Oncofusion

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

Cyclophosphamide - where the KMT2A literature reports two different activated oxazaphosphorines and only one of them is this drug's metabolite

also called cyclophosphamide · CPM · CPA · CTX · Cytoxan · Endoxan · cyclophosphamide monohydrate · 4-hydroperoxycyclophosphamide (4HPC - the in vitro activated form) · oxazaphosphorine (class)

Cyclophosphamide is an alkylating drug: it sticks DNA strands together so the cell cannot copy them. It needs the liver to switch it on, so laboratory studies use a pre-activated version - and two different pre-activated cousins appear in this disease's literature, only one of which is really this drug.

The same thing, for a scientist

Cyclophosphamide is an oxazaphosphorine prodrug requiring hepatic CYP450 4-hydroxylation to 4-hydroxycyclophosphamide, which equilibrates with aldophosphamide and decomposes to phosphoramide mustard (the DNA cross-linker) and acrolein (the urothelial toxin). Interfant-06 gives it **1,000 mg/m2/dose on protocol IB days 1 and 29** with mesna 400 mg/m2 before and at 3 and 6 hours plus hydration at 3,000 ml/m2/24 h, and **500 mg/m2/dose on OCTADA(D) part II days 36 and 49** [E1]. **The identifier trap here is specific and has already produced a near-miss in this beat.** Two activated oxazaphosphorines appear in the KMT2A literature and they are different compounds: **4-hydroperoxy-IFOSFAMIDE**, for which t(4;11) cases were **4.4-fold more resistant** than other 11q23 rearrangements (P = 0.006) [E2], and **4-hydroperoxy-CYCLOphosphamide (4HPC)**, which showed romidepsin synergy in **6 of 6** infant ALL cell lines [E3]. Only the second is this drug's activated form. Ifosfamide is a structural isomer with a different metabolic profile and is **not in Interfant-06 at all**. Attributing the 4.4-fold resistance finding to cyclophosphamide would be a rule-63-class error in chemistry: a same-family compound whose wrong answer looks entirely reasonable.

Go deeper
Where it sits, and the supportive care that comes with it
phasecyclophosphamidesupportive care specified
protocol IB, days 1 and 291,000 mg/m2/dose IV over 1 hhydration 3,000 ml/m2 fluid per 24 h for at least 6 h; mesna 400 mg/m2 IV before and at 3 and 6 hours after the start of the infusion; increase fluid and mesna for microscopic haematuria; furosemide 0.5 mg/kg at 6 and 12 h only if needed for diuresis
OCTADA(D) part II, days 36 and 49500 mg/m2/dose IV over 1 hprotocol states haematuria is unlikely at this dose; if it occurs, hyperhydration plus mesna 500 mg/m2 over 24 h after a 150 mg/m2 loading dose

All from the protocol document 1, with the systemic dose multiplied by 2/3 under 6 months and 3/4 from 6 to 12 months. The second cyclophosphamide dose in protocol IB has its own count thresholds before it can be given (WBC > 1 x10^9/L, neutrophils > 0.3, platelets > 50) 1.

A second interlock worth noting. The protocol directs that if an ARA-C block in protocol IB must be interrupted, the 6-mercaptopurine should be interrupted too, and omitted 6-MP doses made up until the planned cumulative 1,680 mg/m2 is reached 1 - the three drugs of protocol IB are coupled by rule, not merely co-administered [see drug-mercaptopurine].

The two oxazaphosphorines, kept apart

4-hydroperoxy-ifosfamide (4-HOO-ifosfamide), in the infant profile study 2. Two results, and they point in opposite directions:

  • Infants versus older children: no difference - 4-HOO-ifosfamide is in the list of drugs for which no infant/non-infant difference was found, alongside vincristine, anthracyclines, thiopurines and epipodophyllotoxins.
  • Within 11q23: t(4;11)-positive cases were 4.4-fold more resistant than other 11q23 abnormalities (P = 0.006) - one of only two within-11q23 differences that study reports, the other being prednisolone at >7.4-fold.

4-hydroperoxycyclophosphamide (4HPC), in the romidepsin panel 3. Synergy with romidepsin in all six infant ALL cell lines, tied with daunorubicin for the best score, and a significantly enhanced double-strand-break response at the protein level. This is the compound that is cyclophosphamide's activated form.

Why they must not be merged. Ifosfamide and cyclophosphamide are structural isomers with different ratios of activation to the toxic side-products - ifosfamide generates far more chloroacetaldehyde, which is why it, and not cyclophosphamide, causes encephalopathy and Fanconi-type tubulopathy. They are not dose-equivalent, they are not toxicity-equivalent, and ifosfamide is not in this protocol. The 4.4-fold t(4;11) finding is a real and interesting result about the oxazaphosphorine class in the index fusion of this disease, and it is not a cyclophosphamide measurement. This entry records it as class-level, explicitly.

Facts and figures
targetDNA interstrand cross-links formed by phosphoramide mustard after hepatic CYP450 4-hydroxylation. No node exists in this graph for any CYP or for a DNA cross-link repair pathway as a drug target as of cycle 28.
modalitysmall molecule, oxazaphosphorine alkylating PRODRUG requiring hepatic CYP2B6/CYP3A4 activation
in vitro surrogate4-hydroperoxycyclophosphamide (4HPC), a chemically activated form used in culture to bypass hepatic activation
interfant06 positionprotocol IB days 1 and 29 at 1,000 mg/m2/dose; OCTADA(D) part II days 36 and 49 at 500 mg/m2/dose; also part of the busulfan/cyclophosphamide/melphalan conditioning regimen for high-risk patients transplanted in first remission
infant dose adjustmentx 2/3 under 6 months, x 3/4 from 6 through 12 months
identifier trapTWO activated oxazaphosphorines appear in the KMT2A literature and only one is this drug's. 4-hydroperoxy-IFOSFAMIDE: t(4;11) 4.4-fold MORE resistant than other 11q23 (PMID 14712291) - ifosfamide is a structural isomer NOT used in Interfant-06. 4-hydroperoxyCYCLOphosphamide (4HPC): romidepsin synergy 6/6 in infant ALL cell lines (PMID 30679330) - this one IS cyclophosphamide's activated form. Merging them would produce a confident wrong answer that looks entirely reasonable.
kmt2a specific evidenceCLASS-LEVEL AND SPLIT. The one within-11q23 oxazaphosphorine difference (t(4;11) 4.4-fold more resistant) is an IFOSFAMIDE measurement. The one cyclophosphamide-specific measurement is a combination result (6/6 romidepsin synergy). No cyclophosphamide sensitivity comparison between infants and older children exists - the profile study used the ifosfamide analogue.
active metabolites
[
  "4-hydroxycyclophosphamide / aldophosphamide (equilibrium pair)",
  "phosphoramide mustard (the DNA cross-linker)",
  "acrolein (urothelial toxin, the reason for mesna)"
]
key ae
[
  "myelosuppression",
  "haemorrhagic cystitis from acrolein (mesna-preventable)",
  "SIADH and hyponatraemia",
  "gonadal toxicity and infertility, dose-dependent and permanent",
  "secondary malignancy (alkylator class)",
  "cardiotoxicity at conditioning doses"
]
resistance
[
  "aldehyde dehydrogenase (ALDH1A1) detoxification of aldophosphamide - the canonical class mechanism and the basis of ALDH-high haematopoietic stem cell sparing",
  "glutathione S-transferase conjugation",
  "enhanced interstrand cross-link repair",
  "no infant- or KMT2A-specific mechanism identified in this beat; the t(4;11) 4.4-fold result is for ifosfamide"
]
trials
[
  "NCT00550992"
]
How this could bite us back
  • **Cyclophosphamide is permanently gonadotoxic and it is given to infants whose reproductive future is a real endpoint and appears in no trial report here.** High-risk patients receive it twice more in transplant conditioning; nobody in this graph has computed the total alkylator exposure of a child treated on this protocol.
  • The most specific oxazaphosphorine finding in KMT2A-rearranged disease - t(4;11) blasts 4.4-fold more resistant - is about ifosfamide, which is not in this protocol. Reading it as a cyclophosphamide result would be a confident wrong answer that survives every check short of reading the methods, and the compounds differ by one nitrogen mustard arm.
  • The best combination partner found for this drug works by amplifying DNA damage in a disease whose defining lesion is a DNA break at a specific locus. The second-malignancy consequences of adding an HDAC inhibitor to an alkylator in a child with a KMT2A rearrangement have not been assessed.
  • Cyclophosphamide is a prodrug activated by CYP enzymes whose activity in the first year of life is developmentally variable, and the protocol's answer is a fixed dose times 2/3. For every other prodrug in this beat - prednisone, and the asparaginase products - the same structure appears: an activation step nobody measures and a multiplier nobody derived.
  • The three drugs of protocol IB are interlocked by protocol rule: if the cytarabine block is interrupted, mercaptopurine is interrupted too and made up to a cumulative target. Removing or rescheduling cyclophosphamide is therefore not a single-drug decision, and the block's efficacy has never been decomposed.
Still open
  1. Are t(4;11) blasts resistant to activated CYCLOphosphamide as they are to activated ifosfamide? The 4.4-fold finding is the most specific oxazaphosphorine result in this disease and it is for the isomer the protocol does not use. One assay on banked samples would settle it.
  2. How much 4-hydroxycyclophosphamide does an infant form from 1,000 mg/m2? CYP2B6 and CYP3A4 ontogeny across the first year determines it, the dose is then multiplied by 2/3, and no measurement in this population was found.
  3. Is ALDH1A1-mediated detoxification relevant in KMT2A-rearranged infant blasts? High ALDH activity is a defining property of the normal haematopoietic stem cell, and the cell of origin of this disease is a fetal-liver HSC or LMPP.
  4. Cyclophosphamide is given twice as consolidation and again, at far higher dose, in transplant conditioning for the high-risk group. What is an infant's total alkylator exposure by 18 months, and what does it do to fertility in a patient who has not reached puberty?
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 cyclophosphamide 1,000 mg/m2/dose IV over 1 hour on protocol IB days 1 and 29 with hydration of 3,000 ml/m2 fluid per 24 hours for at least 6 hours and mesna 400 mg/m2 IV before and at 3 and 6 hours after the start of the infusion, and 500 mg/m2/dose IV over 1 hour on OCTADA(D) part II days 36 and 49; the second protocol IB dose requires WBC above 1 x10^9/L, neutrophils above 0.3 x10^9/L and platelets above 50 x10^9/L; the protocol states haematuria is unlikely at 500 mg/m2 and specifies mesna 500 mg/m2 over 24 hours after a 150 mg/m2 loading dose if it occurs; the body-surface-area dose is multiplied by 2/3 under 6 months and 3/4 from 6 through 12 months.

    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 two blocks use different mesna regimens (400 mg/m2 x3 in protocol IB, a 150 mg/m2 load plus 500 mg/m2 over 24 h as rescue in OCTADA(D)); this entry reports both as written and does not reconcile them.

    Evidence that argues against this (1)
    • Mesna protects the bladder and not the gonad or the genome. Cyclophosphamide is an alkylating agent with dose-dependent gonadotoxicity and a documented association with secondary malignancy, neither of which the protocol's supportive care addresses and neither of which is reported in the trial's outcomes. Le Deley MC et al. 2003
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    In the MTT assay, 4-hydroperoxy-ifosfamide showed no difference in resistance between 44 infants with ALL and 425 children over 1 year with common/preB ALL, but t(4;11)(q21;q23)-positive cases were 4.4-fold MORE resistant to 4-hydroperoxy-ifosfamide than cases with other 11q23 abnormalities (P = 0.006), alongside being >7.4-fold more resistant to prednisolone (P = 0.033).

    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

    **The compound is 4-hydroperoxy-IFOSFAMIDE, not 4-hydroperoxycyclophosphamide.** Ifosfamide is a structural isomer of cyclophosphamide with a different metabolic profile and is not used in Interfant-06. This entry records the result as a CLASS-LEVEL oxazaphosphorine finding and does not attribute it to cyclophosphamide. The t(4;11) subgroup is a subset of the 34 MLL-rearranged infants in the study, so the comparison is small, and only two of the nine drug classes tested showed a within-11q23 difference, which raises the question of multiplicity.

    Evidence that argues against this (1)
    • The activated form of cyclophosphamide proper, 4-hydroperoxycyclophosphamide, behaved well in KMT2A-rearranged infant ALL cells in a different experiment - synergy with romidepsin in all six lines tested, tied for the best score in that panel. The two oxazaphosphorines therefore have opposite-looking results in this disease, which is itself the reason not to merge them. Cheung LC et al. 2019
  3. E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    Romidepsin showed synergy with 4-hydroperoxycyclophosphamide in all six infant ALL cell lines tested - tied with daunorubicin for the highest score in a panel giving 4 of 6 for vincristine, 3 of 6 for dexamethasone and antagonism in 4 of 6 for methotrexate and L-asparaginase - and significantly enhanced the 4-hydroperoxycyclophosphamide-induced DNA double-strand-break response measured by phospho-H2A.X; the authors' resulting proposal is to schedule romidepsin during the cytarabine- and cyclophosphamide-based phases, 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; the efetch and Europe PMC abstract fields are empty because this is a Haematologica letter. Six cell lines, 2 nM romidepsin with 2 uM 4HPC for 24 hours, Bliss-independence scoring; no primary patient samples and no in-vivo arm for this pair - the in-vivo work used cytarabine. 4HPC is a chemically activated surrogate that bypasses hepatic activation and does not reproduce the metabolite ratios an infant generates.

    Evidence that argues against this (1)
    • A 24-hour cell-line synergy score with a chemically activated prodrug surrogate is several steps from a clinical combination. Hepatic CYP2B6 and CYP3A4 activity, which determine how much 4-hydroxycyclophosphamide an infant actually forms from a 1,000 mg/m2 dose, are developmentally regulated across the first year and are entirely absent from this system. de Kluis T et al. 2025
  4. E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    Cyclophosphamide is not part of the Interfant-06 induction phase and ifosfamide appears nowhere in the protocol: a whitespace-collapsed count over the full 90-page protocol document confirms cyclophosphamide only in protocol IB and OCTADA(D) part II, and the drug classes for which the canonical infant profile study tested an oxazaphosphorine used ifosfamide's activated form rather than cyclophosphamide's.

    What this evidence cannot show

    A bounded absence about one document. Method, 2026-09-20: PDF text extracted with pypdf, lowercased, all whitespace removed before counting because the extractor inserts stray spaces inside words. Positive controls run through the identical query shape on the identical document returned 14 for daunorubicin, 21 for DNR, 9 for vincristine, 8 for mitoxantrone and 8 for leucovorin, so the instrument returns non-zero for terms that are present. This bounds the April 2006 SKION version only.

    Evidence that argues against this (1)
    • Cyclophosphamide IS in Interfant-06 conditioning for transplant, as part of the busulfan/cyclophosphamide/melphalan regimen for high-risk patients in first remission, at doses and in a context entirely different from the 1,000 mg/m2 consolidation dose. A statement about where cyclophosphamide sits in this protocol must name the phase. ClinicalTrials.gov 2007
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