Acute lymphoblastic leukemia (ALL) is the most common malignancy in children accounting
for 25 and 75% of childhood cancers and leukemia, respectively, citied as the major
success stories in the world of oncology where the cure rates have gone up to 80%
(event-free survival [EFS]) from literally zero in the 1950s.[1]
[2]
[3] Prognostic factors play an important role in the strategic standard management of
ALL wherein minimal residual disease (MRD) is now widely regarded as a clinically
significant tool. A meta-analysis has proven that MRD negativity is directly proportional
to the powerful predictors of disease-free survival (DFS) (hazard ratio [HR]: 0.23,
[95% Bayesian credible interval [BCI]: 0.18–0.28] for pediatric patients and 0.28
[95% BCI: 0.24–0.33] for adults) and overall survival (OS) (HR: 0.28, [95% BCI: 0.19–0.41]
and 0.28 [95% BCI: 0.20–0.39] for children and adults with ALL, respectively).[4]
It now provides information depending on when the MRD assessment was performed: after
induction therapy, after consolidation therapy (CT), or before and after stem cell
transplant (SCT) and genomic information for targetable therapies available today,
as shown in [Table 1]. As of today, for the management of ALL, induction therapy to aim complete hematological
recovery and complete remission (CR), followed by CT after attainment of CR, with
standard central nervous system (CNS) prophylaxis, is imperative. It is followed by
SCT in few subsets. Mostly all pediatric and adult ALL guidelines have introduced
informative checkpoints during the management of ALL. For pediatric subgroup, MRD
negativity on day 15 of induction chemotherapy defines excellent outcomes, wherein
in adults, MRD is taken later in the course at 4 weeks of starting induction chemotherapy
and defines better survival rates.[5]
[6]
Table 1
Genetic classification by prognosis of B-cell acute lymphoblastic leukemia
|
Good prognosis
|
Intermediate prognosis
|
Poor prognosis
|
Undetermined prognosis
|
|
Abbreviations: ALL, acute lymphoblastic leukemia; MLL, mixed-lineage leukemia.
at(5; 14); IL3-IGH is a World Health Organization-classified acute leukemia and prognosis
data has not been determined.
|
|
Hyperdiploid karyotypes
|
t(1; 19); TCF3-PBX1
|
Hypodiploid karyotypes
|
t(5; 14); IL3-IGHa
|
|
t(12; 21); ETV6-RUNX1 (TEL-AML1)
|
|
t(9; 22); BCR-ABL
|
|
|
|
Philadelphia-like ALL
|
|
|
|
11q23 MLL arrangements
|
|
Molecular Detection Methods for Minimal Residual Disease
Molecular Detection Methods for Minimal Residual Disease
Molecular detection methods for MRD identify cells either through patterns of phenotypic
markers or differential gene expression through analysis by flow cytometry (FCM),
polymerase chain reaction (PCR), or next-generation sequencing (NGS) ([Fig. 1]).
Fig.1 Detection methods for minimal residual disease. Methods to diagnose minimal residual
disease either through phenotypic marker patterns or differential gene patterns through
analysis by flow cytometry, polymerase chain reaction, real-time quantitative-polymerase
chain reaction, reverse transcription-polymerase chain reaction, or next-generation
sequencing.
An extensive marker screening panel of multiplex PCR assays targeting immunoglobulin/T-cell
receptor (Ig/TR) gene rearrangements of a primary diagnosis sample is used to identify
tumor-specific Ig/TR rearrangements. To discriminate malignant clonal rearrangements
against a polyclonal background, PCR fragments from Ig/TR PCR assays are analyzed.
The most frequently used methods for this fragment analysis are GeneScan or denaturing
high pressure liquid chromatography, followed by heteroduplex analysis, which is comparable
to multiplex PCR.[7]
Sample Prerequisites
Many large-scale studies have confirmed that the bone marrow sample is more informative
than peripheral blood for the detection of MRD.[8]
[9]
[10] There has been a difference of 1–3 log of MRD being lower in a paired peripheral
blood than a bone marrow sample.[11] Therefore, bone marrow assessments might be replaced by analysis of blood samples
in T-ALL but not in BCP-ALL. The difference of residual tumor load is more apparent
in B-ALL as compared with that of T-ALL. Bone marrow aspirate, however, remains the
sample of choice for MRD detection. It is advisable that the first sample of aspirate
should be used for MRD studies. Care should be taken not to dilute it with peripheral
blood, and, usually, a 2 mL, but <5 mL, sample is sufficient to recover cells that
give a sensitivity of B 10–4.[12] Ethylenediaminetetraacetic acid or heparin-anticoagulated samples are good and preferably
to be assayed for MRD detection within 24 to 48 hours.
Minimal Residual Disease in Ph-Negative Acute Lymphoblastic Leukemia and Time Points
Minimal Residual Disease in Ph-Negative Acute Lymphoblastic Leukemia and Time Points
MRD is a time point-dependent variable. MRD levels at different time points have different
prognostic values for relapse: early MRD assessment at the end of induction or early
consolidation identifies patients with a rapid tumor clearance and a very low risk
of relapse representing a good prognosis, whereas any persisting MRD at the end of
CT is associated with a particularly poor prognosis. The Programa para el Tratamiento
de Hemopatias Malignas (PETHEMA) group evaluated the role of MRD (by FCM, cutoff:
5 × 10) in 326 adult high-risk Philadelphia (Ph)-negative ALL patients and confirmed
that the only prognostic factor was represented by MRD persistence after induction
and early consolidation.[13] Similarly, Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) group
in 955 patients assessing the role of SCT post-MRD after induction showed that the
persistence is not abrogated by transplant procedures and that MRD-negative patients
could be spared this approach.[14] The Northern Italian Study Group with MRD post end of induction at week 4 and afterward
10, 16, and 22 weeks to assess liposome-encapsulated cytarabine for CNS prophylaxis
proved profound prognostic effect. The relapse risk (RR) was very low (17% at 5 years)
in the group of week 4 MRD responders and significantly lower (28%) than that in nonresponders
(57%) when week 10 MRD results were examined.[15] The German Multicenter Study Group for Adult ALL for Ph negative patients with SR/HR
features (580 patients) in CR showed MRD after standard induction and consolidation
treatment was the only significant prognostic factor for remission duration and survival
in both risk groups,[16] which has been confirmed later by many trials.[13]
[14]
[15]
Minimal Residual Disease in Ph-Positive Acute Lymphoblastic Leukemia
Minimal Residual Disease in Ph-Positive Acute Lymphoblastic Leukemia
This subset of ALL, defined as a more high-risk group with Ph chromosome-forming breakpoint
cluster region-Abelson gene (BCR/ABLl) rearrangement seen in 20 to 25% of ALL patients,
is more common in adults than the pediatric population and increases in incidence
with increasing age group. This subgroup was always defined as the highest risk group
till tyrosine kinase inhibitors (TKIs) were brought into practice that revolutionized
treatment till the present date where this Ph-positive ALL subset can attain CR in
almost all cases with the TKIs such as imatinib, dasatinib, and ponatinib with or
without chemotherapy as a therapeutic strategy, further improving EFS and also the
OS with the number of Ph-positive patients who could further receive SCT.[17]
[18]
[19]
[20]
[21]
Like in the Ph− subset where MRD reduction serves as a prognostic tool for improved
EFS and OS, the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA)
trial proves again in the Ph-positive subset that MRD reduction correlates with the
EFS and OS irrespective of the inhibitor used[22] and that a very early clearance has a better prognosis.[23] Initially, all Ph-positive patients would be taken for SCT irrespective of the treatment
used, but now, cases that are persistently MRD negative could avoid this and the debate
continues.[24]
[25] For therapeutic purposes too, MRD persistence or positivity or its reversal can
signify the presence of a clone of mutation resistance, like T315-I that warrants
novel TKIs (ponatinib) or combinations with TKI and monoclonal antibodies (blinatumomab).
Today, clinicians can vouch for more from MRD testing where more than one marker could
be identified, like in pediatric ALL pH-positive subsets, 20% or more children could
have significantly higher levels of BCR/ABLl, which is evaluated by estimating both
DNA and RNA fusion levels shown by Hovorkova et al[26] than Ig/TR/TKZF1 deletions, proving that BCR/ABL1 signals could arise from different
hematopoietic progenitors. Similarly, a trial by Cazzaniga et al proved that positive
MRD reports by assessing Ig/TR deletion levels at after induction and consolidation
are strongly prognosticative of relapse,[27] wherein a formal correlation between Ig/TR and BCR/ABL1 proves again a similar conclusion
for relapse with similarity of the technique used (69%). In adult patients, a trial
by Clappier et al[28] found discordance in genomic and RNA-BCR/ABL1 levels because of p210 isoforms, Ikaros
family zinc finger protein 1 (IKZF1) deletions, again proving that signals arise from
different hematopoietic cells than lymphoblasts and represents an altogether different
subset of “CML-like subtype”; however, there is no consensus as to which is suitable
for treatment decisions.
Ph-Like Acute Lymphoblastic Leukemia
Ph-Like Acute Lymphoblastic Leukemia
The rate of CR/CR with incomplete platelet recovery (CRp) was similar in the three
disease subgroups (Ph-like ALL, 89%; Ph-positive ALL, 93%; and B-other, 94%; p = 0.57).[29] However, patients with Ph-like ALL were statistically significantly less likely
to achieve MRD-remission as assessed by FCM (30% for Ph-like ALL vs. 56% for Ph-positive
ALL vs. 87% for B-other; p < 0.001), with achievement of MRD negativity at the time of remission having no impact
on inferior long-term outcomes. In addition, there was no difference in the CR/CRp
rate and MRD remission rate between the Ph-like CRLF2-positive and Ph-like non-CRLF2
groups. Because the majority of patients with Ph-like ALL receive hyper-cyclophosphamide,
vincristine, Adriamycin, and dexamethasone-based treatment, intensification of chemotherapy
treatment is unlikely to benefit adult patients with Ph-like ALL. It remains to be
determined whether addition of novel monoclonal antibodies (such as inotuzumab ozogamicin)
or bispecific antibodies (such as blinatumomab) could improve the outcome of this
group of patients.[30]
Minimal Residual Disease and Stem Cell Transplantation
Minimal Residual Disease and Stem Cell Transplantation
SCT is a procedure still regarded as the one with high mortality and toxicity, which
is performed for ALLs with MRD persistence/positivity and remains a major tool for
decision-making, which could be seen in 20% of patients.[31] Trials have proven the prognostic impact of MRD positive on SCT from time to time[32]
[33] and also the relevance of performing MRD for pretransplant assessment.[34]
[35]
[36] MRD levels of >103 at week 16/22 post consolidation had worst prognosis and posttransplant mortality
with 6-year RR of 64 vs. 23% for MRD <10 shown by Bassan et al.[37] A meta-analysis on 21 reports including >20,000 patients has proven the same MRD
positivity results in posttransplant mortality and reduced relapse-free survival,
EFS, and OS.[38] MRD positivity pretransplant could therapeutically benefit from immunotherapeutic
compounds such as blinatumomab and inotuzumab and possibly chimeric antigen receptor
(CAR)-T-cells in future too, aiming to obtain a MRD-negative status, and also help
in identifying early molecular relapses when done at day 30 as it will help taper
immunosuppression early or preemptively start TKIs in a Ph positive B-ALL.
MRD positivity posttransplant accounts for significantly worse outcomes as compared
with their MRD-negative counterparts[39] but is less commonly practiced as donor chimerism provides risk for early relapse.[40]
Minimal Residual Disease and Novel Markers
Minimal Residual Disease and Novel Markers
Extensive genetics and molecular markers of ALL mandate combining MRD with other markers,
for example, in a 400 young adult cohort of Ph-negative ALL, the GRAAL group identified
a high-risk relapsed population by MRD-del IKZF1 positive, absent NOTCH1/FBXW7 mutation,
N/K-RAS mutation, and/or PTEN gene alteration in T-cell ALL positive.[41] Similarly, in pediatric ALL, presence of IKZF1 intragenic deletion and P2RY8-CRLF2
provides additional prognostic information over MRD alone.[42]
Minimal Residual Disease and Novel Agents
Minimal Residual Disease and Novel Agents
Blinatumomab is a bispecific anti-CD19 and anti-CD3 construct, recruiting cytotoxic
T-cells against CD19 positive blast T-cells, bridging malignant B-cells directly to
CD3 positive T-cells, bypassing T-cell receptor specificity and major histocompatibility
complex class 1 molecules,[43]
[44] and inducing T-cell activation and release of inflammatory cytokines.[45] It has been approved for refractory ALL and more recently for MRD positive patients
(response rates of 43–69%).[46]
[47] Patients with MRD-status have demonstrated a longer median OS and median DFS compared
to MRD-positive counterparts.[48]
[49] In MRD-positive patients, blinatumomab induced a complete MRD response in 78% of
cases and as expected, MRD responders had a longer RFS than nonresponders. A small
fraction of complete MRD responders did not undergo transplant and is still in continuous
CR.[50]
[51]
Inotuzumab ozogamicin—an antibody drug conjugate of monoclonal antibody directed to
CD22 and a cytotoxic agent—is approved as monotherapy of relapsed/refractory adult
CD22-positive ALL patients. Patients treated with inotuzumab ozogamicin reached response
rates ranging from 58 to 81%, with 72 to 78% of these having MRD results below 0.01%[52]
[53] by FCM assessment. While this compound appears to be extremely effective in reinducing
responses, it must be underlined that CR duration is usually short, and therefore
SCT must be performed as soon as possible.
CAR-T-cells are patient-derived or, less frequently, donor-derived normal T-cells
molecularly engineered to express a T-cell receptor-mediating cytotoxicity toward
anti-CD19 (in most cases). After CAR-T-cells are infused into a patient, they act
as a “living drug” against cancer cells: they bind to the target, become activated,
proliferate, and exert their cytotoxic activity. Several groups have shown that most
of the responding patients (both children and adults) become MRD negative (at least
by FCM)[54]
[55] and maintain this status for several months or years.[56]
[57] Data on the prognostic value of MRD in this setting are still preliminary. However,
differently from first-line chemotherapeutic approaches, relapse is observed also
in patients reaching an MRD negativity, mostly because of the loss of CD19. Therefore,
MRD response in this setting seems to be an essential but not sufficient criterion
for the definition of long-term remissions. Higher sensitivities or earlier MRD assessments
might be necessary to identify a subgroup of patients with a particularly rapid and
deep MRD response and a better prognosis.
Conclusions
MRD stands as an independent predictor of DFS and OS for both pediatric and adult
patients of ALL during treatment, and also for pre- and post-SCT settings. Gene fusions
and IG/TR gene molecular rearrangements are used as targets to identify residual leukemic
cells in ALL with/without newer markers for therapeutic actionable purposes and to
also improve the evaluable numbers. Multicolor FCM and real time quantitative-PCR
are broad platforms for MRD assessment and monitoring provided limitations are overcome.
PCR, NGS, and next-generation FCM, making them standard of care is yet to be proven
that could be proven as an important way to identify MRD.
