REVIEW SERIES

Measurable residual disease in acute lymphoblastic

leukemia: methods and clinical context in adult patients
Caner Saygin,1 Joseph Cannova,1 Wendy Stock1 and Lori Muffly2 Correspondence: L. Muffly
[email protected]
1
Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago,
Received: January 7, 2022.
IL and 2Division of Blood and Marrow Transplantation and Cellular Therapy, Stanford Accepted: March 10, 2022.
University, Stanford, CA, USA
https://doi.org/10.3324/haematol.2022.280638

©2022 Ferrata Storti Foundation

Published under a CC BY-NC license

Abstract
Measurable residual disease (MRD) is the most powerful independent predictor of risk of relapse and long-term survival
in adults and children with acute lymphoblastic leukemia (ALL). For almost all patients with ALL there is a reliable method
to evaluate MRD, which can be done using multi-color flow cytometry, quantitative polymerase chain reaction to detect
specific fusion transcripts or immunoglobulin/T-cell receptor gene rearrangements, and high-throughput next-generation
sequencing. While next-generation sequencing-based MRD detection has been increasingly utilized in clinical practice
due to its high sensitivity, the clinical significance of very low MRD levels (<10-4) is not fully characterized. Several new
immunotherapy approaches including blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor T-cell
therapies have demonstrated efficacy in eradicating MRD in patients with B-ALL. However, new approaches to target MRD
in patients with T-ALL remain an unmet need. As our MRD detection assays become more sensitive and expanding novel
therapeutics enter clinical development, the future of ALL therapy will increasingly utilize MRD as a criterion to either
intensify or modify therapy to prevent relapse or de-escalate therapy to reduce treatment-related morbidity and
mortality.

refined by the evaluation of subclinical (not detected mor-

Introduction phologically) measurable (also known as minimal) residual
Multi-agent chemotherapy and risk-adapted protocols disease (MRD), which strongly and independently associ-
revolutionized the management of pediatric acute lym- ates with outcomes across ALL studies and patient co-
phoblastic leukemia (ALL), and transformed this histori- horts.9-12 Furthermore, MRD is increasingly incorporated
cally fatal disease into a curable entity in ~90% of into risk-adapted protocols in adult ALL populations, in
children.1 Although outcomes continue to be inferior for which MRD may inform therapeutic decisions by identify-
adults with ALL relative to children, the successful adop- ing patients at high risk of relapse who may benefit from
tion of pediatric-inspired intensive chemotherapy in the novel treatment approaches and/or allogeneic hemato-
treatment of young adults (<40 years) with ALL has sig- poietic cell transplant (HCT), or patients with chemosen-
nificantly improved overall survival with rates approaching sitive disease at lower risk of relapse who are likely to do
65-80%.2,3 Furthermore, novel immunotherapies such as well in the absence of HCT in first complete remission.13,14
the CD19-directed bi-specific T-cell engager blinatumo- The prognostic significance of MRD in adult ALL has been
mab, the anti-CD22 antibody-drug conjugate inotuzumab shown in both younger adults (<40 years) and older adults
ozogamicin, and chimeric antigen receptor (CAR) T-cell (>40 years).15,16
therapies offer new treatment options for B-lineage ALL MRD is defined as measurable leukemia in a sample that
and are now being explored in the front-line setting.4-6 As is devoid of leukemia cells by morphological assessment
our therapeutic armamentarium expands, accurate risk with a light microscope. It is estimated that a patient
stratification at critical time points is essential to deliver presents with 1012 (a trillion) bone marrow leukemic blasts
optimal treatment to improve outcomes in adults with at diagnosis, and morphological assessment can detect
ALL. After standard multi-agent chemotherapy, the ma- approximately 1% blasts or more (Figure 1). Therefore, MRD
jority of adults will achieve complete morphological re- theoretically represents anything less than 1010 cells (<1%
mission (CR).7,8 However, response assessment is further blasts). In clinical practice, MRD is often used to describe

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Figure 1. Measurable residual disease assessment in acute lymphoblastic leukemia. Schematic representation of disease levels
in acute lymphoblastic leukemia with corresponding measurable residual disease levels. This can resemble an “iceberg”, with
different detection methods offering different levels of sensitivity and breadth of detecting residual disease. MRD: measurable
residual disease; PCR: polymerase chain reaction.

any detectable leukemia below the traditional remission nique which relies on high-throughput next-generation se-
definition of 5% blasts by morphological assessment. quencing (NGS) may offer a more sensitive approach to de-
However, as MRD detection assays have become more tect IG and TCR rearrangements in ALL blasts.18 The main
sensitive, it is generally recognized that an appropriate advantages and disadvantages of these MRD assessment
assay for the detection of ALL MRD in the clinic should be methods are summarized in Table 1.
validated and reproducible at a sensitivity threshold of at
least 10-4, or 0.01% leukemia cells in the bone marrow. The Multicolor flow cytometry
fundamental idea behind MRD interpretation is simple: the MFC is a fast and relatively inexpensive method that is
rate of decline in disease burden in response to systemic broadly applicable to most ALL cases. It distinguishes
therapy is of prognostic value and a measure of risk for leukemic cells based on their aberrant immunophenotype
relapse, and intervening on lower levels of disease should or leukemia-associated immunophenotype. A leukemia-
result in improved outcomes. Herein, we review commonly associated immunophenotype can include antigen over-
used methods of MRD detection in ALL and provide clini- or under-expression, asynchronous antigen expression,
cal context and guidance to practising clinicians on how cross-lineage antigen expression, and ectopic pheno-
to interpret and intervene on MRD in adult ALL. types.16 It is necessary to obtain information about the
immunophenotype at diagnosis in order to track it
throughout the clinical course of an individual patient.
Methods and technical aspects of However, these features may change under therapeutic
pressure, and antigens may be lost or new antigens may
measurable residual disease be over-expressed as the leukemia evolves. To overcome
assessment in acute lymphoblastic this challenge, an alternative flow-based MRD approach
leukemia named “different from normal” has been widely utilized.19
The “different from normal” approach involves a standard-
All MRD detection methods leverage features that are pres- ized panel of several markers that are used to distinguish
ent exclusively in leukemic blasts to differentiate them leukemic cells from normal hematopoietic cells (Figure 2).
from normal cells. Commonly used techniques include The most common markers used to identify leukemic B
multicolor flow cytometry (MFC) to detect leukemic cells lymphoblasts include CD10, CD19, CD45, CD34 and CD38.
by immunophenotypic aberrancies, real-time quantitative Leukemic cells often have high CD10 and low CD38 ex-
polymerase chain reaction (qPCR) for detection of recurrent pression, which may distinguish them from hematogones.
gene fusions (e.g., BCR-ABL1) or rearranged immunoglobulin Aberrant myeloid marker expression (e.g., CD33, CD13, and
(IG) and T-cell receptor (TCR) genes.17 A more recent tech- CD15), or expression of CD9, CD73, and CD81 may also be

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Table 1. Comparison of techniques to measure residual disease in acute lymphoblastic leukemia.

Multi-color flow qPCR for fusion ASO-qPCR for IG/TR High-throughput
cytometry genes genes NGS
Sensitivity 10-4 10-4 to 10-5 10-4 to 10-5 10-6
Applicability >90% 40-50% 90-95% >90%
Advantages - Rapid - Sensitive - Sensitive - Very sensitive
- Relatively inexpensive - Standard primers used - Applicable to most - Applicable to almost
- DfN method does not for specific fusions patients all patients
require access to dia- - Standardized - Clone-unbiased (can
gnostic specimen guidelines in Europe track multiple clones
and evolution)
- Only US FDA-appro-
ved assay (ClonoSEQ)
- Data for MRD use in
peripheral blood
Limitations - Variable sensitivity - Not applicable to all - Time-consuming - Expensive
- Requires technical patients - Expensive - Longer turn-around
expertise - Relies on pre-treat- time than MFC
- Fresh cells required ment sample - Requires diagnostic
- Less standardized - Requires extensive pre-treatment sample
- Immunophenotypic experience and labor
shifts can lead to false
negative results

ALL: acute lymphoblastic leukemia; ASO: allele-specific oligonucleotide; DfN: different-from-normal; FDA: Food and Drug Administration; IG:
immunoglobulin; MFC. multicolor flow cytometry; NGS, next-generation sequencing; qPCR, quantitative polymerase chain reaction; TCR, T-
cell receptor.

helpful to define B-lymphoblasts.20 Clinical laboratories has limitations in these cases. Finally, another recently de-
should be informed if the patient has received CD19-, scribed rare entity called switch ALL (swALL) may pose
CD20- or CD22-targeted therapies, as these markers may challenges for flow-based MRD detection.24 These precur-
no longer be reliable in MRD detection for these patients. sor B-ALL arise from CD2+ lymphoblasts that do not har-
It is important to identify CD19 antigenic escape with flow bor KMT2A rearrangements but have upregulated CEBPα
MRD, since these patients would not benefit from further activity. These cases are characterized by a switch be-
CD19-targeted therapies. In patients receiving anti-CD19 tween precursor B (CD19+ CD14–) and monocytoid (CD19–
agents, other B-cell markers, such as CD20, CD22, and CD14+) immunophenotypes through a transdifferentiation
CD79a, can be used to identify the CD19-negative leuke- mechanism involving alterations in the expression of
mia population, but can also lead to misidentification of CEBPα, PAX5, PU1 and GM-CSFR.25 This disease can be
normal B-cell precursors.21 Therefore, flow MRD should be tracked by using IG gene rearrangements that are pre-
used in conjunction with other methods of MRD evaluation served throughout different switch states.
(such as qPCR or NGS-based methods) for patients who CD34, TdT, CD7, cytoplasmic CD3, and CD1a are commonly
have received anti-CD19 therapies. Clinicians and MRD utilized markers for flow-based MRD detection in T-ALL;
laboratories should also be aware of the rare event of however, MFC for residual T-lymphoblast detection is less
myeloid lineage switch after anti-CD19 therapies, which developed compared to that for B-ALL.26
has been reported in both children and adults treated with Although the sensitivity of MRD MFC from ALL bone mar-
blinatumomab or CAR T-cell therapies.22,23 This is more row aspirate sampling approaches 10-4, results are de-
commonly observed in patients with KMT2A-rearranged pendent upon the quality of sample obtained, and the
ALL, but has also been seen in ALL with BCR-ABL1 trans- laboratory operator’s experience.27 The results of MFC-
location.23 In these cases, flow cytometry may identify based MRD assessment may be optimized by treating
blasts expressing myeloid as opposed to lymphoid samples with EDTA or heparin anti-coagulation and using
markers. Since these cases persistently harbor their cyto- 2-5 mL from the first pull of bone marrow aspirate. Since
genetic rearrangement at the time of myeloid relapse, the degree of cellularity in the sample will affect the re-
complementing flow MRD with reverse transcriptase PCR corded number of events, up to 5 mL may be required for
(RT-PCR)-based MRD assessment can enable accurate di- hypocellular remission samples. There is no evidence for
agnosis of this entity. Similarly, NGS-based MRD assess- unequal distribution of ALL cells in different parts of the
ment may be helpful when flow-based MRD assessment bone marrow compartment as shown in studies of bilat-

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Figure 2. Multicolor flow cytometry as a method of measurable residual disease detection in acute lymphoblastic leukemia. An
example showing multicolor flow cytometry of a bone marrow specimen, obtained after induction with chemotherapy plus
rituximab (anti-CD20) in a patient with B-cell acute lymphoblastic leukemia (B-ALL). The measurable residual disease (MRD)
population (0.2% of total events) is shown in blue, and distinguished from normal B cells and hematogones by its overexpression
of CD10, CD58, and CD34, and underexpression of CD38 and CD81. CD20 expression is lost in leukemic blasts as a result of
rituximab therapy. The radar plot visualization easily distinguishes the B-ALL MRD population.

eral bone marrow sampling for MRD detection.28 Fresh Although MFC may be performed using peripheral blood,
samples sent for MFC should be processed within 24-48 the ability to use blood as a source for MRD detection by
hours of collection, and an advantage of MFC is the rapid MFC is limited by reduced sensitivity.
reporting time for clinical results which may be returned
to the clinician within 3 days of collection. Since MFC as- Real-time quantitative polymerase chain reaction
says try to identify rare events, the number of events Chimeric gene fusions are major oncogenic drivers that
needed to be collected depends on the desired assay sen- can be found in ~40% of B-ALL, and some T-ALL cases.
sitivity and the optimal coefficient of variation as dictated Since these rearrangements are oncogenic, they are often
by Poisson statistics.29 For ALL MRD, the recommended stable throughout the disease course, making them good
current threshold for clinical decisions is 0.01% (10-4) sen- targets for MRD assessment (Figure 3A). RT-PCR can be
sitivity. Thus, to obtain a coefficient of variation of 10%, 106 used to track BCR-ABL1, E2A-PBX1, KMT2A and CRLF2 re-
events must be acquired. Select reference laboratories arrangements in B-ALL, as well as TAL1, TLX1, and TLX3
can reach high sensitivity, as was reported in the fusion transcripts in T-ALL.32 The advantage of fusion
PETHEMA study under the EuroFlow MRD Consortium.13,30 transcript detection is the availability of standardized uni-
By using an optimized erythrocyte bulk-lysis protocol, versal primers specific for each fusion transcript, which
bone marrow samples containing more than 107 cells can simplifies the MRD detection process while offering a
be lysed and resuspended in a small volume of buffer sensitivity of 10-4 to 10-5.33 Quantification of BCR-ABL1
(~100 mL), with which one can achieve the 0.01-0.001% (10- transcripts is relatively straightforward, and p210 tran-
4
to 10-5) sensitivity.19 This bulk-lysis protocol for sample script quantification can be reported using an inter-
preparation was combined with a two-tube next-gener- national standard (IS) because of the work done in
ation flow approach by the EuroFlow Consortium, which chronic myeloid leukemia, while the IS score cannot be
also benefits from an optimized combination of fluoro- applied to the more common (in ALL) p190 transcript, so
chromes and antibody reagents to increase specificity at standard quantification of the BCR-ABL/ABL ratio is used.
very low MRD levels.31 Fusion transcript detection with RT-PCR is an essential

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B C

Figure 3. Molecular methods of measurable residual disease detection in acute lymphoblastic leukemia. (A) Quantitative
polymerase chain reaction (qPCR) and digital droplet PCR (ddPCR) can be used to detect fusion mRNA transcripts in acute
lymphoblastic leukemia (ALL). (B) Allele-specific oligonucleotide qPCR (ASO-qPCR) leverages IG/TR gene rearrangements to
detect measurable residual disease in ALL. It relies on identification of V(D)J sequences in a diagnostic sample, followed by the
design of patient-specific primers for the sequences. (C) High-throughput next-generation sequencing (NGS) also targets
specific V(D)J rearrangements, but it is fast and clone-unbiased as it uses multiplex PCR and does not require development of
patient-specific primers.

method of MRD assessment for patients with BCR-ABL Leukemic blasts arising from these precursor lymphoid
and KMT2A rearrangements, since these patients may ex- cells contain clonal V(D)J rearrangements in more than
perience immunophenotypic shifts or myeloid lineage 80% of cases.33 Given the specificity of the rearranged
switch under the influence of CD19-directed antibody and IG/TR DNA sequence for identification of leukemic cells,
CAR T-cell therapies.23 qPCR-based methods have been developed to track MRD.
Digital droplet PCR (ddPCR) is a third-generation PCR tech- Allele-specific qPCR (ASO-qPCR) is a labor-intense proce-
nology, which is based on separation of the sample into at dure, which relies on the identification of “fingerprint-
least 20,000 water-oil emulsion droplets, followed by PCR like” IG/TR V(D)J sequence(s) in a diagnostic leukemia
amplification in each droplet. This technique is highly sen- sample, followed by development of patient-specific
sitive with improvement in the limit of detection and does primers to assess for the presence of these specific se-
not require a reference curve. Early studies suggest that quence(s) in remission samples (Figure 3B).36 The initial
ddPCR may have utility as a more sensitive MRD detection characterization requires a panel of screening PCR using
tool in Philadelphia chromosome-positive (Ph+) B-ALL.34,35 established primers for the complementarity-determining
During early stages of lymphocyte development, B- and region 3 (CDR3), which is the hypervariable heavy chain
T-cell progenitors undergo somatic recombination of the region of IG/TR genes. This is then followed by Sanger se-
variable (V), joining (J), and in some cases, diversity (D) quencing of products to identify the clones and develop
gene segments of their IG and TCR genes, respectively. patient-specific primers. Depending on template avail-

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ability, primer selection, and the amount of DNA in a given tion tools are included in the assessment.46 The sensitivity
specimen, this patient-specific assay can have a sensi- of NGS MRD approximates 0.0001% (10-6) in the bone mar-
tivity between 10-4 and 10-5.37 IG/TR-based methods are row;47 however, the significance of MRD below the tradi-
applicable to more than 90% of ALL cases, with reduced tional threshold of 10-4 has not been defined across
sensitivity in early T-precursor ALL, since the latter often different clinical scenarios. Similar to flow MRD, the sensi-
arises from more immature progenitors that have not tivity of NGS MRD depends on the amount of input, which
undergone TCR rearrangement. In addition to its laborious is the amount of DNA in this method. The analysis of NGS
and time-consuming methodology, ASO-qPCR may be li- MRD data also requires a bioinformatic pipeline. The U.S.
mited by the loss or emergence of new V(D)J sequences, Food and Drug Administration approved the use of Clo-
leading to false negative MRD results.38 noSEQ NGS technology (Adaptive Biotechnologies, Seattle,
In a study in which both BCR-ABL1 level and IG/TR ASO-qPCR WA, USA) as an MRD assessment method in ALL.48 Studies
were monitored in the bone marrow of Ph+ B-ALL patients, have demonstrated concordance of NGS-based ALL MRD
overall concordance between the two methods was ~70%,39 assessment using bone marrow and peripheral blood; thus,
but IG/TR was found to be more reliable at predicting out- NGS may potentially enable MRD quantification from pe-
comes. However, another study investigating the discrepancy ripheral blood without the need for frequent bone marrow
between the two methods showed that some patients with sampling.49,50 In addition to RT-PCR and ASO-qPCR, NGS-
persistent BCR-ABL1 transcript levels may in fact contain the based MRD may represent another platform to track the
fusion gene in myeloid cells, indicating a chronic myeloid disease in the setting of immunophenotypic shifts or mye-
leukemia-like stem cell disease, which may be missed by loid lineage switch. Since it is an IG/TR-based method, NGS
ASO/qPCR.40 In the GRAAPH-2014 study, 38% of adults with MRD technology may have lower sensitivity in early T-pre-
de novo Ph+ ALL had residual BCR-ABL1 positivity during cursor-ALL, as was the case for ASO-qPCR.
treatment, related to BCR-ABL1 “clonal hematopoiesis”.41 The
presence of BCR-ABL1 clonal hematopoiesis was not associ- Emerging methods
ated with poorer outcome, and patients with residual clonal Routine clinical MRD assessment for a given patient often
hematopoiesis did not benefit from allogeneic HCT. There- includes at least two methods described above (e.g., flow
fore, RT-PCR and IG/TR MRD techniques may complement MRD plus RT-PCR and/or IG/TR-based methods), which pro-
each other and should be monitored simultaneously in Ph+ vide complementary information and are advantageous for
ALL patients in order to guide therapeutic decisions. overcoming the limitations of one method. EuroClonality-
Several studies compared the sensitivity of MFC- and NGS DNA capture (EuroClonality-NDC) is an emerging assay
qPCR-based MRD assessment in peripheral blood versus that is designed as an integrated tool to characterize IG/TR
bone marrow samples. In B-ALL, MRD can be up to three- rearrangements, chromosomal translocations, copy number
logs lower in the peripheral blood than in bone marrow, alterations, and somatic mutations through a standardized
indicating the importance of marrow assessments when pipeline.51 This assay is a robust tool providing a single work-
these methods are used to track MRD.42 In contrast, MRD flow for detection of B- and T-cell clonality, as well as fusion
levels in T-ALL are roughly equivalent between the two transcripts and sequence variants.
sources, which may in part be explained by the thymic ori- As the technology advances, several innovative approaches
gin of this disease.43 are promising to improve the technical aspects of current
MRD assessment methods, including new flow MRD
High-throughput next-generation sequencing methods (e.g., spectral flow cytometry which allows the
Recent advances in NGS have led to a great interest in de- simultaneous analysis of multiple surface markers), novel
veloping NGS-based MRD methods, which can potentially MFC panels, cell-free DNA-based methods, and novel NGS
increase the sensitivity of detection. These assays include strategies (e.g., ALL-specific mutation detection). However,
sequencing the IG/TR gene V(D)J rearrangements as in none of these techniques has robust clinical validation in
ASO-qPCR, but have the capability of simultaneously am- ALL yet, therefore we will not discuss them within the scope
plifying multiple combinations of rearranged IG/TR genes of this review.
by multiplex PCR without the need for patient-specific
primers (Figure 3C).44 Leveraging the high capacity of NGS, Clinical significance of measurable
a larger picture of the IG/TR repertoire can be interrogated
in one experiment, enabling the detection of clones that residual disease in adult acute
are at lower frequency at diagnosis but expand later in the lymphoblastic leukemia
disease trajectory.45 Unlike ASO-qPCR, NGS-based IG/TR
MRD is not clone-biased, meaning that new clones may be Prognostication in ALL has historically relied on baseline
identified as they emerge in remission and relapse samples leukemia characteristics including white blood cell count
if the necessary clonal evolution and new clone identifica- at diagnosis, immunophenotype, and cytogenetic abnor-

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malities.52 Increasingly, studies have demonstrated that tergroup A041501 study (NCT03150693) is evaluating the in-
MRD response to front-line therapy outweighs traditional corporation of inotuzumab ozogamicin into a pediatric in-
prognostic parameters and has emerged as the strongest spired regimen with the aim of early MRD eradication
independent predictor of ALL outcomes.30,53-55 In a meta- following induction therapy to improve event-free survival.
analysis of 39 publications, comprising 13,637 patients of Unfortunately, there is currently no proven MRD “eraser” for
all ages and ALL subtypes, achieving MRD negativity (by patients with T-ALL. For these patients, persistence of MRD
flow or qPCR) was associated with hazard ratios of 0.25 following consolidation or within 3 months of therapy initi-
(0.24-0.33) for event-free survival and 0.28 (0.19-0.41) for ation is a signal to escalate therapy and to strongly consider
overall survival.56 The authors also showed the significant allogeneic HCT in transplant-eligible adults. MRD should be
prognostic utility of MRD at varying time points of assess- assessed at shorter intervals, if possible, in patients with
ment regardless of the MRD detection method used. persistent, or newly detectable MRD, as impending clinical
relapse is often likely.
Monitoring measurable residual disease during front- NGS MRD offers a new and sensitive platform for MRD de-
line multi-agent chemotherapy tection, yet we have limited data for the prognostic signifi-
The addition of MRD to the response assessment following cance of low-level (between 10-4 and 10-6) MRD after
front-line multi-agent chemotherapy is standard of care in multi-agent chemotherapy. The recent GMALL 07/2003 study
the management of ALL.57 Given that the traditional thera- looked at the outcomes of patients who had persistent NGS
peutic course of ALL tends to include up to 12 months of in- MRD below the 10-4 detection level after consolidation ther-
tensive chemotherapy followed by 1.5-2.5 years of apy.61 Patients with undetectable NGS-MRD had significantly
maintenance, understanding the optimal timing and fre- better overall survival when compared to patients with posi-
quency of MRD assessment is important to the delivery of tive NGS-MRD. Similarly, a recent retrospective analysis
high quality ALL care. Few studies have actually reported the looking at 84 Ph-negative B-ALL patients in complete re-
utility of ongoing interval MRD monitoring in patients achiev- mission by flow MRD showed that 38% were MRD-positive
ing MRD-negative response. The German Multicenter Study by the ClonoSEQ NGS method.62 Patients who were MRD
Group on Adult ALL (GMALL) evaluated a cohort of adults negative by the NGS method had significantly lower risk of
with ALL in first complete remission and found that the relapse, which also translated into longer overall survival.
median time between emergence of MRD by ASO-qPCR and Monitoring of MRD for Ph+ ALL may be performed by a variety
clinical relapse was approximately 4 months, suggesting that of methods, but RT-PCR for BCR-ABL1 transcripts is conveni-
the interval between MRD assessment time-points should ent and commonly used in the USA. For adult patients with
likely be 3 months or less, and that ongoing MRD monitoring Ph+ ALL receiving intensive chemotherapy in combination
may provide a potential window for clinical intervention prior with second- or third-generation tyrosine kinase inhibitors,
to fulminant disease relapse.58 Given that the majority of ALL MRD response at 3 months following therapy has been
relapses occur within the first 3 years following diagnosis,59 shown to be highly informative. In a retrospective study con-
MRD monitoring for early relapse detection is likely most ducted by the group at the MD Anderson Cancer Center,
critical during this time period. among 85 adult Ph+ ALL patients who received hyper-CVAD
Typically, MRD is first assessed at the completion of re- plus a tyrosine kinase inhibitor, those who achieved com-
mission induction, which occurs 2-4 weeks following therapy plete molecular remission by RT-PCR at 3 months had an
initiation depending on the treatment regimen utilized. Pa- excellent overall survival even without HCT.63 A phase II study
tients who achieve MRD negativity (at least <10-4) at this early of hyper-CVAD plus ponatinib in Ph+ B-ALL demonstrated
time point are considered to have chemosensitive disease, higher MRD-negative complete remission rates when com-
which is associated with excellent disease-free survival, and pared to the hyper-CVAD plus dasatinib historic control
this information guides the initiation of consolidation ther- group.64 The role of MRD response following chemotherapy-
apy.2 For these patients, we generally recommend that MRD free/light regimens for Ph+ ALL remains unclear but MRD re-
be assessed both following induction therapy and following sponse will likely also be important in identifying patients in
consolidation therapy. For adults who receive a pediatric in- need of intensification and/or additional therapeutic inter-
spired regimen, current data suggest that persistence of ventions.
MRD following consolidation therapy (usually 12-16 weeks
into treatment) may be the time-point with greatest rel- Measurable residual disease and allogeneic hematopoietic
evance for making a change in treatment. Alternative therapy cell transplantation
for MRD persistence includes blinatumomab in patients with Allogeneic HCT is currently indicated for adults with relapsed
B-ALL, given the high rates of reported MRD clearance in the or refractory ALL or patients who demonstrate persistent
BLAST trial among patients with persistent MRD and good MRD following frontline induction/consolidation therapy.
outcomes for responding patients, the majority of whom Several studies have shown that achieving an MRD-negative
proceeded to allogeneic transplant.60 Currently, the U.S. In- state prior to allogeneic HCT improves relapse-free survival

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and overall survival. However, the depth of MRD response sistent MRD. Among children receiving CAR T-cell therapy,
prior to traditional myeloablative allogeneic HCT may be im- real-world datasets have demonstrated improved survival
portant. For example, in an international study of 616 ALL and reduced toxicity following CAR T-cell therapy when ad-
patients who underwent allogeneic HCT, patients with low ministered to patients with lower level/MRD-positive dis-
MRD had similar outcomes to those without MRD pre-trans- ease, as opposed to patients in full clinical relapse. Although
plant.65 MRD positivity after allogeneic HCT was more impor- the MRD-negative response rate following CAR T-cell therapy
tant than pre-HCT MRD positivity, thus close monitoring with in adult ALL is very high (approximated at 81% in a meta-
sensitive techniques is warranted. In a smaller study of adult analysis including 489 adults receiving CAR T-cell therapies
patients with ALL, the presence of pre-transplant MRD at across published trials),74 whether early MRD response to
>10-4 detected by an NGS-based method predicted a 7.7- CAR T-cell therapy translates into long-term durable re-
times higher risk of relapse after transplantation.66 The prog- missions in adult ALL patients remains unclear.
nostic impact of MRD has been demonstrated across Re-emergence or persistence of MRD in patients with Ph+ B-
transplant donor source and conditioning regimens.67,68 It is ALL might be associated with the selection of clones har-
clear that further therapy should be considered to deepen boring ABL1 kinase domain mutations (e.g., T315I).75 It is often
response prior to allogeneic HCT if MRD is present at >10-4; difficult to identify the specific resistance mutation given the
an open question remains whether additional clearance of low level of the transcript in patients without morphological
MRD present at <10-4 is necessary to achieve successful relapse. Blinatumomab has been shown to be effective in
transplant outcomes. eradicating MRD in Ph+ ALL.76 A common strategy would be
Serial tracking of MRD following allogeneic HCT also predicts to combine blinatumomab with ponatinib, since the latter
for impending disease relapse. In a study of 43 adult patients has activity against most of the common resistance muta-
who underwent HCT for ALL, those with detectable qPCR- tions of ABL1.64
based MRD on day +100 had an 80% relapse risk compared
to a 7% risk in patients with undetectable post-HCT MRD.69
In another small study of adult ALL HCT recipients, detection
of MRD by NGS at any level following HCT heralded clinical
Conclusions
relapse; the median time between MRD detection and re- Incorporation of MRD assessment is a fundamental com-
lapse was 3 months.66 Finally, MRD tracking using peripheral ponent of risk-directed therapy in ALL. For almost all pa-
blood following HCT in adult ALL has been shown to be feas- tients with ALL there is a reliable method to assess residual
ible and convenient, to correlate closely with bone marrow disease, which remains the most powerful independent pre-
NGS-based MRD assessment, and to predict for clinical re- dictor of outcomes. Blinatumomab, inotuzumab ozogamicin
lapse.49 and CAR T cells are highly effective immunotherapy ap-
proaches in B-ALL; each is currently being explored alone
Measurable residual disease in relapsed/refractory acute and in combinations as a mechanism for MRD eradication
lymphoblastic leukemia and novel therapies earlier in the course of disease. The development of novel,
In addition to the robust evidence in newly diagnosed ALL, active agents for T-ALL patients with persistent MRD re-
prognostic value of MRD for patients in second complete re- mains an unmet need. As MRD detection assays become in-
mission and beyond has also been demonstrated.70 MRD has creasingly more sensitive and additional novel therapeutics
also played an important role in establishing eligibility and enter clinical development, the future of ALL management
monitoring treatment response in key studies of novel ther- in adults will require nuanced and evidence-based, risk-
apies such as blinatumomab, inotuzumab ozogamicin, and adapted treatment approaches to guide intensification or
CAR T-cell therapy.4,5,71 Not only did MRD-positive ALL define de-escalation of therapy.
the inclusion criteria for the BLAST trial, leading to an ac-
ceptance of MRD as an indication for blinatumomab in B- Disclosures
ALL, but MRD was also shown in secondary analyses to No conflicts of interest to disclose.
predict for relapse-free survival and overall survival in pa-
tients with relapsed/refractory ALL achieving complete re- Contributions
mission following blinatumomab.72 In the landmark phase III All the authors wrote the review article and approved its
INO-VATE trial comparing inotuzumab ozogamicin versus final version.
standard chemotherapy in relapsed/refractory B-ALL, the
MRD-negative complete remission rate, assessed by MFC Acknowledgments
(10-4 sensitivity), was 46% in patients treated with inotuzu- The figures in this review were prepared with BioRender. CS
mab.73 Patients who had MRD response had longer overall is supported by an American Society of Hematology Re-
survival and progression-free survival than patients with per- search Training Award for Fellows.

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REVIEW SERIES C. Saygin et al.

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