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Experimentmoderate confidence 3 of 5: One strong study, or several smaller consistent ones.Q138-kmt2a-aff1-surfaceome

Treat a KMT2A::AFF1 patient-derived xenograft with an NG2-directed agent and read out the CD34+CD19-CD117+ fraction at nadir - does the reservoir the 2006 flow data predicts actually survive?

also called the NG2 reservoir experiment

Give a mouse carrying a child's leukaemia a drug aimed at NG2, then look at what survives. If the surviving cells are the immature ones that lack NG2, the therapy is a debulking agent that needs a partner drug rather than a cure.

The same thing, for a scientist

A 2006 four-colour flow study found the candidate leukaemic stem-cell populations - CD34+CD38- in 11q23 AML and **CD34+CD19-CD117+ in t(4;11) ALL** - uniformly **NG2-negative** across nine patients, three of them t(4;11) [E1]. A 2018 serial-xenotransplantation study in MLL-AF4 leukaemias found NG2 to be a **malleable marker that does not enrich for leukaemia-initiating cells**, and also that high NG2 associates with worse event-free survival and CNS disease [E2]. The two agree that NG2 does not mark the initiating fraction; they disagree about whether NG2-negativity defines a stable population. Neither has been followed by the obvious experiment: **treat and look.** Reagents exist - chondroitinase-ABC and an anti-NG2 monoclonal were both used in vivo in MLL-rearranged B-ALL PDXs and reduced engraftment [E3].

Go deeper
The measurement that decides it

Sort CD34+CD19-CD117+ cells from a KMT2A::AFF1 PDX at diagnosis and measure surface NG2 by quantitative flow against an isotype control, expressed as antibody-binding capacity rather than percent-positive. Then treat and repeat at nadir and at relapse.

The pre-registered scoring: if >= 30% of the CD34+CD19-CD117+ fraction is NG2-positive above isotype at baseline, the reservoir argument fails and NG2 covers the immature compartment after all. If it is below 30% and that fraction is enriched at nadir relative to pre-treatment, the reservoir argument holds and NG2-directed therapy needs a partner 4.

Facts and figures
designKMT2A::AFF1 patient-derived xenograft, at least 3 independent patient samples. Arms: vehicle; anti-NG2 monoclonal; menin inhibitor; the combination. Sort CD34+CD19-CD117+ and bulk blasts at baseline, nadir and relapse.
readoutSurface NG2 by quantitative flow as antibody-binding capacity against isotype, on the sorted CD34+CD19-CD117+ fraction and on bulk; fraction size as a proportion of human CD45+ cells at each timepoint; time to relapse.
controlsIsotype-matched antibody; a CD19-directed arm as the comparator whose escape behaviour is already characterised in this disease; vehicle.
cost tiermed
timeline9-12 months
prespecified scoring>= 30% of the CD34+CD19-CD117+ fraction NG2-positive above isotype at baseline refutes the reservoir argument. Below 30% AND enrichment of that fraction at nadir relative to baseline supports it.
How this could bite us back
  • A mouse cannot report the pericyte toxicity that this beat's normal-tissue analysis flags, so a clean PDX result would be reassuring about efficacy and say nothing about the safety objection.
  • If NG2 is malleable, the nadir readout may capture a transient state rather than a reservoir, and repeating the measurement at relapse is essential rather than optional.
  • Mobilisation of blasts was the mechanism reported for NG2 blockade. In a PDX that reads as reduced engraftment; in a child it could read as CNS seeding.
Still open
  1. Is CD34+CD19-CD117+ still the right phenotype for the initiating fraction in KMT2A::AFF1 ALL? The definition is from 2006 and the experiment is only as good as it.
  2. Would the anti-NG2 monoclonal used in the 2019 PDX work be available for this, or would a new reagent be needed? The published agent is the obvious starting point.
  3. Should the combination arm use a menin inhibitor or a DOT1L inhibitor? Menin inhibition is the clinically available option and this graph holds the better evidence for it.
Every source behind this entry (4)
  1. E1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    Candidate leukaemic stem-cell populations - CD34+CD38- in AML/abn(11q23) and CD34+CD19-CD117+ in ALL/t(4;11) - lacked NG2 expression in all nine patients examined, while NG2 correlated with CD33 on maturing cells.

    PMID 16643437DOI
    What this evidence cannot show

    Nine patients, three with t(4;11), 2006 four-colour flow. Surface-phenotype definitions of leukaemia-initiating cells have been substantially revised since and CD34+CD38- is no longer regarded as reliable.

    Evidence that argues against this (1)
    • A later serial-xenotransplantation study found NG2 malleable and non-enriching for initiating cells, which means NG2-negativity may not define a stable reservoir either. Leukemia 2018
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    In MLL-rearranged infant B-ALL, high NG2 associates with lower event-free survival, more circulating blasts and more frequent CNS disease, and serial xenotransplantation showed NG2 to be a malleable marker not enriching for leukaemia-initiating or CNS-infiltrating cells.

    PMID 28943635DOI
    What this evidence cannot show

    Abstract read in full. A published correction exists for this article, to an author name spelling only.

    Evidence that argues against this (1)
    • Malleability observed under serial xenograft passage may not reflect stability under therapeutic pressure in a patient. Leukemia 2018
  3. E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    NG2 is crucial for MLL-rearranged B-ALL engraftment after intravenous transplantation in PDX models, and in vivo NG2 blockade with chondroitinase-ABC or an anti-NG2 monoclonal antibody caused significant mobilisation of leukaemic cells.

    PMID 30635633DOI
    What this evidence cannot show

    Immunodeficient mice with murine vasculature and pericytes; on-target human-pericyte toxicity cannot be assessed in this system.

    Evidence that argues against this (1)
    • The reported effect is mobilisation rather than killing, and mobilising blasts into the circulation in a disease defined by high circulating blast counts and CNS infiltration is not obviously beneficial. Leukemia 2018
  4. E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.database

    NG2 is bimodal across KMT2A-rearranged TARGET patients, with one of four RNA-seq-profiled MLL-positive samples at 0.01 RPKM, consistent with a non-uniform antigen.

    What this evidence cannot show

    n = 4, bulk tumour expression that cannot distinguish a uniformly low population from a mixture.

    Evidence that argues against this (1)
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