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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3123-3130
Partially Mismatched Pediatric Transplants With Allogeneic
CD34+ Blood Cells From a Related Donor
By
Yoshifumi Kawano,
Yoichi Takaue,
Arata Watanabe,
Osamu Takeda,
Kohji Arai,
Etsuro Itoh,
Yuhju Ohno,
Takanori Teshima,
Mine Harada,
Tsutomu Watanabe,
Yasuhiro Okamoto,
Takanori Abe,
Teruyuki Kajiume,
Takeji Matsushita,
Kazuma Ikeda,
Mikiya Endo,
Yasuhiro Kuroda,
Shigetaka Asano,
Ryuji Tanosaki,
Ken Yamaguchi,
Ping Law, and
John D. McMannis
From the Department of Pediatrics, University of Tokushima,
Tokushima; National Cancer Center Hospital, Tokyo; the Department of
Pediatrics, University of Akita, Akita; the Department of Pediatrics,
University of Hirosaki, Aomori; Kitakyushu Medical Center, Fukuoka; the
Second Department of Internal Medicine, University of Okayama, Okayama;
the Department of Pediatrics, International Medical Center of Japan,
Tokyo; the Division of Blood Transfusion, Kagawa Medical School,
Kagawa; the Department of Pediatrics, Iwate Medical University, Iwate;
the Institute of Medical Sciences, University of Tokyo, Tokyo, Japan;
University of California San Diego, Blood and Marrow Transplantation
Program, San Diego, CA; and Baxter Healthcare Corporation, Irvine, CA.
 |
ABSTRACT |
This was a phase I, multi-center study of 13 pediatric patients
(median age, 11 years) to evaluate toxicity, hematopoietic recovery,
and graft-versus-host disease (GVHD) after allogeneic transplantation
of enriched blood CD34+ cells obtained from genotypically
haploidentical but partially HLA-mismatched related donors (8 parents
and 5 siblings). With regard to rejection, donor HLA disparity was 1 (5), 2 (6), or 3 loci (2). With regard to GVHD, recipient HLA disparity
was 0 (1), 1 (3), 2 (8), or 3 (1). The patients suffered from acute myelogenous leukemia (6), chronic myelogenous leukemia (4), acute lymphoblastic leukemia (2), or hemolytic anemia plus immunodeficiency disorder (1). To reduce the risk of graft failure through the infusion
of a large amount of stem cells, peripheral blood cells (PBC) were
mobilized by recombinant granulocyte colony-stimulating factor (G-CSF; lenograstim, 10 µg/kg/d for 5 days) and
collected by 2 to 5 aphereses. To both enhance engraftment and reduce
GVHD, CD34+ cells were enriched using immunomagnetic
procedures with the Baxter ISOLEX 300 system (Baxter Healthcare Corp,
Irvine, CA) and cryopreserved. After variable cytoreductive regimens, a
median of 7.7 (range, 2.2 to 14) × 106/kg of
CD34+ cells and 1.03 (0.05 to 2.09) × 105/kg CD3+ cells were infused. Using
Center-specific posttransplant supportive care and immunosuppressive
GVHD prophylaxis, two patients experienced early death; one from
veno-occlusive disease at day 17 and one from sepsis at day 18. Nine of
11 patients showed signs of engraftment; however, subsequent rejection
was seen in 4 patients, 2 of whom had autologous recovery. Eight
patients were evaluated in the early phase of marrow recovery. The
median number of days to achieve an absolute granulocyte count of 0.5 × 109/L was 14 (range, 9 to 20) and that to
achieve a platelet count of 20 × 109/L was 17.5 (range,
12 to 23). Donor chimerism persisted in five patients until death or
current survival. All of the surviving patients with
functioning-donor-type hematopoiesis were given total body
irradiation. De novo acute GVHD (grades II and IV) was observed in two
of the eight evaluated patients. Scheduled donor lymphocyte infusion
(DLI), using the CD34 fraction, was administered to four
patients, free of de novo acute GVHD, beginning between 28 to 43 days
after transplant. Three of these patients developed acute GVHD (grades
I, II, and IV). Cytomegalovirus infection was a major infectious
complication but was successfully managed with  -globulin and
gancyclovir treatment with or without additional DLI. Five patients are
currently surviving, free of disease, with a follow-up ranging from 476 to 937 days. Each survivor has functioning hematopoiesis, three of
donor origin and two of autologous origin. In conclusion, our results
show that enriched blood CD34+ cells from a mismatched
haploidentical donor are a feasible alternative source of stem cells,
but do not appear to ensure engraftment. Because none of the patients
who were administered DLI survived, the therapeutic efficacy and safety
of periodic DLI, as an integrated part of such transplants, needs to be
clarified in further studies.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ONE MAJOR LIMITATION of allogeneic bone
marrow transplantation (BMT) is the lack of suitable donors. The
results of transplantation with unrelated marrow grafts are, in
general, inferior to those using matched sibling marrow, with an
increased incidence and severity of graft-versus-host disease (GVHD),
graft rejection, and infections,1-4 although new
immunosuppressive therapies have made the prophylaxis or treatment of
GVHD more effective. In addition, the search for an unrelated matched
donor is time-consuming, and rapid disease progression in some patients
makes this approach impractical. Alternative procedures include
transplanting cells from a related mismatched donor or cord blood
cells. The frequency of serious GVHD is reported to be far lower in
transplants with readily available cord blood cells, but this procedure
is limited by rejection or slow engraftment, possibly due to the small
content of stem cells in the graft.5
The use of an HLA-mismatched related donor avoids the lengthy search
procedure and provides donors for greater than 90% of patients who may
potentially benefit from allogeneic transplantation.6 Depletion of T lymphocytes (T cells) from the graft can reduce the
number of T cells to below the critical threshold needed for the
development of severe de novo GVHD, and this has proved to be the most
effective method for preventing acute GVHD in BMT.7 However, this procedure is associated with an increased incidence of
graft rejection, as well as with an increase in leukemia relapse and
B-cell lymphoproliferative disorders (BLPD).
Mobilized allogeneic peripheral blood cells (PBC) have recently been
shown to be a potential source of stem cells for hematopoietic reconstitution after myeloablative therapy.8,9 Most
importantly, more hematopoietic cells can be collected from blood than
from BM or cord/placenta. Nevertheless, substantially more T cells than
BM are in PBC grafts, which may lead to a higher risk of de novo GVHD.
In a previous study, we showed that negative depletion of T cells is
unsatisfactory with PBC grafts, and is associated with a
significant loss of hematopoietic progenitor cells.10 On
the other hand, recently developed techniques for the positive selection/enrichment of CD34+ cells from marrow or PBC
provide a convenient method for concentrating hematopoietic progenitors
and depleting T cells.11
The present phase I study of 13 children was designed to examine the
safety and effectiveness of blood CD34+ cells enriched from
genotypically haploidentical but partially mismatched related donors.
To prevent serious cytomegalovirus (CMV) infection and BLPD, and to
induce a graft-versus-leukemia (GVL) effect, patients who did not
develop de novo GVHD by day 28 were scheduled to receive periodic
infusion of an escalating dose of thawed donor lymphocytes contained in
CD34 cells, starting from 1 × 105/kg. The primary goal of this study was to examine
whether this new approach could provide a safe and effective graft
while avoiding serious GVHD. We believe that the results of this small
trial may be useful for the development of strategies for the clinical application of enriched CD34+ cells.
| |
MATERIALS AND METHODS |
Human subjects and histocompatibility studies.
Patients who were currently eligible for allogeneic transplant were
enrolled into the study. All of the patients lacked an HLA-identical
sibling or unrelated donor, but did have a readily available healthy
donor within the family who met the donation criteria. In preparation
for the donation, the donor underwent a detailed medical history and
physical examination according to the institution's standard
procedures.
A total of 13 patients, 9 male and 4 female, were treated in
participating institutes with the approval of the respective institutional review boards. The patients ranged in age from 1 to 18 years, with a median of 11 years, and their clinical characteristics are shown in Table 1. The diagnosis
included acute myelogenous leukemia (AML 6; M1 3, M4 1, M5 2), chronic
myelocytic leukemia (CML 4; 2 in the chronic phase and 2 at blastic
crisis), acute lymphoblastic leukemia (ALL, 2), and congenital
immunodeficiency disorder associated with intractable hemolytic anemia
(1). The corresponding donors ranged from 5 to 43 years old (median,
35). HLA-A and -B were typed by serological tests and DRB1 typing was performed using DNA-based techniques. Mixed lymphocyte culture was not
performed for each patient/donor pair. With regard to rejection, donor
HLA disparity was 1 (5), 2 (6) or 3 loci (2). With regard to GVHD,
recipient HLA disparity was 0 (1), 1 (3), 2 (8), or 3 (1).
The administration of human recombinant granulocyte colony-stimulating
factor (G-CSF; lenograstim, Chugai Pharmaceutical Co, Tokyo, Japan) to
healthy donors, collection of PBC, and subsequent transplants with
enriched CD34+ cells were all approved by the institutional
review boards of the participating hospitals. Written informed consent
was obtained from all of the patients and donors or their guardians.
Mobilization and apheresis of PBC.
All cell-preparation procedures, including mobilization,
CD34+ enrichment, and cryopreservation, were performed at
the University of Tokushima as previously published.12 The
donors received G-CSF 5 µg/kg/twice a day, or 10 µg/kg/once a day by subcutaneous injection for 5 days.13
Apheresis was initiated from day 4 to 6 after G-CSF injection, and 300 mL/kg (maximum, 10 L) were processed per session.12
Apheresis was principally continued until a total of >6 × 106 CD34+ cells/kg recipient weight were
collected. The details of the procedure for collecting PBC using the
Fenwal CS 3000 plus (Baxter Limited, Tokyo, Japan) combined with a
small volume collection chamber have been described
previously.12
Isolation and cryopreservation of CD34+
cells.
PBC collected by apheresis were enriched for CD34+ cells
using the ISOLEX-300 system (Baxter Healthcare Corp, Irvine, CA)
according to the manufacturer's suggestions. Briefly, excess platelets
were removed by centrifugation for 20 minutes at 200g at room
temperature. Cells were incubated in phosphate-buffered saline (PBS;
Nissui, Tokyo, Japan) containing 0.5% human  -globulin
(Gammagard; Baxter, Tokyo, Japan) for 15 minutes to block Fc-receptors.
One vial of anti-CD34 monoclonal antibody (MoAb) (9C5, 2 mg) was added
to the cell suspension which contained < 5 × 1010
cells. After 30 minutes of incubation at room temperature with gentle
rotation (4/min), cells were washed three times with PBS containing 1%
human serum albumin (Albumin-Midori; Green Cross Co, Kyoto, Japan).
Sensitized cells were incubated with sheep anti-mouse
IgG1-coated paramagnetic microspheres (Dynabeads; Dynal, Oslo, Norway; 10 mL). Cells rosetted with beads were
captured on permanent magnets and released by chymopapain or peptide
capture included in the kit.
The enriched CD34+ cells were cryopreserved using the
hydroxyethyl starch (HES)/dimethyl sulphoxide (DMSO) method without
controlled-rate cooling.14 Briefly, CD34+ cells
were resuspended in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% autologous serum. Cells were
slowly mixed with an equal volume of a cryoprotectant solution
containing 8% human albumin, 10% DMSO, and 12% HES to give final
concentrations of 5% DMSO and 6% HES. Cells were then transferred to
5-mL polypropylene tubes and placed directly in an electric freezer
that maintained a temperature of  135°C. The non-CD34 cell
fraction was also cryopreserved in tubes as a T-cell source for donor
lymphocyte infusion (DLI) in selected patients.
Hematopoietic progenitor assay and flow cytometry analysis.
The details of the hematopoietic progenitor assays have been reported
elsewhere.15 Briefly, cells were incubated in
methylcellulose medium supplemented with 20% fetal bovine serum (FBS),
450 µg/mL of human transferrin (T-1145; Sigma Chemical Co, St Louis,
MO), 2 U/mL of human recombinant erythropoietin, 1% crystallized
bovine serum albumin (Calbiochem 12657; Hoechst Japan, Tokyo),
interleukin-3 (IL-3; 20 ng/mL; Kirin Brewery Co, Tokyo, Japan), stem
cell factor (20 ng/mL; Kirin Brewery Co), and G-CSF (20 ng/mL). Cells
were placed in 24-well culture plates (Corning 258201; Corning,
NY) in quadruplicate, and incubated in an ESPEC
N2-O2-CO2 BNP-110 incubator (Tabai
ESPEC Co, Osaka, Japan) which maintained a humid atmosphere of 5%
carbon dioxide, 5% oxygen, and 90% nitrogen. After 14 days of incubation, colony-forming units for granulocyte-macrophage (CFU-GM) were scored using an inverted microscope.
Cells expressing the surface CD34 antigen were identified by flow
cytometry analysis as previously reported.16 One hundred microliters of cell suspension were added to a test tube (Falcon 2052;
Becton Dickinson, Lincoln Park, NJ) containing isotype control (phycoerythrin-mouse IgG1) and phycoerythrin-conjugated
CD34 MoAb (Anti-HPCA2 antibody; Becton Dickinson) at a concentration of 1 µg antibody/106 cells. After 30 minutes of incubation
in the dark, cells were washed twice and resuspended in PBS containing
1% bovine serum albumin (BSA). Red blood cells in the sample were
lysed with a solution of 0.826% (wt/vol) NH4Cl, 0.1%
KHCO3, and 0.004% EDTA-4Na. Samples were analyzed with a
FACScan flow cytometer (Becton Dickinson). A total of 20,000 events
were counted to identify the mononuclear cell (MNC) fraction. The flow
cytometric data were analyzed using a gated analysis via a set of
SSMC-FL parameters for CD34+ cells to calculate the
percentage of positive cells. CD3+ cells were also analyzed
using specific MoAb (OKT3; Ortho, Raritan, NJ).
High-dose chemotherapy and transplant procedures.
All of the patients were treated in laminar air flow rooms and
administered oral nonabsorbable antibiotics. Cytoreductive regimens and
immunosuppressive agents are summarized in
Table 2. Enriched CD34+ cells
were thawed and infused into the recipient without manipulation. Prophylaxis against GVHD included cyclosporine A (CyA) beginning on day
0, at a daily dose of 5 mg/kg (10 patients). Conversion to oral CyA was
deferred until signs of gastrointestinal toxicity subsided. Seven of
the 10 patients were also treated with 10 mg/m2 of
methotrexate (MTX) on days 1, 3, and 6. The other two patients received
tacrolimus (FK506) or methylprednisolone (MPSL) instead of CyA with or
without MTX. One patient was not administered prophylactic immunosuppressants. Eleven patients received G-CSF after
transplantation. Support given to the patients included transfusions of
blood products, antimicrobial drugs, nutritional support, and other
measures as determined by clinical conditions and institutional
protocol, and according to the accepted standards of medical care.
To prevent serious viral infection and BLPD, and/or to induce a
GVL effect, patients who did not develop de novo GVHD by day 28 were
scheduled to receive a weekly infusion of an escalating dose of
cryopreserved/thawed donor CD34 cells containing
lymphocytes, starting at 1 × 105/kg in selected
institutes. No attempt was made to further manipulate the
CD34 fraction. The end point was either development
of GVHD or six weekly infusions.
Assessment of engraftment and GVHD.
The early phase of engraftment was defined as trilineage
transfusion-independent recovery of normal peripheral blood cell counts. Neutrophil engraftment day was defined as the third consecutive day on which the absolute granulocyte count (AGC) exceeded 0.5 × 109/L. Platelet engraftment day was defined as the third
consecutive day on which the count exceeded 20 or 50 × 109/L. Sustained and long-term engraftment was evaluated by
cytogenetic markers or by a DNA analysis for variable number tandem
repeats (VNTR). The severity of acute GVHD was graded according to the Seattle score.17
| |
RESULTS |
Donor response to G-CSF administration.
Although some suffered from a flulike syndrome including mild
lumbago, headache, and fatigue, all of the donors generally tolerated
the mobilization regimen well and none were removed from the protocol.
In all of the donors, the white blood cell (WBC) count started to
increase on the second day of G-CSF injection, and reached a peak value
of 45 (range, 21 to 57) × 109/L on day 5. The
percentage of CD34+ cells in peripheral MNC was less than
0.15% in all of the donors before G-CSF treatment, and this increased
to 1.17% (range, 0.04% to 3.08%) on day 4 or 5. The donors then
underwent an average of 3 aphereses (range, 2 to 5), with no immediate
side effects directly related to the procedure. The numbers of MNC,
CFU-GM, and CD34+ cells collected are shown in
Table 3. The target number of
CD34+ cells was enriched in all but two donors (cases 9 and
13) who had <2 × 106/kg CD34+ cells/kg
after enrichment. These donors underwent a second mobilization by G-CSF
2 weeks later, and the combined number of CD34+ cells then
exceeded 2 × 106/kg.
Enrichment of CD34+ cells.
A total of 30 procedures were performed. The entire procedure, after
apheresis to cryopreservation, was completed within 5 hours and the
enrichment results are summarized in Table 3. The median number of
cells subjected to enrichment was 3.8 (range, 1.2 to 10.4) × 1010, and 2.63 (range, 0.14 to 3.65) × 108 CD34+ cells were recovered with a purity of
80% (range, 19% to 98%). The median yield of CD34+ cells
and CFU-GM was 37% and 28%, respectively. Consequently, an average of
7.0 (range, 2.2 to 14) × 106/kg CD34+
cells and 0.97 (range, 0.05 to 2.09) × 105/kg
CD3+ cells were infused (Table 2).
Regimen-related toxicity (RRT) and engraftment.
Engraftment results are shown in Table 2. On the 28th day after
transplantation, sustained engraftment was examined by cytogenetic markers or DNA analysis. With regard to early engraftment, one patient
(case 1) who underwent transplant at refractory relapse of AML with 9.7 × 106/kg CD34+ cells failed to engraft
after a transient increase in the neutrophil count to 1.1 × 109/L. This patient died of RRT on day 43. Case 3 died of
hepatic veno-occlusive disease (VOD) on day 17 and case 12 died of
complicating sepsis at day 18. Two other patients (cases 8 and 10)
developed autologous recovery of hematopoiesis without a transient
increase in donor-type neutrophils and are currently alive, with no
evidence of malignant disease.
Hence, cases 1, 3, 8, 10, and 12 were considered failed engraftment,
and were excluded from the analysis of early engraftment. The median
number of days to achieve an AGC of 0.5 × 109/L and
platelet counts of 20 × 109/L and 50 × 109/L was, respectively, 14 (range, 9 to 20, n = 9), 17.5 (range, 12 to 23, n = 8), and 22 (range, 12 to 36, n = 6). Case 11 developed late rejection of the graft after transient recovery of
hematopoiesis and subsequently developed functioning autologous
recovery, but died of recurrent leukemia.
GVHD and clinical course.
De novo acute GVHD (grades II and III) was observed in 2 (cases 6 and
7) of the 9 evaluated patients (Table 4).
These patients received cells from matched and one-loci mismatched
donors with regard to GVHD, containing 1.03 and 0.02 × 105/kg CD3+ cells, respectively. Both could be
managed without the additional use of immunosuppressants. Consequently,
the addition of T cells to the CD34 fraction was
tested in 4 of the 8 eligible patients. Two of these 4 patients (cases
4 and 5) developed grade II and IV acute GVHD, respectively, after a
single course of DLI. Although both were successfully managed with
additional immunosuppression, case 4 developed recurrence of leukemia
and eventually died on day 277, and case 5 died of sepsis before
achieving a platelet level of 50 × 109/L on day 116.
One of the 4 patients (case 9) who received DLI showed 100% donor-type
reconstitution on 28 day after transplantation. Four courses of weekly
DLI therapy were suspended when the patient developed grade I GVHD.
Four weeks later, DLI therapy was resumed when the patient was found to
be a chimera (87%) and CMV-antigenemia+ with a titer of
>10/5 × 104 cells; both conditions promptly
resolved after two courses of DLI. Because this patient developed
pancytopenia 10 months after transplantation, we reinfused 6.3 × 108/kg of MNC in CD34 fraction. As a
result of this procedure, he developed acute GVHD combined with
pneumonitis and multi-organ failure, and died on day 363 despite
supportive therapies.
The major infectious complication was cytomegalovirus infection, which
was observed in three patients. All events were promptly resolved with
-globulin and gancyclovir treatment with or without additional DLI.
Currently, five patients are surviving with a follow-up ranging from
476 to 937 days; all of these patients, including two cases with
autologous marrow recovery (cases 8 and 10), are completely free of
disease. Case 8 with CML is in complete remission without any sign of
Ph+ cells. None of these patients has developed BLPD or
chronic GVHD.
| |
DISCUSSION |
The primary cause of mortality in transplantation with a mismatched
donor is engraftment failure due to graft rejection mediated by
residual host T cells resistant to conventional conditioning regimens,
which heavily depend on the degree of donor
incompatibility.18 To reduce the rate of graft failure by
eliminating residual T cells, cytoreductive conditioning regimens can
be intensified by increasing the total dose of total body irradiation
(TBI) or by intensifying immunosuppression thereafter.19
However, increasing the intensity of TBI usually leads to increased
RRT.20 The other primary problem in this setting is the
occurrence of GVHD, which is caused by contaminating T cells in the
graft. To prevent this, various strategies have been attempted with
marrow cells. In one study with pediatric patients, BMT with a
mismatched family donor was associated with a decreased incidence of
serious GVHD compared with historical cohorts when ex vivo and in vitro
T-cell depletion by MoAb was applied.6,21 An alternative
approach includes the indirect depletion of T cells by isolating
CD34+ cells. Additional advantages of the enrichment of
CD34+ cells include a reduction in the volume of cells
which may lead to toxic complications at infusion.22
Removal of contaminating erythrocytes may also be important for
preventing hemolysis at thawing, which may cause acute renal failure,
or for ABO-mismatched grafts between donor and patient. However, this
procedure is associated with an increased incidence of graft rejection,
and an increase in leukemia relapse caused by the lack of a GVL effect
and BLPD.
In this study, we intended to reduce the incidence of graft rejection
by increasing the stem cell dose, which can be achieved using mobilized
PBC. This was based on the idea that standard marrow harvest from the
iliac crests results in a suboptimal cell dose for T-cell-depleted
transplants and causes delayed engraftment. Mobilized PBC have been
widely used for allogeneic transplantation. Although ethical concerns,
including oncogenesis, still exist regarding the use of G-CSF for
normal volunteer donors, we believe that the risk of marrow aspiration
under general anesthesia still exceeds that associated with
G-CSF.23
The primary endpoint of this study with a small number of patients was
feasibility. Although this study was contaminated by an inevitable bias
caused by the variation in conditioning and immunosuppressive regimens,
which may affect our assessment of the development of graft rejection
and GVHD, the data we obtained are still informative. To avoid
long-term toxicities in growing children, half of our patients did not
receive a TBI-containing cytoreductive regimen. However, the primary
cause of mortality in this study was a high incidence of graft
rejection, even though there were only two haploidentical-mismatched
transplants with regard to rejection and we infused a median of 7 × 106/kg CD34+ cells; one patient did not
engraft at all and, despite a rapid early recovery of hematopoiesis,
four patients subsequently rejected the graft. All of the surviving
patients with functioning donor-type hematopoiesis had been treated by
TBI, while engraftment failed in most of those who did not receive TBI.
Henslee-Downey et al24 recently published an update of
their pioneering work in transplantation in mismatched family pairs. Seventy-two uniformly treated patients who underwent allogeneic transplant with T-cell-depleted BM obtained from haploidentical related family members were conditioned with a regimen containing TBI
with follow-up immunosuppression. The median number of
CD34+ cells infused was far lower than 1.36 × 106/kg, but the overall probability of engraftment was 88%
at 32 days, although this decreased to below 70% in three-antigen
rejection mismatched pairs. They suggested that more intense
immunosuppression by the use of a higher dose of TBI is crucial for
supporting engraftment, and this appears to have been confirmed in our
current study. Bacigalupo et al25 reported their experience
in 10 patients using a combination of CD34+ cells purified
from BM and G-CSF-primed peripheral blood in HLA-mismatched pairs.
After conditioning with TBI and ALG, the patients received a median of
5.7 × 106/kg CD34+ cells, and all showed
successful engraftment. Considering all of these results, it appears as
though the content of CD34+ cells in the graft is not the
sole factor that ensures stable engraftment after this mode of
transplantation. It has also been reported that the presence of an
optimal dose of T cells in the graft is another primary factor that
ensures stable engraftment.24,26 Because the development of
significant GVHD was not a major obstacle in our study, it is possible
that our procedure removed most of the accessory cell populations,
which are responsible for GVHD and/or for facilitating
engraftment. In the application of a negative purging system to BM
cells,24 it is probable that only accessory cells
responsible for GVHD were removed without the loss of veto cell
activities, which would thereby facilitate engraftment with a far
smaller number of CD34+ cells.
In contrast to our results, the primary problem in previous studies on
transplantation with isolated CD34+ cells has been the
occurrence of GVHD, even in HLA-identical siblings.27 Most
of the patients treated by Bacigalupo et al developed acute GVHD
greater than grade II while on GVHD prophylaxis with CyA after
conditioning with TBI and ALG.25 The incidence of acute
GVHD in the report by Laport et al,28 who used prophylaxis consisting of CyA with or without MTX, was 42% for grade I-II and 21%
for grade III-IV. Henslee-Downey et al24 reported that a
greater number of T cells in the graft was associated with a higher
risk of severe acute GVHD and that the timing of transplant was the
most important factor in improving long-term survival. In their study,
chronic GVHD occurred in 35% of evaluable patients. On the other hand,
in our series, de novo acute GVHD occurred in two patients. Clinically
significant GVHD developed only after the initiation of scheduled
lymphocyte infusion. Thus, our procedure for the purification of
CD34+ blood was successful in terms of T-cell depletion as
a primary endpoint.
Avoidance of clinically significant GVHD, while retaining a GVL effect,
engraftment, and antiviral potential, is of prime importance. Patients
who show stable mixed chimerism after BMT may benefit from allogeneic
cell therapy with immunocompetent lymphocytes and stem
cells.29 Consequently, we added back a low number of
cryopreserved T cells contained in the CD34 fraction
after achieving stable engraftment, as has been tried clinically in
BMT.30 GVHD may be more likely to occur when the cells are
infused in the initial allograft than if the infusion is delayed until
after initial engraftment and hematological recovery. Although
complications including sepsis or viremia due to profound immunodeficiency have been the primary cause of death in many patients
who have undergone allogeneic transplantation with selected CD34+ cells,25,28 we encountered few such
complications. Although the early application of periodic DLI may
become useful for preventing both late graft rejection and CMV
infection, the optimum dose and time schedule of DLI have not yet been
established.
In conclusion, although our findings must be viewed as preliminary
given the small number of children and short follow-up period, we
showed that at least some groups of children at very high risk of
disease without a matched donor may benefit from our procedure. This
gives us the rationale to continue our feasibility study, because our
approach may provide a readily available donor for most candidates for
allogeneic transplant. The incidence of chronic GVHD and the long-term
stability of hematopoiesis will be determined in a longer follow-up of
a larger number of patients.
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted June 17, 1998.
Supported by Grants-in-aid for the Second-Term Comprehensive 10-Year
Strategy for Cancer Control from the Ministry of Health and Welfare.
Address reprint requests to Yoichi Takaue, MD, Department of Medical
Oncology, Hematopoietic Stem Cell Transplant Unit, National Cancer
Center Hospital, 1-1 Tsukiji 5-Chome, Chuo-ku, Tokyo 104, Japan;
e-mail: ytakaue{at}gan2.ncc.go.jp.
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.
| |
ACKNOWLEDGMENT |
The authors thank T. Yasuda for her technical assistance.
| |
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Abstract
Introduction
Abstract