A Phase 1/2 Study of CPX-351 (NSC# 775341) Alone Followed by Fludarabine, Cytarabine, and G-CSF (FLAG) for Children With Relapsed Acute Myeloid Leukemia (AML)
Phase: PHASE1
Conditions: Acute Myeloid Leukemia Post Cytotoxic Therapy, Recurrent Childhood Acute Myeloid Leukemia, Secondary Acute Myeloid Leukemia
Sponsor: Children's Oncology Group

 
 
 
AAML1421 
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Page 1 
Activated: 
04/25/2016      
Version date: 02/22/2018 
Closed: 
06/12/2018 
Amendment: #2 
 
CHILDREN'S ONCOLOGY GROUP 
 
AAML1421 
 
A Phase 1/2 Study of CPX-351 (NSC# 775341; 
 Alone Followed by Fludarabine, 
Cytarabine, and G-CSF (FLAG) for Children with Relapsed Acute Myeloid Leukemia (AML)  
 
IND Sponsor for CPX-351: COG 
 
Phase 1 (Completed 12/2016): Participation Limited to the COG Phase 1 Consortium and 
the following institutions: 
OH006 / Nationwide Children’s Hospital 
CA009 / Children’s Hospital of Los Angeles 
MO024 / Children’s Mercy Hospital and Clinics 
TX011 / University of Texas – Southwestern Medical Center 
MD017 / Johns Hopkins University/Sidney Kimmel Cancer Center 
DE008 / Alfred I DuPont Hospital for Children 
CO011 / Children’s Hospital of Colorado 
CA435 / Valley Children’s Hospital  
MA036 / Dana Farber Cancer Institute 
OH033 / Rainbow Babies and Children’s Hospital 
MS005 / University of Mississippi Medical Center 
MN010 / Children’s Hospitals and Clinics of Minnesota-Minneapolis 
CA078 / Loma Linda University Medical Center 
 
Phase 2: Open to COG Institutions in the USA, Canada, Australia and New Zealand 
 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, AND SHOULD NOT BE COPIED, REDISTRIBUTED OR USED FOR ANY 
OTHER PURPOSE.  MEDICAL AND SCIENTIFIC INFORMATION CONTAINED WITHIN THIS PROTOCOL IS NOT INCLUDED TO 
AUTHORIZE OR FACILITATE THE PRACTICE OF MEDICINE BY ANY PERSON OR ENTITY.  RESEARCH MEANS A 
SYSTEMATIC INVESTIGATION, INCLUDING RESEARCH DEVELOPMENT, TESTING AND EVALUATION, DESIGNED TO 
DEVELOP OR CONTRIBUTE TO GENERALIZABLE KNOWLEDGE.  THIS PROTOCOL IS THE RESEARCH PLAN DEVELOPED 
BY THE CHILDREN’S ONCOLOGY GROUP TO INVESTIGATE A PARTICULAR STUDY QUESTION OR SET OF STUDY 
QUESTIONS AND SHOULD NOT BE USED TO DIRECT THE PRACTICE OF MEDICINE BY ANY PERSON OR TO PROVIDE 
INDIVIDUALIZED MEDICAL CARE, TREATMENT, OR ADVICE TO ANY PATIENT OR STUDY SUBJECT.  THE PROCEDURES IN 
THIS PROTOCOL ARE INTENDED ONLY FOR USE BY CLINICAL ONCOLOGISTS IN CAREFULLY STRUCTURED SETTINGS, 
AND MAY NOT PROVE TO BE MORE EFFECTIVE THAN STANDARD TREATMENT.  ANY PERSON WHO REQUIRES MEDICAL 
CARE IS URGED TO CONSULT WITH HIS OR HER PERSONAL PHYSICIAN OR TREATING PHYSICIAN OR VISIT THE NEAREST 
LOCAL HOSPITAL OR HEALTHCARE INSTITUTION. 
 
 
 
 
 
 
 
 
 
 
 
 
  
 

 
 
 
AAML1421 
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CONTACT INFORMATION 
 
To submit site registration 
documents: 
For patient enrollments: 
Submit study data 
Regulatory documentation must 
be submitted to the CTSU via the 
Regulatory Submission Portal. 
Regulatory Submission Portal: 
(Sign in at www.ctsu.org,            
and select the Regulatory 
Submission sub-tab under the 
Regulatory tab.)  
 
Institutions with patients waiting 
that are unable to use the Portal 
should alert the CTSU Regulatory 
Office immediately at 1-866-651-
2878 to receive further instruction 
and support. 
 
Contact the CTSU Regulatory 
Help Desk at 1-866-651-2878 for 
regulatory assistance. 
Please refer to the patient enrollment 
section of the protocol for instructions 
on using the Oncology Patient 
Enrollment Network (OPEN) which 
can be accessed at 
https://www.ctsu.org/OPEN_SYSTEM/ 
or https://OPEN.ctsu.org. 
 
Contact the CTSU Help Desk with any 
OPEN-related questions at 
ctsucontact@westat.com. 
Data collection for this study 
will be done exclusively through 
Medidata Rave. Please see the 
Data Submission Schedule in the 
CRF packet for further 
instructions.  
 
 
The most current version of the study protocol and all supporting documents must be downloaded from the 
protocol-specific Web page of the CTSU Member Web site located at https://www.ctsu.org. Access to the 
CTSU members’ website is managed through the Cancer Therapy and Evaluation Program - Identity and Access 
Management (CTEP-IAM) registration system and requires user log on with CTEP-IAM username and 
password. Permission to view and download this protocol and its supporting documents is restricted and is 
based on person and site roster assignment housed in the CTSU RSS.  
For clinical questions (ie patient eligibility or treatment-related) contact the Study PI of the Lead Protocol 
Organization. 
For non-clinical questions (ie unrelated to patient eligibility, treatment, or clinical data submission) 
contact the CTSU Help Desk by phone or e-mail:  
CTSU General Information Line – 1-888-823-5923, or ctsucontact@westat.com. All calls and correspondence 
will be triaged to the appropriate CTSU representative.  
The CTSU Website is located at https://www.ctsu.org. 
 
 
 
 
 
 
 
 
 

 
 
 
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TABLE OF CONTENTS 
 
SECTION 
PAGE 
STUDY COMMITTEE 
5 
ABSTRACT 
7 
EXPERIMENTAL DESIGN SCHEMA 
8 
1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
9 
1.1 
Primary Aims 
9 
1.2 
Secondary Aims 
9 
1.3 
Exploratory aims 
9 
2.0 
BACKGROUND 
9 
2.1 
Introduction/Rationale for Development 
9 
2.2 
Preclinical Studies and Pharmacokinetics 
12 
2.3 
Adult Studies 
13 
2.4 
Pediatric Studies 
14 
2.5 
Dosing Rationale 
17 
2.6 
Correlative studies 
18 
3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
22 
3.1 
Study Enrollment 
22 
3.2 
Patient Eligibility Criteria 
25 
3.3 
Definitions 
30 
4.0 
TREATMENT PROGRAM 
31 
4.1 
Overview of Treatment Plan 
31 
4.2 
Cycle 1 
34 
4.3 
Cycle 2 
38 
5.0 
DOSE MODIFICATIONS FOR TOXICITIES 
41 
5.1 
Definition of Dose Limiting Toxicity (DLT) 
41 
5.2 
Dose Modifications Due to Toxicity Related to CPX-351 
41 
5.3 
Modifications and Supportive Care Recommendations for Toxicity Related to 
Protocol Therapy (Cycle 1 and Cycle 2) 
42 
6.0 
DRUG INFORMATION 
44 
6.1 
CYTARABINE:DAUNORUBUCIN LIPOSOME (CPX-351) 
44 
6.2 
CYTARABINE 
47 
6.3 
FILGRASTIM, TBO-FILGRASTIM, FILGRASTIM-SNDZ 
50 
6.4 
FLUDARABINE 
52 
7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
56 
7.1 
End of Therapy & Follow-up 
56 
7.2 
Research Studies for which Patient Participation is Optional 
57 
8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY 
CRITERIA 
58 
8.1 
Criteria for Removal from Protocol Therapy 
58 
8.2 
Off Study Criteria 
58 
9.0 
STATISTICAL CONSIDERATIONS 
58 
9.1 
Sample Size and Study Duration 
58 

 
 
 
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9.2 
Study Design 
59 
9.3 
Methods of Analysis 
61 
9.4 
Evaluability for Response 
62 
9.5 
Evaluability for Toxicity 
62 
9.6 
Correlative Biology Studies 
62 
9.7 
Gender and Minority Accrual Estimates 
64 
10.0 
EVALUATION CRITERIA 
65 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
65 
10.2 
Response Criteria 
65 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS 
66 
11.1 
Purpose 
66 
11.2 
Determination of reporting requirements 
66 
11.3 
Expedited Reporting Requirements – Serious Adverse Events (SAEs) 
67 
11.4 
Specific Examples for Expedited Reporting 
68 
11.5 
Reporting Requirements for Specialized AEs 
70 
11.6 
Exceptions to Expedited Reporting 
70 
11.7 
Reporting Requirements - Investigator Responsibility 
71 
11.8 
General Instructions for Expedited Reporting via CTEP-AERS 
71 
11.9 
Reporting Table for Late Phase 2 and Phase 3 Studies – Table A 
72 
11.10 Protocol Specific Additional Instructions and Reporting Exceptions 
73 
11.11 Reporting of Adverse Events for commercial agents – CTEP-AERS abbreviated 
pathway 
73 
11.12 Routine Adverse Event Reporting 
73 
12.0 
STUDY REPORTING AND MONITORING 
74 
12.1 
CDUS 
74 
12.2 
Data and Safety Monitoring Committee 
74 
13.0 
CYTOGENETIC ANALYSIS GUIDELINES AND REQUIREMENTS 
74 
13.1 
Cytogenetic Analysis Overview 
74 
13.2 
Specimen Collection and Submission 
75 
14.0 
SPECIAL STUDIES SPECIMEN REQUIREMENTS 
77 
14.1 
CPX-351 Pharmacokinetics 
77 
14.2 
Exploratory studies 
79 
APPENDIX I: CTEP AND CTSU REGISTRATION PROCEDURES 
85 
APPENDIX II: SAMPLE SUBMISSION FOR CYTOGENETIC/FISH STUDIES 
88 
APPENDIX III: YOUTH INFORMATION SHEETS 
91 
APPENDIX IV: POSSIBLE DRUG INTERACTIONS 
93 
REFERENCES 
95 
 

  
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INDUSTRY SUPPLIED AGENT: 
 
CPX-351, NSC# 775341, Jazz Pharmaceuticals 
 
 
 
OTHER AGENTS: 
 
Fludarabine, NSC# 312887, Commercial 
 
Cytarabine, NSC# 63878, Commercial 
 
Filgrastim, NSC# 614629, Commercial 
 
 
 
 
IND SPONSOR: COG 
 
 
 
 
 
 
 
 
SEE SECTION 14 FOR SPECIMEN SHIPPING ADDRESSES 
 

  
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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 course 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 
Anthracyclines are an essential element for the treatment of AML in children. Unfortunately, the use of 
anthracyclines is limited by cardiotoxicity that may result in devastating cardiomyopathy, a critical problem 
for some children who are AML survivors. Children’s Oncology Group (COG) clinical trials for newly 
diagnosed AML currently deliver approximately 450 mg/m2 cumulative daunorubicin equivalents for those 
who receive 4 courses of chemotherapy alone, or approximately 300 mg/m2 to those who receive 3 courses 
followed by hematopoietic stem cell transplantation. Without better therapeutic options for children with 
AML, methods to minimize cardiac damage while maintaining anthracycline dose-intensity must be 
identified. 
 
Liposomal delivery systems for anthracyclines are a promising strategy to mitigate cardiotoxicity while 
maintaining efficacy. DaunoXome (DNX), a liposomal anthracycline that has been studied extensively in 
adults and children, has shown superior re-induction rates in adults and children with AML when added to 
a fludarabine/cytarabine/G-CSF backbone. Unfortunately, there is presently not a plan to further develop 
DNX in the United States. 
 
This clinical trial proposes to study CPX-351, a novel liposomal preparation of a fixed 5:1 ratio of 
cytarabine/daunorubicin. Preclinical studies and pharmacokinetics performed in adult trials have 
demonstrated that CPX-351 provides elevated and prolonged encapsulated drug concentrations when 
compared to DNX, and orders of magnitude higher than free drug. Phase 1 trials in adults with AML 
demonstrated safety and tolerability at 100 Units/m2/dose given on Days 1, 3, 5. In addition, adult phase 2 
studies demonstrated a statistically significant advantage in overall survival over standard regimens in 
newly diagnosed and relapsed AML, particularly those with adverse prognostic factors.   
 
AAML1421 is a Phase 1/2 study of CPX-351 for children with AML in first relapse. The primary objectives 
of this study will be to determine the recommended Phase 2 Dose (RPD2) of CPX-351 in children with 
AML and to estimate the response rate (CR+CRp) by determining the best response after up to 2 cycles 
(Cycle 1 = CPX-351 followed by Cycle 2 consisting of FLAG). The phase 1 portion of the study has been 
completed as of 12/12/2016 and dose level #1 (135 Units/m2/dose) was found to be the recommended phase 
2 dose.   This clinical trial is a critically important step in developing this novel liposomal anthracycline 
with the potential to reduce cardiotoxicity while maintaining efficacy for children with AML.  
 
 

  
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EXPERIMENTAL DESIGN SCHEMA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cycle 1 
CPX-351 Days 1, 3, and 5 
135 Units/m2/dose 
Study enrollment 
Cycle 2 
FLAG* 
Intrathecal cytarabine 
Disease 
evaluation 
Meet criteria to begin Cycle 2? 
(see Section 4.2.4) 
*FLAG: Fludarabine and cytarabine, Days 1-5, and filgrastim (G-CSF), Days 1-5 and Day 15 to 
count recovery. 
End of protocol 
therapy 
Off protocol 
therapy 
Yes 
No 

  
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1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
1.1 
Primary Aims 
1.1.1 
To determine a recommended phase 2 dose (RP2D) and the toxicities associated 
with CPX-351 in pediatric and young adult patients with relapsed/refractory AML. 
 
1.1.2 
To estimate the response rate (complete remission (CR) plus complete remission 
with partial platelet recovery (CRp)) after CPX-351 (Cycle 1) followed by FLAG 
(Cycle 2) in children with AML in first relapse. 
 
1.2 
Secondary Aims 
1.2.1 
To estimate the response rate (CR + CRp + CRi) after one cycle of CPX-351.  
 
1.2.2 
To describe the pharmacokinetics of plasma cytarabine and daunorubicin after 
CPX-351 administration to pediatric and young adult patients with 
relapsed/refractory AML. 
 
1.3 
Exploratory aims 
1.3.1 
To describe the response in biomarkers of cardiac injury to a single cycle of CPX-
351. 
 
1.3.2 
To explore the effect of CPX-351 on novel biochemical and imaging markers of 
cardiotoxicity, including plasma microRNAs (miRNA) and myocardial 
deformation. 
 
1.3.3 
To explore the role of rare coding variants as risk factors for anthracycline-induced 
cardiomyopathy. 
 
 
2.0 
BACKGROUND 
2.1 
Introduction/Rationale for Development 
Anthracyclines are essential in the treatment of children with AML. However, it is clear 
that anthracycline-associated cardiotoxicity represents the major adverse sequela for 
survivors of childhood AML. CPX-351 demonstrates at least equivalent response rates to 
both 7+3 (cytarabine 100 mg/m2/dose, Days 1-7; daunorubicin 60 mg/m2/dose, Days 1, 3, 
5) and other anthracycline-containing re-induction regimens in adults with AML. For 
adults with high-risk features, CPX-351 is statistically superior in terms of response rate 
and 1-year overall survival (OS). In children with AML, new therapies are needed to 
maintain or improve outcomes while abrogating the devastating consequences of 
cardiotoxicity for survivors. We propose to study CPX-351 in children with AML in first 
relapse.  
 
Administration of a novel liposomal anthracycline-containing agent in children with AML 
in first relapse is warranted. The prognosis for children with AML in first relapse is poor, 
but historical data consistently demonstrates improved CR rates with anthracycline-
containing re-induction regimens.1,2 The International BFM (i-BFM) group reported in 
children with AML in first relapse, that those who received FLAG plus the liposomal 

  
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anthracycline DaunoXome (DNX) had superior CR rates compared to those who received 
FLAG alone. As a result, administration of DNX is standard for re-induction therapy in 
relapsed disease among the European consortia and DNX is currently being included in 
their clinical trials for children with de novo AML (Dirk Reinhardt, personal 
communication). Recent COG studies for relapsed AML have utilized anthracyclines such 
as idarubicin (AAML06P1, AAML07P1) resulting in a cumulative anthracycline exposure 
of approximately 600 mg/m2 daunorubicin equivalents in combination with either high- or 
standard dosing cytarabine. The OS of patients who experienced bone marrow relapse 
following treatment on COG study AAML0531 revealed that those who relapsed within 
one year had a dismal prognosis, whether they received hematopoietic stem cell transplant 
(HSCT) or not (3 year OS 21% ± 17% with HSCT; 27% ±7% without HSCT p=0.524). 
Although those with a first complete response (CR1) > 1 year demonstrate better OS, they 
still fare poorly overall (3 year OS 51% ± 13% without HSCT; 2 year OS of 20% ± 31% 
with HSCT p=0.008; T. Alonzo, personal communication).  
 
New therapies for children with AML are needed as often cure requires HSCT and comes 
at the expense of high cumulative anthracycline doses. Even with such aggressive 
therapies, OS is poor. CPX-351 offers the potential to maintain or improve survival with 
effective and tested agents, cytarabine and daunorubicin, while potentially reducing acute 
and late cardiac side effects through liposomal encapsulation. While the drugs are not 
novel, this may be the only liposomal anthracycline formulation available to children in 
the United States. With an acceptable toxicity profile, it is conceivable that CPX-351 could 
move quickly into a pediatric phase 3 study. 
 
2.1.1 
Anthracycline Use in Childhood AML  
Cytarabine and anthracyclines have been the cornerstones of AML chemotherapy 
for decades. Anthracyclines are critical for improving survival, particularly 
through intensification of induction therapy.3,4 Currently, the most common 
anthracyclines used for the treatment of de novo pediatric AML in the U.S. are 
daunorubicin and mitoxantrone. Idarubicin is an effective anthracycline but is 
more commonly used in the relapsed setting. Doxorubicin is more commonly used 
in acute lymphoblastic leukemia therapy and in patients with solid tumors. There 
is evidence that daunorubicin demonstrates 20-30% less cardiac toxicity per unit 
dose compared with doxorubicin.5,6, and cumulative anthracycline calculations 
should be made accordingly. Higher cumulative doses with conventional 
preparations result in significant long term toxicity. Currently, COG utilizes 
Medical Research Council (MRC)-based chemotherapy, which has improved 
outcomes for children with AML with a 56% and 66% event free survival (EFS) 
and OS, respectively.7-9 Children with newly diagnosed AML enrolled on COG 
trials (AAML0531, AAML1031) who do not receive HSCT receive a cumulative 
anthracycline dose of approximately 444 mg/m2 (assuming a 1:3 conversion of 
mitoxantrone to daunorubicin equivalent), while those assigned to HSCT on 
AAML1031 (high risk cytogenetic/molecular abnormalities, + MRD) receive 
294 mg/m2. Randomized studies in adults have demonstrated that higher doses of 
anthracyclines lead to superior survival.3,4 In fact, adult cooperative groups in the 
United States (SWOG, MDACC) are increasing cumulative anthracycline doses 
for adolescent/young adult high-risk AML patients in recognition that dose-
intensification improves survival.   
 

  
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Anthracycline use is limited by dose-dependent cardiotoxicity. Anthracycline-
associated cardiac myopathy remains a devastating problem for those children who 
are AML survivors.10,11 Studies of long-term survivors utilizing cardiac MRI 
demonstrate reduced ejection fraction and reduced cardiac mass in patients with 
prior anthracycline exposure.12,13 Emerging data also reveal a significant risk of 
cardiovascular deaths in pediatric AML survivors. Two studies from France and 
England reported a 43.7 standard mortality ratio from heart disease for children 
with AML, the highest among all pediatric cancers.14-16 The literature clearly 
demonstrates that late cardiac effects, such as congestive heart failure, increases 
with higher cumulative doses of anthracyclines.10 One analysis of anthracycline-
induced clinical heart failure (A-CHF) in a cohort of 607 children who had been 
treated with anthracyclines concluded that up to 5% of patients will develop A-
CHF 15 years after treatment, and patients treated with cumulative doses of more 
than 300 mg/m2 are at highest risk. Those receiving cumulative anthracycline 
doses of 600 mg/m2 had a 17.8% risk of developing A-CHF (95% CI, 5.5% to 
30.1%).17 It is important to note that cumulative doses of anthracyclines in AML 
patients are among the highest still administered to children and adults with cancer. 
Without better therapeutic options for children with AML, we must identify 
methods to minimize cardiac damage while maintaining anthracycline dose-
intensity. 
 
2.1.2 
Strategies to Address Cardiotoxicity in Childhood AML 
Rigorous testing of new agents that may mitigate the risk of cardiac toxicity is 
critically important to improving outcomes of children with AML. Previous 
strategies have included alternative derivatives of anthracyclines (mitoxantrone, 
idarubicin) and the use of pre-therapy cardioprotective agents such as dexrazoxane 
(DRZ).15,18 The cardioprotective effect of DRZ was initially attributed to its iron 
chelating effect by ADR-925, an EDTA product of the enzymatic DRZ hydrolysis. 
This theory has been challenged, with studies demonstrating that the inhibition of 
the enzymatic conversion of DRZ to ADR-925 does not abolish the 
cardioprotective effect.19 Another theory to explain the protective effect of DZR 
on cardiac tissue is that its antioxidant properties protect the heart from oxidative 
stress.20-23 However, the use of other antioxidants has not demonstrated protection 
of cardiac tissue from oxidative stress. Most recently, DZR was found to prevent 
the accumulation of anthracycline-induced DNA double strand breaks via its effect 
on the depletion of topoisomerase II α and topoisomerase β. This mechanism is 
also a possible explanation for the reported DZR interference with anthracycline 
pharmacokinetics and efficacy.24,25   
 
Liposomal delivery systems for anthracyclines are a promising strategy to mitigate 
cardiotoxicity while maintaining efficacy.26-28 While data is limited in pediatric 
patients, adult studies have been published that demonstrate evidence of decreased 
cardiotoxicity with liposomal anthracyclines.29,30 DNX is a liposomal 
anthracycline that has been studied extensively in adults and children with newly 
diagnosed and relapsed acute leukemia.2,27,28,31-33 The i-BFM group recently 
published their results of a randomized trial comparing FLAG and FLAG + DNX 
in children with relapsed AML. In this study, DNX was given at a dose of 
60 mg/m2/day on Days 1, 3, and 5 in combination with fludarabine 30 mg/m2/day 
x 5 days followed by cytarabine 2000 mg/m2/day x 5 days. There were no 

  
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statistically significant differences in acute toxicities reported on the 2 arms. Of 
note, Grade 3 to 4 skin toxicity was reported in 4% of patients with FLAG/DNX 
compared with 1% of those treated with FLAG and acute cardiotoxicity ≥ Grade 
3/4 was reported in a total of 5 patients with FLAG/DNX and 1 patient treated with 
FLAG. The study did not report cumulative anthracycline dose prior to enrollment. 
Notably, all cardiotoxicities occurred in the setting of infection. Overall, the CR 
rate (ANC > 1000, platelets > 50,000) after 2 courses was 69% with FLAG/DNX, 
and 59% with FLAG (p=0.07).2 Most recently, DNX was compared to idarubicin 
during induction for children with newly diagnosed AML. In that study, DNX had 
antileukemic activity comparable to idarubicin, demonstrated superior EFS in 
patients with t(8;21), and caused less treatment related toxicity.33 The potential 
advantages of liposomal preparations include: prolonged time in the circulation34 
due to protection of the drug from enzymatic inactivation, altered biodistribution 
of the liposome formulation and potential sparing of normal tissue with a resultant 
reduction in toxicity;35 circumvention of drug efflux transporters responsible for 
drug resistance.36,37 Data presented below suggests that CPX-351 has marked 
pharmacokinetic advantages when compared to DNX. More importantly, there is 
no plan to further develop DNX in the United States, and there are no data on the 
long term benefit of liposomal anthracyclines in children with AML. Developing 
CPX-351 in children with AML is an important step in studying the role of 
liposomal anthracyclines as a cardioprotectant strategy.   
 
2.2 
Preclinical Studies and Pharmacokinetics 
CPX-351 is a liposomal formulation of a fixed combination of cytarabine and daunorubicin 
contained within the liposome at a 5:1 molar ratio. Each unit of CPX-351 contains 1.0 mg 
of cytarabine and 0.44 mg of daunorubicin. Prior studies have demonstrated the importance 
of ratio-dependent synergy and antagonism for combinations of cytarabine and 
daunorubicin. Specifically, chemotherapy combinations in vitro can be synergistic, 
additive, or antagonistic depending on the molar ratio between the two agents.38 
Maintaining a fixed ratio is difficult when using combinations of free drugs given the 
different distribution, metabolism, and elimination properties of the individual drugs. 
Controlling the exposure of drug ratios using liposomal preparations can lead to prolonged 
maintenance of the optimal drug ratio and result in improved efficacy in vivo. In vivo 
delivery of fixed cytarabine:daunorubicin ratios (5:1 molar ratio) shown to be synergistic 
in cell culture results in an increase in antitumor activity in tumor-bearing mice. In 
preclinical models, CPX-351 provided elevated and prolonged drug concentrations that 
were orders of magnitude higher than free drug and also higher daunorubicin 
concentrations compared to Daunoxome (DNX) based on literature reports of the latter. 
These pharmacokinetic advantages have held true in clinical studies of CPX-351 as well. 
When CPX-351 was given Days 1, 3, and 5 in the adult phase 1 study, the clearance of 
cytarabine and daunorubicin was less than 120 mL/h/m2 across all dose levels. This is 
markedly less than the clearance of unencapsulated daunorubicin (38,600 mL/h/m2) and 
cytarabine (134,000 mL/h/m2).39,40 This results in striking differences in plasma 
elimination half-life when comparing CPX-351 with free drug or with DNX. For example, 
the plasma elimination half-life of the daunorubicin component of CPX-351 is 
approximately 24 hours compared to 5-8 hours in DNX. At 48 hours, the plasma 
concentration of the daunorubicin component of CPX-351 is about 15 times greater than 
that of DNX. The plasma half-life of the cytarabine component in CPX-351 at the adult 
MTD is approximately 42.5 hours, significantly longer than unencapsulated cytarabine. Of 

  
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interest, there is almost no distribution phase of CPX-351 suggesting that virtually all 
detectable CPX-351 in the plasma exists in the encapsulated form. In addition, these 
concentrations are maintained in the circulation at the synergistic 5:1 ratio.41-43 Therefore, 
CPX-351 accumulates and persists in the bone marrow at drug concentrations many fold 
higher than free drugs and significantly higher than DNX. Further, liposome encapsulation 
may result in the drug being selectively taken up by the leukemia cells with subsequent 
release of cytarabine and daunorubicin from intracytoplasmic liposomes.43   
 
2.3 
Adult Studies 
2.3.1 
Phase 1 Studies of CPX-351 in Adults 
The first phase 1 study included 48 adult patients with relapsed/refractory leukemia 
(43 patients with AML) who received CPX-351 on Days 1, 3, and 5 of treatment. 
Multiple dose levels between 3 units/m2/dose and 134 units/m2/dose were tested. 
Pancytopenia was universal and was associated with fever, infection, and bleeding 
episodes. Dose-limiting toxicity (DLT) was seen at the 134 units/m2/dose level and 
consisted of congestive heart failure (CHF; 1 patient), hypertensive crisis (1 
patient), and persistent cytopenias beyond 56 days (1 patient). The patient with 
CHF had previously received 365 mg/m2 cumulative anthracycline dose and 
sustained cardiac dysfunction during an episode of acute sepsis in which their LV 
ejection fraction dropped from 52% to 29% but subsequently increased to 47% 
after resolution of the septic episode. Responses were observed in 10 of 43 (21%) 
AML patients treated with CPX-351 including 9 CRs and 1 CRp. PK data from 13 
patients showed a median elimination half-life of 31.1 hours for cytarabine and 
21.9 hours for daunorubicin with the 5:1 ratio maintained for greater than 24 hours. 
Notably, encapsulated drug was detectable at least 7 days following the last dose.44 
Subsequent phase 2 studies have provided additional encouraging data regarding 
the tolerability and response rate to CPX-351.  
 
2.3.2 
Phase 2 Trials of CPX-351 in Adults 
Trial 204 involving elderly patients with de novo AML consisted of a 2:1 
randomization between standard 7+3 treatment and up to 2 courses of induction 
and 2 courses of consolidation therapy with CPX-351 given on Days 1, 3, 5. The 
CR + CRi (complete remission with incomplete blood count recovery) rate was 
67% (N=56) for the patients treated with CPX-351 and 51% (N=21) for those 
treated with 7+3 (P=0.07). Ten patients unresponsive to 1 or 2 courses of 7+3 
induction treatment were crossed over CPX-351 treatment. Of those, 4 achieved 
CR and survived > 12 months. Among those on study with an antecedent 
hematologic disorder and high-risk features, CPX-351 demonstrated significant 
superiority over 7+3 therapy [survival HR=0.40 (p=0.01), EFS HR=0.51, 
(p=0.04), and CR + CRi rate (57.6% vs. 31.6%), p=0.013].45 
 
Trial 205 was a phase 2 study performed in patients 18-65 years of age and in first 
relapse. Patients were randomized 2:1 to receive either CPX-351 or to treatment 
selected 
by 
their 
physician, 
which 
was 
most 
often 
MEC 
(mitoxantrone/etoposide/cytarabine). Those with prior HSCT were eligible 
(22 patients in CPX-351 arm; 7 patients in the other arm). Overall CR+ CRi rates 
were 49.4% in CPX-351 arm and 40.9% in control arm (p=0.5). The bone marrow 
aplasia rate on Days 14-21 was superior in the CPX-351 arm (76% vs. 51%). For 

  
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this study, unfavorable risk leukemia was defined using the European Prognostic 
Index based on length of CR1, cytogenetics, age at relapse, and prior history of 
HSCT. For these patients, the CR+CRi rate was 39.3% in the CPX-351 arm 
compared to 27.6% (p=0.34) in those who received physician-choice 
chemotherapy. The 60-day mortality was approximately equal between the 2 study 
arms (14.8% vs 15.9%). However, the 1-year OS in unfavorable/poor risk patients 
was significantly better in the CPX-351 group with a hazard ratio of 0.55, p=0.02.46  
 
As a result of the improved outcomes for high-risk patients in these phase 2 studies, 
Celator (A Jazz Pharmaceuticals Company) is sponsoring a multi-institution, 
randomized phase 3 study enrolling adults aged 60 to 75 with newly diagnosed, 
high-risk patients with AML. Results of this study were presented at the American 
Society of Clinical Oncology meeting in June of 2016.  Adults with de novo, high-
risk AML/MDS were randomized 1:1 to receive standard therapy with 
cytarabine/daunorubicin (7+3) or CPX-351.  CR rates were superior in the CPX-
351 arm 37.3% vs 25.6% in 7+3 arm (p=0.04). Sixty day mortality rates were 
superior in the CPX-351 arm (21.2% 7+3 vs. 13.7% CPX-351). This was largely 
due to decreased incidence of death due to disease in the CPX-351 arm (3.3% 
CPX-351 vs 13.3% in 7+3 arm). The study met its primary objective, with CPX-
351 demonstrating superior OS compared to 7+3 (median survival 9.56 months vs 
5.95 months, HR=0.69, p=0.005).47  
 
The objectives of this study are to evaluate the safety and efficacy of CPX-351 in 
pediatric AML patients. While in pediatrics, ADE is commonly used for induction 
treatment, the standard-of-care in adult patients is 7+3 (cytarabine plus 
daunorubicin). Given that the adult phase 3 study demonstrates improved efficacy 
and an acceptable toxicity profile, it is likely to become a new standard of care in 
AML. In addition, the use of liposomal encapsulation offers the potential of 
reduced long-term cardiac toxicity. It is important to note that these adult studies 
have not demonstrated a difference between CPX-351 and intensive salvage 
regimens including anthracyclines on cardiac function in patients with prior 
anthracycline exposure. Further study is necessary to fully characterize the 
advantages of liposomal anthracyclines compared to conventional anthracycline 
therapy. 
 
2.4 
Pediatric Studies 
2.4.1 
Phase 1 Trial of CPX-351 in Children 
Cincinnati Children's Hospital is conducting a phase 1 study of CPX-351 in 
children and adolescents (≤ 21 years of age). This is a single institution phase 1 
pilot study that aims to assess the pharmacokinetics, toxicity and tolerability of a 
single course of CPX-351 in pediatric and young adults with relapsed/refractory 
hematologic malignancies. The trial utilizes a rolling six design, with dose level 1 
(DL1) starting at the adult MTD of 100 units/m2/dose once daily on Days 1, 3, and 
5. Dose level 2 will escalate past the adult MTD to 134 units/m2/dose once daily 
on Days 1, 3, 5. The trial was activated 09/09/13 and has accrued 8 patients 
(personal communication, Mike Absalon, MD, study PI). Of the 4 patients enrolled 
on DL1, 1 had AML that relapsed within 6 months of initial diagnosis. This patient 
with AML was taken off study with marrow aplasia on Day 37 to receive HSCT. 
Toxicities on DL-1 have been consistent with intensive chemotherapy reinduction 

  
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regimens for relapsed acute leukemia and there were no DLTs. To date, there have 
been 4 patients enrolled on to DL-2. Two of the 4 patients enrolled at that dose 
level have experienced pain that qualify as DLT per their protocol definition. 
Description of those toxicities are outlined here.  
 
The first patient was a 2 year-old boy with relapsed AML after therapy per COG 
study AAML0531 (arm A) and persistent leukemia following a retrieval attempt 
with a clofarabine based regimen. He had a history of bilateral leg pain for which 
he received gabapentin 100 mg three times daily and oxycodone 1.5 mg once daily 
average during the week prior to initiation of CPX-351. CPX-351 was initiated at 
135 Units/m2/dose (Days 1, 3, 5). On Day 5 of protocol therapy he complained of 
leg pain that was not improved with oxycodone. Morphine 0.5 mg IV was 
administered with temporary relief. On Day 6 he appeared to have abdominal pain, 
leg pain, and a severe headache (holding his head with both hands and crying). 
There was neither trauma nor fall witnessed. He received morphine 0.5 mg 
scheduled every 4 hours with inadequate relief. A CT scan and an MRI/MRV of 
the head were obtained and were non-revealing. On Day 7 he developed fever to 
39.5 and broad spectrum IV antibiotics were initiated. On Day 8 he continued to 
have headache that was refractory to scheduled morphine. Morphine continuous 
infusion was initiated. Due to poor pain control, dexamethasone (approximately 6 
mg/m2/day) and fentanyl by PCA were initiated on Day 9. His headache pain 
appeared to be well controlled by Day 10, and the dexamethasone was weaned 
over the following 3 days. Fentanyl by PCA was discontinued on Day 12 when he 
no longer appeared to be in pain. 
 
A second patient who experienced a DLT was a 16 year-old girl with relapsed 
AML after therapy with COG study AAML1031 (arm A) and persistent leukemia 
following a retrieval attempt with FLAG. She had a history of a generalized pain 
syndrome during chemotherapy and from osteomyelitis isolated to a great toe. Pain 
medications prior to initiation of therapy with CPX-351 were fentanyl patch (25 
mcg/h), gabapentin 300 mg three times daily and hydromorphone 2 mg p.o. prn 
break through pain (usual need 0-1 dose/day). CPX-351 was initiated at 135 
Units/m2/dose (Days 1, 3, 5). On Day 3 of protocol therapy she complained of 
headache with temperature of 38.4. The headache improved with acetaminophen 
and dexamethasone (initiated 4 mg q day). She became afebrile and remained 
afebrile until Day 20. On Day 5 of protocol therapy she complained of severe 
generalized aches stating 10 out of 10 pain that was worse than any she had 
previously experienced. Hydromorphone by patient controlled analgesia (PCA) 
was initiated with initially poor response and titrated upward over the following 5 
days. Methocarbamol was initiated empirically on Day 8 for possible muscle-
related pain. Dexamethasone was discontinued on Day 9. Her pain was thought to 
be very well controlled with hydromorphone PCA, methaocarbamol, fentanyl 
patch, and gabapentin by Day 12 of protocol therapy. The pain was reported as 
being significantly improved by Day 14 and a wean of the hydromorphone by PCA 
initiated. On Day 16 she developed pain related to a localized infection that 
resulted in continuation of narcotic therapy. The infection-related pain was 
qualitatively distinct from the pain syndrome she experienced previously during 
the course.  
 

  
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The plan in the Cincinnati Phase 1 trial of CPX-351 is to follow their dose finding 
guidelines and de-escalate to 100 Units/m2/dose. 
 
Preliminary evaluation of the data from the adult clinical trials demonstrated no 
events of ≥ Grade 3 pain related to CPX-351. There are no known mechanisms for 
pain related to CPX-351 that are published. In each case of the Cincinnati Phase 1 
trial, the pain occurred in patients with pre-existing pain complaints, and was 
managed appropriately by the treating physician. The pain that was determined as 
possibly related to CPX-351 resolved 10 days from the time of the 3rd dose (Day 
5) of study drug. Given the importance of testing CPX-351 at a dose level 
comparable to both conventional and liposomal preparations of anthracyclines 
(See Section 2.5), this study will incorporate specific supportive care guidelines 
and DLT criteria to address this particular toxicity. In AAML1421, there were no 
pain-related DLTs observed among the 6 patients treated at the 135 mg/m2 dose 
level in the dose-finding phase.   
 
2.4.2 
Phase 1 portion of AAML1421 
Six patients were enrolled onto the phase 1 portion of the AAML1421 at DL1 (135 
units/m2/dose on days 1, 3, 5). Each of the patients enrolled has experienced 
expected toxicities commonly seen with intensive reinduction regimens. Four of 6 
patients experienced a documented bacterial infection with isolates identified as E. 
coli, streptococcus mitis, staphylococcus epidermidis, and coagulase negative 
staphylococcus.  One of 6 patients experienced a DLT of grade 3 decrease of 
ejection fraction.  This patient had a history of decreased cardiac function after 
induction therapy for de novo AML (baseline echo EF 64%, SF 44%; end of 
induction 1 EF 51%, SF 26%). At that time patient was started on Lisinopril and 
after HSCT had EF of 55%.  At relapse, the patient had a cardiac MRI and EF was 
58%. Echocardiograms performed as a baseline study for AAML1421 
demonstrated EF 58% and SF 32%. The patient was no longer on Lisinopril. After 
reinduction with CPX-351 on AAML 1421, an echocardiogram performed on Day 
38 showed EF of 58% and SF 32%. The start of cycle 2 was delayed due to count 
recovery and echocardiogram 17 days later showed EF of 36% and SF of 25%. 
Repeat echo 3 days later confirmed Grade 3 decrease in EF (40%). The patient was 
otherwise not ill, no evidence of sepsis.  Lisinopril was started and echo 14 days 
later demonstrated resolution with EF 56% and SF 31.5%.  
 
Therefore, 1/6 patients experienced a DLT and per protocol guidelines the Phase 
II dose for this study is 135 units/m2/dose on days 1, 3, 5. There was one death on 
study of a 21 year old female who passed away approximately 7 weeks after 
initiation of therapy from progressive disease manifested as CNS myeloid sarcoma 
demonstrated on MRI. The patient was CNS negative at the onset of therapy. The 
toxicities and death were not attributed to CPX-351 by the treating physician.  
 
2.4.3 
Options for Development of CPX-351 in Children 
Liposomal anthracycline-based strategies are moving forward in childhood AML. 
Based on available adult and pediatric data, our European colleagues in the MRC, 
i-BFM, and NOPHO are utilizing DNX in their pediatric phase 3 studies (personal 
communication). Given the superior pharmacokinetic properties and favorable 

  
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clinical studies of CPX-351, there is strong rationale to support its development in 
COG.   
 
Results of a randomized Phase III study were presented at the American Society 
of Clinical Oncology meeting in June of 2016.48 Adults with de novo, high-risk 
AML/MDS were randomized 1:1 to receive standard therapy with 
cytarabine/daunorubicin (7+3) or CPX-351.  CR rates were superior in the CPX-
351 arm 37.3% vs 25.6% in 7+3 arm (p=0.04). Sixty day mortality rates were 
superior in the CPX-351 arm (21.2% 7+3 vs. 13.7% CPX-351). This was largely 
due to decreased incidence of death due to disease in the CPX-351 arm (3.3% 
CPX-351 vs 13.3% in 7+3 arm). The study met its primary objective, with CPX-
351 demonstrating superior OS compared to 7+3 (median survival 9.56 months vs 
5.95 months, HR=0.69, p=0.005).48  If CPX-351 demonstrates safety and efficacy 
in our proposed study, the COG Myeloid Disease Committee will have the 
opportunity to incorporate it, along with other novel agents, into the successor 
phase 3 or pilot study for de novo AML. The sponsor has enthusiastically 
supported the development of this agent in the pediatric population. Given the adult 
efficacy and potential beneficial reduction in cardiac toxicity a future randomized 
comparison of CPX-351 to standard of care de novo AML is compelling for the 
following reasons:  
 
1) Phase 1/2 and phase 3 adult studies have demonstrated statistically significant 
benefit for high risk patients.  
2) In AAML1031, mitoxantrone/cytarabine is proving to be a treatment block 
that has longer periods of neutropenia and an increased incidence of infection 
(personal communication, Richard Aplenc, MD). 
3) The use of CPX-351 may be of benefit in addressing cardiotoxicity. It is 
reasonable to hypothesize that novel liposomal delivery systems may at least 
maintain anthracycline efficacy while diminishing the risk for anthracycline-
induced cardiomyopathy. 
4) Eventually, this agent may be able to replace ADE and avoid etoposide 
exposure and the possibility of secondary malignancies.  
5) The i-BFM efficacy data for DNX is compelling; however, DNX is not 
available for future study in the United States.  
 
Thus, AAML1421 will be the first prospective study of short and long-term 
cardiotoxicity with liposomal anthracycline delivery in pediatric patients receiving 
high cumulative doses of anthracyclines 
 
2.5 
Dosing Rationale 
As detailed in the background, the pediatric phase 1 trial of CPX-351 at Cincinnati 
Children's Hospital completed the initial dose level of 100 units/m2/dose on Days 1, 3, 5 
(the adult MTD). There were no DLTs. This finding is consistent with the testing of prior 
liposomal anthracyclines (eg, Doxil, DNX) that have demonstrated safety in children at the 
adult MTD. Enrollment on AAML1421 will begin at DL1, which will be 135 units/m2/dose 
on Days 1, 3, 5.  
 
The rationale for studying this dose level includes:  
 

  
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1) There were no DLTs at 100 units/m2/dose on Days 1, 3, 5 in the aforementioned and 
ongoing pediatric phase 1 study. 
2) The DLTs in the adult study at 134 units/m2 were persistent cytopenia > 56 days (1 
patient), CHF (1 patient), and hypertensive crisis (1 patient). The episode of CHF 
occurred during a period of sepsis and was transient. Hypertensive crises are very rare 
in the pediatric/adolescent and young adult population (AYA) population; therefore 
this toxicity may not be relevant to the eligible patients in this study. The FDA agreed 
to a starting dose of 134 units/m2/dose (our proposal rounds the dose to 135 units/m2) 
upon review of the phase 1 protocol conducted at Cincinnati Children's Hospital.    
3) CPX-351 at a dose of 135 units/m2/dose delivers a total daunorubicin dose of 
approximately 180 mg/m2, which is comparable to the liposomal daunorubicin 
delivered in DNX in the Kaspers et al study.2 Also, this dose is comparable to the 
150 mg/m2 cumulative dose of daunorubicin administered during Induction I of 
AAML0531 and AAML1031, thus providing a dose that is comparable to published 
literature and potentially translatable to a randomized phase 3 study. 
4) If 135 units/m2/dose is not tolerated, there will be a single dose de-escalation to 
100 units/m2/dose (DL0).  
 
The phase 1 portion of AAML1421 was completed in 12/2016 and DL1 (135 
units/m2/dose) was found to be the RP2D. The dose that will be used in the phase 2 
portion of the study will be 135 units/m2/dose given on days 1, 3, and 5. 
 
2.6 
Correlative studies 
2.6.1 
Testing of Cardiac Function and Biomarkers of Cardiac Injury 
Cardiac damage remains a significant dose-limiting toxicity as well as a cause of 
treatment related morbidity and mortality in childhood AML. Echocardiogram 
data are not predictive of those at greatest risk for cardiac damage. The exploratory 
objectives outlined below will evaluate the feasibility of stringent testing of cardiac 
function and biomarkers of cardiac injury to provide a more timely assessment of 
a patient's risk for developing anthracycline-induced cardiomyopathy. These 
studies are important as they have not been studied prospectively in any published 
pediatric studies using liposomal anthracyclines.  
 
The current COG phase 3 AML study, AAML1031, is collecting the most 
comprehensive and complete data set of cardiac function available for children 
with AML. The AAML1031 data will include ejection fraction/shortening fraction 
(EF/SF) values entered into the remote data entry systems at the start of each course 
of chemotherapy for more than 1000 patients. These longitudinal data will not only 
serve as baseline data for patients enrolling on future AML studies and 
demonstrate the feasibility of acquiring serial echocardiogram measurements on 
patients enrolled on COG trials. 
 
The development of clinically evident cardiomyopathy is a relatively rare event 
during the duration of most clinical trial monitoring, and may only become 
symptomatic decades later10,14 or during periods of high stress such as sepsis or 
pregnancy. Biomarkers of cardiac injury may provide a more timely assessment of 
a patient’s risk for development of anthracycline-induced cardiomyopathy. In this 
study we will evaluate the feasibility of incorporating conventional and advanced 
echocardiogram monitoring as well as measurement of potential biomarkers of 

  
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cardiotoxicity in routine assessment of cardiac function. These data will provide 
the foundation for studying, in the next phase 3 AML trial, those markers 
predictive of cardiac morbidity and mortality. Reliable biomarkers for 
cardiotoxicity may allow for the identification of patients most likely to benefit 
from early medical intervention, and permit future evaluations of possible 
cardioprotective strategies. 
 
Potential biomarkers of cardiac injury to be evaluated in this study include 
conventional and advanced echocardiography, serum cardiac troponin-T, N-
terminal probrain natriuretic peptide (NT-proBNP), and high sensitivity C-reactive 
protein (hs-CRP). As an exploratory objective, a peripheral blood microRNA 
profile predictive of myocyte damage will be evaluated following anthracycline 
administration. Additionally, we plan to incorporate whole exome sequencing to 
investigate genetic predisposition to anthracycline-induced cardiomyopathy.  
 
2.6.2 
Echocardiogram, Cardiac Troponin-T, N-terminal probrain natriuretic peptide 
(NT-proBNP), and hs-CRP 
Biomarkers of cardiac injury may provide a more timely assessment of a patient’s 
risk for development of anthracycline-induced cardiomyopathy. Serum levels of 
cardiac troponin-I (cTnI), troponin-T(cTnT), B-type natriuretic peptide (BNP), or 
the amino-terminal fragment of the BNP precursor (NT-proBNP) are associated 
with myocardial ischemia or strain injury and may be predictive of future 
subclinical or symptomatic cardiomyopathy (For reviews see: Dolci et al.49 and 
Christenson et al.50). Lipsultz et al. have described elevations in cTnT and NT-
proBNP among children with high risk ALL who were treated with moderate doses 
of doxorubicin during induction and in post-remission therapy.51 Elevations of 
cTnT at any time during the investigation were seen in nearly half of the children 
that did not also receive dexrazoxane prior to doxorubicin, whereas elevations in 
cTnT were only seen in about 20% of the patients that received dexrazoxane. 
Increases in cTnT were associated with lower LV mass and LV end-diastolic 
posterior wall thickness by echocardiography 4 years later. Differentiating effects 
upon NT-proBNP were also see between the ALL cohorts. Elevations in NT-
proBNP were associated with reduced LV thickness-to-dimension ratio by 
echocardiography. No significant association was noted between hs-CRP and 
changes by echocardiography. Longer follow up is necessary to determine when 
or to what extent these survivors of ALL develop symptomatic cardiac disease but 
this study illustrates how these markers may be used to compare different cardiac 
protection strategies for patients with leukemia in a significantly shorter time 
period. 
 
Patients participating in this study of CPX-351 are likely to have previously 
received significant cumulative doses of anthracyclines and may harbor sub-
clinical or asymptomatic heart disease even prior to treatment with CPX-351. 
Therefore changes in cTnT, NT-proBNP, and hs-CRP will be explored as potential 
biomarkers of cardiotoxicity prior to therapy and following therapy with CPX-351. 
Similarly, conventional echocardiogram parameters, including left ventricular 
ejection and shortening fractions, will be described with characterization of 
baseline abnormalities, as well as the decline from baseline post-CPX-351. 
Further, myocardial deformation, a novel measure of more subtle, regional 

  
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ventricular dysfunction will be explored (as described below). Together these data 
may allow indirect comparison of cardiotoxicity as similarly measured by 
echocardiogram and cardiac biomarkers following other anthracycline-containing 
regimens for hematologic malignancies. Further, it will provide critical 
preliminary data on which to base biomarker and imaging evaluations of 
cardiotoxicity during subsequent studies of CPX-351, such as a randomized phase 
3 study comparing efficacy and cardiotoxicity of this drug with other anthracycline 
regimens used to treat AML.  The optimal timing of cardiac biomarker acquisition 
such as cTnT following anthracycline therapy has not been fully elucidated. 
However, studies assessing cTnT or cTnI elevations following standard 
anthracycline formulations demonstrate troponin elevations as early as 4 hours 
after an anthracycline dose, but more often within several days after anthracycline 
exposure.52 One adult study demonstrated the range of peak troponin 
concentrations being observed between days 6 and 35 of an anthracycline 
containing chemotherapy cycle (mean day 21).53 Additionally, both pediatric and 
adult studies report prolonged troponin release up to 1 month following 
anthracycline exposure.54  Preliminary data from the Cincinnati pediatric phase I 
study of CPX-351 has not demonstrated any elevations in high sensitivity troponin 
at early (24-48 hours after dose 3 of CPX-351) or late (end of cycle) time points. 
[Dr. Michael Absalon, personal communication] While this might reflect the 
absence of significant cardiac injury associated with the liposomal encapsulation 
of daunorubicin, it could also indicate that the longer half-life and sustained plasma 
concentration of the anthracycline component of CPX-351 results in a later rise in 
troponin concentration than seen with standard anthracycline regimens.  Thus, 
serial cardiac biomarker samples will be collected following CPX-351 exposure 
on days 8, 15, 22 and 28-30 of cycle 1. 
 
2.6.3 
Novel Biochemical and Imaging Biomarkers of Cardiotoxicity 
MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression with 
a critical role in modulating cardiac regeneration, repair, and pathologic 
remodeling in response to stress and disease.55 Circulating miRNAs have emerged 
as highly sensitive and specific markers of cardiomyocyte injury and 
cardiovascular disease.56 While the role of miRNAs in modulating anthracycline-
induced cardiomyocyte injury has been demonstrated in animal models,57,58 less is 
known about circulating miRNA profiles in humans receiving anthracyclines. Pilot 
data from a small cohort of children receiving anthracycline chemotherapy 
demonstrates that miR-29b and miR-499 are specifically upregulated by 2-3-fold 
6, 12, and/or 24 hours following a cycle of anthracycline (n=24), while both were 
unchanged in children receiving non-cardiotoxic chemotherapy (n=9). Further, 
plasma expression of these miRNAs was significantly higher 6 and/ or 24 hours 
post-anthracycline in patients with troponin evidence of cardiac injury compared 
to those with no troponin increase.59 These data indicate that these miRNAs reflect 
anthracycline-induced cardiomyocyte injury and warrant further investigation. 
 
To further explore the possibility of using peripheral blood miRNA as a prognostic 
indicator or anthracycline-induced heart injury in children, plasma will be 
collected at baseline, 6 hours following the 3rd dose of CPX-351, and weekly 
through one month after initiation of CPX-351. As above, the longer half-life and 
sustained plasma concentrations of the liposomal daunorubicin component of 

  
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CPX-351 warrant longer monitoring of miRNA as the timing of anthracycline-
induced miRNA elevation may be delayed. The miRNA studies will be conducted 
and funded at Seattle Children’s Hospital/Fred Hutchinson Cancer Research 
Center. 
 
In addition to collecting conventional echocardiogram parameters of LV function 
(SF and EF) we will be requesting DICOM echo images to be sent for central 
analysis of myocardial strain. Myocardial strain is a dimensionless index reflecting 
the total deformation of ventricular myocardium during a cardiac cycle as a 
percentage of its original length and can be readily measured during routine 
echocardiography.60 In contrast to SF and EF, which reflect global left ventricular 
function, myocardial strain, as assessed by speckle tracking echocardiography, is 
able to detect regional defects in myocardial contractility.60 Anthracycline-induced 
reductions in myocardial strain have been demonstrated in children, both during 
and years following anthracycline therapy.60,61 Increasing evidence demonstrates 
that early anthracycline-induced alterations in myocardial deformation precede 
reductions in left ventricular SF or EF.60 Further, reduced peak systolic global 
longitudinal strain (GLS) has been shown to predict subsequent cardiotoxicity in 
adults receiving anthracycline.60 The feasibility of central GLS analysis in the 
setting of this COG trial will be assessed with the goal of evaluating GLS as a 
functional predictor of myocardial damage in the setting of the next COG phase 3 
study in AML. Additionally, change in GLS will provide a functional correlate 
against which to analyze both conventional cardiac biochemical biomarkers and 
plasma miRNAs.  
 
Taken together cardiac biomarkers and advanced cardiac imaging may provide 
valuable information regarding the degree of anthracycline-induced cardiac injury, 
which upon further study may allow risk stratification and facilitate 
cardioprotective intervention in those at high risk for cardiac morbidity.  
 
2.6.4 
Whole Exome Sequencing to Evaluate the Role of Rare Coding Variants as Risk 
Factors For Anthracycline-Induced Cardiomyopathy 
Current research indicates that anthracyclines directly damage cardiac myocytes 
and may cause a relative sarcopenia.62,63 Moreover, anthracyclines suppress 
multiple cardiac transcription factors, and anthracycline uptake into cardiac 
myocytes may be mediated by neuregulin via erbB2 receptors,64 thus varying 
cardiac myocyte anthracycline exposure and subsequent cardiac myocyte damage. 
Early studies demonstrated that increasing anthracycline dose,65 cardiac 
irradiation,66 young age,66 and female gender,67 increase the risk of anthracycline 
associated LV shortening dysfunction. These risk factors have been confirmed in 
more recent studies, which have sought to define genetic risk factors and to 
develop predictive models for the development of anthracycline-associated LV 
shortening dysfunction.68-70 Additional work is needed to define genetic variation 
that may play a role in the development of short term, anthracycline associated 
cardiac toxicity. 
 
 

  
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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.  
 
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.  
Assignment of site registration status in the CTSU Regulatory Support System 
(RSS) uses extensive data to make a determination of whether a site has fulfilled 
all regulatory criteria including but not limited to the following: 
 
An active Federal Wide Assurance (FWA) number 
 
An active roster affiliation with the Lead Network or a participating 
organization 
 
A valid IRB approval 
 
Compliance with all protocol specific requirements. 
 
In addition, the site-protocol Principal Investigator (PI) must meet the following 
criteria: 
 
Active registration status 
 
The IRB number of the site IRB of record listed on their Form FDA 1572 
 
An active status on a participating roster at the registering site. 
 
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.  

  
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Page 23 
 
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. For sites using the CIRB, IRB approval information is 
received from the CIRB and applied to the RSS in an automated process. Signatory 
Institutions must submit a Study Specific Worksheet for Local Context (SSW) to 
the CIRB via IRBManager to indicate their intent to open the study locally.  The 
CIRB’s approval of the SSW is then communicated to the CTSU Regulatory 
Office.  In order for the SSW approval to be processed, the Signatory Institution 
must inform the CTSU which CIRB-approved institutions aligned with the 
Signatory Institution are participating in the study. Other site registration 
requirements (ie, laboratory certifications, protocol-specific training certifications, 
or modality credentialing) must be submitted to the CTSU Regulatory Office or 
compliance communicated per protocol instructions. 
 
3.1.3 
Reservation Requirements 
Prior to obtaining informed consent and enrolling a patient, a reservation must be 
made following the steps below. Reservations may be obtained 24 hours a day 
through the Oncology Patient Enrollment Network (OPEN) system.  
 
Patient enrollment for this study will be facilitated using the Slot-Reservation 
System in conjunction with the Registration system in OPEN. Prior to discussing 
protocol entry with the patient, site staff must use the CTSU OPEN Slot 
Reservation System to ensure that a slot on the protocol is available for the patient. 
Once a slot-reservation confirmation is obtained, site staff may then proceed to 
enroll the patient to this study.  
 
If the study is active, a reservation can be made by following the steps below: 
 
1) Log in to https://open.ctsu.org/open/ using your CTEP IAM user name and 
password.  
2) In order to make a reservation, the patient must have an OPEN patient number. 
Click on the ‘Slot Reservation’ tab to create an OPEN patient number, under 
‘Patients’. 
3) Using the OPEN patient number ‘RESERVE’ a slot for that patient.  
4) On the ‘Create Slot Reservation’ page, select the Protocol Number, enter the 
COG Patient ID, and choose the required stratum (if applicable) in order to 
obtain a reservation. 
 
Refer to the ‘SITE – Slot Reservation Quick Reference’ guide posted under 
the ‘Help’ tab in OPEN for detailed instructions:  
https://www.ctsu.org/readfile.aspx?fname=OPEN/OPEN_SlotReservation_Q
uickReference_SiteUserGuide_102612.pdf&ftype=PDF  
 

  
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3.1.4 
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. To access OPEN, the site user must have an active CTEP-IAM account 
(check at https://ctepcore.nci.nih.gov/iam) and a 'Registrar' role on either the lead 
protocol organization (LPO) or participating organization roster.  Registrars must 
hold a minimum of an AP registration type.  If a DTL is required for the study, the 
registrar(s) must also be assigned the OPEN Registrar task on the DTL. 
 
All site staff will use OPEN to enroll patients to this study. It is integrated with the 
CTSU Enterprise System for regulatory and roster data and, upon enrollment, 
initializes the patient position in the Rave database. OPEN can be accessed at 
https://open.ctsu.org or from the OPEN tab on the CTSU members’ side of the 
website at https://www.ctsu.org. To assign an IVR or NPIVR as the treating, 
crediting, consenting, drug shipment (IVR only), or investigator receiving a 
transfer in OPEN, the IVR or NPIVR must list on their Form FDA 1572 in RCR 
the IRB number used on the site’s IRB approval.  If a DTL is required for the study, 
the IVR or NPIVR must also be assigned the appropriate OPEN-related tasks on 
the DTL. 
 
Prior to accessing OPEN, site staff should verify the following: 
 
 
All eligibility criteria have been met within the protocol stated timeframes. 
 
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. Please print this confirmation for your 
records. 
 
Further instructional information is provided on the CTSU members' web site 
OPEN tab or within the OPEN URL (https://open.ctsu.org). For any additional 
questions 
contact 
the 
CTSU 
Help 
Desk 
at 
1-888-823-5923 
or 
ctsucontact@westat.com. 
 
3.1.5 
Timing 
Patients must be enrolled before treatment begins. The date protocol therapy is 
projected to start must be no later than five (5) calendar days after the date of study 
enrollment. Patients who are started on protocol therapy on a Phase II study 
prior to study enrollment will be considered ineligible. The only exception to 
this is for intrathecal cytarabine, which can be given within 1 week prior to 
administration of CPX-351. If IT cytarabine is given prior to enrollment, a separate 
institutional consent must be obtained. 
 
All clinical and laboratory studies to determine eligibility must be performed 
within 7 days prior to enrollment unless otherwise indicated in the eligibility 
section below. 
 

  
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3.1.5.1 Cytogenetics 
Specimens for cytogenetic analysis are required, and must be obtained 
prior to therapy initiation. It is strongly recommended that these 
specimens be sent to a COG-approved institutional cytogenetics 
laboratory (see Section 13.0). A listing of these laboratories may be 
found on the COG website, as well as methods of attaining COG approval 
for local cytogenetics laboratories without COG approval. For patient with 
refractory disease, the diagnostic specimen may be used. 
 
The documentation of the chromosome analysis results and the 
reports/Forms should be sent to the appropriate reviewer (coordinating 
cytogeneticist) within 2 weeks (14 days) after enrollment (see Appendix 
II). Note: If the cytogenetics findings are available for the initial time of 
diagnosis, please submit the karyotype and/or Final Chromosome Report. 
If the information is not available, please indicate that information in the 
submission form. 
 
Results of cytogenetics are not required to be completed prior to 
enrollment, but samples must be collected prior to therapy initiation and 
submitted to a cytogenetics laboratory. 
 
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 unless otherwise indicated. Exception: the bone marrow 
aspirate may be performed 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 a post-enrollment lab value is outside the limits 
of eligibility, or 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 (SGPT) and serum creatinine. If the recheck is outside 
the limits of eligibility, the patient may not receive protocol therapy and will be 
considered off protocol therapy. 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  
Patients must be ≥ 1 year and ≤ 21 years of age at the time of enrollment. 
 

  
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3.2.2 
Diagnosis  
3.2.2.1 
Patients must have had histologic verification of AML at original 
diagnosis.  
3.2.2.2 
Patient must have one of the following: 
a) Recurrent disease with ≥ 5% blasts in the bone marrow (M2/M3 bone 
marrow), with or without extramedullary disease.   
b) Recurrent disease with an absolute blast count greater than 1,000 per 
microliter in the peripheral blood with or without extramedullary 
disease. 
 
3.2.2.3 
To be eligible for the Dose-Finding Phase: (The Dose-Finding Phase 
Completed in 12/2016)  
Relapse patients 
 
Patients must be in first relapse, and  
 
Patients must not have received prior re-induction therapy. 
 
Refractory patients 
 
Patients must not have received more than one attempt at remission 
induction, which may consist of up to two different therapy courses. 
COG AAML1031 de novo therapy including Induction I and 
Induction II is an example. 
 
Treatment-related AML (t-AML) 
 
Patients must be previously untreated for secondary AML. 
 
3.2.2.4 
To be eligible for the Phase 2 Efficacy Phase: 
Relapse patients: 
 
Patients must be in first marrow relapse, and  
 
Patients must not have received prior re-induction therapy. Donor 
lymphocyte infusion (DLI) is considered a re-induction attempt.  
 
3.2.2.5 
CNS Disease 
Patients must have the status of CNS1 or CNS2 only, and no clinical 
signs or neurologic symptoms suggestive of CNS leukemia, such as 
cranial palsy. See Section 3.3. 
 
3.2.3 
Performance Level  
Patients must have a performance status corresponding to an ECOG scores of 0, 1 
or 2. Use Karnofsky for patients > 16 years of age and Lansky for patients 
 16 years 
of 
age. 
See 
https://www.cogmembers.org/_files/protocol/
Standard/PerformanceStatusScalesScoring.pdf under Standard Sections for 
Protocols. Note: Patients who are unable to walk because of paralysis, but who are 
up in a wheelchair, will be considered ambulatory for the purpose of assessing the 
performance score. 
 

  
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3.2.4 
Prior Therapy 
Patients must have recovered from the acute toxic effects of all prior 
chemotherapy, immunotherapy, stem cell transplant or radiotherapy prior to 
entering this study. All prior treatment-related toxicities must have resolved to 
≤ Grade 2 prior to enrollment. 
 
a. Myelosuppressive 
chemotherapy: 
Must 
not 
have 
received 
myelosuppressive chemotherapy within 3 weeks of entry onto this study 
(excluding hydroxyurea). 
 
Cyto-reduction with hydroxyurea can be initiated and continued for 
up to 24 hours prior to the start of CPX-351. 
b. Biologic (anti-neoplastic agent): At least 7 days since the completion of 
therapy with a biologic agent such as steroids, retinoids. 
Note: For agents that have known adverse events occurring beyond 7 days 
after administration (i.e. monoclonal antibodies), this period must be 
extended beyond the time during which acute adverse events are known to 
occur.  
c. Radiation therapy (RT):  2 weeks for local palliative RT (small port); 
 6 months must have elapsed if prior craniospinal RT or if  50% 
radiation of pelvis;  6 weeks must have elapsed if other substantial BM 
radiation. 
Note: Patients must have received ≤ than 13.6 Gy prior radiation to the 
mediastinum. 
d. Stem Cell Transplant (SCT): No evidence of active graft vs. host disease 
for at least 4 weeks. For allogeneic SCT patients,  3 months must have 
elapsed since transplant.  
 
Must have received no more than 1 prior autologous or allogeneic 
stem cell transplant. 
 
Patients must be off all systemic immunosuppressive therapy for 
at least 2 weeks, excluding hydrocortisone for physiologic cortisol 
replacement. 
e. Intrathecal cytotoxic therapy:  
 
No waiting period is required for patients having received 
intrathecal cytarabine, methotrexate, and/or hydrocortisone.  
 
At least 14 days must have elapsed since receiving liposomal 
cytarabine (DepoCyte) by intrathecal injection. 
f. Growth factors: 
 
Patients must not have received growth factors for 7 days prior to 
CPX-351.  
 
Patients must not have received pegfilgrastim for 14 days prior to 
CPX-351. 
 
3.2.5 
Concomitant Medications Restrictions 
Please see Section 4.1 for the concomitant therapy restrictions for patients during 
treatment. 
 

  
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3.2.6 
Organ Function Requirements 
3.2.6.1 
Adequate Renal Function Defined As: 
- Creatinine clearance or radioisotope GFR  70 mL/min/1.73 m2 or 
- A serum creatinine based on age/gender as follows: 
 
Age 
 
Maximum Serum 
Creatinine (mg/dL) 
 
Male 
Female 
1 to < 2 years 
0.6 
0.6 
2 to < 6 years 
0.8 
0.8 
6 to < 10 years 
1 
1 
10 to < 13 years 
1.2 
1.2 
13 to < 16 years 
1.5 
1.4 
≥ 16 years 
1.7 
1.4 
The threshold creatinine values in this Table were derived from the 
Schwartz formula for estimating GFR71 utilizing child length and stature 
data published by the CDC. 
 
3.2.6.2 
Adequate Liver Function Defined As: 
- 
Direct bilirubin <1.5x upper limit of normal (ULN) for age and 
institution , and 
- 
SGPT (ALT)  3.0x ULN for age and institution (unless it is related 
to leukemic involvement)  
 
3.2.6.3 
Adequate Cardiac Function Defined As: 
- 
Shortening fraction of ≥ 27% by echocardiogram, or 
- 
Ejection fraction of ≥ 50% by radionuclide angiogram or 
echocardiogram. 
- 
Corrected QT (QTcB) interval < 500 msecs. 
 
3.2.6.4 
Central Nervous System Function Defined As: 
- 
Patients with seizure disorder may be enrolled if on anticonvulsants 
and if seizures are well controlled. 
- 
CNS toxicity ≤ Grade 2.  
 
3.2.7 
HIV Disease 
Patients with a known history of HIV are eligible, if they meet all of the following 
conditions: 
 
No history of HIV complications with the exception of CD4 count 
< 200 cells/mm3 
 
No antiretroviral therapy with overlapping toxicity such as myelosuppression 
 
CD4 count > 500 cells/mm3 prior to the diagnosis of relapsed AML 
 
HIV viral loads below the limit of detection  
 
No history of highly active antiretroviral therapy (HAART)-resistant HIV 
 

  
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3.2.8 
Exclusion Criteria 
3.2.8.1 
Patients who have received > 450 mg/m2 daunorubicin equivalents. This 
threshold was chosen because this is the approximate anthracycline 
exposure of patients who have received all 4 courses of chemotherapy 
according to AAML0531 or AAML1031 (300 mg/m2 of daunorubicin 
and 48 mg/m2 of mitoxantrone, using a 3-fold cardiotoxicity multiplier 
for mitoxantrone). Thus, patients who relapse after receiving 
AAML0531/AAML1031 therapy will be eligible for this study, provided 
they have not received any additional anthracyclines. 
NOTE: For the purposes of determining eligibility for this protocol, the 
following cardiotoxicity multipliers will be used to determine 
daunorubicin equivalents: 
 
Doxorubicin: 1 
 
Mitoxantrone: 3 
 
Idarubicin: 3 
 
Epirubicin: 0.5 
 
3.2.8.2 
Patients who are currently receiving another investigational drug. 
 
3.2.8.3 
Patients receiving medications for treatment of left ventricular systolic 
dysfunction. 
 
3.2.8.4 
Patients with any of the following diagnoses: 
 
Acute promyelocytic leukemia (APL) 
 
Down syndrome 
 
Fanconi anemia, Kostmann syndrome, Shwachman syndrome or any 
other known bone marrow failure syndrome 
 
Wilson's disease and any other disorder of copper metabolism 
 
Juvenile myelomonocytic leukemia (JMML) 
 
3.2.8.5 
Patients with documented active, uncontrolled infection at the time of 
study entry.  
 
3.2.8.6 
Patients with known active HBV and HCV infections. 
 
3.2.8.7 
Patients with prior allergy to daunorubicin and/or cytarabine.  
 
3.2.8.8 
Pregnancy and Breast Feeding 
3.2.8.8.1 Female patients who are pregnant are ineligible due to risks 
of fetal and teratogenic adverse events as seen in 
animal/human studies.  
 
3.2.8.8.2 Lactating females are not eligible. 
 
3.2.8.8.3 Female patients of childbearing potential are not eligible 
unless a negative pregnancy test result has been obtained. 
 
3.2.8.8.4 Sexually active patients of reproductive potential are not 
eligible unless they have agreed to use an effective 

  
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Page 30 
contraceptive method for the duration of their study 
participation and for 6 months after the last dose of 
chemotherapy. 
 
3.2.9 
Regulatory Requirements 
3.2.9.1 
All patients and/or their parents or legal guardians must sign a written 
informed consent. 
 
3.2.9.2 
All institutional, FDA, and NCI requirements for human studies must 
be met. 
 
3.3 
Definitions 
CNS disease  
CNS status is defined as: 
CNS 1: In cerebral spinal fluid (CSF), absence of blasts on cytospin preparation, regardless 
of the number of white blood cells (WBCs). 
 
CNS 2: In CSF, presence < 5/μL WBCs and cytospin positive for blasts, or ≥ 5/µL WBCs 
but negative by Steinherz/Bleyer algorithm: 
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; and 
CNS 2c: ≥ 10/μL RBCs; ≥ 5/μL WBCs and cytospin positive for blasts but 
negative by Steinherz/Bleyer algorithm (see below). 
 
CNS3: In CSF, presence of ≥ 5/μL WBCs and cytospin positive for blasts (in the absence 
of a traumatic lumbar puncture) and/or clinical signs of CNS leukemia. 
 
STEINHERZ/BLEYER ALGORITHM FOR EVALUATING TRAUMATIC LUMBAR 
PUNCTURES: 
If the patient has leukemic cells in the peripheral blood and the lumbar puncture is 
traumatic and the cytospin contains ≥ 5 WBC/μL with blasts, the following algorithm 
should be used to distinguish between CNS2 and CNS3 disease: 
 
CSF WBC  
> 2X Blood WBC 
CSF RBC 
   Blood RBC 
 
Therefore, a patient with CSF WBC ≥ 5/μL blasts, whose CSF WBC/RBC is 2X greater 
than the blood WBC/RBC ratio, has CNS disease at diagnosis.  
 
For example, the following patient would be classified as CNS3: CSF WBC = 60/μL; CSF 
RBC = 1500/μL; blood WBC = 46000/μL; blood RBC = 3.0 X 106/μL: 
 
  60    = 0.04  >  2X     46000      = 0.015 
1500                           3.0 X 106 
 
 

  
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4.0 
TREATMENT PROGRAM 
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 
This is a two part study comprised of a Dose-Finding phase to determine the RP2D of 
CPX-351 followed by an Efficacy phase. In both phases of the trial, CPX-351 will be 
administered intravenously over 90 minutes on Days 1, 3, and 5 in Cycle 1 and FLAG will 
be administered in Cycle 2. A diagnostic lumbar puncture will be performed and intrathecal 
chemotherapy will be given prior to each cycle of systemic therapy. Patients without 
evidence of CNS disease (CNS1) do not require additional CNS-directed therapy during 
Cycle 1. Patients with CNS2 disease will receive further intrathecal chemotherapy 
according to the guidelines set in Section 4.2.3. Patients treated on the phase 2, Dose-
Efficacy phase will receive CPX-351 at a dose of 135 units/m2/dose.  Pharmacokinetic 
(PK) sampling during the first cycle of treatment will be optional for the patients enrolled 
in the Efficacy phase of this study (see Section 14). 
 
The use of dexrazoxane will not be allowed on this trial. 
 
Post-treatment therapy: Please note that AAML1421 includes only 2 cycles of therapy.  
Patients in remission following cycle 2 should proceed to transplant or other therapy 
without delay and according to institutional practice. 
 
The phase 1 Dose-Finding portion of the study was completed in 12/2016. The 
recommended Phase 2 dose was found to be 135 Units/m2/ dose. 
DLT assessment for CPX-351 will occur during Cycle 1 of the Dose-Finding phase. 
Patients will be assigned a CPX-351 dose level at the time of enrollment. The starting dose 
(DL1) will be 135 units/m2/dose on Days 1, 3, 5. If 135 units/m2/dose is not tolerated, there 
will be a single dose de-escalation to 100 units/m2/dose (DL0). 
 
CPX-351 
Dose Level 
CPX-351 Dose 
(1 Unit = 1 mg cytarabine and 0.44 mg daunorubicin) 
0 
100 Units/m2/dose  
1 
135 Units/m2/dose  
 
Pharmacokinetic (PK) sampling during the first cycle of treatment will be required for the 
patients enrolled in the Dose Finding phase of this study (see Section 14). 
 
Note: Central venous access in the form of a double lumen Broviac or Hickman, port-a 
cath, or peripherally inserted central catheter is highly recommended. 

  
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4.1.1 
Concomitant Medications and Therapies 
No other cancer chemotherapy or immunomodulating agents may be 
given while the patient is on protocol. 
 
Corticosteroids should not be used as anti-emetic therapy. 
Corticosteroid therapy is not permissible except for the following 
indications:  
 
As treatment or prophylaxis for anaphylactic reactions 
 
As a treatment for symptoms of Ara-C syndrome including fever, 
rash, or conjunctivitis (see Section 5.3.4)    
 
As treatment for rash possibly related to CPX-351 (see Section 
5.3.1) 
 
Suspected or confirmed adrenal insufficiency 
 
Pain management per institutional guidelines. Note, it is not 
recommended to use prolonged courses (> 3 days) of 
corticosteroids for pain management. CPX-351 is a 
myelosuppressive chemotherapeutic agent and extended use of 
corticosteroids may result in increased susceptibility to infections. 
 
During Cycle 1 (CPX-351), the use of filgrastim (G-CSF) or biosimilar 
may be used at the treating physician’s discretion to enhance neutrophil 
recovery when clinically indicated. Routine use of filgrastim in 
clinically well patients awaiting count recovery is not recommended. If 
the treating physician determines that filgrastim use is indicated in 
Cycle 1, the pegylated formulation (pegfilgrastim) should not be used. 
 
 
Necessary supportive measures for optimal medical care will be given throughout 
the study as indicated by the treating physician and the patient’s medical need. For 
example, administration of antiemetics is strongly recommended. Monitoring for 
tumor lysis and preemptive or early intervention is recommended. 
 
It is recommended that the patient’s fluid status and hepatic and renal function be 
carefully monitored during the drug administration period. In this regard, patients 
should be well hydrated during the days of Cycle 1 and Cycle 2 chemotherapy drug 
administration, with the intent of maintaining urine output  3 mL/kg/hr. 
 
If copper toxicity is suspected, check serum copper levels. If abnormal, monitor 
until levels return to normal range. For patients receiving sources of copper such 
as in total parenteral nutrition (TPN), monitor for signs and symptoms of copper 
toxicity. 
 
To prevent drug incompatibility, no other medications should be administered 
concurrently through the same IV line as study drugs. 
 

  
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For 
COG 
Supportive 
Care 
Guidelines 
see: 
https://childrensoncologygroup.org/index.php/cog-supportive-care-guidelines  
under Standard Sections for Protocols. 
 
Antifungal prophylaxis 
Antifungal agents per institutional guidelines should be administered when ANC 
falls below 200/µL and continued until count recovery. 
 
Hospitalization/Hospital Environment 
Hospitalization following each cycle of chemotherapy is strongly recommended 
until the absolute phagocyte count (sum of the neutrophils, bands and monocytes) 
rises for 2 successive days, and the patient is afebrile and clinically stable. An 
additional discharge criterion of an absolute neutrophil count (ANC) of at least 
200/µL is also suggested. 
 
It is recommended that patients should be assigned to rooms with special air 
filtration systems such as high efficiency particulate air filters (HEPA) or clean-air 
rooms with constant positive pressure airflow if at all possible. 
 
Pain Management 
A single-institution, Phase 1 study of CPX-351 was conducted at Cincinnati 
Children’s Hospital that tested the doses prescribed in this protocol. As described 
in the background, 2 patients with pre-existing analgesic requirements experienced 
Grade 3 pain events at the 134 units/m2 dose level that met protocol-derived DLT 
criteria and were considered possibly related to CPX-351. If pain develops in any 
patient after the initiation of CPX-351, it is recommended that the treating 
physician initiate institution-appropriate therapy with intravenous narcotics. 
Corticosteroids for pain are not recommended, but may be used with caution per 
institutional guidelines. For more information, please refer to Section 4.1.1.2.  
 
 

  
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Page 34 
Cycle 1 
4.2 
Cycle 1 
4.2.1 
Therapy Delivery Map – Cycle 1  
Cycle 1 lasts 28 days. This TDM is on 2 pages. 
____________________________               ________________________ 
Patient COG ID number                                        DOB  
   Criteria to start this cycle are found in the patient eligibility criteria found in Section 3.2.  
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
CNS 1 Patients 
Intrathecal Cytarabine 
(IT ARAC) 
Intrathecal (IT) 
 
Age (years)              Dose 
1-1.99  
 
30 mg 
2-2.99  
 
50 mg 
≥ 3 
 
70 mg 
CNS1 Patients:  
 Given at the time of 
diagnostic lumbar puncture 
OR Day 0* 
 Day 28-30$ 
*May be given up to 7 days prior to Day 1.  
$May be given when BM aspirate/biopsy performed at end of Cycle 1.  
CNS 2 Patients# 
Intrathecal Cytarabine 
(IT ARAC) 
Intrathecal (IT) 
 
Age (years)              Dose 
1-1.99  
 
30 mg 
2-2.99  
 
50 mg 
≥ 3 
 
70 mg 
CNS2 Patients#: 
 Given at the time of 
diagnostic lumbar puncture 
OR Day 0* 
 Then give twice weekly 
until CNS is clear.  
 Day 28-30$  
*May be given up to 7 days prior to Day 1.  
Twice weekly IT to begin a minimum of 48 hours following the 3rd dose 
of CPX-351. A minimum of 4 and a maximum of 6 (count includes day 
0 IT) intrathecal treatments may be given.  
$May be given when BM aspirate/biopsy performed at end of Cycle 1. 
See Section 4.2.3 for full details 
CPX-351 
IND # 129443 Do not 
use commercial supply 
IV (central venous 
catheter) over 90 minutes 
135 Units/m2/dose 
1, 3, 5 
Round doses to nearest whole unit. Day 1 dosing should be at least 24 
hours after IT therapy administration.  
 
 
 
Ht _________cm 
 
Wt _________kg 
 
BSA _________m2 
 
Date Due 
Date Given 
Day 
IT ARAC ______mg 
CPX-351      ____Units 
Studies 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
0 
____     mg* 
 
q 
 
 
1 
 
____     Units 
a-p, r, s, t 
 
 
3 
 
____     Units 
 
 
 
5 
 
____     Units 
r, t 
 
 
6 
 
 
 
 
 
7 
 
 
 
 
 
8 
____     mg# 
 
b, f, h, i, j, q, r, s, t 
 
 
11 
____     mg# 
 
q 
 
 
15 
____     mg# 
 
b, f, h, i, j, q, s, t 
 
 
18 
____     mg# 
 
q 
 
 
22 
____     mg# 
 
b, f, h, i, j, q, s, t 
 
 
28 
____     mg$ 
 
l, m, n, p, q, s, t, u 
 
 
For criteria to begin Cycle 2, please see Section 4.2.4. See Section 7.1 for End of Therapy evaluations. 
See Section 5.0 for Dose Modifications for Toxicities and the COG Member website for Supportive Care Guidelines. 
Page 1 of 2 
Days IT 
therapy given 
for CNS2 
patients may 
vary based 
on 
scheduling 

  
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Page 35 
Cycle 1 
 
4.2.2 
Required Observations in Cycle 1 
All baseline studies must be performed prior to starting protocol therapy 
unless otherwise indicated below. Observations a-l and n-q can be performed 
up to 7 days before the start of therapy. “m” can be performed within 2 weeks 
prior to the start of protocol therapy. If performed, the baseline studies need 
not be repeated on Day 1.  
a. History 
b. Physical exam (including VS): Day 1, and then weekly 
c. Ht, Wt, BSA 
d. Performance status 
e. Pregnancy test: Female patients of childbearing potential require a negative 
pregnancy test prior to starting treatment; sexually active patients must use an 
acceptable method of birth control. 
f. CBC, differential, platelets: twice a week until ANC > 1000/µL and PLT > 
100,000/µL and then weekly.   
g. Urinalysis 
h. Electrolytes including Ca++, Mg++, PO4: Day 1, then weekly 
i. 
BUN and Creatinine: Day 1, and then weekly 
j. 
Total and direct bilirubin, AST, ALT: Day 1, and then weekly 
k. Total protein, albumin 
l. 
Echocardiogram or MUGA, and EKG.  
m. Imaging (CT or MRI) of chloroma (only required at baseline if clinically 
indicated, end of cycle only required if chloroma present at baseline) 
n. BMA/clot section or biopsy (see Section 4.2.4).  
o. BMA cytogenetics and FISH (It is recommended that cytogenetics/FISH be 
performed by a COG-approved laboratory (see Section 13.0).  
p. Flow cytometry with minimal residual disease (MRD) determination, 
according to institutional standards. Perform with any subsequent bone 
marrow evaluation during the study. 
q. LP-CSF for cell count, cytospin 
r. Optional Pharmacokinetic study (see Section 14.1) on Day 1, Day 5, and Day 
8, for patients enrolled in the Efficacy phase. 
s. Echo and Blood Biomarkers Optional studies (see Section 14.2.1): Echo: 
Baseline (before CPX-351 infusion, Day 28 (±7 days) but before Cycle 2. 
Blood: Baseline (before CPX-351 infusion), Day 8, Day 15, Day 22 and once 
during Days 28-30. 
t. 
Chemical and Imaging Biomarkers of Cardiotoxicity Optional study (see 
Section 14.2.2): Day 1 before CPX-351 infusion, Day 5, Day 8, Day 15, Day 
22 and once during Days 28-30. 
u. Blood Risk Factors for Cardiomyopathy Optional study (see Section 14.2.3): 
once during Day 28-30. 
This listing only includes evaluations necessary to answer the primary and 
secondary aims. OBTAIN OTHER STUDIES AS REQUIRED FOR GOOD 
CLINICAL CARE.  
 
Comments (Include any held doses, or dose modifications) 
 
Page 2 of 2 

  
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Cycle 1 
4.2.3 
Cycle 1 Treatment Details 
Intrathecal cytarabine: IT 
All patients will receive 2 doses of IT cytarabine using age-based dosing as 
follows: 
 
1) At the time of diagnostic lumbar puncture, or on Day 0 of Cycle 1. Patients 
may be given intrathecal cytarabine up to 1 week prior to Day 1, but at least 
24 hours prior to the start of CPX-351 on Day 1. If IT cytarabine is given prior 
to enrollment, a separate institutional consent must be obtained. 
 
2) At the time the Day 28-30 bone marrow aspirate/biopsy is obtained, or up to 
one week prior to Day 1 of Cycle 2. 
 
Age-based dosing: 
Age (yrs) 
Dose 
1-1.99   
30 mg 
2-2.99   
50 mg 
≥ 3 
 
70 mg 
 
 
For CNS2 patients 
The efficacy of CPX-351 in preventing and treating CNS AML is unknown, 
therefore patients with CNS2 leukemia are required to receive additional 
intrathecal chemotherapy as follows: 
 
 
Patients with CNS2 involvement at screening will receive additional IT therapy.  
This additional therapy may not be given until at least 48 hours following the 3rd 
dose of CPX-351. The additional therapy will consist of IT cytarabine twice 
weekly until the CSF is clear. A minimum of 4 and a maximum of 6 intrathecal 
treatments may be given. This count includes the pre-study/Day 0 dose. Patients 
whose CSF is clear at the second spinal tap will receive 3 intrathecal treatments 
and all other patients will receive 2 intrathecal treatments once the CSF is clear. 
Subjects whose CSF was cleared may then receive subsequent intrathecal 
cytarabine doses at the discretion of the treating physician. Subjects who have 
persistent CNS disease despite 6 doses of intrathecal cytarabine will be off study 
treatment (See Section 8.1). See Section 3.3 for definitions of CSF involvement. 
 
 
For patients who do not have CNS disease at the time of enrollment but who are 
considered high risk for treatment failure in the CNS (examples include: a history 
of CNS leukemia, high WBC at relapse, monocytic leukemia, or KMT2A (MLL)-
rearranged disease) additional intrathecal cytarabine may be given starting a 
minimum of 48 hours following the 3rd dose of CPX-351 at the investigators 
discretion. 
 
 
 
 
 
 
 
 
 

  
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Page 37 
Cycle 1 
CPX-351: IV over 90 minutes 
Days: 1, 3, and 5. 
Dose: 135 Units/m2/dose 
 
CPX-351 should be administered by central venous catheter.  
The dose of CPX-351 can be rounded to the nearest whole unit to avoid decimal 
dosing. 
 
CPX-351 will be provided by Jazz Pharmaceuticals. Do not use commercial 
supply. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
4.2.4 
Response Assessment  
Criteria to Begin Cycle 2 
 
Patients will have a disease assessment on Day 28-30 of Cycle 1. If a bone 
marrow aspirate cannot be evaluated (dry tap, no spicules), a follow-up bone 
marrow aspirate and biopsy must be performed within 2 weeks to assess 
cellularity and response. 
 
If aspirate and/or biopsy results indicate a hypocellular marrow (< 10% 
cellularity on biopsy), then continue to repeat bone marrow aspirate and/or 
biopsy not less than once every 14 days until response can be assessed. 
 
If count recovery is noted within 14 days of an evaluable marrow, there is no 
need to repeat the marrow to confirm response. Once disease response 
assessment can be made after Cycle 1, and non-hematologic toxicities 
probably or definitely related to CPX-351 recover to ≤ Grade 2, then patients 
will receive Cycle 2 of FLAG. 
 
If the bone marrow aspirate and/or biopsy performed after Cycle 1 reveals ≥ 
5% blasts and ≤ 25% blasts, then the patient should proceed to Cycle 2 of 
chemotherapy consisting of FLAG.  
 
If the patient meets criteria for CR, CRp, or CRi after Cycle 1, then the patient 
can proceed to Cycle 2. (See Section 10.2 for definitions of response.) 
 
Note: The patient will be removed from protocol therapy if one of the 
following criteria is met: 
 
Patients with ≥ Grade 2 toxicities not related to CPX-351 lasting > 50 days 
from the start of Cycle 1 that are considered by the investigator to be 
medically significant. 
 
Patients who have > 25% blasts after Cycle 1 will be considered a 
treatment failure and will be removed from protocol therapy.  
 
In the Efficacy phase, if after Cycle 1 (CPX-351) a patient has a 
hypoplastic bone marrow for ≥ 60 days and failure to recover a peripheral 
ANC > 500/µL and a non-transfusion dependent platelet count > 
20,000/µL not due to malignant infiltration or severe infection (defined as 
≥ Grade 3 infection), they will be considered a treatment failure and will 
go off protocol therapy. 
 
In the Dose-Finding phase, patients meeting criteria for a hematologic 
DLT in Cycle 1. The phase 1 Dose-Finding phase completed 12/2016. 
 
 

  
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Page 38 
Cycle 2 
4.3 
Cycle 2  
4.3.1 
Therapy Delivery Map – Cycle 2 
Cycle 2 lasts 28 days. 
______________________                                 ____________________ 
Patient COG ID number                                                    DOB  
Please refer to Section 4.2.4 for criteria to begin Cycle 2. This TDM is on 2 pages. 
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
Filgrastim (G-CSF) or 
biosimilar 
IV or Sub-Q 
5 micrograms/kg/dose  
Daily on Days 1-5; 
restart on Day 15 
until ANC recovery.  
Administer one hour prior to each dose of fludarabine. Continue until 
ANC>500/µL. Record below the last day G-CSF is administered. 
See Section 4.3.3 for administration details. The use of pegfilgrastim 
is not permitted.  
Fludarabine 
(FLUD) 
IV over 30 minutes  
30 mg/m2/dose 
1-5 
See Section 4.3.3 for administration details. 
High Dose Cytarabine  
(HD ARAC) 
IV over 1-3 hours   
2000 mg/m2/dose 
 
 
1-5 
Use eye drops as described in Section 4.3.3.  
To begin 4 hours after the start of the fludarabine infusion. 
Ht _________cm  
Wt _________kg  
BSA _________m2 
Date Due 
Date Given 
Day 
G-CSF  ____ mcg 
FLUD  ____mg 
HD ARAC _____mg 
Studies 
Comments 
 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
1 
____   mcg 
____   mg 
____     mg 
a-k 
 
 
 
2 
____   mcg 
____   mg 
____     mg 
 
 
 
 
3 
____   mcg 
____   mg 
____     mg 
 
 
 
 
4 
____   mcg 
____   mg 
____     mg 
c 
 
 
 
5 
____   mcg 
____   mg 
____     mg 
 
 
 
 
8 
 
 
 
a, c, e, f, g,  
 
 
 
11 
 
 
 
c 
 
 
 
15 
____   mcg 
 
 
a, c, e, f, g,  
 
 
 
18 
 
 
 
c 
 
 
 
22 
 
 
 
a, c, e, f, g,  
 
 
 
25 
 
 
 
c 
Record date of last dose 
of G-CSF. ___/___/___ 
 
 
28 
 
 
 
j, k, l 
See End-of Therapy 
observations in Sect. 7.1. 
See Section 5.0 for Dose Modifications for Toxicities and the COG Member website for Supportive Care Guidelines. 
Page 1 of 2 

  
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Page 39 
Cycle 2 
4.3.2 
Required Observations in Cycle 2 
 
a. Physical exam (including VS): Day 1, and then weekly 
b. Ht, Wt, BSA 
c. CBC, differential, platelets: twice a week until ANC > 1000/µL and PLT 
> 100,000/µL and then weekly.   
d. Urinalysis 
e. Electrolytes including Ca++, Mg++, PO4: Day 1, then weekly 
f. BUN and Creatinine: Day 1, and then weekly 
g. Total and direct bilirubin, AST, ALT: Day 1, and then weekly 
h. Total protein, albumin 
i. 
LP-CSF for cell count, cytospin 
j. 
Echocardiogram or MUGA, and EKG: Prior to Cycle 2 therapy (Echo 
done at the end of Cycle 1 can count as long as it was done within 2 weeks 
prior to the start of Cycle 2), Day 28 (± 7 days) but before any subsequent 
therapy.  
k. BMA/clot section (biopsy if aspirate unsuccessful) 
l. 
Flow cytometry with minimal residual disease (MRD) determination, 
according to institutional standards  
 
 
This listing only includes evaluations necessary to answer the primary and 
secondary aims. OBTAIN OTHER STUDIES AS REQUIRED FOR GOOD 
CLINICAL CARE.  
 
Comments 
(Include any held doses, or dose modifications) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 2 of 2 

  
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Cycle 2 
4.3.3 
Cycle 2 Treatment Details 
Filgrastim (G-CSF) or biosimilar: IV or Sub-Q 
Days: Daily, on Days 1 through 5, one hour prior to each dose of fludarabine.  
Restart on Day 15 and continue until the post-nadir ANC ≥ 500/µL 
Dose: 5 micrograms/kg/dose 
 
Note: Pegfilgrastim cannot be utilized in place of filgrastim or biosimilar. 
 
Fludarabine: IV over approximately 30 minutes 
Days: 1-5. 
Dose: 30 mg/m2/dose 
 
High Dose Cytarabine (HD ARAC): IV infusion over 1-3 hours once daily 
Days: 1-5 
Dose: 2000 mg/m2/dose (begin 4 hours after start of fludarabine) 
 
Please note: Following/during high-dose cytarabine, chemical conjunctivitis may 
occur. Administer steroid eye drops (0.1% dexamethasone or 1% 
prednisolone ophthalmic solution), 2 drops to each eye every 6 hours, 
beginning immediately before the first dose and continuing for 
24 hours after the last dose. If patient does not tolerate steroid eye 
drops, artificial tears may be administered on an every 2-4 hour 
schedule. 
 
See the Parenteral Chemotherapy Administration Guidelines (CAG) on the COG 
website 
at: 
https://cogmembers.org/_files/disc/Pharmacy/ChemoAdmin
Guidelines.pdf for special precautions and suggestions for patient monitoring 
during the infusion. As applicable, also see the CAG for suggestions on hydration, 
or hydrate according to institutional guidelines. 
 
 
 
 
See Section 7. 1 for End of Therapy evaluations. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
 
 
 

  
 
 
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Page 41 
 
5.0 
DOSE MODIFICATIONS FOR TOXICITIES 
5.1 
Definition of Dose Limiting Toxicity (DLT)  
The phase 1 Dose-Finding portion of the study was completed 12/12/2016.   
 
DLT will be defined as any of the following events that occur after the first dose of CPX-
351 during the first cycle of therapy. DLT assessment will occur during the Dose-Finding 
phase. 
 
Non-Hematologic Dose-Limiting Toxicity will be defined as any ≥ Grade 3 non-
hematologic toxicity that occurs after the first dose of CPX-351 that is possibly, probably 
or definitely related to CPX-351, and with the specific exclusion of: 
 
Alopecia 
 
Grade 3 fatigue, anorexia, nausea, vomiting, or diarrhea 
 
Grade 3 or 4 AST or ALT elevation that improves to ≤ Grade 2 within 14 days 
 
Grade 3 or 4 isolated electrolyte abnormalities that resolve, with or without 
intervention, to ≤ Grade 2 within 72 hours. Electrolyte supplementation is 
encouraged. 
 
Tumor lysis syndrome 
 
Grade 3 or 4 fever, febrile neutropenia, or infection with or without hospitalization 
 
Grade 3 rash 
 
Grade 3 mucositis that resolves (with or without supportive care) to ≤ Grade 2 
elevation within 14 days 
 
Early nutritional intervention with TPN or enteral tube feeding for anorexia, nausea, 
or concern for poor nutritional status will not be considered a DLT 
 
Grade 3 troponin elevations without EKG or other evidence of myocardial ischemia 
 
Grade 3 or greater pain due to leukemia, mucositis, typhlitis, infection, or obvious 
injury. 
   
Hematologic Dose-Limiting Toxicity will be defined as failure to recover a peripheral 
ANC > 500/µL and non-transfusion dependent platelet count > 20,000/µL due to 
documented bone marrow aplasia/hypoplasia (i.e., not due to malignant infiltration) for 
greater than or equal to 50 days from the start of the therapeutic cycle. This time period 
reflects approximately between 1 and 2 standard deviations above the approximate median 
time to ANC recovery for patients receiving mitoxantrone/cytarabine on COG study 
AAML0531. Failure to recover peripheral counts due to disease involvement of the bone 
marrow will not be considered dose-limiting. 
 
5.2 
Dose Modifications Due to Toxicity Related to CPX-351  
5.2.1 
 
Only one cycle consisting of three doses of CPX-351 will be administered to 
participants in this study. If ≥ Grade 4 non-hematologic dose limiting toxicity 
probably, or definitely related to CPX-351 occurs prior to completion of all 
planned doses, hold the remaining dose(s). These patients (in both the Dose-
Finding and Efficacy phases) will be removed from protocol therapy. 
 
 

  
 
 
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5.3 
Modifications and Supportive Care Recommendations for Toxicity Related to 
Protocol Therapy (Cycle 1 and Cycle 2) 
5.3.1 
Rash  
A macular-papular rash occurring within one week of treatment initiation occurs 
in the majority of patients treated with CPX-351. Grade 3 rash occurred in 6% of 
adults participating in the phase 1 study. The rash typically responded to topical or 
systemic corticosteroids. Subjects experiencing a rash from CPX-351 should first 
receive topical corticosteroids. Pruritus may be treated with diphenhydramine. If 
extensive or refractory to topical therapy, a 3-5 day course of systemic prednisone 
or methylprednisolone may be used.  
 
5.3.2 
Hand-Foot Syndrome  
Hand-foot syndrome has been reported in patients treated with other liposomal 
chemotherapy products and high-dose cytarabine regimens. Patients who develop 
hand-foot syndrome may receive topical emollients (such as Aquaphor) as well as 
topical or systemic steroids or antihistamines if appropriate. Oral administration of 
vitamin B6 (pyridoxine) can also be used for these patients - BSA <0.5 m2: 50 mg 
per day; BSA 0.5-1.0 m2: 100 mg per day; BSA 1.1- 1.5 m2: 200 mg per day, and 
BSA >1.5 m2: 300 mg per day. 
 
5.3.3 
Neurological Toxicity 
The most common neurotoxicity related to fludarabine or cytarabine 
administration is an acute cerebellar syndrome that may manifest itself as ataxia, 
nystagmus, or dysarthria. However, seizures and encephalopathy have also 
occurred following therapy with high dose cytarabine. Patients experiencing Grade 
3 or 4 cerebellar toxicity should be removed from protocol therapy. 
 
5.3.4 
Ara-C Syndrome including Fever, Rash, or Conjunctivitis 
Do not withhold cytarabine for fever if it is likely to have been caused by the 
cytarabine. However, blood cultures should be obtained even if fever is thought to 
be likely due to cytarabine. Institution of antibiotics for fever associated with 
cytarabine infusion is at the discretion of the treating physician (see fever 
management recommendations in the COG Supportive Care Guidelines). 
 
For rash or conjunctivitis, withhold cytarabine for Grade 3-4 toxicity until resolved 
to ≤ Grade 1. Make up missed doses and consider concurrent treatment with 
hydrocortisone or dexamethasone, and/or with dexamethasone ophthalmic drops 
for conjunctivitis only. 
 
5.3.5 
Renal Toxicity 
Patients with nephrotoxicity secondary to antibiotics, or antifungals, may have 
prolonged excretion of cytarabine leading to more severe marrow and 
extramedullary toxicity.  
 
Patients with a serum creatinine > 2 mg/dL or > 2x normal 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 formula before proceeding with cytarabine therapy. 

  
 
 
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 
If the creatinine clearance is abnormal (< 60 mL/min/1.73m2) then Cycle 2 
high dose cytarabine should be reduced by 50% for each daily dose. With this 
approach, previous research has shown the prevention of subsequent 
neurotoxicity in recipients of high dose cytarabine in the face of renal 
insufficiency.72 
 
 

  
 
 
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6.0 
DRUG INFORMATION  
6.1 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 

  
 
 
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 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
. 
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
 
 
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6.2 
CYTARABINE           
 
 
 
 
 
 
(Cytosine arabinoside, Ara-C, Cytosar®) NSC #63878  
 
(07/13/15) 
 
 
Source and Pharmacology: 
Cytarabine appears to act through the inhibition of DNA polymerase. A limited, but 
significant, incorporation of cytarabine into both DNA and RNA has also been reported. It 
exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) 
and under certain conditions blocking the progression of cells from the G1 phase to the S-
phase. Cytarabine is metabolized by deoxycytidine kinase and other nucleotide kinases to 
the nucleotide triphosphate (Ara-CTP), an effective inhibitor of DNA polymerase. Ara-
CTP is inactivated by a pyrimidine nucleoside deaminase, which converts it to the nontoxic 
uracil derivative (Ara-U). It appears that the balance of kinase and deaminase levels may 
be an important factor in determining sensitivity or resistance of the cell to cytarabine. It 
has an initial distributive phase t½ of about 10 minutes, with a secondary elimination phase 
t½ of about 1 to 3 hours. Peak levels after intramuscular or subcutaneous administration of 
cytarabine occur about 20 to 60 minutes after injection and are lower than IV 
administration. Intrathecally administered doses are metabolized and eliminated more 
slowly with a t½ of about 2 hours. 
 
 

  
 
 
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Page 48 
 
 
Toxicity: (Intravenous, SubQ, IM) 
 
Common 
Happens to 21-100 
children out of every 100 
Occasional 
Happens to 5-20 
children out of every 
100 
Rare 
Happens to < 5 children out of 
every 100 
Immediate: 
Within 1-2 days of 
receiving drug 
Nausea, vomiting, 
anorexia   
 
With High Dose: 
conjunctivitis  
Flu-like symptoms with 
fever, rash  
 
Ara-C syndrome (fever, myalgia, 
bone pain, occasionally chest 
pain, maculopapular rash, 
malaise, conjunctivitis), 
anaphylaxis 
 
With High Dose: 
cardiomyopathies (vasculitis, and 
pericarditis), cerebral and 
cerebellar dysfunction including: 
encephalopathy, aseptic 
meningitis, ataxia, dysphasia, 
nystagmus, a decreased level of 
consciousness, personality 
changes, somnolence, seizures  
Prompt: 
Within 2-3 weeks, 
prior to the next 
course 
Myelosuppression 
(anemia, 
thrombocytopenia, 
leukopenia, 
megaloblastosis, 
reticulocytopenia), 
stomatitis, alopecia 
Diarrhea, hypokalemia, 
hypocalcemia, 
hyperuricemia 
 
With High Dose: 
capillary pulmonary 
leak syndrome (RDS, 
pulmonary edema) 
Hepatotoxicity, sinusoidal 
obstruction syndrome (SOS, 
formerly VOD), urinary retention, 
renal dysfunction, pain and 
erythema of the palms and soles 
Delayed: 
Any time later 
during therapy, 
excluding the above 
conditions 
 
 
Asymptomatic nonoliguric 
rhabdomyolysis 
Unknown 
Frequency and 
Timing: 
Fetal toxicities and teratogenic effects of cytarabine have been noted in humans. It is 
unknown whether the drug is excreted in breast milk. 
 
 
Toxicity: (Intrathecal) 
 
Common 
Happens to 21-100 
children out of every 
100 
Occasional 
Happens to 5-20 
children out of every 
100 
Rare 
Happens to < 5 children out 
of every 100 
Immediate: 
Within 1-2 days of 
receiving drug 
Nausea, vomiting, 
fever, headache 
 
Arachnoiditis 
Rash, somnolence, 
meningismus, convulsions, 
paresis 
Prompt: 
Within 2-3 weeks, 
prior to the next 
course 
 
 
Myelosuppression, ataxia 
Delayed: 
Any time later during 
therapy, excluding the 
above condition 
 
 
Necrotizing 
leukoencephalopathy, 
paraplegia, blindness (in 
combination with XRT & 
systemic therapy) 
 

  
 
 
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Formulation:  
Cytarabine for Injection is available in vials of 100 mg, 500 mg, 1 g, and 2 g containing a 
sterile powder for reconstitution. It is also available at a 20 mg/mL concentration with 
benzyl alcohol (25 mL per vial) or as a preservative free solution (5 mL, 50 mL per vial), 
and at a 100 mg/mL concentration with benzyl alcohol (20 mL vial) or as preservative free 
solution (20 mL vial). Hydrochloric acid and/or sodium hydroxide may be added to adjust 
the pH. Store at 25°C (77°F); excursions permitted to 15°-30°C (59°-86°F). Cytarabine 
solutions should be protected from light.  
 
Guidelines for Administration:  See Treatment and Dose Modification sections of the 
protocol. 
 
IV Infusion: 
Reconstitute the lyophilized powder with Bacteriostatic Water for Injection or NS injection. 
Solution containing bacteriostatic agent should not be used for the preparation of doses 
> 200 mg/m2. May be further diluted with dextrose or sodium chloride containing solutions. 
May give by IV push injection, by IV infusion, or by continuous infusion.  
 
Low Dose (≤ 200 mg/m²/dose): For administration by IV push, reconstitute to a 
concentration of 20-100 mg/mL. 
 
High Dose (≥ 1000 mg/m²/dose): Administer steroid eye drops (dexamethasone or 
prednisolone), 2 drops each eye q6h beginning immediately before the first dose and 
continuing 24 hours after the last dose. If patient does not tolerate steroid eye drops, 
administer artificial tears on a q2-4 hour schedule. 
 
Stability: When reconstituted with Bacteriostatic Water for Injection, cytarabine is stable for 
48 hours at room temperature. Solutions reconstituted without a preservative should be used 
immediately. Discard if solution appears hazy. Diluted solutions in D5W or NS are stable 
for 8 days at room temperature; however, the diluted cytarabine should be used within 24 
hours for sterility concerns.  
 
Intrathecal: 
For intrathecal administration, dilute with 5-10 mL (or volume per institutional practice) 
preservative free 0.9% sodium chloride injection, lactated Ringer’s injection, Elliot’s B 
solution. The volume of CSF removed should be equal to at least ½ the volume delivered. 
 
Patient Age 
(years) 
Recommended 
volume 
10% CSF volume 
CSF Volume * 
1 – 1.99 
5 – 10 mL 
5 mL 
50 + 10 mL (babies) 
2 – 2.99 
5 – 10 mL 
8 mL 
80 + 20 mL (younger children) 
3 – 8.99 
5 – 10 mL 
10 mL 
100 + 20 mL (older children) 
9 or greater 
5 – 10 mL 
13 mL 
130 + 30 mL (adults) 
 
*Rieselbach, R.E. et.al. Subarachnoid distribution of drugs after lumbar injection; N Engl J 
Med. 1962 Dec 20; 267:1273-8 
 
Of Note: Larger volumes approximating at least 10% of the CSF volume, isovolumetric 
delivery, with the patient remaining prone after the procedure may facilitate drug distribution. 
These procedures have not been validated in clinical trials. They are allowed but not mandated 
for patients on COG studies. 

  
 
 
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Intrathecal cytarabine mixed in NS, lactated Ringer’s injection, or Elliot’s B solution is 
stable for 24 hours at 25ºC but contains no preservative and should be administered as soon 
as possible after preparation. 
 
Supplier: Commercially available from various manufacturers. See package insert for 
further information. 
 
6.3 
FILGRASTIM, TBO-FILGRASTIM, FILGRASTIM-SNDZ  
 
(Granulocyte Colony-Stimulating Factor, r-metHuG-CSF, G-CSF, Neupogen®, Granix®, 
Zarxio®) NSC #614629                                                                                     (11/15/16) 
 
 
 
 
 
 
 
 
 
Source and Pharmacology:  
Filgrastim is a human granulocyte colony-stimulating factor (G-CSF), produced by 
recombinant DNA technology. Filgrastim is a 175 amino acid protein with a molecular 
weight of 18,800 daltons manufactured by recombinant DNA technology utilizing E coli 
bacteria into which has been inserted the human granulocyte colony stimulating factor 
gene. It differs from the natural protein in that the N- amino acid is methionine and the protein 
is not glycosylated. G-CSF is a lineage specific colony-stimulating factor which regulates 
the production of neutrophils within the bone marrow and affects neutrophil progenitor 
proliferation, differentiation, and selected end-cell functional activation (including 
enhanced phagocytic ability, priming of the cellular metabolism associated with respiratory 
burst, antibody dependent killing, and the increased expression of some functions 
associated with cell surface antigens). Filgrastim exhibits nonlinear pharmacokinetics with 
clearance dependent on filgrastim concentration and neutrophil count. Filgrastim is cleared 
by the kidney. The elimination half-life is similar for subcutaneous and intravenous 
administration, approximately 3.5 hours. The time to peak concentration when administered 
subcutaneously is 2-8 hours. 
 
Toxicity: 
 
Common 
Happens to 21-100 
children out of every 
100 
Occasional 
Happens to 5-20 children out 
of every 100 
Rare 
Happens to <5 children 
out of every 100 
Immediate: 
Within 1-2 days of 
receiving drug 
 
Local irritation at the injection 
site, headache 
Allergic reactions (more 
common with IV 
administration than 
subq):skin (rash, 
urticaria, facial edema), 
respiratory (wheezing, 
dyspnea) and 
cardiovascular 
(hypotension, 
tachycardia), low grade 
fever  
Prompt: 
Within 2-3 weeks, 
prior to the next 
course 
Mild to moderate 
medullary bone pain 
Increased: alkaline 
phosphatase, lactate 
dehydrogenase and uric acid, 
thrombocytopenia 
Splenomegaly, splenic 
rupture, rash or 
exacerbation of pre-
existing skin rashes, 
sickle cell crises in 
patients with SCD, 
excessive leukocytosis, 
Sweet’s syndrome (acute 

  
 
 
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febrile neutrophilic 
dermatosis) 
Delayed: 
Anytime later during 
therapy 
 
 
Cutaneous vasculitis, 
ARDS 
Late: 
Any time after 
completion of 
treatment 
 
 
MDS or AML (confined 
to patients with severe 
chronic neutropenia and 
long term administration) 
Unknown 
Frequency and 
Timing: 
Fetal toxicities and teratogenic effects of filgrastim in humans are unknown. 
Conflicting data exist in animal studies and filgrastim is known to pass the 
placental barrier. It is unknown whether the drug is excreted in breast milk. 
 
Formulation and Stability:  
Neupogen® supplied as a clear solution of 300 mcg/mL in 1 mL or 1.6 mL vials. 
Neupogen® vials are preservative free single use vials. Discard unused portions of open 
vials. 
Neupogen®, Granix®, and Zarxio® are also available as single use prefilled syringes 
containing 300 mcg/0.5 mL or 480 mcg/0.8 mL of filgrastim for subcutaneous 
administration.  
Store refrigerated at 2º-8ºC (36º-46ºF). Protect from light. Do not shake. Prior to injection, 
filgrastim and filgrastim-sndz may be allowed to reach room temperature for a maximum 
of 24 hours (infusion must be completed within 24 hours of preparation). TBO-filgrastim 
may be removed from 2°C-8°C (36°F-46°F) storage for a single period of up to 5 days 
between 23°C to 27°C (73°F to 81°F). Avoid freezing and temperatures > 30ºC.  
 
For IV use, dilute filgrastim (Neupogen®) and tbo-filgrastim (Granix®) in D5W only to 
concentrations >15 mcg/mL. Filgrastim-sndz (Zarxio®) may be diluted in D5W to 
concentrations between 5 mcg/mL and 15 mcg/mL. At concentrations below 15 mcg/mL, 
human serum albumin should be added to make a final albumin concentration of 0.2% 
(2 mg/mL) in order to minimize the adsorption of filgrastim to plastic infusion containers 
and equipment for all 3 products (communication on file from Teva Pharmaceuticals USA). 
Filgrastim or filgrastim-sndz dilutions of 5 mcg/mL or less are not recommended. TBO-
filgrastim dilutions below 2 mcg/mL are not recommended. Diluted filgrastim biosimilar 
products should be stored at 2º-8ºC (36º-46ºF) and used within 24 hours. Do not shake. 
 
Do not dilute with saline-containing solutions at any time; precipitation will occur. 
 
 
Guidelines for Administration:  
See Treatment, Dose Modifications and Supportive Care sections of the protocol.  
 
Filgrastim biosimilar products should not be administered within 24 hours of (before AND 
after) chemotherapy. 
 
Supplier: Commercially available from various manufacturers. See package insert for 
further information  
 
 
 
 

  
 
 
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6.4 
FLUDARABINE 
 
 
 
 
 
 
 
(Fludara®, fludarabine phosphate, 2-fluoro-ara-AMP) NSC# 312887 
(01/10/18) 
 
 
Source and Pharmacology:  
Fludarabine phosphate is a synthetic purine nucleoside. It differs from the physiologic 
nucleosides, adenosine, in that the sugar moiety is arabinose instead of ribose, and by the 
addition of a fluorine atom to the purine base adenine. Fludarabine is also a fluorinated 
nucleotide analog the antiviral agent vidarabine, (ara-A). The addition of fluorine results 
in increased aqueous solubility and resistance to enzymatic degradation by adenosine 
deaminase. Fludarabine (2-fluoro-ara-A) is commercially available as the monophosphate 
salt (2-fluoro-ara-AMP). The monophosphorylation increases the drug's aqueous solubility 
while maintaining pharmacologic activity. The chemical name for fludarabine phosphate 
is 9H-Purin-6-amine, 2-fluoro-9-(5-0-phosphono β-D-arabino-furanosyl) (2-fluoro-ara-
AMP) and the molecular weight is 365.2.  
 
Fludarabine is a purine antagonist antimetabolite. In vivo, fludarabine phosphate is rapidly 
dephosphorylated to 2-fluoro-ara-A and then it is phosphorylated intracellularly by 
deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This metabolite appears 
to act by inhibiting DNA polymerase alpha, ribonucleotide reductase and DNA primase, 
thus inhibiting DNA synthesis. The mechanism of action of this antimetabolite is not 
completely characterized and may be multi-faceted. 
 
Phase 1 studies in humans have demonstrated that within several minutes after intravenous 
infusion, fludarabine phosphate is converted to the active metabolite, 2-fluoro-ara-A and 
becomes undetectable. Therefore, pharmacokinetics studies have focused on 2-fluoro-ara-
A. Fludarabine phosphate 25 mg/m2 
infused intravenously over 30 minutes to adult cancer 
patients, showed a moderate accumulation of 2-fluoro-ara-A. During a 5-day treatment 
schedule, 2-fluoro-ara-A plasma trough levels increased by a factor of about 2.  
Fludarabine is widely distributed. The volume of distribution at steady state (Vss) reported 
after daily administration of 25 mg/m2 for 5 days to adults averaged at 96-98 L/m2. Tissue 
distribution studies in animals indicate that the highest concentrations of the drug are in 
liver, kidney, and spleen. Although the extent to which fludarabine and/or its metabolites 
distribute into the CNS in humans has not been determined to date, severe neurologic 
toxicity (eg,, blindness, coma) has been reported in patients receiving the drug, particularly 
in high dosages. There is evidence from animal studies that fludarabine distributes into the 
CNS and that a toxic metabolite (2-fluoroadenine, possibly formed by bacteria in the GI 
tract), can be absorbed systematically via enterohepatic circulation and distributed into 
CSF. According to in vitro data, about 19-29% of fludarabine is bound to plasma proteins.  
Following IV administration, fludarabine phosphate is dephosphorylated rapidly to 
fludarabine. Plasma concentrations of fludarabine decline in a linear, dose-independent 
manner. The elimination profile of fludarabine also has been reported to be either biphasic 
or triphasic; however, reported terminal elimination half-lives have been similar. In adult 
cancer patients receiving fludarabine 25 mg/m2 as a 30-minute IV infusion daily for 5 days, 
a terminal half-life of about 20 hours was reported. In a limited number of pediatric 
patients, the plasma concentration profile of fludarabine exhibited both monoexponential 
and biexponential decay, with a mean t1/2 of 10.5 hours in patients with monoexponential 
elimination and a t1/2 of 1.2-1.4 and 12.4-19 hours, respectively, in patients with 
biexponential elimination.  

  
 
 
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Renal clearance accounts for about 40% of the total body clearance of fludarabine. Renal 
elimination appears to become more important at high dosages of the drug. The dose of 
fludarabine needs to be adjusted in patients with moderate renal impairment. 
The use of fludarabine in combination with pentostatin is not recommended due to the risk 
of severe pulmonary toxicity. 
 
Toxicity: 
 
Common 
Happens to 21-100 
subjects out of every 
100 
Occasional 
Happens to 5-20 
subjects out of every 
100 
Rare 
Happens to < 5 subjects out of 
every 100 
Immediate: 
Within 1-2 days 
of receiving drug 
Fever, fatigue, 
weakness, pain, 
nausea, vomiting, 
anorexia, cough, 
dyspnea 
Edema including 
peripheral edema, 
chills, rash, diarrhea, 
rhinitis, diaphoresis, 
malaise, abdominal 
pain, headache, back 
pain, myalgia, 
stomatitis, flu-like 
syndrome 
Anaphylaxis, tumor lysis 
syndrome, dehydration*  
Prompt: 
Within 2-3 
weeks, prior to 
next course 
Myelosuppression 
(anemia, neutropenia, 
thrombocytopenia), 
infection (urinary 
tract infection, herpes 
simplex infection, 
pneumonia, upper 
respiratory) 
Weight loss, 
gastrointestinal 
bleeding, hemoptysis, 
paresthesia, allergic 
pneumonitis, 
bronchitis, 
pharyngitis, visual 
disturbance, hearing 
loss, hyperglycemia  
Sinusitis, dysuria, 
opportunistic infections and 
reactivation of latent viral 
infections like Epstein-Barr 
virus (EBV), herpes zoster and 
John Cunningham (JC) virus 
(progressive multifocal 
leukoencephalopathy 
[PML])L, EBV associated 
lymphoproliferative disorder, 
panycytopenia (can be 
prolonged), pulmonary 
hypersensitivitya (dyspnea, 
cough, hypoxia, interstitial 
pulmonary infiltrate), 
pulmonary toxicity (acute 
respiratory distress syndrome 
[ARDS], pulmonary fibrosis, 
pulmonary hemorrhage, 
respiratory distress, 
respiratory failure), pericardial 
effusion, skin toxicity 
(erythema multiforme, 
Stevens-Johnson syndrome, 
toxic epidermal necrolysis, 
pemphigus), liver failure, 
renal failure, hemorrhage, 
transfusion-associated graft-
versus-host disease has 
occurred following transfusion 
of nonirradiated blood 
products, phlebitis*, sleep 
disorder*, cerebellar 

  
 
 
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syndrome*, depression*, 
mentation impaired*, 
alopecia*, pruritus*, 
seborrhea*, esophagitis*, 
constipation*, mucositis*, 
dysphagia*, hesitancy*, 
cholelithiasis*, abnormal liver 
function tests *, osteoporosis*, 
arthralgia*, abnormal renal 
function test*, proteinuria*, 
epistaxis*, hemorrhagic 
cystitis*, eosinophilia*  
Delayed: 
Any time later 
during therapy, 
excluding the 
above conditions 
 
 
Neurotoxicity (increased with 
high doses): seizures, agitation, 
confusion, weakness, visual 
disturbances, optic neuritis, 
optic neuropathy, photophobia, 
blindness, paralysis, coma, 
death, peripheral neuropathya); 
autoimmune phenomena: 
thrombocytopenia/thrombocyt
openic purpura (ITP), Evans 
syndrome, hemolytic anemia, 
acquired hemophilia 
Late: 
Any time after 
completion of 
treatment 
 
 
Myelodysplastic 
syndrome/acute myeloid 
leukemia (mainly associated 
with prior or concomitant or 
subsequent treatment with 
other anticancer treatments), 
skin cancer (new onset or 
exacerbation) 
Unknown 
Frequency and 
Timing: 
Pregnancy Category D  
Based on its mechanism of action, fludarabine phosphate can cause fetal harm 
when administered to a pregnant woman. Fludarabine phosphate was 
embryolethal and teratogenic in both rats and rabbits. 
 (L) Toxicity may also occur later. 
* Reported in ≤ 3% of subjects.  Since these are not considered life threatening they are not included 
in the consent. 
a These effects were not reported in children. 
 
Formulation and Stability: 
Fludarabine phosphate injection is available as sterile lyophilized powder and in solution. 
Each single-dose vial of powder contains 50 mg of the active ingredient fludarabine 
phosphate, 50 mg of mannitol, and sodium hydroxide to adjust the pH to 7.7. After 
reconstitution, the pH range for the final product is 7.2-8.2. The single-dose solution vial 
contains 25 mg/mL, 2 mL of fludarabine phosphate. It may contain mannitol and is 
preservative-free.  
 
Fludarabine phosphate vials should be stored refrigerated at 2-8ºC (36-46ºF). 
 
Guidelines for Administration: See Treatment and Dose Modification sections of the 
protocol. 

  
 
 
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Fludarabine phosphate powder should be reconstituted with 2 mL of Sterile Water for 
Injection. The solid cake should fully dissolve in 15 seconds or less. The resulting 
concentration is 25 mg/mL. When reconstituted to a final concentration of 25 mg/mL, the 
drug is stable for at least 16 days at room temperature and normal light conditions. The 
manufacturer recommends that the solution be used within 8 hours after reconstitution. 
Prior to administration, fludarabine 25 mg/mL solution or the reconstituted 25 mg/mL 
solution should be further diluted in 100 mL or 125 mL of D5W or NS. Concentrations of 
0.25 to 1 mg/mL have been used in clinical trials. When diluted to a final concentration of 
1 mg/mL, fludarabine is stable for at least 16 days at room temperature and normal light 
conditions. The manufacturer recommends that the diluted solution be used within 8 hours 
after preparation. Parenteral drug products should be inspected visually for particulate 
matter and discoloration prior to administration. 
 
Supplier: Commercially available from various manufacturers. See package insert for 
further information.  
 
 

  
 
 
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7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
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). 
 
7.1 
End of Therapy & Follow-up 
 
STUDIES TO BE OBTAINED 
End of Therapy 
Follow-Up 
Relapse 
Physical Exam 
X 
 
X 
Ht, Wt, BSA 
X 
 
 
Performance status 
X 
 
 
CBC, differential, platelets 
X 
 
 
Electrolytes (Ca++, Mg++, K+), and 
creatinine 
X 
 
 
LP-CSF for cell count, cytospin 
X 
 
X 
Flow cytometry with minimal 
residual disease (MRD) 
determinationa 
Xb 
 
X 
Echocardiogram or MUGA, and 
EKG 
X 
X#  
X 
BMA/clot section (biopsy if 
aspirate unsuccessful) for 
morphologyb  
X 
 
X 
BMA cytogenetics & FISH 
 
 
X@ 
a Perform per institutional standard 
b Need not be repeated if performed at the end of Cycle 2. 
# Echocardiogram or MUGA should be repeated annually from the start of therapy; the EKG should 
be repeated if clinically indicated. 
@ See Section 13.2.6. 
See COG Late Effects Guidelines for recommended post treatment follow-up:  
http://www.survivorshipguidelines.org/ 
 

  
 
 
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Note: Follow-up data are expected to be submitted per the Case Report Forms (CRFs) 
schedule.  
 
 
7.2 
Research Studies for which Patient Participation is Optional 
 
The following table lists samples to be obtained from patients who have provided consent 
for optional correlative studies. 
 
Study 
Baseline 
During Cycle 1 
Sample 
Type/Tube 
Biomarkers of 
cardiac injury 
(Cardiac troponin-T, 
NT-proBNP, hs-
CRP)^ 
See Section 14.2.1 
X 
 Baseline (before CPX-351 
infusion) 
 Day 8* 
 Day 15* 
 Day 22* 
 Day 28-30 (one sample in this 
timeframe) 
3-5 mL blood in a 
gold top tube 
(serum separator 
tube) 
MicroRNA^ 
 
See Section 14.2.2 
X 
 Day 1, before CPX-351 infusion 
 Day 5, 6 hours (± 1 hr) after the 
end of the infusion of the 3rd 
dose of CPX-351  
 Day 8* 
 Day 15* 
 Day 22* 
 Day 28-30 (one sample in this 
timeframe) 
5-10 mL peripheral 
blood in a 
purple/lavender top 
(EDTA) tube 
Whole exome 
sequencing^ 
See Section 14.2.3 
----- 
 Day 28-30 (one sample in this 
timeframe) 
2-4 mL peripheral 
blood in a purple 
top (EDTA) tube 
Pharmacokinetic 
sampling 
 
See Section 14.1 
----- 
 Day 1: 30 minutes after end of 
infusion; Day 5 prior to 
infusion, 30 minutes after end 
of infusion, 6 ± 2 hours from 
start of infusion; Day 8, 72 ± 24 
hours from start of  Day 5 
infusion 
2 mL blood in a 
tube provided in 
PK sampling kit 
(See protocol web 
page for PK 
manual) 
*Samples for Biomarkers of cardiac injury and MicroRNA may be drawn up to 24 hours prior 
to the time point and up to 24 hours after the time point.  
^ See Section 14.2.4 for shipping details. 
 
 
 
 

  
 
 
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8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY 
CRITERIA 
8.1 
Criteria for Removal from Protocol Therapy 
a) Relapsed disease (see Section 10.2). 
b) Treatment failure (see Section 10.2). 
c) Unacceptable toxicity due to protocol therapy (see Section 5.0). 
d) Refusal of further protocol therapy by patient/parent/guardian. 
e) Completion of planned therapy. 
f) Physician determines it is in patient’s best interest. 
g) Development of a second malignancy. 
h) Pregnancy. 
i) Repeat eligibility studies (if required) are outside the parameters required for 
eligibility (see Section 3.2). 
 
Patients who are off protocol therapy are to be followed until they meet the criteria for Off 
Study (see below). Follow-up data will be required unless patient is taken off study. 
 
8.2 
Off Study Criteria 
a) Death. 
b) Lost to follow-up. 
c) Patient enrollment onto another COG study with tumor therapeutic intent (eg, at 
recurrence). 
d) Withdrawal of consent for any further data submission. 
e) The fifth anniversary of the date the patient was enrolled on this study. 
 
 
9.0 
STATISTICAL CONSIDERATIONS 
9.1 
Sample Size and Study Duration 
To complete this study, up to 50 evaluable patients (12 for the Dose Finding portion, 38 for 
the Efficacy portion) are required. Allowing for 10% of patients being inevaluable, an 
enrollment of up to 56 patients is required.  
 
Based on the accrual to the Dose Finding portion of AAML0523 at a similarly restricted 
number of sites, it is estimated that 1.5 patients per month will be enrolled on the Dose Finding 
portion of this study. We estimate that it will require up to 12 months to complete safety and 
DLT assessments for Cycle 1 (CPX-351), allowing 2 months between the first dose of study 
drug and DLT assessment. Based on accrual to recent COG studies for patients with 
relapsed/refractory AML (2.2 patients per month on AAML0523 and 1.8 patients per month 
on AAML07P1 Arm B, run simultaneously), it is estimated that 5 patients overall will accrue 
to this study each month for the Efficacy phase. To evaluate the efficacy of CPX-351, up to 
38 evaluable patients will be enrolled. Therefore, we expect accrual for the Efficacy portion 
study to take approximately 11 months allowing for 2 months between stages 1 and 2 to assess 
response for the last patient treated on stage 1. Allowing for DLT and efficacy assessment, we 
anticipate that 56 patients could be enrolled in approximately 23 months. 

  
 
 
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As described above, we anticipate that 38 patients will be enrolled on the Efficacy phase of 
the study.  In order to more precisely estimate the PK parameters of CPX-351 in children, we 
will extend the PK collection of this study to continue during the Efficacy phase.  In reviewing 
the experience on COG AALL07P1, where optional PK studies were collected in a similar 
manner in the Efficacy phase of that study, samples from 25 of 37 patients (68%) were 
successfully collected.  Therefore, we would expect approximately 25 patients to participate 
in PK collection during the Efficacy phase of AAML1421. In parallel, Jazz Pharmaceuticals 
also has collected PK samples on 16 patients participating in the Cincinnati Phase I/II single-
institution trial (11 at 100 units/m2/dose, 5 at 134 units/m2/dose). They anticipate 5 more 
patients will accrue to that study and provide additional samples  
 
A population PK analysis will be conducted by Jazz Pharmaceuticals using PK data from this 
study, the aforementioned pediatric study in Cincinnati, and future Phase 3 pediatric study to 
better estimate CPX-351 PK parameters in children. We will recruit children with all age 
groups, with an effort to enroll as many young children as possible. We anticipate the PK 
collected on this study will make significant strides in fulfilling their requirements to more 
precisely estimate the PK parameters of CPX-351 in children. 
 
 
9.2 
Study Design 
 
9.2.1 
Dose-Finding Phase 
The phase 1 Dose-Finding portion of the study was completed in 12/2016. 
The Dose-Finding Phase will be conducted using a modified rolling 6 design. Two to six 
patients can be concurrently enrolled onto a dose level, dependent upon (1) the number of 
patients enrolled at the current dose level, (2) the number of patients who have experienced 
DLT at the current dose level, and (3) the number of patients entered but with tolerability data 
pending at the current dose level. Accrual is suspended when a cohort of six has enrolled or 
when the study endpoints have been met. Dose level assignment is based on the number of 
participants currently enrolled in the cohort, the number of DLTs observed, and the number 
of participants at risk for developing a DLT (ie, participants enrolled but who are not yet 
assessable for toxicity). For example, when three participants are enrolled onto a dose cohort, 
if toxicity data is available for all three and there are no DLTs, the recommended dose has 
been reached. If data is not yet available for one or more of the first three participants, or if 
one DLT has been observed, the new participant is entered at the same dose level. Lastly, if 
two or more DLTs have been observed, the dose level is de-escalated. This process is repeated 
for participants five and six. In place of suspending accrual after every three participants, 
accrual is only suspended when a cohort of six is filled. When participants are inevaluable for 
toxicity, they are replaced with the next available participant if escalation or de-escalation 
rules have not been fulfilled at the time the next available participant is enrolled onto the study. 
 
The following table provides the decision rules for enrolling a patient at (i) the current dose 
level (ii) at an escalated dose level, (iii) at a de-escalated dose level, or whether the study is 
suspended to accrual: 
 
 
 
 

  
 
 
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# Pts 
Enrolled 
# Pts with 
DLT 
# Pts without 
DLT 
# Pts with Data 
Pending 
Decision 
2 
0 or 1 
0, 1 or 2 
0, 1 or 2 
Same dose level 
2 
2 
0 
0 
De-escalate* 
3 
0 
0, 1 or 2 
1, 2 or 3 
Same dose level 
3 
1 
0, 1 or 2 
0, 1 or 2 
Same dose level 
3 
0 
3 
0 
Escalate** 
3 
2 
0 or 1 
0 or 1 
De-escalate* 
4 
0 
0, 1, 2 or 3 
1, 2, 3 or 4 
Same dose level 
4 
1 
0, 1, 2 or 3 
0, 1, 2 or 3 
Same dose level 
4 
0 
4 
0 
Escalate** 
4 
2 
0, 1 or 2 
0, 1 or 2 
De-escalate* 
5 
0 
0, 1, 2, 3 or 4 
1, 2, 3, 4 or 5 
Same dose level 
5 
1 
0, 1, 2, 3 or 4 
0, 1, 2, 3 or 4 
Same dose level 
5 
0 
5 
0 
Escalate** 
5 
2 
0, 1, 2 or 3 
0, 1, 2 or 3 
De-escalate* 
6 
0 
0, 1, 2, 3, or 4 
2, 3, 4, 5 or 6 
Suspend 
6 
1 
0, 1, 2, 3 or 4 
0, 1, 2, 3 or 4 
Suspend 
6 
0 or 1 
5 or 6 
0 or 1 
Escalate** 
6 
2 
0, 1, 2, 3 or 4 
0, 1, 2, 3 or 4 
De-escalate* 
* If six patients already entered at next lower dose level, the MTD has been defined. 
** If final dose level has been reached, the recommended dose has been reached. 
 
9.2.2 Efficacy Phase 
The Efficacy Phase is a single arm two-stage design. The following optimal Simon two-stage 
design will be used to test the null hypothesis that the overall response rate is ≤ 40% versus 
the alternative hypothesis that the response rate is ≥ 60%. 
 
 
Cumulative Number 
of Responses 
Decision 
Stage 1: Enter 12 patients 
5 or fewer 
Terminate the trial because 
the therapy is ineffective 
 
6-12 
Inconclusive result, continue 
trial (proceed to Stage 2) 
Stage 2: Enter 26 additional 
patients 
18 or fewer 
Conclude therapy is 
ineffective 
 
19-38 
Conclude therapy is effective 
 
If the therapy is associated with a 40% response rate (CR+CRp) after up to 2 cycles of 
therapy, the therapy will be identified as effective with probability 0.10. If the therapy is 
associated with a 60% response rate, the therapy will be identified as effective with 
probability 0.80. 
 
Results from the CCG 2951 study showed that a combination of mitoxantrone/cytarabine 
(MA) used for induction in pediatric patients with refractory/relapsed AML achieved a 
marrow remission rate of 76% and an overall response (CR + CRp) rate (criteria similar to 

  
 
 
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those used in this study) of 58%.73 Response was assessed after one cycle of MA, however, 
these children received significantly less intensive treatment for their de novo AML than 
those who will relapse after receiving current de novo regimens. In addition to fewer high 
dose cytarabine consolidation cycles, they received a lower cumulative anthracycline 
exposure compared with patients who will relapse following AAML0531 and AAML1031 
(≈ 290 mg/m2 vs. ≈ 440 mg/m2). Thus, a response rate lower than 58% would be expected 
after one cycle for those who now receive anthracycline-containing reinduction regimens. 
However, this response was measured after one cycle of protocol therapy. 
 
More recently, COG AAML0523 tested the combination of clofarabine and cytarabine in 
children with AML in first relapse. In this trial, response was defined as best response (CR 
+ CRp) after up to 2 cycles. Of 48 patients, the overall response rate was 48%.74 Of 23 
responders, 11 (48%) were SD after Cycle 1, then achieved CR or CRp after Cycle 2. This 
supports the importance of receiving two cycles of reinduction therapy in order to 
determine the optimal response to therapy. Importantly, only 1/11 (9%) of patients who 
had > 20% blasts after Cycle 1 achieved CR or CRp in Cycle 2. This is consistent with data 
from the FLAG vs. FLAG/DNX study published by Kaspers et al (Appendix Fig A1, online 
only) demonstrating dismal outcomes and low second remission rates in patients with > 
20% blasts after Cycle 1.2 However, Partial Remission is (PR) is a standard response 
definition for COG leukemia studies and includes blasts percentages between 5 and 25%. 
Therefore, patients who have > 25% blasts after Cycle 1 will be considered non-responders 
and removed from protocol therapy in order to be able to pursue other options.  
 
Kaspers et al. randomized patients to receive either FLAG or FLAG/DNX in children with 
AML in first relapse.2,75 In this trial, the primary endpoint was Day 28 bone marrow status. 
That is, patients who had < 20% blasts at Day 28 were determined to have a good early 
response to therapy. There was not a determination of CR rates after the first cycle of FLAG 
or FLAG/DNX. The secondary endpoint of the study was CR rate after two cycles of 
chemotherapy (FLAG/DNX then FLAG vs. FLAG then FLAG). As mentioned previously, 
the CR rate (ANC > 1000, platelets > 50K) after two cycles was 69% with FLAG/DNX, 
and 59% with FLAG (p=0•07).29 However, their CR definition was different in that study 
(ANC > 1000, platelets > 50K), than what is used for COG relapsed AML studies (ANC > 
1000, platelets > 100K).  
 
9.3 
Methods of Analysis 
The response rate (CR+CRp), best response after up to two cycles, will be determined using 
the approach of Jung and Kim.76 The corresponding 90% confidence interval will be 
calculated using the approach of Koyama and Chen.77 The response rate will be calculated 
as the ratio of the number of patients who demonstrate response after up to two cycles of 
therapy divided by the number of patients evaluable for response. Descriptive statistics will 
be used to summarize the proportion of patients experiencing ≥ Grade 3 non-hematologic 
toxicities, cardiac toxicities, and infections, summarize length of hospitalization time, and 
estimate time to bone marrow and peripheral blood cell count recovery. The impact of the 
timing of relapse on outcomes will be investigated in this study, but these analyses will be 
exploratory. 
 
Pharmacokinetic properties of CPX-351 
Steady state pharmacokinetics of CPX-351 will be obtained in all patients treated in the 
Dose Finding phase of the study. Descriptive statistics will be used to summarize the 
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Area Under the Curve (AUC)). These parameters will be summarized with summary 
statistics, including means, medians, ranges, and standard deviations (if numbers and 
distribution permit). 
 
PK samples will also be collected during the efficacy portion of the study following the 
sparse sampling scheme outlined in Section14.1. Participation in these PK samples will be 
optional for patients treated on the efficacy phase. Descriptive statistics will be used to 
summarize plasma concentrations of total cytarabine and daunorubicin at each time point.  
 
Monitoring of Toxic Death 
Dose modification or termination will be considered if 3 or more of the first 20 patients in 
the Efficacy phase experience a toxic mortality (TM), defined as death within 30 days of 
the last dose of CPX-351 and not associated with disease progression, or if 5 or more of 
the 38 patients in the Efficacy phase experience a TM. Dose modification or termination 
will be considered with probability 0.05 if the true TM rate is 5% and with probability 0.79 
if the true TM rate is 20%. 
 
 
Monitoring of Cardiac Toxicity 
Dose modification or termination will be considered if 3 or more of the first 20 patients in 
the Efficacy phase experience a severe cardiac toxicity, defined as Grade 4 or higher 
ejection fraction probably or definitely related to CPX-351 during the first cycle of 
chemotherapy, or if 5 or more of the 38 patients in the Efficacy phase experience a severe 
cardiac toxicity. Note: Grade 4 or higher ejection fraction related to sepsis will not be 
considered a severe cardiac toxicity for this monitoring rule. Dose modification or 
termination will be considered with probability 0.05 if the true rate is 5% and with 
probability 0.79 if the true rate is 20%. 
 
9.4 
Evaluability for Response  
A patient will be considered evaluable for response if: (1) the patient meets the eligibility 
criteria for the Efficacy Phase; (2) the patient receives at least one dose of CPX-351 at the 
RP2D; and (3) the patient is under follow-up for a sufficient period to evaluate the disease 
at the end of the Cycle 1 or meets the definition of Treatment Failure in Section 10.2. 
Patients who demonstrate a CR or CRp as delineated in Section 10.2 will be considered to 
have experienced a response. All other evaluable patients will be considered non-
responders. A patient who dies as a result of toxicity during Cycle 1 after receiving all or 
part of protocol therapy will be considered a nonresponder. In addition, the proportion of 
patients with CRi will be estimated. 
 
9.5 
Evaluability for Toxicity  
Any patient who receives at least one dose of study drug will be considered evaluable for 
toxicity.  
 
9.6 
Correlative Biology Studies 
9.6.1 
Analytic Plan for Study Objective 1.3.1: Response in Biomarkers of Cardiac Injury 
to a Single Cycle of CPX-351 
The incidence, timing and magnitude of elevations in troponin, NT-BNP, hsCRP 
will be described. Spearman correlation coefficients will be used to correlate the 

  
 
 
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post-treatment values of troponin, NT-BNP, and hsCRP with previous cumulative 
anthracycline dose prior to CPX-351, change in ejection fraction between pre- and 
post-CPX-351 baseline assessed by echocardiogram before and after Cycle 1, and 
change in global longitudinal strain (GLS). A sample size of 30 patients will provide 
80% power (α=0.05) to detect a minimum correlation of 0.49. Additionally, two-
sample t-tests will be used to compare mean post-treatment values of troponin, NT-
BNP, and hsCRP for those with an ejection fraction decrease of less than 10% from 
baseline versus those 10% or greater from baseline. These descriptive analyses will 
provide critical preliminary data on which to base the timing and power 
calculations for subsequent phases of CPX-351 investigations in order to allow 
more definitive assessment of its cardiotoxic effects relative to other anthracycline 
regimens. 
 
9.6.2 
Analytic Plan for Study Objective 1.3.2: Effect of CPX-351 on Novel Biochemical 
and Imaging Markers of Cardiotoxicity, Including Plasma miRNAs and 
Myocardial Deformation   
 
Quantification of change in GLS from baseline 
A paired t-test will be used to compare the mean global longitudinal strain (GLS) 
between the pre-treatment (at relapse) baseline and post-Cycle 1 echocardiogram. A 
sample size of 30 patients will provide 80% power (with 2 sided α=0.05) to detect a 
1.6% reduction in GLS, assuming a GLS standard deviation of 3%.78 
 
Quantification of miRNA change from baseline 
Candidate plasma miRNAs will be quantified at baseline (prior to relapse therapy) 
and Day 5 (6 hours from the 3rd dose of CPX-351), Day 6, and once Day 28-30 post-
CPX-351 using TaqMan microRNA Assays. Plasma miR-29b and -499 fold change 
from pre-treatment baseline will be determined at each post-treatment time point 
using the DDCt method. A sample size of 30 patients will provide 80% power 
(α=0.05) to detect a Ct difference from baseline of 1.7 for miR-29b and 1.6 for miR-
499, assuming Ct variability of 3.2 and 3 standard deviations, respectively, for miR-
29b and miR-499 (based on K. Leger pilot data).  
 
Correlation of GLS and miRNA 
The relationship between 6-hour miR-29b and -499 expression and change in LV 
GLS between the pre-cycle and end of Cycle 1 echocardiograms will be determined 
by calculating a Spearman correlation coefficient. The a priori focus on the 6-hour 
time point as the primary independent variable is based on preliminary data  revealing 
that differences in miRNA expression were greatest 6 hours following anthracycline 
therapy. However, a linear mixed effects model incorporating the additional time 
points will be used to explore the longitudinal relationship with each time point as a 
predictor variable for the measures of early cardiotoxicity. A sample size of 30 
patients will provide 80% power (α=0.05) to detect a minimum correlation of 0.49. 
Power calculations were generated in PASS13. 
 
9.6.3 
Analytic Plan for Study Objective 1.3.3: The Role of Rare Coding Variants as Risk 
Factors for Anthracycline-Induced Cardiomyopathy 
Our computational approach can be summarized as a two-step process with alignment 
of the sequence data using a commercial aligner followed by subsequent statistical 
analyses from post-alignment data. Sequence analysis will follow GATK’s best 

  
 
 
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practices in which duplicate variants from the sequencing process are removed 
(https://www.broadinstitute.org/gatk/guide/best-practices?bpm=DNAseq). 
In 
addition, local realignment is performed on mismatching bases around indels, a 
known artifact often produced by the initial mapping algorithm. Quality scores on 
each individual mapped base are also recalibrated to account for expected errors 
emitted by the sequencing machine. Finally, variations on genomic sites are identified 
relative to a reference genome and scores are assigned to each discovered variant to 
minimize both false positives and negatives. Custom Amazon Machine Image with 
all necessary tools and reference datasets in the Docker format will be developed for 
deployment in the Amazon cloud infrastructure. StarCluster will be used to manage 
the creation of all Amazon EC2 regular instances. Requested data as well as all 
intermediaries and final processed files are stored in Amazon S3 buckets with 
protected access. Finally, processed results will be downloaded to internal servers at 
the Children’s Hospital of Philadelphia (CHOP) for visualization. 
 
9.7 
Gender and Minority Accrual Estimates 
The gender and minority distribution of the study population is expected to be: 
 
Racial Categories 
Ethnic Categories 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
 
Female 
Male 
Female 
Male 
 
American Indian/ Alaska 
Native 
0 
0 
0 
0 
0 
Asian 
1 
4 
0 
0 
5 
Native Hawaiian or Other 
Pacific Islander 
0 
0 
0 
0 
0 
Black or African 
American 
7 
1 
0 
0 
8 
White 
23 
15 
1 
4 
43 
More Than One Race 
0 
0 
0 
0 
0 
Total 
31 
20 
1 
4 
56 
 
This distribution was derived from AAML07P1. 
 
 

  
 
 
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10.0 
EVALUATION CRITERIA 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
This study will utilize version 4.0 of the CTCAE of the National Cancer Institute (NCI) for 
toxicity and performance reporting. A copy of the CTCAE version 4.0 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 v4.0’ is understood to represent the most current version of CTCAE 
v4.0 as referenced on the CTEP website (ie, v4.02 and all subsequent iterations prior to 
version 5.0). 
 
10.2 
Response Criteria   
Response criteria will employ revised AML International Working Group Criteria,79 and 
include:  
 
Overall Response (OR): OR rate is defined as the sum of the number of patients with 
complete remission (CR) plus those with complete remission in the absence of total platelet 
recovery (CRp) divided by the total number of evaluable enrolled patients.  
 
Complete Remission (CR): Attainment of an M1 bone marrow (< 5% blasts) with no 
evidence of circulating blasts or extramedullary disease and with recovery of peripheral 
blood counts (ANC ≥ 1000/µL and platelet count ≥ 100,000/µL). Flow cytometry may also 
be useful to distinguish between leukemia and a regenerating bone marrow. There is no 
requirement for bone marrow cellularity. 
 
CR With Partial Recovery of Platelet Count (CRp): Attainment of an M1 bone marrow 
(< 5% blasts) and no evidence of circulating blasts or extramedullary disease and with 
recovery of ANC ≥ 1000/µL and platelet transfusion independence (defined as: no platelet 
transfusions x 1 week). 
 
Complete Remission with Incomplete Blood Count Recovery (CRi): Attainment of an 
M1 bone marrow (< 5% blasts) and no evidence of circulating blasts or extramedullary 
disease and with ANC < 1000/µL or platelet count < 100,000/µL without platelet 
transfusion independence (defined as: no platelet transfusions x 1 week). 
 
Partial Response (PR): M2 bone marrow (5% to 25% blasts) and at least a 50% decrease 
in bone marrow blast percent from baseline. Bone marrow must have adequate cellularity 
(eg, ≥ 10%, if a biopsy is performed) to determine response. PR status will not be included 
in calculation of response to the regimen. A repeat bone marrow aspiration within 14 days 
may be required to distinguish between a PR and increased blasts caused by bone marrow 
regeneration, and is left at the discretion of the investigator.  
 
Treatment Failure (TF): 
In the Efficacy phase, if after Cycle 1 (CPX-351) a patient has a hypoplastic bone marrow 
for ≥ 60 days and failure to recover a peripheral ANC > 500/μL and a non-transfusion 
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(defined as ≥ Grade 3), they will be considered a treatment failure and will go off protocol 
therapy. 
 
The definition of treatment failure includes: 
a. An increase in the extent of bone marrow infiltration by leukemic cells (absolute 
increase of ≥ 20% blasts) OR 
b. Development of extramedullary disease (EMD) OR 
c. M2 marrow that does not qualify for PR status OR 
d. An M1 marrow with circulating blasts OR  
e. > 25% blasts in the bone marrow after Cycle 1 of therapy.  
 
Relapse: Morphologic relapse after CR/CRp/CRi 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 (eg, bone marrow regeneration after therapy). 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 flow cytometric or molecular analyses suggest relapse (by 
the reappearance of a similar immunophenotype or mutation 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% blasts were observed in the marrow. The reappearance or development of cytologically 
proven extramedullary disease also indicates relapse. Molecular and/or genetic relapse is 
characterized by reappearance of a cytogenetics or molecular abnormality.  
 
Unevaluable: Aplastic or severely hypocellular marrow. In this instance, marrow 
evaluation should be repeated weekly until response determination can be made through at 
least Day 60. 
 
Bone Marrow Classification: 
M1 is  5% blasts 
M2 is 5 to 25% blasts 
M3 is > 25% blasts 
 
 
 
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. Certain adverse events must be reported 
in an expedited manner to allow for timelier monitoring of patient safety and care. The 
following sections provide information about expedited reporting. 
 
11.2 
Determination of reporting requirements 
Reporting requirements may include the following considerations: 1) whether the patient 
has received an investigational or commercial agent; 2) the characteristics of the adverse 
event including the grade (severity), the relationship to the study therapy (attribution), and 
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trial; and 4) whether or not hospitalization or prolongation of hospitalization was associated 
with the event.  
 
An investigational agent is a protocol drug administered under an Investigational New 
Drug Application (IND). In some instances, the investigational agent may be available 
commercially, but is actually being tested for indications not included in the approved 
package label.  
 
Commercial agents are those agents not provided under an IND but obtained instead from 
a commercial source. The NCI, rather than a commercial distributor, may on some 
occasions distribute commercial agents for a trial. 
 
When a study includes both investigational and commercial agents, the following rules 
apply. 
 
Concurrent administration: When an investigational agent is used in 
combination with a commercial agent, the combination is considered to be 
investigational and expedited reporting of adverse events would follow the 
guidelines for investigational agents. 
 
Sequential administration: When a study includes an investigational agent 
and a commercial agent on the same study arm, but the commercial agent is 
given for a period of time prior to starting the investigational agent, 
expedited reporting of adverse events which occur prior to starting the 
investigational agent would follow the guidelines for commercial agents. 
Once therapy with the investigational agent is initiated, all expedited 
reporting of adverse events follow the investigational agent reporting 
guidelines. 
 
11.3 
Expedited Reporting Requirements – Serious Adverse Events (SAEs) 
To ensure compliance with these regulations/this guidance, NCI requires that AEs be 
submitted according to the timeframes in the AE reporting tables assigned to the protocol, 
using the CTEP Adverse Event Reporting System (CTEP-AERS). 
 
Any AE that is serious qualifies for expedited reporting. An AE is defined as any 
untoward medical occurrence associated with the use of a drug in humans, whether or not 
considered drug related. A Serious Adverse Event (SAE) is any adverse drug event 
(experience) occurring at any dose that results in ANY of the following outcomes: 
 
1) Death. 
2) A life-threatening adverse drug experience. 
3) An adverse event resulting in inpatient hospitalization or prolongation of existing 
hospitalization (for ≥ 24 hours). This does not include hospitalizations that are part 
of routine medical practice. 
4) A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions. 
5) A congenital anomaly/birth defect. 
6) Important Medical Events (IME) that may not result in death, be life threatening, 
or require hospitalization may be considered a serious adverse drug experience 
when, based upon medical judgment, they may jeopardize the patient or subject 
and may require medical or surgical intervention to prevent one of the outcomes 
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11.4 
Specific Examples for Expedited Reporting  
11.4.1 SAEs Occurring More than 30 Days After Last Dose of Study Drug 
Any Serious Adverse Event that occurs more than 30 days after the last 
administration of the investigational agent/intervention and has an attribution of a 
possible, probable, or definite relationship to the study therapy must be reported 
according to the CTEP-AERS reporting tables in this protocol.  
 
11.4.2 Persistent or Significant Disabilities/Incapacities 
Any AE that results in persistent or significant incapacity or substantial disruption 
of the ability to conduct normal life functions (formerly referred to as disabilities), 
congenital anomalies or birth defects, must be reported via CTEP-AERS if it 
occurs at any time following treatment with an agent under a NCI, COG, or 
industry sponsor IND/IDE since these are considered to be serious AEs. 
 
11.4.3 Death 
Reportable Categories of Death  
o Death attributable to a CTCAE term.  
o Death Neonatal: A disorder characterized by cessation of life during the first 
28 days of life.  
o Sudden Death NOS: A sudden (defined as instant or within one hour of the 
onset of symptoms) or an unobserved cessation of life that cannot be 
attributed to a CTCAE term associated with Grade 5.  
o Death NOS: A cessation of life that cannot be attributed to a CTCAE term 
associated with Grade 5.  
o Death due to progressive disease should be reported as Grade 5 “Disease 
progression” in the system organ class (SOC) “General disorders and 
administration site conditions”.  Evidence that the death was a manifestation 
of underlying disease (e.g., radiological changes suggesting tumor growth 
or progression: clinical deterioration associated with a disease process) 
should be submitted.  
 
Any death occurring within 30 days of the last dose, regardless of attribution to 
the investigational agent/intervention requires expedited reporting within 
24 hours.  
 
Any death occurring greater than 30 days after the last dose of the investigational 
agent/intervention requires expedited reporting within 24 hours only if it is 
possibly, probably, or definitely related to the investigational agent/intervention. 
 
11.4.4 Secondary Malignancy 
A secondary malignancy is a cancer caused by treatment for a previous 
malignancy (e.g., treatment with investigational agent/intervention, radiation or 
chemotherapy). A metastasis of the initial neoplasm is not considered a secondary 
malignancy.  

  
 
 
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All secondary malignancies that occur following treatment need to be reported via 
CTEP-AERS. Three options are available to describe the event:  
 
Leukemia secondary to oncology chemotherapy  
 
Myelodysplastic syndrome  
 
Treatment related secondary malignancy  
 
Any malignancy possibly related to cancer treatment (including AML/MDS) must 
also be reported via the routine reporting mechanisms outlined in this protocol. 
 
11.4.5 Second Malignancy 
A second malignancy is one unrelated to the treatment of a prior malignancy (and 
is NOT a metastasis from the initial malignancy). Second malignancies require 
ONLY routine reporting via CDUS unless otherwise specified. 
 
11.4.6 Pregnancy, Pregnancy Loss, and Death Neonatal 
NOTE: When submitting CTEP-AERS reports for “Pregnancy”, “Pregnancy loss”, 
or “Neonatal loss”, the Pregnancy Information Form, available at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/Pregna
ncyReportForm.pdf, needs to be completed and faxed along with any additional 
medical information to 301-230-0159. The potential risk of exposure of the fetus 
to the investigational agent(s) or chemotherapy agent(s) should be documented in 
the “Description of Event” section of the CTEP-AERS report. 
 
11.4.6.1 Pregnancy 
Patients who become pregnant on study risk intrauterine exposure of the 
fetus to agents that may be teratogenic. For this reason, pregnancy needs 
to be reported in an expedited manner via CTEP-AERS as Grade 3 
“Pregnancy, 
puerperium 
and 
perinatal 
conditions 
- 
Other 
(pregnancy)” under the Pregnancy, puerperium and perinatal 
conditions SOC. 
 
Pregnancy needs to be followed until the outcome is known. If the baby 
is born with a birth defect or anomaly, then a second CTEP-AERS report 
is required. 
 
11.4.6.2 Pregnancy Loss (Fetal Death) 
Pregnancy loss is defined in CTCAE as “Death in utero”. Any pregnancy 
loss needs to be reported expeditiously, as Grade 4 “Pregnancy loss, 
puerperium and perinatal conditions” SOC. Do NOT report a 
pregnancy loss as a Grade 5 event since CTEP-AERS recognizes any 
Grade 5 event as a patient death. 
 
11.4.6.3 Death Neonatal  
Neonatal death, defined in CTCAE as “Newborn death occurring during 
the first 28 days after birth” should be reported expeditiously, as Grade 

  
 
 
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4 “Death neonatal” under the “General disorders and administration” 
SOC when the death is the result of a patient pregnancy or 
pregnancy in partners of men on study. Do NOT report a neonatal 
death resulting from a patient pregnancy or pregnancy in partners of men 
on study as a Grade 5 event since CTEP-AERS recognizes any Grade 5 
event as a patient death.  
 
11.5 
Reporting Requirements for Specialized AEs 
11.5.1 Baseline AEs 
Although a pertinent positive finding identified on baseline assessment is not an 
AE, when possible it is to be documented as “Course Zero” using CTCAE 
terminology and grade. An expedited AE report is not required if a patient is 
entered on a protocol with a pre-existing condition (eg, elevated laboratory value, 
diarrhea). The baseline AE must be re-assessed throughout the study and reported 
if it fulfills expedited AE reporting guidelines. 
 
a. If the pre-existing condition worsens in severity, the investigator must 
reassess the event to determine if an expedited report is required. 
b. If the AE resolves and then recurs, the investigator must re-assess the event 
to determine if an expedited report is required. 
c. No modification in grading is to be made to account for abnormalities 
existing at baseline. 
 
11.5.2 Persistent AEs 
A persistent AE is one that extends continuously, without resolution between 
treatment cycles/courses.  
ROUTINE reporting: The AE must be reported only once unless the grade 
becomes more severe in a subsequent course. If the grade becomes more severe 
the AE must be reported again with the new grade. 
EXPEDITED reporting: The AE must be reported only once unless the grade 
becomes more severe in the same or a subsequent course. 
 
11.5.3 Recurrent AEs 
A recurrent AE is one that occurs and resolves during a cycle/course of therapy 
and then reoccurs in a later cycle/course. 
ROUTINE reporting: An AE that resolves and then recurs during a subsequent 
cycle/course must be reported by the routine procedures. 
EXPEDITED reporting: An AE that resolves and then recurs during a subsequent 
cycle/course does not require CTEP-AERS reporting unless: 
1) The grade increases OR 
2) Hospitalization is associated with the recurring AE. 
 
11.6 
Exceptions to Expedited Reporting 
An expedited report may not be required for a specific protocol where an AE is listed as 
expected. The exception or acceptable reporting procedures will be specified in the 
protocol. The protocol specific guidelines supersede the NCI Adverse Event Reporting 
Guidelines. These special situations are listed under the CTEP-AERS reporting Table A 
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11.7 
Reporting Requirements - Investigator Responsibility 
Clinical investigators in the treating institutions and ultimately the Study Chair have the 
primary responsibility for AE identification, documentation, grading, and assignment of 
attribution to the investigational agent/intervention. It is the responsibility of the treating 
physician to supply the medical documentation needed to support the expedited AE reports 
in a timely manner.  
 
Note: All expedited AEs (reported via CTEP-AERS) must also be reported via routine 
reporting. Routine reporting is accomplished via the Adverse Event (AE) Case Report 
Form (CRF) within the study database.   
 
11.8 
General Instructions for Expedited Reporting via CTEP-AERS  
The reporting methods described below are specific for clinical trials evaluating 
agents for which the IND is held by COG, an investigator, or a pharmaceutical 
company. It is important to note that these procedures differ slightly from those used 
for reporting AEs for clinical trials for which CTEP holds the IND. 
 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting 
beginning April 1, 2018. All appropriate treatment areas should have access to a copy of 
the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the 
CTEP 
web 
site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications
/ctc.htm. 
 
An expedited AE report must be submitted electronically via CTEP-AERS at:  
https://eapps-ctep.nci.nih.gov/ctepaers 
 
Expedited AE reporting timelines are defined as: 
o 24-Hour; 5 Calendar Days - The AE must initially be reported via CTEP-
AERS within 24 hours of learning of the event, followed by a complete 
expedited report within 5 calendar days of the initial 24-hour report. 
o 7 Calendar Days - A complete expedited report on the AE must be submitted 
within 7 calendar days of the investigator learning of the event.  
 
 
Any event that results in a persistent or significant incapacity/substantial disruption of 
the ability to conduct normal life functions, or a congenital anomaly/birth defect, or is 
an IME, which based upon the medical judgment of the investigator may jeopardize 
the patient and require intervention to prevent a serious AE, must be reported via 
CTEP-AERS if the event occurs following investigational agent administration.  
 
 
Any death occurring within 30 days of the last dose, regardless of attribution to an 
agent/intervention requires expedited reporting within 24 hours via e-mail to the COG 
CTEP-AERS Coordinator and Study Chair.  
 
Any death occurring greater than 30 days of the last dose with an attribution of 
possible, probable, or definite to an agent/intervention requires expedited reporting 
within 24 hours via e-mail to the COG CTEP-AERS Coordinator and Study Chair. 
CTEP-AERS Medical Reporting includes the following requirements as part of the report: 
1) whether the patient has received at least one dose of an investigational agent on this 
study; 2) the characteristics of the adverse event including the grade (severity), the 

  
 
 
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relationship to the study therapy (attribution), and the prior experience (expectedness) of 
the adverse event; 3) the Phase (1, 2, or 3) of the trial; and 4) whether or not hospitalization 
or prolongation of hospitalization was associated with the event.  
 
Fax or email supporting documentation for AEs related to investigational agents to 
COG: Fax # (310) 640-9193; email: COGAERS@childrensoncologygroup.org; Attention: 
COG AERS Coordinator.  
 
 
ALWAYS include the ticket number on all faxed documents. 
 
 
Use the NCI protocol number and the protocol-specific patient ID 
provided during trial registration on all reports. 
 
 
11.9 
Reporting Table for Late Phase 2 and Phase 3 Studies – Table A 
Expedited Reporting Requirements for Adverse Events that Occur on Studies under an 
IND/IDE within 30 Days of the Last Administration of the Investigational 
Agent/Intervention 1 
 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312)  
NOTE: Investigators MUST immediately report to the sponsor (COG) ANY Serious Adverse Events, 
whether or not they are considered related to the investigational agent(s)/intervention (21 CFR 312.64)  
An adverse event is considered serious if it results in ANY of the following outcomes:  
1) Death.  
2) A life-threatening adverse event.  
3) Any AE that results in inpatient hospitalization or prolongation of existing hospitalization for 
≥ 24 hours. This  does not include hospitalizations that are part of routine medical practice. 
4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions.  
5) A congenital anomaly/birth defect.  
6) Important Medical Events (IME) that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they may jeopardize the 
patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed 
in this definition. (FDA, 21 CFR  312.32; ICH E2A and ICH E6.)  
 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI 
via CTEP-AERS within the timeframes detailed in the table below.   
 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 
Timeframes 
Grade 4 & 5 
Timeframes 
Resulting 
in 
Hospitalization 
≥ 24 hrs 
 
7 Calendar Days 
 
 
24-Hour Notification 
5 Calendar Days 
Not resulting in 
Hospitalization 
≥ 24 hrs 
 
Not Required 
 
7 Calendar Days 
NOTE: Protocol specific exceptions to expedited reporting of serious adverse events are found in the 
Specific Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR. Additional Special 
Situations as Exceptions to Expedited Reporting are listed below.  
 
Expedited AE reporting timelines are defined as:  
“24-Hour; 5 Calendar Days” - The AE must initially be reported via CTEP-AERS within 24 hours of 
learning of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour 
notification. 

  
 
 
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“7 Calendar Days” - A complete expedited report on the AE must be submitted within 7 calendar days of 
learning of the AE.  
 
1SAEs that occur more than 30 days after the last administration of investigational agent/intervention and 
have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 5 calendar days for:  
 
All Grade 4, and Grade 5 AEs  
Expedited 7 calendar day reports for:  
 
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
 
Grade 3 adverse events  
 
 
11.10 Protocol Specific Additional Instructions and Reporting Exceptions 
 
Grades 1-4 myelosuppression (anemia, neutropenia, thrombocytopenia) do not 
require expedited reporting.  
 
 
Grades 1-2 AST/ALT elevations do not require expedited reporting. 
 
11.11 Reporting of Adverse Events for commercial agents – CTEP-AERS abbreviated 
pathway 
 
The following are expedited reporting requirements for adverse events experienced by 
patients on study who have not received any doses of an investigational agent on this study. 
Commercial reporting requirements are provided in Table B.  
 
COG requires the CTEP-AERS report to be submitted within 7 calendar days of learning 
of the event. 
 
Table B  
Reporting requirements for adverse events experienced by patients on study who 
have NOT received any doses of an investigational agent on this study. 
 
CTEP-AERS Reporting Requirements for Adverse Events That Occur During 
Therapy with a Commercial Agent or Within 30 Days1 
 
 
Attribution 
Grade 4 
Grade 5 
 
Unexpected 
Expected 
 
Unrelated or Unlikely 
 
 
CTEP-AERS 
Possible, Probable, Definite 
CTEP-AERS 
 
CTEP-AERS 
1This includes all deaths within 30 days of the last dose of treatment with a commercial agent, 
regardless of attribution. Any death that occurs more than 30 days after the last dose of treatment 
with a commercial agent that can be attributed (possibly, probably, or definitely) to the agent and 
is not due to cancer recurrence must be reported via CTEP-AERS. 
 
 
11.12 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. 
 

  
 
 
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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 CTEP-AERS 
reportable events and Grade 3 and higher non-hematologic Adverse Events and all grades 
of the following cardiac events: prolonged QTc interval, ventricular arrhythmia, and left 
ventricular systolic dysfunction.  
 
 
12.0 
STUDY REPORTING AND MONITORING  
The Case Report Forms and the submission schedule are posted on the COG web site with each 
protocol under “Data Collection/Specimens”. A submission schedule is included. 
 
12.1 
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. 
 
12.2 
Data and Safety Monitoring Committee 
To protect the interests of patients and the scientific integrity for all clinical trial research 
by the Children’s Oncology Group, the COG Data and Safety Monitoring Committee 
(DSMC) reviews reports of interim analyses of study toxicity and outcomes prepared by 
the study statistician, in conjunction with the study chair’s report. The DSMC may 
recommend the study be modified or terminated based on these analyses.  
 
Toxicity monitoring is also the responsibility of the study committee and any unexpected 
frequency of serious events on the trial are to be brought to the attention of the DSMC. The 
study statistician is responsible for the monitoring of the interim results and is expected to 
request DSMC review of any protocol issues s/he feels require special review. Any COG 
member may bring specific study concerns to the attention of the DSMC.  
 
The DSMC approves major study modifications proposed by the study committee prior to 
implementation (eg, termination, dropping an arm based on toxicity results or other trials 
reported, increasing target sample size, etc.). The DSMC determines whether and to whom 
outcome results may be released prior to the release of study results at the time specified 
in the protocol document. 
 
 
13.0 
CYTOGENETIC ANALYSIS GUIDELINES AND REQUIREMENTS 
13.1 
Cytogenetic Analysis Overview 
Specimens for cytogenetic analysis are required, and must be obtained prior to 
therapy initiation. It is strongly recommended that all patients enrolled on AAML1421 
have a cytogenetics study performed by a COG-approved laboratory at the time of 
diagnosis and subsequent relapse, should it occur.  
 
The institutional CRA must inform the cytogenetics laboratory that the patient has been 
enrolled in a COG Myeloid study and that the cytogenetics/FISH data must be submitted 

  
 
 
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within 2 weeks after enrollment on the AAML1421 protocol. See Appendix II for 
cytogenetics procedures and for the Sample Authorization Form for reflexive FISH testing.  
 
13.2 
Specimen Collection and Submission 
13.2.1 
It is strongly recommended that specimens for cytogenetic analysis be sent to a COG-
approved institutional cytogenetics laboratory. If a COG institution does not have a COG-
approved laboratory, the institution may send the samples for karyotyping/FISH studies to any 
COG-approved cytogenetics laboratory on a fee-for-service basis. Prior arrangements for 
performing cytogenetics and/or FISH studies should be made with the laboratory. An 
authorization form for the reflexive FISH testing signed and dated (by the physician or 
designees just as with any order) sent to the cytogenetics laboratory with the sample will 
simplify and enhance the ability to obtain FISH testing and results. 
 
13.2.2 
A minimum of 2 mL (optimal volume, 3 mL) of fresh whole bone marrow aspirated 
through a needle into a syringe containing sodium heparin (preservative-free is preferable) 
is recommended in all cases. A first or second draw, or a draw from a repositioned needle, 
is best to ensure a sufficient number of leukemic cells in the aspirate. The specimens should 
be kept at room temperature and transported to the institutional cytogenetics laboratory 
as quickly as possible (always within 24 hours of collection). If shipping is done by 
overnight courier to an approved laboratory, that laboratory should be contacted to obtain 
instructions on transport. Some laboratories request specimens to be transferred in a 15-
mL conical tube filled with RPMI-1640 and 15% heat-inactivated fetal calf serum. 
Specimens should be kept at ambient temperature. 
 
13.2.3 
If bone marrow cannot be aspirated, a bone marrow core biopsy should be submitted. 
 
13.2.4 
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, if more than 
20% circulating blasts are identified, or when a constitutional abnormality (eg, trisomy 21) 
is a possibility. For documenting a constitutional abnormality, a PHA stimulated blood 
study should be performed. 
 
For patients with myeloid sarcoma: 
In addition to the requirements described above, if touch preps are able to be prepared from 
the tumor, the institutional pathologist should distribute/triage order(s) for FISH to be 
performed accordingly (eg, MLL, RUNX1T1-RUNX1, CBFB). 
 
13.2.5 
Results of these studies should be submitted electronically to the corresponding coordinator 
of the Myeloid COG cytogenetics committee for central review (see below). 
 
 
13.2.6 
Requirements for Data Submission 
The following are required for each case: a completed generic COG Cytogenetics/FISH 
Reporting Form (found on the COG Member website) and 2 original karyotypes (different 

  
 
 
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cells), with corresponding full-size metaphase spreads of each abnormal clone or 2 
karyotypes of normal cells with corresponding full-size metaphase spreads in the case of 
normal cytogenetic analysis. This information must be sent to the appropriate coordinating 
cytogeneticist within 2 weeks after enrollment on the AAML1421 protocol and within 
1 month of subsequent relapse, should it occur (see Appendix II). Reports must be sent 
electronically (preferred as PowerPoint presentation). If the laboratory is unable to send an 
electronic file with the documentation, please contact the COG cytogenetics coordinator 
for your area for advice. Reporting forms must be filled out for all cases, whether or not 
the cytogenetic analyses were successful. A separate form is required for each specimen 
(ie, bone marrow and blood) analyzed in each case. Published data on FISH screening 
report an overall incidence of occult aberrations in AML with normal karyotypes between 
3% to 10%.80-82 Thus, for all cases with only normal karyotypes FISH must be performed 
to evaluate for inv(16)/t(16;16), t(8;21), t(15;17) and 11q23. However, if the sample did 
not yield sufficient quantity of metaphases for analysis, -7 and -5/5q- should also be 
performed in addition to the inv(16)/t(16;16), t(8;21), t(15;17) and 11q23. Published data 
indicate that 10%-25% of patients who have one of these recurring translocations may have 
a concomitant deletion encompassing a region adjacent to the breakpoint on one of the 
involved derivative chromosomes. Such deletions may portend a less favorable 
prognosis.83-85 Thus, for all cases in which a recurring chromosomal abnormality [t(8;21), 
inv(16)/t(16;16), 11q23] is identified, it is recommended that FISH be performed with 
probes specific to that rearrangement, to evaluate any variant pattern that might include a 
deletion associated with a gene fusion. FISH forms should also be completed and sent to 
the coordinating cytogeneticist with images documenting the abnormal or normal FISH 
patterns. If the case has a suspected 12p it is recommended to perform FISH using ETV6 
probe. If the laboratory is not able to perform FISH, contact the COG cytogenetic 
coordinators for advice. Any discrepancies in interpretation between cytogenetics/FISH 
results from the laboratory and the coordinator will be discussed between the coordinators, 
with consult of additional cytogenetics committee members as necessary. The COG 
cytogenetics committee decision will prevail. 
Note: Please provide karyotype or Final Chromosome Report findings at initial time 
of diagnosis if available. 
 
 
Cytogenetic Coordinators 
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: 
Betsy Hirsch, Ph.D. 
Susana C. Raimondi, Ph.D. 
Telephone: (612) 273-4952/3171 
Telephone: (901) 595-3537/3536 
E-mail: hirsc003@umn.edu 
E-mail: susana.raimondi@stjude.org 
 
Recommendations for Case Analysis by Institutional, COG-Approved Cytogenetics 
Laboratories (see Appendix II.) 
 
 

  
 
 
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14.0 
SPECIAL STUDIES SPECIMEN REQUIREMENTS 
14.1 
CPX-351 Pharmacokinetics 
The patients participating in the Dose-Finding Phase of the trial are required to provide 
serial blood samples for the assessment of CPX-351 serum concentrations. Patients 
participating in the Efficacy Portion of the protocol may participate in the 
pharmacokinetics study as an optional correlative biology study. Blood samples for the 
Efficacy portion can be collected during Cycle 1 with a sparse sampling scheme as 
described below in Section 14.1.1.  
 
All material necessary for collection and shipping of PK specimens will be provided in a 
kit sent by Celator Pharmaceuticals Inc. (A Jazz Pharmaceuticals Company). Once you 
have a potential patient identified, please order a PK kit. Please refer to the PK manual on 
the protocol page for kit ordering instructions.   
 
 
 
 
 
 
 
 
 
 
 
 
 
14.1.1 Timing of Pharmacokinetic Sampling for the Efficacy Phase 
Blood will be drawn during Cycle 1 only. A blood sample will be collected in a 
tube provided in the kit. Timing of pharmacokinetic sampling will be as follows:  
 
Day 
Time 
Note 
1 
 
30 minutes after end of infusion 
On Day 1 & 5, samples 
should be obtained through 
a central venous catheter 
lumen or peripheral IV that 
is NOT used for the CPX-
351 infusion. 
5 
 
Prior to infusion 
 
30 minutes after end of infusion 
 
6 ± 2 hours from start of infusion 
8 
 
72 ± 24 hours from start of Day 5 infusion 
 
 
 
 
 
 

  
 
 
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14.1.2 Sample Collection and Processing 
 
Collect approximately 2 mL of blood in a tube provided in the kit and 
immediately invert the tube gently 180° and back, 5 times.  
 
Place on crushed ice/water immediately after inverting the tube. Record the 
calendar date and exact time of the blood draw on the AAML1421 PK 
Specimen Shipping Form. 
 
Separate the plasma from the cells by centrifuging under refrigeration (2-8°C) 
for 10 minutes at 1500 x g. 
 
Using a disposable pipette, remove plasma (from the top) without disturbing 
the blood cells. 
 
Label two 3 mL cryovials provided in the kit as detailed below in Section 
14.1.3. Pipette plasma aliquots into each cryovial. Leave approximately 20% 
of dead space in the cryovials to avoid cracking upon freezing. 
 
Place cryovials onto crushed ice/water until placed in the freezer or freeze 
immediately. Freeze at -70°C ± 10°C within 60 minutes of collection. 
 
Plasma will be kept at -70ºC ± 10°C until shipment to the bioanalytical 
laboratory on dry-ice. 
 
14.1.3 Sample Labeling and Shipping 
Each tube should have a freezer-safe label with the following information: 
 
AAML1421  
 
COG patient ID number 
 
COG NCI Site Number  
 
PK time point 
 
Date and start and stop time of CPX-351 infusion. 
 
Sampling date and time  
 
NOTE: Samples should be batched and shipped once all specimens for a patient 
have been collected.  
 
All shipments must be accompanied by a completed AAML1421 PK Specimen 
Shipping Form.  
 
Package the shipping tubes to prevent breakage and contamination in Styrofoam 
boxes provided in the kit containing a generous supply of dry ice. If samples cannot 
be shipped overnight, use a carrier that is capable of replenishing the dry ice in the 
polyfoam box.  
 
Upon shipment of samples, please email Sample.Receipt@inventivhealth.com, 
lavanya.arivelu@inventivhealth.com and Danielle.Morgen@inventivhealth.com   
or fax at 609-951-0080 notifying us that the samples have been shipped and 
provide the number of boxes in shipment and carrier tracking number if available.  
Advise if any special handling is required.  Also, please attach associated 
electronic sample files to the email, if any. 
 
 
 
  

  
 
 
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Samples should be shipped on dry ice, via courier Priority Overnight to: 
 
inVentiv Health Clinical Lab, Inc 
Attn:  Sample Receipt 
301A College Road East 
Princeton, NJ 08540 
Ph: (609) 951-0005 
Fx: (609) 951-0080 
 
Note: The laboratory facilities are closed on Sundays and on certain holidays. To 
ensure a timely delivery, please schedule your shipment no later than 3 days 
before any holiday.  
 
14.2 
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. Thus, results will not be obtained in 
real-time (but rather batched for retrospective review) and results will not be returned to 
the treating physician. The optional studies described below require patient consent. 
 
Please note, if there is an inadequate amount of blood for all optional studies, the studies 
should be prioritized in the following order: 
1. Biomarkers of cardiac injury (see Section 14.2.1) 
2. MicroRNA study (see Section 14.2.2) 
3. Whole exome sequencing (see Section 14.2.3) 
 
Additionally, if patient/parent/responsible party consents, any specimens left over on this 
study after required tests are performed will be banked for future research studies at the 
COG Leukemia Biospecimen Bank. 
 
14.2.1 Echocardiogram, Cardiac Troponin-T, N-terminal Probrain Natriuretic Peptide 
(NT-proBNP), and hs-CRP 
The study of biomarkers for cardiotoxicity will be conducted under the oversight 
of  Dr. Michael Absalon and Cincinnati Children's Medical Center. 
 
For enquiries regarding this study, please contact: 
Michael Absalon, MD, PhD,  
Phone: (513) 636-2061 
Email: michael.absalon@cchmc.org 
 
14.2.1.1 Echocardiogram 
Study participants will undergo standard 2D-echocardiograms prior to 
CPX-351 therapy, Day 28 (± 7 days) of Cycle 1 and must be before 
Cycle 2 begins, Day 28 (± 7 days) of Cycle 2 and must be before any 
subsequent therapy, then yearly from the start of therapy. 
Echocardiographic data will include LV systolic and diastolic function, 
LV shape and size. The echocardiograms are considered as standard of 
care and data will be collected via study case report forms.  
 

  
 
 
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14.2.1.2 Blood biomarkers 
Sample collection and processing 
Samples will be obtained from consenting patients at the following time 
points: 
 
Baseline (before CPX-351 infusion begins)  
 
Day 8* 
 
Day 15* 
 
Day 22* 
 
Day 28-30 of Cycle 1 (one sample in this timeframe) 
 
*Samples for Biomarkers of cardiac injury and MicroRNA may be drawn up to 
24 hours prior to the time point and up to 24 hours after the time point.  
 
At each protocol specified time point, dispense 3 to 5 mL of blood into 
a labeled Serum Separator drawing tube (SST, gold top tube). 
 
Allow the tube to clot for 30-60 minutes in a vertical position. 
Centrifuge at 1300-1800 g for 10-15 minutes at room temperature. Do 
not use brake to stop centrifuge. 
 
Label each cryovial with:  
 
Study number (AAML1421) 
 
COG patient ID number  
 
BPC number 
 
Day of cycle and treatment cycle 
 
Sampling date and time 
 
Type of specimen (serum) 
 
Using a pipette, remove serum from the top of the tube without 
disturbing the blood cells and transfer into the labeled cryovial(s).  
 
Immediately place the cryovial(s) containing the serum sample in a  
-70 ± 10⁰C freezer. 
 
NOTE: Frozen samples should be batched and shipped once all 
specimens for a patient have been collected. 
 
Shipping  
See Section 14.2.4 for shipping details. 
 
NOTE: Each specimen should be sent with a completed AAML1421 
Specimen Transmittal form.  
 
14.2.1.3 Leukemia Bank Sample Processing  
The COG Leukemia Biospecimen Bank will forward specimens 
received for the cardiac troponin-T, N-terminal probrain natriuretic 
peptide (NT-proBNP), and hs-CRP studies to Dr. Absalon in Cincinnati, 
OH. 
 

  
 
 
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14.2.2 Novel Biochemical and Imaging Biomarkers of Cardiotoxicity 
For this study, blood samples and echocardiograms images and reports are 
requested as detailed below. 
 
14.2.2.1 Peripheral blood miRNA study 
To explore the possibility of using peripheral blood miRNA as a 
prognostic indicator or anthracycline-induced heart injury in children, 
plasma will be collected at baseline and at several time points following 
CPX-351 administration. The miRNA studies will be conducted under 
the oversight of Dr. Kasey Leger and Seattle Children’s Hospital. 
 
For enquiries regarding this study, please contact: 
Kasey Leger, MD  
Seattle Children’s Hospital 
Email: kasey.leger@seattlechildrens.org 
 
14.2.2.1.1 
Specimen Time points 
Samples from consenting patients, will be collected at the 
following time points in Cycle 1: 
 
Day 1, prior to CPX-351 infusion 
 
Day 5, 6 (± 1 hr) hours following the end of the CPX-
351 infusion 
 
Day 8* 
 
Day 15* 
 
Day 22* 
 
Day 28-30 (one sample in this timeframe) 
 
*Samples for Biomarkers of cardiac injury and MicroRNA may be drawn up to 
24 hours prior to the time point and up to 24 hours after the time point.  
 
14.2.2.1.2 
Sample collection and processing 
Peripheral blood (5-10 mL) must be obtained in a purple/ 
lavender top tube (EDTA tube). Process within 2 hours of 
collection: 
 
Spin in refrigerated (4oC) centrifuge at 2500g for 10 
minutes 
 
Using sterile pipettes, transfer plasma into cryovials 
in 0.5 mL aliquots.  
 
Samples can be stored at -20ºC for up to 3 days, or 
indefinitely at -80°C until shipped. 
 
14.2.2.1.3 
Labeling 
Label the tubes with: 
 
Study number (AAML1421) 
 
COG patient ID number 
 
BPC number 
 
Date and time of collection 
 
Time point (include treatment cycle and day of cycle) 

  
 
 
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 
Specimen type (plasma) 
 
14.2.2.1.4 
Shipping 
Samples should be sent frozen, on dry ice, using overnight 
delivery.  
 
See Section 14.2.4 for shipping details. 
 
14.2.2.1.5 
Leukemia Bank Sample Processing  
The COG Leukemia Biospecimen Bank will forward 
specimens received for the peripheral blood miRNA 
study to Dr. Leger in Seattle, WA. 
 
14.2.2.2 Myocardial deformation (strain) 
Study participants will undergo a standard 2-dimensional, M-mode, and 
Doppler echocardiogram at baseline and at the end of Cycle 1 and Cycle 
2, then yearly. Please request that both the ejection fraction and 
shortening fraction be calculated for the exam. Echocardiograms will be 
performed per AHA/ACC task force practice guidelines at participating 
institutions.86 All tracings will be sent to Seattle Children’s for central 
review for myocardial deformation and other echocardiographic 
parameters. Myocardial deformation will be measured using Tomtec 
software program, which allows post-hoc strain analysis on standard 
echocardiogram images across vendor platforms. 
 
In addition to the standard echocardiogram report generated by the 
cardiologist at the performing institution, participating institutions are 
requested to send a digital copy of the original tracings from 
echocardiograms to be reviewed for study purposes.   
 
In addition to collecting conventional echocardiogram parameters of LV 
function (SF and EF) we will be requesting DICOM echo images to be 
sent for analysis of myocardial strain. 
 
Following ascertainment of end of Cycle 2 echocardiogram, ship: 
 
de-identified echocardiogram reports and  
 
DICOM echocardiogram images including the baseline 
echocardiogram (prior to protocol therapy), the beginning of 
Cycle 2 and end of therapy echocardiograms. 
 
Note: if a patient goes off protocol therapy at any point during the trial, 
please send the baseline and end of therapy echocardiogram report 
and images. 
 
These files can be burned to a CD and mailed to the address below. 
Multiple studies for the same patient may be submitted on one CD; 
however, please submit only one patient per CD.  These should be 
mailed after Cycle 2 and preferably within 30 days from the end of Cycle 
2. 

  
 
 
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Ship to:  
Kasey Leger, MD  
Seattle Children’s Hospital 
4800 Sand Point Way NE 
M/S MB.8.501 
Seattle, WA 98105 
 
 
 
14.2.3 Evaluation of the Role of Rare Coding Variants as Risk Factors For Anthracycline-
Induced Cardiomyopathy 
For enquiries regarding this study, please contact: 
Richard Aplenc, MD PhD 
Office phone: (267) 426-7252 
Lab phone: 
(267) 426-2288 
Email: 
Aplenc@email.chop.edu 
 
14.2.3.1 Specimens 
2 mL to 4 mL of blood will be collected from consenting patients on 
Day 28-30 of Cycle 1 (one sample in this timeframe) for germline 
whole-exome sequencing. 
 
14.2.3.2 Sample collection and Processing  
Blood must be obtained in purple top tube (EDTA tube). The samples can 
be kept at 4ºC for storage and shipment.  
 
14.2.3.3  Labeling 
Label the tubes with: 
 
study number (AAML1421) 
 
COG patient ID number 
 
BPC number 
 
date and time of collection 
 
time point (include treatment cycle and day of cycle) 
 
source of material (blood)  
 
14.2.3.4 Shipping 
See Section 14.2.4 for shipping details. 
 
14.2.3.5 Leukemia Bank Sample Processing  
The COG Leukemia Biospecimen Bank will forward specimens received 
for the study on evaluation of the role of rare coding variants as risk 
factors for anthracycline-induced cardiomyopathy to Dr. Aplenc in 
Philadelphia, PA. 
 
14.2.4 Instructions for Shipping to the COG Leukemia Biospecimen Bank 
Samples must be shipped with a AAML1421 Specimen Transmittal form.  
 

  
 
 
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Ambient blood in EDTA tube should be placed inside a leak proof biohazard 
envelope with absorbent material and then a pressure resistance Tyvek envelope 
and then shipped at room temperature in a shipping container labeled with an 
Exempt Human Specimen label. Ambient blood may be shipped on Monday 
through Friday for a Tuesday through Saturday delivery. 
 
Serum and plasma must be batch shipped frozen on dry ice. Specimens must be 
placed inside a leak proof biohazard envelope with absorbent material and then a 
pressure resistance Tyvek envelope. Place packaged specimens in an insulated 
shipping container with at least 5 lbs. of dry ice. Complete a dry ice label (UN 
1845) and place the dry ice label and an Exempt Human Specimen label on the 
side of the shipping container. Place a FedEx shipping label on the top of the 
shipping container. Frozen samples may only be shipped on Monday through 
Thursday for a Tuesday through Friday delivery. 
 
NOTE: Specimen collection and shipping supplies are not provided by the 
Leukemia Biospecimen Bank (or the Biopathology Center). Institutions 
are responsible for providing their own collection and shipping supplies. 
 
Samples should be mailed by FedEx Priority Overnight. COG sites may use the 
COG Federal Express account number available at:  
https://members.childrensoncologygroup.org/_files/reference/FEDEXmemo.pdf 
 
Ship to the following address: 
COG Leukemia Biospecimen Bank 
Nationwide Children’s Hospital  
575 Children’s Crossroads, Room WB2255 
Columbus, OH 43215 
Phone: (614)722-2866 
Fax: 
(614)722-2887 
Email: MGLab@nationwidechildrens.org 
 
Call or email the Leukemia Bank before shipping an ambient blood sample 
that will be delivered on a Saturday. 
 
 
 

  
 
 
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APPENDIX I: CTEP AND CTSU REGISTRATION PROCEDURES 
CTEP INVESTIGATOR REGISTRATION PROCEDURES 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register and to renew their registration 
annually. To register, all individuals must obtain a Cancer Therapy Evaluation Program (CTEP) 
Identity and Access Management (IAM) account (https://ctepcore.nci.nih.gov/iam). In addition, 
persons with a registration type of Investigator (IVR), Non-Physician Investigator (NPIVR), or 
Associate Plus (AP) (i.e., clinical site staff requiring write access to OPEN, RAVE, or TRIAD or 
acting as a primary site contact) must complete their annual registration using CTEP’s web-based 
Registration and Credential Repository (RCR) (https://ctepcore.nci.nih.gov/rcr). Documentation 
requirements per registration type are outlined in the table below. 
 
Documentation Required 
IVR 
NPIVR 
AP 
A 
FDA Form 1572 
 
 
Financial Disclosure Form 
 
 
 
NCI Biosketch (education, training, 
employment, license, and certification) 
 
 
 
HSP/GCP training 
 
 
 
Agent Shipment Form (if applicable) 
 
CV (optional) 
 
 
 
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all CTEP 
and CTSU (Cancer Trials Support Unit) websites and applications.  In addition, IVRs and NPIVRs 
must list all clinical practice sites and IRBs covering their practice sites on the FDA Form 1572 in 
RCR to allow the following: 
 
Added to a site roster 
 
Assigned the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN 
 
Act as the site-protocol PI on the IRB approval 
 
Assigned the Clinical Investigator (CI) role on the Delegation of Tasks Log (DTL). 
 
Additional 
information 
can 
be 
found 
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. 
 
 
 

  
 
 
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CTSU REGISTRATION PROCEDURES 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU).  
 
Downloading Site Registration Documents: 
 
Site registration forms may be downloaded from the AAML1421 protocol page located on the 
CTSU members’ website. Permission to view and download this protocol and its supporting 
documents is restricted and is based on person and site roster assignment housed in the CTSU RSS. 
 
Go to https://www.ctsu.org and log in to the members’ area using your CTEP-IAM 
username and password 
 
Click on the Protocols tab in the upper left of your screen 
 
Either enter the protocol # in the search field at the top of the protocol tree, or 
 
Click on the By Lead Organization folder to expand 
 
Click on the COG link to expand, then select trial protocol AAML1421 
 
Click on LPO Documents, select the Site Registration documents link, and download and 
complete the forms provided.  
 
Requirements for AAML1421 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 via the Regulatory 
Submission Portal, where they will be entered and tracked in the CTSU RSS.  
 
Regulatory Submission Portal: www.ctsu.org   (members’ section)  Regulatory Tab  
 Regulatory Submission 
 
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. 
 
When applicable, original documents should be mailed to: 
CTSU Regulatory Office 
1818 Market Street, Suite 1100 
Philadelphia, PA 19103 
 
Checking Your Site’s Registration Status: 
 
You can verify your site registration status on the members’ section of the CTSU website. (Note: 
Sites will not receive formal notification of regulatory approval from the CTSU Regulatory Office.) 
 
Go to https://www.ctsu.org and log in to the members’ area using your CTEP-IAM username 
and password 
 
Click on the Regulatory tab at the top of your screen 
 
Click on the Site Registration tab 
 
Enter your 5-character CTEP Institution Code and click on Go 
 

  
 
 
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Note: The status given only reflects compliance with IRB documentation and institutional 
compliance with protocol-specific requirements as outlined by the Lead Network. It does not reflect 
compliance with protocol requirements for individuals participating on the protocol or the enrolling 
investigator’s status with the NCI or their affiliated networks. 
 
Data Submission / Data Reporting 
 
Data collection for this study will be done exclusively through the Medidata Rave clinical data 
management system. Access to the trial in Rave is granted through the iMedidata application to all 
persons with the appropriate roles assigned in Regulatory Support System (RSS). To access Rave 
via iMedidata, the site user must have an active CTEP-IAM account (check at 
https://ctepcore.nci.nih.gov/iam) and the appropriate Rave role (Rave CRA, Read-Only, CRA (Lab 
Admin, SLA or Site Investigator) on either the LPO or participating organization roster at the 
enrolling site. To the hold Rave CRA role or CRA Lab Admin role, the user must hold a minimum 
of an AP registration type. To hold the Rave Site Investigator role, the individual must be registered 
as an NPIVR or IVR. Associates can hold read-only roles in Rave. If the study has a DTL, 
individuals requiring write access to Rave must also be assigned the appropriate Rave tasks on the 
DTL. 
 
Upon initial site registration approval for the study in RSS, all persons with Rave roles assigned on 
the appropriate roster will be sent a study invitation e-mail from iMedidata. To accept the invitation, 
site users must log into the Select Login (https://login.imedidata.com/selectlogin) using their 
CTEP-IAM user name and password, and click on the “accept” link in the upper right-corner of the 
iMedidata page. Please note, site users will not be able to access the study in Rave until all required 
Medidata and study specific trainings are completed. Trainings will be in the form of electronic 
learnings (eLearnings), and can be accessed by clicking on the link in the upper right pane of the 
iMedidata screen. 
 
Users that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata to 
activate their account. Account activation instructions are located on the CTSU website, Rave tab 
under the Rave resource materials (Medidata Account Activation and Study Invitation 
Acceptance). Additional information on iMedidata/Rave is available on the CTSU members’ 
website under the Rave tab at www.ctsu.org/RAVE/ or by contacting the CTSU Help Desk at 1-
888-823-5923 or by e-mail at ctsucontact@westat.com. 
 

  
 
 
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APPENDIX II: SAMPLE SUBMISSION FOR CYTOGENETIC/FISH STUDIES 
(A COPY OF THIS SECTION MUST BE SENT TO THE RECOMMENDED INSTITUTIONAL, 
COG-APPROVED CYTOGENETICS LABORATORY) 
 
CHROMOSOME ANALYSIS 
 
Bone marrow should be studied by cytogenetics methods in all cases of acute myeloid leukemia. 
 
A back-up blood specimen should be studied when the bone marrow aspirate or bone marrow core 
biopsy is inadequate or unobtainable (in which case short-term unstimulated cultures are 
established to examine spontaneously dividing [presumably leukemic] cells) or when a 
constitutional chromosomal abnormality is a possibility (phytohemagglutinin-stimulated cultures 
should be established to examine presumably nonleukemic [constitutional] lymphocytes). 
 
Short-term (15 - 48 hours) unstimulated cultures are recommended for each bone marrow sample. 
Analysis of direct preparations is successful in some laboratories. Mitogen-stimulated cultures of 
bone marrow samples should not be initiated. 
 
All preparations must be G-banded. (Q- or R-banding will not be accepted as a stand-alone banding 
method.) 
 
Complete analysis of 20 G-banded metaphases is required for each case, except as noted below. 
Complete analysis is defined as follows: the chromosomes in each metaphase cell have been 
counted; each chromosome has been examined to determine whether the banding pattern is normal, 
and all abnormalities present in the cell have been defined. Analysis may be accomplished by 
examining metaphase spreads under the microscope or by imaging. Sometimes analysis of 20 
metaphases is not possible because of poor in vitro growth or a very limited quantity of specimen. 
However, limited characterization of the abnormal clone can still be informative. Such informative 
cases will be considered acceptable. A minimum analysis of 20 metaphases is required for a normal 
case. 
 
Identification of clones will follow the criteria of the Second International Workshop on 
Chromosomes in Leukemia, as stated in the: General Report (Cancer Genet. Cytogenet 2:93-96, 
1980): at least 2 metaphases with identical structural abnormalities or extra chromosomes, or at 
least three metaphases with identical missing chromosomes will constitute a clone. Nonclonal 
abnormalities (excluding random loss) should also be recorded. 
 
Karyotypes are to be designated according to the guidelines described in ISCN 2013, An 
International System for Human Cytogenetic Nomenclature (2013), LG Shaffer, J McGowan-
Jordan and M Schmid (eds); S. Karger, Basel, 2013. 
 
FLUORESCENCE IN SITU HYBRIDIZATION (FISH) 
 
FISH using commercially available probes will be required for the following cases A.) Normal B) 
Insufficient Quantity. C) Abnormal case with an 11q23 or 12p (see below). The laboratory is 
expected to follow the standards and guidelines for FISH put forth by the American College of 
Medical Genetics. If the laboratory is unable to perform FISH tests, contact cytogenetics 
coordinators for advice. 
 
A) For all cases for which G-banding reveals a normal karyotype, FISH with RUNX1T1/RUNX1, 
CBFB, or CBFB-MYH11, PML-RARA and MLL (probes must be performed to rule out a cryptic 

  
 
 
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t(8;21), inv(16) or t(16;16), t(15;17) and 11q23 abnormality. FISH testing should be performed on 
1 day cultures (unstimulated sample).  
 
B) For all cases with failed cytogenetics, FISH with RUNX1T1/RUNX1, CBFB or CBFB-MYH11, 
PML-RARA, MLL, 7 (LSI, D7Z1/D7S486), and 5 (LSI, EGR1/D5S23, D5S721). The threshold for 
a positive FISH finding for –7, -5/5q- will be >30%. If possible, morphologic and 
immunophenotypic data should be obtained as such data can be suggestive of a particular 
abnormality and thereby help the laboratory director to prioritize these studies.  
 
C) When G-banding reveals a known recurring chromosomal rearrangement, specifically one 
involving an t(8;21), inv(16)/t(16;16), or abnormal 11q23, FISH is recommended with a probe set 
targeting the involved loci (ie, RUNX1T1/RUNX1,, CBFB or CBFB-MYH11, MLL) should be 
performed on metaphase or interphase cells to determine whether there is an associated deletion 
involving the regions 3’ or 5’ of the participating genes, and to establish the pattern that can be 
used to monitor this patient’s disease. When G-banding reveals an abnormality of 12p, FISH with 
a probe that can assess ETV6 status (ETV6 breakapart or ETV6-RUNX1) in order to detect ETV6 
rearrangement or deletion. 
 
CYTOGENETIC STUDY/FISH SUBMISSION 
Steps to obtain the FORMS by the institution’s CRA 
1. www.childrensoncologygroup.org 
2. COG members 
3. Committees ►Clinical Research Associates ►Resources ►Data 
Management ►Generic Study Forms 
4. Under COG ►Cytogenetics Reporting/FISH Forms 
Alternatively, the cytogenetics reporting form can be found at : https://cogmembers.org/site/
pages/modal.aspx?mid=9000000007. The COG Forms should be completed by the designated individual 
in the Cytogenetics Laboratory and signed by the Cytogenetics Director. It is highly recommended to scan 
the Forms and e-mail with the appropriate documentation (PowerPoint presentation preferred) to COG 
reviewers for the Myeloid Committee. If the laboratory is unable to send an electronic file with the 
documentation, please contact the COG cytogenetics coordinator for your area for advice 
 
The case should be sent to the appropriate reviewer by Day 14. 
 
Cytogenetic Coordinators 
Please send above materials by e-mail (preferably as 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: 
Betsy Hirsch, Ph.D. 
Susana C. Raimondi, Ph.D. 
Telephone: (612) 273-4952/3171 
Telephone: (901) 595-3537/3536 
E-mail: hirsc003@umn.edu 
E-mail: susana.raimondi@stjude.org 
 
 
 

  
 
 
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CYTOGENETICS REVIEW 
The region’s cytogenetics coordinator will review each case when it is submitted. She/he will determine 
whether each case is adequate in terms of the numbers of metaphase cells analyzed, quality of banding, and 
interpretation of the karyotypes. If the coordinator agrees with the submitting laboratory, the results of the 
study will be entered into the appropriate Rave form. If the coordinator does not agree and the results are 
significant for patient stratification, she/he will send the case to another member of the COG Cytogenetics 
Review Committee for rapid review. If the coordinator does not agree with the submitting laboratory but 
the results are not significant for patient stratification, the case will be taken to the next central review 
session to be reviewed there. If the case is determined as not adequate it will be registered as unknown 
cytogenetics.  
 
(A SIGNED AND DATED COPY OF THIS AUTHORIZATION FORM FOR REFLEXIVE FISH 
TESTING MUST BE SENT TO THE CYTOGENETICS LABORATORY, TOGETHER WITH 
THE BONE MARROW SAMPLE) 
 
AUTHORIZATION FORM FOR REFLEXIVE FISH TESTS 
 
A). REQUIRED FISH if a case is cytogenetically normal or inadequate  
t(8;21)    
 
[RUNX1T1/RUNX1] 
inv(16)/t(16;16)  
[CBFB or CBFB-MYH11] 
t(15;17) 
 
[PML-RARA] 
11q23    
 
[MLL] 
 
B). REQUIRED FISH if a case is cytogenetically inadequate  
-7: 
  
 
[LSI, D7Z1/D7S486] 
-5/5q-:    
 
[LSI, EGR1/D5S23, D5S721] 
t(8;21)    
 
[RUNX1T1/RUNX1] 
inv(16)/t(16;16)  
[CBFB or CBFB-MYH11] 
t(15;17) 
 
[PML-RARA] 
11q23    
 
[MLL] 
 
C). RECOMMENDED FISH if a case has a cytogenetically detectable  
 
11q23  
 
[MLL] 
Abnormality of 12p 
[ETV6 breakapart or ETV6-RUNX1] 
 
 
It is authorized to perform reflexive FISH testing to rule-out cryptic aberrations and/or to evaluate molecular 
deletions. 
 
 
Patient Registration #  __________________________________________ 
 
___________________________________________________________ 
Print Name of Attending Physician 
 
_____________________________________________________________ 
Print Name of Institution and City, State 
 
Signature of attending physician or designee: ________________________  
 
Date: ______________________ 
 
 
 

  
 
 
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APPENDIX III: YOUTH INFORMATION SHEETS 
INFORMATION SHEET REGARDING RESEARCH STUDY 
(for children from 7 through 12 years of age) 
A treatment study of a new drug to treat children with leukemia that has not responded to 
treatment or has come back. 
 
1. We have been talking with you about your illness, acute myeloid leukemia (AML).  Leukemia 
is a type of cancer that grows in the bone marrow. Bone marrow is the spongy tissue inside 
the bones of your body that make blood cells. Relapse means that the cancer has come back 
after treatment. You have received treatment for AML before. We are asking you to take part 
in a research study because the AML has come back.  A research study is when doctors work 
together to try out new ways to help people who are sick. In this study we are trying to learn 
more about how to treat AML that has come back.  
 
2. Children who are part of this study will be given a new drug called CPX-351. Doctors want 
to see if this new treatment will make children with leukemia get better, with fewer heart 
problems later in life. You will also get other drugs used to treat AML. We do not know how 
well the new drug will work in children, teens and young adults. That is why we are doing 
this study.  
 
3. Sometimes good things can happen to people when they are in a research study. These good 
things are called “benefits.” We hope that a benefit to you of being part of this study is your 
cancer will go away. We also hope that the study treatment will help keep more children from 
having heart problems later in life. However, we don’t know for sure if there is any benefit of 
being part of this study. 
 
4. Sometimes bad things can happen to people when they are in a research study.  These bad 
things are called “risks.” One risk to you is that the study treatment may not work as well as 
other treatments to get rid of the cancer for as long as possible. Another risk may be more 
problems, or side effects, from the study treatment. Other things may happen to you that we 
don’t yet know about. 
 
5. Your family can choose to be part of this study or not. Your family can also decide to stop 
being in this study at any time once you start. There may be other treatments for your illness 
that your doctor can tell you about. Make sure to ask your doctors any questions that you 
have. 
 
6. We are asking your permission to collect extra blood. Doctors want to learn more about how 
children’s bodies handle the new drug you will receive. Doctors also want to see if there are 
ways to tell how your heart is affected by the treatment. Most of these samples would be taken 
when other standard blood tests are being performed, but there may be a few extra needle 
sticks. You can still take part in this study even if you don't allow us to collect the extra blood 
samples for research. 
 
 
 

  
 
 
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INFORMATION SHEET REGARDING RESEARCH STUDY 
(for teens from 13 through 17 years of age) 
A treatment study of a new drug to treat children with leukemia that has not responded to 
treatment or has come back. 
 
1. We have been talking with you about your illness, acute myeloid leukemia (AML). Leukemia 
is a type of cancer that grows in the bone marrow. Bone marrow is the spongy tissue inside the 
bones of your body that make blood cells. You have received treatment for AML before. 
Relapse means that the cancer has come back after treatment. After doing tests, we have found 
that the AML has come back.  
 
2. We are asking you to take part in a research study because your AML has come back. A 
research study is when doctors work together to try out new ways to help people who are sick. 
In this study we are trying to learn more about how to treat AML that has come back. Children 
and teens who are part of this study will be given a new drug called CPX-351. Doctors want 
to see if this new treatment will make the leukemia get better, with fewer heart problems later 
in life. You will also get other standard drugs used to treat AML. We do not know how well 
the new drug will work in children, teens and young adults. That is why we are doing this 
study.  
 
3. Sometimes good things can happen to people when they are in a research study. These good 
things are called “benefits.” We hope that a benefit to you of being part of this study is a better 
chance at getting rid of the cancer for as long as possible. We also hope that fewer late effects 
and heart problems will occur. However, we don’t know for sure if there is any benefit of being 
part of this study. 
 
4. Sometimes bad things can happen to people when they are in a research study. These bad things 
are called “risks.” One risk to you is that the study treatment may not work as well as other 
treatments to get rid of the cancer for as long as possible. Another risk may be more problems, 
or side effects, from the study treatment. Other things may happen to you that we don’t yet 
know about. 
 
5. Your family can choose to be part of this study or not. Your family can also decide to stop 
being in this study at any time once you start. There may be other treatments for your illness 
that your doctor can tell you about. Make sure to ask your doctors any questions that you have.  
 
6. We are asking your permission to collect additional blood. Doctors want to learn more about 
how children’s and teens’ bodies handle the new drug you will receive. Doctors also want to 
see if there are ways to tell how your heart is affected by the treatment. Most of these samples 
would be taken when other standard blood tests are being performed, but there may be a few 
extra needle sticks. You can still be treated on this study even if you don't allow us to collect 
the extra blood samples for research.  
 
 
 

  
 
 
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APPENDIX IV: POSSIBLE DRUG INTERACTIONS 
The list below does not include everything that may interact with your chemotherapy. Talk to your doctor 
before starting any new medications, over-the-counter medicines, or herbal supplements and before 
making a significant change in your diet. 
 
To this date, there are no data on specific food or supplement interactions with CPX-351. The drug 
interactions observed with CPX-351 are the same as for each individual components (cytarabine and 
daunorubicin). 
 
Some drugs, food, and supplements may interact with cytarabine (by vein). Examples include: 
 
Drugs that may interact with cytarabine* 
 Clozapine, digoxin, flucytosine, leflunomide 
 
Food and supplements that may interact with cytarabine** 
 Echinacea 
*Sometimes these drugs are used with cytarabine on purpose. Discuss all drugs with your doctor. 
**Supplements may come in many forms, such as teas, drinks, juices, liquids, drops, capsules, pills, or 
dried herbs. All forms should be avoided.  
 
 
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) 
 Some antiepileptics (carbamazepine, phenobarbital, phenytoin, fosphenytoin) 
 Some antiretrovirals (darunavir, lopinavir; nelfinavir, ritonavir, saquinavir, telaprevir, tenofovir, 
tipranavir) 
 Some heart medications (amiodarone, carvedilol, digoxin, dronedarone, nicardipine, propranolol, 
verapamil) 
 Other agents, such as atorvastatin, clozapine, cyclosporine, dexamethasone, ivacaftor, leflunomide, 
natalizumab, nefazodone, progesterone, rifampin, tacrolimus, tofacitinib, and trazodone 
 
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” 
*Sometimes these drugs are used with daunorubicin on purpose. Discuss all drugs with your doctor. 
**Supplements may come in many forms, such as teas, drinks, juices, liquids, drops, capsules, pills, or 
dried herbs. All forms should be avoided.  
 
 
 

  
 
 
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Some drugs, food, and supplements may interact with fludarabine. Examples include: 
 
Drugs that may interact with fludarabine* 
 Clozapine, leflunomide, natalizumab, pentostatin, tofacitinib 
 
Food and supplements that may interact with fludarabine** 
 Echinacea 
*Sometimes these drugs are used with fludarabine on purpose. Discuss all drugs with your doctor. 
**Supplements may come in many forms, such as teas, drinks, juices, liquids, drops, capsules, pills, or 
dried herbs. All forms should be avoided.  
 
 
 
 
 
 
 

  
 
 
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