Vol.
4, 229-234,
January
1998
Telomerase
Clinical
Enzyme
Activity
Multistage
Pathogenesis
Kazuo
Sara
Yashima,
Lauren
S. Gollahon,
M. H. Saboorian,
Adi
and
of Breast
Jerry
Carcinogenesis
multiple
genetic
Maitra,
W. Shay,
and
preceded
Hamon Center for Therapeutic
Oncology
Research
[K. Y., A. F. G.]
and Departments
of Pathology
[S. M., A. M., M. H. S., A. F. G.] and
Cell Biology and Neuroscience
[L. S. G.. J. W. S.], University
of
Texas Southwestern
Medical Center, Dallas, Texas 75235-8593
lesions
years.
For
thelial
that
breast
carcinoma
involves
the identification
was detected
in 1 (14%)
of
of benign breast disease, in 4 (67%) of 6 fibroad.
enomas,
in 11 (92%) of 12 carcinoma
in situ (CIS)
lesions,
and in 16 (94%) of 17 invasive
breast cancers.
There was a
progressive
increase
in the mean tebomerase
levels with
progressive
increase
in severity
of histopathological
change
(P < 0.05).
Almost
all of 67 resection
samples
expressed
hTR, irrespective
of histology.
Expression
was low to moderate
in some
samples
of normal
epithelium
and
nonprolif-
hTR expression
was limited to
epithelial
cells; expression
in stromal cells, including
those in
fibroadenomas,
was negative.
Increased
hTR expression
was
observed
in some
foci of apocrine
metaplasia
and atypical
hyperplasia.
Increased
hTR expression
was also observed
in
all CIS and invasive
lesions, although
considerable
heteroerative
fibrocystic
geneity
was
in CIS
sive
noted.
lesions
up-regulation
tumor
changes.
Focal
up-regulation
was frequently
noted
in the vicinity
of invasive
tumors.
Thus,
of hTR may be a predictive
marker for inva-
necessary
the
The
mechanisms
an
of tumor
molecular
RNA
breast
contains
but
10/6/97.
activated
tissues
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.
‘ Supported
in part by grants from the Geron Corp. (Menlo
Park, CA)
and the Susan 0. Komen Breast Cancer Foundation.
2 To whom
requests for reprints should be addressed, at Hamon Center
for Therapeutic
Oncology
Research,
UT Southwestern
Medical Center,
5323
Harry
Hines
Boulevard,
Dallas,
TX 75235-8593.
Phone:
(214) 648-4921;
Fax:
(214) 648-4924;
E-mail:
gazdar@simmons.
swmed.edu.
and
of
in the devel-
been
tebomerase
TRAP
was
fibroadenomas,
present
assay
and
in a heterogeneous
telomerase
levels may
have
cell
lines,
somatic
and
that
cells
proliferating
in invasive
but also
(12).
and
the
for the
field
RNA
was
because
fraction
the identity
In
activity
However,
extracts,
in CIS
(9-12).
of tebomerase
cancers
by
telomerase
epithelium
cells is unknown.
advance
in the telomerase
coding
samples,
lesions
normal
preneoplastic
as determined
8). In breast
even
biopsy
some
activity,
expression
observed
in invasive
breast
TRAP
assay
uses
whole-tissue
of the gene
5).
in normal
reported
(3,
not only
heterogeneous
ase-expressing
A major
region
synthesizes
In humans,
(2, 3, 6, 7).
weak
lesions,
of
the
tumor
of the telomercame
component
with
the
of human
(hTR; Ref. 5). Although
Northern
analyses
of hTR
not always
parallel
telomerase
enzyme
activity
(13),
observed
good
and tebomerase
concordance
enzyme
for
hTR
(a) it allows
The abbreviations
repeat amplification
in situ hybridization;
genase.
of hTR
activity
(as detected
or a PCR-based
assay
materials,
and
(as
detected
by
by the TRAP
(8). The in situ method
with using a Northern
the use of archival
fresh or fresh frozen
tissues;
expressed
in some surrounding
3
for
template
lymphocytes,
express
activity
activity:
step
and immortal
is inactive
it has
the PCR-based
hybridization
6/I 1/97; revised 10/3/97; accepted
is likely
strategies.
that activation
a small
cancers
assay)
in adult tissues
and tumors
some obvious
advantages,
compared
Received
epi-
invasive
for diagnosis,
which
tebomerase
Refs. 4 and
recently,
also
addition,
in most
cells,
of renewal
More
ISH)
markers
if not obligate,
component
cancer,
for germ
cloning
and
pathogenesis,
of therapeutic
the concept
be a critical,
is expressed
including
cells
involve
CIS,3
of carcinogenesis
understanding
bind to tebomeres
from
DNA repeats
(TTAGGG;
telomerase
lesions
steps
of several
of cancer
(2, 3). Telomerase
is a complex
enzyme
both a catalytic
protein
component
and a RNA com-
that can
tebomeric
cells
preneo-
a period
hyperplasia,
of useful
may
opment
containing
except
over
morphological
atypical
for
tebomerase
ponent.
recognizable
progress
ultimately
for the development
Recent
findings
support
we
development.
cancer,
of the molecular
an RNA-containing
enzyme,
is associated
with cellular immortality
and malignancy.
We investigated
the role of telomerase
during the multistage
pathogenesis
of
breast cancer.
We used the semiquantitative,
PCR-based
telomenc
repeat amplification
protocol
assay for enzyme
activity
(42 specimens
from 42 patients)
and a radioactive
in
situ assay
for expression
of its RNA component
(human
tebomerase
RNA;
hTR) for the identification
of tebomerasepositive cells in archival
resection
samples
(n = 67 from 39
activity
and
by
are
(1). It is believed
that the initial transformation
event
epithelial
cells in the terminal
duct lobular
unit. Elu-
to be
Telomerase,
of morphobogically
develop
hyperplasia,
cidation
tebomerase
the
is a multistep
process
characterized
changes,
and epithebial
cancers
in adults
by a series
plastic
ABSTRACT
Low
229
INTRODUCTION
F. Gazdar
patients).
7 samples
during
Research
Carcinoma’
Milchgrub,
Anirban
Expression
RNA
Cancer
for
has
blot
tebomerase
as opposed
to
(b) because
tebomerase
may be
normal
tissues
such as activated
used are: CIS, carcinoma
in situ; TRAP, telomeric
protocol;
hTR, human
telomerase
RNA; ISH,
GAPDH,
glyceraldehyde-3-phosphate
dehydro-
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1998 American Association for Cancer
Research.
230
Telomerase
Expression
in Breast
Cancer
telomerase
Relative
(units4tg
activity
protein)
It___
100.
0
SurgIcal
Frozen
res.ctions’)
I
0
sections
Mean=21.7
10
.
Fig. I
protein)
Relative
in frozen
telomerase
activity
breast
samples.
Mean=4.34
(units/jig
1
I
Mean=0.77
I
Mean=0.01
0.1
0
0
C00000
lymphocytes
and
the cellular
origin
To further
abnormal,
and
ity, using
using
proliferating
epithelia!
of telomerase
understand
malignant
ISH
MATERIALS
AND
/;;o::c,
assay,
TRAP
(n=17)
it identifies
in the normal,
enzyme
activ-
and hTR
<Q
expression
(rig)
ITAS
A total of 42 surgically
resected
<#{176}
t#’
3.0
3.0
3.0
3.0
,1
,‘Q
s,
$qO”
II
‘I
Protein
method.
, ,,
,
‘f
METHODS
Samples.
Tissue
cells,
Invasive tumor
(n=12)
(n=6)
expression.
the role of telomerase
breast,
we studied
both
the semiquantitative
a radioactive
stem
CIS
Fibroadenoma
BBD
(n=7)
0
3.0
0.3
3.0
0.3
-
sam-
ples (20 frozen
sections
and 22 fresh frozen samples)
for TRAP
assay were obtained
from patients
with invasive
breast
cancer
(n
17),
disease
These
(n
CIS
(n
12),
7) from
=
samples
were
We
analyzed
hTR
The cases were
logical
features
changes
in the
ductal
and
slides
(n
were
67)
were
were
39
of the
Telomerase
six-base
and
this
value
standard
of
cancers
and
their
and fibroadenomas.
examined
(34
with
type.
Patients
8
had
ranged
ductal
S with
carcinomas,
CIS
patterns.
cases,
in age
were
micro-
and
mixed
carcinoma
specimens
multiple
carcinoma
24 were
and
of pathological
of which
from
6
20 to 78.
verified
study.
Assay.
repeat
was
present
Fig. 2 TRAP assay results
carcinoma,
using the internal
values
of telomerase
arbitrary
Extracts
of tissue
specimens
and
as-
were processed
as described
earlier (2,
the signal intensity
of the telomeraseladder
was
normalized
in analyzed
determined
to the
signal
samples.
by
area
obtained
The
in fibroadenoma,
telomerase
ductal
assay
standard
CIS, and invasive
(ITAS).
by expe-
(S. M., M. H. S., and A. F. G.). Appropriate
Board
permission
was obtained
for the
say of telomerase
activity
14, 15). For quantitation,
internal
Five
for ISH.
We
in 3 1 of the
rienced
pathologists
Institutional
Review
tion,
used
lesions
patients
carcinomas,
present
of the comedo
specific,
TX).
extraction.
in breast
Of the carcinomas,
All diagnoses
purposes
expression
carcinomas.
from
lobular
lesions
also
breast
(Dallas,
selected
because
they contained
multiple
historepresenting
the spectrum
of pathological
multistage
pathogenesis
of breast
cancer,
both
fibroadenomas).
2
were
and premalignant
lobular
=
6), or benign
=
Hospital
at -80#{176}Cuntil
cases
epithelium
(n
Memorial
stored
the six fibroadenoma
normal
fibroadenoma
Parkland
integrafor
normalized
the
that
units.
activity
so
obtained
The
activity
of each
of 1 i.g of total
cellular
protein.
ISH for hTR.
previously
(15)
but
sample
ISH for hTR was
with
some
were
expressed
in
was
normalized
to
performed
modifications
as described
in the
washing
steps. After hybridization,
washing
at 50#{176}C
in SX
the tissue was subjected
to stringent
SSC, 10 mM Dli’
for 30 mm, at 65#{176}C
in 50%
SSC,
formamide,
2X
10 mi
DTT
for 20-40
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1998 American Association for Cancer
Research.
mm,
and
Clinical
Cancer
Research
231
a
a
.#{231}
I
.
,..-
,
3 Heterogeneity
of hTR expression
ductal hyperplasia
(H). Both bright-field
are presented.
in normal
Fig.
duct
(D) and
(a) and dark-field
(b)
in
images
Fig. 4 hTR
and dark-field
expression
in apocrine
metaplasia.
(b) images are presented.
Both
bright-field
(a)
RESULTS
washed
and
twice
S mM
at 37#{176}C
in 0.4
EDTA
for
10 mm
RNase
A at 37#{176}C
for 30 mm.
10-20
mm
slides
were
track
emulsion
with
a desiccant
replicate
and 0.1 X SSC
dehydrated
each
for expression
fragment
from
the
was
hybridization
and
field
examination
magnification
pression
readily
magnification;
recognized
into
greater
gene,
GAPDH.
cDNA
in
The
pBR322
Intense
GAPDH
in all sections
were
microscopy,
latter
hTR
expression,
being
more
was
by bright-
but which
by bright-field
examination
usually
was detected
moderate,
examination
present
cxat low
expression
bomerase
benign
breast
nonprobiferative
nomas,
was
of 12 CIS,
In benign
exception,
breast
all positive
activity,
invasive
less
4.34
level
of the
and
units/jig
tumors
telomerase
tumors
at a
in normal
Whereas
illustrated
only
bevels
with
17
and
had
to high
metaplasia
(Fig.
examined
focally,
of
Surgical
39
in
increase
increase
in
in severity
as determined
by
of the
the
TRAP
of hTR,
to moderate
expression
In both
hTR
hTR
examples
disease
samples
(Fig.
in samples
of atypical
was
normal
samples
from
expression
was present
expression
Samples.
histologically
the
high
mean
present
Resection
patients,
bevels
4).
The
to those
Examples
fibrocystic
weak
and
activity
a progressive
nonprobiferative
hTR
1). In CIS
telomerase
2.
in
(44%)
one
of tebomer-
respectively.
0.05,
<
breast
with
(Fig.
similar
sums).
of
of normal
and
of 6 fibroade-
levels
progressive
(P
rank
for
low
relative
was
Te-
samples
of 17 invasive
protein
was
in Fig.
background
patients
crate
Samples.
seven
fibroadenomas,
protein,
There
Expression
epithelium
of
very
of
(8).
test
in
had
change
Kruskall-Wa!lis
and
values
21.7
the mean
are
one
16 (94%)
1.0 unit/jig
invasive
lung
hTR
and
disease
mean
invasive
assay
in Breast
in
samples
than
tumors,
were
Activity
detected
disease
(which
included
mixtures
fibrocystic
disease),
four (67%)
1 1 (92%)
cancers.
ase
Enzyme
activity
of histopathobogica!
by both
expression
detected
examination;
those
studied.
examined
the
signals.
magnification,
than
were
pBluescript.
of weak
disease.
RNA,
expression
(Rock-
the slides
faint
of intact
for hTR
Collection
present
bright-field
or fibrocystic
boxes
Culture
by dark-field
high,
considerably
epithelium
weak,
at high
at low
level
were
dark-field
as follows:
in photoresistant
GAPDH
Type
hybridization,
for the detection
scored
the
for
nuclear
the presence
used
SSC
NTB-2
of a housekeeping
American
signals
sensitive
sample
20 jig/mb
in 2X
Tebomerase
7.5),
mm at 37-50#{176}C, the
in Kodak
for 3 weeks
from
subcloned
Following
bright-field
dipped
(pH
with
washes
for 10-20
at 4#{176}C.
To confirm
from
Zba/HindHI
MD)
treatment
Following
and
also tested
ville,
10 mri Tris-HC1
before
twice
and exposed
slides
obtained
NaCl,
M
observed
22
had
(56%)
3). Modof apocrine
hyperplasia
(the
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1998 American Association for Cancer
Research.
only
232
Telomerase
Expression
in Breast
Cancer
aIt
_
S.
. .
p,..
t,_’-
“4r3
.“.‘‘
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.
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.
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.,;
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,
ft(
.
.
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‘-
.
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,
‘I
Fig. 6 hTR expression
in fibroadenoma
of the breast.
Weak expression
is limited
to the epithelial
elements
(E), whereas
the stromal
elements
(5) are negative.
The weak expression
is better
visualized
on the darkfield image (b) than on the bright-field
image
(a).
Fig. 5 hTR expression
in atypical ductal hyperplasia.
Both bright-field
(a) and dark-field
(b) images
are presented.
There is considerable
up-regulation
of hTR expression
in the atypical
epithelium.
The adjacent
ductal
epithelium
(D) is negative
for expression.
DISCUSSION
nonmalignant
5).
All
samples
five
exclusively
6).
in which
fibroadenomas
expressed
in the epitheliab
cells
Expression
was
limited
centers
of bymphoid
follicles
mature
lymphocytes
were
from
weak
34 patients
to strong.
of
examples
in the
considerable
in some
tion,
of
CIS
and
(Fig.
In breast
be present
fibroadenomas
breast
telomerase
during
noma, the precise
and
lesions
varied
from
of
to
areas
intense
showed
the
of
same
(9-12).
when
replicate
demonstrating
that
When
to areas
expression.
slides
it did
from
noted
lesions
this
was
The
in
fibroadenomas.
in
ent
carcinomas
noted
for
level
were
was
similar
that
mas
(34 units/jig
was
squamous
bower than
pathogenesis
in situ
protein;
carcinomas
in invasive
of lung
level
Ref.
levels
activity,
carcinomas
present
in
hTR
four
breast
to determine
expression
tissues.
activity
in one
(67%)
of
of activity
and
invasive
assay
the
than
were
carcinomas.
levels
in
cases.
lung
the mean
were
con-
in invasive
CIS
in invasive
of the bronchus
six
pres-
demonstrated
(21 .7 units/jig
8). Of interest,
carcicells
malignant
tebomerase
and
higher
breast
to the mean
weak
TRAP
significantly
in invasive
in CIS
expression
an artifact.
mean
the robe of
assay
and used
paraffin-fixed
of in situ
in enzyme
and
TRAP
higher
semiquantitative
in
normal
and understand
benign,
disease
all examples
In
carcinoma
used
in almost
variation
addi-
detected
breast
to
but also
pathogenesis
of breast
of the tebomerase-positive
normal,
Significantly
siderable
cells.
results
semiquantitative
assay
benign
expression
weak
7 and
heterogeneity
not represent
TRAP
of
the
Figs.
the
reported
of histologically
activity
in frozen
samples
localization
in archival,
Whereas
30%;
all
The
in 2 1 of 43 samples
of invasive
were
noted
invasive
telomerase
for cellular
sample
examined
used
been
carcinomas
samples
these
has
invasive
the multistage
identification
We
we
activity
and
occasional
To clarify
is essential.
expres-
However,
was
in almost
was
lesions.
intense
and
varied
expression
adjacent
expression
CIS
and
hTR
uniform.
( 13 of 43 samples,
up-regulation
invasive
carcinoma,
relatively
hTR
Carcino-
tebomerase
in CIS
and
germinal
and fibroadenomas
invasive
was
disease,
not only
cells
and invasive
in Breast
disease
heterogeneity
focal
it occurred
studies,
and
similar
intrasample
cells
cells
and
expression
Fig.
of hTR
Stromal
All CIS
Expression
cells
of the cases
samples,
reproducible
hTR
heterogeneity
intense
levels
cells
tebomerase;
found;
in stromal
present).
negative.
of CIS
one-third
8). The
not
epithelial
of benign
epithelial
morphobogically
about
moderate
but
to
was
lesions;
In all examples
in most
sion
expression
(when
expressed
Heterogeneity
mas.
high
The
protein)
carcinolevels
considerably
lung carcinomas
(8). As in the multistage
cancers,
progressively
increasing
severity
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1998 American Association for Cancer
Research.
in
Clinical
Cancer
a.#{149}
Research
233
,,
0
.
.
‘
.
S
.7
::i.
...
-
1”
8 hTR expression
in ductal CIS (1). Adjacent ducts (D) and stroma
(5) are negative.
Both
bright-field
(a) and dark-field
(b) images
are
presented.
The CIS foci demonstrate
considerable
variation
of expresFig.
hTR expression
in CIS (I) and invasive
carcinoma
(C). Moderate to high expression
is present in the invasive carcinoma,
whereas
there is considerable
variation
of expression
in the CIS foci. Both
bright-field
(a) and dark-field
(b) images
are presented.
Fig.
7
sion,
with
one
invasive
of pathological
and
change
degree
invasive
We
of
is accompanied
tebomerase
enzyme
carcinomas
were higher
used an ISH assay
for
telomerase-positive
cells.
expression
in over
normal
and benign
one-half
breast
Surprisingly,
levels
apocrine
of hTR,
samples
cells
(which
were
cells,
including
cause
weak
ing cells
erative
in apocrine
nonprobiferative
represents
in these
samples.
metaplasia,
was
of lymphoid
was
component
(3),
the finding
fibrocystic
the presence
disease,
cannot
Belevels
regena form
readily
hTR
expression
was
present
in almost
similar
This
up-regulation
squamous
expression
of CIS
focal
was
and
be explained
in squamous
carcinomas
up-regulation
of the
appeared
and
may
represent
the
later
stages
tebomerase
lung
invasive
on
the
basis
up-regulation
was
(8)
loss
and
activity
breast
lesions
(8).
independent
of
is significantly
than
cell
( 16) during
with
their
to
postu-
proliferation
higher
in
adjacent
have
may precede
invasion
(8).
suppressor
Consistent
carcinomas
We
in CIS lesions
of imminent
of a telomerase
of carcinogenesis.
enzyme
CIS
lung
this
theory,
in invasive
respective
CIS
lesions.
Thus,
of
of
cxall
tebomerase
bly in other epitheliab
nomenon.
Low levels
many
to high
not
intense
bated that such intense
up-regulation
invasion
and, thus, may be a predictor
plained.
Moderate
could
addition,
of
areas
invasive
expression
be
of the
noted
probiferat-
to be
In
cells.
one-third
limited
expression
in
breast disease
high
presumably,
expres-
in about
heterogeneity
similar
samples,
epitheliab
observed
cases,
most
positive
in both
of low
considered
which,
In
the
to high
of proliferating
relatively
lesions,
in
noted in a subset
of CIS in the vicinity
of invasive
carcinomas
and in adjacent
foci of corresponding
invasive
tumors.
We have
in stromal
by most
these
up-regulation.
lesions.
was
in morphobogicalby
of proliferation.
follicles
absent
CIS
uniform
heterogeneity
In
marked
and
relatively
However,
noted
showing
carcinomas
was
carcinomas.
the
of fibroadenomas.
is expressed
The
in
hTR
present
Expression
centers
usually
levels
moderate
activity
and low to moderate
hTR
of histologically
normal
and benign
probably
sion
of histologically
fibroadenomas.
was
Expression
epithelia
the
to moderate
expressed
activity
of regenerative
weak
samples
including
hyperplasia.
stromal
and
expression
to germinal
frequency
in CIS lesions.
to precisely
identify
detected
high
present).
telomerase
cells
hTR
focal
and
the
than
hTR
of the
diseases,
ductab
rarely
of tebomerase
some samples
samples
We
activity,
metaplasia
and
of atypical
to epitheliab
by increasing
focus
may
samples
reflect
of normal
“physiological”
control
in breast
carcinomas
(and
possi-
tumors)
appears
to be a two-tiered
pheof activity
and expression
are present
in
and benign
expression
breast
disease.
in proliferating
This
activity
regener-
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1998 American Association for Cancer
Research.
234
Telomerase
Expression
in Breast
ative cells.
In CIS,
regulated
expression
able
and possibly
in atypical
of hTR occurs,
although
heterogeneity
preinvasive
or abrogate
indefinite
in some
lesions
cellular
cell
cases.
tissues
may
senescence,
growth,
for tumor
hyperplasia,
dysthere is consider-
Telomerase
represent
resulting
eventually
up-regulation
was frequently
invasive
tumors.
Whether
marker
Cancer
dysregulation
leading
to malignancy.
noted in CIS foci
such
up-regulation
development
in
a method
to overcome
in a commitment
to
is presently
Focal
investigation.
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Telomerase enzyme activity and RNA expression during the
multistage pathogenesis of breast carcinoma.
K Yashima, S Milchgrub, L S Gollahon, et al.
Clin Cancer Res 1998;4:229-234.
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