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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3156-3162
BCR-ABL Antisense Oligodeoxynucleotide In Vitro Purging and
Autologous Bone Marrow Transplantation for Patients With Chronic
Myelogenous Leukemia in Advanced Phase
By
Paolo de Fabritiis,
Maria Concetta Petti,
Enrico Montefusco,
Maria
Stefania De Propris,
Roberta Sala,
Roberto Bellucci,
Marco Mancini,
Alessandro Lisci,
Francesco Bonetto,
Tim Geiser,
Bruno Calabretta, and
Franco Mandelli
From the Dipartimento di Biotecnologie Cellulari ed Ematologia,
Università "La Sapienza," Rome; Centro Ricerca
Sperimentale, Istituto Regina Elena, Rome, Italy; Lynx Therapeutics
Inc, Hayward, CA; and Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, PA.
 |
ABSTRACT |
BCR-ABL antisense oligodeoxynucleotides (ODN) have provided evidence
of antileukemia effect when tested in vitro against
Philadelphia-positive (Ph-pos) cells and in vivo when injected into
leukemic mice. On the basis of the results obtained in vitro at
diagnosis, eight patients with chronic myelogenous leukemia (CML) were
selected and submitted to autologous bone marrow transplantation (ABMT) with bone marrow (BM) cells purged in vitro with junction-specific (J-sp) BCR-ABL antisense ODN at the time of transformation in accelerated phase or during second chronic phase. Mononuclear BM cells
were treated in vitro for 24 or 72 hours with 150 µg/mL of antisense
ODN yielding a median recovery of 47.6% mononuclear cells, 48.8%
CD34+ cells, and 20.3% clonogenic cells. After a
conditioning regimen including busulphan and etoposide, the reinfused
treated cells allowed engraftment and hematologic reconstitution in all
patients. Evaluation of the antileukemic effect by standard cytogenetic analysis and fluorescence in situ hybridization showed a complete karyotypic response in two cases and a minimal or no response in the
other six. The patient autografted in second chronic phase died in
blast crisis 7 months after ABMT; of the seven patients autografted in
transformation, three developed blast crisis 21 to 39 months after
reinfusion, one died from unrelated BMT complications 30 months after
ABMT, and three are in persistent second chronic phase 14 to 26 months
after autograft. The low toxicity of the protocol and the hemopoietic
reconstitution observed in all patients make this approach feasible;
the marked karyotypic response observed in some patients and the
duration of the second chronic phase show that ODN-mediated BM purging
and autograft is a promising treatment for this high-risk group of CML.
| |
INTRODUCTION |
CHRONIC MYELOGENOUS leukemia is ideally
suited for experimental therapeutic approaches with antisense
ODN1 because the Philadelphia chromosome translocation
results in the generation of BCR-ABL fusion gene2,3 that
encodes for leukemia-specific transcripts. Furthermore, CML remains a
fatal disease that might benefit from experimental approaches. A number of studies have shown that BCR-ABL ODN have antileukemia effects in
vitro and in mice,4,5 suggesting that suppression of
Philadelphia-positive (Ph-pos) cell growth may be due, at least in
part, to a specific mechanism. One of the problems associated with the
systemic administration of ODN in patients with CML may relate to the
nonspecific effects of these compounds and to the possible toxicity
toward normal marrow progenitors that could limit their use as in vitro
purging agents and as drugs for systemic therapy. The application of
antisense ODN in bone marrow (BM) purging before autologous BM
transplantation (ABMT) has been recently reported6,7; in
these studies, however, the c-myb gene was the
target for antisense ODN therapy using CD34+-enriched cells
that were reinfused in patients subjected to high-dose chemotherapy. We
have recently reported that in vitro purging of CML mononuclear cells
with BCR-ABL antisense ODN does not prevent hematologic reconstitution
after ABMT in a patient in accelerated phase.8 In that
study, cells were incubated for 24 hours with the 26-mer
phosphorothioate B2A2 antisense ODN, and the
patient showed marrow engraftment and hematologic reconstitution after a period of time comparable to that necessary for engraftment using
unpurged ABMT.
We now report the results obtained in eight patients with CML
autografted in an advanced phase of the disease. Patients were selected
for purged ABMT on the basis of the clinical status and in vitro tests
performed at diagnosis, which identified as "responders" 62% of
the patients tested.9 In the first three
autografts, cells were incubated with ODN for 24 hours; in the
subsequent five patients the incubation time was prolonged to 72 hours,
based on the evidence that a more pronounced inhibitory effect is
obtained following a more prolonged incubation with ODN and in view of the stable engraftment observed in the first three patients after a
preincubation of 24 hours. The evaluation of Ph-pos cells before and
after ABMT by standard cytogenetics and fluorescence in situ hybridization (FISH) in interphase nuclei together with the clinical follow-up have shown the feasibility and potential benefits of this
combined procedure for patients with CML in advanced phase of the
disease.
| |
PATIENTS AND METHODS |
Patients.
From a group of 35 patients with Ph-pos CML tested in vitro at
diagnosis for inhibition of methylcellulose colony formation with
26-mer BCR-ABL antisense ODN, 8 patients were selected for in vitro
purging with junction-specific antisense ODN followed by ABMT.
Selection was based on the in vitro sensitivity to antisense ODN, which
discriminated between "responders" (cases showing a significative
inhibition of clonogenic Ph-pos cells by the specific antisense ODN as
compared with untreated cells) and "nonresponders" (cases in
which the inhibition of colony formation was not significant), and on
the clinical status (age less than 55 years; good performance status;
no cardiac, renal, or hepatic alterations). Clinical and hematological
data of the patients at diagnosis and at the time of ABMT are reported
in Table 1. Polymerase chain reaction (PCR) analysis revealed the presence of the B2A2
junction in five cases and of the B3A2 junction
in three. All patients received interferon- (IFN-) as standard
treatment after diagnosis, obtaining a 24-months (range, 3 to 52)
median duration of the first chronic phase. ABMT was performed in
accelerated phase in seven cases and in second chronic phase in one; in
all cases, BM harvest and in vitro purging were performed immediately
before starting the conditioning regimen.
Oligodeoxynucleotides.
Phosphorothioate ODN ([S]ODN) were synthesized on a
Lynx-made DNA synthesizer (Lynx Therapeutics, Hayward, CA)
by means of  -cyanoethilphosphoramidite chemistry. The sequences of
the apyrogenic sterile 26-mer B2A2 and
B3A2 antisense ODN used in these studies were
as follows: 5 -CGC TGA AGG GCT TCT TCC TTA TTG AT-3
(B2A2) and 5-CGC TGA AGG GCT TTT GAA CTG TGC
TT-3 (B3A2). Controls included the antisense
not corresponding to the patient m-RNA junction, sense sequences, and a
12-mer ODN containing a two-base mismatched and the TAT motif (5-CTC
TTT CCT TAT-3), postulated to nonspecifically inhibit proliferation of
Ph-pos cells.
In vitro purging and colony assay.
BM (10 to 15 mL/kg body weight) was aspirated from the pelvis under
general anesthesia and collected in transfer packs (Baxter, Deerfield,
IL) containing heparinized medium. After removing plasma and the majority of red cells by centrifugation, mononuclear cells were
separated on a Ficoll density gradient (Nycomed, Oslo,
Norway) and resuspended at a concentration of 1 to 1.5 × 107/mL into 175-cm2 vented tissue culture
flasks (Falcon; Becton Dickinson, Mississauga, Ontario,
Canada), each containing 100 mL of Iscove's medium
supplemented with 10% autologous serum and 150 µg/mL of
B2A2 or B3A2 antisense ODN. Flasks were incubated for 24 or 72 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the incubation
period, adherent and nonadherent cells were poured into 50-mL conical tubes, centrifuged, washed, and cryopreserved in 40% autologous serum
using standard methods.
Control samples (6 × 105 mononuclear cells/mL), either
from the eight CML patients or from normal BM donors, were seeded into 24-well cell culture plates in the same conditioned medium and for the
same incubation period as the marrow used for reinfusion and were
incubated in the absence of ODN or in the presence of sense ODN,
antisense ODN not corresponding to the patient m-RNA junction (J-nonsp
AS) and a mismatched ODN. After incubation, cells were plated in
methylcellulose in the presence of interleukin-3 (IL-3; 20 ng/mL),
granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 ng/mL), and
fetal calf serum (30%). After 12 days, plates were scanned with an
inverted-phase microscope and granulocyte colony-forming unit
(CFU-GM)-derived colonies were counted.
Detection of p210 protein.
For the detection of the BCR-ABL protein, untreated or
B2A2 and B3A2 antisense
ODN-treated cells were lysed and supernatants collected after
centrifugation. Electroblotting to nitrocellulose, probing with the
anti-abl antibody, and protein detection were as
described.9
Conditioning regimen and supportive care.
Conditioning regimen consisted of busulfan (4 mg/kg/d over 4 days) and
VP-16 (20 mg/kg/d over 2 days) followed 48 hours later by reinfusion of
cryopreserved purged cells. Patients were settled in double rooms in a
protected environment and treated with antibiotics, amphotericin,
irradiated blood products, and intravenous nutrition as indicated.
Those seropositive for herpes simplex virus received prophylactic
acyclovir.
Standard cytogenetics.
Cytogenetic evaluation by standard methods was performed on BM cells by
direct technique and short-term culture (24 hours) followed by GTG
banding. The karyotype was expressed according to standard nomenclature
(ISCN 1991).
FISH.
The proportion of Ph-pos cells in the marrow during the follow-up of
the autografted patients was investigated on interphase cells with
FISH, using a mixture of digoxigenin and biotin-labeled cosmid DNA
probes specific for the BCR and ABL genes, respectively (Oncor,
Gaithesburg, MD), according to Arnoldus et al,10 with slight modifications based on the manufacturer's instructions. A
minimum of 500 evaluable cells in interphase were scored for each
sample. The mean percentage of interphase cells with an apparent BCR-ABL fusion signal, evaluated in 15 selected specimens (BM donors
and patients in remission for hematologic diseases other than CML) was
3.5% ± 0.5 and was interpreted due to coincidental overlapping of
the BCR and ABL signal. A BCR-ABL fusion in the test specimens was,
therefore, diagnosed if more than 5.0% (mean ± 3 SD of the
controls).
Criteria for BM engraftment and response.
Peripheral blood and marrow samples were taken at regular intervals
after autografting for morphologic, cytogenetic, and FISH analyses.
Hematologic recovery was defined as the number of days necessary to
reach 0.5 × 109/L neutrophils and 50 × 109/L platelets. Cytogenetic and FISH were comparatively
evaluated at 30, 90, 180, 365, and 730 days after ABMT or until a
second blastic transformation was documented.
| |
RESULTS |
Effect of antisense ODN on mononuclear, CD34+, and
clonogenic CML cells.
Table 2 shows the number of mononuclear,
CD34+, and clonogenic cells obtained and incubated with
antisense ODN, together with the number of cells recovered after
exposure to conditioned medium without ODN and to junction-specific
(J-sp) antisense ODN for 24 or 72 hours. A median of 6.1 × 109 mononuclear cells (range 1.8 to 18), containing a
median of 3.6% CD34+ cells (0.7 to 13) and a median of 4.5 × 106 clonogenic cells (range 0.4 to 136), were treated
in vitro with antisense ODN, yielding a median recovery of 47.6%
(range 30 to 89) mononuclear cells, 48.8% (range 11 to 85)
CD34+ cells, and 20.3% (range 0 to 70) clonogenic cells.
Incubation of cells in culture medium that did not contain ODN produced
a recovery of CD34+ and clonogenic cells similar to the
values found before purging. In some cases, incubation with the
conditioned medium without ODN caused a slight increase of mononuclear
cells (Table 2). As compared with the 24-hour incubation, the 72-hour
incubation produced a further reduction of cell recovery, which was
statistically significant for CD34+ cells
(P = .022) but not for mononuclear and clonogenic cells (Fig
1).
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| Fig 1.
Mononuclear cells, CD34+ cells, and
clonogenic cells recovered after bone marrow purging. Bone marrow
cells were incubated for 24 hours ( ) (n = 3) or 72 hours ( )
(n = 5). The recovery of different cell fractions was calculated as
the proportion of the pretreatment count.
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All patients were also tested for inhibition of methylcellulose colony
formation with several control ODN. Table 3reports the proportion of clonogenic cells (CFU-GM) of the untreated
samples counted after incubation with the J-sp antisense ODN, J-nonsp antisense ODN, sense sequences, and a mismatched TAT containing sequence ODN. As expected, neither sense nor the mismatched sequences caused a significant inhibition of CML clonogenic cells. Incubation with J-nonsp antisense ODN produced a variable recovery of clonogenic cells, greater in all cases than the recovery found after treatment with the J-sp antisense. Moreover, mononuclear cells from five normal
BM samples were incubated with the above ODN under the same conditions
used for CML cells. As previously reported,9,11 no toxic
effect on clonogenic cell growth was found after treatment of normal BM
for 72 hours with B2A2 and
B3A2 antisense ODN, sense sequence ODN, and the
mismatched ODN (data not shown).
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Table 3.
Recovery of Clonogenic Cells (% of Untreated Cells)
After Incubation of Mononuclear Cells With the J-sp Antisense or
Different Control ODN
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As mentioned previously, Ph-pos cells from all cases were tested at
diagnosis for in vitro inhibition of colony formation by the J-sp
antisense ODN. Table 4 reports the number
of colonies/105 mononuclear cells after exposure to J-sp
antisense ODN, J-nonsp antisense ODN, and culture medium without ODN
found at the screening test of each CML patient. All cases showed a
significant inhibition (P < .05) of colony formation
compared with untreated samples and were, therefore, admitted to the
ABMT program. However, incubation with the J-nonsp antisense ODN showed
a number of colonies significantly different in three cases (patient
nos. 4, 6, and 8) and not different (P = not significant) in
five cases, compared with the J-sp antisense treatment, confirming that
a certain degree of nonspecific effect on BCR-ABL-positive CML cells
might be seen using both B2A2 and B3A2 antisense ODN. The incubation with culture
medium without ODN showed, on the contrary, no inhibition in all eight
samples. Finally, when the results of the screening test were compared with those of BM purging, a significant correlation was found, validating the screening test performed during the chronic phase (Fig
2).
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Table 4.
Clonogenic Cells After In Vitro Incubation of Ph-pos
Cells With J-sp Antisense, J-nonsp Antisense, or Culture Medium at the Time of Diagnosis
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| Fig 2.
Correlation of clonogenic cell inhibition after the
screening test at diagnosis and after the large-scale bone marrow
purging.
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BCR-ABL protein expression after treatment with J-sp and J-nonsp
antisense ODN.
Western blot analysis has been performed on untreated and antisense
ODN-treated samples from all patients. Figure
3 shows blots of patient no. 4 who had a
marked decrease in the BCR-ABL protein after treatment with
B2A2 antisense ODN, subsequently confirmed by
the cytogenetic results obtained in samples taken after autograft. Of
the seven remaining cases, one showed a degree of p210 protein
downregulation comparable with the in vitro inhibition of Ph-pos
clonogenic cells (no. 6), two had a modest (nos. 7 and 8), and the
remaining four showed an absence of effect by J-sp and J-nonsp
antisense ODN, including the three cases incubated in vitro for 24 hours, consistent with the relatively long (>48 hours) half life of
the protein.12
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| Fig 3.
Effect of BCR-ABL antisense ODN treatment on p210 levels
in cells from patient no. 4. Lane 1, mononuclear cells incubated for 72 hours with culture medium without ODN (control); lane 2, mononuclear
cells incubated for 72 hours with the J-sp B2A2
antisense ODN; lane 3, mononuclear cells incubated for 72 hours with
the J-nonsp B3A2 antisense ODN.
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Toxicity.
The conditioning regimen was well tolerated in all patients. A mild
degree of nausea and sporadic episodes of vomiting were observed during
the administration of chemotherapy. Two patients suffered from severe
mucositis during aplasia, requiring local or systemic treatment. All
patients developed fever; seven were responsive to broad spectrum
antibiotics. In one patient an enterococcus sepsis was
documented; one patient developed severe pneumonitis with blood culture
positivity for Pseudomonas aeruginosa, Serratia odorifera, and
enterococcus at the same time. No side effects related to the
reinfusion of residual amounts of antisense ODN or long-term toxicities
were observed in this group of patients.
Hematopoietic reconstitution.
The eight patients reached aplasia (<0.1 × 109/L
neutrophils) within 7 days from BM reinfusion. All of them had BM
engraftment, reaching a number of neutrophils >0.5 × 109/L after a median of 26.5 days (range 19 to 56), and
seven patients showed a platelet recovery >50 × 109/L
after a median of 45 days (range 21 to 105). One patient developed blastic crisis and died before platelet reconstitution after 7 months
from ABMT; one patient, who showed no signs of engraftment after 35 days from ABMT, received the reinfusion of back-up peripheral blood
stem cells cryopreserved at diagnosis because of a severe pulmonary
infection and sepsis as described above. When we compared the time
required for hematologic recovery with the duration of the in vitro
incubation (24 hours v 72 hours), no differences were found in
the two groups of patients with regard to both neutrophil and platelet
reconstitution (data not shown).
Cytogenetic and FISH evaluation.
Table 5 summarizes the cytogenetic and FISH
data investigated side by side at various times before and after ABMT.
By cytogenetic analysis, two patients showed complete disappearance of
Ph-pos cells within 90 days from ABMT, although at the subsequent
control (day 180) the leukemic clone reemerged in both of them. One
patient died after 7 months from ABMT in second lymphoid blast
transformation, and one showed a progressive increase in the proportion
of Ph-pos cells, still remaining in second chronic phase with a
proportion of BM ph-negative cells at 2 years of follow-up. The other
six patients showed no significant decrease in the proportion of Ph-pos cells after ABMT (5% to 15% being the range of Ph-negative metaphases found at various controls), not influenced by the starting of maintenance therapy after ABMT.
FISH analysis showed a proportion of rearranged cells comparable with
that found by standard cytogenetics. It is interesting to note,
however, that samples with 100% Ph-pos metaphases at cytogenetics
displayed a proportion of nonrearranged cells (4% to 20% of
interphase cells) by FISH; conversely, when no Ph-pos metaphases were
found at cytogenetics, a proportion of 10% to 30% rearranged cells
was found among the 500 cells evaluated in interphase. As already
reported, FISH analysis appears to be more sensitive than cytogenetics
for monitoring of residual disease in CML.
Follow-up.
Table 6 shows the present clinical
situation of the eight patients after a median follow-up of 27.5 months
from ABMT. The patient autografted in second chronic phase died from
second blast transformation 7 months after reinfusion; three patients
developed blast crisis 21, 21, and 39 months after ABMT, and one
patient died from interstitial pnumonitis after unrelated BMT 30 months after ABMT, while in second chronic phase. The other three cases are
still in second chronic phase 14, 20, and 26 months after autograft.
Four of the eight patients are long survivors after 48, 50, 54, and 86 months from diagnosis. In four patients, the duration of the chronic
phase after autograft has exceeded that of the first chronic phase.
| |
DISCUSSION |
Antisense ODN directed to leukemia-specific transcripts have the
potential to specifically target genes with a pathogenetic role in
leukemogenesis while sparing normal hematopoietic cells. Animal studies
have confirmed the effectiveness of the in vivo treatment of malignant
and nonmalignant disease processes with antisense
ODN.5,13-15 However, for the majority of tumors, including CML, problems of toxicity, uptake, and specificity still need to be
completely solved. The use of ODN for in vitro purging before autograft
partly overcomes these problems. Compared with the dosages necessary
for systemic therapy, the total amount of ODN required for BM purging
is relatively small, the excess of antisense ODN can be washed out
following the in vitro incubation period, and the effects on malignant
and normal clonogenic cells can be monitored with in vitro tests. An
exact quantification of the antileukemia effect exerted by antisense
ODN is, however, complicated by the concomitant effect of the
conditioning regimen.
ABMT has been successfully used in recent years for the treatment of
CML, and both single center and multicenter studies have shown the
feasibility of ABMT in chronic phase CML, although a long-term
advantage for patients receiving an autograft is still to be fully
shown.16-18 Evaluation of the role of autograft in blast-phase CML is more difficult; very few studies have been reported
in this phase of the disease19 at a time when patients are
often in very critical conditions. In an earlier study,20 we treated five patients with CML in blast phase with unpurged autograft; in all cases, a very short second chronic phase was obtained
and all patients died within 6 months from the autograft. The validity
of purging has been investigated mainly in acute leukemias21; the lack of CML-specific antigens on Ph-pos
cells and the involvement of the CD34+ cell compartment in
the disease have hampered the development of immunologic strategies for
ex vivo purging in CML. The use of BCR-ABL antisense ODN may be
considered one of the newest approaches for the treatment of CML, and
the in vitro treatment before autograft appears, at the moment, the
clinical application more likely to produce therapeutic
results.22 We and others have reported that BCR-ABL ODN
inhibit CML colony formation both in blast and in chronic
phase.4,9,11,23 Moreover, BCR-ABL ODN are capable of
suppressing the growth of Ph-pos cells when injected into leukemic mice.5,13 However, nonspecific effects of ODN on
chronic-phase cells and on normal cells have been
reported,24,25 raising concerns on the feasibility of BM
purging before autograft. In the present study we describe the
biological and clinical results obtained in eight patients with CML in
advanced phase autografted with BM cells treated in vitro with BCR-ABL
antisense ODN. Patients were selected on the basis of the in vitro
sensitivity of CML cells to BCR-ABL antisense ODN evaluated at
diagnosis9 and on the basis of clinical criteria.
Twenty-two of the 35 patients tested in vitro showed a significant
reduction of Ph-pos clonogenic cells after treatment with BCR-ABL J-sp
antisense ODN. The validity and reproducibility of the in vitro test
was confirmed by the significant correlation found with the large-scale
BM purging in terms of clonogenic cell inhibition. When compared with
the 24-hour incubation time, the 72-hour incubation of BM cells with antisense ODN induced a further decrease in the number of residual leukemic cells and allowed in some cases a downregulation of the p210
protein. In spite of the small number of cases in the two treatment
groups, the difference was statistically significant for the
CD34+ cell recovery, suggesting that a more prolonged
incubation with BCR-ABL antisense ODN is needed to obtain a marked
inhibition of CML clonogenic cells. These observations are consistent
with the relatively long half-life of the p210 protein.
In general, the overall procedure of ABMT was well tolerated in all
patients. No side effects related to the reinfusion of residual amounts
of antisense ODN were observed, and only one patient developed a severe
pulmonary infection with sepsis that required antibacterial and
antifungal therapy. Although the patient's BM showed initial signs of
engraftment, we decided to reinfuse the untreated mononuclear back-up
cells because of the delay in hematologic reconstitution. All patients
showed neutrophil reconstitution. In one patient, platelet recovery was
not observed because of a second blast transformation developed 7 months after ABMT. A wide range of variation in the number of days to
neutrophil and platelet reconstitution was noted within the eight
patients; however, the intensity of the conditioning regimen currently
used not only for autologous but also for allogeneic BMT makes it
highly likely that BM engraftment was due to the reinfused cells and
that the incubation with the 26-mer antisense ODN, therefore, was not
toxic for the pluripotent stem-cell compartment.
The impact of antisense ODN as in vitro purging agents on the reduction
of Ph-pos cells evaluated after ABMT by standard cytogenetics and by
FISH is of difficult interpretation. Autografts performed in blast
transformation rarely allow a karyotypic response, although sporadic
cases of partial or complete disappearance of Ph-pos cells have been
reported.19 In our study, two patients showed a complete
karyotypic response between 30 and 90 days after ABMT. This result,
however, did not correlate with a better clinical outcome; one patient
(autografted in second chronic phase) in fact developed a second
lymphoid blast transformation and died within 7 months from ABMT, while
the other showed the reappearance of leukemic cells at day 180 from
ABMT. In general, the impact of the purged autograft on the duration of
second chronic phase and survival after autograft is encouraging
(median duration of chronic phase 21 months and survival 27.5 months).
In a recent study,6 CD34+ selected cells or
mononuclear cells were in vitro treated for 24 or 72 hours with
antisense ODN to the c-myb gene and reinfused in patients with
chronic phase or accelerated phase. Despite the very efficient purge,
engraftment in some patients was poor. In our study, all patients
showed hematologic reconstitution and no procedure-related deaths were
observed. Although the rationale for using the c-myb antisense
ODN for in vitro purging has been well shown,26 it cannot
be excluded that prolonged treatment with antisense ODN might partly
affect normal stem/progenitor cells, thus interfering with subsequent
BM engraftment. The selection and in vitro treatment of enriched
CD34+ cells may also reduce the stem-cell compartment.
CD34+ cell loss due to selection of CD34+ cells
and the greater antisense ODN uptake compared with the mononuclear cell
population9 may produce a greater inhibition of
CD34+ cells. Elimination of a greater number of Ph-pos
cells is probably achievable through the in vitro incubation of
CD34+ cells with c-myb antisense ODN,7
but the effects on normal cells need to be further investigated. We
have used an antisense ODN specific for the BCR-ABL junction using the
mononuclear cell compartment as the target of the in vitro incubation.
In addition, after BM reinfusion patients were not treated with growth
factors to accelerate BM engraftment or hematologic recovery. Although the antileukemia effects might be improved, the aim of our procedure was to minimize the nonspecific toxicity of antisense ODN and avoid
manipulations that might prolong the time necessary to engraftment of
BM cells. The data on hematopoietic reconstitution after purging may
serve as a reference for future investigations of antisense ODN in
patients with advanced CML and also assess the nonspecific toxicity of
antisense ODN that might be associated with modifications of the
procedure.
The overall antileukemia effect of the protocol used can probably be
improved. Patients in chronic phase still responding to conventional
therapy may represent a more appropriate target both for a greater
inhibition of Ph-pos cells in vitro by antisense ODN and for a better
activity of the conditioning regimen in vivo. The development of more
specific and active antisense ODN and the optimization of in vitro and
in vivo treatment conditions may produce better results in autografted
CML patients.
| |
FOOTNOTES |
Submitted March 10, 1997;
accepted December 16, 1997.
Supported in part by a grant from Consiglio Nazionale delle Ricerche
(CNR), Special project "Applicazioni Cliniche della Ricerca Oncologica" (ACRO), Roma, Italy.
Address reprint requests to Paolo de Fabritiis, MD, Institute of
Hematology, Via Benevento, 6, 00161 Rome, 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.
| |
ACKNOWLEDGMENT |
We thank Prof R Foà for his helpful comments and for revising the
manuscript.
| |
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Abstract
Introduction
Abstract