Effect of Total Nucleated and CD34
þ
Cell Dose on Outcome
after Allogeneic Hematopoietic Stem Cell Transplantation
Mats Remberger
1
,2
,*
, Johan Törlén
2
, Olle Ringdén
2
, Mats Engström
1
,2
, Emma Watz
1
,3
,
Michael Uhlin
1
,2
, Jonas Mattsson
1
,2
1Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden
2Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Stockholm, Sweden
3Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden
Article history: Received 4 November 2014 Accepted 26 January 2015 Key Words: Cell dose CD34
Hematopoietic stem cell transplantation Graft-versus-host disease Bone marrow
Peripheral blood stem cells
a b s t r a c t
During more recent years only few studies have analyzed the effect of total nucleated cell (TNC) and CD34þ cell dose in allogeneic hematopoietic stem cell transplantation (HSCT). A single-center analysis included 544 patients, 227 with a sibling donor and 317 with an unrelated donor. Most patients (n¼292) were treated with myeloablative conditioning, whereas the remaining patients (n¼ 252) received reduced-intensity conditioning. Bone marrow (BM) (n¼121) and peripheral blood stem cell (PBSC) grafts (n¼423) were analyzed separately. Median TNC and CD34þcell dose was 3.2108/kg versus 11.6108/kg in BM and 3.9 106/kg versus 8.1106/kg in PBSC. In the BM group we found a higher TNC and CD34þ cell dose was associated with a faster neutrophil engraftment (P<.001 andP¼.02). In the PBSC group we found patients given a very high (11106/kg) CD34þcell dose had decreased rates of survival (P¼.001) and increased relapse (P¼.02). A high CD34þcell dose correlated with faster platelet engraftment (P<.01). In HSCT using PBSCs, the CD34þcell doses should be kept below 11106/kg but over 2.5106/kg.
Ó2015 American Society for Blood and Marrow Transplantation.
INTRODUCTION
Hematopoietic stem cell transplantation (HSCT) is a
potentially curative treatment for several hematological
malignancies, bone marrow (BM) failure syndromes, and
some inherited metabolic disorders
[1]
. An adequate number
of nucleated or CD34
þcells in the graft are of utmost
importance to achieve sustained engraftment and good
survival
[2]
. Historically, the approach of more is better has
been applied at many centers. This is 1 contributory rationale
for the increasing use of peripheral blood stem cells (PBSCs)
in HSCT, becasue a PBSC graft contains approximately 5 to 10
times more CD34
þand T cells compared with a BM graft
[3]
.
Higher cell numbers in the graft decrease the risk of rejection
and shorten the phase of aplasia
[4-10]
. However, many
studies have shown the risk for chronic graft-versus-host
disease (GVHD) is increased in patients receiving PBSC
compared with BM, probably because of the higher cell
content
[11-13]
. More recently, only a few studies have
evaluated the effect of cell dose on outcome after HSCT
[4,14-16]
. Hence, it is important to establish which cell dose
is optimal for outcome when using BM or PBSC in the
modern era of HSCT.
METHODS Patients
The effect of the graft cell dose was studied in 544 consecutive HSCT pa-tients transplanted between January 2000 and December 2011 at Karolinska University Hospital, Sweden. Only patients with a malignant disease receiving either BM or PBSCs from a HLA-identical sibling or a HLA-A, -B, and -DR matched unrelated donor were included. Patients transplanted because of solid
tumors (n¼62) were excluded because this was an experimental treatment.
Patient characteristics are displayed inTable 1. Patients receiving a BM
or PBSC graft were analyzed separately because the cell dose differs considerably between these 2 types of grafts and BM is preferentially given to children. The study was approved by the Research Ethics Committee of Karolinska Institutet. The procedures were in accordance with the Helsinki Declaration.
HLA Typing
All patients and donors were typed using PCResequence-specific primer
high-resolution typing for both HLA class I and II alleles[17].
Conditioning Regimen and GVHD Prophylaxis
Reduced-intensity conditioning was given to 252 patients and consisted offludarabine 30 mg/m2
/d for 3 to 6 days in combination with (1)
cyclo-phosphamide 60 mg/kg/d for 2 days (n¼26), (2) 23 Gy total body
irra-diation (TBI) and cyclophosphamide 60 mg/kg/d for 2 days (n¼44), (3) 2 Gy
Financial disclosure:See Acknowledgments on page 892.
*Correspondence and reprint requests: Mats Remberger, Professor,
CAST-unit, Karolinska University Hospital, SE-14186 Stockholm, Sweden.
E-mail address:mats.remberger@ki.se(M. Remberger).
http://dx.doi.org/10.1016/j.bbmt.2015.01.025
1083-8791/Ó2015 American Society for Blood and Marrow Transplantation.
Biology of Blood and
Marrow Transplantation
TBI (n ¼ 19), (4) treosulfan 14 g/m2 for 3 days (n ¼ 24), or (5)
4 mg/kg/d busulfan orally for 2 days (n¼139)[18-20]. Myeloablative
con-ditioning consisted of cyclophosphamide 60 mg/kg/d for 2 days in
combi-nation with (1) 7.5 to 10 Gy single fraction TBI (n¼2), (2) 43 Gy
fractionated TBI (n¼115), or (3) 4 mg/kg/d busulfan for 4 days (n¼175)
[21]. Antithymocyte globulin was given to all patients with an unrelated
donor as part of the conditioning regimen with the last dose on the day
before transplantation[22]. Three hundred twenty-one patients received
Thymoglobulin (Genzyme, Cambridge, MA) at a total dose of 4 to 10 mg/kg, whereas 5 patients received ATG-Fresenius (Fresenius, Bad Homburg, Germany) (25 to 30 mg/kg) and 32 patients received alemtuzumab (30 to 90 mg).
Prophylaxis against GVHD consisted of cyclosporine A (CsA) in
combi-nation with methotrexate (n¼462), prednisolone (n¼1), or
mycopheno-late mofetil (n¼11). Sirolimus and tacrolimus were given to 63 patients, and
2 received tacrolimus and mycophenolate mofetil [23]. Five patients
received CsAþmethotrexate and cyclophosphamide on days 3 and 4 after
HSCT[24]. During thefirst month, blood CsA levels were kept at 100 ng/mL
or 200 to 300 ng/mL when a sibling donor or unrelated donor was used, respectively. In the absence of GVHD, for patients with malignant diseases, CsA was discontinued at 3 to 4 months in HLA-identical transplants and at 6 months in unrelated transplants, whereas it was discontinued after 12 to 24 months in patients with nonmalignant disorders.
Stem Cell Source
BM was the graft source in 121 patients and PBSCs in 423. Before aphaeresis to obtain PBSCs, stem cells were mobilized with subcutaneous
granulocyte colony-stimulating factor (G-CSF) daily for 4 to 6 days[25].
Supportive Care
Supportive care was previously described in detail[26,27].
Statistics
Overall survival (OS) was calculated using the Kaplan-Meier method and compared with the log-rank test. Survival time was calculated from the day of transplantation until death or last follow-up. The incidence of transplant-related mortality (TRM), relapse, and GVHD were obtained using an esti-mator of cumulative incidence curves. Patients were censored at the time of death or last follow-up.
Univariate and multivariate predictive analyses for relapse, TRM, and GVHD were performed with the proportional subdistribution hazard
regression model of Fine and Gray[28], whereas analysis of OS, graft
failure (GF), and engraftment were performed using the Cox proportional
hazards model. Factors with P <.10 in the univariate analysis were
included in the backward elimination multivariate analysis. The main aim
was to evaluate the effect of total nucleated cell (TNC) and CD34þcell dose
on the outcome after HSCT with PBSCs or BM. Analyses were performed using the cmprsk package (developed by Gray, June 2001), Splus 6.2 (Insightful, Seattle, WA), and Statistica software (StatSoft, Tulsa, OK).
RESULTS
Cell Doses
The median TNC dose was approximately 4-fold higher
for patients receiving PBSCs compared with BM, whereas the
CD34
þdose was
z
2-fold higher (
Table 1
). Because of these
differences, further analyses were performed separately
depending on the graft stem cell source, BM or PBSCs.
Bone Marrow
In univariate analysis we found no effect of the TNC and
the CD34
þcell dose on OS (
P
¼
.57 and
P
¼
.86), nonrelapse
mortality (
P
¼
.94 and
P
¼
.74), relapse (
P
¼
.32 and
P
¼
.84),
GF (
P
¼
.32 and
P
¼
.56), acute GVHD (
P
¼
.70 and
P
¼
.23), and
chronic GVHD (
P
¼
.86 and
P
¼
.36). However, a higher TNC
dose was correlated with faster neutrophil engraftment (
P
<
.001). A higher CD34
þcell dose tended to correlate to a faster
neutrophil engraftment (
P
¼
.08). The results from the
uni-variate analysis are displayed in
Supplemental Table 1
.
In the BM cohort the multivariate analysis showed a
correlation between a higher TNC and CD34
þcell dose and a
faster neutrophil engraftment (hazard ratio [HR], 1.07; 95%
con
fi
dence interval [CI], 1.04 to 1.11;
P
<
.001 and HR, 1.05;
95% CI, 1.01 to 1.09;
P
¼
.02, respectively). The complete
re-sults from the multivariate analysis in BM grafts are
dis-played in
Table 2
.
Peripheral Blood Stem Cells
In univariate analysis we found no correlation between
the TNC dose and OS (
P
¼
.77), nonrelapse mortality (
P
¼
.21),
relapse (
P
¼
.91), GF (
P
¼
.32), acute GVHD (
P
¼
.60), chronic
GVHD (
P
¼
.60), or neutrophil and platelet engraftment
(
P
¼
.85 and
P
¼
.37). Furthermore, we found no correlation
between the CD34
þcell dose and GF (
P
¼
.56), nonrelapse
mortality (
P
¼
.22), acute GVHD (
P
¼
.85), chronic GVHD
(
P
¼
.97), or neutrophil engraftment (
P
¼
.37). A higher CD34
þcell dose correlated with a lower OS (
P
¼
.003), higher relapse
(
P
¼
.001), and a trend for faster platelet engraftment
(
P
¼
.10). The results from the univariate analysis are
dis-played in
Supplemental Table 1
.
In the PBSC cohort, the multivariate analysis found a
correlation between a higher CD34
þcell dose and a lower
OS (HR, 1.04; 95% CI, 1.01 to 1.07;
P
¼
.002), higher relapse
incidence (HR, 1.04; 95% CI, 1.01 to 1.09;
P
¼
.03), and a
faster platelet engraftment (HR, 1.02; 95% CI, 1.01 to 1.04;
P
<
.01). The effect of the CD34
þcell dose was studied
more carefully. We found that a very high (
11
10
6/kg)
and a very low (
2.5
10
6/kg) CD34
þcell dose was
associated with worse OS (
P
¼
.001) (
Figure 1
). A very high
(
11
10
6/kg) CD34
þcell dose was associated with an
increased incidence of relapse (
P
¼
.02) (
Figure 2
). The
complete results from the multivariate analysis in PBSC
grafts are displayed in
Table 2
.
BM versus PBSC
Overall, PBSC showed a nonsigni
fi
cantly better OS rate
compared with BM, 62% versus 53% (
P
¼
.22). A
“
rich BM
graft
”
containing more than median TNC dose (
>
3.2
10
8/kg)
Table 1Patient Characteristics Receiving Either BM or PBSCs
BM (n¼121) PBSCs (n¼423)
Median age, yr (range) 14 (<1-64) 48 (<1-69)
Sex (male/female) 76/45 243/180 Diagnose Acute leukemia 66 (55%) 216 (51%) Chronic leukemia 19 (16%) 66 (16%) Lymphoma 12 (10%) 37 (9%) MDS 21 (17%) 72 (17%) Other 3 32 (8%) Late stage (>CR1/CP1) 76 (63%) 224 (53%) Donor Sibling 51 (42%) 176 (42%) MUD 70 (58%) 247 (58%)
Median donor age, yr (range) 29 (.5-63) 38 (16-71)
Female to male 26 (21%) 56 (13%) Median TNC dose,108/kg (range) 3.2 (.8-47) 11.6 (2.0-63.8) Median CD34 dose,106/kg (range) 3.9 (.3-26.6) 8.1 (1.6-56.4) Conditioning MAC/RIC 95/26 (79%/21%) 197/226 (47%/53%) TBI-based 51 (42%) 129 (30%) Chemotherapy-based 70 (58%) 294 (70%) ATG 73 (60%) 285 (67%) GVHD prophylaxis CsAþMTX 106 (88%) 356 (84%) Tacrolimusþsirolimus 11 (9%) 52 (12%) Other 4 (3%) 15 (4%)
MDS indicates myelodysplastic syndrome; CR1/CP1,first complete
remis-sion/first chronic phase; MUD, HLA-A, -B, and -DR matched unrelated donor; MAC, myeloablative conditioning; RIC, reduced-intensity condi-tioning; ATG, antithymocyte globulin; MTX, methotrexate.
was not associated with a better OS rate compared with
PBSC, 47% versus 62% (
P
¼
.07). A similar result was seen
when categorized by CD34 cell dose. However, a PBSC graft
containing less than 11
10
6/kg CD34
þcells had a signi
fi
-cantly better OS rate compared with BM, regardless of cell
dose (66% versus 53%,
P
¼
.03). TRM was 19% using BM and
PBSC. Relapse tended to be lower (26% versus 39%,
P
¼
.06)
and chronic GVHD higher (40% versus 21%,
P
<
.001) using
PBSC compared with BM.
DISCUSSION
Only a few studies have evaluated the effect of cell dose
on clinical outcome after HSCT
[4,14-16]
. Many centers still
rely on the study from Seattle in the late 1970s even though it
only included a small number of patients with severe aplastic
Table 2Results from Multivariate Analysis of Factors Affecting Various Outcome Variables after HSCT with PBSCs and BM
PBSC BM
Characteristic HR, 95% CI, andPValue Characteristic HR, 95% CI, andPValue
Mortality
Age (decades) 1.25, 1.11-1.40,P<.001 Donor age (decades) 1.40, 1.15-1.70,P<.001
CD34þdose (1106) 1.04, 1.01-1.07,P¼.002 Year of HSCT .91, .87-.96,P<.001 Sibling donor .63, .45-.89,P<.01 Late disease 1.59, 1.15-2.19,P<.01 DRI 1.52, 1.19-1.93,P<.001 GF
Sibling donor .12, .02-.61,P¼.01 Major AB0 mismatch 6.38, 1.08-37.6,P¼.04
RIC 5.94, 1.27-27.9,P¼.02
Female to male 10.0, 2.95-24.2,P<.001
TRM
Acute leukemia .47, .30-.73,P<.001 Sibling donor .14, .03-.58,P<.01
Year of HSCT .80, .67-.96,P¼.02 Major AB0 mismatch 3.86, 1.65-9.04,P<.01
DRI 1.59, 1.14-2.21,P<.01 MDS 3.62, 1.49-8.80,P<.01
Relapse
Late disease 1.60, 1.11-2.32,P¼.02 MDS .21, .05-.88,P¼.03
CD34þdose (1106) 1.04, 1.01-1.09,P¼.03
Acute GVHD grades II-IV
ATG .68, .49-.93,P¼.02 Age (decades) .80, .68-.96,P¼.01
RIC .55, .40-.76,P<.001 ATG .54, .30-.97,P¼.04
Late disease 2.13, 1.08-4.18,P¼.03
Acute leukemia .46, .25-.84,P¼.01
Chronic GVHD
Chronic leukemia 1.66, 1.13-2.43,P¼.01 Age (decades) 1.28, 1.05-1.56,P¼.02
Sibling donor 2.07, 1.50-2.85,P<.001 Year of HSCT .95, .90-.98,P¼.02 Neutrophil engraftment MDS .70, .53-.91,P<.01 CD34 cell dose (1106) 1.05, 1.01-1.09,P¼.02 RIC .71, .58-.87,P<.001 TNC dose (1108) 1.07, 1.04-1.11,P<.001 G-CSF 1.93, 1.43-2.63,P<.001 G-CSF 2.59, 1.60-4.14,P<.001 Platelet engraftment CD34þdose (1106) 1.02, 1.01-1.04,P<.01 G-CSF .61, .41-.91,P¼.02 Sibling donor 1.66, 1.36-2.05,P<.001 CsAþMTX .59, .45-.77,P<.001 G-CSF .88, .44-.73,P<.001
DRI indicates disease risk index; Female to male, female donor to male recipient; GF, graft failure; TRM, transplant related mortality.
Figure 1.The effect of the CD34þcell dose (106/kg) on OS in recipients of PBSCs. The various thresholds were determined by quartiles, whereas the very
low dose group (2.5106/kg) was randomly selected.
Figure 2.The effect of the CD34þcell dose (106/kg) on relapse incidence in recipients of PBSCs. The various thresholds were determined by quartiles.
anemia (SAA)
[2]
. To create a contemporary point of reference,
we evaluated the effect of TNC and CD34
þcell dose on
outcome after HSCT at our center during the last 12 years. We
found that a high TNC dose and a high CD34
þcell dose
correlated with faster engraftment of neutrophils in patients
receiving a BM graft. A higher CD34
þcell dose correlated with
faster platelet engraftment in patients receiving a PBSC graft.
Although many studies have shown a correlation
between the CD34
þdose and time to neutrophil engraftment
[4-10]
, some studies analyzing BM and PBSC grafts have
failed to show such a correlation
[10,29-31]
, possibly because
threshold levels differ between the studies probably as a
consequence of diverse patient populations, treatments,
aspiration techniques, and methods of quanti
fi
cation of
CD34
þcells. Another explanation may be that the use of
G-CSF may also contribute to the lack of correlation between
cells infused and neutrophil recovery in other studies. In our
analysis we corrected for the use of G-CSF.
The use of a PBSC graft is associated with faster
engraft-ment compared with BM, and this is often primarily
attrib-uted to the higher cell dose
[3,31-35]
. In patients receiving a
PBSC graft, there was no correlation between cell dose and
time to neutrophil engraftment, in line with many other
studies
[10,29-31]
. This may be because the cell doses in
PBSC grafts are suf
fi
ciently high already and that a further
increase does not reduce time to engraftment. However, a
higher CD34
þdose resulted in faster platelet engraftment,
also in conformity with other studies
[5,8]
. The cell dose
threshold for platelet engraftment may be higher than for
neutrophil engraftment.
In line with previous studies, we were unable to
fi
nd a
correlation between the BM TNC and CD34
þcell dose and
acute GVHD
[33,36]
. It has been reported that a high CD34
cell dose is associated with an increased risk of severe acute
GVHD
[37,38]
.
Although a high CD34
þdose was associated with faster
platelet engraftment, it correlated with reduced OS in the
PBSC group. This is contradictory to most previously
pub-lished results showing that a higher CD34
þcell dose
increased survival
[9,10,29,39-41]
. However, 2 studies
showed increased mortality among patients receiving higher
doses of CD34
þPBSCs
[7,15]
, mainly because of a signi
fi
cantly
higher cGVHD-associated mortality. An optimal CD34
þcell
dose ranging between 4 and 8
10
6/kg was proposed in
some studies
[6,7,15]
. However, Kamel et al.
[4]
suggested a
higher but very narrow optimum CD34
þdose range of 9 to
10
10
6cells/kg.
In our study we propose a wider range of CD34
þcell dose
between 2.5 and 11
10
6/kg. This difference may be due to
patient population, size of study, and methods of quanti
fi
-cation of CD34
þcells. We found that a high CD34
þcell dose
was associated with relapse. In autologous SCT similar results
with a higher relapse incidence among patients receiving the
highest CD34
þcell dose was reported
[42,43]
, probably
because of contaminating malignant cells.
It is unclear why the relapse incidence increased with
increasing CD34
þdoses (
>
11
10
6cells/kg in our analysis)
after allogeneic HSCT. One explanation may be that grafts with
very high numbers of CD34
þcells also are very rich in
regula-tory T cells with the capacity to down-regulate the
antileu-kemic effect. This theory is supported by the fact that the
CD34
þcell dose did not correlated to chronic GVHD in our
study.
In our study, unmanipulated grafts were used and all
donors were HLA-A, -B, and -DR matched. Compared with
many other studies, our cell doses were relatively high, with
at least 10% of patients receiving a very high cell dose. Some
patients received a CD34
þcell dose of up to 50
10
6/kg.
A study from the European Group for Blood and Marrow
Transplantation showed that a
“
rich BM
”
gave better
outcome than PBSCs or less rich BM, linked to a diminution of
the relapse incidence
[44]
. We could not verify these results.
Overall, PBSCs showed a nonsigni
fi
cantly better OS rate
compared with BM, 62% versus 53% (
P
¼
.22). Furthermore, a
PBSC graft containing
<
11.0
10
6/kg CD34
þcells gave better
results than a rich BM.
One drawback may be the heterogeneity of the patients,
including many different diagnoses, both sibling donor and
matched unrelated donor HSCT, and both reduced-intensity
and myeloablative conditioning. However, this is our
clin-ical reality, and we believe our results may help clinicians to
make the right considerations. We propose that when a graft
with a very high cell dose is used, a part may be removed and
frozen for later use as boost, back-up, or donor lymphocyte
infusion. This treatment strategy will improve the clinical
outcome of the HSCT per se and may also save time and
money if a donor lymphocyte infusion or boost is needed.
To conclude, a higher BM TNC and CD34
þdose was
associated with faster neutrophil engraftment. A higher PBSC
CD34
þcell dose was correlated with faster platelet
engraft-ment but increased mortality and relapse.
ACKNOWLEDGMENTS
Financial disclosure:
This study was supported by grants
from the Swedish Children Cancer Foundation (J014/09).
Con
fl
ict of interest statement:
There are no con
fl
icts of
interest to report.
SUPPLEMENTARY DATA
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.bbmt.2015.01.025
.
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