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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1820-1831
Comparative Analysis of Autografting in Chronic Myelogenous
Leukemia: Effects of Priming Regimen and Marrow or Blood Origin of
Stem Cells
By
Catherine M. Verfaillie,
Ravi Bhatia,
Michael Steinbuch,
Todd DeFor,
Betsy Hirsch,
Jeffrey S. Miller,
Daniel Weisdorf, and
Philip
B. McGlave
From the Department of Medicine, Stem Cell Biology Program, Bone
Marrow Transplantation Program, and Department of Laboratory Medicine
at the University of Minnesota, Minneapolis, MN; and The City of Hope
Medical Center, Duarte, CA.
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ABSTRACT |
The aims of this study were (1) to evaluate the effect of
intermediate (cyclophosphamide alone) or intensive (mitoxantrone, cytosine arabinoside, cyclophosphamide) priming on the cytogenetic response in mobilized bone marrow (BM) or peripheral blood (PB) progenitors in patients with chronic myelogenous leukemia (CML), (2) to
determine the incidence of cytogenetic remissions after mobilized
progenitor transplantation in CML, and (3) to determine the effect of
in vivo priming on the ability to select Philadelphia chromosome-negative (Ph-negative)
CD34+HLA-DR cells from mobilized BM or PB
in quantities sufficient for transplantation. Between February 1994 and
March 1997, 44 patients were enrolled in three sequential protocols.
Although the duration of neutropenia after only cyclophosphamide
mobilization was shorter, clinical morbidity for the intermediate and
intensive priming protocols was similar. Cytogenetic responses in
mobilized PB progenitors were similar after mobilization with either
intermediate or intensive chemotherapy. The degree of Ph negativity in
the mobilized product correlated with disease stage at the time of
mobilization (early chronic phase [ECP] > late CP > accelerated
phase). Cytogenetic responses after transplantation with mobilized
progenitors obtained after the different regimens were similar. The
cytogenetic status of the graft predicted the cytogenetic status of
marrow obtained 3 weeks after transplantation whereas cytogenetic
responses 3, 6, and 12 months after transplantation correlated with the
number of BCR/ABL-negative CD34+HLA-DR
cells, but not the number of Ph-negative metaphases in the graft. In
patients with ECP CML, mobilized PB collections yielded significantly more CD34+HLA-DR cells than from steady
state or mobilized BM. CD34+HLA-DR cells
were Ph negative and polyclonal (X-chromosome inactivation) in the
majority of ECP CML patients, before and after mobilization and
irrespective of the mobilization regimen. Because infusion of large
numbers of Ph-negative CD34+HLA-DR cells
predicted superior outcome after transplantation, approaches in which
CD34+HLA-DR cells are selected from
mobilized PB may result in longer lasting and clinically significant
cytogenetic responses after transplantation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
malignancy of the human hematopoietic stem cell.1 The
disease is caused by the Philadelphia chromosome (Ph)2
resulting from a translocation between the c-bcr gene located on
chromosome 22 and the c-abl gene located on chromosome 9.3
The p210BCR/ABL oncoprotein is necessary and sufficient for
malignant transformation.4 p210BCR/ABL has
increased tyrosine kinase activity compared with p145ABL
5 and binds abnormally to the
actin-cytoskeleton6; these characteristics are thought to
be responsible for inducing the malignant behavior of target cells.
CML is a disease that is most frequent during the fifth and sixth
decade of life.7 CML is invariably lethal. After a chronic phase (CP) which lasts 3 to 4 years, the disease transforms into an
accelerated phase and ultimate blast crisis.7 Although
therapy with single-agent chemotherapeutic agents controls symptoms
during the CP,7 disease outcome is not affected. Therapy
with interferon- (IFN-), either alone 8-11 or in
combination with cytosine arabinoside12 can induce
hematologic remissions in >80% of CP patients. In addition, 10% to
25% of patients obtain a major cytogenetic response, which is
associated with a superior survival. However, acquisition of a
hematologic response without major cytogenetic response does not
significantly affect survival.8-11 Ablative chemotherapy
with or without irradiation therapy followed by infusion of normal stem
cells obtained from a closely matched related or unrelated donor
results in 5-year disease-free survival of 30% to
80%.13,14 Success of transplantation is dependent on the
degree of donor match, age of the patient, and the disease stage and
duration at the time of transplantation.15 However, a large
proportion of patients are ineligible for allogeneic transplantation
due to age or lack of a suitably matched donor.
Several lines of evidence suggest that benign Ph-negative stem cells
may coexist with the Ph-positive clone in patients with CML. At
diagnosis, some patients have evidence of Ph-negative hematopoiesis on
routine cytogenetic studies of the bone marrow (BM).16,17
Treatment with IFN-,8-13 busulphan,18 or
intensive chemotherapy19 can result in the reestablishment
of Ph-negative hematopoiesis. Culture of CML BM or peripheral blood
(PB) in long-term culture reveals presence of normal Ph-negative
primitive progenitors.20,21 We and others have shown that
CD34+HLA-DR cells present in
steady-state BM of early CP (ECP) CML patients are highly enriched in
Ph-negative, BCR/ABL mRNA-negative, and polyclonal primitive long-term
culture initiating cells.22-25 This has led to the
hypothesis that autologous transplantation of PB or BM cells may
restore Ph-negative hematopoiesis. Transplantation of autologous PB or
BM cultured in long-term culture20,21 or exposed short term
to IFN- ,26 mafosfamide,27 or anti-myb antisense oligonucleotides28 has resulted in at least
temporary restoration of Ph-negative hematopoiesis. More recently,
several studies have indicated that transplantation of PB cells
obtained after in vivo mobilization with chemotherapy and cytokines may also result in the reestablishment of Ph-negative
hematopoiesis.19,29-35
In this study we sought to compare the effect of priming with
intermediate or high-dose chemotherapy and granulocyte
colony-stimulating factor (G-CSF) or granulocyte-macrophage
colony-stimulating factor (GM-CSF) on the mobilization of Ph-negative
progenitors in BM and PB. We correlated the early course of patients
transplanted with either BM or PB cells obtained after in vivo
chemotherapy priming with patient characteristics, type of mobilizing
chemotherapy, and graft characteristics. We also examined the effect of
priming on the absolute number and clonal origin of
CD34+HLA-DR cells in PB and BM.
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MATERIALS AND METHODS |
Patients
Between February 1994 and March 1997, 44 patients with CML were
enrolled on three sequential autologous transplantation protocols at
the University of Minnesota after informed consent was obtained using
guidelines approved by the Committee on the Use of Human Subjects for
Research at the University of Minnesota. Patient characteristics at the
time of transplantation including age, gender, disease phase, prior
therapy, BM cytogenetics, BCR/ABL status, and white blood cell (WBC)
count are summarized in Table 1. Patients
were classified using the criteria from the National Marrow Donor
Program (NMDP) characterizing CP and accelerated phase
disease. Chronic phase is defined as no significant symptoms after
treatment, no features of accelerated phase or blast crisis, and no
cytogenetic changes other than the Ph chromosome. Accelerated phase is
defined as a rapid doubling of the WBC count, >10% blasts in blood
or marrow, >20% basophils and eosinophils in blood or marrow,
anemia, thrombocytopenia, thrombocytosis, additional cytogenetic abnormalities, splenomegaly not responsive to therapy, and severe myelofibrosis.36
Mobilization Regimens
Between February 1994 and September 1995, 17 patients were mobilized
with a single dose of cyclophosphamide (4 g/m2) and GM-CSF
(250 µg/m2) from day 4 after cyclophosphamide
administration until the end of the PB progenitor cell
(PBPC) collections (protocol CY/GM/BM). When the absolute neutrophil count (ANC) was 500 to
1,000/µL, a BM harvest (1.5 L) followed by three
(range, three to six) PBPC products were obtained. BM and PBPC cells
were cryopreserved without further manipulation and the BM was used as
the graft.
From September 1995 to September 1996, 18 patients received priming
cyclophosphamide (4 g/m2, day 1), mitoxantrone (4 mg/m2, days 1 and 2), and cytosine arabinoside (1 g/m2 twice daily, days 1 and 2) followed by G-CSF (5 µg/kg) from day 4 until the end of PBPC collections as mobilization
(protocol MAC/G/PB). Patients underwent a BM harvest (2 × 108 mononuclear cells [MNC]/kg) before initiation
of the mobilization regimen (cryopreserved as back-up BM stem cells).
Once the ANC reached 500 to 1,000/µL, PBPC containing a minimum of 2 × 106 CD34+ cells/kg were collected and
cryopreserved unmanipulated as graft (median number of collections, 5;
range, 2 to 10). During the same period patients underwent a diagnostic
BM aspirate.
From September 1996 to April 1997, an additional 9 patients received
cyclophosphamide (4 g/m2, day 1) and G-CSF (5 µg/kg) from
day 4 until the end of PBPC collections (protocol CY/G/PB). When the
ANC reached 500 to 1,000/µL, PBPC containing a minimum of 2 × 106 CD34+ cells/kg PBPC (median number of
collections, 4; range, 3 to 5) were collected and cryopreserved
unmanipulated as graft.
The mobilizing chemotherapy was administered on the Inpatient Bone
Marrow Transplantation (BMT) Unit at the University of Minnesota. After
the mobilization chemotherapy, patients were discharged on prophylactic
antibiotics (penicillin, fluconazole, and ciprofloxacin). They were
seen daily in the Outpatient BMT Clinic where PB counts were followed
and patients evaluated for infectious complications. When indicated,
platelet and red blood cell (RBC) transfusions were administered and
patients received intravenous antibiotics for febrile complications
either as inpatients or outpatients, as needed.
Transplantation
All patients were myeloablated with cyclophosphamide (60 mg/kg, days
 7 and 6) and fractionated TBI (165 cGy twice daily; total
dose, 1,320 cGy on days 4, 3, 2, and 1) on
the Inpatient BMT Unit at the University of Minnesota. Between February
1995 and January 1996, patients remained hospitalized until the ANC was
500/µL for 3 consecutive days. After January 1996, patients were
discharged from the hospital whenever possible on day +1 after
transplantation on prophylactic antibiotics. They were followed daily
in the Outpatient BMT Clinic and readmitted to the Inpatient BMT Unit
when febrile. All patients were eligible for additional supportive care
study protocols including, but not limited to, those related to
prophylaxis and treatment of infection, blood product support,
mucositis, and/or nutritional support. After transplantation, patients received either GM-CSF (250 µg/m2, protocol CY/GM/BM) or G-CSF (5 µg/kg; protocols
MAC/G/PB and CY/G/PB) until the ANC was >2,500/µL for 3 consecutive
days.
Posttransplantation Immunotherapy
IFN- was administered once the ANC was >1,000/µL for 3 days
without cytokine support, platelets were >80,000/µL, and patients were RBC transfusion independent. The IFN dose was 0.1 × 106 U/m2 daily subcutaneous
(sc) × 1 week, 0.25 × 106
U/m2 daily sc × 1 week, 0.5 × 106
U/m2 daily sc × 1 week, then escalated by 0.5 MU/m2 every 2 weeks to maximum tolerated dose.
Posttransplantation Follow-Up
Physical examination, analysis of routine chemistry, and PB
studies were performed at 3 weeks and 3, 6, 9, and 12 months after transplantation and annually thereafter during scheduled outpatient visits to the BMT Clinic. BM samples were examined for morphological signs of relapse and for the presence of the Ph chromosome and the
BCR/ABL mRNA by reverse transcription polymerase chain reaction (RT-PCR).
In Vitro Studies
We obtained a PB and BM sample before mobilization, a BM sample when
the WBC count was 700 to 1,000/µL during the recovery phase after
mobilization (CY/GM/BM and MAC/G/PB), and a sample from each PBPC
collection (CY/GM/BM, MAC/G/PB, and CY/G/PB). Each sample was examined
for the presence of the Ph chromosome, the number of colony-forming
cells (CFC) and long-term culture initiating cells (LTC-IC) present,
and the number and clonal origin of
CD34+HLA-DR cells.
Cell selection.
PB and BM MNC were isolated by Ficoll-Hypaque (Sigma Chemical Co, St
Louis, MO) density gradient separation (specific
gravity, 1.077) for 30 minutes at 37°C and
400g. A CD34-enriched population was obtained from PB MNC and
BM MNC using Ceprate avidin-biotin immunoadsorption columns (CellPro
Inc, Bothell, WA) or the MiniMACS selection system (Amgen, Thousand
Oaks, CA). CD34-enriched cells were labeled with
phycoerythrin (PE)-conjugated mouse anti-CD34 antibodies and
fluorescein isothiocyanate (FITC)-conjugated mouse anti-HLA-DR
antibodies (1 mg/106 cells) (Becton Dickinson, San Jose,
CA), incubated for 30 minutes on ice, and then washed with cold
phosphate-buffered saline. Cells were selected on a FACStar-Plus laser
flow cytometry system equipped with a CONSORT 32 computer (Becton
Dickinson) for low forward and side scatter properties and expression
of CD34 and HLA-DR antigens using mouse IgG1-PE and IgG2-FITC
antibodies as control.22,23
Methylcellulose progenitor culture.
As described, 2 × 105 BM MNC or PB MNC, or 2 to 10 × 103 CD34+HLA-DR
cells were plated in methylcellulose containing Iscove's modified Dulbecco's medium (IMDM; GIBCO Laboratories, Grand Island, NY) supplemented with 30% fetal calf serum (FCS; Hyclone,Logan, UT), 3 IU
erythropoietin (Epoietin; Amgen), and 10% supernatant of the carcinoma
cell line 5637. Cultures were incubated in a humidified atmosphere at
37°C and 5% CO2. The cultures were assessed at days 14 to 18 for the presence of burst-forming unit-erythroid, colony-forming unit-granulocyte-macrophage, and mixed colony-forming unit as previously described.37
Long-term cultures.
PB MNC or BM MNC (1 × 106) or
CD34+HLA-DR cells (2 to 10 × 103) were plated in contact with confluent, irradiated
M2-10B4 stromal layers (generous gift from Dr C. Eaves, Vancouver,
British Columbia, Canada), subcultured in 24-well plates in complete
LTC medium (IMDM; GIBCO) with 12.5% FCS, 12.5% horse
serum (Terry Fox Laboratories, Vancouver, Canada), 2 mmol/L glutamine
(GIBCO), 100 U/mL penicillin, 100 U/mL streptomycin (GIBCO), and
106 mol/L hydrocortisone. Cultures were maintained
for 5 weeks in LTC medium in a humidified atmosphere at 37°C and
5% CO2 as described.38 After 5 weeks, adherent
and nonadherent cells were recovered by short-term trypsinization and
replated in methylcellulose culture for 14 to 18 days.
Cytogenetics
BM or PB MNC were cultured overnight and subjected to a 1.5-hour
colcemid incubation followed by lysis with hypotonic potassium chloride and fixation in acid/alcohol as previously
described. Metaphases were analyzed after QFQ or GTG
banding.39
PCR Amplification of BCR/ABL mRNA
Freshly obtained PB- or BM-derived MNC, CD34+ cells, or
sorted CML CD34+HLA-DR cells were
incubated overnight in IMDM + 20% FCS at 37°C in a
fully humidified CO2 incubator, and flash frozen at
70°C. RT-PCR to amplify BCR/ABL mRNA and the
 -actin mRNA as mRNA control were performed as previously
described.22,23,40
Statistics
Spearman's rank order correlation was used to measure the association
between continuous variables in this study. In addition, the
association between cell dose and time to engraftment was evaluated in
a Cox regression analysis to account for two censored patients that
died before being evaluable for engraftment. Estimates of survival and
time to engraftment were calculated by Kaplan Meier estimation.
Univariate comparisons of survival were completed with 95% confidence
intervals (CIs) and the log-rank statistic. In comparing continuous
variables between independent groups and matched groups, the paired and
unpaired t-tests were used, respectively.
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RESULTS |
Transplantations
From February 1995 until April 1997, 44 consecutive patients with CML
that presented to the University of Minnesota for autologous transplantation were enrolled in three sequential autologous
transplantation protocols. Patient characteristics are summarized in
Table 1. There were no significant differences between the three groups for the following parameters: gender, disease stage, cytogenetics at
time of mobilization, or WBC count at time of mobilization. Sixteen
patients were in ECP, which was defined as diagnosed less than 12 months before transplantation. Fifteen patients were in late CP (LCP),
defined as diagnosed more than 1 year before transplantation but with
no clinical or cytogenetic evidence of accelerated phase disease.
Thirteen patients had accelerated phase disease, defined based on
clinical criteria (thrombocytosis, cytopenias, splenomegaly, blast and
basophil percentage, myelofibrosis, or clonal evolution). Because
results for patients in LCP and with accelerated phase disease were not
different, comparisons are reported between patients with ECP disease
and patients with either LCP or accelerated disease combined (advanced
phase [AP CML]).
After priming, the WBC count reached 700 to 1,000 at a median of 15, 18, and 13 days for protocols CY/GM/BM, MAC/G/PB, and CY/G/PB
respectively (Table 2). During the
neutropenic phase after chemotherapy, three, two, and one patients
enrolled on the three sequential protocols were hospitalized because of
infectious complications. Two patients in protocol CY/GM/BM, four
patients in protocol MAC/G/PB, and three patients in protocol CY/G/PB
did not proceed to transplantation. Eight (1, 4, and 3) patients had insufficient CD34+ cells available for transplantation.
These eight patients all had advanced-stage disease. One patient in
protocol CY/GM/BM developed blast crisis immediately after mobilization
and one patient in protocol MAC/G/PB died before transplantation
because of septic shock and diffuse lung damage. One additional patient
enrolled in protocol MAC/G/PB developed pulmonary aspergillosis,
requiring amphotericin-B therapy for 3 months before transplantation.
Fifteen (CY/GM/BM) (93.7%), 14 (MAC/G/PB) (78%), and 6 (CY/G/PB)
(66%) patients proceeded to transplantation. Patients received either
autologous mobilized BM cells and GM-CSF after transplantation (CY/GM/BM) or autologous mobilized PBPC and G-CSF after transplantation (MAC/G/PB and CY/G/PB). WBC (P < .01), RBC (P = .03),
and platelet (P = .01) engraftment was faster for
patients receiving PBPC cells (MAC/G/PB and CY/G/PB) than for patients
receiving BM progenitors (CY/GM/BM) (Table 2).
Median follow-up after transplantation for patients on CY/GM/BM is 27 months (range, 12 to 35 months). Five patients (33%) died 65, 127, 238, 408, and 710 days after transplantation, respectively: one because
of nonengraftment and four in blast crisis. The 1-year actuarial
survival is 80% (95% CI, 60% to 100%). Median follow-up after
transplantation for patients on MAC/G/PB is 12 months (range, 8 to 17).
Four patients (28.5%) died 32, 220, 461, and 560 days after
transplantation, respectively: one because of infection and three in
blast crisis. The 1-year actuarial survival of 86% (95% CI, 68% to
100%) is not significantly different from that observed after
transplantation on protocol CY/GM/BM. Median follow-up for patients on
CY/G/PB is less than 6 months. One patient died in blast crisis (day
117) and one died because of gram-negative sepsis and acute lung injury
occurring after engraftment (day 32).
One-year survival for ECP patients on the three protocols combined was
100% and 2-year survival was 53% (95% CI, 13% to 93%) (
Fig 1A). One-year survival for AP patients was 62%
(95% CI, 40% to 84%) and 2-year survival was 52% (95% CI, 26% to
78%) (Fig 1A). When compared with a historical cohort of 43 patients
who underwent autologous transplantation at the University of Minnesota using IFN--purged BM,26 1- and 2-year survivals were
similar (P = not significant) (Fig 1B).
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| Fig 1.
Survival of autografted CML patients  Cox regression
analysis of survival. (A) Comparison between patients treated on
protocols CY/GM/BM, MAC/G/PB, or CY/G/PB based on disease stage: ECP
CML (diagnosed <1 year before transplantation and in CP), LCP CML
(diagnosed >1 year before transplantation and in CP), and AP CML
(diagnosed >1 year before transplantation and in CP or accelerated
phase). (B) Comparison between patients transplanted at the University
of Minnesota with IFN--purged autologous marrow (1988 to 1994) and
patients transplanted using primed BM or PB cells in protocols
CY/GM/BM, MAC/G/PB, or CY/G/PB.
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Of the seven patients that did not undergo an autograft because of poor
mobilization, two patients underwent an unrelated donor
transplantation. One patient died from toxicity related to the
transplantation procedure. The second patient is alive 2+ months after
transplantation. The other five patients did not receive further
intensive therapy and were maintained on hydroxyurea. Three of the five
patients remain in CP 1 to 2.5 years after mobilization. One of the
five patients has accelerated phase disease and one patient is
requiring intermittent G-CSF to support low neutrophil counts.
After transplantation patients were treated with increasing doses of
IFN- once the ANC was >1,000/µL for 3 days without cytokine support, platelets were >80,000/µL, and patients were RBC
transfusion independent. Twenty-four patients were eligible for IFN-
therapy and had been followed for more than 1 year after
transplantation at the time of evaluation (1-year cytogenetics chosen
to evaluate the possible effect of IFN- on cytogenetics after
transplantation). The IFN- dose was always escalated to the
maximally tolerated dose. Mainly because of hematopoietic toxicity
(thrombocytopenia and/or neutropenia), dose escalation was not
always possible. Patients received a maximum mean dose of IFN- of
1.57 ± 0.28 × 106 U/m2/d (range, 0 to
5.5 × 106 U/m2/d). No correlation between
cytogenetics at 1 year and the dose of IFN- was observed for either
ECP or AP CML patients (r2 = .139 for ECP CML
and r2 = .031 for AP CML).
Composition of the Graft
BM and PB samples obtained before mobilization and at the time the ANC
reached 500 to 1,000 after mobilization were evaluated for the number
of CD34+ cells, CFC, and LTC-IC, as well as for the
presence of Ph-positive metaphases. For ECP CML patients, the number of
CD34+ cells, CFC, and LTC-IC in BM obtained during recovery
after mobilization was significantly lower than in steady-state BM or
in the three to eight combined PBPC collections, and this was
irrespective of the type of mobilization regimen (P < .05)
(data not shown). In contrast, differences were less significant for
patients with AP CML.
Cytogenetic analysis of steady-state BM, and BM or PB obtained after
mobilization is summarized in Figs 2 and
3. In most instances a minimum of 20 metaphases were analyzed. Before mobilization, BM of 18.6% of patients
was partially Ph negative. Three patients treated to partial Ph
negativity with IFN- had accelerated features defined as additional
chromosomal abnormalities in a fraction or all of the persisting
Ph-positive population. In addition, four patients that were 100% Ph
positive also had signs of clonal evolution. When cytogenetics of the
individual PBPC collections were compared, no significant change in the
degree of Ph positivity between the first and last collection was
observed (Fig 3). For the three sequential protocols, mobilization
resulted in a major cytogenetic response in PB progenitors of 20%
(CY/GM/BM), 71% (MAC/G/PB), and 0% (CY/G/PB); a minor cytogenetic
response in 80%, 29%, and 100%; and no cytogenetic response in 0%,
0%, and 0% of ECP CML patients (Fig 2). For AP patients, a major
cytogenetic response was seen in PB progenitors of 25%, 17%, and 0%
of patients; a minor cytogenetic response in 22%, 1%, and 0% of
patients; and no cytogenetic response in 50%, 66%, and 100% of
patients on the three sequential protocols. Cytogenetics were also
available for BM MNC obtained at the time of hematopoietic recovery
from patients on protocols CY/GM/BM and MAC/G/PB. The degree of Ph
positivity in BM and PB MNC was similar (r2 = .73; P < .0001).
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| Fig 2.
Effect of priming on cytogenetics in BM and PB. Marrow
was obtained before priming and PB and BM were obtained after priming
on protocols CY/GM/BM, MAC/G/PB, and CY/G/PB from all ECP and AP CML
patients. Marrow and PB cells were evaluated by metaphase analysis to
detect the number of Ph-positive metaphases. On average, 20 metaphases
from steady-state marrow, mobilized marrow, or PB collections were
analyzed. For PB collections, the average percent of Ph-positive
metaphases in all collections was entered in the figure. Results are
depicted for all samples that were analyzed and divided as follows:
( ), major cytogenetic response, 0% to 30% of metaphases or cells
Ph positive; ( ), minor cytogenetic response, 30% to 90% of
metaphases or cells Ph positive; ( ), no response, 90% to 100% of
metaphases or cells Ph positive.
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| Fig 3.
Contamination of sequential PB collections with
Ph-positive cells remains relatively constant. Marrow was obtained
before priming and PB or BM were obtained after priming on protocols
CY/GM/BM, MAC/G/PB, and CY/G/PB from patients with ECP or AP CML. On
average, 20 metaphases from steady-state marrow, mobilized marrow, or
PB collections were analyzed. Cytogenetic status of all collections are
shown separately. In general, cytogenetic responses did not change
significantly between the first and last PB collections. Marrow and PB
cells were evaluated by metaphase analysis and/or FISH to
detect the number of Ph-positive metaphases.
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Effect of Graft Composition on Engraftment
Patients transplanted with mobilized PBPC on protocols MAC/G/PB or
CY/G/PB had earlier ANC, platelet, and RBC engraftment than patients
receiving mobilized BM progenitors (protocol CY/GM/BM) (Table 2).
Further, we observed a strong correlation between time to ANC and
platelet engraftment and the number of CFC and LTC-IC infused
(Table 3). Although the number of CFC and
LTC-IC present in mobilized BM grafts (CY/GM/BM) was lower than
mobilized PB grafts, this did not reach statistical significance.
Effect of Graft Composition on Cytogenetics After Transplantation
Five (33%) patients on protocol CY/GM/BM obtained a major cytogenetic
response (>70% metaphases Ph negative) 3 weeks after transplantation, four (27%) obtained a minor cytogenetic response (>10% to <70% metaphases Ph negative), and six (40%) had 100% Ph-positive metaphases in recovering BM
(Fig 4). All but one patient had
reoccurrence of 100% Ph-positive metaphases in the BM during the 12 to
24 months after transplantation, and all but one surviving patient are
100% Ph positive at 2 years. Six (43%) patients on protocol MAC/G/BM
obtained a major cytogenetic response, four (29%) obtained a minor
cytogenetic response, and four (29%) recovered 100% Ph-positive BM
(Fig 4). Similar results were observed for patients on CY/G/PB. Two
(33%) patients obtained a major cytogenetic remission, one (17%)
obtained a minor cytogenetic response, and three (50%) continued to be
100% Ph positive after transplantation.
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| Fig 4.
Cytogenetic outcome after transplantation. The BM or PB
inoculum used for grafting as well as BM obtained 3 weeks, 3 months, 6 months, 12 months, and 24 months from all evaluable patients on
protocols CY/GM/BM, MAC/G/PB, and CY/G/PB were evaluated by metaphase
analysis to detect the number of Ph-positive metaphases. On average, 20 metaphases from marrow obtained after transplantation were analyzed.
Patients with ECP CML and AP CML were analyzed separately. Results are
depicted for all samples that were analyzed and divided as follows:
(), major cytogenetic response, 0% to 30% of metaphases or cells
Ph positive; (), minor cytogenetic response, 30% to 90% of
metaphases or cells Ph positive; (), no response, 90% to 100% of
metaphases or cells Ph positive.
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We compared the cytogenetic response of patients transplanted in ECP
versus more advanced CML. Because no significant differences were seen
between patients enrolled in the three sequential protocols, we
evaluated all patients combined. Sixty-seven percent (major cytogenetic
response), 33% (minor cytogenetic response), and 0% (no cytogenetic
response) of ECP patients; 29%, 29%, and 57% of LCP CML patients;
and 11%, 11%, and 78% of AP patients achieved a major, minor, or no
cytogenetic response, respectively. We observed a high correlation
between the Ph status of the inoculum and the Ph status of the
regenerating BM at 21 days (r2 = .71; P < .0001) (Fig 5). This correlation was no
longer present at 3 months (r2 = .37), or later
after transplantation. For instance, in five of five patients who
received a completely or nearly completely Ph-negative graft, a
major/complete cytogenetic response was obtained 3 weeks after
transplantation. However, BM cytogenetics in four of five
patients 6 to 12 months after transplantation were 90% to 100% Ph
positive (Fig 6).
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| Fig 5.
Degree of Ph-positive cell contamination of the graft
predicts cytogenetic response 3 weeks, but not 3 months, after
transplantation. Marrow or PB inoculum and BM obtained 3 weeks and 3 months after autografting was examined by metaphase analysis or FISH to
determine the number of Ph-positive cells present. The number of
Ph-positive cells found in the inoculum was then correlated with the
number of Ph-positive cells present in BM after transplantation. A good
correlation is observed between the cytogenetic response in the
inoculum and at 3 weeks after transplantation. However, no correlation
is observed between the cytogenetic response in the inoculum and 3 months after transplantation.
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| Fig 6.
Cytogenetic relapse observed early after transplantation
even in patients in whom the inoculum was completely or almost
completely Ph negative. Depicted is the posttransplantation cytogenetic
analysis of the BM of five individual patients who received a
Ph-negative inoculum.
|
|
Twelve patients have developed cytogenetic evolution since
transplantation (21 to 720 days after transplantation), and eight patients have proceeded to blast crisis. Somewhat surprisingly, of the
eight patients that died in blast crisis, three were transplanted in
ECP. Two of the three patients received a graft that was less than 30%
Ph positive but developed clonal evolution within 6 months after
transplantation leading to blast crisis at days 222 and 777, respectively. One LCP patient in whom the infused graft contained 60%
Ph-positive metaphases developed blast crisis CML at 3 weeks after
transplantation. Examination of over 100 metaphases did not show
presence of the blast crisis clone in this patient's graft.
Effect of Mobilization on Absolute Number and Clonal Origin of
CD34+HLA-DR Cells
We examined the number and BCR/ABL status of
CD34+HLA-DR cells in steady-state and
mobilized PB and BM obtained from these patients. To compare the number
of CD34+HLA-DR cells in PB or BM, we
extrapolated the actual number of cells recovered by
fluorescence-activated cell sorting (FACS) from the BM sample to 2.5 L
of BM and extrapolated the number of cells recovered by FACS from each
PBPC collection by combining the number of cells/collection in all PBPC
(2 to 8 collections) collections. Because no significant differences
were observed between protocols, data were pooled for ECP patients and
patients with more advanced disease.
In ECP CML patients, 2 ± 0.8 × 106 (0.02 to 9.7 × 106) CD34+HLA-DR
cells were present in 2.5 L steady-state BM. A similar number of
CD34+HLA-DR cells was present in all
PBPC collections combined (2.85 ± 0.7 × 106
[0.08 to 7.6 × 106] [P = .59]). However,
significantly fewer (0.16 ± 0.06 × 106 [0.01 to
0.49 × 106])
CD34+HLA-DR cells were present per 2.5L
BM after mobilization (compared with mobilized PBPC) (P = .04).
We also examined the CD34+HLA-DR cell
populations for contamination with BCR/ABL mRNA-positive cells. We and
others have previously shown that results from fluorescent in situ
hybridization (FISH) and nested RT-PCR for the BCR/ABL gene
rearrangement in CD34+ subpopulations provide similar
results.22,41 In previous studies we also evaluated the
clonal origin of CD34+ HLA-DR cells in
steady-state or mobilized progenitor populations of female CML patients
(X-chromosome inactivation by HUMARA assay). We showed
that RT-PCR-negative populations are polyclonal, giving further
credence to the notion that these cells are highly enriched in
polyclonal, benign, Ph-negative progenitors.42 Steady-state BM CD34+HLA-DR cells from five of seven
evaluated ECP patients were RT-PCR negative. CD34+HLA-DR cells in mobilized BM were
BCR/ABL mRNA-negative in six of eight patients. For mobilized PBPC
collections, presence of BCR/ABL mRNA was analyzed in all available
samples. In five patients, collections obtained on different days were
examined. In four patients, CD34+HLA-DR
cells present in all analyzed PBPC collected were BCR/ABL mRNA negative, whereas in a fifth patient one of the two collections that
was analyzed contained BCR/ABL-positive
CD34+HLA-DR cells.
The minimum dose of CD34+HLA-DR cells
required for timely engraftment in humans is not known. We have
previously postulated that a CD34+HLA-DR
cells dose of 4 × 105/kg may be sufficient for prompt
engraftment. This was based on the relative proportion of
CD34+HLA-DR cells present within the
CD34+ population (<10%) and the minimum dose of 2 × 106 CD34+ cells/kg required to
establish engraftment.43 Therefore, a dose of 4 × 105 CD34+HLA-DR cells/kg,
which is two to four times higher than the calculated minimal
"safe" dose, should suffice for timely engraftment. Three of 13 ECP patients had sufficient CD34+HLA-DR
cells/2.5 L steady-state BM, and in 3 of 3 patients these cells were
BCR/ABL mRNA negative (Fig
7). None of the BM obtained after mobilization
contained >4 × 105
CD34+HLA-DR cells/kg. Mobilized PBPC
collections from 5 of 13 patients had >4 × 105
CD34+HLA-DR cells that were BCR/ABL mRNA
negative in 4 of 5 patients. Recent autografting studies in multiple
myeloma suggest that a minimum dose of 6 × 105
CD34+LinThy1low
cells/kg may be required for timely engraftment.44 Using
these criteria, steady-state BM of 3 patients and mobilized PB from 3 patients would contain sufficient BCR/ABL-negative
CD34+HLA-DR cells for transplant.
View larger version (14K):
[in this window]
[in a new window]
| Fig 7.
Effect of priming chemotherapy on the number and clonal
origin of CD34+HLA-DR cells in marrow and
blood. Marrow was obtained before and BM and PB were obtained after
priming chemotherapy. CD34+HLA-DR cells
were selected using sequential Ficoll-Hypaque separation, column
enrichment, and FACS selection. To calculate the number of
CD34+HLA-DR cells that would be available
for transplantation the following calculations were made: For marrow,
the number of CD34+HLA-DR cells present
per 106 BM cells was multiplied by the calculated number of
cells present in a hypothetical marrow harvest of 2.5 L. For PBPC
collections, the number of CD34+HLA-DR
cells in each PB collection was determined by multiplying the number of
CD34+HLA-DR cells present in
106 PB cells and the total number of cells per collection.
When more than one collection was obtained, the number of
CD34+HLA-DR cells in each collection was
combined to calculate the total number of
CD34+HLA-DR cells available for
transplantation. The number of CD34+HLA-DR
cells available was then divided by the weight of the patient to
determine CD34+HLA-DR cells/kg. ( ),
BCR-ABL negative; ( ), BCR-ABL positive; (), no PCR.
|
|
In contrast to ECP CML, in patients with AP disease, the number of
CD34+HLA-DR cells was highly variable
but did not change markedly when examined in steady-state BM, in BM
obtained at hematopoietic recovery after mobilization, and in the
combined PB collections. As we have previously shown,
CD34+HLA-DR cells present in
steady-state BM from 8 of 10 AP CML patients contained BCR/ABL
mRNA-positive cells. This did not change significantly after
mobilization.
Interestingly, the ability to identify BCR/ABL mRNA-negative
CD34+HLA-DR cells in the mobilized BM or
PB products correlated significantly with the cytogenetic status at
multiple timepoints after transplantation. A significant correlation
was observed between presence of BCR/ABL mRNA-negative
CD34+HLA-DR cells and Ph negativity at 3 weeks (N = 17, r2 = .72, P = .001), 3 months (N = 17, r2 = .65, P = .004), 6 months (N = 12, r2 = .55, P = .049), and 9 months (N = 10, r2 = .58, P = .034) after transplantation.
| |
DISCUSSION |
The goals of the present study were (1) to compare the effect of
intermediate or high-dose chemotherapy priming on the cytogenetic response in mobilized BM or PB progenitors, (2) to determine the incidence of cytogenetic remissions and relapse after mobilized progenitor transplantation in ECP and more AP CML, and (3) to determine
the effect of in vivo priming on the ability to select Ph-negative
CD34+HLA-DR cells from mobilized BM or
PB sufficient for transplantation.
We show that cytogenetic responses observed in PB or BM after priming
with either cyclophosphamide alone or a more intensive regimen of
cyclophosphamide, mitoxantrone, and cytosine arabinoside were
equivalent. The degree of Ph negativity in PB collections collected
after CY/GM, MAC/G, or CY/G mobilization was similar. Likewise, similar
cytogenetic responses were observed in BM or PB after either
mobilization regimen. The strategy to mobilize Ph-negative progenitors
using intensive chemotherapy was first described by the Genoa
group.19 They reported that almost exclusively Ph-negative
cells, and in some cases BCR/ABL mRNA-negative cells, can be mobilized
in the blood in greater than 50% of patients who underwent mobilizing
chemotherapy at diagnosis.19,29,30 Outcomes after
transplant with Ph-negative grafts collected early after diagnosis were
favorable: reestablishment of Ph-negative hematopoiesis in 50% of
patients and persistent Ph-negative hematopoiesis for at least 12 months in greater than 50% of patients. Later reports from the same
group have shown that results for patients who undergo mobilization at
later time points (>1 year from diagnosis) or after treatment with
IFN- are less favorable: partial or no cytogenetic responses are
observed in the mobilized graft; transplantation with such a graft
rarely leads to a complete cytogenetic response even immediately after
transplantation.30 Since then, several other groups have
used chemotherapy to induce Ph-negative hematopoiesis before blood or
BM harvest.31-35,41 Most investigators have noted a
considerable variability in the results obtained. Although chemotherapy mobilization can induce complete or partial cytogenetic responses in a
subset of patients, results are in general not as predictable or as
compelling as those originally described.
We found that mobilization of Ph-negative cells in the blood of
patients with CML is possible. However, we rarely observed a complete
Ph-negative state of the graft and in no patient was the graft BCR/ABL
mRNA negative. Differences between our results and those published by
the Genoa group may be due to differences in the chemotherapy regimens
used. Instead of ICE (ifosfamide, cytarabine, etoposide)
or mini-ICE, we used cyclophosphamide alone or in
combination with mitoxantrone and cytosine arabinoside. The median
duration of neutropenia of 15 to 18 days after mobilization with either
of the priming regimens used in our studies is similar to that reported
for mobilization with the mini-ICE protocol,30,33-35 although shorter than seen after priming with ICE.19,29
This suggests that regimens used in our studies are of similar dose intensity as the mini-ICE regimen and that differences in dose intensity between mobilization regimens used in different studies may
not significantly impact on the Ph status of the mobilized graft. We
cannot exclude that the shorter duration of chemotherapy administration
in our patients (5 to 7 days for ICE and mini-ICE v 2 days for
cyclophosphamide, ARA-C, and mitoxantrone used in our study) may
negatively impact on the type of cells that are mobilized in the blood
when the marrow recuperates. An alternative explanation is that
differences reported by different investigators may be due to patient
selection. The median time from diagnosis for ECP patients enrolled in
our studies was 8 months (range, 7 to 11 months). The best results have
been reported for mobilizations performed immediately after diagnosis.
As the benign hematopoietic clone decreases with time,22 it
is possible that the lower proportion of Ph-negative cells mobilized in
the PB, observed in our studies and reported by other investigators for
patients with ECP CML, may in part be due to the fact that the normal
stem cell pool has decreased considerably even 8 months after
diagnosis.
After transplantation with mobilized progenitors obtained using the
three different mobilization regimens, no significant differences were
observed in cytogenetic response. This is consistent with the
observation that the three mobilization regimens induced similar
degrees of Ph negativity in the graft and that the Ph status 3 weeks
after transplantation is highly correlated with the degree of Ph
negativity in the graft. Of interest, cytogenetic status at 3 or more
months after transplantation did not correlate with the degree of Ph
negativity in the mobilized graft. However, cytogenetic response at 3 to 9 months was highly correlated with the disease stage at time. Thus,
as has been observed for other autograft approaches in CML, outcome
after mobilized PB or BM transplants may depend more on the disease
stage at the time of transplantation than the type of mobilization
regimen used or source of progenitor used (BM or
PB).20,21,26-35 Irrespective of the cytogenetic response in
the graft or at 3 weeks after transplantation, only two patients on the
first two protocols remain in major cytogenetic remission beyond 12 months after transplantation.
Of interest, cytogenetic response at 3 to 9 months was also highly
correlated with our ability to identify and select large numbers of
BCR/ABL mRNA-negative CD34+HLA-DR cells
from mobilized BM or PB. Our studies show that presence of
BCR/ABL-negative CD34+HLA-DR cells in
the graft predicts cytogenetic remission after transplant significantly
better than cytogenetic analysis of the mononuclear cells in the graft.
It is possible that the correlation found between BCR/ABL-negative
CD34+HLA-DR cells in the graft and
cytogenetics after transplant is a manifestation of disease stage at
the time of transplant. Because relapse after autografting may be due
to contamination of the graft, as shown by gene marking
studies,45 and due to persistent disease in the host, as
exemplified by syngeneic or T-cell-depleted
allografts,13-15 cytogenetic relapses observed at 6 months
or later after grafting may be the result of Ph-positive stem cells
contaminating the graft or may be the result of disease persisting in
the host. Relapses occurred significantly later in patients in whom the graft contained a larger population of BCR/ABL-negative
CD34+HLA-DR cells. We hypothesize that
decreased contamination of the graft contributes to the delay in
relapse observed. An alternative or complementary explanation may be
that disease in this group of patients is more chemotherapy and
radiation therapy responsive, resulting in a significantly greater
depletion of the malignant population in the host with the mobilization
and the pretransplant cytoreductive therapy. Stem cell marking studies
at our institution in patients with CP CML are currently underway to
discriminate between these possibilities.
Four patients died early after transplantation due to regimen
associated toxicity (one graft failure and three infectious/toxic deaths). These four patients were either LCP patients or patients with
AP disease: time from diagnosis was 3 to 6 years and two patients were
in AP disease. As has been shown for allogeneic transplant regimens,
time from diagnosis and presence of accelerated features are associated
with increased toxic deaths.13-15 Of note, 27% of patients
developed progressive disease 1 month to 3 years after transplantation.
Sixteen percent of patients progressed to blast crisis CML. Early
progression to blast crisis was independent of patient age, peripheral
WBC count, platelet count, spleen size at the time of transplantation,
or previous therapy. Progression to blast crisis was observed more
often in patients with long-standing disease (LCP) or with accelerated
features. For the three patients transplanted in presumed ECP CML who
progressed to blast crisis within 1 to 3 years after transplant, none
of the characteristics listed above predicted the transformation. In
one late CP CML patient who progressed to blast crisis 3 weeks after
transplantation, the clonal abnormalities observed at the time of blast
crisis could not be detected in the graft inoculum. Progression to
blast crisis was also independent of the type of mobilizing
chemotherapy used. It is possible that the mobilization and preparative
regimen selectively eliminates the CP clone but not subclones with
additional genetic abnormalities, which may be undetectable before
priming. As has been described for autologous transplantations in
lymphoproliferative disorders,46-48 repeated administration
of chemotherapy may induce additional clonal abnormalities in CML. In
CML, the known increased resistance to apoptosis conferred by the
BCR/ABL oncoprotein49,50 may enhance the tendency to
acquire additional genetic abnormalities leading to rapid clonal
expansion and premature blast crisis disease.
We found that use of mobilized PB progenitors rather than mobilized BM
progenitors resulted in earlier engraftment. A strong correlation was
observed between the number of CFC and LTC-IC infused and the time to
ANC, platelet, and RBC recovery. Although differences in CFC and LTC-IC
content between mobilized BM progenitors (protocol CY/GM/BM) and
mobilized PB collections (protocols CY/G/PB and MAC/G/PB) did not reach
statistical significance, there was a trend to higher progenitor
content in the mobilized PB grafts. Thus, slower engraftment in
protocol CY/GM/BM may be caused by the lower progenitor dose infused.
Alternatively, differences in cytokines used for mobilization and
posttransplant support or intrinsic differences between mobilized BM
and PB progenitors may in part be responsible for the differences in
hematopoietic recovery observed between the protocols.51-53
The last objective of the study was to evaluate if in vivo mobilization
would increase the number of patients in whom ex vivo selection of a
BCR/ABL mRNA-negative population of
CD34+HLA-DR cells is feasible. In ECP
CML, the number of CD34+HLA-DR cells in
mobilized BM was lower than in steady-state BM. However, more
CD34+HLA-DR cells could be selected from
the combined mobilized PB collections than from steady-state BM.
Further, irrespective of mobilization, CD34+HLA-DR cells were BCR/ABL mRNA
negative and polyclonal42 in the majority of ECP CML patients. In contrast, the number and clonal origin of
CD34+HLA-DR cells was not significantly
affected by the priming chemotherapy in patients with more advanced
CML. The presence of large numbers of BCR/ABL mRNA-negative
CD34+HLA-DR cells in the graft was
associated with superior cytogenetic response after transplantation
with mobilized grafts. Therefore, we speculate that combined approaches
in which the CD34+HLA-DR cell population
is selected from mobilized PB collections may result in longer lasting
and more significant cytogenetic responses after transplantation.
In conclusion, we describe results of autografting using mobilized PB
or BM progenitors in CML. These studies indicate that neither the
intensity of the mobilizing regimen nor the source of stem cells
significantly affects the cytogenetic remissions obtained immediately
after mobilization or after transplantation. However, cytogenetic
responses in the mobilized product or after transplantation are
significantly correlated with the number of benign primitive
progenitors still present, which is significantly higher in ECP than in
later stages of the disease. We show a correlation between the percent
of Ph-negative committed progenitors present in the inoculum and the
percent of Ph-negative progenitors present 3 weeks after
transplantation. However, this effect is short lived, indicating that
either more primitive progenitors continue to be Ph positive
and/or that disease persisting in the host after the
myeloablative therapy contributes to cytogenetic relapse. Finally, all
patients received IFN- as maintenance therapy after transplantation.
In all but 2 patients, IFN did not prevent resurgence of the
Ph-positive clone. Additional posttransplantation therapies, such as
interleukin-2 with antileukemia lymphoid cell populations54 or continued administration of chemotherapy after transplantion of a
graft modified by a drug resistance gene,55 will need to be
tested to prevent the inexorable progression to cytogenetic relapse
observed after auto-transplantation for CML.
| |
FOOTNOTES |
Submitted November 4, 1997;
accepted April 23, 1998.
C.M.V. is a Scholar of the Leukemia Society of America.
Supported in part by National Institutes of Health Grants PO1-CA-65493
and PO1-CA-21737, the Leukemia Society of America Translational Research Grant 6377-97, and funds from the University of Minnesota Hospitals and Clinics.
Address reprint requests to Catherine M. Verfaillie, MD, Department of
Medicine, Box 806 UMHC, 420 Delaware St SE, Minneapolis, MN 55455;
e-mail: verfa001{at}maroon.tc.umn.edu.
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.
| |
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Abstract
Introduction
Abstract