review

Clinical significance of minimal residual disease in patients

with acute leukaemia undergoing haematopoietic stem cell
transplantation

Dario Campana1 and Wing Leung2,3

1
Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 2Department of Bone
Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, and 3Department of Pediatrics, College of
Medicine, University of Tennessee Health Science Center, Memphis, TN, USA

Summary insights have been gained into the pathogenesis of acute leu-
kaemia. In turn, this has led to a mounting arsenal of novel
In patients with acute leukaemia, the relative risk of relapse
treatment agents. Perhaps more than ever before, it is now
influences the choice between chemotherapy and haemato-
imperative to precisely identify patients who have high
poietic stem cell transplantation (HSCT). The demonstration
chances of cure with standard therapy, those who could be
that minimal residual disease (MRD) is the strongest overall
candidates for experimental therapies, and those for whom
prognostic indicator and can identify patients who are unli-
haematopoietic stem cell transplantation (HSCT) is the only
kely to be cured by standard chemotherapy has added a
possible curative option. In this regard, presenting clinical
powerful new factor to consider when making this decision.
and/or biological features can be helpful but their prognostic
There is substantial data indicating that the likelihood of
accuracy is limited.
relapse after transplant is directly correlated with levels of
Levels of residual disease at any time point during treat-
MRD before transplant. This knowledge can be used to
ment represent an essential parameter for drawing meaning-
adjust the timing of HSCT, and guide the selection of donor,
ful prognostic algorithms and selecting the best available
conditioning regimen, and post-HSCT strategies to maximize
treatment strategies. Achievement of morphological remission
the graft-versus-leukaemia effect. Because MRD emerging
after initial treatment is a prerequisite for cure but patients
post-transplant carries a dire prognosis, its detection can
who are in morphological remission may have varying,
trigger withdrawal of immunosuppression, additional cellular
sometimes high, levels of minimal residual disease (MRD), a
and molecular therapies, or preparations for a second HSCT.
concept proposed well before it was possible to detect it
Although it is not yet clear whether any of these actions will
(Mathe et al, 1966). Initial efforts to measure MRD tried to
significantly improve outcome, it is likely that they will be
exploit possible differences in the growth patterns of normal
most effective for patients with a relatively low tumour bur-
and leukaemia cells but results appeared to be largely subjec-
den, who can be identified only through MRD testing. In
tive and hard to reproduce (Harris & Freireich, 1970; Moore
this article, we review the clinical significance of MRD in the
et al, 1974; Mertelsmann et al, 1981). Cytogenetic studies can
context of autologous and allogeneic HSCT.
identify residual disease in some patients considered to be in
complete clinical and haematological remission, a finding
Keywords: acute lymphoblastic leukaemia, acute myeloid
that correlates with a higher risk of relapse (Freireich et al,
leukaemia, minimal residual disease, haematopoietic stem cell
1992; Chen et al, 2011). This method, however, has a limited
transplantation.
sensitivity, even when it is complemented by fluorescence
in situ hybridization (Bacher et al, 2006).
With the increasing understanding of the genetic lesions
Current approaches to measure MRD in acute
underlying leukaemogenesis and of the factors that influence
lymphoblastic leukaemia (ALL) rely primarily on polymerase
the growth and drug resistance of leukaemic cells, important
chain reaction (PCR) amplification of clonally rearranged
antigen-receptor genes and on flow cytometry (Bruggemann
et al, 2010; Campana, 2012). Measurements of MRD in acute
Correspondence: Dario Campana, Department of Paediatrics, Yong myeloid leukaemia (AML) are based on PCR amplification
Loo Lin School of Medicine, National University of Singapore, of leukaemia-associated genetic abnormalities or on flow
Centre for Translational Medicine, 14 Medical Drive, Level 9-02, cytometric detection of leukaemia-associated immunopheno-
Singapore 117599, Singapore. types (Shook et al, 2009; Buccisano et al, 2012). After HSCT,
E-mail: paedc@nus.edu.sg MRD can also be monitored by analysis of short tandem

ª 2013 John Wiley & Sons Ltd First published online 9 May 2013
British Journal of Haematology, 2013, 162, 147–161 doi:10.1111/bjh.12358
Review

repeats (STR) or variable number tandem repeats (VNTR)
chimerism (Antin et al, 2001). The characteristics of the
methodologies for MRD detection have been extensively
reviewed elsewhere and we refer the readers to the above
articles for a list of relevant publications; Table I summarizes
their main features. We here focus on the clinical importance
of MRD in the context of HCST.
Prognostic significance of MRD prior to HSCT
Acute lymphoblastic leukaemia
The prognostic significance of MRD levels prior to HSCT in
children and adolescents with ALL is well established
(Table II). Knechtli et al (1998a) evaluated MRD by PCR
amplification of antigen-receptor genes in 64 children with
ALL (19 in first remission) 6–81 d prior to HSCT. In this
study, MRD was analysed by using consensus primers to
amplify rearranged immunoglobulin and/or T-cell receptor
(TCR) genes; the PCR products were then separated accord-
ing to their size by polyacrylamide gel electrophoresis and
transferred to a nylon membrane by electroblotting. High-
level MRD (approximately 1–0
1%) was defined when a
clonal band was evident; low-level MRD (0
1–0
001%) when
a band became evident only after probing the membrane
with an oligonucleotide complementary to the junctional
sequence of the predominantly rearranged gene as
determined at diagnosis (Knechtli et al, 1998a). MRD was
‘high’ in 12 patients, ‘low’ in 11 and ‘negative’ in 41; after
excluding eight patients who had an isolated extramedullary
relapse and were MRD-negative in the bone marrow prior to
HSCT, the 2-year event-free survival (EFS) for the three
groups was 0%, 36%, and 73%, respectively (P < 0
001;
Knechtli et al, 1998a). Using a similar methodology, Bader
et al (2002) studied 41 children and adolescents who were in
first or subsequent remission at the time of HSCT: MRD was
high in 17 patients, low in 10 and negative in 14; their 5-year
EFS was 23%, 48% and 78%, respectively (P = 0
022). A
third pioneering study was performed by van der Velden
et al (2001) who targeted antigen-receptor genes with a more
contemporary approach, based on patient-specific primers
complementary to the unique junctional region identified at
diagnosis (also known as allele-specific oligonucleotides,
ASO); MRD was quantified by ‘real-time’ (RQ-) PCR: relapse
post-HSCT occurred in four of the six MRD-positive and
two of the 11 MRD-negative patients (van der Velden et al,
2001). The results of these three studies were combined
with unpublished data in a landmark analysis performed
by the Pre-BMT MRD Study Group including 140 children
with ALL (85 in second or higher remission; Krejci et al,
2003). The 5-year EFS for patients with high, low or
undetectable MRD prior to HSCT was 21%, 41% and
75%, respectively (P < 0
001); MRD was an independent
prognostic factor in a multivariable analysis.

Table I. Methods to monitor minimal residual disease in patients undergoing haematopoietic stem cell transplantation.

PCR of genetic Chimerism assay by

PCR of IG/TCR genes abnormalities Flow cytometry STR/VNTR

Sensitivity 10 4 to 10 5 10 4 to 10 5 10 3 to 10 5 10 2
Applicability B-lineage ALL: c. 95% B-lineage ALL: c. 35% B-lineage ALL: >95% 100%
T-lineage ALL: c. 95% T-lineage ALL: c. 15% T-lineage ALL: >95%
AML: <10% AML: c. 40% AML: c. 95%
Advantages  Sensitivity  Sensitivity  Applicable to most patients  Applicable to all patients
 Specificity  Specificity and  Rapid  No diagnostic sample required
 Accurate quantification stability of marker  Quantitative  Rapid
 Standardized  Fast  Established predictive value  Standardization possible
 Stability of DNA  Relatively cheap  Additional information on  Relatively cheap
 Established normal haematopoiesis  Additive information on
predictive value impending graft rejection
 Stability of DNA
Disadvantages  Not useful for AML  Predictive value less  Potential for phenotypic shifts  Limited sensitivity
 Establishment of PCR established  Relatively expensive  Applicable only after HSCT
conditions labour-intensive*  Variability in transcript:  Extensive expertise required
 Potential for clonal evolution* cell ratio
 Extensive expertise required  Not applicable to
 Relatively expensive most patients
 Instability of RNA

PCR, polymerase chain reaction; IG, immunoglobulin; TCR, T-cell receptor; STR, short tandem repeats; VNTR, variable number tandem repeats;
ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; HSCT, haematopoietic stem cell transplantation.
*These limitations are overcome by newer methods to measure minimal residual disease using deep sequencing of IG/TCR genes, which can also
increase the sensitivity beyond 10 6 (Faham et al, 2012; Wu et al, 2012).

148 ª 2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 162, 147–161
 Table II. Prognostic significance of minimal residual disease prior to haematopoietic stem cell transplantation in acute lymphoblastic leukaemia.

Outcome

MRD Pos

Type No. of MRD Low High

Study of HSCT patients Age group Method Estimate Neg MRD* MRD* P

Knechtli et al (1998a) Allo 64 Paediatric PCR IG/TCR 2-year EFS 73% 36% 0% <0
001
Bader et al (2002) Allo 41 Paediatric PCR IG/TCR 5-year EFS 78% 48% 23% 0
022

ª 2013 John Wiley & Sons Ltd

Dombret et al (2002) Allo 63 Adult RT-PCR BCR-ABL1 3-year incidence 41% 75% 0
01
of relapse
Mortuza et al (2002) Auto 13 Adult PCR IG/TCR Relapses/patients 0/6 6/7 0
005‡
studied
Krejci et al (2003) Allo 140 Paediatric PCR and ASO 5-year EFS 75% 41% 21% <0
001
RQ-PCR IG/TCR
Sramkova et al (2007) Allo 25 Paediatric ASO RQ-PCR IG/TCR Relapses/patients 1/17 6/8 0
001‡

British Journal of Haematology, 2013, 162, 147–161

studied
Spinelli et al (2007) Allo 37 Adult ASO RQ-PCR 3-year incidence 0% 46% 0
027
IG/TCR or RT of relapse
(RQ)-PCR fusion 3-year OS 80% 49% NS
transcripts
Paganin et al (2008) Allo 25 Paediatric ASO RQ-PCR IG/TCR Relapses/patients 2/14 8/11 0
005‡
studied
Bader et al (2009) Allo 91 Paediatric ASO RQ-PCR IG/TCR 4-year incidence 11% 20%/64%† 57% <0
001
of relapse
4-year EFS 64% 48%/19%† 31% 0
036
Patel et al (2010) Allo 36 Adult PCR and ASO Relapses/patients 4/23 2/13 NS
RQ-PCR IG/TCR studied
5-year EFS 50% 52% NS
Patel et al (2010) Auto 25 Adult PCR and ASO 5-year LFS 77% 25% 0
01
RQ-PCR IG/TCR
Giebel et al (2010) Auto 103 Adult Flow cytometry or 5-year LFS 57% 17% <0
001
ASO RQ-PCR
IG/TCR or
RT(RQ)-PCR fusion
transcripts
Elorza et al (2010) Allo 31 Paediatric Flow cytometry 2-year EFS 75% 20% <0
01
Leung et al (2011) Allo 64 Paediatric Flow cytometry 5-year incidence 6% 28% 0
03
of relapse
5-year OS 68% 30% 0
008
Sanchez-Garcia et al (2012) Allo 102 Both Flow cytometry 5-year LFS 66% 43% 0% <0
001
5-year OS 52% 29% 0% <0
001

149
Review
 Review

HSCT, haematopoietic stem cell transplantation; MRD, minimal residual disease; allo, allogeneic; auto, autologous; UCB, umbilical cord blood; PCR, polymerase chain reaction; IG, immunoglobulin
These early findings in childhood ALL were confirmed

genes; TCR, T-cell receptor genes; RT, reverse transcriptase; ASO, allele-specific oligonucleotides; RQ, real time; RT, reverse transcription; EFS, event-free survival; OS, overall survival; LFS, leukae-
0
006

0
05

0
02
and extended in more recent studies (Sramkova et al, 2007;

P
Paganin et al, 2008; Elorza et al, 2010). A prospective study
of 91 children with relapsed ALL who received HSCT in sec-
ond or subsequent remission demonstrated the relationship

MRD*
High between MRD levels prior to HSCT as measured by ASO
RQ-PCR and risk of relapse: 4-year EFS was 31% for patients
MRD Pos

29% with MRD  0
1% (n = 33), 19% for patients with MRD

30%

30%
 0
01–<0
1% (n = 12), 48% for those with MRD detectable
below <0
01% (n = 10) and 64% for patients with undetect-
MRD*

able MRD (n = 36; P = 0
036); cumulative incidence of

Low

relapse was 54%, 64%, 20% and 11%, respectively

(P < 0
001; Bader et al, 2009). In this study, MRD was also
predictive of outcome among patients with different present-
MRD

54%

16%

55%

ing risk features; in a multivariate Cox proportional hazards

Neg

model including sex, age, remission status, time and site of

relapse, immunophenotype, donor type, T-cell depletion,
time to transplantation, and graft-versus-host disease
2-year incidence

(GvHD), MRD was the only independent prognostic factor

of relapse

(Bader et al, 2009). In an analysis of 64 patients with high-

4-year LFS

3-year LFS
Outcome

Estimate

risk ALL who underwent HSCT at St. Jude Children’s

Research Hospital, we found that MRD before transplant as
measured by flow cytometry was a significant and indepen-
dent prognostic factor: 5-year cumulative incidence of relapse
was 28% for the 30 patients who had MRD  0
01% and
or fusion transcripts§

†Two subgroups with relatively low levels of MRD were analysed in this study: <0
01% and 0
01% to <0
1%.

6% for the 34 with MRD <0
01% or undetectable

(P = 0
03); overall survival (OS) rates were 30% and 68%,
Flow cytometry;

Flow cytometry
PCR IG/TCR

respectively (P = 0
008; Leung et al, 2011). Among patients

with detectable MRD, higher levels of MRD predicted a
Method

poorer outcome (Leung et al, 2012).

The prognostic importance of MRD prior to HSCT has
also been demonstrated for adult patients with ALL
(Table II). Detection of BCR-ABL1 transcripts prior to trans-
Age group

plant in patients with Philadelphia (Ph)-positive ALL was a

Paediatric

significant predictor of outcome in an early study: 3-year

‡Calculated by Fisher’s exact test based on the data reported in the article.
Both

incidence of relapse was 75% for patients with detectable

transcripts by semi-quantitative reverse-transcriptase (RT)
PCR (n = 39) and 41% for those with no detectable tran-
scripts (n = 24; P = 0
01; Dombret et al, 2002). More
patients
No. of

recently, it was reported that the degree of response to imati-

170

86

nib prior to transplant in patients with this leukaemia

subtype, as measured by RT(RQ)-PCR MRD monitoring,
was a major determinant of relapse after HSCT (Lee et al,
of HSCT

2012). MRD before transplant evaluated in 37 patients with

Type

UCB

UCB

either Ph-positive or Ph-negative ALL by using RT(RQ)-

mia-free survival; NS, not significant.

PCR targeting BCR-ABL1 or MLL-AFF1 fusion transcripts, or

§PCR methology used not stated.
*Definition varies among studies.

ASO RQ-PCR targeting rearrangements of antigen-receptor

genes yielded prognostic information: cumulative incidence
of relapse was 46% for MRD-positive (n = 25) and 0% for
Bachanova et al (2012)
Table II. (Continued)

MRD-negative patients (n = 12; P = 0
027) after a median

Ruggeri et al (2012)

follow-up of 23 months (Spinelli et al, 2007). In another

study supporting the prognostic significance of pre-HSCT
MRD, this was measured by flow cytometry in 102 children
and adult patients (including 55 older than 14 years of age)
Study

and found to be >0
1% in 18, 0
01 to  0
1% in 12 and

150 ª 2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 162, 147–161

undetectable in 72 patients; leukaemia-free survival (LFS)
rates were 0%, 43% and 66%, respectively (P < 0
001); OS
rates were 0%, 29% and 52% (P < 0
001), with MRD being
the most significantly adverse prognostic factor in a multi-
variable analysis (Sanchez-Garcia et al, 2012).
It should be noted that significant correlations between
MRD and outcome did not emerge in some analyses. Thus,
in a study using PCR amplification of immunoglobulin genes
using either consensus primers to detect the presence of
clonal rearrangements or ASO PCR, MRD was a prognostic
factor overall but was not predictive of relapse among the 19
patients who received allogeneic HSCT in first remission
(Mortuza et al, 2002). Lack of prognostic power was also
noted in a subsequent study from the same group, this time
also including ASO RQ-PCR to detect MRD: among 36
patients with B-lineage ALL receiving allogeneic HSCT in
first remission, 13 were MRD positive and 2 relapsed as
compared to 4 of the 23 patients with MRD negative or
<0
01%; 5-year EFS was 52% vs. 50% (Patel et al, 2010). A
multitude of factors may have caused the discrepancy
between these and other studies, including differences in
cohort composition, pre-transplant chemotherapy and condi-
tioning, donor source and graft-versus-leukaemia effect post-
transplant. The collective results of correlative studies,
however, would indicate that MRD levels prior to transplant
does exert a considerable influence on the success of alloge-
neic HSCT. Recent data with large cohorts of patients under-
going unrelated cord blood transplantation confirmed the
prognostic power of pre-HSCT MRD. One study determined
MRD levels by either flow cytometry or molecular methods
in 170 children with ALL (72 in first remission, 98 in second
or higher remission) and the 4-year LFS was 29% for MRD
positive (n = 74) and 54% for MRD-negative patients
(n = 96; P = 0
006; Ruggeri et al, 2012). Another study used
flow cytometry to study MRD in 86 children and adult ALL
patients: patients with detectable MRD before transplant
(n = 10) had a 2-year relapse rate of 30% and 3-year LFS of
30% as compared to 16% and 55% for the MRD-negative
patients (n = 76; P = 0
02; Bachanova et al, 2012).
Perhaps not unexpectedly, autologous HSCT is associated
with a very poor outcome when the graft is MRD-positive
(Mizuta et al, 1999). Among the 13 adult patients who
received autologous HSCT studied by Mortuza et al (2002),
six of the seven with MRD-positive harvests subsequently
relapsed whereas none of the six who received an MRD-
negative graft did. In the subsequent study by the same
group, MRD positivity prior to autologous HSCT was signif-
icantly associated with a lower rate of LFS at 5-years: 25%
vs. 77% for MRD-negative patients (P = 0
01); of the nine
harvest samples studied, four were positive and three of the
patients who received them relapsed within 9 months from
transplant while none of the five who received MRD-negative
grafts relapsed after a median follow-up of 8
4 years (Patel
et al, 2010). A study of the European Study Group for Adult
ALL analysed data from 103 recipients of autologous HSCT
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161
Review
with Ph-negative ALL (Giebel et al, 2010). The 5-year LFS
was 17% for patients with MRD  0
1% prior to HSCT by
either flow cytometry or ASO RQ-PCR amplification of anti-
gen-receptor genes and 57% for patients with <0
1% or
undetectable MRD (P = 0
0002). The difference was particu-
larly striking for patients with T-lineage ALL: 8% (n = 33)
vs. 62% (n = 13; P = 0
001), and MRD remained the only
predictor in a multivariate analysis including other known
prognostic factors (Giebel et al, 2010).
Acute myeloid leukaemia
In a study of paediatric AML, levels of the WT1 gene were
measured 2 weeks prior to allogeneic HSCT in peripheral
blood of 36 patients who were in morphological remission
(Jacobsohn et al, 2009). Using a cut-off level to define MRD
positivity based on the highest level of WT1 determined in
most healthy individuals by RT-PCR (after normalizing the
values to those obtained in the WT1-positive cell line K562),
11 patients were considered to be MRD-positive: seven
relapsed and two died of transplant-related mortality while
none of the 25 MRD-negative patients relapsed (although
three did not engraft and eight died of transplant-related
mortality); 5-year EFS was 68% vs. 18% (P = 0
007), and
MRD was the only significant prognostic factor in a multi-
variate analysis (Jacobsohn et al, 2009). We used flow cytom-
etry to measure MRD before HSCT in 58 children and
adolescents with AML: 5-year cumulative incidence of relapse
was 37% for the 25 with detectable MRD and 7% for the 33
MRD-negative patients (P = 0
005); OS rates were 47% and
68%, respectively (P = 0
05; Leung et al, 2011).
In the context of autologous HSCT, it is clear that AML
cells present in the inoculum can contribute to relapse (Bren-
ner et al, 1993) and studies have shown significant correla-
tions between levels of MRD as measured by flow cytometry
or PCR amplification of WT1 prior to autologous HSCT
with risk of relapse (Feller et al, 2005; Osborne et al, 2005).
In an early study using flow cytometry, 12 patients had
MRD higher than 0
035% (the best discriminative threshold
according to these investigators) prior to autologous HSCT
and all relapsed at a median of 7 months post-transplant
(Table III). By contrast, among the 19 with lower levels of
MRD, five patients relapsed (median time post-transplant, of
11 months) and 14 remained in remission with a median
follow-up of 56 months (P < 0
001); MRD status was the
strongest prognostic factor in a multivariate analysis (Vend-
itti et al, 2003). Subsequently, this group of investigators
extended their studies to a total of 77 patients who under-
went autologous or allogeneic HSCT: 5-year LFS was 22%
for the 42 who had MRD >0
035% before transplant and
55% for the 35 who had lower MRD (Maurillo et al, 2008).
The prognostic importance of MRD was clearly demon-
strated in a recent study of 99 patients receiving allogeneic
HSCT in first remission: the 24 with detectable MRD by flow
cytometry had a 2-year OS of 30% as compared to 77% for
151
Review

Table III. Prognostic significance of minimal residual disease prior to haematopoietic stem cell transplantation in acute myeloid leukaemia.

Outcome

Type of No. of MRD MRD

Study HSCT patients Age group Method Estimate Neg Pos* P

Venditti et al (2003) Auto 31 Adults Flow cytometry Relapses/patients studied 5/19 12/12 <0
001†
Maurillo et al (2008) Allo/Auto 77 Adult Flow cytometry 5-year LFS 55% 22% 0
004
Jacobsohn et al (2009) Allo 36 Paediatric RT-PCR WT1 1-year incidence of relapse 0% 76% <0
001
5-year EFS 68% 18% 0
007
Leung et al (2011) Allo 58 Paediatric Flow cytometry 5-year incidence of relapse 7% 37% 0
005
5-year OS 68% 47% 0
05
Walter et al (2011) Allo 99 Both Flow cytometry 2-year incidence of relapse 18% 65% <0
001
2-year OS 77% 30% <0
001

HSCT, haematopoietic stem cell transplantation; MRD, minimal residual disease; allo, allogeneic; auto, autologous; RT-PCR, reverse transcrip-
tion-polymerase chain reaction; LFS, leukaemia-free survival; EFS, event-free survival; OS, overall survival.
*Definition varies among studies.
†Calculated by Fisher’s exact test based on the data reported in the article.

the 75 MRD-negative patients; 2-year rates of relapse were
65% and 18%, respectively; MRD remained a significant pre-
dictor of outcome after adjusting for known prognostic vari-
ables, and when analyses were restricted to patients who were
in cytogenetic remission (Walter et al, 2011).
Prognostic significance of MRD after HSCT
Acute lymphoblastic leukaemia
The signal detected by an MRD assay might reveal, directly
or indirectly, the presence of leukaemia stem-cells or simply
originate from end-stage cells lacking self-renewing potential.
If the former is true, one would expect that detection of
MRD post-HSCT will be followed by overt relapse in the
majority of cases unless further therapy is initiated or graft
anti-leukaemia mechanisms prevail. Indeed, most studies
have shown a strong correlation between MRD post-HSCT
and relapse (Table IV).
An early study used consensus primers and PCR amplifi-
cation to detect clonal rearrangements of immunoglobulin
genes in 20 patients with ALL, 17 of whom had been trans-
planted with relapse or refractory disease. Among the 13
MRD-positive patients in the first 100 d after transplant, 11
relapsed and the other two died; there was one relapse
among the seven patients who were MRD-negative, while
two died and the remaining four were alive and well at the
time of the report (Radich et al, 1995). Using consensus
primer-based PCR to target immunoglobulin and TCR genes,
Knechtli et al (1998b) monitored MRD in children with ALL
who received allogeneic HSCT. Among the 68 evaluable
patients with B- or T-lineage ALL, MRD was detected in 36,
either as a consistent positive finding or emerging after an
MRD negative finding: relapse occurred in 28 of these
patients, at 1
5–11 months (median, 3 months) from the first
MRD-positive sample. By contrast, only four of the 32 who
were consistently MRD-negative subsequently relapsed, with
a median follow-up of 43 months (P < 0
0001; Knechtli
et al, 1998b). The authors suggested that technical reasons,
such as poor quality sample, clonal evolution and infrequent
sampling could explain the negative findings in 3 of the 4
patients; the fourth had an isolated testicular relapse
65 months after HSCT. Similar predictive findings were
reported by Uzunel et al (2003) who used ASO-PCR to
monitor MRD in 23 children and nine adults with ALL after
HSCT: eight of the nine MRD-positive patients relapsed
between 0
5 and 30 months from the MRD finding, in con-
trast to six relapses among the 23 MRD-negative patients,
with MRD remaining as the sole significant predictor in a
multivariate analysis. The study of Spinelli et al (2007),
which used RQ-PCR, found that the cumulative incidence of
relapse post-transplant was 80% for the 14 patients who were
MRD-positive on day +100 versus 7% for the 17 MRD-nega-
tive patients (P = 0
0006).
Other investigators used flow cytometry to monitor MRD
post-HSCT (Sanchez et al, 2002). A recent study applied this
method as well as RT(RQ)-PCR amplification of BCR-ABL1
to monitor 139 patients and found that those who were
MRD positive post-transplant had a 2-year EFS of 54% com-
pared to 80% for MRD-negative patients (P < 0
001; Zhao
et al, 2012). Relapse could be predicted by the MRD status
in 22 of 38 patients, and the time from the first MRD posi-
tive result to overt relapse was between 1 and 3 months.
Acute myeloid leukaemia
A few studies reported small cohorts of patients with genetic
subtypes of AML monitored by PCR amplification of fusion
transcripts (Elmaagacli et al, 1998; Bacher et al, 2009), while
others were performed using WT1 as a PCR target (Elmaaga-
cli et al, 2000; Ogawa et al, 2003; Candoni et al, 2011). From
the results of these studies, however, it is difficult to deter-
mine whether MRD monitoring by these methods is predic-
tive of relapse and, if so, whether it would provide timely

152 ª 2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 162, 147–161
 Review

Table IV. Prognostic significance of minimal residual disease after haematopoietic stem cell transplantation (HSCT).

Outcome

Type No. Age MRD MRD

Study of HSCT of patients group Method Estimate Neg Pos* P

Acute lymphoblastic leukaemia

Radich et al (1995) Allo 20 Adult PCR IG Relapses/patients studied 1/7 11/13 0
004†
Knechtli et al (1998b) Allo 68 Paediatric PCR IG/TCR Relapses/patients studied 4/32 28/36 <0
001†
Uzunel et al (2003) Allo 32 Both ASO PCR IG/TCR Relapses/patients studied 6/23 8/9 0
004†
Spinelli et al (2007) Allo 25 Adult ASO RQ-PCR 3-year incidence of relapse 7% 80% <0
001
IG/TCR or RQ-PCR
fusion transcripts
Zhao et al (2012) Allo 139 Both Flow cytometry or 2-year incidence of relapse 8% 54% <0
001
RT(RQ)-PCR 2-year EFS 80% 54% <0
001
BCR-ABL1
Acute myeloid leukaemia
Elmaagacli et al (2000) Allo 38 Adult RT-PCR WT1 Relapses/patients studied 5/24 7/14 NS†
Kwon et al (2012) Allo 21 Adult RT(RQ)-PCR WT1 Relapses/patients studied 0/11 9/10 <0
001†
Diez-Campelo et al (2009) Allo 41 Adult Flow cytometry 4-year EFS 74% 17% 0
01
4-year OS 73% 25% 0
002

HSCT, haematopoietic stem cell transplantation; MRD, minimal residual disease; allo, allogeneic; auto, autologous; PCR, polymerase chain reac-
tion; IG, immunoglobulin genes; TCR, T-cell receptor genes; ASO, allele-specific oligonucleotides; RQ, real time; RT, reverse transcription, EFS,
event-free survival; OS, overall survival; NS, not significant.
*Definition varies among studies.
†Calculated by Fisher’s exact test based on the data reported in the article.

clinical information and/or superior information to chime-
rism studies. To this end, in a recent analysis of 21 patients,
the 11 who had low and stable WT1 levels by RT(RQ)-PCR
post-HSCT also had complete chimerism and remained in
complete remission with a median follow-up of 27 months,
whereas nine of the 10 patients with WT1 overexpression
relapsed (P < 0
001), with the WT1 increase preceding chi-
merism changes (Kwon et al, 2012; Table IV). Other investi-
gators used flow cytometry to monitor MRD post-HSCT.
Diez-Campelo et al (2009) studied MRD in 41 patients with
AML or myelodysplastic syndrome and found that MRD
 0
1% on day 100 post-HSCT provided prognostic informa-
tion: 4-year EFS was 17% as compared to 74% for patients
with lower MRD (P = 0
01); OS was 73% vs. 25%
(P = 0
002); MRD was an independent predictor of outcome
in a multivariate analysis. Of note, Miyazaki et al (2012)
compared results of MRD detection post-allogeneic HSCT by
flow cytometry, RT(RQ)-PCR amplification of WT1 or of
fusion transcripts in a group of 31 patients with AML and 10
with ALL: they found that flow cytometry data predicted out-
come while molecular testing did not. These results recall our
recent experience comparing flow cytometry and PCR ampli-
fication of fusion transcripts to monitor MRD in childhood
AML (Inaba et al, 2012a).
MRD to guide pre- and post-transplant
management strategies
For patients with acute leukaemia who achieve remission,
a key question is whether they should proceed with
chemotherapy or undergo HSCT. In this regard, studies of
MRD have helped identify patients who still harbour high
levels of disease below the detection capabilities of morpho-
logical analysis and have a high risk of relapse if treated with
standard chemotherapy. For example, in our Total XIII study
where MRD was not used to guide treatment, nine children
with ALL had MRD levels  1% at the end of remission
induction therapy (day 42) and a 5-year cumulative inci-
dence of relapse of 72%: only two of nine patients in this
group were alive and in remission at 2
5 and 4
5 years after
diagnosis, and one of them had undergone allogeneic HSCT
because of high-risk presenting features (Coustan-Smith et al,
2000). In the subsequent Total XV study, all patients with
MRD  1% at the end of remission induction therapy
(n = 23, 4
6% of the 498 enrolled in the trial) were eligible
for allogeneic HSCT after receiving reintensification therapy:
12 of the 23 patients were alive and well at the time of the
report (Pui et al, 2009). In a cohort of 31 adult patients with
ALL and detectable MRD at the end of post-remission che-
motherapy, the 5-year LFS was 80% with allogeneic HSCT,
53% with autologous HSCT, and 0% with chemotherapy
(P = 0
003; Laane et al, 2006). In the German Multicentre
Study Group for Adult ALL, the probability of continuous
complete remission at 5 years for patients with Ph-negative
ALL who failed to achieve MRD negative status was signifi-
cantly higher for those who received allogeneic HSCT in first
remission: 66% vs. 12% for those who received chemother-
apy alone (P < 0
0001; Gokbuget et al, 2012).
Children and adolescents with AML and persistent MRD
after remission induction chemotherapy are also at a high

ª 2013 John Wiley & Sons Ltd 153

British Journal of Haematology, 2013, 162, 147–161
Review
risk of relapse (Coustan-Smith et al, 2003; van der Velden
et al, 2010). There is a paucity of studies comparing the rela-
tive benefits of chemotherapy versus HSCT in these patients
but a protocol in which persistent MRD (  0
1%) after three
courses of therapy was an indication for transplant in first
remission yielded cure rates among the highest reported to
date, suggesting a benefit from this strategy (Rubnitz et al,
2010a). In the study by Maurillo et al (2008) with adult
AML patients who had MRD >0
035% after remission induc-
tion therapy, 60 relapses occurred among the 74 patients
who were treated with either chemotherapy alone or autolo-
gous HSCT as compared to 6 relapses (or failures because of
treatment-related mortality) among the 14 patients who
received allogeneic HSCT.
Immunomodulatory strategies post-HSCT may be consid-
ered for patients with high-levels of MRD before transplant
but whether a graft-versus-leukaemia effect can have a signif-
icant clinical impact in ALL is unclear. Lankester et al (2010)
stratified 48 children with ALL according their MRD level:
18 had MRD pre-HSCT of  0
01% and were eligible for
early ciclosporin tapering followed by up to four donor
lymphocyte infusions (DLI). There was no evidence of
benefit for this approach and the EFS for this group was only
19%, although relapses appeared to occur later than expected
or presented at extramedullary sites (Lankester et al, 2010).
There is data suggesting that for patients with emerging
mixed chimerism post-HSCT, reduction of immunosuppres-
sion to increase the graft-versus-leukaemia effect or the initi-
ation of cellular therapy might be beneficial (Formankova
et al, 2003; Bader et al, 2004; Zeiser et al, 2005; Rettinger
et al, 2011; Buckley et al, 2012). For example, in the study
reported by Bader et al (2004), 16 of the 31 patients with
increasing mixed chimerism who received such forms of
immunotherapy reverted to complete chimerism, and 13
were alive and disease-free at the last follow-up (median,
866 d), while 11 of the 15 patients who were not treated
relapsed. Early studies reported a few patients with ALL in
whom MRD post-HSCT remained at low levels or disap-
peared in association with GvHD or DLI (Kanamori et al,
1997; Knechtli et al, 1998b; Miglino et al, 2002; Bradfield
et al, 2004; Dominietto et al, 2007) but the evidence for such
an effect seems more convincing in AML (Zeiser et al, 2005;
Rettinger et al, 2011). A recent study reported 105 patients
with AML who were MRD-positive post-HSCT; 49 received
low-dose interleukin 2 (IL2), and 56 received DLI with or
without IL2. The latter had a significantly lower cumulative
risk of relapse and higher LFS, which was comparable to that
of MRD-negative patients (Yan et al, 2012).
Besides immunomodulatory strategies, other types of
intervention may reduce the adverse impact of MRD post-
HSCT. One study focused on patients with Ph-positive ALL,
using RT-PCR targeting BCR-ABL1 transcripts to monitor
MRD and determine the effects of imatinib post-transplant
(Wassmann et al, 2005). Among 27 patients, MRD became
undetectable in 14 after 0
9–3
7 months and all remained in
154
remission for the duration of the imatinib course, although
three relapsed after imatinib discontinuation (Wassmann
et al, 2005). Pfeifer et al (2012) recently compared the merits
of prophylactic (n = 26) versus MRD-triggered imatinib
(n = 29) post-HSCT. Prophylactic imatinib reduced the
occurrence of molecular relapse but 5-year overall survival
for both groups of patients was high (80% vs. 75%). The
authors noted that patients with early molecular recurrence
and/or higher levels of BCR-ABL1 transcripts appeared to
have limited benefit from imatinib: six of 19 eventually
relapsed and three were switched to dasatinib after imatinib
failed to induce MRD-negativity (Pfeifer et al, 2012). Finally,
Platzbecker et al (2012) recently reported a study in which
azacitidine was given to 20 patients with AML or myelodys-
plastic syndrome and a decrease in donor chimerism to
<80% in peripheral blood CD34+ cells; 16 patients
responded with an increase or stabilization of donor chime-
rism and appeared to have delayed occurrence of relapse.
Practical issues
Minimal residual disease assays have been considerably
refined over the last two decades. Current methods for PCR
amplification of antigen-receptor genes are based on primers
complementary to the leukaemia-specific junctional regions
(Bruggemann et al, 2010). A new development in this area is
the use of consensus primers to universally amplify all rear-
ranged immunoglobulin and TCR gene segments present in a
leukaemic clone followed by high-throughput sequencing.
This approach can identify all clonal gene rearrangements at
diagnosis (or relapse) and monitor their fluctuations during
treatment (Boyd et al, 2009; Faham et al, 2012; Wu et al,
2012). In experiments comparing this assay with flow cytom-
etry and ASO-PCR, we found it to be capable of detecting
MRD as well as the other methods but to have a higher sen-
sitivity: it can potentially detect residual disease at levels
below 1 leukaemic cell in 106 leucocytes if sufficient DNA is
available (Faham et al, 2012). Flow cytometric methods have
also improved with the development of more sophisticated
instruments and the discovery of new markers to distinguish
leukaemic from normal cells (Coustan-Smith et al, 2011).
Finally, the application of single nucleotide polymorphism
arrays and whole-genome sequencing has identified many
previously unknown abnormalities in leukaemia, which could
expand the pool of potential targets for molecular analysis of
MRD, particularly in AML (Ley et al, 2010; Ding et al,
2012). Thus, genes mutated at diagnosis might be used to
track leukaemic cells during follow-up but their suitability
for this purpose remains largely untested.
Figure 1 summarizes the overall application of MRD to
manage patients with paediatric acute leukaemia at St. Jude
Children’s Research Hospital. For patients with ALL, MRD is
currently measured primarily with flow cytometry, reserving
ASO RQ-PCR amplification of antigen-receptor genes for
patients with no suitable MRD markers or whose MRD
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161

Fig 1. Minimal residual disease risk-adapted
approaches before and after HSCT for acute
leukaemia at St. Jude Children’s Research Hos-
pital. MRD, minimal residual disease; HSCT,
haematopoietic stem cell transplantation; IG,
immunoglobulin genes; TCR, T-cell receptor
genes; GvHD, graft-versus-host disease; DLI,
donor lymphocyte infusion; NK cell natural
killer cell.
results are ambiguous. MRD  1% at the end of remission
induction, or persisting MRD post-remission is an indication
for HSCT; chemotherapy pre-HSCT is given with the aim of
reducing MRD levels as much as possible. MRD in patients
with AML is monitored by flow cytometry and by RQ-PCR
targeting fusion transcripts, although the results of the for-
mer are given priority (Inaba et al, 2012b). Patients with
residual disease  5% by flow cytometry after Induction 1 or
MRD  0
1% after Induction 2 are candidates for HSCT.
Patients with MRD  1% at this point receive additional
chemotherapy or natural killer (NK) cell therapy before
HSCT. Those with lower levels of MRD may also receive
additional chemotherapy if the donor is not ready.
Minimal residual disease tests are technically complex and
require highly skilled staff and expensive reagents. Their
systematic use to guide clinical decisions, however, should not
only benefit patients but ultimately save costs, particularly in
the context of HSCT (Goulden et al, 2001). The increasing
use of MRD in the clinic may tempt laboratories without
specific expertise in this area to process samples and release
results. This should be avoided at all costs, as the
consequences of an incorrect result, e.g., a false-positive result
which indicates transplant in a patient who, in fact, is MRD-
negative, could be disastrous. In this regard, centres proficient
in performing MRD testing can generally offer training as well
as distance consultation and quality control. It has been diffi-
cult to draft widely accepted guidelines for MRD testing that
would meet expertise level and quality standards, and be
within the cost limits of most centres. Meeting this objective
is important to facilitate the interpretation of MRD
significance across different studies and implement a more
standardized clinical application of MRD testing.
A problem that may be encountered by transplant centres
is the lack of available diagnostic (or relapse) material to
identify the molecular or immunophenotypic signatures that
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161
Review
can track MRD. The requirement for the sequence of the
leukaemia-specific junctional regions to either amplify (by
ASO PCR) or recognize (after deep sequencing) leukaemic
clones in follow-up samples precludes MRD studies by these
approaches if this material is not available. In principle, one
could perform a clonality analysis using consensus primers
but this approach would have limited sensitivity (Langerak
et al, 2012). Flow cytometric studies of MRD are also best
performed when the immunophenotypic abnormalities
expressed by the leukaemic cells are known, so that tailor-
made antibody panels can be used to track them in remis-
sion samples. Alternatively, broad antibody panels designed
to detect all known abnormalities can be applied; the chances
of success for this approach have been increased by the
wealth of new markers and fluorochromes that can now be
used. In the case of an MRD-negative result, however, the
possibility remains that the leukaemic clone lacks any immu-
nophenotypic abnormality and it remains, therefore, unde-
tectable.
The optimal frequency at which MRD measurements
should be performed remains unclear. MRD prior to HSCT
is typically measured at the end of each block of preceding
chemotherapy; for MRD measurements post-HSCT, the
frequency varies. In fact, the optimal schedule may depend
on the leukaemia subtype. To this end, Ommen et al (2010)
calculated the kinetics of relapse in patients with AML based
on the time relationship between MRD findings and overt
disease recurrence. They found that one test in peripheral
blood every 6 months would be sufficient to detect 90% of
relapses at a median preceding time of 60 d in AML patients
with CBFB-MYH11 AML, but bone marrow testing at more
frequent intervals would be necessary in other subtypes, such
as those with RUNX1-RUNXT1 or NPM1 mutations with or
without FLT3-internal tandem duplications (Ommen et al,
2010). Our approach is to measure MRD monthly during
155
Review

the first 3 months post-HSCT, followed by measurements at
6 and 12 months, but additional measurement are performed
in the case of positive MRD findings or incomplete donor
chimerism.
Conclusions
It has been estimated that less than 1% of the published can-
cer biomarkers enter clinical practice (Kern, 2012). MRD
clearly belongs to this select group; it is the most useful
prognostic parameter overall in acute leukaemia. Because
HSCT is effective but also carries the risk of considerable
toxicity, it should be reserved for patients who are unlikely
to be cured with a less intensive and toxic treatment. Thus,
modern treatment protocols rely on MRD to identify such
patients. As discussed in this article, data suggest that opting
for HSCT may improve the outcome for these very-high risk
patients (Laane et al, 2006; Maurillo et al, 2008; Pui et al,
2009; Rubnitz et al, 2010a; Leung et al, 2011; Buckley et al,
2012; Gokbuget et al, 2012), but additional analyses are
required.
Minimal residual disease levels prior to HSCT are directly
related to risk of relapse post-HSCT, particularly in ALL
(Krejci et al, 2003; Bader et al, 2009; Leung et al, 2012). It
should be noted, however, that high levels of MRD pre-
transplant do not completely preclude success (Schneider
et al, 2001; Bader et al, 2009; Leung et al, 2012). Moreover,
we found that the adverse prognostic impact of any given
level of MRD was lower in children with ALL or AML trans-
planted in recent years (2000–2007) as compared to earlier
cohorts (Leung et al, 2012). For example, in our study of
patients with persistent MRD at <5% at the time of HSCT
treated in the more recent era, the 5-year OS rate was 67%
for patients with AML and 49% for ALL (Leung et al, 2012).
Whether treatment intensification prior to HSCT in efforts
to reduce MRD levels diminishes the risk of relapse post-
HSCT or simply adds toxicity with marginal benefits remains
to be determined (Goulden & Steward, 2002; Buckley et al,
2012). To this end, targeted therapies without cross-resis-
tance or overlapping toxicity may play a crucial role, as
suggested by the experience with blinatumomab, indicating
that patients who achieved MRD-negativity with this agent
had an excellent outcome post-allogeneic HSCT (Handgret-
inger et al, 2011; Topp et al, 2011). Recent data demonstrat-
ing clinical responses in patients receiving autologous T cells
genetically modified to express anti-CD19 chimeric receptors
(Porter et al, 2011) provide further indication of the anti-
leukaemic potential of immune cells, and suggest new ways
to reduce MRD levels pre-HSCT, or perhaps replace HSCT
altogether.
While DLI can be effective, it also has a high risk of
GvHD (Kolb et al, 1995). There is considerable evidence that
NK cell alloreactivity can suppress relapse post-HSCT with-
out GvHD (Ruggeri et al, 2002; Giebel et al, 2003; Leung
et al, 2004; Cooley et al, 2010), which has led to the practice
of selecting donors with a NK receptor profile that predicts
the strongest alloreactive potential. There are also data
indicating that allogeneic NK cell infusions can have an anti-
leukaemia effect, particularly in AML (Miller et al, 2005;
Rubnitz et al, 2010b). NK cells can be effectively expanded
and activated (Fujisaki et al, 2009; Lapteva et al, 2012; Ru-
jkijyanont et al, 2013), and also redirected against different
leukaemia cell types using clinically applicable methods (Imai
et al, 2005; Shimasaki et al, 2012). To this end, NK cells
expressing a chimeric receptor that recognizes the CD19
molecule and delivering activation signals through CD3f and
4-1BB markedly enhanced NK cell killing of CD19+ ALL and
lymphoma cells, even in autologous setting (Imai et al, 2005;
Shimasaki et al, 2012). More recently, we found that a recep-
tor containing NKG2D (a NK activating receptor) and the
signalling molecules CD3f and DAP10 significantly improved
NK cell activity against a variety of cancer cell types (Chang
et al, 2013). Antibodies targeting markers expressed by
leukaemic cells (e.g., CD19, CD33) could be used to trigger
antibody-dependent cytotoxicity, while antibodies against NK
inhibitory receptors could further augment NK cell activity
(Chan et al, 2012). We expect that MRD-directed NK cell
therapy will increasingly be used to reduce leukaemia burden
before HSCT or eradicate MRD post-HSCT.
In summary, MRD has had a major impact on many criti-
cal facets of the clinical management of patients with acute
leukaemia, including HSCT. MRD monitoring helps refining
the eligibility criteria for HSCT, and can guide treatment
choices before and after HSCT. As new targeted agents and
cell-based therapies become available, MRD should provide
an essential tool for selecting the best candidates for these
therapies and measuring their effects.
Acknowledgements
This work was supported in part by the National Medical
Research Council of Singapore grant 1299/2011, the Viva
Foundation for Children with Cancer, the National Institutes
of Health Cancer Center Support (CORE) grant P30
CA021765, the Assisi Foundation of Memphis, and the
American Lebanese Syrian Associated Charities (ALSAC).

References
Antin, J.H., Childs, R., Filipovich, A.H., Giralt, S.,
Mackinnon, S., Spitzer, T. & Weisdorf, D. (2001)
Establishment of complete and mixed donor
chimerism after allogeneic lymphohematopoietic
transplantation: recommendations from a work-
shop at the 2001 Tandem Meetings of the Inter-
national Bone Marrow Transplant Registry and
the American Society of Blood and Marrow
Transplantation. Biology of Blood and Marrow
Transplantation, 7, 473–485.
Bachanova, V., Burke, M.J., Yohe, S., Cao, Q.,
Sandhu, K., Singleton, T.P., Brunstein, C.G.,
Wagner, J.E., Verneris, M.R. & Weisdorf, D.J.
(2012) Unrelated cord blood transplantation in
adult and pediatric acute lymphoblastic
leukemia: effect of minimal residual disease on

156 ª 2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 162, 147–161

relapse and survival. Biology of Blood and Mar-
row Transplantation, 18, 963–968.
Bacher, U., Kern, W., Schoch, C., Schnittger, S.,
Hiddemann, W. & Haferlach, T. (2006) Evalua-
tion of complete disease remission in acute mye-
loid leukemia: a prospective study based on
cytomorphology, interphase fluorescence in situ
hybridization, and immunophenotyping during
follow-up in patients with acute myeloid leuke-
mia. Cancer, 106, 839–847.
Bacher, U., Badbaran, A., Fehse, B., Zabelina, T.,
Zander, A.R. & Kroger, N. (2009) Quantitative
monitoring of NPM1 mutations provides a valid
minimal residual disease parameter following
allogeneic stem cell transplantation. Experimen-
tal Hematology, 37, 135–142.
Bader, P., Hancock, J., Kreyenberg, H., Goulden,
N.J., Niethammer, D., Oakhill, A., Steward, C.G.,
Handgretinger, R., Beck, J.F. & Klingebiel, T.
(2002) Minimal residual disease (MRD) status
prior to allogeneic stem cell transplantation is a
powerful predictor for post-transplant outcome
in children with ALL. Leukemia, 16, 1668–1672.
Bader, P., Kreyenberg, H., Hoelle, W., Dueckers,
G., Handgretinger, R., Lang, P., Kremens, B.,
Dilloo, D., Sykora, K.W., Schrappe, M., Niemey-
er, C., Von Stackelberg, A., Gruhn, B., Henze,
G., Greil, J., Niethammer, D., Dietz, K., Beck,
J.F. & Klingebiel, T. (2004) Increasing mixed
chimerism is an important prognostic factor for
unfavorable outcome in children with acute
lymphoblastic leukemia after allogeneic stem-cell
transplantation: possible role for pre-emptive
immunotherapy? Journal of Clinical Oncology,
22, 1696–1705.
Bader, P., Kreyenberg, H., Henze, G.H., Eckert, C.,
Reising, M., Willasch, A., Barth, A., Borkhardt,
A., Peters, C., Handgretinger, R., Sykora, K.W.,
Holter, W., Kabisch, H., Klingebiel, T. & von
Stackelberg, S.A. (2009) Prognostic value of
minimal residual disease quantification before
allogeneic stem-cell transplantation in relapsed
childhood acute lymphoblastic leukemia: the
ALL-REZ BFM Study Group. Journal of Clinical
Oncology, 27, 377–384.
Boyd, S.D., Marshall, E.L., Merker, J.D., Maniar,
J.M., Zhang, L.N., Sahaf, B., Jones, C.D., Simen,
B.B., Hanczaruk, B., Nguyen, K.D., Nadeau,
K.C., Egholm, M., Miklos, D.B., Zehnder, J.L. &
Fire, A.Z. (2009) Measurement and clinical
monitoring of human lymphocyte clonality by
massively parallel VDJ pyrosequencing. Science
Translational Medicine, 1, 12ra23
Bradfield, S.M., Radich, J.P. & Loken, M.R. (2004)
Graft-versus-leukemia effect in acute lympho-
blastic leukemia: the importance of tumor
burden and early detection. Leukemia, 18, 1156–
1158.
Brenner, M.K., Rill, D.R., Moen, R.C., Krance,
R.A., Mirro, J. Jr, Anderson, W.F. & Ihle, J.N.
(1993) Gene-marking to trace origin of relapse
after autologous bone-marrow transplantation.
Lancet, 341, 85–86.
Bruggemann, M., Schrauder, A., Raff, T., Pfeifer,
H., Dworzak, M., Ottmann, O.G., Asnafi, V.,
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161
Baruchel, A., Bassan, R., Benoit, Y., Biondi, A.,
Cave, H., Dombret, H., Fielding, A.K., Foa, R.,
Gokbuget, N., Goldstone, A.H., Goulden, N.,
Henze, G., Hoelzer, D., Janka-Schaub, G.E.,
Macintyre, E.A., Pieters, R., Rambaldi, A., Riber-
a, J.M., Schmiegelow, K., Spinelli, O., Stary, J.,
von Stackelberg, A., Kneba, M., Schrappe, M. &
van Dongen, J.J. (2010) Standardized MRD
quantification in European ALL trials: proceed-
ings of the Second International Symposium on
MRD assessment in Kiel, Germany, 18-20 Sep-
tember 2008. Leukemia, 24, 521–535.
Buccisano, F., Maurillo, L., Del Principe, M.I., Del
Poeta, G., Sconocchia, G., Lo-Coco, F., Arcese,
W., Amadori, S. & Venditti, A. (2012) Prognos-
tic and therapeutic implications of minimal
residual disease detection in acute myeloid leu-
kemia. Blood, 119, 332–341.
Buckley, S.A., Appelbaum, F.R. & Walter, R.B.
(2012) Prognostic and therapeutic implications
of minimal residual disease at the time of trans-
plantation in acute leukemia. Bone Marrow
Transplantation, doi: 10.1038/bmt.2012.139
Campana, D. (2012) Should minimal residual dis-
ease monitoring in acute lymphoblastic leukemia
be standard of care? Current Hematologic Malig-
nancy Reports, 7, 170–177.
Candoni, A., Toffoletti, E., Gallina, R., Simeone,
E., Chiozzotto, M., Volpetti, S. & Fanin, R.
(2011) Monitoring of minimal residual disease
by quantitative WT1 gene expression following
reduced intensity conditioning allogeneic stem
cell transplantation in acute myeloid leukemia.
Clinical Transplantation, 25, 308–316.
Chan, W.K., Kung Sutherland, M., Li, Y., Zalevsky,
J., Schell, S. & Leung, W. (2012) Antibody-
dependent cell-mediated cytotoxicity overcomes
NK cell resistance in MLL-rearranged leukemia
expressing inhibitory KIR ligands but not acti-
vating ligands. Clinical Cancer Research, 18,
6296–6305.
Chang, Y.H., Connolly, J., Shimasaki, N., Mimura,
K., Kono, K. & Campana, D. (2013) A chimeric
receptor with Nkg2d specificity enhances natural
killer cell activation and killing of tumor cells.
Cancer Research, 73, 1777–1786.
Chen, Y., Cortes, J., Estrov, Z., Faderl, S., Qiao,
W., Abruzzo, L., Garcia-Manero, G., Pierce, S.,
Huang, X., Kebriaei, P., Kadia, T., De Lima, M.,
Kantarjian, H. & Ravandi, F. (2011) Persistence
of cytogenetic abnormalities at complete remis-
sion after induction in patients with acute mye-
loid leukemia: prognostic significance and the
potential role of allogeneic stem-cell transplanta-
tion. Journal of Clinical Oncology, 29, 2507–
2513.
Cooley, S., Weisdorf, D.J., Guethlein, L.A., Klein,
J.P., Wang, T., Le, C.T., Marsh, S.G., Gera-
ghty, D., Spellman, S., Haagenson, M.D., Lad-
ner, M., Trachtenberg, E., Parham, P. &
Miller, J.S. (2010) Donor selection for natural
killer cell receptor genes leads to superior sur-
vival after unrelated transplantation for acute
myelogenous leukemia. Blood, 116, 2411–
2419.
Review
Coustan-Smith, E., Sancho, J., Hancock, M.L.,
Boyett, J.M., Behm, F.G., Raimondi, S.C., Sandl-
und, J.T., Rivera, G.K., Rubnitz, J.E., Ribeiro,
R.C., Pui, C.H. & Campana, D. (2000) Clinical
importance of minimal residual disease in child-
hood acute lymphoblastic leukemia. Blood, 96,
2691–2696.
Coustan-Smith, E., Ribeiro, R.C., Rubnitz, J.E.,
Razzouk, B.I., Pui, C.H., Pounds, S., Andrean-
sky, M., Behm, F.G., Raimondi, S.C., Shurtleff,
S.A., Downing, J.R. & Campana, D. (2003) Clin-
ical significance of residual disease during treat-
ment in childhood acute myeloid leukemia.
British Journal of Haematology, 123, 243–252.
Coustan-Smith, E., Song, G., Clark, C., Key, L.,
Liu, P., Mehrpooya, M., Stow, P., Su, X., Shurt-
leff, S., Pui, C.H., Downing, J.R. & Campana, D.
(2011) New markers for minimal residual dis-
ease detection in acute lymphoblastic leukemia.
Blood, 117, 6267–6276.
Diez-Campelo, M., Perez-Simon, J.A., Perez, J.,
Alcoceba, M., Richtmon, J., Vidriales, B. & San
Miguel, J. (2009) Minimal residual disease mon-
itoring after allogeneic transplantation may help
to individualize post-transplant therapeutic
strategies in acute myeloid malignancies. Ameri-
can Journal of Hematology, 84, 149–152.
Ding, L., Ley, T.J., Larson, D.E., Miller, C.A.,
Koboldt, D.C., Welch, J.S., Ritchey, J.K., Young,
M.A., Lamprecht, T., McLellan, M.D., McMich-
ael, J.F., Wallis, J.W., Lu, C., Shen, D., Harris,
C.C., Dooling, D.J., Fulton, R.S., Fulton, L.L.,
Chen, K., Schmidt, H., Kalicki-Veizer, J., Mag-
rini, V.J., Cook, L., McGrath, S.D., Vickery,
T.L., Wendl, M.C., Heath, S., Watson, M.A.,
Link, D.C., Tomasson, M.H., Shannon, W.D.,
Payton, J.E., Kulkarni, S., Westervelt, P., Walter,
M.J., Graubert, T.A., Mardis, E.R., Wilson, R.K.
& DiPersio, J.F. (2012) Clonal evolution in
relapsed acute myeloid leukaemia revealed by
whole-genome sequencing. Nature, 481, 506–
510.
Dombret, H., Gabert, J., Boiron, J.M., Rigal-Huguet,
F., Blaise, D., Thomas, X., Delannoy, A., Buzyn,
A., Bilhou-Nabera, C., Cayuela, J.M., Fenaux, P.,
Bourhis, J.H., Fegueux, N., Charrin, C., Boucheix,
C., Lheritier, V., Esperou, H., MacIntyre, E.,
Vernant, J.P. & Fiere, D. (2002) Outcome of
treatment in adults with Philadelphia chromo-
some-positive acute lymphoblastic leukemia–
results of the prospective multicenter LALA-94
trial. Blood, 100, 2357–2366.
Dominietto, A., Pozzi, S., Miglino, M., Albarracin,
F., Piaggio, G., Bertolotti, F., Grasso, R., Zupo, S.,
Raiola, A.M., Gobbi, M., Frassoni, F. &
Bacigalupo, A. (2007) Donor lymphocyte
infusions for the treatment of minimal residual
disease in acute leukemia. Blood, 109, 5063–5064.
Elmaagacli, A.H., Beelen, D.W., Kroll, M., Trzen-
sky, S., Stein, C. & Schaefer, U.W. (1998) Detec-
tion of CBFbeta/MYH11 fusion transcripts in
patients with inv(16) acute myeloid leukemia
after allogeneic bone marrow or peripheral
blood progenitor cell transplantation. Bone
Marrow Transplantation, 21, 159–166.
157
Review
Elmaagacli, A.H., Beelen, D.W., Trenschel, R. &
Schaefer, U.W. (2000) The detection of wt-1
transcripts is not associated with an increased
leukemic relapse rate in patients with acute
leukemia after allogeneic bone marrow or
peripheral blood stem cell transplantation. Bone
Marrow Transplantation, 25, 91–96.
Elorza, I., Palacio, C., Dapena, J.L., Gallur, L., de
Toledo, J.S. & de Heredia, C.D. (2010) Relation-
ship between minimal residual disease measured
by multiparametric flow cytometry prior to allo-
geneic hematopoietic stem cell transplantation
and outcome in children with acute lymphoblas-
tic leukemia. Haematologica, 95, 936–941.
Faham, M., Zheng, J., Moorhead, M., Carlton,
V.E., Stow, P., Coustan-Smith, E., Pui, C.H. &
Campana, D. (2012) Deep-sequencing approach
for minimal residual disease detection in acute
lymphoblastic leukemia. Blood, 120, 5173–5180.
Feller, N., Jansen-van der Weide, M.C., van der
Pol, M.A., Westra, G.A., Ossenkoppele, G.J. &
Schuurhuis, G.J. (2005) Purging of peripheral
blood stem cell transplants in AML: a predictive
model based on minimal residual disease
burden. Experimental Hematology, 33, 120–130.
Formankova, R., Sedlacek, P., Krskova, L., Rihova,
H., Sramkova, L. & Star, J. (2003) Chimerism-
directed adoptive immunotherapy in prevention
and treatment of post-transplant relapse of
leukemia in childhood. Haematologica, 88, 117–
118.
Freireich, E.J., Cork, A., Stass, S.A., McCredie,
K.B., Keating, M.J., Estey, E.H., Kantarjian,
H.M. & Trujillo, J.M. (1992) Cytogenetics for
detection of minimal residual disease in acute
myeloblastic leukemia. Leukemia, 6, 500–506.
Fujisaki, H., Kakuda, H., Shimasaki, N., Imai, C.,
Ma, J., Lockey, T., Eldridge, P., Leung, W.H. &
Campana, D. (2009) Expansion of highly cyto-
toxic human natural killer cells for cancer cell
therapy. Cancer Research, 69, 4010–4017.
Giebel, S., Locatelli, F., Lamparelli, T., Velardi, A.,
Davies, S., Frumento, G., Maccario, R., Bonetti,
F., Wojnar, J., Martinetti, M., Frassoni, F.,
Giorgiani, G., Bacigalupo, A. & Holowiecki, J.
(2003) Survival advantage with KIR ligand
incompatibility in hematopoietic stem cell trans-
plantation from unrelated donors. Blood, 102,
814–819.
Giebel, S., Stella-Holowiecka, B., Krawczyk-Kulis,
M., Gokbuget, N., Hoelzer, D., Doubek, M.,
Mayer, J., Piatkowska-Jakubas, B., Skotnicki,
A.B., Dombret, H., Ribera, J.M., Piccaluga, P.P.,
Czerw, T., Kyrcz-Krzemien, S. & Holowiecki, J.
(2010) Status of minimal residual disease deter-
mines outcome of autologous hematopoietic
SCT in adult ALL. Bone Marrow Transplanta-
tion, 45, 1095–1101.
Gokbuget, N., Kneba, M., Raff, T., Trautmann, H.,
Bartram, C.R., Arnold, R., Fietkau, R., Freund,
M., Ganser, A., Ludwig, W.D., Maschmeyer, G.,
Rieder, H., Schwartz, S., Serve, H., Thiel, E.,
Bruggemann, M. & Hoelzer, D. (2012) Adult
patients with acute lymphoblastic leukemia and
molecular failure display a poor prognosis and
158
are candidates for stem cell transplantation and
targeted therapies. Blood, 120, 1868–1876.
Goulden, N. & Steward, C. (2002) Clinical rele-
vance of MRD in children undergoing allogeneic
stem cell transplantation for ALL. Best Practice
& Research. Clinical Haematology, 15, 59–70.
Goulden, N., Oakhill, A. & Steward, C. (2001)
Practical application of minimal residual disease
assessment in childhood acute lymphoblastic
leukaemia annotation. British Journal of Haema-
tology, 112, 275–281.
Handgretinger, R., Zugmaier, G., Henze, G.,
Kreyenberg, H., Lang, P. & von Stackelberg,
A. (2011) Complete remission after blinatumo-
mab-induced donor T-cell activation in three
pediatric patients with post-transplant relapsed
acute lymphoblastic leukemia. Leukemia, 25,
181–184.
Harris, J. & Freireich, E.J. (1970) In vitro growth
of myeloid colonies from bone marrow of
patients with acute leukemia in remission. Blood,
35, 61–63.
Imai, C., Iwamoto, S. & Campana, D. (2005)
Genetic modification of primary natural killer
cells overcomes inhibitory signals and induces
specific killing of leukemic cells. Blood, 106,
376–383.
Inaba, H., Coustan-Smith, E., Cao, X., Pounds,
S.B., Shurtleff, S.A., Wang, K.Y., Raimondi, S.C.,
Onciu, M., Jacobsen, J., Ribeiro, R.C., Dahl,
G.V., Bowman, W.P., Taub, J.W., Degar, B.,
Leung, W., Downing, J.R., Pui, C.H., Rubnitz,
J.E. & Campana, D. (2012a) Comparative analy-
sis of different approaches to measure treatment
response in acute myeloid leukemia. Journal of
Clinical Oncology, 30, 3625–3632.
Inaba, H., Bhojwani, D., Pauley, J.L., Pei, D.,
Cheng, C., Metzger, M.L., Howard, S.C., Rub-
nitz, J.E., Sandlund, J.T., Ribeiro, R.C., Leung,
W., Campana, D., Pui, C.H. & Jeha, S. (2012b)
Combination chemotherapy with clofarabine,
cyclophosphamide, and etoposide in children
with refractory or relapsed haematological
malignancies. British Journal of Haematology,
156, 275–279.
Jacobsohn, D.A., Tse, W.T., Chaleff, S., Rade-
maker, A., Duerst, R., Olszewski, M., Huang,
W., Chou, P.M. & Kletzel, M. (2009) High WT1
gene expression before haematopoietic stem cell
transplant in children with acute myeloid leu-
kaemia predicts poor event-free survival. British
Journal of Haematology, 146, 669–674.
Kanamori, H., Sasaki, S., Yamazaki, E., Ueda, S.,
Hattori, M., Fukawa, H., Tamura, T., Harano,
H., Matsuzaki, M., Ogawa, K., Mohri, H. & Ok-
ubo, T. (1997) Eradication of minimal residual
disease during graft-versus-host reaction
induced by abrupt discontinuation of immuno-
suppression following bone marrow transplanta-
tion in a patient with Ph1-ALL. Transplant
International, 10, 328–330.
Kern, S.E. (2012) Why your new cancer biomarker
may never work: recurrent patterns and remark-
able diversity in biomarker failures. Cancer
Research, 72, 6097–6101.
British Journal of Haematology, 2013, 162, 147–161
Knechtli, C.J.C., Goulden, N.J., Hancock, J.P.,
Grandage, V.L.G., Harris, E.L., Garland, R.J.,
Jones, C.G., Rowbottom, A.W., Hunt, L.P.,
Green, A.F., Clarke, E., Lankester, A.W., Cor-
nish, J.M., Pamphilon, D.H., Steward, C.G. &
Oakhill, A. (1998a) Minimal residual disease sta-
tus before allogeneic bone marrow transplanta-
tion is an important determinant of successful
outcome for children and adolescents with acute
lymphoblastic leukemia. Blood, 92, 4072–4079.
Knechtli, C.J., Goulden, N.J., Hancock, J.P., Harris,
E.L., Garland, R.J., Jones, C.G., Grandage, V.L.,
Rowbottom, A.W., Green, A.F., Clarke, E.,
Lankester, A.W., Potter, M.N., Cornish, J.M.,
Pamphilon, D.H., Steward, C.G. & Oakhill, A.
(1998b) Minimal residual disease status as a pre-
dictor of relapse after allogeneic bone marrow
transplantation for children with acute lympho-
blastic leukaemia. British Journal of Haematology,
102, 860–871.
Kolb, H.J., Schattenberg, A., Goldman, J.M., Her-
tenstein, B., Jacobsen, N., Arcese, W., Ljungman,
P., Ferrant, A., Verdonck, L. & Niederwieser, D.
(1995) Graft-versus-leukemia effect of donor
lymphocyte transfusions in marrow grafted
patients. European Group for Blood and Mar-
row Transplantation Working Party Chronic
Leukemia. Blood, 86, 2041–2050.
Krejci, O., van der Velden, V., Bader, P., Kreyen-
berg, H., Goulden, N., Hancock, J., Schilham,
M.W., Lankester, A., Revesz, T., Klingebiel, T. &
van Dongen, J.J. (2003) Level of minimal resid-
ual disease prior to haematopoietic stem cell
transplantation predicts prognosis in paediatric
patients with acute lymphoblastic leukaemia: a
report of the Pre-BMT MRD Study Group. Bone
Marrow Transplantation, 32, 849–851.
Kwon, M., Martinez-Laperche, C., Infante, M., Car-
retero, F., Balsalobre, P., Serrano, D., Gayoso, J.,
Perez-Corral, A., Anguita, J., Diez-Martin, J.L. &
Buno, I. (2012) Evaluation of minimal residual
disease by real-time quantitative PCR of Wilms’
tumor 1 expression in patients with acute
myelogenous leukemia after allogeneic stem cell
transplantation: correlation with flow cytometry
and chimerism. Biology of Blood and Marrow
Transplantation, 18, 1235–1242.
Laane, E., Derolf, A.R., Bjorklund, E., Mazur, J.,
Everaus, H., Soderhall, S., Bjorkholm, M. &
Porwit-MacDonald, A. (2006) The effect of
allogeneic stem cell transplantation on outcome
in younger acute myeloid leukemia patients with
minimal residual disease detected by flow
cytometry at the end of post-remission chemo-
therapy. Haematologica, 91, 833–836.
Langerak, A.W., Groenen, P.J., Bruggemann, M.,
Beldjord, K., Bellan, C., Bonello, L., Boone, E.,
Carter, G.I., Catherwood, M., Davi, F., Delfau-
Larue, M.H., Diss, T., Evans, P.A., Gameiro, P.,
Garcia Sanz, R., Gonzalez, D., Grand, D.,
Hakansson, A., Hummel, M., Liu, H., Lombardia,
L., Macintyre, E.A., Milner, B.J., Montes-Moreno,
S., Schuuring, E., Spaargaren, M., Hodges, E. &
van Dongen, J.J. (2012) EuroClonality/BIOMED-
2 guidelines for interpretation and reporting of
ª 2013 John Wiley & Sons Ltd

Ig/TCR clonality testing in suspected lymphopro-
liferations. Leukemia, 26, 2159–2171.
Lankester, A.C., Bierings, M.B., Van Wering, E.R.,
Wijkhuijs, A.J., de Weger, R.A., Wijnen, J.T.,
Vossen, J.M., Versluys, B., Egeler, R.M., van Tol,
M.J., Putter, H., Revesz, T., van Dongen, J.J.,
van der Velden, V.H. & Schilham, M.W. (2010)
Preemptive alloimmune intervention in high-
risk pediatric acute lymphoblastic leukemia
patients guided by minimal residual disease level
before stem cell transplantation. Leukemia, 24,
1462–1469.
Lapteva, N., Durett, A.G., Sun, J., Rollins, L.A.,
Huye, L.L., Fang, J., Dandekar, V., Mei, Z., Jack-
son, K., Vera, J., Ando, J., Ngo, M.C., Coustan-
Smith, E., Campana, D., Szmania, S., Garg, T.,
Moreno-Bost, A., Vanrhee, F., Gee, A.P. &
Rooney, C.M. (2012) Large-scale ex vivo expan-
sion and characterization of natural killer cells
for clinical applications. Cytotherapy, 14, 1131–
1143.
Lee, S., Kim, D.W., Cho, B.S., Yoon, J.H., Shin,
S.H., Yahng, S.A., Lee, S.E., Eom, K.S., Kim,
Y.J., Chung, N.G., Kim, H.J., Min, C.K., Lee,
J.W., Min, W.S. & Park, C.W. (2012) Impact of
minimal residual disease kinetics during imati-
nib-based treatment on transplantation outcome
in Philadelphia chromosome-positive acute lym-
phoblastic leukemia. Leukemia, 26, 2367–2374.
Leung, W., Iyengar, R., Turner, V., Lang, P., Bad-
er, P., Conn, P., Niethammer, D. & Handgretin-
ger, R. (2004) Determinants of antileukemia
effects of allogeneic NK cells. Journal of Immu-
nology, 172, 644–650.
Leung, W., Campana, D., Yang, J., Pei, D., Cou-
stan-Smith, E., Gan, K., Rubnitz, J.E., Sandlund,
J.T., Ribeiro, R.C., Srinivasan, A., Hartford, C.,
Triplett, B.M., Dallas, M., Pillai, A., Handgretin-
ger, R., Laver, J.H. & Pui, C.H. (2011) High
success rate of hematopoietic cell transplantation
regardless of donor source in children with very
high-risk leukemia. Blood, 118, 223–230.
Leung, W., Pui, C.H., Coustan-Smith, E., Yang, J.,
Pei, D., Gan, K., Srinivasan, A., Hartford, C.,
Triplett, B.M., Dallas, M., Pillai, A., Shook, D.,
Rubnitz, J.E., Sandlund, J.T., Jeha, S., Inaba, H.,
Ribeiro, R.C., Handgretinger, R., Laver, J.H. &
Campana, D. (2012) Detectable minimal resid-
ual disease before hematopoietic cell transplan-
tation is prognostic but does not preclude cure
for children with very-high-risk leukemia. Blood,
120, 468–472.
Ley, T.J., Ding, L., Walter, M.J., McLellan, M.D.,
Lamprecht, T., Larson, D.E., Kandoth, C., Pay-
ton, J.E., Baty, J., Welch, J., Harris, C.C., Lichti,
C.F., Townsend, R.R., Fulton, R.S., Dooling,
D.J., Koboldt, D.C., Schmidt, H., Zhang, Q.,
Osborne, J.R., Lin, L., O’Laughlin, M., McMich-
ael, J.F., Delehaunty, K.D., McGrath, S.D., Ful-
ton, L.A., Magrini, V.J., Vickery, T.L., Hundal,
J., Cook, L.L., Conyers, J.J., Swift, G.W., Reed,
J.P., Alldredge, P.A., Wylie, T., Walker, J., Kal-
icki, J., Watson, M.A., Heath, S., Shannon,
W.D., Varghese, N., Nagarajan, R., Westervelt,
P., Tomasson, M.H., Link, D.C., Graubert, T.A.,
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161
DiPersio, J.F., Mardis, E.R. & Wilson, R.K.
(2010) DNMT3A mutations in acute myeloid
leukemia. New England Journal of Medicine, 363,
2424–2433.
Mathe, G., Schwarzenberg, L., Mery, A.M., Cattan,
A., Schneider, M., Amiel, J.L., Schlumberger,
J.R., Poisson, J. & Wajcner, G. (1966) Extensive
histological and cytological survey of patients
with acute leukaemia in “complete remission”.
British Medical Journal, 5488, 640–642.
Maurillo, L., Buccisano, F., Del Principe, M.I., Del,
P.G., Spagnoli, A., Panetta, P., Ammatuna, E.,
Neri, B., Ottaviani, L., Sarlo, C., Venditti, D.,
Quaresima, M., Cerretti, R., Rizzo, M., De Fab-
ritiis, F.P., Lo Coco, F., Arcese, W., Amadori, S.
& Venditti, A. (2008) Toward optimization of
postremission therapy for residual disease-posi-
tive patients with acute myeloid leukemia. Jour-
nal of Clinical Oncology, 26, 4944–4951.
Mertelsmann, R., Moore, M.A., Broxmeyer, H.E.,
Cirrincione, C. & Clarkson, B. (1981) Diagnostic
and prognostic significance of the CFU-c assay
in acute nonlymphoblastic leukemia. Cancer
Research, 41, 4844–4848.
Miglino, M., Berisso, G., Grasso, R., Canepa, L.,
Clavio, M., Pierri, I., Pietrasanta, D., Gatto, S.,
Varaldo, R., Ballerini, F., Verdiani, S., Casarino,
L., DeStefano, F., Sessarego, M., Dominietto, A.,
Raiola, A.M., Bregante, S., di Grazia, C., Gobbi,
M. & Bacigalupo, A. (2002) Allogeneic bone
marrow transplantation (BMT) for adults with
acute lymphoblastic leukemia (ALL): predictive
role of minimal residual disease monitoring on
relapse. Bone Marrow Transplantation, 30, 579–
585.
Miller, J.S., Soignier, Y., Panoskaltsis-Mortari, A.,
McNearney, S.A., Yun, G.H., Fautsch, S.K.,
McKenna, D., Le, C., Defor, T.E., Burns, L.J.,
Orchard, P.J., Blazar, B.R., Wagner, J.E., Slung-
aard, A., Weisdorf, D.J., Okazaki, I.J. & McGl-
ave, P.B. (2005) Successful adoptive transfer and
in vivo expansion of human haploidentical NK
cells in cancer patients. Blood, 105, 3051–3057.
Miyazaki, T., Fujita, H., Fujimaki, K., Hosoyama,
T., Watanabe, R., Tachibana, T., Fujita, A., Mat-
sumoto, K., Tanaka, M., Koharazawa, H., Tagu-
chi, J., Tomita, N., Sakai, R., Fujisawa, S.,
Kanamori, H. & Ishigatsubo, Y. (2012) Clinical
significance of minimal residual disease detected
by multidimensional flow cytometry: serial
monitoring after allogeneic stem cell transplan-
tation for acute leukemia. Leukemia Research,
36, 998–1003.
Mizuta, S., Ito, Y., Kohno, A., Kiyoi, H., Miyam-
ura, K., Tanimoto, M., Takamatsu, J., Naoe, T.,
Morishima, Y., Ueda, R. & Saito, H. (1999)
Accurate quantitation of residual tumor burden
at bone marrow harvest predicts timing of sub-
sequent relapse in patients with common ALL
treated by autologous bone marrow transplanta-
tion. Nagoya BMT Group.. Bone Marrow Trans-
plantation, 24, 777–784.
Moore, M.A., Spitzer, G., Williams, N., Metcalf, D.
& Buckley, J. (1974) Agar culture studies in 127
cases of untreated acute leukemia: the prognos-
Review
tic value of reclassification of leukemia accord-
ing to in vitro growth characteristics. Blood, 44,
1–18.
Mortuza, F.Y., Papaioannou, M., Moreira, I.M.,
Coyle, L.A., Gameiro, P., Gandini, D., Prentice,
H.G., Goldstone, A., Hoffbrand, A.V. & Foroni,
L. (2002) Minimal residual disease tests provide
an independent predictor of clinical outcome in
adult acute lymphoblastic leukemia. Journal of
Clinical Oncology, 20, 1094–1104.
Ogawa, H., Tamaki, H., Ikegame, K., Soma, T.,
Kawakami, M., Tsuboi, A., Kim, E.H., Hosen,
N., Murakami, M., Fujioka, T., Masuda, T.,
Taniguchi, Y., Nishida, S., Oji, Y., Oka, Y. &
Sugiyama, H. (2003) The usefulness of monitor-
ing WT1 gene transcripts for the prediction and
management of relapse following allogeneic stem
cell transplantation in acute type leukemia.
Blood, 101, 1698–1704.
Ommen, H.B., Schnittger, S., Jovanovic, J.V.,
Ommen, I.B., Hasle, H., Ostergaard, M.,
Grimwade, D. & Hokland, P. (2010) Strikingly
different molecular relapse kinetics in NPM1c,
PML-RARA, RUNX1-RUNX1T1, and CBFB-
MYH11 acute myeloid leukemias. Blood, 115,
198–205.
Osborne, D., Frost, L., Tobal, K. & Liu Yin, J.A.
(2005) Elevated levels of WT1 transcripts in
bone marrow harvests are associated with a high
relapse risk in patients autografted for acute
myeloid leukaemia. Bone Marrow Transplanta-
tion, 36, 67–70.
Paganin, M., Zecca, M., Fabbri, G., Polato, K., Bi-
ondi, A., Rizzari, C., Locatelli, F. & Basso, G.
(2008) Minimal residual disease is an important
predictive factor of outcome in children with
relapsed ‘high-risk’ acute lymphoblastic leuke-
mia. Leukemia, 22, 2193–2200.
Patel, B., Rai, L., Buck, G., Richards, S.M., Mortu-
za, Y., Mitchell, W., Gerrard, G., Moorman,
A.V., Duke, V., Hoffbrand, A.V., Fielding, A.K.,
Goldstone, A.H. & Foroni, L. (2010) Minimal
residual disease is a significant predictor of
treatment failure in non T-lineage adult acute
lymphoblastic leukaemia: final results of the
international trial UKALL XII/ECOG2993. Brit-
ish Journal of Haematology, 148, 80–89.
Pfeifer, H., Wassmann, B., Bethge, W., Dengler, J.,
Bornhauser, M., Stadler, M., Beelen, D., Vucinic,
V., Burmeister, T., Stelljes, M., Faul, C., Dreger,
P., Kiani, A., Schafer-Eckart, K., Schwerdtfeger,
R., Lange, E., Kubuschok, B., Horst, H.A.,
Gramatzki, M., Bruck, P., Serve, H., Hoelzer, D.,
Gokbuget, N. & Ottmann, O.G. (2012)
Randomized comparison of prophylactic and
minimal residual disease-triggered imatinib after
allogeneic stem cell transplantation for BCR-
ABL1 positive acute lymphoblastic leukemia.
Leukemia, doi: 10.1038/leu.2012.352.
Platzbecker, U., Wermke, M., Radke, J., Oelschlae-
gel, U., Seltmann, F., Kiani, A., Klut, I.M.,
Knoth, H., Rollig, C., Schetelig, J., Mohr, B.,
Graehlert, X., Ehninger, G., Bornhauser, M. &
Thiede, C. (2012) Azacitidine for treatment of
imminent relapse in MDS or AML patients after
159
Review
allogeneic HSCT: results of the RELAZA trial.
Leukemia, 26, 381–389.
Porter, D.L., Levine, B.L., Kalos, M., Bagg, A. &
June, C.H. (2011) Chimeric antigen receptor-
modified T cells in chronic lymphoid leukemia.
New England Journal of Medicine, 365, 725–733.
Pui, C.H., Campana, D., Pei, D., Bowman, W.P.,
Sandlund, J.T., Kaste, S.C., Ribeiro, R.C.,
Rubnitz, J.E., Raimondi, S.C., Onciu, M., Cou-
stan-Smith, E., Kun, L.E., Jeha, S., Cheng, C.,
Howard, S.C., Simmons, V., Bayles, A., Metzger,
M.L., Boyett, J.M., Leung, W., Handgretinger,
R., Downing, J.R., Evans, W.E. & Relling, M.V.
(2009) Treating childhood acute lymphoblastic
leukemia without cranial irradiation. New Eng-
land Journal of Medicine, 360, 2730–2741.
Radich, J., Ladne, P. & Gooley, T. (1995) Polymer-
ase chain reaction-based detection of minimal
residual disease in acute lymphoblastic leukemia
predicts relapse after allogeneic BMT. Biology of
Blood and Marrow Transplantation, 1, 24–31.
Rettinger, E., Willasch, A.M., Kreyenberg, H.,
Borkhardt, A., Holter, W., Kremens, B., Strahm,
B., Woessmann, W., Mauz-Koerholz, C., Gruhn,
B., Burdach, S., Albert, M.H., Schlegel, P.G.,
Klingebiel, T. & Bader, P. (2011) Preemptive
immunotherapy in childhood acute myeloid leu-
kemia for patients showing evidence of mixed
chimerism after allogeneic stem cell transplanta-
tion. Blood, 118, 5681–5688.
Rubnitz, J.E., Inaba, H., Dahl, G.V., Ribeiro, R.C.,
Bowman, W.P., Taub, J.W., Pounds, S., Raz-
zouk, B.I., Lacayo, N., Cao, X., Meshinchi, S.,
Degar, B., Airewele, G., Raimondi, S.C., Onciu,
M., Coustan-Smith, E., Downing, J.R., Leung,
W.H., Pui, C.H. & Campana, D. (2010a) Mini-
mal residual disease-directed therapy for child-
hood acute myeloid leukemia: results of the
AML02 multicenter trial. The Lancet Oncology,
11, 543–552.
Rubnitz, J.E., Inaba, H., Ribeiro, R.C., Pounds, S.,
Rooney, B., Bell, T., Pui, C.H. & Leung, W.
(2010b) NKAML: a pilot study to determine the
safety and feasibility of haploidentical natural
killer cell transplantation in childhood acute
myeloid leukemia. Journal of Clinical Oncology,
28, 955–959.
Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K.,
Shlomchik, W.D., Tosti, A., Posati, S., Rogaia,
D., Frassoni, F., Aversa, F., Martelli, M.F. &
Velardi, A. (2002) Effectiveness of donor natural
killer cell alloreactivity in mismatched hemato-
poietic transplants. Science, 295, 2097–2100.
Ruggeri, A., Michel, G., Dalle, J.H., Caniglia, M.,
Locatelli, F., Campos, A., de Heredia, C.D.,
Mohty, M., Hurtado, J.M., Bierings, M., Bitten-
court, H., Mauad, M., Purtill, D., Cunha, R.,
Kabbara, N., Gluckman, E., Labopin, M., Peters,
C. & Rocha, V. (2012) Impact of pretransplant
minimal residual disease after cord blood trans-
plantation for childhood acute lymphoblastic
leukemia in remission: an Eurocord, PDWP-
EBMT analysis. Leukemia, 26, 2455–2461.
Rujkijyanont, P., Chan, W.K., Eldridge, P.W., Loc-
key, T., Holladay, M., Rooney, B., Davidoff,
160
A.M., Leung, W. & Vong, Q. (2013) Ex vivo acti-
vation of CD56+ immune cells that eradicate neu-
roblastoma. Cancer Research, 73, 2608–2618.
Sanchez, J., Serrano, J., Gomez, P., Martinez, F.,
Martin, C., Madero, L., Herrera, C., Garcia,
J.M., Casano, J. & Torres, A. (2002) Clinical
value of immunological monitoring of minimal
residual disease in acute lymphoblastic leukae-
mia after allogeneic transplantation. British Jour-
nal of Haematology, 116, 686–694.
Sanchez-Garcia, J., Serrano, J., Serrano-Lopez, J.,
Gomez-Garcia, P., Martinez, F., Garcia-Castel-
lano, J.M., Rojas, R., Martin, C., Rodriguez-
Villa, A., Molina-Hurtado, J.R., Alvarez, M.A.,
Casano, J. & Torres-Gomez, A. (2012) Quantifi-
cation of minimal residual disease levels by flow
cytometry at time of transplant predicts out-
come after myeloablative allogeneic transplanta-
tion in ALL. Bone Marrow Transplantation, 48,
396–402.
Schneider, M., Hettinger, K., Matthes-Martin, S.,
Konrad, M., Peters, C., Gadner, H. & Panzer-
Grumayer, E.R. (2001) Influence of transplanta-
tion regimen on prognostic significance of
high-level minimal residual disease before
allogeneic stem cell transplantation in children
with ALL. Bone Marrow Transplantation, 28,
1087–1089.
Shimasaki, N., Fujisaki, H., Cho, D., Masselli, M.,
Lockey, T., Eldridge, P., Leung, W.H. & Cam-
pana, D. (2012) A clinically adaptable method
to enhance the cytotoxicity of natural killer cells
against B-cell malignancies. Cytotherapy, 14,
830–840.
Shook, D., Coustan-Smith, E., Ribeiro, R.C., Rub-
nitz, J.E. & Campana, D. (2009) Minimal resid-
ual disease quantitation in acute myeloid
leukemia. Clinical Lymphoma & Myeloma, 9
(Suppl. 3), S281–S285.
Spinelli, O., Peruta, B., Tosi, M., Guerini, V., Salvi,
A., Zanotti, M.C., Oldani, E., Grassi, A., Inter-
mesoli, T., Mico, C., Rossi, G., Fabris, P., Lamb-
ertenghi-Deliliers, G., Angelucci, E., Barbui, T.,
Bassan, R. & Rambaldi, A. (2007) Clearance of
minimal residual disease after allogeneic stem
cell transplantation and the prediction of the
clinical outcome of adult patients with high-risk
acute lymphoblastic leukemia. Haematologica,
92, 612–618.
Sramkova, L., Muzikova, K., Fronkova, E., Krejci,
O., Sedlacek, P., Formankova, R., Mejstrikova,
E., Stary, J. & Trka, J. (2007) Detectable mini-
mal residual disease before allogeneic hemato-
poietic stem cell transplantation predicts
extremely poor prognosis in children with acute
lymphoblastic leukemia. Pediatric Blood & Can-
cer, 48, 93–100.
Topp, M.S., Kufer, P., Gokbuget, N., Goebeler, M.,
Klinger, M., Neumann, S., Horst, H.A., Raff, T.,
Viardot, A., Schmid, M., Stelljes, M., Schaich,
M., Degenhard, E., Kohne-Volland, R., Brugge-
mann, M., Ottmann, O., Pfeifer, H., Burmeister,
T., Nagorsen, D., Schmidt, M., Lutterbuese, R.,
Reinhardt, C., Baeuerle, P.A., Kneba, M., Einsel-
e, H., Riethmuller, G., Hoelzer, D., Zugmaier,
British Journal of Haematology, 2013, 162, 147–161
G. & Bargou, R.C. (2011) Targeted therapy with
the T-cell-engaging antibody blinatumomab of
chemotherapy-refractory minimal residual dis-
ease in B-lineage acute lymphoblastic leukemia
patients results in high response rate and pro-
longed leukemia-free survival. Journal of Clinical
Oncology, 29, 2493–2498.
Uzunel, M., Jaksch, M., Mattsson, J. & Ringden,
O. (2003) Minimal residual disease detection
after allogeneic stem cell transplantation is
correlated to relapse in patients with acute
lymphoblastic leukaemia. British Journal of
Haematology, 122, 788–794.
van der Velden, V., Joosten, S.A., Willemse, M.J.,
Van Wering, E.R., Lankester, A.W., van Dongen,
J.J. & Hoogerbrugge, P.M. (2001) Real-time
quantitative PCR for detection of minimal resid-
ual disease before allogeneic stem cell transplan-
tation predicts outcome in children with acute
lymphoblastic leukemia. Leukemia, 15, 1485–
1487.
van der Velden, V.H., Sluijs-Geling, A., Gibson,
B.E., te Marvelde, J.G., Hoogeveen, P.G., Hop,
W.C., Wheatley, K., Bierings, M.B., Schuurhuis,
G.J., de Graaf, S.S., Van Wering, E.R. & van
Dongen, J.J. (2010) Clinical significance of flow-
cytometric minimal residual disease detection in
pediatric acute myeloid leukemia patients trea-
ted according to the DCOG ANLL97/MRC
AML12 protocol. Leukemia, 24, 1599–1606.
Venditti, A., Maurillo, L., Buccisano, F., Del Poeta,
G., Mazzone, C., Tamburini, A., Del Principe,
M.I., Consalvo, M.I., De Fabritiis, P., Cudillo,
L., Picardi, A., Franchi, A., Lo-Coco, F. & Ama-
dori, S. (2003) Pretransplant minimal residual
disease level predicts clinical outcome in patients
with acute myeloid leukemia receiving high-dose
chemotherapy and autologous stem cell trans-
plantation. Leukemia, 17, 2178–2182.
Walter, R.B., Gooley, T.A., Wood, B.L., Milano, F.,
Fang, M., Sorror, M.L., Estey, E.H., Salter, A.I.,
Lansverk, E., Chien, J.W., Gopal, A.K., Appel-
baum, F.R. & Pagel, J.M. (2011) Impact of
pretransplantation minimal residual disease, as
detected by multiparametric flow cytometry, on
outcome of myeloablative hematopoietic cell
transplantation for acute myeloid leukemia.
Journal of Clinical Oncology, 29, 1190–1197.
Wassmann, B., Pfeifer, H., Stadler, M., Bornhauser,
M., Bug, G., Scheuring, U.J., Bruck, P., Stelljes,
M., Schwerdtfeger, R., Basara, N., Perz, J.,
Bunjes, D., Ledderose, G., Mahlberg, R.,
Binckebanck, A., Gschaidmeier, H., Hoelzer, D.
& Ottmann, O.G. (2005) Early molecular
response to posttransplantation imatinib deter-
mines outcome in MRD+ Philadelphia-positive
acute lymphoblastic leukemia (Ph+ ALL). Blood,
106, 458–463.
Wu, D., Sherwood, A., Fromm, J.R., Winter, S.S.,
Dunsmore, K.P., Loh, M.L., Greisman, H.A.,
Sabath, D.E., Wood, B.L. & Robins, H. (2012)
High-throughput sequencing detects minimal
residual disease in acute T lymphoblastic
leukemia. Science Translational Medicine, 4,
134ra63.
ª 2013 John Wiley & Sons Ltd

Yan, C.H., Liu, D.H., Liu, K.Y., Xu, L.P., Liu, Y.R.,
Chen, H., Han, W., Wang, Y., Qin, Y.Z. &
Huang, X.J. (2012) Risk stratification-directed
donor lymphocyte infusion could reduce relapse
of standard-risk acute leukemia patients after
allogeneic hematopoietic stem cell transplanta-
tion. Blood, 119, 3256–3262.
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 147–161
Zeiser, R., Spyridonidis, A., Wasch, R., Ihorst, G.,
Grullich, C., Bertz, H. & Finke, J. (2005) Evalua-
tion of immunomodulatory treatment based on
conventional and lineage-specific chimerism
analysis in patients with myeloid malignancies
after myeloablative allogeneic hematopoietic cell
transplantation. Leukemia, 19, 814–821.
Review
Zhao, X.S., Liu, Y.R., Zhu, H.H., Xu, L.P.,
Liu, D.H., Liu, K.Y. & Huang, X.J. (2012)
Monitoring MRD with flow cytometry: an
effective method to predict relapse for ALL
patients after allogeneic hematopoietic stem cell
transplantation. Annals of Hematology, 91, 183–
192.
161