Vol. 4, 2187-2194,
September
Expression
Profile
of
by in Situ
Assessed
for Patients
Toshifumi
Clinical
1998
with
Hideki
Sakai,
Yutaka
Saito
Hybridization
Untreated
Takehiko
TSUTUSakI,2
Hiroshi
Prostate-specific
Kanetake,
Antigen Is a Novel
Prostate
0.001),
and
levels PSA and
Messenger
Prognostic
Research
2187
RNA
Marker
Cancer1
Koji,
Departments of Urology (T. T., H. S., H. K., Y. S.] and Histology Cell Biology IF. K.], Nagasaki University School of Medicine, Nagasaki 852-8501, Japan
Cancer
compared of PSA mRNA
with tumors
those Our
expressing low that analysis of in biopsy specimens
results
suggested
in specific areas untreated prostate cancer may of prognosis of prostate cancers.
expression
of patients good
with mRNA.
with
assessment
provide
a
INTRODUCTION PSA3
ABSTRACT
amino
The present tionship between prostate-specific
study
was
to define
undertaken
histological grade antigen (PSA) mRNA
(Gleason
the rein-
grade)
expression
and and to
of PSA mRNA expression as a possible for untreated prostate cancers. The primary grade areas of 104 prostatlc biopsy spedmens were anal for the expression ofPSA mRNA and Its protein by nonradioactive in situ hybridization and immunohistochemistry,
marker
respectively.
formed expression the
to
A multivariate
examine the correlation and several dlinicopathological
iminunostaining
mens. The percentage increased significantly
age analysis
survival
level of PSA of spedmens with
19 and
is secreted into
blood
protein
was
PSA parameters, in biopsy
in men
tumor
marker
mRNA e.g.,
degree
of differentiation that
of prostate
and poorly
cancer-specific
higher levels (representing death curves
high area
area
of PSA higher
of tumors
survival,
untreated
patients
with
expression in the higher grade of either primary or secondary were at high risk for cancer-related
mRNA grade
of PSA mRNA expression in the higher of tumors had a significantly poorer prognosis
PSA
groups,
12).
However,
degree
For
other
in patients
by 3.5
Stamey ng/ml
10 times
with benign including
ours,
the
prostatic reported
On the other hand, Hakalahti there was no difference in the level as well
as
et al.
(9)
for every higher
amount
cm3
than
that
of PSA
generally
in
decreases
tissue. A number of rethat poorly differentiated
between
of prostate
differentiated
as well
lower intensities of staining for PSA moderately differentiated tumors (10-
correlation
of differentiation
In
cancer, valuable
with prostate
volume,
example,
at least
of cancer
countries.
with prostate as the most
tumor
increases
a level
is in the
carcinomas
as by radioactive
PSA
synthesis
and
the
cancers is not well defined. et aL (10) demonstrated that of PSA
mRNA
as assessed
between
well
by Northern
blot-
ISH. Qui et al. (5) reported
previ-
expression detected by radioactive ISH was and malignant tissue samples, but the cxlower in cancer tissues than in benign epitheiurn, and
ously that PSA mRNA present in both benign pression
grade (P
(7).
tumors show significantly compared with well and
ting,
(P = 0.017). Furthermore, in cancer-specific survival based on PSA mRNA expression status, patients with levels
serum
with
of the prostate of PSA causes
many
in BPH. However, the relative cancer tissue, as measured by IHC,
observed prostate search
and
PSA levels
proportionately
cancer,
to analyze
grade)
intensity
between
States
(7, 8). Serum
increase
positive for PSA mRNA histological grade. Im-
part
and management of patients concentration is considered
cancer reported
cells
a small
in the liver (1-6). is one of the most frequent
per-
sped-
glycoprotein with 237 localized on chromosome
epitheial only
in the United
the screening serum PSA
in comparison
correlation
plasma;
and catabolized Prostate cancer
death
single-chain by a gene
by glandular
seminal
for PSA mRNA showed a the signal intensity in both primary and secondary grade areas of each specimen and the histological grade (P < 0.0001). Only 26.0% of spedmens positive for PSA protein were also positive for PSA mRNA (and vice versa, 6.7%). Other tumors were either positive for both (66.3%) or negative for both (1.0%). When the Cox’s proportional hazards regression model was used significant
of the signal
advanced
between
analysis
33,000 is encoded
Mr
that
mainly
the level
evaluate prognostic
is a
acids
=
was
that PSA mRNA expression tended to decrease in high-grade tumors, although this was statistically insignificant. Unfortunately, both studies did not assess the retention of hybridizable RNA in each
specimen,
too small entiation Received 3/30/98; accepted 6/19/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by Grant-in-Aid 06404059 from the Miistiy of Education, Science, Sports and CUlture of Japan. 2 To whom requests for reprints should be addressed, at Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Phone: 81-95-849-7340; Fax: 81-95-8497343; E-mail:
[email protected].
and
to define of prostate
In the present mRNA
in 109 cases
the number the relationship
of specimens between
in both
studies
the degree
was
of differ-
cancer and level of PSA mRNA expression. study, we investigated the expression of PSA with
benign
and
malignant
prostatic
tissue
The abbreviations used are: PSA, prostate-specific antigen; ISH, in situ hybridization; IHC, immunohistochemistry; BPH, benign prostatic hyperplasia T-T, thymine-thymine; ACS, automated chemibuminescence system. 3
2188 Expression
of PSA mRNA
samples
using
dimerized inalby
in Prostate
nonradioactive
ISH
oligodeoxynucleotide developed
sensitive
all specimens
laboratory
with
an excellent
used
in the
pression
Our
results
of PSA
marker
for
This was
were
orig-
45 bases
to be a very
637-681,
was
found (13).
examined
sufficient
RNA by ISH of 285 rRNA localized immunohistochemically that
in prostate
the
presence
cancers
(14).
In in
of high
may
cx-
be useful
complementary
as a
known
sequences.
thesized 3’-ends with
T-T
dimer.
AND
specimens with
were
prostate
versity
age ofthese
patients
In addition,
tate
June
from
performed at either
digital
18 gauge
needle ofthe
or transrectal a biopsy
biopsies
approach
specimen
tissue
was
not always
sections
were
coated
slides.
Informed
before
biopsy. All patients
were
given
with
mounted
gen. Mean endocrine
had
a luteinizing
evaluated
(histological level
PSA clinically
Tandem-R and Eiken
ACS-PSA Refs.
the
16),
PSA
Concentration.
Serum
by three
assay
different
PSA
cutoff
with
immuno-
described ized
and
Examination
were
stained
grade
of malignancy
with
H&E
was
determined
grading system (16), and secondary grades Higher
primary
grade
indicated
or secondary
Grading.
for pathological in each
40% tion.
Biopsy
specThe
using
the
a specimen grade.
of a high
grade
all
pretreated
with
citrate
20X
(pH 7.0)]
Tris-HCI
solution,
testicular
DNA
deionized
formamide.
at 37#{176}Cwith
(pH
500 p.g/ml
(Sigma
Chemical
The mem-
1 pjg/mi
T-T dimer-
tRNA,
and the sites
T-T
in the
sented
were PSA
salmon
testicular
of peroxidase
After as
were
visualHC1,
with
Sections
PSA
After
oligo-DNAs
medium
fixation
for PSA
as above.
formamide
stained were
hybridization
15 mm.
in 4X SSC until used for hybridizacarried out for 12 h at 37#{176}C with 2
50%
of peroxidase
to were This
in PBS for 5 rain, the sections were glycine in PBS for 30 mm and kept in
antisense
were
according
21). Briefly, sections standard procedures.
K at 37#{176}C for
hybridization
washed
was performed
(13, using
with 0.3% H202 in methanol for 15 peroxidase, 0.2 N HC1 for 20 rain,
proteinase
dimerized
in 2X
Then,
SSC,
followed
visualized was
and the
as described considered
slides by 2X
immunohistochemically,
signal
dis-
the
above. positive
when
the
black deposits in individual cells exceeded the level. When staining was almost similar to that of control,
the other hand, when the signal only.
pg/m1
3,3’-diaminobenzidine-4
ISH
Nonradioactive
solved
sites
125
and 40% deionized formamide. were stained immunochemically
described previously and rehydrated
pjg/mi
the sample
a strong
and
of strongly clear
Experiments mRNA
conducted antisense
was
considered
staining was classified was present but limited
A classification
ity of PSA of
10 mrs
and NiSO4(NH)2SO4.
Control
and the tissue area containing was selected for further investi-
containing
containing
deionized formamide Hybridization was
The
ng/
examination. sample
(21),
with 4% paraformaldehyde immersed in 2 mg/nil
were
17-19).
Pathological
yeast
a solution
ISH.
SSC. were
15.0
a
Japan).
specified,
M sodium
1 X Denhardt’s
overnight
previously
using
H2O2, Cod2
(ACS-PSA,
value,
Tokyo,
pg/m1
250
negative
ng/ml;
The
using
solution,
(normal
