Bone Marrow Transplantation (2010), 1–11
& 2010 Macmillan Publishers Limited All rights reserved 0268-3369/10

www.nature.com/bmt

ORIGINAL ARTICLE

Disease stage stratiﬁed effects of cell dose in unrelated BMT for
hematological malignancies: a report from Japan marrow donor program
Y Inamoto1, K Miyamura2, S Okamoto3, H Akiyama4, H Iida5, T Eto6, Y Morishima7, K Kawa8,
A Kikuchi9, Y Nagatoshi10, J Tanaka11, T Ashida12, M Hirokawa13, M Tsuchida14 and S Mori15
for the Japan Marrow Donor Program1
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of
Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan; 3Department of Medicine, Keio University School of
Medicine, Tokyo, Japan; 4Hematology Division, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; 5Department of
Hematology, Meitetsu Hospital, Nagoya, Japan; 6Department of Hematology, Hamanomachi Hospital, Fukuoka, Japan;
7
Department of Hematology and Cell Therapy, Aichi Cancer Center, Nagoya, Japan; 8Department of Pediatrics, Osaka Medical
Center and Research Institute for Maternal and Child Health, Osaka, Japan; 9Department of Pediatrics, Graduate School of
Medicine, the University of Tokyo, Tokyo, Japan; 10Department of Pediatrics, National Kyushu Cancer Center, Fukuoka, Japan;
11
Department of Hematology and Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 12Division of
Hematology, Department of Internal Medicine, Kinki University School of Medicine, Osaka, Japan; 13Akita University Hospital,
Clinical Oncology Center, Akita, Japan; 14Department of Pediatrics, Ibaragi Children’s Hospital, Ibaragi, Japan and 15Hematopoietic
Stem Cell Transplantation Unit, National Cancer Center Hospital, Tokyo, Japan

Cell dose is one of the major factors that can be manipulated in unrelated BMT. However, regarding diseasestage-stratiﬁed effects of cell dose, data are limited.
We analyzed the registry data from 3559 patients withacute leukemia, CML and myelodysplastic syndrome who
received T-cell replete unrelated BMT through the Japan
Marrow Donor Program. Adjusted effects of cell dosewere evaluated for various outcomes separately according
to disease stages and children or adults. Acute GVHD andnonrelapse mortality were not affected by cell dose.
Among children, a cell dose lower than 3.0  108/kg was
associated with lower engraftment rates in advanced-stage
diseases. Among adults, a cell dose of 3.4  108/kg or
higher was associated with lower relapse rates and better
survival rates only in early-stage diseases, whereas
cell dose below 2.3  108/kg was associated with lower
engraftment rates in advanced-stage diseases. In conclusion, effects of cell dose may differ among disease stages.
A cell dose of 3.4  108/kg or higher is recommended only
for adults with early-stage diseases. With the number ofpatients available for analysis in this study, we could notshow any signiﬁcant beneﬁts associated with 4.6  108/kg
or higher in children.
Bone Marrow Transplantation advance online publication,
8 November 2010; doi:10.1038/bmt.2010.281
Keywords: allogeneic; cell dose; disease stage; unrelated

Correspondence: Dr Y Inamoto, Clinical Research Division, Fred
Hutchinson Cancer Research Center, 1100 Fairview Avenue North,D5-290, PO Box 19024, Seattle, WA 98109-1024, USA.
E-mail: yinamoto@js3.so-net.ne.jp
Received 29 March 2010; revised 21 June 2010; accepted 12 August 2010

Introduction
Allogeneic hematopoietic cell transplantation has been
established as a curative therapy for hematologicalmalignancies.1,2 Because of the better understandingof the signiﬁcance of HLA allele compatibility and theadvances in supportive care, the results of BMT fromunrelated donors are improving.3–5
Cell dose is one of the major factors that can be
manipulated by physicians and affect transplant outcomes.6–8 Historically, its importance for engraftment andhematological recovery has been documented in patientswith aplastic anemia.9,10 Several subsequent studies showedthat cell dose was also associated with better survival due todecreased nonrelapse mortality (NRM) in hematologicalmalignancies. However, other important factors, such as
patient age, disease, conditioning, GVHD prophylaxis,
ABO compatibility, donor characteristics and HLA matching, also affect the transplant outcome.11,12 Therefore,
the actual effect of cell dose should be conﬁrmed after
adjustment for all of these factors with a sufﬁcient numberof patients.
On the other hand, the GVL effect may work differently
according to disease stages. Rocha et al.13 showed that celldose was associated with decreased relapse rates in AML in
ﬁrst CR, whereas no signiﬁcant associations between celldose and relapse rates were observed in other studies,including various diseases.7,8,11 These conﬂicting results
suggested that the cell dose effect is worth analyzing
separately according to disease stages.
In this report, we examined adjusted effects of cell doseon various transplant outcomes according to disease stages
and children or adults using the detailed registry data of
3559 patients who received T-cell replete unrelated BMT
through the Japan Marrow Donor Program.

Cell dose effects in unrelated BMT
Y Inamoto et al

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Patients and methods
Patients
The data set consisted of 5071 unrelated BMTs facilitatedby the Japan Marrow Donor Program between 1993 and2005. Of these 5071 patients, 3559 with AML, ALL, CML
and myelodysplastic syndrome who received their ﬁrst
T-cell replete myeloablative transplantation with GVHD
prophylaxis containing calcineurin inhibitor without
antithymocyte globulin were selected for this study. The
patients and donors were all Japanese. Informed consent
for this registry study was obtained from patients anddonors in accordance with the declaration of Helsinki. This
study was approved by the data management committees ofJapan Marrow Donor Program.
Transplantation procedure
Patients were conditioned with various regimens determined by each transplant center. The proportions of TBI
regimen were assessed from the database. Red cells and/or
plasma removal from the graft was performed for ABOmajor and/or -minor mismatched transplantation. All
grafts were BM because the donation of PBSCs fromunrelated donors is not yet approved in Japan. GVHD
prophylaxis was categorized into either a CsA-based or
tacrolimus-based prophylaxis.
HLA matching
HLA-A, -B and -DRB1 alleles were identiﬁed by highresolution DNA typing as described previously.3,4 As ourprevious study showed that a single-allele mismatch at
DRB1 locus had no impact on engraftment, acute andchronic GVHD, NRM, relapse and OS in the Japanese
population,4 it was considered as a HLA-matched transplantation in this study.
Deﬁnition of disease stage and outcomes
Early stage was deﬁned as the status of the ﬁrst and second
CR of AML and ALL, the ﬁrst chronic phase of CML andrefractory anemia of myelodysplastic syndrome, whereas
advanced stage was deﬁned as other status. For cytogenetic
categorization, patients were divided into three categories:
good risk (AML with t(15;17), inv16 or t(8;21)), intermediate risk (other than good or poor risk) or poor risk
(ALL with t(9;22) or t(4;11), CML with additional
abnormalities other than t(9;21) or myelodysplastic syndrome with complex or chromosome 7 abnormalities).14
Engraftment was deﬁned as an ANC of more than 500/ml
for 3 consecutive days in the peripheral blood, andanalyzed among all patients. Acute GVHD was gradedby established criteria.15 Chronic GVHD was assessed inpatients surviving beyond day þ 100, and was classiﬁed aslimited or extensive according to the Seattle criteria.16
Statistical analysis
Cell dose was deﬁned as harvested total nucleated cell dose.
Analysis was performed separately for disease stages, andchildren or adults. Children were deﬁned as patients who
were aged 12 years or younger for two reasons. One reason
was because cell dose per patient body wt had a stronger
linear correlation with age at these ages. Another reason
Bone Marrow Transplantation

was because patients aged 12 years or younger were usuallytreated with children’s protocols. To determine the impactsof low and high cell doses on the outcomes in the current
practices, cut-off points were set at upper and lower 25% of
the cell dose separately in children and adults. Patientcharacteristics and causes of NRM were tested for
associations using the w2-test for discrete variables, andthe Spearman rank correlation test for continuous variables. Cumulative incidences of NRM, relapse and GVHD
were estimated by Gray’s method. Relapse was considered
as a competing risk in NRM, deaths without relapse as acompeting risk in relapse, and deaths without GVHD as acompeting risk in GVHD. OS was calculated using the
Kaplan–Meier method and P-values were calculated usinga Log-rank test. Multivariate analyses were performed
using logistic regression model for engraftment, the Cox
proportional hazard regression model for OS, and themultivariate proportional hazard modeling of subdistribution functions in competing risks for NRM, relapse and
GVHD.17 Variables considered in the analysis were celldose, patient age (linear), ABO incompatibility (none,
major or minor), disease stage (early or advanced),
cytogenetics (good, intermediate or poor), the number of
HLA-mismatched loci, patient sex, donor sex, female tomale transplantation, conditioning (TBI regimen, antithymocyte globulin regimen, and reduced-intensity regimen),
GVHD prophylaxis (CsA-based or tacrolimus-based),donor age (linear), year of transplant (categorical) andpreceding grades II–IV acute GVHD (only for chronic
GVHD analysis). Cell dose was kept in the ﬁnal model eventhough it was not statistically signiﬁcant. All statistical tests
were two-sided, and P-values less than 0.05 were consideredsigniﬁcant. Analysis was performed using STATA (Stata
Statistical Software: Release 10.0., Stata Corporation,
College Station, TX, USA) and R version 2.10.0 (TheR Foundation for Statistical Computing, Vienna, Austria).

Results
Patient characteristics
The number of patients with AML, ALL, CML andmyelodysplastic syndrome were 1205 (34%), 1140 (32%),755 (21%) and 459 (13%), respectively. The medianvolumes of harvested marrow for child and adult recipientswere 426 mL (range, 83–1045) and 850 mL (range, 220–
1500), respectively (Po0.0001). The median numbersof harvested cells for child and adult recipients were3.63  108/kg (range, 0.58–13.7) and 2.92  108/kg (range,
0.16–12.1), respectively (Po0.0001). Cut-off points
were set at 3.0 and 4.6  108/kg for children, and 2.3
and 3.4  108/kg for adults. Patient characteristics weresummarized in Tables 1 and 2. Recipient age, recipient–donor gender compatibility, recipient body wt, GVHD
prophylaxis and the year of transplantation showedstatistically signiﬁcant differences according to cell dosein children. Recipient age, recipient–donor gender compatibility, recipient body wt, ABO mismatch, disease type inearly-stage malignancy, GVHD prophylaxis and the year oftransplantation showed statistically signiﬁcant differences
according to cell dose in adults.

Cell dose effects in unrelated BMT
Y Inamoto et al

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Table 1

Patient characteristics in children

Characteristic

P

Cell dose
o3.0  10 /kg (n ¼ 140)
8

No.

8

3.0–4.6  10 /kg (n ¼ 248)

%

No.

%

8

X4.6  10 /kg (n ¼ 128)
No.

%

Recipient age, years
Median
Range

9
0–12

8
0–12

5
0–12

o0.001

Donor age, years
Median
Range

35
21–50

34
20–50

32
20–50

0.20

Sex (recipient/donor)
Male/male
Female/female
Male/female
Female/male

33
41
50
16

Recipient body wt, kg
Median
Range

27
5–72

ABO mismatch
Match
Major mismatch
Minor mismatch

96
29
15

69
21
11

154
55
39

62
22
16

66
37
25

52
29
20

24
29
36
11

71
65
58
54

29
26
23
22

25
5–49

47
23
25
33

37
18
20
26

0.001

o0.001

17
4–44

0.063

Disease
Early-stage malignancy
AML
ALL
CML
MDS
Advanced-stage malignancy
AML
ALL
CML
MDS

18
62
7
4

20
68
8
4

53
107
14
4

30
60
8
2

23
52
10
4

26
58
11
4

10
28
4
7

20
57
8
14

18
37
1
14

26
53
1
20

9
18
2
10

23
46
5
26

Cytogenetics
Good risk
Intermediate risk
Poor risk
Not available

4
110
18
8

3
79
13
6

17
189
25
17

7
76
10
7

8
98
17
5

6
77
13
4

0.55

Conditioning
TBI regimen
Non-TBI regimen

122
18

87
13

209
39

84
16

102
26

80
20

0.25

GVHD prophylaxis
Cyclosporin-based
Tacrolimus-based

44
96

31
69

100
148

40
60

71
57

55
45

o0.001

No. of HLA mismatch by DNA typing
095
1 locus40
2 or more loci
5

68
29
4

190
52
6

77
21
2

90
33
5

70
26
4

0.39

Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

13
28
39
21

44
67
87
50

18
27
35
20

31
50
32
15

24
39
25
12

0.009

0.50

0.51

18
39
54
29

Abbreviation: MDS ¼ myelodysplastic syndrome.

Engraftment
Engraftment was achieved in 500 of 516 (97%)
child patients and 2882 of 3043 (95%) adult patients.
Multivariate analysis showed that o3.0  108/kg was
associated with lower engraftment rates in children withadvanced-stage diseases (odds ratio, 0.15; 95% conﬁdence
interval (CI), 0.03–0.74; P ¼ 0.02) and o2.3  108/kg
was associated with lower engraftment rates in adults
with advanced-stage diseases (odds ratio, 0.60; 95% CI,
0.37–0.97; P ¼ 0.039).
Bone Marrow Transplantation

Cell dose effects in unrelated BMT
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Table 2

Patient characteristics in adults

Characteristic

P

Cell doseo2.3  10 /kg (n ¼ 755)
8

No.

%

8

2.3–3.4  10 /kg (n ¼ 1519)
No.

%

8

X3.4  10 /kg (n ¼ 769)
No.

%

Recipient age, years
Median
Range

34
13–65

34
13–66

32
13–62

0.0076

Donor age, years
Median
Range

34
20–51

34
20–68

34
20–51

0.42

Sex (recipient/donor)
Male/male
Female/female
Male/female
Female/male

309
179
188
79

Recipient body wt, kg
MedianRange

61
29–120

ABO mismatch
Match
Major mismatch
Minor mismatch

401
191
163

53
25
22

800
417
302

53
27
20

355
271
143

46
35
19

41
24
25
10

666
287
253
313

44
19
17
21

59
25–112

336
132
91
210

44
17
12
27

o0.001o0.00155
23–90

o0.001

Disease
Early-stage malignancy
AML
ALL
CML
MDS
Advanced-stage malignancy
AML
ALL
CML
MDS

187
148
89
48

40
31
19
10

347
281
248
62

37
30
26
7

149
155
135
34

32
33
29
7

104
62
59
58

37
22
21
20

189
129
124
139

33
22
21
24

98
61
62
75

33
21
21
25

Cytogenetics
Good risk
Intermediate risk
Poor risk
Not available

54
615
54
32

7
81
7
4

116
1215
105
83

8
80
7
5

45
622
58
44

6
81
8
6

0.59

Conditioning
TBI regimen
Non-TBI regimen

634
121

84
16

1245
274

82
18

621
148

81
19

0.25

GVHD prophylaxis
CsA-based
Tacrolimus-based

337
418

45
55

833
686

55
45

418
351

54
46

o0.001

No of HLA mismatch by DNA typing
0584
1 locus158
2 or more loci
13

77
21
2

1183
306
30

78
20
2

608
146
15

79
19
2

0.90

Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

9
21
44
26

227
500
509
283

15
33
34
19

113
293
230
133

15
38
30
17

o0.001

0.002

0.83

70
158
329
198

Abbreviation: MDS ¼ myelodysplastic syndrome.

Acute and chronic GVHD
The cumulative incidences of grades II–IV acute GVHD
in children and adults were 50 and 43%, respectively.
Bone Marrow Transplantation

Multivariate analysis showed no statistically signiﬁcantassociation of cell dose with incidences of grades II–IV
acute GVHD in children and adults.

Cell dose effects in unrelated BMT
Y Inamoto et al

5
Table 3

Variables associated with relapse in (a) children and (b) adults

Variable

Early-stage disease
Univariate
HR

95% CI

Advance- stage disease
Multivariate

PHR

95% CI

Univariate
P(a)n ¼ 358
Cell dose (  108/kg)
3.0–4.6
o3.0
X4.6

1.00
1.06
1.22

(0.60–1.87)(0.70–2.14)

1.00
0.84
0.48

1.00
0.99
1.20

(0.56–1.75)(0.69–2.09)

1.00
0.98
0.52

Recipient age
Linear

0.95(0.90–1.01)

0.14

0.99

(0.92–1.07)

0.83

Donor ageLinear

1.01(0.99–1.04)

0.37

0.96

(0.92–0.99)

Cytogenetics
Intermediate risk
Good risk
Poor risk

1.00
Unevaluablea
1.43

o0.001(0.76–2.69)

1.00
Unevaluablea
0.27

o0.001
1.42

1.00
1.71
(0.76–2.65)

ABO mismatch
Match
Major mismatch
Minor mismatch

1.00
1.11
0.80

(0.64–1.91)(0.40–1.61)

HLA mismatch
Match
Mismatch

1.00
0.95

Recipient sex
Male
FemaleDonor sex
Male
FemaleHR

95% CI

Multivariate
PHR

95% CIP(0.54–1.95)(0.53–1.72)

0.93
0.87

n ¼ 158

1.18
0.98(0.66–2.14)(0.54–1.81)

0.02

0.96

(0.92–0.99)(0.8–3.67)
0.27

0.16
0.78

(0.27–2.24)

0.64

0.72
0.54

1.00
0.48
0.66

(0.24–0.94)(0.33–1.31)

0.031
0.23

(0.61–1.48)

0.81

1.00
0.63

(0.38–1.04)

0.072

1.00
0.97

(0.61–1.55)

0.90

1.00
0.92

(0.56–1.52)

0.76

1.00
1.11

(0.70–1.76)

0.67

1.00
0.99

(0.61–1.63)

0.98

Female donor to male recipient
No
1.00
Yes
1.20

(0.72–2.02)

0.48

1.00
1.17

(0.69–2)

0.56

Conditioning
Non-TBI regimen
TBI regimen

1.00
0.62

(0.36–1.06)

0.08

1.00
0.67

(0.38–1.21)

0.18

GVHD prophylaxis
CsA-based
Tacrolimus-based

1.00
0.91

(0.57–1.45)

0.68

1.00
1.02

(0.62–1.67)

0.93

Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

1.00
0.86
1.02
0.72

(0.44–1.70)(0.53–1.96)(0.32–1.61)

0.67
0.95
0.42

1.00
1.29
1.20
0.99

(0.64–2.6)(0.61–2.39)(0.4–2.44)

0.47
0.60
0.98

(b)
n ¼ 1883
Cell dose (  108/kg)
2.3–3.4
o2.3
X3.4

1.00
1.13
0.61

(0.85–1.49)(0.43–0.85)

0.41
0.0042

Recipient age
Linear

0.99(0.98–1.00)

Donor ageLinear0.99

Cytogenetics
Intermediate risk
Good risk
Poor risk

1.00
0.97
1.43

0.57
0.96

1.03
0.95

0.021

1.00
0.48
0.25

(0.23–0.98)

0.043

n ¼ 1160
1.00
1.09
0.60

1.00
1.20
0.91

(0.94–1.55)(0.70–1.18)

0.14
0.48

1.00
1.21
0.90

(0.94–1.56)(0.70–1.17)

0.13
0.44

0.28

0.99

(0.98–1.00)

0.015

0.99

(0.98–1.00)

0.0088(0.97–1.00)

0.088

0.99

(0.98–1.00)

0.20(0.60–1.58)(0.91–2.24)

0.91
0.12

1.00
1.33
1.00

(0.89–1.99)(0.66–1.51)

0.16
0.98

(0.82–1.44)(0.43–0.85)

0.56
0.004

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Cell dose effects in unrelated BMT
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Table 3

Continued

Variable

Early-stage disease
Univariate

Advance- stage disease
Multivariate

HR

95% CIP

ABO mismatch
Match
Major mismatch
Minor mismatch

1.00
1.10
0.97

(0.83–1.46)(0.70–1.36)

HLA mismatch
Match
Mismatch

1.00
0.92

Recipient sex
Male
FemaleDonor sex
Male
FemaleUnivariate
HR

95% CIPHR

95% CI

P

0.52
0.88

1.00
0.70
0.77

(0.55–0.90)(0.59–1.02)

0.0045
0.07

1.00
0.71
0.76

(0.56–0.92)(0.58–1.01)

0.0081
0.055(0.70–1.22)

0.57

1.00
0.73

(0.57–0.92)

0.00931.00
0.73(0.57–0.93)

0.01

1.00
1.11

(0.87–1.43)

0.40

1.00
1.08

(0.87–1.33)

0.47

1.00
1.05

(0.81–1.35)

0.72

1.00
0.90

(0.73–1.13)

0.37

Female donor to male recipient
No
1.00
Yes
0.87

(0.62–1.22)

0.41

1.00
0.81

(0.61–1.09)

0.17

Conditioning
Non-TBI regimen
TBI regimen

1.00
1.36

(0.95–1.95)

0.10

1.00
1.08

(0.82–1.42)

0.58

GVHD prophylaxis
CsA-based
Tacrolimus-based

1.00
1.50

(1.17–1.92)

0.00141.00
1.07(0.87–1.31)

0.53

Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

1.00
1.20
1.59
2.02

(0.77–1.86)(1.05–2.43)(1.27–3.19)

0.42
0.03
0.0028

1.00
1.06
1.24
1.19

(0.74–1.52)(0.87–1.76)(0.81–1.76)

0.74
0.23
0.37

HR

1.49

95% CI(1.16–1.91)

P

Multivariate

0.0017Abbreviations: CI ¼ conﬁdence interval; HR ¼ hazard ratio.
aHazard ratio was unevaluable because of no events.

The cumulative incidences of limited or extensive
chronic GVHD in children and adults were 34 and 45%,
respectively. Multivariate analysis in children showed astatistically signiﬁcant association of o3.0  108/kg withhigher incidences of chronic GVHD in advanced-stage
diseases (hazard ratio, 2.46; 95% CI, 1.17–5.17; P ¼ 0.017).
Multivariate analysis in adults showed no statisticallysigniﬁcant association of cell dose with incidences ofchronic GVHD.

NRM
The cumulative incidences of NRM at 5 years in children andadults were 21 and 39%, respectively. Multivariate analysis
showed no statistically signiﬁcant association of cell dose withincidences of NRM in children (Supplementary Table S1a)
and adults (Supplementary Table S1b). Causes of NRM
according to cell dose were not statistically different inchildren. As a cause of NRM in adults, the proportions of
idiopathic pneumonia syndrome were statistically different
according to cell dose (13, 14 and 23% for o2.3, 2.3–3.4 and43.4  108/kg, respectively; P ¼ 0.002).
Bone Marrow Transplantation

RelapseThe cumulative incidences of relapse at 5 years in children
and adults were 27 and 25%, respectively. Multivariate
analysis in children showed no statistically signiﬁcantassociation of cell dose with incidences of relapse(Table 3a). Multivariate analysis in adults showed astatistically signiﬁcant association of 43.4  108/kg withlower incidences of relapse in early-stage diseases (hazard
ratio, 0.60; 95% CI, 0.43–0.85; P ¼ 0.004) (Table 3b).
Results were similar when CML in chronic phase was
excluded from analysis in adults (data not shown).
OS
The median follow-up periods among survivors were 57
months (range, 9–140 months) in children and 55 months(range, 3–147 months) in adults. The OS rates at 5 years
among children with early-stage diseases were 67, 75 and
68% for o3.0, 3.0–4.6 and 44.6  108/kg, respectively(P ¼ 0.74; Figure 1a). The OS rates at 5 years amongchildren with advanced-stage diseases were 31, 36 and
40% for o3.0, 3.0–4.6 and 44.6  108/kg, respectively

Cell dose effects in unrelated BMT
Y Inamoto et al

7Child early stage disease

a 100

b 100
80
Overall survival (%)

Overall survival (%)

80

60

40

>4.6 × 108/kg

20

0

1

2

3

c 100

60

40

>4.6 × 108/kg

20

3.0–4.6 × 108/kg≤3.0 × 108/kg

0

3.0–4.6 × 108/kg

P = 0.74

4
5
6
7
8
9
Years after transplantation

10

11

12

0

13

P = 0.46≤3.0 × 108/kg

0
1

2

3

4

5

6

7

8

9

10

11

12

13

11

12

13

Years after transplantation

d 100

Adult early stage disease80

Adult advanced stage disease80
Overall survival (%)

Overall survival (%)

Child advanced stage disease60

40>3.4 × 108/kg

20

40>3.4 × 108/kg
2.3–3.4 × 108/kg≤2.3 × 108/kg

20

2.3–3.4 × 108/kg

P = 0.0029≤2.3 × 108/kg

0

60

0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

0

1

2

3

Years after transplantation

P = 0.27

4
5
6
7
8
9
Years after transplantation

10

Figure 1 Kaplan–Meier estimates of OS according to cell dose: (a) among children with early-stage diseases; (b) among children with advanced-stage
diseases; (c) among adults with early-stage diseases; and (d) among adults with advanced-stage diseases.

(P ¼ 0.46; Figure 1b). The OS rates at 5 years among
adults with early-stage diseases were 54, 57 and 65% for
o2.3, 2.3–3.4 and 43.4  108/kg, respectively (P ¼ 0.0029;
Figure 1c). The OS rates at 5 years among adults
with advanced-stage diseases were 26, 28 and 31% for
o2.3, 2.3–3.4 and 43.4  108/kg, respectively (P ¼ 0.27;
Figure 1d).
Multivariate analysis in children showed no statistically signiﬁcant association of cell dose with survival
rates (Table 4a). Multivariate analysis in adults showeda statistically signiﬁcant association of 43.4  108/kg with
better survival rates only in early-stage diseases (hazard
ratio, 0.74; 95% CI, 0.62–0.90; P ¼ 0.002) (Table 4b).

Discussion
This study showed that effects of cell dose on transplant
outcomes were different among disease stages. Amongchildren, we could not show any statistically signiﬁcanteffects of cell dose except the lower engraftment rates andhigher incidences of chronic GVHD associated with
o3.0  108/kg in advanced-stage diseases. Among adults,
cell dose 43.4  108/kg was associated with decreased
relapse rates and better survival rates in early-stage
diseases, whereas cell dose was not associated withoutcomes except the lower engraftment rates witho2.3  108/kg in advanced-stage diseases.
Although many studies reported that higher cell dose
improved OS rates,8,11,12,18,19 effects of cell dose on relapse
and NRM rates were not consistent among studies
probably because of the differences in diseases, stages andtransplant procedures. Furthermore, it is not practical toanalyze child and adult patients together because biology of
disease, treatment protocols and harvested total nucleatedcells per body wt are likely to differ between them.
Therefore, we investigated cell dose effects separately
according to disease stages and children or adults, andextended analysis to various outcomes.
Although several studies showed that engraftment rates
were improved with higher cell dose,6,11 our results did notshow any statistically signiﬁcant merits with high cell dose
both in children and adults. Low cell dose was associatedwith worse engraftment rates in advanced-stage diseases inboth children and adults. Effects of low cell dose would beparticularly great in advanced-stage diseases consideringthat graft failure occurs more frequently in advanced-stage
diseases.7
Effects of cell dose on relapse rates were controversial.
Although several studies did not show any effects of celldose on relapse rates,7,8,11 the results of our study
supported those by Rocha et al.13 among patients with
AML in the ﬁrst CR, and those by Barrett et al.20 after
Bone Marrow Transplantation

Cell dose effects in unrelated BMT
Y Inamoto et al

8
Table 4

Variables associated with OS in (a) children and (b) adults
Early stage disease (n ¼ 358)

Variable

Univariate
HR

95% CI

Advanced stage disease (n ¼ 158)

Multivariate
PHR

95% CI

Univariate
P

Multivariate

95% CIPHR

95% CI

P

1.00
1.59
0.99

(0.85–2.95)(0.63–1.56)

0.14
0.96

1.00
1.39
0.87

(0.87–2.20)(0.53–1.43)

0.17
0.59

1.00
0.57

(0.35–0.91)

0.02

1.00
1.15
0.94

(0.97–1.37)(0.80–1.12)

0.11
0.52

1.00
1.04
1.61

(0.77–1.40)(1.26–2.07)

0.80
o0.001HR(a)n ¼ 358
Cell dose (  108/kg)
3.0–4.6
o3.0
X4.6

1.00
1.15
1.18

(0.72–1.85)(0.74–1.89)

0.56
0.49

Recipient age
Linear

1.01(0.95–1.07)

0.86

1.04

(0.98–1.10)

0.20

Donor ageLinear

1.02(1.00–1.05)

0.11

1.01

(0.98–1.04)

0.41

Cytogenetics
Intermediate risk
Good risk
Poor risk

1.00
0.75
1.09

(0.27–2.06)(0.60–1.96)

0.58
0.79

1.00
1.18
1.20

(0.55–2.56)(0.60–2.39)

0.67
0.61

ABO mismatch
Match
Major mismatch
Minor mismatch

1.00
1.40
1.49

(0.88–2.22)(0.89–2.51)

0.15
0.13

1.00
0.87
0.71

(0.54–1.39)(0.41–1.25)

0.55
0.24

HLA mismatch
Match
Mismatch

1.00
1.72

(1.30–2.27)

o0.0011.00
1.11(0.77–1.60)

0.58

Recipient sex
Male
Female1.00
1.04(0.70–1.54)

0.86

1.00
1.25

(0.85–1.85)

0.25

Donor sex
Male
Female1.00
1.26(0.85–1.87)

0.25

1.00
0.72

(0.49–1.07)

0.10

Female donor to male recipient
No
1.00
Yes
1.10

(0.71–1.70)

0.68

1.00
0.63

(0.40–0.99)

0.05

Conditioning
Non-TBI regimen
BI regimen

1.00
1.01

(0.59–1.72)

0.98

1.00
1.26

(0.74–2.15)

0.40

GVHD prophylaxis
CsA-based
Tacrolimus-based

1.00
1.07

(0.71–1.60)

0.75

1.00
0.83

(0.56–1.22)

0.34

Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

1.00
0.74
0.59
0.69

(0.44–1.25)(0.34–1.03)(0.35–1.36)

0.27
0.06
0.29

1.00
1.10
0.87
0.90

(0.65–1.87)(0.51–1.49)(0.46–1.76)

0.73
0.61
0.76

n ¼ 158
1.00
1.09
1.18

1.00
1.72

(0.68–1.75)(0.74–1.89)(1.30–2.27)

0.73
0.48

o0.001(b)n ¼ 1883
Cell dose (  108/kg)
2.3–3.4
o2.3
X3.4

1.00
1.05
0.75

(0.88–1.25)(0.62–0.90)

0.59
0.002

1.00
1.06
0.74

(0.89–1.26)(0.62–0.90)

0.54
0.002

1.00
1.10
0.94

(0.93–1.31)(0.79–1.11)

0.25
0.47

Recipient age
Linear

1.01(1.01–1.02)

o0.0011.01(1.01–1.02)

o0.0011.00(1.00–1.01)

0.61

Donor ageLinear

1.01(1.00–1.02)

0.01

1.01

(1.00–1.02)

0.02

1.00

(0.99–1.01)

0.42

Cytogenetics
Intermediate risk
Good risk
Poor risk

1.00
0.79
1.09

(0.59–1.06)(0.82–1.45)

0.12
0.56

1.00
1.05
1.59

(0.78–1.41)(1.24–2.04)

0.75
o0.001Bone Marrow Transplantationn ¼ 1160

Cell dose effects in unrelated BMT
Y Inamoto et al

9Table 4

Continued
Early stage disease (n ¼ 358)

Variable

Univariate

Advanced stage disease (n ¼ 158)

Multivariate

Univariate

Multivariate

HR

95% CIPHR

95% CIPHR

95% CIP

ABO mismatch
Match
Major mismatch
Minor mismatch

1.00
1.16
1.08

(0.98–1.37)(0.89–1.31)

0.08
0.42

1.00
1.18
1.12

(1.00–1.40)(0.92–1.36)

0.05
0.26

1.00
1.10
1.11

(0.94–1.30)(0.93–1.33)

0.23
0.26

HLA mismatch
Match
Mismatch

1.00
1.41

(1.22–1.63)

o0.0011.00
1.38(1.19–1.60)

o0.0011.00
1.34(1.18–1.53)

o0.001

Recipient sex
Male
Female1.00
0.88(0.75–1.02)

0.08

1.00
0.96

(0.83–1.10)

0.55

Donor sex
Male
Female1.00
1.00(0.86–1.16)

0.97

1.00
0.96

(0.83–1.11)

0.56

Female donor to male recipient
No
1.00
Yes
1.11

(0.93–1.34)

0.25

1.00
1.06

(0.89–1.27)

0.50

Conditioning
Non-TBI regimen
TBI regimen

1.00
0.90

(0.74–1.08)

0.26

1.00
1.00

(0.83–1.19)

0.97

GVHD prophylaxis
CsA-based
Tacrolimus-based

1.00
1.04

(0.90–1.20)

0.60

1.00
0.85

(0.74–0.97)

0.02Year of transplantation
1993–1996
1997–2000
2001–2003
2004–2005

1.00
0.75
0.82
0.92

(0.60–0.93)(0.66–1.02)(0.72–1.19)

0.009
0.072
0.54

1.00
0.77
0.70
0.66

(0.62–0.95)(0.56–0.87)(0.51–0.85)

0.014
0.001
0.001

1.00
0.79
0.80
0.85

(0.63–0.99)(0.64–1.00)(0.65–1.11)

0.04
0.053
0.23

HR

95% CI

P

1.00
1.31

(1.15–1.50)

o0.001

1.00
0.79
0.72
0.68

(0.63–0.98)(0.58–0.90)(0.53–0.88)

0.032
0.005
0.003

Abbreviations: CI ¼ conﬁdence interval; HR ¼ hazard ratio.

identical twin BMT. Interestingly, our results showed lower
relapse rates not associated with higher incidences of acute
GVHD, which was also observed in the studies by Rocha
et al.13 and by Barrett et al.20 GVL effect is inﬂuenced bydisease types and stages possibly because of the differencesin expression of tumor Ags, co-stimulatory molecules,
resistance to killing and growth patterns.21,22 It has been
demonstrated that the GVL effect works more efﬁciently
for minimal residual disease than for active disease.23,24
Therefore, it is reasonable that decreased relapse rates with
X3.4  108/kg was limited to early-stage diseases. Although
it may be argued that patients with CML in chronic phasegreatly inﬂuence the outcomes,25 the results were similareven if these patients were excluded from analysis.
What are effector cells of cell dose effect? Calculated withthe published data,26 1  108/kg nucleated BM cells include
8  106/kg T cells, 3  106/kg B cells and 2  106/kg nature
killer cells. Considering the cell dose used in adaptive
immunotherapies with these cells,27–29 this number ofT cells can alter the outcome but that of nature killer cells
will not. Therefore, we speculated that T cells would be themost likely population affecting relapse rates. As theregistry did not have data as to graft composition during

the study period, we could not conﬁrm this hypothesis in
our data. Using total nucleated cells as the surrogate forcell dose may have limitations because some studies showedthat more speciﬁc fractions, such as CD34 þ cell dose also
predicted transplant outcomes.30,31 Future studies analyzing the effect of subpopulations in grafts are warranted.
Many previous studies reported that higher cell dose
decreased NRM, particularly related to infection.7,8,12,32
However, no signiﬁcant effects of cell dose on NRM rates
were observed in our study. To address this discrepancy, we
performed a further analysis on causes of NRM according
to cell dose, which showed no signiﬁcant differences in the
proportions of deaths from infection both in children andadults. This would partly account for the discrepancy.
In light of the study which reported that 7  107/kg
nucleated cells are enough to induce GVHD after donor
leukocyte infusion,33 higher cell dose may result inincreased incidences of GVHD. However, most of theprevious studies showed that cell dose had no effect onacute GVHD or that higher cell dose decreased acute
GVHD.7,8,18 They speculated a possible effect of accessory
cells, such as MSCs, and a possibility that higher cell dose
decreased early post transplant infections that might
Bone Marrow Transplantation

Cell dose effects in unrelated BMT
Y Inamoto et al

10amplify GVHD. Our results were compatible with thesereports. We could not explain why o3.0  108/kg resulted
in increased incidences of chronic GVHD among children
with advanced-stage diseases.
There are two possible explanations for the discrepancy
observed with regard to the effect of cell dose on OS inchildren and adults. First, a much greater volume of
harvested marrow for adults as compared with children(almost twice the volume) might bring about higher
contamination of peripheral blood and increase the doseof graft T cells to produce the different effects.34 Second,cell dose effect might be already saturated in children
because most children received much more cell dose thanadults (7  107/kg more at median). Different analyticalpower between children and adults would not account forthe discrepancy as the point estimate of hazard ratio inchildren with early-stage diseases was more than 1.0 with
44.6  108/kg (Table 4a).
In summary, our results suggested a strategy todetermine an optimal cell dose of BMT according todisease stages to maximize the efﬁcacy of BMT and
minimize the risk of donors, although these results should
be interpreted with caution because of their retrospective
nature. In terms of overall beneﬁts, cell dose of 3.4  108/kg
or higher is recommended only for adults with early-stage
diseases. With the number of patients available for analysis
in our study, we could not show any signiﬁcant beneﬁts
associated with 4.6  108/kg or higher in children.

Conﬂict of interest
The authors declare no conﬂict of interest.

Acknowledgements
This work was supported in part by a Grant-in-Aid from the
Ministry of Health, Labour and Welfare of Japan.

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