. A portion of the cohort (34
patients--39%) was treated with neoadjuvant androgen ablation and
were defined as hormone-treated. Patients who did not receive
neoadjuvant therapy were defined as hormone naive. Additionally,
biochemical relapse was examined. Relapse was defined as an
elevation in the serum PSA level in a patient who had previously
demonstrated an undetectable PSA level post-prostatectomy. That is,
only patients who had an undetectable PSA level after surgery were
included in the cohort, as this indicated that the surgical
resection was complete and the patient was free of disease.
Patients who had PSA relapse were classified as treatment failures
with tumor recurrence.
[0240] Immunohistochemistry. An avidin-biotin immunoperoxidase
assay was performed on formalin-fixed, paraffin-embedded tissue
sections. Deparaffinized sections were treated with 1%
H.sub.2O.sub.2 in order to block endogenous peroxidase activity.
Sections were subsequently immersed in boiling 0.01% citric acid
(pH 6.0) in a microwave oven for 15 minutes to enhance antigen
retrieval, allowed to cool, and incubated with 10% normal horse
serum (Organon Tecknika Corp, Westchester, Pa.), to block
non-specific tissue immunoreactivities. A well characterized
antibody to p16 (Ab-1, Oncogene Research Products, Cambridge,
Mass.; 2 ug/ml final concentration) was then incubated overnight at
4.degree. C. Biotinylated horse anti-mouse IgG antibodies (Vector
Laboratories, Inc., Burlingame, Calif.; 1:25 final dilution) were
utilized as the secondary reagents. This was followed by
avidin-biotin immunoperoxidase complexes (1:25, Vector
Laboratories, Inc.) for 30 minutes. Diaminobenzidine was used as
the final chromogen and hematoxylin was used as the nuclear
counterstain. Immunoreactivities, assessed in tissue sections from
a single representative block in each case, were classified as a
continuum of data from undetectable levels or (0%) to homogenous
staining levels (100%). Data was independently obtained by two
observers, with minor inter-observer variability which was resolved
by review of the problem cases. Tumors were grouped into two
categories defined as follows: Group A (.ltoreq.5% nuclear
immunoreactivity in tumor cells) and Group B (>5% nuclear
immunoreactivity in tumor cells).
[0241] In Situ Hybridization. Primers specific for the exon
1.alpha. sequence of the INK4A gene were utilized to create
digoxigenin-labeled probes for in situ hybridization. Probes were
cloned into a PCR-Script recombinant plasmid (Stratagene, La Jolla,
Calif.). Plasmid DNA (1 ug) was linearized using BamHI and XhoI.
Antisense and sense riboprobes were generated from in vitro
transcription of the linearized DNA using T7 and T3 RNA
polymerases, respectively. Transcription was sustained for 2 hours
at 37.degree. C. in 1.times. transcription buffer (Boehringer
Mannheim, Indianapolis, Ind.), 20 U of RNAse inhibitor, 10 mmol/L
each of ATP, GTP, CTP, 6.5 mmol/L UTP and 3.5 mmol/L
digoxigenin-UTP. Deparaffinized tissue sections were rinsed in
water and PBS for 10 minutes. The slides were digested with
Proteinase K (50 ug/ml) for 18 minutes at 37.degree. C. in PBS, and
post-fixed at 4 C. in a freshly prepared solution of 4%
paraformaldehyde in PBS for 5 minutes. Prehybridization was done
for 30 minutes at room temperature (RT) in 50% formamide and
2.times. sodium chloride/sodium citrate (SSC). The hybridization
buffer consisted of 50% deionized formamide (v/v), 10% dextran
sulphate (50% stock solution), 2.times.SSC (20.times. stock
solution), 1% SDS (10% stock solution), and 0.25 mg/ml of herring
sperm DNA (10 mg/ml).
[0242] Hybridization was performed overnight at 45.degree. C.
applying 10 pmol/L digoxigenin-labeled riboprobe in 50 ul of
hybridization buffer per section under a coverslip. The coverslips
were removed and the slides were washed in pre-warmed 2.times.SSC
for 20 minutes at 42.degree. C. twice, followed by washes in
pre-warmed 1.times.SSC and 0.5.times.SSC at 42.degree. C. for 20
minutes. After these washes the slides were incubated in normal
sheep serum diluted in buffer pH 7.5 and successively in the same
buffer with anti-digoxigenin-AP antibody (Boehringer Mannheim) at a
dilution of 1:500 for 1 hour at RT. The visualization was
accomplished by nitro-blue tetrazolium
5-bromo-4-chloro-3-indoylphosphate- . The slides were
counterstained with methyl green and mounted.
[0243] INK4A exon-1.alpha. transcript levels were examined in a
subgroup of 21 cases. Consecutive tissue sections were used to
analyze p16 protein by immunohistochemistry and INK4A exon-1.alpha.
transcript levels by in situ hybridization. As in the
immunohistochemical analysis, tumors were grouped into two
categories defined by the absence (.ltoreq.5% tumor cells with
cytoplasmic staining) or presence (>5% tumor cells with
cytoplasmic staining) of transcripts.
[0244] Statistical Methods. The statistical analyses of the data
from the 88 primary prostate cancer patients were conducted as
follows. The response variable, time to PSA relapse, was defined as
the time from radical prostatectomy to the time of first detectable
PSA measurement. Patients who did not achieve a non-measurable PSA
after radical prostatectomy were excluded from the analysis.
Patients who were still alive at the time of analysis without
relapse were censored at the date of last follow-up. The baseline
variables examined were PSA measurement at time of diagnosis,
hormone status, Gleason score (hormone nave patients only), stage
of disease, and percent p16 expression.
[0245] Associations between p16 expression and different
categorical variables (hormone status, tumor grade, tumor stage,
and pretreatment PSA levels) were assessed by Mantel-Haenszel
chi-square test Continuous variables, such as pre-treatment PSA,
not known to follow a particular distribution were compared between
two or more groups using Wilcoxon non-parametric tests.
[0246] The Cox proportional hazards model was used to examine the
multivariate relationship between PSA relapse-free time from
prostatectomy and the baseline variables listed above. The final
model was determined using the "all subsets" procedure in SAS PHREG
and the Score criterion (26). As normal and benign tissues showed
little to no p16 expression, positive expression was described as
>5% nuclear expression. This cutpoint was specified a priori and
used for the subsequent statistical analysis. Immunohistochemical
and in situ hybridization studies were completed, analyzed, and
recorded blind to clinical information. Kaplan-Meier estimates of
relapse-free survival stratified by p16 classification were
evaluated. The LIFETEST procedure in SAS was used to generate the
Kaplan-Meier estimates and the resulting survival curves (26, 27).
The Log rank test was used to test the hypothesis of no survival
differences between p16 positive and p16 negative populations.
EXPERIMENTAL RESULTS
Experimental Results for the Fourth Series of Experiments
[0247] The normal human prostate displayed undetectable levels of
p16 protein and INK4A exon 1.alpha. transcripts in ductal and
acinar epithelial cells. A lack of p16 immunoreactivity in these
cells was observed in hormone-treated and hormone naive cases.
Fibromuscular stroma cells also showed undetectable exon 1.alpha.
transcripts levels. A similar negative pattern of p16 expression
was observed upon the examination of prostatic tissue affected with
benign hyperplasia (FIG. 10).
[0248] To determine the frequency and potential clinical
implications of p16 alterations in prostate cancer, we analyzed a
cohort of 88 primary prostate carcinomas. Two patterns of p16
protein expression were noted. We observed that 50 of the 88 cases
(57%) had very low (.ltoreq.5% nuclear immunoreactivity; 7 cases)
or undetectable (43 cases) levels of p16 protein expression (Group
A) (FIG. 11A). In a subgroup of these cases we also performed in
situ hybridization assays, which revealed that all cases had
undetectable INK4A exon 1.alpha. transcripts (FIG. 11B). However,
we noted that 38 of the 88 cases (43%) displayed nuclear staining
with anti-p16 specific antibodies (Group B) (FIG. 1C).
Immunoreactivities in tumor cells were further stratified into
three categories: 6% to 29% nuclear staining (n=11 cases); 30% to
59% nuclear staining (n=15 cases); and 60% to 100% nuclear staining
(n=12 cases). In a subset of these patients, we also conducted in
situ hybridization assays with the INK4A exon 1.alpha. specific
probe. All cases displaying positive immunoreactivities also
displayed moderate to high levels of exon 1.alpha. transcripts
(FIG. 11D).
[0249] Table 3 summarizes the associations between p16 phenotypes
and clinicopathological variables, which were assessed by
Chi-square analyses. Immunohistochemical detection of p16 was not
associated with Gleason grade, described as either low (Gleason
grade 4-6) or high (Gleason grade 7-10) (P=0.153). Similarly, no
association was observed between p16 nuclear expression and
pathologic stage, defined as organ-confined (T.sub.1, T.sub.2) and
non organ-confined (T.sub.3, T.sub.4, or lymph node+) (P=0.087).
However, there was a strong association between p16 nuclear
expression and pre-treatment PSA levels, based on cutoff points of
<4, 4-10, and >10 ng/ml (P=0.018). A similar result was
observed when PSA was assessed as a continuous variable (P=0.01).
In addition, we noted a significant correlation between p16 nuclear
expression and the use of neoadjuvant androgen ablation
(P=0.001).
3TABLE 3 Association of p16 Immunoreactivity with Tumor Grade,
Hormonal Status, Tumor Stage, and Preoperative PSA Levels P16
Immunoreactivity (& of patients) Number of P- Subjects
.ltoreq.5% 5% value All Subjects 88 50 (57%) 38 (43%) Gleason
Grade* 82 <7 30 (37) 24 (80) 6 (20) p = 0.153 .gtoreq.7 18 (22)
11 (61) 7 (39) unable to evaluate 34 (41) Hormonal Status 88
hormone naive 54 (61) 40 (74) 14 (26) p = 0.001 hormone-treated 34
(39) 10 (29) 24 (71) Pathologic Stage 88 T.sub.2 53 (60) 34 (64) 19
(36) p = 0.087 .gtoreq.T.sub.3 35 (40) 16 (46) 19 (54) Pretreatment
88 PSA (ng/ml) <4.0 18 (20) 14 (78) 4 (22) 4-10 29 (33) 19 (66)
10 (34) p = 0.018 >10 41 (47) 17 (41) 24 (59) *Grading is based
on the radical prostatectomy specimen. Patients who had received
neoadjuvant androgen therapy were unable to be graded consistently.
Six patients did not have Gleason grade information and were
excluded from this analysis.
[0250] A strong association was also found between p16 nuclear
overexpression and tumor recurrence, as defined by biochemical
(PSA) relapse. Increasing p16 expression correlated with an
increased relative hazard of relapse, suggesting a continuous
relationship of the data. Overall, tumor recurrence was observed in
34 of 88 cases (39%). Thirteen of 50 cases (26%) with
undetectable-to-low p16 expression (Group A) developed tumor
recurrence. However, tumor recurrence was observed in 21 of 38
cases (55%) with p16 nuclear overexpression (Group B) (P=0.002)
(FIG. 12). Nevertheless, in a multivariate analysis adjusted for
tumor grade, pre-treatment PSA, and pathologic stage,
overexpression of p16 did not contribute prognostic information
over pre-treatment PSA, the strongest independent predictor of
tumor recurrence.
Experimental Discussion for the Fourth Series of Experiments
[0251] Normal prostate tissues display undetectable levels of p16
protein and INK4A exon 1.alpha. transcripts. It has been reported
that p16 expression is low to undetectable in most normal human
tissues analyzed (15, 28, 29). In support of these observations,
there are relatively low and near-constant levels of p16 protein
and mRNA throughout the cell cycle of normal lymphocytes in culture
(30). The lack of p16 expression in hyperplastic glands parallels
that of normal prostatic tissue. Based on these data, it is our
hypothesis that these negative p16 phenotypes reflect basal
physiologic levels of p16.
[0252] Primary prostatic adenocarcinomas revealed two distinct p16
phenotypes. Most tumors were found to have undetectable or very low
levels of p16 protein expression (Group A--57% of cases). This was
associated with low levels or absence of INK4A exon 1.alpha.
transcripts. Another group of tumors showed elevated p16 protein
expression (Group B--43%) which was consistently associated with
increased INK4A exon 1.alpha. transcripts. These findings suggest
an upregulation of the INK4A-.alpha. gene, resulting in p16 protein
overexpression. Patients in Group B had a more aggressive course,
demonstrated by high levels of pre-treatment PSA (P=0.018) and a
sooner time to biochemical (PSA) relapse (P=0.002). A worse
prognosis for Group B is also revealed by the trending association
of p16 overexpression with higher pathologic stage.
[0253] The negative phenotype observed in Group A might correspond
to the normal physiologic state, reflecting low-to-undetectable p16
levels. Alternatively, it could be related to mutations affecting
the INK4A gene, especially homozygous deletions, or methylation of
the INK4A exon 1.alpha. promoter region. Nevertheless, it has been
reported that these events are infrequent in prostate cancer
(17-23). Furthermore, it appears the tumors with INK4A mutations
have a more aggressive clinical course (31-33). Contrary to this,
in the present study, we observed that Group A patients had a less
aggressive behavior than Group B patients. For these reasons, we
hypothesize that the negative phenotype observed in Group A is more
likely a reflection of the normal physiologic state.
[0254] The up-regulation of the INK4A-.alpha. gene, resulting in
the overexpression of p16 protein, may develop through different
mechanisms. An association between increased p16 transcript and
protein levels occur in tumor cell lines and certain primary
neoplasms that lack functional pRb (1, 34-37). Moreover,
p16-mediated inhibition of cell cycle progression appears to be
dependent upon functional pRb (38, 39). These data support an
association between p16 and pRb, where absence of functional pRb
limits p16 activity and possibly promotes INK4A-.alpha.
upregulation. Alternatively, enhanced activation of the
INK4A-.alpha. gene may occur. E2F1, a direct activator of the INK4A
exon 1.beta. promoter, does not appear to directly activate
INK4A-.alpha. transcription (40). However, evidence does exist for
an indirect effect, as E2F1 overexpression has been reported to
markedly increase p16 transcripts and p16-related CKI activity
(41). Overexpression of cyclin D1 and/or of Cdk4 may also influence
p16 expression, through a compensatory feedback loop where
deregulation of cyclin D/Cdk4 complexes results in increased levels
of p16 protein (28, 42). In summary, it appears that an altered RB
axis could trigger p16 overexpression in certain systems.
[0255] Cellular stress produced by replicative senescence (43-45),
hyperthermia (46), and UV irradiation (47) has been reported to
trigger p16 overexpression. In the present study, another type of
cellular stress, androgen ablation, may account in part for this
observed phenomenon. A subset of patients were treated with
neoadjuvant androgen ablation, a strategy reported to decrease the
incidence of positive surgical margins after prostatectomy (48). In
the present study, p16 overexpression was observed in 71% of
hormone-treated versus 26% of hormone-naive patients (P=0.001).
These data suggest that p16 expression may be enhanced by androgen
depletion. Androgens are known to modulate the expression of other
CKI's such as p27 and p21 (49). In addition, it has been reported
that the presence of androgens triggers downregulation of p16 in
LNCaP cells (50), a finding consistent with our observation of p16
overexpression in cases of androgen ablation. It is also possible
that the association between p16 expression and androgen ablation
may, in part, reflect staging bias by clinicians. In this setting,
patients thought to have advanced disease may have been treated
with neoadjuvant therapy.
[0256] Based on the the above referred data, it is our working
hypothesis that p16 overexpression in prostate cancer represents an
altered phenotype, which identifies a subgroup of patients with a
higher likelihood of post surgical failure and tumor recurrence. In
support of this postulate, a preliminary report in prostate cancer
has demonstrated an association between p16 overexpression and poor
outcome, as related to biochemical failure (51). In addition, p16
overexpression has been associated with tumor progression and a
poor prognosis in ovarian (52) and breast cancers (37). Though p16
acts as a negative cell cycle regulator, specific mechanisms may
contribute to its altered expression, overcoming p16-mediated tumor
suppressor activities. Ongoing studies may elucidate mechanisms of
p16 overexpression relative to androgen depletion and/or
alterations in the RB axis.
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