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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2189-2196
Innovative Two-Step Negative Selection of Granulocyte
Colony-Stimulating Factor-Mobilized Circulating Progenitor Cells:
Adequacy for Autologous and Allogeneic Transplantation
By
Alessandro Rambaldi,
Gianmaria Borleri,
Gianpietro Dotti,
Piermario Bellavita,
Ricardo Amaru,
Andrea Biondi, and
Tiziano Barbui
From the Divisione di Ematologia e Centro Trasfusionale, Ospedali
Riuniti Bergamo, Bergamo, Italy; and Clinica Pediatrica
Università di Milano, Monza, Italy.
 |
ABSTRACT |
A major obstacle in purifying either autologous or allogeneic
hematopoietic stem cells from granulocyte colony-stimulating factor
(G-CSF) mobilized circulating progenitor cells (CPC) is represented by
the huge cellularity present in each apheretic product. To obtain a
significant debulking of unwanted cells from the leukapheresis, we
developed a modified protocol of immune rosetting whereby human
ABO-Rh- compatible red blood cells (RBCs) are treated with chromium
chloride and then coated with murine monoclonal antibodies (MoAbs)
against leukocyte antigens. When experiments were performed with
leukaphereses obtained from normal donors or from T-cell acute
lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine
MoAbs against human mature myeloid cells (CD11b) and T cells (CD6);
whereas, in the case of patients with B-precursor ALL, B-cell
non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were
coated with anti-CD11b only. After incubation with CPC, rosetting cells
(myeloid precursor cells, granulocytes, monocytes, and T cells) were
removed by Ficoll-Hypaque density gradient centrifugation with a blood
cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a
significant reduction of the initial cellularity was consistently
obtained (range, 72% to 97%), whereas the median absolute recovery of
the CD34+ cells was above 85% (range, 64 to 100), with a
10-fold relative enrichment ranging from 3% to 41%. In a second step,
CPC can be further purged of contaminating T or B cells by incubation
with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a
SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to
four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+
cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At
the end of the procedure the T- or B-cell depleted fraction retained a
high proportion of the initial hematopoietic CD34+ stem
cells, with a median recovery above 70% (range 48% to 100%) and an
unmodified clonogenic potential. In five patients (two follicular NHL
and three ALL) the purified fraction of stem cells was found disease
free at the molecular level as assessed by polymerase chain reaction
(PCR) analysis of the t(14;18) chromosome translocation or
clono-specific DNA sequences of IgH or T-cell receptor  and  chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a
highly efficient purging of T or B cells from G-CSF expanded and
mobilized CPCs. This approach might lead to the eradication of the
neoplastic clone in the autologous stem cell inoculum as well as for
T-cell depletion during allogeneic transplantation.
| |
INTRODUCTION |
IN THE NORMAL bone marrow the
CD34+ cell fraction contains virtually all the
hematopoietic progenitor cells (HPCs).1 After priming with
hematopoietic growth factors (G-CSF or GM-CSF), either alone or in
combination with high-dose chemotherapy, mobilized circulating
progenitor cells (CPCs) expressing the CD34 antigen on the cell surface
can be easily collected from peripheral blood in quantities
approximately 10-fold higher than previously obtained from bone
marrow.2 The purification of human stem cells has clinical
relevance for autologous3 and allogeneic
transplantation4 and for the usage of the stem cells as
vehicles for gene transfer.5,6 High-dose chemotherapy and
autologous transplantation are increasingly being used to treat
patients with hematologic malignancies and solid tumors even though the
neoplastic contamination of CPCs7-9 may contribute to
subsequent relapses.10 Because many tumor cell types
including multiple myeloma (MM), lymphomas, breast and ovarian cancer,
and neuroblastoma do not usually express the CD34 antigen,
CD34+ positive selection may be effective for the purging
of the autologous grafts. Different commercial devices are now
available for laboratory and clinical scale enrichment of these cells
by immunoaffinity columns or immonomagnetic beads, and preliminary
clinical results have already been obtained by using purified
CD34+ cells for autologous
transplantation.8,9,11-15 More recently, some investigators
started to analyze the feasibility of using purified CD34+
cells for allogeneic transplantation.4,16,17 In fact,
allogeneic CPC preparations may provide the possibility to obtain
T-cell depleted fractions of stem cells while preventing an
unacceptable loss of hematopoietic progenitors. Therefore,
CD34+ cell selection can be used as an efficient method for
preventing graft versus host disease (GVHD) without an increased risk
of graft failure or rejection.18 Unfortunately, at least
two major concerns are opposing a wide clinical use of purified
CD34+ cells. The immune reconstitution can be severely
compromised if inadequate numbers of T and B cells are present in the
graft, a problem that deserves particular attention, especially in the case of patients with nonhematologic malignancies. Moreover, most acute
leukemia cell types express the CD34 antigen, thus preventing the use
of this method of purification in the autologous setting.
The aim of this study was to develop an efficient, reproducible, and
relatively inexpensive method for clinical scale preparation of CPC for
transplantation procedures. Furthermore, the selective- and
lineage-specific elimination of the neoplastic fraction from the
autologous graft as well as the normal T cells from the allogeneic graft was the aim of the same procedure. This novel methodology consists of two distinct steps: in the first step, 85% of the initial
cellularity (granulocytes, monocytes, and eventually T cells) is
allowed to form rosettes with chromium chloride-treated human
ABO-Rh-compatible red blood cells (RBCs) coated with murine monoclonal
antibodies (MoAbs) antihuman CD11b (and CD6 when T-cell depletion is
needed) and then removed by gradient sedimentation, without a
significant loss of the CD34+ cells present in the input.
Subsequently, in step two, this debulked apheresis is very efficiently
purged of unwanted B or T cells by the use of lineage-specific
monoclonal microbeads.
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MATERIALS AND METHODS |
Mobilization and harvesting of autologous or allogeneic CPCs.
Autologous CPCs were collected in patients with non-Hodgkin's lymphoma
(NHL), acute lymphoblastic leukemia (ALL), and MM, after different
consolidation protocols of high-dose chemotherapy followed by rhG-CSF
(Filgrastim; Roche, Milan, Italy) administration (5 µcg/kg/day), as previously described.19 Leukapheresis was performed as soon as white blood cells (WBCs) were at least 3.0 × 109/L and peripheral blood CD34+ cells were  0.5%. Ten liters of blood were processed daily through an indwelling
central venous catheter (Groshong CV Catheter; Band Inc, Salt Lake
City, UT) by using a cell separator COBE spectra (COBE, Lakewood, CO).
Allogeneic CPCs were collected from HLA-identical, MLC-negative normal
siblings upon treatment with 2 × 5 µg/kg/day of rhG-CSF
administered subcutaneously every 12 hours for 5 or 6 consecutive days.
The procedure of leukapheresis was started on day 5 (after the ninth
dose of G-CSF) by using a COBE spectra cell separator. Ten liters of
blood were processed daily by using the cubital vein in all donors, as
previously described.20 The absolute number of
CD34+ cells, lymphoid cells (T, B, and NK cells), and
mature myeloid cells (granulocytes and monocytes) were evaluated daily
in the peripheral blood by flow cytometry with fluorescein
isothiocyanate conjugated (FITC) murine MoAbs against human CD34, CD3,
CD19, CD56, CD11b, CD14, CD16, or negative control and a FACScan
analyzer (Becton Dickinson, Mountain View, CA). In vitro colony assay
for evaluation of erythroid and myeloid colony forming units was
performed as previously described.19,20 Informed consent
was obtained from the patients and the donors by using forms approved
by the Institutional Review Board. A fully detailed explanation of the potential risks and benefits concerning the collection of G-CSF mobilized CPCs for autologous and allogeneic transplantation was given
to normal donors and the patients. Normal donors and patients were more
than 18 years of age. The experimental use of apheresis products for
immune rosetting and immunomagnetic depletion of T or B cells was begun
when unmodified preparations of CPC (containing 6 × 106 CD34+ cells/kg of recipient body weight)
were already harvested and cryopreserved.
Immune rosettes.
The method is based on a previously published procedure based on the
ability to couple murine MoAbs to RBCs by chromium
chloride.21 The following mouse hybridoma cell lines were
obtained from American Type Culture Collection (ATCC, Rockville, MD):
OKM1 (IgG2b, ATCC CRL 8026) reactive with human granulocytes,
monocytes, NK cells and committed myeloid precursor cells (CD11b
antigen); and T12 (IgM, clone 3Pt12B8; ATCC HB8136) reacting with human
T cells and some B cells (CD6 antigen). Partially purified preparations of these MoAbs were obtained by ammonium sulphate precipitation of
spent culture supernatants of each hybridoma. ABO-Rh-compatible, irradiated (25 Gy), and filtered human red blood cells (HRBCs) were
obtained from the Blood Bank. For coating with MoAbs, 150 mL of packed
HRBCs were washed three times in normal saline (centrifugation at 3000 RPM for 5 minutes at room temperature) with a COBE 2991 apparatus and a
blood cell processor set. After the third wash, 30 mL (3 mg/mL) of
partially purified MoAbs (CD11b and eventually CD6 in the case of
normal donors and T-ALL patients) were added at the same blood
processor set. Thereafter, and under continuous agitation, 250 mL of a
0.1% solution of chromium chloride (CrCl3 .6H2O; Sigma, St.Louis, MO; prepared in normal saline from
a 1% [W/V] stock solution with the pH adjusted to 5.0 with 10 N
NaOH) were added dropwise over a 15-minute time period. After
incubation for 5 additional minutes at room temperature the reaction
was stopped by the addition of 300 mL of phosphate-buffered saline (PBS) supplemented with 2.5% Human Serum Albumin (PBS-HSA).
Apparently, the isotype of the antibodies (IgM or IgG) does not affect
the coupling process to RBCs. MoAb-coated HRBCs were washed twice, resuspended in 100 mL of PBS 2.5% HSA, and mixed with 11 to 58 × 109 white blood cells (200 mL final volume, hematocrit less
than 5%) obtained from leukaphereses of G-CSF-treated individuals. Rosette formation was performed within the same blood cell processor set by two centrifugation steps at 3000 RPM for 30 minutes. At the end
cells were resuspended in 400 mL of PBS 2.5% HSA, transferred, and
layered onto the top of 200 mL Ficoll Hypaque (by using a second blood
cell processor set), and centrifuged for 45 minutes at 3000 rpm.
Nonrosetting cells were harvested at the Ficoll interface and washed
twice with PBS-HSA. After 5 minutes of incubation with hypotonic
NH4 Cl buffer (NH4 Cl 8.99 gr/L,
KHCO3 1gr/L, Na4 EDTA 0.037 gr/L, pH 7.3) to
lyse residual erythrocytes, cells were washed with PBS-HSA, resuspended
in RPMI 1640 10% FCS, counted, and stained with MoAbs for FACS
analysis. The described procedure required approximately 3 hours of
work performed by one operator and the estimated cost (including
production and purification of antibodies, chemical reagents, tissue
culture media, two blood cell separation sets, and 1 U of filtered,
irradiated, RBCs) was 400 US dollars.
Immunomagnetic purging of T or B cells.
To obtain a high degree of T- or B-cell depletion, partially purified
hematopoietic progenitors (0.5 to 13 × 109 cells)
obtained by immune rosetting were labelled with primary unconjugated
MoAbs reacting against T cells (anti-CD2 antigen, clone 35.1; IgG2a,
ATCC HB222; and anti-CD7, clone WT1, IgG1) or with a panB MoAb (clone
HD37, anti CD19, IgG1; kindly provided by Dr. Moldenhauer, Heidelberg,
Germany). In MM patients (two cases), cells were incubated with
anti-CD19 and anti-CD5622 (clone N901, IgG1;
kindly provided by Dr JD Griffin, Boston, MA). Thereafter, an indirect
labelling was performed with goat antimouse magnetic microbeads,
according to the manufacturer instructions (Miltenyi Biotec,
Germany). After labelling, cells were washed as above, resuspended in
PBS-HSA (30 mL final volume), and layered, by the use of a peristaltic
pump, onto the top of a type-D depletion column (composed of
ferromagnetic fibers) inserted within a SuperMACS apparatus (Miltenyi
Biotec). Unstained cells were eluted with PBS-HSA from the column kept
within the magnetic field of the cell separator. The T- or B-cell
depleted fraction of HPCs was finally resuspended in autologous plasma,
counted, phenotypically analyzed, and cryopreserved. The time required
to complete this negative depletion was 3 hours and the estimated cost
of two vials of goat antimouse magnetic microbeads and one type-D
depletion column, necessary for each procedure, was 1,500 US dollars.
Molecular evaluation of minimal residual disease (MRD).
In patients with ALL, MRD was evaluated by molecular analysis of
junctional regions of rearranged T-cell receptor (TCR) or genes. Specific patterns of recombination of TCR or genes were
identified at diagnosis by Southern blot analysis, according to
standard techniques.23 The TCR and gene
rearrangements were subsequently amplified by polymerase chain reaction
(PCR) with V or V family primers and J or J general
primers, respectively. The PCR products were cloned in pMos vector
(Amersham, Buckinghamshire, UK) sequenced to define the precise
nucleotide sequence of the junctional regions. Based on these sequence
data, leukemia-patient-specific oligonucleotide probes were
synthesized to analyze subsequent marrow or leukapheretic peripheral
blood samples. Reaction products were identified by hybridization
studies with DNA oligonucleotide probes labeled with [ - 32 P] ATP. In one case of B-precursor ALL, MRD monitoring
was performed by amplification of the CDRIII IgH with VH and JH
consensus primers, as described.24 Two follicular lymphoma
patients bearing the t(14;18) chromosome abnormality were evaluated by
PCR analysis of BCL2-IgH rearrangement, according to published
methods.25
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RESULTS |
Effect of immune rosetting on overall recovery and composition of G-CSF
mobilized CPC.
In normal donors receiving G-CSF (10 µg/kg/d) for 5 to 6 days, the procedure of CPC mobilization and collection was safe and always well tolerated. The apheretic products were debulked of committed myeloid cells and T lymphocytes by immune rosetting with
anti-CD11b and -CD6 antibodies. The experimental procedure of immune
rosetting was always carried out on the second or third harvest after a
sufficient amount of unmodified CPCs were used or stored in liquid
nitrogen. As shown in Table 1, 18 experiments were performed with a median starting cellularity of 41.3 × 109 (range, 29.7 to 58.2) nucleated cells. After
immune rosetting, the median reduction of the initial cellularity was
91% (range, 72 to 97) and the cell loss was mainly due to a marked
(more than 85%) depletion of myeloid cells and T lymphocytes. A
parallel enrichment of CD34+ cells was obtained (more than
10-fold) with a median recovery of HPCs of 92% (range, 67 to 100).
Similar results were obtained by using autologous CPCs collected in
T-ALL patients after high-dose chemotherapy and G-CSF administration.
Due to the effect of high-dose chemotherapy, the starting cellularity,
as well as the absolute number of CD3+ cells, was lower
than that observed in the apheresis obtained from normal allogeneic
donors, whereas the amount of CD34+ cells was significantly
higher (Table 1). Nonetheless, the debulking effect of the procedure
(88% overall cell loss, with more than 84% of mean CD3+
cell loss), as well as the absolute recovery and the enrichment fold of
CD34+ cells, was remarkably similar. In patients with B
precursor ALL and B-NHL the apheretic products were debulked by immune
rosetting with RBCs coated with CD11b only. Also, in this case, the
initial cellularity and the absolute number of T and B lymphocytes were less abundant as compared to normal donors. As expected, the loss of
CD3+ cells was lower, even though the overall reduction
obtained by immune rosettes was similar.
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Table 1.
Effect of Immune Rosettes on Cellularity of
Leukapheresis Obtained From Normal Donors, T- or B-Precursor Acute
Lymphoblastic Leukemia (ALL), B-Non-Hodgkins' Lymphoma (B-NHL) and
Multiple Myeloma (MM) Patients
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Highly efficient depletion of T cells by anti-CD2-CD7 or B cells by
anti-CD19 magnetic microbeads.
To purge the contaminating normal or neoplastic T lymphocytes still
present in the apheretic products after the debulking procedure with
immune rosettes, partially purified allogeneic (9 experiments from
normal donors) or autologous (7 experiments from T-ALL patients) CPCs
were incubated with a mixture of anti-CD2 and -CD7 MoAbs and indirectly
stained with goat antimouse magnetic microbeads. After loading onto a
D-type depletion column, normal or leukemic contaminating T cells were
significantly removed as judged by staining with anti-CD3 MoAbs
(Table 2) and other T-cell-specific antigens like CD5, CD4, and CD8 (data not shown). The overall T-cell
depletion obtained by the two combined procedures allowed a final 3 to
4 logs reduction of the T-cell content. Despite such aggressive removal
of T cells, the median overall recovery of CD34+ cells was
above 70% in both autologous and allogeneic CPCs (Table 2). Similar
experiments were performed to remove B cells in CPCs obtained from
patients with B-precursor ALL, B-NHL, and MM. As shown in
Table 3, the percent and the absolute
number of CD19+ cells detectable after this purification
approach were very limited. Again, the absolute recovery of
CD34+ cells from the initial leukapheresis was excellent,
with a mean value above 80%.
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Table 2.
T-Cell Depletion by Immune Rosettes and T-cell-specific
Magnetic Microbeads (CD2 + CD7) Obtained From Normal Donors and
T-ALL Patients
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Table 3.
B-Cell Depletion by Immune Rosettes and B-Cell-Specific
Magnetic Microbeads (CD19) of Leukaphereses Obtained From B-NHL,
B-precursor ALL, or MM Patients
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Evaluation of MRD.
A PCR-based quantitation of MRD was performed on CPC samples obtained
from five patients before and after depletion of contaminating tumor B
or T lymphocytes by purging with lineage-specific immunobeads. Molecular analysis was performed by demonstration of chimeric genomic
products generated by the t(14;18) chromosomal translocation or by
analysis of leukemia-specific DNA sequence, amplified from the
rearranged TCR , , or IgH chain genes. As shown in
Table 4, after the purification procedure,
CD19-purged HPCs obtained from two follicular NHLs and two B-precursor
ALLs were judged as PCR negative within the sensitivity limits of our
assays (between 10-4 to 10-5). Similarly, after
purging with T-cell-specific microbeads the PCR evaluation of DNA
samples from a T-ALL patient showed the absence of the
leukemic-specific clone (Table 4 and Fig
1). Interestingly, in this case the amount of MRD was very limited and
not detectable if searched within the whole unmodified apheretic
product (Fig 1). However, the leukemic contamination was clearly shown
in the unwanted, wasted, T-cell fraction retained within the magnetic column. Similar results were obtained in all the analyzed cases in
which the neoplastic B-lymphoid cells were similarly trapped within the
depletion column (Table 4).
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Detection of MRD by PCR analysis of V 1-J 1 junction of the TCR gene in a T-ALL patient. DNA samples were
extracted from bone marrow aspirates performed at the onset of the
disease (Diagnosis), at the end of induction chemotherapy when it was considered in complete hematologic remission (CR), from unmanipulated G-CSF mobilized CPC obtained after a consolidation course with high
dose chemotherapy (Apheresis), from cells coated by anti-CD2 and -CD7
magnetic microbeads and retained within the depletion column (Unwanted
cells), from the purified T-cell depleted fraction of circulating
progenitor cells (Purified CPC), and from the peripheral blood
lymphocytes of a normal donor (PBL). The sensitivity of the PCR
reaction was checked by serial log dilution of the patient's DNA
obtained at diagnosis with DNA from a normal donor (lower lane of the
figure). PCR products were blotted onto nylon membranes and hybridized
with the indicated clonospecific probe labeled with ( - 32 P) ATP.
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Transplantation of T- or B-cell purged autologous and allogeneic
CPC.
We performed autologous transplantation in three patients by using CPCs
highly purified by immune rosetting and immunomagnetic purging. In the
case of the T-ALL patient presented in Fig 1, after a myeloablative
therapy with high-dose Ara-C (2 g/m2 × 2/d for 6 days) and fractionated total body irradiation (TBI, 12cGy), the
purified, leukemia-free fraction of HPCs obtained at the end of the
purification procedure (6.8 × 106 /kg
CD34+ cells) was autografted. The neutrophil engraftment
(more than 1.5 × 109/L) was observed after 12 days
and more than 20 and 100 × 109 /L platelets were
counted at days +15 and + 30, respectively. Interestingly, the
molecular evaluation of the bone marrow 100 days after transplantation
did not show persistence of leukemic cells within the patient (data not
shown). Similar results were obtained in two MM patients resistant to
conventional chemotherapy and who underwent transplantation after a
high-dose Melphalan (200 mg/m2) conditioning regimen, with
CD19 and CD56 purged stem cells.26,27 The numbers of
infused CD34+ cells were 8.3 and 10 × 10 6 /kg CD34+ cells, respectively. A rapid
hematologic engraftment was observed in both patients, because more
than 1.5 × 109/L neutrophils were counted at days +10
and +11 and more than 20 × 109/L platelets were
counted at day +14 (Table 5).
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Table 5.
Hematologic Reconstitution After Autologous
Transplantation On Lineage-Specific Purging With Magnetic
Microbeads
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Because the use of unmodified allogeneic CPC has been possibly
associated with increased chronic GVHD28 in six acute
leukemia patients undergoing transplantation from HLA-identical
siblings, allogeneic CPCs were extensively T-cell depleted by immune
rosetting and immunomagnetic purging. However, to prevent an increased
rate of graft failure and leukemia relapse,29 escalating
moderate30 amounts of donor T lymphocytes (from a minimum
of 2.5 to a maximum of 50 × 106 /kg) were rescued
from the rosetted cells (by hypotonic lysis with NH4 Cl
buffer, as described in Materials and Methods), enumerated by
immunophenotyping with anti-CD3 antibody, and added back to the stem
cell fraction just before cryopreservation. Only two apheretic
procedures were necessary to infuse more than 5 × 106
/kg CD34+ cells and despite the in vitro manipulation, a
prompt hematologic reconstitution was observed in each patient (Table
6). According to previously published
experience,16,17 a conventional GVHD prophylaxis with a
combination of Methotrexate and Cyclosporine A was
performed20,31 and neither acute (more than grade I) or
chronic GVHD was observed in these patients.
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Table 6.
Effect of T-Cell Dose on the Quality and Timing of
Short-Term Hematological Engraftment after CPC Allogeneic
Transplantation
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| |
DISCUSSION |
The manipulation of specific cell subpopulations of marrow or
peripheral blood origin has become an interesting way to increase the
applicability and reduce the toxicity of hematopoietic transplantation. However, the manipulation of CPCs obtained from G-CSF treated normal
donors or patients is hampered by the huge cellularity present in the
apheretic products, which often exceeds the absolute number of 50 × 109 cells, and by the fact that under the
stimulatory effect of G-CSF, mature myeloid cells (mostly granulocytes)
acquire different characteristics of cell density preventing their
sedimentation on normal Ficoll gradients. Therefore, two main
approaches to the CD34+ purification have been taken either
by positive or by negative selection. Indeed, in the perspective of
genetic manipulation, positive selection of CD34+ cells is
likely to be the ideal option to ensure that the selected gene is
transduced only in the small target population of pluripotent progenitor cells.32 Although transplantation of positively
selected CD34+ cells purified by immunoaffinity columns or
immunomagnetic bead adsorption has been shown as a feasible
procedure,4,9,11-15 the specific depletion of unwanted
cells seems preferable for several reasons including the preservation
of the manipulation of CD34+ cells from binding with murine
MoAbs and the need of their subsequent detachment by using chemical or
physical methods. Moreover, most acute leukemias of both myeloid and
lymphoid origin are positive for CD34 antigen expression thus reducing
in this setting the clinical applicability of methods that rely only on
the positive selection of the stem cell fraction. On the contrary, the
selective elimination of residual neoplastic cells detectable in the
stem cell fraction obtained from some patients with MM,33
follicular lymphoma,34 and breast cancer35
could be achieved by the use of MoAbs either in association with
complement,36 conjugated to toxic compounds,37
or by the use of magnetic microbeads proven to eradicate the neoplastic
clone at the molecular level.25,38 However, it has to be
mentioned that positive selection of CD34+ cells, combined
with negative depletion steps, could also achieve high levels of
purity,39 even though purging strategies based only on
depletion techniques could avoid an extensive removal of T cells from
the autograft of patients with B-lymphoproliferative disorders and
solid tumors, thus reducing the risk of long-lasting immunodeficiency.
In this manuscript, we described a simple and reproducible method based
on a two-step negative selection of cytokine mobilized circulating
progenitor cells. With the first step of immune rosetting and
subsequent Ficoll gradients we were able to obtain a drastic reduction
of the massive starting cellularity of the leukapheretic products. The
use of human RBCs suitable for transfusion and the fact that the whole
procedure was performed by using clinical grade devices without the use
of tubes or pipettes reduces the risk of microbiological contaminations
and represents a crucial advantage of this approach. Most importantly,
the consistent reduction of the mature myeloid cells without a
significant loss of CD34+ cells allows a subsequent further
effective purification of the human hematopoietic stem cells either by
positive selection of the CD34+ cells or by further purging
of the contaminating T- or B-cell fraction depending on the specific
medical need. The results we obtained showed that we can specifically
and reproducibly remove from the graft several logs of normal T cells
as well as of contaminating tumor cells as assessed by PCR analysis.
The infusion of sibling-matched allogeneic hematopoietic stem cells
purified according to this method, along with the add back
of escalating amounts of T cells, was followed by a rapid hematologic engraftment, thus confirming the lack of any toxicity associated with the purification procedure.
In the setting of autologous transplantation, our data showed that an
efficient purification of neoplastic B- or T-lymphoid cells could be
reached and in the five cases analyzed at the molecular level, a
disappearance of the neoplastic clone could be shown. Several obvious
advantages are potentially associated with the use of purified stem
cells for autologous hematopoietic transplantation in acute
leukemia40 and lymphoma.41 We know that many
patients can eventually relapse and die from their disease because of
the infusion of tumor cells along with the autologous
graft.42,43 Indeed, MRD persistence after autologous
transplantation in NHL patients is associated with poor outcome, thus
suggesting that neoplastic contamination of the graft should be
eradicated for cure.41 The possibility of eradicating the
residual leukemic cells in the apheretic product represents an
important result because very little data about effective immunologic
purging of ALL are available so far. Obviously, whereas these data
represent a clear demonstration of the technical feasibility of a rapid and reproducible two-step purging procedure of cytokine-mobilized HPCs,
the clinical outcome of autologous and allogeneic transplants in the
diverse proposed clinical settings waits for future validation.
| |
FOOTNOTES |
Submitted June 12, 1997;
accepted October 28, 1997.
Supported in part by grants from the Associazione Italiana per la
ricerca contro il cancro (AIRC); the Consiglio Nazionale per le
Ricerche (CNR) Target Project on Biotechnology; the Associazione Paolo
Belli, Lotta alla Leucemia; and Fondazione Tettamanti.
Address correspondence to Alessandro Rambaldi, MD, Division of
Hematology, Ospedali Riuniti di Bergamo, Largo Barozzi, 1, 24100 Bergamo, 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.
| |
REFERENCES |
1.
Krause DS,
Fackler MJ,
Civin CI,
May S:
CD34: Structure, biology, and clinical utility.
Blood
87:1,
1996[Free Full Text]
2.
To LB,
Haylock DN,
Simmons PJ,
Juttner CA:
The biology and clinical use of blood stem cells.
Blood
89:2233,
1997[Free Full Text]
3.
Lowemberg B,
Voogt P:
Autologous stem cell transplantation and purging.
J Clin Oncol
14:2194,
1996[Medline]
[Order article via Infotrieve]
4.
Link H,
Arseniev L,
Bahre O,
Kadar JG,
Diedrich H,
Poliwoda H:
Transplantation of allogeneic CD34+ blood cells.
Blood
87:4903,
1996[Abstract/Free Full Text]
5.
Dilloo D,
Rill D,
Entwistle C,
Boursnell M,
Zhong W,
Holden W,
Hommaday M,
Inglis S,
Brenner M:
A novel Herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.
Blood
89:119,
1997[Abstract/Free Full Text]
6.
Ward M,
Richardson C,
Pioli P,
Smith L,
Podda S,
Goff S,
Hesdorffer C,
Bank A:
Transfer and expression of human multiple drug resistance gene in human CD34+ cells.
Blood
84:1408,
1994[Abstract/Free Full Text]
7.
Shpall EJ,
Jones RB:
Release of tumors cells from bone marrow.
Blood
83:623,
1994[Free Full Text]
8.
Brugger W,
Bross KJ,
Glatt M,
Weber F,
Mertelsmann R,
Kanz L:
Mobilization of tumors cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors.
Blood
83:636,
1994[Abstract/Free Full Text]
9.
Lemoli RM,
Fortuna A,
Motta MR,
Rizzi S,
Giudice V,
Nannetti A,
Martinelli G,
Cavo M,
Amabile M,
Mangianti S,
Fogli M,
Conte R,
Tura S:
Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: Positive selection and transplantation of enriched CD34+ cells to remove circulating tumors cells.
Blood
87:1625,
1996[Abstract/Free Full Text]
10.
Rill DR,
Santana VM,
Roberts WM,
Nilson T,
Bowman LC,
Krance RA,
Heslop HE,
Moen RC,
Ihle JN,
Brenner MK:
Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells.
Blood
84:380,
1994[Abstract/Free Full Text]
11.
Gorin NC,
Lopez M,
Laporte JP,
Quittet P,
Lesage S,
Lemoine F,
Berenson RJ,
Isnard F,
Grande M,
Stachowiak J,
Labopin M,
Fouillard L,
Morel P,
Jouet JP,
Noel-Walter MP,
Detourmignies L,
Aoudjhane M,
Bauters F,
Najman A,
Douay L:
Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma.
Blood
85:1647,
1995[Abstract/Free Full Text]
12.
Shpall EJ,
Janes RB,
Bearman SJ,
Franklin WA,
Archer PG,
Curiel T,
Bitter M,
Claman HN,
Stemmer SM,
Purdy M,
Myers SE,
Hami L,
Taffs S,
Heimfeld S,
Hallagan J,
Berenson RJ:
Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: Influence of CD34-positive pheripheral-blood progenitores and growth factors on engraftment.
J Clin Oncol
12:28,
1994[Abstract]
13.
Berenson RJ,
Besinger WI,
Hill RS,
Andrews RG,
Garcia-Lopez J,
Kalamasz DF,
Still BJ,
Spitzer G,
Buckner CD,
Bernstein ID,
Thomas ED:
Engraftment after infusion of CD34+ cells in patients with breast cancer or neuroblastoma.
Blood
84:1241,
1994
14.
Berger KM,
Rapatel C,
de Lumley L,
Lutz P,
Plantaz D,
Vannier JP,
Bergeron C,
Mechinaud F,
Favrot M,
Bonhomme J,
Travade P,
Demeoco F:
CD34+ cell immunoselection from G-CSF-alone-primed peripheral blood in children with low body mass.
Br J Haematol
91:431,
1995[Medline]
[Order article via Infotrieve]
15.
Alcorn MJ,
Holyoake TL,
Richmond L,
Pearson C,
Farrel E,
Kyle B,
Dunlop DJ,
Fitzsimons E,
Steward WP,
Pragnell IB,
Franklin IM:
CD34-positive cells isolated from cryopreserved pheripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.
J Clin Oncol
14:1839,
1996[Abstract/Free Full Text]
16.
Bensinger WI,
Buckner CD,
Shanon-Dorcy K,
Rowley S,
Appelbaum FR,
Benyunes M,
Clift R,
Martin P,
Derimer T,
Storb R,
Lee M,
Schiller G:
Transplantation of allogeneic CD34+ pheripheral blood stem cells in patients with advanced hematologic malignancy.
Blood
88:4132,
1996[Abstract/Free Full Text]
17.
Urbano-Ispizua A,
Rozman C,
Martinez C,
Marin P,
Briones J,
Rovira M,
Feliz P,
Viguria MC,
Merino A,
Sierra J,
Mazzara R,
Carrerar E,
Montserrat E:
Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood.
Blood
89:3967,
1997[Abstract/Free Full Text]
18.
Delain M,
Cahn JY,
Racadot E,
Flesch M,
Plouvier E,
Mercier M,
Tiberghien P,
Pavy JJ,
Deschaseaux M,
Deconinck E,
Couteret Y,
Brion A,
Herve P:
Graft failure after T cell depleted HLA identical allogeneic bone marrow transplantation: Risk factors in leukemic patients.
Leuk Lymphoma
11:359,
1993[Medline]
[Order article via Infotrieve]
19.
Cortelazzo S,
Viero P,
Bellavita P,
Rossi A,
Buelli M,
Borleri GM,
Marchioli S,
Bassan R,
Comotti B,
Rambaldi A,
Barbui T:
Granulocyte colony stimulating factor following pheripheral blood progenitor cells transplant in non-Hodgkin's lymphoma.
J Clin Oncol
13:935,
1995[Abstract]
20.
Rambaldi A,
Viero P,
Bassan R,
Buelli M,
Rossi A,
Bellavita P,
Spinelli O,
Amaru R,
Micheletti M,
Borleri G,
Cortelazzo S,
Comotti B,
Barbui T:
G-CSF-mobilized pheripheral blood progenitor cells for allogeneic transplantation of resistant or relapsing acute leukemias.
Leukemia
10:860,
1995
21.
Anderson KC,
Griffin JD,
Bates MP,
Slaughenhoupt BL,
Schlossman SF,
Nadler LM:
Isolation and characterization of human B lymphocyte enriched population. I. Purification of B cells by immune rosette depletion.
J Immunol Methods
61:283,
1983[Medline]
[Order article via Infotrieve]
22.
Van Camp B,
Durie BGM,
Spier C,
De Waele M,
Van Riet I,
Vela E,
Fruttiger Y,
Richter L,
Grogan TM:
Plasma cells in multiple myeloma express a natural killer cell-associated antigen: CD56 (NKH-1; Leu-19).
Blood
76:377,
1990[Abstract/Free Full Text]
23.
Biondi A,
Rambaldi A:
Polymerase chain reaction (PCR) approach for the evaluation of minimal residual disease in acute leukemia.
Stem Cells
12:394,
1994[Abstract]
24.
Steward CG,
Potter MN,
Oakhill A:
Third complementary determining region (CDRIII) sequence analysis in childhood B-lineage acute lymphoblastic leukemia: Implication for the design of oligonucleotide probes for use in monitoring minimal residual disease.
Leukemia
6:1213,
1992[Medline]
[Order article via Infotrieve]
25.
Gribben JG,
Saporito L,
Barber M,
Blake KW,
Edwards RM,
Griffin JD,
Freedman AS,
Nadler LM:
Bone marrow of non-Hodgkin's lymphoma patients with a bcl-2 translocation can be purged of ploymerase chain reaction-detecable lymphoma cells using monoclonal antibodies and immunomagnetic beads depletion.
Blood
80:1083,
1992[Abstract/Free Full Text]
26.
Bergsagel PL,
Smith AM,
Szczepeck A,
Mant MJ,
Belch AR,
Pilarski LM:
In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56 and monotypic Ig light chain.
Blood
85:436,
1995[Abstract/Free Full Text]
27.
Szczepek AJ,
Bergsagel PL,
Axelsson L,
Brown CB,
Belch AR,
Pilarski LM:
CD34+ cells in the blood of patients with multiple myeloma express CD19 and IgH mRNA and have patients-specific IgH VDJ gene rearrangements.
Blood
89:1824,
1997[Abstract/Free Full Text]
28.
Majolino I,
Saglio G,
Scimé R,
Serra A,
Cavallaro AM,
Fiandaca T,
Vasta S,
Pampinella M,
Catania P,
Indovina A,
Marcenò R,
Santoro A:
High incidence of chronic GVHD after primary allogeneic peripheral blood stem cells transplantation in patients with hematologic malignancies.
Bone Marrow Transplant
17:555,
1996[Medline]
[Order article via Infotrieve]
29.
Marmont A,
Horowitz MM,
Gale RP,
Sobocinski K,
Ash RC,
Bekkum DW,
Champlin RE,
Dicke KA,
Goldman RA,
Herzing RH,
Hong R,
Masaoka T,
Rimm AA,
Ringden O,
Speck B,
Weiner RS,
Bortin MM:
T-cell depletion of HLA identical transplant in leukemia.
Blood
78:2120,
1991[Abstract/Free Full Text]
30.
Verdonck LF,
Dekker AW,
de Gast GC,
van Kempen ML,
Lokhorst HM,
Nieuwenhuis HK:
Allogeneic bone marrow transplantation with a fixed low number of T cells in the marrow graft.
Blood
83:3090,
1994[Abstract/Free Full Text]
31.
Storb R,
Deeg HJ,
Whithead J,
Appelbaum F,
Beatty P,
Bensinger WI,
Buckner CD,
Clift R,
Doney K,
Farewell V,
Hansen J,
Hill R,
Lum R,
Martin P,
McGuffin R,
Sanders J,
Stewart P,
Sullivan K,
Witherspoon R,
Yee J,
Thomas ED:
Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute-graft-versus-host disease after marrow transplantation for leukemia.
N Engl J Med
314:729,
1986[Abstract]
32.
Dunbar CE,
Cottler-Fox M,
O'Shaunessy JA,
Doren S,
Carter C,
Berenson R,
Brown S,
Moen RC,
Greenblatt J,
Stewart FM,
Leitman SF,
Wilson VH,
Cowan K,
Young NS,
Nienhuis AW:
Retrovirally marked CD34-enriched peripheral blood and marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85:3048,
1995[Abstract/Free Full Text]
33.
Willems P,
Croockewit R,
Raymarkers R,
Holdrinet G,
van der Bosh G,
Huys E,
Mesink E:
CD34+ selection from myeloma peripheral blood cell autografts contain residual tumor cells due to impurity, not to CD34+ myeloma cells.
Br J Haematol
93:613,
1996[Medline]
[Order article via Infotrieve]
34.
Macintyre EA,
Belanger C,
Debert C,
Canioni D,
Turhan AG,
Azagury M,
Hermine O,
Varet B,
Flandrin G,
Schmitt C:
Detection of clonal CD34+19+ progenitors in bone marrow of BCL2-IgH-positive follicular lymphoma patients.
Blood
86:4691,
1995[Abstract/Free Full Text]
35.
Mapara MY,
Korner IJ,
Hildebranndt M,
Bargou R,
Krahl D,
Reichardt P,
Dorken B:
Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cell from leukapheresis products in breast cancer patients: Comparison of immunocytochemical tumor cell staining and reverse transcriptase-polimerace chain reaction.
Blood
89:337,
1997[Abstract/Free Full Text]
36.
Anderson KC,
Ritz J,
Takvorian T,
Coral F,
Daley H,
Gorgone BC,
Freedman AS,
Canellos GP,
Schlossman SF,
Nadler LM:
Hematologic engraftment and immune reconstitution posttransplantation with anti-B1 purged autologous bone marrow.
Blood
69:597,
1987[Abstract/Free Full Text]
37.
Uckun FM,
Reaman GH:
Immunotoxins for treatment of leukemia and lymphoma.
Leuk Lymphoma
18:195,
1995[Medline]
[Order article via Infotrieve]
38.
Di Nicola M,
Siena S,
Corradini P,
Bregni M,
Milanesi M,
Magni M,
Ruffini PA,
Ravagnani F,
Tarella C,
Gianni AM:
Elimination of bcl-2-positive follicular lymphoma cells from blood transplants with high recovery of haematopoietic progenitors by the Miltenyi CD34+ cell sorting system.
Bone Marrow Transplant
18:1117,
1996[Medline]
[Order article via Infotrieve]
39.
Bertolini F,
Thomas T,
Battaglia M,
Gibelli N,
Pedrazzoli P,
Robustelli della Cuna G:
A new two-step procedure for 4.5 log depletion of T and B cells in allogeneic transplantation and of neoplastic cells in autologous transplantation.
Bone Marrow Transplant
19:615,
1997[Medline]
[Order article via Infotrieve]
40.
Soiffer RJ,
Roy DC,
Gonin R,
Murray C,
Anderson KC,
Freedman AS,
Babinowe SAN,
Roberts MJ,
Spector N,
Pesek K,
Mauch P,
Nadler LM,
Ritz J:
Monoclonal antibody-purged autologous bone marrow transplatation in adults with acute lymphoblastic leukemia at high risk of relapse.
Bone Marrow Transplant
12:243,
1993[Medline]
[Order article via Infotrieve]
41.
Gribben JG,
Freedman AS,
Neuberg D,
Roy DC,
Blake KW,
Woo SD,
Grossbard ML,
Rabinowe SN,
Coral F,
Freeman GJ,
Ritz J,
Nadler LM:
Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma.
N Engl J Med
325:1525,
1991[Abstract]
42.
Brenner MK,
Rill DR,
Moen RC,
Krance RA,
Mirro J,
Anderson WF,
Ihle JN:
Gene-marking to trace origin of relapse after autologous bone-marrow transplantation.
Lancet
341:85,
1993[Medline]
[Order article via Infotrieve]
43.
Deiseroth AB,
ZU Z,
Claxton D,
Hanania EG,
Fu S,
Ellerson D,
Goldberg L,
Thomas M,
Janicek K,
Anderson WF,
Hostor J,
Korbling M,
Durett A,
Monn R,
Berenson R,
Hoinfeld S,
Hamer J,
Calvert L,
Tibbits P,
Talpaz M,
Kantarjian H,
Champlin R,
Reading C:
Genetic marking shows that Ph+cells present in autologous transplants of chronic myelogenous leukemias (CML) contribute to relapse after autologous bone marrow in CML.
Blood
83:3068,
1994[Abstract/Free Full Text]
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Abstract
Introduction
Abstract