ARTICLE
Auteur(s) :, Nicolas
Mounier*, Jérome Larghero, Julien Manson, Pauline
Brice, Isabelle Madelaine-Chambrin, Josette Brière, Marjane
Ertault, Christophe Hennequin, Jean-Michel Miclea, Marc Benbunan,
Jean-Pierre Marolleau, Christian Gisselbrecht
Institut universitaire d’hématologie, Hôpital Saint-Louis,
Inserm ERM 0220, 1, avenue Claude-Vellefaux, 75010 Paris,
France
After fifteen years of clinical trial research, autologous
stem-cell transplantation (ASCT) is now widely used for lymphoma as
consolidation in chemosensitive patients either in frontline or in
relapse [1-5]. Today, with ASCT, bone marrow transplantation has
been replaced by peripheral blood stem cells (PBSC) especially
because the latter lead to quicker engraftment, with reduced
morbidity and duration of hospital stay [6-9]. The use of
granulocyte-colony stimulating factor (G-CSF), given after PBSC
infusion, improves neutrophil recovery. Both the rapidity and
stability of engraftment correlate with the number of progenitor
cells in the autograft. Granulocyte/macrophage-colony forming unit
(GM-CFU) and CD34+ cells are the main variables used to evaluate
the number of progenitors upon which the decision to perform ASCT
is based [10-13]. These cell counts are usually quantified on fresh
PBSC leukapheresis components before cryopreservation. There is a
strong positive correlation between the number of CD34+ cells in
the graft and the quality of the engraftment [14]. A number of
CD34+ cells below 2.106/kg has been shown to identify
patients at high risk of slow engraftment and an optimal number
around 5.106/kg has been proposed to accelerate
engraftment 15,16. To date, more than 90 percent of PBSC
transplant patients have been engrafted without delay, but some
patients failed to achieve complete hematologic recovery after one
year, suggesting that the low morbidity incidence of the ASCT
procedure could still be improved. Recently, Nieboer et al. [17]
found that reinfusing more CD34+ cells can also accelerate
long-term hematologic recovery.As PBSC transplantations are
performed with cells cryopreserved in liquid nitrogen, the number
of post-thaw CD34+ cells has been proposed as a useful check on the
quality of the reinfused components and a potential prognostic
factor of engraftment [18]. However, the respective impacts of
prefreeze and post-thaw numbers of CD34+ cells on long term
hematologic recovery have not been studied.This prompted us to
perform a cohort study of lymphoma patients treated by ASCT with
PBSC, to evaluate the occurrence of incomplete long term
hematologic recovery and assess its prognostic factors.
Patients and methods
Patient selection
Using the database of the Société Francaise de Greffe de Moelle,
the patients transplanted in our department were identified.
Non-lymphoma patients were excluded from the present study. Files
were reviewed and data on hematologic recovery were collected by
means of a standard form. From November 1, 1995 to November 1,
2000, a total of 173 consecutive lymphoma patients achieved either
complete remission or unconfirmed complete remission and were
consolidated by high-dose chemotherapy and ASCT with unpurged PBSC.
We only included in the present analysis the 133 patients who were
alive without relapse or treatment one year after ASCT. Of these
133 patients, we report here the results obtained for 106 for whom
hematologic data were available. The remaining 27 patients shared
the same characteristics and successfully engrafted but they were
not included in the present analysis because they had missing
values in their blood counts (i.e. platelets or leucocytes
subpopulations) 100 days or one year after ASCT.
Stem cell collection
PBSC were collected from 96 patients by leukapheresis during the
hematological recovery phase after the last inductive cycle and
from the remaining ten after an additional mobilization regimen
followed by daily subcutaneous administration of 5 μg per kg G-CSF.
At the time of hematologic recovery, peripheral blood CD34+ cell
counts were determined daily and leukapheresis was started when
counts exceeded 10/μL. Leukapheresis procedures were continued for
2 to 4 consecutive days, depending on the number of CD34+ cells
harvested. The targeted number of CD34+ cells was
3.106/kg, but, the minimum required was fixed at
2.106/kg.
Cryopreservation and thawing procedures
Excess plasma was removed from PBSC components by centrifugation in
600-mL blood transfer bags at 650 g for 10 minutes. The volume of
the residual cell pellet was adjusted with human serum albumin
between 1995 and 1998 and with Elohes® (Fresenius Kabi,
France) between 1998 and 2000. Precooled freezing solution was
added slowly to the cell components with continual mixing to
achieve a final concentration of 10 % DMSO. Cell
concentrations before freezing were less than 3.108 per
mL. Cells were cooled at 1°C per minute to -40 °C with compensation
for heat of superfusion, and then at 10°C per minute to -100°C,
using a rate-controlled freezer (Nicool, Air Liquide, France).
Frozen cells were stored in liquid nitrogen until reinfusion.
Before reinfusion, HPC bags were thawed in a 37°C water bath,
washed twice in a glucose solution (B-Braun, France) and finally
suspended in albumin. The cell suspension was then rapidly infused
into the patient through a central venous catheter at about
20 mL per minute.
Transplantation modalities
Patients were eligible for ASCT if they reached complete remission
after induction treatment as appropriate for their lymphoma type,
and after PBSC had been collected. All patients undergoing ASCT
were required to have adequate pulmonary, liver and renal
functions. All patients had an indwelling central venous catheter
and were housed in laminar air-flow rooms for the duration of
aplasia. All received the entire collected dose of CD34+ cells and
were given G-CSF (5 μg/kg/day), starting one day after stem
cell reinfusion, and antiviral prophylaxis. None had erythropoietin
support. Prevention of veno-occlusive disease with heparin
(1 mg/kg) was used for patients receiving total body
irradiation conditioning regimen (TBI). Patients who developed
fever and whose absolute granulocyte count was less than
0.5 G/L were treated with intravenous broad spectrum
antibiotics. All patients received irradiated packed red blood
cells and platelet products to maintain a hemoglobin level >
8 g/dL and a platelet count > 20 G/L.
CD34+ cell evaluation
Automated cell counts and CD34+ cell quantification were performed
both before and after thawing. Quantification was performed
following dual-platform Ishage guidelines [19] with total white
blood cell count obtained on an automated hematology analyzer
(Sysmex, Roche). For cell labeling, at the first and last steps in
the procedure, one million PBSC were incubated for 10 minutes
at room temperature with 20 μL anti-CD34-phycoerythrin and
anti-CD45-fluorescein isothiocyanate monoclonal antibodies (Becton
Dickinson, France). CD34+ cell quantification was performed on
viable whole population stained with propidium iodide. This
dual-platform strategy has been used for all patients included in
this study. We assume that a single-platform gating strategy
eliminates the need for white blood cell count on an automated
analyzer and could potentially increases the analytical precision
of the methodology. However, these different techniques, as well as
the microvolume fluorimetry method for CD34+ cell enumeration, have
been shown to give equivalent results [20]. Immunofluorescence
analysis was performed using a 5-parameter FACSscan (Becton
Dickinson Immunocytometry Systems, San Jose, CA). The total number
of CD34+ cells obtained at the end of this analysis corresponded to
the number infused into the patient.
Evaluation of GM-CFU
Cells were seeded at 1.104 per plate in small Petri
dishes (diameter 35 mm), and cultured in a volume of 1 ml
standard methylcellulose complete medium containing 0.9 %
methylcellulose, 30 % FCS, 1 percent BSA, 100 μM
2-mercaptoethanol, 2 mMl-glutamine, and 10 %
WBC-conditioned agar medium (Methocult H4431, StemCell
Technologies, Vancouver, Canada). All cultures were plated in
triplicate, incubated at 37°C in humidified air with 5-percent
CO2 and scored on day 14, using an inverted microscope.
Colonies composed of more than 50 nonerythroid cells were scored as
GM-CFU.
Statistical analysis
The main end points of the study were the occurrence of
engraftment, defined by the end of aplasia with granulocyte >
0.5.109/l, and the long term hematologic recovery,
defined by a normal blood count with a hemoglobin level
(13 g/dl for males, 12 g/dl for females) leukocytes >
4.109/l and platelets > 150.109/l. These
end points were calculated as a cumulative probability, and the
effects of potential risk factors were examined using the Cox
proportional-hazards model [21]. The thresholds for the continuous
biologic variables were checked by including cubic smoothing
splines in the risk function of the Cox model 21.
Demographic and baseline laboratory data were compared among
subpopulations by Fisher’s exact test for nominal variables and
Wilcoxon’s rank test for continuous variables. The independence of
prefreeze and post-thaw variables was explored by the correlation
coefficient test. Differences between the results of comparative
tests were considered significant if the two-sided P value was less
than 0.05.
All statistical analyses were performed using SAS 8.2 software
(SAS Institute, Cary, NC) and Splus 2000 software (MathSoft,
Cambridge, MA).
Results
Patient characteristics
The study population of 106 patients comprised 59 males and 47
females. Their median age was 43 years (range 17 to 66). The
cohort included 94 (89 %) cases of non-hodgkin lymphoma and 12
(11 %) of Hodgkin lymphoma. Main clinical characteristics are
shown in table 1( Table 1 ). The study
population of 106 patients included 82 (78 %) with
disseminated disease (stage 3-4). Fifty seven patients (54 %)
received frontline (i.e. in first CR) ASCT, 42 were in first
relapse (i.e second CR) and 7 in second relapse. At relapse, 85
(80 %) had stage 3-4 disease with extranodal localizations in
58 (55%). Treatment characteristics are given in table 2(
Table 2 ). The median number of
chemotherapy cycles (i.e. number of individual course) was 7 (range
3 to 23). Twelve patients received oral route chemotherapy and 16
localized radiation therapy. At time of leukapheresis, none of the
patients had marrow involvement. Graft characteristics are given in
the bottom section of table 2. We confirm the significant
correlation between the numbers of GM-CFU and prefreeze CD34+ cells
(r = 0.81, p < 0.001) however it was lower with post-thaw CD34+
cells (r = 0.66, p < 0.001). There was no significant
correlation with the post-thaw GM-CFU. As shown in ( figure 1 ), we observed a
significant correlation between the numbers of prefreeze and
post-thaw CD34+ cells (r = 0.77, p < 0.001). However, the
correlation is worse in the values below 10.106/kg than
in the values above suggesting that this correlation should be
further investigated by multivariate analysis.
Table 1 Patient characteristics at diagnosis of
lymphoma
|
Characteristics
|
N = 106
|
%
|
|
Non Hodgkin lymphoma
|
94
|
89
|
|
DBLC
|
42
|
41
|
|
Follicular
|
25
|
23
|
|
Lymphocytic
|
13
|
12
|
|
Non anaplastic T
|
6
|
6
|
|
Mantle Cells
|
5
|
5
|
|
Lymphoblastic
|
3
|
2
|
|
Hodgkin lymphoma
|
12
|
11
|
|
Stage III-IV
|
82
|
78
|
|
Performance status 2-4
|
6
|
5
|
|
Extranodal involvement > 1 site
|
24
|
22
|
|
Bone-marrow involvement
|
46
|
43
|
Table 2 Patient characteristics at PBSC grafting
|
Characteristics
|
N = 106
|
%
|
|
Transplantation
|
|
|
|
First line
|
57
|
54
|
|
Relapse
|
49
|
46
|
|
Number of chemotherapy regimens
|
|
|
|
1
|
47
|
44
|
|
2
|
37
|
35
|
|
3
|
16
|
15
|
|
4-6
|
6
|
6
|
|
Conditioning regimen
|
|
|
|
Including TBI
|
36
|
34
|
|
BEAM
|
58
|
55
|
|
CBV
|
9
|
8
|
|
PBSC graft
|
Median
|
Range
|
|
Prefreeze CD34+ (106/kg)
|
6.1
|
1.9-55.2
|
|
Post-thaw CD34+ (106/kg)
|
5.6
|
1.2-55.2
|
|
Prefreeze TNC (108/kg)
|
30.1
|
5.0-195.3
|
|
Post-thaw TNC (108/kg)
|
16.5
|
3.5-77.9
|
|
Prefreeze GM-CFU (104/kg)
|
32.3
|
2.1-784.3
|
|
Post-thaw GM-CFU (104/kg)
|
6.6
|
1.1-166.5
|
Engraftment
Four parameters were assessed by complete blood count each day:
platelets > 20 G/L, platelets > 50 G/L, granulocytes
> 0.5 G/L and white cells > 1 G/L for at least 48 hours.
The median numbers of days required to achieve a granulocyte count
above 0.5 G/L and a platelet count above 20 G/L were
respectively 10 days (range 7-18) and 11 days (range 5- 37). The
median numbers of days required to achieve a white cell count above
1 G/L and a platelet count above 50 G/L were respectively 10
days (range 7-18) and 14 days (range 7- 58).
Granulocyte recovery was not significantly affected by age, sex,
the extension of the disease, the number of disease phases or the
type of conditioning regimen. However, large numbers of CD34+ cells
(both at prefreeze and post-thaw) were predictive of fast
granulocyte recovery. For CD34+ cell numbers, the smoothing spline
curve (( figure
2 )) which estimates the functional form of its prognostic
value, show how the relative risk (RR) of granulocyte recovery
increase as the CD34+ cell numbers increase. This curve has a
monotonic pattern which enabled us to determine a single cut-off
point in order to categorize the covariate. In ( figure 2 ), for the
prefreeze number of CD34+ cells, the best point was estimated at
5.106/kg because the curve break the zero line at
5.106/kg. It indicated that patients with more than
5.106/kg have a time to granulocyte engraftment (above
0.5 G/L) equal or below the median of the study population (10
days). Using the same approach, the cut-off point was also set at
5.106/kg for the post-thaw numbers of CD34+ cells. In
addition, neither the number of Total Nucleated cells (TNC) or of
GM-CFU was significant.
The results for platelet recovery were identical: CD34+ cell
numbers (both at prefreeze and postthaw) were the only two
variables significantly correlated to platelet recovery.
In multivariate analysis, the number of prefreeze CD34+ cells
was the only variable significantly correlated to granulocyte and
platelet recovery. No other clinical or graft parameters were
significantly predictive of the rapid engraftment (i.e. age, sex,
the extension of the disease, the number of disease phases or the
type of conditioning regimen).
Long term hematologic recovery
Three parameters (platelet count, white cell count and hemoglobin
level) were studied 100 days and one year after ASCT. Long
term hematopoietic recovery could not be evaluated in 8 patients
because of incomplete blood counts.
table 3( Table 3 ) shows the results
for these 3 parameters. After one year, the hemoglobin level was
normal (13 g/dl for males, 12 g/dl for females) in 46 (47%)
patients, the leukocyte count (4.109/l) in 75
(77 %) and the platelets count (150 109/l) in 59
(60 %). Thirty-two patients (33 %) had a normal blood
count, 31 (32 %) had 2 hematologic lineages completely
reconstituted, 22 patients (22 %), 1 lineage and 13
(13 %) none. The results of the univariate analyses according
to the main hematologic characteristics are given in table 4( Table 4 ). Age ≥ 50, marrow involvement and
prefreeze CD34+ cell number also had a significant adverse effect
on the probability of achieving a normal blood count. Examination
of ( figure 3 )
demonstrate the same effect for CD34+ cell numbers with a best
cut-off point estimated at 5.106/kg. The probability of
achieving a normal blood count is still raising for values above
5.106/kg but its gain is much lower between 5 and
10.106/kg than between 2 and 5.106/kg. In the
multivariate analysis, the only independent predictive factor for a
normal blood count after one year was a prefreeze CD34+ cell number
above 5.106/kg (RR = 3.06 [1.44–6.48], p < 0.003).
The RR for a post-thaw number of CD34+ cells above
5.106/kg was 2.54 [0.87-7.36] (p =0.08). This could be
explained by confounding variables such as the higher correlation
observed between GM-CFU and prefreeze CD34+ cells versus post-thaw
CD34+ cells. These findings were the same in a subset analysis for
patients with higher initial number of venous CD34+ cells collected
only once, suggesting that an optimal long-term hematologic
recovery after ASCT required a number of prefreeze CD34+ cells of
at least 5.106/kg.
In summary, both for the engraftment and long term hematologic
recovery, the number of prefreeze CD34+ cells was the only
significant variable in multivariate analysis. Moreover, the impact
of long term hematologic recovery seems to benefit on survival with
a trend in favor of patients who achieve a normal blood count at
one year (2-yr OS94 versus 79 %, p = 0.06).
Table 3 Long term hematologic recovery of 98 lymphoma
patients after ASCT
|
100 days post-ASCT
|
1 year post-ASCT
|
|
Hemoglobin (g/dL, median, [range])
|
11.8 [8.7-14.9]
|
12.9 [7.7-15.8]
|
|
White cells (G/L, median, [range])
|
4.3 [1.2-11]
|
5.0 [2.0-13.5]
|
|
Granulocyte (G/L)
|
2.7 [0.7-6.1]
|
3.0 [1.2-10.3]
|
|
Lymphocyte (G/L)
|
1.1 [0.1-3.3]
|
1.4 [0.5-4.3]
|
|
Platelets (G/L)
|
161 [26-610]
|
177 [35-410]
|
|
Return to normal value (%)
|
|
|
|
Hemoglobin level
|
22
|
47
|
|
White cell count*
|
58
|
77
|
|
Platelet count
|
57
|
60
|
|
3 lineages
|
12
|
33
|
|
2 lineages
|
30
|
32
|
|
1 lineage
|
35
|
22
|
|
0 lineage
|
19
|
13
|
Table 4 Univariate analysis of hematologic recovery one
year after ASCT according to the main hematologic characteristics
of 98 lymphoma patients
|
Characteristics
|
0-1 lineage
|
2-3 lineages
|
p-value
|
|
n = 35
|
n = 63
|
|
|
Age (median, [range])
|
51 [20-66]
|
40 [17-59]
|
0.001
|
|
Stage III-IV (%)
|
74
|
87
|
0.13
|
|
Extranodal involvement >1 site (%)
|
23
|
27
|
0.65
|
|
Marrow involvement (%)
|
79
|
21
|
0.03
|
|
TBI containing regimen (%)
|
37
|
30
|
0.48
|
|
Disease phases (median, [range])
|
2 [1–3]
|
1 [1–3]
|
0.40
|
|
Chemotherapy regimens (median, [range])
|
2 [1–3]
|
1 [1–6]
|
0.05
|
|
Prefreeze CD34+ (106/kg, median, [range])
|
4.75 [1.9-25.4]
|
6.53 [2.6-43.4]
|
0.03
|
|
Post-thaw CD34+ (106/kg median, [range])
|
4.90 [1.14-24.2]
|
5.63 [2.1-55.3]
|
0.13
|
Discussion
To ensure the good quality and safety of the final cell component
of the PBSC graft, international guidelines and regulations have
been issued [22]. The wish to provide a quality-assurance program,
including the principles of good manufacturing practice, which
applied to all phases of cell collection, processing and
reinfusion, led us to evaluate the long term hematologic recovery
and assess its prognostic factors.
To limit bias, we only included in the present analysis patients
who were given unpurged PBSC and G-CSF. No patients required
multiple mobilizations. As TNC and GM-CFU have been reported to
have a poor correlation with engraftment kinetics [23], a special
attention was paid to prefreeze and post-thaw CD34+ cell numbers
and to patient clinical characteristics. In the present study, we
confirmed previous reports showing that after ASCT with PBSC,
granulocyte and platelet recovery are significantly affected by the
number of prefreeze CD34+ cells [14, 24, 25]. As the quantity of
reinjected CD34+ cells is the best and even the only predictive
factor of engraftment, we confirmed the general feeling among
physician that the optimal number is 5.106/kg which
allows faster engraftment and fewer platelet transfusions [15,
16].
Few studies have been published on the long term hematologic
recovery. In the present study, we found that one year after ASCT,
only 33 % of patients had a normal complete blood count, and
that the hemoglobin level was only normal in 47 %. Theses
findings are the same in the recent paper by Nieboer et al., where
the autograft was not washed and no G-CSF was given, suggesting a
limited bone-marrow reserve after ASCT [17]. Most of authors agree
that the best predictive factor of a normal hematologic
reconstitution is the quantity of reinfused CD34+ cells. In
particular, patients who received more than 5.106
CD34+/kg had a better blood count after the engraftment [26, 27].
Like Rossi et al., we found that age and initial marrow involvement
had an effect on this count [28], but we confirmed, in multivariate
analysis that the only independent predictive factor for a normal
count was a prefreeze number of CD34+ cells above
5.106/kg.
However, it is still not clear whether CD34+ cells in the PBSC
should be quantified before or after cryopreservation. If done
after cryopreservation, quantification would include the effects of
freezing and thawing and therefore should better reflect the
composition of the graft. Thus, a recent study by Feugier et al.
showed that the post-thaw CD34+ cell number is an independent
prognostic factor of the engraftment and could be used to check the
quality of the freezing and thawing procedures in PBSC
transplantation [18]. In the present study, we observed a
significant correlation between prefreeze and post-thaw CD34+ cell
numbers, but only the prefreeze number retained prognostic value,
both for engraftment and long term hematologic recovery. To explain
our findings, there are very few studies concerning the effects of
post-thaw parameters on hematologic recovery. Although no standard
technique for PBSC freezing is accepted by all centers, the
differences between freezing procedures are generally minor. The
problem is that post-thaw manipulations, which remove not only the
DMSO but also damaged cells and residual reagents from any
prefreeze treatment, may substantially reduce the number of viable
infused cells [29]. In this short serie (n = 37), as in the present
study, the autograft was washed but patients did not receive G-CSF.
One of the most interesting hypotheses in this connection concerns
the presence of apoptotic cells within the graft [30]. The
leukapheres, cycles of freezing/thawing and the various stages of
PBSC transformation might indeed degrade the cells without killing
them. The method of quantifying living CD34+cells does not take
account of apoptosis. Reinfused dead cells will obviously not be
able to multiply and the same applies to apoptotic cells. De Boer
et al have pooled the aliquots of each leukapheresis to count CD34+
cells and they found a large proportion of apoptotic cells in
post-thaw CD34+ cells [30]. This might be a reason why, in our
study, the prefreeze CD34+ cells proved to be a better prognostic
factor of engraftment and long term hematologic recovery than the
number of post-thaw CD34+ cells.
Conclusion
One year after ASCT with PBSC, only one third of our lymphoma
patients achieved a normal blood count. At present, the number of
prefreeze CD34+ cells seems to be the best prognostic factor for
long term hematologic recovery. Pending the development of new
tools assessing post-thaw graft cell apoptosis, an optimal long
term hematologic recovery after ASCT seems to require a number of
prefreeze CD34+ cells of at least 5.106/kg.
Acknowledgments
The authors thank Mathilde Dreyfus for editing the English and
Sylvie Corre for secretarial assistance. They also thank the two
referees for their helpful comments and suggestions.
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