Cytometry Part B (Clinical Cytometry) 84B:359369 (2013)

Review Article

Detection of Minimal Residual Disease in

Pediatric Acute Lymphoblastic Leukemia
Giuseppe Gaipa,1* Giuseppe Basso,2 Andrea Biondi,1 and Dario Campana3
1

M. Tettamanti Research Center, Pediatric Clinic University of Milano Bicocca, Monza, Italy
Laboratorio di Oncoematologia Pediatrica, Department of Pediatrics, University of Padova, Padova, Italy
3
Department of Pediatrics, National University of Singapore, Singapore

Minimal residual disease (MRD) is a powerful predictor of the overall response to treatment in childhood
acute lymphoblastic leukemia (ALL). The most reliable and validated methods to assess MRD in ALL are
flow cytometric (FCM) analysis of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigenreceptor gene rearrangements. Results of studies correlating MRD
with clinical outcome and technical improvements in FCM technology support the implementation of
MRD studies by this method in the clinic. Gene expression profiling of leukemic and normal cells has
identified new MRD markers, which can be incorporated to improve the applicability and sensitivity of
FCM-based MRD monitoring. The combined use of MRD and emerging information on genetic lesions of
C 2013 International Clinical
ALL offers the possibility of further refining risk-assignment approaches. V
Cytometry Society
Key words: childhood; acute lymphoblastic leukemia; minimal residual disease; flow cytometry; RQ-PCR

How to cite this article: Gaipa G, Basso G, Biondi A, Campana D. Detection of Minimal Residual Disease in
Pediatric Acute Lymphoblastic Leukemia. Cytometry Part B 2013; 84B: 359369.

INTRODUCTION
Most children with acute lymphoblastic leukemia
(ALL) achieve complete remission with current treatment regimens but leukemia relapse, the main cause of
treatment failure, still occurs in a significant proportion
of patients (1). Periodic assessment of treatment
response provides an indication of the sensitivity of leukemic cells to chemotherapy and of the overall treatment effectiveness. Historically, treatment response has
been monitored by morphological examination of bone
marrow aspirates, a task which is a fundamental component of the clinical care of patients with ALL (2).
Because this approach has a limited sensitivity and specificity, it can result in failure to detect residual leukemic
cells, potentially leading to under-treatment and an
increase risk of relapse. Conversely, misclassification of
normal cells as ALL blasts may result in over-treatment
and an increase in the risk of treatment-related
morbidity.
Over the last 23 decades, much effort has been
made to develop accurate and sensitive methods that
could determine response to treatment more precisely
than morphology, and to detect residual leukemia persisting in patients considered to be in morphologic

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V

remission, i.e. minimal residual disease (MRD) (3,4).

Many studies have demonstrated that MRD is a powerful
predictor of clinical outcome in childhood ALL (512),
supporting the use of a more stringent definition of
hematological remission based on MRD assays rather
than morphology (13).
METHODOLOGIES FOR MRD DETECTION
To be informative, MRD assays for ALL should allow
to the detection of one leukemic cell among 10,000 normal cells or more. They should also reliably discriminate
leukemic and normal cells, and allow a timely output of
Correspondence to: Giuseppe Gaipa, M. Tettamanti Research Center, University of Milano Bicocca, Via Pergolesi, 33 Monza 20052,
Italy. E-mail g.gaipa@hsgerardo.org
Grant sponsor: Comitato M.L.Verga and Fondazione Tettamanti, Fondazione Citt
a della Speranza, Fondazione Cariparo, Associazione Gian
Franco Lupo, Associazione Italiana per la Ricerca sul Cancro, Fondazione Cariplo, and Ministero dellIstruzione, Universita e Ricerca
(MIUR).
Received 31 October 2012; Revised 2 April 2013; Accepted 23
March 2013
Published online 26 June 2013 in Wiley Online Library (wileyonline
library. com).
DOI: 10.1002/cyto.b.21101

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Table 1
Main Features and Applicability of the Methods Widely Used for Study MRD in Pediatric ALL
Method

Target

Applicability

Sensitivity
24

Flow cytometry

Leukemia-associated
immunophenotypes

95% or more

10

Real-time
quantitative
(RQ) PCR

Fusion gene
transcripts

40%

1024

Ig/TCR gene
rearrangements

9095%

1025

the results. Finally, they should be robust enough to support consistent MRD estimates when applied in different
laboratories (1418). Currently, the most reliable methods to study MRD in ALL are flow cytometric (FCM)
analysis of leukemia-associated immunophenotypes (i.e.,
aberrant phenotypes expressed in leukemic cells but
not in normal bone marrow or peripheral blood cells),
and polymerase chain reaction (PCR) amplification of
antigen receptor gene rearrangements (2,1423); gene
fusion transcripts can also be used as PCR targets in
some cases (24). The main features of these techniques
are outlined in Table 1.
A BRIEF HISTORY OF THE DEVELOPMENT OF MULTICOLOR
FLOW CYTOMETRY IMMUNOPHENOTYPING
Antigen expression is assessed by quantification of the
signal emitted by fluorochrome-conjugated-specific monoclonal antibodies (MoAb). Fluorescein excited by an argon
laser was initially used in the Herzenberg laboratory, followed by two-color fluorescence detection (25).
Then, additional antibody-conjugated fluorochromes
were developed to increase the number of colors
(26). From the 1980s, the capacity of several molecules
to absorb and transfer energy to other fluorescent molecules was exploited to produce tandem dyes such as PETexas Red, PE-Cy5, PE-Cy5.5, PE Cy7, and Alexa (27,28),
thus many laboratories were soon routinely performing
four-color experiments. Further advances rapidly led to
the possibility of studying a great number of functionally
distinct lymphocyte populations in human peripheral
blood by the simultaneous detection of six to eight different cell surface antigens (29). The introduction of violet lasers (405 nm) and of nanocrystals (called quantum
dots) or organic polymers capable of conducting electrons (30) led to the current polychromatic flow cytometry (PFC) capable of analyzing up to 18 colors in a single cell (31).
The development of PFC required a parallel advancement in hardware, software, and reagents. Engineering
advances in optics and signal processing (digital electronics) are areas of active development (26,32). Hardware improvements are supported by software development aiming at rapidly processing the bulk of raw data,

Advantages

Disadvantages

Widely applicable; rapid;

accurate and direct
MRD quantification
Rapid; reflects the
leukemic clone size
Widely applicable; highly
sensitive; accurate
MRD quantitation

Operator-dependent; need for

further standardization
Uncertain quantitation of MRD;
not completely standardized;
potential false-positive results
due to RNA degradation.
Laborious and costly;need for
experienced personnel and
standardization; oligoclonality
and clonal evolution may
produce false-negative results

to correct fluorescence spill-over between dyes (i.e.,

compensation), as well as to display and analyze
n-parameters and store them. Different software packages are now available, including FACSDivaTM [Becton
Dickinson], KaluzaTM [Beckman Coulter], FlowJoTM
[www.treestar.com], CytobankTM [www.cytobank.org],
SPICETM [http://exon.niaid.nih.gov/spice/], SamSPECTRALTM [R-package], FLAMETM, SPADETM [www.cytospade.org], FlowTypeTM [R-package], FlowCAPTM [flowcap.flowsite.org], GemStoneTM [www.vsh.com], InfinicyteTM
[www.infinicyt.com].
The increased number of fluorochromes did impact
on the quality of PFC analysis. For example, Bj
orklund
and collaborators used eight-color PFC to better define
the CD341 subsets in comparison with the previous
three-color method and proposed this approach to study
MRD in acute myeloid leukemia (AML) (33). Rare circulating progenitor cells of hematopoietic and/or endothelial origin could be detected with high sensitivity in
human peripheral blood or cord blood by using PFC
(34,35).The ability to measure more markers simultaneously can allow full antibody panels to be fitted into one
to two tubes, and the collection of meaningful information even from small samples. To this end, a 10-color
panel has been reported to detect leukemia and lymphoma cells in several types of small cell samples, a
development that could significantly improve diagnostic
procedures (36). Finally, the simultaneous analysis of
several parameters can also help to determine with
greater accuracy the presence of functionally important
cell subsets, such as those with stem cell features
(32). These advantages notwithstanding, several studies
demonstrated that MRD monitored by four-color FCM
can still be clinically informative (11,37,38).
PFC may introduce the potential for technical problems. For example, setting up multicolor FCM for MRD
analysis is not simply achieved by adding new reagents
to existing panels but requires careful testing, validation,
and quality control (26,39,40). In this process, antibody
clones must be carefully selected, as several clones recognizing the same marker may differ significantly in
their reactivity (e.g., CD34, a key marker for MRD detection) (41).

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MRD IN PEDIATRIC ALL

SOME CONSIDERATIONS REGARDING FLUOROCHROMES

Desirable fluorochromes for leukemia immunophenotyping are as follows: (i) biologically inert, (ii) have high
cell-associated fluorescence intensities, (iii) exhibit little
spectral overlap amongst each other, and (iv) can be easily conjugated to antibodies (26).
PE-conjugates are generally brighter than FITCconjugates, while PE-Cy5 and APC reagents show an
intermediate level of brightness. Other available fluorochromes and tandems include APC-Cy5, PerCP-Cy5,
APC-Cy7, and PerCP-Cy7. In general, the brightest fluorochrome should be used for detection of the weakest
marker. A surface molecule whose expression is
unknown is initially best studied with an antibody conjugate that uses one of the brightest fluorochrome (e.g.,
PE, PE-Cy5, or APC). The choice of fluorochrome for the
other markers depends on instrument characteristics. In
general, PerCP-conjugated MoAbs have some limitations
related to their low sensitivity and the restricted number
of commercially available MoAbs conjugated to this fluorochrome. PE-Cy5 tandem conjugates are widely available but have some nonspecific binding, especially to
monocytic cells (42). Another issue to be considered is
the possible spectral overlap between the various dyes
in the staining combinations. Although hardware and
software compensation can partially solve overlapping
problems, a careful choice of reagents can minimize the
requirement for such procedures. APC reagents are
quite sensitive; the combined use of APC with PE-Cy7
allows limited fluorescence compensation and reduces
nonspecific binding. However, the introduction of new
generation fluorochromes may also implicate compensation complications and attention should be drawn to
obtain optimal instrument setting; for example DiGiuseppe and collaborators calculated compensation values
for AmCyan and other fluorochromes using the beadsbased set provided by the manufacturers protocol, and
using unstained and singly stained lymphocytes as compensation controls. Improved compensation of the
violet-excited dye (i.e. AmCyan) was obtained when
cells rather than the commercial beads were used as
compensation controls (43).
IMMUNOLOGICAL MARKERS OF MRD IN ALL: EVOLUTION
AND DISCOVERY
It has been known for more than three decades that
ALL cells have immunophenotypic features that distinguish them from normal bone marrow and peripheral
blood cells. Initial studies were performed by two-color
immunofluorescence microscopy (2). With the advent of
FCM, it soon became clear that the features of this technology, such as the potential for detecting multiple
parameters in a large number of cells while quantifying
antigen expression, were particularly well suited to
MRD studies.
Two conceptually separate approaches are applied to
the selection of markers for MRD studies in T-lineage
and B-lineage ALL. In T-lineage ALL, the main task

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361

consists of identifying cells with immunophenotypic features of immature T cells. Normal thymocytes may
express identical cell marker profiles but they are normally confined to the thymic microenvironment. Hence,
the detection of cells with immunophenotypic features
of immature T cells (e.g., simultaneous expression of terminal deoxynucleotidase transferase and CD3) in the
blood or marrow of patients with T-lineage ALL indicates
MRD. In B-lineage ALL, simple identification of immature
B cells is generally not sufficient (see below for an
exception to this rule) because normal immature B cells
are a constituent of the normal bone marrow cell population and can be particularly abundant in bone marrow
samples recovering from chemotherapy (44,45). Therefore, B-lineage ALL cells must be distinguished from normal B-cell progenitors by virtue of cell markers that
become aberrantly expressed during leukemogenesis
(4649). However, if the follow-up bone marrow specimen does not contain lymphoid progenitors (a scenario
that presents itself during the first 23 weeks of
remission-induction therapy) then the mere presence of
cells with features of immature B cells can be an indication of residual disease. Indeed, in bone marrow samples collected from 11 patients with T-lineage ALL after
2 weeks of remission induction therapy, CD191 cells
expressing CD10 and/or CD34 were either undetectable
or represented <0.01% of bone marrow mononuclear
cells in all patients (50). Moreover, MRD levels measured
by these three markers correlated well with those measured by more complex FCM analysis or PCR amplification of antigen-receptor genes (50). This exception notwithstanding, all other MRD studies in B-lineage ALL
require the identification of the aberrant immunophenotypes expressed by each leukemia, which is typically
undertaken at diagnosis. Some investigators studied
MRD in follow-up samples without prior knowledge of
the immunophenotype of the leukemic cells, a process
that involves searching for aberrant profiles by using a
very large panel of antibody combinations (51). This
approach may be the only option when patients are
referred from another center for continuation therapy or
transplant, but a negative MRD result would be clearly
difficult to interpret.
Aberrant immunophenotypes can be found in the
majority of patients. For example, in the St Jude Total
XV study, 482 of 492 (98%) enrolled patients with newly
diagnosed ALL had leukemic cells at diagnosis that
expressed leukemia-associated immunophenotypes that
would allow the detection of MRD at the 0.01% level at
any time point during therapy, including at times of vigorous bone marrow cell recovery (52). However, the differences between ALL and normal cells are often subtle
and discerning them takes much experience. Logically,
the greater the immunophenotypic difference between
the two cell types the easier it should be to recognize
ALL cells. In this context, the identification of new
markers to be incorporated into MRD panels should be
beneficial by expanding the multidimensional approach
to MRD data analysis and taking advantage to the

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FIG. 1. Markers that can be used to monitor MRD in B-lineage ALL.

Black dots correspond to markers that denote immature B-lymphoid
cells but cannot distinguish between normal and leukemic cells. The
remaining dots indicate markers that can be used to define leukemiaassociated immunophenotypes: markers identified in early studies
(1st generation) are shown in the gray dots; those identified more
recently through gene expression array comparisons (62) are in white.
Examples of antibody combinations and their fluorochromes which can
be used to study MRD are shown in the table.

capabilities of contemporary instruments which have at

least doubled the number of parameters that could be
traditionally measured.
The task of identifying new markers of ALL was traditionally based on observations made by directly comparing the immunophenotype of normal and leukemic cells
(48,53). This approach was also used in more recent
studies which identified CD81 (54), CD49f (55), and
CD11b (56) as potentially useful markers. Another strategy is to compare the gene expression profiles of normal and leukemic CD191 CD101 cells and search for
differentially expressed genes. In an early study, this
approach led to the identification of CD58 as a useful
marker (57), a finding which was confirmed by several
other studies (5861). More recently, a study compared
genome-wide gene expression of leukemic cells from
270 patients with B-lineage ALL to that of purified normal CD191CD101 B cell progenitors from four healthy
donors (62). Among the differentially expressed genes,
30 were selected for further studies by FCM in 200 Blineage ALL and 61 non-leukemic bone marrow samples
(62). Twenty-two of the 30 markers (CD44, BCL2,
HSPB1, CD73, CD24, CD123, CD72, CD86, CD200,
CD79b, CD164, CD304, CD97, CD102, CD99, CD300a,
CD130, PBX1, CTNNA1, ITGB7, CD69, CD49f) were differentially expressed in about 80% of ALL cases (62).
Notably, some of the markers, such as CD304 (63) and
CD123 (64), were independently found to be differentially expressed in ALL by other investigators. Studies
using paired diagnosis-relapse samples demonstrated the
stability of the new markers, which have allowed MRD
monitoring by FCM in more than 150 consecutive
patients with a potential sensitivity of 1 in 100,000 (62).
Figure 1 summarizes first and second generation markers
for MRD studies in ALL; examples of the immunophenotypic differences between normal and leukemic cells
detected by some of the new markers are shown in
Figure 2.

STANDARDIZATION OF FLOW CYTOMETRIC METHODS FOR

MULTICENTRIC MRD STUDIES
Early efforts in standardization of flow cytometric protocols for MRD detection in ALL were initially carried
out in the context of BIOMED-1 Concerted Action by
six different European laboratories (47). In that study,
characterization of normal bone marrow-derived precursor-B cell subpopulations by three-color immunophenotyping provided templates as a frame of reference for
discriminating normal from malignant precursor-B cells.
An extensive standardization of four-color FCM MRD
protocol for multicentric applications was performed by
four laboratories within the AIEOP-BFM-ALL 2000 study
(23). This addressed several technical issues including
methodological harmonization regarding sample preparation, reagent selection and instrument set-up, intra and
inter-laboratory quality monitoring, as well as specific
personnel training focused on FCM data interpretation.
Despite differences in cytometers and software usage,
blinded inter-laboratory tests of list-mode data interpretation gave highly concordant results (intraclass correlation coefficient [ICC], 0.979). The reproducibility in
inter-laboratory sample exchange was also assessed:
among 63 follow-up samples, exchanged between two
centers, the positive/negative concordance as well as
the quantitative reproducibility of MRD-values, were
very high (23). Concordance between all four centers in
the analysis of artificial mixtures of normal and leukemic
cells was also high (ICC: 0.98): of 164 MRD-values available, there were five false values each among 113 positive and 51 negative values, resulting in a sensitivity of
95.6% and a specificity of 90.2% (23). The application of
such a standardized multicentric FCM protocol to study
MRD has yielded significant correlations with treatment
outcome in pediatric ALL. Thus, Ratei et al. (65) showed
that flow cytometric blast reduction rates on day 8 and
day 15 had a predictive impact on the remission status
after induction therapy in the AIEOP-BFM ALL 2000 protocol, and Basso et al. demonstrated feasibility and

FIG. 2. Immunophenotypic differences between normal and leukemic

cells. Flow cytometric dot plots show the immunophenotype of gated
CD191 CD101 cells from normal bone marrow (BM) samples (top
row) and from diagnostic samples of B-lineage ALL (bottom row) as
defined by some of the recently identified markers. Dashed lines
enclose areas were most normal CD191 CD101 cells are located.

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MRD IN PEDIATRIC ALL

robustness of this approach in refining MRD-based stratification with measurements on day 15 (37). The feasibility of standardized multicentric MRD assessment has
also been shown by others groups (66,67). Six-eightcolor-FCM MRD techniques are currently employed for
leukemia immunophenotyping. Advances in algorithm
and methods for analyzing complex data should further
facilitate MRD data analysis and foster standardized analytical approaches (68). The recently reported efforts of
the EuroFlow group should contribute to standardize
leukemia immunophenotyping in a major way, and also
have an impact on MRD protocols (69).
MOLECULAR METHODS FOR MRD DETECTION IN ALL
AntigenReceptor Gene Rearrangements
Somatic rearrangement of Ig and TcR gene loci occurs
during early differentiation of any B and T cell, by joining the germline variable (V), diversity (D), and joining
(J) gene segments (3). By this process, each lymphocyte
gets a specific combination of V-(D-) J segments that
encodes the variable domains of Ig or TcR molecules.
The uniqueness of each rearrangement further depends
on random insertion and deletion of nucleotides at the
junction sites of V, (D), and J gene segments, making
the junctional regions of Ig and TcR genes as
fingerprint-like sequences. This combined sequence
constitutes a specific signature of each lymphocyte.
Because of the clonal origin of the neoplasm, each
malignant lymphoid disease will represent the expansion
of a clonal population with a specific Ig/TcR signature
(70).
Virtually all B-lineage ALL patients have rearranged
Immunoglobulin heavy chain (IgH) genes (70). In addition, rearrangements of the Ig Kappa deleting element
(Kde) occur at a relatively high frequency (approximately 60%) (71). Cross-lineage incomplete TCRD rearrangements occur in more than 40% of all patients (40%
Vd2-Dd3, 19% Dd2-Dd3 and 13% showed both) (72).
Complete TCRD rearrangements (Vd-Jd) are very rare in
B-lineage ALL. Detection of TCRG rearrangements
occurs in more than 50% of the patients with B-lineage
ALL (72). Cross-lineage TCRB recombinations occur in a
small percentage (1520%) of patients with B-lineage
ALL. The success rate of detecting appropriate MRDPCR targets is lower in T-ALL compared to patients with
B-lineage ALL. The addition of TCRB gene rearrangements to MRD-PCR target identification increases the
availability of targets in T-ALL. Moreover, the junctional
regions of (complete) TCRD rearrangements, similar to
IGH in B-lineage ALL, frequently include extensive Nnucleotide insertions, thus enabling the design of highly
specific ASO primers. In contrast, the junctions of
TCRG and incomplete IGH rearrangements are commonly smaller resulting in a significantly lower ratio of
sensitive targets (about 75%).
For MRD studies, the specific rearrangements must be
sequenced in each case upfront. Ig/TcR gene rearrangements can be used as universal PCR-MRD targets. In

Cytometry Part B: Clinical Cytometry

combination with fluorescent probes, allele-specific oligonucleotide (ASO) primers can be designed complementary to the patient- and tumor-specific junctional
region sequence of each target. A large standardization,
quality control and guidelines on RQ-PCR analysis and
interpretation have been assessed within the EuroMRD group (previous European Study Group for MRD
detection in ALL, ESG-MRD ALL) (16). More recently,
the possibility to use high-throughput sequencing (HTS)
for disease-specific IGH sequence quantification was
proposed and it is likely that new and fast technologies
for rapid sequencing will broaden the availability of
MRD quantification B and T cell malignancies (73).
However, the predictive value of MRD was also demonstrated in childhood T-lineage ALL (74,75), infant ALL
(76), in mature-B ALL (77), as well as in patients with
intra chromosomal amplification of chromosome 21
(78). It is likely that in a near future PCR studies in ALL
MRD will benefit of more rapid and sensitive
approaches such as next-generation sequencing technologies (73,79).
Gene Fusions
Somatic chromosome aberrations can be used as PCR
targets for MRD detection because they are tumorspecific and stable during the disease course. The most
widely used for MRD detection are gene fusions, such as
BCR-ABL1, MLL-AFF1, TCF3-PBX1, and ETV6-RUNX1,
resulting in the expression of aberrant mRNA transcripts. Recurrent abnormalities suitable for routine
MRD studies in clinical samples are present in approximately 40% of children with ALL. Among more recently
identified abnormalities that could, in principle, be used
for monitoring MRD, are gene fusions involving CRLF2
(e.g., IGH@-CRLF2 and P2RY8-CRLF2). A consensus on
the reagents and minimal requirements for MRD monitoring by RQ-PCR of fusion transcripts has been reached
by several European laboratories collaborating on a
Europe Against Cancer program (80). Currently the use
of RQ-PCR of fusion genes is limited to the Ph1 ALL
(81).
CLINICAL SIGNIFICANCE OF MRD IN PEDIATRIC ALL
Many studies have demonstrated the prognostic significance of MRD levels in pediatric ALL. Such studies have
focused on newly diagnosed patients (58,8289),
patients with first relapse leukemia (9,9092), and
patients undergoing allogeneic hematopoietic stem cell
transplant (HSCT) (9396). They unanimously showed
that MRD is a powerful predictor of outcome, leading to
the adoption of MRD as a critical risk-assignment criteria
(12,52).
Recent studies have provided strong evidence that
MRD can also be informative among patients with specific subtypes of ALL. Thus, Conter et al. (12) found that
MRD was predictive of outcome among patients with
the TEL-AML1, high hyperdiploidy, or BCR-ABL1 molecular abnormalities. Schrappe et al. (75) found that MRD
measurements on days 33 and 78 of treatment allowed

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the delineation of distinct prognostic subgroups among

patients with T-ALL. Bowman et al. (97) reported that
MRD measurements in peripheral blood on day 8 and in
the bone marrow on day 29 predicted outcome in children with B-lineage ALL receiving augmented therapy
because of high-risk presenting clinical features. Finally,
van der Velden et al. (76) found that MRD levels at the
end of induction and consolidation could predict outcome in infants with ALL.
One of the main clinical uses of MRD monitoring is to
measure the kinetics of initial leukemia cytoreduction,
which, in turn, is a predictor of response overall. To this
end, it is becoming clear that studies of MRD at early
time points during therapy can be extremely powerful.
Early studies demonstrated that MRD testing after 23
weeks of remission induction therapy could predict outcome (7,98). More recently, Sutton et al. (85) measured
MRD during treatment in 108 patients and found that
measurements on day 15 were strong predictors of
relapse risk. A study of the Associazione Italiana Ematologia Oncologia Pediatrica assessed MRD in bone marrow samples from 830 children with ALL collected on
day 15 of treatment (37). MRD levels 10%, seen in
11% of patients, were associated with a 5-year relapse
rate of 47.2%, 0.1% to < 10% levels (47%) a rate of
17.5% and <0.1% levels (42%) with a rate of 7.5%. In a
multivariate analysis, MRD was the strongest prognostic
factor when compared to presenting features, and
retained prognostic significance when MRD detected by
PCR at later time points was added to the model. MRD
studies at these early time points are technically less
challenging that those are later time points because of
the paucity of normal lymphoid progenitors present in
bone marrow (these cells are eliminated by remission
induction chemotherapy) (50). Hence, the demonstration of their clinical utility is of particular practical
importance.
FLOW CYTOMETRY VERSUS PCR: CONCORDANCE,
CORRELATIONS, AND COMPLEMENTARY USE
Several studies have compared MRD estimates
obtained by FCM and PCR (99104). Most of these
reports considered a cut-off of 0.01% for positivity, and
showed a degree of concordance of >90%. Most of discrepancies were due to FCM-negative/PCR positive
results occurring at the lowest level of MRD. Discrepancies (either qualitative or quantitative) may be explained
by different factors affecting either PCR or FCM, such as
quality of clonal PCR-markers (22), nonspecific amplification of normal DNA (105), oligoclonality and/or clonal
evolution (106111), age-related (47) or therapy-related
bone marrow B-cell precursor regeneration status
(112,113), immunophenotypic changes between diagnosis and relapse (114,115) or drug-induced immunophenotypic modulation (59). This latter phenomenon has
been described during early phases of treatment in BFMoriented protocols, when steroids components are
largely predominant, in particular down modulation of
CD10 and CD34, and up modulation of CD19, CD20,

CD45RA, CD11a was observed, whereas CD58 was stable (59). Despite this, the accuracy of MRD detection
was not negatively affected, as residual modulated blasts
were located outside regions of residual normal B cells
from the same follow-up sample. Finally, Dworzak et al.
demonstrated that drug-induced up modulation of CD10
and CD34 were reversible after stop of steroid containing chemotherapy (116); therefore, the leukemiaassociated immunophenotypes seen at diagnosis remain
an important frame of reference even though transient
changes may occur during the early phase of treatment.
However, prevalence and inter-individual variations of B
cell precursor subpopulations have been described so
far in pediatric bone marrow during and after chemotherapy (112,113).
Table 2 summarizes the results obtained in some of
the studies comparing FCM and PCR in the simultaneous detection of MRD. Variations in sample collection
may affect the concordance between the two methods.
Both FCM and PCR samples should be collected from
the same bone marrow aspirate; MRD levels in a second
bone marrow aspiration through the same needle could
be lower because of hemodilution (117). The use of
mononucleated cells from the same density centrifugation separation should increase the concordance
between the two methods. However, several studies
have compared PCR and FCM in MRD detection by
using mononuclear cells and total nucleated cells,
respectively (see Table 2). Among these, Th
orn et al.
(118) adopted a virtual Ficoll gate when they analyzed
FCM data files in order to mimic the procedure of density gradient centrifugation. The calculated fraction of
mononuclear cells was determined by applying a mathematical formula and then used to extrapolate the MRD
values that would be found in Ficoll-separated cells. One
study found that the impact of using either monucleated
or total nucleated cells on the concordance of FCM
with PCR-results was minimal, and that concordance
rates may be influenced by the time point at which
MRD is measured (104).
In the final analysis, the choice of the MRD method to
be used is primarily dependent on the expertise and
resources available, and on clinical trial design. To this
end, for risk assignment strategies relying on early evaluations of treatment response, i.e. day 15, FCM should be
preferable as current PCR methods to detect antigen
receptor genes require a longer (typically 3 weeks)
preparation time.
CONCLUSIONS
MRD studies have now been incorporated into clinical
protocols where they are used to assess risk of relapse
and rationalize treatment decisions. MRD studies at early
time points can identify patients with good treatment
response, who may be curable with less intensity therapy. Current clinical trials are testing this possibility
(50). Another attractive application of MRD is in the
context of trials of novel agents or treatment regimens,
where MRD measurements can provide a rapid

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365

97.1%
93.3%
90.0%
80.0%
0.01%
0.01%
0.1%
0.01%
4
3
3/4
4
10 210
1024
1023
1024

10
1024
1023
1024

89.0%
0.01%
3/4

25
23

102321025
1024

1
10
4
3

Cytometry Part B: Clinical Cytometry

Kerst et al. (102)

Ryan et al.(103)
Th
orn et al. (118)
Gaipa et al. (104)

22
Malec et al. (101)

MNC, mononucleated cells; TNC, total nucleated cells.

MNC
MNC
FCM: TNC, PCR: MNC
FCM: TNC, PCR: MNC
105
151
726
2,701
30
29
228
1115

2
FCM: TNC, PCR: MNC
71

1
3
FCM: TNC, PCR: MNC
MNC
69
227

AIEOP-BFM-2000
St.Jude Total XIII;
XIV; XV.
NOPHO 92 and
NOPHO 2000
Not reported
UK-ALL 97/99
NOPHO 2000
AIEOP-BFM 2000

69
1375

FCM: TNC, PCR: MNC

16
ALL-BFM-95

mayer,
Dworzak and Gru
Leukemia and
Lymphoma 2003
Veltroni et al. (58)
Neale et al. (100)

84

ACKNOWLEDGMENTS
We thank Ms. Elaine Coustan-Smith for helpful discussions and providing Figure 2, Dr. Barbara Buldini, Dr.
Andrea Zangrando, and Mr. Oscar Maglia for their support in this study. We also thank the I-BFM-ALL-FCMMRD-Study Group.

24

95.6%
96.7%
4
4
1024
1025
Not reported
1024

Any level
0.01%

83.0%
3
Not reported
Not reported

0.01%

78.0%
3
102321025
FCM: TNC, PCR: MNC
89
NOPHO 92

23

assessment of efficacy. As a caveat to this use, a recent

trial demonstrated a significantly better EFS for ALL
relapsed patients treated in induction with mitoxantrone
vs idarubicine but no apparent difference in MRD at the
end of induction (119). Whether a single MRD positive
finding during or off treatment is sufficient to define
relapse and initiate retrieval therapy, and whether it
would be of benefit to initiate treatment at such stage
instead of waiting for overt relapse remains unclear. The
finding of MRD, however, should trigger frequent
monitoring.
In sum, MRD studies are a key tool towards the development of personalized treatment for children with ALL
and FCM has significantly contributed to widen the
applicability of this valuable test.

LITERATURE CITED

Malec et al. (101)

Study

6 (early and
late remission
induction)
>2

102321025

Any level

Concordance
rate
Cut-off
for
positivity
FCM
colors
(n)
PCR
sensitivity
FCM
sensitivity
N of time
points
studied
Source of starting
material
No. of
samples
No. of
patients
studied
Clinical
protocol

Table 2
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