Review Article
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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GAIPA ET AL.
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
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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GAIPA ET AL.
363
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
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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GAIPA ET AL.
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
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
22
Malec et al. (101)
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
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
LITERATURE CITED
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
Studies Directly Comparing MRD Detection by Flow Cytometry (FCM) and Polymerase Chain Reaction (PCR) Amplification of Antigen Receptor Genes
366
GAIPA ET AL.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
367
368
GAIPA ET AL.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
369
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