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Explainermoderate confidence 3 of 5: One strong study, or several smaller consistent ones.Q34-mrd-monitoring

Sequencing-based MRD: better than flow in paediatric B-ALL, and applied to infant KMT2A-rearranged disease exactly once

also called NGS-MRD · HTS-MRD · immunosequencing MRD · clonoSEQ · IGH/TRG sequencing MRD

Instead of designing one test per patient, this method sequences all the antibody-gene rearrangements in a marrow sample at once and counts how many belong to the leukaemia. It finds more leftover disease than flow cytometry does, and in the one infant case where it has been used it showed the leukaemia's clones surviving all the way through a change of cell type.

The same thing, for a scientist

High-throughput sequencing of IGH and TRG rearrangements replaces patient-specific ASO-PCR with amplicon sequencing of the whole repertoire, identifying the index clone from the diagnostic sample and counting it thereafter. In 619 paired pretreatment and end-of-induction marrows from Children's Oncology Group AALL0331 and AALL0232, HTS identified 55 (38.7%) more patients as MRD-positive at 0.01% than flow, those discrepant patients did worse than flow-negative patients, and the increased analytic sensitivity identified 19.9% of standard-risk patients with no detectable MRD at any level whose 5-year EFS was 98.1% and OS 100% [E1]. **In infant KMT2A-rearranged ALL it has been reported once.** A PubMed search combining infant terms, KMT2A or MLL, residual disease terms and every sequencing term returns **2 records**, of which one is a partner-gene discovery paper and the other is a single case report [E2][E3]. That case is nonetheless informative: clonoSEQ IGH/IGL sequencing in an infant with KMT2A::EPS15 B-ALL found three dominant sequences present in the original B-ALL, in the blinatumomab-induced lineage-switched AML **and** in the post-transplant relapsed B-ALL, with four additional sequences at relapse of which three had been present at low abundance at diagnosis [E3]. That is simultaneously a demonstration that a sequencing-based Ig readout survives a lineage switch that flow did not, and a demonstration of exactly the oligoclonality and subclonal selection that make a single-target ASO assay unsafe here.

Go deeper
The bounded absence, with the queries

Searched 2026-09-20. Multi-word phrases quoted and bounded with [tiab] throughout, and querytranslation read on every result.

queryrecordswhat they are
(infant[tiab] OR infants[tiab]) AND (KMT2A[tiab] OR MLL[tiab]) AND ("minimal residual disease"[tiab] OR "measurable residual disease"[tiab]) AND ("next-generation sequencing"[tiab] OR "high-throughput sequencing"[tiab] OR "clonoSEQ"[tiab] OR "immunosequencing"[tiab])2one partner-discovery paper 2, one diagnostic assay paper
(infant[tiab] OR infants[tiab]) AND leukemia[tiab] AND "clonoSEQ"[tiab]1the lineage-switch case report 3
infant[tiab] AND ("clonoSEQ"[tiab] OR "next-generation sequencing"[tiab] OR "high-throughput sequencing"[tiab]) AND "residual disease"[tiab]2a 2025 review naming NGS-MRD as a future direction, and 2

Bounded conclusion: no prospective or retrospective cohort study of sequencing-based MRD in infant KMT2A-rearranged ALL is indexed in PubMed. The absence is bounded by these symbol and phrase sets, includes the brand name clonoSEQ and the retired symbol MLL, and does not cover conference abstracts, which are not indexed. It is an absence of published cohorts, not proof that nobody has run the assay.

Bounded rejection. Three record classes were read and excluded: the KMT2A partner-gene discovery literature using targeted NGS on DNA and RNA, which uses sequencing to find the rearrangement and then tracks MRD by other means 2; the general paediatric B-ALL HTS-MRD literature, which is on-beat as a performance benchmark 1 but contains no KMT2A-rearranged or infant subgroup analysis; and the adult IGH repertoire literature, which is cited in concept-proximal-vdj-bias-and-ig-mrd-in-kmt2a-aff1 for mechanism rather than for MRD performance.

What the single infant case shows

A 6-month-old girl with t(1;11)(p32;q23)/KMT2A::EPS15 B-ALL, refractory to ALL-directed chemotherapy, lineage-switched to AML after blinatumomab; myeloid-directed chemotherapy cleared the AML by flow while a residual CD19-positive B-ALL population persisted at 0.14%; after bridging blinatumomab and transplant she relapsed with CD19-positive B-ALL one month post-transplant. clonoSEQ IGH/IGL analysis found three dominant sequences present at all three disease states, and four extra sequences at relapse, three of which had been present at low abundance in the diagnostic sample 3.

Three distinct lessons, each of which generalises further than a case report normally would because each is about the assay rather than the patient:

  1. A sequence-based Ig readout is lineage-independent in practice. The same IGH/IGL clones were found in the AML. Flow could not follow the clone; the sequence could. This is a genuine advantage over flow that the phenotype-based assay cannot acquire.
  2. Low-abundance diagnostic subclones become relapse clones. Three of the four new relapse sequences were already there at diagnosis below the level at which a single-target assay would have been built. An ASO-PCR designed against the dominant clone would have been designed against a clone that was not the one that came back.
  3. The oligoclonality that repertoire sequencing reveals is the oligoclonality that ASO-PCR silently assumes away. This is the clinical instance of the pattern concept-mrd-monitoring-ig-tcr-rearrangement records from the diagnostic side.
Why it has not been adopted here, and what it would take

Three obstacles, only one of which is technical.

The target problem is the same one. HTS-MRD reads Ig/TCR rearrangements. Everything that makes Ig/TCR a poor target in infant KMT2A-rearranged disease - absent, oligoclonal, unstable, proximally biased V usage with short CDR3 - applies to the sequencing version of it. Sequencing mitigates oligoclonality, because it tracks every clone rather than one, and mitigates instability partially, because clonally related sequences sharing a DNJ-stem can be recognised as one family 4. It does not create a target where none exists.

The comparator is unusually strong. In most of paediatric ALL, HTS-MRD competes against flow, and wins 1. In infant KMT2A-rearranged ALL the incumbent is a genomic fusion-junction assay that is fusion-specific by construction and has no physiological background. The case for switching is therefore much weaker here than elsewhere, which is a coherent reason for the literature gap rather than an oversight.

Sample volume. An infant marrow is small and is already being divided between morphology, flow, cytogenetics, FISH, breakpoint characterisation and biobanking.

The experiment that would settle it is cheap: run HTS-MRD retrospectively on the stored Interfant-06 marrows that already have paired KMT2A-breakpoint and Ig/TR results, and ask whether HTS recovers the 19 patients the Ig/TR assay missed 5. The samples, the comparator and the outcomes all already exist.

Facts and figures
assay classamplicon high-throughput sequencing of IGH/IGK/IGL/TRG/TRB repertoire
analytegenomic DNA
targetindex clone(s) identified from the diagnostic repertoire
cheapest decisive studyrun HTS-MRD retrospectively on stored Interfant-06 marrows with paired KMT2A-breakpoint and IG/TR results and ask whether it recovers the 19 IG/TR-negative, KMT2A-positive patients
paediatric b all benchmark
{
  "study": "COG AALL0331 + AALL0232",
  "paired_samples": 619,
  "extra_mrd_positive_patients": 55,
  "extra_pct": 38.7,
  "threshold": "0.01%",
  "source_pmid": "29284596"
}
bounded absence
{
  "searched": "2026-09-20",
  "queries": [
    {
      "q": "(infant[tiab] OR infants[tiab]) AND (KMT2A[tiab] OR MLL[tiab]) AND (\"minimal residual disease\"[tiab] OR \"measurable residual disease\"[tiab]) AND (\"next-generation sequencing\"[tiab] OR \"high-throughput sequencing\"[tiab] OR \"clonoSEQ\"[tiab] OR \"immunosequencing\"[tiab])",
      "records": 2
    },
    {
      "q": "(infant[tiab] OR infants[tiab]) AND leukemia[tiab] AND \"clonoSEQ\"[tiab]",
      "records": 1
    },
    {
      "q": "infant[tiab] AND (\"clonoSEQ\"[tiab] OR \"next-generation sequencing\"[tiab] OR \"high-throughput sequencing\"[tiab]) AND \"residual disease\"[tiab]",
      "records": 2
    }
  ],
  "conclusion": "no cohort study of sequencing-based MRD in infant KMT2A-rearranged ALL is indexed in PubMed; the only infant application is one case report",
  "not_covered": [
    "conference abstracts",
    "unindexed registry or vendor reports"
  ]
}
only infant application
{
  "pmid": "33749383",
  "design": "single case report",
  "finding": "three dominant IGH/IGL sequences shared across diagnostic B-ALL, lineage-switched AML and post-transplant relapsed B-ALL; 3 of 4 new relapse sequences present at low abundance at diagnosis",
  "title": "Lineage Switch in an Infant B-Lymphoblastic Leukemia With t(1;11)(p32;q23); KMT2A/EPS15, Following Blinatumomab Therapy.",
  "authors": "Du J, Chisholm KM, Tsuchiya K, et al.",
  "venue": "Pediatr Dev Pathol",
  "year": 2021,
  "title_provenance": "Title/authors/venue backfilled from PubMed esummary on 2026-09-20 because this citation stored a bare PMID with no title, which made it unscoreable by verify-citations.py. BACKFILLING IS NOT VERIFICATION: it confirms the PMID resolves to a real paper, not that this is the right paper for the claim. That still requires reading."
}
why not adopted
[
  "the Ig/TCR target problem is not solved by sequencing it",
  "the incumbent comparator here is a fusion-specific genomic assay, not flow",
  "infant marrow volume is already divided across many assays"
]
How this could bite us back
  • Sequencing-based MRD reclassifies patients upward: 38.7% more MRD-positive at the same threshold in paediatric B-ALL. Deployed in infants, where intensification means myeloid-style blocks or transplant in a child under one year, a more sensitive assay converts directly into more toxic therapy for patients whose benefit from it has not been shown at that sensitivity.
  • The one infant case shows relapse clones present at diagnosis below the level any single-target assay would have picked. That is an argument for sequencing, but it is also an argument that MRD negativity by any method is a statement about the clone that was measured.
  • Sequencing the Ig repertoire in a disease whose founding cell may be pre-VDJ is a more sophisticated version of the same category error. No amount of sequencing depth creates a rearrangement the cell never made.
  • The proprietary assay is the one with clinical validation. A recommendation to use sequencing-based MRD in infant leukaemia is in practice a recommendation to use one vendor's test, which is a different kind of claim from a recommendation to use qPCR.
Still open
  1. Would sequencing-based Ig MRD recover the 19 Interfant-06 patients whose disease the ASO-PCR Ig/TR assay missed at the cut-off? The samples, the comparator assay and the outcomes all exist; the study has not been done.
  2. Does marker-DNJ-stem tracking, which captures a whole family of clonally related sequences, neutralise the ongoing-recombination problem in KMT2A::AFF1, or does it merely make the instability visible?
  3. In an infant marrow of limited volume, what is the minimum DNA input at which HTS-MRD reaches 10^-5, and is that compatible with the other assays competing for the same aspirate?
  4. Is there any setting in which HTS-MRD would beat the genomic breakpoint assay in this disease, rather than beating the assays the breakpoint assay already beats?
Every source behind this entry (5)
  1. E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    In 619 paired pretreatment and end-of-induction bone marrow samples from Children's Oncology Group AALL0331 and AALL0232, high-throughput sequencing of IGH and TRG versus flow cytometry gave similar 5-year event-free and overall survival at a 0.01% MRD threshold but HTS identified 55 (38.7%) more patients as MRD-positive, those discrepant patients had worse outcomes than flow-negative patients, and HTS identified 19.9% of standard-risk patients with no MRD at any detectable level whose 5-year EFS was 98.1% and OS 100%.

    PMID 29284596DOI
    What this evidence cannot show

    Paediatric B-ALL across two risk-stratified COG trials; AALL0232 enrolled only patients with day-29 flow MRD below 0.1%, so the cohort is selected on the comparator's own result. No infant or KMT2A-rearranged subgroup is reported. The 98.1% EFS in the no-detectable-MRD group is a retrospectively identified subgroup and has not been used prospectively to de-escalate therapy.

    Evidence that argues against this (1)
    • The same study found HTS and flow gave similar 5-year EFS and OS for MRD-positive and MRD-negative patients at the 0.01% threshold, so on the primary prognostic comparison the two methods performed alike and the gain is confined to reclassification at the margin. Wood B et al. 2018
  2. E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    Targeted next-generation sequencing on both DNA and RNA, with cytogenetic and FISH prescreening, characterised three novel KMT2A rearrangements in childhood acute leukaemia (KMT2A-BTK, KMT2A-NUTM2A and KMT2A-PRPF19) and the authors state that these characterisations allow tracing the dynamics of minimal residual disease in the given patients.

    BTK, NUTM2A, and PRPF19 Are Novel KMT2A Partner Genes in Childhood Acute Leukemia. · Zerkalenkova E, Lebedeva S, Borkovskaia A, et al. · Biomedicines 2021
    PMID 34440129DOI
    What this evidence cannot show

    Three patients, and the MRD statement describes what the characterisation enables rather than reporting MRD results. This is sequencing used to establish a target, not sequencing used as the MRD assay, so it does not count as an application of NGS-MRD.

    Evidence that argues against this (1)
    • The same approach at a larger scale is the recombinome network's, where KMT2A-targeted NGS achieves near 100% breakpoint discovery and the resulting MRD assay is a patient-specific qPCR rather than a sequencing readout - so sequencing's established role in this disease is target discovery, not MRD quantitation. Meyer C et al. 2023
  3. E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    In a 6-month-old infant with t(1;11)(p32;q23)/KMT2A-EPS15 B-ALL who lineage-switched to AML after blinatumomab and later relapsed with CD19-positive B-ALL one month after allogeneic transplant, next-generation sequencing of IGH/IGL by clonoSEQ detected three dominant sequences present in the original B-ALL, the lineage-switched AML and the post-transplant relapsed B-ALL, with four additional sequences at relapse of which three had been present at low abundance in the original diagnostic sample; the authors state this is the first report of a lineage-switch case using clonoSEQ to define clonality across all three disease states.

    PMID 33749383DOI
    What this evidence cannot show

    A single case report. It shows what the assay can do in one patient and quantifies nothing. Myeloid-directed chemotherapy cleared the AML by flow while a 0.14% CD19-positive B-ALL population persisted, so flow and sequencing are not being compared under controlled conditions. The clonoSEQ assay is proprietary and the reported sequence counts are not independently reproducible from the paper.

    Evidence that argues against this (1)
    • The persistence of the same IGH/IGL clones through the switch is also consistent with the switched population being a minor non-switched contaminant, and the paper's own observation of a residual 0.14% CD19-positive population at the myeloid phase means B-lineage cells were demonstrably present throughout - so the sequencing result does not prove the myeloid cells themselves carried those rearrangements. Du J et al. 2021
  4. E4strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    High-throughput sequencing with clonally related IGH sequences identified by a shared DNJ-stem, applied to 280 adults with newly diagnosed BCP-ALL, identified IGH clonal evolution at diagnosis in one third of patients and allowed the whole family of clonally related sequences including lowly abundant members to be captured under a marker DNJ-stem.

    PMID 37143651DOI
    What this evidence cannot show

    Adults, and a methodological framework introduced by the authors rather than an independently validated standard. Whether marker-DNJ-stem tracking improves clinical MRD performance has not been tested prospectively against conventional ASO-PCR.

    Evidence that argues against this (1)
    • Capturing the whole clonally related family solves evolution within a lineage of rearrangements but not absence: in infants the underlying issue includes targets that never existed, and no bioinformatic framework recovers a rearrangement the founding cell never made. Agraz-Doblas A et al. 2019
  5. E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    In Interfant-06, 19 patients were MRD-positive above protocol cut-offs by the KMT2A genomic breakpoint target and not by IG/TR targets, of whom 9 relapsed (6-year cumulative incidence of relapse 48.7%), against 12 patients positive by IG/TR and not KMT2A of whom 1 relapsed (CIR 8.3%, P = .02).

    PMID 33405950DOI
    What this evidence cannot show

    Read in full text. Post-hoc subgroup analysis on small numbers with wide intervals; the comparison exists only in patients who had both targets. Whether sequencing-based Ig MRD would recover those 19 patients is a hypothesis, not a result - the study used ASO-PCR.

    Evidence that argues against this (1)
    • Overall agreement between the two targets in the same study was 94% with no significant discrepancy at end of induction, so the 19 patients are a small tail and a sequencing assay would have to recover them specifically rather than improve average agreement. Stutterheim J et al. 2021
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