Abstract
Introduction
The prognosis of myeloid leukaemia associated with Down syndrome (ML-DS) is excellent with reduced-intensity chemotherapy; however, that of refractory or relapsed ML-DS remains dismal. These contrasting outcomes highlight the importance of risk-stratified therapy, based on reliable prognostic factors, to prevent treatment-related mortality and relapse. In addition, novel therapeutic strategies capable of overcoming the poor prognosis of high-risk patients are eagerly anticipated. Based on the results of molecular analyses, in vitro functional assays and anecdotal case reports, azacitidine (AZA), a hypomethylating agent, is a promising candidate for novel therapeutic strategies for high-risk ML-DS.
Methods and analysis
In this multicentre, single-arm phase II clinical trial, the Japan Children’s Cancer Group AML-D24, we will stratify patients with newly diagnosed ML-DS according to age at diagnosis, GATA1 mutation status and treatment response. Patients <24 months of age at the diagnosis with a GATA1 mutation who achieve morphological complete remission and are negative for flow cytometric measurable residual disease at the end of induction will be classified as the low-risk (LR) group and receive less intensive chemotherapy. The other participants, stratified into the non-LR group, will be treated with AZA-combined chemotherapy. The objective of this trial is to evaluate the efficacy and safety of AZA-combined chemotherapy in children with non-LR ML-DS. The primary endpoint is the 3-year event-free survival in the non-LR group. The planned enrolment is 145 patients, including 47 non-LR patients. The study period consists of a 7-year accrual period (including an enrolment suspension period for interim analysis) followed by a 3-year follow-up period for a total duration of 10 years.
Ethics and dissemination
This study was approved by the National Hospital Organization Review Board for Clinical Trials (Nagoya, Japan) on 12 May 2025 and was registered in the Japan Registry of Clinical Trials (https://jrct.mhlw.go.jp/latest-detail/jRCTs041250031). Written informed consent will be obtained from all patients and/or their guardians. The results of this study will be disseminated through publication in peer-reviewed journals and presentation at national and international scientific conferences.
Trial registration number
jRCTs041250031.
Keywords: Leukaemia, Paediatric oncology, Clinical Protocols
STRENGTHS AND LIMITATIONS OF THIS STUDY.
This nationwide phase II trial is the first in the world to evaluate the efficacy and safety of azacitidine-combined chemotherapy for high-risk cases of myeloid leukaemia associated with Down syndrome (ML-DS).
The risk stratification scheme is refined by incorporating age at the diagnosis, morphological response to the induction therapy, flow-cytometric measurable residual disease after the induction therapy and GATA1 mutation status.
Cell-free DNA, genomic and epigenomic analyses will be performed to evaluate their potential usefulness for precisely predicting patient outcomes.
Because of the limited number of patients with newly diagnosed ML-DS, a randomised controlled trial is not feasible.
Introduction
Children with Down syndrome (DS) are 10–20 times more likely to develop acute leukaemia than are those without DS. In particular, acute myeloid leukaemia harbouring GATA1 mutations frequently occurs during the first 4 years of life and is referred to as myeloid leukaemia associated with DS (ML-DS). Because leukaemic blasts in ML-DS are highly sensitive to chemotherapeutic agents, previous studies have reported excellent outcomes for children with ML-DS, with 3-year overall survival (OS) of 89.7% in the Japan Children’s Cancer Group (JCCG) AML-D11 study and 5-year OS of 89‐93% in the Children’s Oncology Group (COG) and Berlin-Frankfurt-Munster (BFM) studies.1–3 In contrast, the prognosis of patients with refractory or relapsed ML-DS remains dismal, with a 3-year OS rate of only 20%–25%.4 5 These contrasting outcomes highlight the importance of risk-stratified chemotherapy, based on reliable prognostic factors, to prevent treatment-related mortality and relapse. In addition, novel therapeutic strategies capable of overcoming the poor prognosis of high-risk (HR) patients are eagerly anticipated.
Previous studies have reported various factors that predict outcomes in patients with ML-DS, such as karyotypes, older age and morphological response; however, these factors have shown poor reproducibility.6–9 In a retrospective analysis of two consecutive trials in Japan, JCCG AML-D05 and D11, which had identical risk stratification and treatment regimens, we found that age ≥24 months at the diagnosis was associated with a poorer prognosis,10 which was inconsistent with the results of other clinical trials.2 11 Recently, flow cytometric measurable residual disease (FCM-MRD) at the end of induction (EOI) has been identified as a powerful prognostic factor in both the COG AAML0431 and the JCCG AML-D11 trials.1 3 In the COG AAML0431 trial, 21 (14.4%) of 146 evaluable patients were positive for FCM-MRD (>0.01%) at EOI and had poorer outcomes (5-year event-free survival (EFS), 76.2% vs 92.7% in those with negative EOI-FCM-MRD).1 Similarly, in the JCCG AML-D11 trial, 5 (7.7%) of the 65 patients were positive for EOI-FCM-MRD (≥0.05%), and their 3-year EFS was inferior to that in patients with negative EOI-FCM-MRD (60.0% vs 93.3%).3 Based on these findings, the JCCG AML Committee has adopted EOI-FCM-MRD as a stratification factor in the successor trial, AML-D16 (trial registration number: jRCTs041190047). To further refine the risk stratification, we are conducting a multicentre phase II clinical trial, AML-D24, in which patients <24 months of age at the diagnosis and negative for EOI-FCM-MRD are stratified into the low-risk (LR) group. In contrast, patients who are ≥24 months of age at the diagnosis or who show a poor response to initial induction therapy (either morphologically or by FCM-MRD) are stratified into the non-LR group and considered candidates for treatment intensification. We also include wild-type GATA1 as a factor indicating non-LR because GATA1 mutations are highly characteristic of ML-DS.12 When this novel risk stratification from the AML-D24 trial was applied to cohorts of the AML-D11 trial, the 3-year EFS of patients in the LR group (ie, those <24 months of age, harbouring GATA1 mutation and with negative EOI-FCM-MRD) was 97.9%, whereas that of the non-LR group was 73.7%.
Because the vulnerability of patients with DS limits further intensification of cytotoxic chemotherapeutic agents, the introduction of non-conventional therapies such as molecular-targeted agents is anticipated. Among these, azacitidine (AZA), a hypomethylating agent, is a promising candidate as a novel therapeutic strategy in patients with ML-DS, as frequent alterations in genes regulating epigenetic modifications have been identified.13 14 Moreover, AZA has demonstrated encouraging efficacy against ML-DS cell lines by inducing cell cycle arrest, apoptosis and differentiation.15 An anecdotal report suggested its anti-leukaemic effect16 and a retrospective case series confirmed its safety in refractory or relapsed ML-DS.17 Based on this evidence, we incorporated a 5-day AZA regimen, for the first time in the world, into the AML-D16 backbone chemotherapy for the non-LR group in the AML-D24 trial to improve the outcome of these patients without increasing toxicity.
Methods and analysis
Protocol version
This manuscript is based on Protocol Version 1.1, dated 28 April 2025.
Purpose and study design
This study is a multicentre, non-blinded, single-arm phase II clinical trial. The purpose of this study is to examine the efficacy and safety of AZA-combined chemotherapy for non-LR children with ML-DS according to risk stratification based on age at the diagnosis, GATA1 mutation status and treatment response including FCM-MRD status at the end of the initial remission induction therapy.
Trial oversight
This is an investigator-initiated trial sponsored and coordinated by the JCCG. The JCCG AML Committee serves as the trial management group and is responsible for the study design and oversight of trial conduct, meeting monthly. Day-to-day trial management is undertaken by the principal investigator (PI), Daisuke Hasegawa (Department of Pediatrics, St. Luke’s International Hospital, 9-1 Akashi-cho, Chuo-ku, Tokyo 104-8560, Japan; email: hasedai1313@gmail.com), with support from the JCCG data centre for data collection and management, central monitoring and coordination of the statistical analyses. The PI has overall responsibility for the trial, including interpretation of the study data and the decision to publish the study results. Independent safety oversight is provided by the Data and Safety Monitoring Committee (DSMC).
Endpoints
Primary endpoint
3-year EFS in the non-LR group (ie, standard-risk (SR)+HR+MRD-HR).
Secondary endpoints
3-year OS, cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) in the non-LR group.
3-year OS, EFS, CIR and NRM in the entire cohort (ie, LR+SR+HR+MRD-HR).
3-year OS, EFS, CIR and NRM in each risk group.
Remission and positivity of FCM-MRD (defined as ≥0.05% and leukaemic cell clustering3) after initial induction therapy in the entire cohort.
Remission and positivity of FCM-MRD after initial induction therapy in patients aged ≥2 years receiving AZA-combined induction therapy.
Remission and positivity of FCM-MRD after the initial induction therapy in patients aged <2 years receiving induction therapy without AZA.
Positivity of FCM-MRD after intensification therapies 1 and 2 and at the end of therapy in the MRD-HR group.
Remission after induction therapies 2 and 3, and positivity of FCM-MRD after the initial induction therapy, induction therapy 2, induction therapy 3, and at the end of therapy in the HR group.
Incidence of myelosuppression, adverse events, treatment reductions and deaths during the initial induction therapy in patients <2 years of age and in patients ≥2 years of age.
Incidence of myelosuppression, adverse events, delay in starting the subsequent treatment course and treatment reduction during each treatment course in each risk group.
Eligibility criteria
Inclusion criteria
Newly diagnosed ML-DS including mosaic DS.
Age 4 months to <18 years at the diagnosis.
Patients registered in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG)-CHM-14 and with a JPLSG number.
Written informed consent from patient and/or guardian.
Exclusion criteria
Severe intracranial haemorrhage (grade ≥3 according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0).
Uncontrolled diabetes.
Intractable heart failure (presence of congenital heart disease itself is not an exclusion criterion).
Uncontrolled infection.
Pregnant or suspected to be pregnant.
History of congenital or acquired immunodeficiencies.
Central nervous system (CNS) leukaemia (diagnostic lumbar puncture is performed only in cases strongly suspected of CNS infiltration).
Acute promyelocytic leukaemia.
BCR::ABL1-positive AML.
Acute leukaemias of ambiguous lineage including mixed phenotype acute leukaemia.
Myeloid sarcoma.
History of previous chemotherapy (except chemotherapy for transient abnormal myelopoiesis) or radiation therapy.
Inappropriate status judged by the PI and coinvestigators.
Central review for diagnosis and treatment evaluation
Diagnostic immunophenotyping: National Center for Child Health and Development.
FCM-MRD: Mie University, Kyoto University and Kindai University.
GATA1 mutation analysis: Hirosaki University.
GATA1-MRD: Hirosaki University.
Cell-free DNA analysis: National Cancer Center Research Institute (Tokyo, Japan).
Genomic analysis: National Cancer Center Research Institute (Tokyo, Japan).
DNA methylation analysis: National Research Institute for Child Health and Development.
Treatment procedures
Because GATA1 mutation analysis is performed centrally and the results become available during induction therapy, GATA1 mutation status is not used for stratification of induction therapy itself.
In this phase II study, children with newly diagnosed ML-DS will receive risk-adapted multiagent chemotherapy and the risk classification will be determined according to the stratification factors shown in table 1. An overview of the treatment regimens is provided in figure 1 and table 2.
Table 1. Risk stratification.
| Risk group | Definition |
|---|---|
| Low risk (LR) | Patients <2 years old at diagnosis with GATA1 mutation, achieving CR and negative FCM-MRD after Induction 1 (BMA-2) |
| Standard risk (SR) | Patients who do not meet the criteria for LR, MRD-HR or HR |
| MRD high risk (MRD-HR) | Patients with CR after Induction 1 and positive FCM-MRD (BMA-2) |
| High risk (HR) | Patients without CR after Induction 1 (BMA-2) |
Patients who achieve CR but have unevaluable FCM-MRD, and those with negative FCM-MRD and negative or indeterminate GATA1 mutation status by Sanger sequencing, are classified as SR.
BMA, bone marrow aspiration; CR, complete remission; FCM, flow cytometry; MRD, measurable residual disease.
Figure 1. Treatment schema of the AML-D24 study. The SR group includes patients with negative or indeterminate GATA1 mutation by Sanger sequencing, and those whose FCM-MRD is unevaluable. AZA, azacitidine; BMA, bone marrow aspiration; C, cytarabine; cC, continuous cytarabine; CET, cytarabine, etoposide, pirarubicin; CT, cytarabine, pirarubicin; CR, complete remission; E, etoposide; FCM, flow cytometry; hC, high-dose cytarabine; HR, high risk; LR, low risk; ML-DS, myeloid leukaemia of Down syndrome; MRD, measurable residual disease; MRD-HR, MRD high risk; SR, standard risk; T, pirarubicin; V, vincristine.
Table 2. Treatment protocol.
| Treatment element/drug | Single or daily dose | Days of application per element |
|---|---|---|
| CET | ||
| Cytarabine (1-hour IV) | 100 mg/m2 per day | 1–7 |
| Etoposide (2-hour IV) | 150 mg/m2 per day | 3–5 |
| Pirarubicin (1-hour IV) | 25 mg/m2 per day | 1, 2 |
| AZA-CET | ||
| Azacitidine (10-minute IV) | 75 mg/m2 per day* | 1–5 |
| Cytarabine (1-hour IV) | 100 mg/m2 per day | 6–12 |
| Etoposide (2-hour IV) | 150 mg/m2 per day | 8–10 |
| Pirarubicin (1-hour IV) | 25 mg/m2 per day | 6, 7 |
| CT | ||
| Cytarabine (1-hour IV) | 100 mg/m2 per day | 1–7 |
| Pirarubicin (1-hour IV) | 25 mg/m2 per day | 1, 2 |
| AZA-CT | ||
| Azacitidine (10-minute IV) | 75 mg/m2 per day* | 1–5 |
| Cytarabine (1-hour IV) | 100 mg/m2 per day | 6–12 |
| Pirarubicin (1-hour IV) | 25 mg/m2 per day | 6, 7 |
| AZA-cCVT | ||
| Azacitidine (10-minute IV) | 75 mg/m2 per day* | 1–5 |
| Cytarabine (24-hour continuous IV) | 100 mg/m2 per day | 6–12 |
| Vincristine (IV push) | 1 mg/m2 per day† | 7 |
| Pirarubicin (1-hour IV) | 40 mg/m2 per day | 6 |
| AZA-hCE | ||
| Azacitidine (10-minute IV) | 75 mg/m2 per day* | 1–5 |
| Cytarabine (2-hour IV) | 1 g/m2 per dose | 6–10 (every 12 hours) |
| Etoposide (2-hour IV) | 100 mg/m2 per day | 7–9 |
The dose of azacitidine is 2.5 mg/kg per day in patients weighing <10 kg or <1 year of age.
Max dose is 1 mg/body per day.
AZA, azacitidine; AZA-cCVT, azacitidine + continuous cytarabine + vincristine + pirarubicin; AZA-CT, azacitidine + cytarabine + pirarubicin; AZA-hCE, azacitidine + high-dose cytarabine + etoposide; CET, cytarabine + etoposide + pirarubicin; CT, cytarabine + pirarubicin; IV, intravenous.
Induction 1
Patients <2 years of age will receive CET (cytarabine, etoposide, pirarubicin), whereas those ≥2 years of age will receive AZA-CET, consisting of CET plus AZA (75 mg/m²/day or 2.5 mg/kg/day in patients weighing <10 kg or <1 year of age, days 1–5) (table 2). Bone marrow assessment will be performed after Induction 1 at bone marrow aspiration (BMA)−2 to determine complete remission (CR).
Postinduction therapy
After induction 1, the patients will be stratified according to risk factors (table 1).
LR patients will receive three courses of intensification therapy (CET, CT and CT) identical to the LR regimen in the AML-D16 trial.
SR patients will receive four courses of AZA-combined intensification therapy (AZA-CET, AZA-CT, AZA-CET and AZA-CT).
Patients in the LR or SR groups whose FCM-MRD at BMA-2 is positive will be reclassified as MRD-HR after the first intensification course and receive four courses of intensive AZA-combined therapy (AZA-hCE, AZA-cCVT, AZA-hCE and AZA-cCVT).
HR patients will receive AZA-cCVT and AZA-hCE as Induction 2 and 3, respectively. After CR is confirmed, patients will receive three additional courses of AZA-combined therapy (AZA-cCVT, AZA-hCE and AZA-cCVT).
Follow-up and study status
The planned study period consists of a 7-year accrual period (including an enrolment suspension period for interim analysis) followed by a 3-year follow-up period for a total duration of 10 years.
Patient enrolment was initiated on 27 June 2025 and is scheduled to end in June 2032. Data collection will end in June 2035.
Sample size considerations
The planned enrolment is 145 patients with ML-DS overall including 47 non-LR patients. Based on the results of the AML-D11 trial, the threshold 3-year EFS for non-LR patients is set at 70%. The expected 3-year EFS for non-LR patients in this trial is set at 85%. Using the hypothesis test described in the statistical analysis section, recruiting 47 non-LR patients is enough to attain a one-sided α of 0.05 and β of 0.2.18
Data collection and management
Trial data will be collected at participating sites using case report forms and managed by the JCCG data centre. Data will be entered into a validated data capture system, with range and consistency checks and query management to ensure data quality.
Statistical analysis
The full analysis set includes all registered patients who fulfil the eligibility criteria, excluding those with serious protocol violations.
For the primary endpoint, Kaplan-Meier curves regarding EFS (defined as the time from the start of treatment until induction failure (for the HR group), relapse, secondary malignancy or death) will be calculated for non-LR patients. To determine whether the 3-year EFS exceeds the predefined threshold of 70%, a one-sided test will be performed using the arcsine square-root transformed Kaplan-Meier estimate of the 3-year EFS and the Greenwood SE. For this one-sided test, α is set at 0.05.
The following secondary endpoints will be calculated for the full analysis set, as well as for appropriate subgroups:
3-year EFS.
3-year OS: OS is defined as the time from the start of treatment until death. The 3-year OS will be estimated in the same manner as the EFS.
3-year CIR: The CIR will be estimated using the time from the achievement of CR until relapse and described using the cumulative incidence function. NRM and secondary malignancies are considered as competing risk events.
3-year NRM: The NRM will be estimated using the time from the achievement of CR until NRM and described using the cumulative incidence function. Relapse and secondary malignancies are considered as competing risk events.
Remission (binary endpoint): The remission will be described with the proportion and exact (Clopper-Pearson) CI. The same will apply hereafter for the binary endpoints.
FCM-MRD positivity (binary endpoint).
Incidence of myelosuppression, adverse events, treatment reduction and deaths during initial induction therapy (binary endpoint).
Incidence of myelosuppression, adverse events, delay in starting the subsequent treatment course and treatment reduction during each treatment course (binary endpoint).
Unless otherwise stated, the level for the CI is set at 95%.
The efficacy of AZA-combined therapy will be confirmed if the primary hypothesis test on the 3-year EFS for the non-LR group is significant and if the point estimate of 3-year EFS for the SR and MRD-HR groups exceeds the predefined threshold of 80% and 60%, respectively.
All statistical analyses will be performed using SAS V.9.4 (SAS Institute).
Interim analysis and monitoring
No interim analysis for efficacy is planned. Sequential monitoring will be conducted for safety purposes. All deaths and CTCAE grade 4 or higher serious adverse events occurring during the trial will be reported to the PI. Discontinuing patient enrolment will be considered if the lower limit of the 95% CI for the mortality rate of patients administered AZA exceeds 5%.
In addition, to minimise anticipated adverse events related to AZA, patient enrolment will be temporarily suspended once 10 patients eligible for AZA-combined therapy have completed their first AZA treatment course, at which point an interim analysis for safety will be conducted. The results of this analysis will be submitted to the DSMC for review and recommendation on whether to continue, modify or terminate the study.
To ensure that the study is being conducted safely according to its protocol and that accurate data are being collected, regular monitoring will be conducted throughout the study primarily based on case reports, with monitoring reports prepared annually for all registered patients.
An independent DSMC, comprising haematologists/oncologists and a biostatistician who are independent of the study team and have no competing interests, oversees participant safety. The DSMC reviews the pre-planned safety interim analysis (performed after the first 10 patients eligible for AZA-combined therapy have completed their first AZA course) and meets at least annually and on an ad hoc basis in response to the event of a serious safety-related issue, providing recommendations to continue, modify or terminate the trial. Central monitoring is performed by the JCCG data centre using data reported through case report forms, with monitoring reports prepared annually for all registered patients.
Participating institutions
As of 30 November 2025, 90 institutions are participating in this study. All institutions belonged to the JCCG study group.
Ethics and dissemination
The AML-D24 study was approved by the National Hospital Organization Review Board for Clinical Trials (Nagoya, Japan; approval No. C2025-001) on 12 May 2025 and was registered in the Japan Registry of Clinical Trials on 30 May 2025 (jRCTs041250031). Written informed consent will be obtained from all patients and/or their guardians using the participant information sheet and informed consent form (see online supplemental file 1). Assent will also be obtained from participants when the investigator and the parent(s) or legal guardian(s) consider the participant capable of understanding the study and providing assent (see online supplemental file 2).
The results of this study will be disseminated through publication in peer-reviewed journals and presentation at national and international scientific conferences.
Confidentiality
Participants are identified by a unique subject number. Directly identifying information is retained at the participating site; the data centre handles only data coded with these unique subject identifiers. Access to trial data is restricted to authorised personnel, and data are stored and processed in accordance with applicable Japanese laws and regulations (including the Act on the Protection of Personal Information) and the Clinical Trials Act (Act No. 16 of 14 April 2017).
Data sharing
The datasets that will be generated and/or analysed during the current study will not be publicly available because they will contain potentially identifying information on a rare patient population (children with ML-DS). After publication of the primary study results, de-identified individual participant data underlying the reported results may be made available on reasonable request to the corresponding author, subject to approval by the JCCG, execution of a data sharing agreement, and compliance with applicable Japanese laws and regulations and the scope of participant consent.
Supplementary material
Acknowledgements
The authors thank all the JCCG investigators involved in the AML-D24 trial; Nohana Takeuchi, Mei Shiratani, Shizuka Nanno and Ikuko Miwa from the JCCG data center for their support as data managers in protocol creation and trial data management.
Footnotes
Funding: This study is supported by grants for Practical Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED) (grant number 25ck0106043h0001 to DH).
Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (https://doi.org/10.1136/bmjopen-2025-115972).
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient consent for publication: Not applicable.
Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
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