Why and how to quantify minimal residual disease in acute lymphoblastic leukemia? Leukemia

Abstract

Several studies have demonstrated that monitoring of minimal residual disease (MRD) in childhood and adult acute lymphoblastic leukemia (ALL) significantly correlates with clinical outcome. MRD detection is particularly useful for evaluation of early treatment response and consequently for improved front-line therapy stratification. MRD information is also significant for children undergoing allogeneic hematopoietic stem cell transplantation and those with relapsed ALL. Currently, three highly specific and sensitive methodologies for MRD detection are available, namely multiparameter flow cytometric immunophenotyping, real-time quantitative polymerase chain reaction (RQ-PCR)-based detection of fusion gene transcripts or breakpoints, and RQ-PCR-based detection of clonal immunoglobulin and T-cell receptor gene rearrangements. In this review, characteristics, pitfalls, advantages and disadvantages of each MRD technique are critically discussed. The special emphasis is put on interlaboratory standardization, especially in view of the results obtained within the European collaborative BIOMED-1, BIOMED-2, and Europe Against Cancer projects and recent developments by European Study Group on MRD detection in ALL and EuroFlow Consortium. Standardized MRD techniques form the basis for stratification of patients into the risk groups in new treatment protocols mainly in childhood ALL. Only the results of these studies can answer the question whether MRD-based treatment intervention is associated with improved outcome.

Full text

CONCISE REVIEW
Why and how to quantify minimal residual disease in acute lymphoblastic leukemia?
T Szczepan
´ski
1,2
1
Department of Pediatric Hematology and Oncology, Silesian Medical Academy, Zabrze, Poland and
2
Department of
Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Several studies have demonstrated that monitoring of minimal
residual disease (MRD) in childhood and adult acute lympho-
blastic leukemia (ALL) significantly correlates with clinical
outcome. MRD detection is particularly useful for evaluation
of early treatment response and consequently for improved
front-line therapy stratification. MRD information is also
significant for children undergoing allogeneic hematopoietic
stem cell transplantation and those with relapsed ALL.
Currently, three highly specific and sensitive methodologies
for MRD detection are available, namely multiparameter flow
cytometric immunophenotyping, real-time quantitative poly-
merase chain reaction (RQ-PCR)-based detection of fusion
gene transcripts or breakpoints, and RQ-PCR-based detection
of clonal immunoglobulin and T-cell receptor gene rearrange-
ments. In this review, characteristics, pitfalls, advantages and
disadvantages of each MRD technique are critically discussed.
The special emphasis is put on interlaboratory standardization,
especially in view of the results obtained within the European
collaborative BIOMED-1, BIOMED-2, and Europe Against Can-
cer projects and recent developments by European Study
Group on MRD detection in ALL and EuroFlow Consortium.
Standardized MRD techniques form the basis for stratification
of patients into the risk groups in new treatment protocols
mainly in childhood ALL. Only the results of these studies can
answer the question whether MRD-based treatment interven-
tion is associated with improved outcome.
Leukemia (2007) 21, 622–626. doi:10.1038/sj.leu.2404603;
published online 15 February 2007
Keywords: acute lymphoblastic leukemia; minimal residual
disease; flow cytometric immunophenotyping; real-time polymerase
chain reaction; immunoglobulin and T-cell receptor genes; fusion
genes
The rationale for detection of MRD in ALL
The concept of minimal residual disease (MRD) detection in
acute lymphoblastic leukemias (ALL) is inherently associated
with the progress in treatment of these malignancies.
1
More than
80% of childhood and 35% of adult ALL patients can be cured
with modern chemotherapy supplemented with hematopoietic
stem cell transplantation (HSCT) in high risk patients (reviewed
by Hoelzer et al.
2
). Still, a substantial number of ALL patients
relapse and the prediction of relapse with conventional
prognostic factors such as age, blast count at diagnosis,
immunophenotype at diagnosis, presence of chromosome
aberrations, response to steroid prophase and classical clinical
risk group assignment is far from optimal. Also microarray-based
gene expression profiling could not identify gene signatures,
typically associated with high risk of relapse. Tracing residual
leukemic cells during early phases of treatment provides
prognostic information superior to all known classical prog-
nostic factors. Several prospective studies in childhood ALL
demonstrated that the most relevant information comes from
detection of MRD in bone marrow at the early phases of
treatment, particularly at the end of induction treatment
(reviewed by Szczepan
´ski et al.
3
and Cazzaniga and Biondi
4
).
Children with undetectable MRD at the end of induction have
an excellent prognosis and are good candidates for treatment
de-intensification or at least should not be subjected to further
treatment intensification, particularly not to HSCT.
5–9
In child-
hood ALL, a group of ultrafast responders could be identified
with successful clearance of MRD in bone marrow even within
first 2 weeks of the induction.
10,11
In contrast, children with
high MRD levels at the end of induction treatment are in urgent
need for treatment intensification or even for novel treatment
approaches, particularly when such high MRD levels persist into
the consolidation treatment.
5–9
Children with high-risk primary
ALL and children with relapsed ALL planned for allogeneic
HSCT can also profit from MRD monitoring.
12–17
It is now
generally accepted that one of the prerequisites for successful
allo-HSCT is maximal reduction of MRD before start of the
transplant procedure. Patients with high pretransplant MRD
levels are at very high risk for ALL relapse.
12,14,15
Currently, the
ongoing studies aim at assessment of possible approaches to
lower pretransplant MRD levels and/or attune graft-versus-host
reaction according to post-transplant MRD levels by modifying
the immunosuppression to improve the outcome after HSCT.
Finally, it was recently demonstrated that treatment stratification
of standard risk adult ALL patients can be substantially improved
when including MRD information.
18
In conclusion, based on the above-summarized significant
results, several prospective treatment protocols, mainly in
childhood ALL have been initiated including MRD-based
treatment intervention as an essential part. Only the results of
these studies can answer the question whether MRD-based
treatment intervention is associated with improved outcome.
The methods for quantification of MRD in ALL
Optimal MRD techniques should be characterized by patient
specificity (or at least leukemia specificity), satisfactory sensi-
tivity (at least 10
4
, i.e., one malignant cell among 10 000
normal cells), applicability for the vast majority of patients under
the study, feasibility (easy standardization and rapid collection
of results for clinical application) as well as intralaboratory and
interlaboratory reproducibility. Another prerequisite for reliable
MRD technique is precise quantification of MRD levels. The
stringent criteria described above are for the greater part met by
three approaches, namely multiparameter flow cytometric
immunophenotyping, real-time quantitative polymerase chain
Received 7 January 2007; accepted 10 January 2007; published
online 15 February 2007
Correspondence: Dr T Szczepan
´ski, Department of Pediatric Hema-
tology and Oncology, Silesian Medical Academy, Ul. 3 Maja 13/15,
41-800 Zabrze, Poland.
E-mail: szczep57@poczta.onet.pl
Leukemia (2007) 21, 622–626
&2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00
www.nature.com/leu

reaction (RQ-PCR)-based detection of fusion gene transcripts or
breakpoints, and RQ-PCR-based detection of clonal immuno-
globulin (Ig) and T-cell receptor (TCR) gene rearrangements
(Table 1).
19–21
Quantification with multiparameter flow cytometric
immunophenotyping
Precise quantification is an inherent feature of flow cytometry,
which measures single cells. Up till now, immunophenotypic
MRD detection in ALL was based on 3–4 color flow cytometry.
This methodology relies on tracing the leukemia-specific
immunophenotypes as the result of cross-lineage antigen
expression, maturational asynchronous expression of antigens,
antigen overexpression, absence of antigen expression, ectopic
antigen expression, and various combinations of above-men-
tioned features bringing the ALL blasts into the ‘empty spaces’
between normal lymphoid differentiation (summarized by
Campana
19
and Szczepan
´ski et al.
20
). Still the detection limit
of this technique is not lower than 10
3
–10
4
, which never-
theless should be sufficient for identifying high-risk ALL patients.
Another pitfall of flow cytometry is the modulation of antigen
expression occurring during the treatment, which can change
the leukemia-specific immunophenotype into a phenotype that
resembles that of normal lymphoid precursors.
22,23
Moreover,
immunophenotypic changes might occur between diagnosis
and relapse.
24
Therefore, following of at least two leukemia-
specific immunophenotypes per patient has been recommended
to prevent false-negative results. It is striking that the most signi-
ficant MRD studies in ALL employing flow cytometric immuno-
phenotyping were based on a single expert laboratory.
7,9
This
raises the issue of interlaboratory standardization of flow
cytometric reagents and procedures, which is essential for
international multicenter treatment protocols. With the advent
of bench top X6-color flow cytometers, higher sensitivity for
MRD monitoring should be achieved, but with the increase of
technical complexity the need for interlaboratory standardiza-
tion becomes even more urgent. In Europe this need for
innovation and standardization is supported by the European
Commission via the EuroFlow Consortium aiming at standardi-
zation of ‘Flow cytometry for fast and sensitive diagnosis and
follow-up of hematological malignancies’.
25
RQ-PCR-based quantification of leukemia-associated
fusion genes
Leukemia-associated fusion genes resulting from chromosomal
translocations are directly linked to leukemogenesis and there-
fore represent very good and stable disease-specific markers.
After numerous single-center studies, the uniform primers and
Table 1 Characteristics of the techniques currently employed for MRD detection in ALL
Flow cytometric
immunophenotyping
PCR analysis of chromosome
aberrations (mainly detection of fusion
gene transcripts)
PCR analysis of Ig/TCR genes
(junctional region specific approach)
Sensitivity 10
3
–10
4
10
4
–10
6
10
4
–10
5
Applicability
Precursor-B-ALL 495% 40–45%
a
90–95%
T-ALL 495% 15–35%
b
90–95%
Advantages Applicable for most patients
Relatively cheap
Rapid: 1–2 days
Relatively easy and cheap
Sensitive and leukemia-specific
Stable target during disease course
Rapid: 2–3 days
Suitable for monitoring of uniform
patient groups (e.g. BCR-ABL+ ALL)
Applicable for virtually all patients, if
IGH,IGK-Kde, TCRG, and TCRD gene
rearrangements are used as targets
Sensitive and patient specific
Rapid during follow-up: 2–3 days
(if junctional region is identified and
if RQ-PCR is used)
Disadvantages Limited sensitivity
Need for preferably two aberrant
immunophenotypes per patient,
because of chance of
immunophenotypic shifts
Drug-induced modulation of the
immunophenotype might influence
the levels of antigenic expression
Useful in only a minority of patients
Not patient-specific – cross-
contamination of PCR products might
lead to false-positive results (even at
diagnosis)
Differences in fusion transcript
expression levels between the patients
Stability of fusion gene transcripts
decreases over time
Time consuming at diagnosis:
identification of the junctional regions
and sensitivity testing
Relatively expensive
Need for preferably two PCR targets
per patient, because of chance of
clonal evolution
Two sensitive targets (p10
4
) available
in B80% of patients
Recent developments and
standardization in European
networks
X6-color cytometry gives
promises of increased sensitivity
and specificity; currently under
development in the European
EuroFlow Consortium
Largely standardized thanks to pan-
European collaboration within the
BIOMED-1 (fusion transcript detection)
and EAC project (RQ-PCR)
Methods for identification of fusion
gene breakpoints at the DNA level
provide patient-specific targets
Target identification standardized
within the European BIOMED-1 and
BIOMED-2 networks
RQ-PCR for MRD detection
standardized by the European Study
Group for MRD detection in ALL
(ESG-MRD-ALL)
Abbreviations: B-ALL, B-cell acute lymphoblastic leukemia; EAC, Europe Against Cancer; Ig, immunoglobulin; IGH, immunoglobulin heavy chain
gene; IGK, immunoglobulin kappa light chain gene; MRD, minimal residual disease; RQ-PCR, real-time quantitative polymerase chain reaction;
PCR, polymerase chain reaction; T-ALL, T-cell acute lymphoblastic leukemia; TCR, T-cell receptor; TCRD, T-cell receptor delta gene, TCRG, T-cell
receptor gamma gene.
a
In childhood ALL this particularly concerns t(12;21)(TEL-AML1) and in adult ALL particularly t(9;22)(BCR-ABL).
b
This mainly concerns del(1)(p32 p32) with SIL-TAL1 fusion and t(5;14) with aberrant HOX11L2 expression, together occurring in 25–35% of
childhood T-ALL and in 15-20% of adult ALL (Graux
48
).
Why and how to quantify MRD
T Szczepan
´ski et al
623
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protocols for reverse transcription (RT)-PCR for identification of
the most frequent fusion transcripts in ALL: t(1;19)(q23;p13) with
the E2A-PBX1 fusion gene, t(4;11)(q21;q23) with the MLL-AF4
fusion gene, the two main types of t(9;22)(q34;q11) with
BCR-ABL fusion genes, t(12;21)(p13;q22) with the TEL-AML1
fusion gene, and the intrachromosomal microdeletion on 1p32
with the SIL-TAL1 fusion gene, were standardized within the
European BIOMED-1 and Europe Against Cancer networks.
26–28
However, these leukemia specific markers can be identified in
not more than half of ALL patients, which limits their application
as MRD markers for general cohorts of ALL patients. Still, several
translocations are significant prognostic markers and identify
‘homogenous’ ALL subgroups. Both in childhood and adult ALL,
t(9;22) with BCR-ABL fusion gene is associated with a dismal
outcome, although rare cases with a very good sensitivity to
chemotherapy could be also identified in this generally drug-
resistant subgroup of ALL.
29
With the addition of targeted
treatment such as imatinib mesylate to current chemotherapy
regimens for BCR-ABL
þ
ALL, BCR-ABL transcripts have become
the first-choice marker for MRD monitoring by RT-RQ-PCR.
30,31
In contrast, TEL-AML1 fusion gene occurring in approximately
25% of children with ALL identifies a subgroup with a very good
prognosis for which the advantage of MRD monitoring is not
yet fully proven.
The protocols for detection and quantification of fusion gene
transcripts based on RT-RQ-PCR employing TaqMan technology
have been developed thanks to European EAC network.
27
These
MRD assays are characterized by reproducibly high sensitivity
of 10 plasmid molecules or 10
4
RNA cell line dilution for the
majority of the targets. The EAC study also selected appropriate
reference genes to correct variations in RNA quality and
quantity and to calculate the sensitivity of each measurement.
28
These ‘control’ genes are characterized by highly stable
expression in blood and bone marrow of normal and patient
samples.
Because of the high sensitivity of PCR techniques, cross-
contamination of RT-PCR products between patient samples is a
major pitfall in RT-PCR-mediated MRD studies, resulting in up
to 20% of false-positive results.
32
Such cross-contamination is
difficult to recognize, as fusion gene transcripts, although
leukemia-specific, are not patient-specific markers. Also the
levels of fusion gene transcripts can vary significantly between
patients. This is in contrast to PCR products obtained from
genomic breakpoint fusions, which can be identified by use
of patient-specific oligonucleotide probes in RQ-PCR assays.
Unfortunately, in most translocations occurring in ALL the
breakpoints are widely over multiple intronic sequences and
their precise identification is rather complex. Nevertheless, the
group of Marschalek was able to develop a single standardized
approach to identify the vast majority of breakpoint fusions in
the many different chromosome translocations involving the
MLL gene on chromosome 11q23. The DNA breakpoints of MLL
fusions were shown to be highly specific and sensitive RQ-PCR
markers for MRD detection in MLL-rearranged infant and adult
ALL.
33,34
Such DNA-based approaches for precise breakpoint
identification and MRD monitoring should ideally be developed
for the other major ALL subgroups with specific chromosome
translocations.
RQ-PCR-based quantification of junctional regions of Ig
and TCR gene rearrangements
RQ-PCR-based detection and quantification of junctional
regions of clonal Ig and TCR gene rearrangements is by far the
most widely employed strategy of MRD monitoring in ALL.
Although this MRD strategy is the most laborious, expensive and
time consuming, it is the most reproducible approach not only
within the same laboratory but also between different labora-
tories. The junctional regions of clonal Ig and TCR gene
rearrangements are fingerprint-like sequences for each lymphoid
malignancy and can be identified in the vast majority of
ALL patients using the standardized primer sets established
through the European collaboration within the BIOMED-1 and
-2 frameworks.
35–37
The immunobiologic studies identified and
characterized oligoclonality and clonal evolution of Ig/TCR
gene rearrangements between diagnosis and relapse.
38–42
Therefore, it is widely accepted that preferably at least two
Ig/TCR targets should be followed per patient.
Although initial MRD studies employing different semiquan-
titative approaches, revealed significant results,
5,8,43
only the
introduction of RQ-PCR was the major step towards the wider
dissemination of this MRD strategy.
21,44
This issue of Leukemia
contains an excellent summary of Van der Velden et al.
45
on
interpretation of RQ-PCR data of MRD monitoring by Ig/TCR
gene rearrangements based on the experience of 30 MRD-PCR
laboratories forming the European Study Group on MRD
detection in ALL (ESG-MRD-ALL). This international initiative
has resulted in significantly improved standards, definitions
and guidelines for RQ-PCR-based MRD detection via Ig/TCR
gene rearrangements. Based on extensive experimentation, this
study clearly established definitions of MRD positivity and MRD
quantitative range, within which MRD levels can be expressed
in a reproducible way. As shown by earlier reports, this
quantitative range is usually until 10
4
and 10
5
, depending
on the type of gene rearrangement and the size of junctional
region. Another important guideline concerns the discrimination
between low MRD positivity outside the quantitative range and
false positivity resulting from nonspecific background amplifica-
tion. Clearly, some Ig/TCR gene rearrangement junctions
(particularly of TCRG), although unique for a particular patient,
are so similar to rearrangements in the normal Ig/TCR repertoire
that nonspecific amplification might occur from non-leukemic
lymphocytes at low or even moderate levels. Therefore, the
sensitivity of this MRD approach cannot easily be further
improved. Realizing this, the studies demonstrating frequent
MRD positivity in remission samples of children with ALL at the
levels of 10
7
should be interpreted with extreme caution.
Finally, the report contains additional criteria for interpreta-
tion of RQ-PCR data, which take into account whether the
clinical protocol aims at therapy intensification or treatment
reduction. The conclusion that the ESG-MRD-ALL guidelines for
interpretation of RQ-PCR MRD data enable uniform interpreta-
tion of MRD data between different MRD laboratories is
therefore fully justified.
45
Choice of MRD technique
The three major MRD techniques provide information expressed
as seemingly identical MRD levels. Nevertheless, while flow
cytometry relies on protein expression, fusion genes are
generally detected at messenger RNA levels, and Ig/TCR
junctions are RQ-PCR targets at the DNA level. As summarized
in Table 1, the three methodologies also differ in their sensitivity
and applicability. Therefore, MRD data obtained with different
techniques are hardly interchangeable. Small single-center
studies claimed that results of MRD detection by flow cytometry
and quantitative PCR of patient-specific Ig/TCR gene rearrange-
ments are largely comparable.
46,47
Indeed in 70–80% of
samples with MRD levels 410
3
both techniques seem to give
comparable results (p3-fold difference). However, in samples
Why and how to quantify MRD
T Szczepan
´ski et al
624
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with MRD levels p10
3
, many discrepancies between the two
techniques have been found; this hampers the recognition of
low-risk patients.
46,47
Consequently, usage of different MRD
techniques for different patients within the same treatment
protocol should be avoided.
What would be the future of MRD studies in ALL?
The story of MRD is one of the most exciting examples of
translational research, where complicated basic research was
transferred into high-technology laboratory diagnostics. It is
beyond doubt that MRD diagnostics will be included in all ALL
treatment protocols, as MRD data provide so far the most
optimal reflection of the in vivo response to treatment, which
gives the clinician a better knowledge and control of the clinical
course in individual patients. It is fair to assume that such
individualized medicine will finally translate into improved
outcome of ALL patients. This is already obvious in case of
high-risk ALL patients, particularly those undergoing allogeneic
HSCT and still remains to be proved for general cohorts of
patients. Introduction of novel, preferably targeted therapy will
create additional applications of MRD monitoring. Still ongoing
international collaborative efforts are necessary to ensure that all
diagnostic MRD laboratories speak the same ‘MRD language’.
Acknowledgements
The author is supported by scientific Grants number 2P054 095 30
and 3PO5E 094 25 from the Polish Ministry of Science and Higher
Education.
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