Oncofusion

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Drugmoderate confidence 3 of 5: One strong study, or several smaller consistent ones.Q51-backbone

Methotrexate - 5 g/m2 through kidneys that are still maturing, and the one backbone drug the canonical infant sensitivity study never tested

also called methotrexate · MTX · HD-MTX · amethopterin · methotrexate sodium · intrathecal methotrexate · IT MTX · 4-amino-10-methylfolic acid

Methotrexate blocks the folate chemistry cells need to make DNA. Infants get it in three completely different ways - a huge intravenous dose with a rescue drug, small weekly tablets for two years, and injections into the spinal fluid - and the huge dose leaves the body through kidneys that are only just finishing their development.

The same thing, for a scientist

Methotrexate is a folate antagonist inhibiting dihydrofolate reductase; its polyglutamated metabolites additionally inhibit thymidylate synthase and de-novo purine synthesis. Interfant-06 uses it three ways [E1]: **high-dose, 5,000 mg/m2 over 24 hours on MARMA part I days 1 and 8** with leucovorin rescue 15 mg/m2 at 42, 48 and 54 hours; **oral, 20 mg/m2 weekly** through maintenance to 104 weeks; and **intrathecal, dosed by age band** (6 mg under 1 year, 8 mg from 1 year) at induction days 1 and 29, protocol IB day 24, MARMA days 2 and 9, MAE day 1, OCTADA(D) and maintenance weeks 1 and 15. **There is essentially no KMT2A-specific methotrexate evidence and the absence has a specific cause:** the canonical in-vitro infant ALL drug-resistance profile - the study that produced the >580-fold prednisolone and 1.9-fold cytarabine numbers - **did not test methotrexate at all** [E2]. The one agent-level datum found in KMT2A-rearranged infant cells is negative: romidepsin **antagonised** methotrexate in four of six infant ALL cell lines [E3]. The infant-specific story is renal: HD-MTX is cleared by glomerular filtration, GFR matures steeply across the first year [E4], and the protocol handles this with age-referenced renal thresholds and level-driven rescue rather than with an age-specific dose [E1].

Go deeper
Three drugs in one, by route
routedosewhere
high-dose IV5,000 mg/m2 over 24 hMARMA part I, days 1 and 8
oral20 mg/m2 once weeklymaintenance, to 104 weeks from diagnosis
intrathecal6 mg if age <1 y, 8 mg if >=1 yinduction days 1 and 29 (plus days 8 and 22 if CNS-positive, then weekly until clear), protocol IB day 24, MARMA days 2 and 9, MAE day 1, OCTADA(D), maintenance weeks 1 and 15

All from the protocol document 1. The systemic doses are multiplied by 2/3 under 6 months and 3/4 from 6 to 12 months; the intrathecal dose is not reduced at all - intrathecal drugs are explicitly exempt and are banded by age instead. So an infant of 4 months receives two-thirds of the calculated systemic methotrexate and the full age-band intrathecal dose.

The HD-MTX supportive-care package is the most prescriptive in the protocol 1: co-trimoxazole stopped 48 hours before and until the level falls below 0.2 uM; pre-hydration with glucose 5%/NaCl 0.45% plus sodium bicarbonate at 125 ml/m2/hour for 6 hours, urine pH held above 7.0 with bicarbonate raised to 75 mmol/L if it is not; levels at T24 and T48 and every 6-24 hours until below 0.2 uM; leucovorin 15 mg/m2 at T42, T48 and T54, continued 6-hourly if the T48 level is above 0.2 uM.

The renal-maturation problem, and how the protocol dodges it

High-dose methotrexate is eliminated almost entirely by the kidney, and glomerular filtration is the single most steeply maturing pharmacokinetic process of the first year of life 4. A 5 g/m2 infusion is the largest single exposure in the whole protocol and is the one whose clearance depends most on the organ that is least mature.

The protocol's answer is not an age-specific dose. It is 1:

  • a renal gate before the dose - if creatinine is above the upper normal limit for age or has risen more than 30% from baseline, measure GFR; if GFR is below the upper normal limit for age, consider a 50% dose reduction; if GFR is below 30 ml/min/1.73m2, omit methotrexate;
  • and level-driven rescue after it, extending leucovorin until the plasma level clears.

This is a genuinely good design: it replaces a guessed age multiplier with a measured renal function and a measured drug level, which is exactly what drug-vincristine shows is missing everywhere else in this protocol. It is worth naming as the counter-example, because the 2/3 and 3/4 multipliers elsewhere have no such instrumentation.

What it does not do is tell anyone what exposure an infant actually achieves. The protocol targets a safe trough, not a therapeutic AUC, so HD-MTX in infants is dosed for toxicity avoidance and monitored for toxicity avoidance, with no efficacy target anywhere in the loop.

The KMT2A evidence, which is an absence with a named cause

The study every other entry in this beat leans on - 44 infants against 425 older children, MTT assay - reports results for prednisolone, L-asparaginase, cytarabine, 2-chlorodeoxyadenosine, vincristine, anthracyclines, thiopurines, epipodophyllotoxins and 4-hydroperoxy-ifosfamide 2. Methotrexate is not in the list. That is the likely reason there is no infant-versus-older-children methotrexate sensitivity number anywhere: the assay that generated all the others did not include it. A short-term MTT in folate-containing medium is a poor instrument for an antifolate, which is the standard methodological reason.

So the honest statement is: this is not a drug that has been tested in KMT2A-rearranged infant ALL and found no different. It is a drug that has not been tested. Those are different, and this graph has a standing rule about confusing them.

The one measurement that does exist points the wrong way for combination design: romidepsin antagonised methotrexate in four of six infant ALL cell lines, alongside antagonising L-asparaginase in four of six, which is why the authors' scheduling proposal names protocol IB and the second half of OCTADAD - the cytarabine and cyclophosphamide phases - and avoids MARMA 3.

Facts and figures
targetDHFR (dihydrofolate reductase), with polyglutamate metabolites additionally inhibiting thymidylate synthase and de-novo purine synthesis. No gene node for DHFR or TYMS exists in this graph as of cycle 28.
modalitysmall molecule, folate antagonist antimetabolite
interfant06 positionHIGH DOSE 5,000 mg/m2 IV over 24 h, MARMA part I days 1 and 8, with leucovorin 15 mg/m2 at T42/T48/T54; ORAL 20 mg/m2 weekly through maintenance to 104 weeks; INTRATHECAL 6 mg (<1 y) or 8 mg (>=1 y) at induction days 1 and 29, protocol IB day 24, MARMA days 2 and 9, MAE day 1, OCTADA(D) and maintenance weeks 1 and 15
infant dose adjustmentsystemic doses x 2/3 under 6 months and x 3/4 from 6 through 12 months; INTRATHECAL doses exempt and banded by age instead
renal gatecreatinine above the age-specific upper normal limit or a >30% rise from baseline triggers a GFR measurement; GFR below the age-specific upper normal limit -> consider 50% dose reduction; GFR below 30 ml/min/1.73m2 -> omit methotrexate
kmt2a specific evidenceESSENTIALLY NONE, AND UNTESTED RATHER THAN NULL. The canonical infant in-vitro profile study did not include methotrexate among the nine drug classes it assayed. The only agent-level measurement in KMT2A-rearranged infant cells is a negative combination result (romidepsin antagonism in 4 of 6 lines).
interacting supportive careco-trimoxazole must be stopped 48 hours before HD-MTX until the plasma level falls below 0.2 uM
key ae
[
  "mucositis",
  "myelosuppression",
  "nephrotoxicity and delayed clearance",
  "hepatotoxicity",
  "acute and subacute neurotoxicity including stroke-like syndrome",
  "leukoencephalopathy (route- and dose-dependent, and additive with intrathecal exposure)",
  "chemical arachnoiditis via the intrathecal route"
]
resistance
[
  "reduced folate carrier (SLC19A1) loss impairing uptake",
  "impaired polyglutamation by folylpolyglutamate synthase",
  "DHFR amplification",
  "no infant- or KMT2A-specific mechanism identified in this beat"
]
trials
[
  "NCT00550992"
]
How this could bite us back
  • **Five grams per square metre is given to a baby whose kidneys are still maturing, and the whole safety system is a measured level plus a rescue drug.** If the level is not taken, or the rescue is late, the outcome is renal failure and mucosal destruction in a patient with no marrow.
  • The protocol instructs that co-trimoxazole - the standard Pneumocystis prophylaxis - be stopped around every high-dose methotrexate course. That is a deliberate, repeated interruption of infection prophylaxis in the population whose commonest treatment-related cause of death is infection.
  • Methotrexate is given systemically and intrathecally in overlapping windows, and the neurotoxicity of the two routes is additive. In an infant that toxicity manifests as a developmental outcome measured years later, which no trial in this graph reports.
  • This is the least-evidenced drug in the beat and it delivers the protocol's single largest exposure. Any proposal to de-escalate the backbone will find no data here in either direction, which makes methotrexate the easiest thing to keep by default and the hardest to defend.
  • The most promising combination partner found for this backbone antagonises methotrexate in two-thirds of the cell lines tested. A combination strategy built on the synergy phases implicitly concedes the phase that carries CNS control.
Still open
  1. What high-dose methotrexate exposure does an infant actually achieve at 5 g/m2 multiplied by 2/3? There is no efficacy target in the protocol, no infant clearance measurement in the literature this beat searched, and the monitoring loop is designed entirely around avoiding toxicity.
  2. Is KMT2A-rearranged infant ALL more or less methotrexate-sensitive than other ALL? Nobody has measured it. The study that measured everything else did not assay this drug, and that is a testable gap rather than a settled null.
  3. Intrathecal methotrexate is dosed by age band and is exempt from the systemic 2/3 reduction, so a 4-month-old receives a full 6 mg into a CSF volume that is a fraction of an older child's. What is the intrathecal exposure per unit CSF volume across the first year, and does it track the leukoencephalopathy risk?
  4. If HDAC inhibitors antagonise methotrexate in four of six infant ALL lines, does that hold in vivo? The scheduling proposal that follows from it would exclude romidepsin from MARMA, which is the phase containing the largest single drug exposure in the protocol.
Every source behind this entry (4)
  1. E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

    Interfant-06 gives methotrexate as high-dose 5,000 mg/m2 IV over 24 hours on MARMA part I days 1 and 8 with leucovorin rescue 15 mg/m2 PO or IV at 42, 48 and 54 hours continued 6-hourly if the T48 level exceeds 0.2 uM; as oral 20 mg/m2 weekly in maintenance to 104 weeks; and intrathecally at 6 mg for age under 1 year or 8 mg from 1 year at multiple timepoints; supportive care requires stopping co-trimoxazole 48 hours before until the level is below 0.2 uM, pre-hydration and alkalinisation to urine pH above 7.0, and levels at T24 and T48; if creatinine exceeds the age-specific upper normal limit or rises more than 30% from baseline the GFR is measured, with a 50% dose reduction considered if GFR is below the age-specific upper normal limit and methotrexate omitted if GFR is below 30 ml/min/1.73m2.

    What this evidence cannot show

    A trial protocol document, not a peer-reviewed publication, and specifically the SKION (Dutch) national version of April 2006; the trial was amended during its course (asparaginase provisions in MARMA and OCTADA(D) among others), so later versions may differ. Doses given are PLANNED doses; delivered cumulative dose per patient is lower wherever a course was omitted, delayed or curtailed by a stopping rule, and the trial report does not publish delivered dose per patient. Retrieved and text-extracted by this beat on 2026-09-20 (90 pages, 194,979 characters); page numbers are the PDF's own. The intrathecal doses quoted are from the protocol IB section; the protocol states intrathecal doses 'according to age as indicated in the schemes' elsewhere and this beat verified the numbers at one phase. The renal gate is worded as advice ('it is advised', 'consider') rather than as a mandatory rule.

    Evidence that argues against this (1)
    • A level-driven rescue protects against toxicity and says nothing about whether the infant achieved an antileukaemic exposure. No efficacy target for methotrexate exposure exists anywhere in this protocol, and the systemic dose is still multiplied by the unjustified 2/3 or 3/4 age factor before any of the monitoring begins. de Kluis T et al. 2025
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    The canonical in-vitro drug-resistance profile of infant ALL - 44 infants against 425 children over 1 year with common/preB ALL - reports results for prednisolone, L-asparaginase, cytarabine, 2-chlorodeoxyadenosine, vincristine, anthracyclines, thiopurines, epipodophyllotoxins and 4-hydroperoxy-ifosfamide, and does not report methotrexate.

    In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype. · Ramakers-van Woerden NL, Beverloo HB, Veerman AJ, Camitta BM, Loonen AH, van Wering ER, et al. · Leukemia 2004
    PMID 14712291DOI
    What this evidence cannot show

    A bounded absence about one study, established by reading its abstract, which enumerates the drug classes tested and their results. It is possible that methotrexate was assayed and not reported; this beat did not obtain the full text. The general methodological reason antifolates are omitted from short-term MTT panels - folate in the culture medium and the need for prolonged exposure - is standard practice rather than a statement from this paper.

    Evidence that argues against this (1)
    • The absence of a methotrexate result in this one study is not the absence of the whole literature. Age-related pharmacogenomic work across 767 children and 309 adults with B-ALL found seven drugs differing in ex-vivo activity between children and adults - asparaginase, prednisolone, mercaptopurine, dasatinib, nelarabine, daunorubicin and inotuzumab - and identified subclusters within KMT2A-rearranged ALL linked to age and cytotoxic drug resistance. Methotrexate is again not among the seven. Yoshimura S et al. 2024
  3. E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    Romidepsin ANTAGONISED methotrexate in four of six infant ALL cell lines and L-asparaginase in four of six, while synergising with daunorubicin and 4-hydroperoxycyclophosphamide in all six, with vincristine in four of six and with dexamethasone in three of six; the authors' resulting proposal is to schedule romidepsin during the cytarabine- and cyclophosphamide-based phases of the Interfant backbone, namely protocol IB and the second half of OCTADAD.

    PMID 30679330DOI
    What this evidence cannot show

    Read from the PMC HTML full text on 2026-09-20 (efetch and Europe PMC both return an empty abstract because this is a Haematologica letter). Six cell lines, Bliss-independence scoring with Chalice software; no primary patient samples, and the in-vivo arm tested only cytarabine. Antagonism in a 24-72 hour cell-line assay is weak evidence about a 24-hour 5 g/m2 infusion with leucovorin rescue.

    Evidence that argues against this (1)
    • Cell-line synergy and antagonism scores are poor predictors of clinical combination effects, and an antagonism result for an antifolate is particularly hard to interpret because HDAC inhibition changes the expression of folate transporters and of thymidylate synthase, effects that would not be captured by a fixed-ratio Bliss analysis at a single timepoint. Cheung LC et al. 2019
  4. E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    The pharmacokinetics of drugs used to treat infant ALL are dominated by rapid first-year developmental change, with renal maturation, body composition, blood composition and the ontogeny of drug-metabolising enzymes and transporters as the key factors, and current dosing guidelines are extrapolated from paediatric or adult standards by body-surface-area or age adjustment rather than derived for infants.

    Effect of developmental changes on pharmacokinetics of drugs used in the treatment of infant acute lymphoblastic leukaemia-A comprehensive review. · de Kluis T, Brigitha LJ, Hanff LM, Huitema ADR, van der Sluis IM, Diekstra MHM · Br J Clin Pharmacol 2025
    PMID 41139514DOI
    What this evidence cannot show

    A narrative review, cited for the framing and for the identification of renal maturation as the dominant process, not for any number. Reviews are weak evidence for specifics. It does not report an infant methotrexate clearance measurement, and this beat found none.

    Evidence that argues against this (1)
    • The same review's logic - that immaturity means altered clearance and therefore dose adjustment - is exactly what failed when tested directly for vincristine, where body-surface-area-normalised clearance in neonates and infants did not differ from older children's and the customary reduction produced significantly lower exposure. Developmental reasoning predicts a direction and has been wrong about it. Barnett S et al. 2022
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