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Applying molecular measurable residual disease testing in acute myeloid leukaemia

Published:December 08, 2022DOI:https://doi.org/10.1016/j.pathol.2022.11.003

      Summary

      Molecular testing in acute myeloid leukaemia (AML) has continued to dramatically advance in recent years, facilitating the ability to detect residual disease at exponentially lower levels. With the advent of the recently updated ELN consensus recommendations, there is increasing complexity to ordering and interpreting measurable residual disease (MRD) assays in AML. We outline the technology itself in conjunction with the relevant testing timepoints, clinically significant thresholds and potential prognostic and therapeutic significance of MRD testing for the major molecular targets in AML. This practical overview should assist haematologists in incorporating molecular MRD assays routinely into their personalised AML clinical management.

      Keywords

      Introduction

      Acute myeloid leukaemia (AML) is a cancer characterised by clonal expansion of myeloid precursors, resulting in impaired haematopoiesis. The molecular landscape and spectrum of mutations, along with the clonal architecture at single cell resolution, is being rapidly unravelled.
      • Döhner H.
      • Weisdorf D.J.
      • Bloomfield C.D.
      Acute myeloid leukemia.
      • Papaemmanuil E.
      • Gerstung M.
      • Bullinger L.
      • et al.
      Genomic classification and prognosis in acute myeloid leukemia.
      The Cancer Genome Atlas Research Network
      Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.
      Despite initial response to intensive chemotherapy, the relapse risk remains substantial for the majority of patients with AML. Thus, there is a need for accurate and dynamic monitoring of measurable residual disease (MRD), which functions as a reservoir for future relapse.
      The detection of sub-microscopic levels of leukaemia cells following therapy, regardless of methodology, is strongly prognostic for earlier relapse and shorter survival.
      • Short N.J.
      • Zhou S.
      • Fu C.
      • et al.
      Association of measurable residual disease with survival outcomes in patients with acute myeloid leukemia: a systematic review and meta-analysis.
      Testing for MRD is widely accepted as standard of care for patients with AML, with consensus recommendations detailing the approach, technical standards and reporting requirements for MRD assessment recently updated by the European LeukemiaNet (ELN).
      • Heuser M.
      • Freeman S.D.
      • Ossenkoppele G.J.
      • et al.
      2021 update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.
      This paper discusses a process for clinical implementation of MRD monitoring, with emphasis on relevant testing timepoints, clinically significant thresholds, prognostic significance and potential therapeutic intervention, that we hope will assist the haematology community in incorporating molecular MRD testing into the routine care of patients with AML.

      Technical overview

      Haematologists have an expanding armamentarium for monitoring disease burden in patients with AML; each method associated with advantages and disadvantages (Table 1). With morphology limited by a 5% sensitivity threshold, MRD assays are defined by a limit of disease detection of at least 10−3 and offer substantially deeper insight into the dynamic changes associated with response to treatment. Positive MRD test results have significant prognostic ramifications, although as explored below, relapse isn't always inevitable. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is the most common and sensitive molecular MRD assay, utilising RNA transcripts associated with certain AML subtypes (e.g., mutated NPM1 or core binding factor fusions) to achieve high sensitivity, reproducibility and to facilitate detection of fusion genes without requiring the location of intronic breakpoints. A distinct advantage of RT-qPCR is that results are often expressed relative to a housekeeping gene, commonly ABL1, to account for variability in RNA quality and PCR amplification efficiency.
      Table 1Genetic testing platforms in AML
      Method overviewLimit of detectionMRD assayDNA/RNA/cellAdvantagesDisadvantages
      KaryotypingCulture of leukaemic blasts followed by microscopic assessment of banded metaphase chromosomes to detect aneuploidies and structural rearrangements at a resolution of ≈5Mb.≈2.5–5%NoCell
      • Relatively cheap and widely available
      • Provides genome wide assessment, including clonal heterogeneity
      • 50% of AML patients present with normal karyotype
      • Requires actively dividing cells
      • Requires significant time, labour and technical expertise
      • Least sensitive technique
      Fluorescent in situ hybridisation (FISH)Hybridisation of fluorescently-labelled specific DNA sequence probes with patient DNA to detect copy number variations and rearrangements. Resolution of 100kb–1Mb.≈1%NoCell (targeted regions)
      • Quick turnaround time
      • Does not require cultured cells
      • Poorer sensitivity than other techniques
      • Requires baseline abnormal karyotype or cryptic rearrangements
      • Requires multiple probes at diagnosis to comprehensively assess disease
      Multiparameter flow cytometry (MFC)Fluorochrome-conjugated antibodies to identify leukaemia associated (LAIP) or difference from normal (DfN) patterns of protein expression on/within leukaemic blasts10−3–10−4 (greater sensitivity can be achieved with a method that targets leukaemic stem cells)
      • Li S.Q.
      • Xu L.P.
      • Wang Y.
      • et al.
      An LSC-based MRD assay to complement the traditional MFC method for prediction of AML relapse: a prospective study.
      YesCell
      • Sensitive
      • Quick turnaround time
      • Widely applicable
      • Increasingly available
      • High level of technical expertise required
      • Not fully standardised
      • Impacted by immunophenotypic shifts if employing LAIP approach
      Quantitative reverse transcription polymerase chain reaction (RT-qPCR)PCR amplification with mutation-specific primer/probe mix to detect mutated gene transcripts quantified in real time relative to a housekeeping gene10–5YesRNA (cDNA)
      • Widely available
      • Well standardised
      • Very sensitive
      • Applicable to 40–50% patients with appropriate molecular targets (less as patient ages)
      • Limited to single gene per assay
      • Limited to specific mutation types with commercially available primer/probe sets (unless design and validate personalised assays)
      • Sample should reach lab within 48–72 hours to minimise RNA degradation
      Droplet digital polymerase chain reaction (ddPCR)Random partitioning of sample into thousands of droplets then PCR amplification with both wild type and mutation-specific probes followed by fluorescent end-point absolute quantification.10–4YesDNA (less commonly RNA)
      • Sensitive
      • Quick turnaround
      • Less technical expertise required
      • Only applicable to 40–50% patients with appropriate molecular targets
      • Usually single gene per assay
      • Limited to specific mutation types with commercially available primer/probe sets
      Massively parallel sequencing (MPS)Standard workflow is library preparation (DNA fragmentation, adaptor ligation, amplicon or capture enrichment of targeted genes, PCR amplification) then massively parallel sequencingCommon platforms 1–2% (lower with improvements such as unique molecular identifiers to correct for intrinsic PCR error rate)Not commonlyDNA (less commonly RNA)
      • Increasingly available
      • Multiple genes assessed simultaneously
      • Low sensitivity (in absence of technological and bioinformatic enhancement)
      • Expensive
      • Not standardised
      • May have slow turnaround
      ChimerismQuantifies short tandem repeats (STR) or indel polymorphisms that differ between donor and recipient0.1–1.0% depending on platform employedNoDNA
      • Available and applicable to all transplant patients
      • Recipient-derived genetic material doesn't equate with persistence of leukaemia cells
      • STR method is labour intensive
      Molecular MRD testing requires knowledge of the gene or fusion genes involved as well as details of the specific mutation/breakpoints. This ensures the correct primer/probe system is employed. Performing the incorrect assay may lead to a false negative result with significant clinical impact. In RT-qPCR, each patient sample is run in triplicate along with positive (target mutation present), negative (target mutation absent) and no template (no cDNA) controls to minimise the chance of false positive and negative results. As RNA-based results with RT-qPCR represent gene expression, they are not interchangeable with DNA-based molecular assays [such as droplet digital PCR (ddPCR) and massively parallel sequencing (MPS) platforms]. Molecular testing also differs from the phenotypic aberrations underlying multiparameter flow cytometry (MFC-MRD) results. MFC-MRD requires analysis of >500,000 CD45+ cells and comprises two major strategies: leukaemia-associated immunophenotype (LAIP) which defines a unique diagnostic phenotype for ongoing monitoring, and different from normal (DfN) which searches for aberrant immunophenotypes outside the spectrum of normal recovering bone marrow.
      Molecular MRD results may be expressed as absolute copy number, logarithmic changes or relative to the patient's pre-treatment diagnostic result. Serial monitoring should ideally be performed from the same tissue source employing the same assay methodology. A negative molecular result should be interpreted within the context of the sample quality (i.e., haemodilute aspirate, delayed transportation for RNA based assays, marrow hypocellularity), homogeneity of leukaemia distribution and sensitivity of the assay (Table 1).

      Major MOLECULAR targets

      NPM1

      Nucleophosmin (NPM1) is a nucleolar shuttling protein involved in cell division, ribosome biogenesis and DNA repair. It is the most commonly mutated gene in AML, usually characterised by a 4 bp duplication or insertion resulting in a frameshift impacting key tryptophan residues at positions 288 and 290 leading to loss of a nucleolar import signal and cytoplasmic delocalisation.
      • Falini B.
      • Brunetti L.
      • Sportoletti P.
      • et al.
      NPM1-mutated acute myeloid leukemia: from bench to bedside.
      Screening tests include melt curve or fragment analysis. This is followed by Sanger sequencing or MPS to identify the precise mutation type. Mutation Type A (duplication TCTG) is the most common variant, found in 75–80% of cases, followed by mutations B and D, each identified with 5% frequency.
      • Falini B.
      • Brunetti L.
      • Sportoletti P.
      • et al.
      NPM1-mutated acute myeloid leukemia: from bench to bedside.
      With more than 100 NPM1 variants documented, identifying the mutation type is crucial to subsequent monitoring.
      In the recently updated 2022 ELN AML recommendations, mutated NPM1 conveys a favourable prognosis in the absence of FLT3 internal tandem duplications (ITD). All other FLT3-ITD and NPM1 variant combinations are considered intermediate risk.
      • Döhner H.
      • Wei A.H.
      • Appelbaum F.R.
      • et al.
      Diagnosis and management of AML in adults: 2022 ELN recommendations from an international expert panel.
      Adverse cytogenetic features, such as monosomy 7 or deletion 17p override the favourable impact of NPM1
      • Angenendt L.
      • Röllig C.
      • Montesinos P.
      • et al.
      Chromosomal abnormalities and prognosis in NPM1-mutated acute myeloid leukemia: a pooled analysis of individual patient data from nine international cohorts.
      and co-occurrence of NPM1, FLT3-ITD and DNMT3A mutations also identifies a poor prognosis subset.
      • Papaemmanuil E.
      • Gerstung M.
      • Bullinger L.
      • et al.
      Genomic classification and prognosis in acute myeloid leukemia.
      NPM1 mutations are well suited for MRD monitoring, as they are almost never associated with pre-leukaemic clonal haematopoiesis. They are also a stable marker of disease following intensive chemotherapy, with 91–99% of patients relapsing with the same NPM1 variant.
      • Cocciardi S.
      • Dolnik A.
      • Kapp-Schwoerer S.
      • et al.
      Clonal evolution patterns in acute myeloid leukemia with NPM1 mutation.
      ,
      • Ivey A.
      • Hills R.K.
      • Simpson M.A.
      • et al.
      Assessment of minimal residual disease in standard-risk AML.
      For rare variants, an alternative to RT-qPCR is MPS for MRD monitoring.
      • Blombery P.
      • Jones K.
      • Doig K.
      • et al.
      Sensitive NPM1 mutation quantitation in acute myeloid leukemia using ultradeep next-generation sequencing in the diagnostic laboratory.
      ddPCR is also likely to be applicable in the near future.
      The most widely accepted landmark for assessing NPM1 MRD, derived from the NCRI AML17 trial, is after two cycles of intensive chemotherapy (Table 2). Patients that were MRD negative in peripheral blood (PB) had a 3 year survival of 75% vs 24% if MRD was positive and 3 year relapse risk of 30% vs 82%.
      • Ivey A.
      • Hills R.K.
      • Simpson M.A.
      • et al.
      Assessment of minimal residual disease in standard-risk AML.
      The majority of patients in this trial had received two cycles of cytarabine with daunorubicin. Whether MRD positivity at this timepoint should prompt treatment modification to alternative induction and consolidation regimens requires further clinical investigation.
      Table 2Clinically applicable molecular MRD testing in AML
      TargetPrognostically significant timepointsPrognostically significant thresholds
      NPM1After 2 cycles of intensive therapy+ vs – (PB)
      Completion of therapy<3–4 log10 reduction relative to level at diagnosis (BM) or >1–2% relative to ABL1 (BM)
      q3 months (BM) or q4–6 weeks (PB) for 2 years post-therapy>1–2% relative to ABL1 (BM)
      q3 months post-transplant>1% relative to ABL1 (especially if >10%) (BM) or ≥0.02% VAF by DNA deep sequencing (BM)
      At any timeConversion from – to + (confirmed on a repeat sample) or ≥1 log10 rise
      CBFB::MYH11After 2 cycles of intensive therapy
      Of note, the standard induction cycle in this CBF-2006 trial included two extra doses of daunorubicin 35mg/m2 and six extra doses of cytarabine 1000mg/m2.26AML, acute myeloid leukaemia; BM, bone marrow; MFC, multiparameter flow cytometry; MRD, measurable residual disease; PB, peripheral blood; VAF, variant allele frequency.
      <3 log10 reduction relative to level at diagnosis (BM)
      Completion of therapy and q3 months (BM) or q4–6 weeks (PB) for 2 years post-therapy>50 copies/105 ABL1 (0.05%; BM) or >10 copies/105 ABL1 (0.01%; PB)
      3 months post-transplant<3 log10 reduction relative to level at diagnosis (BM)
      At any timeConversion from – to + (confirmed on a repeat sample) or ≥1 log10 rise
      RUNX1::RUNX1T1After 2 cycles of intensive therapy
      Of note, the standard induction cycle in this CBF-2006 trial included two extra doses of daunorubicin 35mg/m2 and six extra doses of cytarabine 1000mg/m2.26AML, acute myeloid leukaemia; BM, bone marrow; MFC, multiparameter flow cytometry; MRD, measurable residual disease; PB, peripheral blood; VAF, variant allele frequency.
      <3 log10 reduction relative to level at diagnosis (BM)
      Completion of therapy and q3 months (BM) or q4–6 weeks (PB) for 2 years post-therapy>500 copies/105 ABL1 (0.5%; BM) or >100 copies/105 ABL1 (0.1%; PB)
      3 months post-transplant<3 log10 reduction relative to level at diagnosis (BM)
      At any timeConversion from – to + (confirmed on a repeat sample) or ≥1 log10 rise
      KMT2A::XAt any timeConversion from – to + (confirmed on a repeat sample)
      FLT3-ITDPre-transplant>0.001% VAF by DNA deep sequencing (BM or PB)
      CEBPANot established; use alternative marker or MFC-MRD
      IDH1/2Not definitively established; use alternative marker or MFC-MRD
      DNMT3A, TET2, ASXL1No role in MRD testing; use alternative marker or MFC-MRD
      a Of note, the standard induction cycle in this CBF-2006 trial included two extra doses of daunorubicin 35mg/m2 and six extra doses of cytarabine 1000mg/m2.
      • Jourdan E.
      • Boissel N.
      • Chevret S.
      • et al.
      Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia.
      AML, acute myeloid leukaemia; BM, bone marrow; MFC, multiparameter flow cytometry; MRD, measurable residual disease; PB, peripheral blood; VAF, variant allele frequency.
      NPM1 mutations should additionally be measured in bone marrow (BM) at the end of consolidation and then every 3 months for 2 years post-consolidation. MRD can alternatively be performed on peripheral blood every 4–6 weeks during follow-up,
      • Heuser M.
      • Freeman S.D.
      • Ossenkoppele G.J.
      • et al.
      2021 update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.
      this need for increased frequency reflecting the 1-log10 reduction in sensitivity in PB compared with BM.
      • Ivey A.
      • Hills R.K.
      • Simpson M.A.
      • et al.
      Assessment of minimal residual disease in standard-risk AML.
      Approximately 25% of patients remain MRD positive at the end of chemotherapy,
      • Ivey A.
      • Hills R.K.
      • Simpson M.A.
      • et al.
      Assessment of minimal residual disease in standard-risk AML.
      of whom nearly half may remain relapse-free at 12 months.
      • Tiong I.S.
      • Dillon R.
      • Ivey A.
      • et al.
      Clinical impact of NPM1-mutant molecular persistence after chemotherapy for acute myeloid leukemia.
      Patients may manifest low level residual disease (MRD-LL) with <1–2% NPM1 mutant transcripts relative to ABL1, which is associated with low risk of relapse. Levels exceeding 1–2% in BM at end of treatment or during follow-up are generally considered an indicator of treatment failure requiring therapeutic response.
      • Krönke J.
      • Schlenk R.F.
      • Jensen K.O.
      • et al.
      Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group.
      ,
      • Shayegi N.
      • Kramer M.
      • Bornhäuser M.
      • et al.
      The level of residual disease based on mutant NPM1 is an independent prognostic factor for relapse and survival in AML.
      Patients failing to achieve at least a 4.4-log10 reduction in NPM1 mutant transcripts at end of treatment also have a significantly increased risk of relapse.
      • Tiong I.S.
      • Dillon R.
      • Ivey A.
      • et al.
      Clinical impact of NPM1-mutant molecular persistence after chemotherapy for acute myeloid leukemia.
      CR with molecular relapse is defined by the ELN as either conversion from MRD negative to positive status (confirmed on a repeat sample within 4 weeks) or a 1-log10 increase between positive samples. Molecular relapse is frequently followed by haematological relapse within 3–4 months in the absence of intervening therapy.
      • Heuser M.
      • Freeman S.D.
      • Ossenkoppele G.J.
      • et al.
      2021 update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.
      In patients who are destined to fail initial therapy, accurate quantitation of MRD provides the opportunity to intervene before overt relapse has occurred. It is important to perform log calculations only between samples from the same tissue source.
      The optimal management of molecular relapse remains uncertain. Approaches include proceeding directly to allogeneic stem cell transplant, with good outcomes demonstrated when transcript levels are <1% in BM and <0.2% in PB among patients known to be negative for FLT3-ITD at diagnosis,
      • Dillon R.
      • Hills R.
      • Freeman S.
      • et al.
      Molecular MRD status and outcome after transplantation in NPM1-mutated AML.
      salvage chemotherapy, venetoclax-based therapy,
      • Tiong I.S.
      • Dillon R.
      • Ivey A.
      • et al.
      Venetoclax induces rapid elimination of NPM1 mutant measurable residual disease in combination with low-intensity chemotherapy in acute myeloid leukaemia.
      azacitidine
      • Platzbecker U.
      • Middeke J.M.
      • Sockel K.
      • et al.
      Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an open-label, multicentre, phase 2 trial.
      or targeted therapy if concurrent mutations such as FLT3 are present. Pre-emptive therapy has been shown to improve MRD clearance
      • Tiong I.S.
      • Loo S.
      • Abro E.U.
      • et al.
      A prospective phase 2 study of venetoclax and low dose Ara-C (VALDAC) to target rising molecular measurable residual disease and early relapse in acute myeloid leukemia.
      and relapse-free survival (10.6 vs 0.7 months in one study).
      • Tiong I.S.
      • Dillon R.
      • Ivey A.
      • et al.
      Clinical impact of NPM1-mutant molecular persistence after chemotherapy for acute myeloid leukemia.
      Whether survival outcomes are improved by pre-emptive therapy, what that therapy should be and what therapeutic endpoint should be targeted requires future prospective validation. For patients with persistent MRD preceding allogeneic transplantation, myeloablative conditioning may improve post-transplant outcomes, compared to reduced intensity conditioning.
      • Hourigan C.S.
      • Gale R.P.
      • Gormley N.J.
      • et al.
      Measurable residual disease testing in acute myeloid leukaemia.
      Patients should also be monitored for at least 2 years post-transplant. Interventions have been suggested at BM MRD levels >1% and definitely >10% since these patients imminently relapse.
      • Shayegi N.
      • Kramer M.
      • Bornhäuser M.
      • et al.
      The level of residual disease based on mutant NPM1 is an independent prognostic factor for relapse and survival in AML.
      ,
      • Spyridonidis A.
      How I treat measurable (minimal) residual disease in acute leukemia after allogeneic hematopoietic cell transplantation.
      DNA-based deep targeted MPS that demonstrates persistent mutant NPM1 ≥0.02% variant allele frequency beyond 3 months has also been associated with increased post-transplant relapse.
      • Delsing Malmberg E.
      • Johansson Alm S.
      • Nicklasson M.
      • et al.
      Minimal residual disease assessed with deep sequencing of NPM1 mutations predicts relapse after allogeneic stem cell transplant in AML.
      Management of post-transplant molecular relapse has yet to be standardised, with options including weaning of immunosuppression, donor lymphocyte infusions
      • Hofmann S.
      • Götz M.
      • Schneider V.
      • et al.
      Donor lymphocyte infusion induces polyspecific CD8(+) T-cell responses with concurrent molecular remission in acute myeloid leukemia with NPM1 mutation.
      and/or hypomethylating agent therapy.
      • Platzbecker U.
      • Middeke J.M.
      • Sockel K.
      • et al.
      Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an open-label, multicentre, phase 2 trial.
      Venetoclax combinations have also been administered in case reports,
      • Dillon R.
      • Potter N.
      • Freeman S.
      • et al.
      How we use molecular minimal residual disease (MRD) testing in acute myeloid leukaemia (AML).
      with preliminary responses reported. The clinical efficacy of pre-emptive strategies in the post-transplant setting are also being further investigated.

      Core-binding factor leukaemias

      Core-binding factor (CBF) leukaemias are defined by the presence of either inv (16)(p13; q22)/t (16; 16) or t (8; 21)(q22; q22), leading to the formation of CBFB::MYH11 and RUNX1::RUNX1T1 fusion genes, respectively. These fusions result in loss of function of the CBF heterodimeric transcription factor complex necessary for normal haematopoiesis.
      • de Bruijn M.F.T.R.
      • Speck N.A.
      Core-binding factors in hematopoiesis and immune function.
      They are both associated with a favourable prognosis.
      • Döhner H.
      • Wei A.H.
      • Appelbaum F.R.
      • et al.
      Diagnosis and management of AML in adults: 2022 ELN recommendations from an international expert panel.
      MRD assessment is usually performed by RT-qPCR, with the ELN guidelines recommending repeat testing in PB after completion of two cycles of chemotherapy, in the BM at the end of consolidation and in PB every 4–6 weeks for 2 years after treatment completion.
      • Heuser M.
      • Freeman S.D.
      • Ossenkoppele G.J.
      • et al.
      2021 update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.
      It is important to recognise that patients may have low level persistent molecular disease without relapsing (less commonly in CBFB::MYH11), linked to quiescent leukaemic stem cells and/or non-leukaemic cells.
      • Yin J.A.
      • Frost L.
      Monitoring AML1-ETO and CBFbeta-MYH11 transcripts in acute myeloid leukemia.
      In CBFB::MYH11, mutation types A, D and E account for approximately 95% of cases. The risk of relapse among patients who remain MRD positive after induction therapy is ∼40–50%, indicating half of these patients may never relapse.
      • Jourdan E.
      • Boissel N.
      • Chevret S.
      • et al.
      Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia.
      ,
      • Rücker F.G.
      • Agrawal M.
      • Corbacioglu A.
      • et al.
      Measurable residual disease monitoring in acute myeloid leukemia with t(8;21)(q22;q22.1): results from the AML Study Group.
      In contrast, MRD status after intensive chemotherapy cycle 4 and during follow-up is informative, with a clinically relevant threshold of 10 copies/105 ABL1 (=0.01%) in PB and 50 copies/105 ABL1 (=0.05%) in the BM, discriminating 5 year estimated relapse risk of 97% vs 7% and 100% vs 10%, respectively.
      • Yin J.A.
      • O'Brien M.A.
      • Hills R.K.
      • et al.
      Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial.
      In this same MRC AML-15 trial, molecular persistence of RUNX1::RUNX1T1 after cycle 4 and during follow-up was also prognostic if exceeding 100 copies/105 ABL1 (=0.1%) in PB and 500 copies/105 ABL1 (=0.5%) in BM, associated with 5 year estimated relapse risk of 100% vs 7% in both instances. Another study identified that a 3-log reduction at the end of consolidation was prognostic, with relapse-free survival 61.1% vs 33.7%.
      • Puckrin R.
      • Atenafu E.G.
      • Claudio J.O.
      • et al.
      Measurable residual disease monitoring provides insufficient lead-time to prevent morphologic relapse in the majority of patients with core-binding factor acute myeloid leukemia.
      Although interventions at these thresholds may be considered,
      • Dillon R.
      • Potter N.
      • Freeman S.
      • et al.
      How we use molecular minimal residual disease (MRD) testing in acute myeloid leukaemia (AML).
      there is broader agreement that intervention at molecular relapse represents a more biologically rational alternative.
      • Lane S.
      • Saal R.
      • Mollee P.
      • et al.
      A >or=1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse.
      There is no consensus on how to manage molecular relapse in CBF leukaemia. Therapeutic options include salvage chemotherapy with/without gemtuzumab,
      • Hospital M.A.
      • Prebet T.
      • Bertoli S.
      • et al.
      Core-binding factor acute myeloid leukemia in first relapse: a retrospective study from the French AML Intergroup.
      azacitidine (two CBF patients were part of the RELAZA2 trial)
      • Platzbecker U.
      • Middeke J.M.
      • Sockel K.
      • et al.
      Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an open-label, multicentre, phase 2 trial.
      or direct allogeneic transplant. Post-transplant levels should also be monitored as per the ELN follow-up recommendations. In the post-transplant setting, a significant threshold of >1% has been suggested.
      • Spyridonidis A.
      How I treat measurable (minimal) residual disease in acute leukemia after allogeneic hematopoietic cell transplantation.
      Failure to achieve a 3-log10 reduction in transcript levels by 3 months following transplantation is also prognostic.
      • Tang F.F.
      • Xu L.P.
      • Zhang X.H.
      • et al.
      Monitoring of post-transplant CBFB-MYH11 as minimal residual disease, rather than KIT mutations, can predict relapse after allogeneic haematopoietic cell transplantation in adults with inv(16) acute myeloid leukaemia.
      ,
      • Qin Y.Z.
      • Wang Y.
      • Xu L.P.
      • et al.
      The dynamics of RUNX1-RUNX1T1 transcript levels after allogeneic hematopoietic stem cell transplantation predict relapse in patients with t(8;21) acute myeloid leukemia.
      In these instances and/or a 1-log10 rise in transcript expression, tapering of immunosuppression and donor lymphocyte infusion should be considered.
      • Wang Y.
      • Wu D.P.
      • Liu Q.F.
      • et al.
      In adults with t(8;21)AML, posttransplant RUNX1/RUNX1T1-based MRD monitoring, rather than c-KIT mutations, allows further risk stratification.

      KMT2A rearrangements

      KMT2A (previously MLL) rearrangements are also amenable to molecular monitoring although this is limited by the large number of fusion partners (ddPCR MRD assays are available for fusion partners ELL, MLLT1, MLLT3, AFDN, AFF1 and MLLT10). The most common t(9;11)(p21.3;q23.3)/KMT2A::MLLT3 fusion is associated with an intermediate prognosis whilst all other KMT2A rearrangements are deemed adverse risk as per ELN recommendations.
      • Döhner H.
      • Wei A.H.
      • Appelbaum F.R.
      • et al.
      Diagnosis and management of AML in adults: 2022 ELN recommendations from an international expert panel.
      In t (9; 11), MRD negativity after induction by RT-qPCR or double induction by MFC has prognostic relevance.
      • Scholl C.
      • Schlenk R.F.
      • Eiwen K.
      • et al.
      The prognostic value of MLL-AF9 detection in patients with t(9;11)(p22;q23)-positive acute myeloid leukemia.
      ,
      • Freeman S.D.
      • Hills R.K.
      • Virgo P.
      • et al.
      Measurable residual disease at induction redefines partial response in acute myeloid leukemia and stratifies outcomes in patients at standard risk without NPM1 mutations.
      One approach has been to consider avoiding allogeneic transplantation in patients achieving early MRD negativity.
      • Dillon R.
      • Potter N.
      • Freeman S.
      • et al.
      How we use molecular minimal residual disease (MRD) testing in acute myeloid leukaemia (AML).
      Alternatively, the GIMEMA AML1310 trial implemented a protocol in intermediate risk AML [incorporating t (9; 11) patients] of autologous transplant if MRD negative and allogeneic transplant if MRD positive after one cycle of consolidation. This resulted in no difference in 2 year overall survival or disease-free survival, although MFC-MRD rather than RT-qPCR was employed.
      • Venditti A.
      • Piciocchi A.
      • Candoni A.
      • et al.
      GIMEMA AML1310 trial of risk-adapted, MRD-directed therapy for young adults with newly diagnosed acute myeloid leukemia.
      Patients should be monitored carefully post-transplant as any persistence or re-emergence of KMT2A rearrangement on RT-qPCR was associated with near universal relapse.
      • Liu J.
      • Wang Y.
      • Xu L.P.
      • et al.
      Monitoring mixed lineage leukemia expression may help identify patients with mixed lineage leukemia--rearranged acute leukemia who are at high risk of relapse after allogeneic hematopoietic stem cell transplantation.
      One suggested approach is routine BM assessment every 3 months with any confirmed positivity prompting intervention.
      • Spyridonidis A.
      How I treat measurable (minimal) residual disease in acute leukemia after allogeneic hematopoietic cell transplantation.

      FLT3

      FMS-like tyrosine kinase 3 (FLT3) is a transmembrane ligand-activated receptor tyrosine kinase involved in survival of haematopoietic progenitors, proliferation and differentiation. Constitutively activating mutations are present in approximately 30% of newly diagnosed AML and occur as internal tandem duplications (ITDs) of the juxtamembrane and/or first tyrosine kinase domain (25%), mutations in the second tyrosine kinase domain (FLT3-TKD) usually impacting codons 835 and/or 836 (5–7%), and the recently recognised trinucleotide deletions in the juxtamembrane domain predominantly involving codons 572–575.
      • Young D.J.
      • Nguyen B.
      • Zhu R.
      • et al.
      Deletions in FLT-3 juxtamembrane domain define a new class of pathogenic mutations: case report and systematic analysis.
      The prognostic significance of FLT3-TKD mutations at diagnosis is unclear, and their role in MRD assessment is yet to be defined. As per ELN, all FLT3-ITD mutations are now classified as intermediate prognosis in patients without additional adverse mutations or cytogenetics.
      • Döhner H.
      • Wei A.H.
      • Appelbaum F.R.
      • et al.
      Diagnosis and management of AML in adults: 2022 ELN recommendations from an international expert panel.
      Whilst previously stratified into four subgroups by the presence or absence of co-existing NPM1 mutations and by the mutant-to-wild-type allelic ratio,
      • Döhner H.
      • Estey E.
      • Grimwade D.
      • et al.
      Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.
      this has been simplified in the latest ELN classification in the era of FLT3 inhibitors. ITD insertion site is also prognostic, with an adverse impact of insertion into the TKD1 domain.
      • Rücker F.G.
      • Du L.
      • Luck T.J.
      • et al.
      Molecular landscape and prognostic impact of FLT3-ITD insertion site in acute myeloid leukemia: RATIFY study results.
      • Schlenk R.F.
      • Kayser S.
      • Bullinger L.
      • et al.
      Differential impact of allelic ratio and insertion site in FLT3-ITD-positive AML with respect to allogeneic transplantation.
      • Kayser S.
      • Schlenk R.F.
      • Londono M.C.
      • et al.
      Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is associated with resistance to chemotherapy and inferior outcome.
      FLT3-ITD mutations are commonly diagnosed by fragment analysis. Mutant and wild type peak heights or area under the peaks are used for calculating the allelic ratio.
      • Thiede C.
      • Steudel C.
      • Mohr B.
      • et al.
      Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.
      Any molecular FLT3 assay faces the challenges of heterogeneity in ITD size and site, the possibility of multiple co-existing ITD mutations and bioinformatic alignment difficulties. These issues are being addressed and MPS assays with increasing sensitivity have been developed.
      • Levis M.J.
      • Perl A.E.
      • Altman J.K.
      • et al.
      A next-generation sequencing-based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations.
      ,
      • Blätte T.J.
      • Schmalbrock L.K.
      • Skambraks S.
      • et al.
      getITD for FLT3-ITD-based MRD monitoring in AML.
      The use of FLT3 as an MRD marker is currently evolving. Whilst molecular persistence likely represents residual disease, in many cases mutations aren't stable with loss of FLT3-ITD seen in 25% of patients in earlier studies
      • Cloos J.
      • Goemans B.F.
      • Hess C.J.
      • et al.
      Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.
      ,
      • Kottaridis P.D.
      • Gale R.E.
      • Langabeer S.E.
      • et al.
      Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors.
      and up to 46% in the era of FLT3 inhibitors
      • Schmalbrock L.K.
      • Dolnik A.
      • Cocciardi S.
      • et al.
      Clonal evolution of acute myeloid leukemia with FLT3-ITD mutation under treatment with midostaurin.
      (possibly confounded by the lack of sensitivity with conventional testing). Advancement in assays may alter this, as suggested by the observation that FLT3-ITD levels >0.001% detected by deep targeted MPS prior to transplant are associated with significantly increased risk of relapse and inferior survival.
      • Loo S.
      • Dillon R.
      • Ivey A.
      • et al.
      Pre-transplant FLT3-ITD MRD assessed by high-sensitivity PCR-NGS determines post-transplant clinical outcome.
      Until this is confirmed by additional studies, caution should be taken before employing FLT3-ITD as a MRD marker.
      • Heuser M.
      • Freeman S.D.
      • Ossenkoppele G.J.
      • et al.
      2021 update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.

      CEBPA

      The CCAAT/enhancer binding protein alpha (CEBPA) gene encodes a transcription factor critical to regulation of granulopoiesis and is mutated in 10–15% of AML patients. Previously only biallelic CEBPA mutations, usually characterised by an N-terminal frameshift mutation and a C-terminal inframe insertion/deletion on separate alleles, were deemed to portend a favourable prognosis.
      • Döhner H.
      • Estey E.
      • Grimwade D.
      • et al.
      Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.
      Recent studies have demonstrated that prognosis is dependent primarily on the presence of a monoallelic CEBPA inframe mutation in the C-terminal DNA-binding bZIP domain, regardless of the presence or absence of a concomitant mutation in the N-terminal transactivation domain.
      • Taube F.
      • Georgi J.A.
      • Kramer M.
      • et al.
      CEBPA mutations in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome.
      ,
      • Wakita S.
      • Sakaguchi M.
      • Oh I.
      • et al.
      Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia.
      Approximately 5–10% of these patients in fact harbour a germline CEBPA variant which confers predisposition to AML (confirmed through testing non-haematopoietic tissue).
      • Tawana K.
      • Wang J.
      • Renneville A.
      • et al.
      Disease evolution and outcomes in familial AML with germline CEBPA mutations.
      Molecular assays are difficult due to the GC-rich content, repeat regions and heterogeneous mutations spanning the whole intronless gene. Screening assays include fragment analysis or inclusion in MPS diagnostic panels. Whilst MFC-MRD has been employed, to our knowledge no molecular MRD assays in CEBPA have been reported and will face the challenges outlined above.

      IDH1/2

      Isocitrate dehydrogenase (IDH) genes, IDH1 and IDH2, are mutated in approximately 8% and 12% of AML patients, respectively, and their prognostic significance remains uncertain.
      • DiNardo C.D.
      • Ravandi F.
      • Agresta S.
      • et al.
      Characteristics, clinical outcome, and prognostic significance of IDH mutations in AML.
      Mutations predominantly occur at position R132 within IDH1 and R140 or R172 within IDH2, leading to production of the oncometabolite 2-hydroxyglutarate causing hypermethylation, differentiation blockade and self-renewal. They can be diagnosed through mutation-specific PCR, melt curve analysis, ddPCR and/or MPS.
      MRD testing is achievable through ddPCR (at a sensitivity of 0.1%) whereas RT-qPCR is not well established and routine MPS currently lacks necessary sensitivity. Studies have demonstrated IDH mutated cells can persist following intensive chemotherapy, and in that context IDH1/2 mutations (particularly IDH2-R140) are more likely to represent markers of pre-leukaemic clonal haematopoiesis than actual driver mutations.
      • Wiseman D.H.
      • Williams E.L.
      • Wilks D.P.
      • et al.
      Frequent reconstitution of IDH2(R140Q) mutant clonal multilineage hematopoiesis following chemotherapy for acute myeloid leukemia.
      Although persistent IDH1/2 mutations may be associated with increased risk of relapse, the rationale for IDH1/2 MRD testing to justify intervention is tempered by the lack of correlation with timing of relapse and mutational burden.
      • Ok C.Y.
      • Loghavi S.
      • Sui D.
      • et al.
      Persistent IDH1/2 mutations in remission can predict relapse in patients with acute myeloid leukemia.
      Applying MRD testing for IDH1/2 mutations therefore is still developing and an alternative molecular marker or MFC-MRD should be used when available.

      DNMT3A, TET2 and ASXL1

      DNMT3A, TET2 and ASXL1 (known collectively as ‘DTA’) mutations have been shown to persist after chemotherapy with no clinical impact.
      • Jongen-Lavrencic M.
      • Grob T.
      • Hanekamp D.
      • et al.
      Molecular minimal residual disease in acute myeloid leukemia.
      They are commonly eliminated following allogeneic transplant;
      • Ivey A.
      • Hills R.K.
      • Simpson M.A.
      • et al.
      Assessment of minimal residual disease in standard-risk AML.
      however, even persistence post-transplant has not been linked with relapse.
      • Heuser M.
      • Heida B.
      • Büttner K.
      • et al.
      Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations.
      Therefore, DTA mutations should not be considered as MRD markers but rather indicators of pre-leukaemic clonal haematopoiesis.

      Lower intensity regimens

      Whilst MRD has been studied predominantly in younger patients receiving intensive chemotherapy as outlined above, its applicability to lower intensity venetoclax regimens is emerging.
      • Maiti A.
      • DiNardo C.D.
      • Wang S.A.
      • et al.
      Prognostic value of measurable residual disease after venetoclax and decitabine in acute myeloid leukemia.
      Analysis of VIALE-A patients demonstrated improved event-free survival and overall survival in patients attaining MRD negativity assessed through MFC. Nearly half of these patients only achieved this response at the end of cycle 7 or beyond, indicating the benefit of testing is not fully apparent at earlier time points.
      • Pratz K.W.
      • Jonas B.A.
      • Pullarkat V.
      • et al.
      Measurable residual disease response and prognosis in treatment-naïve acute myeloid leukemia with venetoclax and azacitidine.
      The role of molecular MRD testing and the ability of MRD results to influence therapeutic intensity in this less fit cohort requires further investigation.

      Conclusion

      Molecular testing in AML has rapidly advanced over the last decade. MRD testing of established targets utilising appropriate assays at defined landmarks offers prognostic information beyond pre-treatment stratification and may guide early therapeutic intervention. As more recurrent translocations are identified, the number of potential targets for MRD study will likely grow (e.g., DEK::NUP214 and NUP98 fusions). Through ongoing technological advancement, improved standardisation and further prospective studies, the clinical utility of molecular MRD testing is likely to strengthen and expand to include more AML patients. The enhanced prognostication afforded by MRD monitoring will play an increasingly central role in guiding therapeutic decisions in the future, with the ultimate goal of a reduction in the risk of relapse and improved overall survival.

      Conflicts of interest and sources of funding

      AHW has served on advisory boards for Novartis, Astra Zeneca, Astellas, Janssen, Amgen, Roche, Pfizer, AbbVie, Servier, Gilead, BMS, Shoreline, Macrogenics and Agios; receives research funding from Novartis, AbbVie, Servier, Janssen, BMS, Syndax, Astex, Astra Zeneca, Amgen; serves on speaker's bureaus for AbbVie, Novartis, BMS, Servier, Astellas; is an employee of the Walter and Eliza Hall Institute (WEHI). WEHI receives milestone and royalty payments related to the development of venetoclax, and current and past employees of WEHI may be eligible for financial benefits related to these payments; AHW receives such a financial benefit. HJI has served in an advisory capacity for Phebra and Syros. MK declares no conflicts of interest. No funding was received to assist with the preparation of this manuscript.

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