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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2234-2239
CLINICALOBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Engraftment, clinical, and molecular follow-up of patients with
multiple myeloma who were reinfused with highly purified CD34+
cells to support single or tandem high-dose chemotherapy
Roberto M. Lemoli,
Giovanni Martinelli,
Elena Zamagni,
Maria Rosa Motta,
Simonetta Rizzi,
Carolina Terragna,
Roberto Rondelli,
Sonia Ronconi,
Antonio Curti,
Francesca Bonifazi,
Sante Tura, and
Michele Cavo
From the Institute of Hematology and Medical Oncology "L. & A. Seragnoli" and Department of Pediatrics, University of Bologna,
Bologna, Italy.
 |
Abstract |
Eighty-two patients with advanced multiple myeloma (MM) were
enrolled in 2 sequential clinical studies of 1 or 2 courses of myeloablative therapy with stem cell support. Conditioning regimens consisted of high-dose melphalan (MEL) with or without total body irradiation (TX1 = 35) and MEL as the first preparative regimen, followed within 6 months by busulfan and melphalan (TX2 = 47). On
the basis of adequate stem cell harvest, 31 patients (TX1 = 13;
TX2 = 18) were transplanted with highly purified CD34+ cells. Positively selected stem cells did not adversely affect hematopoietic reconstitution compared with unmanipulated peripheral blood stem cell.
Overall, the complete remission (CR) rate of evaluative patients was
13.8% and 41% for single and double autotransplant, respectively
(P = .04). Moreover, 3 patients undergoing TX2 achieved molecular remission and 2 remain PCR-negative after 36 and 24 months
from autograft. The median event-free survival (EFS) durations for TX1
and TX2 were 17 and 35 months, respectively (P = .03). Actuarial 3-year overall survival for patients treated with 1 or 2 transplants are 76% and 92%, respectively (P = NS). On
multivariate analysis, superior EFS was associated with low  2
microglobulin (2-M) level at diagnosis and TX2, whereas overall
survival was correlated with 2-M. Positive selection of CD34+
cells did not influence the achievement of clinical or molecular CR, as
well as remission duration or survival of MM patients. Thus, whereas multiple cycles of high-dose therapy may be beneficial for patients with myeloma, the clinical impact of tumor cell purging remains highly questionable.
(Blood. 2000;95:2234-2239)
© 2000 by The American Society of Hematology.
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Introduction |
High-dose therapy followed by autologous stem cell
transplantation has been shown to improve complete remission (CR) rate, event-free survival (EFS), and overall survival (OS) in patients with
multiple myeloma (MM) when compared with conventional chemotherapy in a
randomized study.1 However, the lack of plateau in the survival curves of MM patients with long-term follow-up indicates that
it may be unlikely to cure the disease by autologous stem cell
transplantation. In this regard, the reinfusion of myeloma cells
contaminating autologous grafts may contribute to disease relapse as
demonstrated in other hematologic malignancies and solid
tumors.2,3 The issue of myeloma cell contamination in the
leukapheresis products collected after mobilization protocols has
recently been addressed.4-7 These studies have consistently shown the presence of 104 to 109 tumor cells
per autograft by immunophenotyping and molecular analysis of patient
specific Ig heavy chain (IgH) complementary determining region III (CDR
III).5,7 Moreover, an inverse correlation between plasma
cell concentration in peripheral blood stem cell (PBSC) collections and
disease-free survival has recently been demonstrated with more than
2 × 105 tumor cells per liter predicting for early
relapse.8
Positive selection of hematopoietic CD34+ progenitor cells has been
shown as a feasible approach for removing 2.5 to 4.5 log of myeloma
cells from PBSC collections with a limited loss of normal stem
cells.5,7 Recently, several phase II studies and 1 phase
III randomized trial have shown that transplantation of positively
selected CD34+ cells results in rapid and stable recovery of
hematopoiesis, with no difference with respect to patients receiving
unmanipulated PBSC.5,7,9
However, the clinical role of purging to prevent disease relapse or
progression remains to be determined at this stage. Whereas, indirect
evidence of the therapeutic benefit of purging has been accumulating
over the years for different malignancies,2,3,10,11 conclusive data are not currently available for MM. In fact, it may
well be that residual chemotherapy-resistant cells are responsible for
the clinical outcome and circulating myeloma cells may simply reflect
overall tumor burden.
In this study, we report the engraftment, clinical, and molecular
follow-up of 82 patients with advanced stage MM submitted to single
( = 35) or double ( = 47) transplantation of autologous stem cells.
Among these 2 sequential cohorts of patients, we could separately
analyze individuals who, on the basis of adequate stem cell
mobilization, received 1 ( = 13) or 2 ( = 18) reinfusions of highly
enriched CD34+ cells.
The results presented here suggest that reinfusion of positively
selected CD34+ cells provides effective hematopoietic support for 1 or
2 subsequent courses of high-dose therapy. However, removal of tumor
cells does not seem to improve the serologic or molecular CR rate, EFS,
and overall survival of MM patients.
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Patients and methods |
Patients
Eighty-two advanced stage MM patients aged between 18 and 65 years
underwent autologous stem cell transplantation at the Institute "Seragnoli" from January 1994 to December 1998 and their clinical characteristics are shown in Table 1. Data
were analyzed as of May 30, 1999. Up to December 1995, 31 individuals
were submitted to a single autograft (see below) and were reinfused
with either unmanipulated PBSC ( = 18) or positively selected CD34+
cells ( = 13). From January 1996, 47 patients were administered 2 subsequent lines of high-dose chemotherapy ("tandem" or
"double" transplantation) (see below) supported by untreated PBSC
( = 29) or enriched CD34+ cells ( = 18). Because of the lack of
adequate stem cell mobilization, in the same period, 4 additional
patients were submitted to a single course of myeloablative
chemotherapy with PBSC support. The diagnosis of MM was made using
standard criteria12 and the patients were staged according
to Durie and Salmon classification.13 Eligibility criteria
included: Karnofsky status greater than 70%, creatinine clearance
greater than 60 mL/min, cardiac ejection fraction and pulmonary
function tests greater than 50% of predicted values; bilirubin and
transaminases less than 2 times the normal upper limit; white blood
cell and platelet counts greater than 3 × 109/L and
100 × 109/L, respectively; negative serology for
human immunodeficiency virus (HIV) and hepatitis B surface antigen and
no evidence of active infection. The protocol was approved by the
University Hospital ethical committee and each patient gave written
informed consent.
Peripheral blood stem cell mobilization and collection
As previously reported,5 patients were treated with
cyclophosphamide (Cy; 7 g/m2), followed by the subcutaneous
administration of 5 µg/kg of granulocyte colony-stimulating factor (G-CSF) starting on day +2 from chemotherapy and continuing until the completion of PBSC collection. Once the CD34+
cell count was greater than 20 000/ mL of peripheral blood (PB),
patients underwent leukaphereses using a Cobe Spectra (Cobe, Lakewood,
CO). On the basis of our previous experience,5 to obtain a
target cell dose of at least 2 × 106 CD34+ for each
reinfusion, we established 4 or 8 × 106/kg CD34+
cells for single or tandem autograft, respectively, as the minimum
number of hematopoietic cells, to be collected over 2 days, for
proceeding to ex vivo manipulation. The patients ( = 46) who failed
to mobilize an adequate number of CD34+ cells to start the selection
process were reinfused with unmanipulated PBSC. Five additional
patients did not have their CD34+ stem cells enriched because of the
loss of availability of the clinical grade device used in this study
(Ceprate SC Concentrator, see below). During the study period, 12 additional patients (11%) mobilized less than
2 × 106 CD34+ cells per kilogram and were not
submitted to stem cell transplantation, whereas 10 patients were
enrolled in a pilot trial on selection and transplantation of CD34+
B-lineage negative cells.14
Stem cell enrichment and cryopreservation
One or 2 leukapheresis products were processed by the Ceprate SC
Concentrator (CellPro, Bothell, WA) to positively select CD34+ cells as
previously described,5 while an additional apheresis was
stored as an unmanipulated back-up. The CD34+ cell fraction was
recovered and resuspended in phosphate-buffered saline (PBS) containing
7.5% dimethyl sulfoxide (DMSO) and 4% human serum albumine (HSA) to a
final volume of 4.5 mL. Unmanipulated PBSC collections were stored in
10% DMSO. The cells were then cryopreserved using a controlled-rate
freezing method and kept at  196°C. At the time of
reinfusion, CD34+ cells were rapidly thawed in a water bath at 37°C
and diluted by slowly adding 4.5 mL of heparinized PBS. The cell
suspension was further diluted with PBS to 30 mL of final volume for
each vial and reinfused via a central line.
Cell phenotype analysis and colony assay
Flow cytometric analysis was carried out by direct
immunofluorescence on blood cells from the apheresis products before
and after CD34+ cell separation. The following human MoAb was used: anti-CD34 (HPCA-2) phycoerythrin (PE) (Becton Dickinson, Palo Alto,
CA). Isotype control IgG1-PE was also purchased from Becton Dickinson.
Data acquisition and analysis were assessed on a FACScalibur instrument
by CellQuest software (Becton Dickinson) as earlier reported.5
Samples of PB cells were evaluated in tissue culture assay to determine
myeloid progenitor cell growth as previously described.5 Briefly, 50 × 104 unseparated cells or 1000 to 5000 purified hematopoietic progenitors were plated in duplicate in culture
medium consisting of 1 mL of Iscove's modified Dulbecco's medium
(IMDM), supplemented with 24% fetal calf serum (FCS; Sera Lab, Crawley
Down, Sussex, UK), 0.8% BSA (Sigma), 10 4 mol/L
2-mercaptoethanol (Sigma). To measure the optimum clonogenic efficiency, 10% (vol/vol) of a selected lot of
phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM) was
added. Methylcellulose final concentration was 1.1%.
Granulocyte-macrophage colony-forming unit (CFU-GM) was scored after 14 days of incubation at 37°C in a fully humidified 5%
CO2 atmosphere.
Stem cell transplantation
From January 1994 to December 1995, MM patients were enrolled in a
clinical trial of positive selection of CD34+ cells to support a single
course of high-dose chemotherapy (TX1). Untreated or CD34+-selected
cells were reinfused on day 0 after a preparative regimen consisting of
high-dose melphalan (MEL; 200 mg/m2; = 29) administered
intravenously at day 1 or melphalan (140 mg/m2) at
day 3 and total body irradiation (10 Gy; = 6) at day 1. G-CSF was administered at 5 µg/kg/d subcutaneously from day +6 until
absolute neutrophil count (ANC) reached more than
0.5 × 109/L for 3 consecutive days. From January
1996, we started a pilot trial of "tandem" transplantation of
purified CD34+ cells for advanced stage MM patients (TX2). The first
conditioning regimen was MEL 200 mg/m,2 as reported
previously. The second course of myeloablative therapy was scheduled
within 6 months from MEL and consisted of busulfan (12 mg/kg total
dose) given orally 4 times daily from day 4 to day 2 and
melphalan 120 mg/m2 on day 1 (Bu/Me). Stem cell
reinfusion and G-CSF treatment were the same as above.
Patients were nursed in single or double rooms in reverse isolation and
received antimicrobial prophylaxis consisting of oral nystatin and
ciprofloxacin. Packed red blood cells (RBCs) and single-donor platelet
transfusions were administered to maintain a hemoglobin level more than
8 g/dL and a platelet count greater than
10 × 109/L. Patients were treated with broad
spectrum antibiotics when fever developed and ANC was less than
0.5 × 109/L. Amphotericin B was added if the
patients had persistent fever after 4 to 7 days of intravenous
antimocrobial therapy. No prophylactic antipneumocystis and antiviral
therapy was administered. The severity of side effects that occurred in
the peritransplant period was assessed according to the World Health
Organization (WHO) scoring system. Documented infection was defined in
febrile patients as the occurrence of a single blood culture that was
positive for any microorganism. Patients with invasive infection
required histologic documentation or culture.
Patients who achieved CR or partial remission (PR) (see below) after
completion of the transplantation program received  -interferon (-IFN) subcutaneously (3 × 106 IU/m2
3 times a week) beginning at the time of full hematologic recovery and
continued until evidence of disease progression.
The primary end point of the clinical study was time to hematopoietic
reconstitution, which was defined as the number of days taken to
achieve an ANC greater than 0.5 × 109/L (first of 3 consecutive days) and an unsupported platelet count greater than 20 and
50 × 109/L. Secondary end points were the
evaluation of tumor cell purging, incidence of serologic and molecular
CR (see below), rate of EFS , and OS.
Response criteria
Assessment of tumor response to transplantation was generally
performed 30 days after reinfusion of autologous stem cells and was
planned every 3 months thereafter. MM patients were considered responsive to treatment if they showed at least 50% reduction in tumor
mass. PR was defined as a tumor mass reduction of at least 75%. CR
required the absence of monoclonal gammopathy in serum and urine by
immunofixation (IF) analysis, and a normal BM aspirate and biopsy. In
all cases, these findings had to be present on at least 2 occasions 3 months apart. New lytic lesions and/or any increase in plasma cell
infiltration in BM and monoclonal gammopathy were considered as disease
progression or relapse. Patients with at least 2 negative IF analysis
were monitored for the presence of residual disease at the molecular level.
Molecular analysis of minimal residual disease (MRD)
BM and PB specimens were prepared for DNA and RNA analysis as
described.14,15 The molecular study was performed at
diagnosis on BM samples, on unmanipulated apheresis products, after
CD34+ cell selection, and during clinical follow-up every 3 months for the first 6 months, twice a year for the first 2 years, and once a year
thereafter. Molecular CR was defined as PCR-negative results in at
least 2 subsequent evaluations.
VDJ gene rearrangement amplification was performed with a panel of VH
family-specific primers,4 together with a JH consensus primer.14,15 To determine the VH segment used in VDJ gene
rearrangement, 7 amplifications were performed for each patient. The
reaction mixture (50 µL) contained 200 µmol/L of dNTPs,
1 × PCR buffer (10 mmol/L BME, 6.7 µmol/L EDTA pH 8, 67 µmol/L Tris pH 8.8, 170 mg/mL BSA), 7.7 mmol/L MgCl2, 50 pmol/L of each primer, 2% DMSO and 0.3 U Taq DNA polymerase
(Boehringer Mannheim, Monza, Italy). Thirty cycles of amplification
were performed. Denaturation at 95°C for 30 seconds, annealing at
61°C for 40 seconds and extension at 72°C for 50 seconds were
then followed by a 7-minute final extension at 72°C.
An aliquot of 15 µL was analyzed on ethidium bromide stained 3%
agarose gel: a single, discrete band of approximately 300 base pairs
(bp) was obtained at diagnosis. A 30 µL aliquot of the amplification
product corresponding to the VH family-specific gene rearrangement was
loaded on a 1.25% low-melt preparative grade agarose gel (BioRad,
Segrate, Italy). The 300-bp band was excised from the gel and purified
with a Gel Nebulizer Micropure Separator (Amicon, Milan, Italy),
according to the manufacturer's instructions. An aliquot of purified
DNA was directly sequenced with the family-specific VH primer using the
Thermo Sequenase DNA cycle-sequencing kit (Amersham, Milan, Italy).
Sequence analysis was performed using the PC-GENE software
(IntelliGenetics). To generate patient-specific amplifications,
patient-specific primers were designed on the CDRII and CDRIII region identified.
Clonally expanded B cells were detected by amplifying 1 µg of DNA or
10 µL of cDNA using the patient-specific primers directed to the
CDRII and CDRIII regions. Fifty cycles of amplification were performed
consisting of denaturation at 96°C for 30 seconds, annealing at the
best tested temperature for 30 seconds, and extension at 72°C for
40 seconds, followed by a 7-minute final extension at 72°C. The
reaction mixture (50 µL) contained 200 µmol/L dNTPs, 1 × PCR buffer, 500 mmol/L KCl,100 mmol/L Tris pH 8.3, 2.5 mmol/L MgCl2, 50 or 100 pmol/L of patient-specific primers,
and 1 U of AmpliTaq Gold (Parkin Elmer, Milan, Italy). An aliquot of 15 µL was analyzed on agarose gel as reported above. A rearrangement band of approximately 150 bp was obtained from each patient. The sensitivity of each set of primers (1:105-106)
was assessed on the DNA or RNA from the patient initial marrow specimen
serially diluted in an appropriate amount of DNA or RNA from normal PB cells.
Statistical analysis
The results are presented as median values and ranges where
applicable. EFS and OS among categorical prognostic variables measured
before start of therapy were compared using the log-rank test.16 To avoid bias in favor of patients submitted to
tandem transplant, landmark analysis was used to compare EFS and OS
between the 2 groups.17 The analysis was performed by
determining the time (6 months) within which 90% of the patients
received the second cycle of high-dose therapy. Only the individuals
event-free and alive at that landmark were compared in the 2 studies.
The probabilities of neutrophil and platelet recovery and achievement of CR and PR of the different series of patients were compared by means
of the Kaplan and Meier method. Cox regression was used to examine
continuous and categorical univariate and multivariate effects of
prognostic features on EFS and OS.18 Variables measured after start of therapy were incorporated in the Cox models as time-dependent covariates.
| |
Results |
Between January 1994 and December 1998, 82 patients with advanced MM
were considered eligible for 1 or 2 courses of high-dose chemotherapy
supported by stem cell transplantation and their clinical
characteristics are reported in Table 1. On the basis of adequate stem
cell mobilization, 31 patients (37.8%) were submitted to positive
selection and transplantation of CD34+ cells, whereas the remaining 51 individuals were reinfused with unselected PBSC. Overall, the median
interval between diagnosis and transplantation was 14 months (range
4-78). All individuals had received 1 or more lines of treatment before
transplant and none of them was in CR at time of study. The median
duration of alkylating agents therapy was 9 months (0-18). Thirty-one
patients (37.8%) showed responsive disease at the time of
autotransplant (greater than 50% reduction of tumor mass), whereas 51 patients (62.1%) were enrolled in the trial with refractory or
progressive disease. Although this was a prospective nonrandomized
study, the 2 groups of patients (TX1 and TX2) were well balanced for
the clinical parameters considered (Table 1).
Positive selection of CD34+ cells, engraftment results, and
toxicity
A median of 2 aphereses (range 1-2) were performed. Similar to our
previous results,5 stem cell processing resulted in the
median recovery of 65% (range 10%-100%) of the initial content of
CD34+ cells with a purity of 89% (range 48%-98%). Clonogenic assays
demonstrated the recovery of 44.5% (range 6%-100%) of CFU-C. Overall, 31 patients were reinfused after MEL with a median of 4.6 × 106 purified CD34+ cells/kg (range 1.6-10.4)
and 12.1 × 104 CFU-C/kg (range 1-64) and showed a
rapid and sustained reconstitution of hematopoiesis. None of the
patients required reinfusion of unselected back-up cells. The median
time to an ANC greater than 0.5 × 109/L and to 20 and 50 × 109 platelet per liter was 11, 12, and 15 days, respectively. Transfusion requirement was minimal and the median
time to hospital discharge after reinfusion was only 13 days (range
11-28). When analyzed separately, no difference was found between
patients who received MEL as the only preparative regimen ( = 13) and
individuals for whom MEL represented the first of 2 sequential lines of
myeloablative chemotherapy ( = 18). Bu/Mel conditioning regimen was
administered at a median time of 4 months (range 3-15) from MEL. The
second course of high-dose chemotherapy (supported with a median of
5.5 × 106 enriched CD34+ cells/kg; range 2.8-11.9)
did not result in a delayed hematopoietic recovery, compared with TX1
(median time of 10.5 days to ANC greater than
0.5 × 109/L; range 9-12, and median time of 11 days
to 20 × 109 platelet/L; range 10-100). Hospital
discharge occurred at a median of 14 days (range 12-21) after transplant.
We then compared the engraftment of MM patients reinfused with selected
CD34+ cells with that of patients who received unmanipulated PBSC
containing a median of 3.7 (range 1.3-9.7; TX1) and 6.4 (range 2.5-13.2; TX2) × 106 CD34+ cells/kg. As reported in
Figure 1, the time to hematologic reconstitution was not statistically different in the 2 groups of
patients, both after the first and the second autograft. Long-term complete hematopoietic reconstitution as defined as ANC and platelet count greater than 2.5 and 100 × 109/L,
respectively, was documented in all patients.
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| Fig 1.
Kaplan-Meier plot of probability of recovery of
neutrophils to 0.5 × 109/L (A) and
recovery to an unsupported platelet count of
50 × 109/L (B) in patients undergoing 1 (TX1) or 2 (TX2) courses of high-dose chemotherapy with selected or
unselected CD34+ cells.
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Tandem transplant procedure either supported by CD34+ cells or by
unselected PBSC was well tolerated and severe extrahematologic toxicity
(more than grade 2 according to WHO scoring system) was mainly limited
to oral mucositis. There were 2 treatment-related deaths (4.2%)
(veno-occlusive disease-VOD = 1 and interstitial pneumonia = 1)
after Bu/Mel and reinfusion of PBSC. Other toxicities included
hemorrhagic cystitis (4 patients), pulmonary toxicity requiring steroid
therapy in 1 patient, and severe life-threatening VOD (data not shown).
One patient who was reinfused with purified CD34+ cells after
TBI-containing conditioning regimen (TX1) died in the peritransplant
period because of interstitial pneumonia.
Clinical outcome and molecular follow-up
PBSC collections and CD34+ cell fractions were analyzed for the
presence of myeloma cells by PCR reaction for CDRIII. Consistent with
our previous results,5 we found that all the leukaphereses and 22/31 of the selected cell products did contain residual clonal B
cells (data not shown).
Eight of 13 patients who received 1 course of myeloablative therapy and
CD34+ selected cells were responsive to transplantation (more than 50%
reduction of tumor burden). However, none of them achieved clinical and
serologic CR. Conversely, 5/18 (28%) patients with advanced disease
who underwent double autotransplant achieved CR. Only 2/5 patients were
reinfused with tumor-free autografts (Figure
2) and only 1 showed a transient molecular
remission. To date, 4 individuals are still in clinical and serologic
CR, despite detection of molecular MRD.
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| Fig 2.
Molecular follow-up of myeloma patients who achieved
serologic and clinical CR after TX1 or TX2.
Nos. 1 to 8 and 9 to 13 represented double autotransplants supported
with PBSC or CD34+ cells, respectively, whereas Nos. 14 and 15 were
patients who were submitted to a single course of high-dose therapy
with unmanipulated stem cells.
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Pooling together PBSC and CD34+ cells-supported transplants, CR and PR + CR rates for evaluative patients who were administered 1 or 2 lines
of high-dose chemotherapy, respectively, are 13.8%, 41%
(P = .04) and 44%, 71.8%. Figure
3 shows a Kaplan-Meier type plot for the
probability of achieving CR (A) and CR + PR (B) for TX1 and TX2.
Moreover, molecular CR was only observed in 3 MM patients submitted to
tandem autotransplantation (CD34+ = 1; unmanipulated PBSC = 2) and
2 of these individuals ( = PBSC) remain PCR-negative after 24 and 36 months from completion of the program (Figure 2).
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| Fig 3.
Kaplan-Meier plot of probability of achieving CR (A) and
CR + PR (B) for TX1 and TX2.
The time, in months, is calculated from the date of the (first)
transplant. CD 34+-selected transplants were pooled together with
unmanipulated autografts. Disease complete response is significantly
correlated with TX2 (see text). One single patient (TX2) reached CR
after 32 months from transplant after IFN treatment.
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Prognostic factors
On univariate analysis of 10 pretreatment variables (Table 1), 2 (-2 microglobulin level at diagnosis  4 mg/L and responsive disease at time of transplant) had significant (P < .05)
association with EFS and OS, whereas age, sex, disease stage, M
component isotype, C-reactive protein level, BM plasmacytosis, time
from diagnosis to transplant, and reinfusion of selected CD34+ cells did not have any correlation. Only -2 microglobulin level maintained independent significance on multivariate analysis for both EFS (relative risk = 0.5, P = .005) and OS (relative
risk = 0.6, P = .02). Moreover, to assess the potential
impact of response and treatment (ie, TX1 and TX2) on EFS and OS, a
time-dependent covariate analysis was conducted accounting for a second
cycle of high-dose therapy, time to high-dose therapy, CR, time to CR,
CR + PR, and time to CR + PR. A statistically significant association
was only found between EFS and any second transplant (relative
risk = 0.3, P = .02).
Figure 4 compares the probability of EFS
(A) and OS (B) for MM patients submitted to single or tandem autograft
according to landmark analysis. The median follow-up from transplant is 34 (range 5-84) and 28 (range 2-53) months for patients receiving TX1
or TX2, respectively, and their median EFS durations were 17 and 35 months (P = .03). Actuarial 3-year OS for patients treated with 1 or 2 transplants are 76% and 92%, respectively
(P = NS).
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| Fig 4.
Probability of EFS (A) and OS (B) for patients undergoing
1 or 2 courses of myeloablative therapy assessed according to landmark
analysis (see "Patients and methods").
Also in this case, selected and unselected transplants were pooled
together. Superior EFS is significantly associated with TX2.
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| |
Discussion |
Positive selection of CD34+ cells is widely applied to remove
CD34-cancer cells from autologous grafts. This is based on early results 5,7,9,19-22 showing the ability of selected cells to restore autologous hematopoiesis after myeloablative therapy as well
as untreated PBSC. However, whether purified stem cell can be used to
support multiple lines of high-dose therapy and whether extensive
purging of myeloma cells would represent a significant improvement in
the patient outcome has yet to be demonstrated. In this regard, Vescio
et al9 showed the same probability of progression-free
survival at 1 year of MM patients reinfused with selected and
unselected stem cells in a randomized study mainly focused on
engraftment capacity of enriched CD34+ cells.
In this paper, we report a single Center experience of 82 patients with
intermediate to advanced-stage MM who were enrolled from January 1994 to December 1998 in 2 sequential phase II trials to determine (1) the
feasibility of positive selection of CD34+ cells in pretreated myeloma
patients; (2) the indirect purging of tumor cells; and (3) the ability
of positively selected CD34+ cells to support the hematopoietic
reconstitution after 1 or 2 courses of myeloablative chemotherapy. Of
note, the choice of a more aggressive therapy ("tandem
transplant") from 1996 was mainly based on the promising results
reported by the University of Arkansas group23 and the lack
of plateau in the survival curve of MM patients submitted to a single
course of high-dose therapy. Thus, we reasoned that by coupling
effective ex vivo purging with the delivery of 2 lines of myeloablative
chemotherapy we could benefit myeloma patients with otherwise poor
prognosis. We set 4 or 8 × 106cells/kg (single or
double autotransplant, respectively) as the minimum number of CD34+
elements to be collected from PB to proceed to stem cell enrichment.
This was based on our and others previous experience5,7,9,24 showing delayed platelet engraftment after the reinfusion of less than 2 × 106 CD34+
cells/kg and prediction of approximately 50% loss of progenitor cells
during processing. Notably, only 40% of heavily pretreated myeloma
patients met the requirements for stem cell selection. Thus, autologous
transplantation and stem cell selection should be planned shortly after
diagnosis to avoid long-term exposure to alkylating agents that prevent
optimal mobilization of CD34+ cells.
In this paper, we confirmed that reinfusion of positively selected
CD34+ cells does not adversely affect the ability of hematopoietic stem
cells of restoring BM function after 1 course of myeloablative therapy.
Moreover, we demonstrated that purified CD34+ cells could be used to
support multiple cycles of high-dose chemotherapy ("tandem" transplantation) as well as unmanipulated PBSC. Despite maintenance treatment with -IFN, we did not observe any late engraftment failure
or delayed infections in these patients. The absence of late viral or
fungal infections is important as extensive removal of T and B cells
from autografts may increase the risk of long-lasting immunodeficiency
in MM patients undergoing 2 or more courses of high-dose chemotherapy.
In this regard, the randomized phase III trial9 did not
show any difference between patients reinfused with selected CD34+
cells or unselected PBSC as for the number and the type of infections
within the first 3 months from transplantation. Furthermore, TBI- or
cyclophosphamide-containing preparative regimens25 may be
more immunosuppressive than MEL and Bu/Mel used in our study.
As previously reported,5 PCR-based monitoring of MRD
allowed the detection of myeloma cells in the majority of CD34+ cell products. This finding may be due to either CD34 tumor cells contaminating the CD34+ cell population or to CD34+CD19+ myeloma elements.26 Thus, strategies to achieve tumor-free
autografts have recently been developed by purification of
CD34+Thy1+Lin cells27 or by positive/negative
selection of CD34+ B-lineage negative progenitors.14
However, when CD34 selected autotransplants were analyzed as
part of a multivariate analysis for EFS and OS, we did not find any
clinical advantage in favor of tumor cell purging. This was a
prospective nonrandomized study and may not be sufficiently powered to
assess small but still clinically significant differences. However,
despite the consistent involvement of PBSC collections from large
numbers of malignant cells in myeloma patients with advanced disease,
the high cost of stem cell selection, the relatively low percentage of
patients who could be submitted to this procedure, and the potential of
delayed platelet engraftment in case of reinfusion of less than
2 × 106 CD34+ cells/kg suggest that positive
selection of CD34+ cells should not be routinely performed until an
update of the phase III trial, comparing selected versus unselected
cells, will conclusively assess the role of tumor cell purging in MM.
Noteworthy, tandem autotransplant (regardless of whether enriched stem
cell were reinfused) resulted in a CR rate of 41% which was
significantly better than that of a single course of myeloablative therapy. Moreover, molecular follow-up demonstrated that 3 patients achieved at some point PCR negativity and 2 of them remain in molecular
and clinical CR after 2 and 3 years from transplantation. These results
extend early observations from Corradini et al28 and
Bjorkstrand et al29 who reported few cases of molecular CR
in patients undergoing double but not single
autotransplant.28 Although a longer follow-up and a larger
number of patients are needed to determine the role of molecular
evaluation in predicting relapse in myeloma, in other B-cell
malignancies, PCR negativity has been correlated with a better clinical
outcome.30
The impact of the second transplant and other prognostic features on
EFS and OS of patients with intermediate and advanced stage MM was also
evaluated. On multivariate analysis, TX2 and 2 microglobulin level
at diagnosis were associated with superior EFS. Again, this was not a
randomized study and the results should be taken with caution. However,
these data may confirm previous results indicating the clinical benefit
of multiple cycles of high-dose therapy with stem cell support for
pretreated MM patients.23 In this respect,
Palumbo et al31 have recently demonstrated the superiority
of 2 to 3 cycles of submyeloablative doses of melphalan in comparison
with standard chemotherapy in elderly myeloma patients. The issue of 1 versus 2 autotransplants for MM patients at diagnosis is currently
being addressed in 2 randomized studies.32,33
In conclusion, whereas the clinical impact of tumor cell removal from
autografts remains highly questionable at this stage, PBSC-supported
MEL-based tandem transplant may be an attractive alternative for
myeloma patients with symptomatic disease.
| |
Footnotes |
Submitted July 29, 1999; accepted December 1, 1999.
Supported by the Italian Association for the Research Against Cancer
(AIRC), Milan, Italy; MURST; Project 60% and 40%; and University of
Bologna (Funds for selected research topics).
Reprints: Roberto M. Lemoli, Institute of Hematology and
Medical Oncology "Seragnoli," Via Massarenti, 9-40100 Bologna, Italy; e-mail: rmlemoli{at}med.unibo.it
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Attal M, Harousseau JL, Stoppa AM, et al.
A prospective randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma.
N Eng J Med.
1996;335:91[Abstract/Free Full Text].
2.
Brenner MK, Rill DR, Moen RC, et al.
Gene-marking to trace the origin of relapse after autologous bone-marrow transplantation.
Lancet.
1993;341:85[Medline]
[Order article via Infotrieve].
3.
Deisseroth AB, Zu Z, Claxton D, et al.
Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML.
Blood.
1994;83:3068[Abstract/Free Full Text].
4.
Corradini P, Voena C, Astolfi M, et al.
High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood harvests and after autografting.
Blood.
1995;85:1596[Abstract/Free Full Text].
5.
Lemoli RM, Fortuna A, Motta MR, et al.
Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells.
Blood.
1996;87:1625[Abstract/Free Full Text].
6.
Gazitt Y, Tian E, Barlogie B, et al.
Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma following treatment with cyclophosphamide and GM-CSF.
Blood.
1996;87:805[Abstract/Free Full Text].
7.
Schiller GJ, Vescio RA, Freytes C, et al.
Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma.
Blood.
1995;86:390[Abstract/Free Full Text].
8.
Gertz MA, Witzig TE, Pineda AA, Greipp P, Kyle RA, Litzow MR.
Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation.
Bone Marrow Transplant.
1997;337:19.
9.
Vescio R, Schiller G, Stewart AK, et al.
Multicenter Phase III trial to evaluate CD34+ selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma.
Blood.
1999;93:1858[Abstract/Free Full Text].
10.
Gribben JG, Freedman AS, Neuberg D, et al.
Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma.
N Eng J Med.
1991;325:1525[Abstract].
11.
Laporte JP, Douay L, Lopez M, et al.
One hundred twenty-five adult patients with primary acute leukemia autografted with marrow purged by mafosfamide: a 10-year single institution experience.
Blood.
1994;84:3810[Abstract/Free Full Text].
12. Chronic Leukemia-Myeloma Task Force, National Cancer Institute.
Proposed guidelines for protocol studies-II. Plasma cell myeloma.
Cancer Chemother Rep. 1973;4(part 3):724.
13.
Durie BGM, Salmon SE.
A clinical staging system for multiple myeloma: correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival.
Cancer.
1975;36:842[Medline]
[Order article via Infotrieve].
14.
Lemoli RM, Martinelli G, Olivieri A, et al.
Selection and transplantation of autologous CD34+B-lineage negative cells in advance phase multiple myeloma patients: a pilot study.
Br J Haematol.
1999;107:419-428[Medline]
[Order article via Infotrieve].
15.
Martinelli G, Terragna C, Lemoli RM, et al.
Clinical and molecular follow up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of hematopoietic CD34+ stem cells.
Haematologica.
1999;84:397[Abstract/Free Full Text].
16.
Mantel N.
Evaluation of survival data and two new rank order statistics arising in its consideration.
Cancer Chemother Rep.
1966;50:163[Medline]
[Order article via Infotrieve].
17.
Anderson JR, Cain KC, Gebler RD.
Analysis of survival by tumor response.
J Clin Oncol.
1983;1:710[Abstract].
18.
Cox DR.
Regression models and life-tables (with discussion).
J Royal Stat Soc, Series B.
1972;34:187.
19.
Shpall EJ, Jones RB, Bearman SI, et al.
Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral blood progenitors and growth factors on engraftment.
J Clin Oncol.
1994;12:28[Abstract].
20.
Berenson RJ, Bensinger WI, Hill RS, et al.
Engraftment after infusion of CD34+ marrow cells in patients with breast cancer and neuroblastoma.
Blood.
1991;77:1717[Abstract/Free Full Text].
21.
Brugger W, Henschler R, Heimfeld S, Berenson RJ, Mertelsmann R, Kanz L.
Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP 16, ifosfamide, carboplatin and epirubicin.
Blood.
1994;84:1421[Abstract/Free Full Text].
22.
Gorin NC, Lopez M, Laporte JP, et al.
Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma.
Blood.
1995;85:1647[Abstract/Free Full Text].
23.
Vesole DH, Barlogie B, Jagannath S, et al.
High-dose therapy for refractory multiple myeloma: improved prognosis with better supportive care and double transplants.
Blood.
1994;84:950[Abstract/Free Full Text].
24.
Schiller G, Vescio R, Freytes C, et al.
Autologous CD34-selected blood progenitor cell transplants for patients with advanced multiple myeloma.
Bone Marrow Transplant.
1998;21:141[Medline]
[Order article via Infotrieve].
25.
Rossi JF, Legouffe E, Feguex N, et al.
Autologous transplantation (AT) of CD34+ peripheral blood progenitor cells (PBPC) after double (D) high dose chemotherapy (HDC) in multiple myeloma (MM) is followed by severe immunodeficiency (ID) and high production of interleukin-6 (IL-6) related-C reactive protein (CRP) requiring additive immunotherapy [abstract].
Blood.
1996;88(suppl 1):142a.
26.
Szczepek AJ, Bergsagel PL, Axelsson L, Brown CB, Belch AR, Pilarski LM.
CD34+ cells in the blood of patients with multiple myeloma express CD19 and IgH mRNA and have patient-specific IgH VDJ gene rearrangements.
Blood.
1997;89:1824[Abstract/Free Full Text].
27.
Tricot G, Gazzit Y, Leemhuis T, et al.
Collection, tumor contamination and engraftment kinetics of highly purified hematopoietic progenitor cells to support high-dose therapy in multiple myeloma.
Blood.
1998;91:4489[Abstract/Free Full Text].
28.
Corradini P, Voena C, Tarella C, et al.
Molecular and clinical remissions in multiple myeloma: role of autologous and allogeneic transplantation of hematopoietic cells.
J Clin Oncol.
1999;17:208[Abstract/Free Full Text].
29.
Bjorkstrand B, Ljungman P, Bird JM, Samson D, Garthon G.
Double high-dose chemotherapy with autologous stem cell transplantation can induce molecular remissions in multiple myeloma.
Bone Marrow Transplant.
1995;15:367[Medline]
[Order article via Infotrieve].
30.
Gribben JG, Neuberg D, Freedman AS, et al.
Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increase risk of relapse after autologous bone marrow transplantation for B-cell lymphoma.
Blood.
1993;81:3449[Abstract/Free Full Text].
31.
Palumbo A, Triolo S, Argentino C, et al.
Dose-intensive melphalan with stem cell support (MEL 100) is superior to standard treatment in elderly myeloma patients.
Blood.
1999;94:1248[Abstract/Free Full Text].
32.
Attal M, Payen C, Facon T, et al.
Single versus double transplant in myeloma: a randomized trial of the "InterGroup Francais du Myeloma" (IFM) [abstract].
Blood.
1997;90:418a.
33.
Tosi P, Cavo M, Zamagni E, Ronconi S, Benni M, Tura S.
A multicentric randomized clinical trial comparing single vs double autologous peripheral blood stem cell transplantation for patients with newly diagnosed multiple myeloma: results of an interim analysis. [abstract]
Blood.
1999;94:715a.
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Abstract
Introduction