Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1950-1956
Granulocyte Colony-Stimulating Factor Enhances Bone Marrow Stem Cell
Damage Caused by Repeated Administration of Cytotoxic Agents
By
Ronald van Os,
Simon Robinson,
Tara Sheridan,
John M.K. Mislow,
Donald Dawes, and
Peter M. Mauch
From the Joint Center for Radiation Therapy, Department of Radiation
Oncology, Harvard Medical School, Boston, MA.
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ABSTRACT |
Despite the increasing use of cytokines to circumvent the acute
dose-limiting myelotoxicity of cancer treatment, little is known about
the combined effects of cytotoxic agents and cytokines on the primitive
stem cells responsible for long-term hematopoiesis. In an experimental
model, we administered cytotoxic agents that have variable effects on
primitive stem cells in C57BL/6 (B6)-mice. Mice received six
every-other-week doses of cyclophosphamide (CY, 84 mg/kg),
VP-16 (24 mg/kg) + cisplatinum (2.4 mg/kg),
carboplatinum (50 mg/kg), chlorambucil (12 mg/kg), BCNU
(13.2 mg/kg), or TBI (80 cGy). Granulocyte
colony-stimulating factor (G-CSF; 250 µg/kg/day) was administered
subcutaneously twice daily on days 3 to 6 after each dose of the
cytotoxic agent. Comparison with animals receiving the cytotoxic agent
alone was made to investigate the effects of G-CSF on long-term
hematopoiesis. Hematopoiesis was measured 20 weeks after the last dose
of the cytotoxic agent by assessment of peripheral blood counts, marrow
cellularity, progenitor cell content (colony-forming units-spleen;
CFU-S), and primitive stem cell number (long-term repopulating ability
and day 28 and day 35 cobblestone area-forming cell [CAFC]
frequencies). Exposure to cytotoxic agents alone resulted in a
significant decrease in primitive stem cells (as measured by
repopulating units [RU] and day 28 and day 35 CAFC content) in
animals given carboplatinum, chlorambucil, BCNU, and TBI, but not in
animals treated with cyclophosphamide or VP-16 and cisplatinum. The
addition of G-CSF resulted in a significant decrease in stem cell
content when compared with no G-CSF administration in animals treated
with chlorambucil, BCNU, or TBI. Thus, G-CSF administered after
repeated exposure to cytotoxic agents, appeared to damage the primitive
stem cell compartment when used in combination with agents known to
damage primitive stem cells. These results, although obtained in an
experimental model, should raise concerns for the indiscriminate use of
G-CSF in the clinic.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE MAJORITY OF PATIENTS undergoing
autologous transplantation for lymphoma and multiple myeloma receive
stem cells previously exposed to cytotoxic agents. These patients are
at risk for impaired hematopoiesis. The risk appears to correlate with
the intensity and number of cycles of prior multiagent
chemotherapy.1-3 The greater the exposure to cytotoxic
agents, the higher the risk.
There has been increasing use of cytokines to circumvent the acute
dose-limiting myelotoxicity of many cancer treatments and bone marrow
transplantation (BMT).4,5 However, little is known about
the effects of cytokines on the primitive stem cells, which provide for
long-term hematopoietic support. In theory, the use of cytokines could
be either beneficial or detrimental. Excessive stimulation of primitive
stem cell proliferation by cytokines may lead to loss of primitive stem
cells and premature bone marrow failure (as with kit-ligand [KL]
given before and after 5-fluorouracil [5-FU]6,7), whereas
selective stimulation of later progenitors may have no effect, or even
a protective effect on primitive stem cells. A previous study in mice
has suggested that administration of granulocyte-macrophage
colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating
factor (G-CSF) to speed recovery from repeated doses of high dose
cyclophosphamide (350 mg/kg) appears to damage primitive stem
cells.8 This study demonstrated that primitive stem cell
function was impaired in groups given G-CSF or GM-CSF when assessed by
serial transplantation into lethally irradiated recipients 7 weeks
after the last cycle of cyclophosphamide.
The current experiments were designed to further study the effect of
G-CSF on hematopoietic stem cell populations after repeated drug
exposure and to explore potential mechanisms of cytokine-induced primitive stem cell damage. A number of different cytotoxic agents were
evaluated to determine whether the effects of G-CSF were independent of
the direct effect of cytotoxic agents on the marrow.
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MATERIALS AND METHODS |
Mice.
Male C57BL/6J (B6)-mice purchased from Jackson Laboratories (Bar
Harbor, ME) were used as recipients of cytotoxic agents and cytokines
and for competitive repopulation studies. Male congenic C57BL/6J-Gpi-1a/Gpi-1a (B6-Gpi-1a)
mice, purchased from Jackson Laboratories, were used as a source of
normal untreated marrow in the competitive repopulation assays.
Cytotoxic agent studies.
Experiments were designed to expose groups of 3-month old male B6-mice
to six every-other-week intraperitoneal (IP) doses of each cytotoxic
agent (in a total volume of 0.2 mL/mouse). rhuG-CSF (Amgen Inc,
Thousand Oaks, CA) was administered twice daily on days 3 to 6 after
cytotoxic agent administration as shown in
Fig 1. The dose of each cytotoxic agent was
determined as the amount which resulted in a fractional day 8 colony-forming units-spleen (CFU-S) survival of 0.37 (Do) at 24 hours
after a single IP administration.9 This equivalent endpoint
for each agent was selected because the effects on progenitor cells
appears to account for the dose-limiting toxicity of cytotoxic agents
in the clinic.
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| Fig 1.
Design of repeated cytotoxic agent and G-CSF
administration. Mice were given six 2-week cycles of cytotoxic agents,
half of the mice received G-CSF twice daily on days 3 through 6 at 250 µg/kg/day. No treatment was given on days 7 through 14.
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The cytotoxic agents used were cyclophosphamide (Bristol Myers,
Princeton, NJ; 84 mg/kg), VP-16 (etoposide; Bristol Myers; 24 mg/kg)
combined with cisplatinum (Bristol Myers; 2.4 mg/kg), chlorambucil
(Sigma, St Louis, MO; 12 mg/kg), carboplatinum (Sigma; 50 mg/kg), BCNU
(1,3-bis(2-chloroethyl)-1-nitrosourea; Bristol Myers; 13.2 mg/kg), and
total body irradiation (TBI; 80 cGy given at a dose-rate of 0.93 Gy/minute from a 137Cs-source (Gamma Cell 40, Atomic Energy
of Canada, Ottawa, Canada). Saline was used in control mice.
Cyclophosphamide was chosen because of its limited effect on primitive
stem cells at conventional doses. Less is known about the effects of
VP-16 and cisplatinum on primitive stem cells; this combination was
selected because of its common use in the clinic. Carboplatinum and
chlorambucil have intermediate effects on primitive stem cells; BCNU
and TBI were selected because these agents are known to permanently
damage the stem cell compartment.10-12 Because doses of the
cytotoxic agents were chosen to have mild acute effects on the
hematopoietic system, no severe neutropenia was observed as a result of
these treatments. Hence, very few deaths occurred before the time of scheduled sacrifice (20 weeks after the last dose of each cytotoxic agent). Only three of 194 mice died after completion of six cycles, but
before 20 weeks (one mouse treated with BCNU alone, one with BCNU+G-CSF, and one mouse treated with chlorambucil
[CAM] alone). In addition, no differences were found in
white blood cell (WBC) counts at the time of the next cytotoxic agent
administration to indicate that there was no ongoing neutropenia (data
not shown). The objective of this study was to compare G-CSF effects
under conditions of stem cell damage without acute drug-related
lethality.
Half of the mice were treated with rhuG-CSF (250 µg/kg/day,
administered in two equal doses, subcutaneously, for 4 days 8 to 10 hours apart, starting 2 days after each cytotoxic agent). The rhG-CSF
doses used in mice are usually up to 50-fold higher than in humans, at
least in part due to limited cross-species reactivity. Studies in mice
have shown that 20-fold higher dose of G-CSF (when compared with human
studies) given for up to 14 days are required for enhanced neutrophil
recovery after 5-FU13,14 or TBI.15
After the sixth dose of each cytotoxic agent, animals were rested to
allow recovery of peripheral blood counts before testing for
hematopoietic reserve. Although full recovery of counts was anticipated
at approximately 1 month after administration of each cytotoxic agent,
we chose a longer interval for assessment of hematopoietic stem cell
reserve to assess for permanent damage. Previous murine studies have
shown that after BMT it can take up to 4 months for stable
hematopoiesis to occur.16,17 Therefore, we chose a time of
20 weeks after administration of the last cytotoxic agent to analyze
animals for WBC counts, marrow cellularity, marrow progenitor cell
content (day 8 CFU-S content), and primitive stem cell content
(long-term competitive repopulating ability [LTRA] in vivo and day 28 and day 35 cobblestone area-forming cell [CAFC] content in vitro).
Using these endpoints we could measure if there was permanent damage to
the marrow.
Competitive repopulation in vivo.
The competitive repopulation (CR) assay measures the long-term
repopulating ability of a test cell population relative to normal bone
marrow cells in vivo.18 For determination of CR, varying
numbers of bone marrow or blood test cells (5 × 105
to 4 × 106) from B6-Gpi-1b mice were
mixed with a constant number (5 × 105 to 1 × 106) of control B6-Gpi-1a marrow cells. The
mixtures were injected into groups of five to six lethally irradiated
B6-Gpi-1b recipients. Absence of endogenous marrow
repopulation was determined by injecting one group (Gpi-1b)
with control cells only (Gpi-1a). Recipients were killed at
6 months after BMT and the ratios of test (Gpi-1b) to
control (Gpi-1a) cells determined by electrophoresis of
peripheral blood erythrocytes.19 The number of repopulating
units (RU), a measure of long-term repopulating cells, was calculated
according to the formula:
where
C is the number of RU in control (Gpi-1a) marrow and 1 RU
is defined as the repopulating ability of 105 normal bone
marrow cells.20
CAFC assay in vitro.
In vitro determination of hematopoietic stem and progenitor cell
frequencies was performed by limiting dilution analysis (LDA) of CAFC
in microcultures according to methods previously
described,21-23 with some modifications.7
Cultures were scored at days 28 and 35 by scanning each well under an
inverted microscope for the presence of cobblestone areas (CA). CA are
colonies of immature hematopoietic cells (at least six cells per
colony) residing within a preestablished FBMD-1 (provided
by Dr Steve Neben, Genetics Institute, Cambridge, MA) stromal layer.
The proportion of negative wells at each dilution was used in a
Poisson-based LDA calculation to determine the CAFC
frequency.23,24 It has been reported that in vitro CAFC
(day 28 or day 35) show considerable overlap in function with in vivo
repopulating cells, but the precise relation between these subsets is
not known.22,23,25 In the current experiments, we chose to
use both the in vivo and in vitro assays.
Statistics.
To test differences between treatment groups for statistical
significance, P values were calculated with the Student's
t test assuming unequal variances of the two variables. The
Poisson-based LDA calculation for CAFC frequencies also provides a
95%, 99%, and 99.9% confidence interval.23,24 These
intervals were used to determine significant differences as P < .05 (P > .01), P < .01 (P > .001) and
P < .001, respectively. Cytotoxic agent-treated mice were compared with saline-treated controls and G-CSF-treated mice
were compared with mice receiving the cytotoxic agent alone.
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RESULTS |
Blood and marrow counts and progenitor cell and stem cell content in
mice previously exposed to cytotoxic agents.
B6 mice that received six cycles of cyclophosphamide,
VP-16+cisplatinum, carboplatinum, chlorambucil, BCNU, TBI, or saline (controls) were killed at 20 weeks after completion of the cytotoxic agent administration for assessment of long-term hematopoietic parameters. Results of WBC, bone marrow cellularity (BMC), and CFU-S
content per hind limb (HL) are listed in
Table 1. Stem cell numbers (CAFC-28/HL,
CAFC-35/HL, and RU/HL) are shown in Table
2. There was no difference in long-term hematopoiesis in mice receiving
cyclophosphamide or VP-16 and cisplatinum compared with saline
controls.
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Table 1.
Hematological Parameters and Hematopoietic Progenitor
Numbers at 20 Weeks After Completion of a Six-Cycle Administration
of Cytotoxic Agents
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Table 2.
Hematopoietic Stem Cell Numbers at 20 Weeks After
Completion of a Six-Cycle Administration of Cytotoxic Agents
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Carboplatinum- and chlorambucil-treated mice had moderate decreases in
long-term hematopoietic stem cell reserve as demonstrated by decreases
in marrow cellularity (carboplatinum, P = .04), CFU-S/HL (carboplatinum, P = .03), CAFC-28/HL (carboplatinum, P < .05, and chlorambucil: P < .05), CAFC-35/HL
(carboplatinum, P < .05, and chlorambucil, P < .05), and RU/HL (chlorambucil, P < .01) when measured 5 months after the last dose of carboplatinum or chlorambucil.
BCNU- and TBI-treated mice had the greatest decrease in long-term
hematopoietic stem cell reserve as demonstrated by decreases in nearly
all hematopoietic parameters including WBC (TBI, P = .03),
marrow cellularity (BCNU, P = .02), CFU-S/HL (BCNU, P < .01, and TBI, P < .01), CAFC-35/HL (BCNU, P < .01), and RU/HL (BCNU, P < .01, and TBI, P < .01)
when measured 5 months after the last dose of BCNU or TBI. Animals
showed decreases in primitive stem cell content before manifesting
decreases in blood or marrow cellularity.
Blood and marrow counts and progenitor cell and stem cell numbers in
mice previously exposed to cytotoxic agents: The effect of G-CSF
administration.
The addition of G-CSF to control, cyclophosphamide-treated, VP-16 + cisplatinum-treated, and carboplatinum-treated mice had only a very
modest effect on long-term hematopoietic stem cell reserve (see
Tables 3, 4,
and 5). G-CSF administration did not result in a
significant decrease in progenitor or primitive stem cell content
compared with the cytotoxic agent administration without G-CSF.
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Table 3.
Hematological Parameters and Hematopoietic Progenitor
Numbers at 20 Weeks After Completion of a Six-Cycle Administration
of Cytotoxic Agents. Effect of G-CSF
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Table 4.
Hematological Parameters and Hematopoietic Progenitor
Numbers at 20 Weeks After Completion of a Six-Cycle Administration
of Cytotoxic Agents. Effect of G-CSF
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Table 5.
Hematopoietic Stem Cell Numbers at 20 Weeks After
Completion of a Six-Cycle Administration of Cytotoxic Agents. Effect of
G-CSF
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However, G-CSF administration appeared to adversely effect marrow stem
cell reserve when administered after chlorambucil, BCNU, or TBI,
cytotoxic agents that by themselves have a detrimental effect on marrow
reserve. G-CSF + chlorambucil administration resulted in a loss of
long-term hematopoietic stem cell reserve as demonstrated by decreases
in CAFC-28/HL (P < .001) and RU/HL (P < .01)
compared with chlorambucil alone. An even greater loss in stem cell
reserve was seen with G-CSF administered after BCNU or TBI, as
demonstrated by decreases in marrow cellularity (TBI, P = .05),
CFU-S/HL (BCNU, P < .01, and TBI, P < .01),
CAFC-28/HL, (BCNU, P < .001, and TBI, P < .001), CAFC-35/HL (TBI, P < .05), and RU/HL (BCNU, P < .01, TBI, P = .01) compared with BCNU or TBI alone.
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DISCUSSION |
G-CSF increasingly has been used to reduce the severity of
treatment-related neutropenia after high dose chemotherapy or
BMT26,27; reviewed by Welte et al.28 G-CSF is
thought to act mainly on granuloid precursors; little is known about
the long-term effects of G-CSF on the more primitive hematopoietic stem
cells.
In one of the few reports evaluating the effects of G-CSF on
hematopoietic stem cells, Hornung and Longo8 demonstrated enhancement of primitive stem cell damage by the addition of G-CSF to
high dose cyclophosphamide (350 mg/kg) given in six every other weekly
cycles. Seven weeks after the last dose of cyclophosphamide, marrow was
obtained for serial transplantation into lethally irradiated recipients. After three serial transplants, mice given G-CSF (or GM-CSF) and high dose cyclophosphamide demonstrated long-term marrow
stem cell damage by deficiency in generating hematopoietic progenitors
and CFU-S, and in marrow repopulating ability compared with mice
treated with cyclophosphamide alone. In the current study,
cyclophosphamide administered at lower doses (84 mg/kg) with or without
G-CSF did not appear to damage primitive stem cells. This is in
agreement with previous reports that cyclophosphamide has a greater
detrimental effect on progenitor cells than on the more primitive stem
cells.9,29-31 The fourfold higher doses used in the Hornung
study resulted in high animal mortality when repeated in our laboratory
(unpublished data) suggesting that cyclophosphamide may result in a
loss of marrow reserve only at doses that cause life-threatening
myelosuppression.
Rather than studying the effect of dose intensification, we varied the
use of G-CSF after administration of cytotoxic agents given at a
constant dose. In this way we could study the direct effect of the
G-CSF rather than the combined effect of the dose intensification
combined with the G-CSF. The agents and doses chosen were equally toxic
to the CFU-S day 8 population, but varied in their effects on the more
primitive stem cells. As progenitor and peripheral blood numbers are
dose-limiting for the clinical use of cytotoxic agents, while primitive
stem cell numbers appear important for long-term marrow reserve, the
experiments were designed to model clinical practice while testing for
the long-term consequences of treatment. Progenitor and stem cell
numbers were compared at 20 weeks after the end of six consecutive
2-week cycles of cytotoxic agents to provide a long-term measure of
marrow damage.
This study demonstrates that G-CSF administered after multiple doses of
cytotoxic agents appears to impair long-term hematopoiesis and marrow
stem cell reserve. G-CSF had no significant adverse effect when
administered to control, cyclophosphamide or VP-16, and
cisplatinum-treated mice; animals that had demonstrated little detriment in long-term repopulating ability without the use of G-CSF.
G-CSF added to carboplatinum also showed no additional adverse effect
despite the moderate stem cell deficit seen with this cytotoxic agent
alone. However, when G-CSF was given after chlorambucil, BCNU, or TBI,
all agents known to damage primitive stem cells, additional loss in
primitive stem cell capacity was seen. These effects were most apparent
at the primitive stem cell level (CAFC-28/35 and RU), indicating a
permanent loss in marrow stem cell reserve with the addition of G-CSF
to each cytotoxic agent. This deficit also led to reduced progenitor
cell numbers, but had less effect on WBC and marrow counts suggesting
that peripheral blood and marrow cellularity were maintained even when
there was long-term loss of marrow reserve. The results confirm that
normal peripheral counts are a poor indicator of marrow stem cell
reserve after exposure to cytotoxic agents.32
There is evidence that although the marrow stem cell compartment has
considerable reserve potential, it is limited in proliferative and
self-renewal capacity. Over 30 years ago, there was data to suggest
that stem cells have limited doubling capacity.33,34 Experimentally, limited self-renewal and proliferative capacity of stem
cells has been demonstrated through serial transplantation of marrow
into lethally irradiated recipients35-37; through exposure
of marrow to radiation,38,39 to combinations of cytokines
and cytotoxic agents,6,8 or to cytotoxic agents that damage
early stem cells40-43 and through the ability of
proliferative stress to considerably reduce marrow
reserve.32,39,44 Based on this evidence, the most likely
mechanism for G-CSF-induced damage of primitive stem cells after
multiple doses of cytotoxic agents is the increased proliferation of
stem cells in response to G-CSF at the expense of self-renewal.
G-CSF-driven differentiation of progenitor cells into mature granuloid
lineages would leave the subsequent recovery of the precursor pool to
be recruited from primitive stem cells. In the current experiments,
where the cytotoxic agent dose was calculated to be equally toxic to
the progenitor cell pool (CFU-S), the adverse effect of G-CSF was seen
only in animals with cytotoxic agent-induced damage to primitive stem
cells. This suggests that mice with limited marrow reserve are the most
susceptible to the repeated use of G-CSF; this appears to result from
increased proliferative stress on an already damaged primitive stem
cell compartment.
Other possible mechanisms for G-CSF-induced stem cell damage include:
(1) G-CSF may damage primitive stem cells directly. This appears
unlikely because when G-CSF was combined with cyclophosphamide, VP-16+cisplatinum or saline, no permanent reduction in stem cell numbers was observed compared with the use of these agents without G-CSF. (2) G-CSF may cause damage to stromal cells leading to a
reduction in the supportive capacity of the hematopoietic
microenvironment, or it may reduce adherence of stem cells to the
stroma leading to their release into the circulation. Migration of stem
cells from the marrow to the peripheral blood and/or spleen by
the combined effects of cytotoxic drugs and G-CSF45 would
have affected our primitive stem cell measurements, which were
restricted to the bone marrow. However, 20 weeks after a mobilization
protocol, blood and marrow stem cell levels will most certainly have
returned to normal levels. It has been shown that marrow stem cell
numbers are normal 2 to 7 weeks after mobilization protocols including
G-CSF in mice or baboons.45-47 (3) G-CSF may activate the
cycling of primitive stem cells making them more susceptible to the
effects of a subsequent cytotoxic agent exposure. This appears to occur
with cytotoxic agents that affect cycling cells, such as the
combination of 5-FU and kit-ligand.6,7,48,49 There is less
support for this possible mechanism in the current study because the
drugs that showed the largest effects on stem cells (BCNU, TBI) are not
cell cycle-specific.
Additional work is needed to further characterize the mechanisms for
and the circumstances under which cytokines such as G-CSF (cytokine
dose, number and duration of cycles) damage primitive hematopoietic
stem cells. However, data from these experiments suggest that G-CSF,
when administered after multiple courses of a cytotoxic agent, may
damage marrow self-renewal capacity. This damage appears to occur under
circumstances where the self-renewal capacity of the marrow is already
compromised.
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FOOTNOTES |
Submitted January 5, 1998;
accepted April 17, 1998.
Supported by the Grant No. RO1 CA 10941-26 and P50 HL54785-01 from the
National Institutes of Health, Bethesda, MD.
Address reprint requests to Ronald van Os, PhD, Joint Center for
Radiation Therapy, Department of Radiation Oncology, Harvard Medical
School, 330 Brookline Ave, Boston, MA 02215; email:
RVANOS{at}speedy.jcrt.harvard.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors would like to thank Dr Samuel Hellman (A.N. Pritzker
Distinguished Professor, University of Chicago) for critically reviewing the manuscript.
| |
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Abstract
Introduction
Abstract
