Native E. coli asparaginase - the second-largest in-vitro resistance signal in infant ALL, and a different product from the pegylated one four doses later
also called asparaginase · L-asparaginase · L-ASP · ASP · native E. coli asparaginase · Asparaginase medac · colaspase · crisantaspase (Erwinia - a DIFFERENT enzyme, see caveats) · Erwinase · Erwinia chrysanthemi asparaginase
Asparaginase is a bacterial enzyme, not a chemical, that destroys an amino acid leukaemia cells cannot make for themselves. Infant leukaemia cells are twelve times more resistant to it than older children's - the second-biggest difference of any drug measured.
The same thing, for a scientist
Asparaginase is an enzyme drug that hydrolyses circulating L-asparagine (and, to a lesser extent, glutamine), starving lymphoblasts that lack asparagine synthetase. Interfant-06 induction uses **native E. coli asparaginase (Asparaginase medac) 10,000 U/m2/dose** IV over 1 hour or IM on days 15, 18, 22, 25, 29 and 33 [E1]; the pegylated product is a **different entry** and is used later [see `drug-pegaspargase`]. **The KMT2A/infant evidence is real and is the second-largest effect in the beat:** infant ALL blasts are **12-fold** resistant to L-asparaginase against 425 older children with common/preB ALL (P = 0.001), behind only the >580-fold prednisolone figure and in the same experiment that found them 1.9-fold MORE sensitive to cytarabine [E2]. Independently, asparaginase is one of seven drugs whose ex-vivo activity differs between children and adults with B-ALL, and KMT2A-rearranged ALL contains age-linked subclusters associated with cytotoxic drug resistance [E3]. **And there is a negative combination result:** romidepsin antagonised L-asparaginase in four of six infant ALL cell lines [E4].
Three enzymes, one word - and the protocol switches between them by rule
'Asparaginase' names at least three distinct biological products with different dosing units, schedules, half-lives and immunogenicity: native E. coli asparaginase, PEGylated E. coli asparaginase (pegaspargase), and Erwinia chrysanthemi asparaginase (crisantaspase/Erwinase), which is a different enzyme from a different organism used when the E. coli product is not tolerated. Interfant-06 uses all three, by rule 1:
- Induction: native E. coli, 10,000 U/m2/dose, six doses (days 15, 18, 22, 25, 29, 33).
- MARMA and OCTADA(D): pegaspargase 2,500 U/m2 (Oncaspar). If pegaspargase is unavailable, native E. coli 5,000 U/m2 every 3 days for 9 doses is substituted.
- After severe anaphylaxis: E. coli 10,000 U/m2 is replaced by pegaspargase 2,500 U/m2 every 2 weeks, or Erwinase 20,000 U/m2 three times per week.
- Discontinued for clinically evident pancreatitis confirmed by amylase or ultrasound; withheld for significant haemorrhagic or thrombotic complications pending coagulation work-up.
The protocol also records a substitution fact worth keeping: citing the COALL group, one dose of 45,000 U/m2 native E. coli asparaginase gives asparagine and glutamine depletion of equal duration to one dose of pegaspargase 1. That is an 18-fold ratio in units for equal depletion - which is why a unit count is not an asparaginase exposure, and why 'the protocol gives asparaginase' is not a quantity.
This is the same trap as prednisone versus prednisolone, in a biologic. Two entries, because they are two products.
The 12-fold resistance, and what it probably is
Infant ALL blasts were 12-fold more resistant to L-asparaginase than 425 older children with common/preB ALL (P = 0.001), and proB ALL of all ages showed the same relative resistance 2. The authors' reading is that the proB immunophenotype carries most of it, with the role of MLL not excluded - the same conclusion they reach for the steroid result.
The standard mechanistic account of asparaginase resistance is asparagine synthetase (ASNS) expression, either in the blast or in bone-marrow mesenchymal cells supplying asparagine in trans. This beat found no measurement of ASNS in KMT2A-rearranged infant ALL and does not assert one; the resistance is a measured phenotype with an unmeasured mechanism in this disease, which is the opposite situation to prednisolone, where four mechanisms are reported and the phenotype is what they explain.
The clinical consequence is real. Asparaginase is one of the most important drugs in paediatric ALL, and the disease with the worst outcome is twelve-fold resistant to it in vitro while receiving it on a schedule designed for children who are not.
Toxicity: the infant evidence is an exclusion
The largest analysis of asparaginase-associated toxicity in children - 4,925 patients on COG AALL0232 and AALL0434 receiving a single pegaspargase dose of 2,500 IU/m2 without a maximum - found increased risk of at least one toxicity in patients aged 10 years or older and in those with obesity plus high body surface area, and found no association between the toxicities and end-of-induction MRD positivity 5. Its eligibility floor is age 1, so it says nothing about infants and its age signal runs in the opposite direction from this beat's population.
The practical infant toxicity picture therefore comes from the infant trials themselves, and it is dominated by infection rather than by asparaginase: in the most recent prospective infant ALL study, grade 3-4 infection occurred in 44% of patients during induction and all four treatment-related deaths were infectious, on a backbone that included pegaspargase 2,500 units/m2 6. Asparaginase's own signature toxicities - pancreatitis, thrombosis, hypersensitivity, hepatotoxicity, hypertriglyceridaemia - are in that study's adverse-event profile (hypertriglyceridaemia was the most frequent reinduction event at 23%) but are not separately attributed to it.
| target | circulating L-asparagine (and L-glutamine) - hydrolysed extracellularly, starving lymphoblasts that lack asparagine synthetase. The resistance gene of record for the class is ASNS; no ASNS node exists in this graph as of cycle 28 and no ASNS measurement in KMT2A-rearranged infant ALL was found by this beat. |
|---|---|
| modality | bacterial enzyme (biologic), native Escherichia coli L-asparaginase |
| interfant06 position | remission induction only: 10,000 U/m2/dose IV over 1 hour or IM on days 15, 18, 22, 25, 29 and 33 (six doses). Pegaspargase is used in MARMA and OCTADA(D) - see drug-pegaspargase. |
| interfant06 dose | 10,000 U/m2/dose |
| infant dose adjustment | x 2/3 under 6 months, x 3/4 from 6 through 12 months |
| in vitro resistance infant vs older children fold | 12 |
| kmt2a specific evidence | GENUINE PHENOTYPE, UNMEASURED MECHANISM. 12-fold in-vitro resistance in infant ALL (second-largest effect after prednisolone) and the same relative resistance in proB ALL of all ages; asparaginase is one of seven age-differential drugs in a 1,076-patient ex-vivo panel. No ASNS or mesenchymal-supply measurement in this disease was found. |
substitution rules
[ "pegaspargase unavailable in MARMA/OCTADA(D) -> native E. coli 5,000 U/m2 every 3 days x 9", "severe anaphylaxis -> pegaspargase 2,500 U/m2 every 2 weeks OR Erwinase 20,000 U/m2 three times weekly", "protocol-cited COALL equivalence: one 45,000 U/m2 native dose = one pegaspargase dose for duration of asparagine and glutamine depletion (an 18-fold unit ratio)" ]
key ae
[ "hypersensitivity and anaphylaxis", "pancreatitis", "thrombosis and haemorrhage", "hepatotoxicity and hypoalbuminaemia", "hypertriglyceridaemia", "hyperglycaemia (the protocol notes this is usually steroid-driven in induction)", "silent inactivation by neutralising antibody" ]
resistance
[ "asparagine synthetase expression in the blast (the class mechanism; NOT measured in KMT2A-rearranged infant ALL by any study found here)", "asparagine supplied in trans by bone-marrow mesenchymal cells", "neutralising antibodies causing silent inactivation - the reason activity-level monitoring exists", "the proB immunophenotype, which carries the 12-fold in-vitro resistance at any age" ]
trials
[ "NCT00550992", "NCT00075725", "NCT00408005", "NCT02553460" ]
- **Asparaginase gives an infant a 12-fold-resistant leukaemia the full adult-derived schedule of a drug that causes pancreatitis, thrombosis and anaphylaxis.** The toxicity is delivered whether or not the leukaemia is killed, and the resistance was measured before the protocol was written.
- The obvious response - more asparaginase, or activity-guided intensification - lands in a population whose induction deaths are infectious and whose hepatic protein synthesis this drug already suppresses. The largest toxicity dataset in children excluded infants by its age floor, so the safety case for intensifying would have to be built from scratch.
- 'Asparaginase' in this protocol means three different enzymes with an 18-fold unit ratio between two of them for equal depletion. Any graph claim, dose comparison or exposure calculation that does not name the product is meaningless, and the literature routinely does not.
- Anaphylaxis to the E. coli product forces a switch to Erwinia asparaginase, which has a shorter half-life and a global supply history of shortages. A resistance-driven strategy that raises hypersensitivity rates would increase demand for the one product that has repeatedly been unavailable.
- If the resistance belongs to the proB immunophenotype rather than to the fusion, then KMT2A-germline proB infants - the group with much better outcomes - share it, and the drug is not where the difference between those groups lies. Intensifying it would add toxicity to both and change neither.
- What drives the 12-fold asparaginase resistance of infant and proB ALL blasts? ASNS is the obvious candidate and has not been measured in KMT2A-rearranged infant ALL by any study this beat found. This is a one-experiment question on existing sample banks.
- Should asparaginase be dosed to activity level in infants? Fixed-unit dosing is already being abandoned in older children in favour of trough-activity targeting, and the population with the largest resistance signal is the one still receiving a fixed schedule designed for children who are not resistant.
- Is the resistance a property of the proB phenotype or of the fusion? The cited study cannot separate them, and the answer decides whether an asparaginase strategy should be aimed at KMT2A-rearranged disease or at CD10-negative proB disease of any genotype.
- Does asparaginase contribute to the infection mortality that dominates infant induction? Hypogammaglobulinaemia and suppressed hepatic protein synthesis are plausible routes and no infant study has attributed toxicity by agent.
- E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
Interfant-06 induction gives native E. coli asparaginase (Asparaginase medac) 10,000 U/m2/dose IV over 1 hour or IM on days 15, 18, 22, 25, 29 and 33; if pegaspargase is unavailable in MARMA or OCTADA(D), native E. coli asparaginase 5,000 U/m2 every 3 days for 9 doses is substituted, the protocol noting from COALL data that a single 45,000 U/m2 dose of native E. coli asparaginase gives asparagine and glutamine depletion of equal duration to one pegaspargase dose; after severe anaphylactic reactions E. coli 10,000 U/m2 is replaced by pegaspargase 2,500 U/m2 every 2 weeks or Erwinase 20,000 U/m2 three times per week; asparaginase is discontinued for clinically evident pancreatitis confirmed by raised amylase or ultrasound, and the protocol notes that hyperglycaemia in induction is usually due to steroids rather than to asparaginase.
Interfant-06 treatment protocol for infants under 1 year of age with acute lymphoblastic leukaemia - International collaborative treatment protocol, SKION version 01 (April 2006): chapter 5 chemotherapy schedule and chapter 7 modification for toxicity · Interfant-06 study group / SKION (Stichting Kinderoncologie Nederland, Dutch Childhood Oncology Group) · Interfant-06 study protocol document (90 pp.) 2006What 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. Asparaginase provisions were among the parts of Interfant-06 amended during the trial, so the substitution rules quoted here are those of the April 2006 SKION version and may not describe what a given patient received. The 45,000 U/m2 equivalence is attributed by the protocol to the COALL group without a citation and this beat did not verify it against a primary source.
Evidence that argues against this (1)
- Equal duration of asparagine depletion is not equal antileukaemic effect, and depletion duration is the surrogate the field uses precisely because the effect endpoint is unavailable. Contemporary practice has moved to individualised dosing against trough asparaginase ACTIVITY levels rather than fixed units, which is a tacit statement that fixed-unit equivalences do not deliver equivalent exposure. Brigitha LJ et al. 2025
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In the MTT assay, 44 infants with ALL were 12-fold more resistant to L-asparaginase than 425 children over 1 year with common/preB ALL (P = 0.001), the second-largest difference in that study after more than 580-fold for prednisolone; proB ALL of all ages showed the same relative resistance to L-asparaginase and prednisolone, and age was not related to resistance within the proB or within the MLL-rearranged groups.
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 2004What this evidence cannot show
In-vitro LC50 on bulk primary blasts. Asparaginase in culture is a particularly indirect assay because the drug acts by depleting a medium nutrient rather than by entering the cell, so an MTT LC50 measures sensitivity to asparagine withdrawal in a defined medium and not sensitivity to the enzyme in a patient. No mechanism was measured. The authors attribute much of the effect to the proB immunophenotype.
Evidence that argues against this (1)
- Because age did not affect resistance within the proB or MLL-rearranged groups, the finding is not 'infants are asparaginase-resistant' but 'proB and MLL-rearranged blasts are, at any age'. Any intervention framed around infancy rather than around phenotype is aimed at the wrong variable, and the same caution applies to the steroid result from the same study. Ramakers-van Woerden NL et al. 2004
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Across 767 children and 309 adults with newly diagnosed B-ALL profiled for ex-vivo sensitivity to 21 drugs, asparaginase was one of seven drugs (with prednisolone, mercaptopurine, dasatinib, nelarabine, daunorubicin and inotuzumab ozogamicin) showing differential activity between children and adults, six of the seven being explained by age-related differences in molecular subtype; transcriptomic profiling uncovered subclusters within KMT2A-rearranged ALL linked to age and to cytotoxic drug resistance.
Impact of Age on Pharmacogenomics and Treatment Outcomes of B-Cell Acute Lymphoblastic Leukemia. · Yoshimura S, Li Z, Gocho Y, Yang W, Crews KR, Lee SHR, et al. · J Clin Oncol 2024What this evidence cannot show
The age contrast is children versus adults, not infants versus older children, and the youngest patients are not separately analysed in the abstract. The KMT2A-rearranged subcluster finding is transcriptomic and its link to drug resistance is an association across the cohort. Ex-vivo sensitivity of primary blasts is the same class of assay as E2 with the same limits.
Evidence that argues against this (1)
- Six of the seven age-differential drugs were explained by subtype composition rather than by age itself, which argues that age-based dose and schedule reasoning - including the 2/3 and 3/4 multipliers in Interfant-06 - is capturing biology that belongs to the leukaemia rather than to the patient. Ramakers-van Woerden NL et al. 2004
- E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
Romidepsin ANTAGONISED L-asparaginase in four of six infant ALL cell lines, alongside antagonism with methotrexate in four of six, while synergising with daunorubicin and 4-hydroperoxycyclophosphamide in all six.
Romidepsin enhances the efficacy of cytarabine in vivo, revealing histone deacetylase inhibition as a promising therapeutic strategy for KMT2A-rearranged infant acute lymphoblastic leukemia. · Cheung LC, Cruickshank MN, Hughes AM, Singh S, Chua GA, Ford J, et al. · Haematologica 2019What this evidence cannot show
Read from the PMC HTML full text on 2026-09-20; the abstract fields are empty because this is a Haematologica letter. Six cell lines, Bliss-independence scoring, no primary samples, no in-vivo arm for this pair. An asparaginase combination result in a defined culture medium is doubly indirect, since the drug's action depends on medium asparagine content.
Evidence that argues against this (1)
- Cell-line combination scores predict clinical outcomes poorly in both directions, and antagonism with an enzyme drug whose effect is nutrient depletion is especially sensitive to assay medium. The result is used here to constrain a scheduling proposal, not to claim that HDAC inhibition would harm asparaginase efficacy in a patient. Cheung LC et al. 2019
- E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In 4,925 patients aged 1 to 30 years on Children's Oncology Group trials AALL0232 and AALL0434, each receiving a single induction dose of pegaspargase 2,500 IU/m2 without a maximum dose, increased risk of at least one asparaginase-associated toxicity was seen in patients aged 10 years or older (P = .002) and in those with obesity together with high body surface area (P < .0001) but not with high body surface area alone; asparaginase-associated toxicities were not associated with end-of-induction MRD positivity.
Toxicity from asparaginase during acute lymphoblastic leukemia induction: a report from the Children's Oncology Group. · Orgel E, Maese LD, Devidas M, Militano O, Rau RE, Angiolillo AL, et al. · Blood Adv 2026What this evidence cannot show
**The eligibility floor is age 1, so this study contains no infants** and its age signal points away from this beat's population. It concerns pegaspargase rather than the native E. coli product used in Interfant-06 induction. Obesity and high body surface area are not meaningful exposures in an infant cohort. Cited here to bound what is known, not to transfer a risk estimate.
Evidence that argues against this (1)
- That asparaginase toxicity was not associated with end-of-induction MRD in children aged 1-30 does not license the reassuring reading in infants. Interfant's asparaginase is a different product on a different schedule, and the infant population's dominant induction toxicity is infection, which this analysis did not examine. Gruber TA et al. 2026
- E6strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In a multicentre single-arm phase 1/2 study of 50 infants (median age 171 days, 35 with KMT2A rearrangement) on a backbone of dexamethasone, mitoxantrone and pegaspargase 2,500 units/m2 with bortezomib and vorinostat, grade 3-4 infection occurred in 44% of patients during induction and febrile neutropenia in 42%, hypertriglyceridaemia was the most frequent reinduction grade 3-4 event at 23%, and all four treatment-related deaths were infectious.
Bortezomib and vorinostat in combination with mitoxantrone, dexamethasone, and pegasparaginase during induction and reinduction for infants with acute lymphoblastic leukaemia: a multicentre single-arm phase 1/2 study. · Gruber TA, Jeha S, Deyell RJ, Lewis V, Chang BH, Lowe EJ, et al. · Lancet Haematol 2026What this evidence cannot show
Single-arm, n = 50, primary endpoint tolerability, and the backbone includes two investigational agents so no toxicity can be attributed to asparaginase specifically. Hypertriglyceridaemia is a characteristic asparaginase effect but is not attributed to it in the report. The authors state the adverse events were consistent with previous infant ALL studies.
Evidence that argues against this (1)
- Asparaginase contributes to infection risk indirectly through hypogammaglobulinaemia and through hepatic protein-synthesis suppression, so attributing the infectious deaths entirely to myelosuppression from the cytotoxics may understate its role. Equally, no component of this backbone can be separated from the others in a single-arm study. Orgel E et al. 2026
- ← confers resistance toASNS - the resistance gene for a backbone drug, never measured in this disease
THE RESISTANCE GENE FOR THIS DRUG: asparaginase exploits low ASNS, and ASNS induction via GCN2 is how resistance emerges.
- part of →Interfant-06 — 651 infants, and the definitive answer that myeloid-style consolidation does not help
native E. coli 10,000 U/m2 on induction days 15/18/22/25/29/33
remission induction
induction enzyme therapy against a 12-fold resistant blast
12-fold in-vitro resistance in infant ALL; same relative resistance in proB ALL at any age
the two resistant poles of the infant in-vitro profile: >580-fold and 12-fold in one experiment
ANTAGONISED in 4 of 6 infant ALL cell lines
same catalytic enzyme, different product: 2,500 U/m2 vs 10,000 U/m2, and a protocol-cited 18-fold unit ratio for equal depletion
predicts re-sensitisation: menin inhibition lowers the ASNS that asparaginase exploits. PubMed has ZERO records for menin AND asparaginase, parsed and run twice
thrombosis is among its dose-limiting toxicities, conventionally via antithrombin III depletion; any CBS contribution would be additional to that and is unevidenced
asparagine depletion is the canonical GCN2-activating stimulus and asparaginase is backbone therapy in KMT2A-rearranged ALL; no experiment has combined it with a menin inhibitor, and ASNS falls 1.72 log2 under one
- ← confers resistance toCBS (P35520) - the largest fall in the whole panel (-4.07), a token that collides with Cajal bodies AND CTCF binding sites, and a germline phenotype whose hallmark is thrombosis
bite-back: CBS deficiency causes homocystinuria whose hallmark is thromboembolism, and asparaginase has thrombosis among its dose-limiting toxicities - the combination this graph proposes would stack two prothrombotic mechanisms