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Effects
of Recombinant
Human
Hematopoietic
By Ulrich
progenitor
blood
and
cell
bone
levels
recombinant
human
ing-factor
(rG-CSF)
in a phase
number
of circulating
macrophage,
vated
two
with
days
after
of different
oral
remained
G
clinical
up
to
and
100-fold
often
of progenitor
The
abso-
after
remained
The
cells
in vitro
as the
native
bial
thereby
leading
infections.’5
receiving
cytotoxic
effects
G-CSF
is a proliferative
but
deplete
nisms
progenitor
cell
numbers
From
the
Cancer
of
Medical
the
when
C
(G-
progeni-
with
no capacity
for
in vivo might
thereunless
depletion.
To
other
monitor
mecha-
these
possi-
we analyzed
progenitor
cell levels in
and bone marrow
of patients
who were
Research
Unit,
Research,
The
Royal
Walter
and
Melbourne
Eliza
Hall
Hospital;
the
Clinical
Research
Programme,
Melbourne
Tumour
Biology
Ludwig Institute for Cancer Research. Royal Melbourne
Hospital;
and the Department
of Clinical Haematology
and OncolBranch,
ogy.
Royal
Children’s
Hospital.
Victoria.
Australia.
Submitted
April 25, /988; accepted August 22, 1988.
Supported
in part by the Deutsche
Forschungsgemeinschaft
(Grant Du 152/I-I),
the Institut
National
de la Sante et de la
Recherche
MEdicale,
the Carden Fellowship
Fund of the AntiCancer
Council
of Victoria,
Research
Council,
Canberra,
Address
reprint
Research
Unit.
Research,
Royal
requests
The
Walter
Melbourne
and the National
Australia.
to
and
Ulrich
Eliza
Health
and
D#{252}hrsen, MD,
Hall
Institute
Hospital
P0 3050.
article
were defrayed
of
Victoria,
Medical
Cancer
Medical
Austra-
ha.
The publication
costs
ofthis
charge payment.
This article
“advertisement”
in accordance
indicate
© 1988
by Grune
2074
& Stratton,
by page
must therefore
be hereby marked
with 18 U.S.C. section 1734 solely to
this fact.
0006-4971/88/7206/01
in part
Inc.
1O$3.OO/O
treatment.
30
treated
variable
after
and
did
In
then
treatThe
to stimu-
significantly
days
rG-CSF.
doses
exceeded
gig/kg/d
of rG-CSF
dur-
after
treat-
progenitor
of rG-CSF
or 60
nG-CSF
cells
nine
or
with
progenitor
change
& Stratton,
rG-CSF
decreased.
patients
and
with
Metcalf
granulocyte-macrophage
equaled
elsewhere.
on
slightly
not
but
by Grune
1988
Donald
marrow
rG-CSF
low
and
was
factor
rG-CSF
gig/kg/d.
the same
for granulocyte
cells
of G-CSF
ble effects
of G-CSF,
the peripheral
blood
Institute
stimulus
transit
for
that
by
very
in humans.’’9
these
are
Administration
compensate
shown
vitro
melphalan
resistance
to microwith cancer patients
have
can be observed
of bone
were
hormone.6
to enhanced
clinical
trials
chemotherapy
of G-CSF
ton cells,
self-renewal.
fore
Recent
in
was
patients
with
Investigations
in animals
have indicated
that G-CSF
is
capable
of inducing
a significant
increase
in total granulocytes both in healthy
animals8’#{176} and in animals
with congenital’ ‘ or drug-induced
defects
of the granulocyte-macrophage
system,’2’4
lation
Morstyn,
marrow
most
levels
and
mammalian
cells,7
and
the
bacterially
synthesized
recombinant
molecule
(rG-CSF)
found
to have the same
of activities
in
ment
CSF)
is a proliferative
stimulus
for precursors
of
neutrophils
in vitro.’2
Human
G-CSF
has been purified
from
various
sources,35
cDNAs
cloned
and expressed
in bacterial6
range
in the
but
dcrelative
factor
George
four
of progeni-
COLONY-stimulating
cells
ing
in periph-
frequency
tor
Factor
Patients
colony-stimulating
lineages
of therapy.
unchanged.
RANULOCYTE
The
Kannourakis,
responsiveness
of granulocyte-
megakaryocyte
cessation
types
trial.
in Cancer
ment
patients
stimulat-
cells
rG-CSF
the
frequency
blood
I/Il
George
in the
cancer
Colony-Stimulating
Cells
Boyd,
monitored
of 30
increase
treatment
Janis
granulocyte-colony
and
a dose-related
of
were
progenitor
erythroid.
showed
days
Villeval,
marrow
receiving
lute
Progenitor
D#{252}hrsen,Jean-Luc
Hematopoietic
peripheral
Granulocyte
cell
at or below
pretreatment
was
10
values
given.
Inc.
in a phase
1/11 clinical
trial
reported
‘ 7,18
MATERIALS
Criteriafor
Eligibility
and
AND
METHODS
Description
of Patients
The criteria
for patient
eligibility
were reported
in detail elsewhere.”’8
In brief, patients
had histologic evidence
of metastatic
cancer with a life expectancy of at least 2 months. No chemotherapy
or radiotherapy
had been administered
in the 6 weeks
preceding
treatment
with rG-CSF, and radiotherapy
was limited to less than
50% of the bone marrow. The type of tumor had to justify treatment
with
melphalan,
and
significant
nonmalignant
disease
such
as can-
diac, respiratory,
renal, or hepatic dysfunction
was excluded.
Pretreatment
hemoglobin
levels were greater than 10 g/dL, polymorphonuclear
cell counts greater
than 1,500/giL,
and platelet counts
greater than 100,000/giL.
Before initiation of treatment the patients
gave informed
consent.
The study was performed
at the Royal Melbourne
Hospital
under
the ethical guidelines of the National
Health and Medical Research
Council of Australia
and the Food and Drug Administration
of the
United
States
and
aforementioned
comprised
a total
requirements
(Table
Properties
of rG-CSF
rG-CSF
was supplied
174-amino
acid
protein
of 30 patients
by Amgen
who
fulfilled
the
I).
Corp.
Thousand
is a nonglycosylated
Oaks,
single-chain
CA. The
polypep-
tide with a molecular
weight of about I 8.6 kilodaltons
that is
produced by Escherichia
cohi and purified via a series of chromatographic
steps.6 The final product
was formulated
in an aqueous
buffer as a sterile solution at a concentration
of 0.25 mg/mL,
and
each batch was demonstrated
to be biologically
active and free from
pyrogens before release.
Study
Protocol
and Drug
Dosage
Each patient received two cycles of rG-CSF.
The first
cycle
consisted of a five-day treatment
period with rG-CSF alone followed
by a therapy-free
interval of three days’ duration.
The second cycle
aimed
at investigating
the effects
of rG-CSF
during
drug-induced
interval day. After a
single intravenous
(IV) injection of the alkylating
agent melphalan
(25 mg/m2),
nG-CSF treatment
continued
for nine days at the same
dose level as that used in the first cycle.
myelosuppnession
Administration
and
began
of rG-CSF
on the
fourth
was by either
the subcutaneous
(SC)
or IV route, and doses varied from 0.3 to 60 gig/kg body weight/d.
In
general,
groups of three patients were treated with each dose and
route of administration.
In individual
patients the dose of rG-CSF
was not varied. For doses of 0.3 and 1 gig/kg, the SC administration
Blood,
Vol 72, No 6 (December),
1988: pp 2074-208
1
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
G-CSF
TREATMENT
AND
PROGENITOR
CELLS
2075
Table
.
Daily
Dose
Age,
0.3 gig/kg
1
gig/kg
Sex
Origin
63,
M
Rectum
66,
M
66, F
58,
M
60, M
57.
3 gig/kg
Characteristics
and Dosage
of rG-CSF
SC Administration
of
rG-CSF
1 . Patient
M
and Type
Age, Sex
Origin
and Type
of Tumor
CT
RT
+
+
-
Pleura (undifferentiated)
Lung (undifferentiated
Ca)
+
-
+
-
Lung (squamous
+
+
68,
+
+
51, M
Lung (adeno-Ca)
-
-
-
-
64,
F
Kidney
-
-
-
-
66,
F
Ovary
+
+
-
-
60,
M
-
-
+
-
69,
F
Colon (adeno-Ca)
Bone marrow (multiple myeloma)
+
+
+
-
+
+
+
+
cell Ca)
Oropharynx (squamous cell Ca)
Lung (squamous cell Ca)
Lung (squamous
Unidentified
M
RT
+
74, M
4 1,M
CT
(adeno-Ca)
78. F
6 1, M
64, M
46.
IV Administration
of Tumor
cell Ca)
(adeno-Ca)
Rectum (adeno-Ca)
Colon (adeno-Ca)
Lung (small cell Ca)
Lymph
node (non-Hodgkin’s
lympho-
M
Lung (small
cell Ca)
(Ca)
(adeno-Ca)
ma)
10 gig/kg
64.
F
70. M
68, M
Abdomen
(carcinoid)
Lymph node (non-Hodgkin’s
ma)
Lung (adeno-Ca)
lympho-
-
-
62,
F
Stomach
+
-
56,
M
Unidentified
-
-
30 gig/kg
Abbreviations
: CT. previou
s chemotherapy;
RT, previous
radiothera
75, M
Parotid
Unidentified
F
of a single daily injection, and 10 gig/kg was administered
as a continuous
SC infusion over the entire treatment
period by using
Cormed infusion pumps (Medical
Specialities,
East Kew, Victoria,
Australia).
The intermediate
dose of 3 gig/kg
was administered
either as an SC bolus injection
or as a continuous
infusion.
For IV
administration,
the daily amount
of nG-CSF
was divided into two
doses administered
at 12-hour intervals in a volume of 100 mL of 5%
glucose as a 20- to 30-minute
infusion via a central venous line.
Doses administered
IV ranged from 1 to 60 gig/kg/d,
thus allowing a
comparison
with the SC administration
at the 1-, 3-, and 10-gig/kg
levels (Table 1).
Collection
Marrow
and Processing
ofBlood
(adeno-Ca)
(adeno-Ca)
66.
M
Bone marrow (multiple
Lung (adeno-Ca)
69.
M
Colon
45,
F
Unidentified
46,
M
Rectum (adeno-Ca)
(adeno-Ca)
(undifferentiated)
myeloma)
-
-
-
-
-
+
-
-
+
+
-
-
-
-
+
-
-
+
py; Ca, carcinoma.
consisted
Bone
(adeno-Ca)
62,
6 1,M
60 gig/kg
(adeno-Ca)
and
Samples
Peripheral
blood was obtained
on the day preceding
the first
treatment
cycle, after four days of rG-CSF
treatment,
two days after
the end of the first cycle, and on the last day of the second nG-CSF
course,
ie, nine days after the administration
of melphalan.
To
minimize
possible interference
of accessory cells with colony formation by progenitor
cells,#{176}
samples were fractionated
by density
gradient centrifugation
and removal of adherent cells before culture.
An aliquot of the blood sample was used to obtain a full blood count,
and the remaining
volume was subjected
to gradient
centnifugation
(30 minutes at 400 g) using Ficoll-Hypaque
(density, 1.077 g/mL;
Pharmacia,
mc, Uppsala,
Sweden).
This fractionation
step was
routinely
monitored
by absolute
cell counts
and cytocentnifuge
preparations
of cells from the interface and pellet that were stained
with May-Grunwald-Giemsa
and then typed morphologically.
The
interface mononuclear
cell fraction
was washed, suspended
in Dulbecco’s modified Eagle’s medium containing
10% fetal calf serum
(FCS),
and incubated
in 90-mm
plastic Petni dishes for one hour at
37#{176}C
to remove adherent cells.#{176}
The proportion of nonadherent
cells
was determined
for each specimen
from cell counts before and after
depletion of adherent
cells and the nonadhenent
cell fraction used for
progenitor
cell assays.
Bone
the day
days of
ent cells
marrow specimens were obtained
by sternal aspiration
on
before initiation
of the first nG-CSF
cycle and after four
treatment.
Gradient
centrifugation
and depletion
of adherwere performed
as described
for peripheral
blood cells.
Progenitor
Cell
progenitor
Assays
cell assays were performed
in 1-mL cultures
in
35-mm
plastic
Petri dishes.
Peripheral
blood cells were usually
cultured at 2 x i0 cells/dish
and bone marrow
cells at 5 x i0
cells/dish.
The cultures
were incubated
in a fully humidified
atmosphere of 5% CO2 in air at 37#{176}C
for up to 14 days.
Granulocyte-macrophage
and early erythnoid
progenitor
cells
were grown in agan cultures
prepared
by adding
1 vol of doublestrength
Iscove’s modified Dulbecco’s
medium and 1 vol of FCS to 2
vol of 0.6% agan.’ Gnanulocyte-macrophage
progenitor
cells were
stimulated
by the addition
to each dish of (a) 500 units of purified
rG-CSF (specific activity,
2 x i0 U/mg)
dissolved in a volume of
0.1 mL of saline, (b) 1,500 units of purified recombinant
granulocyte-macrophage
colony-stimulating
factor (nGM-CSF)
(specific
activity,
2 x i0 U/mg),
(c) a mixture of 500 units of rG-CSF
plus
1,500 units of rGM-CSF,
on (d) 0.1 mL of normal saline for the
detection of spontaneous
colony development.
(Fifty units pen milliliter
is defined
as the concentration
of a CSF stimulating
the
formation of half-maximal
numbers of colonies in conventional
agar
cultures of bone marrow
cells.’) Since the responsiveness
to GMCSF of bone marrow
cells from different
individuals
varied
widely
(see Results),
a higher concentration
ofGM-CSF
was used to ensure
maximal
stimulation
of progenitor
cells. For each stimulus,
cultures
were prepared
in quadruplicate
and duplicate
dishes scored routinely
after 5, 7, and 14 days of incubation
by using a dissection microscope
at 35 x magnification.
Clones
of three to 40 cells were scored as
clusters and clones of more than 40 cells as colonies.’ Cultures
were
then fixed with 2.5% glutaraldehyde,
transferred
to glass slides, and
stained with Luxol-fast
blue and hematoxylin
for differential
colony
counts.’
All
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2076
Early
erythroid
recombinant
progenitor
cells were stimulated
by 2 units of
(rEpo, supplied by Amgen) plus 0.1 mL
erythropoietin
of human placental
conditioned
medium
(HPCM)
to stimulate
the development
of maximal
numbers
of colonies.2’
Cultures
were scored after 14 days of incubation
and then fixed and
stained
for differential
colony counts
as before.
Discrimination
between pure erythroid
and mixed erythroid
colonies
was based on
of a preparation
nuclear
morphology.
Late erythroid
progenitor
cells (colony-forming
units, erythroid;
CFU-E)
were assayed in methylcellulose
cultures by using 2 U/mL
of rEpo.’ The cultures were scored after seven days of incubation
by
using an inverted microscope and CFU-E identified as hemoglobinized clones of eight cells or more.
Megakaryocytic
colonies were grown by a plasma clot technique
using
4 x i0
peripheral
blood
cells or 2 x i0
bone
marrow
cells
per
1-mL
dish.22 After 1 1 days of incubation
the cultures were dried,
labeled
with a monoclonal
mouse antibody
against the glycoprotein
lIb/Illa
complex (antibody
WM 18, kindly provided by Dr M.
Berndt,
Sydney,
Australia),23
and stained
with a fluoresceinconjugated
sheep
antimouse
immunoglobulin
antibody
(Silenus
Laboratories,
Hawthorn,
Victoria, Australia).
Megakanyocyte
cobnies were identified
under the fluorescence
microscope
as clones of
three
cells
or more
Calculation
with
intense
ofProgenitor
surface
Cell
labeling.
Numbers
per
In most of the data, levels of progenitor
number
per l0
nonadherent
Blood
Volume
cells are expressed
mononuclear
cells.
To calculate
as the
the rise
in absolute numbers of progenitor
cells induced by rG-CSF treatment (see Fig 4), the numbers of progenitor cells were corrected for
changes
cell
in total
mononuclear
differential
and
counts
nonadherent
cells determined
and
cells.
changes
With
the cell fractions
the
in the
on the basis of white
ratio
exception
between
of
one
adherent
patient
after
during
tamed
72%
cell numbers
samples
before
(one
tailed)
and after
was used
treatment
to compare
with rG-CSF.
Before
blood
therapy
cells
during
rG-CSF
py,
Treatment
consisted
clear
composed
cells
nating
to
30
two
to
g/kg/d
of
fraction
to
the
rG-CSF,
days
of rG-CSF
days
after
immature
the
blood
morphology
in this
100%]),
the
although
cell
before
during
dominant
fractionated
patients
the
therapy,
±
of therapy,
in
patients
rG-CSF.
and
posttreatment
and 84%
cells
from
observed
and
60 tg/kg/d
two
patients
with
of
of
of theratreatment,
contaminating
no consistent
influence
on
74%
(range,
19%
±
39%
to
values
the respective
81%
were
(50%
to 100%).
The
pretreat-
18%
±
(39%
to
fluctuations
in
of adherent
cells in samples
collected
at
from
the same
patient
did not generally
percentage
different
times
a two-
to threefold
difference,
was not correlated
Frequencies
ofBlood
with
and
any
variability
of rG-CSF
the dose
Early
(after
to 100%];
± 3.8%
[88%
proportion
of
progenitor
progenitors)
CSF
admin-
rGM-CSF
rose
(Fig
two
rG-CSF
(P
1). In most
days
after
<
day
mature
of
.01)
rG-
during
patients,
this effect
discontinuation
of
stimulated
by either
rG-CSF
of G-CSF-responsive
and GMcells both rose to a similar
extent
the levels
progenitor
therapy.
granulocyte,
With
all doses of rG-CSF
adminmacrophage,
granulocyte-macrophage,
CFCs
showed,
overall,
(Fig 2).
frequencies
three
significantly
In cultures
treatment.
or rGM-CSF,
CSF-responsive
of circulating
(CFCs)
(presumptively
less
to stimulation
by a mixture
with rG-CSF
demonstrable
eosinophil
Cells
The frequency
cells.
cells
responsive
and
colony
patients
formation
treated
with
1 . 5 per
In
of rG-CSF,
the
10
no significant
was monitored
change
with
3 or 10 tg/kg/d
mononuclear
nonadherent
therapy
to 8.5 during
rG-CSF
the cessation
of therapy.
The
SC-treated
receiving
patient
rG-CSF
frequency
of megakaryocyte
per
10 cells
rG-CSF
in addition
monocytes.
Progenitor
14 colony-forming
from
up
mononuclear
exhibited
days
end
(before
cells
of rG-CSF
SC or 30 g/kg/d
IV, respectively.
In the patient
receiving
the lower
SC dose,
circulating
megakaryocyte
CFCs
rose
5% contami-
[83%
96.8%
was
administered
samples
6.8%
was
15%
±
the
observed
four
the
In bone marrow,
with
from
lympho-
unchanged
a small
precursors
lymphocytelike
of
virtually
cells
ment
exceed
after
and
types
therapy
as compared
with 66% ± 24% (25% to
and 72% ± 23% (16%
to 100%) after treat-
Megakaryocyte
gradient
mononu-
injected
percentage
cessation
some
less than
patients
94.0%
with
fraction.
ment
100%)
of
Neutrophilic
cell
minority
had
numbers
and nonadherent
cells within
the
In peripheral
blood,
the propor-
adherent
of nonadherent
100%)
100%)
a
rG-CSF
between
mononuclear
of peripheral
of cells
and
from
remained
granulocytic
from
fraction
exclusively
In blood
granulocytes.
cells
four
almost
or monocyte
cytelike
the
rG-CSF,
with
only
conand
fraction
sampled
did not change
major
after
third
of progenitor
actual
rG-CSF.
the
days
doses,
the
unexpectedly
band forms,
the
7.0%;
±
two
rG-CSF
from
frequencies
to
with
93.3%
5.5%;
±
ratio
lower
collected
the
were
95.3% ± 3.7%),
with
mononuclear
cells.
tion
case,
therapy
treatment,
94.1%
the
with
of the high-density
cell
the density
gradient
granulocytes
in relative
from the interface
of the Ficoll
± 4.0%
(range,
87% to 100%)
of
eosinophilic
istered,
Samples
with
collected
of 95.8%
In this
significantly
and
ofCell
fraction
corrected
according
cells cultured.
The composition
from
the pellet
following
RESULTS
Fractionation
observed
cell
and after rG-CSF
therapy
to 75% mature
granulocytes,
metamyelocytes.
cells were
mononuclear
rG-CSF
t test for paired
progenitor
patient
treatment
was still
Analysis
The
as that
low-density
(see
from
treatment.
Statistical
composition
the
but
ET AL
istered.
the interface
were comparable
in
mononuclear
cell composition
before
and after treatment
with
rG-CSF.
The changes
in the absolute
numbers
of mature granulocytes and gnanulocyte-macrophage
progenitor
cells (see Fig 4) were
expressed
as the ratio between the absolute
numbers
per unit blood
volume
by comparing
samples from individual
patients
before and
Results),
same
DUHRSEN
Cultures
were 7, 66.5,
IV, treatment
(no samples
and 41.5.
induced
progenitor
were
cells
before
administration
and 9.5 after
respective
values in the other
analyzed
In the
a rise
patient
in the
4.5 to 26
cells
from
after
withdrawal
therapy).
stimulated
by
rEpo
plus
HPCM
consistently
showed
a comparable
rise in the number
of hemoglobinized
day
14 CFCs
following
the administration
of rG-CSF.
of
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
G-CSF
TREATMENT
AND
PROGENITOR
CELLS
2077
a’
a
a’
E
ci
0
C
U,
a,
a’
0
C
0
-a
ci
V
0
E
z
-101
23456
-101
Days
.
initiation
23456
-10
of G-CSF treatment
14 CFCs in peripheral
blood (closed
marrow
(open
symbols.
broken
lines) before,
during,
and after treatment
with different
doses of
rG-CSF.
Each point represents
mean values from duplicate
agar
cultures
stimulated
by 500 units of rG-CSF
plus 1 .500 units of
rGM-CSF.
Each panel contains
data from three
patients
(circles,
squares.
triangles)
treated
at the same
dose level of rG-CSF.
Asterisks
denote
patients
with
previous
chemotherapy
and/or
radiotherapy.
Fig
1
Frequency
after
symbols,
solid
of day
lines)
Differential
and
colony
counts
that
indicated
affected
both pure erythroid
(Fig 2). These latter
colonies
morphology
before
and after
the
increase
and mixed
erythroid
colonies
did not vary significantly
in
treatment
with rG-CSF
and
contained
predominantly
macrophage
cells. The
or eosinophilic
elements
in addition
to erythroid
characteristic
feature
of erythroid
progenitors
in
blood,
with
cultures
following
a higher
treatment
The
increased
crab blood
either
to
altered
proportion
did
with
levels
enhancing
effects
the latter
possibility,
pretreatment
and
change
than
major
in
cells
have been due
CFCs
or an
with
inhibitory
formation
in vitro.
experiments
were
posttreatment
in periph-
detected
rG-CSF
might
of circulating
accessory
mixing
blood
samples
and
scored
after
prepared
from
five
each
Clusters
cells.
days
of
alone.
by G-CSF
5 clones)
cells
the
ratio
between
in
fact
later
developed
into
day
14
lineage,
population
(day
blood
and
late
of
day
14 CFCs
and
day 5 clones
was 40.9%
± 28.4%
(analysis
of 168 cultures),
which suggests
that a significant
fraction
of the day 5 clones
only
a
myelocytes,
only 6.3%
±
3.9% (analysis
of 98
at day 5. In contrast,
in cultures
of
tion after
stimulated
incubation
at day 14 was
that of clusters
erythroid
did not deviate
of colonies
devel-
progenitor
and
by
from
colony
numbers
in the combined
cultures
significantly
from the sum of the numbers
in cultures
by promyelocytes
between
day 14 CFCs and day 5 clones
not changed
significantly
by treatment
To exclude
performed
formed
or
SC and mononuuntreated
healthy
with rG-CSF
the
Late
colonies
peripheral
cells
are
this parameter
can be used to assess the number
of
precursor
cells.24’25 In bone marrow
cultures
the frequency
cultures)
increase
patient treated
with 10 gig/kg/d
ofrG-CSF
clear
nonadherent
blood
cells
from
an
donor.
Before,
during,
and after treatment
oping
of G-CSF treatment
Fig 2.
Frequency
of different
types of day 14 CFCs in peripheral blood (closed
symbols,
solid lines) and bone marrow
(open
symbols,
broken lines) from two patients
before.
during. and after
treatment
with rG-CSF.
Circles.
data from a patient
treated
with
10 gig/kg/d
of rG-CSF
IV; squares.
data from a patient
treated
with
3 gig/kg/d
SC. Abbreviations:
G-CFC.
granulocyte
colonyforming
cells; GM. granulocyte-macrophage;
M. macrophage;
Eo.
eosinophil;
E. pure erythroid;
Mix. mixed erythroid.
G-. GM-. M-,
and Eo-CFCs
were enumerated
in cultures
stimulated
by 500 units
of rG-CSF
plus 1 .500 units of rGM-CSF.
and E- and Mix-CFCs
were
enumerated
in cultures
stimulated
by 2 units of rEpo plus I 0%
HPCM.
primarily
rG-CSF.
on colony
and
frequently
colonies
in the
of progenitor
of
less
of mixed
not
after therapy
with
increased
numbers
proportion
using
granulocytic
of marrow,
Days after initiation
bone
colony
seven
days
size,
cell
of incubation
size,
colonies.
and
were
(Fig
3). The
was particularly
presence
striking
ratio
hemoglobinizaused
tens to identify
late progenitor
cells (CFU-E).
Late granulocyte
and erythroid
progenitors
both
remarkable
increase
in numbers
following
rG-CSF
tration
CFU-E
The
in blood cultures
was
with rG-CSF.
In the
as parameshowed
a
adminis-
of significant
numbers
of
since this type of progenitor
cell was usually
undetectable
in the blood before treatment.
These
peripheral
blood
CFU-E
were
usually
larger
and
frequently
less intensely
hemogbobinized
than were the CFU-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
DUHRSEN
2078
100
ET AL
0
ca
30
a
10
‘-a’
2
03
.E
2
P
10
:
:
S
10
3
0#{149}3
Subcutaneous
1
administration
3
10
Intravenous
30
60
administration
Dose of G-CSF injected (ug/kg/day)
V
Dose-related
changes
in the ratio
(four days after
treatment
v pretreatment)
of blood progenitor
cells (top
panels) and blood neutrophils
(bottom
panels)
induced
by treatment
with
rG-CSF.
Open
circles.
patients
with
no history
of
previous
chemotherapy
and/or
radiotherapy;
closed
circles.
patients
with previous
chemotherapy
and/or
radiotherapy.
Left
panels. data from patients receiving
SC bolus injections
(0.3 to 3
gig/kg/d)
or continuous
SC infusions
of rG-CSF
(3 to 10 gig/kg/d).
Absolute numbers of progenitor
cells before rG-CSF
therapy
were
6.6 ± 4.3 x 1O’/L of blood (means and SD) and absolute numbers
of neutrophils.
6.1 ± 3.1 x 10’/L
of blood. Right panel, data from
patients
receiving
IV infusions.
Pretreatment
values for progenitor
cells were 9.8 ± 10.1 x 10’ and for neutrophils.
5.4 ± 3.0 x 10/L.
Progenitor
cells were
enumerated
in cultures
stimulated
by 500
units of rG-CSF
plus 1 .500 units of rGM-CSF
after
14 days of
incubation.
Fig
4.
rG-CSF
.5
:bg
E
z
1 0 1 2 3 6
Days
after
initiation
treatment
of G-CSF
Fig 3.
Frequency
of day 5 cluster-forming
cells (top panels)
and CFU-E (bottom
panels)
in peripheral
blood (closed
symbols.
solid lines) and bone marrow
(open symbols.
broken lines) before.
during.
and after treatment
with different
doses of rG-CSF.
Each
panel contains
data from three
patients
(circles.
squares.
trianglee) treated
at the same dose level of rG-CSF.
Asterisks
denote
patients
with previous
chemotherapy
and/or
radiotherapy.
contrast,
considerable
(P < .01).
not
E
developing
in
slightly
earlier
Dose-response
bone
marrow
maturational
relationship.
cultures,
thus
suggesting
a
stage.
The administration
of rG-
CSF
was followed
by a dose-dependent
increase
in the
numbers
of neutrophilic
granulocytes
in peripheral
blood.’7”8
We compared
the effect of individual
doses of rG-CSF
on
mature
neutrophils
with
a combination
of rG-CSF
elevate
related.
maximal
the
capacity
no further
with
rG-CSF
day
14 CFCs
increase
the average
was
8.2
of administration
progenitor
the rise
observed
cell
in
at higher
±
calculated
7.7
x
numbers
showed
neutrophils,
3- and
10-gig/kg
of
I I -fold
the
from
erythroid
was dose
of delivery,
of rG-CSF,
treatment
For the SC
blood.
total
blood
a similar
dose dependency
to
pronounced
increases
being
(P
<
.01).
In
of
and platelets
therapy.’7”8
initiation
therapy
of rG-CSF
numbers
colonies
(mainly
patients
treated
high
days
of rG-CSF
progenitor
progenitor
doses
did
cultures.
blood
cells
frequently
of clusters
granubocytic).
with
blood
megakaryocyte
in unstimulated
prepared
from
of low
four
in number
as their counterparts
lineage.
In contrast,
the
Colony
formation
lated
agar
cultures
tion
of circulating
after
cell numbers
increased
up
with 10 gig/kg/d
of rG-CSF
and
bers of mature
erythrocytes
significantly
during
rG-CSF
of
a
in neutrophil
rise
calculated
numbers
1 to 5 x 106/L.
Circulating
was
of circulating
CFCs
numbers
did
numbers
increase
to
Before
levels
an
of rG-CSF
in calculated
dose
higher,
ranged
relationship
and the lowest doses
efficient
in inducing
blood
progenitor
In patients
treated
by
number
104/L
the increase
mature
at the
IV doses.
exceed
therapy,
100-fold.
dose-response
of rG-CSF,
proved
increase
in the
While the maximal
stimulated
the levels
of mature
blood
granulocytes
Both
for the SC and the IV routes
effects were observed
at 10 gig/kg/d
with
route
that
clear-cut
showed the same increase
the granulocyte-macrophage
that on day 14 CFCs
and rGM-CSF.
4 demonstrates
Figure
no
observed
after IV infusion
IV administered
rG-CSF
(usually
In blood
of rG-CSF
showed
not
cells
cells
in
numchange
Unstimubefore
the
the forma-
macrophage)
cultures
to
or
from
SC or IV
and
some
(10 to
60 gig/kg/d)
the frequency
of spontaneously
developing
colonies
increased
five- to 20-fold
after therapy,
but again
granulocytic
colonies
were by far the most frequent
colony
type.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
G-CSF
TREATMENT
Frequencies
AND
ofBone
PROGENITOR
Marrow
CELLS
2079
Progenitor
Table
Cells
In contrast
to the consistent
changes
in the number
of
peripheral
blood CFCs, the behavior
of the marrow
progenimore
variable.
was associated
ton cells was
with rG-CSF
of
day
CFCs,
14
megakaryocyte
Concentrations
Numbers
which
affected
both
lineages
progenitor
cell
the
From
(Figs
.
Daily
1 to 3). Likewise,
Dose
of pretreatment
values
in two patients
receiving
10 or 30
of rG-CSF,
respectively.
There was no consistent
between
irradiation
and
bone
marrow
a history
CFCs
after
precursors
an increase
not change
In Vitro
treatment
(decrease
in frequency
noticeably
during
Responsiveness
in the
treatment
or
frequency
of
(Fig
in
3).
number
was more
in 74%
common
Spontaneous
bone marrow
treatment
with
ofProgenitor
Doses
Marrow
and After
Cells
Treatment
of rG-CSF
EC,, (U/mL)
rG-CSF
for
With
Posttreatment
Stimulation
rGM-CSF
Pretreatment
Posttreatment
198
0.3gig/kgSC
91
99
181
lgig/kglV
42
62
9
91
69
94
31
33
12
27
20
2
5
2
4
IV
18
Data on each
different
of
for
colony
cultures
and
did
rG-CSF.
Cells
3gig/kgSC
30 gig/kg
1). While
progenitor
cells tended to show
rG-CSF
therapy
as their
more
(61% of patients,
Fig
formation
in unstimulated
CFU-E
cluster
cytotoxic
reduction
rG-CSF
late granulocyte-macrophage
the same changes
during
immature
patients),
of prior
the observed
of Bone
Before
Stimulation
for
Pretreatment
gig/kg/d
correlation
in Cultures
Different
(U/mL)
With
of
rG-CSF
(EC,,)
and rGM-CSF
of Half-Maximal
to 30% or 67%
dropped
levels
With
of rG-CSF
Development
Six Patients
EC
granulocyte-
in Units
the
of Colonies
Obtained
In 70% of patients,
treatment
with a decrease
in the number
and erythroid
macrophage
2.
Stimulating
patient.
and rGM-CSF
6
line refer
Serial
were
to results
twofold
used
when
dilutions
to stimulate
using
marrow
agar cultures
bone marrow cells from patients
of treatment
with
Cultures
9
cells
known amounts
of
nonadherent
rG-CSF.
5
152
containing
before
stimulated
from
a
of rG-CSF
5 x
10
and after four days
by rG-CSF
were
scored
after 7 days and cultures stimulated
by rGM-CSF
after 14 days of
incubation. Fifty units per milliliter is the concentration of CSF required for
half-maximal stimulation of normal human marrow cells.
to rG-CSF
and rGM-CSF
To investigate
changes
in the
rG-CSF
cells
induced
to CSFs,
induce an increase
in the number
the expense
of the granulocytic
six patients
before
were stimulated
and after
in agar
G-CSF
induced
in experimental
whether
treatment
with
responsiveness
of progenitor
bone marrow
cells obtained
from
four days
of rG-CSF
treatment
cultures
with
rG-CSF
or rGM-CSF.
serial
twofold
dilutions
of known
amounts
The CSF concentrations
of
stimulating
the development
of half-maximal
numbers
of colonies
determined
graphically
(effective
concentration,
[EC50])
and
marrow
cells.
used
to characterize
the
Untreated
bone
marrow
cells
from
showed
little variation
in responsiveness
to
illustrated
by similar
EC50 values
before
(Table
2). In contrast,
responsiveness
to
widely
from
more
shallow
patient
different
rG-CSF,
rG-CSF
rGM-CSF
in general
to patient,
CSF
titration
curve.
In
were
cells
50%
tion’2”3
associated
individual
patients
which is
therapy
varied
with
occurred
response
(Table
curves
2). In
remained
a significant
cells to the
of
change
in the
CSFs had not
the dosetherapy.
addition,
the shape
of
uninfluenced
by rG-CSF
the
Nine
ofBlood
Progenitor
Myelosuppression
days
after
Cells
melphalan
0.3 to 3 gig rG-CSF/kg/d
During
treatment,
had
no detectable
patients
receiving
progenitors
in
equaled
before
or slightly
initiation
of the
those
exceeded
first
treatment
in the same
other
cycle
with
On the basis
the
administration
of the known
of G-CSF
actions
regulator
in vivo
would
or
the
initiation
by
cells
number
of circulating
of
in
cells
in most
patients,
progenitor
and,
the
accessory
administration
increase
of
the
a slight
reduction
in the frequency
of bone marrow
progeniton cells. The increase
in the absolute
numbers
of circulating
CFCs affected
all types and maturational
stages of progenitor cells
assayed
and was already
rG-CSF.
Although
subsets
in
peripheral
therapy.
Thus,
clones, between
onies,
and
colonies
the
the
frequency
relative
blood
the ratios
eosinophil
between
remained
interpretation
demonstrable
the frequency
in bone
frequency
were
marrow,
characterof progenitor
cell
after
retained
erythroid
and
pure
virtually
mixed
unchanged.
This
argues
that
increased
the
increase
in peripheral
further
investigation.
mechanisms
at low doses
of CFCs in
between
day 14 CFCs
and granulocyte-macrophage
in vitr”2’4’6’26
to
either
here,
the
pronounced
cells were simply
the consequence
CFCs from the marrow.
be expected
G-CSF
of other
hematopoietic
regulators
by
or some compensatory
homeostatic
mechanism.
The
of G-CSF
an
progenitor
after
G-CSF
administragranulocyte-macrophage
megakaryocyte
CFCs
and
rG-CSF.
DISCUSSION
showed
hematopoietic
production
the
patients
unexpectedly
of
pluripotent
stem cells.’2 Assuming
that the direct
actions
of
G-CSF
in vivo are not different
from
those
in vitro,
this
observation
suggests
the interaction
of G-CSF
with some
blood approached
istic features
of
the peripheral
blood.
In patients
receiving
10 gig rG-CSF/
kg/d
or higher,
progenitor
cells were detectable,
and with
doses of 30 and 60 gig rG-CSF/kg/d,
progenitor
cell numbers
number
in spleen
and bone marrow
that
not only
included
but also erythroid
and
of IV-injected
Frequencies
Drug-Induced
in mice
total
In the patients
described
rG-CSF
was followed
by a
patients,
however,
the EC50 values
both for rG-CSF
and rGM-CSF
were in the same range before and after the administration
rG-CSF,
which
indicates
that
responsiveness
of the progenitor
a
CFCs
in
granubocytes
at
pool.
While
major increases
in circulating
granulocytes
animals8”5
and patients
receiving
chemo-
investigations
increase
of the
responsiveness
therapy,’’9
of mature
progenitor
circulating
Possibilities
day 5
col-
erythroid
against
progenitor
of unselective
responsible
for
blood progenitor
rG-CSF
and
release
of
the rG-CSF-induced
cell numbers
require
include
the
release
of
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
DUHRSEN
2080
existing
marrow
progenitor
cells, increased
production
of
progenitor
cells from stem cells in the marrow
or spleen,
or a
combination
of these.
Information
on the physical
nature,
antigens,
and
in this context.
surface
useful
Following
reaches
peak
preliminary
treatment
rG-CSF
pearance
usually
duration
levels
data
status
of these
cells
would
be
in fractionated
marrow,
blood may not account
Successful
hematopoietic
ing
stem
cytotoxic
blood CFC numbers
decrease
of 1 week’s
that
cycling
that
eral
with
2 to 3 weeks
suggest
alkylating
that
after
treatment
high
can
Our
doses
of
nant
cells.3”36
of stem
therapy,
but
of hematopoietic
patients
and
The
this
needs
confirmation
course
significance
of
One
of control
analysis.
the
in
frequency
of
bone
to assess
because
of a lack
cell counts
and the possibility
of
of
dilution
of marrow
aspirates
by peripheral
blood.
sections
from
rG-CSF-treated
monkeys
have demhypercellularity,’4
thus suggesting
that part of the
decreased
frequency
present
study
increased
of
might
be
proportion
than
to a reduction
cells.
In
of
that
CFCs
artificial
more
was observed
and due simply
mature
absolute
in the
in the
to an
granulocytes
rather
numbers
of progenitor
some patients
after
rG-CSF
treatment
the
of CFCs in fractionated
blood actually
exceeded
at
least
frequency
for
and
major
cells
maneuver
stem
after
from
of the
cells
with
harvest
and
following
the infusion
peripheral
technique
blood.3’
is the
in the circulation.33’37
cells
the
before
period
reduced
derived
limitations
of plunipotent
stem
then injection
of donors
cells,
anesthesia
the neutropenic
progenitor
of the
general
even if the bone marrow
is
or infiltrated
with malig-
is possibly
behavior
decrease
CFCs
is difficult
information
on total marrow
marrow
a variable
Marrow
onstrated
by analysis
need
Furthermore,
transplantation
may be capable
of leading
to a more
rapid
reapof progenitor
cells in the circulation
after chemoa time
the
circumvent
marrow
dilution
by periphin these patients.
in patients
receiv-
treatment
has been reported
when using
from peripheral
blood,3#{176}’35a procedure
allow the collection
of stem cells
damaged
by previous
radiotherapy
initial
increase
therapy.27’29
with
that
agents,
show a very pronounced
followed
by a gradual
myeboablative
cells harvested
so variable
for decreases
reconstitution
ET AL
parallel
rG-CSF
scarcity
Should
the
that of progenitor
might be a useful
of peripheral
blood
cells
for
transplantation.
ACKNOWLEDGMENT
The
authors
J. Keech,
Sister
wish
to thank
Professor
and the staff
R. Fox,
of the Haematology
Melbourne
Timpano
for
providing
antiplatelet
excellent
Australia,
technical
for
of the
blood and
marrow
specimens
used in these studies;
Dns L. Souza and K. Alton
from Amgen for supplying
the nG-CSF and rEpo; L. Di Rago and J.
Royal
Hospital,
Dr L. Campbell,
Department
providing
assistance;
Dr
M.
Berndt
and Dns G. Johnson,
for many helpful discussions.
A.W.
antibodies;
gess, and W. Vainchenker
for
Bur-
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I . Metcalf
D: The Hemopoietic
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New York, Elsevier,
1984
2. Metcalf
D, Nicola
NA: Proliferative
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granulocyte
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poietic
cells. J Cell
3. Nicola
analogue
human
munine
4.
leukaemic
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I 16:198,
on normal
mouse
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granulocyte
1983
CG, Metcalf
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D: Identification
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1985
Begley
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5. Nomuna
Y, Asano
granulocyte
colony-stimulating
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Proc
M, Kubota N, Tamura
M, Ono
and characterization
of human
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(G-CSF).
EMBO J 5:871,
H, Imazeki
M, Ueyama
colony-stimulating
1985
82:1526,
I, Oheda
5: Purification
LM, Boone TC, Gabrilove
J, Lai PH, Zsebo KM,
DC, Chazin yR. Bruszewski
J, Lu H, Chen KK, Barendt
J, Platzer E, Moore MAS, Mertelsmann
R, Welte K: Recombinant
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7. Nagata 5, Tsuchiya
M, Asano 5, Kazino Y, Yamazaki
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0, Hirata Y, Kubota N, Oheda M, Nomuna H, Ono M:
Molecular
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6. Souza
Metcalf
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control of normal
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mice
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1988 72: 2074-2081
Effects of recombinant human granulocyte colony-stimulating factor on
hematopoietic progenitor cells in cancer patients
U Duhrsen, JL Villeval, J Boyd, G Kannourakis, G Morstyn and D Metcalf
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