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Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1073-1090
REVIEW ARTICLE
Autologous Stem Cell Transplantation in Acute Myelocytic Leukemia
By
N.C. Gorin
From the Department of Hematology and Centre de Recherche
Claude-Bernard sur la Thérapie Cellulaire, Hôpital
Saint-Antoine AP-HP, Paris, France.
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INTRODUCTION |
IN THE PAST 30 YEARS, the treatment of
acute myelocytic leukemia (AML) has considerably evolved and improved.
The general evolution in the therapeutic strategy has invariably been
on the direction of more aggressive treatment being administered early, as soon as a first complete remission (CR1) has been achieved. The
rationale has been to provide maximum antitumor effect with so-called
consolidation (or intensification) at a stage of minimal residual
disease in an effort to eradicate the very last leukemic cells in the
patient's body. However, it is important to remember that total
leukemia eradication is not mandatory, because indirect evidence has
shown that cure of the disease is achievable if reduction of the tumor
burden can reach a level low enough to be controlled by the patient's
own immune system. Such a level can today be obtained, although still
unpredictably, by modern conventional chemotherapy or high-dose
myeloablative regimens with or without total body irradiation (TBI)
followed by stem cell transplantation. Numerous retrospective data
exist from single institutions and from international registries on the
use of conventional chemotherapy alone or allogeneic or autologous
blood or marrow stem cell transplantation (ABMT). Recently, results of
randomized studies comparing chemotherapy alone to allogeneic and
autologous stem cell transplantation have become available. In
parallel, prognostic factors have been identified, among which age,
response to induction therapy in reaching CR1, and cytogenetics have
proved major. Nonetheless, the choice of the therapeutic strategy has
reached no consensus. Other important questions regarding ABMT, such as
the role of purging the autograft from residual tumor cells and the
potential benefit of autologous peripheral blood (PB) over
marrow stem cells, are still being investigated.
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BACKGROUND: FROM ALLOGENEIC BMT AND STEM CELL CRYOPRESERVATION TO
MODERN ABMT IN ACUTE MYELOCYTIC LEUKEMIA |
The history of Autologous stem cell transplantation probably started
technically with the demonstration in 1955, by Barnes and
Loutit,1 that bone marrow could be successfully
cryopreserved, after initial works by Polge et al2 on
preservation of bull sperm. Improvements in cryopreservation techniques
continued untill the early 1980s.3-7 Several preclinical
models in mice,8 rabbits,9 monkeys,10,11 and dogs12-15 demonstrated the
ability of frozen marrow to engraft and reconstitute hematopoiesis. In
the dog model, the minimum dose to obtain engraftment was defined in
particular and the kinetics of engraftment were shown to be correlated
to the dose of marrow infused.14,15 Interestingly, the
correspondence with the human situation was almost perfect. In 1975, the medical ground for ABMT was ready.
The introduction and the development of ABMT for AML first took
advantage of lessons from allogeneic BMT. We reported our first
successful ABMT in 1977,16 in a patient with AML in first relapse. Marrow collected in first remission and stored in liquid nitrogen was infused after a myeloablative regimen; the patient engrafted and entered a second remission. Several teams started cryopreserving CR1 marrow to treat patients in
relapse.17-19 The demonstration that results achieved with
allogeneic BMT were considerably better when delivered early in
CR20 suggested that a similar approach should be followed
with ABMT. The interest in ABMT grew substantially in Europe and North
America in view of the major advantages of ABMT, ie, the absence of the necessity to find an HLA-identical family donor, the absence of development of a potentially lethal graft-versus-host disease, and the
possibility of applying the treatment to older patients. It appeared
later that the absence of graft-versus-leukemia, an initially
unrecognized companion of graft-versus-host disease, could be in some
circumstances an inconvenience.21 Another major impediment
of ABMT, considered from the very beginning, was the risk to reinfuse
to the patients with the graft leukemic clonogenic cells that would
contribute to or initiate leukemia recurrence. From 1982, several
techniques to purge the graft from residual tumor cells have been
established.
ABMT is now almost exclusively used to consolidate patients in first or
subsequent remission; some teams have systematically applied in vitro
purging techniques to the graft,22-36 whereas others
relying on agressive in vivo chemotherapy courses administered before
marrow harvesting and referring to it as in vivo purging have
not.37-50 However, arguments have accumulated in the past 10 years in favor of in vitro purging, and the demonstration has been
brought forward that infused leukemic clones at least can contribute to relapse51; nonetheless, the clinical
demonstration that in vitro purging improves the outcome is still
lacking in the absence of prospective randomized studies, so that the
field remains controversial. In 1985 and later, PB collected at time of
recovery from chemotherapy induced aplasia was shown to contain a large
number of hematopoietic progenitors able to reconstitue hematopoiesis
more rapidly than marrow.52,53 Techniques of mobilization
with chemotherapy and cytokines were developed. In the past 5 years,
there has been a considerable interest in PB autografting for AML.
However, the initial hope that the level of tumor contamination of PB
would be less than marrow or even nil has not been
substantiated.54,55 As of December 1997, the registry of
the European Cooperative Group for Blood and Marrow transplantation
(EBMT) contained information on a total of 65,000 transplants, of which
4,000 are ABMT for AML.
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CLINICAL EXPERIENCE WITH AUTOLOGOUS BONE MARROW TRANSPLANTATION FOR
AML |
ABMT for Patients in Relapse
In the early days of ABMT, from 1977 to 1983, marrow was collected in
patients with AML in CR1 to be reinfused at time of relapse,16-19,38 after myeloablation obtained with TBI or
chemotherapy combinations. Marrow purging was not available. Most
patients did not receive maintenance therapy posttransplantation.
Results of these early trials indicated a high rate of CR2 (~75%),
with, however, no long-term survivors. Of interest was the observation that many patients experienced CR2 of longer duration than CR1, a
phenomenon called at that time inversion. These early trials, although
disappointing, did demonstrate the feasibility of ABMT in AML as well
as the sensitivity of blast cells to high-dose cytotoxic therapy with
the possibility of achieving a much higher remission rate than obtained
at that time with conventional salvage regimens. In a recent
reevaluation of this approach, the Seattle team56,57 has
obtained a relapse-free survival of 41% at 2 years, with the longest
survivor at 12 years. With a few exceptions,58 ABMT at
relapse is no longer used.
ABMT for Adult Patients in Remission
Tables
1-4 summarize the experience of several
centers. Two pretransplant sets of regimens have mainly been used, the
same as for allogeneic stem cell transplantation: one is
cyclophosphamide (CY; 120 mg/kg) and TBI, originally
developed in Seattle,20 with possible variations according
to whether TBI is delivered in a single fraction (10 Gy)23,
fractionated (12 Gy),47 or even hyperfractionated (doses up to 13.2 Gy)23 and according to whether CY is administered
before or after TBI, with a separation of 48 hours between both. The other is the Busulfan-CY combination, with a total dose of 16 mg/kg
busulfan over 4 days and either 200 mg/kg CY over the next 4 days, as
originally designed by the Baltimore team,59 or 120 mg/kg
over the next 2 days, as modified by the Columbus team60 in
an effort to reduce toxicity. Of interest is that all of these regimens, when tested in the Brown Norway myelocytic leukemia rat
model, have induced a 8- to 10-log leukemia cell kill.61 High-dose etoposide (VP16) at 60 mg/kg has been more
recently introduced by the Stanford team to be combined with
TBI62 or Busulfan.27-28,63
A third regimen, the BAVC (800 mg/m2 carmustine [BCNU],
450 mg/m2 each of amsacrine [M-AMSA], and VP16, and 900 mg/m2 cytosine-arabinoside [ARA-C]) has been
introduced by the Roma team and, because of its reduced toxicity, has
been found particularly useful in older patients and/or in
CR2.43,48
For adult patients autografted in CR1 with unpurged marrow (Table 1),
reported leukemia-free survival range from 26% to 53%. Three studies
have some peculiarities.
The London University College study39 planned two
autografts. However, a minority of patients reached second
intensification. In these patients, the leukemia-free survival was
67%, but the benefit of the second intensification remains unclear
because of a selection bias, with patients reaching the second
intensification being more likely to belong to the good prognosis
group.
A prospective study at City of Hope (Duarte, CA) evaluated a single
cure of high-dose ARA-C consolidation therapy as a modality of in vivo
purging before marrow collection with no subsequent in vitro
purge.41 Sixty consecutive patients were included. The
leukemia-free survival was 49% and the relapse incidence was 44% by
intention to treat. In patients actually autografted, the leukemia-free
survival and relapse incidence were 61% and 33%, respectively, with a
plateau after 30 months.
In the Bologna study,42 ABMT was performed in late CR1 and
comparison with historical patients treated by CT and still in CR at
the same time was in favour of ABMT.
For adult patients autografted in CR1 with purged marrow (Table 2),
reported leukemia-free survival range from 41% to 80% and relapse
rates from 19% to 48%. Purging has consisted almost exclusively of
cyclophosphamide derivatives (4 hydroperoxycyclophosphamide [4HC] or
mafosfamide). Figure 1 shows, as an
example, the plateau achieved for leukemia-free survival at 70% and
relapse incidence at 27% in 50 consecutive patients (median follow-up,
7 years) autografted in first CR in San Francisco and
Berkeley28 with marrow purged with 4HC following the
original pretransplant regimen combining busulfan and etoposide
introduced by the Stanford team.63 In patients in second
remission, the same team reports leukemia-free survival and relapse
incidence of 52% and 35%.
View larger version (11K):
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[in a new window]
| Fig 1.
Leukemia-free survival and relapse rate for 50 patients
transplanted in first remission with marrow purged with 4HC, following a preparative regimen combining busulfan (16 mg/kg) plus etoposide (60 mg/kg). (Reprinted with permission from Linker et
al.28)
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The Parma team25 has claimed better results when adjusting
the dose of mafosfamide to the individual sensitivity of the normal
colony-forming unit (CFU)-blast compartment. Only the Manchester team22 has used long-term culture conditions that they have shown to be deleterious to survival of leukemic colony-forming units-leukemic (CFU-L).
Few teams have had the simultaneous experience of autografting with
purged or unpurged marrow.45,63 Within a phase 2 trial exploring the value of their preparative regimen, the Stanford team63 compared small numbers of patients autografted in
CR1 receiving either unpurged marrow or marrow purged with 4HC. They initially reported a leukemia survival of 57% and a relapse incidence of 28% in the group receiving purged marrow versus a leukemia-free survival of 32% and a relapse incidence of 62% with unpurged marrow. However, with a longer follow-up, the difference never reached significance. The kinetics of engraftment were slower in the purged group.
Results of ABMT in CR2 (Table 3) indicate leukemia-free survival from
21% to 52% and relapse incidence from 30% to 63%. Purging, when
used, has been performed essentially with cyclophosphamide derivatives.
One notable exception has been the use of CD14+ CD15
monoclonal antibodies by the Pittsburg team.33,34 Results have been better in patients experiencing the longest duration of CR1.
Some teams have claimed outcome equivalent to allogeneic BMT.
Fewer series of ABMT are available in children. Some of them in
addition combine different pretransplant regimens64-66 and mix unpurged and purged autografts (Table 4). For patients autografted in CR1, the leukemia-free survival range from 41% to 87% and the retrospective analysis of the Italian registry is in favor of TBI. In
CR2, reported leukemia-free survival rates are 36% and 41.5%. Results
of CCSG and EBMT are presented as equivalent to allogeneic BMT.
Analyses of all these results are, of course, difficult, because they
are retrospective and concern small series of patients and selection
bias have likely taken place, at least in some institutions. Comparison
with conventional chemotherapy is impossible, because ABMT has
concerned only patients in CR. One may argue though that the clinical
setting of ABMT is comparable to allogeneic BMT and propose that
results achieved with ABMT indeed convey the same information
(including the pitfalls) as those reported at the same periods for
allogeneic BMT. Whatever the scientific significance of these results,
they have demonstrated the feasibility of ABMT in CR1 and CR2. The high
rate of long-term disease-free survivors observed in almost all
institutions has generated hope and justified prospective controlled
studies.
Kinetics of Engraftment
The very first clinical studies had already outlined that the kinetics
of engraftment after marrow autografting were particularly slow in AML,
contrasting with much more rapid hematopoietic recoveries in acute
lymphocytic leukemia (ALL), lymphomas, and solid
tumors.67 With unpurged marrow in patients in CR1, recovery
of leukocytes to 109/L or polymorphonuclears (PMN) to 0.5 × 109/L occur around days 30 through 4568
and recovery of platelets to 50 × 109/L around days
70 through 100, with very wide ranges and extremes up to 1 year for
leukocytes and PMN and up to 2 years for platelets. Therefore, the
classical definition of engraftment failure, ie, no PMN recovery by day
28, cannot be applied in this situation, in which delayed engraftment
is a more appropriate term. Although no randomized study is available
to compare the kinetics of engraftment with purged and unpurged marrow,
purging with CY derivatives is believed to further delay
engraftment.30,36,45,69-71 In the experience of the ECOG
group with 4HC purged marrow, recoveries of PMN greater than 0.5 × 109/L and platelets greater than 20 × 109/L have been observed at median days 32 and
64. In our own experience29,30 with mafosfamide purged
marrow, recoveries of PMN and platelets to 50 × 109/L
have been observed at days 30 and 90, and about 15% of our patient
population has needed platelet support for more than 1 year. It has
been shown that more rapid recoveries are obtained for younger
patients, if PB counts on day of harvest are normal,71 when
the transplant is performed in CR1 rather than in CR2, if in CR1
earlier after obtention of remission, and after pretransplant regimens
that do not include TBI. This has been linked to the infusion of higher
doses of stem cells,4,69,71-73 less damage by
previous chemotherapy, and less stroma disruption, respectively. Engraftment failure accounts for approximately 10% of the cases. If
other causes have been ruled out, such as defective cryopreservation and/or occurrence of complications in the posttransplant
course, such as cytomegalovirus infection or liver veino occlusive
disease, it has been essentially related to the infusion of poor
marrow, in particular when collected in patients heavily pretreated
with mitoxantrone and/or high-dose ARA-C.74 In our
own experience30 with marrow purged by mafosfamide,
engraftment of both neutrophils and platelets has been more rapid when
purging has been adapted to the individual sensitivity of
colony-forming unit-granulocyte-macrophage (CFU-GM),
which has resulted in less patients receiving grafts with no detectable
CFU-GM after purging; the transplant-related mortality has been
significantly lower and the leukemia-free survival has been
significantly higher in those in whom the dose of CFU-GM per kilogram
harvested has been above the median value of 5.2 × 104/kg. Along the same line, one study in particular has
correlated better kinetics of engraftment and also better outcome
posttransplant to better development of progenitors from the graft in
long-term culture assays.75 However, most studies
evaluating the content of AML marrow collected in CR have rather found
a markedly defective in vitro growth of hematopoietic progenitors and a
severe functional defect of marrow stroma as compared with normal
marrow or marrow of patients with other diseases, which may explain the
delay to engraft observed, specifically in AML.76 The
simultaneous search for the highest dose of stem cells and the lowest
tumor contamination achieved by previous in vivo purging is in fact a
combination of two conflicting goals.
Speeding up engraftment is, therefore, an active field of
investigation. Some teams have used granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-1 (IL-1) after ABMT despite the theoretical concern about a potential stimulation of leukemic clones, with no major
improvement of the kinetics. A possible reduction of neutropenia with
IL-1 has been counterbalanced by side effects.77 The use of
so-called primed marrow, ie, marrow collected after 2 to 5 days of
G-CSF or GM-CSF administration, which accelerates engraftment in
patients autografted for solid tumors, has not been tested in AML. IL-3
followed by GM-CSF for delayed engraftment has been of limited
value.78 In fact, because the major problem in patients
autografted for AML is platelet reconstitution, results of trials
testing the administration posttransplant of megacaryocyte growth and
development factor (MGDF) or thrombopoietin (TPO) are eagerly waited
for. Inhibitors of hematopoiesis that prevent normal progenitors from
entering cell cycle can be used in vitro to protect the normal stem
cell compartment when purging with alkylating agents such as
mafosfamide. Amifostine in particular accelerates engraftment in
patients with breast cancer autografted with marrow purged with
4HC79; it is presently being evaluated in AML. The only
effective measure to accelerate significantly engraftment in AML, as in
other diseases, is the use of PB stem cells. Several
teams80,81 have reported rapid recoveries on day 12 for PMN
and day 25 for platelets; a recent pair-matched analysis from EBMT
comparing PB with marrow autografts has shown recoveries of PMN and
platelets to occur significantly more rapidly with PB at day 13 versus
29 for PMN and at day 42 versus 60 for platelets. However, even with
PB, delays in platelet recovery do occur and extreme values have ranged up to 2 years. However, the benefit brought by the use of PB has been
counterbalanced by the concern that mobilization may increase blood
contamination46,54,55,80,82; reports of increased relapse
incidence have generated caution and interest in adding several
consolidation chemotherapy courses before leukaphereses to take
advantage of in vivo purging and also interest in in vitro purging. It
is presently unknown whether these manipulations performed in an effort
to reduce tumor contamination will or will not slow down the kinetics.
Monitoring of the serum concentrations of hematopoietic growth factors
during myeloablation and hematopoietic recovery83 has shown
a sharp decrease of leukemia inhibitory factor (LIF) and
an increase of IL-3 during the pretransplant regimen administration, possibly related to induction of stem cell cycling and differenciation. In the posttransplant period, IL-6, G-CSF, and IL-8 have increased from
days 6 through 9 and peaked in parallel to neutrophil recovery. The
peak was found higher in patients receiving PB. These observations have
so far had no therapeutic implications.
Prognostic Factors Other Than Purging
Because, with a few exceptions, single-institution studies have
concerned populations of patients too small to enable multivariate analyses, most identified prognostic factors have first come out of
international retrospective surveys. Results from registries are
criticized as relying on nonconsecutive reporting of poorly controlled
data and should be interpreted with great caution. Nonetheless, the
relevance of the prognostic factors listed below has later been
confirmed in several prospective randomized studies.
Results of ABMT in CR1 are superior in children and in younger adults,
but ABMT can still be considered in older patients.84 The
outcome of 111 patients older than 50 years of age was compared with
the outcome in 786 younger patients (median age, 35 years). The relapse
incidence was identical in both groups, but the transplant-related mortality was higher in older patients, so that, in the end, the leukemia-free survival was significantly lower. Regimens, such as the
BAVC, with a better tolerance may be favored in older patients.
Results of ABMT in CR1 are better in rapid remitters.85
They are also better by cytogenetics in the standard risk group as
opposed to the poor prognostic group, which includes all abnormalities of chromosomes 5 and/or 7 and hypodiploidy.86 In
fact, by multivariate analyses, cytogenetics at diagnosis were found to
be the strongest prognostic indicator for relapse and leukemia-free
survival, not only after ABMT, but also after allogeneic BMT, as
previously shown with conventional chemotherapy.
Results in CR2 are better in patients who experienced longer CR1
durations.85
Cyclophosphamide and TBI was found equivalent to the
Busulfan-cyclophosphamide combinations,87 both for ABMT and
allogeneic BMT, with the exception of more cases of liver
veino-occlusive disease with BU-CY. A randomized comparison of CY and
FTBI at 1,320 cGY versus the original BU-CY, before autografting with marrow purged with 4HC,88 has similarly shown no difference in CR1 but has suggested a trend in favor of TBI in more advanced disease.
The question of whether the dose of ARA-C administered before the
transplant period to the patients had an impact on the outcome post-ABMT was considered of importance in view of the results of the
CALGB randomized trial on the role of intensive postremission chemotherapy, which had shown a better survival in patients receiving the highest dose arm.89 A recent retrospective analysis on
1,629 patients autografted as well as allografted in CR1 concluded in the absence of any impact in both situations, leading to the
speculation that high-dose therapy administered with ABMT would replace
or erase the need for previous high-dose ARA-C.90
The timing of the transplant also is important. Results are
significantly better in patients autografted late, when in
CR.85 This has been mainly explained by a selection bias,
with patients transplanted late having spontaneously pretransplant a
higher probability of being cured. However, alternatively, one can
argue that late autografts may have taken advantage of more
consolidation chemotherapy administered before marrow collection,
leading to a better in vivo purge of the graft.
Most relapses occur during the first year after transplant and, in the
end, patients with AML autografted in CR1 or CR2 who are still disease
free 2 years after ABMT have a more than 80% chance of being
cured.91 Late relapses, after 24 months, are quite uncommon
with purged marrow.85,92,93
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IN VITRO PURGING |
The amount of theoretical, experimental, preclinical, and clinical work
published in the past 15 years on purging has been considerable.
Purging in AML has mostly been performed using cyclophosphamide derivatives. Cyclophosphamide itself has no antitumor action. It is
metabolized in the liver to generate the active catabolites such as
4HC. Mafosfamide is an analog that generates in vitro free 4 hydrocyclophosphamide.
Data have accumulated in favor of purging for ABMT in acute leukemia,
most of all AML, at the same time when similar observations have been
gathered for non-Hodgkin's lymphomas,94 chronic myelocytic leukemia,95 and, more recently, solid tumors.54
However, it has been argued that in vivo purging obtained by agressive
chemotherapy delivered before stem cell collection could bypass the
need for additional in vitro treatment. Also, as seen above, because
the kinetics of engraftment after ABMT for AML may be slower with
purged marrow, it has been proposed that any advantage of purging in
terms of a reduction in the relapse incidence could be obliterated by
the toxicity of a longer aplasia duration.
Rationale
The Brown Norway rat model of human AML, extensively studied by the
Johns' Hopkins group,96-98 has provided the first
rationale for purging. In this model, the dose of leukemic cells that,
when infused to the animals, kills 50% of them (LD 50), is 25. With the average weight of an animal of 250 g, this was postulated to
translate in the human situation into a LD 50 around 10,000 leukemic
cells for an adult of 70 kg. Leukemic rats submitted to TBI, followed
by infusion of syngeneic marrow purposedly contaminated with 1%
leukemic cells, to mimic the human situation, survived, attesting to
succesful engraftment; however, they did not develop recurrent lethal
disease only if the graft had been treated in vitro with 4HC at a dose
greater than 60 nmol/L, attesting to the succesful purging above this
4HC dose threshold. Later experiments using the IPC 81 subline grown in
culture from the original rat leukemia predicted that a similar purging
approach with human marrow would result in CFU-GM residual values
around 1%.98
Numerous studies in humans using immunophenotyping,99,100
serial karyotyping,101 growth of leukemic progenitors
CFU-L,102 and, more recently, molecular detection of tumor
cells by polymerase chain reaction
(PCR)103-105 have clearly shown that positive
detection of residual tumor cells in the marrow of patients with AML in CR predicts for relapse; some of these studies have inversely correlated the level of minimal residual disease to the probability of
cure. The Johns' Hopkins group in particular has been able to grow
CFU-L from the majority of their patients autografted in CR2 or CR3
with marrow purged by 4HC.102 They compared the sensitivity
of CFU-L and normal CFU-GM to 4HC and identified two groups of patients
according to whether the sensitivity of CFU-L was higher (sensitive
group) or identical to (resistant group) the sensitivity of CFU-GM. The
relapse incidence was 15% in the sensitive group and 80% in the
resistant group. They proposed that a better purging had been obtained
in the sensitive group. However, an alternate explanation whereby CFU-L
sensitivity to 4HC in fact was but a surrogate marker of the
sensitivity of the disease to chemotherapy in general could not be
ruled out.
The most important demonstration that leukemic cells harvested with the
autologous graft at least can contribute to relapse has come from gene
marking studies.51,106 The neomycin resistance gene in a
retroviral vector has been used to mark autologous unpurged marrow
infused to patients; in the few patients who relapsed, the resurgent
blast cells contained the neomycin-resistance gene marker, clearly
indicating that they originated from the graft. Similar observations
were made with neuroblastomas, chronic myelocytic leukemia, and solid
tumors.
One aspect of purging that has been overlooked for many years is the
contribution of freezing. AML-CFU are more sensitive to
cryopreservation than their normal counterparts. Freezing induces a
one-log reduction in clonogenic AML-CFU.107
Furthermore, cells fragilized by in vitro treatment are more sensitive
to freezing and thawing.108 Also, it is unclear whether
dimethylsulfoxide infused to the patients with the autograft, in case
it is not washed before hand, has any antileukemic
impact.109
All things considered, calculations extrapolating from the rat leukemia
model to a human autograft contaminated by 0.5% leukemic cells, taking
into account the fact that 1% only of leukemic cells are indeed
clonogenic and freezing is the obligatory first step of purging, would
indicate that a single log tumor reduction in the graft may in fact be
of considerable importance. These theoretical considerations will have
to be reevaluated in view of the recent identification of the
AML-initiating cell (SLIC) by transplantation of human leukemic cells
into SCID and NOD-SCID mice.110 Because the frequency of
SLIC in PB of leukemic patients has been shown by limiting dilution
assays to be around 1/250,000, ie, more than a 1,000fold lower than
the AML-CFU, the necessary reduction by purging would need to be less
than estimated from AML-CFU data, but the sensitivity of SLIC to
Cyclophosphamide derivatives is unknown.
Although the major action of cyclophosphamide derivatives is tumor
cytotoxicity, other actions also have been claimed, in particular an
increase in natural killer (NK) cell population and
activity111 as well as induction of apoptosis in leukemic cells that can be further enhanced by IL-3 and IL-6.112 An
increase in NK cell activity has been documented in AML patients after ABMT with marrow purged by mafosfamide,113 suggesting that
purging with mafosfamide may add to tumor destruction in the graft some secondary antitumor immunomodulation.
Purging With Cyclophosphamide Derivatives and Doses of Marrow
Infused
In the context of purging, an intriguing observation made at several
centers has been the relationship existing between the doses of marrow
infused and the outcome of the transplanted patients. This observation
has conveyed the message that what is done to the graft itself may have
an impact posttransplant.
(1) The Baltimore team generated a clinical program for AML at high
risk of relapse. Patients in CR2 and beyond were intensified by the
BU-CY combination followed by infusion of autologous marrow purged in
vitro by 4HC at the constant dose of 100 µg/mL.36,59,71 The probability for the patients to remain in CR was correlated to the
residual fraction of the normal progenitors CFU-GM in the graft
after in vitro treatment.114 It was 40% versus 18% for CFU-GM residual surviving fractions in the autograft below or above the
1% median value, respectively.
Our own group designed in 1982 a program of high-dose intensification
with cyclophosphamide and TBI, followed by reinfusion of autologous
marrow purged by mafosfamide.29,30,115-117 By multivariate analysis, the leukemia-free survival was significantly higher in
patients who received richer bone marrow, as evaluated before purging.
In a more recent update on 234 patients, we also found, as in
Baltimore, the relapse incidence to be correlated to the CFU-GM
residual fraction after purging. It was 50% versus 31% for patients
receiving autografts with CFU-GM residual fraction after purging above
or below the median, respectively. The speculation was that less
residual CFU-GM meant more agressive purging, which in turn resulted in
less relapses.
(2) The Roma team had a different experience with unpurged marrow. The
leukemia-free survival was lower in patients receiving doses of marrow
evaluated in nucleated cells per kilogram above the median value. They
concluded that more leukemic cells had probably been reinfused with the
higher unpurged marrow doses.
These observations may be taken as indicating that better results
indeed are achieved with the highest doses of stem cells provided that
high levels of contamination by tumor cells should be avoided; this
would, in theory, correspond to AML treated with CY derivatives.
Conversely, this explanation would account for the absence of such a
dose effect both in AML in the absence of purging and also in ALL
despite attempts at purging, because ALL progenitors have been shown to
be highly resistant. A similar observation has been reported in
allografted recipients, with a better leukemia-free survival in those
receiving the higher doses of marrow.118 The intervention
of a stem cell competition effect has been postulated, whereby an
expanded normal stem cell pool would express a growth advantage
and/or a higher resistance (eg, to inhibitors of leukemic
origin) when faced by a minimal residual tumor
population.119
(3) Normal progenitors and, by implication, CFU-L experience individual
levels of sensitivity to mafosfamide. We adapted the dose of
mafosfamide for purging to the individual sensitivity of each patient
normal CFU-GM in an effort to determine the highest possible dose of
mafosfamide and ensure maximal tumor reduction.29,30 The
dose selected was the LD95 defined as the dose sparing 5% CFU-GM,
evaluated 15 days before marrow collection, on a small aspirate from
which aliquots had been taken and incubated with increasing doses of
mafosfamide. With this approach, the doses of mafosfamide used for
marrow incubation ranged from 15 µg to 170 µg/2 × 107 buffy coat cells/mL. This individual sensitivity also
was taken into account by the Parma team, who adjusted the dose of
mafosfamide from 61 to 146 µg/mL to reach a 50% inhibition of the
normal CFU-Blast compartment. Not only did this team report on better
leukemia-free survival in their patients autografted with marrow
treated at adjusted levels of mafosfamide over constant dose purging
and over no purging,25 but, interestingly, patients
receiving adjusted dose mafosfamide purged marrow were the only ones to
respond to recombinant GM-CSF,70 a finding that was
explained by dose adjustment sparing the more primitive CFU-Blast
compartment.
International Registry Retrospective Analyses on Purging With
Cyclophosphamide Derivatives
Several retrospective analyses on purging have been
performed.85,92 The most important one involved 59 European
teams that had reported 919 autografts for consolidation of AML up to
December 31, 1989. Marrow was purged with mafosfamide in 269 patients. The outcome posttransplant was correlated to the initial response to CR
induction: inital rapid responders had a better leukemia-free survival
(53% v 42%) and lower relapse incidence (46% v 57%)
than did slow responders. Multivariate analysis in several populations showed significantly the efficacy of marrow purging in AML in CR1. In
patients autografted after TBI, the relapse incidence with purged
marrow was 29% versus 50% and 16% versus 60% when considering only
those autografted within 6 months of CR. In slow responders, the
results were 20% versus 61%, significantly in favor of purging,
whereas the relapse incidence were similar in rapid responders. The
relapse patterns were different in that the plateau for persisting
remission started at 23 months with purged marrow and at 32 months with
unpurged marrow. In CR2, marrow purging was also associated with a
better leukemia-free survival in patients receiving TBI, but the
relapse incidence was not significantly different. It was concluded
that purging was most likely to bring benefit to a specific category of
patients, ie, those transplanted early (Fig
2) and slow responders, in whom the probability that leukemic cells
might still persist in the graft at time of collection was higher.
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| Fig 2.
Relapse rates of patients autografted in first remission,
after TBI, with either purged or nonpurged bone marrow. (A) Patients autografted within 6 months of obtaining remission. (B) Patients autografted later than 6 months after remission. (A) P = .02. (B) P = not significant. (Reprinted with permission from Gorin et al.85)
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These data were later confirmed by an IBMTR-ABMTR retrospective study
on patients with AML autografted in the United States. In this last
study, the kinetics of engraftment were slower in those autografted
with purged marrow, both for neutrophils and platelets, but the relapse
incidence at 3 years was lower in CR1 (40% v 70%) and in CR2
(50% v 90%) and likewise the leukemia-free survival better in
CR1 (50% v 30%) and in CR2 (40% v 15%; unpublished data).
Future Direction: Cytoprotective Agent-Mafosfamide Combination
A recent approach to prevent toxicity of purging with mafosfamide
towards the normal stem cell pool has been the use of cytoprotective agents. Amifostine, a phosphorylated aminothiol prodrug is more selectively transformed into its active cytoprotective metabolite in
normal tissues that have a higher concentration of alkaline phosphatase
and a neutral pH environment.120
The Denver team79 has investigated the impact of amifostine
on the protection against 4HC in the context of purging for autologous
BMT in breast cancer. Leukocyte engraftment was achieved 10 days
earlier and the average number of platelet transfusions and days of
antibiotic therapy were reduced by 50%.
The ability of amifostine to selectively protect normal bone marrow
progenitor cells versus leukemic progenitor cells from the cytotoxic
effect of mafosfamide has been evaluated in vitro.121 Amifostine pretreatment has resulted in a statistically significant protection of CFU-GM and burst-forming unit-erythroid (BFU-E) and quite
unexpectedly was found to increase the cytotoxicity of mafosfamide on
the fresh human leukemia progenitor cells, with, in the end, an
estimated 6 log therapeutic ratio.
A prospective multicentric randomized study of the role of amifostine
in the context of purging with mafosfamide is presently ongoing.
Other Purging Means
Cyclophosphamide derivatives represent more than 90% of the global
experience in the field of purging for ABMT in AML. Other drugs have
been occasionally proposed and used, with no demonstrated advantage:
these include VP-16,31,122 alkyl
lysophospholipids,123 and Simvastatin, an
antithypercholesterolemic drug that inhibits HMGCoA.124
Antimyeloid monoclonal antibodies, CD14 and CD15 (PM-81 and AML 2-23)
with complement, have been tested in CR2/CR3 patients and shown to
allow rapid hematologic engraftment without toxicity.33,125 A trial with anti-CD33 antibody, eliminating virtually all committed myeloid progenitors, on the other hand, has resulted in a significant engraftment delay.126 Enhancement of in vitro
immunological antitumor activity has been obtained using several
approaches: the use of bispecific monoclonal antibodies anti-CD13 and
anti-CD3,127 selective targeting of lymphokine-activated
killer cells by CD3 monoclonal antibody against the
interferon-inducible Fc receptor on AML blast cells,128
and the combination of monoclonal antibodies wth low-dose
4HC.129 None of these has been tested in a clinical situation.
The Manchester group developed in 1986 a technique of long-term culture
that, when applied to AML marrow in relapse, appeared to selectively
destroy the leukemic clones to the advantage of the normal
hematopoietic progenitors. This has then been used to purge marrow
collected in CR1.130 In a small series of 26 patients
(Table 2), the leukemia-free survival has been as high as 80%, but a
selection bias cannot be ruled out. A similar approach has been used
with success by the Vancouver group in chronic myelocytic leukemia,131 in which the conditions of the long-term
culture have favored the survival and development of the Phi-negative compartment.
IL-2 to generate in vitro LAK cells with antileukemic
activity132 has unfortunately resulted in the destruction
of immature hematopoietic progenitors, causing failure to engraft.
Finally, leukemic cells have a higher heat sensitivity than the normal
hematopoietic subset.133 The therapeutic gain can be
augmented by preincubation with the tetrapeptide AcSDKP (Goralatide). A
hyperthermic purging protocol with incubation of the marrow at 43°C
for 90 minutes has been proposed. Combination with cyclophosphamide derivatives would be a further logical step.
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IMMUNE THERAPY AND ABMT |
Detection of antileukemic specific activity has been observed
spontaneously post-ABMT.134,135
After anecdotal reports of complete or partial remission obtained with
IL-2 in patients with advanced AML, IL-2 has also been used post-ABMT
in an effort to stimulate immune functions and NK cell
activity.134,135 Some pilot studies have concerned patients relapsing post-ABMT; other studies have used IL-2 after ABMT to maintain CR in higher risk patients. Except for the Seattle
study136 in which a leukemia-free survival of 71% at 4 years has been reported in a small number of patients autografted
either in relapse or in CR2, the general experience has remained
unconvincing.137,138 An interim analysis of a randomized
study of linomide (a potent inductor of endogenous IL-2 secretion and
NK cell activation) post-ABMT in CR1 has failed to show any difference
in the two arms. Cyclosporine A at low doses post-ABMT has been
administered in an effort to generate graft-versus-host
disease/graft-versus-leukemia and indeed has induced cutaneous
manifestations of graft-versus-host disease in most patients, but with
no demonstration of any antileukemic effect.139
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PB STEM CELL TRANSPLANTATION FOR AML |
Despite the original assumption that PB would be less contaminated than
marrow and even might be free of tumor, early experiences with PB
transplants were disappointing and, in fact, suggested that, at least
after induction, a large amount of leukemic clones were mobilized.
Massive reinfusion of these cells to patients led to early
relapses.55,80,82 Leukaphereses after consolidation rather
than after induction were recommended.
The first retrospective EBMT studies comparing marrow to PB autografts
for AML in CR1 indicated at 8 years a leukemia-free survival of 51% in
1,279 recipients of marrow and of 44% in 100 recipients of PB with no
significant difference (unpublished data).
However, when discriminating between purged and unpurged marrow,
results with purged marrow were significantly superior to PB, with a
leukemia-free survival of 57% in 251 purged marrow autografts versus
44%; the relapse incidence was 50% versus 37% (P = .0006).
These results did not differ from the original single institution
retrospective comparison of purged marrow versus PB observed by the
Heidelberg group in 1989.24
There was no difference whether the mobilization had been achieved
using chemotherapy, growth factors, or both. However, the number of
chemotherapy courses precollection shown was important, but only in
rapid responders. Patients receiving leukaphereses collected after a minimum of two chemotherapy courses had a
significantly lower relapse incidence (20% v 62%,
P = .008) and a significantly better leukemia-free
survival (69% v 35%, P = .02) than patients receiving leukaphereses collected after only one chemotherapy course.
(manuscript submitted).
The conclusion of these studies was that autografting with PB may do as
well as purged ABMT, provided that it takes advantage of in vivo
purging represented by additional courses of chemotherapy administered
to the patients before collection. Recent results from
several institutions that follow this therapeutic strategy look
promising, with reported leukemia-free survival as high as 71% at 5 years in CR1 and only a slight engraftment delay.81 Alternatively, because it has been clearly shown that stem cell mobilization techniques also mobilize tumor cells, PB purging is being
considered.140
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COMPLICATIONS OF ABMT FOR AML |
Complications of autografting for AML depend on several variables,
among which the population age, the nature of the pretransplant regimen
used, and the source of stem cells infused (which influences the
duration of aplasia) are essential. As predicted from theory, graft-versus-host disease has almost never been observed, despite a few
anecdotal reports. Apart from this, the nature of the complications observed after ABMT in AML has not differed from complications of
allogeneic BMT; however, reported incidences have usually been lower.
Early complications have included mucositis (up to 100% with high-dose
melphalan and etoposide), bacterial and fungal sepsis, pulmonary
aspergillosis, cytomegalovirus infection and disease (up to 20% with
TBI and BU-CY), and liver veno-occlusive disease (up to 20% with
BU-CY). Transplant-related mortality has varied from as low as 5% in
single experimented institutions to an unexpected high of 12% in the
large MRC 10 randomized trial, which included small and very small
centers. In a large recent retrospective EBMT study,47 the
transplant-related mortality has been 13% and 8%, respectively, in
adults and children autografted in CR1 and 20% and 12% in CR2. Late
complications combine sterility, cataracts, and secondary malignancies.
Sterility is almost constant after TBI or BU-CY,141
although successful pregnancies have been occasionally reported.
However, alternate chemotherapy combinations may offer better chances,
although this has to be balanced with a lower antileukemic
effect.142 Cataracts are directly correlated to the use of
TBI. Several studies have indicated that TBI fractionation decreases
its incidence. In a recent retrospective study143 of 1,063 patients transplanted after TBI, which included 490 AML, the risk was found to be significantly lower after ABMT as compared with allogeneic BMT and also lower in younger patients, in cases with
heparin administration (as administered in some centers for prevention
of liver veno-occlusive disease), and in the absence of long-term
steroid administration.
In 1985144 and later in 1993,101 we reported on
the occurrence of multiple chromosome abnormalities in patients with
acute leukemia autografted with marrow purged by mafosfamide after TBI:
30% of our patients autografted with marrow purged with mafosfamide
had complex chromosome abnormalities detectable at some time, as late as 88 months posttransplant, in a minority of cells, which either persisted or were only transient, and appeared not to be related to the
original leukemic clone. These observations have so far not been
correlated to any unfavorable outcome. Because we did not find similar
abnormalities in patients autografted with purged marrow but after
non-TBI pretransplant regimens, we postulated that these rare abnormal
circulating cells were endogenous progenitor cells that had survived
the previous repeated aggression of chemotherapy induction and
consolidation courses and the pretransplant regimen including TBI.
Similar findings have been reported by others in AML145 and
patients with non-Hodgkin's lymphoma,146 as well as after allogeneic transplantation, especially with T-cell-depleted marrow, supporting the same explanation. Myelodysplastic syndromes post-ABMT have been described as a late complication, occuring with a frequency of up to 14.5% at 5 years, in patients with non-Hodgkin's
lymphoma.147 Occurence of myelodysplastic syndromes or
secondary leukemia in patients autografted for AML has been mentioned
in meetings but so far not published. It is possible that some have
been confused with recurrence of the original disease. The recent
introduction of high-dose etoposide in pretransplant regimens may major
the risk.148
| |
THE AML-3 EXCEPTION |
Old data,149 collected before the introduction of
all-trans retinoic acid (ATRA), had indicated in patients
autografted for an acute promyelocytic leukemia (M3) defined by
cytology a leukemia-free survival at 7 years of 48% in CR1 and 31% in
CR2. Results achieved with allo-BMT were not superior because of a very
high transplant-related mortality of 42%, as opposed to 18% for ABMT.
A more recent analysis86 on 999 patients with evaluable
cytogenetics has found the t(15,17) translocation to be the strongest favorable prognostic factor for the outcome post-ABMT, with a leukemia-free survival of 56% and an overall survival of 68% at 5 years.
Meanwhile, however, the whole field of AML3 has changed since
1990150 with the advent of ATRA, its use in combination
with chemotherapy for induction of remission,150-152 the
detection of the PML/RAR fusion transcript by PCR and the
demonstration of its prognostic relevance for detection of minimal
residual disease,104,105 and the design of a better
treatment strategy. In the recent AIDA trial,104 induction
with ATRA and Idarubicin followed by 3 consolidation courses has
resulted in 95% CR and a leukemia-free survival of about 80% at 2 years. Moreover, at the end of the consolidation period, 98% of the
patients had undetectable PML/RAR transcripts. Therefore, stem cell
transplantation cannot be recommended as part of the upfront therapy of
M3. In contrast, in the minority of patients who still relapse,
induction of a CR2 again with ATRA and chemotherapy is easily
achievable and ABMT should be seriously considered. It has been
proposed that patients in CR2 remaining PCR positive proceed to
allo-BMT if possible, whereas those reaching PCR negativity be
intensified with ABMT.105 Purging the autograft in M3 might
take advantage of several peculiarities: CD34+ selection,
because a majority of M3 leukemic progenitors have been reported to be
CD34 (in contrast to other types), and in vitro
incubation with ATRA, not taking into account more classical means such
as monoclonal antibodies or cyclophosphamide derivatives. The unique
possibility for monitoring minimal residual disease by PCR further
makes studies and protocols along these lines appealing; unfortunately,
the predicable small patients accrual has so far generated little enthusiasm.
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THE PLACE OF ABMT IN THE THERAPEUTIC STRATEGY OF AML |
Several retrospective studies comparing chemotherapy with allo-BMT and
ABMT have been performed in the past 15 years. In a first joint
EORTC-EBMT analysis, 236 patients treated according to the AML 5 and 6 EORTC chemotherapy protocols were compared with 453 allografted and 182 autografted patients registered by EBMT.153 At 6 months
posttransplant, allografted recipients had a significant better
leukemia-free survival than the chemotherapy group and the autografted
group was in between, with no significant advantage, however, over
chemotherapy. In a retrospective analysis of their own single
institution experience, the Genoa team154 has compared the
outcome of 159 AML patients allografted or autografted with unpurged
marrow in CR1 after TBI. At 8 years, results have been identical, with
leukemia-free survival rates of 52% and 49%, respectively. Of
interest was the finding that the relapse incidence after ABMT (39%)
was identical to the incidence in allotransplant recipients who
developed no chronic graft-versus-host disease (37%), but was higher
than in those with chronic graft-versus-host disease (30%). Two recent
EBMT retrospective studies compared the outcome of patients with AML
after ABMT; the first one to allo-BMT with an HLA-identical
sibling47 and the second one to allo-BMT with an
HLA-identical volunteer donor.155 Results indicated a
significant higher transplant-related mortality for allografting and a
significant higher relapse rate for autografting.
In terms of leukemia-free survival, results were significantly superior
for patients allografted with an HLA-identical sibling marrow in CR1
but not in CR2. Results of ABMT and unrelated marrow transplants did
not differ, an observation also made by the Seattle team.156
Table 5 lists the results of prospective
randomized studies completed: some have consisted of two arms and
compared allogeneic BMT with ABMT68,157-159 or ABMT with
conventional chemotherapy.64 Others with three arms have
compared the three treatment modalities.160-163 In all,
except the ECOG study,162 marrow used for ABMT was
unpurged. Results are given in the majority by intention to treat. As
an example, in the study conducted in Boston, all patients entering CR1
were offered allogeneic BMT or auto-BMT after a consolidation course
using high-dose ARAC (6 g/m2 total). The actuarial
leukemia-free survival in the two groups of patients actually
transplanted was identical (62%). The contribution of high-dose ARAC
was felt to be important. Of all the studies, the two biggest by
patient accrual and number of institutions involved have been the
international AML8 EORTC-GIMEMA trial161 and the UK MRC 10 trial.163
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Table 5.
Prospective Randomized Studies Comparing Transplants to
Conventional Chemotherapy for the Treatment of AML
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The AML8 trial compared in 941 patients 10 to 45 years of age in CR1
allogeneic BMT (in those with an HLA-identical sibling) with either
autologous BMT or a second course of high-dose cytarabine and
daunorubicin in the others. The relapse incidence was the highest in
the intensive chemotherapy group and the lowest in the allogeneic
transplantation group; the transplant-related mortality was the highest
after allogeneic transplantation and the lowest after chemotherapy. In
the end, the leukemia-free survival was significantly better after both
transplant modalities, over chemotherapy with values at 5 years of 55%
for allogeneic BMT, 48% for ABMT, and only 30% for conventional
chemotherapy. Another finding of the trial was that many patients who
relapsed in the conventional arm could be rescued by ABMT, which in the
end resulted in a similar overall survival in the three groups.
The MRC10 trial recruited 1966 patients from 163 institutions. Patients
reaching CR1 received high-dose consolidation before going either to
allogeneic transplantation if they had an identical family donor or
being randomized to additional high-dose chemotherapy with no further
treatment (STOP) or ABMT. The overall survival of the population at 7 years was 40%. Prognostic factors defined three risk categories. The
good-risk group had favorable cytogenetics. The poor-risk group
cumulated unfavorable cytogenetics to poor blast clearance after the
first induction course. All other patients belonged to the so-called
standard group. Having a donor for an allogeneic transplant was
significantly better in term of leukemia-free survival in the standard
group but not in the others. In all categories, ABMT was superior to
the STOP arm, with, at 7 years, a significantly lower relapse incidence
(37% v 58%) and a significantly higher leukemia-free survival
(54% v 40%; Fig 3). Because the
transplant-related mortality in the ABMT arm was high, it was felt that
some improvement might be searched in this direction in the future. Two
other multicentric trials, one in children64 and the other
one in adults,160 failed to show any advantage of ABMT over
conventional chemotherapy. In the first one (POG), a lower relapse
incidence in the ABMT arm was counterbalanced by a higher
transplant-related mortality. In the second one (GOELAM), results of
allogeneic BMT, ABMT, or intensive chemotherapy consolidation were
identical. The investigators attributed their findings to an unexpected
high relapse rate in the allo BMT arm and the use of high-dose ARAC (24 g/m2) in the chemotherapy arm, which was felt to reproduce
results achieved with the higher dose arm in the CALGB ARAC dose
study.89 They concluded that intensive chemotherapy
consolidation with high-dose ARAC was similar to ABMT.
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| Fig 3.
MRC-10 Study. Leukemia-free survival of patients
randomized to ABMT or STOP arm. (Reprinted with permission from Burnett
et al, "Randomized comparison of autologous bone marrow
transplantation to intensive chemotherapy for acute myeloid leukaemia
in first remission: Results of MRC-AML 10 trial," volume 351, page
705. © by The Lancet Ltd, 1998.163)
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In summary, with all trials considered, comparisons of allogeneic BMT
to ABMT have been in favor of the first transplant modality. Comparisons of ABMT to conventional chemotherapy have shown in most
instances a significantly reduced relapse incidence in the ABMT arm.
Also, in most studies, ABMT has been associated with a better
leukemia-free survival, except when a high transplant-related mortality
has suppressed the benefit resulting from the reduced relapse
incidence.64,163 No study has shown an advantage of conventional chemotherapy over ABMT. This indicates that ABMT represents the highest available antitumor consolidation regimen, which
is limited, however, by its feasibility and toxicity, as recently
confirmed by a metaanalysis of seven of the randomized trials conducted
between 1984 and 1995.164 It is possi |
Introduction
References