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1993 82: 1410-1414
Collection of peripheral blood hematopoietic progenitors (PBHP) from
patients with severe aplastic anemia (SAA) after prolonged
administration of granulocyte colony-stimulating factor
A Bacigalupo, G Piaggio, M Podesta, MT Van Lint, M Valbonesi, G Lercari, PG Mori, M Pasino, E
Franchini and L Rivabella
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Collection of Peripheral Blood Hematopoietic Progenitors ( P B H P ) From
Patients With Severe Aplastic Anemia (SAA) After Prolonged Administration
of Granulocyte Colony-Stimulating Factor
By A. Bacigalupo,
G. Piaggio, M. Podesth, M.T. Van Lint, M. Valbonesi, G. Lercari, P.G. Mori, M. Pasino, E. Franchini,
L. Rivabella, 0. Figari, G. Sogno, M.R. Raffo, and A.M. Marmont
The aim of this study was t o test whether prolongedadministration of granulocyte colony-stimulating factor (G-CSF)
would allow the collection by leukapheresisof PBHP in patients with SAA. For this purpose, nine SAA patients, 7 t o
46 years old, six of whom were enrolled at diagnosis of
their disease and three after previous immunosuppression
had failed, were treated with antilymphocyte globulin
(ALG) (day 1 t o 5 ) , cyclosporin A (5 mg/kg/d orally) (day 6
to 90) and G-CSF 5 Ng/kg/d (day 6 to 90).A total of 40
leukaphereses were performed, (range 2 t o 7 per patient),
between days
10 and
168 from G-CSF treatment.
White blood cell count at the time of harvest ranged from
1.2 to 18.1 X 109/L. Results can be summarized as follows: the median number of cells collected per patient was
5.0 X 108/kg (range 2.6 t o 18.7). the median number of
CD34+ cells was 1.8 X 106/kg (range 0.27 to 3.8) and
the median number of colony-forming units granulocytemacrophage (CFU-GM) was 3.9 X 104/kg (range 0 t o 39).
Twenty leukaphereses performed between days 33 and
77 of G-CSF treatment grew granulocyte macrophages
and erythroid colonies in vitro. No colony growth was obtained from 20 leukaphereses performed before day 33
or after day 80. In six patients the total number of CFUG M recovered were in the range described for autologous
peripheral blood stem cell grafts. (2.6 t o 39 X 104/kg). In
conclusion, this study suggests that circulating hematopoietic progenitors can be recovered after ALG priming and
after at least 1 month of G-CSF treatment in a proportion of
patients with SAA. Whether these cells will be suitable for
autologous transplantation remains t o be determined.
0 1993 by The American Society of Hematology.
T
preservation. If the numbers and quality of PBHP are found
suitable, the use of cryopreserved PBHP for autotransplant
in SAA patients not responding to ALG may be considered.
We report our initial experience with nine SAA patients
who were treated with ALG for 5 days, followed by cyclosporin A (CsA) and granulocyte (G-CSF) for 3 months.
+
+
HIS WORK was prompted by the study of Haas et al,’
which showed that granulocyte-macrophage colonystimulating factor (GM-CSF) priming could mobilize a sufficient number of PBHP to allow autologous transplantation in lymphomas with chemoradiotherapy-induced
hypoaplasia. In fact, although it is now recognized that large
numbers of PBHP can be recovered after high-dose chemoand more so with the use of growth factors,46
little has been done on patients with a reduced marrow reservoir, such as those described by Haas et al, who were reported as “not eligible for marrow harvest.”’
The second important assumption is that the majority of
patients with acquired SAA have residual hematopoietic
progenitors, and that the number of these is usually underestimated by current in vitro culture
This assumption seems to be supported by hematologic reconstitution following antilymphocyte globulin (ALG) treatment in
the majority of SAA p a t i e n t ~ , ‘ and
~ ‘ ~by enhanced growth
in the presence of stem cell factor (SCF).I4 If many SAA
patients have residual stem cells, it may be possible to mobilize hematopoietic progenitors with the use of in vivo
growth factors and collect them by leukapheresis for cryo-
From the Divisione di Ematologia e Centro Trasfusionale, Ospedale San Martino, and the Emato-Oncologia Pediatrica e Centro
Trasfusionale. Ospedale Gaslini, Genova, Italy.
Submitted August 14, 1992; accepted April 29, 1993.
Supported by the Associazione Ricerca Trapinnto Midollo Osseo
(ARITMO). and Associazione Italiana Ricerca contro il Cancro
(AIRC).
Address reprint requests to A. Bacigalupo, MD, Divisione Ematologia 2, Ospedale San Martino. 16132 Genova, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8205-0019$3.00/0
1410
+
+
+
+
MATERIALS AND METHODS
Patients. Nine patients were admitted with the diagnosis of acquired aplastic anemia: main clinical data are outlined in Table 1.
All nine were transfusion dependent at the time of G-CSF treatment according to an ongoing European Group for Bone Marrow
Transplantation (EBMT) trial designed for patients presenting with
less than 0.5 x lo9 polymorphonuclear neutrophil leukocytes
(PMN)/L. This protocol includes horse ALG (Merieux, Lyon,
France), 15 mg/kg/d on days 1 through 5 ; 6-methylprednisolone, 5
mg/kg/d intravenously (IV) on days I through 5,2.5 mg/kp/d IV on
days 6 through 10, then tapering the dose until discontinuation on
day +30,; CsA, 5 mg/kg/d orally from day +6 to day +90; and
G-CSF 5 pg/kg/d (IV for 15 days, then subcutaneous)from day +6
to day +90. One patient (no.2) was not eligible for this study (PMN
0.9 X 109/L)and therefore received the same regimen without GCSF. He underwent a leukapheresisimmediately after ALG administration and, because of deteriorating peripheral blood (PB)
counts, was started on G-CSF on day +5 1.
Leukapheresis. Each patient underwent two to seven leukaphereses using a continuous flow cell separator Fenwal CS 3000
(Deerfield, USA).
Cell surface markers. PB cells recovered from leukapheresis
were processed with a workstation Coulter Q-Prep (Coulter, Hieleah, FL), without further separation. Cell surface antigens were
detected by a direct immunofluorescenceusing a panel of conjugated fluorescein isothiocyanate (FITC)/phosphatidylethanolamine (PE) MoAbs: Cyto-stat Coulter Clone CD3 (Coulter), CD4,
CD8, for T lymphocytes; CD19 and HLA-DR for B lymphocytes;
CD33 for myelomonocytic cells (Coulter); and CD34 HPCA-2 for
progenitor cells (Becton Dickinson, Mountain View, CA). Fluorescence was analyzed with a Coulter EPICS Profile I1 (Coulter). Isotypically matched mouse Igs, directly conjugated to FITC or PE, were
used as negative controls in all experiments.
E / d , Vol82, No 5 (September 1). 1993: pp 1410-1414
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PB HEMATOPOIETIC PROGENITORS IN APLASTIC ANEMIA
141 1
Table 1. Clinical Data of Patients Studied
Patient
No.
Age
Sex
Ws)
Cause
1
2
3
M
hepat
idiop
4
M
5
6
7
8
9
M
M
46
11
12
17
45
18
23
4
10
M
F
M
M
M
idiop
idiop
idiop
idiop
idiop
idiop
idiop
PB counts
at Treatment
Interval
Between
Diagnosis
and G-CSF
Treatment
PMN
x 109/L
Plt
RBC
Plt
Present
Status
30 d
60 d
1 Yr
4 yrs
120d
60 d
150 d
60 d
15 d
0.28
0.38
0.40
0.70
0.03
0.03
0.20
0.29
0.30
5
8
12
15
6
15
18
5
10
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
A W Tx-l
Dead
Dead
Tx-D
A W Tx-l
Tx-D
Tx-D
A W Tx-l
Tx-D
Transfusions
at Treatment
Abbreviations: d. days; Plt, platelets; A W , alive and well; Tx-D, transfusion dependent; Tx-I, transfusion independent.
In vitro colony growth. PB cells recovered from leukapheresis
( lo5) were plated unfractionated in 1 mL of Iscove's modifed-Dul-
becco's medium (IMDM) containing 30% fetal calf serum (FCS), in
0.9% methylcellulose, for colony-forming unit granulocyte-macrophage (CFU-GM) growth with 100 ng/mL of recombinant human
(rh)GM-CSF (Sandoz, Basel, Switzerland). In the attempt to improve colony formation, PB cells from three leukaphereses were
kept in liquid culture ( lo6 cells/mL) for 5 days in the presence of
SCF (Genzyme, Boston, MA), 100 ng/mL.
Cells from the first 18 leukaphereses were further separated as
follows: after a Ficoll Hypaque gradient (ICN Flow, Costa Mesa,
CA) (1,077 g/cm2) and plastic adherence (2 hours at 37"C), cells
were T depleted (with CAMPATHIM, 1 pg/106 cells plus autologous complement 20%, or E-rosetting with neuraminidase-treated
sheep RBCs) and plated for CFU-GM growth as described above.
CD34+ cells were positively selected by incubating 10 X lo6 Tcell-depleted cells with CD34 MoAb (HPCA-2, Becton Dickinson)
for 36 at 4°C and then for 36 at 4°C with antimouse Ig-coated
Dynabeads (Dynal, Oslo, Norway): thereafter the CD34-negative
and CD34-positive fractions were plated in 0.9% methylcellulose
for colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) growth with IMDM containing 30%
FCS, 2/10-4 mol/L Hemin, 5/10-5 beta mercaptoethanol, 1% bovine serum albumin (Fraction V; Sigma, St Louis, MO), in the
presence of 2U/mL rh erythropoietin (rhEpo; Cilag, Milan, Italy),
100 ng of rhGM-CSF (Sandoz, Basel, Switzerland), and 100 ng/mL
rh interleukin-3 (provided by Sandoz) with or without SCF (20
ng/mL) (Genzyme).
The cultures were incubated for 14 days in a 3 7 T , 5% CO, humidified incubator. Colonies were counted with an Olympus IM
inverted microscope (Olympus, Lake Success, NY).
Cryopreservation. PB cells were cryopreserved in culture medium (TC 199) containing 10%dimethyl sulfoxide and 5% autologous heparinized plasma by means of a freezing flow rate unit
(Planer 203). Cryopreserved samples were stored in gas phase of
liquid nitrogen.
RESULTS
Patients. All patients completed the designed course of
treatment (Table I). Two patients (no. 2 and 3) died on day
150 and +200 of infection: we were unable to collect from
these patients PB cells capable of colony formation in vitro.
The other seven patients are surviving between day 100 and
day 365 after G-CSF treatment: four are transfusion dependent and three are transfusion independent. The number of
+
patients is too small to draw any conclusions on the effect of
G-CSF administration on hematologic response.
Leukapheresis. A total of 40 leukaphereses were performed. There were no major problems in performing the
procedures also with low leukocyte counts. Leukaphereses
were performed, when possible, at intervals of 7 to 15 days.
However, there were variations because ofthe fact that these
were all referred patients, often not available in Genova for
the procedure. The median number of cells collected per
procedure was 5.0 X 108/kg(range 2.6 to 18.7) (Table 2).
Cell phenotype. The surface phenotype of recovered
cells was expressed as medians (range) as follows: CD3,55%
(13%to 80%);CD4, 30.5% (4%to 39%);CD8,26% (17%to
62%);CD19, 13% (3.3% to 28%);DR, 12% (3.2% to 31%);
CD33+34-, 10% (0.3% to 30%); CD33-34+, 0.2% (0% to
1%); CD33+34+,0.3% (0%to 4.4%).
We saw no major increment of circulating CD34' cells
during treatment with G-CSF. The median number of
CD34' cells collected per leukapheresis was 1.8 X 106/kg
(range 0.27 to 3.8) (Table 2).
Table 2. Summary of Collected PB Cells, CFU-GM Growth, and
C D 3 4 Positivity in Nine Patients Undergoing Leukaphereses After
G-CSF Treatment
Patient
No.
1
2
3
4
5
6
7
8
9
Median
Range
No. of
Cells
X 108/kg
No. of
Colonies
x l@/kg
No. CD34'
Cells
x 10e/kg
Autologous
Hematologic
Recovery
5.0
5.49
2.95
10.4
2.95
2.66
2.66
13.8
18.7
39.1
0
0
13.0
3.9
2.6
0.93
26.2
17.1
1.8
3.84
1.34
3.1
3.78
1.13
0.27
nt
nt
Yes
No
No
No
Yes
No
No
5.0
(2.6-1 8.7)
3.9
(0-39)
1.8
(.27-3.8)
Yes
No
Autologous hematologic recovery indicates patients who have recovered autologous hematopoiesis and have become transfusion independent.
Abbreviation: nt, not tested.
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BACIGALUPO ET AL
1412
Colony formation in vitro. Twenty leukaphereses performed before day +30 or after day +80 grew no colonies
(Fig 1). Twenty leukaphereses performed between day +33
and day +77 yielded a large number of GM and erythroid
colonies (Table 2; Figs 1 and 2). PB cells from two aphereses
(patient no. 1 ) also grew large numbers of burst-forming
units erythroid (BFU-E): 17 and 37 X 104/kg(Fig 3). In the
last two patients (no. 8 and 9) who underwent 6 to 7 leukaphereses in the suggested appropriate "window" at 7 to 10day intervals, the number of CFU-GM recovered was very
high (1 7 and 26 X 104/kg (Table 2 ) . Samples from four
leukaphereses were thawed and plated in vitro for colony
formation: CFU-GM recovery was 36% when compared
with top colony formation before cryopreservation.
T-cell depletion and colonyformation. T-cell depletion
(TCD) did not seem to enrich for hematopoietic progenitors, and indeed had a detrimental effect on three leukaphereses from which large number of colonies had been
grown from unseparated cells. Two of these cell populations
had been treated with CAMPATH- 1M and one with E-rosetting. There was one exception: the last leukapheresis
from patient no. I grew no colonies before or after TCD but
did grow 35 mixed colonies (CFU-GEMM) from IO5
CD34' positively selected cells.
DISCUSSION
In this study, we have shown that prolonged administration of G-CSF and CsA to patients with aplastic anemia,
following treatment with ALG, can result in mobilization of
large numbers of hematopoietic progenitors. We believe
this finding confirms that some patients with SAA still have
a stem cell reservoir in spite of pancyt~penia.'~~''
This is
supported by previous work from Torok Storb' who first
showed an increased number of BFU-E in the PB after ALG
therapy. Our finding is also in keeping with data obtained in
animals showing that treatment with G-CSF alone mobilized pluripotent stem cells in the PB which could then be
successfully used for autografts.16Indeed, in six patients the
total number of CFU-GM recovered from the PB was in the
range reported to allow hematopoietic reconstitution if infused after ablative ~hemoradiotherapy.~
This may also suggest that the "response" of SAA patients to ALG with or
without G-CSF may be dependent on circulation in the PB
and re-seeding of hematopoietic progenitors: in patients in
whom the accessory cell network has been modified by socalled immunesuppression, this leads to hematologic recovery. In patients in whom the marrow microenvironment is
still abnormal, re-seeding of hematopoietic progenitors
fails. It is in the latter patients that harvesting and cryopreserving hematopoietic progenitors from PB can offer an additional chance of treatment if the underlying disease can be
cured, for example, by high-dose cyclophosphamide.
However, there are still several open questions: the timing
of cell collection, the quality of cells collected, and their
ability to allow sustained hematologic recovery if used for
autologous transplantation. As to the first question, our
data would indicate that circulation of hematopoietic progenitors in PB occurs in the second and part of the third
month of G-CSF treatment. As more patients are enrolled
in the study we may find colony growth also beyond the
third month, but for the time being we would advise performing leukapheresis every 10 days between day +30 and
day +80 of G-CSF treatment.
As to the quality of cells collected we know they can form
granulocyte-macrophage and erythroid colonies in vitro in
large numbers. We also know that CFU-GM growth is a
functional test predicting engraftment after autologous PB
cell transplants," although information concerning the association between CFU-GM growth and engraftment has
been obtained in patients with malignancy and a functioning bone marrow such as lymphomas, myelomas, and solid
t ~ m o r s . An
~ - ~exception is the work by Haas et al,' who
"mobilized" PBHP from patients with chemoradiotherapyinduced cytopenia and used them successfully for autologous grafts.
Finally, will these cells be capable of sustained hematologic recovery if infused after high-dose cyclophosphamide.
CFU-GM xl0^4/kg
14
12 -
"
m
"
x
'1
2
t
"
t
t
Fig 1. Numbers of CFU-GM colonies X 104/kg
grown for single leukaphereses, plotted against
time (days) of G-CSF treatment. There is no colony
30 or after dav 80. Conformation before dav
+
+
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
PB HEMATOPOIETIC PROGENITORS IN APLASTIC ANEMIA
1413
t
2. CFU-GM colonies
from patient no. 4: PB cells colFig
lected by apheresis on day
55 of G-CSF treatment.
+
There are several in vitro studies suggesting that hematopoiparticularly clonality, may not necessarily indicate an inetic progenitors from SAA patients are abnormal: in particutrinsic defect, or a preleukemic disorder.2' In keeping with
lar Marsh et all" recently showed with long-term cultures
this view is a recent contribution showing the lack of point
that they have a reduced capacity of proliferation and surmutation of N-rus oncogene in marrow cells from SAA pavival also when grown on normal stromal layers: we do not
tients.22We favor this view, and regard the high incidence of
know if this is caused by a functional and possibly reversible
leukemia and myelodysplasia seen in long-term survivors
defect or an intrinsic abnormality. The finding of a high
after ALG23as the expression of long-lasting stressed hemaproportion of female patients with clonal h e m a t o p o i e s i ~ ' ~ ~topoiesis.
~~
If we were capable of modifying the accessory cell
would argue in favor of the latter hypothesis.
circuits, as known to occur after high-dose cyclophosphaHowever, a recent review has pointed out that abnormalimide, and if we then reinfused autologous hematopoietic
ties of hematopoietic progenitors from SAA patients, and
progenitors, this might allow better hematologic recovery
P-, i
*
*'
I
I
I
I
Fig 3. BFU-E colonies from
patient no. 1 : PB cells collected
by apheresis on day
33 of GCSF treatment.
+
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
BACIGALUPO ET AL
1414
than seen with ALG alone, and thus reduce the risk of late
clonal disease.
Although this is only a working hypothesis, we believe our
present findings warrant further investigations in this area.
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