Clinical implications of chimerism after allogeneic hematopoietic stem cell transplantation in children with non-malignant diseases.
ABSTRACT The effects of chimerism on outcomes following allogeneic hematopoietic stem cell transplantation (HSCT) are unclear and may differ between diseases. We retrospectively evaluated the association between chimerism and transplant outcomes in children with nonmalignant diseases.
Chimerism was evaluated using short-tandem repeat polymerase chain reaction (STR-PCR) in 48 patients, with mixed chimerism (MC) defined as greater than 1% recipient cells.
The only variable exerting a significant influence on patients' chimerism status was the number of infused CD34+ cells. MC was detected in 23 transplants (9 showing transient MC; 10 with sustained low levels [≤30%] of autologous cells; and 4 with high-level MC [>30%]). The degree of STR-PCR at 28 days after HSCT was significantly higher in patients with high-level MC than those with transient or low-level MC. All patients with transient or low-level MC successfully maintained engraftment and showed a clinical response to HSCT, whereas 2 of the 4 patients with high-level MC experienced graft failure. The incidences of grades II-IV acute and chronic graft-versus-host disease (GVHD) were significantly higher in patients with complete donor chimerism (CC) than MC. We observed no significant survival differences between CC and MC groups. However, the survival rate was lower in patients with high MC than those with low-level or transient MC (P=0.03).
In non-malignant diseases, MC may indicate a tolerant state with a decreased incidence of GVHD. However, high-level MC may signify an increased risk of graft failure and a lower survival rate.
- SourceAvailable from: templehealth.org[show abstract] [hide abstract]
ABSTRACT: Twenty-one patients with hematologic malignancies were treated with the fludarabine (120-125 mg/m(2)) and cyclophosphamide (120 mg/kg) nonmyeloablative conditioning regimen. Graft versus host disease (GVHD) and graft rejection prophylaxis was with tacrolimus and mycophenolate mofetil. Thirteen of the 21 patients (62%) had mixed chimerism (< or = 90% donor cells) at day 60 and 11 (52%) of these patients had mixed chimerism which persisted until day 100. Immunosuppression was discontinued in 12 of 13 patients and two of them converted to full chimerism by day 100. Eight patients received a donor lymphocyte infusion (DLI) and five of them converted to full donor chimerism with DLI alone. Two patients were given GM-CSF in addition to a DLI with conversion to full donor chimerism. Three patients (14%) had graft failure requiring a second transplant using fludarabine (125 mg/m(2)) and melphalan (140 mg/m(2)). With a median followup of 2.8 years, 15 patients are alive - one with disease and 14 with no disease. Two patients died of acute GVHD, one of chronic GVHD, and three due to progressive disease. We conclude that the nonmyeloablative fludarabine/cyclophosphamide regimen results in a significant incidence of mixed chimerism and graft rejection but is well tolerated. We suggest a more intense regimen, such as fludarabine and melphalan, be used in patients with a high risk of early disease progression to establish early engraftment and graft versus tumor effect.American Journal of Hematology 06/2007; 82(6):419-26. · 4.00 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow-up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.Blood 02/1990; 75(1):296-304. · 9.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Six hundred and eighteen patients with acquired aplastic anaemia grafted from an HLA-identical sibling donor between 1976 and 1990 in eight European centres were reported to the Working Party for Severe Aplastic Anaemia (SAA) Registry and were evaluable for analysis of the incidence of graft failure/rejection and the outcome of second bone marrow transplants (BMT). The number of patients experiencing graft rejection declined significantly over the study period from 32% to 8% (p < 0.0001). This coincided with the introduction of cyclosporine to the conditioning regimen for BMT. The graft rejection rate in the post-hepatitis SAA group was significantly lower than in the group with idiopathic SAA (4% vs 20%) (p = 0.001). The use of irradiation in the conditioning regimen significantly reduced the number of patients experiencing graft rejection (7% vs 21%) (p = 0.004). Age, sex and severity of disease did not influence the rate of sustained engraftment. Of the 85 patients experiencing graft rejection, 41 received a second transplant: their survival is 33% vs 8% for patients not transplanted a second time (p = 0.003). The major factor predicting the outcome of second BMT for SAA was the interval from first BMT. Patients receiving a second BMT within 60 days from the first BMT had a significantly poorer outcome.Bone Marrow Transplantation 04/1994; 13(3):233-7. · 3.54 Impact Factor
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V O LU M E 46ㆍ N U M BER 4ㆍ D ecem ber 2011
THE KOREAN JOURNAL OF HEMATOLOGY
O R I G I N A L A R T I C L E
Clinical implications of chimerism after allogeneic hematopoietic
stem cell transplantation in children with non-malignant diseases
Meerim Park1, Kyung Nam Koh2, Jong Jin Seo2, Ho Joon Im2
Department of Pediatrics, 1College of Medicine, Chungbuk National University, Cheongju, 2Asan Medical Center Children’s Hospital,
University of Ulsan College of Medicine, Seoul, Korea
p-ISSN 1738-7949 / e-ISSN 2092-9129
Korean J Hematol 2011;46:258-64. Korean J Hematol 2011;46:258-64.
Received on November 30, 2011
Revised on December 5, 2011
Accepted on December 7, 2011
The effects of chimerism on outcomes following allogeneic hematopoietic stem cell trans-
plantation (HSCT) are unclear and may differ between diseases. We retrospectively eval-
uated the association between chimerism and transplant outcomes in children with non-
Chimerism was evaluated using short-tandem repeat polymerase chain reaction
(STR-PCR) in 48 patients, with mixed chimerism (MC) defined as greater than 1% recipient
The only variable exerting a significant influence on patients’ chimerism status was the
number of infused CD34＋ cells. MC was detected in 23 transplants (9 showing transient
MC; 10 with sustained low levels [≤30%] of autologous cells; and 4 with high-level MC
[＞30%]). The degree of STR-PCR at 28 days after HSCT was significantly higher in patients
with high-level MC than those with transient or low-level MC. All patients with transient
or low-level MC successfully maintained engraftment and showed a clinical response to
HSCT, whereas 2 of the 4 patients with high-level MC experienced graft failure. The in-
cidences of grades II-IV acute and chronic graft-versus-host disease (GVHD) were sig-
nificantly higher in patients with complete donor chimerism (CC) than MC. We observed
no significant survival differences between CC and MC groups. However, the survival
rate was lower in patients with high MC than those with low-level or transient MC
In non-malignant diseases, MC may indicate a tolerant state with a decreased incidence
of GVHD. However, high-level MC may signify an increased risk of graft failure and a lower
Non-malignant disease, Allogeneic hematopoietic stem cell
Ho Joon Im, M.D., Ph.D.
Department of Pediatrics, Asan Medical
Center Children’s Hospital, University of
Ulsan College of Medicine, Pungnap-dong,
Songpa-gu, Seoul 138-736, Korea
Ⓒ2011 Korean Society of Hematology
Allogeneic hematopoietic stem cell transplantation
(Allo-HSCT) is a well-established treatment for several non-
malignant diseases, including severe aplastic anemia (SAA),
inherited bone marrow failure (BMF) syndromes, im-
munodeficiencies, and metabolic disorders . Although
complete donor hematopoiesis is a desirable outcome of al-
lo-HSCT in malignant disorders, complete replacement of
the recipient’s hematopoietic system is not considered neces-
sary to improve the underlying disease state in patients with
non-malignant disorders. This coexistence of host and donor
hematopoietic cells is known as mixed chimerism (MC).
Rates of graft failure and rejection are high in patients
with non-malignant diseases [2, 3]. MC has been linked
to graft rejection [4, 5], suggesting that early testing for
posttransplant chimerism may help to identify patients at
increased risk of graft rejection, who may therefore require
immunotherapy . Although patients with a significant
Korean J Hematol 2011;46:258-64.
Chimerism in non-malignant diseases 259
Table 1. Characteristics of patients and donors.
Follow-up duration, months, median (range)
Median age, years, median (range)
No. CD34×106/kg infused, median (range)
Bone marrow failure (BMF)
Severe aplastic anemia
Other diseases in BMFa)
Chronic granulomatous disease
Severe combined immunodeficiency
Matched related (8/8)
1 mismatch (7/8)
2 mismatches (6/8)
≥3 mismatches (≤5/8)
Matched unrelated (8/8)
1 mismatch (7/8)
2 mismatches (6/8)
≥3 mismatches (≤5/8)
Stem cell source
a)Other diseases in BMF: 2 pure red cell anemia, 2 congenital
amegakaryocytic thrombocytopenia, 1 Kostmann disease, and 1
congenital dyserythropoietic anemia. b)Neurodegenerative disea-
ses: 2 Krabbe disease and 1 adrenoleukodystrophy.
proportion of host cells are known to be at increased risk
of rejection in cases of malignant disease, only a few studies
have focused exclusively on children with non-malignant
diseases [7, 8]. Furthermore, the utility of chimerism in pre-
dicting subsequent transplant outcomes in non-malignant
diseases has not yet been established.
The main aims of this study were to identify the variables
affecting chimerism and to determine the impact of MC
on transplantation outcomes in patients undergoing al-
lo-HSCT for non-malignant diseases.
MATERIALS AND METHODS
1. Patients and donors
Between April 2000 and March 2011, 48 children under-
went 50 allo-HSCTs for non-malignant diseases at the Asan
Medical Center Children’s Hospital in Seoul, Korea. In this
study, we analyzed the initial HSCT data from all patients.
Median age at first HSCT was 8.4 years (range, 0.6-20.5
years), and the median duration of follow-up was 41 months
(range, 8-138 months). Diseases were classified as BMF or
non-BMF. All donor-recipient pairs were fully typed for
HLA-A, -B, -C, and -DR using high-resolution molecular
typing. As a surrogate marker of iron overload at the time
of transplantation, serum ferritin was routinely measured
as a part of the pretransplant work-up before the beginning
of the conditioning regimen using a 2-site sandwich immuno-
assay with direct chemiluminescence (ADVIA Centaur,
Siemens). All patients were stable, without fever or other
events, suggesting that pretransplant ferritin levels are a good
estimator of iron status and are not significantly affected
by an inflammatory state. Data were analyzed in October
2011. The demographic and clinical characteristics of patients
and donors are summarized in Table 1. This retrospective
study was approved by the institutional review board at
the Asan Medical Center in Seoul, Korea.
2. Graft-versus-host disease
Acute and chronic graft-versus-host diseases (GVHD)
were graded according to the established criteria .
3. Analysis of chimerism
Whole blood samples were collected from individual do-
nors and recipients at 1 (day 28), 2, 3, 6, and 12 months
after allo-HSCT. In cases where MC was detected, chimerism
was tested at more frequent intervals. DNA was prepared
from whole blood samples using the QIAamp Blood Kit
(Qiagen, Hilden, Germany) and quantified spectrophoto-
metrically. Polymorphic short-tandem repeat (STR) markers
were amplified using the AmpFlSTR Profiled Plus PCR am-
plification kit (Applied Biosystems, Foster City, CA, USA).
Amplified fragments were analyzed using an ABI PRISM
310 Genetic Analyzer (Applied Biosystems), and peak areas
were quantified using Genescan software (Applied Biosys-
tems). The percentages of donor and recipient DNA were
calculated from individual proportions of donor and recipient
peak areas . Unless otherwise stated, the degrees of
STR-PCR and MC were based on host portions.
4. Definition of chimerism and responses
Patients were stratified individually based on serial
STR-PCR analyses, with chimerism defined as described pre-
viously [7, 8]. Patients with samples showing 1% or less
autologous signals after HSCT were classified as having ach-
ieved complete donor chimerism (CC). We selected a cutoff
of 1%, since microsatellite and several tandem repeat meth-
ods have a sensitivity limit of 1-5%. MC was defined as
having a recipient fraction of 1% to 99% at any time after
HSCT. Patients initially classified as having MC who turned
into CC were classified in the transient MC group, while
those showing sustained autologous signals greater than 1%
Korean J Hematol 2011;46:258-64.
260Meerim Park, et al.
Fig. 1. Chimeric status of patients with high-level mixed chimerism.
were categorized in the sustained MC group. In the sustained
MC group, patients consistently displaying autologous signals
that did not exceed 30% were considered to have low-level
MC, while those displaying a significant increase in autolo-
gous cells, up to 30%, were considered to have high-level
MC. Patients with more than 99% recipient signals were
defined as having attained complete recipient chimerism
(RC) or autologous reconstitution.
Since there is no clear consensus on the definition of
clinical remission in patients with non-malignant diseases,
we assessed the response relative to correction of the under-
lying disease using disease-specific measures. These included
normal hemograms along with transfusion independence in
patients with BMF syndromes, immune reconstitution and
lack of infection in patients with immunodeficiencies, en-
zyme concentrations and the results of brain imaging and
neurologic examinations in patients with metabolic dis-
orders, and absence of symptoms and discontinuation of
pretransplant-specific therapies in patients with autoimmune
disorders . A complete response (CR) was defined as
a normal hemogram with eradication of the underlying
disease. A partial response (PR) was defined as the presence
of some positive effects of HSCT, but not fulfillment of the
criteria for CR (e.g., patients with BMF that displayed some
improvement, but were still transfusion dependent). Disease
progression was defined as the absence of positive effects
Graft failure comprises 2 clinical entities: (1) failure to
achieve an absolute neutrophil count of 0.5×109/L or less
and marrow hypoplasia for less than 60 days with or without
the existence of donor-type hematopoiesis, and (2) complete
loss of donor-type hematopoiesis occurring anytime after
transplantation. Late graft failure was defined as the loss
of the graft after engraftment during follow-up.
The probabilities of overall survival (OS) and event-free
survival (EFS) were estimated using the Kaplan-Meier
method. An event was defined as graft failure or death either
due to treatment toxicity or disease progression. Fisher’s
exact test was used to compare differences in categorical
variables affecting chimerism and response to HSCT between
groups, whereas the Mann-Whitney U-test was used to com-
pare continuous variables. We also attempted to model rela-
tionships between 2 continuous variables by linear
regression. For multivariate analysis, the logistic regression
model was used. Rreceiver operating characteristic (ROC)
plot analysis was performed to determine the threshold value
of CD34＋ cells for predicting chimerism. Probabilities of
acute and chronic GVHD were calculated using the cumu-
lative-incidence-function method; death without GVHD was
the competing event. All statistical analyses were performed
using the SPSS statistical program (SPSS Inc., Chicago, IL,
USA) or R 2.10.1 software (The R Foundation for Statistical
Computing, 2007). All P-values were 2-sided, with P＜0.05
considered statistically significant.
Forty-eight patients underwent allo-HSCT, and 2 required
a second transplant due to graft failure after the first HSCT.
At a median follow-up of 41 months, the 3-year OS and
EFS rates were 91.5±4.1% and 85.3±5.1%, respectively. Four
patients died, 1 due to graft failure and the other 3 due
to transplant-related complications, including veno-occlu-
sive disease, hemorrhage, multi-organ failure, and infection.
During follow-up, 23 patients (48%) showed sustained
CC and 23 (48%) showed MC. Peak MC in the groups of
patients with transient (N=9), low-level (N=10), and high-
level (N=4) MC occurred at a median of 3, 3.5, and 2.3
months, respectively. Patients with transient MC achieved
CC at a median of 7.7 months after HSCT. All 4 patients
with high-level MC showed more than 30% autologous sig-
nals within 2 months after HSCT. The chimeric status of
patients with high-level MC is shown in Fig. 1.
Two of the 4 patients with high-level MC subsequently
experienced graft failure after HSCT (Table 2). One of these
patients (UPN 88) achieved CR after the second HSCT, but
died due to transplant-related complications resulting in mul-
tiple organ failure after 5 months. The second patient (UPN
127) received 2 donor lymphocyte infusions (DLIs), but did
not achieve stable engraftment, and subsequently died due
to infection and pulmonary hemorrhage.
RC was detected on day 28 after HSCT in 2 patients,
1 with hemophagocytic lymphohistiocytosis (HLH) and 1
with SAA. Although the patient with HLH experienced graft
failure, BM showed autologous recovery, and the patient
Korean J Hematol 2011;46:258-64.
Chimerism in non-malignant diseases 261
Table 2. Characteristics of patients with high-level mixed chimerism.
68% (2 mo)
96% (3 mo)
66% (2.5 mo)
69% (1 mo)
No CR (＋91)
MMUD Yes (61 d)
Yes (61 d)
All time variables are presented on the basis of the transplantation date.
a)Patient 88 achieved stable engraftment after the second HSCT, but died of transplant-related complications that resulted in multiple organ
failure 5 months after the second HSCT. b)Although patient 127 received a second DLI, he did not achieve engraftment and died of
transplant-related complications 81 days after HSCT.
Abbreviations: PRCA, pure red cell anemia; SAA, severe aplastic anemia; KD, Kostmann disease; MRD, matched related donor; MMUD,
mismatched unrelated donor; MMRD, mismatched related donor; Cy, cyclophosphamide; ATG, antithymocyte globulin; TBI, total body
irradiation; Bu, busulfan; TCD, T-cell depletion; DLI, donor lymphocyte infusion; CR, complete remission; ND, not done.
Fig. 2. Receiver operating characteristic curve used to determine the
optimal CD34＋ cell number cut-off value for predicting chimerism.
remained in CR at data cut-off. The SAA patient achieved
neutrophil engraftment on day 11 (10% autologous portion
of MC on day 20), but neutropenia occurred at day 14,
and STR-PCR revealed 100% host signal at day 28. Owing
to graft failure, the patient underwent a second HSCT and
achieved CC with stable engraftment.
2. STR-PCR at day 28 post-HSCT
We observed a linear relationship between the degree
of day-28 STR-PCR and pretransplant ferritin concentra-
tions. Higher ferritin concentrations were significantly asso-
ciated with a higher recipient proportion of MC by day
28 after HSCT (r2=0.2, P=0.03). At day 28 after HSCT,
STR-PCR values were significantly higher in patients with
high-level MC than those with transient or low-level MC
(50.8% vs. 3.7%, P＜0.01).
We observed a linear relationship between the day-28
STR-PCR and time to engraftment. Specifically, a high pro-
portion of autologous cells at day 28 after HSCT were sig-
nificantly associated with late neutrophil (r2=0.24, P＜0.01)
and platelet (r2=0.15, P=0.02) engraftment. No differences
were evident in the day-28 STR-PCR between patients with
PR and CR. However, STR-PCR at day 28 was significantly
lower in the response than the non-response group (6.1%
vs. 66.0%, P＜0.01).
3. Variables affecting chimerism
The variables tested for effects on chimerism included
patient age, disease category, numbers of infused CD34＋
and CD3＋ cells, source of stem cells, donor type, degree
of HLA match, pretransplant serum ferritin concentration,
and intensity of the conditioning regimen. Increasing MC
was only observed in patients with BMF. The number of
infused CD34＋ cells was significantly higher in the CC
group than in the MC group (13.2×106/kg vs. 7.2×106/kg,
respectively; P＜0.01). Similarly, we observed a marked in-
crease in the number of infused CD3＋ cells in the CC
group compared to the MC group (4.92×108/kg vs. 2.61×
108/kg, respectively; P=0.02). The only variable with a sig-
nificant influence on patients’ chimerism status was the num-
ber of infused CD34＋ cells, as determined by multivariate
analysis. Using ROC curves, the optimal cut-off value of
CD34＋ cells for predicting the chimerism status was esti-
mated to be 8.35×106/kg (70.6% sensitivity, 69.2% specific-
ity), indicating that patients who received less than 8.35×
106/kg of CD34＋ cells are at higher risk of developing MC
4. Chimerism and transplantation outcomes
The relationship between chimerism status and clinical
response is shown in Table 3. All patients with CC and
low-level/transient MC showed clinical responses to HSCT.
Among the 23 patients with posttransplant CC, 20 achieved
CR, including 1 who experienced late graft failure 39 months
after HSCT, but achieved CR after immunosuppressive
therapy. The remaining 3 patients achieved PR, 1 each with
adrenoleukodystrophy, Krabbe disease, and SAA. Among the
23 patients with posttransplant MC, 21 showed clinical re-
Korean J Hematol 2011;46:258-64.
262 Meerim Park, et al.
Table 3. Posttransplantation outcome according to chimerism status.
a)Although the patient maintained CR with CC for 39 months after transplantation, he showed bone marrow hypoplasia in spite of having CC
and was rescued by immunosuppressive therapy. b)Died of transplant-related complications with partial or complete response to the
transplantation. c)Died of graft failure.
Abbreviations: CC, complete donor chimerism; MC, mixed chimerism; RC, recipient chimerism; CR, complete response; PR, partial response;
NR, no response; TPL, transplantation; DLI, donor lymphocyte infusion.
Fig. 3. Kaplan-Meier curves showing overall survival in high-level mixed
chimerism and transient or low-level mixed chimerism groups.
Fig. 4. Kaplan-Meier curves showing event-free survival in high-level
mixed chimerism and transient or low-level mixed chimerism groups.
sponses to HSCT. Two patients with high-level MC experi-
enced graft failure. The incidence of CR in the transient,
low-level, and high-level MC subgroups was 89% (8/9), 80%
(8/10), and 50% (2/4), respectively (P=0.03). The incidence
of graft failure was significantly higher in the high-level
MC subgroup than the low-level and transient MC subgroups
(P=0.03). Higher ferritin concentrations before HSCT (P＜
0.01) and lower numbers of infused CD34＋ cells (P=0.01)
were additionally associated with graft failure.
The incidences of grades II?IV acute and chronic GVHD
were significantly higher in CC patients than in MC patients
(56.5% vs. 13.0%, P＜0.01; and 47.8% vs. 21.7%, P=0.04,
respectively). No significant differences in survival were evi-
dent between the CC and MC groups. However, the OS
rate was significantly lower in patients with high-level MC
than in patients with low-level and transient MC (50.0%
vs. 93.8%, P=0.03; Fig. 3). The EFS rate of patients with
high-level and low-level/transient MC was 87.5% and 50.0%,
respectively (P=0.05; Fig. 4). The causes of death in the
high-level MC group included graft failure and multi-organ
failure after second HSCT.
This study presents a retrospective analysis of chimerism
status and the effects of chimerism on clinical outcomes
in children undergoing HSCT for non-malignant diseases.
Allo-HSCT was considered the only curative approach for
non-malignant diseases in our patients. In our cohort of
48 patients receiving 50 allo-HSCTs, 5 showed graft failure.
The overall survival rate was 91.5%, similar to that reported
in earlier studies involving patients with non-malignant dis-
eases [2, 12].
Unlike hematologic malignancies, where 100% donor en-
graftment is always desired, the number of donor cells re-