Transplantation of CD34+ Peripheral Blood Progenitor Cells After HighDose Chemotherapy for Patients With Advanced Multiple Myeloma
By Gary Schiller, Robert Vescio, Cesar Freytes, Gary Spitzer, Firoozeh Sahebi, Myung Lee, Chun Hua Wu, Jin Cao,
Jong C. Lee, Charlie H. Hong, Alan Lichtenstein, Michael Lill, Jeff Hall, Ronald Berenson, and James Berenson
A major potential problem ofautologous transplantation in
the treatmentof advanced malignancy is the infusion
of tumor cells. A multi-institutional study of purified CD34-selected peripheral blood progenitor cell (PBPC) transplantation was conducted in 37 patients with advanced multiple
myeloma receiving myeloablative chemotherapy. Fourteen
days after intermediate-dose cyclophosphamide, prednisone, and granulocyte colony-stimulating factor (G-CSF), a
median of 3 (range, 2 t o 5) 10-Lleukaphereses yielded 9.8 x
108/kg (range, 3.7 t o 28.3) mononuclear cells. The adsorbed
(column-bound) fraction contained 5.9 x lo6cells/kg (range,
1.6 to 25.5) with 4.65 x lo6CD34 cells/kg (range, 1.2 t o 23.3).
Using Poisson distribution analysis of positive polymerase
chain reactions with patient-specific complementarity-determining region 1 (CDR1) and CDR3 Ig-gene primers, tumor
was detected in leukapheresis products from 8 of 14 unselected patients and ranged from 1.13 x 10‘ t o 2.14 x 10’
malignant celldkg. After
CD34 selection, residual tumor was
detected in only three patients’ products. Overall, a greater
than 2.7- t o 4.5-log reduction in contaminating multiple myeloma cells was achieved. CD34 PBPCs were infused 1 day
after busulfan (14 mglkg) and cyclophosphamide (120 mg/
kg), and granulocyte-macrophage colony-stimulating factor
was used until hematologic recovery. The median time to
both neutrophil and platelet recovery was 12 days (range,
11 t o 16 days and 9 t o 52 days, respectively). The median
numberoferythrocyte
andplatelet transfusions was 7
(range, 2 t o 37) and 3 (range, 0 t o 85). respectively. Patients
receiving fewer than 2 x lo6 CD34 cells/kg had significantly
prolonged neutropenia, thrombocytopenia, and an increased red bloodcell and platelettransfusion requirement.
Thus, CD34selection of PBPCs markedly reduces tumor contamination in multiple myeloma and provides effective hematopoietic support for patients receivingmyeloablative
therapy.
0 1995 by The American Societyof Hematology.
D
apy-responsive disease, have achieved high response rates
and improved long-term myeloma-free survival.” In a recent
study of untreated intermediate- to high-stage multiple myeloma, patients assigned to high-dose chemotherapy and BM
transplantation achieved complete response and progressionfree survival that was statistically significantly better than
that achieved by patients assigned to conventional chemotherapy.” A potential problem, unresolved by theuse of
autologous BM support, is the presence of clonogenic plasma
cells in the BM product.
One approach to improve the efficacy of autologous transplantation in multiple myeloma has been through the use
of autologous peripheral blood progenitor
In a
previous study, we showed that peripheral blood progenitor
cells effectively restore hematopoiesis after high-dose chemotherapy,I6but this product may also be contaminated with
clonogenic multiple myeloma cell^.'^"' Thus, although effective as a source of hematopoietic support, tumor contamination exists in mobilized peripheral blood and may, like BM,
contribute to early relapse.
A way to reduce the risk of tumor cell contamination in
the autograft is through purification of the progenitor cell
material. This may be achieved either by purging with a
battery of B-cell antigens” or by positive selection for hematopoietic progenitor cells.20~z1
To improve upon the results
we achieved using peripheral blood progenitor cells, we evaluated a system of positive progenitor cell selection. The
CEPRATE system (Cellpro, Bothell, WA) is a continuousflow column-selection system that relies on the high affinity
between avidin and biotin to select antibody-labeled hematopoietic progenitor cells.22~2’
The positive selection of cells
expressing the CD34 antigen has beenused to provide a
source of hematopoiesis with a minimal likelihood of reinfusing tumor cells and has been used in clinical studies of
BMT for breast carcinoma and other malignancies.” In the
present study, we used the CEPRATE column to select for
purified peripheral blood progenitor cells to support patients
receiving myeloablative preparative chemotherapy for ad-
ESPITE THE SENSITIVITY of multiple myeloma to
alkylator-based chemotherapy, the disease remains
uniformly fatal with a median survival of 30 months using
standard drug regimens.’ Consequently, dose-intensive chemotherapywithmarrow
transplantation has beenusedin
the hopes of inducing complete remission and prolonged
progression-free s u r v i ~ a l . ~Although
-~
allogeneic transplantation may be curative for some patients,8 most cannot
receive this therapy because of advanced age at presentation
and subsequent high treatment-related mortality. As a result,
several large trials of high-dose chemotherapy and autologous bone marrow transplantation (ABMT) have been reported for patients with advanced multiple myel~ma.~”’
Initial patients were eligible for treatment based on resistance
to conventional chemotherapy and although cytoreduction
was achieved, progression-free survival was b~ief.~.’,’~
Favorable prognostic factors for sustained response included chemotherapy-sensitive disease and shorter duration of primary
treatment. Based on these results, current trials of ABMT,
which include recently diagnosed patients with chemotherFrom the Department of Medicine, Division of Hematology/Oncology, and Jonsson Comprehensive Cancer Center, UCLA School
of Medicine, Los Angeles, CA: the Department of Veterans Affairs,
West Los Angeles,CA;CellPro, Bothell, WA; Audie Murphy VA
Hospital, University of Texas San Antonio, San Antonio; and the
Department of Oncology, St Louis University, St Louis, MO.
Submitted October 27, 1994; accepted February 2, 1995.
Supported in part by Public Health Service Grant No. 5 MO1
RR00865-20.
Address reprint requests toGary Schiller, MD,Department of
Medicine, Division of Hematology/Oncology, University of California Los Angeles School of Medicine, L o s Angeles 90024-1678.
The publication costsof 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 facf.
0 1995 by The American Society of Hematology.
0006-4971/95/8601-0024$3.00/0
390
Blood, VOI 86, NO 1 (July I ) , 1995: PP 390-397
TRANSPLANTATION OF CD34' PBPC
vanced-stage multiple myeloma, anddefined a threshold
dose required for sustained engraftment.
To determine residual myeloma cell contamination in the
leukapheresis and CD34-selected stem-cell products, we
used a novel approach to quantify tumor cell contamination.
The Ig heavy-chain sequence can be used as a tumor-specific
marker in multiple myeloma because of the unique rearrangements of multiple Ig heavy-chain variable (VH), diversity, and joining-region (JH) gene segmentsz4 Further
sequence specificity is generated by the insertion of nongermline nucleotidesz5and the process of somatic mutation.*'j
In multiple myeloma, somatic mutation occurs frequently
especially in the unique regions of the VH sequences
that are termed the complementarity-determining regions
(CDRS).~'.~~
Using a PCR assay with patient-specific CDR
primers, tumor contamination was assessed in the leukapheresis product before and after CD34 selection. Quantitative
determination was achieved using Poisson distribution analysis of positive polymerase chain reactions (PCRs).
MATERIALS ANDMETHODS
Thirty-seven subjects, 34 to 65 years old (median, 51 years), with
multiple myeloma were consecutively enrolled from July 1993 to
September 1994. Data were analyzed as of September 28, 1994. The
study design was approved by the Institutional Review boards of all
participating institutions and by the Food and Drug Administration
under an investigational device exemption (IDE); all patients gave
written informed consent to participate.
The diagnosis of multiple myeloma was made using the criteria
and staging of Dune and Salmon.29Patients were entered if they
had evidence of advanced multiple myeloma characterized by one
of the following features at diagnosis or thereafter: (1) serum IgG
greater than 5 g/dL, IgA greater than 3 g/dL, or urine M component
greater than 4 g/24 hours; (2) osteolytic bone lesions or radiographic
evidence of diffuse osteoporosis; and (3) P2-microglobulin greater
than 3 m@. Patients with nonsecretory myeloma were eligible if
there was evidence of extensive marrow plasmacytosis. Patients with
major cardiac, pulmonary, gastrointestinal, neurologic, or hepatic
disease, serum creatinine greater than 2 mg/dL, age greater than 70
years, or Karnofsky performance status less than 70%were excluded.
Patients were also excluded from study for disease defined as refractory to conventional chemotherapy or initially responsive and subsequently progressive on chemotherapy. Patients whohad received
more than 1 year of alkylator-based Chemotherapy, regardless of
disease response, were also ineligible for entry.
Cytapheresis and cryopreservation. Autologous blood progenitor cells were procured after treatment with 2.5 g cyclophosphamide
intravenously on day 0, 2 mgkg prednisone orally daily for 4 days
(days 0 to 3), and 10 ygkg granulocyte colony-stimulating factor
(G-CSF; Amgen, Thousand Oaks, CA) subcutaneously daily starting
1 day after cyclophosphamide.16Blood progenitor cell apheresis was
begun 14 days after cyclophosphamide treatment. Continuous flow
leukapheresis was performed with a Cobe Spectra (Cobe, Lakewood,
CO). Blood volume processed per run was 10 L at a flow rate of
50 to 70 mL/minute. After this procedure, mononuclear cells were
collected and incubated with biotinylated antibody 12-8 (a CD34
antibody) in RPM1 with 0.1% human serum albumin and washed in
phosphate-buffered saline (PBS) to remove unbound antibody." The
antibody-treated cells were passed successively through a 15 X 2.5
cm Chromaflex column containing 20 mL avidin-biogel until a minimum of 1.0 X IO6 CD34' cellskg were obtained. Cells were cryopreserved in 10% dimethyl sulfoxide by control-rated freezing and
391
stored in the gas phase of liquid nitrogen. A back-up BM harvest
sample was cryopreserved in all patients.
CD34 enumeration. Cell counts were performed using a Coulter
counter (Coulter Electronics, Hialeah, FL). A total of 0.5 to 1 X IO6
cells were obtained from the leukapheresis product, the flow-through
fraction from the CEPRATE column, and from the adsorbed fraction.
Cells were incubated on ice in the dark for 30 minutes with 20 yL of
anti-CD34 monoclonal antibody (MoAb; HPCA2) conjugated with
phycoerythrin (Becton Dickinson, Palo Alto, CA) or the appropriate
isotype control (murine IgGl conjugated with phycoerythrin) (Becton
Dickinson). Samples were run on a FACScan flow cytometer (Becton-Dickinson) and 50,000 events were acquired. Data were analyzed
using Lysis I1 software (Becton Dickinson). Dead cells were excluded on the basis of forward- and side-scatter analysis. CD34'
cells were defined with histogram analysis using the whole live-cell
population. Analysis gates were set using the CD34 bright cluster
obtained after immunoadsorption. The percentage positive cells in
the isotype control were subtracted from the CD34' percentage to
give the final percentage of CD34+ cells.
Conditioning regimen for transplantation and maintenance.
Busulfan (0.875 mgkg orally four times daily for 4 days on days
-7 to -4) and cyclophosphamide (60 mgkg intravenously over 1
hour daily for 2 days on days -3 and -2) were given to all patients."
Bladder imgation with normal saline was infused at 1,000 mL/h at
the start of cyclophosphamide and was continued for 24 hours after
the last dose; mesna wasnot used. Patients with neutrophils less
than 0.5 x 109/L were placed in reverse isolation in single rooms
and received nystatin and either norfloxacin or ciprofloxacin. Those
with a temperature greater than 38.5"C received imipenem or cefoperazone-sulbactam or other broad-spectrum antibacterial antibiotics.
Patients with documented or suspected fungal infection received
either empiric amphotericin B or fluconazole. Pentoxifylline was not
routinely used. Random or single donor platelet transfusions were
givento maintain platelets 2 10 X 1 0 y L Redblood cells were
transfused to maintain a hematocrit 2 27%. Forty-eight hours after
completing pretransplant conditioning, purified peripheral blood progenitor cells were rapidly thawed and given intravenously, after
which all patients received daily 500 yg granulocyte-macrophage
colony-stimulating factor (GM-CSF Leukine, Immunex, Seattle,
WA) intravenously or subcutaneously until neutrophil recovery (definedas absolute neutrophil count [ANC] greater than 1.000 for
3 consecutive days). Patients who achieved a complete or partial
remission after the autologous transplantation received a interferon
subcutaneously (3 X IO6 IU/m2 three times per week as tolerated)
and decadron orally (20 mg/mz daily for 4 days every 4 weeks)
beginning 100 days after transplantation and continued for 1 year
or until there was evidence of progressive disease.
Response criteria. Patients surviving greater than 1 month posttransplantation were evaluated for response by skeletal radiographs,
urine and serum electrophoresis, BM biopsy and serum creatinine,
albumin, and P2-microglobulin. These were done pretransplantation
andmonthly for 4 months and then every 2 months thereafter or
until disease progression or relapse. Complete remission was defined
as normal BM cellularity, less than 5% plasma cells, no paraprotein
in serum and urine by immunoelectrophoresis or immunofixation,
and no evidence of progressive bony lesions. Partial remission was
defined as a 275% reduction from diagnosis of BM plasmacytosis
andserum or urine paraprotein levels, without progressive bony
lesions. A minimal response was defined as any reduction in BM
plasmacytosis with a reduction in serum or urine paraprotein by less
than 75%. Disease progression was defined as the day when BM
recurrence and/or new lytic bone disease on radiograph and progressive M component (>25% increase) were detected.
Survival data were computed from the transplantation day. Comparisons of patient features were performed using the Wilcoxon rank
392
sum test.” Comparison of the frequency of freedom-from-progression and survival between groups was made with the Fisher’s exact
test.31Comparison of means were calculated by chi-square test. Patients were analyzed for survival and progression-free survival from
the time of transplantation using the product-limit method of Kaplan
and Meier.” Summary estimates include actuarial survival fractions
at 95% confidence intervals. Analyses were performed using the
P values were two-sided throughout.
BMDP statistical pa~kage.~’
Sample preparation for tumor detection. Autograft tumor contamination was assessed in 14 of 37 patients transplanted. To determine the Ig gene sequence expressed by the myeloma cells in each
patient, total RNA was extracted” from BM mononuclear cells isolated after density centrifugation. The earliest BM specimen obtained
from each patient was used for this initial procedure (nine obtained
at the time of BM harvest and five obtained 1 to 5 months before
BM harvest). The myeloma Ig VH gene sequence was determined as
previously described.34Briefly, cDNA was created using Moloneymurine leukemia virus reverse transcriptase (GIBCO, Gaithersburg,
MD), BM RNA and Ca or Cy consensus primers (constant-region
a or y consensus primers). Because the VH gene sequences have
been grouped into six families on the basis of sequence homology,
the cDNA was aliquoted into six different reaction tubes. A consensus primer specific for one of the six VH gene familiess5 was then
added and 32 cycles of PCR amplification were performed using
Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) and the hot
start technique.36This allowed for detection of the monoclonal VH
gene product in most cases, yet does not yield a product using normal
BM RNA. Detectable PCR product was excised from a low melt
agarose gel and ligated into a pCR-Script SK(+) (Stratagene, La
Jolla, CA) or pCR I1 (Invitrogen, San Diego, CA) vector. The PCR
insert was then sequenced using the Sequenase I1 kit (US Biochemicals, Cleveland, OH) according to manufacturer’s instructions with
’’S a deoxyadenosine triphosphate (crdATP; Amersham, Arlington
Heights, IL). To be certain that the sequence obtained was from the
myeloma clone and not from normal plasma or B cells, a minimum
of three identical sequences were needed. The myeloma VH gene
sequence was then compared with known germline VHandJH
gene^^^.'^ using the DNAsis program (Hitachi, San Bruno, CA). For
use in the tumor detection assay, patient-specific oligonucleotide
primers were prepared complementary to the most unique sequences
in the CDRl or CDR2 (sense) and CDR3 (antisense).
PCR conditions. Sample DNA was isolated3’ from an aliquot of
the BM mononuclear sample described above, the CD34-selected
autograft, and leukapheresis product before CD34 selection. DNA
(0.6 pg = 100,000 cells) was amplified after the addition of SO pmol
of each primer, 200 mmol/L of each deoxynucleotide, and 2.5 U of
Taq polymerase in a final volume of 100 pL. Sixty cycles of the
PCR were performed to increase the sensitivity of the assay. Fifteen
microliters of the PCR product was then electrophoresed through a
1.6% agarose gel. The agarose gel was then soaked in SYBR green
stain (CYBERGREEN Molecular Probes, Eugene, OR) for 60 minutes and PCR products were visualized by exposure to UV light.
PCR conditions using the patient-specific primers were optimized
for each patient using the PCR Optimizer kit (Invitrogen) with BM
DNA serially diluted 1:10 with placental DNA and annealing temperatures of either 55°C or 60°C.
Tumor contamination assessment. Once the optimum annealing
temperature and buffer composition were determined, all subsequent
PCRs for that patient’s samples were run under identical conditions.
DNA from the sample (BM, leukapheresis, or CD34-selected product) was then serially diluted in 0.5-log increments with placental
DNA to maintain a final DNA quantity of 0.6 Kg per reaction. Five
replicate samples were processed at each serial dilution concentration
and all reactions were done concurrently. Original sample contamination was then calculated based upon the Poisson distribution of
SCHILLER ET AL
positive reactions at each sample dilution?’ Control PCR reactions
were performed using 0.6 pg of sample DNA with @-actinprimers
(Stratagene).
Sensitivio of the PCR assay to detect tumor cells. The efficacy
of the CEPRATE immunoadsorption column was assessed in 14
patients by comparing the degree of tumor contamination of the
autograft product before and after CD34 selection. The IgVH sequence of the myeloma cells was determined in each of these patients
by reverse transcription PCR of BM RNA with a VH family and an
Ig constant-region primer. We confirmed that the sequence obtained
was from the myeloma cells because each PCR yielded product
when only one ofthe six VH family primers wasused (VHl, 4
patients; VH2, 1 patient; VH3, 7 patients; VH4, 1 patient; VHS, l
patient). In addition, multiple clones (range, 3 to 6) were sequenced
after insertion of the PCR product into a vector. These sequences
were identical in every case and corresponded to the VH gene family
identified in the initial PCR.
Oligonucleotide primers that were complementary totheCDR
sequences were then designed for use in the tumor-detection PCR
assay. These CDR primers should detect all malignant cells in a
sample because the VH sequence lacks clonal diversity in multiple
myel~ma.*~
Tumor
~ * ~ contamination was quantitated by the serial
dilution of sample DNA with placental DNA in 0.5-log increments
until product was no longer detectable by PCR amplification. Five
replicate reactions were done at each serial dilution to improve assay
sensitivity and improve the quantitative properties of the assay. Specificity was confirmed by the absence of product when placental or
a different patient’s leukapheresis DNA was substituted for sample
DNA.
Under optimal conditions, the PCR should detect a single copy
of the target gene after 40 cycles of PCR!’ To maximize sensitivity,
we increased the number of PCR cycles to 60 because this has
occasionally increased our ability to detect the tumor VH gene product. Because quantitation of tumor contamination is not based on
the relative intensity of the PCR band, but only on whether it is
present or notin each tube, we wanted to assure that we would
detect any amplified product with this technique. First, each PCR
reaction was optimized using the patient’s diluted BM sample with
the PCR Optimizer kit (Invitrogen), which varies Mg+’, pH conditions, and annealing temperatures. This was performed until the PCR
amplified the greatest amount of VH product on serially diluted
myeloma BM samples without additional nonspecific amplified PCR
bands. Finally a more sensitive stain for DNA, SYBR Green, was
used that identifies smaller quantities of the PCR product than ethidium bromide. By using these PCR conditions with the very specific
CDR primers, we can detect one target gene copy within each PCR
tube that contains DNA from 100,000cells. Therefore, the likelihood
of a PCRtube yielding a positive PCR result is basedupon the
presence of at least one target gene copy in the reaction mixture at
that particular dilutional concentration. Because five reactions are
done at each serial dilution, the Poisson distribution4’ can then be
applied to the positive PCR results from all of the serial dilutions
for each sample to determine the percentage of tumor contamination
in the undiluted BM, leukapheresis, or CD34-selected material.
To verify the validity of this technique, sample DNA from the
myeloma IM-9 cell line4’ was serially diluted with placental DNA
to a total amount of 0.6 pg (DNA from =100,OOO cells) in 0.5-log
increments. Using I”9-specific CDR primers, the IM-9 VH gene
product was detectable in all sample reactions (S replicates at each
dilution) when diluted s100,000-fold and in two reactions after
300,000-fold dilution. No product was detectable with further dilution. Therefore, thepure IM-9 cell line sample had a calculated
tumor Contaminationbased upon Poisson distribution statistics which
are within 0.22 log of the actual cell purity of the undiluted IM-9
cells. Myeloma cell contamination of the BM sample from the 14
TRANSPLANTATION OF CD34+ PBPC
patients analyzed in this study was calculated using this PCR assay
and then compared with a cytospin of the same sample. These samples were obtainedafter the patients hadreceived multiple courses of
chemotherapy. Thus,BM contamination was low, and was calculated
using Poisson distribution analysis to range from 0.1% to 5.4% in
these patients using the PCRand Poisson distribution analysis. In
all cases, these results closely matchedthepercentage of plasma
cells identifiedby microscopic examination of these BM samples
validating assay sensitivity andaccuracy. Specifically, in thethree
cases with greater than 2% plasma cells, the calculated percentage
from the PCR assay was within 0.3 log ofthe proportion determined
by cytological examination. In the cases with 5 1% plasma cells,
the percentage on the cytospins also closely matched the percentage
determined by Poisson distribution analysis, although an exact logarithmic difference could not be assessed because of the infrequency
of tumor cells in these latter specimens.
RESULTS
Overall response and survival. Thirty-seven patients
with chemotherapy-responsive or stable multiple myeloma
were entered into study; clinical characteristics are shown
in Table 1. Twenty-three were menand 14 were women.
Paraproteins were detected in 33 of 37 patients: IgG in 27,
IgA in 4, and light chains only in 2. At diagnosis, five patients were Durie-Salmon stage IA, 13 stage IIA, 13 stage
IIIA, and six stage IIIB. Before treatment, 35 patients had
lytic bone lesions or osteoporosis on radiographs. All patients had received chemotherapy and/or steroids before
transplantation, which included infusional vincristine, doxorubicin, and oral dexamethasone (VAD) ( l 1 patients), VAD
plus alkylating agents (9 patients), alkylating agents (3 patients), dexamethasone alone (4 patients), and other treatments (10 patients).
Median duration of follow-up for surviving patients from
the time of transplantation was 7.8 months (range, 1.4 to
14.5 months). Of 34 evaluable patients, complete and partial
remissions were achieved in 5 (14.7%) and 28 (82.4%) respectively; one patient achieved a minimal response. Three
patients have subsequently progressed and two have died
(one on day +l39 and one on day +337). Disease stage,
type of myeloma, and pretransplantation chemotherapy were
analyzed and found to have no effect on response or progression-free survival. Actuarial progression-free and overall
survival 1 year from transplantation are 67% +- 19% and
68% -t- 21%, respectively (Fig 1).
Hematologic recovery and toxicity. A median of three
collections (range, 2 to 5) was required to procure 9.8 X lo8
mononuclear cellskg (range, 3.7 to 28.3 X lo8cellskg) and
CD34% ranged from less than 1% to 15% (median 1%). A
median of 5.9 X lo6 adsorbed cellskg (range, 1.6 X 10'kg
to 25.5 X 106kg) and 4.65 X lo6 CD34 cellskg (range, 1.2
to 23.3 X 106kg)was obtained. CD34 purity after processing
ranged from 27% to 91% (median, 77%). All 37 patients
achieved neutrophil amounts greater than 0.5 X 109/Lat a
median of 12 days after transplantation (range, 11 to 16
days). Thirty-four patients achieved an untransfused platelet
count greater than 20 X 109/L at a median of 12 days after
transplantation (range, 9 to 52). Three patients did not
achieve complete platelet recovery because of treatmentrelated mortality on days 28, 38, and 52. Three patients
393
who achieved early platelet recovery developed late platelet
transfusion-dependence because of infection, in two of
whom infection and thrombocytopenia have now resolved.
The median numbers of units of erythrocytes and platelets
transfused to date are 7 (range, 2 to 37) and 3 (0 to SS),
respectively. No patient required reinfusion of back-up BM.
Six patients received fewer than 2 X lo6 CD34+ cellskg
and had a statistically significantly increased red blood cell
and platelet transfusion requirement, and a prolonged median
time to platelet recovery (P = .005, P = .002, and P < .001,
respectively) (Table 2).
There were five treatment-related deaths, one caused by
viral pneumonia (day +38), two caused by hepatic venoocclusive disease (day +28 and day +52), two caused by
late pulmonary toxicity attributed to the conditioning regimen (day +l77 and day +217). Other toxicities consisted
of hemorrhagic cystitis (4 patients), and nonfatal interstitial
pneumonitis (4 patients), nonfatal hepatic veno-occlusive
disease (4 patients) and cardiac dysrhythmia (1 patient).
Twenty-two of 37 patients developed mild to moderate
(grades 1 to 2) mucositis, nausea and vomiting, and 9 patients
hadmore severe (grades 3 to 4) gastrointestinal toxicity.
Seven patients had nonfatal bacteremia.
Three patients sustained oral herpes simplex infection and
one patient had infection with herpes zoster.
Purging effectiveness using CD34 selection. Because the
sensitivity of the assay is 0.001% per reaction (1:1OO,OOO)
and five replicate reactions are done at each concentration,
the overall sensitivity of the PCR assay is 0.0002%
(1:500,000). Before CD34 selection, tumor cells were detectable in the leukapheresis product in eight of 14 patients
(Table 3) (Fig 2). Calculated tumor contamination ranged
from 0.23% to 0.0015% in these eight patients. Tumor contamination of the CD34-selected autograft product obtained
from the same day as the leukapheresis sample wasthen
assessed. Myeloma cell contamination remained detectable
in only three of the 14 patients (Table 3) (Fig 2). Moreover,
CD34-selected autograft tumor cell contamination in these
three patients was reduced (0.019%, 0.0030%, and 0.0005%)
and the sample with the greatest contamination also had the
highest initial contamination of the leukapheresis product
before CD34 selection (0.23%). Positive controls using pactin primers with undiluted specimen DNA yielded the appropriately sized PCR product of similar band intensity in
each case indicating that the sample DNA was of sufficient
quality to permit PCR amplification.
The number of myeloma cells in the unselected leukapheresis product ranged from less than 1.60 X 103kg to 2.14 X
106kg (calculated by multiplying the percentage contamination by mononuclear cell numberkg). A minimum of 1.13
X io" multiple myeloma cells/kg were present in the leukapheresis products from the eight patients with detectable
disease contamination. In the 14 patients analyzed, multiple
myeloma contamination after CD34 selection ranged from
less than 6 to 2 X lo3 cellskg. This reduction in tumor
contamination after CD34-selection results from both the
increased purity (ie, lack of tumor cells) of the autograft
product and the median 2.2-log reduction in the total number
of cells. In the 8 of 14 patients with detectable leukapheresis
SCHILLER ET AL
394
Table 1. Patient Characteristics at the Time of Enrollment
~
~~~
At Enrollment
UPN
Age
(yrl
321
324
323
327
331
60
59
56
59
65
368
45
369
44
335
43
341
345
44
Myeloma Type
Stage at
Diagnosis*
II
II
I
II
I
II
II
42
I1
111
I
343
55
IllB
357
48
358
57
365
45
Ill
II
Ill
376
401
394
398
399
428
405
427
419
430
452
339
461
468
478
479
480
481
482
483
491
501
489
41
51
39
55
57
42
58
52
64
44
41
52
63
34
43
57
47
55
37
48
45
64
61
Interval
DX to TX
IllB
IllB
II
I1
II
I
111
IllB
lllB
111
Ill
111
111
111
II
Ill
II
Ill
II
111
I
IllB
111
Previous Treatment
VAD, Decadron
Decadron
a-IFN, VAD, Decadron
MP
VBMCP,
HD Decadron, a-IFN
VAD
VBMCP, a-IFN
VAD
VBMCP, Decadron
VACOP-B,
M-BACODZ, Decadron
MP, VAD
VAD, VBMCP
MP, a-IFN, Decadron
VAD, IFEX, VP16,
Decadron
VAD
VAD, CTX, Decadron
MP, VAD
VAD
MP, VAD
VAD
MP, VAD
HD Decadron
VAD
MP, VAD
MP, Decadron
VAD
MP, VAD
VAD
VAD
MP, VAD, Decadron
CTX, Decadron, MP
Decadron
VAD
MP, VAD
MP, VAD
VAD
MP
has)
Highest Pre-TX
Plasma
02-Microglobulin
9.5
4
21
5.5
45.5
5.6
2.9
2.4
1.4
1.4
7.5
15
5
8.5
47
2.6
1.6
2.9
2.6
1.2
4
10.17
14
17
9
3.4
1.7
2.5
5
23
10
6.5
8
9.5
8.5
5
4.5
9
11.5
10.5
12
4
16
17
5
8
12
9.5
10
5
7.5
55%
40%
5%
28%
-
>30%
50%
>80%
0.86 95%
Diffuse
atypical PC
2.2
1.48
1.4
3.5
1.4
1.5
1.3
5.2
2.7
20%
1%
85%
95%
40%
Modt
Atypical
0.5
1.39
4.98
2.5
2.4
ND
Urine
M-Protein
Bone
Lesions
0.97
NA
0.7
0.16
NA
1.32
1.49
NA
1.8
2.4
M-Protein
(g/dLl
0.44
ND
1.7
2.7
4.
2.1
2.7
3.4
24.7
2.7
1.4
3.6
2.2
Marrow %
Cells
DX
0.89
2.1 1
NA
1 .o
1.4
90%
15%
Modt
Packed
2.46
0.69
90%
30%
>90%
30%
9%
10%
3.0
1.69
3.2
1.4
2%
1.06
2.28
16%
2.9
1.49
1.3
1.18
25%
35%
33%
46%
70%
50%
+
1.49
1.15
ND
0.112
Abbreviations: UPN, unique patient number; a-IFN, a interferon; MP, melphalan, prednisone; VBMCP, vincristine, carmustine melphalan,
cyclophosphamide, prednisone; HD, high dose; VACOP-B, etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone;
MBACODZ, 2
cycles of methotrexate, bleornycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone;IFEX, ifosfamide; VP16, etoposide; CTX, cyclophosphamide; PC, plasmacytosis; Mod t, moderate increase; ND, n o data; NA, not applicable.
* Durie Salmon stage.
product myeloma contamination, the log reduction in autograft tumor contamination by the CD34-selection procedure
ranged from more than 2.7 to more than 4.5 (Table 4).
DISCUSSION
Thirty-seven patients with multiple myeloma received
pretransplantation conditioning with busulfan and cyclophosphamide followed by the infusion of CD34-selected peripheral blood progenitor cells. All patients were determined
not to have chemotherapy-resistant disease because there
was no evidence of progressive multiple myeloma from conventional chemotherapy given just before transplantation.
Thus, conventional treatment was used to identify a group
of patients likely to derive benefit from high-dose chemothe rap^.^ Patients whohad received morethan 1 year of
alkylator-based chemotherapy were ineligible, regardless of
response, because of concern over residual marrow function
and impaired progenitor cell mobilization.I6 Peripheral blood
progenitor cells were then purified by positive selection for
the CD34 antigen. Mobilization was achieved via a combina-
395
TRANSPLANTATION OF CD34' PBPC
1f-h
'
0.7
04.
0
~
,
l00
Table 3. Contamination of BM, Leukapheresit Product and
Adsorbed Cell Product in Multiple Myeloma Patients
7
'
3.9
,
200
300
400
6
DAYS
2.0
Fig 1. Progression-free survivalfor
M M patients
undergoing
CD34-selected peripheral blood progenitor cell transplantation for
multiple myeloma.
tion of chemotherapy, corticosteroids, and hematopoietic
growth factor. Despite purification of these early progenitor
cells and, thus, a marked decrease in the number of mononuclear cells infused, reconstitution of hematopoiesis was both
rapidand complete. A threshold dose of 2 X IOh CD34'
cellskg was defined, below which both transfusion support
and engraftment were significantly prolonged. Complete and
partial remissions were achieved in 33 of 34 evaluable patients (97%).
Using PCR analysis with patient-specific Ig-gene primers,
we found tumor contamination in the leukapheresis specimensfrommorethanhalf
of the patients studied. Other
investigators have also detected tumor cells in similar clinical specimens.'" However, the frequency of patients with
contamination of leukapheresis products was lower using
less sensitive tumor detection techniques. The use of Poisson
distribution analysis of multiple PCR reactions allows for a
much more sensitive and accurate assessment of tumor burden. Using this technique. we showed a marked (>2.7- to
>4.5-log) reduction in the total number of tumor cells after
CEPRATE-CD34 selection of these clinical leukapheresis
specimens. These findings suggest that CD34' selection of
peripheral blood progenitor cells produces a product that
%BM
OhLeukapheresis
UPN
Contamination
Contamination
321
327
339
343
357
419
430
452
461
481
482
483
491
501
1.1
0.9
l .o
1.1
2.5
2.0
5.4
1 .o
0.2
0.0015
0.0375
0.0054
%Adsorbed
Contamination
<0.0002
<0.0002
0.0005
<0.0002
<0.0002
<0.0002
0.0017
<0.0002
0.2300
<0.0002
<0.0002
0.0015
.c0.0002
0.0086
c0.000
0.0190
0.1
co.ooo2
<0.0002
<0.0002
<0.0002
0.2
0.0100
<0.0002
<0.0002
<0.0002
0.0030
Calculations are based on the Poisson distribution of positive PCR
reactions using 0.6 pg of DNA per reaction with patient-specific primers. Serial dilution of the sample DNA with placental DNA was done
in 0.5-log increments and all amplification reactions were done in
replicate five times. Concentrations of less than 0.0002 were based
on negative results with an assay sensitivity of 0.001% per reaction.
Abbreviation: UPN, unique patient number.
achieves promptand complete engraftment with minimal
tumor contamination.".23 Minimal contamination remained
only in those specimens heavily contaminated withmyeloma. suggesting that further cytoreduction couldyield
specimens free of residual myeloma. The useofpositive
progenitor cell selection in transplantation is predicated on
M
LK Only
LK 1:3
LK 1:lO M P
Table 2. Time t o Neutrophil andPlatelet Recovery and Transfusion
Requirements as a Function of CD34 Peripheral Blood Progenitor
Cell Number Infused
CD34-PBPCkg
<2 x 108
Patients (no.)
Time to ANC > 500 m m
Median (days)
12
Time to Platelet > 20,000 m m
12
Median (days)
Red blood cell transfusions (no.)
Platelet transfusions (no.)
6
>2 x 108
31
.05
14
21
19
30
P
6
2
<.001
,005
,002
Fig 2. Myeloma cell contaminationof the autograft productsfrom
patient 339. Sample DNA from the leukapheresis ILK) product and
from theadsorbed cell (AD) product after CD34 selection was serially
diluted with the
placental DNA t o maintain a final quantity of 0.6 p g
per reaction. The PCR was performed usingpatient-specific VH gene
CDR primers. Five replicate samples were processed at each dilutional concentration. Controls using placental (P) DNA only are also
shown. M, molecular weight marker 123 bp. The expected VH gene
product is 260 bp.
396
SCHILLER ET AL
Table 4. Tumor Cell Reduction by CD34 Affinity-Cell Processing of
Leukapheresis Products
UPN
Leukapheresis
Contamination
Cells/kg*
321
327
339
419
452
461
481
491
29,550
480,000
127,440
16,660
2,139,000
11,325
243,380
90,000
Adsorbed Cell
Contamination
Cells/kg*
Tumor Cell Log
Reduction by
CD34 Selection
<20
<l6
83
132
2,090
>3.2
24.5
3.2
>2.7
3.0
>3.2
>4.2
2.8
<8
<l4
250
Abbreviation: UPN, unique patient number.
*Tumor cell contamination in cells/kg was calculated by multiplying the percentage contamination by the number of mononuclear
cells/kg collected.
dose-intensive therapy in similar chemotherapy-sensitive
multiple myeloma patients followed by transplantation of
both purged and unpurged autologous BM or stem cells have
shown similar
In conclusion, peripheral blood progenitor cells are an
effective form of purified hematopoietic support achieving
substantial reduction in myeloma cell contamination. At infused cell doses of greater than 2 X lo6 CD34’ cellskg,
this product provides safe, rapid, and sustained hematologic
recovery in patients receiving myeloablative chemotherapy.
Whether this form of purified stem cell support will produce
improved progression-free survival in multiple myeloma will
require further trials. A multi-institutional phase I11 study
comparing CD34 selected versus unselected PBPC transplantation as treatment of multiple myeloma has been initiated to answer this question.
ACKNOWLEDGMENT
the concept that malignant cells in multiple myeloma do not
express the CD34 antigen; this was previously shown in
five patients with advanced multiple myeloma.34An initial
collection of CD34’ cells was obtained by passage of BM
mononuclear cells through the CEPRATE LC34-BIOTIN
system. In comparison with the starting sample, this process
reduced the concentration of tumor cells by 1.5 to 2.5 logs
when assayed using DNA PCR with patient-specific CDR
primers. When these cells were further purified using fluorescence-activated cell sorting, no residual myeloma could
be detected, thus confirming that CD34+ cells are not part
of the malignant plasma cell clone.
Other forms ofBM or peripheral blood progenitor cell
purification are less feasible for use in transplantation for
multiple myeloma. No single antigen has been identified that
is specific for the malignant cells. Consequently, the negative
selection of tumor cells by means of antibody-purging requires a panel of MoAbs to be successful and may still fail
to identify all malignant cells.12 The low rate of plasma cell
replication makes the elimination of malignant cells in the
autograft by exposure to cytotoxic agents problematic. Furthermore, extensive exposure to previous chemotherapy may
render any form of chemotherapy purging hazardous because
of a well-recognized risk of delayed
In the
treatment of multiple myeloma, prolonged pancytopenia has
beennotedwith 4-hydroperoxycyclophosphamide” andin
both studies using MoAb purging of auto graft^,'^'^' with median times to neutrophil engraftment of 19, 21, and 25 days,
respectively. Therefore, because of the inherent methodologic difficulties of antibody purging, and the risks associated
with in vitro exposure to cytotoxic agents, we chose to purify
peripheral blood progenitor cells by positive selection. The
procedure, which yielded an average of 4.65 X IO6 CD34’
cellskg, produced a progenitor cell material capable of rapid
engraftment, with a median time to neutrophil and platelet
recovery of 12 days after infusion.
The l-year progression-free survival in our study was 67%
? 19%, a very encouraging result, but one that will require
prolonged follow-up given the group of patients studied,
which consisted of patients with chemotherapy-responsive
intermediate- to high-stage multiple myeloma. Studies of
We thank Linda Rodman for her help in preparing the typescript.
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