Risk-Stratified Therapy for Acute Myeloid Leukemia in Down Syndrome
Phase: PHASE3
Conditions: Acute Myeloid Leukemia, Down Syndrome, Myelodysplastic Syndrome, Myeloid Leukemia Associated With Down Syndrome, Myeloproliferative Neoplasm
Sponsor: Children's Oncology Group



 
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AGENT 
                 NSC#  
IND# 
 
Cytarabine  
 
# 063878 
Commercial 
 
Daunorubicin 
 
# 82151  
Commercial 
 
Asparaginase (Erwinia)  
#106977 
Commercial 
 
Etoposide 
 
# 141540 
Commercial 
 
Asparaginase (E.coli) 
# 109229 
Commercial 
 
Mitoxantrone 
 
# 301739 
Commercial 
SEE SECTIONS 13 and 14 FOR SPECIMEN 
SHIPPING ADDRESSES. 
Thioguanine 
 
# 000752 
Commercial 
 
 
 
 

 
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The Children's Oncology Group has received a Certificate of Confidentiality from the federal government, 
which will help us protect the privacy of our research subjects. The Certificate protects against the 
involuntary release of information about your subjects collected during the cycle of our covered studies. 
The researchers involved in the studies cannot be forced to disclose the identity or any information collected 
in the study in any legal proceedings at the federal, state, or local level, regardless of whether they are 
criminal, administrative, or legislative proceedings. However, the subject or the researcher may choose to 
voluntarily disclose the protected information under certain circumstances. For example, if the subject or 
his/her guardian requests the release of information in writing, the Certificate does not protect against that 
voluntary disclosure. Furthermore, federal agencies may review our records under limited circumstances, 
such as a DHHS request for information for an audit or program evaluation or an FDA request under the 
Food, Drug and Cosmetics Act. The Certificate of Confidentiality will not protect against mandatory 
disclosure by the researchers of information on suspected child abuse, reportable communicable diseases, 
and/or possible threat of harm to self or others. 
 
ABSTRACT 
Acute Myeloid Leukemia in children with Down syndrome (DS) is a distinct form of myeloid leukemia 
(DS AML) that is characterized by a young age of onset (< 4 years of age), a frequently megakaryoblastic 
blast morphology and immunophenotype, a common prodrome of myelodysplastic syndrome, and a high 
probability of event-free survival (EFS) for the majority of children compared to AML in children without 
DS. DS AML blasts typically contain somatic mutations of the gene encoding the hematopoietic 
transcription factor GATA1 and are hypersensitive to selected chemotherapeutic agents such as cytarabine, 
which has historically been used at high doses for the treatment of AML. Recent studies, therefore, have 
pursued reduced treatment intensity for children with DS AML to reduce the morbidity associated with its 
treatment.  
 
COG study A2971 used therapy based on CCG 2891 but eliminated dexamethasone as well as etoposide 
and, despite this reduction in treatment, maintained favorable survival outcomes (5-year EFS 79%). 
AAML0431, building on A2971, focused on the reduction of the cumulative dose of daunorubicin (by 25%) 
and of intrathecal chemotherapy (from 7 to 2 doses) and achieved a 3-year EFS of 90%. These results 
demonstrate that the 85-90% of children with DS AML have a highly favorable prognosis and may therefore 
benefit from further reduction of treatment intensity. Therefore, given the hypersensitivity of DS AML 
blasts to cytarabine and the profound neutropenia and infections associated with its use, the elimination of 
high-dose cytarabine therapy (HD Ara-C) is the next logical step in reducing treatment intensity for the 
majority of children with DS and AML. As described below, children with DS AML will be stratified based 
on the level of MRD at the end of induction to receive reduced intensity therapy versus standard therapy. 
Using this approach, we aim to maintain the overall excellent outcome for children with DS AML, yet 
reduce morbidity in the 85-90% with a favorable prognosis. AAML0431, the level of minimal residual 
disease (MRD) measured by multi-parameter flow cytometry in the bone marrow at the end of this first 
cycle of induction therapy, predicted outcome. Therefore, as described below, MRD will be used to stratify 
patients with DS AML to reduced intensity versus standard therapy. 
 
All patients will first receive a common cycle of induction therapy that includes a continuous infusion of 
standard-dose cytarabine, bolus infusion of daunorubicin and oral 6-thioguanine (TAD). Following interim 
analysis, patients in the standard risk group (MRD ≤ 0.05% at the end of Induction I) who received reduced 
intensity therapy consisting of two additional induction cycles of TAD and two cycles of intensification 
therapy with standard-dose cytarabine and etoposide with the elimination of HD Ara-C were found to have 
an inferior 2 year event-free survival (EFS) compared to patients who received HD Ara-C on AAML0431. 
Thus, following amendment 4A, the standard risk arm of this study is permanently closed. Patients enrolled 
after amendment 4A who are assigned to the standard risk arm after Induction I will be removed from study.   

 
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Patients in the high risk group (MRD ≥ 0.05% at the end of Induction I) will remain on protocol therapy 
and be treated with intensified therapy consisting of cycles of mitoxantrone and cytarabine (MA), etoposide 
and cytarabine (AE) and Capizzi II (HD Ara-C and asparaginase) as used for high risk AML in children 
without DS.  
 
The exploratory aims of this study include the analysis of a comparison of different methods to detect MRD 
in DS AML (flow cytometry, PCR-based and sequencing-based detection of clone-specific GATA1 
mutations) with regard to feasibility and analytical characteristics. Finally, a bank of viably cryopreserved 
DS AML bone marrow samples collected at end of induction and relapse, and corresponding non-tumor 
DNA collected at end of Induction I, will be established.  
 
In sum, study AAML1531 will tailor the intensity of AML treatment for children with DS (under 4 years 
of age) to the risk of relapse as measured by MRD after the first cycle of treatment. The study aims to 
maintain the excellent survival outcome, to reduce toxicity of therapy for the majority of patients with 
favorable disease and to improve survival for the minority of high risk patients who may benefit from 
therapy intensification.  
 
 

 
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1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
1.1 
Primary Aims 
1.1.1  To determine the 2-year event-free-survival (EFS) for children with standard risk 
DS AML (MRD-negative after one cycle of induction therapy) after elimination 
of HD Ara-C from the treatment regimen. 
 
1.1.2  To determine the 2-year EFS for children with high risk DS AML (MRD-positive 
after one cycle of induction therapy) after intensification of treatment equivalent 
to that used for high risk AML in children without DS. 
 
1.2 
Exploratory Aims 
 
1.2.1 
To compare the feasibility and analytical characteristics of flow cytometry, PCR 
and targeted error-corrected sequencing of GATA1 mutations as methods to detect 
MRD in DS AML. 
 
1.2.2 
To establish a DS AML cell bank of viably frozen bone marrow samples collected 
at the end of induction and corresponding non-tumor DNA samples collected at 
end of Induction 1.  
 
 

 
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2.0 
BACKGROUND 
2.1 
Outcomes of AML in Children with Down Syndrome  
During the early 1990s, it was recognized that children with Down syndrome (DS) and 
AML (DS AML) have a significantly higher EFS and lower relapse rate compared to 
children without DS. The landmark study by the Pediatric Oncology Group POG 8498 
demonstrated both the feasibility of clinical trials in children with DS AML and 
unexpectedly superior outcomes (EFS 100% at 4 years).1 DS AML is now recognized as a 
clinically and biologically distinct form of leukemia. It is characterized by a 150-fold 
increased incidence,2 younger age of onset (under 4 years of age)2,3, predominant 
megakaryoblastic blast phenotype, high prevalence of antecedent myelodysplastic 
syndrome4 and significantly increased sensitivity to cytotoxic drugs such cytarabine and 
anthracyclines (see Section 2.3). Somatic mutations of the gene encoding the hematopoietic 
transcription factor GATA1 were identified as a specific disease mechanism underlying DS 
AML5-7 as well as the observed hypersensitivity of DS AML blasts to selected 
chemotherapeutic agents.8 This biologically distinct character of DS AML is further 
supported by the observation that DS AML evolves from a fetal accumulation of 
megakaryoblasts - termed transient myeloproliferative disorder (TMD), transient leukemia 
or transient abnormal myelopoiesis. TMD is detectable in 10-30% of DS newborns and 
spontaneously resolves in the vast majority of cases. In approximately 20% of cases, TMD 
is followed within the first 4 years of life by the onset of DS AML, which typically harbors 
a concordant, clone-specific mutation of GATA1.9 
 
Based on these observations, the extrapolation to children with DS of AML treatment 
approaches that were optimized in the overall pediatric population has been replaced by the 
design of specific treatment protocols for DS AML.10-12 In view of the excellent survival 
of patients with DS AML, early attempts at reducing the intensity of treatment were 
initiated and proved successful.10-12 However, despite the hypersensitivity of DS AML 
blasts to cytarabine, the use of HD Ara-C continues to be carried over from the treatment 
of AML in the general population to treatment protocols for DS AML.1,3,4,13-15 The excellent 
survival of children with DS AML, now approaching 90%, suggests the feasibility of 
reducing the intensity of treatment for the majority of patients. In contrast, a small 
proportion of patients with DS AML do not benefit from current treatment approaches and 
experience poor survival due to refractory or relapsed disease.16-18 For this small, but 
important group, intensification of treatment may be necessary. 
 
2.2 
Rationale for Study Design - Introduction of Risk-Adapted Therapy for DS AML 
In contrast to other contemporary treatment approaches to pediatric leukemia, current 
protocols for DS AML lack a risk-adapted stratification of treatment intensity. As a result, 
cycles of high-dose chemotherapy such as HD Ara-C continue to be administered to all 
patients with DS AML. 
 
The majority of patients with DS AML have prognostically very favorable disease and do 
not benefit from high-intensity chemotherapy. Maximizing dose intensity has proved a 
successful strategy for the treatment of AML in the general pediatric population.4,19 In 
contrast, a highly dose-intense AML chemotherapy protocol resulted in a 32% induction 
death rate and decreased EFS in children with DS.4 Given both the decreased survival of 
children with DS treated with intensive AML therapy protocols, which were optimized in 
the non-DS pediatric population, and the hypersensitivity of DS AML blasts to cytarabine 
and anthracyclines, COG has pursued the rational development of treatment protocols 

 
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specific for DS AML. Initial efforts successfully reduced anthracycline exposure while 
maintaining high EFS. Prompted by the hypersensitivity of DS AML blasts to cytarabine, 
we hypothesize that use of HD Ara-C, a treatment element which was developed for AML 
in the overall pediatric population but has remained part of treatment protocols for DS AML, 
substantially contributes to adverse events of AML therapy in children with DS without 
improving EFS for these patients. 
 
Therefore, we propose to eliminate HD Ara-C from the treatment of AML in the majority 
of children with DS who have standard risk disease. To guard against any decrease of the 
current favorable EFS, the proposed reduction of treatment intensity will be limited to 
children with DS AML lacking known risk factors for an adverse outcome (DS AML 
standard risk group, SR). Age under 4 years and negative minimal residual disease (MRD) 
in the bone marrow as determined by multi-parameter flow cytometry at the end of the first 
induction cycle identified a prognostically highly favorable subset of DS AML patients in 
study AAML0431.20 Approximately 85% of DS- AML patients are expected to fall into this 
SR group and to have a highly favorable outcome.10-12 For these patients, treatment intensity 
will be reduced by discontinuing the administration of HD Ara-C (See Table 1 below). 
 
At the same time, treatment studies consistently identify a small subset of patients who do 
not achieve long term survival with current treatment protocols for DS AML due to relapse 
or refractory disease.3,11,16-18,21 Survival of patients with relapsed or refractory DS AML is 
poor even following treatment with hematopoietic stem cell transplantation (HCT; EFS 
26%17; overall survival, OS, 21% ).16 Subsequent relapse rather than toxicity of HCT was 
a major barrier to successful outcomes.16,18 This suggests treatment intensification in this 
group may provide a benefit of improved disease control that counterbalances the risk 
associated with intensive treatment. Early response to treatment (as measured by MRD 
in the bone marrow after the first cycle of induction therapy) will be used to identify this 
high risk group. 
 
In sum, patients will be stratified according to relapse risk into one of two arms. Arm A 
will include patients with standard risk DS AML (approximately 85% of all patients) and 
administer treatment of reduced intensity (without HD Ara-C) compared to predecessor 
study AAML0431. Arm B will include the approximately 15% of patients with high risk 
DS AML who will receive intensified treatment compared to predecessor study 
AAML0431 (equivalent to the high risk arm of AAML1031). 
 
Outcomes of this study will be compared to AAML0431 from the start of Induction II since 
both studies include the same treatment during Induction I. 
 
 

 
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continuous infusion of anthracyclines could not be shown to provide a benefit of lower 
cardiotoxicity compared to bolus infusion.30-33 Therefore, in study AAML1531 
daunorubicin will be administered as short minute infusion, rather than the previously used 
continuous infusion over 96 hours. The cumulative dose of anthracycline in the standard 
risk group (240 mg/m2) remains identical to that of AAML0431, while patients in the high 
risk group will receive 272 mg/m2. The use of dexrazoxane will be allowed in the current 
study as per institutional preference (refer to Section 4.1.7 for additional information).  
 
2.6 
Poor Outcome of Patients with Relapsed/Refractory DS AML 
While the majority of DS AML patients (approx. 85%) have a highly favorable outcome 
across a range of treatment regimens developed by the COG, Berlin-Frankfurt-Münster 
(BFM), and Japanese study groups,10-12 a lack of response to DS-specific AML therapy 
is associated with a low probability of long-term survival. Three-year OS was 25.9% 
among 29 children with relapsed and refractory DS AML.17 Allogeneic HCT was 
successful only in 2 of 8 recipients.17 Similar outcomes were reported by a registry-based 
study with a 3-year OS of 21% among 21 children with DS AML undergoing HCT.16 Of 
note, subsequent relapse (cumulative incidence of relapse, CIR, 62%) rather than 
treatment-related toxicity (CI TRM 25%) was the predominant cause of treatment 
failure.16 These observations support the clinical study of an intensification of treatment 
compared to current DS AML-specific protocols for the high risk group of DS AML. 
 
2.7 
Justification and Safety of Intensified Treatment for High Risk DS AML 
Several study groups have gained experience with more intensive therapy for DS AML as 
proposed by this study for the 10-15% of patients in the high risk group. BFM study AML-
BFM 93 used mitoxantrone and Ara-C (HAM, Ara-C 3g/m2 q12h on day 1-3; mitoxantrone 
10 mg/m2 on day 4, 5) for the treatment of DS AML and reported five early deaths among 
51 children with DS AML, one prior to start of chemotherapy, two during induction, one 
after HAM and one on day 40 due to intracranial hemorrhage.12  
 
Intensification of treatment using the proposed cycles AE and MA was also used for older 
patients with DS AML (> 4 years of age) on AAML0531. Of 6 eligible patients, one patient 
without reported adverse events during the first two cycles subsequently relapsed and went 
off study. A total of 4 patients received AE (Intensification I) without Grade 4 or 5 adverse 
events. During the subsequent MA cycle, one each among these 4 patients developed Grade 
4 infection/Grade 3 hypotension, Grade 3 infection/Grade 3 mucositis and Grade 3 
infection and one had no reported adverse events (Alan Gamis, Todd Alonzo, personal 
communication). One patient subsequently died after the fifth cycle of therapy 
(Intensification III/Capizzi II HD Ara-C) due to colitis and systemic inflammatory response 
syndrome in the setting of relapse and pancytopenia. 
 
Administration of reduced doses of mitoxantrone and cytarabine (hAM; Ara-C 1g/m2 q12 
on Day 1-3 and mitoxantrone 7 mg/m2 on Day 3, 4) is being used in study BFM DS-ML 
2006 by the BFM study group for all patients with DS AML. This treatment element, 
however, is equivalent in intensity to predecessor study AAML0431 (cumulative drug 
doses BFM DS-ML 2006 v. AAML0431: Cytarabine 27.4 v. 27.8 g/m2; doxorubicin 
equivalents 226 v. 240 mg/m2; etoposide 450 v. 740 mg /m2) and, therefore, appears more 
appropriate for patients with standard risk DS AML rather than the group of high risk 
patients selected for an intensified approach.  
 

 
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Based on these reports, MA as currently used for high risk non-DS AML in study 
AAML1031, will be used for intensification of treatment for DS-AML. The intensive 
therapy proposed for the high risk group of DS AML in this study is expected to be 
associated with increased morbidity, but not with excessive mortality. Given the overall 
poor prognosis of relapsed DS AML, the risks associated with intensification of treatment 
for high risk DS AML are expected to be balanced by the benefit of improved disease 
control. Since intermediate steps short of introduction of MA (and AE) will not achieve 
the intended net intensification of treatment for high risk DS AML, we will adopt cycles 
MA (Ara-C 1 g/m2 q12 h on day 1-4, mitoxantrone 12 mg/m2 on day 3-6) and AE (Ara-C 
1g/m2 q12h on day 1-5, etoposide 150mg/m2 on day 1-5) from the treatment protocol for 
high risk non-DS AML, which was associated with a 22.5% rate of febrile neutropenia and 
1.6% rate of non-leukemic death in study AAML0531 in children without DS34 and retain 
a cycle of Capizzi II from the treatment of DS AML used in predecessor study AAML0431, 
which was associated with a 29.7% rate of > Grade 3 febrile neutropenia (see Section 2.4, 
Table 2 ) but no treatment-related mortality.35    
 
To guard against excessive toxicity, the proposed approach will take advantage of the 
experience of studies CCG 2891, NOPHO-9336 and BFM DS-ML 2006 and allow recovery 
from myelosuppression prior to and after cycles including HD Ara-C and mitoxantrone. 
Monitoring rules for excessive toxicity and an interim safety analysis after the first 6 
patients on the high risk arm have completed treatment with MA will be included to meet 
the objective of safety while pursuing adequate intensification of treatment for high risk 
disease.  
 
The favorable outcomes of DS AML (EFS 80% or greater) have been achieved by different 
study groups with a variety of treatment approaches. Studies by COG and legacy groups, 
for example, have used 6 cycles in A2971 and AAML0431. A Japanese study group11 
achieved similar results with only 5 cycles. Interim data for the ongoing DS AML study 
DS-ML 2006 by the BFM study group presented at the COG Fall Meeting in 2014 showed 
an OS of 90% with treatment using 4 cycles of chemotherapy (D. Reinhardt, pers. 
communication). Outcomes appear to be a function of treatment intensity (in the context 
of increased sensitivity of DS AML blasts to cytarabine, anthracyclines, and etoposide) 
rather than of the number of treatment cycles. The same conclusion appears to follow from 
the successful reduction of treatment cycles for non-DS AML from 5 in AAML0531 to 4 
on AAML1031 in keeping with the approach of the MRC in the UK.37 Thus, on 
AAML1531, standard risk DS AML patients will receive 5 cycles of less intensive therapy 
and high risk DS AML patients will receive 4 cycles of more intensive therapy.  
 
2.8 
Impact of This Study on Trial Design for DS AML 
Children with DS AML represent an ideal group to pursue a strategy of dose reduction, 
particularly of cytarabine, based on a strong biological, pharmacological rationale and 
feasibility. Two previous COG studies initiated this effort. A2971 broke ground by using 
induction therapy cycles of lower intensity (TAD) and AAML0431 reduced the dose of 
anthracycline (to 240 mg/m2), eliminated CNS-directed consolidation therapy and 
collected first data on the prognostic value of MRD in DS AML in a blinded fashion. Both 
studies, however, retained cycles of HD Ara-C, which originate from the optimization of 
AML treatment approaches in the general pediatric population and continue to be 
administered to children with DS AML. The hypothesis that HD Ara-C is not required for 
the majority of patients with DS AML to maintain the high survival but contributes to 
avoidable infectious toxicity, prolonged hospitalization and increased resource utilization 

 
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needs to be tested. AAML0431 retrospectively demonstrated the prognostic value of MRD 
in DS AML. AAML1531 now introduces the prospective use of MRD for the stratification 
of treatment intensity for DS AML according to the risk for relapse. 
 
In summary, the proposed study AAML1531 builds on the success of the initial steps taken 
by two previous COG studies, A2971 and AAML0431; implements MRD-based risk 
stratification of DS AML; and by eliminating HD Ara-C advances AML therapy for children 
with DS in the direction of treatment reduction for the majority of patients. In addition, for 
the first time, this protocol provides an intensified therapeutic strategy targeted to high risk 
patients with DS AML, for whom there are currently neither specific treatment studies nor 
treatment recommendations. Thus, AAML1531 achieves risk-adapted stratification of 
treatment for DS AML as a rational and necessary next step of trial design for this vulnerable 
population. 
 
2.9 
Interim Analysis of Patients Treated on the Standard Risk Arm 
According to the study analysis plan, an interim analysis of treatment outcomes for lack of 
efficacy on the Standard Risk (SR) arm (Arm A) was performed after 50% of the expected 
number of EFS events had been observed (after 8 of 16 events). Based on a model 
generated using data of SR patients in the historical control (predecessor study 
AAML0431) and the amount of follow-up available at the time of data cut off (30 June 
2018), 2.4 EFS events were expected in SR patients compared to 8 EFS events observed. 
The projected 2-year EFS for SR patients on AAML1531 is significantly lower than on 
AAML0431 (85.6% compared to 93.5%, p 0.0002). As of October 5, 2018, 9 of 109 SR 
patients on AAML1531 have experienced a relapse at 136 to 327 days after study entry. 
Four of these relapses occurred on therapy (1 during Intensification I, 3 during 
Intensification II) and 5 during the first 6 months of follow up after completion of protocol 
therapy. Of the 9 relapsed SR patients, 8 have died.   
 
These data show lack of sufficient efficacy of AAML1531 protocol therapy for SR patients. 
Based on this information, post-Induction I treatment on the SR Arm A of AAML1531 
was permanently closed. 
 
2.10 
Risk Stratification in DS AML 
2.10.1 Prognostic Value of Minimal Residual Disease in Pediatric AML 
Early response to treatment as assessed by flow cytometric measurement of MRD 
in the bone marrow after induction therapy has been shown to be an independent 
and powerful prognostic factor in pediatric leukemia. In contemporary trials for 
pediatric acute lymphoblastic leukemia for example, MRD has become an 
indispensable part of risk stratification.38 Similarly, MRD has been found to predict 
relapse in pediatric non-DS AML.39,40 Particularly in the large subset of 
intermediate risk AML, MRD allowed the distinction of standard and high risk 
groups whereas the use of morphology alone proved insufficient41 (and 
S. Meshinchi, personal communication). The addition of flow cytometric MRD 
measurement to standard morphological bone marrow evaluation of early 
treatment response improved prognostication in study CCG 2961. The 3-year OS 
of patients in morphological remission after Induction I was 41 ± 26% if MRD was 
positive (> 0.1%), compared to 69 ± 10% for those who were MRD-negative.42 A 
strong prognostic impact of MRD was also apparent in the recent study AAML03P1. 
Among patients in morphological remission at the end of Induction I, 46 (24%) of 188 
were MRD-positive and experienced an increased relapse risk (RR) at 3 years 

 
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(60 ± 16%, P < 0.001) and lower OS (56 ± 16%, P = 0.002) compared with those who 
were MRD-negative (RR 29 ± 8%; OS 80 ± 8%).41 This study not only demonstrated 
the prognostic value of MRD in non-DS AML over morphology alone but also showed 
that 30% of patients deemed not to be in morphological remission were in fact MRD-
negative and had a good outcome.41 MRD was analyzed retrospectively in the recently 
closed successor study AAML0531. The positive predictive value of MRD for relapse 
was 52.5% and the negative predictive value was 86.2% demonstrating that MRD is 
an important prognostic marker in pediatric AML.34 
 
2.10.2 Prognostic Value of Early Response to Treatment in DS AML 
2.10.2.1 Evaluation of early response of DS AML to treatment by morphology  
A2971 indicated a prognostic role for early response to induction 
chemotherapy. Patients with < 5% blasts in the bone marrow (by 
morphology) on day 14 of induction therapy (n = 77) showed a trend to 
increased 5-year DFS compared to those (n = 27) with > 5% blasts (86% 
vs. 72%, P = 0.12).43 Both 5-year EFS and DFS were significantly higher 
for patients in morphological remission at the end of Induction I. The 
morphological blast percentage proved prognostic. EFS was 88 ± 9% for 
patients with 0-1% blasts (n = 53) compared with 73 ± 11% for those with 
> 1% blasts (n = 63; P = 0.032). Similarly, patients with 0-5% blasts 
(n = 107) at the end of Induction I had an EFS of 83 ± 7% compared to 
only 44 ± 33% (P < .001) for those with > 5% blasts (n = 9). DFS was 
90 ± 9% for patients with 0-1% blasts (n = 52) vs. 73 ± 11% for those with 
> 1% blasts (n = 63, P = 0.015). The corresponding DFS using 5% blasts 
at the end of induction as a cutoff was 83 ± 7% (0-5% blasts, n = 106) and 
44% ± 33% (> 5% blasts, n = 9; P < 0.001, T. Alonzo, personal 
communication). These data suggest that, as in non-DS AML, early 
response to therapy is prognostic in DS AML, although morphology alone 
may not be sufficiently sensitive. 
 
2.10.2.2  Evaluation of Early Response of DS AML to Treatment by MRD 
Data collected from 146 patients in the recently completed study 
AAML0431 provide direct evidence for the prognostic value of MRD 
measured by flow cytometry after Induction I in DS AML. The unique 
immunophenotype of DS AML blasts permitted their detection by multi-
parameter flow cytometry at a level as low as 0.01% (D. Campana, 
personal communication). EFS among 125 patients who were MRD-
negative (MRD < 0.05%) was significantly higher than in 21 patients 
(14%) who were MRD-positive (93% v. 76% at 2 years; log-rank 
P = .007). This difference extended to OS (2-yr OS 96% v. 76%, log-rank 
P < 0.001), indicating that the prognostically unfavorable group of DS 
AML patients can be identified by MRD and that this cohort may benefit 
from intensification of therapy. Complying with the FDA requirement that 
the MRD cut-off be above the threshold of sensitivity of the assay, 
AAML1531 will use a cutoff of 0.05%. Based on the data of AAML0431, 
this cut-off safely captures the large majority of MRD-negative patients 
for the proposed reduction in therapy (only 5 patients had MRD levels 
between 0.01 and 0.1%, with 2 of these patients relapsing, 1 electively 

 
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withdrawing at the end of induction, and 2 patients remaining in 
remission). 
 
While MRD measured in the bone marrow by flow cytometry at the end 
of Induction I is prognostic and provides superior detection of persistent 
disease compared to morphology alone, this approach does have 
limitations. On AAML0431, 14 patients relapsed and of the 12 patients for 
whom MRD data was available, only 7/12 (58%) were MRD positive at 
the end of Induction I, suggesting that MRD by flow cytometry is unable 
to detect all high risk patients. The reasons for this may be related to the 
flow cytometry method used to identify residual clones, namely the 
leukemia-associated immunophenotype (LAIP) at diagnosis, which is 
susceptible to immunophenotypic gains or losses in the major clone as well 
as the emergence of initially undetected minor clones.41,44 A newer flow 
cytometry methodology based on the detection of “different from 
normal”, which is expected to have improved accuracy and sensitivity, 
will be employed in the current study. The same methodology is currently 
used on AAML1031 to detect MRD in non-DS AML. In addition, the 
proposed study will evaluate alternative DNA-based methods to measure 
MRD such as digital PCR and targeted sequencing. The presence of clone-
specific somatic mutations of the GATA1 gene in DS AML blasts provides 
a particularly suitable target for the detection and quantification of MRD 
for response assessment in clinical trials. Currently there is no validated 
PCR or sequencing assay that could serve as the gold standard for the 
quantitative detection of cells harboring clone-specific GATA1 mutations. 
The value of these molecular methods to detect MRD in DS AML will be 
determined in Exploratory Aim 1.2.2. 
2.10.3 Age  
Age at diagnosis was prognostic in a multivariate analysis of outcomes for 161 DS 
AML patients treated on CCG 2891 with standard timed induction. Age 2 years or 
older was associated with an odds ratio of 4.9 (P = 0.006) for relapse. Children 
older than 4 years (n = 9), in particular, had a significantly lower 6-year-EFS of 
33 ± 31%.3 This finding was confirmed by study A2971, in which patients younger 
than 4 years of age has a significantly higher 5-year-EFS than older children 
(81 ± 7% v. 33 ± 38%) although there were only six children older than 4 years of 
age.10 Consistent with a prognostic impact of age in DS AML, a relapse rate of 
40% was observed in a cohort of 10 children with DS AML 4 years of age or older 
at diagnosis.45 In addition, blasts in 8 of these 10 older children with DS and AML 
lacked a GATA1 mutation. Taken together, the poor outcome and lower prevalence 
of somatic GATA1 mutations suggest that older children have a disease more in 
keeping with pediatric non-DS AML. Children with DS and AML aged 4 years 
and older, therefore, are not eligible for enrollment on the current study.  
 
2.10.4 Blast Cytogenetics 
The impact of cytogenetic abnormalities on outcome in DS AML is less well 
defined than in non-DS AML. Monosomy 7, an accepted marker of high risk 
pediatric and adult AML in the absence of DS, also occurs in DS AML (3.5 -
10%).3,11,46,47 An international retrospective study of 451 DS AML patients found 
monosomy 7 was associated with a moderately worse outcome (7-year EFS 67%), 

 
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but this was not statistically significant.47 Combining data reported for CCG 2891 
and A2971 with a Japanese cohort,11 only 11 of 16 patients (69%) with DS AML 
and monosomy 7 were in continuous remission. These data suggests a potentially 
inferior outcome for patients with DS AML and monosomy 7 compared with 
overall outcomes for DS AML after treatment with CCG-based (EFS 79%)10 and 
MRC-based protocols (DFS 83%).21 However, as these data are limited and involve 
a very small number of patients, DS AML patients enrolled on AAML1531, whose 
blasts are found to have monosomy 7 will not be assigned to the high risk arm 
based on this cytogenetic finding alone. Instead they will be stratified like patients 
without this cytogenetic abnormality according to the MRD-based assessment of 
their early response to treatment.  
 

 
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3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
3.1 
Study Enrollment 
3.1.1 
Patient Registration 
Prior to enrollment on this study, patients must be assigned a COG patient ID 
number. This number is obtained via the Patient Registry module in Open once 
authorization for the release of protected health information (PHI) has been 
obtained. The COG patient ID number is used to identify the patient in all future 
interactions with COG. If you have problems with the registration, please refer to 
the online help. For additional help or information, please contact the CTSU Help 
Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
In order for an institution to maintain COG membership requirements, every patient 
with a known or suspected neoplasm needs to be offered participation in APEC14B1, 
Project:EveryChild A Registry, Eligibility Screening, Biology and Outcome Study. At 
this time, however, enrollment onto APEC14B1 is NOT a prerequisite to 
enrollment on AAML1531. 
 
A Biopathology Center (BPC) number will be assigned as part of the registration 
process. Each patient will be assigned only one BPC number per COG Patient ID. 
For additional information about the labeling of specimens please refer to the 
Pathology and/or Biology Guidelines in this protocol. 
 
Please see Appendix I for detailed CTEP Registration Procedures for Investigators 
and Associates, and Cancer Trials Support Unit (CTSU) Registration Procedures 
including: how to download site registration documents; requirements for site 
registration, submission of regulatory documents and how to check your site’s 
registration status. 
 
3.1.2 
IRB Approval 
Each investigator or group of investigators at a clinical site must obtain IRB 
approval for this protocol and submit IRB approval and supporting documentation 
to the CTSU Regulatory Office before they can be approved to enroll patients. For 
CTEP and Division of Cancer Prevention (DCP) studies open to the National 
Clinical Trials Network (NCTN) and NCI Community Oncology Research 
Program (NCORP) Research Bases after March 1, 2019, all U.S.-based sites must 
be members of the NCI Central Institutional Review Board (NCI CIRB). In 
addition, U.S.-based sites must accept the NCI CIRB review to activate new 
studies at the site after March 1, 2019. Local IRB review will continue to be 
accepted for studies that are not reviewed by the CIRB, or if the study was 
previously open at the site under the local IRB. International sites should continue 
to submit Research Ethics Board (REB) approval to the CTSU Regulatory Office 
following country-specific regulations.  
 
Sites participating with the NCI CIRB must submit the Study Specific Worksheet 
for Local Context (SSW) to the CIRB using IRBManager to indicate their intent 
to open the study locally. The NCI CIRB’s approval of the SSW is automatically 
communicated to the CTSU Regulatory Office, but sites are required to contact the 
CTSU Regulatory Office at CTSURegPref@ctsu.coccg.org to establish site 

 
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preferences for applying NCI CIRB approvals across their Signatory Network. Site 
preferences can be set at the network or protocol level. Questions about 
establishing site preferences can be addressed to the CTSU Regulatory Office by 
email or calling 1-888-651-CTSU (2878). 
 
Sites using their local IRB or REB, must submit their approval to the CTSU 
Regulatory Office using the Regulatory Submission Portal located in the 
Regulatory section of the CTSU website. Acceptable documentation of local 
IRB/REB approval includes: 
 
Local IRB documentation; 
 
IRB-signed CTSU IRB Certification Form; and/or 
 
Protocol 
of 
Human 
Subjects 
Assurance 
Identification/IRB 
Certification/Declaration of Exemption Form. 
 
In addition, the Site-Protocol Principal Investigator (PI) (i.e. the investigator on 
the IRB/REB approval) must meet the following criteria in order for the processing 
of the IRB/REB approval record to be completed: 
 
Holds an active CTEP status; 
 
Rostered at the site on the IRB/REB approval (applies to US and Canadian 
sites only) and on at least one participating roster; 
 
If using NCI CIRB, rostered on the NCI CIRB Signatory record; 
 
Includes the IRB number of the IRB providing approval in the Form FDA 
1572 in the RCR profile; and 
 
Holds the appropriate CTEP registration type for the protocol.  
 
Additional Requirements 
Additional requirements to obtain an approved site registration status include: 
 
An active Federal Wide Assurance (FWA) number; 
 
An active roster affiliation with the Lead Protocol Organization (LPO) or 
a Participating Organization (PO); and 
 
Compliance with all protocol-specific requirements (PSRs). 
 
For information about the submission of IRB/REB approval documents and other 
regulatory documents as well as checking the status of study center registration 
packets, please see Appendix I. 
 
Institutions with patients waiting that are unable to use the Portal should alert the 
CTSU Regulatory Office immediately at 1-866-651-2878 in order to receive 
further instruction and support. For general (non-regulatory) questions call the 
CTSU General Helpdesk at: 1-888-823-5923. 
 
Note: Sites participating on the NCI CIRB initiative and accepting CIRB 
approval for the study are not required to submit separate IRB approval 
documentation to the CTSU Regulatory Office for initial, continuing or 
amendment review. 
 
 

 
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3.1.3 
Study Enrollment 
Patient enrollment will be facilitated using the Oncology Patient Enrollment 
Network (OPEN). OPEN is a web-based registration system available on a 24/7 
basis. OPEN is integrated with CTSU regulatory and roster data and with the Lead 
Protocol Organization (LPOs) registration/randomization systems or the Theradex 
Interactive 
Web 
Response 
System 
(IWRS) 
for 
retrieval 
of 
patient 
registration/randomization assignment. OPEN will populate the patient enrollment 
data in NCI’s clinical data management system, Medidata Rave. 
 
Requirements for OPEN access:   
 
A valid CTEP-IAM account; 
 
To perform enrollments or request slot reservations:  Must be on an LPO 
roster, ETCTN corresponding roster, or participating organization roster 
with the role of Registrar. Registrars must hold a minimum of an Associate 
Plus (AP) registration type; 
 
If a Delegation of Tasks Log (DTL) is required for the study, the registrars 
must hold the OPEN Registrar task on the DTL for the site; and 
 
Have an approved site registration for the protocol prior to patient 
enrollment. 
 
To assign an Investigator (IVR) or Non-Physician Investigator (NPIVR) as the 
treating, crediting, consenting, drug shipment (IVR only), or receiving investigator 
for a patient transfer in OPEN, the IVR or NPIVR must list the IRB number used 
on the site’s IRB approval on their Form FDA 1572 in RCR. If a DTL is required 
for the study, the IVR or NPIVR must be assigned the appropriate OPEN-related 
tasks on the DTL. 
 
Prior to accessing OPEN, site staff should verify the following: 
 
Patient has met all eligibility criteria within the protocol stated timeframes; 
and  
 
All patients have signed an appropriate consent form and HIPAA 
authorization form (if applicable). 
Note:  The OPEN system will provide the site with a printable confirmation of 
registration and treatment information. You may print this confirmation for your 
records.  
 
Access OPEN at https://open.ctsu.org or from the OPEN link on the CTSU 
members’ website. Further instructional information is in the OPEN section of the 
CTSU website at https://www.ctsu.org or https://open.ctsu.org. For any additional 
questions, 
contact 
the 
CTSU 
Help 
Desk 
at 
1-888-823-5923 
or 
ctsucontact@westat.com. 
 
3.1.4 
Timing  
Study enrollment must take place within five (5) calendar days of beginning 
protocol therapy. If enrollment takes place before starting therapy, the date 
protocol therapy is projected to start must be no later than five (5) calendar days 
after enrollment. 
 

 
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NOTE: the above timing does not apply to the diagnosis of Down syndrome, 
only the clinical and laboratory studies. 
 
3.1.5 
Inclusion of Women and Minorities 
Both men and women of all races and ethnic groups are eligible for this study.  
 
3.2 
Patient Eligibility Criteria 
Important note: The eligibility criteria listed below are interpreted literally and cannot 
be waived. All clinical and laboratory data required for determining eligibility of a 
patient enrolled on this trial must be available in the patient's medical/research record 
which will serve as the source document for verification at the time of audit.  
 
All clinical and laboratory studies to determine eligibility must be performed within 
7 days prior to enrollment (except studies confirming the diagnosis of Down 
syndrome) unless otherwise indicated. Bone marrow evaluations must be completed 
within 14 days prior to enrollment. Laboratory values used to assess eligibility must 
be no older than seven (7) days at the start of therapy. Laboratory tests need not be 
repeated if therapy starts within seven (7) days of obtaining labs to assess eligibility. 
If laboratory values are > 7 days old, then the following laboratory evaluations must 
be re-checked within 48 hours prior to initiating therapy: CBC with differential, 
bilirubin, ALT and serum creatinine. Imaging studies, if applicable, must be obtained 
within 2 weeks prior to start of protocol therapy (repeat the tumor imaging if 
necessary). 
 
3.2.1 
Age at Diagnosis 
Children with Down syndrome > 90 days and < 4 years of age at diagnosis of AML 
or Myelodysplastic Syndrome (MDS) (see Appendix III). 
 
NOTE: The presence of myeloblasts in infants with DS during the first 90 days of life 
is consistent with transient myeloproliferative disease (TMD) rather than DS AML.  
 
3.2.2 
Diagnosis 
3.2.2.1 Patients must have constitutional trisomy 21 (Down syndrome) or trisomy 
21 mosaicism (by karyotype or FISH). 
 
3.2.2.2 Patient has one of the following:  
 
 
Patient has previously untreated de novo AML and meets the criteria for 
AML with ≥ 20% bone marrow blasts as set out in the WHO Myeloid 
Neoplasm classification (see Appendix II).   
o Attempts to obtain bone marrow either by aspirate or biopsy must 
be made unless clinically prohibitive. In cases where it is clinically 
prohibitive, peripheral blood with an excess of 20% blasts and in 
which adequate flow cytometric and cytogenetics/FISH testing is 
feasible can be substituted for the marrow exam at diagnosis. 
 
 
Patient has cytopenias and/or bone marrow blasts but does not meet the 
criteria for the diagnosis of AML (WHO Myeloid Neoplasm classification, 

 
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see Appendix II) because of < 20% marrow blasts) and meets the criteria for 
a diagnosis of MDS (see Appendix III).  
 
 
For patients who do not meet criteria for AML or MDS as outlined above. 
Patient has a history of Transient Myeloproliferative Disorder (which may or 
may not have required chemotherapy intervention) and: 
i) is > 8 weeks since resolution of TMD with ≥ 5% blasts in the 
peripheral blood, OR 
ii) has an increasing blast count (≥ 5%) in serial bone marrow aspirates 
performed at least 4 weeks apart. 
 
3.2.3 
Prior Therapy 
Children who have previously received chemotherapy, radiation therapy or any 
anti-leukemic therapy are not eligible for this protocol, with the exception of 
cytarabine for the treatment of TMD (see Section 3.2.5.2 for timing restriction). 
 
3.2.4 
Organ Function Requirements 
There are no minimal organ function requirements for enrollment on this study.  
 
Note: See Section 5.0 for dose adjustment in case of significant organ dysfunction. 
Previous cardiac repair with sufficient cardiac function as outlined in Section 5.0 
is not an exclusion criteria. 
 
3.2.5 
Exclusion Criteria 
3.2.5.1 
Patients with promyelocytic leukemia (FAB M3)  
3.2.5.2 
Prior therapy 
Patients ≤ 30 days from the last dose of cytarabine used for treatment of 
TMD. 
 
3.2.6 
Regulatory Requirements  
3.2.6.1 
Each patient’s parents or legal guardians must sign a written informed 
consent. 
3.2.6.2 
All institutional, FDA, and NCI requirements for human subjects 
research must be met. 
 
3.3 
Definitions 
3.3.1 
Transient myeloproliferative disease (TMD)  
A disorder of fetal hematopoiesis defined by the presence of circulating non-
lymphoid, non-erythroid blasts in the blood of infants (< 90 days of age) with 
constitutional trisomy 21 or trisomy 21 mosaicism. 
Note: the resolution date of TMD is defined as the date when blasts, abnormal 
blood counts and clinical signs of TMD were first documented as resolved. 
 

 
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3.3.2 
CNS Leukemia  
3.3.2.1 CNS disease at diagnosis is defined as:  
 
Any number of blasts on a cytospin prep in an atraumatic 
(< 100 RBCs) lumbar puncture.  
 
Blasts in a traumatic tap in which the WBC/RBC ratio in the CSF is 
twice that in the peripheral blood.  
 
Clinical signs of CNS leukemia (such as facial nerve palsy, brain/eye 
involvement or hypothalamic syndrome). Extra-ocular orbital masses 
are not considered CNS leukemia. 
 
Radiographic evidence of an intracranial, intradural mass consistent 
with a chloroma. 
 
See Appendix V for more details.  
 
3.3.2.2 Method of Evaluating Initial Traumatic Lumbar Punctures: 
If the patient has leukemic cells in the peripheral blood and the lumbar 
puncture is traumatic and contains blasts, the following algorithm should 
be used to diagnose CNS disease: 
 
CSF WBC > 2X 
Blood WBC 
CSF RBC 
Blood RBC 
 
A patient with CSF blasts, whose CSF WBC/RBC is 2X greater than the 
blood WBC/RBC ratio, has CNS disease at diagnosis. For example: CSF 
WBC = 60/μL; CSF RBC = 1,500/μL; blood WBC = 46,000/μL; blood 
RBC = 3 X 106/μL: 
 
  60   = 0.04 
> 2X 
 46,000  = 0.015 
1,500 
3 X 106 
 
3.3.3 
Initial CBC 
The first WBC at the treating COG institution. 
 
3.3.4 
High Risk MDS/AML of Down syndrome 
Minimal residual disease (MRD) ≥ 0.05% in the bone marrow at the end of 
Induction I.  
 
3.3.5 
Standard Risk MDS/AML of Down syndrome 
Minimal residual disease (MRD) < 0.05% in the bone marrow at the end of 
Induction I. 
 
Note: For definitions of response criteria, please see Section 10.2 
 
 

 
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4.0 
TREATMENT PLAN 
Timing of protocol therapy administration, response assessment studies, and surgical 
interventions are based on schedules derived from the experimental design or on established 
standards of care. Minor unavoidable departures (up to 72 hours) from protocol directed therapy 
and/or disease evaluations (and up to 1 week for surgery) for valid clinical, patient and family 
logistical, or facility, procedure and/or anesthesia scheduling issues are acceptable (except 
where explicitly prohibited within the protocol). 
 
4.1 
Overview of Treatment Plan 
See the Parenteral Chemotherapy Administration Guidelines (CAG) for children on the 
COG 
website 
at: 
https://www.cogmembers.org/_files/disc/Pharmacy/ChemoAdmin
Guidelines.pdf for special precautions and suggestions for patient monitoring during the 
infusion. As applicable, see the CAG for suggestions on hydration, or hydrate according to 
institutional guidelines. 
 
For COG Supportive Care Guidelines see: 
https://childrensoncologygroup.org/index.php/cog-supportive-care-guidelines under 
Standard Sections for Protocols. 
 
IMPORTANT: An aliquot of the diagnostic bone marrow sample must be sent to the 
laboratory of Dr. M. Loken at Hematologics, Inc. to facilitate later MRD 
measurement (for shipping information see Section 14.2). If a bone marrow sample 
cannot be obtained, please send a blood sample (see Section 14.1). 
 
4.1.1 
Induction I (All Patients) 
All patients enrolled on AAML1531 receive Induction I as the first treatment cycle 
with intrathecal cytarabine, continuous infusion of cytarabine (96 hours), bolus 
infusion of daunorubicin and oral 6-thioguanine (6-TG) twice daily for 4 days 
(TAD). This cycle has a minimum duration of 28 days. 
 
4.1.2 
End of Induction I Bone Marrow Aspirate (BMA) 
1) End of Induction I BMA should be obtained between Induction Day 28 to 
Day 49 as guided by count recovery. 
 
2) If the marrow is hypoplastic and/or there is little or no evidence of normal 
hematopoiesis, a repeat marrow should be performed after a further 7–21 days 
(based upon the peripheral blood count recovery and the clinician’s judgment) 
and remission status assessed at this later time point. If the bone marrow 
remains hypoplastic, then marrow studies should be repeated every 1-3 weeks 
(based upon the peripheral blood count recovery and the clinician’s judgment) 
until an accurate bone marrow status is ascertained.  
 
3) Patients with an MRD value < 0.05% (MRD-negative) will be removed from 
study and should receive the remainder of therapy per standard of care. 
Patients with an MRD value ≥ 0.05% (MRD-positive) will continue treatment 
on the High Risk Arm (Arm B). All patients will need to wait for the MRD 

 
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assessment prior to proceeding to Induction II (partial response and refractory 
disease are described below).  
4.1.3 
Risk Group Assignment 
After recovery from the Induction I, patients are assigned to either the Standard or 
High Risk Group based on the level of MRD in the bone marrow aspirate 
(approximately Day 28). If there is no sign of count recovery a bone marrow 
aspirate should be performed no later than Day 49 from the start of Induction I to 
rule out the presence of refractory MDS/AML.  
 
4.1.4 
Standard Risk Patients 
Standard risk patients are those with an MRD level of < 0.05% at the end of 
Induction I. Arm A for Standard Risk patients closed to accrual and treatment 
with amendment #4A. Patients enrolled after amendment #4A who are Standard 
Risk after Induction I will be removed from study and will continue treatment off 
protocol per standard of care. No further data will be collected on these patients.  
 
4.1.5 
High Risk Patients 
High risk patients are those with a MRD level of ≥ 0.05% in the bone marrow at 
the end of Induction I. They are treated according to Arm B. Following Induction 
I they receive a cycle of mitoxantrone and cytarabine (MA) as Induction II; a cycle 
of cytarabine and etoposide (HR-AE) as Intensification I; a cycle of Capizzi II (HD 
Ara-C and Asparaginase) as Intensification II for a total of 4 cycles of intensive 
therapy. No additional intrathecal therapy will be given after Induction I.  
 
Patients in the High Risk Group (Arm B) will have a repeat bone marrow aspirate 
and MRD measurement performed at the end of Induction II (MA) as part of 
standard clinical care for patients with previously positive MRD. The MRD result 
at the end of Induction II will be reported. However, MRD data obtained at the end 
of Induction II will not be used for treatment decisions but will aid in evaluating 
the performance of the MRD test. HR patients in a morphologic remission but 
MRD-positive should remain on protocol therapy. 
 
4.1.6 
Peripheral Blood Count Recovery 
1) If the bone marrow after Induction I shows a complete response (CR, see 
Section 10.2), Induction II will be administered when the ANC ≥ 1000/μL and 
platelets ≥ 100,000/μL. A CBC should be repeated every 4 days until counts 
are adequate. 
 
2) For all cycles, if the counts have not recovered by Day 49, repeat the bone 
marrow studies. A bone marrow biopsy is encouraged. 
 
3) If the platelet count is rising progressively to 75,000/μL but has not reached 
100,000/μL by three weeks after the ANC has reached 1,000/μL, patients may 
proceed to the next cycle of therapy.  
 
4) Note: Patients with a Partial Response (PR) or Refractory Disease (RD) (> 5% 
leukemic blasts confirmed by MRD) should start Induction II (MA) on the 
High risk Arm (Arm B) regardless of blood counts. Patients who have a 

 
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positive MRD (> 0.05% leukemic blasts) at the end of Induction I should start 
Induction II (MA) on the High risk Arm (Arm B) no later than Day 56 even if 
they have not met criteria for count recovery.    
 
4.1.7 
Use of Cardioprotectant (Dexrazoxane) 
The use of dexrazoxane in association with anthracyclines will be left to 
institutional preference and per institutional guidelines. Therefore, is neither 
mandated nor prohibited by the AAML1531 protocol. Data on dexrazoxane use 
and cardiac outcomes (shortening fraction and ejection fraction) will be 
prospectively collected during chemotherapy and follow-up.  
 
NOTE: As per the product monograph ZINECARD® (dexrazoxane) is indicated 
for reducing (preventing) the incidence and severity of cardiotoxicity associated 
with doxorubicin administration for the treatment of metastatic breast cancer in 
patients who have already experienced a partial response or at least maintained 
stable disease.48 This is based on efficacy for cardioprotection in metastatic breast 
cancer patients who had already received at least 300 mg/m2 of doxorubicin.49 
 
 
 
 

  
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Induction I (ALL PATIENTS) 
4.2.2 
Required Observations in Induction I 
All baseline studies (a-m below) must be performed prior to starting 
protocol therapy unless otherwise indicated below.  
 
a. Physical exam, CBC with differential & platelets, twice weekly while in the 
hospital.  
b. Creatinine, BUN, weekly while in the hospital. 
c. Electrolytes (Ca++, Mg++, PO4), weekly while in the hospital. 
d. AST, ALT, bilirubin (unconjugated and conjugated), weekly while in the 
hospital. 
e. Height, weight. 
f. ECG. 
g. ECHO or MUGA. 
h. Unilateral bone marrow aspirate (BMA); biopsy if unable to obtain a BMA  
i. 
Bone marrow baseline immunophenotype (Immunophenotyping including 
CD41 and/or CD61, CD33, CD34, CD14, CD7 and Gly-A is strongly 
recommended). 
j. 
CSF cell count and cytospin. 
k. BMA for MRD, submit sample to Hematologics (peripheral blood may be 
used for baseline sample, see Section 14.1). 
l. 
Optional bone marrow sample for GATA1 mutation analysis (see 
Section  14.1). 
m. Optional bone marrow sample for banking (see Section 14.1). 
 
OBTAIN OTHER STUDIES AS REQUIRED FOR GOOD CLINICAL CARE.  
 
COMMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 2 of 2 

  
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Induction I (ALL PATIENTS) 
4.2.3 
Induction I Treatment Details (All Patients) 
Criteria to start Induction I are the patient eligibility criteria described in 
Section  3.2.  
 
 
 
Intrathecal Cytarabine: 
The dose of intrathecal (IT) cytarabine may be given at the time of the diagnostic 
lumbar puncture. If IT cytarabine is not given at time of diagnostic LP, then 
administer on Day 1 of Induction I. If IT cytarabine is given prior to diagnosis, a 
separate institutional consent must be obtained. 
 
Age-based dosing: 
Age (months):   
Dose:  
0 to  
< 13 months  
20 mg 
13 to 
< 25 months  
30 mg 
25 to  < 36 months  
50 mg 
≥ 36 to < 48 months 
70 mg 
 
For CNS positive patients: IT cytarabine will be given twice weekly until the CSF 
is clear plus two additional intrathecal treatments (for a minimum of 4 and 
maximum of 6 doses). Patients with persistent CNS leukemia after 6 doses of IT 
cytarabine will be taken off protocol therapy. 
 
 
 
Cytarabine: Continuous IV infusion for 96 hours 
Days: 1-4. 
Age-based dosing: 
 
 
Age (months):   
Dose: 
< 36 months            
6.67 mg/kg/24 hrs 
 
 
≥ 36 months             
200 mg/m2/24 hrs 
 
 
DAUNOrubicin: Slow IV push or infusion over 1-15 minutes 
Days: 1-4. 
Age-based dosing: 
 
 
Age (months):   
Dose: 
< 36 months            
0.67 mg/kg/ day 
 
 
≥ 36 months             
20 mg/m2/ day 
 
It is suggested that DAUNOrubicin be administered through the tubing of rapidly 
infusing solution of D5W or 0.9% NaCl and that it is infused into a large vein. 
Protect from sunlight.  
 
Thioguanine: PO BID 
Days: 1-4. 
Age-based dosing: 
 
 
Age (months):   
Dose: 
< 36 months            
1.65 mg/kg/dose BID 
≥ 36 months             
50 mg/m2/dose BID 
 
 
 
Administer dose in AM and at bedtime. Round thioguanine doses to nearest 10 mg 
by quartering tablets. The totally daily dose may be divided unequally in the 
morning and evening to minimize rounding. 
 
See Section 5.0 for Dose Modifications based on Toxicities. 

  
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Induction I (ALL PATIENTS) 
 
Duration Induction I 
The duration of Induction I is a minimum of 28 days. Patients enrolled after 
amendment 4A who are Standard Risk (MRD < 0.05%) after Induction I will be 
removed from study (See Section 8.2).  High risk patients (MRD ≥ 0.05%) 
continue with Induction II of Arm B when ANC ≥ 1000/µL and a platelet count 
≥ 100,000/µL (see Section 4.1.6 for exceptions).  
 

  
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Induction II (High Risk - Arm B) 
4.3.2 
Required Observations in Induction II (High Risk-Arm B)  
 
a. Physical exam, CBC with differential & platelets, twice weekly while in the 
hospital. 
b. Creatinine, BUN, weekly while in the hospital. 
c. Electrolytes (Ca++, Mg++, PO4), weekly while in the hospital. 
d. AST, ALT, bilirubin (unconjugated and conjugated), weekly while in the 
hospital. 
e. Height, weight. 
f. Creatinine clearance or GFR if serum creatinine >2 mg/dL (177 µmol/L) or 
> 2x normal for age. 
g. ECG prior to Induction II. 
h. ECHO or MUGA prior to Induction II. 
i. 
BMA and MRD sample for all patients. Note: MRD results will be reported 
to the study site but not be used for treatment stratification at this time point. 
See Section 14.2 for details. 
 
OBTAIN OTHER STUDIES AS REQUIRED FOR GOOD CLINICAL CARE.  
 
COMMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Induction II (High Risk - Arm B) 
4.3.3 
Induction II Treatment Details (High Risk-Arm B) 
Induction II will be administered to high risk DS AML patients when 
ANC ≥ 1000/µL and platelets ≥ 100,000/µL, or in the setting of a PR or RD any 
time after Day 28 of Induction I. Exception: if the platelet count is rising 
progressively to 75,000/μL but has not reached 100,000/μL by 3 weeks after the 
ANC has reached 1,000/μL, patients may proceed to the next cycle of therapy. 
 
Hospitalization is mandatory for the duration of Induction II until count recovery. 
Dexrazoxane may be administered at the discretion of the treating institution prior 
to Mitoxantrone. Information whether or not dexrazoxane was used must be 
recorded.  
 
The BMA and MRD test will be administered upon count recovery from Induction 
II, and no later than 49 days from the start of Induction II if there is no sign of 
count recovery. 
 
The duration of Induction II is a minimum of 28 days. 
 
High Dose Cytarabine: IV infusion over 1-3 hours, twice a day     
Days: 1-4. 
All patients: 33 mg/kg/dose q12 hours (i.e. 66 mg/kg/day, divided.) 
 
Note: Administer the diluted solution at Hours 0-3 and Hours 12-15. 
 
Administer steroid eye drops such as 0.1% dexamethasone or 1% prednisolone 
ophthalmic solution, 2 drops in each eye every 6 hours beginning immediately 
before the first dose of cytarabine and continuing until 24 hours after the last dose. 
If the patient does not tolerate steroid eye drops, administer artificial tears on an 
every 2-4 hour schedule.  
 
MitoXANTRONE:  
IV over 15 to 30 minutes  
Days: 3-6. 
All patients: 0.4 mg/kg/day 
 
Administer through the tubing of a rapidly infusing solution of D5W or 0.9% 
NaCl. Avoid extravasation; the use of a central line is suggested.  
 
Note: On the Days 3 and 4, mitoXANTRONE should be given 8 hours after the 5th 
and 7th high dose cytarabine infusions are completed. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
 
Patients who do not achieve a complete morphological remission after the end of 
Induction II will go off protocol therapy. 
 
Intensification I will be administered to high risk DS AML patients with a 
morphologic CR (see Section 10.2) when ANC ≥ 1000/µL and platelets 
≥ 100,000/µL. See Section 4.1.6 for exceptions.

  
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Intensification I (High Risk -Arm B) 
4.4.3 
Intensification I (High Risk-Arm B) 
Patients who do not achieve a complete morphological remission by the end of 
Induction II will be off protocol therapy  
 
Intensification I will be administered to high risk DS AML patients when ANC 
≥ 1000/µL and platelets ≥ 100,000/µL. Exception: if the platelet count is rising 
progressively to 75,000/μL but has not reached 100,000/μL by 3 weeks after the 
ANC has reached 1,000/μL, patients may proceed to Intensification I. Intensification 
I is a minimum of 28 days. Hospitalization is mandatory for the duration of 
Intensification I until count recovery.  
 
High Dose Cytarabine: IV over 1-3 hours, twice a day 
Days: 1-5. 
All patients: 33 mg/kg/dose every 12 hrs (i.e. 66 mg/kg/day, divided) 
 
Administer steroid eye drops such as 0.1% dexamethasone or 1% prednisolone 
ophthalmic solution, 2 drops in each eye every 6 hours beginning immediately 
before the first dose of cytarabine and continuing until 24 hours after the last dose. 
If the patient does not tolerate steroid eye drops, administer artificial tears on an 
every 2-4 hour schedule.  
 
Etoposide (ETOP): IV infusion over 90-120 minutes  
Days: 1-5.  
All patients : 5 mg/kg/day 
 
Etoposide doses should immediately follow the 1st, 3rd, 5th, 7th, and 9th dose of 
cytarabine. 
 
Infuse diluted solution (concentration ≤ 0.4 mg/mL). The use of an in-line filter 
during the infusion is suggested. Slow rate of administration if hypotension occurs.  
 
 
See Section 5.1.2 on Allergy to Etoposide. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
 
It is required that patients have an ANC ≥ 1000/µL and a platelet 
count ≥ 100,000/µL before proceeding with Intensification II. See Section 4.1.6 for 
exceptions. 
 

  
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Intensification II (High Risk -Arm B) 
4.5.2 
Required Observations in Intensification II (High Risk-Arm B)  
 
a. Physical exam, CBC with differential & platelets, twice weekly while 
in the hospital. 
b. Creatinine, BUN, weekly while in the hospital.  
c. Electrolytes (Ca++, Mg++, PO4), weekly while in the hospital. 
d. AST, ALT, bilirubin (unconjugated and conjugated), weekly while in 
the hospital. 
e. Height, weight. 
f. Creatinine 
clearance 
or 
GFR 
if 
serum 
creatinine >2 mg/dL 
(177 µmol/L) or > 2x normal for age. 
 
OBTAIN OTHER STUDIES AS REQUIRED FOR GOOD CLINICAL CARE.   
 
COMMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Intensification II (High Risk -Arm B) 
4.5.3 
Intensification II Treatment Details (High Risk-Arm B) 
Intensification II will be administered to high risk DS AML patients when ANC 
≥ 1000/µL and platelets ≥ 100,000/µL. Exception: if the platelet count is rising 
progressively to 75,000/μL but has not reached 100,000/μL by 3 weeks after the 
ANC has reached 1,000/μL, patients may proceed to Intensification II 
Intensification II is a minimum of 28 days. Hospitalization is mandatory for the 
duration of Intensification II until count recovery.  
 
High Dose Cytarabine: IV infusion over 3 hours, every 12 hours Days: 1, 2, 8 
and 9. 
All patients: 100 mg/kg/dose, q12 hours (i.e. 200 mg/kg/day, divided). 
 
 
 
 
Note: Administer the diluted solution at Hours 0-3 and Hours 12-15. 
 
Administer steroid eye drops such as 0.1% dexamethasone or 1% prednisolone 
ophthalmic solution, 2 drops in each eye every 6 hours beginning immediately 
before the first dose of cytarabine and continuing until 24 hours after the last dose. 
If the patient does not tolerate steroid eye drops, administer artificial tears on an 
every 2-4 hour schedule.  
 
Asparaginase (E. coli): IM (preferred) or IV over at least 30 minutes 
through the tubing of a freely running IV of a normal saline or D5W 
infusion 
Days : 2 and 9. 
All patients : 200 international units/kg/dose  
 
Note: Administer at hour 18 on Days 2 and 9 (i.e. 6 hours after the start of the 4th 
and 8th dose of cytarabine). 
 
OR 
 
Asparaginase (Erwinia): IM or IV in 100 mL of normal saline over 1-2 hours 
Days: 2 and 9. 
All patients: 830 international units/kg/dose  
 
Notes:  
 
Administer at hour 18 on Days 2 and 9 (i.e. 6 hours after the start of the 
4th and 8th dose of cytarabine). 
 
Some product lots are not for IV administration; refer to manufacturer for 
batch-specific information.  
 
If Asparaginase (E.coli or Erwinia) is not available, pegaspargase should not be 
given. In this case asparaginase should be omitted. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
Protocol therapy is completed after blood count recovery (ANC ≥ 1000/µL and 
platelets ≥ 100,000/µL and ≥ 7 days from the last platelet transfusion) after 
Intensification II. 

  
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5.0 
DOSE MODIFICATIONS FOR TOXICITIES 
5.1 
Allergy 
5.1.1 
Allergy to Asparaginase 
 
5.1.1.1 Local Reactions (Inflammation at Injection Site, Swelling): 
Continue asparaginase (E.coli or Erwinia) administration in the presence 
of Grade 1 allergic reaction (transient flushing or rash; drug fever < 38°C). 
Premedication with antihistamines in the absence of prior hypersensitivity 
has been discouraged in the past since antihistamine use may mask the 
appearance of systemic allergy and fail to alert the provider of the presence 
of asparaginase neutralizing antibodies. Asparaginase activity assays are 
now commercially available and may help determine if neutralizing 
antibodies are present, thus the use of premedication is left to the discretion 
of the provider.   
 
5.1.1.2 Anaphylaxis/Systemic Allergic Reactions: 
Discontinue asparaginase (E.coli or Erwinia) if the patient develops a 
systemic 
allergic 
reaction 
(urticaria, 
wheezing, 
laryngospasm, 
hypotension, etc.). Should an allergy be diagnosed after the first dose 
given on Day 2 during Intensification II (for high risk patients), then the 
dose due on Day 9 should not be administered (unless the patient 
previously received E. Coli and Erwinia is available, in this case Erwinia 
dosing can be substituted for Day 9 asparaginase).  
 
5.1.2 
Allergy to Etoposide 
Etoposide allergic reactions may be managed with pre-medications such as 
diphenhydramine 1 mg/kg (maximum dose 50 mg) IV, ranitidine 1 mg/kg IV 
(maximum dose 50 mg), hydrocortisone 1-4 mg/kg IV, and by slowing the rate of 
the infusion. Etoposide phosphate may be substituted for etoposide for reactions 
that cannot be controlled with pre-medication and the slowing of the etoposide 
infusion rate. 
 
Etoposide phosphate is rapidly converted to etoposide in vivo and provides total 
drug exposure, as represented by AUC (0-infinity) that is statistically 
indistinguishable from that measured for etoposide at equimolar doses. As such 
etoposide phosphate may be substituted at the same dose and at the same rate. 
 
5.1.3 
ARAC syndrome: 
ARAC Syndrome: Do not withhold cytarabine for fever if it is likely to have been 
caused by the cytarabine. Obtain blood cultures if a central line is present. For 
Grade 3-4 rash or conjunctivitis, withhold cytarabine until toxicity resolved. 
 
 

  
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5.2 
Cardiac Toxicity 
5.2.1 
Left Ventricular Systolic Dysfunction  
Daunorubicin and mitoxantrone will be held if there is evidence of cardiac disease 
by echocardiogram or MUGA (shortening fraction < 28% or EF < 55%). Cardiac 
examination with echocardiogram (or MUGA) is required prior to the start of all 
chemotherapy cycles containing daunorubicin or mitoxantrone, at the end of 
protocol therapy, and in follow up. Please see Section 7.1 for long-term follow-up 
monitoring.  
 
Do not re-start anthracyclines if held for left ventricular shortening dysfunction 
that is not associated with a microbiologically proven bacteremia or sepsis. If the 
left ventricular shortening dysfunction occurred in the setting of clinical sepsis 
even if cultures are negative, then anthracyclines may be reinstituted at the treating 
clinician’s discretion once the shortening fraction has returned to ≥ 28% or 
EF ≥ 55%.  
 
5.3 
CNS Events (Bleed, Thrombosis or Infarction) 
Hold asparaginase. Treat with FFP, factors or anticoagulation as appropriate. Resume at 
full dose when all symptoms have resolved (and evidence of recanalization in case of 
thrombosis by CT/MRI). Consider evaluation for inherited predisposition to thrombosis. 
 
5.4 
Coagulopathy 
If symptomatic, omit Day 9 asparaginase (E.coli or Erwinia) and consider factor 
replacement (FFP, cryoprecipitate). Do not hold asparaginase (E.coli or Erwinia) for 
abnormal laboratory findings without clinical symptoms. 
 
5.5 
Hepatic Toxicity 
5.5.1 
Transaminases 
If the ALT or AST are > 10x ULN, attempts should be made to identify the cause. 
In most cases, the therapy may proceed without modification. 
 
5.5.2 
Hyperbilirubinemia 
 
If the conjugated/direct bilirubin is > 3 mg/dL and is not a result of the leukemia, 
modify the doses of daunorubicin, etoposide, and mitoxantrone as follows below. 
For all cases in which the direct bilirubin is elevated at the point in time that the 
next cycle is to begin, consider delaying the cycle for 1 week to determine whether 
the direct bilirubin falls to an acceptable level. 
 
 
 
 
Asparaginase 
Asparaginase has been associated with hepatic toxicity but dosing guidelines for 
hepatic toxicity are not available. Thus, asparaginase administration (E.coli or 
Erwinia) in the setting of hepatic toxicity is at the clinician’s discretion. 
 
Daunorubicin, Etoposide and Mitoxantrone 
Dosage adjustments should be made as indicated in the table below: 

  
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for age should be hydrated orally or intravenously. Following hydration, the patient 
must have a creatinine clearance ≥ 60 mL/min/1.73m² as measured preferably by 
a nuclear GFR scan, timed urine collection for creatinine clearance, or calculated 
by the Schwartz formula50  before proceeding with HD cytarabine therapy (doses 
of > 1 g/m2). If the CrCl is abnormal (< 60 mL/min/1.73m2) then high dose 
cytarabine should be reduced from twice daily to once daily dosing, at the same 
previously prescribed doses (e.g., 50% daily dose reduction). With this approach, 
previous research has prevented subsequent neurotoxicity in recipients of high 
dose cytarabine in the face of renal insufficiency.51 
 
5.12.2  Etoposide  
In patients with impaired renal function, the following initial dose modification of 
etoposide should be considered based on measured or calculated creatinine 
clearance: for CrCl > 60 mL/min/1.73m2 give full dose, for CrCl of 
15-60 mL/min/1.73m2 give 75% of the dose (25% dose reduction). Subsequent 
doses should be based on patient tolerance and clinical effect.  
 
5.13 
Thrombosis 
Discontinue asparaginase and treat with appropriate antithrombotic therapy, as indicated.  
 
 
6.0 
DRUG INFORMATION  
Please see Appendix IV for drug interactions associated with the drugs used in this study. 
 
See the consent document for toxicities. All other information is available on the COG website in 
the 
Commercial 
Agent 
Monographs 
(https://www.cogmembers.org/_files/disc/pharmacy/
CommercialAgentsMonographs.pdf) and is provided under Standard Sections for Protocols at: 
https://cogmembers.org/site/pages/default.aspx?page=Prot_reference_materials.  
 
 

  
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9.0 
STATISTICAL CONSIDERATIONS 
9.1 
Statistical Design 
This study is a non-randomized study of children with DS and AML. The primary endpoint 
of interest will be the event-free survival (EFS) from the end of Induction I to failure to 
achieve remission at the end of Induction II, relapse, occurrence of a second malignancy or 
death. 
 
Other outcome measures will include average total number of days per patient spent on 
protocol therapy, early death rates, overall survival from on study, treatment related 
mortality, relapse risk, percentage of patients experiencing Grade 3 or higher toxicity, time 
to count recovery, duration of hospitalization, and infection rates. 
 
9.2 
Patient Accrual and Expected Duration of Trial 
AAML1531 will be compared against a fixed 2-year EFS of 93.5% for SR patients and 
76% for HR patients which were observed for comparable patients treated on the prior 
COG study of DS AML patients (AAML0431). For SR patients, assuming a null EFS of 
93.5% at 2 years, there is 95% power to detect an alternative EFS of 87% at 2 years with 
1-sided testing at the 10% level of statistical significance if there are 200 SR patients 
who continue to Induction II. For HR patients, assuming a null EFS of 76% at 2 years, 
there is 80% power to detect an alternative EFS of 88% at 2 years with 1-sided testing at 
the 10% level of statistical significance if there are 41 high risk patients who continue to 
Induction II. The necessary sample size was computed using a binomial test. The final 
analysis of EFS will occur after a minimum of 2 years after the last patient is enrolled. 
 
* Replaced with Amendment #6 
Up to 3% of patients enrolled are expected to be found ineligible, up to 3% are expected 
to go off protocol therapy at the end of Induction I, and approximately 83% of patients 
are expected to be SR. Therefore, enrollment of up to 256 patients will be required to 
assure that there are 200 SR patients who continue to Induction II and there are 41 HR 
patients continue to Induction II. Based on an accrual estimate of 60 patients per year, 
accrual of 256 patients is expected to be completed in approximately 4.3 years. 
 
* Updated with Amendment #4A 
As of October 22, 2018, 26 of the 143 patients enrolled are HR patients who continued 
to Induction II. Up to 3% of patients enrolled are expected to be found ineligible, up to 
3% are expected to go off protocol therapy at the end of Induction I, and approximately 
83% of patients are expected to be SR. Therefore, enrollment of up to 256 patients will 
be required to assure that there are 41 HR patients who continue to Induction II. Based 
on an accrual estimate of 60 patients per year, accrual of an additional 113 patients is 
expected to be completed in approximately 2 years. 
 
* Updated with Amendment #6 
As of August 25, 2020, 32 of the 222 patients enrolled are HR patients who continued to 
Induction II and 5 patients have not been risk classified yet. Assuming 10% of enrolled 
patients will be HR, enrollment of up to 312 patients will be required to assure that there 
are 41 HR patients who continue to Induction II. Based on an accrual estimate of 60 
patients per year, accrual of an additional 90 patients is expected to be completed in 
approximately 1.5 years. 

  
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9.3 
Statistical Analysis Methods 
9.3.1 
Analysis Plan 
The Kaplan-Meier method will be used to estimate 2-year EFS from the end of 
Induction I along with 95% log-minus-log transformed confidence limits 
separately for HR and SR patients at end of Induction I. To compare the Kaplan 
Meier estimate of 2-year EFS (KM2) with fixed values, we will use the test 
statistic 
ln(−ln(KM2))−ln(−ln(0.935))
𝐸𝑠𝑡𝑖𝑚𝑎𝑡𝑒𝑑𝑆𝐷[ln(−ln(𝐾𝑀2))] for SR and 
ln(−ln(KM2))−ln(−ln(0.76))
𝐸𝑠𝑡𝑖𝑚𝑎𝑡𝑒𝑑𝑆𝐷[ln(−ln(𝐾𝑀2))] for HR 
patients and compare these with a standard normal distribution.52 
 
* Updated with Amendment #4A 
AAML1531 was temporarily closed to accrual on October 1, 2018, due to results 
of an interim analysis identifying a significantly lower EFS for SR patients than 
that expected based on comparative data from AAML0431. Post-Induction I 
treatment on the SR Arm A was permanently closed on October 18, 2018. All 
patients enrolled prior to amendment #4A who were assigned to the SR arm were 
to be taken off protocol therapy but may stay on study. These SR patients 
enrolled prior to amendment #4A who are taken off protocol therapy due to the 
closure of treatment on the SR arm will be censored in future EFS analysis at the 
time they were taken off protocol therapy. Patients enrolled after amendment 
#4A who are assigned to the SR arm will be removed from study and continue 
treatment off protocol per standard of care. No further data will be collected on 
these patients.  
 
9.3.2 
Interim Monitoring 
Standard Risk Patients - lack of efficacy 
Monitoring for insufficient EFS from the end of Induction I of the treatment for 
SR patients on this study will utilize monitoring based on the Lan-DeMets 
criterion with an -spending function t2 (truncated at 3 standard deviations) and 
10% type I error. Formal monitoring analyses of differences in the number of 
EFS events observed in the available follow-up and the expected number of 
events under the null hypothesis for the available follow-up will be performed 
after approximately 50% of the expected number of EFS events have been 
observed (after 8 of 16 total events for SR) which is anticipated to occur after 
approximately 156 patients are assigned to the SR arm. For interim monitoring, 
we will conduct a Woolson one-sample log rank test for SR to compare the 
observed EFS with AAML0431 EFS for patients who continue to Induction II 
treated as fixed and known. EFS for AAML0431 will be characterized by the 
following separate cure model for SR patients: 
 
S(t) = 0.905 + (0.095)*exp(-0.00127*t) t measured in days 
 
A p-value that is less than the boundary value of 0.025 for 50% information time 
will result in rejection of the hypothesis and the possible suspension of 
enrollment to the stratum will be identified to the COG DSMC. 
 
* Updated with Amendment #4A 
 
 
 
Interim monitoring has been completed for standard risk patients.  
 

  
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High Risk Patients - efficacy 
Monitoring for improvement in EFS from the end of Induction I of the treatment 
for HR patients on this study will utilize monitoring based on the Lan-DeMets 
criterion with an -spending function t2 (truncated at 3 standard deviations) and 
10% type I error. Formal monitoring analyses of differences in the number of 
EFS events observed in the available follow-up and the expected number of 
events under the null hypothesis for the available follow-up will be performed 
after approximately 50% of the expected number of EFS events have been 
observed (5 to 6 of 11 total events for HR) which is anticipated to occur after 
approximately 16-21 patients are assigned to the HR arm. For interim 
monitoring, we will conduct a Woolson one-sample log rank test for HR patients 
to compare the observed EFS with AAML0431 EFS for patients who continue 
to Induction II treated as fixed and known. EFS for AAML0431 will be 
characterized by the following cure model for HR patients: 
 
S(t) = 0.75 + (0.25)*exp(-0.0037*t) t measured in days. 
 
A p-value that is less than the boundary value of 0.025 for 50% information time 
will result in rejection of the hypothesis and the possible suspension of 
enrollment to the stratum will be identified to the COG DSMC. 
 
High Risk Patients – toxic deaths 
No toxic deaths were observed on AAML0431. Possible suspension of 
enrollment to the High Risk stratum will be considered if 3 or more toxic deaths, 
i.e. deaths as first event while on protocol therapy or within 30 days of going off 
protocol therapy, are observed on the High Risk arm. For a true toxic death rate 
of 2%, there is a 4.9% chance of observing 3 or more toxic deaths. For a true 
toxic death rate of 10%, there is a 79.1% chance of observing 3 or more toxic 
deaths. 
 

  
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10.0 
EVALUATION CRITERIA  
10.1 
Common Terminology Criteria for Adverse Events (CTCAE)  
This study will utilize version 5.0 of the CTCAE of the National Cancer Institute (NCI) for 
toxicity and performance reporting. A copy of the CTCAE version 5.0 can be downloaded 
from the CTEP website  
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm). 
Additionally, toxicities are to be reported on the appropriate case report forms. 
 
Please note: ‘CTCAE v5.0’ is understood to represent the most current version of CTCAE 
v5.0 as referenced on the CTEP website (i.e., v5.02 and all subsequent iterations prior to 
version 6.0). 
 
10.2 
Response Criteria for Patients with Acute Myeloid Leukemia and Myelodysplastic 
Syndrome of DS 
If the bone marrow sample used for the assessment of response is aplastic or severely 
hypocellular, the bone marrow aspirate and complete response assessment (including 
MRD) should be repeated every 7 to 21 days until a response evaluation can be made. 
 
10.2.1 Complete Response (CR)  
The bone marrow is cellular with regenerating normal hematopoietic cells. There 
are  5% blast cells by morphology and there is no evidence of extramedullary 
disease (EMD). 
 
10.2.2 Partial Response (PR)  
If there is neither a Complete Response (CR) nor Refractory Disease (RD).  
 
10.2.3 Refractory Disease (RD)  
Two consecutive bone marrow evaluations (separated by at least 2 weeks) that are 
at least moderately cellular and contain ≥ 5% leukemic blasts by morphology and 
MRD or evidence of extra-medullary disease at the end of Induction II. In patients 
with myelodysplastic syndrome, there are > 5% malignant blasts.  
 
10.2.4 Relapse  
Morphologic relapse after CR is defined as a reappearance of leukemic blasts in 
the peripheral blood or ≥ 5% blasts in the bone marrow not attributable to any other 
cause (e.g. bone marrow regeneration) after documented CR. In the setting of 
recent treatment, if there are no circulating blasts and the bone marrow contains 
5% to 20% blasts, a repeat bone marrow performed at least a week later is 
necessary to distinguish relapse from bone marrow regeneration. Should local 
flow cytometric analyses suggest relapse (by the reappearance of a similar 
immunophenotype to the original leukemia) in the presence of <5% blasts, or ≥ 5% 
blasts in a regenerating marrow, a repeat bone marrow(s) performed at least a 
week later is necessary to confirm relapse by morphologic methods. In such 
instances the date of recurrence is defined as the first date that more than 5% 
leukemic blasts were observed in the marrow.  

  
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Cytogenetic relapse is characterized by reappearance of a cytogenetic or molecular 
abnormality after documented CR. A bone marrow examination performed at least 
a week later is necessary to confirm cytogenetic relapse.  
 
Extramedullary disease relapse is defined as appearance of cytologically proven 
extramedullary disease after documented CR.  
 
10.2.5 Unevaluable 
The bone marrow is aplastic or severely hypocellular (with any blast percentage). 
In this instance, bone marrow evaluation should be repeated every 7 to 21 days 
until a response determination can be made. 
 
 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS 
11.1 
Purpose 
Adverse event data collection and reporting, which are required as part of every clinical 
trial, are done to ensure the safety of patients enrolled in the studies as well as those who 
will enroll in future studies using similar agents.  
 
11.2 
Determination of Reporting Requirements 
Reporting requirements may include the following considerations: 1) the characteristics of 
the adverse event including the grade (severity); 2) the relationship to the study therapy 
(attribution); and 3) the prior experience (expectedness) of the adverse event. 
  
Commercial agents are those agents not provided under an IND but obtained instead from 
a commercial source. In some cases an agent obtained commercially may be used for 
indications not included in the package label. In addition, NCI may on some occasions 
distribute commercial supplies for a trial. Even in these cases, the agent is still considered 
to be a commercial agent and the procedures described below should be followed. 
 
Determine the prior experience Expected events are those that have been previously 
identified as resulting from administration of the agent. An adverse event is considered 
unexpected, for reporting purposes only, when either the type of event or the severity of 
the event is not listed in:  
 
 
the current known toxicities for each commercial agent as provided in the Drug 
Information for Commercial Agents Used by the Children’s Oncology Group 
posted on the COG website; or 
 
the drug package insert. 
 
Secondary Malignancy 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (eg, 
treatment with investigational agent/intervention, radiation or chemotherapy). A metastasis 
of the initial neoplasm is not considered a secondary malignancy.  
 
All secondary malignancies that occur following treatment need to be reported via CTEP-
AERS. Three options are available to describe the event:  

  
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11.4 
Routine Adverse Event Reporting 
Note: The guidelines below are for routine reporting of study specific adverse events on 
the COG case report forms and do not affect the requirements for CTEP-AERS reporting. 
 
The NCI defines both routine and expedited AE reporting. Routine reporting is 
accomplished via the Adverse Event (AE) Case Report Form (CRF) within the study 
database. For this study, routine reporting will include all toxicities reported via CTEP-
AERS and all Grade 3 and higher non-hematologic Adverse Events, and all grades of the 
following cardiac Adverse Events: prolonged QTc interval and left ventricular systolic 
dysfunction. 
 
11.5 
Syndrome Reporting 
Unless otherwise specified in this protocol, syndromes should be reported as a single event 
using the CTCAE term for the composite syndrome, and not as the individual events that 
make up the syndrome. For example, Tumor Lysis Syndrome should be reported under the 
composite definition rather than reporting the component events (hyperkalemia, 
hyperphosphatemia, hypocalcemia, hyperuricemia) separately. 
 
12.0 
RECORDS AND REPORTING 
See the Case Report Forms posted on the COG web site with each protocol under “Data 
Collection/Specimens”. A submission schedule is included. 
 
12.1 
Clinical Data Update System (CDUS) 
This study will be monitored by the Clinical Data Update System (CDUS). Cumulative 
CDUS data will be submitted quarterly to CTEP by electronic means. Reports are due 
January 31, April 30, July 31 and October 31. This is not a responsibility of institutions 
participating in this trial. 
 
 
13.0 
PATHOLOGY GUIDELINES AND SPECIMEN REQUIREMENTS 
13.1 
Diagnostic Specimen Requirements for Central Review 
1. 
One Wright & Giemsa stained and 6 unstained bone marrow aspirate smears. 
2. 
Flow cytometry report.  
3. 
Bone marrow pathology report.  
4. 
Cytogenetic report and reports for any molecular testing for chromosomal 
translocations  
5. 
Results of a complete blood and differential count (manual or automated) 
obtained on the same day as the bone marrow aspirate 
6. 
One H&E stained and 6 unstained sections of bone marrow core biopsy (if 
obtained). 
7. 
One H&E stained and 6 unstained sections of bone marrow clot section (if 
available). 
8. 
One Wright & Giemsa stained and 2 unstained peripheral blood smears (if 
available). 
9. 
Specimen Transmittal Form. 

  
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For patients with myeloid sarcoma (chloroma), the following materials must also be 
submitted for central pathology review:  
 
1) 
One H&E stained slide and 6 unstained recuts from the biopsy material of the 
myeloid sarcoma, if obtained. Slides must be sent from a representative block 
with remaining diagnostic material. 
2) 
Immunohistochemistry stained slides that were used to establish the diagnosis 
(this is strongly recommended, but not required; these special stained slides will 
be sent back at the conclusion of review, if requested by contributor). 
3) 
A copy of pathology report (including results of special stains, flow cytometry 
and cytogenetics, if performed). 
 
Please label all materials for Central Review with the subject’s COG identification number, 
the institutional surgical pathology identification number (SPID) and the block number 
from the corresponding report. 
 
13.1.1 Specimen Shipment for Central Review  
Please send all the above mentioned materials by mail or using your 
institution’s courier account to: 
Biopathology Center – AAML1531 
Nationwide Children’s Hospital 
700 Children's Drive, WA1340 
Columbus, OH 43205 
Phone: (614) 722-2865 
Fax: 
(614) 722-2897 
Email: BPCParaffinTeam@nationwidechildrens.org 
 
The Biopathology Center will forward the materials to: 
 
 
 
 
 
 
 
 
13.2 
Local Cytogenetic Analysis and Data Submission to Central Laboratory 
13.2.1 Specimen Collection for Local Cytogenetics Analysis 
Collect approximately 5 mL of bone marrow for cytogenetics in a sodium heparin 
tube (green top vacutainer) or utilize transport media provided by your 
cytogenetics laboratory. It is best to use the specimen from the first or second draw 
for cytogenetics analysis in order to capture the dividing abnormal cells. It is 
recommended that cytogenetics laboratories keep leftover cytogenetic pellets in 
order to evaluate equivocal results. 
 

  
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Please note: Peripheral blood (3-5 mL) collected in sodium heparin should be 
submitted as a back-up to the bone marrow when the marrow sample is suboptimal 
or unobtainable. 
 
It is required that a specimen be sent to your institutional cytogenetics laboratory 
at study entry. Submission of an additional specimen is recommended at relapse, 
but is not required. A case will be considered normal when a +21, mosaic +21 or 
Robertsonian translocation is present. A case will be considered abnormal when 
an acquired clonal chromosomal abnormality is present, in addition to the 
constitutional +21, mosaic +21, or Robertsonian translocation. 
 
13.2.2 Data Submission to Central Laboratory Following Local Cytogenetics Analysis 
Submit the following to one of the central COG Cytogenetics Laboratories listed 
below after completion of local cytogenetic studies: 
1) 
COG Cytogenetics Reporting Form (CYTOGFRM FISHFRM.pdf available 
from Generic Forms on COG Web site).  
2) 
If abnormal, two different abnormal karyotypes from each cell line. 
3)  If normal, two normal karyotypes. 
4)  If FISH was performed, please send the following: 
a) Images to document the findings, and a  
b) COG FISH Reporting Form (CYTOGFRM FISHFRM.pdf available 
from Generic Forms on COG web site).  
 
Please send above materials by e-mail (preferably as a PowerPoint file) to the 
following COG Cytogenetics Laboratories:  
 
WEST OF MISSISSIPPI RIVER 
(INCLUDE MINNESOTA AND 
WISCONSIN), AUSTRALIA, NEW 
ZEALAND, WESTERN CANADA 
EAST OF MISSISSIPPI RIVER  
(EXCLUDE MINNESOTA AND 
WISCONSIN), EUROPE, EAST 
CANADA 
 
SEND TO:  
 
SEND TO: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
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14.2  
Destination 1 (MRD for Michael Loken’s Laboratory) 
14.2.1 Forms 
Utilize AML Specimen Transmittal forms specific for AAML1531.  
 
14.2.2 Labeling and Packaging 
 
Label the tubes with COG ID number, date and time of collection, time 
point (include treatment cycle and day of cycle), and source of material 
(e.g., bone marrow).  
 
Specimens should be placed inside a leak proof biohazard envelope with 
absorbent material and then a pressure resistance Tyvek envelope.  
 
14.2.3 Shipping 
MRD samples should be mailed by FEDERAL EXPRESS PRIORITY 
OVERNIGHT. COG sites may use the COG Federal Express account number 
available at:  
https://members.childrensoncologygroup.org/_files/reference/FEDEXmemo.pdf  
 
Include a COG Specimen Transmittal form and ship at room temperature to: 
 
 
 
 
  
  
 
 
 
Lab contact information: 
Phone: (800) 860-0934 or (206) 223-2700 
Fax: (206) 223-5550 
Weekends and After Hours: (206) 264-4459 
Email: clientservices@hematologics.com 
 
Weekend Specimens: The lab is staffed 6 days a week. For Saturday delivery, 
please use a Saturday delivery sticker and check the Saturday delivery box on the 
address label. Both sticker and checked box are necessary to insure proper 
handling.  
 
Please allow 2-4 business days for samples to be processed. Results from end of 
Induction 1 will be communicated by fax and entered into RAVE. 
 
 

  
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14.3 
Destination 2 (COG Leukemia Biospecimen Bank, BPC) 
14.3.1 Forms 
Utilize AML Specimen Transmittal forms specific for AAML1531.  
 
14.3.2 Labeling and Packaging 
 
Ship at room temperature. 
 
Label the tubes with COG ID number, patient name, date of birth, date and 
time of collection, time point (include treatment cycle and day of cycle), 
and source of material (i.e., bone marrow).  
 
Specimens should be placed inside a leak proof biohazard envelope with 
absorbent material and then a pressure resistance Tyvek envelope as per 
IATA regulations. 
 
14.3.3 Shipping 
COG Leukemia Biospecimen Bank 
Nationwide Children’s Hospital  
700 Children’s Drive, C0825 
Columbus, OH 43205 
 
Leukemia Bank contact information: 
Phone: (614) 722-3270 
Fax: 
(614) 722-2856 
Email: MGLab@nationwidechildrens.org 
 
Call or email the Leukemia Bank only when shipping a sample to be delivered on 
Saturday. 
 
Samples should be mailed by FEDERAL EXPRESS PRIORITY OVERNIGHT. 
COG sites may use the COG Federal Express account number available at:  
https://members.childrensoncologygroup.org/_files/reference/FEDEXmemo.pdf 
 
Samples should be sent as soon as possible and preferably less than 24 hours from 
sample collection; except samples collected on weekends or holidays should be 
shipped the first working day following collection.  
 
Samples may be shipped on Monday through Friday for Tuesday through Saturday 
delivery. If specimen is not shipped on the day of collection, please store in a 
refrigerator until shipment. 
 
14.4 
Banking Specimens 
If the patient consents, any specimens left over on this study after required tests are 
performed will be banked for future research studies, as detailed in Section 14.1. Samples 
should be labeled and shipped as detailed in Section 14.3. 
 

  
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15.0 
DESCRIPTION AND ANALYSIS OF EXPLORATORY STUDIES  
The exploratory studies detailed in this section are for research purposes only, and results will not 
be used to make clinical treatment decisions. With the exception of the MRD results after 
Induction I, all other results will not be obtained in real-time (but rather batched for retrospective 
review,) and results will not be returned to the treating physician. 
 
Please note, if there is an inadequate amount of bone marrow for all studies, the studies should be 
prioritized in the following order: 
 
1. Bone marrow for Minimal Residual Disease Testing (See Section 14.2) 
2. Bone marrow for GATA1 (See Section 14.3) 
3. Bone marrow for viable cell banking (See Section 14.3) 
 
15.1 
Exploratory Aims 
15.1.1 To determine the extent to which elimination of HD Ara-C from treatment of 
standard risk DS AML decreases adverse events and resource utilization in 
children with DS. With amendment #4A, this exploratory aim is complete. 
 
Comparison will be made to predecessor study AAML0431, which included 
treatment with HD Ara-C. Since the first cycle of induction therapy is identical in 
both studies, comparison will be made for the time interval from end of Induction 
I to the completion of protocol therapy. Specifically, we will determine if 
elimination of HD Ara-C from treatment of standard risk DS AML results in: 
 
15.1.1.1  A significant decrease of the number of days per patient spent on 
protocol therapy compared to predecessor study AAML0431. 
Subjects who are MRD negative following Induction I, will continue 
on Arm A for SR patients with the elimination of HD Ara-C. We 
hypothesize that the elimination of the cycle of HD Ara-C will result 
in overall shorter treatment duration by reducing the total number of 
cycles of therapy as well as less cumulative myelotoxicity that might 
contribute to delays in starting subsequent cycles. A two-sample t-test 
will be used to compare average number of days per patient spent on 
protocol therapy for standard risk patients on AAML1531 compared 
to patients treated on AAML0431. 
 
15.1.1.2  A significant decrease of the average number of days of hospitalization 
per patient compared to predecessor studies AAML0431 and A2971. 
We similarly anticipate that the duration of hospitalization throughout 
protocol therapy will be reduced. The number of days in hospital will 
be determined for patients treated on AAML1531 Arm A and 
compared with the predecessor study A2971. A2971 patients with DS 
AML less than 4 years of age stayed a mean of 35.1 days in hospital 
(standard deviation 18.9) after the start of Induction II through the 
remainder of protocol therapy.10 Data regarding days of hospitalization 
during protocol treatment were not collected for study AAML0431. 
These data, however, are available for a subset of patients who were 

  
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treated at centers contributing data to the Pediatric Health Information 
Systems (PHIS) database. Data from AAML0431 will be merged with 
PHIS resource utilization data as previously described.53 Based on this 
prior experience, approximately 40% of patients enrolled on 
AAML0431 will have PHIS data available. Two-sample t-test will be 
used to compare average number of days of hospitalization per patient 
for standard risk patients on AAML1531 compared to patients treated 
on AAML0431 and A2971.  
 
15.1.1.3  A significant decrease of the number (per patient) and rate (per duration 
of treatment) of sterile site infections compared to the predecessor study 
AAML0431. 
In addition, we hypothesize that the elimination of HD Ara-C will lead 
to less morbidity compared to patients whose treatment historically 
included this cycle. On the predecessor study, AAML0431, the 
greatest number of adverse events occurred during the administration 
of HD Ara-C (27.5% of the total reported), with 65% of adverse events 
classified as Grade 3 or greater (6.5% were Grade 4). These included 
Grade 3 or greater sterile site bacterial infection in 23% of patients and 
febrile neutropenia in 30% of patients, resulting in the need for 
continuous hospitalization.35 We anticipate fewer sterile site infections 
for patients with DS AML being treated on AAML1531 without HD 
Ara-C compared with the predecessor study, AAML0431, which 
included the use of HD Ara-C. The number (per patient) and rate (per 
duration of treatment) of sterile site infections of patients treated for 
DS AML on Arm A will be compared to AAML0431 after the start of 
Induction II. Poisson regression will be used to compare rate (per 
duration of treatment) of sterile site infections for standard risk 
AAML1531 patients with AAML0431 patients.  
 
15.1.1.4 A significant decrease of resource utilization by AML treatment 
compared to the predecessor study AAML0431. 
Given our anticipation of fewer sterile site infections and shorter 
hospitalization, we hypothesize that the elimination of HD Ara-C on 
AAML1531 will ultimately result in decreased resource utilization 
(after the start of Induction II on AAML1531 Arm A), as sepsis is the 
single most important factor determining length of stay and hospital 
costs in the treatment of children with leukemia.29 Resource utilization 
will be compared between patients on AAML0431 and AAML1531 at 
PHIS sites by comparing rates of inpatient resource utilization 
including antimicrobial usage, ICU level care resources, standard 
pharmaceutical supportive care measures, and blood products. PHIS 
adjusted inpatient costs will also be compared between patients 
enrolled on AAML0431 and AAML1531.54,55 These measurements of 
resource utilization and standardized costs will be compared between 
the current treatment protocol (Arm A, no HD Ara-C cycle) and 
predecessor study AAML0431, which included the use of HD Ara-C.  
 

  
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These data on duration of therapy, length of hospital admission and 
sterile site infections are routinely submitted for patients with AML. 
Analysis of these exploratory objectives is expected to measure 
potential gains made for SR DS AML patients by decreasing the 
numbers of adverse events through a reduction in treatment intensity. 
 
15.1.2 To compare feasibility and analytical characteristics of flow cytometry, PCR and 
targeted error-corrected sequencing of GATA1 mutations as methods to detect 
MRD in DS AML (Exploratory aim 1.2.1) 
 
Background. MRD. The assessment of early response to treatment by measuring 
minimal residual leukemic disease (MRD) in the bone marrow has provided a new, 
independent and powerful prognostic marker for children with leukemia.38,41 In 
clinical practice, the blasts of acute lymphoblastic leukemia (ALL) can be detected 
by multi-dimensional flow cytometry based on patterns of surface and cytoplasmic 
proteins that are either different from normal hematopoietic cells41 or associated 
with a patient’s specific leukemia.56 Alternatively, malignant lymphoblasts can be 
quantified at submicroscopic levels by DNA-based polymerase chain reactions 
designed to amplify an individual patient’s clone-specific rearrangement of 
antigen receptor (immunoglobulin and T cell receptor) genes.57 In contrast, 
detection of MRD in acute myeloid leukemia (AML) relies predominantly on 
multi-dimensional flow cytometry58 due to the absence of widely prevalent, clone-
specific molecular lesions.59 The blasts of AML in young children with Down 
syndrome (DS), however, typically contain somatic, clone-specific mutations of 
the gene encoding the hematopoietic transcription factor GATA1 on the X 
chromosome.7 These mutations consist of short insertions, deletions and point 
mutations clustering within and adjacent to exon 2.6,7,60 As a result, measurement 
of MRD in DS AML is uniquely feasible both by multi-dimensional flow 
cytometry and DNA-based methods.  
 
Methods. Multi-dimensional flow cytometry has the advantage that it can be 
performed rapidly, but its sensitivity and specificity is limited in DS AML.35 Of 
note, interpretation of flow cytometric MRD data in DS AML is complicated by 
the observation that normal bone marrow cells in DS may express patterns of 
surface markers that are absent from normal non-DS bone marrow (M. Loken, 
personal communication). These patterns must not be confused with evidence of 
residual leukemia (when defined as marker pattern that is different from normal). 
DNA-based methods, such as quantitative real time PCR can detect individual 
patients’ clone-specific GATA1 mutations in exon 2 using either mutation-specific 
primers (and a shared probe) or a mutation-specific probes (and shared primers) 
with high sensitivity and specificity but can encounter limitations to feasibility due 
to constraints on the design of primers and probes in a short exon. Digital PCR 
technology, with its increased options for primer design, and massively parallel 
sequencing, which circumvents the constraints of designing mutation-specific 
oligonucleotide primers and probes, provide promising new approaches to the 
detect GATA1 mutations and measure MRD in DS AML.  
 
Hypotheses. We hypothesize that measurement of MRD in DS AML by digital 
PCR to detect clone-specific GATA1 mutations is feasible and both more sensitive 
and specific than multi-dimensional flow cytometry. We hypothesize further that 

  
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measurement of MRD in DS AML by error-corrected sequencing (ECS) of exon 2 
of GATA1 to detect clone-specific mutations is as sensitive and specific as digital 
PCR but more cost-effective (since there is no need for mutation-specific primer 
and probe design). ECS has shown sensitivity for single base substitutions to 
1:10,000, validated by digital PCR61 and comparable to flow cytometry or RT-
PCR.61  
 
Aims   
1. To determine the feasibility, sensitivity and specificity of digital PCR as a 
method to detect MRD in DS AML (by amplifying each patient’s clone-
specific GATA1 mutation) compared to flow cytometry.  
 
2. To determine the feasibility, sensitivity, specificity and cost of targeted, error-
corrected sequencing (of a region encompassing exon 2 of GATA1) as a 
method to detect MRD in DS AML compared to digital PCR and flow 
cytometry. 
 
Experimental Plan. DNA will be extracted from the diagnostic bone marrow 
samples of the patients with DS AML accrued by AAML1531. Of the 240 
evaluable patients expected to enroll on AAML1531, 228 (95%) are anticipated to 
have diagnostic bone marrow available for analysis. Somatic GATA1 mutations 
will be determined for all samples. MRD will be measured on Day 28 after the 
start of the first cycle of induction chemotherapy (Induction I). During this interval, 
a patient-specific GATA1 mutation probe will be designed and tested for sensitivity 
and specificity in serial dilutions of diagnostic bone marrow DNA. Suitable probes 
will achieve >10-4 GATA1 mutation-positive blasts in a background of normal bone 
marrow DNA. In parallel to MRD measurement by multi-dimensional flow 
cytometry, for patients who consent to this optional study, bone marrow samples 
obtained on Day 28 will be assessed for MRD by digital PCR (using the prepared 
probe and standard primer combinations) as well as ECS of exon 2 of GATA1 
(using high-fidelity PCR amplification across the 240 base pair exon, which can 
be individually barcoded and multiplexed for simultaneous amplification from 
multiple patients). 
 
Analysis Plan. To determine feasibility, the proportion of cases will be recorded 
for which the design of a sufficiently sensitive primer and probe combination for 
digital PCR was successful by Day 28. Similarly, the proportion of cases will be 
determined for which the requirements for DNA quality and multiplex 
amplification were met. Library preparation, sequencing and bioinformatic 
analysis will follow previously established protocols.62 Positive and negative 
percent agreement of MRD results obtained on Day 28 will be compared between 
flow cytometry, digital PCR and ECS for each pair of detection methods. 
Corresponding 95% confidence intervals will be calculated for the percent positive 
and negative agreement for each pair of detection methods. McNemar’s test will 
be used to compare percent positive and negative agreement for two detection 
methods relative to the third detection method. MRD results obtained with each 
method will be correlated with the probability of disease-free survival and the 
cumulative incidence of relapse.  
 

  
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Relevance. This direct comparison of methods is expected to determine the 
optimal method to measure treatment response by MRD and predict relapse risk in 
DS AML in the context of risk-based treatment assignment by flow cytometry 
based MRD. Results, therefore, will establish the foundation of accurate risk-based 
stratification of treatment intensity for future therapeutic trials in DS AML. 
 
15.1.3 To establish a DS AML cell bank of viably frozen bone marrow samples and 
corresponding non-tumor DNA samples collected at end of induction. 
 
Background. The exploratory aim 1.2.2 is to establish a biobank of viably frozen 
bone marrow samples from DS AML patients collected at the end of Induction I 
for future research. 
 
Experimental Plan. Bone marrow cells will be collected at end of Induction 1 and 
viably cryopreserved for those patients who consent to this optional study. Bone 
marrow samples collected at the time point of MRD analysis, which in the majority 
of cases will be obtained at the time of morphological remission, will provide non-
tumor DNA for genomic analyses of DS AML blasts.  
 
Relevance. The repository of viably cryopreserved bone marrow samples of 
DS AML, remission samples and other sources non-tumor DNA will provide 
essential support for the objectives of this study and serve as a vital resource 
for future mechanistic studies in DS AML. 
 

  
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 
Addition to a site roster 
 
Assign the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN 
 
Act as the site-protocol Principal Investigator (PI) on the IRB approval 
 
Assign the Clinical Investigator (CI) role on the Delegation of Tasks Log (DTL). 
 
In addition, all investigators acting as the Site-Protocol PI (investigator listed on the IRB approval), 
consenting/treating/drug shipment investigator in OPEN, or as the CI on the DTL must be rostered 
at the enrolling site with a participating organization.  
 
Additional 
information 
is 
located 
on 
the 
CTEP 
website 
at 
https://ctep.cancer.gov/investigatorResources/default.htm. For questions, please contact the RCR 
Help Desk by email at RCRHelpDesk@nih.gov. 
 
CTSU REGISTRATION PROCEDURES 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU).  
 
Requirements For AAML1531 Site Registration: 
 
• 
IRB approval (For sites not participating via the NCI CIRB; local IRB documentation, an IRB-
signed CTSU IRB Certification Form, Protocol of Human Subjects Assurance 
Identification/IRB Certification/Declaration of Exemption Form, or combination is accepted ) 
 
Submitting Regulatory Documents: 
Submit required forms and documents to the CTSU Regulatory Office using the Regulatory 
Submission Portal on the CTSU website.  
To access the Regulatory Submission Portal log in to the CTSU members’ website, go to the 
Regulatory section and select Regulatory Submission. 
Institutions with patients waiting that are unable to use the Regulatory Submission Portal should 
alert the CTSU Regulatory Office immediately at 1-866-651-2878 in order to receive further 
instruction and support. 
 
Checking Your Site’s Registration Status 
Site registration status may be verified on the CTSU members’ website.  
 
Click on Regulatory  at the top of the screen; 
 
Click on Site Registration; and  
 
Enter the site’s 5-character CTEP Institution Code and click on Go. 
o Additional filters are available to sort by Protocol, Registration Status, Protocol Status, 
and/or IRB Type. 
Note: The status shown only reflects institutional compliance with site registration requirements as 
outlined within the protocol. It does not reflect compliance with protocol requirements for 
individuals participating on the protocol or the enrolling investigator’s status with NCI or their 
affiliated networks. 
 

  
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Data Submission / Data Reporting 
Medidata Rave is a clinical data management system being used for data collection for this 
trial/study. Access to the trial in Rave is controlled through the CTEP-IAM system and role 
assignments.  
Requirements to access Rave via iMedidata: 
 
A valid CTEP-IAM account; and  
 
Assigned a Rave role on the LPO or PO roster at the enrolling site of: Rave CRA, Rave 
Read Only, Rave CRA (LabAdmin), Rave SLA, or Rave Investigator.  
Rave role requirements: 
o Rave CRA or Rave CRA (Lab Admin) role must have a minimum of an Associate Plus 
(AP) registration type; 
o Rave Investigator role must be registered as an Non-Physician Investigator (NPIVR) 
or Investigator (IVR); and 
o Rave Read Only role must have at a minimum an Associates (A) registration type. 
Refer to https://ctep.cancer.gov/investigatorResources/default.htm for registration types and 
documentation required. 
 
Data Quality Portal 
The Data Quality Portal (DQP) provides a central location for site staff to manage unanswered 
queries and form delinquencies, monitor data quality and timeliness, generate reports, and review 
metrics.  
 
The DQP is located on the CTSU members’ website under Data Management. The Rave Home 
section displays a table providing summary counts of Total Delinquencies and Total Queries. DQP 
Queries, DQP Delinquent Forms and the DQP Reports modules are available to access details and 
reports of unanswered queries, delinquent forms, and timeliness reports. Review the DQP modules 
on a regular basis to manage specified queries and delinquent forms. 
 
The DQP is accessible by site staff that are rostered to a site and have access to the CTSU 
website. Staff that have Rave study access can access the Rave study data using a direct link on the 
DQP. 
To learn more about DQP use and access, click on the Help icon displayed on the Rave Home, 
DQP Queries, and DQP Delinquent Forms modules. 
 
Note: Some Rave protocols may not have delinquent form details or reports specified on the 
DQP. A protocol must have the Calendar functionality implemented in Rave by the Lead Protocol 
Organization for delinquent form details and reports to be available on the DQP. Site staff should 
contact the LPO Data Manager for their protocol regarding questions about Rave Calendaring 
functionality.   
 
 
 

  
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APPENDIX II: WHO CLASSIFICATION OF AML63 
Acute myeloid leukemia and related neoplasms 
Acute myeloid leukemia with recurrent genetic abnormalities 
AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 
APL with t(15;17)(q22;q12); PML-RARA 
AML with t(9;11)(p22;q23); MLLT3-MLL 
AML with t(6;9)(p23;q34); DEK-NUP214 
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 
AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 
Provisional entity: AML with mutated NPM1 
Provisional entity: AML with mutated CEBPA 
Acute myeloid leukemia with myelodysplasia-related changes 
Therapy-related myeloid neoplasms 
Acute myeloid leukemia, not otherwise specified 
AML with minimal differentiation 
AML without maturation 
AML with maturation 
Acute myelomonocytic leukemia 
Acute monoblastic/monocytic leukemia 
Acute erythroid leukemia 
Pure erythroid leukemia 
Erythroleukemia, erythroid/myeloid 
Acute megakaryoblastic leukemia 
Acute basophilic leukemia 
Acute panmyelosis with myelofibrosis 
Myeloid sarcoma 
Myeloid proliferations related to Down syndrome 
Transient abnormal myelopoiesis 
Myeloid leukemia associated with Down syndrome 
Blastic plasmacytoid dendritic cell neoplasm 
 
 
 

  
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APPENDIX IV: POSSIBLE DRUG INTERACTIONS 
The lists below do not include everything that may interact with chemotherapy. You should be 
encouraged to talk to your child’s doctors before starting any new medications, using over-the-counter 
medicines, or herbal supplements and before making a significant change in the diet. 
 
Cytarabine (IV) 
 
Some drugs, food, and supplements may interact with cytarabine (be vein). Examples include: 
Drugs that may interact with cytarabine 
 
Clozapine, natalizumab, flucytosine, leflunomide 
 
Food and supplements that may interact with cytarabine 
Echinacea 
 
 
Daunorubicin 
 
Some drugs, food, and supplements may interact with daunorubicin. Examples include: 
Drugs that may interact with daunorubicin 
 Some antibiotics and antifungals (clarithromycin, erythromycin, itraconazole, ketoconazole, 
rifampin) 
 Some antiepileptics (carbamazepine, phenobarbital, phenytoin, fosphenytoin) 
 Some antiretrovirals (lapatinib, lopinavir; nelfinavir, ritonavir, saquinavir, telaprevir, tipranavir) 
 Some heart medications (amiodarone, carvedilol, digoxin, dronedarone, quinidine, propafenone, 
verapamil) 
 Some chemotherapy (be sure to talk to your doctor about this) 
 Ado-trastuzumab emtansine, bevacizumab, trastuzumab, taxane derivatives) 
 Other agents, such as atorvastatin, clozapine, cyclosporine, dexamethasone, ivacaftor, leflunomide, 
lumacaftor, natalizumab, nefazodone, progesterone, ranolazine, rifampin, tacrolimus, tofacitinib, and 
trazodon 
 
Food and supplements that may interact with daunorubicin 
 
Echinacea 
 
Grapefruit, grapefruit juice, Seville oranges, star fruit 
 
St. John’s Wort 
 
Drinks, food, supplements, or vitamins containing “flavonoids” or other “antioxidants” 
 
 

  
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Etoposide 
 
Some drugs, food, and supplements may interact with etoposide. Examples include: 
Drugs that may interact with etoposide 
 
Antibiotics  
o Clarithromycin, erythromycin, nafcillin, rifapentin, rifampin, telithromycin 
 
Antidepressants and antipsychotics  
o Clozapine, nefazodone 
 
Antifungals  
o Fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole 
 
Arthritis medications  
o Leflunomide, tofacitinib 
 
Anti-rejection medications 
o Cyclosporine 
 
Antiretrovirals and antivirals  
o Atazanavir, darunavir, delaviridine, efavirenz, etravirine, fosamprenavir, indinavir, 
lopinavir, nelfinavir, nevirapine, ritonavir, saquinavir, Stribild®, telaprevir,  
 
Anti-seizure medications  
o Carbamazepine, fosphenytoin, phenobarbital, phenytoin, primidone 
 
Heart medications  
o Amiodarone, dronenarone, verapamil 
 
Some chemotherapy (be sure to talk to your doctor about this) 
 
Many other drugs, including the following: 
o Aprepitant, atovaquone, bosentan, deferasirox, ivacaftor, lomitapide, mifepristone, 
modafinil, natalizumab, pimozide 
 
Food and supplements that may interact with etoposide 
 Echinacea 
 Glucosamine 
 St. John’s Wort 
 Grapefruit, grapefruit juice, Seville oranges, star fruit 
 
 
 

  
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Mitoxantrone 
 
Some drugs, food, and supplements may interact with mitoxantrone. Examples include: 
Drugs that may interact with mitoxantrone 
 Aripiprazole 
 Clozapine 
 Cyclosporine 
 Eltrombopag 
 Leflunomide  
 Natalizumab 
 Tofacitinib  
 
Food and supplements that may interact with mitoxantrone 
 Echinacea 
 
 
Thioguanine 
 
Some drugs, food, and supplements may interact with thioguanine. Examples include: 
Drugs that may interact with thioguanine 
 Arthritis medications: leflunomide, tofacitinib 
 Other medications, such as adalimumab, allopurinol, azathioprine, certolizumab pegol, clozapine, 
etanercept, golimumab, infliximab, natalizumab, olsalazine, sulfasalazine 
 
Food and supplements that may interact with thioguanine 
 Echinacea 
 
 
 

  
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APPENDIX V: DEFINITION OF CNS LEUKEMIA 
 
 
 - CNS 1: In cerebral spinal fluid (CSF), absence of blasts on cytospin preparation, regardless of the number 
of white blood cells (WBCs). 
 
- CNS 2a: < 10/µL RBCs; < 5/µL WBCs and cytospin positive for blasts. 
- CNS 2b: ≥ 10/µL RBCs; < 5/µL WBCs and cytospin positive for blasts.   
- CNS 2c: ≥10/µL RBCs; ≥ 5/µL WBCs and cytospin positive for blasts but negative by Steinherz/Bleyer 
algorithm. 
 
- CNS 3a: < 10/µL RBCs; ≥ 5/µL WBCs and cytospin positive for blasts. 
- CNS 3b: ≥ 10/µL RBCs, ≥ 5/µL WBCs and cytospin positive by Steinherz/Bleyer algorithm. 
- CNS 3c: Clinical signs of CNS leukemia (such as facial nerve palsy, brain/eye involvement or 
hypothalamic syndrome). 
 
 
 
 
 
 
 

  
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