640
Effect of
peripheral-blood progenitor cells mobilised by filgrastim
(G-CSF) on platelet recovery after high-dose
chemotherapy
haemopoietic growth factor granulocyte
colony-stimulating factor (G-CSF; filgrastim)
substantially shortens the period of severe
neutropenia that follows high-dose chemotherapy
and autologous bone-marrow infusion by
stimulating granulopoiesis. Filgrastim also increases
numbers of circulating progenitor cells. We have
studied the ability of filgrastim to mobilise peripheralblood progenitor cells and assessed their efficacy
when infused after chemotherapy on recovery of
neutrophil and platelet counts.
17 patients with non-myeloid malignant disorders
received filgrastim (12 µg/kg daily for 6 days) by
The
follows
marrow
high-dose chemotherapy and autologous boneinfusion can be substantially shortened by
with G-CSF .3GM-CSF is also beneficia1.5-7
However, neither growth factor affects the duration of
severe thrombocytopenia. This feature is consistent with
their lack of action on megakaryocyte colonies in vitro and
on platelet production in vivo.1,2
In early studies, an unexpected finding was the high
numbers of circulating progenitor cells in patients who
received G-CSF or GM-CSF.8,9 We have examined the
ability of G-CSF (filgrastim) to mobilise peripheral-blood
progenitor cells; we have also assessed the feasibility of
collection and efficacy of these cells when infused after
treatment
high-dose chemotherapy.
continuous subcutaneous infusion. Numbers of
granulocyte-macrophage progenitors
in
blood increased a median of 58-fold over
pretreatment values, and numbers of erythroid
progenitors increased a median of 24-fold. Three
leucapheresis procedures collected a mean total of
33 (SEM 5·7) × 104 granulocyte-macrophage
progenitors per kg body weight. After high-dose
chemotherapy in 14 of the patients (busulphan and
cyclophosphamide), these cells were used to
augment autologous bone-marrow rescue and posttransplant filgrastim treatment. Platelet recovery was
in these patients than in controls
who received the same treatment apart from the
infusion of peripheral-blood progenitors; the platelet
count reached 50 × 109/l a median of 15 days after
infusion of haemopoietic cells in the study patients
compared with 39 days in controls (p = 0·0006). The
accelerated neutrophil recovery associated with
filgrastim treatment after chemotherapy was
maintained.
This method may be widely applicable to aid both
neutrophil and platelet recovery after high-dose
chemotherapy; it will allow investigation of
significantly faster
peripheral-blood progenitor-cell allotransplantation.
Introduction
The
Patients and methods
peripheral
haemopoietic growth factors, granulocyte colonystimulating factor (G-CSF) and granulocyte-macrophage
colony-stimulating factor (GM-CSF), were first identified
by their ability to stimulate the clonal growth of
haemopoietic progenitor cells in vitro.The purification and
subsequent molecular cloning of these factors has allowed
study of their effects in various clinical settings.2 The
recombinant human G-CSF and GM-CSF produced in
Escherichia coli now have the approved names filgrastim and
ecogramostim. The period of severe neutropenia that
Eligible patients were those with non-myeloid malignant
disorders of poor prognosis; some were in remission after initial
chemotherapy but had poor prognostic features (acute
lymphoblastic leukaemia and non-Hodkgin lymphoma) and others
had had an inadequate response to, or relapse after, chemotherapy
(acute lymphoblastic leukaemia, non-Hodgkin lymphoma,
Hodgkin’s disease, and genn-cell tumour). The study took place
under the ethical guidelines of the National Health and Medical
Research Council of Australia and the US Food and Drug
Administration. All patients gave informed consent. Recruitment
started in December, 1989.
All 17 study patients (table I) completed the leucapheresis phase
of the study. After an initial leucapheresis, filgrastim (Amgen,
Thousand Oaks, California, USA) was given as a continuous
subcutaneous infusion (12 fig/kg daily) for 6 days, by way of a 23
gauge needle connected to a Conned infusion pump (Medina, New
York, USA). Leucapheresis was repeated on days 5, 6, and 7 with a
’Fenwal CS-3000’ cell separator (Baxter, Deerfield, Illinois, USA);
we used a modified mononuclear-cell collection program with the
red-cell interface set at 020 units. Each leucapheresis continued
until at least 7 litres of blood had been processed. The mononuclearcell product was further processed to reduce the volume and the
final cell suspension was cryopreserved.10
The bone marrow of all patients was harvested and cryopreserved
before they underwent chemotherapy; 3 patients did not receive
high-dose chemotherapy (1 refused and 2 had progressive disease).
High-dose chemotherapy consisted of oral busulphan (4 mgkg
daily) on days - 7, - 6, - 5, and - 4 and intravenous
ADDRESSES: Departments of Clinical Haematology and Medical
Oncology (W P. Sheridan, FRACP, Prof R. M. Fox, PhD) and
Diagnostic Haematology (C. G. Begley, PhD, D. Maher, FRACP, K. M.
McGrath, FRCPA), Royal Melbourne Hospital, Victoria; Walter and
Eliza Hall Institute of Medical Research, Melbourne (C G.
Clinical
Bone
Marrow
Begley);
Haematology and
Transplantation Unit, Royal Adelaide Hospital, South Australia
(C A. Juttner, FRACP, L. Bik To, MD); Clinical Haematology and Bone
Marrow Transplantation Unit, Alfred Hospital, Prahran,
Victoria (J. Szer, FRACP); and Ludwig Institute for Cancer
Research, Melbourne Tumour Biology Branch, Victoria,
Australia
(G. Mostyn, PhD). Correspondence to Dr William P Sheridan,
Department of Clinical Haematology and Medical Oncology, Royal
Melbourne Hospital, c/o Post Office, Royal Melbourne Hospital, Victoria
3050, Australia
641
TABLE I-PATIENT CHARACTERISTICS
’Before high-dose chemotherapy.
ALL=acute lymphoblastic leukaemia.
cyclophosphamide (60 mg/kg daily) on days -3 and - 2.
Cryopreserved bone marrow and peripheral-blood mononuclearcell preparations obtained by leucapheresis were infused on day 0.
Filgrastim was given after the cell infusions as previously
described.3
Clinical status was assessed and complete blood count, including
differential white-cell count, done daily during the leucapheresis
phase and again after bone-marrow/peripheral-blood progenitorcell infusion until recovery. Bone-marrow samples were examined
and scored as previously described.3 Standard criteria were used for
starting and stopping parenteral antibiotics, administration of
platelet and red-cell transfusions, use of parenteral nutrition, and
discharge from hospital.’," Filgrastim administration was
continued at outpatient visits if necessary.
Haemopoietic progenitor cells were assayed in bone-marrow
samples and in peripheral blood and leucapheresis product obtained
before and after filgrastim treatment. Progenitor cells (colonyforming cells) were assayed by counting of colonies after 14 days’
culture.8,12 The number of progenitors per ml of sample was
calculated as: frequency (per 105 mononuclear cells) x sample
mononuclear-cell count.
The results of the study patients were compared with those of two
groups of control patients who met the same eligibility criteria and
were treated in two previous studies. Control group I (patients
treated between July, 1988, and February, 19903) received the
same
high-dose chemotherapy, autologous bone-marrow
transplantation, and filgrastim after chemotherapy but no
filgrastim-mobilised peripheral-blood progenitor cells. Control
group II (patients treated between February, 1987, and June, 1988)
received the same high-dose chemotherapy and autologous bonemarrow transplantation but no filgrastim. All clinical-care policies
were maintained unchanged throughout the study and control
periods.
Comparisons among groups were made with standard statistical
tests by a computer statistics program (NCSS, Kaysville, Utah,
USA). Student’s t test was used for continuous variables, Peto’s
generalisation of Wilcoxon’s rank-sum test for time-dependent
variables, and Fisher’s exact test for proportions. Results are given
as mean and SEM or median (for time-dependent variables and fold
increases).
Results
As expected, during the 6 days of filgrastim treatment for
collection of peripheral-blood progenitor cells, the total
white-cell count rose from 5-3 (0-6) x 109/1 to 36-4
(46) x 109/1. These cells were predominantly mature and
band neutrophils. Before filgrastim treatment, the number
of
granulocyte-macrophage progenitor cells in the
peripheral blood was low (0-04 [0’01] x 103/ml). However,
after 5 days of treatment, the numbers had increased to 1-7
Student’s t test); the median
(03)x103/ml (p<0001.
increase over baseline numbers was 58-fold (fig 1).
Fig 1-Haemopoietic progenitor cells in peripheral blood ()
and leucapheresis product (D) of study patients before and
during filgrastim treatment.
The response of circulating erythroid progenitors to
filgrastim was similar to that of granulocyte-macrophage
progenitors; numbers in peripheral blood rose from 0 020
(0-005) x 103fmI to 09 (0-2) x 103/ml (p < 0-005, fig 1).
Baseline leucapheresis before filgrastim treatment yielded
200 ml product containing 15 (06) x 103 granulocytemacrophage progenitors/ml. Despite the peripheral-blood
neutrophilia induced by filgrastim, the leucapheresis
product on days 5-7 was consistently 80-90% mononuclear
cells. The leucapheresis product reflected the peripheralblood increase in progenitor cells; the product on day 5
contained 46 (9) x 103 granulocyte-macrophage progenitors
a median increase from baseline of
per ml (p< 0.001),
63-fold (fig 1). The rise in the number of mononuclear cells
collected was less extreme (07 [0’1] x 1010 at baseline to 2-4
[0’4] x 1010 on day 5). Three consecutive daily collections
produced a total of 5-6 (07) x 1010 mononuclear cells and 21
(3-8) x 106 granulocyte-macrophage progenitors, which is
equivalent to 83 (09) x 10g and 33 (5-7) x 104 per kg ideal
body weight, respectively (fig 2). There was substantial
variation in the yield among patients, which was attributed
to differences in previous chemotherapy and radiotherapy.
The yield of granulocyte-macrophage progenitors after
filgrastim treatment was not correlated with the numbers in
peripheral blood at baseline.
There were similar changes in erythroid progenitor
numbers. Baseline leucapheresis yielded 10 (0-5) x l03fmI
compared with 31 (7) x 103/ml on day 5 of treatment
(p < 0005); the median increase was 24-fold (fig 1). Thus
642
1
L
3
4
t)
b
f
9
o
U 10 12 13
14 15
16/
Patient number
Fig 2-Total granulocyte-macrophage progenitors obtained by
leucapheresis.
filgrastim mobilised large numbers of peripheral-blood
progenitor cells for collection by leucapheresis even in this
group of heavily pretreated patients.
The median time to recovery of a platelet count of at least
50 x 109/1, independent of platelet transfusion, was
significantly shorter in the patients who received filgrastimmobilised peripheral-blood progenitor cells than in controls
(p = 0-0006, multiple-group Peto-Wilcoxon test; table II,
fig 3). A spontaneous increase in platelet count to 50 x 109/1
was seen as early as day 10. After day 12, the mean platelet
count in study patients was greater than that in control
patients (fig 4). Study patients received fewer units of
platelets than did the controls (table II). Time to a platelet
count of 50 x 109/1 independent of platelet transfusions was
weakly correlated with the number of granulocytemacrophage progenitors collected (r2 0’50).
Aspirates or biopsy samples of bone marrow were
available from most patients after bone-marrow infusion
(study group median 15 days; control group I 14 days;
control group II 21 days). In accord with the rapid
platelet-count recovery in the study group, megakaryocyte
=
numbers
above the normal range in 7 of 111
with
none of 18 patients in control
study patients compared
I
Fisher’s
exact
group (p < 0°001,
test) and 1 of 9 in control
II
group
(p < 0-03).
The median time to neutrophil recovery (> 0-5 x 109/1)
was 9 days in study patients (table II). Thus, with use of
were
within
or
filgrastim-mobilised peripheral-blood progenitor-cells,
neutrophil recovery was maintained but the
severe thrombocytopenia was significantly
accelerated
period of
Day after mfusion
Fig 3-Probability of recovery of platelet count of at least
50x109/I after high-dose chemotherapy and infusion of
haemopoietic cells.
- =study group (n=14), ---=control group I (n=25);
= control group(n=13) p<0 0005 for difference between study
II,
group and control group I; p < 0.002 for study group vs control group
no significant difference between the control groups.
shorter. Duration of hospital stay was short in the study
group, and other features showed that the low hospital
morbidity associated with the use of filgrastim3 was
maintained
(table II).
Filgrastim was well tolerated. Mild bone pain occurred in
14 patients in control group I but in none of the study
patients (p< 0-001, Fisher’s exact test). No other adverse
effects attributable to filgrastim were reported.
Adverse effects of leucapheresis among the 17 study
patients consisted of citrate-induced perioral paraesthesias
(2 patients), skin infection related to the venous access
catheter (1 patient), and symptomless falls in platelet count
in all patients. Platelet counts did not change significantly
during filgrastim treatment before leucapheresis, but fell
after each leucapheresis in proportion to the pretreatment
platelet count. No clinically evident haemorrhages occurred
during the leucapheresis phase.
1 patient in the study group died soon after
transplantation from pulmonary haemorrhage syndrome
associated with sepsis and inadequate engraftment (day 21).
This patient had the lowest number of granulocyte-
TABLE II-ENGRAFTMENT INDICES AND HOSPITAL MORBIDITY
*Two-group Peto/Wilcoxon test, because of the
tMultlple-group Peto/Wilcoxon test
Vv
many compansons, individual p values should be
interpreted with caution
643
Day
after infusion
Fig 4-Mean (SEM) platelet count after high-dose
chemotherapy and infusion of haemopoietic cells.
 = study group (n = 14); . = control group I (n=25); 0 = control
groupII (n=13).
macrophage progenitors collected (0-5 x 104/kg) and had
previously received 17 courses of chemotherapy for
Hodgkin’s disease (12 anticancer agents in 4 regimens
during 5 years). 2 of 38 controls died early: 1 died on day 14
from sepsis and the other on day 21 from pulmonary
haemorrhage syndrome. There were no episodes of late
engraftment failure in the study group, as would be expected
since the treatment included infusion of both bone marrow
and peripheral-blood progenitor cells. Hepatic venoocclusive disease developed after chemotherapy in 3 of 14
study patients compared with 12 of 38 controls. No patient
had busulphan-induced convulsions.
Discussion
Our main finding was that filgrastim successfully
mobilised peripheral-blood progenitor cells, which could be
readily collected by leucapheresis; when they were used to
infusion
after
high-dose
augment bone-marrow
of
severe
the
period
thrombocytopenia was
chemotherapy,
The
shortened.
study was suggested by the
significantly
numbers
of peripheral-blood
that
increases
G-CSF
finding
in
animals
have
confirmed this
cells.8,B
Studies
progenitor
of
G-CSF
and
have shown
unexpected biological property
that the number of more primitive bone-marrowreconstituting cells in the blood is also increased by
G-CSF.14
The acceleration of platelet recovery meant that the study
group needed fewer platelet transfusions than did the
controls. At the time of engraftment there was significantly
greater megakaryocyte cellularity in the study group than in
controls. Improved platelet recovery has the potential for
important benefits--eg, less exposure to blood products,
lower risk of HLA alloimmunisation, lower hospital costs,
and fewer outpatient attendances for platelet transfusions.
The findings should, however, be confirmed in a
randomised clinical trial.
The accelerated recovery of peripheral-blood platelet
counts was unexpected because G-CSF does not stimulate
megakaryocyte growth in vitro or increase platelet numbers
in vivo. Administration of high numbers of peripheralblood progenitor cells collected after chemotherapy priming
(with or without colony-stimulating factors) is also
associated with faster platelet recovery than is autologous
bone-marrow transplantation alone.15-1? The accelerated
platelet recovery associated with peripheral-blood
progenitor-cell infusions probably reflects the activity of
megakaryocyte progenitor cells released non-specifically by
filgrastim or chemotherapy. The target-cell populations and
mechanisms of release are unknown.
The number of progenitor cells collected by leucapheresis
after filgrastim treatment seems to be at least ten times
greater than the number collected after ecogramostim
treatment,18 although no direct comparisons have been
reported. The yield of granulocyte-macrophage progenitors
did, however, vary substantially. We speculate that this
variation was attributable to differences in haemopoieticstem-cell reserve due to differing amounts of previous
chemotherapy. Better results and higher yields of
peripheral-blood progenitor cells would be likely if
filgrastim administration and leucapheresis were
undertaken before there had been substantial damage to
haemopoietic-stem-cell reserve. This possibility could be
investigated in patients about to undergo high-dose
chemotherapy as initial medical management of cancer-for
example, in investigational protocols for women with stage
II breast cancer and adverse prognostic factors.19 Since
severe neutropenia and thrombocytopenia can now be
systematic investigation of high-dose
chemotherapy is possible.
Other reported strategies to achieve consistent
amplification of peripheral-blood progenitor cells include
the addition of a haemopoietic growth factor (GM-CSF) to
high-dose cyclophosphamide 17 or etoposide 20 and use of a
disease-specific chemotherapy regimen with GM-CSF .21
However, rapid engraftment with peripheral-blood
progenitor cells mobilised by a haemopoietic growth factor
alone, without chemotherapy, has not previously been
reported. The synergy among the various growth factors in
vitroZZ-Z5 suggests that use of filgrastim with one of the other
factors might further enhance peripheral-blood progenitorcell numbers. Human stem-cell factor might also be useful Zs
Autografting studies with peripheral-blood progenitor
cells do not prove long-term haemopoietic reconstitution
from this source. Our study did not attempt to address this
question, since bone marrow was infused as well as
peripheral-blood progenitor cells. After bone-marrow
allografts, at least 17 % of patients show mixed haemopoietic
chimerism;Z6 the proportion rises to 90% when graftversus-host effects are eliminated by T-cell depletion .27
Thus, some haemopoietic stem cells seem to survive
myeloablative regimens. Nevertheless, the persistence of
normal blood counts for long periods after severely
myelotoxic regimens suggests that peripheral-blood
progenitor cells may contain adequate numbers of marrowreconstituting cells. is Unequivocal evidence to support this
suggestion could be obtained by means of peripheral-blood
progenitor-cell allotransplantation and assessment of longterm donor engraftment with genetic markers. Such
allotransplantation, previously impossible because
mobilisation depended on the use of cytotoxic
chemotherapy in healthy donors, can now be done with
filgrastim mobilisation.
overcome,
We thank Dr P. Rowlings, Dr M. Green, Dr J. Cebon, Dr A. Boyd, Mr D.
Watson, Ms S. Grogan, Mr G. Duggan, Ms J. Bayly, Ms M. Flux, Ms E. de
Luca, Ms S. Hauser, Mr D. N. Haylock, Mrs P. G. Dyson, Ms N. Messino,
644
and staff of the blood banks (Royal Melbourne and Alfred Hospitals) and
Leukaemia Research Unit (Institute for Medical and Veterinary Science);
Institute for Drug Technology, Melbourne, for study monitoring; and Dr K.
Alton, Dr L. Souza, and Dr M. Vincent (Amgen, Thousand Oaks, California,
USA), Dr A. Burgess (Ludwig Institute), and Prof D. Metcalf (Walter and
Eliza Hall Institute). This work was supported by grants from the
Anti-Cancer Council of Victoria, the Victorian Health Promotion
Foundation, the Anti-Cancer Foundation of the Universities of South
Australia, the National Health and Medical Research Council of Australia,
Baxter Healthcare, Deerfield, Illinois, and Amgen, Thousand Oaks,
California.
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From The Lancet
Pilot fatigue
Between 80% and 90% of civil aeroplane accidents in the United
States are ascribed to "pilot error". That is to say, the expected
hazards of flying, unexpectedly bad weather, low cloud, fog, icing,
lightning, and other acts of God, as well as all the mechanical
defects-engine failure and the rest-account for only a little more
than a tenth of all aeroplane crashes. McFarland says that "in the
early stages of the war the British pilots were evidently losing more
planes in landing accidents in returning home from abroad than
during actual combat with the enemy". Whether this was so or not,
anyone who has experienced the weariness produced by eight or ten
hours of operational flying knows that many of these crashes were
also due to pilot error, for the inefficiency of fatigue must have
played an important part.... In civil aviation many of the causes of
fatigue can be modified, but in war flying some must of necessity
remain unchanged. Fatigue in war flying arises from physical and
psychological stress. Some of the physical causes of fatigue can be
lessened, although the requirements of operational flying and the
design of combat planes present many difficulties. For instance,
engine noise, vibration, glare from the fuselage, the discomfort of
cramped positions, cold, difficult instrument reading at night, and
even uncomfortable clothes all play a part and must be considered
individually in relation to the pilot and to his duties. Other causes,
often overlooked-irregular working hours, long periods of duty,
curtailed sleep, or unaccustomed hours of sleep with consequent
alteration in feeding and living habits-must add considerably to
the fatigue factors in a pilot’s life. The physiological effects of
prolonged flying at very high altitudes-30 000 ft or more-or of
repeated ascents and descents are not yet fully understood, but it is
known that intellectual efficiency is diminished at heights over
12 000 ft, largely because of the relative anoxia.... Now
physiological evidence shows that fatigue of the degree met in man
cannot be related to any peripheral changes; it is central in origin,
and more psychological than physiological too. Thus, although the
pilot does not perform much physical work, and although
intellectual work appears to require no extra energy in terms of
calories, he can still become exhausted with fatigue.
(Feb 21,1942)
George Morstyn