- Email: [email protected]
Molecular Minimal Residual Disease Monitoring in Acute Myeloid Leukemia
Accepted Manuscript Molecular Minimal Residual Disease Monitoring in Acute Myeloid Leukemia: Challenges and Future Directions Adrian Selim, Andrew S. Moore PII:
S1525-1578(17)30485-3
DOI:
10.1016/j.jmoldx.2018.03.005
Reference:
JMDI 689
To appear in:
The Journal of Molecular Diagnostics
Accepted Date: 27 March 2018
Please cite this article as: Selim A, Moore AS, Molecular Minimal Residual Disease Monitoring in Acute Myeloid Leukemia: Challenges and Future Directions, The Journal of Molecular Diagnostics (2018), doi: 10.1016/j.jmoldx.2018.03.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular minimal residual disease monitoring in acute myeloid leukaemia: challenges and future directions Adrian Selim* and Andrew S. Moore*†‡
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From the The University of Queensland Diamantina Institute,* Translational Research Institute, Brisbane; the Oncology Services Group,† Children’s Health Queensland Hospital and Health Service, Brisbane; and the UQ Child Health Research Centre,‡ The University of
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Queensland, Brisbane, Australia
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Correspondence: Andrew S. Moore Children’s Health Queensland Research Directorate Level 7, Centre for Children’s Health Research
62 Graham St, South Brisbane, Queensland, 4101, Australia
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Email: [email protected]
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Running Head: Molecular MRD in AML
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Funding: A.S.M. is supported by the Children’s Hospital Foundation, Queensland. Disclosures: None declared.
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ACCEPTED MANUSCRIPT Abstract
The ability to sensitively monitor minimal residual disease (MRD) has played a key role in improving the management and outcomes for a number of leukaemias, particularly acute promyelocytic leukaemia and childhood acute lymphoblastic leukaemia. In contrast, MRD
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monitoring in acute myeloid leukaemia (AML) has been limited by variable assay methodologies and a relative paucity of patient-specific MRD markers. Inter- and intra-tumor genetic heterogeneity pose significant challenges for the identification of molecular markers
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suitable for MRD monitoring in AML, particularly for those cases without structural
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chromosomal rearrangements associated with fusion genes. Furthermore, the need to discriminate which mutations may be suitable for MRD monitoring creates additional complexity. The mainstay of current molecular MRD monitoring is real-time quantitative PCR, targeting fusion genes, mutations, and gene over-expression. New technologies, particularly next-generation sequencing approaches, offer new ways to overcome these
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limitations. Here, we review the techniques available for molecular MRD monitoring in AML
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and discuss their utility in clinical practice.
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ACCEPTED MANUSCRIPT Introduction
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by diverse genetic abnormalities and variable morphology, immunophenotypes, and clinical outcomes1. Genetic heterogeneity exists not only between different AML cases but within each individual case
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and mutations in AML may pre-date leukaemia-initiating events, occur after initiation, and contribute to the founding AML clone, or occur sub-clonally after leukaemogenesis2. Thus, the clonal composition of the leukaemia can vary significantly, as sub-clones (along with
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their late acquired mutations) are subjected to natural and treatment-induced selection
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pressures, expanding, diminishing, or even being cleared3-5. These factors present challenges when selecting genetic abnormalities as markers of post-treatment minimal residual disease (MRD) in AML. Despite this, the impetus for MRD monitoring in AML is ever-present, as countless studies have established the role of MRD as a dynamic predictor of relapse risk and overall survival, often stronger than any pre-treatment variables1. Moreover, the ultimate aim
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of MRD monitoring in leukaemia is to be able to utilize this dynamic marker to personalize therapy, such that treatments and their associated toxicities are curbed in low-risk patients,
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and intensified in those at high risk of relapse.
The characteristics of a suitable MRD marker can be defined by ‘3 Ss’; a marker must be
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‘specific’, ‘sensitive’, and ‘stable’. Moreover, some would argue for a fourth characteristic, ‘stemness’, whereby a marker is present only in those cells capable of self-renewal and hence relapse, and not in bulk leukaemic cells1. Although such markers are being actively pursued in the context of multicolor flow cytometric MRD determination6, it remains a largely philosophical ideal in the molecular context. The terms ‘sensitive’ and ‘specific’ can be a source of confusion. As characteristics of the MRD marker, a highly sensitive marker is one that is present in all leukemic cells, and a highly specific marker is one that is only present in leukaemic cells while being absent in non-leukemic/pre-leukemic cells. A lack of sensitivity
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will produce false negative MRD, whereas a lack of specificity will produce false positives. The second use of the term sensitivity is the analytical sensitivity, a performance characteristic of the assay, more appropriately described as the lower limit of detection (LOD). A commonly accepted benchmark in LOD in AML MRD assays is 10-3 (0.1%),
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meaning one leukemic cell (or equivalent) in 1,000 cells measured, although some molecular MRD assays have LODs of 10-5 to 10-7. The theoretical benefit of utilizing more sensitive assays is an ability to identify more patients at risk of relapse, and to quantitatively track
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minute changes in low-level MRD. Despite a lack of strong evidence for MRD-directed therapeutic approaches in non-APL AML, by employing a sequential MRD surveillance
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strategy, one could expect better outcomes when therapy is initiated at the time of ‘molecular relapse’ rather than frank haematological relapse. Risk-adapted treatment protocols taking into account sequential MRD results have shown favorable results in paediatric AML (AML027), although the study relied on comparison to historical cohorts. Hopefully, with the
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results of prospective, randomized clinical trials assessing the value of MRD-directed therapy, such as the United Kingdom’s NCRI AML17 trial, which randomized patients with informative molecular markers to “MRD monitoring” vs “no MRD monitoring” strategies,
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the question of whether modification of post-remission treatment intensity based on MRD
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status in AML will be answered8.
The mainstay of molecular MRD monitoring at present are real-time quantitative PCR (RQPCR)–based methods, which can be employed when a suitable target is present, afford high analytical sensitivity, and, to a significant extent, allow for standardization between different laboratories. Targets for RQ-PCR as well as other molecular methods can be divided into three categories: i) chimeric fusion gene transcripts; ii) gene mutations; and iii) overexpressed genes.
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PCR-based assays of fusion genes and NPM1 mutations have the potential to collectively monitor approximately 60% of AML presenting in children and younger adults; however, this proportion steadily declines with increasing age, encompassing only 32% of those over 609 . PCR of over-expressed genes, most commonly WT1, can be applied in a significant
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proportion of AML cases (ranging from 13% to 73% in different studies depending on whether PB or BM is used and the threshold for defining over-expression10, 11), but with its limited dynamic range, is generally restricted to those who lack other molecular markers.
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The nucleic acid source for molecular assays may be genomic DNA or RNA, each with a set
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of advantages and disadvantages. DNA is more stable and easier to extract than RNA, making it favorable for collection, processing, transport, and storage. In the case of fusion genes, breakpoints at the gDNA level are unique, providing patient-specific markers of MRD, reducing the risk of cross-contamination between different samples in the laboratory, and essentially eliminating the possibility of amplifying non-leukaemic clones12,
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. Moreover,
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DNA provides an absolute quantitation of the number of leukaemic cells in a sample, as opposed to RNA-based assays, which are also affected by mRNA expression levels and
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mRNA stability. On the other hand, use of DNA requires sequencing and individualized primer/probe design, significantly increasing complexity, time of implementation, and cost,
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compared with RNA transcripts which lack intronic segments and are often restricted to a small number of common variants amenable to standardized primer/probe sets for screening. Moreover, gene over-expression assays utilize the differential expression of genes between normal and leukaemic cells and must therefore be RNA-based. These considerations have resulted in limited interest in DNA-based monitoring in routine practice, with RNA-based RQ-PCR methods predominating.13 Sample quality is particularly important for RNA, and quantitation of a control gene in parallel with the target of interest provides insight into sample quality and reverse transcriptase efficiency. 14 Overall, the accuracy of an MRD test is
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determined by the diagnostic sensitivity and specificity of the marker, the prevalence of the leukemic cell in the sampled tissue (ie, bone marrow or peripheral blood), the sample size collected (as well as its quality), and the lower limit of detection of the assay. 15 RQ-PCR–based MRD approaches are highly amenable to standardization, as demonstrated
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over a decade ago by the Europe Against Cancer collaboration, whereby quantitative assays for nine of the most common fusion genes in leukemias were standardized across 26
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laboratories, and later for WT1 assays by the European LeukemiaNet.10, 16
As with any clinical laboratory testing, cost, throughput, and turn-around-times are critical
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considerations. Compared with multiparametric flow cytometry–based MRD, in which each sample is assayed individually, with results potentially available within one day, most molecular MRD tests must be batched to be economically viable.
Here we review the three classes of molecular targets mentioned above, including factors
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which affect their identification, as well as their suitability for MRD monitoring, and discuss future directions in molecular MRD monitoring with a particular focus on next-generation
Fusion genes
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sequencing (NGS) applications. A summary is presented in Table 1.17-23
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The monitoring of fusion genes is possible in AML cases characterized by a recurrent chromosomal rearrangement, which allows fusion gene transcripts such as RUNX1RUNX1T1, CBFB-MYH11, DEK-NUP214, NUP98-NSD1, and a myriad of KMT2A (MLL) fusions to be quantitated, most commonly by RNA-based RQ-PCR. These rearrangements have provided the most robust molecular MRD markers, and have allowed monitoring for approximately 50% of children, 30% of adults aged 15 to 60, and 10% of adults aged >60.23 Assay sensitivities are among the highest available, often approaching 10-5. From a practical viewpoint, the number of probe/primer pairs required to screen for these abnormalities is
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variable, and determined by the level of conservation of breakpoints for each respective rearrangement. For example, there is only a single type of RUNX1-RUNX1T1 transcript, greatly simplifying its screening. On the other hand, in screening for its CBFB counterpart (CBFB-MYH11), three different reverse primers along with a common forward primer and
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probe are required to identify the three commonest break-points to cover approximately 95% of cases.16
In current routine practice, a subset of AML cases with fusion events suitable for MRD
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monitoring will be missed, including cytogenetically cryptic, uncommon, and novel fusions.
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That is because we currently rely upon the diagnostic karyotype ± a limited FISH and/or a screening PCR panel for their identification, thereby limiting detection based on prior knowledge and common abnormalities. Moreover, in many cases the diagnostic services required to identify or confirm rearrangements by FISH, PCR, or sequencing and/or identify breakpoints to design PCR-based MRD assays are unavailable. As an example, there are at
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least 28 different rearrangements of the NUP98 gene in haematopoietic malignancies.24 In AML, the commonest of these is the NUP98-NSD1 fusion, which is enriched in paediatric AML cases, and produces a cytogenetically cryptic t(5;11), with frequency reported to range
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from 3.6% to 7.4% in unselected paediatric AML series25. Traditionally, to identify a specific
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NUP98 rearrangement, it first needs to be screened for by a FISH break-apart probe, then confirmation of the partner gene would require further FISH analysis if a suitable probe was available, PCR if a putative gene was suspected, or if truly novel, would require sequencing. Other factors also reduce FISH probe hybridization such as deletion within the target region, as was seen in two out of 22 NUP98 rearranged cases in one study, in which the NUP98 FISH probe failed, prompting RNA sequencing for further characterization25. It is therefore evident that in order to identify as many fusion events suitable for MRD monitoring as possible, an agnostic method with a high sensitivity for fusion gene detection is highly
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desirable. One promising approach is RNA-based NGS assays which are discussed in detail later.
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Gene Mutations Currently only one recurrent mutation that of the NPM1 gene has emerged as a suitable MRD marker in AML. The commonest mutation in adult AML, NPM1 mutations (NPM1mut), are
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present in 30% to 35% of all AML cases and 50% to 60% of AML with normal karyotype26. Ivey et al elegantly showed that monitoring NPM1mut MRD by RQ-PCR using a mutation-
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specific primer with a common primer and probe afforded a median sensitivity of 10-5, reliably predicted relapse on sequential monitoring, was stably present at relapse in 99% of cases and gave highly informative information to guide the decision to undertake stem cell transplantation.20
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To understand what constitutes a suitable gene mutation for MRD purposes, a basic understanding of AML leukaemogenesis is required. The majority of mutations in an AML genome are actually random events that occurred within a haematopoietic stem/progenitor
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cell prior to the acquisition of a leukaemia initiating event, or in many cases a few initiating,
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or so-called driver mutations. Subsequently, the progenitor acquires a number of cooperating mutations, and gives rise to the founding clone that is detected at diagnosis.2 Further cooperating mutations do occur and can contribute to disease progression/relapse but often only in a subset of leukemic cells, giving rise to sub-clones. Therefore, a mutation suitable for MRD monitoring is one occurring between the leukaemia-initiating event and the emergence of the founding clone, visually represented in Figure 1. Related to this concept is that of the variant allele frequency (VAF) of a mutation, which is the fraction of tumor cells that harbor a specific mutation, assuming a relatively pure
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leukemic sample and a diploid genome, variations of which reflect intra-tumoral heterogeneity.27 The archetypal initiating events are the aforementioned recurrent structural rearrangements which produce fusion genes, and are present in almost all leukaemic cells, resulting in a VAF of approximately 0.5. On the other hand, it is challenging to identify
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driver mutations in cases such as AML with normal karyotype. In this setting, the VAF can be helpful, as initiating/driver mutations should have VAF approaching 0.5 for heterozygous mutations (sensitive), persist within sub-clones, and therefore detectable at relapse (stable).
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However, for some early driver mutations, specificity becomes a problem, due to the existence of the mutation in a pre-leukaemic state. This is particularly relevant for mutations
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in epigenetic modifiers, such as DNTM3A and IDH1/2 which are common in adult AML. In NPM1-mutated cases, Ivey et al found many cases of persistence of DNMT3A and IDH mutations at high levels in patients who achieved NPM1-MRD negativity and maintained long-term remission.20 Furthermore, in five of 15 de novo AML cases, Wong et al
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demonstrated “rising clones” in one or more remission samples that were not detected by enhanced exome sequencing at diagnosis, enriched for mutations in TP53, TET2, DNMT3A, and ASXL1, all of which were shown to be present in rare cells before therapy by more
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sensitive techniques28. These findings are in line with our current understanding of clonal haematopoiesis of indeterminate potential (CHIP), and the use of these mutations in the MRD
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context would therefore lead to false positivity in many instances.20,29 Unfortunately, many of the most common recurrent mutations in AML such as those within tyrosine kinase genes such as FLT3 and KIT, or in the RAS/MAPK/MEK pathway such as NRAS and PTPN11, are often present at lower VAF, suggesting that these mutations are often late, sub-clonal events.3, 5 Taking the above factors into account, it is becoming increasingly clear that to identify gene mutations suitable for monitoring in AML other than NPM1, a patient-specific approach is
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required, which can only be afforded by NGS-based technologies. With the above issues limiting the use of many recurrent mutations in AML, it is important to note that when a leukaemia-specific mutation is defined as a mutation occurring between the leukaemiainitiating event and the emergence of the founding clone, the mutation need not be common,
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discussion of genomic DNA-based NGS below.
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a driver mutation, or even pathogenic. Identifying such mutations is outlined further in the
Gene Overexpression
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In patients who lack a leukaemia-specific target, quantitation of gene transcripts overexpressed in AML has aimed to provide a ‘universal’ MRD target. The best characterized of these is WT1, which is overexpressed in the majority of AML cases.20 The sensitivity of assays taking this approach is limited by baseline levels of normal expression in
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peripheral blood and bone marrow. In the case of WT1, unlike most molecular assays, peripheral blood is more sensitive than bone marrow due to its lower baseline WT1 expression. The performance of early WT1 assays was limited by factors which reduced their
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specificity and sensitivity, which led to the European LeukaemiaNet (ELN) collaborative effort to select an assay with optimal performance10. It was noted that mutations within the
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WT1 gene, occurring in 10% of AML patients, tended to cluster within the 3’ regions of the gene, leading to false negativity of assays that amplified this region. Other assays had problems with cross-reactivity with genomic DNA. Finally, the best performing of nine WT1 assays was selected. From a cohort of 129 uniformly treated adults, only 46% of patients had sufficiently high diagnostic WT1 expression levels in PB to allow discrimination of at least a 2-log reduction in expression. Despite this limitation, the ELN study and others have demonstrated increased relapse risk for those with sub-optimal reductions in or re-emergence
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of WT1 transcripts at early time-points, pre-HSCT and post-HSCT.11, 30,31 Moreover, Marani et al also demonstrated that incorporation of WT1 log reduction was able to further refine relapse risk in patients with MRD detectable by flow cytometry post-induction, highlighting that orthogonal MRD methods may be complementary when used together rather than
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provide the same information.
Another contemporary approach to improve the performance of MRD detection by overexpression was evaluated by Steinbach et al, who measured the expression of seven
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genes known to be overexpressed in AML (including WT1), along with three housekeeping
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genes, on the same TaqMan Low Density Array (TLDA)32. Although WT1 was the best performing single marker of MRD positivity, of the 42 follow-up samples that were classified as MRD positive, WT1 was only positive in 20 (48%) of the cases, which means that the gene panel allowed for detection of more than twice the number of MRD-positive cases than WT1
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alone. Moreover, the TLDA provided for a simple, fast, and standardized methodology.
Future directions in molecular MRD monitoring
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Droplet digital PCR
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Droplet digital PCR (ddPCR) is a recently commercialized technology that allows precise quantification of target nucleic acids in a sample by first partitioning it into nanoliter-sized, highly uniform, water-in-oil droplets33. This limiting dilution results in either one or zero target molecules in most reactions. After end-point PCR using fluorescently quenched oligonucleotide probes, measurement of fluorescence of each partition individually allows absolute nucleic acid quantification by Poisson statistical analysis of the number of positive and negative reactions34-36. Although the technology may not play a role in the identification of suitable markers for MRD, several characteristics of ddPCR, compared to conventional
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RQ-PCR make it very promising for use in MRD monitoring. Most notable of these is the capacity for absolute quantification independent of a reference standard, robustness to variations in PCR efficiency, and overall simplicity34. There is growing interest in the use of ddPCR for MRD measurement in many haematological malignancies. One such example is
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in chronic myeloid leukaemia (CML), in which monitoring BCR-ABL1 fusion transcripts by RQ-PCR has become standard of care. In this disease, where second generation tyrosine kinase inhibitors (TKI’s) are resulting in deeper molecular responses, the lower limit of
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detection (LOD) of the RQ-PCR method may be insufficient, especially when determining which patients may be eligible for TKI cessation37. Several groups have now demonstrated
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improved assay sensitivity as well as superior precision with ddPCR of BCR-ABL1 transcripts compared to a conventional RQ-PCR assay37,38. In fact, the LOD can be adjusted to the precise requirement of any application by modifying the amount of input RNA and the number of replicates39, making ddPCR an exciting addition to the MRD armamentarium.
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Recently, a novel application of ddPCR for MRD monitoring of NPM1mut AML was reported40. In this study, common forward primer and probes were used as previously described37, along with a degenerate reverse primer capable of generating oligonucleotides
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with all possible tetranucleotide insertions in exon 12 position 863, characteristic of 95% of
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NPM1 mutations. The multiplex assay was tested against mutation-specific ddPCR assays using a multitude of common and rare mutations from patient and cell line samples, with overall excellent concordance between the two assays as well as with RQ-PCR. This could potentially benefit clinical laboratories in testing of rare NPM1 mutations, obviating the need for patient-specific assays, as well as in testing for patients for which the mutant sequence is unknown. In the post allogeneic transplant setting, Brambati et al published their experience performing ddPCR MRD monitoring for DNMT3A and IDH1/2 mutations41. In their series of 89 patients,
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at least one of these mutations was present in 33.7% of the cases. By performing monthly BM evaluation in the first three months, then once every three months for the first year and yearly thereafter, six of the nine patients who relapsed had MRD positivity in the BM immediately preceding relapse, with a median time from first positivity to relapse of 60 days.
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Conceivably, this provides a feasible time frame for pre-emptive therapy and the high sensitivity (significantly greater than molecular chimerism studies and FISH) should allow
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more patients to be treated with low level disease.
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Next-Generation Sequencing (NGS)
Due to rapidly improving technology and falling cost, the prospect of using NGS for both identification and monitoring of MRD targets is becoming increasingly promising. Again, both genomic DNA and RNA can be utilized and each can be subjected to comprehensive or
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targeted NGS assays. With their inherent strengths and limitations, selection of the correct platform and subsequently the correct mutation(s) to monitor is pivotal.
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Genomic DNA sequencing
Klco et al demonstrated the potential to use genomic mutation clearance to define groups
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with differing relapse risk. Although not designed for highly sensitive molecular MRD detection, the study illustrated some of the factors that must be considered in setting up an NGS-based MRD assay.42 For 25 adult AML cases, cryopreserved diagnostic and postinduction samples were subjected to enhanced exome sequencing. From another 25 patients who had previous exome or genome sequencing, DNA extracted from day 30 FFPE BM tissue was used to generate short amplicons (using AmpliseqTM) for deep sequencing of a mean of 12.1 mutations from each case. Although the enhanced exome sequencing provided a mean coverage of leukaemia-associated variants at day 30 of 256X, the deep sequencing of
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the FFPE BM tissue, which was of significantly poorer quality, afforded a mean coverage on day 30 of 14,780X, more than 50 times greater. Factors influencing the sensitivity of an NGS-based MRD assay include sequencing coverage, DNA quantity, and the type of target mutation23. At diagnosis, when identifying
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leukaemia-specific mutations, use of a comprehensive sequencing platform such as wholeexome sequencing may be suitable, since, while read depth of individual mutations may be low, breadth of coverage may allow many mutations with high VAF to be detected. On
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follow-up studies on the other hand, read depth in the regions of interest must be increased
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substantially to allow for highly sensitive MRD detection, meaning that the sequencing must be targeted.
Another important consideration when deciding which NGS platform is to be used, is the type of mutations to be identified. For example, if the intention is to detect single nucleotide
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polymorphisms (SNPs) and small insertions/deletions (indels) in coding regions, wholeexome sequencing (WES) will provide greater coverage than whole-genome sequencing (WGS). Conversely, if fusion genes are the primary target, WGS is more suited than WES,
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which can only detect these structural variations in the subset of rearrangements with breakpoints in or near exons, significantly reducing sensitivity for fusion gene detection.43
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Moreover, fusion events are identified by WGS only through intensive computational analysis, and the large amount of sequencing needed may be restrictive in terms of cost and throughput44. On the other hand, RNA sequencing can be used to interrogate gene fusions as long as they are sufficiently expressed, with the added capacity for targeted assay development, making these the most promising NGS-based methods for identifying as well as potentially monitoring MRD of fusion genes. 12, 43
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Most studies in NGS-based MRD have focused on the monitoring of mutations in the genes most commonly mutated in the AML genome, which has highlighted several shortcomings to this approach. Firstly, as mentioned previously, common mutations often include those which are often present in pre-leukaemic clones (eg, TP53, DNMT3A, and ASXL1), rendering them
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unsuitable as MRD markers, unless monitoring in the context of allogeneic haematopoietic stem cell transplant (allo-HSCT), where tracking the clearance of this pre-leukaemic clone as a marker of recipient haematopoietic origin may be useful. Secondly, other common,
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recurrently mutated genes in AML (eg, FLT3, PTPN11, and NRAS) are often sub-clonal and thus unreliably present at relapse, as noted in several studies.3,
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Furthermore, although
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mutations in epigenetic regulators are among the commonest mutations in adult AML, studies in childhood AML have demonstrated them to be rare, and in general childhood cases display a relative paucity of mutations, making identification of mutations for MRD even more difficult in this population.3, 46 Thus, it is evident that in many cases NGS is needed to define
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patient- and leukaemia-specific markers. An example of this approach was published by Malmberg and colleagues, who performed WES on FACS-sorted leukaemic cells and lymphocytes from 17 AML patients.23 In doing so, they were able to filter both germline and
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early HPSC passenger mutations, and subsequently removed mutations with 95% CI of VAF
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below 0.5, in effect selecting leukaemia-specific mutations as defined earlier. This approach yielded a median of 11 leukaemia-specific mutations per case, with DNMT3A mutations being excluded. Using targeted deep sequencing in one patient and selecting three SNP mutations and a type A NPM1 mutation, they were able to monitor MRD in subsequent samples with high sensitivity (significance threshold set at VAF 0.027% for SNPs and 0.006% for NPM1), and a high level of correlation was demonstrated between mutational loads of each target mutation at the various time-points, as well as with concurrent NPM1mut DNA-based RQ-PCR measurements. Furthermore, all mutations were present at high
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mutational load at relapse. This approach may prove to be a realistic alternative in those patients without other sensitive MRD markers. Finally, one remaining question to be addressed is whether NGS-based MRD has advantages over, or is able to provide additional information to MRD detected by RQ-PCR or other
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methods. One study which illustrates this potential, involved comparison of MRD detection in NPM1mut AML by targeted deep sequencing and flow cytometry in 22 samples from six patients.22 The first finding here was increased sensitivity, with a limit of detection of
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approximately 0.001% MRD in cell dilution studies, an order of magnitude greater than
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observed in concurrent flow cytometry studies, and the detection of MRD in all clinical samples which were negative by flow cytometry. Secondly, the specificity of leukaemic blast detection is potentially much greater by NGS, owing to immunophenotypic overlap with regenerative blasts in flow cytometry, which could lead to either over or under-estimation of MRD, when compared with the non-limiting specificity of NPM1 mutations in this study,
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with no positive reads in the PB of 20 normal blood donors. Moreover, in addition to the effect of specificity, quantitation of bone marrow MRD is also conceivably more accurate by
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NGS as the DNA of early erythroid precursors is included, whereas this is not possible by flow cytometry due to erythroid lysis protocols typically used. NGS-based identification of
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NPM1 mutant sequences for subsequent MRD monitoring is more streamlined than the multistep conventional approaches. However, possibly the most striking finding seen in this study was NPM1-mutant clonal heterogeneity, seen in two of the six patients studied. In one of these patients, although still detectable, the level of the index mutation was overtaken by an alternative NPM1 mutation, which would not be reflected by a mutant-specific PCR assay (although potentially quantitated accurately by the multiplex ddPCR assay described earlier40) and therefore lead to falsely reduced quantitation of disease burden.
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As mentioned earlier, RNA-based NGS approaches offer the potential to be both high yield in terms of fusion gene detection, and streamlined – that is, in one step identifying the partner genes as well as provide the specific breakpoint required for design of PCR-based MRD
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detection. Alternatively, the identified fusion could be monitored by a targeted sequencing assay. Whole transcriptome sequencing can be used to identify both known and novel fusion transcripts through computational tools44. However, target-enrichment strategies may further
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enhance detection sensitivity while reducing the cost of sequencing47. Amplicon-based target
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enrichment strategies require prior knowledge of fusion partners for opposing primer design and are therefore unable to identify novel fusion genes. This and other limitations in cancer fusion gene detection prompted the development of the Anchored Multiplex PCR (AMP) targeted sequencing method, which, from small amounts of input RNA is able to uncover unknown sequences adjacent to a known nucleic acid sequence by using a one-sided nested
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primer approach, enabling fusion detection with only one partner gene targeted 48. AMPenriched targeted RNA sequencing (Archer® FusionPlex®, ArcherDx, Boulder, CO) has
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recently been evaluated for MLL-fusion breakpoint identification in childhood leukaemia cases, and compared to long-distance inverse-PCR (LDI-PCR), a gold-standard technique
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which provides patient-specific genomic DNA breakpoints for subsequent MRD monitoring by RQ-PCR21, 49. Of the 39 samples tested harboring nine different MLL fusions, all but one fusion identified by LDI-PCR were detected by AMP, one fusion unable to be characterized by LDI-PCR was identified by AMP, and furthermore, AMP identified multiple MLL-fusion transcript isoforms in 47% of the patients. Importantly, implementation of LDI-PCR for MLL breakpoint identification is still a decision based on prior knowledge or suspicion of an MLL rearrangement usually by cytogenetics/FISH, whereas AMP-enriched sequencing could be applied to unselected patients to identify MLL or any fusion genes expressed in the leukaemia
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for subsequent MRD monitoring. The study provided strong evidence to support the potential use of AMP-enriched targeted RNA sequencing for highly sensitive MRD detection – by performing serial dilutions of MV4-11 (MLL-AFF1) cells in Kasumi-1 cells from 10-2 to 10-7, and subjecting the extracted RNA to AMP-enrichment, the MLL fusion was detectable down
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to the 10-7 dilution. Moreover, as an NGS assay, leveraging the base pair resolution to
identify sequence variants (point mutations, small insertions/deletions) of relevance to AML is also promising. A pitfall of this technology however, is a significant artefactual false-
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positive fusion call rate, requiring rigorous review of breakpoints and extensive test
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validation.50
Conclusion
MRD monitoring in haematological malignancies is a burgeoning field as it uniquely
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provides a dynamic assessment of relapse risk independent of baseline prognostic evaluation. The experience in both acute promyelocytic leukaemia and childhood acute lymphoblastic leukaemia have demonstrated that MRD-based risk adapted therapies can improve outcomes
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in those diseases; however, we eagerly await similar evidence in AML51,52. However, the inter- and intra-tumor genetic heterogeneity in AML has posed challenges in defining
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specific, sensitive, and stable molecular MRD markers for all patients, and with many different assays required, standardization has also proven challenging. As outlined in this review, improving technology, particularly through next-generation sequencing, coupled with an improved understanding of AML leukaemogenesis, is leading to new methods to both identify and monitor suitable MRD targets in this challenging malignancy.
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ACCEPTED MANUSCRIPT Figure Legend
Figure 1. Over time, haematopoietic stem/progenitor cells (HSPC) acquire passenger mutations (P), most of which are benign and may be found in mature progeny. Later, this HSPC acquires a leukaemia-initiating mutation (i). If this initiating/early driver mutation commonly exists in a pre-
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leukaemic state (such as DNMT3A mutations), the mutation may potentially be present in mature progeny. The progenitor acquires further co-operating mutations (c), as well as passenger mutations (p), which gives rise to the founding AML clone (X). Subsequent co-operating and passenger
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mutations (c1, c2, p1, p2) occur in subsets of leukaemic cells, giving rise to sub-clones (1 and 2). Thus, suitable mutations for minimal residual disease monitoring are those that are acquired between the
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leukaemia-initiating event and the emergence of the founding clone (with the exception of mutations commonly present in the pre-leukaemic state). Adapted from Welch et al2, with
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permission from Elsevier.
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ACCEPTED MANUSCRIPT Table 1. Characteristics of selected molecular MRD assays. Assay
Nucleic
Analytical Sensitivity
Reference
0.025-0.05%
Hokland et al (2012)17
0.001%
Willekens et al (2016)18
0.025-0.05%
Hokland et al (2012)17
acid
RNA
Fusion gene DEK-NUP214 RUNX1-RUNX1T1 MLL-MLLT3
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RNA
Gene-
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RQ-PCR
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source
overexpression
>1:100 in 23% *
WT1
Ommen (2016)19
>1:200 in 7 % *
>1:100 in 59% †
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Steinbach 7 gene set
Ommen (2016)19
DNA or
Gene Mutation
RNA NPM1mut
Ivey et al (2016)20
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0.001%‡
RNA or
at least equivalent to
DNA
RQ-PCR
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ddPCR
NGS-based
Fusion gene
RNA
10-7 §
Afrin et al (2018)21
NPM1mut DNA
0.001%
Salipante et al (2014)22
Patient-specific SNVs DNA
0.025%
Malmberg et al (2016)23
MLL-AFF1
Gene mutations
DNA
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ACCEPTED MANUSCRIPT *Adult AML, compared with median normal expression. †Childhood AML, compared with 90% upper normal limit. ‡Median sensi^vity for 27 different NPM1 muta^ons.
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§Cellular dilution of MV4-11 cell line in Kasumi-1 cell line. Anchored Multiplex PCR–enriched RNA sequencing (Archer® FusionPlex®Heme panel).
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ACCEPTED MANUSCRIPT
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HSPC
1
Founding clone
P + i
P
?
P + i + c + p (X)
P + i
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P
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X + c1 + p1
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Mature progeny
Subclones
2 X + c2 + p2
P = passenger mutations in HSPC i = initiating mutation(s) c = co‐operating mutations in founding clone p = passenger mutations in founding clone X = P + i + c + p c1= cooperating mutations in subclone 1 c2= cooperating mutations in subclone 2 p1 = passenger mutations in subclone 1 p2 = passenger mutations in subclone 2
S1525-1578(17)30485-3
DOI:
10.1016/j.jmoldx.2018.03.005
Reference:
JMDI 689
To appear in:
The Journal of Molecular Diagnostics
Accepted Date: 27 March 2018
Please cite this article as: Selim A, Moore AS, Molecular Minimal Residual Disease Monitoring in Acute Myeloid Leukemia: Challenges and Future Directions, The Journal of Molecular Diagnostics (2018), doi: 10.1016/j.jmoldx.2018.03.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular minimal residual disease monitoring in acute myeloid leukaemia: challenges and future directions Adrian Selim* and Andrew S. Moore*†‡
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From the The University of Queensland Diamantina Institute,* Translational Research Institute, Brisbane; the Oncology Services Group,† Children’s Health Queensland Hospital and Health Service, Brisbane; and the UQ Child Health Research Centre,‡ The University of
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Queensland, Brisbane, Australia
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Correspondence: Andrew S. Moore Children’s Health Queensland Research Directorate Level 7, Centre for Children’s Health Research
62 Graham St, South Brisbane, Queensland, 4101, Australia
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Email: [email protected]
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Running Head: Molecular MRD in AML
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Funding: A.S.M. is supported by the Children’s Hospital Foundation, Queensland. Disclosures: None declared.
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ACCEPTED MANUSCRIPT Abstract
The ability to sensitively monitor minimal residual disease (MRD) has played a key role in improving the management and outcomes for a number of leukaemias, particularly acute promyelocytic leukaemia and childhood acute lymphoblastic leukaemia. In contrast, MRD
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monitoring in acute myeloid leukaemia (AML) has been limited by variable assay methodologies and a relative paucity of patient-specific MRD markers. Inter- and intra-tumor genetic heterogeneity pose significant challenges for the identification of molecular markers
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suitable for MRD monitoring in AML, particularly for those cases without structural
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chromosomal rearrangements associated with fusion genes. Furthermore, the need to discriminate which mutations may be suitable for MRD monitoring creates additional complexity. The mainstay of current molecular MRD monitoring is real-time quantitative PCR, targeting fusion genes, mutations, and gene over-expression. New technologies, particularly next-generation sequencing approaches, offer new ways to overcome these
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limitations. Here, we review the techniques available for molecular MRD monitoring in AML
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and discuss their utility in clinical practice.
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ACCEPTED MANUSCRIPT Introduction
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by diverse genetic abnormalities and variable morphology, immunophenotypes, and clinical outcomes1. Genetic heterogeneity exists not only between different AML cases but within each individual case
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and mutations in AML may pre-date leukaemia-initiating events, occur after initiation, and contribute to the founding AML clone, or occur sub-clonally after leukaemogenesis2. Thus, the clonal composition of the leukaemia can vary significantly, as sub-clones (along with
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their late acquired mutations) are subjected to natural and treatment-induced selection
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pressures, expanding, diminishing, or even being cleared3-5. These factors present challenges when selecting genetic abnormalities as markers of post-treatment minimal residual disease (MRD) in AML. Despite this, the impetus for MRD monitoring in AML is ever-present, as countless studies have established the role of MRD as a dynamic predictor of relapse risk and overall survival, often stronger than any pre-treatment variables1. Moreover, the ultimate aim
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of MRD monitoring in leukaemia is to be able to utilize this dynamic marker to personalize therapy, such that treatments and their associated toxicities are curbed in low-risk patients,
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and intensified in those at high risk of relapse.
The characteristics of a suitable MRD marker can be defined by ‘3 Ss’; a marker must be
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‘specific’, ‘sensitive’, and ‘stable’. Moreover, some would argue for a fourth characteristic, ‘stemness’, whereby a marker is present only in those cells capable of self-renewal and hence relapse, and not in bulk leukaemic cells1. Although such markers are being actively pursued in the context of multicolor flow cytometric MRD determination6, it remains a largely philosophical ideal in the molecular context. The terms ‘sensitive’ and ‘specific’ can be a source of confusion. As characteristics of the MRD marker, a highly sensitive marker is one that is present in all leukemic cells, and a highly specific marker is one that is only present in leukaemic cells while being absent in non-leukemic/pre-leukemic cells. A lack of sensitivity
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will produce false negative MRD, whereas a lack of specificity will produce false positives. The second use of the term sensitivity is the analytical sensitivity, a performance characteristic of the assay, more appropriately described as the lower limit of detection (LOD). A commonly accepted benchmark in LOD in AML MRD assays is 10-3 (0.1%),
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meaning one leukemic cell (or equivalent) in 1,000 cells measured, although some molecular MRD assays have LODs of 10-5 to 10-7. The theoretical benefit of utilizing more sensitive assays is an ability to identify more patients at risk of relapse, and to quantitatively track
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minute changes in low-level MRD. Despite a lack of strong evidence for MRD-directed therapeutic approaches in non-APL AML, by employing a sequential MRD surveillance
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strategy, one could expect better outcomes when therapy is initiated at the time of ‘molecular relapse’ rather than frank haematological relapse. Risk-adapted treatment protocols taking into account sequential MRD results have shown favorable results in paediatric AML (AML027), although the study relied on comparison to historical cohorts. Hopefully, with the
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results of prospective, randomized clinical trials assessing the value of MRD-directed therapy, such as the United Kingdom’s NCRI AML17 trial, which randomized patients with informative molecular markers to “MRD monitoring” vs “no MRD monitoring” strategies,
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the question of whether modification of post-remission treatment intensity based on MRD
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status in AML will be answered8.
The mainstay of molecular MRD monitoring at present are real-time quantitative PCR (RQPCR)–based methods, which can be employed when a suitable target is present, afford high analytical sensitivity, and, to a significant extent, allow for standardization between different laboratories. Targets for RQ-PCR as well as other molecular methods can be divided into three categories: i) chimeric fusion gene transcripts; ii) gene mutations; and iii) overexpressed genes.
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PCR-based assays of fusion genes and NPM1 mutations have the potential to collectively monitor approximately 60% of AML presenting in children and younger adults; however, this proportion steadily declines with increasing age, encompassing only 32% of those over 609 . PCR of over-expressed genes, most commonly WT1, can be applied in a significant
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proportion of AML cases (ranging from 13% to 73% in different studies depending on whether PB or BM is used and the threshold for defining over-expression10, 11), but with its limited dynamic range, is generally restricted to those who lack other molecular markers.
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The nucleic acid source for molecular assays may be genomic DNA or RNA, each with a set
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of advantages and disadvantages. DNA is more stable and easier to extract than RNA, making it favorable for collection, processing, transport, and storage. In the case of fusion genes, breakpoints at the gDNA level are unique, providing patient-specific markers of MRD, reducing the risk of cross-contamination between different samples in the laboratory, and essentially eliminating the possibility of amplifying non-leukaemic clones12,
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. Moreover,
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DNA provides an absolute quantitation of the number of leukaemic cells in a sample, as opposed to RNA-based assays, which are also affected by mRNA expression levels and
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mRNA stability. On the other hand, use of DNA requires sequencing and individualized primer/probe design, significantly increasing complexity, time of implementation, and cost,
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compared with RNA transcripts which lack intronic segments and are often restricted to a small number of common variants amenable to standardized primer/probe sets for screening. Moreover, gene over-expression assays utilize the differential expression of genes between normal and leukaemic cells and must therefore be RNA-based. These considerations have resulted in limited interest in DNA-based monitoring in routine practice, with RNA-based RQ-PCR methods predominating.13 Sample quality is particularly important for RNA, and quantitation of a control gene in parallel with the target of interest provides insight into sample quality and reverse transcriptase efficiency. 14 Overall, the accuracy of an MRD test is
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determined by the diagnostic sensitivity and specificity of the marker, the prevalence of the leukemic cell in the sampled tissue (ie, bone marrow or peripheral blood), the sample size collected (as well as its quality), and the lower limit of detection of the assay. 15 RQ-PCR–based MRD approaches are highly amenable to standardization, as demonstrated
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over a decade ago by the Europe Against Cancer collaboration, whereby quantitative assays for nine of the most common fusion genes in leukemias were standardized across 26
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laboratories, and later for WT1 assays by the European LeukemiaNet.10, 16
As with any clinical laboratory testing, cost, throughput, and turn-around-times are critical
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considerations. Compared with multiparametric flow cytometry–based MRD, in which each sample is assayed individually, with results potentially available within one day, most molecular MRD tests must be batched to be economically viable.
Here we review the three classes of molecular targets mentioned above, including factors
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which affect their identification, as well as their suitability for MRD monitoring, and discuss future directions in molecular MRD monitoring with a particular focus on next-generation
Fusion genes
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sequencing (NGS) applications. A summary is presented in Table 1.17-23
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The monitoring of fusion genes is possible in AML cases characterized by a recurrent chromosomal rearrangement, which allows fusion gene transcripts such as RUNX1RUNX1T1, CBFB-MYH11, DEK-NUP214, NUP98-NSD1, and a myriad of KMT2A (MLL) fusions to be quantitated, most commonly by RNA-based RQ-PCR. These rearrangements have provided the most robust molecular MRD markers, and have allowed monitoring for approximately 50% of children, 30% of adults aged 15 to 60, and 10% of adults aged >60.23 Assay sensitivities are among the highest available, often approaching 10-5. From a practical viewpoint, the number of probe/primer pairs required to screen for these abnormalities is
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variable, and determined by the level of conservation of breakpoints for each respective rearrangement. For example, there is only a single type of RUNX1-RUNX1T1 transcript, greatly simplifying its screening. On the other hand, in screening for its CBFB counterpart (CBFB-MYH11), three different reverse primers along with a common forward primer and
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probe are required to identify the three commonest break-points to cover approximately 95% of cases.16
In current routine practice, a subset of AML cases with fusion events suitable for MRD
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monitoring will be missed, including cytogenetically cryptic, uncommon, and novel fusions.
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That is because we currently rely upon the diagnostic karyotype ± a limited FISH and/or a screening PCR panel for their identification, thereby limiting detection based on prior knowledge and common abnormalities. Moreover, in many cases the diagnostic services required to identify or confirm rearrangements by FISH, PCR, or sequencing and/or identify breakpoints to design PCR-based MRD assays are unavailable. As an example, there are at
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least 28 different rearrangements of the NUP98 gene in haematopoietic malignancies.24 In AML, the commonest of these is the NUP98-NSD1 fusion, which is enriched in paediatric AML cases, and produces a cytogenetically cryptic t(5;11), with frequency reported to range
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from 3.6% to 7.4% in unselected paediatric AML series25. Traditionally, to identify a specific
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NUP98 rearrangement, it first needs to be screened for by a FISH break-apart probe, then confirmation of the partner gene would require further FISH analysis if a suitable probe was available, PCR if a putative gene was suspected, or if truly novel, would require sequencing. Other factors also reduce FISH probe hybridization such as deletion within the target region, as was seen in two out of 22 NUP98 rearranged cases in one study, in which the NUP98 FISH probe failed, prompting RNA sequencing for further characterization25. It is therefore evident that in order to identify as many fusion events suitable for MRD monitoring as possible, an agnostic method with a high sensitivity for fusion gene detection is highly
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desirable. One promising approach is RNA-based NGS assays which are discussed in detail later.
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Gene Mutations Currently only one recurrent mutation that of the NPM1 gene has emerged as a suitable MRD marker in AML. The commonest mutation in adult AML, NPM1 mutations (NPM1mut), are
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present in 30% to 35% of all AML cases and 50% to 60% of AML with normal karyotype26. Ivey et al elegantly showed that monitoring NPM1mut MRD by RQ-PCR using a mutation-
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specific primer with a common primer and probe afforded a median sensitivity of 10-5, reliably predicted relapse on sequential monitoring, was stably present at relapse in 99% of cases and gave highly informative information to guide the decision to undertake stem cell transplantation.20
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To understand what constitutes a suitable gene mutation for MRD purposes, a basic understanding of AML leukaemogenesis is required. The majority of mutations in an AML genome are actually random events that occurred within a haematopoietic stem/progenitor
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cell prior to the acquisition of a leukaemia initiating event, or in many cases a few initiating,
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or so-called driver mutations. Subsequently, the progenitor acquires a number of cooperating mutations, and gives rise to the founding clone that is detected at diagnosis.2 Further cooperating mutations do occur and can contribute to disease progression/relapse but often only in a subset of leukemic cells, giving rise to sub-clones. Therefore, a mutation suitable for MRD monitoring is one occurring between the leukaemia-initiating event and the emergence of the founding clone, visually represented in Figure 1. Related to this concept is that of the variant allele frequency (VAF) of a mutation, which is the fraction of tumor cells that harbor a specific mutation, assuming a relatively pure
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leukemic sample and a diploid genome, variations of which reflect intra-tumoral heterogeneity.27 The archetypal initiating events are the aforementioned recurrent structural rearrangements which produce fusion genes, and are present in almost all leukaemic cells, resulting in a VAF of approximately 0.5. On the other hand, it is challenging to identify
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driver mutations in cases such as AML with normal karyotype. In this setting, the VAF can be helpful, as initiating/driver mutations should have VAF approaching 0.5 for heterozygous mutations (sensitive), persist within sub-clones, and therefore detectable at relapse (stable).
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However, for some early driver mutations, specificity becomes a problem, due to the existence of the mutation in a pre-leukaemic state. This is particularly relevant for mutations
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in epigenetic modifiers, such as DNTM3A and IDH1/2 which are common in adult AML. In NPM1-mutated cases, Ivey et al found many cases of persistence of DNMT3A and IDH mutations at high levels in patients who achieved NPM1-MRD negativity and maintained long-term remission.20 Furthermore, in five of 15 de novo AML cases, Wong et al
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demonstrated “rising clones” in one or more remission samples that were not detected by enhanced exome sequencing at diagnosis, enriched for mutations in TP53, TET2, DNMT3A, and ASXL1, all of which were shown to be present in rare cells before therapy by more
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sensitive techniques28. These findings are in line with our current understanding of clonal haematopoiesis of indeterminate potential (CHIP), and the use of these mutations in the MRD
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context would therefore lead to false positivity in many instances.20,29 Unfortunately, many of the most common recurrent mutations in AML such as those within tyrosine kinase genes such as FLT3 and KIT, or in the RAS/MAPK/MEK pathway such as NRAS and PTPN11, are often present at lower VAF, suggesting that these mutations are often late, sub-clonal events.3, 5 Taking the above factors into account, it is becoming increasingly clear that to identify gene mutations suitable for monitoring in AML other than NPM1, a patient-specific approach is
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required, which can only be afforded by NGS-based technologies. With the above issues limiting the use of many recurrent mutations in AML, it is important to note that when a leukaemia-specific mutation is defined as a mutation occurring between the leukaemiainitiating event and the emergence of the founding clone, the mutation need not be common,
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discussion of genomic DNA-based NGS below.
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a driver mutation, or even pathogenic. Identifying such mutations is outlined further in the
Gene Overexpression
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In patients who lack a leukaemia-specific target, quantitation of gene transcripts overexpressed in AML has aimed to provide a ‘universal’ MRD target. The best characterized of these is WT1, which is overexpressed in the majority of AML cases.20 The sensitivity of assays taking this approach is limited by baseline levels of normal expression in
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peripheral blood and bone marrow. In the case of WT1, unlike most molecular assays, peripheral blood is more sensitive than bone marrow due to its lower baseline WT1 expression. The performance of early WT1 assays was limited by factors which reduced their
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specificity and sensitivity, which led to the European LeukaemiaNet (ELN) collaborative effort to select an assay with optimal performance10. It was noted that mutations within the
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WT1 gene, occurring in 10% of AML patients, tended to cluster within the 3’ regions of the gene, leading to false negativity of assays that amplified this region. Other assays had problems with cross-reactivity with genomic DNA. Finally, the best performing of nine WT1 assays was selected. From a cohort of 129 uniformly treated adults, only 46% of patients had sufficiently high diagnostic WT1 expression levels in PB to allow discrimination of at least a 2-log reduction in expression. Despite this limitation, the ELN study and others have demonstrated increased relapse risk for those with sub-optimal reductions in or re-emergence
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of WT1 transcripts at early time-points, pre-HSCT and post-HSCT.11, 30,31 Moreover, Marani et al also demonstrated that incorporation of WT1 log reduction was able to further refine relapse risk in patients with MRD detectable by flow cytometry post-induction, highlighting that orthogonal MRD methods may be complementary when used together rather than
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provide the same information.
Another contemporary approach to improve the performance of MRD detection by overexpression was evaluated by Steinbach et al, who measured the expression of seven
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genes known to be overexpressed in AML (including WT1), along with three housekeeping
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genes, on the same TaqMan Low Density Array (TLDA)32. Although WT1 was the best performing single marker of MRD positivity, of the 42 follow-up samples that were classified as MRD positive, WT1 was only positive in 20 (48%) of the cases, which means that the gene panel allowed for detection of more than twice the number of MRD-positive cases than WT1
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alone. Moreover, the TLDA provided for a simple, fast, and standardized methodology.
Future directions in molecular MRD monitoring
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Droplet digital PCR
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Droplet digital PCR (ddPCR) is a recently commercialized technology that allows precise quantification of target nucleic acids in a sample by first partitioning it into nanoliter-sized, highly uniform, water-in-oil droplets33. This limiting dilution results in either one or zero target molecules in most reactions. After end-point PCR using fluorescently quenched oligonucleotide probes, measurement of fluorescence of each partition individually allows absolute nucleic acid quantification by Poisson statistical analysis of the number of positive and negative reactions34-36. Although the technology may not play a role in the identification of suitable markers for MRD, several characteristics of ddPCR, compared to conventional
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RQ-PCR make it very promising for use in MRD monitoring. Most notable of these is the capacity for absolute quantification independent of a reference standard, robustness to variations in PCR efficiency, and overall simplicity34. There is growing interest in the use of ddPCR for MRD measurement in many haematological malignancies. One such example is
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in chronic myeloid leukaemia (CML), in which monitoring BCR-ABL1 fusion transcripts by RQ-PCR has become standard of care. In this disease, where second generation tyrosine kinase inhibitors (TKI’s) are resulting in deeper molecular responses, the lower limit of
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detection (LOD) of the RQ-PCR method may be insufficient, especially when determining which patients may be eligible for TKI cessation37. Several groups have now demonstrated
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improved assay sensitivity as well as superior precision with ddPCR of BCR-ABL1 transcripts compared to a conventional RQ-PCR assay37,38. In fact, the LOD can be adjusted to the precise requirement of any application by modifying the amount of input RNA and the number of replicates39, making ddPCR an exciting addition to the MRD armamentarium.
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Recently, a novel application of ddPCR for MRD monitoring of NPM1mut AML was reported40. In this study, common forward primer and probes were used as previously described37, along with a degenerate reverse primer capable of generating oligonucleotides
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with all possible tetranucleotide insertions in exon 12 position 863, characteristic of 95% of
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NPM1 mutations. The multiplex assay was tested against mutation-specific ddPCR assays using a multitude of common and rare mutations from patient and cell line samples, with overall excellent concordance between the two assays as well as with RQ-PCR. This could potentially benefit clinical laboratories in testing of rare NPM1 mutations, obviating the need for patient-specific assays, as well as in testing for patients for which the mutant sequence is unknown. In the post allogeneic transplant setting, Brambati et al published their experience performing ddPCR MRD monitoring for DNMT3A and IDH1/2 mutations41. In their series of 89 patients,
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at least one of these mutations was present in 33.7% of the cases. By performing monthly BM evaluation in the first three months, then once every three months for the first year and yearly thereafter, six of the nine patients who relapsed had MRD positivity in the BM immediately preceding relapse, with a median time from first positivity to relapse of 60 days.
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Conceivably, this provides a feasible time frame for pre-emptive therapy and the high sensitivity (significantly greater than molecular chimerism studies and FISH) should allow
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more patients to be treated with low level disease.
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Next-Generation Sequencing (NGS)
Due to rapidly improving technology and falling cost, the prospect of using NGS for both identification and monitoring of MRD targets is becoming increasingly promising. Again, both genomic DNA and RNA can be utilized and each can be subjected to comprehensive or
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targeted NGS assays. With their inherent strengths and limitations, selection of the correct platform and subsequently the correct mutation(s) to monitor is pivotal.
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Genomic DNA sequencing
Klco et al demonstrated the potential to use genomic mutation clearance to define groups
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with differing relapse risk. Although not designed for highly sensitive molecular MRD detection, the study illustrated some of the factors that must be considered in setting up an NGS-based MRD assay.42 For 25 adult AML cases, cryopreserved diagnostic and postinduction samples were subjected to enhanced exome sequencing. From another 25 patients who had previous exome or genome sequencing, DNA extracted from day 30 FFPE BM tissue was used to generate short amplicons (using AmpliseqTM) for deep sequencing of a mean of 12.1 mutations from each case. Although the enhanced exome sequencing provided a mean coverage of leukaemia-associated variants at day 30 of 256X, the deep sequencing of
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the FFPE BM tissue, which was of significantly poorer quality, afforded a mean coverage on day 30 of 14,780X, more than 50 times greater. Factors influencing the sensitivity of an NGS-based MRD assay include sequencing coverage, DNA quantity, and the type of target mutation23. At diagnosis, when identifying
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leukaemia-specific mutations, use of a comprehensive sequencing platform such as wholeexome sequencing may be suitable, since, while read depth of individual mutations may be low, breadth of coverage may allow many mutations with high VAF to be detected. On
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follow-up studies on the other hand, read depth in the regions of interest must be increased
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substantially to allow for highly sensitive MRD detection, meaning that the sequencing must be targeted.
Another important consideration when deciding which NGS platform is to be used, is the type of mutations to be identified. For example, if the intention is to detect single nucleotide
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polymorphisms (SNPs) and small insertions/deletions (indels) in coding regions, wholeexome sequencing (WES) will provide greater coverage than whole-genome sequencing (WGS). Conversely, if fusion genes are the primary target, WGS is more suited than WES,
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which can only detect these structural variations in the subset of rearrangements with breakpoints in or near exons, significantly reducing sensitivity for fusion gene detection.43
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Moreover, fusion events are identified by WGS only through intensive computational analysis, and the large amount of sequencing needed may be restrictive in terms of cost and throughput44. On the other hand, RNA sequencing can be used to interrogate gene fusions as long as they are sufficiently expressed, with the added capacity for targeted assay development, making these the most promising NGS-based methods for identifying as well as potentially monitoring MRD of fusion genes. 12, 43
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Most studies in NGS-based MRD have focused on the monitoring of mutations in the genes most commonly mutated in the AML genome, which has highlighted several shortcomings to this approach. Firstly, as mentioned previously, common mutations often include those which are often present in pre-leukaemic clones (eg, TP53, DNMT3A, and ASXL1), rendering them
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unsuitable as MRD markers, unless monitoring in the context of allogeneic haematopoietic stem cell transplant (allo-HSCT), where tracking the clearance of this pre-leukaemic clone as a marker of recipient haematopoietic origin may be useful. Secondly, other common,
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recurrently mutated genes in AML (eg, FLT3, PTPN11, and NRAS) are often sub-clonal and thus unreliably present at relapse, as noted in several studies.3,
42,45
Furthermore, although
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mutations in epigenetic regulators are among the commonest mutations in adult AML, studies in childhood AML have demonstrated them to be rare, and in general childhood cases display a relative paucity of mutations, making identification of mutations for MRD even more difficult in this population.3, 46 Thus, it is evident that in many cases NGS is needed to define
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patient- and leukaemia-specific markers. An example of this approach was published by Malmberg and colleagues, who performed WES on FACS-sorted leukaemic cells and lymphocytes from 17 AML patients.23 In doing so, they were able to filter both germline and
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early HPSC passenger mutations, and subsequently removed mutations with 95% CI of VAF
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below 0.5, in effect selecting leukaemia-specific mutations as defined earlier. This approach yielded a median of 11 leukaemia-specific mutations per case, with DNMT3A mutations being excluded. Using targeted deep sequencing in one patient and selecting three SNP mutations and a type A NPM1 mutation, they were able to monitor MRD in subsequent samples with high sensitivity (significance threshold set at VAF 0.027% for SNPs and 0.006% for NPM1), and a high level of correlation was demonstrated between mutational loads of each target mutation at the various time-points, as well as with concurrent NPM1mut DNA-based RQ-PCR measurements. Furthermore, all mutations were present at high
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mutational load at relapse. This approach may prove to be a realistic alternative in those patients without other sensitive MRD markers. Finally, one remaining question to be addressed is whether NGS-based MRD has advantages over, or is able to provide additional information to MRD detected by RQ-PCR or other
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methods. One study which illustrates this potential, involved comparison of MRD detection in NPM1mut AML by targeted deep sequencing and flow cytometry in 22 samples from six patients.22 The first finding here was increased sensitivity, with a limit of detection of
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approximately 0.001% MRD in cell dilution studies, an order of magnitude greater than
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observed in concurrent flow cytometry studies, and the detection of MRD in all clinical samples which were negative by flow cytometry. Secondly, the specificity of leukaemic blast detection is potentially much greater by NGS, owing to immunophenotypic overlap with regenerative blasts in flow cytometry, which could lead to either over or under-estimation of MRD, when compared with the non-limiting specificity of NPM1 mutations in this study,
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with no positive reads in the PB of 20 normal blood donors. Moreover, in addition to the effect of specificity, quantitation of bone marrow MRD is also conceivably more accurate by
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NGS as the DNA of early erythroid precursors is included, whereas this is not possible by flow cytometry due to erythroid lysis protocols typically used. NGS-based identification of
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NPM1 mutant sequences for subsequent MRD monitoring is more streamlined than the multistep conventional approaches. However, possibly the most striking finding seen in this study was NPM1-mutant clonal heterogeneity, seen in two of the six patients studied. In one of these patients, although still detectable, the level of the index mutation was overtaken by an alternative NPM1 mutation, which would not be reflected by a mutant-specific PCR assay (although potentially quantitated accurately by the multiplex ddPCR assay described earlier40) and therefore lead to falsely reduced quantitation of disease burden.
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As mentioned earlier, RNA-based NGS approaches offer the potential to be both high yield in terms of fusion gene detection, and streamlined – that is, in one step identifying the partner genes as well as provide the specific breakpoint required for design of PCR-based MRD
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detection. Alternatively, the identified fusion could be monitored by a targeted sequencing assay. Whole transcriptome sequencing can be used to identify both known and novel fusion transcripts through computational tools44. However, target-enrichment strategies may further
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enhance detection sensitivity while reducing the cost of sequencing47. Amplicon-based target
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enrichment strategies require prior knowledge of fusion partners for opposing primer design and are therefore unable to identify novel fusion genes. This and other limitations in cancer fusion gene detection prompted the development of the Anchored Multiplex PCR (AMP) targeted sequencing method, which, from small amounts of input RNA is able to uncover unknown sequences adjacent to a known nucleic acid sequence by using a one-sided nested
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primer approach, enabling fusion detection with only one partner gene targeted 48. AMPenriched targeted RNA sequencing (Archer® FusionPlex®, ArcherDx, Boulder, CO) has
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recently been evaluated for MLL-fusion breakpoint identification in childhood leukaemia cases, and compared to long-distance inverse-PCR (LDI-PCR), a gold-standard technique
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which provides patient-specific genomic DNA breakpoints for subsequent MRD monitoring by RQ-PCR21, 49. Of the 39 samples tested harboring nine different MLL fusions, all but one fusion identified by LDI-PCR were detected by AMP, one fusion unable to be characterized by LDI-PCR was identified by AMP, and furthermore, AMP identified multiple MLL-fusion transcript isoforms in 47% of the patients. Importantly, implementation of LDI-PCR for MLL breakpoint identification is still a decision based on prior knowledge or suspicion of an MLL rearrangement usually by cytogenetics/FISH, whereas AMP-enriched sequencing could be applied to unselected patients to identify MLL or any fusion genes expressed in the leukaemia
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for subsequent MRD monitoring. The study provided strong evidence to support the potential use of AMP-enriched targeted RNA sequencing for highly sensitive MRD detection – by performing serial dilutions of MV4-11 (MLL-AFF1) cells in Kasumi-1 cells from 10-2 to 10-7, and subjecting the extracted RNA to AMP-enrichment, the MLL fusion was detectable down
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to the 10-7 dilution. Moreover, as an NGS assay, leveraging the base pair resolution to
identify sequence variants (point mutations, small insertions/deletions) of relevance to AML is also promising. A pitfall of this technology however, is a significant artefactual false-
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positive fusion call rate, requiring rigorous review of breakpoints and extensive test
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validation.50
Conclusion
MRD monitoring in haematological malignancies is a burgeoning field as it uniquely
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provides a dynamic assessment of relapse risk independent of baseline prognostic evaluation. The experience in both acute promyelocytic leukaemia and childhood acute lymphoblastic leukaemia have demonstrated that MRD-based risk adapted therapies can improve outcomes
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in those diseases; however, we eagerly await similar evidence in AML51,52. However, the inter- and intra-tumor genetic heterogeneity in AML has posed challenges in defining
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specific, sensitive, and stable molecular MRD markers for all patients, and with many different assays required, standardization has also proven challenging. As outlined in this review, improving technology, particularly through next-generation sequencing, coupled with an improved understanding of AML leukaemogenesis, is leading to new methods to both identify and monitor suitable MRD targets in this challenging malignancy.
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ACCEPTED MANUSCRIPT Figure Legend
Figure 1. Over time, haematopoietic stem/progenitor cells (HSPC) acquire passenger mutations (P), most of which are benign and may be found in mature progeny. Later, this HSPC acquires a leukaemia-initiating mutation (i). If this initiating/early driver mutation commonly exists in a pre-
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leukaemic state (such as DNMT3A mutations), the mutation may potentially be present in mature progeny. The progenitor acquires further co-operating mutations (c), as well as passenger mutations (p), which gives rise to the founding AML clone (X). Subsequent co-operating and passenger
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mutations (c1, c2, p1, p2) occur in subsets of leukaemic cells, giving rise to sub-clones (1 and 2). Thus, suitable mutations for minimal residual disease monitoring are those that are acquired between the
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leukaemia-initiating event and the emergence of the founding clone (with the exception of mutations commonly present in the pre-leukaemic state). Adapted from Welch et al2, with
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permission from Elsevier.
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ACCEPTED MANUSCRIPT Table 1. Characteristics of selected molecular MRD assays. Assay
Nucleic
Analytical Sensitivity
Reference
0.025-0.05%
Hokland et al (2012)17
0.001%
Willekens et al (2016)18
0.025-0.05%
Hokland et al (2012)17
acid
RNA
Fusion gene DEK-NUP214 RUNX1-RUNX1T1 MLL-MLLT3
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RNA
Gene-
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RQ-PCR
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source
overexpression
>1:100 in 23% *
WT1
Ommen (2016)19
>1:200 in 7 % *
>1:100 in 59% †
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Steinbach 7 gene set
Ommen (2016)19
DNA or
Gene Mutation
RNA NPM1mut
Ivey et al (2016)20
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0.001%‡
RNA or
at least equivalent to
DNA
RQ-PCR
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ddPCR
NGS-based
Fusion gene
RNA
10-7 §
Afrin et al (2018)21
NPM1mut DNA
0.001%
Salipante et al (2014)22
Patient-specific SNVs DNA
0.025%
Malmberg et al (2016)23
MLL-AFF1
Gene mutations
DNA
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ACCEPTED MANUSCRIPT *Adult AML, compared with median normal expression. †Childhood AML, compared with 90% upper normal limit. ‡Median sensi^vity for 27 different NPM1 muta^ons.
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§Cellular dilution of MV4-11 cell line in Kasumi-1 cell line. Anchored Multiplex PCR–enriched RNA sequencing (Archer® FusionPlex®Heme panel).
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ACCEPTED MANUSCRIPT
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HSPC
1
Founding clone
P + i
P
?
P + i + c + p (X)
P + i
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P
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X + c1 + p1
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Mature progeny
Subclones
2 X + c2 + p2
P = passenger mutations in HSPC i = initiating mutation(s) c = co‐operating mutations in founding clone p = passenger mutations in founding clone X = P + i + c + p c1= cooperating mutations in subclone 1 c2= cooperating mutations in subclone 2 p1 = passenger mutations in subclone 1 p2 = passenger mutations in subclone 2