Download Loss of Cyclin-dependent Kinase Inhibitor p27Klpl Is a Novel

[CANCER RESEARCH 58. 542-548.
February 1. 1998)
Loss of Cyclin-dependent
Kinase Inhibitor p27Klpl Is a Novel Prognostic Factor in
Localized Human Prostate Adenocarcinoma1
John Tsihlias,2 Linda R. Kapusta,2"3 Gerrit DeBoer, Izabella Morava-Protzner, Ingrid Zbieranowski,
Nandita Bhattacharya, G. Charles Catzavelos, Laurence H. Klotz, and Joyce M. Slingerland3
Department of Pathology [L R. K.. I. M-P., G. C. C.I, Cancer Biology Research ¡J.T., N. B.. J. M. S.], and Division of Urology []. T.. L H. K.¡.Sunnyhrook Health Science
Center: Department of Politologi: Women's College Hospital ¡I.Z.]; and Departments of Medical Oncology ¡J.M. SJ and Clinical Trials and Epidemiology ¡G.D./. TorontoSunnvbrook Regional Cancer Center. University of Toronto, Toronto, Ontario, M4N 3M5, Canada
ABSTRACT
p27Klpl is a cyclin-dependent
kinase inhibitor
that negatively
regulates
cell proliferation by mediating cell cycle arrest in G,. This study was
undertaken to assess the prognostic value of p27Klpl in localized human
prostate cancer. Archival material from 113 radical prostatectomy spec
imens obtained between 1985 and 1993 was stained immunohistochemically for p27Kipl protein using a commercially available antibody. Patient
charts were reviewed for preoperative serum prostate-specific antigen,
clinical and pathological staging, Gleason tumor grade, time to biochem
ical and clinical recurrence, and survival. Strong p27Klpl staining was
uniformly seen in benign prostatic epithelial components in all tumor
sections. p27Kipl staining was reduced in most prostate cancers and was
variable in prostatic intraepithelial neoplasia. Decreased p27Kipl staining
«25'r of nuclei stained positive for p27KIpl) correlated with seminal
vesicle involvement (/' = 0.0032) and with higher Gleason grade
I/' = 0.0114). On univariate analysis, low p27Kipl predicted an increased
risk of treatment failure in the node-negative cohort (/' = 0.0037) and in
the subset who did not receive neoadjuvant hormonal therapy (/' = 0.049).
Low p27Klpl expression was an independent predictor of treatment failure
on multivariate analysis of lymph node negative prostate cancers follow
ing radical retropubic prostatectomy (n = 102; /" = 0.047). Seminal vesicle
involvement (/' = 0.034) and positive surgical margins (/' = 0.047) were
also independent prognostic factors for disease recurrence. In patients
who received preoperative neoadjuvant hormonal therapy, low p27Klpl in
the pathological specimen was an even stronger predictor of outcome than
it was in the entire group (n = 23, P = 0.015).
INTRODUCTION
Prostate carcinoma is the most common solid malignancy and the
second most common cause of cancer-related death in men. There
were 337,000 estimated new cases of prostate cancer and 45,400
estimated prostate cancer-related deaths in North America in 1996 (1,
2). Many of these cancers are slow-growing, well-differentiated tu
mors that do not affect longevity. Others are rapidly growing and
aggressive (3-5). Traditional parameters used to predict extent of
disease and natural history include: clinical stage, tumor grade, and
serum PSA.4 Published nomograms allow more accurate prediction of
based on these factors is difficult. A more accurate prognostic marker
for this patient group would permit assignment of more aggressive
treatment to those most likely to fail and would justify expectant
management (deferral of active treatment) in those likely to have
indolent disease.
There is increasing evidence that cell cycle regulators are disrupted
in human cancers (7). The cell cycle is governed by cdks, the activities
of which are regulated by binding of positive effectors, the cyclins (8,
9); by negative regulators, the cdk inhibitors (10); and by phosphorylation and dephosphorylation events (11, 12). The cdks integrate
mitogenic and growth-inhibitory signals and coordinate cell cycle
transitions (13, 14). Passage through G, into S phase is regulated by
the activities of cyclin D-, cyclin E-, and cyclin A-associated kinases.
B-type cyclin-associated kinases regulate G2-M. Two families of cdk
inhibitors have been identified. INK4 family members pl5INK4B,
p!6INK4A, p 18, and pl9 bind cyclin D-dependent kinases, specifically,
cdk4 and cdko. Kinase inhibitor protein (KIP) family members, in
cluding p21cipl, p27Kipl, and p57Kip2, bind and inhibit their targets.
The cdk inhibitors regulate entry into and progression through the cell
cycle by modulating the activity of cdks in response to mitogenic and
antimitogenic stimuli. The cyclins, cdks, and cdk inhibitors are fre
quently altered in cancer or disrupted secondarily by other oncogenic
events (7).
p27Klpl is a cdk inhibitor that regulates progression from G, into S
phase by binding to the cyclin E-cdk2 complex and inhibiting this
kinase (15). Regulation of this protein appears to occur primarily at
the posttranslational level by ubiquitin-mediated degradation (16).
p27Klpl binds and inhibits its target cdks in a stoichiometric manner.
Thus, the availability of p27Klpl is critical in the regulation of cyclin/
cdk activity. Although mutations in the p27Ktpl gene are rare in
human tumors (17-20), decreased p27Klpl protein has potential im
portance as a prognostic factor in breast (21, 22), colon (23), and
gastric carcinoma (24), and its reduction may be associated with
tumor progression. Here, we demonstrate that the loss of p27Klpl
appears to be of prognostic
carcinoma.
value in primary resectable
prostate
pathological stage using these three parameters (6). Most patients,
however, fall in a middle zone where meaningful prognostication
PATIENTS
Received 9/16/97; accepted 12/2/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked atlvertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was funded by the Canadian Breast Cancer Research Initiative. J. T. is a
Fellow of the
Fox Run, and
Cancer Care
Ontario.
2 The first
-1Requests
National Cancer Institute of Canada, supported by funding from the Terry
was also supported by the Toronto-Sunnybrook Regional Cancer Center and
Ontario. J. M. S. is a Clinician Investigator, supported by Cancer Care
two authors contributed equally to this work.
for reprints should be addressed to either Dr. L. Kapusta. Department of
Pathology, or Dr. J. Slingerland. Cancer Biology Research, Sunnybrook Health Science
Center, 2075 Bayview Avenue, Toronto, Ontario, Canada, M4N 3M5. Phone: (416) 4804120 or (416) 480-6100. ext. 3494; Fax: (416)480-5703;
E-mail:joyce.slingerland@
utoronto.ca.
4 The abbreviations used are: PSA, prostate-specific antigen; cdk, cyclin dependent
kinase; INK4, inhibitor of cdk4; TNM. tumor-node-metastasis;
CAS, Cell Analysis
Systems; NHT, neoadjuvant hormonal therapy; KIP, kinase inhibitor protein.
AND METHODS
Patient Population. The study group consisted of 113 patients who had
undergone radical prostatectomy, performed by one of two surgeons, at Sun
nybrook Health Science Center between January 1986 and April 1993 and for
whom clinical follow-up data were available. Paraffin blocks from the embed
ded radical prostatectomy specimens were selected on the basis of having both
benign and malignant histology present within the same tumor section. The
clinical database prepared from patient records included preoperative PSA,
clinical and pathological staging, Gleason tumor grade, time to treatment
failure, and survival. The patients were staged according to the TNM system
(25). Treatment failure was defined as any one of the following: institution of
hormonal therapy postoperatively. failure of the PSA to return to undetectable
levels postoperatively (<0.2 /xg/liter; Hybritech assay), or any new PSA
elevation above undetectable levels on two successive readings during post
operative follow-up. Patients who had lymph node metastasis at the time of
542
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LOSS OF P27IÃœPI:A PROGNOSTIC
FACTOR
surgery were treated routinely with postoperative (adjuvant) hormonal therapy.
Because institution of hormone therapy is within our definition of treatment
failure, node-positive eases were excluded from the prognostic factor analysis.
Immunohistochemistry.
Expression of p27Klpl protein was determined
IN PROSTATE
CANCER
Table 1 Clinical and pathnloaical
Parametern(%)Preoperative
(80.5)163420796501794816422694
factorsPreoperative
PSA*<44-1010-20>20Clinical
immunohistochemically.
Paraffin sections of tumor blocks were deparaffinized
with xylene. rehydrated, and microwaved for 10 min in 10 mM citrate buffer
(pH 6.0). Sections were blocked with 3% hydrogen peroxide in methanol
followed by normal horse serum (1:20 dilution) in 0.1 M PBS at pH 6.0 and
then incubated overnight at 4°Cwith anti-p27Kipl monoclonal antibody (Trans-
(27)12(12)11
T-stage'TibTicT2aT2bT2cNeedle
(10)9(8)56
duction Laboratories, Lexington, KY) diluted 1:1000 (0.25 ng/ml) in PBS.
Slides were then reacted with biotin-labeled anti-mouse IgG and incubated
with preformed avidin-biotin-peroxidase
complex (Vector Laboratories,
Burlingame. CA). Metal-enhanced diaminobenzidine substrate (Pierce, Rock-
(50)25
(22)12(11)57(66)22(25)8(9)24(21)89
grade2-678-10Neoadjuvant
biopsy Gleason
ford, IL) was then added in the presence of horseradish peroxidase. Sections
were counterstained with hematoxylin, dehydrated, and mounted. The degree
of p27Klpl staining was scored independently by two pathologists (L. R. K. and
I. M-P.). All sections selected contained both benign and malignant histology,
which allowed comparison of immmunohistochemical
staining of p27Klpl in
therapyYesNoPostoperative
hormonal
(79)49
benign and malignant tissue. Negative controls using isotype-specific polyclonal mouse immunoglobulin showed no staining. Furthermore, the pattern of
p27 staining in tumor samples obtained with the monoclonal antibody from
Transduction Laboratories was confirmed using three different polyclonal
p27Klpl sera (one from Santa Cruz Biotechnology, Santa Cruz, CA; and two
factorsGleason
tumor2-678-10Extra
grade from resected
(43)45
(40)19(17)76
extensionYesNoMargins
capsular
kindly provided by T. Hunter, the Salk Institute. La Jolla. CA; Ref. 26). p27
staining with the Santa Cruz antibody was lost when the antibody was preadsorbed with blocking p27 peptide (Ref. 21 and data not shown). Between 15
and 20 high-power fields of tumor were scored for the percentage of nuclei
showing positive p27Klpl staining. The scoring system used was as in our
previous
studies (21): 1, 0-25%;
2, 25-50%;
3, 50-75%;
data
p27"22(19.5)26652368396422069718418410123192
(%)113(100)18(19)40(42)26
p2791
(67)37
(33)70
positiveYesNoSeminal
(62)43
(38)21
involvementYesNoLymph
vesicle
and 4, >75%
(19)92(81)11(10)102
positive nuclear staining.
Image Analysis. Image cytometric analysis was performed with the CAS
200 (Becton Dickinson Cellular Imaging Systems, San Jose, CA), a videobased interactive image cytometer (27). using the Quantitative Nuclear Anti
gen Program software (Version 3.06; CAS Inc., Becton Dickinson Cellular
Imaging Systems). Four negative fields on each slide were analyzed to estab
lish the nuclear and antibody threshold optical densities by which the image
analyzer could discriminate positive nuclear staining from background. To
provide a second independent method to verify nuclear p27Klpl staining scores,
involvementYesNoCurrent
node
(90)104(92)3(3)6(5)<25%
statusAliveDead
unknown)Dead
(cancer or cause
(other cause)Total
1Less than 25% of nuclei stained positively for p27 lp on immunohistochemislry.
" In ng/liter.
' Clinical T-stage per the TNN staging system (see text for details).
image analysis was performed on selected cases scored previously by percent
age of positive nuclei. Three cases in each of the p27Klpl staining categories
(categories
1-4) were studied by image analysis. Between 20 and 30 nuclei
from normal prostate epithelial cells and 50 nuclei from carcinoma cells were
selected randomly and outlined on the video screen. Total nuclear area,
percentage positive nuclear area, and the sum of the optical densities were
quantified for each case, for both benign and malignant nuclei. A minimum
total nuclear area of 5000 /¿m2was measured for each case by the CAS image
analyzer.
Statistical Methods. Time to treatment failure and its dependence on
putative prognostic factors was investigated by the log-rank method (28) and
Cox's proportional hazards regression analysis (29). Stepwise selection of
variables was used to determine the best predictors. Kaplan-Meier curves were
constructed (30) to show the probability of remaining free of failure as a
function of time after diagnosis. Correlation analysis between prognostic
factors was done using Spearman and Pearson coefficients.
tered preoperatively to 24 of 113 patients in a variable manner. The
drug regimens used were cyproterone acetate (14 patients), diethylstilbesterol (5 patients), leuprolide plus nilutamide (2 patients), finasteride (2 patients), and flutamide alone ( I patient). Duration of hor
monal treatment was also variable, with median duration of 4 weeks,
a mean of 6.1 weeks, and a range of 1-26 weeks. Overall survival was
similar to that observed in other contemporary studies on outcomes of
radical prostatectomy (33-35).
Immunohistochemistry Results and Image Analysis. Strong ex
pression of p27K'pl was consistently noted in virtually all nuclei of
benign prostatic epithelium, and there was a consistent but variable
degree of reduction in both the intensity of p27K'pl staining and the
number of nuclei stained in prostate carcinoma (Fig. 1). Although the
nuclei of prostatic intraepithelial neoplasia tended to be p27Klpl
RESULTS
Clinical and Pathological Cohort Data. Median patient age was
65 years (range, 39-75 years), and median follow-up was 4.6 years
(range, 0.75-11.4). The clinical and pathological data are summarized
in Table 1. Gleason scores were stratified into three groups, scores
2-6, score of 7, and scores 8-10, based on the observation that a
Gleason score of 7 confers a significantly worse prognosis than does
a score of 6 (31, 32). The preoperative Gleason score was known for
87 patients; preoperative serum PSA was available in 96 patients. All
other data were available for the entire cohort (n = 113). The majority
of patients were clinical stage T2 (n = 93), and the remainder were T,
(n = 20). Neoadjuvant hormonal therapy (NHT) had been adminis-
positive, there was a variable reduction in intensity of staining.
Image analysis provided a validation of the visual scoring system
used for quantitation of p27Klpl nuclear staining by immunohistochemistry. Image
highest values of
range, 144-186;
each category of
analysis of benign nuclei consistently showed the
summated absorbance in all cases studied (n = 12;
mean, 154.4). Over 150 nuclei were evaluated in
p27Klpl staining (percentage nuclear staining cate
gory 1, 2, 3, or 4). Of malignant tumor nuclei, cases scored as 1
(<25% nuclei staining) had the lowest summated absorbance values
(range, 40-46; mean, 42.6), whereas cases scored as 4 (>75% nu
clear staining) had the highest value for malignant nuclei (range,
107-117; mean, 112.0). Cases in the intermediate range, scored as 2
543
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LOSS OF P27Klpi: A PROGNOSTIC
FACTOR
IN PROSTATE
CANCER
K**»**^
Fig. I. p27K'pl nuclear staining of prostale adenocarcinoma. Paraffin-embedded
material from
radical prostatectomy specimens was stained for
p27KlP' and counterstained with hematoxylin. The
~c-
percentage of malignant nuclei with positive nu
clear staining was scored independently by two
pathologists into four categories. Large arrow
heads, benign prostatic glandular epithelium; small
arrows, prostatic adenocarcinoma. A. adenocarcinoma of prostate, Gleason score 4. with no nuclear
staining for p27Klp', category 1 (0-25% nuclei
staining positive). B, adenocarcinoma of prostate.
Gleason score 3, with p27Klpl nuclear staining cat
egory 2 (25-50% nuclei positive). C, adenocarci
noma of prostate. Gleason score 3. with p27Klpl
nuclear staining category 3 (50-75% nuclei posi
tive). D, adenocarcinoma of prostate, Gleason score
3 with high nuclear p27Klpl staining, category 4
(>75% nuclear staining). Magnification (A-D),
X400.
%*A
•¿*'
A
»
Table 2 Correlation
of p27K'1'' with other prognostic parameters"
p27K""
n
Univariate analysis of lymph node-negative cases (n = 102) by Cox
regression or by the log-rank method identified the following as
prognostic for risk of treatment failure: seminal vesicle involvement
(P = 0.0030), low p27Kipl staining (<25% positive nuclei;
P = 0.0037), postoperative Gleason grade (0.0094), and positive
surgical margins (P = 0.030; Table 3A).
Correlation coefficient
All113)Seminal
patients (n =
involvementGleason
vesicle
tumorMargins
grade from resected
positivePreoperative
coefficient)Extracapsular
PSA ' (Pearson
extensionNHTLymph
involvementAge
node
coefficient)Preoperative,
(Pearson
Table 3 Univariate analysis for time to treatment failure
Prognostic marker
89)Seminal NHT excluded (n =
involvementGleason
vesicle
tumorMargins
grade from resected
positivePreoperative
coefficient)Eixtracapsular
PSA* (Pearson
P"
Node-negative cases (n = 102)*
140.0930.1850.1440.550.51
Seminal vesicle involvement
0.0030
<25% nuclei p27 positive
0.0037
Gleason grade from resected tumor (2-6, 7, 8-10)
0.0094
extensionLymph
Margins positive
0.030
involvementAge
node
Needle biopsy gleason grade (2-6. 7. 8-10; n = 78)
0.051
(Pearson coefficient)113113113%11311311311389898973898989-0.340-0.202-0.201-0.198-0.153+0.146-0.065-0.055-0.309-0.267-0.179-0.157-0.156-0.064-0.0710.00020.0320.0330.0540.1070.1230.500.560.00
Neoadjuvant hormonal therapy
0.25
" Low p27Klpl, <25% nuclear staining; high p27Klpl, s25% nuclear staininig; SpearClinical T-stager
0.26rf
Preoperative PSA' (<4, 4-10, 10-20, >20; n = 86)
0.35d
man rank correlation coefficient, except where otherwise noted.
* In fig/liter.
Extracapsular extension
0.38
Age
0.42
Node-negative cases, excluding NHT (n = 79)
Gleason grade from resected tumor (2-6, 7, 8-10)
Needle biopsy Gleason grade (2-6. 7. 8-10; n = 58)
Seminal vesicle involvement
<25% nuclei p27 positive
Margins positive
Extracapsular extension
Preoperative PSA' (<4, 4-10, 10-20, >20; n = 64)
Clinical T-stage'
(25-50% nuclear staining) or 3 (50-75% nuclear staining), had summated absorbance values between those of groups 1 and 4: cases
scored as 2 had a range of 58-71 and a mean of 63.0, and cases scored
as 3 had a range of 83-92 and a mean of 88.3.
Prognostic Role of p27Kipl. In a correlation analysis between
prognostic factors, low 27Klpl was strongly associated with seminal
vesicle involvement and with increased Gleason grade (P = 0.0002
and P = 0.032 respectively; Table 2). A trend toward increasing PSA
was observed when p27Kipl was low (P = 0.054). Low p27Kipl did
Age
" From score )f or log-rank test.
* Sample size, except when indicated otherwise.
' Clinical T-stage per the TNM staging system (see text for details).
J P for linear trend.
not correlate significantly with extracapsular extension, lymph node
involvement, use of NHT, or patient age (Table 2).
0.0027
0.012
0.044
0.049
0.059
0.30
O^
0.65d
0.74
' In
544
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LOSS OF P27KIHI: A PROGNOSTIC
FACTOR
IN PROSTATE
CANChK
only those tumors showing low p27Klpl nuclear staining (<25%
nuclei positive) had a distinctly worse prognosis over the follow-up
period than did the remainder of the cohort (P = 0.0037; Fig. 25,
Kaplan-Meier curves). The probabilities of being free of treatment
failure at 5 years were 38 ± 23% for low-p27Klpl tumors and
72 ±10% for the high-p27Kipl tumors.
Multivariate analysis of node-negative tumors (n = 102) identified
low p27Klpl as a novel independent prognostic factor for time to
treatment failure (P = 0.047; risk ratio = 2.08; Table 4). Seminal
01
9
234567
10
11
vesicle involvement and positive surgical margins were also of inde
pendent prognostic value, with risk ratios of 2.29 (P = 0.034) and
2.07 (P = 0.047). respectively (Table 4). Confirmation of these
observations in other larger data sets is desirable because the 95%
confidence intervals are broad.
Effects of NHT. The effect of NHT on time to treatment failure did
not reach statistical significance (P = 0.25; Table 3A). A trend toward
increased p27Klpl staining was observed in tumors from patients who
12
received NHT. Only 2 of 24 tumors (8.3%) resected following androgen ablation therapy had low p27Klpl (<25% positive nuclei),
100 :
whereas 20 of 89 tumors (22.5%) that did not receive NHT had low
p2yKipi yjjjj, (Jifferengg was nO( statistically significant (P = 0.15;
two-tailed Fisher's exact test). NHT had a significant effect on the
80
distribution of grade in the resected specimens. Among the 89 patients
who did not receive NHT, Gleason scores of 2-6, 7, and 8-10 were
observed in 44 (49%), 34 (38%), and 11 (12%) patients, respectively.
In the 24 patients who received NHT, 5 (21%) had Gleason scores of
2-6, 11 (46%) had Gleason scores of 7, and 8 (33%) had Gleason
scores of 8-10. This trend toward increased Gleason grade following
NHT was statistically significant ()f for trend = 8.7; P = 0.003).
Interestingly, when node-negative cancers treated with preoperative
NHT were analyzed separately, low p27Klp' in the resected tumor was
60
40 :
20
25% p27
p = 0.0037
01
23456789
10
11
an even more significant predictor of reduced time to treatment failure
than it was in the group as a whole (P = 0.015; n = 23). When the
patients who received NHT were removed from the analysis, low
p27Kipl still predicted reduced time to relapse (P = 0.049; Table 3B
and Fig. 2C, Kaplan-Meier curve). On multivariate analysis of the
cases with negative lymph nodes who did not receive NHT (n = 79),
only Gleason grade proved to be a statistically significant prognostic
factor for relapse-free rate (/> = 0.0034; risk ratio = 2.10).
12
Years
100 -
80 ;
DISCUSSION
>25%p271 '-m—
«---h
6J0'S0)i1
+H
¡
Established modalities for managing localized prostate cancer in
clude radical prostatectomy, radical radiation therapy, and surveil
lance. Untreated low-grade cancers in men with life expectancies of
less than 10-15 years are not likely to affect longevity (3). When
radical therapy is not given to patients with tumors of moderate or
high grade, there is a loss in life years of 4-5 and 6-8 years,
respectively (3). Radical prostatectomy has a low mortality but sig
nificant morbidity, with impotence resulting in 50-70% and stress
incontinence in 2-6% of cases (34-36). In tumors treated by radical
H<25%p27p
:g2040
;1
01
= 0.049
23456789
10
11
12
Years
Fig. 2. A, Kaplan-Meier curve of time to treatment failure (n = 102). B, Kaplan-Meier
curves of time to treatment failure, comparing patients with <25% p27Klpl nuclear
staining to those with >25% p27Kipl nuclear staining (n = 102; P = 0.0037). C,
Kaplan-Meier curve in patients with node-negative cancers, excluding those who received
NHT, comparing <25% p27Klpl nuclear staining to those with 225% p27Klpl nuclear
staining (n = 79; P = 0.049).
Table 4 Multivariate analysis for lime to treatment failure
Model for treatment failure
Risk ratio (CD"
Node-negative cases (n = 102)
(1.07-4.92)
Seminal vesicle involvement
2.08 (1.01-4.27)
<25% p27 positive nuclei
(1.01-4.24)2.10''
2.07
Margins positive
NHT recipients excluded (n = 79)
(1.28-3.44)0.034
Gleason grade from resected
tumor (2-6. 7. 8-10)2.29
" CI. 95% confidence interval for the relative risk ratio.
h From Wald x~' Per step.
The Kaplan-Meier curve of time to treatment failure for nodenegative study patients showed a 5-year actuarial failure-free rate of
66% (±9.6%, 95% confidence interval; Fig. 14). When the diseasefree rate was plotted versus p27Klpl staining scores of 1, 2, 3, or 4,
0.047
0.0470.0034
545
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LOSS OF P27lap': A PROGNOSTIC FACTOR IN PROSTATE CANCER
prostatectomy, increasing grade predicts a shorter disease-free sur
vival (37). When the cancer is organ confined, however, disease-free
survival is excellent, regardless of tumor grade (38). Because radical
prostatectomy is unlikely to be curative in locally advanced disease, a
marker that accurately predicts whether or not cancer is organ-con
fined would allow better treatment assignment.
Low p27Klpl correlated with seminal vesicle involvement and with
positive surgical margins in the entire cohort but not with extracapsular extension. Of 19 tumors with poorly differentiated disease
(Gleason sum of 8-10), 7 had low p27Kipl. Pathological staging
revealed that none of these seven were organ confined. Of the re
maining 12 high-grade tumors without low p27Klpl, 3 were organ
confined and 1 was specimen confined. Thus, low p27Klpl in poorly
differentiated
tumors suggests a low likelihood of organ-confined
cancer. For such individuals, radical surgery is unlikely to be curative,
and the increased risks associated with surgery may not be warranted.
Conversely, higher p27Klpl staining (scores of 2-4) may better iden
tify the subset of tumors likely to be organ confined. In this study,
p27K'p' staining was performed on cancers after surgical resection.
Evaluation of p27Klpl staining of preoperative needle biopsy material
and the cell cycle (49-51). Preliminary studies suggest a role for
p27K'P' ¡nandrogen-regulated prostate epithelial proliferation and
differentiation. Chen et al. (52) studied the androgen-mediated effects
on normal prostate in rats. Castration resulted in atrophy of the gland
due to apoptosis and arrest of surviving cells. Readministration of
testosterone induced epithelial cell proliferation, followed by cellular
differentiation and an increase in p27K'pl. It was postulated that the
increased p27K'pl was playing a role in slowing proliferation as the
cells underwent differentiation.
Clinical trials of NHT prior to radical prostatectomy for clini
cally localized disease consistently show reduced rates of positive
surgical margins in the treated study arms. With the immaturity of
follow-up to date, none of these studies has shown a significant
improvement in disease-free survival (53-56). In our retrospective
analysis, there was a trend toward increased time to treatment
failure following NHT. Although our numbers were small, tumors
from patients who received preoperative NHT expressed higher
levels of p27Klpl protein than did those from untreated patients. As
reported by others (57-59), NHT appeared to cause a shift toward
a higher Gleason grade. However, the "high grade" of such tumors
for presurgical prognostication of organ confinement should be un
dertaken in a larger study.
In lymph node-negative tumors, low p27Klpl was an independent
marker for shorter time to failure. p27K'pl staining may assist in
is likely an artifact resulting from the distortion of tumor histology
following androgen ablation. NHT has been shown to reduce the
proliferative activity of tumors (58). The trend we observed toward
increased p27Klpl staining following NHT is in keeping with the
stratifying patients for adjuvant treatment after radical prostatectomy.
Adjuvant external-beam radiotherapy lowers local failure rates after
reduction in proliferation observed by others. Of note, patients
whose tumor p27Klpl remained low after NHT had a shorter
relapse-free survival (P = 0.015). Thus, low p27Kipl levels after
radical prostatectomy; however, indications for this additional treat
ment are not always clear (39, 40). Low p27Klpl may prove to have
NHT may identify de novo hormone-resistant
tumors or tumors
additional predictive value with regard to radiotherapy when it is
considered together with other known risk factors for local recurrence
after radical prostatectomy.
p27Klpl staining has been evaluated in previous studies by visual
unlikely to have a durable response to androgen ablation. Further
more, our data raise the provocative possibility that androgen
ablation therapy may be affecting the cell cycle through inhibition
of p27Klpl degradation pathways, leading to increased p27Klpl-
scoring of the nuclear staining of tumor cells (21-23, 41). Our image
analysis provided a second method of quantitating p27Klpl nuclear
mediated inhibition of cyclin/cdks. Effects of hormone withdrawal
on the cell cycle may precede the induction of apoptosis (60-63)
staining. The image analysis, although considerably more time con
suming, validates the visual scoring method, which can be more
readily applied to routine histological material.
The abundance of p27Klpl is important in the regulation of cyclin/
cdk activity. p27Klpl binds and inhibits a broad range of cyclin/cdk
complexes. When cultured cells exit the cell cycle, p27Kipl mRNA
levels remain constant, whereas p27Klpl protein increases, suggesting
that p27K'pl is largely posttranslationally regulated (42). Important
or be entirely independent of it. Unraveling the pathway for
antiandrogen mediated effects on prostate cell cycle kinetics may
lead ultimately to the discovery of new treatments for prostate
cancer and, in particular, for hormone-refractory advanced prostate
involvement of the ubiquitin pathway has been demonstrated (16).
Studies in normal human oral mucosa and human skin keratinocytes
demonstrate an inverse relationship between cell proliferation and
p27Kipl protein levels (43).5 A reduction in the level of p27Kipl could
contribute to resistance to growth inhibitors, deregulation of cell
proliferation, and oncogenic change (1.2. 44). Indeed, multiple organ
hyperplasia, including hyperplasia of the prostate, and pituitary tu
mors develop in p27Klpl knockout mice, indicating p27Klpl is an
cancer.
In conclusion, we found that low p27Klpl protein in lymph nodenegative human prostate cancer is of independent prognostic value for
treatment failure after radical prostatectomy. Although the risk ratio
was 2.08, the confidence intervals were broad, reflecting the small
sample size in this study. The validity of our observations is supported
by a similar and independent observation that low p27Klpl staining
predicts poor outcome for patients with organ-confined prostate can
cer made by another group (64). Among node-negative patients,
p27Klpl was of even greater predictive value in the subset who
received NHT. When NHT patients were excluded from the analysis,
p27KlP' was not an independent prognostic factor in the multi variate
important inhibitor of cell proliferation (45-47).
A relationship between low p27Klpl levels and aggressive behavior
has been demonstrated recently in various malignancies (21-24).
Unlike other cdk inhibitor genes, such as p/5INK4B and p76INK4A, the
/?27Klpl gene is rarely mutated in human cancers (17-20). Instead, loss
of p27Klp[ appears to occur through accelerated degradation by the
ubiquitin-proteasome pathway (21, 23, 48).
The increase of p27K'pl protein and/or activity by inhibitory cytokines (e.g., transforming growth factor-ß) suggests that this cdk
inhibitor represents an important link between extracellular regulators
s J. M. Slingerlund. unpublished observations.
model. However, with the small number in this subgroup, our study
may have lacked the power necessary to detect a significant result.
Further studies of the prognostic value of p27Klpl in preoperative
biopsy specimens may ultimately strengthen the clinical decision that
aggressive local therapy, such as radical prostatectomy, should be
assigned. Further investigation may establish p27Klpl as an accurate
predictor of pathological stage prior to treatment or as a marker of the
need for adjuvant treatment postoperatively. Larger studies comparing
p27K'p' levels in preoperative needle biopsies and in surgically re
sected prostate cancers following NHT may allow early detection of
tumors that are refractory to androgen ablation therapy. Finally,
p27Klpl and the cellular machinery that regulates its protein levels
546
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LOSS OF P27K1PI: A PROGNOSTIC
may represent a novel target for therapeutic intervention in prostate
cancer.
IN PROSTATE
CANCER
27
ACKNOWLEDGMENTS
We are grateful to M. Viscardi for valuable help in preparation
manuscript.
FACTOR
of the
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Loss of Cyclin-dependent Kinase Inhibitor p27Kip1 Is a Novel
Prognostic Factor in Localized Human Prostate
Adenocarcinoma
John Tsihlias, Linda R. Kapusta, Gerrit DeBoer, et al.
Cancer Res 1998;58:542-548.
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