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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4489-4495
Collection, Tumor Contamination, and Engraftment Kinetics of Highly
Purified Hematopoietic Progenitor Cells to Support High Dose
Therapy in Multiple Myeloma
By
G. Tricot,
Y. Gazitt,
T. Leemhuis,
S. Jagannath,
K.R. Desikan,
D. Siegel,
A. Fassas,
S. Tindle,
J. Nelson,
C. Juttner,
A. Tsukamoto,
J. Hallagan,
K. Atkinson,
C. Reading,
R. Hoffman, and
B. Barlogie
From the Division of Hematology/Oncology and the Arkansas Cancer
Research Center, University of Arkansas for Medical Sciences, Little
Rock, AR; and Systemix Inc, Palo Alto, CA.
 |
ABSTRACT |
Unfractionated peripheral blood stem cell (PBSC) grafts contain
measurable quantities of myeloma cells and are therefore a potential
source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+
Thy1+ Lin peripheral blood
cells are substantially enriched for stem cell activity, yet contain
virtually no clonal myeloma cells. A study was performed in patients
with symptomatic myeloma, who had received 12 months or less of
preceding standard chemotherapy, to evaluate the feasibility of large
scale purification of primitive hematopoietic stem cells in order to
study engraftment kinetics posttransplantation and the degree of tumor
cell contamination of this cell population, based on polymerase chain
reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage
colony-stimulating factor (GM-CSF). A combination of elutriation and
chemical lysis was used to deplete PBSC collections of monocytes,
granulocytes, erythrocytes, and platelets. Subsequently,
CD34+ Thy1+ Lin
progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of  7.2 × 105 cells/kg to support tandem transplants. After high
dose melphalan (200 mg/m2) eight engrafted successfully,
although granulocyte (absolute neutrophil count [ANC]
>0.5 × 109/L, 16 days) and platelet recovery
(platelets > 50 × 109/L, 39 days) was substantially
delayed when compared with unmanipulated PBSC grafts; one patient
required infusion of a reserve graft because of lack of evidence of
engraftment by day +28. Three patients proceeded to a second graft
with high dose melphalan and total body irradiation; two required
infusion of a reserve graft and both died of infectious complications;
one showed delayed, but complete, engraftment after this myeloablative
regimen. Two of the nine evaluable patients attained a clinical
complete remission (CR). The grafts from three patients were tested for
tumor contamination and contained no detectable clonal myeloma cells.
Larger quantities of purified cells may be required to resolve the
problem of delayed engraftment.
| |
INTRODUCTION |
BOTH A FRENCH RANDOMIZED
study1 and a pair-mate analysis between our total therapy
patients and comparable patients treated with standard therapy on
protocols of the Southwest Oncology Group2 indicate that
autotransplants not only effect higher complete remission (CR)
rates, but also significantly extend event-free survival
(EFS) and overall survival (OS). In newly diagnosed patients, tandem
transplants can induce molecular remissions based on quantitative polymerase chain reaction (PCR) amplification assays of
patient-specific complementarity-determining region III (CDR III) DNA
sequences on bone marrow (BM) samples, able to detect one clonal B cell in a background of 105 normal cells.3,4
Using either sensitive immunofluorescence techniques or PCR for CDR
III, myeloma cells have been detected in virtually all peripheral blood
stem cell (PBSC) products.5-7 Most patients have between
0.1% and 1% clonal B cells in their PBSC collections,6 resulting in the infusion of approximately 0.5 to 5 × 108 myeloma cells with each transplant. These quantities
are probably sufficient to contribute to relapse after transplantation,
as has been shown with autotransplants in acute myeloid
leukemia8 and neuroblastoma.9 In an attempt to
obtain tumor-free grafts, bone marrows of myeloma patients have been
purged with 4-hydroperoxycyclophosphamide (4HC)10 or with
monoclonal antibodies,11 but these manipulations have
resulted in delayed engraftment. In contrast, purified
CD34+ cells reconstitute hematopoiesis rapidly and
reliably12-15 and the CD34 antigen is thought not to be
expressed on myeloma cells.16 However, with the currently
available techniques, the purity of the CD34+ selected
cells varies between 30% and 90%,12-15 still leaving detectable levels of myeloma cells in these
preparations.7,16 Further purification of these selected
CD34+ cells by fluorescence-activated cell sorting (FACS)
appears to completely eliminate all clonal B cells,16,17
suggesting that residual myeloma cells in CD34+ selected
grafts are due to contaminating CD34-negative cells. Other
investigators have, however, reported that cells clonally related to
the myeloma cells can be found within the highly purified CD34+ fraction.18,19
Based on these data, we decided to investigate whether a subpopulation
of CD34 cells, CD34+ Thy1+
Lin, purified by FACS, would provide us with
tumor-free grafts. CD34+ Thy1+
Lin are substantially enriched for stem cell
activity20,21 and give rise to myeloid, B-, and T-cell
lineages.20,22 Our preclinical studies have shown that
purified CD34+ Thy1+
Lin stem cells do not contain clonal myeloma cells,
as measured with PCR for CDR III.6 A clinical study was
started using high dose cyclophosphamide (HD-CTX) plus
granulocyte-macrophage colony-stimulating factor (GM-CSF) to mobilize
PBSCs for selection of CD34+ Thy1+
Lin cells in support of tandem high dose
chemotherapy/radiotherapy regimens. The aims of our study were to
evaluate in how many patients adequate amounts of these
CD34+ Thy1+ Lin
cells could be collected, what their purity and recovery rates were,
whether large scale purified cells were tumor-free, and what the
engraftment kinetics of these cells were posttransplantation.
| |
MATERIALS AND METHODS |
Patients.
Fifteen patients with symptomatic multiple myeloma (MM)
and  12 months of preceding standard dose chemotherapy were enrolled in the study. Patients were between 15 and 70 years of age and their BM
contained < 30% plasma cells (percent of all cells) before peripheral stem cell mobilization. The required Zubrod performance score was 0 or 1. Other requirements were: creatinine 2 mg/dL; forced
vital capacity and diffusing capacity for carbon monoxide 50% of
predicted; systolic ejection fraction 50%, direct bilirubin 1.5
mg/dL and transaminases two times the upper limit of normal; absolute
neutrophil count (ANC) 1.5 × 109/L and platelets
150 × 109/L; negative serology for human
immunodeficiency virus (HIV) and hepatitis B surface antigen and no
evidence of active infection. Patients with a second malignancy, which
was active or had been diagnosed within 5 years before study entry, as
well as patients with recent chemotherapy (4 weeks) or excessive
prior local radiotherapy, which would not allow total body irradiation
of 1,125 cGy, were excluded. Five patients had stem cells purified, but
had not received a transplant with these selected stem cells at the
time the study was terminated. However, four of these patients had
adequate numbers of selected cells for two autotransplants, while one
patient failed to meet the criteria to start the selection process (see
below).
Therapy.
Mobilization of PBSCs was attained by administration of HD CTX 6 g/m2, followed by GM-CSF (250 mg/m2) until
completion of the collections.23 Apheresis was started when
the ANC count was >0.5 × 109/L and
platelets approached or exceeded 50 × 109/L. A sample
of the apheresis product was analyzed for cell count and
CD34+ Thy1+ Lin
cell content. The apheresis product was sent immediately by overnight mail after each collection to Systemix, Inc, Palo Alto, CA, if the
following criteria were met: (1) total number of WBC 109;
(2) percentage of CD34+ Thy1+
Lin cells 1.5%; (3) percentage of viable cells
70%; and (4) the red blood cell (RBC) contamination of
the undiluted sample 0.9 × 106/µL.
Patients who were unable to meet these criteria during 3 consecutive
days were considered failures. After obtaining adequate numbers of
selected CD34+ Thy1+
Lin cells, defined as 3.6 × 105/kg for each transplant (see Discussion), additional
unfractionated PBSC were collected as back-up in case of delayed
engraftment with the selected cells (ANC count < 0.5 × 109/L by day +28 posttransplantation). A dose of 3 × 108/kg mononuclear cells/kg had to be available as
a reserve graft. Nine patients proceeded with the first transplant
after melphalan 100 mg/m2 daily for 2 days (days  3
and 2); the selected cells were given after 1 day of rest (day
0).2 Patients with sustained engraftment not requiring a
reserve graft were eligible for a second autotransplant. The
preparative regimen for the second transplant consisted of melphalan
140 mg/m2 (day 4) followed by fractionated total
body irradiation (1,125 cGy) in nine fractions over 3 days on days
3, 2, and 1. Selected cells were infused the day
after completion of the radiotherapy (day 0). All patients were treated
in a high efficiency particulate air-filtered room with prophylactic
antimicrobial, antifungal, antipneumocystis, and antiviral therapy. The
second transplant was scheduled 4 to 6 months after the first.
Response criteria.
Patients were evaluated for response at 6 weeks and 3 months after
their autotransplant. A partial remission (PR) required a tumor mass
reduction of at least 75%, including a Bence-Jones proteinuria to less
than 100 mg/day and BM aspirate and biopsy with no more than 5% plasma
cells. A CR was defined by the absence of monoclonal gammopathy in
serum and urine, using immunofixation analysis, and a normal BM
aspirate and biopsy. With both PR and CR, these findings had to be
present on at least two occasions with a minimum interval of 2 months.
New lytic lesions (but not compression fractures), hypercalcemia, or
other new evidence of disease constituted relapse or progressive
disease. EFS and OS were both calculated from the time of the first
transplant. Events included disease recurrence and death from any
cause; in case of OS, deaths from disease or other causes were
considered events.
Flow sorting of plasma cells and generation of the CDR III primer
for allele-specific oligonucleotide (ASO)-PCR.
Ficoll-Hypaque-separated BM mononuclear cells (BMMCs) were stained for
the CD38 and CD45 antigens (CD45 fluorescein isothiocyanate [FITC];
CD38-phycoerythrin [PE], Becton Dickinson [BD], Mountain View, CA).
The CD38bright CD45low cells were sorted using
a FACStar Plus cell sorter (BD) as described previously.6
DNA extraction, generation of the CDR III primers, and radiolabeled
ASO-PCR reaction were performed. Dilution curves with normal peripheral
blood lymphocyte DNA between 10% and 0.001% myeloma cell DNA were
generated for each patient. Autoradiograms were scanned
densitometrically and quantitation of myeloma cells in PBSC collections
was performed by log linear regression, as described before.
Stem cell purification.
On receipt at SyStemix, each day's collection was analyzed for cell
number, viability, and stem cell phenotype and was processed using
standard operating procedures in accordance with good manufacturing practices (GMP). Each incoming apheresis product was enriched for
mononuclear cells by counterflow centrifugal elutriation using a
Beckman model J6-MI centrifuge equipped with a standard rotor and large
Sanderson elutriation chamber (Beckman Instruments, Palo Alto, CA).
After mixing, the unfractionated apheresis samples were injected into
the elutriator at a flow rate of 25 mL/minute. The cells were
elutriated at 22°C with a rotor speed of 2,000 RPM in
phosphate-buffered saline (PBS) containing 0.5% human serum albumin
(HSA; Alpha Therapeutics, Los Angeles, CA), 0.1% dextrose, and 0.3 mmol/L EDTA. Cell fractions eluting at 48 to 70 mL/minute (Fr 48-70 cells) were pooled and subsequently treated with 5 mmol/L phenylalanine
methyl ester hydrochloride (PME; Terumo Medical Corp, Elkton, MD) to
remove residual monocytes and immature granulocytes.24,25 The Fr 48-70 cells were resuspended to a concentration of 2 × 107 cells/mL in Minimal Essential Medium (MEM; Gibco, Grand
Island, NY) and mixed with an equal volume of freshly prepared 10 mmol/L PME in MEM, plus 200 U/mL recombinant DNAse (Benzonase; Nycomed Pharma, Copenhagen, Denmark). The PME solution was adjusted to pH 7.4 with 0.5 mol/L NaOH before mixing with the cells. The cells were
incubated for 40 minutes at room temperature, then 30 minutes at
37°C. The PME-treated cells were centrifuged through 20% Percoll (Pharmacia, Uppsala, Sweden) at 2,000g to remove debris.
Residual erythrocytes were lysed with 150 mmol/L ammonium chloride
lysis buffer, pH 7.5, for 5 minutes at 4°C and washed in PBS
containing 1% HSA. Before staining, the cells were incubated with
0.1% human immunoglobulin (Gammimune; Bayer, Inc, Berkeley, CA) for 10 minutes at 4°C. The cells were stained at a concentration of 2 × 107 cells/mL for 20 minutes at 4°C with a panel
of SyStemix-manufactured monoclonal antibodies that recognize CD34,
Thy1, CD14, and CD15 antigens (2.5 µg/mL
CD34-Sulforhodamine; 2.5 µg/mL Thy1-Biotin; 1 µg/mL
CD14-FITC; 1 µg/mL CD15-FITC). Without washing, streptavidin (Societa
Prodotti Antibiotici; Milano, Italy) was added to the cells at a final
concentration of 0.15 mg/mL and incubated for an additional 20 minutes.
Cells were washed twice in cold PBS with 1% HSA; 2 × 107/mL cells were resuspended and incubated for 20 minutes
at 4°C with 5 µg/mL PE-biotin (SyStemix-manufactured). In
preparation for sorting, the cells were washed once with cold PBS with
1% HSA and resuspended to 2 × 107 cells/mL in the
same buffer solution plus 100 U/mL Benzonase. Cells were sorted using a
SyStemix-manufactured dual laser high-speed fluorescence-activated cell
sorter. FITC and PE were excited by an argon laser emitting 488 nm
light and sulforhodamine (SRG) was excited by a rhodamine 6G dye laser
emitting 590 nm light. Forward angle and side scatter sort windows were
set up to exclude very small or very large cells and cell clusters.
Selection criteria for CD34 (SRG) and the lineage markers CD14 and CD15
(Lin-FITC) were based on the fluorescence of unstained cells
(autofluorescence), and the selection criteria for the Thy1
(PE) were based on the background fluorescence of cells stained with
anti-Thy1-biotin and biotin-PE with no streptavidin. Cells
were sorted at 4°C at a rate of 15,000 to 20,000 cells per second.
The CD34+ Thy1+
Lin cells were sorted directly into a culture tube
containing a small volume of SyStemix-manufactured culture media.
Cryopreservation.
After sorting, the purified HSC suspension was washed free of sorting
sheath fluid, counted with a hemacytometer, and resuspended in
SyStemix-manufactured cryopreservation media containing 2% hetastarch,
4% HSA, and 7.5% dimethyl sulfoxide (DMSO) as
cryoprotectants, and aliquoted into four cryovials (two per
transplant). Cells were frozen using a programmable step-down freezer
(Cryomed #110; Forma Scientific, Marietta, OH) with a freezing profile
previously optimized for HSC survival. The step-down freezer is
programmed to freeze at 1°C/minute until the sample reaches
45°C, then at 10°C/minute until the sample
reaches 140°C. After the product temperature reached
140°C, the vials were transferred to the liquid phase of a
liquid nitrogen cryostorage tank until they were shipped to the
University of Arkansas for Medical Sciences in a liquid nitrogen dry
shipper (CP65; Taylor-Wharton Scientific, Harrisburg, PA). The selected
cells were stored at the University of Arkansas in the liquid phase of
nitrogen until the time of transplantation, when the selected cells
were rapidly thawed in a warm water bath, diluted in heparinized
medium, and infused via a central venous line.
| |
RESULTS |
Mobilization phase.
Fifteen patients received HD CTX 6 g/m2 and GM-CSF 250 mg/m2 for stem cell mobilization with the intent to select
CD34+ Thy1+ Lin
cells, but five patients never received a transplant with selected stem
cells, due to early termination of the study (see Materials and
Methods). Clinical data and outcome of the remaining 10 patients are
summarized in Table 1. One patient failed
to meet the minimum criteria on 3 consecutive days to proceed with the
selection procedure (see Materials and Methods). This patient had
received seven cycles of the M2 protocol [vincristine,
1,3-bis(2-chloroethyl)nitrosourea (BCNU), melphalan,
cyclophosphamide, and prednisone] before the mobilization phase. He
had a total of 5.37 × 106/kg CD34+ cells
collected and proceeded with high dose melphalan supported by an
unselected peripheral stem cell transplant. The median number of
CD34+ cells collected in the remaining nine patients was
30.5 × 106/kg (range, 9.2 to 84.7),
obtained during one apheresis procedure in one patient, two procedures
in two patients, three in five patients, and four in one
patient. The median number of CD34+
Thy1+ Lin cells collected
was 10.5 × 106/kg (range, 8.5 to 19). The median
recovery rate of CD34+ cells was 6% (range, 3% to 13%).
The median purity of CD34+ Thy1+
Lin cells was 91%; this percentage is a
conservative estimate, as the remaining events were almost entirely due
to contaminating RBC and cell debris. The median cell viability was
98% after sorting and 86% after thawing before infusion
(Table 2).
Tumor contamination of the selected stem cells.
The selected cells of three of the nine patients were analyzed for the
presence of myeloma-related clonal B cells using PCR reaction for CDR
III. With a sensitivity to detect one tumor cell in 100,000 nonclonal
cells, no evidence of clonal B cells was detected in the FACS-sorted
cells (Fig 1).
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| Fig 1.
ASO-PCR on PBSC harvest, purified CD34+
Thy1+ Lin cells and
posttransplant BM in three patients (A, B, and C). The first 4 or 5 lanes represent the dilution curves for each patient. The subsequent lanes represent DNA from unsorted as well as purified
CD34+ Thy1+ Lin
cells. The last lane shows the degree of tumor contamination in the
posttransplant BM.
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Engraftment kinetics, immunologic recovery, and toxicities.
The nine patients with adequate quantities of CD34+
Thy1+ Lin cells received
high dose melphalan at 200 mg/m2 as preparative regimen for
their first transplant. The median number of sorted cells infused was
4.6 × 105/kg (range, 3.6 to 8.5). One patient failed
to show signs of engraftment (ANC >0.5 × 109/L and
platelets >20 × 109/L) by day 28 and received a
nonselected PBSC graft on day 31. He reached an ANC of 0.5 × 109/L by day 37 and platelets > 20 × 109/L untransfused by day 44. The times to ANC and platelet
recovery for the other eight patients are provided in
Table 3. Figure 2 shows the engraftment
kinetics of these eight patients, as well as those of a control group
of patients, mobilized with an identical regimen and no more than 12 months of prior standard chemotherapy. For patients who received
selected CD34+ Thy1+
Lin cells, the median times to ANC 0.5 × 109/L and 2.5 × 109/L were 16 and > 78 days, respectively; the median times to platelets 20, 50, and 100 × 109/L were 21, 39, and 43 days, respectively. Five
patients required more than 100 days posttransplantation to recover
their granulocyte count to 2.5 × 109/L
and/or their platelets to 100 × 109/L. None
of the eight evaluable patients had recovered an absolute CD4 count of
more than 100/µL by day +100. Four patients received a delayed second
transplant with unmanipulated stem cells 5, 6, 7, and 8 months,
respectively, after their first transplant with CD34+
Thy1+ Lin cells; the CD4
count in these patients remained less than 100/µL up to the time of
their second transplant. Toxicities and transfusion requirements were
assessed on the nine patients who received selected cells. Seven
patients developed fever > 101°F, but only one had a proven
septicemia with streptococcus pneumoniae. Other moderate to severe
toxicities observed were mucositis in four patients, diarrhea in seven
patients, nausea and vomiting in eight patients, and esophagitis in one
patient. The median numbers of RBC and platelet
transfusions required after transplantation were four (range, 0 to 14)
and 6 (range, 4 to 18),
respectively.
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| Fig 2.
Depicts the time to recover granulocytes to 0.5 × 109/L and platelets to 50 × 109/L for the
eight patients who received CD34+
Thy1+ Lin cells compared with
that of 128 control patients with 12 months of preceding standard
chemotherapy who received unmanipulated PBSC mobilized with the same
regimen.
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The first three patients enrolled in this study received a second
transplant after melphalan 140 mg/m2 and total body
irradiation of 1,125 cGy. The number of sorted cells infused/kg were
1.6, 2.8, and 8.2 × 105, respectively; the viability
of the infused cells was 78%, 56%, and 67%, respectively. Patient
no. 1 developed a life-threatening enterocolitis with staphylococcus
aureus and Clostridium difficile on day +14, requiring two surgical
interventions. Because of these problems and the slow recovery after
his first transplant, the patient received additional unselected PBSC
on day +15. He recovered an ANC of 0.5 and 2.5 × 109/L on day +23 and 24, respectively. However, he
developed multiorgan failure on day +22, requiring mechanical
ventilation. He eventually died on day +91 of overwhelming septicemia.
Patient no. 2 required infusion of nonselected PBSC on day +34 because
of failure to engraft. On day +36, he developed a right upper lobe
infiltrate, treated with broad-spectrum antibiotics. He recovered an
ANC of 0.5 and 2.5 × 109/L on day 42 and 44, respectively. Because of progressive pneumonia, requiring mechanical
ventilation on day +45, an open lung biopsy was performed on day +46,
showing diffuse alveolar damage with no causative infectious agent. He
eventually died on day +56 of adult respiratory distress syndrome
(ARDS) and pulmonary hemorrhage. The third patient showed
slow recovery after his second transplant with selected cells, but did
not require infusion of nonselected mobilized PBSC. The times to ANC > 0.5 and > 2.5 × 109/L were 14 and 30 days,
respectively; the times to platelets > 20, > 50 and > 100 × 109/L untransfused were 39, 39, and 62 days, respectively.
This patient is alive 18+ months after his second transplant. Because
of the difficulties encountered with these second transplants, the
study was discontinued. After the first transplant with selected cells, two patients achieved a complete and two a partial remission. Three
patients progressed at 7, 10, and 11 months, respectively, including
one CR and one PR patient. Two patients died after their second
transplant, as described above. The projected EFS and OS at 24 months
is 60% and 80%, respectively.
| |
DISCUSSION |
The minimum quantities of highly purified and viable FACS-sorted
hematopoietic cells (7.2 × 105/kg) required to
support tandem transplant were obtained in nine of 10 patients who had
received only limited standard chemotherapy before stem cell
mobilization (12 months). The purity and viability of the selected
cells before freezing was excellent (>90%). However, the recovery of
CD34+ cells was low. Although only one patient required
infusion of unselected mobilized PBSC as back-up after the first
transplant with high dose melphalan, the median times to granulocyte
recovery to > 0.5 × 109/L (16 days) and platelets > 50 × 109/L (39 days) were considerably longer
than the 10 and 12 days, respectively, observed in our historical
control group of patients with 12 months of prior chemotherapy
receiving unselected cells (Fig 2). The delayed engraftment could
theoretically be the result of the immature phenotype of the selected
cells, requiring a longer time to provide sufficient quantities of
granulocytes and platelets. Alternatively, it could be due to
administration of inadequate amounts of hematopoietic stem cells. It
remains unknown how many CD34+
Thy1+ Lin cells are required
for full engraftment after transplantation. We and others have shown
that after unselected PBSC transplants, prompt engraftment occurs with
administration of 2 × 106/kg CD34+
cells.23,26,27 The same number of CD34+ cells
selected with an avidin-biotin immunoadsorption method also provides
rapid hematologic recovery posttransplantation.12-14 Thy1 expression has been detected on approximately 25% of
adult BM CD34+ cells.28 Based on these data, we
assumed that a dose of 0.5 × 106/kg FACS-sorted
CD34+ Thy1+ Lin
cells would allow prompt engraftment. Taking into account an average
90% viability and a 90% purity of the selected cells, a cell dose of
3.6 × 105 pure and viable CD34+
Thy1+ Lin cells/kg should
have been sufficient for a single transplant. Thy1
expression, however, of mobilized CD34+ PBSC is higher than
on BM CD34+ cells (median, 25% to 50%),29,30
with the highest percentages of CD34+
Thy1+ Lin cells present
during the first 2 days of collections.31,32 It appears
much more plausible that the delayed engraftment observed in our study
was due to infusion of inadequate quantities of purified stem cells
based on the following circumstantial evidence. First, in a study by
Schiller et al14, transplantation of < 2 × 106/kg selected CD34+ PBSC after high dose
chemotherapy for advanced myeloma resulted in significantly prolonged
neutropenia and thrombocytopenia with median times to ANC > 0.5 × 109/L and platelets > 20 × 109/L of 14 and 21 days versus 12 days for both parameters
when 2 × 106/kg selected cells were infused. The
engraftment times of patients with insufficient CD34+ cells
in that study were comparable with our findings. Second, in lethally
irradiated mice, rapid and sustained hematopoietic recovery was
observed when > 200 purified SCA1+
Thy1+ Lin cells were
transplanted and very little difference in engraftment kinetics was
observed between mice receiving purified BM stem cells and those
administered unmanipulated BM containing identical numbers of
SCA1+ Thy1+
Lin cells.33 Even when purified stem
cells were mixed with SCA1 BM cells in a
competitive repopulation assay, most of the early myeloid cells were
derived from the SCA1+
Thy1+ Lin cells, indicating
their superiority over SCA1 cells, even
in the early phases of myeloid reconstitution.33 Third, if
delayed engraftment was caused by the immaturity of the purified stem
cells, a swift increase in granulocyte and platelet counts to normal
levels would have been expected, once engraftment had been established.
This was not the case in our study.
Because of the delayed engraftment observed in our patients, the
question arises whether the recovery in peripheral counts posttransplantation was effected by the infused progenitor cells or was
the result of endogenous BM recovery, as melphalan 200 mg/m2 is probably not a myeloablative regimen. No data are
available on recovery of BM function after administration of such a
high dose of melphalan without stem cell rescue. However, when
melphalan 140 mg/m2 was administered to 56 patients with
GM-CSF support, but without a stem cell graft, the median time to ANC > 0.5 × 109/L was 23.5 days (range, 21 to
28),34 considerably longer than the 16 days (range, 13 to
26) observed in our study. No data on platelet recovery were available
in this French study. In our patient no. 3, a day +15 BM showed 50%
cellularity with all hematopoietic lineages well represented and with
only 5% plasma cells. Such a high cellularity is not expected after
high dose melphalan without stem cell rescue. This same patient showed
full engraftment, although delayed, after his second autotransplant
with selected cells when he received a truly myeloablative conditioning
regimen with melphalan 140 mg/m2 and total body irradiation
of 1,125 cGy. Therefore, it appears likely that the purified
hematopoietic stem cells contributed to a large extent to hematopoietic
recovery, although we cannot completely exclude that in some patients
hematologic recovery might have been due at least in part to endogenous
BM recovery.
Although the viability of the sorted cells was high before freezing and
before administration of the first autotransplant, cell viability in
two of the three patients receiving a second autotransplant was low
(56% and 67%). It is unclear whether the decrease in viability with
the second transplant is the consequence of increased fragility of the
purified cells because of the long sorting process, or due to the
storage of only small quantities of cells for several months.
Finally, the clinical impact of tumor-free grafts on the outcome of MM
patients remains questionable at this time. An average unmanipulated
PBSC graft contains approximately 108 myeloma
cells.6 Only one third of all patients (untreated and
treated, including refractory MM) referred to our center and scheduled
to receive tandem transplants achieve a CR.7 The other
patients not attaining a CR still have at least 109 myeloma
cells left after tandem transplants.35 This is
approximately 10 to 100 times more than the average amount of myeloma
cells infused with each transplant.6 Unless our high dose
chemotherapy can reduce the tumor load to < 106 myeloma
cells, ie, a 7 log reduction of malignant cells from the time of
diagnosis,35 stem cell selection is unlikely to be
beneficial. Such a level of tumor reduction is unlikely to occur in > 15% of patients, ie, half of the patients who attain a CR. Moreover,
stem cell selection processes deplete not only tumor cells, but also T
cells, which will prolong immune reconstitution posttransplantation, as
suggested by our study; this will be even more pronounced if highly
immunosuppressive preparative regimens are used containing total body
irradiation.36 It appears much more likely that
posttransplantation immunologically-based strategies such as idiotype
vaccination, dendritic cell infusions, or humanized monoclonal
antibodies targeting the clonogenic myeloma cells, will ultimately
improve the outcome in MM. These interventions should affect both
myeloma cells remaining in the patient posttransplantation and myeloma
cells reinfused with the graft.
| |
FOOTNOTES |
Submitted August 27, 1997;
accepted January 27, 1998.
Supported in part by Grant No. CA 55819 from the National Cancer
Institute, Bethesda, MD.
Address reprint requests to G. Tricot, MD, PhD, Bone Marrow/Stem Cell
Program, University of Maryland Cancer Center, Room 922C, 22 S Greene
St, Baltimore, MD 21201-1595.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the many physicians who referred
patients for the study, the technical expertise of Sandy Mattox and
Dwayne Bracy, the expert and compassionate care of the bone marrow
transplant staff, and the diligent secretarial assistance provided by
Conelia Williamson and Michele Mullins.
| |
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Abstract
Introduction
Abstract