S. Coon. IV,. Abdul. Haleem,. Samuel. Taylor. IV,. James. Hutchinson,. William. Panje ..... Park,. IL). Both newly diagnosed. (16 patients) and locally recurrent. (10.
Vol.
1, 527-537,
Ma’s’ 1995
Cell
Kinetics
Valery
M.
of Head
Kotelnikov,2
Abdul
Haleem,
James
Hutchinson,
David
D. Caldarelli,
and
Clinical
John
Samuel
Harvey
and
S. Coon
Taylor
Neck
The
Luke’s
Medical
Center,
Chicago,
Illinois
initiation
for
selecting
lifenative
rate,
measured
cell
the
head
of labeled
poorly
[labeling 2.7%
poorly
index
and
(P
potential
(47.3
±
mm.
To
5.1-21.5
doubling
times
track
the
biopsy
was
fate
label,
demonstrated peated in the these
second cells
most
7-14
cell
death
may
proliferative between
had
rate
cells
of tumor
patients,
after On
be major
potential
2242
iododedid
to the dilu-
second
biopsy
resulting
from
re-
of cells
25%
suggesting
7 labeled
cells
to
Thus,
the
cell differentiation, cell
may
explain
doubling
times
loss
and from
the
the difference and
the actual
growth.
West
Harrison
cells
cells
(LI).
section
Street,
Suite
108,
at Rush
Chicago,
IL
Cancer 60612-
decade
studies The
This
problem
concerns
the
depends
on the
mathematical
validity
an alternative
methodology
tumor
cell
the actual
loss
volume
factor.
estimated
to have
but yet have In the study
labeling
been
of cells
(11,
3 The abbreviations oxyunidine; IdUrd, mous cell carcinoma; Drug Administration; time; T, cell cycle
(2,
3,
is not
17).
Begg
is the
(l8)
admixture
biopsy,
resulting
of tumor
in
BndUnd-
if an immunocells
to measure
in LI.
a tumor et
Bennett
tumors FCM LI is nearly in a biopsy section. The of
only
T
the
can
measurement
applied
(16,
be confirmed
in humans
doubling
not been described IdUnd
time time.
factor
by
17). using
85
and
The
directly
for
(19,
we used
and
BndUnd labeled
and
than
is due to neck
20).
experimental
studied
here of
head
above
is shorten
difference
93%
documented
mentioned
which
in human
between
visualization
16, one
be avoided
doubling
well
with
widely
methodology
method
studies
This be
was tumors
(18).
kinetic potential
tumor
method
first
accuracy
of T measurements
an in vivo
disaggregated
of human
of FCM
the
introduced
using of
BndUnd-labeled
instead
have on have
underestimate
tumor
can
kinetics
et a!. (12)
proportion
problem of
cell
this
recent
second
(20),
proportion
this
problems.
which
pathways
for a high
FCM
a!. (1 1) demonstrated that for diploid four times lower than LI measured
the cell
pertreatment
parameters
of the
is used
was
pro-
proliferative
during
methods
in the disaggnegated
biopsy
The
the
[3H]thymidine
in a variety
determination
nohistoehemical
Institute. be addressed,
last
by major
led to a calculated
migrated
foci.
two
detection
The
prethenapy
radiotherapy,
1 1). Begg
of its popularity
histochemical
that
iododeoxyuri-
to necrotic of tumor
This
Received 8/16/94; accepted 1/20/95. 1 Supported by funds from Rush Cancer 2 To whom requests for reprints should Institute, 3824.
in the
a
treatment
the
tumor
with
cycle
kinetics
indicated
inaccurate labeled
to
predicting
that
subsequent
the
cell
In spite
of nontumon
patients
Due
13-15).
mea-
patients
with
During
to study
neck
(10,
cell
BndUnd
cells.
as
h
(2,
nate prior
regrow
these
cells
for measuring
out
180
In general,
of
pointed
calcu-
and labeling
injection
optimal
90 and the
(6-9).
were
18.8-84.5
however,
tumor routes
time The
These
label,
Day and
LIs
patient trials
parameter
for
be responsible
DNA
a method
11,
an inverse and
clinical
during
rapidly
the
as a major
presented
of head
in vitro
of S-phase
applied
to
may
recent
of
accelerate
cells
studies
FCM3
tumor
and one the
incorporating
transition,
short
in
1.5).
biopsies.
undiluted
compartment. very
24.8%
after
pattern
In two
of tumors
of cell cycle
and
in four
days
the
labeled
dine/bromodeoxyuridine. arrest
cells, infusion.
biopsy
parts
LI
from
of labeled
had not divided
keratinized
The
between
between
divisions.
LIs of two
ranged
a “fragmented”
cell
±
in
cells
used
an
and
failures.
either
used
reported
and
been
may
negnowth
proportion
The
of G2 was
obtained
treatment
of the
after
duration
h (12.8
utilized
55.9%.
tumor,
S-phase
from
oxyundine/bromodeoxyundine tion
and
This
5).
of treatment
percentage
In one well-differentiated
6.7). The duration
not receive
0.135). 39.4
tumors
(1)
importance
has
neoplastie
Earlier
in well-differentiated and 23.8 ± 2.1%
mucoepidermoid
ranged
lated
SE)
were
29.1 %, respectively.
surements
repeat
(LI)]
(4,
evidence
these
be
plays
disease
of proliferative
regimens the
cancers the
S-phase
of two
may
to
of
a!.
knowledge
addition
the
sequential
The
lymphoepithelioma
lymphoma.
differentiated
in 26 squa-
received
studied.
±
SCCs SCCs
rate were
bromodeoxyuridine,
and
(mean
±
in a high-grade
a malignant
22 of which
and
differentiated
2.5%
proliferative
Patients
biopsied
differentiated
3.0%
cell cancer,
(SCCs).
was
cells 20.4
was
moderately was
neck
of iododeoxyuridine the tumor
SCCs
tumor
and
carcinomas
infusions which
of
et
results
radiotherapy In
neck
course
of
of radiotherapy
outcome.
mitting
with
preliminary
that
and
the
percentage
suggested
the
rate
mous
The
have
of head
Chauvel
between
survival. 3)
rate
in determining
of treatment.
relationship
ABSTRACT We
role
outcome
Griem,
Rush Cancer Institute [V. M. K., A. H., S. T., H. D. P.], Department of Pathology [i. S. C.], Department of Otolaryngology [J. S. C., i. H., W. P., D. D. C.], and Department of Radiation Oncology [K. G.],
patients
proliferative
important
D. Preisler
Rush-Presbyterian-St. 60612-3833
527
INTRODUCTION
IV,
Panje,
Katherine
Research
Cancers1
IV,
William
Cancer
SCCs
Cell
loss
tumors
in humans. the method
and cells,
subsequent a method
of in vivo immuwhich
used are: FCM, flow cytometry; BrdUrd, bromodeiododeoxyunidine; LI, labeling index; SCC, squaNCI, National Cancer Institute; FDA, Food and T, S-phase duration time; T,,, potential doubling time; T02, duration of G2.
528
Cell Kinetics
of Head and Neck
Table
Cancers
Summary
I
of clinical
and histopathol
ogical
data of patients
give n IdUrd
and BrdUnd
for cell kin etics studies
Sample Histology
code
Moderate
Maxillary
High High
Neck Larynx
Low Moderate Low Moderate Moderate Moderate Low Low Low Moderate
Floor of Tonsil Larynx Larynx Larynx Floor of Floor of Larynx Base of Floor of Epiglottis
Moderate Moderate
Esophagus Base of tongue
II III
Low Low Moderate
Larynx Vocal cord Maxillary sinus
Recurrent II IV
Moderate Moderate
Tonsil Soft palate
IV IV
Moderate
Larynx
Recurrent
HN-5
Mucoepidermoid
HN- 1 1 HN-22 HN-35
Lymphoepithelioma Mucoepidermoid Lymphoma
High High
Base of tongue Nasopharyngeal
IV II
Low
Base of tongue
Recurrent
High
Pharynx
II
successfully
used
and solid
tumors
Hoshino
et a!. (22).
This
cycle
parameter
information
Moderate
to study
cell
technique
not
determinations,
regarding
proliferation
in our laboratory
the
(21) only
but
in human
received
in that of
and
provides
also
morphological
and gives
aspects
precise
over
valuable
end
of
tumor
to understand The
results
the routes of this
head and neck tumors integrity of the tumor proliferative
of cell
study
loss
confirm
from
that
a tumor.
a high
of these
IdUrd
proportion
of of the of the
tumors.
MATERIALS The
AND
IdUrdlBrdUrd
study
was
Board
of the Rush
BrdUrd
METHODS protocol
reviewed
were
and
University,
supplied
(H&N
approved
1 392)
by the
the NC!,
by the NC!.
utilized
Informed
the local Institutional Review Board, NC!, and tamed from every patient before the administration
Review and
required
by
FDA was of !dUrd
oband
BrdUrd.
sections
Both
IL)
newly
patients)
or West
diagnosed
patients
(16
were
clinical
and
histological
tumors
which
were
as having
a malignant
Suburban
eligible
patients) for
and study.
information
studied. neoplasm
study were diagnosed Luke’s Medical Cen-
Hospital locally Table
regarding
In all cases before
(Oak
patients study.
was
on Alcian
of
sections
IdUrd
only
stained
with
patients
the same
a second
the
No therapy
removed
were
patients
was given
in
the
used
biopsy.
In
to these
the
10 cases
of
obtained
for
received
Department
was
and a Two-
Pathology
immunohistochemical
biopsy
iv.
At the
3 h, dehydrated
coverslips
by
from
IdUrd
given
methacrylate.
Seven
followed
obtained
for
blue
When
was
surgically
in glycol
processing.
paraffin
day.
each
the infusions.
solution
embedded
placed
same
day,
between
the tumor
in Bouin’s
finally
infusion
methods. 7-14
patients
days
after
between
the
biopsies.
Immunochistochemical labeling
have
Park,
IL).
procedures
been
described
for
in detail
earlier
Single-labeling Procedure. rehydrated in distilled water with
binding mAb
3%
for 45 sites
3D9
H202 mm
for the
and
for 30 mm, 4 N HCI
then for
anti-!dUrdlBrdUrd
(a gift from
Dr. G. L. Mayers,
(10 the
lated
the patients
and
reagent enzyme
were
diagnosed patients
In short
double they
Elite
antimouse
!gG
(1 :200,
(a mixture of avidin-DH 1 :50). These reagents ABC
Kit
(Vector,
30
mm),
treated
specimens
They
are
are
with
then
Pronase
20 mm
to expose
antibodies.
The
Roswell
1 provides
Nineteen
and
(23).
The tissue for 10 mm.
rial Institute, Buffalo, NY; 1:200), which IdUrd and BrdUrd, is applied for 30 mm
recurrent
single
are as follows.
(1 mg/ml)
ten (Chicago,
the
interval
mm-thin
incubated Patients who participated in this in Rush-Presbyterian-St.
on
a 0.5-h
and
first
All patients and treated either
BndUrd
immunohistochemistry
IdUrd
consent
II IV II
in this
Institutional
and the FDA.
Recurrent
tongue mouth
with
methanol,
two
mouth mouth
and
fixed
the first one.
Recurrent IV II Recurrent IV III Recurrent
on the same
was
also
mouth
infused
of the last infusion
In four
Recurrent
were
30 mm
were
sinus
Recurrent Recurrent
immunohistochemical
grow rapidly and that maintenance tissue is essential to the analysis
characteristics
both
BndUrd
portion
and provides a unique opportunity to track the fate of tumor cells if serial biopsies are obtained. The latter may
help
Stage
SCC 5CC 5CC SCC 5CC SCC 5CC SCC SCC 5CC SCC 5CC 5CC SCC 5CC SCC SCC 5CC 5CC 5CC 5CC 5CC
leukemias
growth labeled
Localization
HN-4 HN-6 HN-7 HN-8 HN-l2 HN-l4 HN-15 HN- I 7 HN- 1 8 HN-19 HN-21 HN-25 HN-28 HN-29 HN-3 1 HN-32 HN-33 HN-34 HN-37 HN-39 HN-40 HN-47
has been
cell
Grade
Park
the mouse
Memo-
is specific for both followed by biotinyfollowed
by
the
ABC
solution, 1 :50, and biotinylated were supplied in the Veetastatin
Burlingame,
CA).
At
the
final
steps
Clinical
specimens
are
stained
nahydroehlonide
with
0.025%
in 0.003%
H202
nis’ hematoxylin
solution.
Paraffin
same
that
way
except
specimens
we
used
anti-BndUnd 3D9
were
used
As
we used
!dUnd
only.
antibody
There
with
was
are
40
labeling
several
rat
tumor
of
of the
cell
cycle,
which
according
anti-BndUnd
To we
apply
allows
to
procedure
(Sena
Labs,
Crawley
529
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labeled
.
I
14
instead
samples
no cross-reactivity
In
procedure.
cells
for
-
‘,.‘_.‘
in the
mm.
below)
demonstrate control
and BndUnd-labeled
stained
for
kit (see
Procedure.
staining
IdUnd-
treated
Han-
IdUrd.
of the
chemical
to
from
Double-labeling parameters
different
with
are lasts
nat ABC
a negative
sections
sections
Research
tet-
countenstained
application
a slightly
and
only.
body,
HCI
antibody
antibody
BrdUnd
the
3,3’-diaminobenzidine
and
Cancer
.
rat
King-
-
Fig.
I
SCC
-
-
.,.
Peripheral labeling (LI = 12.1%). x 200.
of
.
the
.‘
tumor
island
in well-differentiated
dom) diluted 1 :200 and a Veetastatin Elite rat ABC kit. 3D9 antibody (1:20) in PBS containing 0.5% Tween 20 and 1.5% normal
horse
followed
result,
then
three
labeled
types
with
nuclei),
Labeled
gold
Cells
Counting sections
of viable
and
labeled
normal
nuclei
Four
to six fields
As
with
BndUnd and
a
those
only
BndUnd
low,
studied
to calculate Necrotic are
ative
rate.
tissue,
from islands
cells of Cell
for
cycle.
the
nonlinear
It was
age
assumed
distribution
to be 0.8
(11,
acetone
and
20).
Frozen absolute
of Cytokeratin
sections
cold
are fixed
methanol.
LSAB
Kit
human
cytokenatin
The
(DAKO
staining
as
and
antibody the
in cold
is performed
Corporation) 10
K10
sequentially
using
monoclonal
mouse
(DAKO-CK1O,
chromogen.
DAKO
anti-
DE-K10)
The
with
countenstain
is ethyl
the
labeling
areas
predominantly
cells
are
values
of
tumor only
prolifen-
basal
the
central
layers
Time
(golden using
were (blue
used. Three nuclei) and
and golden/blue the
nuclei)
formula:
tumor
Nd0Ub,C
cells labeled labeled with of two
±
with IdUnd
=
NdOUb,C
±
the
BrdUrd
sum
BndUnd only, only, and T
injections.
T,
X
BrdUrd
LT/NIdUrd
(A)
of double-labeled NIdUrd
is the
5
the
interval
of the tumor
was
cells
and
number
of
cells
between
the
start
calculated
from
lost
their
did
not
-
X X TjLI
(B)
cells K10-positive
the
island.
to any of
apparent
high-grade
vessels
stroma
(Figs.
5 and
between
tumor
5).
within
islands
the
sample
Fig.
In the other in different correlation
cells
cells
In these which had
chaotic (Fig. were
4).
In
grouped
cells
of blood and
infiltrating
and
tumor
interstitial
different
degrees
of
HN-8, HN-18, and HN-29, there in the proportion of cells labeled this
In this
21 tumors, areas was between
the
which
panenehyma
demonstrated
8 illustrates
HN-6.
was
endothelial
tumor
6). Lymphoid
labeling (Fig. 7). In samples HN-5, HN-6, was considerable heterogeneity fields.
Labeled both
3).
pattern
labeled
for of
cells
labeling
of The
positive
SCCs
anatomical
tumors
(Fig.
common
the
nest 1).
regions
(Fig.
In high-grade
structure,
were
shows
all
few
blood
cells
strongly
tumors
S-phase
correlate
tissue
were
labeled
within
located the
(Fig.
contained
around
tumor
other
various
were
structures
islands
distributed
while
island
organ-specific
in adjacent
cells
islands
kenatinizing
In
be showing
labeled
of tumor
2).
to
contained
tumor
regions
SCCs
tumor
in Tumors
found
panenchyma
layers
(Fig.
vessels
labeled T
some
of these
scattered
were
tumor
of
K10
the
some
In
consisted
22.4-80%.
the equation:
in
in basal
island
eases
tissue
T
clusters
pants
Cells
cells
patterns.
island
were
sections IdUnd only
in
cytokenatin
interstitial
are counted.
is calculated
on
field.
and
the
pearls
evenly
in every
average
of DNA-synthesizing
DNA-synthesizing
tumor
cells of
kenatin
double-stained labeled with
T
and
Distribution
viable
representing
tumor
RESULTS
eyepiece
of labeled
assessment
and IdUndfBrdUnd The
cells)
proportions
with
of the
of labeled
are counted
viable
necrotic
X 10 square
maximum,
the
distribution
containing
tumor
Cycle
For these studies hundred to 500 cells are counted.
cells
without
In tumor
BndUnd
of the section
minimum,
determine
LI (percentage
a 10
intermediate
fields
of nonkeratinized
points
factor
the cell
Immunohistochemistry
(gold (nuclei
tissue,
The
The
(2000-5000
to
interstitial tissue.
tumor
excluded
Calculation
examined
using
fields
and
the
where
through
13-15,
sections
staining.
in the section:
IdUnd
first
assessed.
500-1000
high,
tissue
are
tumor
in selected From
with
The
final
X is a connection
diaminobenzidine
is determined
cells.
LI.
(both
where
of cells
blue).
non-neoplastie
is then
cells)
gnaticule
with
mAb
for
nuclei), with
and
stained
proportion
tumor
labeled
both
mouse
can be identified (blue
is
rabbit
Denmark).
substrate
This
with
green.
All areas,
only
those
with
blue
label.
sections
APAAP
Copenhagen, fast
of cells
IdUnd
and
stained
with
second
of the
and
Corporation,
treated
as the
treatment
immunoglobulin
DAKO
are
is applied
sequential
anti-mouse
from
serum
by
the
sort
specimen
however, practically L!s
of
variation
the
LI
ranged
in
the
from
the proportion of the same. Fig. 9
determined
in two
dif-
530
Cell Kinetics
of Head
and Neck
Cancers
.
Pt,
.
s
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Fig. 2 Cytokeratin K10 staining of well-differentiated tral pant of the tumor island is K10 positive, while layers are K10 negative. X 200.
5CC.
The
cen-
basal (proliferating)
Fig. (LI
Random
4
distribution x 200.
51.6%).
=
of labeled
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along
ferent
areas
differentiated
the tumor
of the
same
embedded in plastic was originally sent embedded
5CC (LI
island.
-.
=
19.9%).
Labeled
cells are
tumor:
one
sample
of the
while the other of Pathology
tumor
5
SCCs
ately
differentiated
higher,
Average as well as minimum for tumors labeled with both IdUrd
and maximum and BrdUrd
values of LIs are summarized
the BndUrd
effectively
On average was
DNA synthesis during the IdUrd infusion or before the BrdUnd infusion. Those labeled entered S-phase either during the interval on infusion.
an infusion minimal,
Hence,
time
the difference 23.6
SE, P = 0.66), indicating first label the LI increases
LIldUrd
of 90 mm
± 3.6%
between versus
that during by only
BrdUrd
±
was
rate
high,
of morphologically
20.4
SCCs
± 2.7%
One
well-differenti-
(range,
had similar
21.6
± 3.3%
the succeeding 2%.
39.4%
and
14.5-27.6).
LIs of 23.8
of a malignant late with either
Moder-
± 2.1%
average
tumors
the
(range,
appeared
grade (3.0% and differentiated tumors,
significant
and relapsed In 10 samples
(mean
±
cells
of the interstitial
1 h after
the
was
detected.
The
± SE, 4.1
difference (25.2
±
a high tissue, LI
of
± 1.3%).
LI of 22
was
LIs
2.5%, to
and
correlate
39.4% for well-differrespectively). The LI
lymphoma was 24.8%. LI values clinical stage or site of the tumors.
2.2%)
(mean
The
lymphoepithelioma
mucoepidermoid
with the morphological entiated and poorly
and 30-mm
respectively.
55.9%,
was 23.2 ± 2.0%. The LI of a high-grade
statistically
represents
of label. the 90-mm
SCCs
in two
in Table 2. Data for LIs of cells labeled with IdUnd or BrdUrd only are also in Table 2. Cells labeled with IdUrd only represent
during
proliferative
ated
a small blood vessel.
16.2-35.1). There is no significant difference between these LIs (P = 0.135). The LIs of two poorly differentiated SCCs were
in paraffin.
cells which ended during the interval with BrdUnd only
:-
I
A group oflabeled cell nuclei around cell is also labeled. X 200.
The
was
specimen and was
LIs
label
Fig.
endothelial
in our laboratory to the Department
L..:&_
)
‘t
X 100.
.
‘:I
t t.
:
:
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I
Moderately
::
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Fig. 3 scattered
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.
‘,
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a
,.
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.
between
did not comeThere was no
primary
(24.4
5.2%) 5CC LIs (P = 0.31). level of infiltration by mononuclear which these
separates cells
ranged
tumor from
cell islands, 0-13.2%
±
Clinical
:I’.w1.iP
‘
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.
9
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.
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.,
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Fig.
6
Labeled
tumor.
endothelial
cells
in the
stromal
compartment
of
the
X 400.
Duration
of S-Phase
and Potential
Doubling
labeling
cannot
3. i’s durations
be done).
ranged
from
12.1 h (mean doubling times
± SE, ranged
43.2
±
h (mean
between detected
The
data
are presented
h, with
5.1-21.5
in Table
a median
value
of
12.8 ± 1.5). The calculated potential from 18.8-84.5 h, with a median of
SE,
47.2
± 6.7).
There
was
no
99.6%
of the
HN-15
and HN-33
0 LI was
13 after
first
biopsy.
BrdUrd,
duration
of G2 (TG2)is
longer
tumors
between
the
of IdUrd
three patients formed 85-100
(not mm
approximately
3 h after
the start
label
In these
biopsies
(IdUmd).
beled
with
these
tumors
IdUrd
90 mm infusion
(the and
time the
interval
biopsy).
In
included in Table 3) the biopsy was penafter the infusion of BrdUrd; in other words, of the infusion some
but not BrdUrd.
must
be less
than
of the first
mitotic
Hence,
figures
TG2 in some
Tumor
the
infusion
were
of IdUrd
of four tumors, and
BrdUrd,
taken
the
following
of Label
Most
onstrated
of
the
a fragmented
from
Resulting
labeled
nuclei
infusions In the
after
pattern,
indicating
that
biopsy
the
nuclei
was
Day
0 biopsy
after
being cells
(Table
indistinguishable which initially with
from
suggests labeled
the appearance
that these or underwent
fragmented
label
4) the LI in the first
(Fig.
biopsy
Of the labeled cells, label and in 26.5%
patient
L12 was
HN-25,
higher
In patient
to have
the
pearls
were
of Cell
Nuclei
in Necrotic
(Patient
HN-12),
present.
In
unlabeled
contained
the
nuclei
necrotic
were
noted
Nontumor
area,
labeled
(Fig.
12).
Cells.
Dividem-
four
second
biopsies.
Labeled
fragmented
mononuclear
leukocytes
were
has been
recognized
in than
HN-12
Tumor the most in general In their cycle
cell
growth
kinetics
important determinants (24) and of head and comprehensive
parameters
for
review 198
SCCs
of curability neck tumors Sasaki based
et a!. (6) mainly
as one
of human in particular on the
but
of cell
kinetics
in 5CC
in Table
5.
of
tumors (25).
reported
cell
measure-
in humans (12, we summarize
(21.7%),
in all
DISCUSSION
a new generation of cell kinetics studies To facilitate the discussion of our results,
Lii
cells
present
in the second biopsy the label was undiluted. In than
and
la-
73.5%
(39.4%)
In the
of necrosis
FCM. Later, nucleotides
and 29.1%
corn-
Foci. areas
ment of [3H]thymidine LI in vitro and duction of BrdUrd and other halogenated
25.9%
di-
(Fig.
labeled
in the
was
second biopsy. had fragmented
lOb).
divisions
not
patient
polyrnorphonuclear
had not divided
fewer
two
appeared
of one
labeled
of the label
cells
cells
in the other
1 lb).
(Fig.
cell
in the
biopsy
and
initially
IdUnd/
while
Debris
biopsy
pyknotic
beled cells had undergone one on more divisions between the first and second biopsies (Fig. 10). In contrast, in some cells the whole nucleus remained stained. The pattern of the label in these
the
nuclei
were
all cells
second
Labeled
Cell
second
of
all of the
pearls
phenomena
Repeated
in the
infusion
on in the
of Label from the Basal Layer of the Tumor to Its Central Cornifying Structures (Pearls). Two
Labeled
Dilution
19.4%
almost
cycle,
of cells
Day
HN-33,
versus
the
studied,
one division 25%
9.3%
after
tumors
In patient
BrdUrd
observed.
sions.
weeks
2
of the at least
12.9%.
the LI was
of patients HN-15,
of
cells
days
9 LI was
biopsy In patient
Migration
Island
of
7-14
first
second label.
vided.
second biopsy
Day
approximately
1 la).
in the Absence
the
through
in the
HN-33) contained pearl-like immediately after IdUrd/
Treatment In the second
P, tumor
of the tumors (Patients HN-25 and structures. In the biopsy obtained
3 h.
Cells
and
in two
pressed
Fate of Proliferating
(5).
la-
DNA
were
cells
had fragmented
the infusion,
Within
correlation
labeled
19.9%
Day
passed
than
stroma
i. a tumor
the proportion of cells with undiluted label (23.8%) was essentially the same as that in patient HN-12. In contrast, 93.8 and
the LI and T1,,, values. Labeled mitotic figures were not in any specimen. This suggests that the minimum beginning
r
Time
In 12 patients with SCC, full cell cycle parameters were calculated (in others either only IdUrd was infused or viable tumor tissue was present only in paraffin sections in which double
:i
cells within
7
t,’l
‘V
:1VVV..-
c_%
Labeled mononuclear parenchyma. x 400. Fig.
4ri
W#{225} ‘:r
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:
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.
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Cancer
the introstimulated 26, 27). the data
532
Cell Kinetics
of Head and Neck
Cancers
80
70 60 50 40 -J
30 20 10 0 3
5
7
9
11
13
15
17
field
represents
19
21
23
Field# Fig.
8
Variation
of LIs in adjacent
microscopic
fields
in high-grade
SCC
HN-6.
Each
Table
40
Sample code 30
0
LI minimum
LI maximum
20.2 55.9 16.2 15.7 27.7 26.4 22.5 29.4 35.1 18.7 27.6 24.6 14.5 16.2 26.6 26.6 19.4 16.9
19.2 19.2 13.7 6.5 25.2 17.3 14.8 22.5 28.2 9.2 22.6 20.8 12.6 7.1 19.9 18.8 17.8 16.5
22.1 80 17.2 32.2 30.6 29.5 26.2 34.1 55.2 27.6 33.1 28.3 16.4 40.2 30.5 33.3 21.2 17.5
32.8 14.1 18.4 32.3 29.1 2.5 3
22 10.7 7.1 27.2 16.7 0.6
42 19.6 25.4 58 41.4 4.8
24.8
23.7
26
HN-12
20
HN-14
a.
HN-lS”
1
HN-17’ HN-18 HN-l9 HN-21 HN-25 HN-28 HN-29 HN-31
10 p=0.004
0
HN-32’
0
10
30
20
40
Paraffin Fig. 9 Correlation tumor. One fragment one in paraffin.
between LIs counted in different areas of the same of tumor was embedded in plastic and the other
HN-33 HN-346 HN-37’ HN-39’ HN-40’ HN47C
HN-5 HNllC HN-22 HN35b
Proportion tion
of S-phase
other
cells
studies
occurring
hand,
wide
In this
varying
study
of these (LI
discriminate cells are
tumors
is why
the
that
cells
in S-phase
varies
range,
0-13.2%).
It
is only
application
other
tissue
cells,
of
of all FCM
head
S-phase
that
among
evident
results
and
that
neck
in an
other
LI or BrdUrd
only)
n.d.’ 51.6
15.5 nd. 25.9 22.4 19.9 29.4 33.5 16.7 25 21.7 13.2 15.2 24.5 26.6 nd. nd. 32.8 14.1 18.4 32.3 23.8 2.5 2.5 nd.
nd. , not done. I) The tumor was removed 3-3.5 h after the infusion (samples are not included in calculations of 1 and C Tumors labeled with IdUrd only. a
of BndUnd
cells
nonmalignant
these cells and tumor cells The proportion of tetnaploid 4%
the
not only
but
widely is
in most
a naturally On
of
and
frac-
(IdUrd
observations.
proportion
tumor
that to
in 5CC.
for our
stromal
of mononuclear
between tetnaploid.
ploidy)
the of
due
rates
The
than
partly
reasons
we found
proportions
studied
be
of proliferative
admixture
(LIs).
is higher
may
underestimates
the
Cells
study
This
be technical
FCM of
5).
range
may
First,
cells.
in our
(Table
there
because
age
of DNA-synthesizing
cells.
+ BrdUrd)
LI average
HN-4 HN-6 HN-7 HN-8
.
tumor
LIs in head and ne ek tumors
2
LI (IdUrd
.
r=0.85,
300-500
SCCs
inevitable
the
contains
timation
percent-
sions
cannot
which
unless (and
tumor higher
14).
That
undenes-
LI
5),
are
disaggregated
the use of in vitro than
may
in which
S-phase
when
data cells
tumor
cell
suspen-
(1 1, 18).
lower
This
to the
values
studied
Second,
the tumors FCM
(18).
of are
be
the
those case
S fractions
of
other
only
2.6%
labeling
obtained in Hemmer’s
were
groups, (Table
5).
the
in LI estimates
in vivo
studies
(7,
study
as well
(Table
lower
in comparison
(7)
significantly
with
results
from
median
Conversely,
10,
percentage
of
LIs reported
by
Clinical
Table
Cell kinetics
3
Patient
parameters
in patients neck
with SCC of head and
T
code
12.2
18.8
HN-7 HN-12 HN-14 HN-18
5.1 14.8 17.3 21.5
26.3 45.7 61.8 51.3
8.5
40.7
9.9 6.2 10.7 15.8 11.9 20.5
31.7 22.9 64.8 83.2 38.8 84.5
12.8 1.5
47.2 6.7
HN-19 HN-21 HN-25 HN-28 HN-29 HN-31 HN-33
Mean SEM
.d
.
.
T
.--. .-..
,,
.
,
which
used
in vivo
significantly higher. LI values may S-phase
cells
labeling
be affected
to the DNA
procedures
(10,
by the duration
label
in
.
.
-..
/
of
case
LIs
of
BrdUrd)
cells
is on
with
stained
with
average
mAb
2.0%
BrdUnd-specific
3D9
higher
antibody
(for
both
that
of cells
than
nucleotides
the
tumor
specimen
previous 4-6
studies
(2, 3, 1 1, 13,
16) biopsies
BndUrd injection. Many this period of time, producing
during resulting
in an overestimation
Wilson
et a!. (13)
tions
to obtain
between
more
the labeling
imen
therefore
realistic
and
when
divide cells cells. correc-
there
is a lag
of the spec-
heterogeneity
within
individual
cell kinetics may be another potential when we compared LIs of different embedded
in plastic
ment (Fig.
of Pathology), 9). Similarly,
cant
differences
different
Cell Cycle series (Table
source regions
laboratory)
and
with
respect
to
of error. However, of the same tumor in paraffin
the
from
cell
kinetic
the same
Parameters.
parameters
tumor. The duration
measured
in
methodology.
of the S-phase LIs
(Table
in 5CC
of the
An indirect
head
and
approach
neck
using
to approximate
a constant
of S-phase
counts mm
postinfusional (28).
a, Day 0 in one cell
the duration
availability
obtained
by
and
by Begg et a!. (2) T measurements
by
us
of
other
the average 10.8
=
This
h (see
is very
double-labeling
shorten S-phase duration (see, for example, Ref.
tumors
close
to
methodology 1 1; Table 5). using different
in SCC 18). The
in companduration of
(21-23).
is an excellent
reported
methodology
by
of Begg than
correlation
authors
et a!.
(12),
used
the Ti,,,, in our
study
(about
discrepancy
in biopsy sections as compared (see above). Nevertheless, both neck
actual were
by these
be explained
tumor assumed
11,
obtained
who
sured FCM
versus
of T. values 15)
This
may
reported
3,
is shorter
and
those
(2,
days)
(20).
cells,
21.6/2.0
and Bennett et a!. (Ref. made by other authors
solid
there
and
the head
every Given
SCCs reported here is also shorter than the T. by the same double-labeling method in human leu-
and While
us using
90-mm
words,
the S-phase.
by tumor
1 h X
and Methods”).
results
in
be
30- and
In other
enters
transition
would
1 in “Materials
S-phase determined
between
is 2.0%.
cell population
the
in our
double-labeling
difference
rate of cell cycle
duration
short 4 This time is slightly longer if one the drug which in humans is 10-20
is to use the difference 2). This
2% of the tumor
kemias of S-phase
range between 5.1 and 21.5 h, with a median of 12.1 h 5). This is the first attempt to assess the duration of
S-phase
11;1.
-
X 400.
methods suggested ison to other tumors
(Depart-
we found that LIs were highly correlated Wilson et a!. (13) also did not find signifiin
samples
(our
tumors
:
,
of the label after repeated cell divisions. label; b, Day 7 biopsy: fragmented label,
the label is undiluted.
Equation
for study. The
Fig. 10 Dilution biopsy: undiluted
hour
special
acquisition
,
.
at least
of S-phase
to make
LI values
of the tumor
taken
initially labeled cells two labeled daughter
of the proportion
proposed
,
:,
/‘
b
immediately
were
.
--:
be because of the movement of labeled cells from S and later because of the division of labeled cells. In
h after
‘
,
1.
-
,-.
after the administration of a DNA label. The longer the interval between the end of the infusion and the biopsy, the less accurate the LI will into G2-M
I
#{149}1-.
and
observations
obtain
/‘
stained
These
to
2).
IdUrd
demonstrate that prolonged infusions of halogenated may seriously affect the results of LI determinations. It is important
(Table
‘‘.
P
1;
(_
(‘.)
was
0.5 h4 when one of the halogenated nucleotides was injected and 1.5 h when both IdUrd and BndUrd were administered. In fact, the
,
1 1, 19) are
in our
-
- --..-.
a
of exposure
which
vivo,
.
10#{149}
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533
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/
.
T1,,,
HN-6
Cancer
authors
by higher
doubling
(4-5
LI values
the 2
days). mea-
to the values obtained by methods demonstrate very times
to be 1-6
which months
for 5CC and
longer
of
534
Cell
Kinetics
of Head
and
Table
Neck
Cancers
Labeling
4
patterns
in serial
samples
from
Tr,,
Sample
Histology
HN-12 HN-l5 HN-25 HN-33
SCC,M
ldUrd/BrdUrd
Infusions
SCC,M SCC,M SCC,W
IdUrd/BrdUrd IdUrd/BrdUrd IdUrd/BrdUrd
patients
who
(h)
did
not receive
therapy
between
two
biopsies Cells with fragmented/ unfragmentedl label (%)
Days between biopsies
LII
LI2
45.7
25.9
22.9 84.5
19.9 21.7 19.4
29.1 12.9 39.4 9.3
73.5/26.5 93.8/6.2 76.8/23.8 99.6/0.4
7 9 9 13
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,,‘ -
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1
:.,:
tween
and
T
b
was
should
go through
SCCs
45.7
human (19)
5CC. reported
did not find after
the
the average labeled
in biopsies
the
infusion,
and
3 h.
Fate above,
the labeled
there
actual
of
the
the minimum
of tumor
figures
TG,
Tumor doubling
Bneseiani
but
tumors
Cells.
between time.
in
et a!.
the T, The
we
obtained
90 mm
detected
labeled
3e h after
in these
of G2
as 8.1 h. Since
in samples infusion,
difference
volume
method,
approximately
Proliferating
is a large
duration
to be as long
IdUnd
removed
the
mitoses
TG,
mitotie
beginning
mitoses
the
Using
concerning
in
difference
the
start
is between
As
of 1.5
discordance
for here
difference
25%
that
the initial
infusion
of
only
these
DNA DNA
provide
tumor
of cells
from
appeared remained
of egress
kenatinizing
undiluted
of a tumor
that
is via
SCCs
direct
experimental
20).
(Fig.
studies
deto
time.
(Table
One pool.
4) approxi-
stopped
proliferating
suggested 7-9 these
cells
1 la).
Our
contribute
by the fact days
after
the
cells
did
not
differentiation.
proliferating
island
and neck
(19,
the proliferating
was
indicating
head
doubling
HN-33
This
To
of a
93% The
to have
synthesis.
of the
period.
concept
which
and
IdUnd/BndUnd,
the
in human
85 and
and
volume
9-day
introduced factor
HN-12
label
a
phenomena
T)
in the volume
the in
calculations.
three
of the cells
a tripling
the
(20)
theoretical
between
on the margin
on
samples
demonstrate
undergo mitosis. A second mode differentiated
cells
with
This
in serial
in tumors
completing
labeled
to be between
is the egress
after
the
9-fold
Steel
For mately
of the
week
based
phenomenon example,
time
In wellwere
One
located
week
later
labeled cells had migrated into the pearls (Fig. 1 lb). The phenomenon of migration of cells from undifferentiated regions of
mentioned
of a tumor
scnibed
if one
cycle
increase
estimated
performed
is evident
cell
The
in a single
cell loss factor.
was
time
4).
divisions
Similarly,
this
evidence the
three
should
doubling
(Table
h. Hence,
HN-25
studies
tumor
HN-12
tumor
spontaneous
Fig. 11 Migration of the label to cornified pearls. a, Day 0 biopsy: labeled cells are limited to the periphery of the tumor island; b, Day 7 biopsy: peripheral cells are unlabeled, the pearl contains compressed remnants of labeled cell nuclei. X 400.
actual
tumor
explain
reports
focus of necrosis X 400.
I’
-
tumor
few
.,t
#{149}.
of the tumor.
are
‘
4
#{149} #{149} ,.
--
12 Labeled fragments of cell nuclei in the 9 biopsy. V, viable tumor tissue; N, necrosis.
considers
There
,
..
o4’-’
-
.t,t(
v
.
..---.
Fig. Day
-
,
*:;. 4%,
.
#{149}
“
4
5,
-
,
.
and
a tumor
be-
beaning
to pearl
structures
transplantable
5CC
was (29).
previously
reported
in rats
Clinical
Table No. of patients
Cell
ki neties
[3H]thymidine in labeled mitoses [3H]thymidine in [3H]thymidine in Static cytometry [3H]thymidine in
16
data)
of SCC S-phase mean
Methods
5
198 (pooled 88 37
5
vivo, curve
an d neck
%,
vivo
vitro vitro
Research
T,
h,
mean,
(range)
2.5 (2.1-3.6)
23.5 (18-34)
1.9
16.1
Reference 19 10 6 32
11.2
1
(2.3-22.4) 40.4 (primary) 24.5 (metastatie) 17.2 2.6
FCM
68 33
FCM BrdUrd
1 68
Xenograft in nude mice BrdUrd in vivo, FCM
82
BrdUrd
FCM
535
(literature)
TJT,#{216}, days, mean, (range)
17.5 (11-36) 17.0 (2.0-30.0) 12.1 ± 3.9 18.0 (2-48)
28
in vitro,
of head
cells, (range)
Cancer
9 8 7
(0-23.2)
123
in
vivo,
FCM,
22
IdUrd and biopsy
in
8.0 (1.2-30.0)
6.2 (1.2-40.9) 5.7
13.7 (7.3-37.5) 9.9
15
5.2 (2.1-15.0) 2.0
9.3 (6.4-18.0) 12.8
3
(0.8-3.5)
(5.1-21.5)
6.8 (FCM)
vivo,
23.7
(7.4-S
1 .6)
13.
All
of these
It is
important
have
0
long loss
cell
cycle
and
provide
7-14
One
of labeled days after
whose label had the same 0), these cells presumably diluted nuclei nified
cells that were present in the the infusion of IdUrdfBrdUrd.
second I, cells
intensity as the label in the first biopsy (Day did not divide (arrested in G2); II, cells with
is time
(fragmented) label, which divided one or more times; III, labeled of necrotic cells; IV, labeled compressed nuclei within the conpearls. IUdR, iododeoxyunidine; BrdU, bromodeoxyurdine.
BndUnd to
interval
infusion
this
method
significant (12,
of
the
second found become elsewhere
mode
biopsy
is via
of the patient
in necrotic necrotic indicate
that the number toxic therapy.5
of egress foci, (Fig. that of these
cell
HN-12
indicating
For example,
death.
many that
labeled the
labeled
nuclei cells
in the
were had
originally
12). Additional studies to be published some tumors contain apoptotie cells and cells
increases
markedly
during
cyto-
only
single-
and
the
between
cells
would and
of
point
leave
label
of the infusion
during
the
(true period
16). to
to
and
with
BrdUnd
during
is a modifi-
the
interval).
the
circulating
which
were
+ cells
labeled
after
of time labeled
during
1 h
of the infusion
label.
which
only,
is 1 h (30
being
in S-phase
mm),
both
period
S-phase
For with
IdUnd
which Once
leaving
tech-
(30).
with
infusions,
a
G2-M
(including
labeled
two
(30
to
Quastlen
of the duration
LI)
permit
move
of cells
the
According
double-labeling
of cells of
of Begg
h) between
in our study
Cells,
30-mm
infusions
enough
proportion
independent
infusion
and
BrdUrd.
BrdUrd
cells
S-phase
all cells
T,1
of
clarification.
13,
long
used
ofthe
plus
availability
would
list
Proliferation
(4-6
(12,
be
labeled
the proportion
the
and
biopsy
the
points
be the same
this
to
and
require
interval
cells).
infusion
IdUrd,
the
the methodology
by Wimber
use
which
Cell
[‘4CJ-thymidine
the cells
the starting
infusion
5 Mundle, A. Raza, V. Kotelnikov, N. Wood, I. Coon, J. Hutchinson, W. Panje, D. Caldarelli, S. Taylor, K. Gniem, S. Shott, and H. Preisler,
and
IdUnd
must
double-labeled
mm of IdUnd with
we
add
IdUrd
which
using
the method and
for
Disturbances
between
of
BndUrd-labeled
described
of
IdUnd are
interval
[3H]-
study
of
the
Studies
of
in our
issues
the tumor
In contrast,
16).
cation
nique
the
fraction
(20).
Aspects
a prolonged
and
in Fig.
differentiation
responsible
pool
Infusions
between
recommend
and
tumor.
Vivo
of the tumor.
calculation A third
in
study
graphically
death
for the difference
the
are two methodological
et a!. (12) types 7-14
of
Methodological Using
and biopsy Fig. 13 Four biopsy obtained
basis
time
This
mechanisms
demonstrated
a further
Some
cell
proliferative
transition
Studies
that as
a tumor’s
11
are presented
stress
recognized
from
doubling
There
phenomena to
been
cell
actual
Day
33 2
1 1.2 (4.8-33.2)
IUdR/BrdU Day
17-19
4.7 (1.4-16.9)
14.9 (biopsy) 9.2 (2.1-28.0)
FCM
BrdUrd
3.1
12.7 (2.4-30.5)
FCM
BrdUrd in vivo, biopsy BrdUrd in s’ivo,
31
22-25
enter which
The
second
at the
starting
the cell cycle the
tumor
S
manuscript
in preparation.
is excised.
To
provide
important
that
zT
better
not
precision
be longer
for
than
the
T calculation,
the duration
of 02
it is (30).
536
Cell Kinetics
of Head
According
to the
between use
a T
The
longer
we
than
with
cells.
In this way
several
high,
exclusion This
which
demonstrated
layers
will
the tumor
results
cancers
treatment
are
the
struc-
of
cy-
In normal
epithelium
K10 (31).
from
cells
13.
from
Lewis,
animals
(29).
tumors
after
By definition
the potential
The
of the in vivo valuable
cell
to assess
labeled
the
rethese
doubling
opinion
tumors
data
described
time
that
that
require
above
also
IdUrd/BrdUrd
double-labeling
kinetics
allows
data, cells
cells
within
in serial
of
head
intensive suggest
that
the tumor,
be
biopsies.
We
thank
Erzsebet
Horvath
and
Jennifer
Hartman
for
skillful
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