1. Art and Diseño

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Differential Mobilization of Myeloma Cells and Normal Hematopoietic Stem
Cells in Multiple Myeloma After Treatment With Cyclophosphamide and
Granulocyte-Macrophage Colony-Stimulating Factor
By Yair Gazitt, Erming Tian, Bart Barlogie, Chris L. Reading, David H. Vesole, Sundar Jagannath, Judith Schnell,
Ron Hoffman, and Guido Tricot
Peripheral blood stem cells (PBsCs) mobilized with highdose chemotherapy and hematopoietic growth factors are
now widelyused t o support myeloablative therapyof multiple myeloma and effect complete remissions in up t o 50%
of patients with apparent extension of event-free and overall
survival. Because tumor cells are present not only in bone
marrow, but also in virtually allPBSC harvests, it is conceivable that autografted myeloma cells contribute t o relapse
after autotransplants. In this study, the kinetics of mobilization of normal hematopoietic stem cells were compared
with those of myeloma cells present in PBSC harvests of 12
patientsafter high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor administration.
CD34+ and CD34+Lin-Thy+ stem cell contents were
measured by multiparameter flow cytometry, and myelomacells
were quantitated by
immunostaining for therelevant Ig light
chain and by a quantitative polymerase chain reaction for
the myeloma-specific CDRlllsequence. Results indicated
marked heterogeneity in the percentages of mobilized stem
cells among different patients(0.1% t o 22.2% for CD34' cells
and 0.1% t o 7.5% for CD34+Lin-Thy+cells, respectively). The
highest proportions ofhematopoietic progenitor cells were
observed early during apheresis, with 9 of 12 patients mobilizing adequate amounts of CD34' cells for 2 autotransplants
(>4 x 106/kg) within the first 2 days, whereas peak levels
(percent and absolute numbers) of myeloma cells were present on days 5 and 6 (0.5% t o 22.0%). During the lastdays of
collection, mobilized tumor cells exhibited more frequently
high labeling index values (1% to 10%; median, 4.4%) and an
immature phenotype (CD19'). The differential mobilization
observed between normal hematopoietic stem
cells and myeloma cells can be exploited t o reduce tumor cell contamination in PBSC harvests.
0 1996 by The American Societyof Hematology.
A
completion of cyclophosphamide treatment and continued daily until
the completion of the PBSC collections. A Quinton double lumen
catheter was inserted in the subclavian vein for stem cell apheresis.
Apheresis procedures were performed on a Cobe Spectra Blood
Cell Processor (Lakewood, CO) and PBSCs were collected once the
leukocytes count exceeded 0.5 X 1Oy/Land platelets approached 50
X 10yL(untransfused). Collections continued until at least 6 X lo*
mononuclear cellsikg were obtained (usually within 4 to 6 days) or
for a maximum of 14 days. However, there was no set target for
CD34' or for CD34+Lin-Thy+ cells. All patients signed an informed
consent form approved by the local Institutional Review Board.
Flow sorting of plasma cellsand generation of the CDRIIIprimer
for ASO-PCR. Ficoll-Hypaque-separated BM mononuclear cells
(BMMCs) were stained for the CD38 and CD45 antigens (CD45fluorescein isothiocyanate [F'ITC]; CD3S-phycoerythrin [PE]; Becton Dickinson [BD], Mountain View, CA). The CD38b"gh'CD451""
cells were sorted using a FACStar Plus cell sorter (BD) as described
previously.'3.18DNA extraction, generation of the CDRIII primers,
and radiolabeled ASO-PCR reaction were performed as previously
de~cribed.'~."-~'
Dilution curves of myeloma cell DNA between 10%
and 0.001% myeloma cell DNA were generated for each patient by
diluting myeloma cell DNA into normal peripheral blood lymphocytes DNA, as previously described.l3.Iy Autoradiograms were
scanned densitometrically and quantitation of myeloma cells in
UTOLOGOUS TRANSPLANTATION is now frequently used in the treatment of patients with multiple
myeloma (MM)."4 Complete remissions (CRs) are observed
in up to 50% of patients with newly diagnosed MM, but,
unfortunately, relapses remain a pr~blem.',~"
Compared with
standard autologous bone marrow (BM) transplants, peripheral blood stem cells (PBSCs) procured after the administration of high-dose cyclophosphamide and/or hematopoietic
growth factors significantly reduce the duration ofBM
aplasia and hence treatment-related morbidity and mortality.'"'
Because MM is a disease considered to be restricted to
the BM, the concentration of myeloma cells in PBSCs was
felt to be negligible. However, we and others have documented tumor cells in PBSC harvests with a frequency of
0.01% to greater than
This observation prompted
the exploration of hemopoietic stem cell selection as a means
of reducing tumor cell ~ontamination.'~
We have focused
on a CD34+ subcompartment that expresses Thy+ and Lin(CD34', Thy+, Lin-) and is composed of cells capable of
unlimited self-renewal and multilineage differentiation.14"'
This fraction contains less than 0.001%myeloma cells, based
on polymerase chain reaction (PCR) amplification of the
CDRIII sequence.13
In the current study, high-dose cyclophosphamide and
granulocyte-macrophage colony-stimulating factor (GMCSF) were administered for PBSC mobilization. Marked heterogeneity was observed (>50-fold) among different patients in the number of stem cells mobilized, peaking during
the first 3 days of apheresis, whereas myeloma cells were
present then in low numbers but increased by more than 2
logs on days 5 to 6.
MATERIALS AND METHODS
Mobilization and procurement of PBSCs. Patients with recently
diagnosed (N = 5) or previously treated myeloma (N = 7) received
6 g/m' of cyclophosphamide over 12 hours. GM-CSF was initiated
at a dose of 250 pglm2 subcutaneously within 24 hours after the
Blood, Vol 87, No 2 (January 15), 1996: pp 805-811
From the Department of Medicine, Division of Hematology-Oncology, University of Arkansas for Medical Sciences, Little Rock,
AR; and SyStemix, Palo Alto, CA.
Submitted June 15, 1995; accepted August 30, 1995.
Supported by National Institutes of Health Grants No. POI-CA
55819 and CA 539340.
Address reprint requests to Yair Gazitt, PhD, Department of Medicine, Division of Hematology-Oncology, University of Arkansas for
Medical Sciences, 4301 WMarkham Slot 508,Little Rock, AR 72205.
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.
0 19% by The American Society of Hematology.
0006-4971/96/8702-0025$3.00/0
805
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806
PBSCcollections was performed by log linearregressionas previously described."."
Quantitation of CD34'Lin-Thy' stem cells. Stem cells were de(1 0' cells) for CD34
tected by staining samples from apheresed blood
using anti CD34-PE(HPCA-2;BD)andforThy(CD,90)
using
biotin-labeled GM 201 (SyStemix, Palo Alto, CA), followed
by RED
FITC670-streptavidin(Systemix). Lin' cellswerestainedwith
conjugated antibodies to CD14,CDIS, CD2, CD16, CD19, and antiglycophorin (all from BD). Isotypic controls corresponding to the
antibody isotypes and the proper fluorochrome were used to deterIgG l-biotin and avidin-RED670 were
mine the background staining.
used as a control for the Thy antibody. Propidium iodide was added
to all tubes to exclude dead cells. Tubes were run on a FACSCAN
BOW cytometer (BD). Six parameters (FS, SS, PI, FITC, PE, and
Red 670) were examined in listmode using the ListView software
(Phoenix Flow Systems, San Diego,
CA). The cells were sequentially
gated from viable nucleated cells to the lymphoblastoid region and
then to each of the two color combinations,
ie. CD34 versus Lin
and CD34 versus Thy. Each fluorochrome
was colorgated. Fifty
thousand cells were analyzed.
Sruiningfor myeloma cells. Staining of myeloma cells was per(50,000 cellsfromapheresissamples)after
formedoncytospins
ethanol fixation (30 minutes at 4°C). Slides were stained for both K
and X light chains of human Ig (rabbit antibody-Rhodamine conjugate; Dako, Carpinteria, CA). Plasma cells were identified by positivestainingforthepertinent
light chainand by theirlymphoid/
plasmacytoid morphology. Positive staining for the irrelevant light
chain was always less than 10% of the relevant light chain. At least
S00 cells were scored.
Staining of immuture myeloma cells und determination of plusma
cell labeling index (PCLI). Severalmethodsfordetermination
of
PCLI were tested using different anti BrdU antibodies and different
cell denaturationprotocolsasdescribed
previously.2' The best
results were obtained using the procedure described by Lokhorst et
al." Briefly, samples from apheresed blood (10' cells) were allowed
to incorporate bromodeoxyuridine ( I 0 pmol/L BrdU for 1 hour at
37°C). Subsequently, aliquots of 50,000 cells were cytospun, fixed
with ethanol, air-dried, andtreated with 2 N HCI (30 minutes at room
temperature) to denatureDNA.Afterneutralization
in phosphatebuffered saline (PBS), slides were stained for
K and A light chains
as described above and for BrdU with anti-BrdU antibodies (mouse
monoclonal; Dako), followedby antimouse-FITC (Jackson Immunochemicals, West Grove, PA). Double-stained cells were counted as
cycling p k m d cells. Cycling plasma cells were
identified by positive
staining for the relevant light chain (red),
by positive staining for
BrdU (green), and by their lymphoidlplasmacytoid appearance. Positive staining for the irrelevant light chain was less than 10% of the
relevant light chain. At least 500 plasma cells were scored. Mouse
lgGI was used as a control for background staining for the BrdU
antibody. This procedure wasused successfully in our laboratory for
more than 1 year to monitor more than 400 patients with significant
correlationbetweendiseaseactivityandPCLI(Gazitt,manuscript
in preparation).
Immature plasma cells were detected by cytoplasmic staining for
K/A lightchain(rhodamine-conjugatedantibodies),
as described
above,andfor
CD19' usingFITC-anti-CD19antibody(mouse,
monoclonal; BD). Most CDIY' cells were small lymphocytoid,with
predominantmembranestaining
for CD19. In 2 patients. CD19'
cells had the appearance of plasmacytoid cells with
weak staining
for cytoplasmic CD19 antigen and positive staining for the relevant
light chain. Positive stainingof CD19' cells with the irrelevant light
less than 1070 of therelevantlightchain.
At
chainwasalways
least S00 plasma cells were scored. Mouse FITC-IgGI was used as
isotypic control for background staining for the CD19 antibody.
GAZITT ET AL
RESULTS
The time courseof stem cell mobilization for 4 representative patients is depicted in Fig I . Maximum proportions of
CD34' and CD34'Lin-Thy' were observed on days 1 to 3,
ranging from 1.8% to 22.2% and 0.48% to 8.2%.respectively. Figure 2 portrays the cumulative amount of CD34'
stem cells in the 12 MM patients studied. Sufficient quantities of CD34+ cells perkilogram fortwo autotransplants
(>4 X 10') were obtained after two collections in 9 of the
12 patients. Eight patients mobilized greater than 10 X loh/
kg of CD34' cells within the first 3 days of collection. Three
patients did not mobilize well.
The proportions of myeloma cells in PBSC harvests of 4
patients, including the percentages of light chain restricted
B cells (PLC), their labeling index (PCLI), and the proportion of immature myeloma cells(CD19') are depicted in Fig
3. In all 4 patients, mobilization of myeloma cells peaked
on days S and 6, with PCLI values ranging between 3.1 %
and 7.5% on these days. Maximum concentrations of light
chain-restricted CD19' cells (presumedto represent preplasmacytic myeloma cells) usually coincided with peak values
of PCLI. The cumulative amountof myeloma cells in the I2
myeloma patientsis depicted in Fig 4. Ten patientsmobilized
greater than S X 107/kg and 6 greater than 1 X lORkgmyeloma cells during the 6 days of apheresis. One patient did
not mobilize significant amounts of myeloma cells. In the
other 1 1 patients, greater than 75% of myeloma cells were
collected during the last 2 days of apheresis.
Figure S summarizes the proportions of CD34'.
CD34'Lin Thy*, and myeloma cells duringthe consecutive
days of PBSC apheresis. Median percentages of maximum
CD34' and CD34'Lin-Thy' cells were 5.5% (range, 0.2%
to 22.2%) and 2.7% (range. 0.1% to 8.2%), respectively.
Both cell populationswere largest on days 1 to 3, with a
subsequent significant decline reaching a minimum by day
6 ( P < .001). In contrast, only 2 of 12 patients had ~ 2 . 5 %
myeloma cells by immunocytochemistry in their PBSC collections on days 1 and 2, compared with 1 I of 12 patients
by day 6. A median of 4.4% myeloma cells (range, 1.6%to
10.9%) werecycling, most frequentlyon days 4 to 6 of
PBSC collection. Similarly, CD19'lightchain-restricted
cells peaked on day 6, with a median of 5.1% (range, 0%
to 16%;results not shown). Theleast overlap between hematopoietic progenitor and myeloma cell mobilization was observed on days I and 2.
PCR-basedquantitation of minimalresidual disease is
more sensitive (2 to 3 logs) than immunofluorescence-based
quantitation of tumor cells. Therefore, quantitation of myeloma cells in PBSC collections was also performed by PCR
amplification of the clone-specific CDRIII sequence. Representativeresults from 6 patients (B.D., H.R., B.L., L.J.,
M.G.. and D.P.) are shown in Fig 6. The dilution curves
(left panels) indicate a sensitivity of detection of I myeloma
cell in 10,000 to100,000nonmyeloma cells. A daily increase
in the number of myeloma cells in PBSC collections (right
panels) was seen. Densitometric scanning of the autoradiograms and quantitation by log-linear regression in 3 patients
(B.D., H.R., and B.L.) showed a low content of myeloma
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DIFFERENTIAL MOBILIZATION OF MYELOMA AND STEMCELLS
807
Patient B.D.
Patient B.L.
12.5
12 5
UCD%+
ICD%+/Thy+
10 0
Aphemia
Patient
H.R
Patient M.G.
20
m
b-
10
e
5
Fig 1. Mobilization of stem
cells in PBSC harvests of MM patients. Percentages of hematopoietic
progenitor
cells during
consecutive days of apheresis in
4 patients.
E
O5
od
Aphemsii
Aphereais
cells during the first days of apheresis (<0.01%, 1.2%, and
0.1%, respectively), with subsequent daily increases of up
to one log, reaching 6.5%, 16.9%, and 22%, respectively,
on day 6. Two patients (L.J. and D.P.) had already high
levels of myeloma cells on day 1 (4.4% and 9.6%, respec-
tively), with no obvious change during subsequent days of
apheresis. One further patient (M.G.) had a low baseline
level of myeloma cells (0.15%), with no further increase
during subsequent days of apheresis. A significant correlation was observed between the levels of myeloma cell measured by the PCR method and by immunofluorescence with
R values of 0.93 to 0.97.
KINETICS OF MOBILIZATION OF CD%+ STEMCELLS
4b
=DAY1
EDAYZ
[IDDAY3
DISCUSSION
mDAY4
Mobilization of PBSCs with high-dose chemotherapy
alone or in combination with hematopoietic growth factors
results in a 10- to 100-fold increase in the number of circulating hematopoietic progenitor ~ e l l s ~ and
~ . ’ effects
~
prompt,
complete and durable engraftment after myeloablative treatment.”” Probably not unexpectedly, this approach also leads
to mobilization of tumor ~ e l l s , ’ ’ ~including
~~
myeloma
P8
IQ
0
+
C.E.
R.S.
H.R.
C.I.
D.P.
C.G.
M.oG.
L.J.
B.L.
B.D.
M.G.
B.S.
Fig 2. Cumulative number of CD34’ stem cells in MM patients.
Absolute daily amounts of CD34+ stem cells per kilogram were
calculated from the total WBC count per collection, the percentage of
CD34+ cells, and the patient‘s body weight.
Myeloma cells are present in virtually all mobilized PBSC
harvests with a frequency of 0.01% to greater than 10%.l3
Depletion of myeloma cells can be achieved by positive
selection for CD34+cells” or, morecompletely, by selection
for CD34+Thy+Lin- stem cells.’3
This is the first detailed study assessing, simultaneously,
the kinetics of mobilization of both hematopoietic progenitor
cells and myeloma cells, including their proliferative capacity and the immature myeloma cell phenotype. The highest
proportions of progenitor cells (CD34’ and CD34+ThytLincells) were observed during the first 2 days of apheresis,
when the total white blood cell (WBC) count was still low
(0.5 to 2 X 109/L). More than 4 X 106/kg CD34+ cells,
sufficient for two auto transplant^,^ were collected during the
first 2 days in 9 of the 12 patients. Two of the three patients
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GAZllT ET AL
808
Patient B.D.
Patient B.L.
"1
301
151
OPL
I PCCI L
C lD 1 9 K
=PLC
I P CI LClD 1 9 K
Apheresis
Apheresis
Patient H.R.
Patient M.G.
Fig 3. Mobilization of my-
Apheresis
eloma cells in P B X harvests of
MM patients. Percentages of
plasma
cells,
light-chain
restricted CD19+ cells, and cycling
plasma cells during consecutive
days of apheresis.Plasmacells
of
are expressed as a percentage
the total number of cells in the
daily collections. PCLl and CD19
cells are expressed as a percentage
of
the
total
number
of
plasma cells scored.
O P LI PCCI LClD 1 9 K
Aphemk
whodidnot
mobilize adequate amounts of CD34+ cells
had prolonged prior treatment with alkylating agents (> 12
months), resulting in permanent stem cell damage, as reported before.' The proportions of CD34' cells in these 3
patients were comparable to those observed in peripheral
blood of unmobilized MM patients (<l%).
In contrast to stem cells, the highest concentrations of
myeloma cells (>75% of total tumor cells) were collected
during days 5 and 6 of apheresis, when the percentage and
absolute number of progenitor cells were already rapidly
declining. Hence, an ideal window for PBSC collection with
KINETICS OF MOBILIZATION OF MYELOM4CELLS
'"1
M.G. C.I.
hl0.G.
B.L. D.P.
R.S.
B.O.
C.E.
C.G.
HR. 8.5. L.J.
Fig 4. Cumulative number of myeloma cells in MM patients. Absolute daily amounts of myeloma cells per kilogram were calculated
from the total WBCcountpercollection,
the percentage of light
chain-restricted myeloma cells, and the patient's body weight.
high progenitor cell content and minimal myeloma cell contamination exists during the first few days after W C and
platelet counts start to recover.
Moreover, during the last days of apheresis, a significant
proportion of myeloma cells was cycling and exhibited a
more immature phenotype (CD19+). Both immunocytochemical and PCR-based methodologies detected comparable amounts of myeloma cells in the daily collections (R
values of 0.93 to 0.97). The more sensitive PCR-based assay
showed a daily increase in concentration of myeloma cells
by 1 log in several patients.
Whether mobilization of myeloma cells is the result of
cyclophosphamide treatment or the administration of hematopoietic growth factor remains unknown. We are currently
conducting a study comparing the yield of CD34+ cells and
degree of myeloma cell contamination in MM patients mobilized with either G-CSF or G-CSF plus cyclophosphamide.
A direct comparison between G-CSF and cyclophosphamide
would have been preferable, but cyclophosphamide as a sole
mobilizing agent results in lower quantities of CD34+ cells
per kilogram, CD34+CD38- cells per kilogram, and CFUGM per kilogram when compared with G-CSF alone." The
fact that most of the multilineage hematopoietic growth factors are capable of mobilizing hematopoietic stem cells as
well as
cells12.13.27.28.31-34confirms the notion that cytokines are not cell-type specific, although the differential mobilization observed between hematopoietic progenitor cells
and myeloma cells suggests a greater sensitivity of hematopoietic progenitor cells to cytokines compared with myeloma
cells.
The molecular basis of homing of hematopoietic progenitor cells and tumor cells to the BM and their release into the
peripheral blood is not well under~tood.3~
Sustained high
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DIFFERENTIALMOBILIZATION
OF MYELOMAANDSTEM
809
CELLS
levels of circulating growth factors may downregulate the
expression of adhesion molecules such as VLA-4, ICAM- I ,
and ELAM-l on the surface of stem cells, endothelial cells,
and myeloma cells. In support of this hypothesis is the recent
observation ofhigh
levels of circulating hematopoietic
RD.
H.R.
A
B.L.
r
a
V
APHERESIS
D.P.
Y r
L-
W
IO ' IO IO ' 10' IO '
I
I
2 1 .
I
. -= *
.a
.-
l
M Y 2
..
4
.
f
.-
MMY Y4 5
M Y 3
-
M Y 6
APHERESIS
*
2
;
;
.
MM
YM
Y
2Y
3 4
*
?"
j
*:
MV5
.
M Y 6
Fig 5. Comparison between mobilization of stem cells and myeloma cells in 12 patients. Stem cells were measured by flowcytometry and myeloma cells by immunocytochemistry as described in the
Materials and Methods.A significant decrease ( P < .001) in the median concentrationof CD34' cells from 5.5% on day 2 t o 0.8% on day
6 (Fig 3A) concurs with a decrease in CD34'Lin-Thy' cells from a
median of2.7% on day 1t o 0.29% on day 6 (Fig 38). Medianpercentages of myeloma cells were low ondays l through 3 10.3546, 0.650/0,
and 1.846, respectively) and increased markedly ondays 4 through 6
(4.2%. 5.7%. and 11.2%; Fig 3C).
Fig 6. ASO-PCR amplification of theCDRlll band the dailyin PBSC
collections of6 MM patients. The left panel shows the dilution
curve
of DNA (10% t o 0.001%1 from purified myeloma cells with normal
lymphocyte DNA. Lanes d, through ds represent DNA from PBSC
harvests of these patients duringconsecutive days of collection.
growth factors in umbilical cord blood"and
their effect
on the expression of the adhesion molecules, ELAM-l and
ICAM-I.37Umbilical cord blood induces rapid hematopoietic engraftment after myeloablative treatment. Interestingly,
CD34' cells express increased levels of L-selectin after
PBSC mobilization strategies, which correlated withthe
speed of hemopoietic engraftment.3x
While depletion methods for myeloma cells from PBSCs
are currently being
we recommend that, in
the absence of such methods, large volume exchanges early
after leukocyte and platelet recovery are perf~rmed.'"~~'
Our
conclusions are supported by the absence of breast cancer
cells, as measured by sensitive immunocytochemistry and
clonogenic assays, in single day large volume PBSC collections.."
ACKNOWLEDGMENT
The authors thank Christina Bewley for her dedicated secretarial
assistance.
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Differential mobilization of myeloma cells and normal hematopoietic
stem cells in multiple myeloma after treatment with
cyclophosphamide and granulocyte-macrophage colony-stimulating
factor
Y Gazitt, E Tian, B Barlogie, CL Reading, DH Vesole, S Jagannath, J Schnell, R Hoffman and G
Tricot
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