AML08: A Phase II Randomized Trial of Clofarabine Plus Cytarabine Versus Conventional Induction Therapy And A Phase II Study Of Natural Killer Cell Transplantation In Patients With Newly Diagnosed Acute Myeloid Leukemia
Phase: PHASE3
Conditions: Acute Myeloid Leukemia
Sponsor: St. Jude Children's Research Hospital

 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
SJCRH 
NCT00703820 (AML08) 
Initial version, dated:  04/03/2008, Submitted to CPSRMC 04/07/08.  Resubmitted: 4/25/08 and 5/13/2008.  Resubmitted to IRB 
5/28/2008 and June 12, 2008.  (IRB Approved: June 14, 2008) Activation Date:  August 1, 2008 
Amendment 1.0, dated: August 28, 2008 (IRB Approved:  September 23, 2008) 
Amendment 2.0, dated: December 24, 2008 (IRB Approved: February 13, 2009)  
Amendment 3.0, dated: June 26, 2009 (IRB Approved: August 11, 2009).  Activated: August 17, 2009 
Amendment 4.0, dated:   January 21, 2010 (IRB approved: March 11, 2010).  Activated:  March 19, 2010 
Revision 4.1, dated April 13, 2010 (IRB approved: April 27, 2010).  Activated: May 12, 2010 
Revision 4.2, dated May 17, 2010 (IRB approved: June 1, 2010).  Activated: June 7, 2010 
Letter of Amendment #1: Deletion of Mylotarg, dated June 22, 2010 (IRB Approved:  June 23, 2010). Activated: July 12, 2010 
Amendment 5.0, dated July 8, 2010 (IRB Approved: August 26, 2010).  Activated: August 31, 2010 
Amendment 6.0, dated April 05, 2011(IRB Approved: May 26, 2011).  Activated: June 8, 2011 
Amendment 7.0, dated August 22, 2011(IRB Approved: October 3, 2011).  
Revision 7.1, dated October 05, 2011 (IRB Approved:  October 24, 2011) 
Revision 7.2, dated February 01, 2012 (IRB Approved:   February 8, 2012).  Activated: March 1, 2012 
Amendment 8.0, dated October 1, 2012 (IRB Approved: January 18, 2013) Activated: M arch 6, 2013 
Revision 8.1, dated March 15, 2013 (IRB approved March 21, 2013) Activated: March 27, 2013  
Amendment 9.0, dated June 13, 2013.  Resubmitted to IRB July 12, 2013 (IRB approved:  July 15, 2013).   
Revision 9.1, dated July 18, 2013.  (IRB approved: July 19, 2013) Activated: August 28, 2013 
Amendment 10.0:  February 10, 2014.  (IRB approved: February 12, 2014) Activated: February 18, 2014  
Letter of Amendment #4, dated May 29, 2015 (IRB approved: May 29, 2015) Activated: June 1, 2015   
Revision 10.1: June 2, 2016. (IRB approved: June 7, 2016) Activated: July 5, 2016 
 
AML08: A PHASE III RANDOMIZED TRIAL OF CLOFARABINE PLUS CYTARABINE 
VERSUS CONVENTIONAL INDUCTION THERAPY AND A PHASE II STUDY OF NATURAL 
KILLER CELL TRANSPLANTATION IN PATIENTS WITH NEWLY DIAGNOSED ACUTE 
MYELOID LEUKEMIA 
 
Principal Investigator 
 
Jeffrey E. Rubnitz, MD, PhD1 
 
Other St. Jude Investigators 
 
Sima Jeha, MD1 
Stanley Pounds, PhD4 
Aditya Gaur, MD, MBBS5 
Ching-Hon Pui, MD1 
Randall Hayden, MD3 
Susana Raimondi, PhD3 
Hiroto Inaba, MD, PhD1 
Raul Ribeiro, MD1 
John Choi, MD, PhD3 
Brandon Triplett, MD2 
 
St. Jude Children's Research Hospital 
262 Danny Thomas Place 
Memphis, Tennessee  38105-2794 
Telephone:  (901) 595-3300 
 
Additional contact information for St. Jude Principal Investigator: 
 
 
 
 
Departments of 1Oncology, 2Bone marrow Transplant & Cellular Therapy, 3Pathology, 4Biostatistics, 
5Infectious Diseases 
 
 
 
Contents of this document may not be extracted without permission from the Principal Investigator. 

 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
External Co-Investigators (Collaborating Institutions): 
 
Stanford University Medical Center 
Pediatric Hematology/Oncology 
Packard Children’s Hospital 
725 Welch Road 
Palo Alto, CA 94304 
 
Gary Dahl, MD 
Norman Lacayo, MD 
 
Cook Children’s Medical Center 
Hematology/Oncology 
901 Seventh Avenue 
Suite 220 
Fort Worth, TX 76104 
 
Paul Bowman, MD 
Ken Heym, M.D. 
 
Children’s Hospital of Michigan 
3901 Beaubien Blvd. 
Detroit, MI 48201 
 
Jeffrey Taub, MD 
 
Dana-Farber Cancer Institute and 
Children’s Hospital, Boston 
Pediatric Oncology 
44 Binney Street 
Boston, MA 02115 
 
Barbara Degar, MD 
 
 
University of Chicago 
Pediatric Hematology/Oncology MC4060 
University of Chicago 
5841 S. Maryland Avenue 
Chicago, IL 60637 
 
John Cunningham, M.D. 
 
Rady Children’s Hospital San Diego 
3020 Children’s Way 
San Diego, CA 92123 
 
Deborah E. Schiff, M.D. 
National University Health System, 
Singapore 
Paediatric Hematology/Oncology/BMT 
University Children's Medical Institute 
5 Lower Kent Ridge Road 
Singapore 119074 
Republic of Singapore 
 
Allen Eng-Juh Yeoh, M.D. 
 
 
External Scientific Collaborators 
 
National University of Singapore 
 
Dario Campana, MD, PhD 
Elaine Coustan-Smith, MSc 
 
 
 

 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
 
 
AML08: A PHASE III RANDOMIZED TRIAL OF CLOFARABINE PLUS 
CYTARABINE VERSUS CONVENTIONAL INDUCTION THERAPY AND A PHASE II 
STUDY OF NATURAL KILLER CELL TRANSPLANTATION IN PATIENTS WITH 
NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA 
 
Letter of Amendment 
 
January 9, 2014 
 
 
This letter is being sent as clarification that collaborating sites may choose to not participate in 
the vorinostat exploratory objective of this study, as proposed with amendment 9.0. 
 
If the decision is made that a site will opt out, then all applicable patients at that site will receive 
standard therapy for Induction II with LD-ADE alone. 
 
Since this objective is exploratory and does not require a specific number of patients, this will 
not adversely affect the study analyses. 
 
This information will be forthcoming with the next protocol amendment; however, sites may 
choose to opt out at any time. 
 

 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
 
 
 
AML08: A PHASE III RANDOMIZED TRIAL OF CLOFARABINE PLUS 
CYTARABINE VERSUS CONVENTIONAL INDUCTION THERAPY AND A PHASE II 
STUDY OF NATURAL KILLER CELL TRANSPLANTATION IN PATIENTS WITH 
NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA 
 
Letter of Amendment 
 
November 20, 2013 
 
 
Rationale 
 
This letter is being sent as clarification that if Erwinia asparaginase is given (see Section 4.5 of 
protocol), it may be administered according to local institutional guidelines, either IV or IM. 
 
Currently, the protocol and consent state IV administration.  These will be revised to IV or IM 
with the next protocol amendment. 
 
Collaborating sites may follow local administration guidelines for the administration of this 
commercially available agent.  The following St. Jude guidelines may be used, but are not required: 
 
Administration 
 
For IV administration, Erwinia asparaginase will be diluted in 50 mL NS to infuse over 30 
minutes to 1 hour via syringe. Erwinia asparaginase should be administered to run concurrently 
with IVF. (NS at TKO is sufficient.) 
 
Erwinia asparaginase should be administered at least 2 hours after an LP/IT since some cases of 
over sedation were reported when given “simultaneously” with sedation and/or an LP/IT at 
other institutions.   Further, it is desirable to avoid asparaginase directly before an IT because 
asparaginase could possibly interfere with the efficacy of methotrexate. 
 
Erwinia asparaginase may also be administered IM. 
 
Monitoring and concerns for anaphylaxis 
 
Obtain vital signs pre infusion, and then remain at the patient’s bedside for the first 5 minutes. 
Visually observe the patient at 10 minutes, and directly observe and obtain vital signs at 15 
minutes and at the end of the infusion. Repeat vital signs at discharge. Patients must remain in 

 
 
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Protocol document date: 06-02-2016 
the Medicine Room for 1 hour after administration to be observed for adverse effects. Keep NS 
at TKO during the 1 hour post watch time unless otherwise ordered.  To prepare for 
anaphylaxis: 
 
• Have oxygen, suction and pulse oximetry at bedside during and after infusion 
• Have readily available* the following medications:  
 
o Diphenhydramine 1 mg/kg (max 50 mg) for IV administration 
o Epinephrine (1:1000) 0.01 mL/kg (max 0.3 mL) for SQ administration 
o Hydrocortisone 100 mg/m2 for IV administration 
o NS for IV administration 
 
*Readily available means in the general area – such as the emergency medication box in the 
medicine room or inpatient areas. 
 
 
 
 
 
 
 
 
 
 

 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 

 
 
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IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Table of Contents 
 
1.0 
OBJECTIVES ......................................................................................................... 1 
1.1 
Primary objective ................................................................................................ 1 
1.2 
Secondary objective ............................................................................................ 1 
2.0  
BACKGROUND AND RATIONALE ................................................................... 2 
2.1 
Background and rationale for therapy................................................................. 2 
2.2 
Background and rationale for correlative and biologic studies ........................ 20 
3.0  
ELIGIBILITY CRITERIA AND STUDY ENROLLMENT ............................... 27 
3.1 
Diagnostic criteria ............................................................................................. 27 
3.2 
Inclusion criteria – all participants .................................................................... 28 
3.3 
Exclusion criteria – all participants ................................................................... 28 
3.4 
Criteria for randomization................................................................................. 28 
3.5 
Enrollment on study .......................................................................................... 29 
4.0 
TREATMENT PLAN ........................................................................................... 29 
4.1 
General overview .............................................................................................. 29 
4.2 
Induction therapy (2 courses) ............................................................................ 31 
4.3  
Consolidation I for patients who will not receive SCT ..................................... 37 
4.4 
Consolidation therapy for HR patients who will undergo SCT ........................ 37 
4.5 
Consolidation II (HD-AraC/Asp) ..................................................................... 38 
4.6 
NK cell therapy ................................................................................................. 39 
4.7 
CNS therapy ...................................................................................................... 42 
5.0 
SUPPORTIVE CARE GUIDELINES .................................................................. 43 
5.1 
Prophylaxis and treatment of metabolic derangement ...................................... 43 
5.2 
Treatment of hyperleukocytosis ........................................................................ 43 
5.3 
Prevention and treatment of complications related to tumor lysis .................... 43 
5.4 
Prophylaxis for Pneumocystis jiroveci pneumonia ........................................... 43 
5.5 
Prophylaxis for fungal infections (REQUIRED) .............................................. 43 
5.6 
Prophylaxis for viridans streptococcal infection (REQUIRED) ....................... 44 
5.7 
Management of febrile neutropenia .................................................................. 45 
5.8 
Management of Capillary Leak Syndrome related to clofarabine .................... 46 
5.9 
Management of hypertension related to sorafenib ............................................ 46 
5.10 
Drug interactions ............................................................................................... 46 
5.11 
Growth factors .................................................................................................. 47 
5.12 
Conjunctivitis prophylaxis ................................................................................ 47 
5.13 
Treatment modifications ................................................................................... 47 
6.0 
DRUG INFORMATION ...................................................................................... 48 
7.0  
EVALUATIONS, TESTS, AND OBSERVATIONS .......................................... 49 
7.1 
Required evaluations for diagnosis and response (all patients) ........................ 49 
7.2 
Additional required evaluations for patients receiving NK cells ...................... 50 
7.3 
Long-term follow-up evaluations ..................................................................... 50 
7.4 
Routine tests ...................................................................................................... 51 
7.5 
Research tests .................................................................................................... 51 
8.0  
EVALUATION CRITERIA ................................................................................. 51 
8.1 
Response criteria ............................................................................................... 51 
8.2 
Toxicity evaluation criteria ............................................................................... 52 
9.0 
OFF THERAPY AND OFF-STUDY CRITERIA ................................................ 52 
9.1 
Criteria for removal from protocol therapy (off therapy) ................................. 52 
9.2 
Criteria for removal from study (off study) ...................................................... 52 
9.3 
NK cell donors .................................................................................................. 52 

 
 
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9.4 
NK cell recipients ............................................................................................. 52 
10.0 
BIOLOGIC STUDIES .......................................................................................... 53 
10.1 
NK cell receptor study ...................................................................................... 53 
10.2 
Minimal residual disease studies ...................................................................... 53 
10.3 
Pharmacokinetic studies .................................................................................... 53 
10.4 
Pharmacogenetic studies ................................................................................... 53 
10.5 
In vitro drug sensitivity assays .......................................................................... 54 
10.6 
Histone acetylation studies ............................................................................... 54 
10.7 
Other biology studies ........................................................................................ 54 
11.0 
SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS ............. 54 
11.1 
Adverse event monitoring and reporting .......................................................... 54 
11.2 
Reporting adverse events and deaths to St. Jude IRB ....................................... 54 
11.3  Reporting requirements from St. Jude to FDA ................................................. 57 
11.4  Reporting to St. Jude Regulatory Affairs Office (RAO) .................................. 57 
11.5 
Reporting from St. Jude to Sanofi Group Pharmacovigilance .......................... 57 
11.6 
Other reporting mechanisms ............................................................................. 59 
11.7 
Process for reporting AEs to/from St. Jude and collaborating sites ................. 59 
12.0 
DATA COLLECTION, STUDY MONITORING AND CONFIDENTIALITY . 61 
12.1 
Data collection and management ...................................................................... 61 
12.2 
Study monitoring .............................................................................................. 61 
12.3 
Confidentiality .................................................................................................. 62 
13.0 
STATISTICAL CONSIDERATIONS.................................................................. 62 
13.1 
Design and analysis for primary objective ........................................................ 62 
13.2 
Monitoring rules................................................................................................ 64 
13.3 
Comparison and stratification for amendment 6 ............................................... 67 
13.4 
Analyses for secondary objective ..................................................................... 67 
13.5 
Analyses for exploratory objectives .................................................................. 68 
14.0 
OBTAINING INFORMED CONSENT ............................................................... 69 
14.1 
Consent/Assent at Induction and Post-Induction .............................................. 69 
14.2 
Consent at the age of majority .......................................................................... 70 
14.3 
Consent when English is not the primary language .......................................... 70 
15.0 
DATA SAFETY MONITORING BOARD.......................................................... 70 
16.0 
REFERENCES ..................................................................................................... 72 
 
Appendices: 
 
Appendix I: Drug Information 
Appendix II: Criteria for GVHD 
Appendix III: Specimen Submission Guidelines 
Appendix IV: Molecular Diagnostics for Fever and Neutropenia (St Jude patients only) 
Appendix V: Randomization form 
Appendix VI:  Treatment Schema 
 
Attached supporting documents:   
 
Therapeutic Cell Infusion 
Informed Consent Statements 

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1.0 
OBJECTIVES 
 
The overall objective of this protocol is to improve the cure rate of acute myeloid leukemia 
(AML).  
 
1.1 
Primary objective 
 
To compare the immunologic complete response rate after one course of therapy in patients 
who receive cytarabine + daunorubicin + etoposide (ADE) with that in patients who 
receive clofarabine + cytarabine (Clo/AraC). 
 
 
1.2 
Secondary objective 
 
To estimate the event-free survival (EFS) of standard risk (SR) patients who receive 
chemotherapy alone and the EFS of SR patients who receive chemotherapy followed by 
natural killer (NK) cell transplantation. 
 
 
1.3 
Exploratory objectives 
 
1.3.1  To genotype natural killer (NK) cell receptors and measure their expressions at 
diagnosis and after induction therapy, and to explore the associations of these 
features with treatment outcome. 
 
1.3.2  To assess the prognostic value of levels of minimal residual disease in peripheral 
blood at day 8 of induction I. 
 
1.3.3  To validate new markers and methods for minimal residual disease (MRD) 
detection. 
 
1.3.4 To identify new prognostic factors by applying new technologies to study patient 
material.  
 
1.3.5 To identify pharmacogenetic, pharmacokinetic and pharmacodynamic predictors for 
treatment-related outcomes in the context of the systemic therapy used in the 
protocol. 
 
1.3.6 To describe the impact of antibiotic and antifungal prophylaxis on invasive 
bacterial and fungal infections, febrile neutropenia, hospitalization, and antibiotic 
resistance. 
 
1.3.7 To explore the feasibility and toxicity of administering vorinostat in combination 
with chemotherapy in selected high-risk patients. 
 

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2.0  
BACKGROUND AND RATIONALE 
 
2.1 
Background and rationale for therapy 
 
2.1.1 Introduction 
 
Although the treatment outcome for children with leukemia has improved dramatically 
during the past 20 years, the cure rate for AML continues to lag behind that achieved for 
children with acute lymphoblastic leukemia (ALL). Currently, greater than 80% of children 
with ALL are cured, compared to only about 50% of those with AML.1-5 Most patients 
with AML achieve remission after 1 to 2 courses of induction therapy, but approximately 
half of these patients suffer relapse of their disease. In addition, despite improvements in 
supportive care, treatment-related morbidity and mortality remain significant problems.6,7 
Therefore, the overall goal of our AML program is to develop novel therapies that 
overcome drug resistance, decrease relapse rates, and reduce the short- and long-term 
adverse effects of treatment. The present study will assess the feasibility and efficacy of a 
novel form of therapy – haploidentical NK cell transplantation – in patients with standard-
risk AML. In addition, we will investigate the efficacy of clofarabine + cytarabine 
(Clo/AraC) in newly diagnosed patients with AML and attempt to optimize outcome 
through the use of MRD-adapted therapy and further improvements in supportive care.  
 
Initially, AML08 included 5 courses of chemotherapy for the following reasons: a) the data 
from the MRC AML12 trial had not yet been published, b) the COG trial used 5 courses, 
and c) five courses was, at that time, considered “standard of care.” The MRC AML12 
results have now been published (J Clinical Oncology 28:586-595, 2010). Briefly, the 
results show that among 992 patients randomized to receive 4 or 5 courses of 
chemotherapy, there were no differences in OS (52% vs. 53%) or relapse (54% vs. 49%). 
Among the 270 children randomized to 4 vs. 5 courses, there were no differences in relapse 
(36% vs. 36%), DFS (63% vs. 62%), or OS (74% vs. 74%). Based, in part, on these results, 
the upcoming COG trial will administer 4 courses of chemotherapy. Because there appears 
to be no benefit to course #5 (consolidation III), but some risks (mortality, morbidity, and 
quality of life), this amendment will eliminate consolidation III.  
 
2.1.2 Overview of clofarabine 
 
Clofarabine (2-chloro-9-[2’-deoxy-2’-fluoro-β-D-arabinofuranosyl]adenine; Cl-F-ara-A; 
CAFdA) is a rationally designed, second generation purine nucleoside analogue. 
Clofarabine was designed as a hybrid molecule to overcome the limitations and incorporate 
the best qualities of both fludarabine (F-ara-A) and cladribine (2-CdA, CdA) both of which 
are currently approved by various regulatory authorities for treatment of hematologic 
malignancies. Because clofarabine has a chloro- group at the 2-position of adenine, its 
chemical structure is more closely related to 2-CdA than to F-ara-A. Halogenation at the 
2-position of adenine renders this class of compounds resistant to intracellular degradation 
by the enzyme adenosine deaminase. Substitution of a fluorine at the C-2’-position of the 
arabinofuranosyl moiety of clofarabine increases its stability in gastric acid and decreases 
its susceptibility to phosphorolytic cleavage by the bacterial enzyme Escherichia coli 
purine nucleoside phosphorylase in the gastrointestinal tract, both of which may lead to 
enhanced oral bioavailability.8,9 Clofarabine was approved in December 2004 by the 

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IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
United States Food and Drug Administration (US FDA) for the treatment of pediatric 
patients with relapsed or refractory acute lymphoblastic leukemia (ALL) after at least 2 
prior regimens based on the induction of complete responses. 
 
Mechanism of action 
 
The precise mechanism of action of clofarabine on dividing and non-dividing cells is 
unknown. Like the other nucleoside analogues (cytarabine, ara-A [vidarabine], cladribine, 
fludarabine), clofarabine must be converted to the 5’-triphosphate form by deoxycytidine 
kinase (dCK) to be active within cells. Clofarabine is more efficient as a substrate for 
purified recombinant dCK, exceeding cladribine and the natural substrate, deoxycytidine.10 
Evidence suggests that the primary cytotoxic effect of clofarabine is due to its inhibition of 
DNA synthesis. The triphosphate form of clofarabine is an inhibitor of both DNA 
polymerase α and ribonucleotide reductase.11 These effects lead to depletion of 
intracellular deoxynucleotide triphosphate pools, and inhibition of elongation of DNA 
strands during synthesis.12 With respect to inhibition of ribonucleotide reductase, 
clofarabine and cladribine are superior to fludarabine.10 With respect to inhibition of DNA 
polymerase α, clofarabine and fludarabine are similar and both are superior to cladribine.10 
Thus, in comparison to cladribine and fludarabine, clofarabine more completely inhibits 
both ribonucleotide reductase and DNA polymerase α, versus one or the other. 
 
Unlike fludarabine, clofarabine is active in non-dividing cells and in cells with a low 
proliferation rate. Clofarabine can induce the apoptotic pathway as part of its cytotoxic 
effect on cells.9 Clofarabine has been shown to disrupt the integrity of mitochondria in 
primary chronic lymphocytic leukemia (CLL) cells. The damage leads to release of pro-
apoptotic mitochondrial factors.13 These effects are postulated to induce apoptosis in 
indolent, non-dividing CLL cells. This result was not seen with fludarabine and may 
explain, at least in part, the enhanced cytotoxicity of clofarabine though the physiologic 
and clinical implications of these observations remain uncertain and under continued 
investigation. 
 
Pharmacokinetics of clofarabine in adult patients with AML 
 
Pharmacokinetic data were collected from 13 adult patients with refractory or relapsed 
AML in an open-label study in which they were treated with clofarabine 40 mg/m2/day IV 
infusion over 1 hour for 5 consecutive days (Clinical Report – Protocol CLO-221, February 
13, 2004). Stationary pharmacokinetics were observed between Days 1 and 5, and plasma 
concentrations declined rapidly thereafter and exhibited biphasic kinetics. The estimated 
terminal half-life was approximately 6 hours and ranged from 4.1 to 8.6 hours. Consistent 
with this short half-life, pre-dose concentrations on Day 2 were about 10% or less of 
maximal concentrations at the end of infusion. After 4 days of dosing, pre-dose 
concentrations averaged 13.8 ng/mL and ranged from 4.0 to 23.1 ng/mL. Because of the 
short half-life of clofarabine, there was little-to-negligible accumulation with once daily 
dosing of clofarabine at 40 mg/m2 by 1-hour IV infusion.   
 

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Clinical experience with clofarabine 
 
The maximum tolerated dose (MTD) of clofarabine IV in adult patients has been 
determined to be 40 mg/m2/day for 5 consecutive days, which is lower than the tolerable 
daily dose for pediatric patients, 52 mg/m2/day.  
 
Relapsed, refractory adult AML 
 
Phase I trials were initiated in 1999 and the first study was a traditional dose-escalation 
study where the objective was to establish the MTD in adult patients with solid tumors or 
hematologic malignancies.14  The starting dose was 15 mg/m2 administered daily for 5 days 
based on an animal study in which this dose was safe with no observable toxicities.  
However, 2 of the first few patients on study experienced myelosuppression and required 
dose de-escalations before the MTD of 2 mg/m2 was identified in patients with solid 
tumors. Dose escalation in patients with hematologic malignancies increased to 55 mg/m2, 
at which point patients experienced dose-limiting toxicities (DLTs) of reversible 
hepatotoxicity, and the MTD was determined to be 40 mg/m2/day. Among the 32 patients 
diagnosed with acute leukemia, 2 patients achieved a CR and 3 achieved CRp for an 
overall response rate of 16%. Clofarabine pharmacokinetics were dose proportional across 
all the doses studied, but intracellular clofarabine triphosphate (which had large interpatient 
variability) began to show saturation at doses greater than about 20 mg/m2/day.  
 
In a Phase II study reported by Kantarjian et al,15 62 adult patients with relapsed or 
refractory acute leukemia received clofarabine 40 mg/m2 IV once daily for 5 days every 3 
to 6 weeks. Twenty (20) patients achieved a CR, 9 achieved a CRp, and 1 achieved a 
partial response for a total response rate of 48%. The predominant toxicities were 
reversible liver dysfunction (as indicated by elevated ALT and AST and 
hyperbilirubinemia), skin rashes, palmar-plantar erythrodysesthesia syndrome, and 
mucositis. No correlation was observed between plasma clofarabine concentrations and 
intracellular clofarabine triphosphate concentrations, although it was observed that 
responders showed an accumulation of intracellular clofarabine triphosphate on Day 2 
compared to non-responders.  
 
In contrast, a multi-center Phase II trial (CLO221) initiated in 2002 in adult patients with 
relapsed or refractory acute myelogenous leukemia received clofarabine 40 mg/m2 once 
daily for 5 days every 28 days for 2 cycles with subsequent cycles being dosed with 30 
mg/m2.16,17 Only 1/40 (3%) patients achieved a CR of 20.4 weeks duration. Nausea, 
vomiting, headache, diarrhea, anorexia, dermatitis, and stomatitis were the most frequently 
drug-related reported AEs. Drug-related renal toxicities were reported for 10% of the 
patients; however, these patients either had a concurrent clinical condition associated with 
renal toxicity or at least 1 concomitant medication known to increase the potential for renal 
toxicity. Pharmacokinetic data available for 33% of the patients indicate clofarabine had a 
high tissue distribution with minimal accumulation and rapid elimination (primarily as 
unchanged drug) in the urine. These seemingly discrepant results must be considered in the 
context of the patient populations treated; in the CLO-221 experience, approximately 25% 
of patients were refractory to prior therapy, whereas no subgroup of patients with primary 
refractory disease was reported in the prior experience conducted and reported by 
Kantarjian and colleagues.15 

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Based upon the single-agent activity observed with clofarabine in several populations of 
patients with relapsed or refractory acute leukemias, efforts have been undertaken to 
combine clofarabine with other anti-leukemic agents. Faderl and colleagues conducted a 
Phase I/II study in adult patients with first relapse or first salvage of primary refractory 
AML or ALL.18 Patients were treated with doses of clofarabine of 15-40 mg/m2/day by 1-
hour IV infusion daily for Days 2 through 6 followed 4 hours later by cytarabine 
1 g/m2/day by 2-hour IV infusion daily for Days 1 through 5. Cytarabine alone was 
administered on Day 1 at the same dose. Thirty-two patients were treated; 12 in the Phase I 
portion and 20 in the Phase II portion at 40 mg/m2/day. The Phase II dose of 40 mg/m2/day 
was based on the absence of DLT at this dose in combination; this dose was not exceeded 
since it had been determined as the MTD from single-agent studies with clofarabine. The 
median patient age in this study was 60 (range: 18-84 years) and the majority of patients 
had AML (78%). Efficacy data are available for 32 patients; of these 7/32 (22%) achieved 
a complete response and an additional 5/32 (16%) achieved a complete response with 
inadequate platelet recovery for an overall response rate of 38% (12/32 patients). In the 25 
patients with AML, CR was 28% and CRp 12% for an ORR of 40%. Two of four patients 
with MDS had a CRp.  The most frequently reported drug-related AEs (i.e., those occurring 
in ≥20% of patients) were nausea, diarrhea, vomiting, dermatitis, flushing, palmar-plantar 
erythrodysesthesia syndrome, and headache. Changes in post-baseline chemistry 
parameters were mild to moderate in the majority of patients and were reversible if not 
attributable to the disease. Bone marrow function was suppressed, resulting in neutropenia, 
lymphocytopenia, anemia, and thrombocytopenia. Plasma pharmacokinetic data are 
available for 9 patients and are consistent with those in patients with refractory or relapsed 
AML dose with clofarabine as a single agent, suggesting that no gross pharmacokinetic 
interaction was occurring between cytarabine and clofarabine.  
 
A Phase II open label study of the same combination was conducted in patients with de 
novo AML, AML evolved from MDS, and relapsed AML.   Patients were treated with 
clofarabine 40 mg/m2/day by 1-hour IV infusion followed four hours later by cytarabine 
1 g/m2/day by 2-hour IV infusion daily for Days 1 through 5.19 The median patient age in 
this study was 67 (range: 38 - 82 years) and 61% (n=14) had received at least one prior 
cytotoxic regimen. Two early deaths were observed due to disease progression and sepsis. 
Grade 4 neutropenia developed in all patients and most patients had some degree of edema 
or third spacing syndrome. Other AE’s included reversible acral rash. Efficacy data are 
available for 21 evaluable patients; of these 14/21 (67%) achieved a complete response and 
an additional 2/21 (9%) achieved a partial response for an overall response rate of 76% 
(16/21 patients). Complete cytogenetic remissions occurred in 9/20 (45%) patients. Durable 
remissions and low toxicity allowed some patients to proceed to non-myeloablative 
allogeneic stem cell transplantation.   
 
In an on-going open-label Phase I/II study, patients with previously untreated and 
refractory AML received therapy with a combination of clofarabine and daunorubicin.20 
Patients were enrolled into five treatment cohorts containing 6 patients each which were 
expanded based on toxicity scoring. All patients received 50 mg/m2/day daunorubicin on 
days 1, 3 and 5. Cohorts 1, 2, 3, and 4 received clofarabine doses 15 mg/m2/day, 20 
mg/m2/day, 25 mg/m2/day, and 30 mg/m2/day, respectively on days 1 – 5 of the dosing 
regimen. Cohort 5 received the feasible dose of clofarabine derived from cohorts 1 to 4 in 
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Three induction deaths were reported in the third cohort, and as a result, 20 mg/m2/day was 
the dose of clofarabine chosen to combine with daunorubicin and GO. AST and ALT 
toxicities > grade 2 were not observed in any of the patients. Grade 4 cardiac toxicity was 
observed in 2 patients at the 25 mg/m2/day cohort. One patient dosed at 30 mg/m2 
experienced grade 3 elevations in bilirubin. Another patient dosed at the same dose level 
had grade 4 renal toxicity. In the triplet combination, 2 patients experienced grade 3 nausea 
and one other patient experienced grade 4 cardiac toxicity. Efficacy data are available for 
37 patients; of these 24/37 (65%).achieved a complete response. Overall survival at 1 year 
was 62% for the 37 patients enrolled into this study. 
 
In an open-label Phase I study, patients with AML received salvage therapy with a 
combination of clofarabine and idarubicin or clofarabine, idarubicin, and cytarabine.21 Four 
and three dose levels of each combination, respectively, were administered to patients. 
Clofarabine 22.5 mg/2/day for 5 days and idarubicin 12 mg/m2/day for 3 days resulted in 
grade 3 AEs of diarrhea, mucositis, esophagitis and rash which required dose de-escalation. 
When clofarabine 22.5 mg/m2/day for 5 days, idarubicin 8 mg/m2/day for 3 days, and 
cytarabine 1 g/m2/day for 5 days was administered, grade 3 diarrhea, acute renal failure and 
elevated bilirubin was observed and required dose de-escalation. Ultimately, the MTDs 
were determined to be, for the two drug combination, clofarabine 22.5 mg/m²/day x 5 days 
plus idarubicin 10 mg/m² x 3 days, and for the three drug regimen, clofarabine 22.5 
mg/m²/day x 5 days plus idarubicin 6 mg/m²/day x 3 days plus 0.75 mg/m²/day x 5 days. 
Dose limiting toxicities with the two drug regimen were elevated SGPT and bilirubin, and 
headache. For the three drug regimen, DLTs were increased bilirubin, diarrhea, mucositis, 
and decreased left ventricular ejection fraction. An overall response rate of 22% (3 CR and 
2 CRp) was observed with the combination of clofarabine and idarubicin and an overall 
response rate of 48% (10 CRs) was observed with the three drug combination of 
clofarabine, idarubicin, and cytarabine.  
 
Previously untreated adult AML 
 
Faderl and colleagues conducted a Phase II study in patients ≥50 years with previously 
untreated AML with cytarabine 1 g/m2 alone on Day 1, clofarabine administered by IV 
infusion over 1 hour followed 4 hours later by cytarabine 1 g/m2/day administered by IV 
infusion over 2 hours on Days 2 through 5, and clofarabine alone administered by IV 
infusion over 1 hour on Day 6 repeated every 4 to 6 weeks.22 The most frequently reported 
AEs reported during induction included myelosuppression, fever of unknown origin, 
infection, diarrhea, nausea, vomiting, headaches, skin rashes (including palmar plantar 
erythrodysesthesia), facial flushing, and transient abnormal liver function. During the 
consolidation phase, infections were reported for more than 50% of the patients and grade 
3 or higher myelosuppression was reported for 98% of the patients. A total of 9 patients 
died during the study: 1 each from acute renal failure and bullous skin rash, 
Stevens-Johnson syndrome and sepsis, bacterial sepsis and multisystem organ failure, 
fungal sepsis, complications of neutropenia and sepsis, and 4 due to sepsis-related 
complications with 2 progressing to multi-organ failure. The overall response rate was 60% 
(52% CR and 8% CRp).  
 
In a prospective randomized Phase II study presented at the 2005 annual meeting of the 
American Society of Hematology,23 older adult patients (≥60 years with newly diagnosed 

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AML) were treated with clofarabine 30 mg/m2/day IV for 5 consecutive days or 
clofarabine 30 mg/m2/day IV for 5 consecutive days in combination with low dose ara-C 
(LDAC) 20 mg/m2/day SC for 14 consecutive days. Based on a Bayesian randomization 
schema 76 patients were enrolled, of whom 16 were treated with clofarabine alone and 60 
were treated with the combination of clofarabine and LDAC. The median patient age was 
71 years (range: 60-83 years). Five of the 16 (31%) patients treated with clofarabine alone 
achieved a complete response and 38/60 (63%) patients treated with the combination 
therapy achieved a complete response. Both treatment regimens were tolerated in this 
patient population without a distinction in terms of toxicity. Interestingly, the early 
mortality rate was lower in the combination arm (17%) compared with the clofarabine 
alone arm (31%); however, conclusions regarding the early mortality rate are premature 
based on the limited number of patients treated with single-agent clofarabine. In an 
additional open-label, dose-escalation Phase I/II study reported at the  2005 annual meeting 
of the American Society of Hematology, elderly patients ≥60 years with de novo AML 
received cytarabine 100 mg/m2/day continuous infusion for 7 days starting on Day 1 and 
clofarabine at a starting dose of 30 mg/m2/day for 5 days starting on Day 2 with subsequent 
dose escalation based on response and toxicity.24 Preliminary data are available for 4 
patients; 2 died from drug-related infections and 2 achieved a CR.  Grade 3 and 4 AEs 
included fever and neutropenia (2 patients), renal toxicity (2 patients), and capillary leak 
syndrome (1 patient). The DLTs were observed at the first dose level, therefore, the 
protocol was amended to allow for dose de-escalation to clofarabine 22.5 mg/m2/day as 
well as to limit age to 60-75 years inclusive. As a result of the observed toxicity profile, 
routine use of aggressive pre-hydration and antibiotic/antifungal prophylaxis is now 
mandatory in this study.  
 
The safety and efficacy of single agent clofarabine in previously untreated AML patients 
was reported in a single center Phase II study of 30 older adult patients (>65 years old) who 
were considered unfit for chemotherapy.25  Overall response rate was 56%, with 43% CR 
and CRp 13%. The most common ≥ Grade 3 toxicities were transient increases in bilirubin 
and SGPT, hand foot syndrome, and rash. Based on these encouraging results, a multi-
center study was performed in a similar patient population and results were presented at 
ASH 2006.26 Patients were considered unfit for standard chemotherapy based on age, 
comorbidity, and performance status. Patients were treated with clofarabine at a dose of 30 
mg/m2/day for 5 days, repeated every 28 – 42 days. Sixty-two percent of patients were age 
70 or older, 69% of patients had intermediate cytogenetics, 30% had adverse cytogenetics, 
and 31% had a prior hematologic disorder. Overall response rate was 44% (21% CR, 23 % 
CRi). The CR/CRi rate of 57 % and 50% in patients 70 – 74 years and > 75 years of age, 
respectively. The CR/CRi rate was 49% in patients with adverse cytogenetics. One year 
survival was 18% in non-responders as compared with a 32% observed in responders (CR 
+ CRi). 
 
Pediatric leukemia 
 
In parallel to the adult program, a pediatric program was initiated in 2000.  In the Phase I 
study in pediatric patients with hematologic malignancies, 25 patients were treated in 
cohorts of escalating doses up to 70 mg/m2, a dose at which 1 patient had grade 4 
hyperbilirubinemia and grade 3 elevated transaminases, and 1 had a grade 3 skin rash; the 
MTD was determined to be 52 mg/m2.27 Of the 13 patients treated at 52 mg/m2, grade 2 to 

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grade 3 increases in bilirubin and liver transaminases were observed. A total of 5 patients 
achieved a CR and 3 achieved a PR for an overall response rate of 32%. Clofarabine 
plasma concentrations were generally smaller in the pediatric population than the adult 
population when administered the same dose, but there did not appear to be much 
difference in intracellular clofarabine triphosphate concentrations. Thus, the MTD was 
determined to be 52 mg/m2 and the recommended Phase II dose.   
 
Due to the results observed in the Phase I studies in pediatric patients with hematologic 
malignancies, interest by investigators and physicians, with support from regulatory 
authorities, led to the initiation of 2 parallel Phase II trials in 2002 in patients with either 
relapsed or refractory ALL or relapsed or refractory AML.28,29 Both studies evaluated 
clofarabine 52 mg/m2 administered once daily over a 2-hour IV infusion for 5 days every 2 
to 6 weeks. In the 61 patients enrolled in the relapsed or refractory ALL study (61% males: 
39% females, 1 to 20 years old), the overall remission rate (CR+CRp) was 20%; 30% 
(18/61) of patients showed a response (7CR, 5CRp, 6PR). Responses were noted in 15 of 
50 (30%) patients with B- lineage ALL, 2 of 6 (33%) with T-cell ALL. Responders 
received a median of 3 prior induction regimens; 50% (9/18) had prior HSCT and 50% 
(9/18) were refractory to the preceding induction regimen. Response rate in refractory 
patients was 26% (9/35). After clofarabine treatment, 10 patients proceeded to transplant 
(including 8 responders). Six of 10 patients who received a transplant were alive at last 
follow up (survival range: 30.1+ - 145.1+ weeks). Response duration in 6 patients with CR 
or CRp who did not receive a transplant ranged from 4.3 to 58.6 weeks; 2 patients 
maintained CR for 47.9 and 58.6 weeks after clofarabine therapy. Median overall survival 
for the patients who achieved at least a PR was 66.6 weeks compared to 12.9 weeks for all 
patients.28,29 In 42 pediatric patients with relapsed or refractory AML, the response rate was 
26% (1 CRp, 10 PR). Responders had received a median of 2 prior induction regimens, 
36% (4/11) had prior HSCT and 55% (6/11) patients were refractory to the preceding 
induction regimen. Response rate in refractory patients was 21% (6/28). One patient who 
had received 5 prior induction regimens achieved CRp. 13 (31%) patients (1CRp, 6PR, 
3NE, 3TF) underwent HSCT after completing clofarabine therapy, 5 of whom were alive at 
last follow-up (survival range: 62.7+ 160.1+ weeks). Many patients proceeded to HSCT 
as soon as a donor was identified without waiting for the patient to go into remission, 
making remission difficult to assess. Median overall survival for patients who achieved at 
least a PR was 32.1 weeks compared to 23.4 weeks for all patients.29 
 
Among the 113 pediatric patients with ALL and AML, the most frequently reported drug-
related AEs were vomiting (66% ALL and 65% AML) and nausea (58% ALL and 
70% AML). Other drug-related AEs reported by at least 10% of the pediatric patients 
overall included febrile neutropenia (31% ALL and 28% AML), pyrexia (21% ALL and 
26% AML), pruritus NOS (24% ALL and 20% AML), dermatitis NOS (24% ALL and 
17% AML), headache NOS (18% ALL and 35% AML), diarrhea NOS (21% ALL and 
22% AML), anxiety NEC (16% ALL and 7% AML), fatigue (15% ALL and 13% AML), 
mucosal inflammation NOS (16% ALL and 15% AML), and flushing (12% ALL and 
11% AML). Anorexia occurred in 12% ALL and 9% AML and palmar-plantar 
erythrodysesthesia syndrome in 12% ALL and 9% AML. 
 

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Study and dose rationale 
 
The safety profile of clofarabine appears acceptable within the target populations studied to 
date in the clinical studies summarized in Section 2.4. Clofarabine has demonstrated anti-
cancer activity through inhibition of DNA synthesis and repair, induction of apoptosis, and 
possibly through other mechanisms. Numerous responses have been observed after 
treatment with clofarabine in heavily pre-treated relapsed/refractory patients with ALL or 
AML.   
 
The rationale for combining clofarabine with cytarabine is based on the ability of 
clofarabine to modulate Ara-CTP levels.11,12 Clofarabine inhibits ribonucleotide reductase, 
resulting in decreased levels of deoxynucleotides, which then leads to a decrease in the 
feedback inhibition of deoxycytidine kinase, the rate limiting enzyme in Ara-CTP 
synthesis. Thus, the combination of clofarabine with cytarabine should lead to increased 
levels of intracellular Ara-CTP. A phase I/II study of this combination supported this 
strategy and demonstrated that this combination is safe and active in adults with relapsed 
and refractory AML.18 Recently, the Children’s Oncology Group has activated 
AAML0523, “A Phase I/II Study of Clofarabine in Combination with Cytarabine in 
Pediatric Patients with Refractory/Relapsed Leukemia.”  
 
Although three-drug induction therapy with cytarabine, an anthracycline, and etoposide is 
used in many contemporary clinical trials, we have demonstrated that it is safe and 
effective to initiate therapy with cytarabine and cladribine. In the St. Jude AML97 trial, 
patients initially received a 5-day course of cytarabine + cladribine, followed by induction 
with daunorubicin, cytarabine, and etoposide (DAV).2 MRD-negativity was achieved in 
51% of patients after the block of cytarabine + cladribine and in 61% of patients after the 
first course of DAV.30 In addition, 90% of patients achieved morphologic CR after DAV 
#1. For the entire cohort of patients treated on AML97, the 3-yr EFS was 49%. 
Importantly, after an amendment to AML97 that removed the early use of autologous SCT 
and introduced MRC-based consolidation therapy, the 3-yr EFS estimate is 57%, similar to 
the preliminary results of AML02. Together, these data demonstrate that early use of 
cytarabine + cladribine is safe, effective at reducing tumor burden, and contributes to an 
overall outcome that is similar to that achieved on other contemporary trials.  
 
2.1.3 Rationale for the use of high-dose cytarabine 
 
Clinical trials have demonstrated that increases in the intensity of induction therapy, either 
through intensification of timing3,31 or through dose intensification32-34 lead to improvements 
in the outcome of AML. A Southwest Oncology Group (SWOG) study that randomly 
assigned adults with AML to groups that received daunorubicin with low dose cytarabine 
(LDAC) or with high dose cytarabine (HDAC) demonstrated a superior relapse-free survival 
for the HDAC arm (33% vs 21%, p=0.049).33 Similarly, an Australian Leukemia Study 
Group (ALSG) trial that randomized patients to receive daunorubicin and etoposide with 
LDAC or HDAC showed that the estimated duration of remission was 45 months with 
HDAC and only 12 months with LDAC (p=0.0005). Furthermore, 49% of patients remain 
relapse free after achieving CR with HDAC compared to 24% after LDAC.32 
 

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On the recently completed St. Jude AML02 trial, pediatric patients with AML were 
randomly assigned to induction therapy with daunorubicin, etoposide and either low dose 
cytarabine (LD-ADE) or high dose cytarabine (HD-ADE). An interim analysis performed 
12/5/07 revealed no statistical difference in day 22 MRD, EFS, or OS between the two 
arms, but there were trends toward better outcome among patients treated on the HD-ADE 
arm. Among patients treated on the HD-ADE arm, 31 of 86 (36%) were MRD+ at day 22, 
compared with 41 of 94 (44%) on the LD-ADE arm (p=0.27). The 3-year EFS and OS 
estimates were 59% ± 9% and 71% ± 8% for patients treated on the HD-ADE arm and 57% 
± 8% and 63% ± 8% for patients treated on the LD-ADE arm (p=0.77 for EFS and 0.57 for 
OS). There were no significant differences in toxicity between the HD-ADE and LD-ADE 
arms. Therefore, the standard arm of the present trial will include the use HDAC during 
induction I.  
 
2.1.4 Rationale for the addition of gentuzumab ozogamicin 
 
Gentuzumab ozogamicin (GO) is a humanized anti-CD33–calicheamicin conjugate that 
was approved for use in AML by the FDA in May 2000.35,36 In a phase I study, toxicity 
was primarily myelosuppression and several patients had clearing of their blood and bone 
marrow blasts.37 A phase II study demonstrated a response rate of approximately 30% after 
the administration of two doses of GO, 9 mg/m2 given two weeks apart.38 In that study, 
23% of patients developed grade 3 or 4 hyperbilirubinemia and 17% developed elevated 
liver transaminases. Hepatic veno-occlusive disease (VOD) has also been reported, 
primarily, but not exclusively, in patients who received GO after SCT.39-41  
 
It is likely that GO will be most beneficial in combination with conventional 
chemotherapy. In this regard, investigators from the Medical Research Council (MRC) 
demonstrated that it was feasible to give GO at a dose of 3 mg/m2 in combination with 
daunorubicin and cytarabine (DA) or fludarabine, cytarabine, GCSF, and idarubicin 
(FLAG-Ida).42 The complete remission (CR) rate among patients who received DA + GO 
or FLAG-Ida + GO was 91%. At the 2006 meeting of the American Society of 
Hematology, Burnett et al presented preliminary results of the MRC AML15 trial. In this 
trial, 1115 patients were randomly assigned to chemotherapy (DA or ADE or Ida-FLAG) ± 
GO (3 mg/m2) during induction. The CR rate was 85%, with no differences in induction 
deaths or refractory disease between the chemotherapy alone and the chemotherapy + GO 
arms. With a median follow up of 15 months, the 3-year DFS estimates were 51% for 
patients who received GO and 40% for patients who did not (p=0.008). The 3-year OS 
estimates were 53% and 46% respectively (p=0.4). The Children’s Oncology Group (COG) 
recently completed a pilot study of 341 patients (AAML03P1) to test the feasibility of 
administering ADE + GO as Induction I to children with AML. The CR rate was 84% after 
one course of therapy, with a toxic mortality rate of only 1.5%. The preliminary results, 
with a median follow up only 226 days, indicates that the EFS (p=0.035) and OS (p=0.016) 
were both slightly better than that of CCG2961.  
 
Based on the promising results from the MRC and COG, GO will be given to standard and 
low risk patients on the present trial. However, because of the potential increased risk of 
fatal VOD, high risk patients, who will undergo SCT, will not receive GO.   
 

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Update June 22, 2010: A confirmatory, post approval clinical trial was begun by Wyeth 
(now Pfizer) in 2004. The trial was designed to determine whether adding Mylotarg to 
standard chemotherapy demonstrated an improvement in clinical benefit (survival time) to 
AML patients. The trial was stopped early when no improvement in clinical benefit was 
observed, and a greater number of deaths occurred in the group of patients who received 
Mylotarg compared with those receiving chemotherapy alone. 
 
As a result of the FDA Safety Report describing this trial, and the FDA’s recommendation 
that Mylotarg not be commercially available to new patients, the AML08 trial was 
amended as of June 22, 2010 to delete the use of Mylotarg. This will only affect 
consolidation I MAG in LR and SR patients not scheduled to receive SCT. 
 
2.1.5 Rationale for the use of NK cell transplantation 
 
Natural killer (NK) cells can kill target cells without the need for prior sensitization or 
activation.43 The ability of NK cells to perceive alterations in the expression of MHC class 
I on host cells was reported by Karre and colleagues more than a decade ago.44 They 
observed that mouse tumors lacking MHC antigens (“missing self”) were more readily 
killed by NK cells than tumors expressing normal levels of class I. Many human leukemia 
or cancer cells were found to have very low levels of MHC expression, thereby avoiding 
the recognition by T cells. In humans, NK cells are regulated by killer immunoglobulin-
like receptors (KIRs) that are encoded by a gene family on chromosome 19q13 and 
recognize specific HLA class I alleles.43,45-47 KIRs includes receptors with activating as 
well as inhibitory potential, receptors that have no known ligand, and receptors with 
specificity for MHC class I such as HLA-A, -B  and -C. The receptors specific for MHC 
class I molecules on target cells inhibit NK effector functions such as cytotoxicity and 
cytokine production. Each NK cell generally expresses at least one inhibitory receptor that 
recognizes a self HLA molecule, thus preventing autoimmunity. Fourteen members of the 
KIR family have been identified on human NK cells thus far.48 Clinical data suggest that 
KIR2DL2 and KIR2DL3 that recognize an epitope shared by HLA-C group 1 allotypes, 
KIR2DL1 that recognizes an epitope shared by HLA-C group 2 allotypes, and KIR3DL1 
that recognizes an epitope shared by HLA-Bw4 allotypes are important determinants of 
anti-leukemic effect.46,49-51 
 
In hematopoietic stem cell transplantation (SCT), the NK cells of the donor may exert 
potent anti-leukemia effects if the cognate MHC class I epitope is absent on the patient’s 
leukemia cells for the donor’s inhibitory KIRs. This potency has been demonstrated in both 
mouse models and in clinical transplantation.46,49-51 In a study of 57 adult patients with 
AML, none of the 34 patients who received a KIR-ligand mismatched haploidentical SCT 
had a disease relapse.52 In an analysis of 130 patients with hematologic malignancies 
undergoing unrelated donor SCT, those with KIR-ligand incompatibility had higher 
probabilities of overall survival and disease free survival and lower rates of transplant-
related mortality and relapse.50 Moreover, none of the 13 patients with myeloid 
malignancies who received SCT from a KIR-ligand incompatible donor relapsed; all are 
alive and disease free after SCT. In another study of 51 patients at the St. Jude Children’s 
Research Hospital with direct measurement of the donor KIR repertoire, patients with a 
KIR matched donor had a 4-fold higher risk of relapse than those with a KIR mismatched 
donor.46,53 In contrast to KIR mismatch, KIR-ligand mismatch was not a significant factor 

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for the prediction of relapse. The absence of beneficial effects of KIR-ligand mismatch was 
observed in many studies.54-57 Taken together, these data underscore the importance of 
direct assessment of donor KIR repertoire rather than donor KIR-ligands. 
 
The anti-leukemic effect of NK cells is not limited to HLA-non-identical SCT; this effect 
has also been demonstrated for patients undergoing SCT from HLA-identical sibling 
donors who, by definition, were always KIR-ligand compatible.51 Using donor KIR 
genotyping, this study demonstrated that KIR mismatch was an independent predictor of 
relapse among patients with AML and myelodysplastic syndrome (MDS).51  
 
In addition to the anti-leukemia effects, NK cells are capable of reducing the incidence of 
graft rejection and graft versus host disease (GVHD) in animal models; the former is in 
part based on the mechanism of donor NK cells against patient’s T cells, and the latter on 
donor NK cells against patient’s antigen-presenting cells.52 Taken together, adoptive 
transfer of NK cells from the hematopoietic stem cell donor may have multiple advantages, 
including reduction in relapse, graft rejection, GVHD, and viral infections.58 
 
Donor lymphocyte infusion (DLI) containing all types of lymphocytes (B, T, and NK cells) 
has been commonly used after SCT for the prevention or treatment of relapse, graft 
rejection, and viral infections. However, the potential clinical benefits of DLI are often 
outweighed by the risk of T cell-mediated GVHD. This is particularly problematic in 
haploidentical transplants, in which as few as 3 × 104 T cells may cause fatal GVHD.59 
Thus, we sought to develop a novel clinical scale isolation method to obtain highly purified 
NK cells for clinical use. Using the St. Jude NKCELL protocol, we performed 12 
consecutive purification of NK cells from 12 normal volunteers.58 The system uses a two-
step procedure. First, mononuclear cells obtained by leukapheresis are depleted of T cells by 
CD3+ cell depletion using the CliniMACS. Second, the CD3-depleted product is enriched 
for CD56+ cells using the CliniMACS.  The final products contained a median of 1.6 x 108 
mononuclear cells and 91% CD3-CD56+ cells.58 In addition, the final products had minimal 
contamination with T cells (at or below the lower limit of detection in 10 of the 12 products) 
or B cells (median 0.2%).58 The median recovery of 160 million NK cells can provide more 
than 2 million NK cells per kg body weight for an adult of average size or greater than 10 
million NK cells per kg body weight for a pediatric patient younger than 3 years. The 
number of T cells infused will be fewer than 1×103/kg, which is more than 10-fold below 
the threshold number (3-5x104/kg) for the development of GVHD.59,60 We also found that 
the expression of KIRs, adhesion molecules, intracellular cytokines, perforin, and granzyme 
B in NK cells was not significantly different before and after cell purification.61 Extensive 
proliferative capacity and potent antitumor activity of the NK cells were demonstrated by 
using an immunodeficient mouse model.61 In addition, GVHD developed in all mice 
transplanted with unpurified mononuclear cells, but in none of the 10 mice transplanted with 
purified NK cells.61 
 
Based on these preclinical data, we initiated a clinical study of HLA-haploidentical NK cell 
transplantation for infants with leukemia who had received stem cell transplantation from 
the same donor (Protocol Mnemonic: INF-T2, FDA IDE 11533). Nine patients have been 
enrolled thus far. All the NK-cell grafts contained less than 5x104/kg T cells and no patient 
developed GVHD. The long-term efficacy of this treatment is not yet known because of the 
small number of patients with short follow-up. 

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Recently, Miller et al62 demonstrated the successful transfer and expansion of 
haploidentical NK cells in the non-SCT setting with minimal toxicity and no GVHD. In 
this study, 19 adult patients with high risk AML (primary refractory disease, relapsed 
disease not in remission, secondary AML, or relapsed disease after SCT) received 
cyclophosphamide (60 mg/kg x 1 or 2 doses), fludarabine (25 mg/m2 daily x 5 doses), IL-2 
(10 million units per dose x 6 to 9 doses), and an infusion of 2 x 107 CD3-depleted NK cell 
product (NK cells enriched to approximately 40%). Eight of 15 AML patients showed at 
least 1% engraftment at day 7 or later after the infusion. In addition, 5 patients achieved 
CR, including 3 of 4 with KIR-ligand mismatched donor. Interestingly, 2 patients with 
refractory AML also achieved CR despite receiving NK cells from a KIR-ligand 
compatible donor. These data suggest that factors other than KIR are important 
determinants of anti-leukemic effects and patients with KIR compatible donor may also 
benefit from NK cell transplantation. 
 
Based on these results, we undertook a pilot study of haplo-identical NK cell 
transplantation for patients with AML (Protocol Mnemonic: NKAML, FDA IDE 11533). 
As of December 1, 2007, we have enrolled 11 patients on the NKAML protocol, including 
8 who were in CR. The median NK cell dose was 27 x 106/kg (range, 5 to 80 x 106/kg) and 
all patients showed evidence of engraftment (median 2% at day 2 and 6% at day 7). The 
procedure has been tolerated well, with only one SAE to date (prolonged pancytopenia 
without infection). All 8 patients who were in CR at the time of enrollment remain in CR. 
 
2.1.6 Rationale for hematopoietic stem cell transplantation (SCT) 
 
Studies performed more than 20 years ago demonstrated that allogeneic stem cell 
transplantation (SCT) is a feasible and effective alternative to chemotherapy as post 
remission treatment for AML.63-65 Many subsequent studies have demonstrated that 
relapse-free survival rates for AML patients undergoing matched related donor SCT are 
better than those of patients who receive only chemotherapy.66-70 Some studies have also 
shown an overall survival advantage associated with SCT; however, others have shown no 
such advantage, primarily because of transplant-related mortality.71,72 On the MRC AML10 
trial, SCT did reduce the risk of relapse, but neither autologous or allogeneic SCT produced 
an overall survival advantage.4 Similarly, the CCG-213 trial failed to show a superiority of 
SCT over chemotherapy when results were analyzed by the intent to treat method.73,74 In 
contrast, the largest and most recent randomized comparison of SCT versus chemotherapy, 
the CCG-2891 trial, demonstrated that for patients who achieved CR, survival in the 
allogeneic SCT group was significantly superior to that in the autologous SCT and 
chemotherapy arms.3 Because of the controversies regarding the role of SCT in AML,75,76 
this protocol will include SCT only for high-risk patients. 
 
2.1.7 Rationale for the use of sorafenib 
 
Aberrant activation of receptor tyrosine kinases and effectors of activated signal 
transduction pathways involved in cellular differentiation, proliferation and survival 
represent therapeutic targets in AML.77 Oncogenic mutations in N-RAS and K-RAS, leading 
to activation of the Ras/Raf/mitogen-activated protein (MAP)/extracellular signal-regulated 
kinase (ERK) kinase/ERK mitogen-activated protein kinase signal transduction pathway, 
are observed in approximately 17% of children with AML.78,79 Activating mutations in 

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FLT3 and c-kit are found in about 20% and 7% of pediatric patients with AML, 
respectively, and confer a worse prognosis.80-82 Inhibitors of FLT3 are currently being 
evaluated in patients with relapsed or refractory AML containing FLT3 mutations.83 
However, therapy with highly selective tyrosine kinase inhibitors may be hampered by the 
activation of multiple signal transduction pathways in leukemia cells and redundancy and 
between the pathways.77  
 
Sorafenib (BAY 43-9006) is an oral bisarylurea derivative that was initially developed as a 
potent inhibitor of Raf-1 kinase (IC50 = 6 nmol/L) but was later found to inhibit wild-type 
and mutant B-Raf (IC50 = 22 and 38 nmol/L, respectively) in vitro as well.84-86 The Raf 
proteins are an integral component of the Ras/Raf/MAP/ERK signaling cascade with a 
well-established role in cellular proliferation and survival.87 Sorafenib shows broad activity 
against various tumor cell lines in vitro and in xenograft models. In addition to its effect on 
Raf, sorafenib also potently inhibits receptor tyrosine kinases, including vascular 
endothelial growth factor receptor (VEGFR)-1/2/3, murine platelet-derived growth factor 
receptor (PDGFR)-ß, c-kit, and FLT3 (IC50 = 33 nmol/L).84-86 Thus, the anticancer activity 
of sorafenib is likely due to its effects on multiple targets.  
 
Sorafenib recently received Food and Drug Administration (FDA) approval for the 
treatment of adult renal cell carcinoma and hepatocellular carcinoma based on results from 
a phase III studies.88 The approved adult dose is 400 mg orally BID. Diarrhea, rash, fatigue, 
and hand–foot skin reactions were the most common adverse events associated with 
sorafenib. Hypertension and cardiac ischemia were rare serious adverse events that were 
more common in patients receiving sorafenib than in those receiving placebo.  
 
The Ras/Raf/Mek/ERK pathway has been shown to be potentially involved in AML 
pathophysiology.89 In a recent study, markedly elevated phospho-ERK levels were found in 
83.3% of the AML samples and treatment with a MEK inhibitor resulted in significantly 
decreased phospho-ERK levels.90,91 Sorafenib also has potent activity against kinases 
reported to potentially be contributing to AML physiopathology. Importantly, sorafenib 
inhibited FLT3-ITD kinase activity and cell signaling and demonstrated potent antitumor 
efficacy in a FLT3-ITD leukemia tumor xenograft model.92 Sorafenib was nearly 10 times 
more potent at inhibiting phosphorylation of FLT3, Stat5 and ERK1/2 in FLT3-ITD 293 
cells than in FLT3-WT 293 cells. Sorafenib also potently inhibited the enzymatic activity 
of FLT3 activation loop mutant D835Y, suggesting that this compound may have activity 
against multiple mutant forms of FLT3.  
 
Evidence of single-agent activity against AML with FLT3-ITD has been recently 
reported.93 In this study, sorafenib effectively induced growth arrest and apoptosis in Ba/F3 
cells containing FLT3-ITD and prolonged survival in a mouse model of FLT3-ITD-
positive AML. In a phase I clinical trial in patients with refractory or relapsed AML, 
sorafenib reduced the blast percentages in blood and bone marrow of patients with FLT3-
ITD. In this study, sorafenib was tolerated well, with no dose-limiting toxicities reported.  
 
The Children’s Oncology Group is currently conducting a Phase I study of sorafenib in 
children with refractory solid tumors or leukemias (ADVL0413). This trial was recently 
amended to exclude patients with abnormal liver function tests and to provide guidelines 
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follow the guidelines of the COG trial, as described below (section 4) and will use the 
current dose level of the COG trial of 200 mg/m2/dose BID (adult equivalent dose of  360 
mg BID for a BSA of 1.8 m2).  
 
2.1.8 Rationale for the use of valproic acid 
 
Beginning with amendment 9.0, vorinostat will be 
substituted for valproic acid 
 
In contrast to Down syndrome (DS) children with acute 
megakaryocytic leukemia (M7), who have EFS rates of 
80-100%, non-DS children with M7 AML have much 
inferior EFS rates.94-97 In a previous report from St. Jude, 
the EFS rate of de novo non-DS M7 AML patients treated 
with chemotherapy alone was 0%, while the EFS rate on 
the CCG-2861/2891 studies was 21%,  significantly 
poorer than that for the non-DS AML group overall.94,95 In 
the AML02 trial, approximately 10% of patients were 
classified as M7, and their 3-year EFS estimate was 44% 
± 12%. Of the 18 M7 cases without the t(1;22), only 6 are 
alive, all after SCT. In this trial, no M7 patients without the t(1;22) have been cured by 
chemotherapy alone.  
 
Histone deacetylase (HDAC) inhibitors, including the anti-epileptic agent, valproic acid, 
are currently being evaluated in the treatment of a variety of cancers.98-102 The activity of 
valproic acid alone and in combination with chemotherapy agents has been evaluated in 
vitro against the non-DS M7 cell line, Meg-01 
(Jeff Taub, unpublished observations). Meg-01 
cells incubated with various concentration of 
valproic acid for 48h were harvested and 
subjected to flow cytometry analysis to determine 
viable cells and Western blotting to determine 
apoptosis. Valproic acid killed Meg-01 cells in a 
dose-dependent manner (Figure 1, panel A) by 
promoting the cells undergoing apoptosis as 
indicated by cleavage of caspases 8, 9, and 3 and PARP on the Western blots (Figure 1, 
panel B). 
 
When valproic acid was incubated simultaneously with cytarabine, there was synergistic 
cytotoxicity against Meg-01 cells as determined by standard isobologram. Meg-01 cells 
with a dose range of valproic acid with up to >30-fold greater sensitivity to ara-C in the 
presence of 1.5 mM valproic acid than in the absence of valproic acid (Figure 2, panel A). 
Synergistic cytotoxicity with daunorubicin was also observed at higher valproic acid doses 
(1.5 mM) (Figure 2, panel B). 
 
The incorporation of valproic acid as a new agent in the treatment of M7 AML has several 
potential advantages including: i) widespread use in the pediatric population with known 
toxicity, ii) measurement of therapeutic levels are readily available in the clinical setting, 
A
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Figure 2. VPA sensitizes Meg-01 cells to ara-C and 
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and iii) clinically achievable trough levels (used in the treatment of pediatric patients with 
epilepsy103 correspond to in vitro concentrations demonstrating anti-leukemia activity 
either as a single agent or in combination with ara-C. An oral rapid loading dose schedule 
of valproic acid after 3 days achieved a median trough level of 101 mg/L (equivalent to 
~0.7 mM) in 35 pediatric patients.103 Two patients with serum levels of 140 and 146 mg/L, 
developed elevated ammonia levels with nausea and fatigue, which resolved in both 
patients when the valproic acid dose was reduced. 
 
Amendment 9.0 – vorinostat HDAC inhibitor 
 
Although valproic acid has modest activity as a histone deacetylase inhibitor, more potent 
agents are now available. Vorinostat (suberoylanilide hydroxamic acid) inhibits human 
class 1 and 2 histone deacetylases, is orally bioavailable, and has limited single-agent 
activity in AML (Garcia-Manero et al, Blood 111:1060-1066, 2008). In a Phase II trial that 
included 75 adult patients with AML or MDS, vorinostat (500 mg po TID) was given for 
three days prior to idarubicin (12 mg/m2/day x 3 days) and cytarabine (1.5 g/m2/day x 3-4 
days) (Garcia-Manero et al, J Clin Oncol 30:2204-2210, 2012). This regimen was well 
tolerated and resulted in an overall response rate of 85%. Vorinostat has also been safely 
administered for seven days prior to the combination of cytarabine and etoposide (Gojo et 
al, Clin Cancer Res 19:1838-1851, 2013). We have recently given vorinostat (100 
mg/m2/dose po TID x 3 days) prior to the combination of idarubicin and cytarabine in 
pediatric patients with relapsed AML (J Rubnitz, H Inaba, personal observations) without 
excessive toxicity. 
 
Because vorinostat is a more potent HDAC inhibitor than valproic acid and can be safely 
combined with chemotherapy, vorinostat will be given prior to Induction II to high-risk 
patients with wild-type FLT3 as of amendment 9.0.  Collaborating institutions may elect to 
opt out of treatment with vorinostat, and give standard Induction therapy with LD-ADE 
(without vorinostat). 
 
2.1.9 Rationale for risk group assignment 
 
As discussed above, several studies have demonstrated that non-Down syndrome patients 
with megakaryoblastic leukemia have significantly worse outcomes than patients with 
other subtypes of AML.94-96 The EFS estimates for patients with megakaryoblastic 
leukemia treated on the CCG 2891 trial or on St. Jude trials were only 22% and 14%, 
respectively.94,95 In the St. Jude study94 and in a report from the European Group for Blood 
and Marrow Transplantation,104 patients who underwent SCT in 1st CR had a better 
outcome than those who received chemotherapy. However, a study by French investigators 
suggested that children with megakaryoblastic leukemia and the t(1;22), but without Down 
syndrome, had a better outcome than similar children without this karyotypic abnormality, 
indicating that this subgroup may not need transplantation.96 Based on these results, 
patients with M7 AML will be candidates for SCT only if their leukemic blasts do not carry 
the t(1;22).  
 
Conventional cytogenetic studies have demonstrated that the karyotype of leukemic blasts 
is one of the best predictors of outcome.105,106 An analysis of over 1600 patients enrolled on 
the MRC AML 10 trial revealed that the t(8;21) and inv(16) were each associated with a 

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favorable outcome (5-year OS estimates 69% and 61%, respectively), whereas a complex 
karyotype, -5, del(5q), monosomy 7 (-7), and abnormalities of 3q predicted a poor 
outcome.105 Based on these observations, the MRC investigators proposed a 
cytogenetically-based risk classification system that is used by many cooperative groups 
today.105 Among the 340 patients in the MRC study who were less than 15 years old, those 
with a favorable karyotype had a 3-year survival estimate of 78%, compared to 55% for the 
intermediate risk group and 42% for the adverse risk group. The poor results associated 
with -7 have been confirmed by a large international collaborative study.107 This study, 
which included 172 patients with -7 (with or without other abnormalities) and 86 patients 
with del(7q) (also with or without other changes), demonstrated lower CR rates and worse 
outcome for patients with -7 compared to those with del(7q). The outcome for patients who 
had del(7q) and a favorable genetic alteration had a good outcome, suggesting that the 
presence of the del(7q) did not change the impact of the favorable feature. By contrast, 
patients with -7 and inv(3), -5/del(5q), or +21, had a dismal outcome (5-year OS 5%) that 
was not impacted by SCT.107 On AML08, patients with core-binding factor (CBF) 
leukemia [t(8;21), inv(16), or t(16;16)] will be considered low risk, whereas patients whose 
blasts contain -5 or -7 will be candidates for SCT. 
 
Other important cytogenetic abnormalities include rearrangements of the MLL gene, which 
are seen in up to 20% of cases of AML.108-110 Although some studies suggest that the 
t(9;11) confers a favorable outcome,109 most investigators consider patients whose 
leukemic cells contain 11q23 abnormalities to be at intermediate risk of relapse.105  
 
During the past 10 years, molecular studies have demonstrated heterogeneity within 
cytogenetically defined subgroups of AML and have also identified new, prognostically 
important subgroups. Mutations of c-kit, ras, and FLT3 have been detected in childhood 
and adult AML cases; c-kit mutations may be particularly important in cases of CBF 
leukemia.82,111-114 Several studies of adult patients with t(8;21) demonstrated that patients 
with c-kit codon 816 mutations had significantly higher relapse rates and worse outcome 
than patients with t(8;21) and wild type c-kit.111-113 Mutations of c-kit also appear to confer 
worse outcome among patients with inv(16) in some, but not all, studies.115 c-kit mutations 
have been detected in 3% to 11% of pediatric AML cases, but their prognostic impact is 
uncertain.114,116 In one study, 37% of CBF cases had c-kit mutations, but there was no 
difference in outcome between cases with and without mutations.114 In contrast, 
investigators from the Japanese Childhood AML Cooperative Study Group found c-kit 
mutations in 8 of 46 patients with t(8;21) and demonstrated that these mutations were 
associated with significantly worse relapse rates, DFS, and OS.82 
 
The impact of FLT3 mutations in childhood and adult AML has been established by dozens 
of studies. In one of the first studies reported, the estimated 5-year OS rate was only 14% 
for adult patients with internal tandem duplications (ITD) of FLT3, whose presence  was 
the strongest prognostic factor in multivariate analysis.117 A subsequent study of 854 
patients treated in the MRC AML trials demonstrated that a FLT3-ITD was present in 27% 
of cases and was associated with an increased relapse risk and worse probabilities of DFS, 
EFS, and OS.118 Other reports have confirmed the presence of FLT3-ITD in 20% to 30% of 
adult AML cases, but some studies suggest that the negative impact of this alteration on 
survival may depend on the absence of the wild type allele or the ratio of the mutant to the 
wild type allele.119-121  

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Studies of childhood AML indicate that FLT3-ITD occurs in only 10%-15% of cases, but is 
still associated with a poor outcome.80,81,122,123 An analysis of 91 pediatric patients with 
AML treated in CCG trials demonstrated an 8-year EFS estimate of only 7% for patients 
with FLT3-ITD, while a study of 234 patients treated on Dutch AML protocols showed a 5-
year EFS of only 29%.81,122 In both studies, multivariate analysis demonstrated that FLT3-
ITD was the strongest predictor of relapse. A more recent study of 630 patients treated on 
contemporary CCG trials confirmed the poor outcome of patients with FLT3-ITD and 
demonstrated that survival decreased with increasing allelic ratios (AR) of FLT-ITD to 
FLT3 wild type.80 Patients with ITD AR greater than 0.4 had a significantly worse 
progression free survival than those with lower AR (16% versus 72%). Furthermore, 
investigators from the CCG compared the outcome of patients in whom the FLT3-ITD was 
present in CD34+/CD33- precursors to patients in whom the mutated gene was present 
only in the more mature CD34+/CD33+ progenitors.124 Outcome was dramatically worse 
in patients in whom the mutation was present in the less mature precursors, confirming the 
heterogeneity within FLT3-ITD-positive patients and suggesting that only a subset of these 
patients have a poor prognosis.  
 
Other molecular alterations implicated as prognostic factors in AML include expression of 
ATP-binding cassette transporters,125-127 CEBPA mutations,128,129 DCC expression,130 
VEGF secretion,131 expression of apoptosis-related genes,132-134 expression of BAALC,135 
expression of ERG,136,137, NPM1 mutations,138-140 partial tandem duplications (PTD) of the 
MLL gene,141,142 and global gene expression patterns.143-147 However, data on the 
importance of these factors in childhood AML is not yet mature enough to allow us to use 
these factors in current risk group assignment.  
 
In adults with AML, several studies have demonstrated that mutations of the CEBPA gene 
are associated with a favorable prognosis, especially among patients with normal 
karyotypes and wild-type FLT3.128,129,148,149 Renneville et al148 detected CEBPA mutations 
in 8% (53/638) of cases of adult AML and demonstrated that patients with mutations had 
better DFS (50% vs. 24%, p=0.03) and OS (47% vs. 31%, p=0.11) than patients without 
mutations. However, abnormal karyotype or FLT3-ITD predicted worse outcome among 
patients with CEBPA mutations. Outcome was particularly good for patients with normal 
karyotype, CEBPA mutations, and wild type FLT3 (64% OS and 62% DFS).  
 
Ho et al150 recently reported the results of the first evaluation of the prognostic significance 
of CEBPA mutations in childhood AML. Among 847 AML cases treated on CCG/COG 
protocols, 4.5% (38 of 847) had CEBPA mutations, including 17% of cases with normal 
karyotypes. Patients with CEBPA mutations had better EFS (70% vs. 38%, p=0.015) and 
lower cumulative incidence of relapse (CIR, 13% vs. 44%, p=0.007) than patients without 
mutations. FLT3-ITDs were detected in only 2 cases with CEBPA mutations. In a 
multivariate analysis, CEBPA mutation was an independent favorable prognostic factor for 
EFS (HR=0.36, p=0.024), In addition, the outcome of patients with CEBPA was similar to 
that of patients with CBF leukemia (EFS, 78% vs. 59%; OS, 78% vs. 74%; CIR, 7% vs. 
28%). 
 
Like CEBPA, mutations of the NPM1 gene are associated with a favorable outcome among 
adult cases of AML with normal karyotype and wild-type FLT3.138,139,149,151,152 Brown et 
al140 demonstrated that NPM1 mutations were present in 8% (23/295) of children with 

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AML treated on the POG 9421 study and were associated with FLT3-ITD and normal 
karyotype. In children lacking FLT3-ITD, there was a favorable impact of NPM1 mutations 
on outcome (EFS, 69% vs. 35%, p=0.51). The relatively small group of children with 
normal karyotype, wild-type FLT3, and NPM1 mutation appear to have an outcome similar 
to that of children with CBF leukemia. Investigators from the BFM/DCOG study groups 
also demonstrated that NPM1 mutations occur in about 8% of children with AML (25/297) 
and in 20% (20/100) of those with normal karyotypes.153 Among patients with normal 
karyotypes, NPM1 mutations were an independent prognostic factor for EFS (80% vs. 
39%, p=0.01) and OS (85% vs. 60%, p=0.06). 
 
Because the favorable impact of CEBPA and NPM1 mutations previously demonstrated in 
adult AML studies have now been confirmed, at least retrospectively, in pediatric AML 
trials, all patients enrolled on AML08 will be screened for these genetic abnormalities. 
Patients who have normal karyotypes and mutations of CEBPA or NPM1 and wild type 
FLT3 will not be eligible for stem cell transplantation, even if a matched sibling donor is 
available. 
 
2.1.10 Rationale for the use of minimal residual disease studies 
 
Many studies have demonstrated the prognostic importance of early response to therapy as 
assessed by morphologic examination of the bone marrow154 or by minimal residual 
disease (MRD) studies using flow cytometric detection of aberrant 
immunophenotypes.30,155-158 In all of these studies, patients who had slow clearance of 
leukemic blasts had higher relapse rates and worse outcome than patients who had rapid 
clearance of MRD. In our ongoing AML02 trial, 172 of 182 (94.5%) patients had AML 
cells with aberrant phenotypes at diagnosis (sensitivity was 10-3 in 90 cases and 10-4 in 82 
cases). The success rate of MRD assays in this multicenter study was high, with 1296 of 
1313 (98.7%) of samples received having adequate viability and cellularity for informative 
analysis. At day 22, 69 of 167 (41.3%) had ≥ 0.1% MRD. MRD positivity at day 22 was 
significantly associated with FAB type, cytogenetics, and FLT3 status. There was no 
significant difference in the rates of MRD positivity between samples obtained from 
patients treated at St Jude and those treated at other institutions, further supporting the 
adequacy of the MRD analysis performed on shipped material. The three-year survivals 
according to day 22 MRD level were as follows: MRD negative, 79% ± 9%, MRD 0.1% to 
1%, 73% ± 22%, and MRD ≥ 1%, 43% ± 12% (p=0.003). In AML08, we will continue 
using MRD as determined by flow cytometry for risk assignment. In addition, we will 
explore whether day 8 MRD in peripheral blood is a practical approach that might allow 
early therapeutic intervention in future studies. 
 
2.1.11 Rationale for the use of prophylactic antimicrobials 
 
Patients with AML who receive intensive chemotherapy are at high risk for bacterial sepsis 
and are especially susceptible to viridans streptococcal infections (VSI).159,160 We recently 
reviewed the charts of 66 patients who were treated at St. Jude on the AML02 protocol 
from October 2002 to June 2006. Overall, patients who received any prophylactic antibiotic 
regimen had an 86% (95% CI: 66-95%) reduction in VSI (P<0.001) and a 76% (95% CI: 
62-85%) reduction in all bacterial infections (P<0.001), compared to those who did not 
receive prophylactic antibiotics. Oral cephalosporins were ineffective at preventing overall 

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bacterial infections (P=0.97) or VSI (P=0.70). Intravenous cefepime completely prevented 
VSI. Furthermore, there was an 84% (95% CI: 52-94%) reduction in all bacterial infections 
(P<0.001). However, because we observed the emergence of life-threatening resistant 
gram-negative infections in two patients receiving cefepime, we instituted the prophylactic 
use of intravenous vancomycin with oral ciprofloxacin. The use of a vancomycin regimen 
resulted in an 84% (95% CI: 66-92%) decrease in total bacterial infections (P<0.001) and a 
97% (95 CI: 78-100%) decrease in VSI (P<0.001). Importantly, fungal infection rates were 
similar in patients who received prophylactic antibiotics compared to those who did not 
(0.9 vs. 1.0 infection per 1000 patient days). Patients who received prophylactic antibiotics 
had shorter lengths of hospital stay (LOS), with a median (range) of 7 days (0-39) vs. 15 
days (0-56) for each course of chemotherapy. Patients who received a vancomycin regimen 
had a 46% (95% CI: 28-59%) reduction in LOS (P<0.001) and those who received 
intravenous cefepime a 31% (95% CI: 9-47%) reduction (P=0.008) compared to patients 
who did not receive prophylactic antibiotics. Overall, there was only one death from 
septicemia; this occurred in a patient who was not receiving prophylactic antibiotics. Based 
on these results, prophylactic antibiotics will be used for all patients on AML08. 
 
2.1.12 CNS therapy 
 
On AML02, treatment of clinical and subclinical CNS leukemia initially consisted of 
intrathecal cytarabine, which was used successfully on the POG 9421 and CCG 2961 
protocols. However, among the first 26 patients enrolled on AML02, 3 suffered relapses. 
The protocol was then amended to include triple intrathecal therapy with methotrexate, 
hydrocortisone, and cytarabine (MHA). Among the subsequent 170 patients enrolled, only 
1 suffered an isolated CNS relapse. Thus, we will continue to use IT MHA on AML08. 
 
2.1.13 Treatment of patients less than 10 kg 
 
To reduce the potential for cardiac toxicity, dosing of daunorubicin and mitoxantrone for 
patients less than 10 kg will be based on weight rather than body surface area. Similarly, 
for infants < 1 month of age, or for infants < 3 months of age who were born significantly 
prematurely defined as < 36 weeks gestation, dosing of cytarabine, clofarabine, 
asparaginase, and etoposide, will be based on weight. 
 
2.2 
Background and rationale for correlative and biologic studies 
 
2.2.1 Minimal residual disease studies 
 
MRD studies provide powerful prognostic information, but it is likely that their 
informative power could be increased by improving their sensitivity. Indeed, current 
methods can detect 1 leukemic cell in 10,000 normal cells in approximately half of patients 
with AML; in the remaining patients, the maximum sensitivity is 1 in 1000. We postulate 
that sensitivity could be improved by the availability of additional markers. To this end, we 
performed comparative studies of gene expression of AML cells and normal CD34+ 
CD13+ cells to identify new markers of AML to be used for MRD studies. These studies 
identified several promising candidates. A second approach to improve the sensitivity of 
MRD studies by flow cytometry is to analyze more parameters simultaneously. In AML02, 
we used 4-color flow cytometry but a 3-laser flow cytometer capable of visualizing 9 or 

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more fluorochromes is now available in the laboratory. In AML08 we plan to compare the 
MRD results obtained with these new markers and methodologies to the standard marker 
combinations used in AML02. Moreover, we intend to explore the notion that AML cells 
with immunophenotypic features of immaturity (e.g., CD34+ CD38- cells, cells expressing 
ALDH) have increased drug resistance as compared to more mature AML cells. Therefore, 
we will measure the prevalence of these AML subsets among AML cells before and during 
treatment, a possibility that has been opened by the availability of 9-plus color instruments. 
Finally, we will use the same approach and commercially available anti-phosphoprotein 
antibodies to determine the effect of sorafenib on cell signaling pathways of AML cells.   
 
2.2.2 NK cell receptor studies 
 
The normal physiological roles of NK cells are to control infection and prevent cancer. In 
fact, NK cells are the only immune cells that have been shown by prospective cohort study 
in healthy persons to have immunosurveillance capability against human cancer.161 In a 
prospective study of more than 3000 healthy volunteers who were followed for 11 years, 
the risk of cancer was associated with decreased NK cell cytotoxicity against K562 
leukemia cells, decreased expression of the NK receptor NKRP1, and decreased production 
of cytokines such as TNFα and IFNγ in NK cells.  
 
Three recent studies of adult leukemia have demonstrated a direct link between leukemia 
and NK cell. In one study of patients with acute myeloid leukemia, the majority (16 of 18) 
of blood samples showed defective expression and function of NK cell – triggering 
receptors (NCRs) (dull).162 The expression of NK cell surface receptors was low, and the 
cytolytic activity against autologous leukemia cells, autologous B lymphoblasts, and NK 
cell–sensitive cell lines was weak. The abnormal NCR (dull) phenotype was confirmed in 
another study of 71 patients with acute myeloid leukemia and was found to be present in 
various morphologic and genotypic subtypes of leukemia.163 In the third study of 25 AML 
and 14 ALL cases, the expression of HLA class I was frequently downregulated and that of 
the NK cell receptor ligands PVR and Nectin-2 were upregulated.164 Together, these results 
suggest that NK receptor – ligand interaction may be crucial for the development of 
leukemia.  
 
The normalization of NK cell receptor expression may be of prognostic value as NK cells 
may be important for the control of leukemia relapse and infection. These roles of NK cells 
have been shown by killer cell inhibitory receptor (KIR)-mismatched allogeneic stem cell 
transplantation.46 Longitudinal study of adult AML patients showed that the NCR (dull) 
phenotype acquired during leukemia development was reversible in patients achieving 
complete remission after induction chemotherapy.163 Reversibility of the NCR (dull) 
phenotype after complete remission suggested that leukemia cells might be involved in 
NCR down-regulation. Alternatively, the recovery of normal NCRs may allow the recovery 
of normal NK cell function that contributes to the acquisition of remission status. 
Interestingly, a correlation was found between the NCR (dull) phenotype and poor survival 
in AML patients after chemotherapy,163 suggesting that NK-deficient activation caused by 
NCR down-regulation could play a role in treatment outcome. Therefore, we will study NK 
cell receptor expression at diagnosis and before reinduction to elucidate the role of NK cell 
in the development and treatment response of childhood ALL. 
 

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2.2.3 Biological prognostic factors 
 
To assess the prognostic importance of specific genetic features, all patients in this trial 
will be studied by conventional cytogenetics and screened at diagnosis for the presence of 
the AML1-ETO, MLL-AF9 and MYH11-CBFβ  fusion transcripts. In addition, cases with 
11q23 rearrangements will be screened for MLL-AF4, MLL-AF6, MLL-AF10, MLL-ELL, 
and MLL-ENL (as indicated), and cases with M7 morphology will be screened for OTT-
MAL. Cases without one of the known genetic abnormalities will be analyzed by 
fluorescence in situ hybridization (FISH) for presence of MLL rearrangements and by RT-
PCR for presence of cryptic NUP98-NSD1. FLT3-ITD and FLT3 point mutations will be 
detected by PCR and direct sequencing. Wild-type to mutant ratios will be determined in 
order to assess whether it adds prognostic information as previously demonstrated in adult 
de novo AML.121 Leukemic cells that express fusion transcripts will also undergo RT-PCR 
during and after therapy. In addition to FLT3-ITD, other genes known to be mutated and 
associated with outcome as well as novel candidate genes will/may be sequenced after 
whole genome amplification of genomic DNA in order to assess the frequency of mutation 
and the association of genotype with outcome.  
 
2.2.4 High-resolution single nucleotide polymorphism (SNP) array and gene expression   
 
Currently available risk stratification features, such as leukocyte count, morphology, 
karyotype, MRD, standard genetic features, and gene expression profiles alone probably do 
not correctly molecularly and prognostically classify all AML patients. Previous studies 
have shown that gene expression profiling using DNA microarrays is a powerful approach 
to molecularly classify acute leukemias and to distinguish subtypes of AML.145,165 
However, a predictive signature for outcome in pediatric AML still remains to be defined. 
We will continue our effort to identify signatures predicting outcome based on gene 
expression profiles which will be obtained for all cases with sufficient material.  
 
At St. Jude, a high resolution study of DNA copy number abnormalities and loss-of-
heterozygosity in acute lymphoblastic leukemia using SNP microarrays identified a high 
frequency of recurring deletions in ALL, most notably involving genes regulating B-
lymphoid development in B-progenitor ALL.166 Presence of some of the deletions was 
found to be associated with outcome. A pioneer study of 110 cases of pediatric AML from 
different St. Jude AML protocols showed that high-resolution SNP arrays identified 
numerous cryptic copy number abnormalities and copy-neutral loss of heterozygosity 
(LOH) or uniparental disomy in pediatric de novo AML. Because of the small number of 
samples in this pioneer cohort, recurring lesions in 5-10% of the cases may have been 
missed and association with outcome could not be studied. Similar frequencies of cryptic 
copy number abnormalities were observed in adult MDS and t-AML samples (Gondek et 
al. Blood 2008;111:epub) using SNP array as complementary approach to conventional 
cytogenetic analysis. We propose to obtain gene expression profiles and high-resolution 
SNP array data for patients enrolled in AML08 (Affymetrix 500K 6.0). Two vials with 2x 
107 cells should be reposited for this purpose for each patient. We will explore the 
prognostic value of integrated analysis of somatic copy number abnormalities, copy-neutral 
LOH, gene expression profiles, sequence mutation and MRD. Profiles will be analyzed to 
determine whether they have a relationship with any morphologic, genotypic, 
immunophenotypic or clinical characteristic.  

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Most studies assess leukemic blasts in bulk by genome-wide expression or SNP array 
profiling. There is evidence for heterogeneity within each sample based on cytogenetic and 
FISH data. Some of the lesions and aberrant gene expression profiles might be only present 
in a subset of the blasts. For example, we identified cases in our pioneer AML cohort for 
which gains of one copy of a genetic locus was identified by SNP array analysis of bulk 
cells while FISH showed that a high level gain was only present in <30% of the cells. In a 
comparative study of copy number abnormalities at relapse and diagnosis in 17 pediatric 
AML cases, about 30% of cases had novel lesions at relapse which had not been detected at 
diagnosis (ASH abstract #984). These lesions might be present but not detectable at 
diagnosis because they might occur only in a subset of cells. Some of these subpopulations 
may represent leukemia-initiating or stem cells within each tumor bulk which maintain 
propagation of the disease. Studying cells with CD34+CD38-, a population shown to be 
enriched for leukemia-initiating cells, may provide information which cannot be obtained 
from the bulk of cells. A recent study revealed that within single tumor samples 
CD34+CD38- had a different gene expression profile than CD34+CD38+.167 We propose 
to analyze gene expression, copy number and LOH within the subpopulations of single 
AML tumor samples to determine whether the genetic heterogeneity of the tumor 
contributes to their resistance to therapy and to the relapse of disease.  
 
To investigate why patients fail to respond to Induction I, we plan to assess whether 
residual blasts late in or after Induction I have characteristic genetic features or expression 
profiles, different to those at time of diagnosis. Comparison between blasts at diagnosis and 
residual blasts after Induction I for each case individually would allow determine whether 
the treatment selected for a genetic feature or profile. To identify lesions with predictive 
value, we would also compare the constellation of genetic lesions at diagnosis in cases with 
failure to respond to Induction I to cases that undergo a complete response. To address this 
question, we propose to sort blasts from samples obtained from blood at day 8 or from 
marrow at Day 22 and extract DNA and RNA from the blast population to analyze gene 
expression, copy number and LOH. 
 
2.2.5 Phospho-signaling network analysis 
 
Recently, a quantitative method to profile aberrant phospho-signaling networks has been 
established in primary AML leukemia cells. Phospho-signaling has been shown to provide 
prognostic information as a summary read-out which might be a more accurate classifier 
than any single genetic or phenotypic feature identified so far.168 Using this flow-based 
approach, the signaling response can be determined in different subpopulations by co-
staining with surface markers and also after in vitro exposure to inhibitors. We would like 
to explore within AML08 whether characterization of the phospho-signaling networks can 
be used to classify within known genetic subtypes and to predict response to therapy. 
 
Phospho-flow profiling will not be prospectively performed on all AML08 samples. 
Instead, all samples will be banked and selected samples will later be studied by Dr. 
Downing’s laboratory at St. Jude and by Dr. Lacayo’s laboratory at Stanford University. 
Dr. Lacayo’s laboratory proposes to interrogate signaling mechanisms in training and 
validation set of banked AML patient samples from Pediatric Oncology Group (POG) 
studies and from the present study. Maps of signaling profiles for groups and individual 
AML patients will be created using 100 available banked samples from the POG 9421 

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study and additional samples from AML08. Dr. Lacayo will test the hypothesis that 
patients with similar signaling profiles will have similar responses to therapy and that gene 
expression differences will correlate with specific patterns of signaling in AML. If a profile 
of signaling is significantly associated with clinical outcome, they will then validate this 
panel in real-time with a second (banked samples) and third set (prospectively collected) of 
additional AML patient specimens. Using bioinformatic techniques the network signaling 
can be inferred by studying key nodal points. The analysis of POG 9421 samples will 
include a matrix of 196 data points. From this data matrix we will identify 5-10 nodal 
points that can be studied using banked AML08 samples. This approach will reduce the 
required number of cells to 5-10x106 viable cells in DMSO, from 50 additional banked 
samples. Dr. Lacayo will initially study a total of 25 AML08 banked samples for a pilot 
study, with release of 25 additional samples after review of data generated.  
 
2.2.6 Pharmacokinetic studies 
 
Sorafenib pharmacokinetics - Note: sorafenib pharmacokinetic studies were completed 
with LOA #4, May 29, 2015 
 
After oral administration, maximum sorafenib plasma concentrations are reached between 
6 and 12 hours, and the terminal half-life ranges from 20 to 40 hours.169 Consequently, 
steady-state is reached after approximately 7 days with the achievement of concentrations 
4- to 6-fold (approximately 1-log) higher than those on day 1 with twice daily dosing. 
Sorafenib is metabolized primarily by cytochrome P-450 3A4 (CYP3A4) with involvement 
of UGT-1A9 and shows extensive binding in human plasma (>99.5%). Like most drugs 
that are metabolized by CYP3A4, sorafenib exhibits wide pharmacokinetic variability with 
a coefficient of variation for AUC ranging from 50-90%. Associations between sorafenib 
exposure and side effects and outcome remain to be performed. The pharmacokinetics of 
sorafenib have not been evaluated in children. Therefore, it will be important to understand 
the disposition of this agent in the setting of pediatric AML and associations with toxicity 
and efficacy. Furthermore, availability of this information will allow evaluation of the 
possible associations of common germline variants in genes encoding transporters and 
enzymes and sorafenib pharmacokinetics. 
 
L-carnitine analysis in urine 
 
L-carnitine (vitamin Bt) is an essential nutrient, particularly in infants, and is involved in the 
mitochondrial oxidation of long-chain fatty acids, it stimulates mitochondrial metabolism, 
decreases oxidative stress, and inhibits apoptosis by preventing ceramide formation. 
Carnitine elimination takes place in the kidney mainly by glomerular filtration and secretion 
at the proximal tubules, and may return to the systemic circulation via a process of drug 
reabsorption. Our recent studies have indicated that the solute carrier OCTN2 is a key 
regulator of this reabsorption and that the function of OCTN2 can be substantially 
diminished by a number of commonly used anticancer drugs, including epipodophyllotoxins 
and anthracyclines. The subsequent loss of l-carnitine in the urine is potentially an important 
cause of secondary l-carnitine deficiency, which phenomenon will likely contribute to a 
variety of treatment related side effects. 
 
Under amendment 4.0, this study will not be done. 

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Etoposide and daunorubicin pharmacokinetics 
 
Pharmacokinetic studies of etoposide and daunorubicin will be performed in patients with 
urinary collection for l-carnitine concentrations. Availability of this information will allow 
evaluation of the possible associations of urinary l-carnitine loss with the pharmacokinetic 
profiles of etoposide and daunorubicin.  
 
Under amendment 4.0, this study will not be done. 
 
2.2.7 Pharmacogenetic studies 
 
A growing body of research is highlighting the role that variations in genes encoding drug-
metabolizing enzymes and drug transporters play in explaining the variability seen in 
treatment-related outcomes of several important anticancer agents. Drug metabolizing 
enzymes and transporters are localized to the intestines, liver, and kidneys and contribute to 
systemic pharmacokinetic variability, but they are also localized in cancer cells and can 
contribute to intracellular pharmacokinetic variability and drug action. For example, in 
childhood AML, studies have shown that overexpression of cellular drug efflux 
transporters from the ABC family such as BCRP (ABCG2) and MRP3 (ABCC3) is 
associated with a poor prognosis.170 Cellular uptake carriers from the SLC family are also 
under expressed in AML. From microarray analysis of blasts cells from over 200 patients 
enrolled on six previous frontline AML trials at St. Jude, including AML02, we have some 
evidence that under expression of the uptake carriers ENT1 (SLC29A1), CNT3 
(SLC28A3), and OCTN1 (SLC22A4) and overexpression of the efflux transporters p-
glycoprotein (ABCB1) and MRP1 (ABCC1) are associated with worse long-term outcome. 
We also know that drugs used in AML therapy such as cytarabine, clofarabine, 
daunorubicin, and etoposide are substrates for several efflux transporters (e.g., p-
glycoprotein, BCRP, MRP2, MRP4), uptake carriers (e.g., ENT1, CNT3, OCTN1, 
OCTN2) and/or drug metabolizing enzymes (e.g., CYP3A4). We propose to evaluate 
associations between genetic polymorphisms in drug-metabolizing enzymes and drug 
transporters and treatment-related outcomes, including both toxicity and efficacy. 
 
Pharmacogenetics of nucleoside analogs  
 
Cytarabine and clofarabine, nucleoside analogs that are used in the treatment of AML are 
prodrugs and must be converted to the triphosphate form by deoxycytidine kinase (DCK) 
for their anti-leukemic effect. 
 
The genes of relevance to activation of cytarabine and clofarabine include equilibrative 
nucleoside transporter (hENT1), critical for intracellular uptake of ara-C. Once inside the 
cell, cytarabine and clofarabine are activated to their active tri-phosphate forms (ara-CTP 
or clofarabine-TP) by a series of intracellular enzymes, with deoxycytidine kinase (DCK) 
catalyzing the first rate limiting metabolic step. Cytidine deaminase (CDA) and 5’-
Nucleotidase (NT5C2) are the primary inactivating enzymes. The primary mechanism of 
resistance appears to be the insufficient intracellular levels of the active triphosphate 
metabolite ara-CTP; this may be due to: a) inefficient cellular uptake due to low levels of 
the transporter, hENT1; b) reduced levels of the activating enzymes, primarily DCK; c) 
increased levels of inactivating enzymes, such as NT5C2 and CDA; and d) increased 

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cellular dCTP pools, that can compete with DNA incorporation of ara-CTP or clofarabine-
TP and also inhibit DCK activity. The intracellular dCTP pools, in turn, are regulated by 
ribonucleotide reductase (RRM1 and RRM2). 
 
We have shown that genetic variations in several of these genes integral to activation of 
these nucleoside analogs results in changes in expression or activity of proteins that affect 
drug uptake, activation and degradation. Previous unpublished analysis of data on pre-
treatment gene expression profiles in 40 patients from AML97 have indicated that 
expression of the ENT1 and NT5C2 genes is associated with the clinical outcome. We have 
recently reported novel coding SNPs and other regulatory  SNPs in DCK that affect its 
function and expression, respectively are were also associated with the leukemic blast ara-
CTP  levels in the patients enrolled in AML97 protocol undergoing treatment with ara-C.171 
Although the sample size was very less, interestingly the two AML patients heterozygous 
for coding changes in DCK also relapsed despite having favorable cytogenetic abnormalities 
[inv16 and t(8;21), respectively].171 We have also identified coding and regulatory genetic 
polymorphism in NT5C2 that are associated with its mRNA expression, sensitivity to ara-C 
in lymphoblast cell lines and with blast ara-CTP levels in AML patients.  
 
Thus, the observed inter-patient variation in the anticancer efficacy of 
cytarabine/clofarabine could be, in part, due to inter-patient variation in the expression 
and/or activity of proteins that affect drug uptake, activation and degradation. Further, 
since the activated triphosphate form of clofarabine inhibits DNA polymerases, inhibits 
ribonucleotide reductase, and disrupts mitochondria, genetic variation in RRM1 and RRM2 
could have an influence on its therapeutic efficacy. Therefore, we propose to further 
explore whether genetic polymorphisms in the key candidate genes (DCK, hENT1, CDA, 
NT5C2, RRM1, RRM2 etc.) in the metabolic pathway of nucleoside analogs are associated 
with early blast clearance, treatment response or toxicity in AML patients. 
 
2.2.8 Infectious disease aims 
 
As described in section 2.1.11, a retrospective review of prophylactic antibiotics used in a 
subset of patients treated on AML02 demonstrated that certain regimens dramatically 
reduced the incidence of bacterial infections. In AML08, we will prospectively collect 
information and describe the impact of antibiotic/antifungal prophylaxis on the outcome 
measures summarized below. These include variables that are routinely collected as part of 
pediatric oncology protocols (CTCAE v 3.0) permitting cross-protocol comparisons as well 
as some data that will be collected specifically for this protocol. Monitoring for 
antimicrobial resistance using stool surveillance cultures will be done only on St. Jude 
participants since such surveillance is part of standard of care only at this institution. 
Where possible, comparisons will be made with similar immunosuppressed patients based 
on historical information or from other cooperative group protocols.  Outcome measures 
for descriptive analysis in the study population include: 
 
• Invasive bacterial infections 
• Invasive fungal infections 
• Fever with neutropenia episodes 
• Rates of antimicrobial resistance (invasive infections) 
• Hospitalization days 

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We will also measure the impact of antibiotic and antifungal prophylaxis in terms of rates of 
antimicrobial resistance (information for invasive infections from all participating sites and 
stool surveillance cultures from St. Jude data only). 
 
A large number of episodes of fever with neutropenia remain with unclear etiology. We 
suspect that many such “fever of unknown origin” episodes are due to viral infections. The 
ability to diagnose viral respiratory infections significantly improves with use of a PCR 
based approach over conventional diagnostic techniques such as direct fluorescent assay or 
culture. Asymptomatic infection in immunocompromised patients due to respiratory 
viruses has been previously demonstrated.172,173 However, the contribution of such 
asymptomatic viral infection to fever with neutropenia in patients being treated for an 
underlying malignancy remains unclear.174,175 Therefore, we propose the routine use of 
respiratory PCRs176 for influenza A, influenza B, respiratory syncytial virus, human 
parainfluenza virus 1-3, human  adenovirus, human rhinovirus, coronavirus, enterovirus  
and human metapneumovirus (MultiCode-PLx Respiratory PCR panel; EraGen 
Biosciences, Madison, WI) on nasal wash specimens collected at admission for febrile 
neutropenia (all patients regardless of the presence of  respiratory symptoms).  
 
Under amendment 4.0, the respiratory PCR panel on nasal wash samples will not be done. 
Note that this research aim has been deleted in amendment 4.0.   
 
3.0  
ELIGIBILITY CRITERIA AND STUDY ENROLLMENT 
 
According to institutional and NIH policy, the study will accession research participants 
regardless of gender and ethnic background.  Institutional experience confirms broad 
representation in this regard. 
 
3.1 
Diagnostic criteria 
 
Patients must have one of the following three characteristics: 
 
1. Acute myeloid leukemia fulfilling the criteria of the WHO Classification 
 
2. <20% marrow myeloblasts and evidence of a clonal de novo AML genetic 
abnormality [e.g., t(8;21), inv(16), t(9;11)] 
 
3. Myeloid sarcoma (also referred to as extramedullary myeloid tumor, 
granulocytic sarcoma, or chloroma), with or without evidence of a leukemic 
process in the bone marrow or peripheral blood, with confirmation of 
myeloid differentiation. 
 
Patients with secondary AML following treatment of primary malignancy are 
eligible.  
 

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3.2 
Inclusion criteria – all participants 
 
3.2.1 Age ≤ 21 years at time of study entry. 
3.2.2 No prior therapy for this malignancy except for one dose of intrathecal therapy 
and the use of hydroxyurea or low-dose cytarabine (100-200 mg/m2 per day 
for one week or less) for hyperleukocytosis 
3.2.3 Written informed consent according to institutional guidelines 
3.2.4 Female patients of childbearing potential must have a negative pregnancy 
test within 2 weeks prior to enrollment 
3.2.5 Male and female participants must use an effective contraceptive method 
during the study and for a minimum of 6 months after study treatment. 
 
3.3 
Exclusion criteria – all participants 
 
3.3.1 Down syndrome 
3.3.2 Acute Promyelocytic Leukemia (APL) 
3.3.3 Juvenile Myelomonocytic Leukemia (JMML) 
3.3.4 Fanconi anemia (FA) 
3.3.5 Kostmann syndrome 
3.3.6 Shwachman syndrome 
3.3.7 Other bone marrow failure syndromes 
3.3.8 Use of concomitant chemotherapy, radiation therapy, or immunotherapy 
other than as specified in the protocol. 
3.3.9 Use of investigational agents within 30 days or any anticancer therapy for 
this malignancy within 2 weeks before study entry with the exception of IT 
therapy, hydroxyurea, or low-dose cytarabine as stated above. The patient 
must have recovered from all acute toxicities from any previous therapy. 
3.3.10 Systemic fungal, bacterial, viral, or other infection not controlled (defined as 
exhibiting ongoing signs/symptoms related to the infection and without 
improvement, despite appropriate antibiotics or other treatment). 
3.3.11 Pregnant or lactating patients. 
3.3.12 Any significant concurrent disease, illness, or psychiatric disorder that would 
compromise patient safety or compliance, interfere with consent, study 
participation, follow up, or interpretation of study results.   
 
3.4 
Criteria for randomization 
 
Participants must meet the following criteria to qualify for HD-ADE versus Clo/AraC 
randomization. Participants who do not meet these criteria may still be enrolled, but will be 
treated on HD-ADE arm and will NOT be randomized. 
 
3.4.1 Normal creatinine for age 
3.4.2 Serum bilirubin ≤ 1.5 x upper limit of normal (ULN). 
3.4.3 Aspartate transaminase (AST/SGOT) and alanine transaminase 
(ALT/SGPT) ≤ 2.5 x ULN 
3.4.4 Alkaline phosphatase ≤ 2.5 x ULN 
 

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3.5 
Enrollment on study 
 
3.5.1 St. Jude Children’s Research Hospital 
 
A member of the study team will confirm potential participant eligibility as defined in 
Section 3.1-3.2, complete and sign the ‘Participant Eligibility Checklist’.  The study team 
will enter the eligibility checklist information into the Patient Protocol Manager (PPM) 
system. Eligibility will be reviewed, and a research participant-specific consent form and 
assent document (where applicable) will be generated. The complete signed consent/assent 
form(s) must be faxed or emailed to the CPDMO at 
to complete the enrollment 
process. 
 
The CPDMO is staffed 7:30 am-5:00 pm CST, Monday through Friday. A staff member is 
on call Saturday, Sunday, and holidays from 8:00 am to 5:00 pm.  Enrollments may be 
requested during weekends or holidays by calling the CPDMO “On Call” cell phone 
 or referencing the “On Call Schedule” on the intranet). 
 
3.5.2 Collaborating Sites 
 
Collaborating Site research participants should be registered at St. Jude within 24 hours of 
enrollment at the site.  The completed Eligibility Checklist and entire signed Informed 
Consent should be faxed to 
.  Please call 
 if confirmation of the 
enrollment information is needed. The Protocol Eligibility Coordinator will then register 
the research participant in the Patient Protocol Manager (PPM) system.   
 
4.0 
TREATMENT PLAN 
 
4.1 
General overview 
 
Treatment will be based on cytogenetic and molecular characteristics, morphology, and 
response to therapy as assessed by flow cytometry. Risk groups are defined below. The 
general treatment plan will consist of chemotherapy for LR patients, chemotherapy ± NK 
cell therapy for SR patients, and chemotherapy + SCT for HR patients. HR patients who do 
not have a suitable stem cell donor or who decline SCT will be eligible for NK cell therapy, 
but will be analyzed separately. The definition of suitable stem cell donor and the 
conditioning regimens used for SCT will be determined by local institutional protocols or 
guidelines. In addition, although we do not recommend SCT for SR patients, the role of 
SCT for SR patients who have a matched sibling donor is controversial and is widely 
recommended by many pediatric oncologists and by the current COG protocol. Therefore, 
SR patients who have matched sibling donors may proceed to SCT after Induction II at the 
discretion of the treating physician. However, because of the favorable prognosis recently 
described for patients with wild-type FLT3 and mutations of NPM1 or CEPBA, these 
patients will not be candidates for SCT, even if a matched sibling donor is available.  
 
Note about administration guidelines for all treatment phases: The timing and duration for 
administration for all commercially available agents are provided in the treatment phase 
sections as guidelines only. Variations in the timing and duration of chemotherapy 
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acceptable, as long as the treating investigator and/or PI determines that there was no 
impact on patient safety.   
 
These variations will not be considered protocol deviations, as long as the total dose is 
given within 10% of protocol specified dose 
 
To reduce the potential for cardiac toxicity, dosing of daunorubicin and mitoxantrone for 
patients less than 10 kg will be based on weight rather than body surface area. Similarly, 
for infants < 1 month of age, or for infants < 3 months of age who were born significantly 
prematurely defined as < 36 weeks gestation, dosing of cytarabine, clofarabine, 
asparaginase, and etoposide, will be based on weight. 
 
NK cell therapy for collaborating site patients, except Singapore, will be done at St. Jude. 
The patient, one parent, and the NK donor will need to travel to Memphis and stay for 
about 5 weeks. St. Jude will cover travel costs for the patient, donor and one parent, 
housing for up to 4 family members, and all medical costs during this time. St. Jude will 
also cover the travel costs for the NK cell donor. 
 
Guidance during times of drug shortages and unavailability: 
 
Treating investigators are urged to consult with the PI or co-PI and use their best clinical 
judgment in optimizing therapeutic intent and ensuring patient safety in managing the 
protocol-specified therapy. Although these decisions may constitute “Protocol Violations,” 
they are unavoidable and made in consideration of the best interest of an individual 
patient. These will not be considered monitoring/audit findings if appropriately 
documented. All protocol deviations must be noted in the research database and the 
alterations in therapy due to the agent shortage will be captured. This should be 
accomplished by entering “dose modified” and details noted in the comments field. These 
deviations will also be noted in the Deviation Log with the notation “Drug 
substitution/reduction due to unavoidable drug shortage/unavailability”. 
 
4.1.1 Low-risk (LR) criteria (not eligible for SCT or NK cell therapy) 
 
Core binding factor (CBF) leukemia [t(8;21)/AML1-ETO or inv(16)/t(16;16)/CBFβ-
MYH11] and MRD < 0.1% at day 22, regardless of other genetic features. Patients with 
CBF leukemia who have MRD ≥ 0.1%, but less than 5%, at day 22 or who have increasing 
levels of fusion transcript will be considered SR and thus eligible for NK cell therapy.  
 
4.1.2 Standard-risk (SR) criteria (eligible for NK cell therapy) 
 
• CBF leukemia with MRD ≥ 0.1%, but less than 5%, at day 22 or increasing 
levels of fusion transcript 
• FLT3-ITD and MRD < 0.1% at day 22  
• Absence of low-risk or high-risk features 
 

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4.1.3 High-risk (HR) criteria (candidates for SCT; eligible for NK cell therapy) 
 
• t(6;9), t(8;16), t(16;21), -7, -5, or 5q- 
• FAB M0 or M6 
• FAB M7 without t(1;22) 
• Treatment-related (secondary) AML 
• RAEB-2 or AML arising from prior MDS 
• FLT3-ITD and MRD ≥ 0.1% at day 22 
• All other patients with poor response to therapy (must have one of the following 
features) 
- MRD ≥ 5% at day 22 
- MRD ≥ 0.1% after Induction II 
 
4.2 
Induction therapy (2 courses) 
 
All patients will receive two courses of induction therapy that will include one course of 
either high dose cytarabine, daunorubicin, and etoposide (HD-ADE) or one course of 
clofarabine and cytarabine (Clo/AraC), followed by one course of low dose cytarabine, 
daunorubicin, and etoposide (LD-ADE). Patients will be randomly assigned to receive one 
of the following induction regimens.   
 
4.2.1 Induction I: HD-ADE 
 
- Cytarabine: 3 g/m2 (100 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 3 hours q12 hours x 6 doses (days 1, 3, 5)  
- Daunorubicin: 50 mg/m2 (1.67 mg/kg for patients less than 10 kg) IV over 6 
hours on days 2, 4, 6 (3 doses) 
- Etoposide: 100 mg/m2 (3.3 mg/kg for infants < 1 month of age, or for infants < 
3 months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 4 hours on days 2-6 (5 doses) 
 
4.2.2 Induction I: Clo/AraC 
 
- Clofarabine: 52 mg/m2 (1.7 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 2 hours on days 1-5 (5 doses). 
- Cytarabine: 1 gram/m2 (33 mg/kg for infants < 1 month of age, or for infants < 
3 months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 2 hours on days 1-5 (5 doses; each dose to start 4 hours after 
the start of clofarabine) 
 
Clofarabine will be supplied by Genzyme. 
 
Special considerations for patients with hyperleukocytosis: 
 
In patients with leukocyte counts greater than 100 x 109/L or symptoms of hyperviscosity, 
leukapheresis or exchange transfusion should be performed according to local institutional 

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guidelines. In addition, patients with leukocyte counts greater than 50 x 109/L, especially 
those with M4, M4Eo, or M5 morphology, are at risk for severe cardiopulmonary and renal 
complications associated with rapid cell lysis during the initiation of chemotherapy with 
nucleoside analogues, including cytarabine and clofarabine.177 Therefore, the 
administration of hydroxyurea (10 to 20 mg/kg/day PO; maximum 1000 mg PO BID) or 
cytarabine (100 mg/m2/dose IV every 12 hours) should be considered in such cases. 
Hydroxyurea or low-dose cytarabine may be given prior to Induction I at the discretion of 
the treating physician.  
 
Special considerations for administration of clofarabine 
 
Clofarabine is excreted primarily by the kidneys.  Therefore, drugs with known renal 
toxicity should be avoided during the 5 days of clofarabine treatment in each cycle. 
Additionally, the liver is a known target organ for clofarabine toxicity.  Therefore, 
concomitant use of medications known to induce hepatic toxicity should be avoided. 
Hepatic and renal function should be assessed prior to and during treatment with 
clofarabine and it is recommended that the patient’s fluid status and hepatic and renal 
function be carefully monitored during the drug administration period. All patients should 
receive hydration each day of clofarabine treatment, giving careful consideration to the 
cardiac and renal function of the patient. To the extent possible, use of nephrotoxic (e.g., 
vancomycin, amphotericin B, etc.) and hepatotoxic (e.g., voriconazole, cyclosporine, etc.) 
agents is to be avoided during clofarabine administration.  
 
Prophylactic steroid administration has been administered by some investigators and has 
been reported to minimize the occurrence and/or severity of the following potential 
clofarabine-related toxicities: nausea, vomiting, skin rash/desquamation, and capillary leak 
syndrome. Therefore, methylprednisolone, 0.5 to 1 mg/kg/dose IV, should be given prior to 
each dose of clofarabine.  
 
4.2.3 Evaluation of response and criteria for starting Induction II 
 
All patients will undergo bone marrow aspirate and LPIT at day 22 (see Section 7.1) and 
will start Induction II at approximately day 29. 
 
• Patients with MRD ≥ 0.1% or with definitive leukemic blasts by morphology may 
start Induction II prior to day 29 regardless of blood counts. 
• In patients with MRD < 0.1% and hypocellular marrows, Induction II may be 
delayed until there are signs of hematopoietic recovery, but should not start later 
than day 36. A second bone marrow aspirate may be performed if clinically 
indicated.  
 
4.2.4 Induction II: LD-ADE 
 
• Cytarabine: 100 mg/m2 (3.3 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 30 minutes q12 hours on days 1-8 (16 doses) 
• Daunorubicin: 50 mg/m2 (1.67 mg/kg for patients less than 10 kg) IV over 6 hours 
on days 2, 4, 6 (3 doses) 

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• Etoposide: 100 mg/m2 (3.3 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 4 hours on days 1-5 (5 doses) 
 
4.2.5 Induction II for patients with FLT3-ITD: LD-ADE + sorafenib 
 
Sorafenib, a commercially available inhibitor of FLT3, will be incorporated into the 
treatment regimen of patients whose leukemic blasts carry FLT3-ITD.  Patients must have 
total bilirubin, SGOT (AST), and SGPT (ALT) < two times the upper limit of normal 
(ULN) for age to be eligible to receive sorafenib.  
 
Patients with FLT3-ITD will take sorafenib, 400 mg/m2 per day, orally in two divided 
doses (200 mg/m2/dose BID) starting one day after the completion of Induction II and 
continuing for 21 days. The maximum dose of sorafenib will be 400 mg PO BID. If 
needed, the sorafenib 200 mg tablet will be cut (halved or quartered) and the dose will be 
rounded up to the nearest 50 mg as shown in the table below. Sorafenib may be taken with 
or without food. If taken with food, sorafenib should be taken with a moderate to low fat 
meal. If a patient has vomiting within 30 minutes of taking sorafenib, the drug may be re-
administered. 
 
Sorafenib 400 mg/m2/day rounded to the nearest 50 mg divided in two doses 
BSA Range 
Calculated Daily Dose Actual Daily Dose AM Dose 
PM Dose 
0.18 – 0.31 m2 
72 -124 mg 
100 mg 
50 mg 
50 mg 
0.32 – 0.43 m2 
125 -174 mg 
150 mg 
100 mg 
50 mg 
0.44 – 0.56 m2 
175 – 224 mg 
200 mg 
100 mg 
100 mg 
0.57 - 0.68 m2 
225 – 274 mg 
250 mg 
150 mg 
100 mg 
0.69 – 0.81 m2 
275 – 324 mg 
300 mg 
150 mg 
150 mg 
0.82 – 0.93 m2 
325 – 374 mg 
350 mg 
200 mg 
150 mg 
0.94 – 1.06 m2 
375 – 424 mg 
400 mg 
200 mg 
200 mg 
1.07 – 1.18 m2 
425 – 474 mg 
450 mg 
250 mg 
200 mg 
1.19 – 1.31 m2 
475 – 524 mg 
500 mg 
250 mg 
250 mg 
1.32 – 1.43 m2 
525 – 574 mg 
550 mg 
300 mg 
250 mg 
1.44 – 1.56 m2 
575 – 624 mg 
600 mg 
300 mg 
300 mg 
1.57 – 1.68 m2 
625 – 674 mg 
650 mg 
350 mg 
300 mg 
1.68 – 1.81 m2 
675 – 724 mg 
700 mg 
350 mg 
350 mg 
1.82 – 1.93 m2 
725 – 774 mg 
750 mg 
400 mg 
350 mg 
≥ 1.94 m2 
≥ 775 mg 
800 mg* 
400 mg 
400 mg 
*Maximum dose. (400 mg BID is the FDA approved adult dose)  
 
Patients with FLT3-ITD who do not experience toxicity related to Sorafenib after Induction 
II may also receive a 21-day course of sorafenib after subsequent courses of chemotherapy. 
 
Voriconazole and other azoles should be avoided during sorafenib administration. 
Micafungin should be used for fungal prophylaxis in patients who are receiving sorafenib.  
 
Sorafenib will be dose-reduced by 50% or discontinued in patients who experience the 
following adverse events if such events are felt to be probably or definitely related to 

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sorafenib. The treating physician, in consultation with the PI, will determine whether to 
reduce the dose or discontinue sorafenib. 
 
Note that all dose reductions will be made by deleting the evening dose, thereby reducing 
the daily dose by approximately 50%. 
 
 Any grade 4 non-hematological toxicity 
 Any grade 3 non-hematological toxicity with the exception of 
- Grade 3 nausea and vomiting of less than < 5 days duration 
- Grade 3 transaminases that return to levels that meet initial eligibility 
criteria within 7 days of sorafenib interruption and that do not recur upon re-
challenge with sorafenib  
- Grade 3 fever or infection < 5 days duration 
- Grade 3 hypokalemia, hypophosphatemia, hypomagnesemia, or 
hypocalcemia responsive to oral supplementation 
 Any grade 2 non-hematological toxicity that persists for ≥ 7 days and is considered 
sufficiently medically significant or sufficiently intolerable by patients that it 
requires treatment interruption 
 Hypertension defined as: 
- A diastolic blood pressure >25 mmHg above the 95th percentile for age and 
gender confirmed by repeated measurement 
- In patients on antihypertensive therapy, a diastolic blood pressure ≤ 25 
mmHg above the 95th percentile for age and gender for > 14 days 
- Grade ≥ 4 hypertension 
 
Suggested Dose Modifications for Sorafenib for Hand-Foot Skin Reaction and skin rash 
Hand-foot skin reaction 
Grade 
Occurrence 
Suggested Dose Modification 
Grade 1 
Numbness, dysesthesia, 
paresthesia, tingling, 
painless swelling, erythema 
or discomfort of the hands 
or feet which does not 
disrupt the patient’s normal 
activities 
Any  
Promptly institute supportive measures  such as topical therapy 
for symptomatic relief and continue sorafenib treatment  
Grade 2 
Painful erythema and 
swelling of the hands or 
feet and/or discomfort 
affecting the patient’s 
normal activities 
 
First  
Promptly institute supportive measures such as topical therapy 
for symptomatic relief and decrease sorafenib by 50% 
• 
If toxicity resolves to grade 0–1 after dose reduction, may 
increase sorafenib back to full dose. 
• 
If toxicity develops rapidly within several days or does not 
resolve to grade 0–1 despite dose reduction, interrupt 
sorafenib treatment for a minimum of 7 days and until 
toxicity has resolved to grade 0–1.   
• 
When resuming treatment after dose interruption, resume 
sorafenib at reduced dose  
• 
If toxicity is maintained at grade 0–1 at reduced dose for a 
minimum of 7 days, may increase back sorafenib to full dose. 
Second  
Discontinue sorafenib 

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Suggested Dose Modifications for Sorafenib for Hand-Foot Skin Reaction and skin rash 
Grade 3 
Moist desquamation, 
ulceration, blistering or 
severe pain of the hands or 
feet, or severe discomfort 
that causes the patient to 
be unable to work or 
perform activities of daily 
living 
First 
occurrence 
Institute supportive measures such as topical therapy for 
symptomatic relief and interrupt sorafenib treatment for a 
minimum of 7 days and until toxicity has resolved to grade 0–1 
• 
When resuming treatment after dose interruption, decrease 
sorafenib by 50%. 
• 
If toxicity is maintained at grade 0–1 at reduced dose for a 
minimum of 7 days, may increase back sorafenib to full dose. 
Second  
Discontinue sorafenib  
Skin rash 
Grade 
Occurrence 
Suggested Dose Modification 
Grade 1 
Macular or papular 
eruption or erythema 
without associated 
symptoms 
Any  
Promptly institute supportive measures  such as topical therapy 
for symptomatic relief and continue sorafenib treatment  
Hand-foot skin reaction 
Grade 
Occurrence 
Suggested Dose Modification 
Grade 2 
Macular or papular 
eruption or erythema with 
pruritus or other associated 
symptoms; localized 
desquamation or other 
lesions covering <50% of 
body surface area (BSA) 
First  
Promptly institute supportive measures such as topical therapy 
for symptomatic relief and decrease sorafenib by 50%  
• 
If toxicity resolves to grade 0–1 after dose reduction, may 
increase sorafenib back to full dose. 
• 
If toxicity develops rapidly within several days or does not 
resolve to grade 0–1 despite dose reduction, interrupt 
sorafenib treatment for a minimum of 7 days and until 
toxicity has resolved to grade 0–1.  
• 
When resuming treatment after dose interruption, resume 
sorafenib at reduced dose 
• 
If toxicity is maintained at grade 0–1 at reduced dose for a 
minimum of 7 days, may increase back sorafenib to full dose. 
Second  
Discontinue sorafenib 
Grade 3 
Severe, generalized 
erythroderma or macular, 
papular or vesicular 
eruption; desquamation 
covering ≥50% BSA 
First  
Institute supportive measures such as topical therapy for 
symptomatic relief and interrupt sorafenib treatment for a 
minimum of 7 days and until toxicity has resolved to grade 0–1 
• 
When resuming treatment after dose interruption, decrease 
sorafenib by 50% 
• 
If toxicity is maintained at grade 0–1 at reduced dose for a 
minimum of 7 days, may increase back sorafenib to full dose. 
Second  
Discontinue sorafenib  
See Section 5.9 for management of hypertension related to sorafenib 
 
 

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4.2.6 Induction II for other HR patients: LD-ADE + vorinostat* 
 
Patients with M7 AML without t(1;22) and other HR patients without FLT3-ITD will be 
treated with a combination of vorinostat and LD-ADE. Vorinostat (100 mg/m2/dose PO 
TID) will be given for 3 days (Days -2, -1, 0) prior to the initiation of Induction II 
chemotherapy.  
 
 Days -2, -1, 0: Vorinostat 100 mg/m2/dose PO TID 
 Days 1-8: LD-ADE as described above 
 
Histone acetylation will be assessed in vivo as a measure of the effects of vorinostat. 
Peripheral blood samples (collected in 3 mL of preservative-free heparin) will be drawn as 
follows:  
 
1. Prior to the first dose of vorinostat on Day -2 
2. On Day 1 prior to the initiation of chemotherapy 
 
Suggested dosing table for vorinostat* capsule doses at 300 mg/m2/day, given TID 
BSA range (m2) 
Calculated dose range 
Capsules per day 
(100 mg/capsule) 
Dose divisions 
am/mid-day/pm 
(number of capsules) 
0.9 to 1.15 
270 mg to 345 mg 
3 
1/1/1 
1.16 to 1.50 
348 mg to 450 mg 
4 
2/1/1 
1.51 to 1.83 
453 mg to 549 mg 
5 
2/2/1 
1.84 to 2.17 
552 mg to 651 mg 
6 
2/2/2 
2.18 to 2.49 
654 mg to 747 mg 
7 
3/2/2 
≥ 2.5  
 
8 
3/3/2 
 
Patients with a BSA less than 0.9 m2 and patients who cannot swallow whole capsules 
should take vorinostat liquid 100 mg/m2/dose, rounded to the nearest 5 mg.  
 
A suspension can be prepared locally by mixing 20 mL of OraPlus with the contents of 
twenty 100 mg vorinostat capsules in a 4 ounce glass bottle. After shaking for up to 3 
minutes to disperse, add an additional 20 mL of OraSweet. Shake the container to disperse, 
resulting in a final concentration of 50 mg/mL. The suspension should be stored at room 
temperature for a maximum of 2 weeks. 
 
*Note:  Collaborating sites may elect to not participate in vorinostat exploratory objective 
of this study.  If the decision is made that a site will opt out, then all applicable patients at 
that site will receive standard therapy for Induction II with LD-ADE. 
 
Reference:  Fouladi M, Park JR, Stewart CF, et al, "Pediatric Phase I Trial and 
Pharmacokinetic Study of Vorinostat: A Children's Oncology Group Phase I Consortium 
Report," J Clin Oncol, 2010, 28(22):3623-9. [PubMed 20606092] 
 

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4.2.7 Special subgroup 
 
HR patients with MRD < 0.1% may proceed directly to SCT after Induction I if a suitable 
donor is available and the transplant can be performed without delay. Based on the results of 
AML02, this will apply to about 1% of patients.  
 
4.3  
Consolidation I for patients who will not receive SCT 
 
All LR patients, those SR patients who will not receive SCT, and HR patients who do not 
have a suitable donor, will receive mitoxantrone, and cytarabine (MA) as consolidation I.  
Criteria for starting Consolidation I: ANC ≥ 300/µl and rising and platelet count ≥ 30,000/µl 
and rising. 
 
4.3.1 MA (mitoxantrone/Ara-C) 
 
- Mitoxantrone: 12 mg/m2 (0.4 mg/kg for patients less than 10 kg) IV over 1 hour 
on days 3-5 (3 doses) 
- Cytarabine: 1 g/m2 (33 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 2 hours every 12 hours on days 1-4 (8 doses) 
 
4.4 
Consolidation therapy for HR patients who will undergo SCT 
 
HR patients are eligible to undergo SCT after Induction II. HR patients who have < 0.1% 
MRD and for whom a donor has been identified should proceed directly to SCT after 
Induction II. HR patients who have ≥ 0.1% MRD or for whom a donor workup is still in 
progress are eligible to receive therapy prior to SCT as follows:  
 
4.4.1 HR patients who have < 0.1% MRD and are awaiting SCT 
 
4.4.1.1 HR patients who have <0.1% MRD, but for whom a SCT donor is not yet available 
(expected time from count recovery after Induction II to SCT greater than about 4 weeks), 
should receive a third course of chemotherapy consisting of mitoxantrone and cytarabine 
(MA) identical to consolidation I above  
 
- Mitoxantrone: 12 mg/m2 (0.4 mg/kg for patients less than 10 kg) IV over 1 hour on 
days 3-5 (3 doses) 
 
- Cytarabine: 1 g/m2 (33 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 2 hours every 12 hours on days 1-4 (8 doses) 
 

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4.4.1.2 HR patients who have <0.1% MRD who will undergo SCT less than 4 weeks after 
Induction II, may receive low-dose cytarabine and oral thioguanine, rather than MA, at the 
discretion of the treating physician.  
 
- Cytarabine: 75 mg/m2 (2.5 mg/kg for infants < 1 month of age or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation)  IV over 30 minutes on days 1-4 and 15-18 
 
- Thioguanine: 60 mg/m2 orally at bedtime on days 1-28 
 
4.4.2 HR patients who have ≥ 0.1% MRD after Induction II 
 
Because the optimal therapy for patients who will undergo SCT, but who have MRD ≥ 
0.1% is unknown, this protocol will allow several therapeutic options. The therapy for each 
patient will be decided by the treating physician, with consultation from the local SCT 
team and the PI.  
 
Option 1: SCT 
 
Patients with low levels of MRD (e.g., < 1%) and for whom a SCT donor is ready, may 
proceed directly to SCT. 
 
Option 2: NK cell therapy, followed by SCT 
 
Patients who have a KIR-mismatched family member who is greater than 18 years old may 
undergo NK cell transplantation as described below. These patients will then undergo SCT 
after recovery from NK cell transplantation. 
 
Option 3: Conventional therapy, followed by SCT 
 
Patients who have ≥ 0.1% MRD are eligible to receive conventional chemotherapy with 
mitoxantrone and cytarabine as described above (section 4.4.1.1) prior to SCT.  
 
4.5 
Consolidation II (HD-AraC/Asp) 
 
Criteria for starting Consolidation II: ANC ≥ 300/µl and rising and platelet count ≥ 
30,000/µl and rising. 
 
- Cytarabine 3 g/m2 (100 mg/kg for infants < 1 month of age, or for infants < 3 
months of age who were born significantly prematurely defined as < 36 weeks 
gestation) IV over 3 hours every 12 hours on days 1, 2, 8, 9 (8 doses) 
- E. coli L-asparaginase 6000 (200 Units/kg for infants < 1 month of age, or for 
infants < 3 months of age who were born significantly prematurely defined as < 36 
weeks gestation) Units/m2 IM 3 hours after 4th and 8th doses of cytarabine.  

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*When E. coli L-asparaginase is no longer available, each dose during consolidation II 
should be replaced with: 
 
- Erwinia asparaginase 25,000 Units/m2 (833 Units/kg for infants < 1 month of age, 
or for infants < 3 months of age who were born significantly prematurely defined as 
< 36 weeks gestation) IV or IM (according to institutional standard) over 1 hour, 3 
hours after the 4th and 8th doses of cytarabine. 
 
Each institution should follow local administration guidelines for this commercially available 
agent.  The following suggested guidelines may be used, but are not required. 
 
Administration 
 
For IV administration, Erwinia asparaginase will be diluted in 50 mL NS to infuse over 30 
minutes to 1 hour via syringe. Erwinia asparaginase should be administered to run 
concurrently with IVF. (NS at TKO is sufficient.) 
 
Erwinia asparaginase should be administered at least 2 hours after an LP/IT since some 
cases of over sedation were reported when given “simultaneously” with sedation and/or an 
LP/IT at other institutions.   Further, it is desirable to avoid asparaginase directly before an 
IT because asparaginase could possibly interfere with the efficacy of methotrexate. 
 
Erwinia asparaginase may also be administered IM. 
 
Monitoring and concerns for anaphylaxis 
 
Obtain vital signs pre infusion, and then remain at the patient’s bedside for the first 5 
minutes. Visually observe the patient at 10 minutes, and directly observe and obtain vital 
signs at 15 minutes and at the end of the infusion. Repeat vital signs at discharge. Patients 
must remain in the Medicine Room for 1 hour after administration to be observed for 
adverse effects. Keep NS at TKO during the 1 hour post watch time unless otherwise 
ordered.  To prepare for anaphylaxis: 
 
• Have oxygen, suction and pulse oximetry at bedside during and after infusion 
• Have readily available* the following medications:  
 
o Diphenhydramine 1 mg/kg (max 50 mg) for IV administration 
o Epinephrine (1:1000) 0.01 mL/kg (max 0.3 mL) for SQ administration 
o Hydrocortisone 100 mg/m2 for IV administration 
o NS for IV administration 
 
*Readily available means in the general area – such as the emergency medication box 
in the medicine room or inpatient areas. 
 
4.6 
NK cell therapy  
 
Criteria for starting NK cell therapy: ANC ≥ 300/µl and rising and platelet count ≥ 30,000/µl 
and rising. 

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Standard risk patients who have a KIR-mismatched family member who is greater than 18 
years old will undergo NK cell transplantation. In addition, HR patients who do not have a 
suitable stem cell donor or who decline SCT will be eligible for NK cell therapy if they 
have a KIR-mismatched family member, but will be analyzed separately. All other patients 
will receive no further therapy.  
 
4.6.1 Donor selection and testing 
 
Patients will receive NK cell transplantation from an adult family member who shares at 
least one HLA-haplotype with the recipient. The donor will be selected such that there is 
donor KIR – recipient KIR-ligand mismatch. Specific exclusion criteria include pregnancy 
and any other medical condition that, in the opinion of an independent physician, precludes 
performance of an apheresis procedure. 
 
Donors will undergo screening, examination, and testing as described in 21 CFR 1271 and 
the Guidance "Eligibility Determination for Donors of Human Cells, Tissues, and Cellular 
and Tissue-Based Products (HCT/Ps)" to determine eligibility.  
 
Histocompatibility testing, including HLA and KIR genotyping, are performed in a CLIA-
certified, ASHI- and CAP-inspected laboratory of the Department of Pathology, SJCRH. 
Molecular techniques will be utilized to characterize donor and recipient HLA Class I gene 
content to the level sufficient to determine the ligand repertoire for KIR recognition. 
 
4.6.2 NK cell collection and selection 
 
On day -1 the donor will undergo apheresis once. The cells obtained will be purified for 
CD56+ cells by the two-step procedure described previously.58 The CliniMACS selection 
column will be operated using the Standard Operating Procedures of the Human 
Applications Laboratory. For this protocol, our goal is to infuse immediately after 
processing on Day 0 all the NK cells collected to give >2x106 CD56+ cells/kg of recipient 
body weight, but allowing for a CD3+CD56− cell dose of no greater than 0.05x106 /kg. The 
maximum dose of cells that would be infused is 400 x 106 CD56+ cells/kg. The minimum 
dose that would be infused is 0.1 x 106 cells/kg. The lot release criteria of the NK cell 
product is detailed in the table below. 
 
Parameter 
Specification 
CD56+ purity 
≥ 70% CD56+ cells 
CD3+ log depletion 
≥ 2.5 logs 
Viability 
≥ 70% 
Gram Stain 
No organisms seen 
*Calcofluor White stain 
No fungal elements seen 
*Sterility 
Negative 
*Endotoxin 
≤ 2 EU/ml 
*Product can be released for immediate infusion with results pending. Final review must 
include complete results 

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4.6.3 Quality assurance for cellular products 
 
The Department of Therapeutic Production and Quality has established an independent 
division of Quality Assurance (QA). This group is responsible for the management of 
Quality Control, Quality Assurance and Quality Improvement processes for the Human 
Applications Laboratory. Production and QA Systems that are in place include: 
 
• Standard Operation Procedures for production and quality processes 
• Documentation of Donor Eligibility 
• Documentation of processes captured in Batch Records 
• In-process quality control testing including sterility 
• Release Specifications established for all products 
• Out of Specification Reporting and Investigation Process 
• Authorization by QA for the release of all products after review of records and 
release specification test results 
• Product labeling procedures with multiple person review 
• Variance Management Process 
• Personnel Competence and Proficiency Program 
• Inventory control and documentation of product history through patient infusion 
 
Test results that are out of specification for products that are needed on a clinically urgent 
basis will be evaluated by the laboratory medical director. The patient's physician or 
attending transplant physician will be informed of the test result prior to infusion of the 
product. Notification regarding positive sterility results before or after infusion will be 
given to the primary attending physician and patient and/or parent/guardian. Notification to 
the FDA and St. Jude IRB will be given and will include testing results or adverse events 
and any required intervention. An investigation following TPQ/HAL SOPs will be 
completed, reviewed by TPQ Quality Assurance and outcomes of the investigation 
reported. 
 
4.6.4 Treatment schema 
 
• Day -7: Cyclophosphamide 60 mg/kg IV over 1 hour. Patients should receive IV 
hydration at 150 ml/m2/hr for 2 hours prior to cyclophosphamide and 24 hours after 
cyclophosphamide. Mesna 15 mg/kg/dose IV should be given immediately before 
cyclophosphamide and at 3, 6, and 9 hours after cyclophosphamide. 
• Days -6 through -2: Fludarabine 25 mg/m2/day IV over 30 minutes (5 doses) 
• Days -1, +1, +3, +5, +7, +9: IL-2 1 million units/m2 given subcutaneously 
• Day -1: Donor pheresis 
• Day 0: NK cell infusion  
 
NK cells may be infused in either the inpatient or outpatient setting by a physician, 
Physician Assistant, Nurse Practitioner, or qualified RN. Careful monitoring and 
supportive care during NK cell infusion will be guided in part by the Standard Operating 
Procedures for Lymphocytes Infusions in the St. Jude Nursing Policy & Procedure Manual 
(See Attached Nursing Policy and Procedure: Therapeutic Cell Infusion).  
 

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No steroids, including the use of hydrocortisone as pre-medication, may be given to 
patients during the 3 days prior to the NK cell infusion or during the first 7 days after the 
infusion. 
 
MESNA dose and administration may vary based on physician recommendation and 
institutional standards. These variations will not be considered treatment deviations. 
 
4.7 
CNS therapy 
 
All patients should undergo lumbar puncture and receive an age-appropriate dose of 
intrathecal (IT) chemotherapy at the time of diagnosis. Triple intrathecal therapy with 
methotrexate, hydrocortisone, and cytarabine (MHA) will be used for all CNS therapy at 
the doses shown below. However, patients who receive IT cytarabine prior to enrollment 
on AML08 will be eligible for treatment on AML08 and should not receive an additional 
dose of IT MHA at the time of enrollment. 
 
To reduce the risk of neurotoxicity, we recommend separating IT therapy from high-dose 
cytarabine (≥ 1 g/m2) by approximately 24 hours. If systemic therapy needs to begin 
urgently (e.g., in cases with hyperleukocytosis), the initial dose of IT therapy may be 
delayed as the discretion of the treating physician.  
 
IT MHA Dosing 
Patient Age  Methotrexate Hydrocortisone Cytarabine Volume 
< 1 year 
6 mg 
12 mg 
18 mg 
6 ml 
1-2 years 
8 mg 
16 mg 
24 mg 
8 ml 
2-3 years 
10 mg 
20 mg 
30 mg 
10 ml 
> 3 years 
12 mg 
24 mg 
36 mg 
12 ml 
 
Leucovorin rescue (5 mg/m2 per dose; 5 mg maximum per dose) will be given orally or 
intravenously at 24 and 30 hours after each IT MHA treatment. Leucovorin dosing and 
intrathecal volume may be adjusted according to local institutional guidelines.  
 
Patients with no evidence of CNS disease (CNS1: no leukemic blast cells on CSF cytospin) 
will receive 4 total doses of intrathecal therapy, given at approximately one month intervals 
or at the beginning of each of the first 4 courses of chemotherapy. IT therapy will not be 
given before NK cell therapy. 
 
Patients with overt CNS leukemia (CNS3: ≥ 5 leukocytes per µl of CSF and the presence 
of leukemic blast cells on CSF cytospin) will receive weekly intrathecal therapy until the 
CSF is free of blast cells (minimum number of doses, 4). These patients will then receive3 
additional doses of intrathecal therapy (minimum total number of doses, 7) at 
approximately 1-month intervals (generally given with each subsequent course of 
chemotherapy).  IT therapy will not be given before NK cell therapy. 
 
Patients with < 5 leukocytes per µl of CSF and the presence of leukemic blast cells on CSF 
cytospin (CNS2) will receive weekly intrathecal therapy until the CSF is free of blast cells. 
These patients will then receive 3 additional doses of intrathecal therapy at approximately 

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1-month intervals (generally given with each subsequent course of chemotherapy). IT 
therapy will not be given before NK cell therapy. 
 
Patients who are unable to undergo lumbar puncture and receive intrathecal therapy prior to 
starting induction I should be treated as CNS2 unless they have overt CNS leukemia (CNS3). 
 
5.0 
SUPPORTIVE CARE GUIDELINES 
 
5.1 
Prophylaxis and treatment of metabolic derangement 
 
Care should be taken to prevent hyperuricemia and hyperphosphatemia in patients with 
large tumor burdens. Such patients should receive IV hydration at 3000 ml/m2/day before 
the initiation of therapy, oral phosphate binders (aluminum hydroxide, calcium carbonate, 
or sevelamer), and recombinant urate oxidase or allopurinol as needed. 
 
5.2 
Treatment of hyperleukocytosis 
 
In patients with WBC > 100 x 109/L or symptoms of hyperviscosity, leukapheresis or 
exchange transfusion may be used according to local institutional guidelines. The 
administration of hydroxyurea (10 to 20 mg/kg/day PO; maximum 1000 mg PO BID) or 
cytarabine (100 mg/m2/dose IV every 12 hours) is also allowed. 
 
5.3 
Prevention and treatment of complications related to tumor lysis 
 
Patients with AML, especially those with high leukocyte counts and M4, M4Eo, or M5 
morphology, are at risk for severe cardiopulmonary complications during the initiation of 
chemotherapy.177 Although the mechanism of these complications is unknown, it is 
believed that rapid cell lysis may lead to a systemic inflammatory response syndrome 
(SIRS). We have also observed that this inflammatory response may respond to treatment 
with steroids and may be prevented by steroids. Therefore, for patients with elevated 
leukocyte counts, investigators should consider methylprednisolone, 0.5 to 1 mg/kg/dose 
IV every 12 hours, starting prior to the first dose of chemotherapy and continuing for 3 to 5 
days. In addition, hydroxyurea, or low-dose cytarabine may also be given prior to induction 
therapy to reduce tumor burden and reduce the risk of SIRS. Hydroxyurea or low-dose 
cytarabine may be given prior to Induction I at the discretion of the treating physician.  
 
5.4 
Prophylaxis for Pneumocystis jiroveci pneumonia 
 
All patients should receive TMP/SMZ (trimethoprim 150 mg/m2/day and sulfamethoxazole 
750 mg/m2/day in 2 divided doses on Monday, Tuesday, and Wednesday of each week). 
For patients who cannot tolerate TMP/SMZ, daily oral atovaquone or monthly aerosolized 
pentamidine may be substituted. 
 
5.5 
Prophylaxis for fungal infections (REQUIRED) 
 
Patients with AML are at especially increased risk for fungal infections, most commonly 
candidiasis and aspergillosis. Although there is no national standard for antifungal 
prophylaxis of these patients, effective regimens include voriconazole and posaconazole. 

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Micafungin and caspofungin are acceptable alternatives, but fluconazole and itraconazole 
are not recommended because of lack of activity against Aspergillus. Antifungal 
prophylaxis should be initiated at the completion of induction I and continue until count 
recovery after the final course of chemotherapy. Prophylaxis should be given daily except 
on days that patients are receiving daunorubicin, etoposide, or mitoxantrone.  
 
Acceptable antifungal prophylaxis options are listed below, in order of preference. 
Currently, we recommend voriconazole, as we prefer to reserve posaconazole for 
breakthrough infections. All patients on AML08 must receive prophylactic antifungal 
therapy, although the agent used may be based on local institutional guidelines. Please call 
the PI if you wish to use an antifungal agent other than one listed below.  
 
Voriconazole and other azoles should be avoided during sorafenib administration. 
Micafungin may be used for fungal prophylaxis in patients who are receiving sorafenib.  
 
• Voriconazole PO 
 
≥ 1 – 11 years: Loading dose: Not recommended 
Maintenance Dose: 7 mg/kg q12h (rounded up to 50 or 100 mg dose increments.) 
 
≥ 12 years, < 40 kg:  Loading dose: 200 mg PO q12h day 1 
Maintenance Dose: 100 mg PO q12h 
 
≥12 years, ≥ 40 kg: 
Loading dose: 400 mg PO q12h day 1 
Maintenance Dose: 200 mg PO q12h 
 
• Posaconazole 
 
Patients > 13 years old: 200 mg PO 3 times/day with meals 
Patients < 13 years old: dose not established 
 
• Micafungin:  
 
< 40 kg: 1 mg/kg/day IV (max 50 mg/day) 
> 40 kg: 50 mg/day IV 
 
• Liposomal amphotericin B IV: 3-5 mg/kg/day 
 
• Caspofungin: 1 mg/kg/day IV (max 50 mg/day) 
 
5.6 
Prophylaxis for viridans streptococcal infection (REQUIRED) 
 
All patients should have a dental evaluation at the time of diagnosis. During and following 
each course of chemotherapy, we recommend oral rinsing with 10-15 ml undiluted 
chlorhexidine (Peridex) for 30 seconds 2-4 times per day. For infants and small children, a 
soft cloth saturated with rinse can be rubbed on gum and at tooth line. 
 

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Because patients with AML are at high risk for bacterial sepsis, especially sepsis due to 
viridans streptococci, all patients on AML08 must receive prophylactic antibiotics (see 
section 2.1.13 for rationale). Prophylactic antibiotics should be started when the ANC < 
1000 and falling or predicted to fall, and continue until the ANC > 100 and rising. 
Antibiotics may be given by the parents or other caregivers at home, according to local 
institutional guidelines.  
 
Acceptable prophylactic regimens, in order of preference, include the following: 
 
• Vancomycin 400 mg/m2/dose IV every 12 hours (maximum 1 gram per dose) plus 
ciprofloxacin 250-350 mg/m2/dose PO every 12 hours (maximum 500 mg per dose) 
• Cefepime 1500 mg/m2/dose IV every 12 hours (maximum 2 gram per dose) 
• Vancomycin 400 mg/m2/dose IV every 12 hours (maximum 1 gram per dose) plus 
cefepime 1500 mg/m2/dose IV every 12 hours (maximum 2 gram per dose). Note 
that this regimen is not recommended by the PI, but is acceptable if preferred by the 
local institution or treating physician.  
• Please call the PI if you wish to use a prophylactic regimen other than one listed 
above. Because local infection rates, organisms, and susceptibilities vary, other 
prophylactic regimens will be allowed. However, regimens should be uniform at 
each collaborating site and data on each regimen will be captured to explore the 
efficacy of each regimen.  
 
5.7 
Management of febrile neutropenia 
 
Episodes of fever and neutropenia should be managed according to institutional guidelines. 
Patients with fever (defined as any oral temperature ≥ 38.3° C or an oral temperature of ≥ 
38.0° C that persists for one hour) and neutropenia (defined as ANC < 500 cells/µL) should 
be given IV antibiotics immediately. We recommend the following guidelines: 
 
• Patients who develop fever while receiving prophylactic vancomycin and 
ciprofloxacin: 
 
o Begin cefepime (1500 mg/m2/dose IV every 8 hours) 
o Increase vancomycin to 400 mg/m2/dose IV every 8 hours 
o Discontinue ciprofloxacin 
 
• Patients who develop fever while receiving prophylactic cefepime: 
 
o Begin vancomycin (400 mg/m2/dose IV every 8 hours) and meropenem (20 
mg/kg/dose IV every 8 hours) 
o Discontinue cefepime 
 
Patients who have suspected catheter-related infection, have evidence of sepsis (including 
shock, hypotension, rigors, septic emboli, unexplained respiratory distress or hypoxemia, 
or poor peripheral perfusion), or are  known to be colonized by Pseudomonas aeruginosa, 
should also receive tobramycin, 60 mg/m2/dose IV every 6 hours. 
 

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Patients who have severe abdominal pain or radiographic findings suggesting typhlitis, 
severe abdominal pain with evidence of sepsis, focal findings suggestive of intra-
abdominal infection on physical examination, or known or suspected infection with 
Bacillus cereus should receive meropenem, 20 mg/kg/dose IV every 8 hours, instead of 
cefepime. 
 
5.8 
Management of Capillary Leak Syndrome related to clofarabine 
 
In pediatric studies, during or shortly after clofarabine administration a few patients 
developed signs and symptoms consistent with capillary leak syndrome. In these heavily 
pretreated patients, it has been difficult to separate potential drug-related cases of capillary 
leak syndrome from concurrent medical conditions such as infection/sepsis, progressive 
disease, or other underlying problems resulting from prior anti-leukemic therapies.  
 
For these reasons, during and after each dose of clofarabine investigators are to assess 
patients for the onset of the following signs or symptoms ≥ grade 2: 
 
• Tachypnea or other evidence of respiratory distress; 
• Unexplained hypotension; and/or 
• Unexplained tachycardia. 
 
If one or more of these signs or symptoms occurs during study drug infusion, clofarabine 
administration is to be interrupted or held as clinically indicated. It is recognized that the 
total infusion time for this clofarabine dose in this circumstance may exceed 1 hour. Thus, 
if the patient’s condition stabilizes or improves, clofarabine administration may resume. 
Pretreatment with steroids (methylprednisolone, 0.5 to 1 mg/kg/dose IV) is recommended 
for all subsequent doses during the remainder of that treatment cycle.   
 
5.9 
Management of hypertension related to sorafenib 
 
A diastolic blood pressure (DBP) equal to the 95th percentile for age and gender will be 
defined as the upper limit of normal (ULN). Patients with elevated DBP at any time should 
have blood pressure measurements performed twice weekly until the DBP is less than the 
ULN. Patients with Grade 2/3 hypertension that is well controlled with oral medications 
may continue therapy. Sorafenib should be discontinued for persistent, symptomatic, or 
Grade 4 hypertension.  
 
If the DBP remains above the ULN for age and gender on recheck, then start single agent 
antihypertensive therapy and monitor blood pressure at least every 3 days. If the DBP 
drops below the ULN for age within 14 days, continue single agent antihypertensive 
therapy. However, if the DBP remains above the ULN for age for more than 14 days after 
the institution of antihypertensive, sorafenib should be discontinued.  
 
5.10 
Drug interactions 
 
Efforts should be made to avoid the use of enzyme-inducing anticonvulsants, such as 
phenytoin, phenobarbital, and carbamazepine. Because azole antifungals such as 
fluconazole, itraconazole, voriconazole, and posaconazole may have inhibitory effects on 

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drug-metabolizing enzymes, these agents should be held on days that patients are receiving 
daunorubicin, etoposide, or mitoxantrone.  
 
5.11 
Growth factors 
 
Prophylactic use of hematopoietic growth factors (GM-CSF or G-CSF) is not 
recommended. However, GM-CSF (250 µg/m2/day) or G-CSF (5-10 µg/kg/day) should be 
considered for patients who have documented or suspected fungal infections or bacterial 
sepsis. 
 
5.12 
Conjunctivitis prophylaxis 
 
Dexamethasone ophthalmic solution (0.1%), 2 drops to both eyes four times per day, or 
artificial tears (e.g., hydroxymethylcellulose, hypromellose, polyvinyl alcohol), 2 drops to 
both eyes every 2-6 hours, should be used during HDAC administration and for 24 hours 
after completion to prevent conjunctival irritation. 
 
5.13 
Treatment modifications 
 
5.13.1 Renal impairment 
 
Patients who have received amphotericin B or nephrotoxic antibiotic regimens for at least 7 
days, and patients in whom serum creatinine is greater than two times normal for age, 
should have their glomerular filtration rate (GFR) measured before they receive HDAC. In 
patients with a GFR ≤ 60 ml/min per 1.73m2, we recommend decreasing the dosage of 
cytarabine from 3 g/m2 to 2 g/m2 every 12 hours. No decrease is necessary for standard 
dose cytarabine (≤ 1g/m2/day) regimens. 
 
5.13.2 CNS toxicity 
 
Patients with grade 3 or greater cerebellar toxicity should not receive additional HDAC. 
These cases should be discussed with the PI to determine further management. 
 
5.13.3 Cardiac toxicity 
 
Patients with clinical evidence of congestive heart failure should receive no additional 
daunorubicin or mitoxantrone. In the event of a fractional shortening < 28%, consideration 
will be given to discontinuing administration of daunorubicin and mitoxantrone. 
 
5.13.4 Hepatic toxicity 
 
Patients who develop veno-occlusive (VOD) disease of the liver (manifested by greater 
than 5% increase in weight and a total bilirubin greater than 5 mg/dL) during induction I or 
induction II should not receive treatment with GO. The treatment of patients who are 
candidates for GO but who have developed VOD should be discussed with the Principal 
Investigator. These patients are eligible to remain on study. 
 

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Daunorubicin, mitoxantrone, and etoposide doses should be adjusted in patients who 
develop increased direct bilirubin levels during therapy.  
 
Daunorubicin and mitoxantrone should be adjusted as follows: 
 
• Direct bilirubin 2-4 mg/dl: 50% dose decrease 
• Direct bilirubin 4-6 mg/dl: 75% dose decease 
• Direct bilirubin > 6 mg/dl: hold dose 
 
Similar dose reductions of etoposide should be considered in patients with elevated 
bilirubin, especially in the presence of serum albumin levels less than 2.5 g/dl.  
 
At the time of diagnosis, elevated bilirubin levels may reflect leukemic infiltrate, rather 
than toxic liver damage. In such cases, full doses of chemotherapy may be given at the 
discretion of the treating physician.  
 
5.13.5 Dosing of infants 
 
To reduce the potential for cardiac toxicity, dosing of daunorubicin and mitoxantrone for 
patients less than 10 kg will be based on weight rather than body surface area. Similarly, 
for infants < 1 month of age, or for infants < 3 months of age who were born significantly 
prematurely defined as < 36 weeks gestation, dosing of cytarabine, clofarabine, 
asparaginase, and etoposide, will be based on weight. 
 
6.0 
DRUG INFORMATION 
 
See Appendix I for Information on all agents to be used in this protocol. 
 

A M L 0 8  
Pa ge 4 9  of 8 2  
 
 
Re visi o n 1 0. 1 , dated : 0 6- 0 2- 2 0 1 6 
  
  
  
 
I R B A p pr o val date: 0 6- 0 7- 2 0 1 6  
 
Pr ot oc ol d oc u me nt date: 0 6- 0 2- 2 0 1 6 
7. 0  
E V A L U A TI O N S, T E S T S, A N D O B S E R V A TI O N S  
 
7. 1 
Re q uire d e v al u ati o ns f or di a g n osis a n d res p o ns e ( all p atie nts)  
 
Baseli ne  Da y 2 2 of I n d I 
Pri or t o eac h 
c o urse of 
t hera p y 
E n d of 
t hera p yg 
At ti me of 
rela pse 
C B C D  
X  
X  
X  
X  
X  
C M P  
X  
 
X  
X  
X  
E C H O or M U G A  
X  
 
X e  
X e  
 
E K G  - R E Q UI R E D  
X  
 
X e  
X e  
 
B M A a  
X a  
X f 
X f 
X f 
X f 
B o ne marr o w bi o ps y b  
X b  
 
 
 
 
Peri p heral bl o o d  M R D  
 
Da ys 8 a n d 2 2  
 
 
 
Cell c o u nt a n d c yt os pi n 
of C S F c 
X 
X 
X 
 
X 
Pre g na nc y test d  
X  
 
 
 
 
H L A t y pi n g of patie nt, 
pare nts, a n d f ull si bli n gs  
X h 
 
 
 
 
N K t y pi n g of pare nts i 
 
 
Pri or t o c o ns I  
 
 
N K cell rece pt or st u dies  
X  
 
Pri or t o c o ns I  
 
 
P har mac o ge netic st u dies  
X  
 
Pri or t o c o ns I   
 
 
C B C D, c o m plete bl o o d c o u nt wit h differe nti al; C M P, c o m plete met a b olic p a nel, i ncl u di n g electr olytes, B U N, 
cre ati ni ne, A S T, A L T, bilir u bi n. D aily L F Ts ( A L T, A S T, t ot al a n d direct bilir u bi n) a n d ser u m a m m o ni a levels d uri n g 
v ori n os t at a d mi nistr ati o n f or p atie nts receivi n g v ori n ost at d uri n g I n d ucti o n II.  
a At t he ti me of e nr oll me nt, a b o ne m arr o w as pir ate s h o ul d be o bt ai ne d f or m or p h ol o gic, i m m u n o p he n oty pic 
(i ncl u di n g t he i de ntific ati o n of a le uke mi a-ass oci ate d p he n oty pe f or M R D st u d ies), m olec ul ar, a n d cyt o ge netic 
a n alyses. F or p atie nts wit h elev ate d le uk ocyte c o u nts a n d hi g h bl ast perce nt a ges, a n d p atie nts t o o ill t o u n der g o b o ne 
m arr o w as pir ate, all di a g n ostic st u dies m ay be perf or me d o n bl o o d r at her t h a n b o ne m arr o w.  
b B o ne m arr o w bi o psies m ay be o mitte d i n c ases i n w hic h a di a g n ostic b o ne m arr o w as pir ate is n ot perf or me d.  
c Ex a mi n ati o n of t he C S F s h o ul d be perf or me d wit h every d ose of i ntr at hec al t her a py  
d A p plies o nly t o fe m ale p atie nts of c hil d be ari n g p ote nti al  
e E C H O or M U G A,  a n d E K G s h o ul d be perf or me d pri or t o c he m ot her a py c o urses t h at i ncl u de d a u n or u bici n or 
mit ox a ntr o ne. P atie nts w h o h ave a n or m al b aseli ne E C H O or M U G A a n d w h o receive cl of ar a bi ne a n d cyt ar a bi ne i n 
I n d ucti o n I d o n ot nee d a c ar di ac ev al u ati o n pri or t o I n d ucti o n II. T he e n d of t her a py c ar di ac ev al u ati o ns m ay be 
perf or me d a nyti me wit hi n t he first ye ar after c o m pleti o n of t her a py.    
f All s u bse q ue nt b o ne m arr o w as pir ates s h o ul d be se nt f or m or p h ol o gic a n d M R D a n alyses o n all p atie nts. I n a d diti o n, 
f or p atie nts wit h le uke mi a -s pecific f usi o n tr a nscri pts, all s u bse q ue nt b o ne m arr o w as pir ates s h o ul d be se nt f or R T -
P C R a n alysis.  
g T he ti me of ne utr o p hil a n d pl atelet rec overy after c o ns oli d ati o n II ( or after N K cell t her a py  w here a p plic a ble) will be 
c o nsi dere d t he e n d  of t her a py.  
h H L A ty pi n g of p atie nts s h o ul d i ncl u de allele level res ol uti o n f or H L A - A, B, C w, a n d D R1. H L A ty pi n g m ay be d o ne 
at St. J u de at n o c ost t o p artici p a nt, f a mily or c oll a b or ati n g i nstit uti o ns.  
i F or p atie nts w h o are p ote nti al c a n di d ates f or N K cell t her a py (see secti o n 4. 1), N K  a n d H L A  ty pi n g m ust be o bt ai ne d 
fr o m b ot h p are nts pri or t o c o ns oli d ati o n I.  
 
See secti o n 1 0. 3 f or P h ar m ac oki netic St u dies  
See secti o n 1 0. 5 f or Hist o ne acetyl ati o n St u dies  
 

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7.2 
Additional required evaluations for patients receiving NK cells 
 
7.2.1 Pre-study evaluation for recipients 
 
• Pre-transplant chimerism  
• NK cell phenotyping  
 
7.2.2 Pre-study evaluation for donors 
 
• HLA typing 
• Pre-transplant chimerism  
• NK cell phenotyping  
 
7.2.3 Evaluations during treatment 
 
• BMA for morphology, MRD, and RT-PCR (if applicable) 1 month after NK cell 
infusion 
• NK cell chimerism studies  should be performed on days 7, 14, 21, and 28 
 
Note: because NK cell studies are performed only on weekdays (Monday through Friday), 
these tests may be sent within 48 hours of the time they are due (e.g., if day 2 falls on a 
Sunday, the tests may be drawn on day 3). Post-transplant chimerism studies should also be 
performed after day 28 in cases of persistent chimerism. 
 
7.2.4  NK cell recipients will be monitored for a period of 45 days after the NK cell 
infusion for GVHD. GVHD will be evaluated and graded using the criteria found in 
Appendix II of this protocol. 
 
7.3 
Long-term follow-up evaluations 
 
The time of neutrophil and platelet recovery after consolidation II or NK cell infusion will 
be considered the end of therapy. 
 
7.3.1 CBC with differential and platelet count 
 
As clinically indicated 
 
7.3.2 Bone marrow examinations 
 
Bone marrow aspirates for MRD (and RT-PCR if applicable) should be performed every 4 
months for one year (i.e., 3 additional evaluations following the end of therapy evaluation). 
If there is evidence of an aberrant phenotype or leukemia-specific fusion transcript at one 
year, bone marrow examinations should continue to be performed every 4-6 months or as 
clinically indicated. 
 
7.3.3 Echocardiograms and EKG should be performed yearly. 

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7.4 
Routine tests 
 
All studies listed in Table 7.1 and 7.3 are considered part of the routine care of AML 
patients, with exception of NK typing, NK cell receptor studies, pharmacokinetic, 
pharmacogenetic, and histone acetylation studies. 
 
7.5 
Research tests 
 
NK typing studies, NK cell receptor (chimerism and phenotyping) studies, 
pharmacokinetic, pharmacogenetic, and histone acetylation studies, high-resolution single 
nucleotide polymorphism (SNP) array and gene expression analysis, phospho-flow 
profiling, in vitro drug sensitivity assays, and minimal residual disease studies are 
considered research tests in this study. 
 
8.0  
EVALUATION CRITERIA 
 
8.1 
Response criteria 
 
The response after each course of chemotherapy will be determined by the examination of 
the bone marrow before the next course of chemotherapy is begun. If two bone marrow 
examinations are performed after a course of therapy, the second examination will be used 
to classify the response to that course. For the purposes of this protocol, MRD-negative is 
defined as < 0.1% blasts with a leukemia-associated phenotype detected by flow cytometry. 
Because morphologic examination of the bone marrow during periods of hematopoietic 
recovery after intensive chemotherapy may be unreliable, response will be based on blast 
percentage by flow cytometry (MRD). Blast percentages determined by morphology will 
be used in cases that are not evaluable by flow cytometry. If the blast percentage is less 
than 5% in such cases, they will be classified as Complete Response, MRD not evaluable.  
 
8.1.1 Complete response, MRD-negative 
• < 0.1% blasts by flow cytometry 
 
8.1.2 Complete response, MRD-positive 
• 0.1% to ≤ 5% blasts by flow cytometry 
 
8.1.3 Partial response 
• 5% to 25% blasts by flow cytometry 
 
8.1.4 No response 
• > 25% blasts in the bone marrow by flow cytometry 
 
8.1.5 Relapse 
• Subsequent appearance, after achievement of CR, of ≥ 5% blasts in the bone 
marrow by flow cytometry 
 

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8.2 
Toxicity evaluation criteria 
 
This study will utilize the CTCAE Version 3.0 for toxicity and performance reporting.  A 
copy of the CTCAE version 3.0 can be downloaded from the CTEP home page 
(http://ctep.info.nih.gov). 
 
9.0 
OFF THERAPY AND OFF-STUDY CRITERIA 
 
9.1 
Criteria for removal from protocol therapy (off therapy) 
 
• Patients who have ≥ 5% MRD after two courses of therapy may be removed 
from protocol therapy and entered onto protocols for relapsed or refractory 
disease at the investigator’s discretion. Alternatively, these patients may 
proceed directly to SCT 
• Relapse at any site 
• Second malignancy 
• Refusal of therapy  
• Development of unacceptable toxicity during therapy 
• Completion of planned therapy 
• Major deviation in protocol therapy not approved by the PI 
• Enrollment on another therapeutic study or treatment plan 
 
9.2 
Criteria for removal from study (off study) 
 
• Death 
• Lost to follow-up 
• Withdrawal of consent for further participation or data submission 
• Found, after enrollment, to be ineligible 
 
9.3 
NK cell donors 
 
NK cell donors will be followed for adverse events and serious adverse events on the day 
of apheresis and for seven days following apheresis. Donors will be removed from therapy 
(off therapy) and from study (off study) on the eighth day after apheresis.  
 
9.4 
NK cell recipients 
 
Patients who receive NK cell infusions will receive no further therapy, other than 
supportive care, after the sixth dose of IL-2, which is given on day +9 after the infusion. 
Thus, they will essentially be off therapy at that time. We will, however, follow these 
patients until all toxicities have resolved to less than grade 2.  
 

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10.0 
BIOLOGIC STUDIES 
 
10.1 
NK cell receptor study 
 
Peripheral blood will be obtained prior to Induction I and prior to Consolidation I for 
immunophenotyping and genotyping for NK cell receptors as described previously.46,164 
We will assess NK cell receptors including KIRs, NCRs, NKG2D, DNAM-1, 2B4, and 
NTBA. 
 
10.2 
Minimal residual disease studies 
 
This study will apply immunologic and molecular methods as performed in AML02. As in 
AML02, immunologic methods will be used for clinical purposes, but molecular results will 
be reported as well. All participants registered in AML08 whose leukemic cells are amenable 
to our assays will be eligible for testing. Peripheral blood samples (5-10 ml) will be taken 
only on days 8 and 22. Bone marrow samples will be analyzed at all time points. 
 
10.3 
Pharmacokinetic studies 
 
Note: Pharmacokinetic studies were completed with LOA #4, 5/29/2015 
 
In order to study the pharmacokinetics of sorafenib, 7 blood samples (3 mL each) will be 
collected in a green top tube prior to the first dose on day 1, then serial blood sampling on 
day 8 (at steady-state) at pre-treatment, then at 2, 4.5 and 7.5 hours. Weekly pre-treatment 
troughs before the morning dose will be obtained on days 15 ± 2 days and 21 ± 2 days for 
the first cycle. Samples for sorafenib pharmacokinetics should be obtained even if 
sorafenib is discontinued prior to the day of sampling. All pre-treatment trough samples 
should be obtained within a ± 3 hour window (9-15 hours after the previous dose but before 
the subsequent dose). Total sorafenib will be quantitated using a validated LC-MS-MS 
assay and unbound sorafenib will be measured using a validated micro-equilibrium dialysis 
method in the Pharmacokinetics Shared Resource with Dr. Sharyn Baker as the 
pharmacokinetic investigator for these studies. Pharmacokinetic data will be analyzed 
using model-independent and modeling techniques. See appendix II for sample processing 
and shipping instructions.   
 
10.4 
Pharmacogenetic studies  
 
Note: Pharmacogenetic studies were completed with LOA #4, 5/29/2015 
 
We will collect peripheral blood samples at diagnosis and at the start of consolidation (or at 
the time of count recovery after Induction II) for extraction of germline DNA. Assessment 
of germline polymorphisms in drug metabolizing enzymes and drug transporters will be 
performed by Dr. Sharyn Baker and genes involved in the activation pathway of nucleoside 
analogs will be performed by Dr. Jatinder Lamba using a multiplex assay such as a 48-plex 
format (DNAPrint genomics). 
 

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10.5 
In vitro drug sensitivity assays 
 
Drug sensitivity assays completed with LOA #4, 5/29/2015 
 
As part of an ongoing project to develop new drugs for AML in Dr. Sharyn Baker’s 
laboratory, we plan to evaluate the activity of novel agents in diagnostic blasts in a short-
term co-culture system to mimic the bone marrow microenvironment. For example, new 
drugs to be tested include receptor tyrosine kinase inhibitors, STAT inhibitors, PI3K/mTOR 
inhibitors, IGF1-R inhibitors, HDAC inhibitors, and HSP90 inhibitors. 1 x 107 cells will be 
aliquoted and frozen until the time of in vitro testing. Blast cells will be thawed and plated on 
mesenchymal stromal cells, co-cultured overnight and treated with drug for 72 hours. Cell 
viability and apoptosis will be assessed by flow cytometry in the Pharmaceutical Sciences 
department. If enough cells are available a second aliquot of 1 x 107 cells will be used to 
assess changes in gene expression before and after drug treatment using the latest microarray 
technology in the Hartwell center. For example, we are interested in changes in genome wide 
changes in methylation status after ex vivo treatment of blasts cells with HDAC inhibitors.  
 
10.6 
Histone acetylation studies 
 
Histone acetylation studies completed with Revision 10.1, 6/2/16 
 
For patients who receive vorinostat (see section 4.6), histone acetylation will be assessed in 
peripheral blood samples (collected in 3 mL of preservative-free heparin) on days -2, 1, and 6 
of Induction II as described in section 4.2.6. Histone H3 and H4 acetylation will be analyzed 
by Western blot using antibodies directed against acetylated histone H3 or H4.100  
 
10.7 
Other biology studies  
 
High-resolution single nucleotide polymorphism (SNP) array and gene expression analysis 
will be performed as described in section 2.2.4. Phospho-flow profiling will be performed 
as described in section 2.2.5. 
 
11.0 
SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS 
 
11.1 
Adverse event monitoring and reporting 
 
This study will utilize the CTCAE Version 3.0 for toxicity and performance reporting.  A 
copy of the CTCAE version 3.0 can be downloaded from the CTEP home page 
(http://ctep.info.nih.gov).  Additionally, the toxicities are to be reported on the appropriate 
data collection forms or electronic data entry screens. 
 
11.2 
Reporting adverse events and deaths to St. Jude IRB 
 
Only “unanticipated problems involving risks to participants or others” referred to hereafter 
as “unanticipated problems” are required to be reported to the St. Jude IRB promptly, but 
in no event later than 10 working days after the investigator first learns of the unanticipated 
problem.  Regardless of whether the event is internal or external (for example, an IND 
safety report by the sponsor pursuant to 21 CFR 312.32), only adverse events that 

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constitute unanticipated problems are reportable to the St. Jude IRB.  As further described 
in the definition of unanticipated problem, this includes any event that in the PI’s opinion 
was: 
• Unexpected (in terms of nature, severity, or frequency) given (1) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document, as well as other 
relevant information available about the research; (2) the observed rate of 
occurrence (compared to a credible baseline for comparison); and (3) the 
characteristics of the subject population being studied; and 
• Related or possibly related to participation in the research; and 
• Serious; or if not serious suggests that the research places subjects or others at a 
greater risk of harm (including physical, psychological, economic, or social harm) 
than was previously known or recognized.  
 
Unrelated, expected deaths do not require reporting to the IRB (e.g., deaths off therapy and 
not related to protocol therapy).  Though death is “serious”, the event must meet the other 
two requirements of “related or possibly related” and “unexpected/unanticipated” to be 
considered reportable. 
 
Deaths meeting reporting requirements are to be reported immediately to the St. Jude IRB, 
but in no event later than 48 hours after the investigator first learns of the death.  For the 
purposes of this study, this includes all deaths of patients on active protocol therapy or 
within 30 days of receiving protocol therapy. 
 
The following definitions apply with respect to reporting adverse experiences:  
 
Serious Adverse Event: Any adverse event temporally associated with the subject’s 
participation in research that meets any of the following criteria: 
• results in death; 
• is life-threatening (places the subject at immediate risk of death from the event as it 
occurred); 
• requires inpatient hospitalization or prolongation of existing hospitalization; 
• results in a persistent or significant disability/incapacity; 
• results in a congenital anomaly/birth defect; or 
• any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention to 
prevent one of the other outcomes listed in this definition (examples of such events 
include:  any substantial disruption of the ability to conduct normal life functions, 
allergic bronchospasm requiring intensive treatment in the emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, 
or the development of drug dependency or drug abuse), a congenital anomaly/birth 
defect, secondary or concurrent cancer, medication overdose, or is any medical 
event which requires treatment to prevent any of the medical outcomes previously 
listed.   
 

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Unexpected Adverse Event:  
• Any adverse event for which the specificity or severity is not consistent with the 
protocol-related documents, including the applicable investigator brochure, IRB 
approved consent form, Investigational New Drug (IND) or Investigational Device 
Exemption (IDE) application, or other relevant sources of information, such as 
product labeling and package inserts; or if it does appear in such documents, an 
event in which the specificity, severity or duration is not consistent with the risk 
information included therein; or  
• The observed rate of occurrence is a clinically significant increase in the expected 
rate (based on a credible baseline rate for comparison); or  
• The occurrence is not consistent with the expected natural progression of any 
underlying disease, disorder, or condition of the subject(s) experiencing the adverse 
event and the subject’s predisposing risk factor profile for the adverse event.  
 
Internal Events: Events experienced by a research participant enrolled at a site under the 
jurisdiction of St. Jude IRB for either multicenter or single-center research projects. 
 
External Events: Events experienced by participants enrolled at a site external to the 
jurisdiction of the St. Jude Institutional Review Board (IRB) or in a study for which St. 
Jude is not the coordinating center or the IRB of record. 
 
Unanticipated Problem Involving Risks to Subjects or Others: An unanticipated 
problem involving risks to subjects or others is an event which was not expected to occur 
and which increases the degree of risk posed to research participants.  Such events, in 
general, meet all of the following criteria:  
 
• unexpected;  
• related or possibly related to participation in the research; and  
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized.  An unanticipated problem involving risk to subjects or 
others may exist even when actual harm does not occur to any participant.  
 
Consistent with FDA and OHRP guidance on reporting unanticipated problems and 
adverse events to IRBs, the St. Jude IRB does not require the submission of external 
events, for example IND safety reports, nor is a summary of such events/reports required; 
however, if an event giving rise to an IND safety or other external event report constitutes 
an “unanticipated problem involving risks to subjects or others” it must be reported in 
accordance with this policy.  In general, to be reportable external events need to have 
implications for the conduct of the study (for example, requiring a significant and usually 
safety-related change in the protocol and/or informed consent form). 
 
Although some adverse events will qualify as unanticipated problems involving risks to 
subjects or others, some will not; and there may be other unanticipated problems that go 
beyond the definitions of serious and/or unexpected adverse events. 
 

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Examples of unanticipated problems involving risks to subjects or others include: 
 
• Improperly staging a participant’s tumor resulting in the participant being assigned 
to an incorrect arm of the research study; 
• The theft of a research computer containing confidential subject information 
(breach of confidentiality); and  
• The contamination of a study drug.  Unanticipated problems generally will warrant 
consideration of substantive changes in the research protocol or informed consent 
process/document or other corrective actions in order to protect the safety, welfare, 
or rights of subjects or others. 
 
11.3  Reporting requirements from St. Jude to FDA 
 
Any unexpected fatal or unexpected life-threatening event (e.g. progressive multifocal 
leukoencephalopathy or pulmonary toxicity as described on page 47) judged by the PI to 
possibly be due to the investigational agent, will be reported to the FDA by telephone or 
fax as soon as possible but no later than seven calendar days after notification of the event 
and followed by a written safety report as complete as possible within eight additional 
calendar days (i.e. full report 15 calendar days total after notification of event). 
 
Unexpected, non-fatal and non-life-threatening SAEs, which occur in on-study participants 
during the time periods specified in Section 9.1 that are considered due to or possibly due 
to the investigational agent, will be reported to the FDA by written safety report as soon as 
possible but no later than 15 calendar days of the notification of the occurrence of the 
event. Expected SAEs, even unexpected fatal SAEs, considered by the PI to be not related 
to the study, will be reported to the FDA in the Annual Review Report along with non-
serious AEs.  All FDA correspondence and reporting will be conducted through the St. 
Jude Office of Regulatory Affairs. 
 
11.4  Reporting to St. Jude Regulatory Affairs Office (RAO) 
 
Copies of all correspondence to the St. Jude IRB, including SAE reports, are provided to 
the St. Jude Regulatory Affairs office by the St. Jude study team.  FDA-related 
correspondence and reporting will be conducted through the Regulatory Affairs office. 
 
11.5 
Reporting from St. Jude to Sanofi Group Pharmacovigilance 
 
The following reporting requirements apply only to participants treated with clofarabine 
during Induction I, and are applicable only during the time period from first dose of 
clofarabine until recovery from Induction I and start of Induction II.  
 
Serious adverse event (SAE): any untoward medical occurrence that at any dose:  
 
• Results in death, 
• Is life threatening, (Note: the term “life-threatening” refers to an event/reaction in 
which the patient was at risk of death at the time of the event/reaction; it does not 
refer to an event/ reaction which hypothetically might have caused death if it were 
more severe), 

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• Requires inpatient hospitalization or results in prolongation of existing 
hospitalization, 
• Results in persistent or significant disability/incapacity, 
• Is a congenital anomaly/birth defect, or 
• Is a medically important event or reaction. Medical and scientific judgment should 
be exercised in deciding whether other situations should be considered serious, such 
as important medical events that might not be immediately life-threatening or result 
in death or hospitalization, but might jeopardize the patient or might require 
intervention to prevent one of the other outcomes listed in the definition above. 
 
Related Adverse Event, i.e. Adverse Drug Reaction (ADR): There is a reasonable 
possibility according to the IST/ISS sponsor that the product may have caused the event. 
 
Unexpected Adverse Drug Reaction: An adverse reaction, the nature or severity of which is 
not consistent with the applicable product information (e.g., Investigator's Brochure for an 
unapproved investigational medicinal product or package insert/summary of product 
characteristics for an approved product). An expected ADR with a fatal outcome should be 
considered unexpected unless the local/regional product labeling specifically states that the 
ADR might be associated with a fatal outcome. 
 
AESI: An adverse event of special interest (AESI) is an adverse event (serious or non-
serious) of scientific and medical concern specific to the Sponsor’s product or program, for 
which ongoing monitoring and rapid communication by the Investigator to the Sponsor 
may be appropriate. Such events may require further investigation in order to characterize 
and understand them. AESIs may be added or removed during a study by protocol 
amendment. 
 
St. Jude PI and study team (Investigator-sponsored trial or IST) responsibilities include: 
 
• The IST/ISS sponsor warrants that the study will be performed in compliance with 
all applicable local and international laws and regulations, including without 
limitation ICH E6 guidelines for Good Clinical Practices.  
• The IST/ISS sponsor shall be responsible for the respect of all obligations required 
by applicable local and international laws and regulations.  
• The sponsor shall be responsible for ensuring submission of required expedited and 
periodic reports to the appropriate Health Authority (HA), the Ethics Committee 
and investigators of each country participating in the IST/ISS (based on applicable 
regulations). 
• The IST/ISS sponsor is responsible for providing any “Dear Investigator Letter” 
(DIL) for new safety finding received from Sanofi group entity to the investigators 
and Ethics Committee in each country participating in the study. 
• The sponsor must report the following information in English to the Sanofi group 
entity Pharmacovigilance contact: 
 
1. Routine transmission of: Only related SAEs must be transmitted within 1 working day of 
the Investigator’s awareness or identification of the event. 

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2. Other events or periodic reports (e.g. Development Safety Update Report (DSUR)), 
submitted to Regulatory Authority must be transmitted at the time of submission. 
3. Other significant safety issues or findings in a study pertaining to safety of product 
must be transmitted within 1 working day. (e.g., Data Safety Monitoring Board 
recommendations). 
4. The study report of any IST/ISS must contain a section describing safety review and 
conclusion. 
5. The reference safety information to be used by the IST/ISS sponsor for evaluation of 
expectedness of adverse events shall be the current approved product label available in 
the country. 
 
Sanofi Group Entity Pharmacovigilance Contact: 
 
IST/ISS Investigators will notify Sanofi via fax or email, attention: Sanofi 
Pharmacovigilance (PV): 
 
Fax: 
   
E-mail:  
 
 
11.6 
Other reporting mechanisms 
 
Copies of all correspondence to the St. Jude Institutional Review Board (IRB), including 
SAE reports, are provided to the St. Jude Office of Research Compliance and Regulatory 
Affairs (ORCRA). All FDA related correspondence and reporting will be conducted 
through the ORCRA. Continuing review reports of protocol progress and summaries of 
adverse events will be filed with the St. Jude IRB, Institutional Biosafety Committee (IBC), 
and FDA at least annually. 
 
11.7 
Process for reporting AEs to/from St. Jude and collaborating sites 
 
Adverse events from collaborating sites will also be reviewed by the PI and discussed in 
study team meetings as described above.  SAE reports from collaborating sites for AEs that 
are serious, unexpected, and at least possibly related to protocol treatment or interventions 
will be reported to site IRB and the St. Jude IRB within the reporting requirements 
described above.  The PI will determine if this is an event that will need to be reported 
expeditiously to all participating sites, considering the following criteria: 
 
• Is the AE serious, unexpected, and related or possibly related to participation in the 
research? 
• Is the AE expected, but occurring at a significantly higher frequency or severity 
than expected? 
• Is this an AE that is unexpected (regardless of severity that may alter the IRB’s 
analysis of the risk versus potential benefit of the research and, as a result, warrant 
consideration of substantive changes in the research protocol or informed consent 
process/document? 
 
With the submission of the “Reportable Event” in St. Jude TRACKS application, the PI 
will indicate if all sites should be notified to report to their IRBs, and if the protocol and/or 

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consent should be amended (consent will be amended if event is information that should be 
communicated to currently enrolled subjects).   
 
Generally, only events that warrant an amendment to the protocol and/or consent will be 
reported expeditiously to all sites.  However, any event may be reported expeditiously to all 
sites at the discretion of the PI.  
 
A cumulative summary of Grade 3-5 AEs and expected/unrelated deaths that occur more 
than 30 days after protocol treatment will be reported to all sites with study progress report 
at the time of continuing review.  
 
For collaborating sites: Serious AND unexpected events are to be reported to the St. Jude 
PI (Dr. Jeffrey Rubnitz) within 72 hours via phone call or email.  Unexpected deaths must 
be reported to the St. Jude PI via phone call or email within 24 hours of the event.  A 
written report must follow.  In addition, the study coordinator 
 should 
also be contacted regarding the event.  
 
Jeffery Rubnitz 
Leukemia/Lymphoma Division 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place 
Memphis, TN 38105 
Phone: 
 
Email: 
 
 
 
Leukemia/Lymphoma Division 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place 
Memphis, TN  38105 
Phone: 
 
Email: 
 
 
For the purposes of this protocol, the following events will NOT be considered serious nor 
unexpected adverse events: 
 
• Hospitalization for treatment related febrile neutropenia 
• Hospitalization for expected complications of treatment or expected toxicities of the 
commercially available agents used in this study (except for grade 4 non-
hematologic toxicities) 
• Hospitalization for treatment of expected signs or symptoms of disease 
complications or progression of disease 
• Death not related to therapy (e.g. disease progression) > 30 days after last dose of 
protocol therapy 
 

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12.0 
DATA COLLECTION, STUDY MONITORING AND CONFIDENTIALITY 
 
12.1 
Data collection and management 
 
Data Management will be supervised by the Director of Clinical Trials Management, 
Oncology Programs, working with Dr. Rubnitz or his designee. All protocol-specific data 
and all grade 3-5 non-hematological adverse events will be recorded by the clinical 
research associates into the CRIS database, ideally within 2-4 weeks of completion of study 
phase. All questions will be directed to the attending physician and/or PI and reviewed at 
regularly-scheduled working meetings. The attending physicians (or their designees) are 
responsible for keeping up-to-date roadmaps in the patient’s primary SJCRH medical chart. 
 
Regular (at least monthly) summaries of toxicity and protocol events will be generated for 
the PI and the department of Biostatistics to review.  Biostatistics will work with the PI in 
generating the progress reports for the IRB and the DSMB.  
 
12.2 
Study monitoring 
 
The Eligibility Coordinators will verify 100 % of the informed consent documentation on 
all participants and verify 100% of St. Jude participants’ eligibility status within 10 
working days of the completion of enrollment. 
The study team will meet at appropriate intervals to review case histories or quality 
summaries on participants. 
 
The Clinical Research Monitor will assess protocol and regulatory compliance as well as 
the accuracy and completeness of all data points for the first two participants then 15% of 
study enrollees every six months. Accrual will be tracked continuously for studies that have 
strata.   All SAE reports will be monitored for type, grade, attribution, duration, timeliness 
and appropriateness on all study participants semi-annually. 
 
The monitor will also verify 100% of all data points on the first two participants and on 
15% of cases thereafter.  Protocol compliance monitoring will include participant status, 
eligibility, the informed consent process, demographics, staging, study objectives, 
subgroup assignment, treatments, evaluations, responses, participant protocol status, off-
study, and off-therapy criteria.  The Monitor will generate a formal report which is shared 
with the Principal Investigator (PI), study team and the Internal Monitoring Committee 
(IMC). Monitoring may be conducted more frequently if deemed necessary by the CPDMO 
or the IMC. 
 
Continuing reviews by the IRB and CT-SRC will occur at least annually.  In addition, SAE 
reports in TRACKS (Total Research and Knowledge System) are reviewed in a timely 
manner by the IRB/ OHSP. 
 
Monitoring of this protocol is considered to be in the High-Risk 3 (HR-3) category, 
according to the St. Jude Data Safety and Monitoring Plan (DSMP).  The Monitoring Plan 
is outlined in a separate document from this protocol, but has been submitted for review 
and approval by the Clinical Trials Scientific Review Committee and the Institutional 
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St. Jude affiliates and domestic collaborating study sites will be monitored on-site by a 
representative of St. Jude at intervals specified in the Data and Safety Monitoring Plan.  
International collaborators will be monitored by a Contract Research Organization (CRO) 
under contractual agreement with St. Jude. 
 
12.3 
Confidentiality 
 
Study numbers will be used in place of an identifier such as a medical record number. No 
research participant names will be recorded on the data collection forms. The list containing 
the study number and the medical record number will be maintained in a locked file and will 
be destroyed after all data have been analyzed. The medical records of study participants 
may be reviewed by the St. Jude IRB, FDA, and St. Jude clinical research monitors. 
 
13.0 
STATISTICAL CONSIDERATIONS 
 
13.1 
Design and analysis for primary objective 
 
The primary objective of this study is to compare two initial courses of therapy in terms of 
the proportion of patients who have MRD levels of less than 0.1% (MRD-) as measured by 
flow cytometry. The two courses to be compared are HD-ADE and Clo/AraC.  HD-ADE 
was studied as an initial course in the AML02 protocol. As of February 2008, 57 of 91 
(62.6%) MRD-evaluable patients on AML02 had no detectable MRD after treatment with 
one course of HD-ADE. 
 
In AML02, we observed a strong correlation between MRD levels at day 22 and event-free 
survival.  In AML02, the 3-year EFS of MRD- and MRD+ AML02 patients are 74.3% (+/- 
7.0% SE) and 47.2% (+/- 9.2% SE), respectively (p = 0.001). Clearly, MRD after one 
course is strongly correlated with long-term prognosis.  As such, MRD serves as a useful 
measure of treatment efficacy. 
 
The MRD- rate among AML02 patients varies substantially by risk-group. When classified 
according to the risk definitions of AML08, 21 of 23 (91.3 %) low-risk patients, 27 of 41 
(65.9%) standard-risk patients, and 9 of 27 (33.3%) high-risk patients were MRD- after one 
course. Therefore, the statistical analysis for the primary objective of this protocol will be 
stratified by risk group.   
 
In this study, patients will be randomized to receive HD-ADE or Clo/AraC as their initial 
course of therapy and MRD will be evaluated at day 22 of this course. The design calls for 
enrollment of up to 240 MRD-evaluable patients (Table 13.1). The design allows for 4 
interim analyses and one final analysis. In each analysis, an exact risk-stratified Mantel-
Haenzel test will be used to test the null hypothesis that the ratio of the odds of MRD- on 
Clo/AraC to the odds of MRD- on HD-ADE is equal to one. The risk-stratification 
categories for the analysis are defined in section 4.1 of the protocol.  MRD-status will not 
be considered in assigning subjects to risk-strata for purposes of this statistical analysis. In 
the four interim analyses, the result will be considered statistically significant if the p-value 
is less than 0.005. If an interim analysis gives a significant p-value at the 0.005 level, we 
will temporarily cease accrual and consult the St. Jude DSMB on how to best proceed. In 

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the final analysis, the result will be considered statistically significant if the p-value is less 
than 0.0429. The level of the final analysis was determined using the Haybittle-Peto 
method as implemented in East software, windows version 5.2. 
 
We performed simulations to further evaluate the level and power of the selected design. 
The simulation is based on one million replications. With risk-specific MRD-rates equal to 
those observed on the HD-ADE arm of AML02, the approximate level is 0.0396 (99.9% CI 
= 0.0389 – 0.0402). The design provides a power of approximately 0.8013 (99.9% CI = 
0.7999 – 0.8026) to declare statistical significance if the odds of MRD- of the Clo/araC are 
0.4 times that of the HD-ADE arm. Assuming an equal odds ratio for all risk groups, an 
odds ratio of 0.4 corresponds to MRD- rate of 62.6% in the HD-ADE arm and an MRD- 
rate of 45.0% in the Clo/AraC arm. For the Clo/AraC arm, this corresponds to assumed 
MRD- rates of 80.7%, 43.5%, and 16.7% among low, standard, and high risk patients, 
respectively. 
 
Also, the design provides power of approximately  0.8297 (99.9% CI = 0.8284 – 0.8309) to 
detect the setting in which the odds of MRD- on HD-ADE are 2.5 times the odds of MRD- 
on HD-ADE. An odds ratio of 2.5 corresponds to an MRD- rate of 62.6% in the HD-ADE 
arm and an MRD- rate of 78.1% in the Clo/AraC arm.  For the Clo/AraC arm, this 
corresponds to assumed MRD- rates of 96.3%, 82.8%, and 55.5% among low, standard, 
and high risk patients, respectively.  
 
Table 13.1. Statistical Characteristics of the Design. The table gives the number of subjects 
in each arm at each interim analysis (n/arm), the p-value threshold used in each interim 
analysis (α), the cumulative level, i.e., the cumulative probability of a Type I error over the 
interim analyses (Cum. Level), the cumulative power (Cum. Power) over the interim 
analyses for an odds ratio of 2.5, and the cumulative power over the interim analyses for an 
odds ratio of 0.4. Estimates of level and power are based on simulations with one million 
replications. 
 
  
  
Cum. 
Cum. Power 
Cum. Power 
n/arm 
a 
Level 
(OR = 2.5) 
(OR = 0.4) 
48 
0.005 
0.002861 
0.044709 
0.038920 
96 
0.005 
0.005752 
0.162844 
0.145386 
144 
0.005 
0.008301 
0.316510 
0.287142 
192 
0.005 
0.010505 
0.472528 
0.435433 
240 
0.0429 
0.039559 
0.829673 
0.801282 
 
 
 
 
 
 
Accrual targets are based on the number of MRD-evaluable patients. As of February 2008, 
23 of 209 (11%) patients treated on AML02 were not evaluable for MRD after their first 
course of therapy. If the same proportion of patients is not evaluable for MRD in AML08, a 
total of 270 patients will need to be randomized to obtain 240 MRD-evaluable patients. At 
the accrual rate of 40 patients per year observed on AML02, roughly 7 years are required to 
accrue 270 eligible patients. 
 
The randomization will be stratified according to risk features of the disease. For patients 
for whom karyotype is available, the randomization will be stratified by inv(16) vs. t(8,21) 
vs. 11q23 vs. M7 vs. others. If the karyotype is not available, the randomization will be 

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stratified primarily by FAB type: M2 with Auer rods vs. M4Eo vs. M5 vs. M7 vs. others. 
The randomization is stratified to maintain a balance between the two arms with respect to 
factors that are known to affect the secondary outcome measures such as CR rate and EFS. 
 
The randomization will be performed using a program that implements the block-
randomization scheme proposed by Zelen.179 The program resides on our network and has 
been routinely used for randomization since 1992. The Pharmacy will be provided access 
to the program and will be responsible for randomizing patients. The system stores all 
required data for randomization in a secure Access database. Once a patient is randomized, 
all related data are frozen in the database and cannot be changed. All eligible patients who 
are registered and randomized will be included in the analyses consistent with the intent-to-
treat principle. 
 
13.2 
Monitoring rules 
 
13.2.1 Monitoring for unequal survival in the two arms 
 
We will compare the event-free and overall survival of the two arms concurrently with 
each interim analysis. At each interim analysis, we will perform the exact log-rank test at 
the 0.005 level to compare event-free and overall survival. If the results are significant, we 
will temporarily cease accrual and seek the guidance of the St. Jude DSMB in determining 
whether to continue the study. Additionally, a level of 0.0429 will be used in the final 
analysis of the secondary objective of comparing survival distributions.  This will result in 
a Type I error rate of less than or equal to 0.05 according to the Haybittle-Peto method as 
implemented in East software, windows version 5.2. For these stopping rules, EFS is 
defined as the time elapsed from the date of study enrollment to death, relapse, or diagnosis 
of a second malignancy, with event-free patients censored at last follow-up. OS is defined 
as the time elapsed from enrollment date to date of death, with living patients censored at 
last follow-up. 
 
Table 13.2 gives simulation-based estimates of the probability of rejecting the null 
hypothesis and the expected enrollment of the study of the EFS monitoring rule for specific 
values of the 3-year EFS. Each simulation transformed the Kaplan-Meier EFS of the HD-
ADE arm of AML02 by raising it to the power that gives the value of the 3-year EFS for 
each arm to be simulated. Data was generated from the transformed survival curve for the 
simulation. The simulation used one million independent realizations for each set of EFS-
values for HD-ADE and Clo/AraC. The simulation indicates that the monitoring rule has a 
Type I error probability of less than 0.05 (see probability of rejection in highlighted cells).  
 
Table 13.3 shows simulation-based estimates for the OS monitoring rule.  The simulation 
was performed using the historical OS experience in the HD-ADE arm of AML02 in an 
analogous manner as described above. One-million independent realizations were 
generated and the simulation indicates that the Type I error probability is less than or equal 
0.05.  
 

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Table 13.2.  Simulation Estimates of the Statistical Characteristics of the EFS monitoring rule.  For each scenario, the 
table provides simulation estimates of the probability of rejecting the null hypothesis of equal EFS at some point in the 
study [Pr(Rej)] and the expected number of subjects enrolled [E(n)] on the study prior to rejection of the null 
hypothesis as part of the monitoring. 
3-yr EFS 
HD-ADE 
45% 
50% 
55% 
60% 
65% 
Clo/araC 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
30% 
0.431 
237 
0.716 
209 
0.907 
178 
0.982 
150 
0.998 
129 
35% 
0.165 
257 
0.401 
240 
0.687 
213 
0.893 
182 
0.978 
153 
40% 
0.048 
265 
0.155 
258 
0.382 
241 
0.671 
215 
0.886 
184 
45% 
0.022 
267 
0.046 
265 
0.150 
258 
0.375 
242 
0.666 
216 
50% 
0.046 
265 
0.021 
267 
0.045 
265 
0.148 
259 
0.376 
243 
55% 
0.150 
258 
0.046 
265 
0.021 
267 
0.045 
266 
0.150 
259 
60% 
0.374 
242 
0.149 
258 
0.045 
266 
0.020 
267 
0.044 
266 
65% 
0.667 
216 
0.376 
243 
0.151 
259 
0.044 
266 
0.019 
268 
70% 
0.889 
184 
0.678 
216 
0.388 
242 
0.156 
258 
0.045 
266 
75% 
0.980 
154 
0.901 
182 
0.701 
214 
0.412 
241 
0.167 
258 
80% 
0.998 
130 
0.986 
151 
0.921 
178 
0.743 
210 
0.453 
238 
 
Table 13.3.  Simulation Estimates of the Statistical Characteristics of OS monitoring rule.  The table provides 
simulation estimates of the probability of rejecting the null hypothesis of equal EFS [Pr(Rej)] and the expected number 
of subjects enrolled on the protocol prior to rejection of the null hypothesis [E(n)] under the monitoring rule. 
3-yr EFS 
HD-ADE 
45% 
50% 
55% 
60% 
65% 
Clo/araC 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
Pr(Rej) 
E(n) 
30% 
0.404 
241 
0.685 
217 
0.887 
189 
0.975 
163 
0.997 
142 
35% 
0.154 
259 
0.374 
244 
0.654 
221 
0.870 
193 
0.970 
166 
40% 
0.045 
266 
0.144 
259 
0.354 
246 
0.637 
223 
0.863 
195 
45% 
0.020 
267 
0.043 
266 
0.138 
260 
0.348 
247 
0.633 
224 
50% 
0.043 
266 
0.020 
268 
0.042 
266 
0.137 
260 
0.350 
247 
55% 
0.138 
260 
0.042 
266 
0.019 
268 
0.041 
266 
0.138 
260 
60% 
0.347 
247 
0.137 
260 
0.041 
266 
0.018 
268 
0.041 
266 
65% 
0.633 
224 
0.350 
247 
0.138 
260 
0.041 
266 
0.017 
268 
70% 
0.865 
195 
0.643 
224 
0.360 
246 
0.143 
260 
0.041 
267 
75% 
0.972 
167 
0.879 
194 
0.667 
222 
0.382 
245 
0.154 
260 
80% 
0.997 
144 
0.979 
165 
0.901 
190 
0.707 
219 
0.421 
243 
 
13.2.2 Monitoring for deaths due to causes other than disease progression 
 
It is important to monitor the study for an unacceptable rate of death due to causes other 
than disease progression (i.e., infection, toxicity, transplant-related complications, etc.). On 
the AML02 study, as of February 2008, patients randomized to HD-ADE had a cumulative 
incidence of deaths due to causes other than disease progression of 5.7% (± 2.5% SE) at 1 
year, 8.6% (±3.2% SE) at 2 years, and 10.4% (± 3.6% SE) at 3 years post-enrollment. 
Patients randomized to LD-ADE had a similar outcome with a cumulative incidence of 
2.1% (± 1.5% SE) at 1 year, 10.2% (± 3.5% SE) at 2 years, and 11.9% (± 3.9% SE) at 3 

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years post-enrollment. We wish to monitor the AML08 study to ensure that the rate of 
deaths due to causes other than disease progression is similar to or less than that of the 
AML02 study. 
 
Therefore, concurrently with each interim analysis, we will compute the cumulative 
incidence of death due to causes other than disease progression for each arm of the AML08 
study. If the cumulative incidence estimate for either arm is greater than 10% at 1 year or 
15% at 2 years, then we will temporarily halt accrual while determining whether the study 
should continue. Gray’s method will be used to compute cumulative incidence estimates.180 
 
13.2.3 Monitoring for futility 
 
In the spring of 2013, the St. Jude DSMB recommended that stopping rules for futility be 
incorporated into the protocol. In response to their request, Dr. Cheng Cheng developed the 
futility stopping rules shown in Table 13.4. The stopping rule will be evaluated 
concurrently with the fourth planned interim analyses of the primary MRD endpoint (i.e., 
when 192 evaluable patients have been enrolled). At that time, two one-sided tests of the 
odds ratio will be performed and futility will be declared if the p-value of each one-sided 
test crosses the boundary indicated in Table 13.4. The St. Jude DSMB will be notified of 
the result of this analysis. If futility is declared, we will consult the St. Jude DSMB to 
determine subsequent actions to be taken. The futility analysis design is powered for each 
H1 at the effect size OR=2.5 and 0.4 respectively. Tables 13.5 and 13.6 show the statistical 
properties of the rule determined by EAST 5. 
 
Table 13.4.  Futility Analysis Stopping Rules 
Sample size = 192 
Declare futility if  
H1: OR>1 
P>=0.177 
H1: OR<1 
AND P>=0.184 
 
Table 13.5.  Statistical properties of the futility analysis with OR=2.5. 
Information 
Fraction 
Cumulative 
Accrual 
Beta 
Spent 
Boundary 
to Reject 
H1 
Boundary Crossing Probabilities 
Under 
H0 
Under 
H1 
Under 
H1/2 
0.600 
144.000 
0.067 
0.473 
0.527 
0.067 
0.317 
0.800 
192.000 
0.132 
0.177 
0.309 
0.064 
0.232 
1.000 
240.000 
0.200 
0.059 
0.113 
0.068 
0.158 
 
Table 13.6.  Statistical properties of the futility analysis with OR=0.4. 
Information 
Fraction 
Cumulative 
Accrual 
Beta 
Spent 
Boundary 
to Reject 
H1 
Boundary Crossing Probabilities 
Under 
H0 
Under 
H1 
Under 
H1/2 
0.600 
144.000 
0.043 
0.518 
0.482 
0.043 
0.264 
0.800 
192.000 
0.093 
0.184 
0.344 
0.050 
0.238 
1.000 
240.000 
0.149 
0.057 
0.122 
0.054 
0.169 
 

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13.3 
Comparison and stratification for amendment 6 
 
Amendment 6 eliminates the 5th course of therapy from AML08 on the basis of a 
randomized MRC study showing no statistically or clinically meaningful difference in EFS 
or OS according to completion of 4 or 5 courses of chemotherapy. Similar results were 
obtained in a retrospective comparison between those completing 4 or 5 courses of therapy 
among AML02 patients in remission following 4 courses of therapy. 
 
At the conclusion of the AML08 study, we will use the log-rank test to compare EFS and 
OS of patients enrolled before amendment 6 to that of patients enrolled after amendment 6. 
If a significant difference is observed, then we will report the finding, stratify comparisons 
of EFS and OS between the Clo/AraC and HD-ADE arms according to this amendment, 
and include the amendment as a predictor in Cox regression analyses. 
 
Amendment 6 only impacts therapy given after the primary endpoint (Day 22 MRD) is 
evaluated, so there is no need to modify statistical analysis plans for the primary objective. 
 
13.4 
Analyses for secondary objective 
 
Brief analysis strategies for each secondary objective are provided below. Several 
secondary objectives involve extensive multiple-testing. Multiple-testing issues should be 
addressed via control or estimation of the false discovery rate (FDR). Published 
recommendations developed at St. Jude will be used to select the most appropriate FDR 
method for each analysis.181,182 
 
Objective 1.2.1 To describe the event-free survival (EFS) of standard-risk patients who 
receive chemotherapy relative to that of standard-risk patients who chemotherapy followed 
by an infusion of natural killer (NK) cells from a KIR-mismatched donor. 
 
For this objective, we will compute Kaplan-Meier estimates for each group and compare 
those estimates with the exact log-rank test. For this analysis, event-free survival will be 
defined as the time elapsed from the completion of the final course of chemotherapy to 
death or diagnosis of relapse or a second malignancy, with event-free patients censored at 
last follow-up. 
 
To date, ten AML patients have received NK cell therapy during first remission under the 
NKAML protocol. Most of these patients had KIR-mismatched donors and to date none 
have experienced relapse. The therapy is promising but the outcome is not yet well 
characterized.  Additionally, biologic studies suggest that NK cell therapy should be most 
effective in the KIR-mismatch setting. Therefore, there is interest to gain more experience 
with NK-cell therapy and better characterize prognosis of patients receiving this therapy.   
 
Based on experience of AML02 patients, we expect that 28% of enrolled patients will be 
standard-risk and complete 5 courses and be available to contribute to this objective. Thus, 
we anticipate a total of 76 patients to contribute to this objective and expect one-half (38) 
patients to have a KIR-mismatched donor and receive NK-cell therapy. In the HD-ADE 
arm of AML02, 24 patients enrolled prior to July 1, 2007 met the requirements to 

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contribute to this component of AML08. As of February 2008, the estimated 3-year event-
free survival of this group was 74.7% (± 15.3% SE). 
 
13.5 
Analyses for exploratory objectives 
 
Objective 1.3.1 To genotype natural killer (NK) cell receptors and measure their 
expressions at diagnosis and after induction therapy, and to explore the associations these 
features with treatment outcome. 
 
We will use arm-stratified exact chi-square tests to explore the association of the NK cell 
receptor genotypes with response to induction therapy (CR vs. no CR or MRD+ vs. MRD-). 
Additionally, we will explore the possibility of genotype-treatment interactions with logistic 
regression models. 
 
We will explore the association of NK cell receptor genotype and expression with event-
free and overall survival using Cox models, a rank-based test that accounts for censoring, 
or the log-rank test.183,184 
 
Objective 1.3.2 To explore the prognostic value of levels of minimal residual disease in 
peripheral blood at day 8 of induction I. 
 
We will use logistic regression models to explore the association of day 8 MRD in 
peripheral blood with response and post-induction I MRD. We will use Cox regression 
models and Jung’s procedure to explore the association of day 8 MRD in peripheral blood 
with event-free and overall survival. The analysis will account for initial arm. We will also 
account for subsequent therapy if sample sizes allow. 
 
Objective 1.3.3 To validate new markers and methods for minimal residual disease (MRD) 
detection. 
 
We will compute the correlation of MRD measurements using new markers with the MRD 
measurements determined in the same way as in AML02. Additionally, we will use Cox 
regression, a rank-based procedure, or the log-rank test to explore the prognostic value of 
MRD measured with the new markers.183,184  
 
Objective 1.3.4 To identify new prognostic factors by applying new technologies to study 
patient material 
 
We will use SNP microarrays to identify genomic lesions. Summary signals will be 
computed for each probe set and then reference-normalized using the method described by 
Mullighan et al.166 The summary signal profile for each tumor will be compared to a 
control signal profile obtained from one or multiple control samples by subtraction or 
taking the ratio. The resulting signal difference will be segmented using circular binary 
segmentation or Bayesian change-point analysis. Segments will be then characterized as 
showing evidence of amplification, evidence of deletion, or no evidence of alteration. The 
inferred copy number states will be compared across different cell subpopulations from the 
same individual’s tumor and across samples from different subjects. Additionally, we will 

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use log-rank tests, a rank-based procedure, or Cox models to explore the association of 
identified lesions with event-free and overall survival.183,184 
 
We will use mRNA microarrays to compare the expression of leukemic blasts prior to 
therapy to that of residual blasts following induction I. We will measure expression at 
diagnosis and post-therapy in each subject with sufficient material for the assays. For each 
pair of arrays from the same subject, we will compute the log-ratio of expression after 
treatment to that prior to treatment. Within each arm, we will apply the signed-rank test to 
the expression change values to test for significant expression changes. Additionally, we 
will compare the expression changes across arms with the rank-sum test. Also, we will use 
linear models to explore the possibilities of that the differences in expression response to 
therapy between the two arms vary according to disease subtype. Finally, we will use a 
rank-based procedure or Cox regression to explore the association of expression with 
event-free survival or overall survival.183,184 
 
Objective 1.3.5 To identify pharmacogenetic, pharmacokinetic and pharmacodynamic 
factors that associate with clinical outcomes in the context of the systemic therapy used in 
the protocol. 
 
We will use logistic regression models to explore the association of PK variables (AUC, 
clearance, etc.), PD variables (IC50, etc.) and pharmacogenetic (PG) variables with toxicity 
and response to therapy (MRD+/- or CR). We will use Cox models or a rank-based 
procedure to explore the association of PK, PD, and PG variables with event-free and 
overall survival.183,184 We will use correlation coefficients and linear models to explore the 
association of PK variables with PD variables. We will use ANOVA models or the 
Kruskall-Wallis test to explore the association of PG variables with PD and PK variables.   
 
Objective 1.3.6 To describe the impact of antibiotic and antifungal prophylaxis on invasive 
bacterial and fungal infections, febrile neutropenia, hospitalization, and antibiotic resistance.  
 
We will use Gray’s method estimate the cumulative incidence of each type of infection in 
the protocol.180 Additionally, we will use Gray’s test to compare the cumulative incidence 
of infection of patients assigned to HD-ADE to that of patients assigned to Clo/AraC. 
 
14.0 
OBTAINING INFORMED CONSENT 
 
14.1 
Consent/Assent at Induction and Post-Induction 
 
The process of informed consent for AML08 will follow institutional policy. The informed 
consent process is an ongoing one that begins at the time of diagnosis and ends after the 
completion of therapy. Informed consent should be obtained by the attending physician or 
his/her designee, in the presence of at least one non-physician witness. Initially, informed 
consent will be sought for the institutional banking protocol (research study), blood 
transfusion and other procedures as necessary. After the diagnosis of AML is established, 
we will invite the patient to participate in the AML08 protocol. After completion of 
induction, we will seek informed consent for their participation in risk-directed post-
induction therapy. 
 

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Throughout the entire treatment period, participants and their parents receive constant 
education from health professionals at SJCRH and are encouraged to ask questions 
regarding alternatives and therapy. All families have ready access to chaplains, 
psychologists, social workers, and the St. Jude ombudsperson for support, in addition to 
that provided by the primary physician and other clinicians involved in their care. 
 
We will also obtain verbal assent from children 7 to 14 years old and written assent for all 
patients >14 years of age. Patients who reach the age of majority while on study will be re-
consented for continued participation on AML08, according to Cancer Center and 
institutional policy. 
 
14.2 
Consent at the age of majority 
 
The age of majority in the state of Tennessee is 18 years old. Research participants on 
active therapy must be consented at the next clinic visit after their 18th birthday. 
Participants, who have reached age of majority and have completed all protocol-directed 
therapy, will be re-consented with a separate consent specifically for this purpose (AOM 
consent). Participants, who reach age of majority after the 5 year protocol required follow-
up, will be followed for survival and late effects as per the SJLTFU. A waiver for AOM 
consent will be requested for these participants at St. Jude. 
 
If an affiliate site is located in a state where a different age of majority applies, that location 
must consent the participants according to their local laws. Collaborating sites may follow 
this guidance for AOM, or may follow institutional policy (and will provide institutional 
policy to St. Jude). 
 
14.3 
Consent when English is not the primary language 
 
When English is not the patient, parent, or legally authorized representative’s primary 
language, the Social Work department will determine the need for an interpreter. This 
information will be documented in the participant’s medical record. Either a certified 
interpreter or the telephone interpreter’s service will be used to translate the consent 
information. The process for obtaining an interpreter and for the appropriate use of an 
interpreter is outlined on the Interpreter Services, OHSP, and CPDMO websites. 
Collaborating Sites will follow institutional policy for consenting non-English speaking 
participants (and will provide institutional policy to St. Jude). 
 
15.0 
DATA SAFETY MONITORING BOARD 
 
The protocol progress will be reviewed and monitored by the St. Jude Children’s Research 
Hospital Data Safety Monitoring Board (DSMB). Data summaries will be provided to the 
DSMB by the Department of Biostatistics after review by the Principal Investigator. The 
data will include patient accrual, demographic summaries, grade 3/4 toxicities, major 
adverse events (i.e. deaths, relapses, second malignancies) and results of interim and final 
analyses as specified in the protocol. The data are retrieved from the database and are 
reviewed at every AML08 meeting, which is generally held twice a month, and are 
reflected in the minutes, which are provided to the biostatistician. When the appropriate 
number of patients have failed or accrued, triggering interim or final analysis, the 

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biostatistician will be informed by the PI. Should a safety stopping rule be exceeded, the 
protocol will be temporarily closed until the DSMB can review the situation. Reporting 
will comply with Cancer Center and St. Jude institutional guidelines for monitoring. 

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16.0 
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157. Langebrake C, Creutzig U, Dworzak M, et al. Residual disease monitoring in childhood acute 
myeloid leukemia by multiparameter flow cytometry: the MRD-AML-BFM Study Group. J 
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158. Kern W, Voskova D, Schoch C, et al. Determination of relapse risk based on assessment of 
minimal residual disease during complete remission by multiparameter flow cytometry in 
unselected patients with acute myeloid leukemia. Blood 2004;104:3078-3085. 
159. Okamoto Y, Ribeiro RC, Srivastava DK, et al. Viridans streptococcal sepsis: clinical features 
and complications in childhood acute myeloid leukemia. J Pediatr Hematol Oncol 
2003;25:696-703. 
160. Gamis AS, Howells WB, DeSwarte-Wallace J, et al. Alpha Hemolytic Streptococcal Infection 
During Intensive Treatment for Acute Myeloid Leukemia: A Report From the Children's 
Cancer Group Study CCG-2891. J Clin Oncol 2000;18:1845-1855. 
161. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K. Natural cytotoxic activity of 
peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general 
population. Lancet 2000;356:1795-1799. 
162. Costello RT, Sivori S, Marcenaro E, et al. Defective expression and function of natural killer 
cell-triggering receptors in patients with acute myeloid leukemia. Blood 2002;99:3661-3667. 
163. Fauriat C, Just-Landi S, Mallet F, et al. Deficient expression of NCR in NK cells from acute 
myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in 
NCRdull phenotype induction. Blood 2007;109:323-330. 
164. Pende D, Spaggiari GM, Marcenaro S, et al. Analysis of the receptor-ligand interactions in the 
natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence 
for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112). Blood 
2005;105:2066-2073. 
165. Ross ME, Mahfouz R, Onciu M, et al. Gene expression profiling of pediatric acute 
myelogenous leukemia. Blood 2004;104:3679-3687. 
166. Mullighan CG, Goorha S, Radtke I, et al. Genome-wide analysis of genetic alterations in 
acute lymphoblastic leukaemia. Nature 2007;446:758-764. 
167. Gal H, Amariglio N, Trakhtenbrot L, et al. Gene expression profiles of AML derived stem 
cells; similarity to hematopoietic stem cells. Leukemia 2006;20:2147-2154. 
168. Irish JM, Hovland R, Krutzik PO, et al. Single cell profiling of potentiated phospho-protein 
networks in cancer cells. Cell 2004;118:217-228. 
169. Strumberg D, Clark JW, Awada A, et al. Safety, pharmacokinetics, and preliminary antitumor 
activity of sorafenib: a review of four phase I trials in patients with advanced refractory solid 
tumors. Oncologist 2007;12:426-437. 
170. Steinbach D, Legrand O. ABC transporters and drug resistance in leukemia: was P-gp nothing 
but the first head of the Hydra? Leukemia 2007;21:1172-1176. 
171. Lamba JK, Crews K, Pounds S, et al. Pharmacogenetics of deoxycytidine kinase: 
identification and characterization of novel genetic variants. J Pharmacol Exp Ther 
2007;323:935-945. 
172. Peck AJ, Englund JA, Kuypers J, et al. Respiratory virus infection among hematopoietic cell 
transplant recipients: evidence for asymptomatic parainfluenza virus infection. Blood 
2007;110:1681-1688. 
173. Debiaggi M, Canducci F, Sampaolo M, et al. Persistent symptomless human 
metapneumovirus infection in hematopoietic stem cell transplant recipients. J Infect Dis 
2006;194:474-478. 
174. Antoniadou A, Giamarellou H. Fever of unknown origin in febrile leukopenia. Infect Dis Clin 
North Am 2007;21:1055-90, x. 

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175. Engelhart S, Glasmacher A, Exner M, Kramer MH. Surveillance for nosocomial infections 
and fever of unknown origin among adult hematology-oncology patients. Infect Control Hosp 
Epidemiol 2002;23:244-248. 
176. Freymuth F, Vabret A, Cuvillon-Nimal D, et al. Comparison of multiplex PCR assays and 
conventional techniques for the diagnostic of respiratory virus infections in children admitted 
to hospital with an acute respiratory illness. J Med Virol 2006;78:1498-1504. 
177. Hijiya N, Metzger ML, Pounds S, et al. Severe cardiopulmonary complications consistent 
with systemic inflammatory response syndrome caused by leukemia cell lysis in childhood 
acute myelomonocytic or monocytic leukemia. Pediatr Blood Cancer 2005;44:63-69. 
178. Panetta JC, Wilkinson M, Pui CH, Relling MV. Limited and optimal sampling strategies for 
etoposide and etoposide catechol in children with leukemia. J Pharmacokinet Pharmacodyn 
2002;29:171-188. 
179. Zelen M. The randomization and stratification of patients to clinical trials. J Chronic Dis 
1974;27:365-375. 
180. Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a competing 
risk. Ann Stat 1988;16:1141-1154. 
181. Cheng C, Pounds S. False discovery rate paradigms for statistical analyses of microarray gene 
expression data. Bioinformation 2007;1:436-446. 
182. Pounds SB. Estimation and control of multiple testing error rates for microarray studies. Brief 
Bioinform 2006;7:25-36. 
183. Cox DR. Regression models and life-tables. J R Stat Soc B 1972;34:187-220. 
184. Jung SH, Owzar K, George SL. A multiple testing procedure to associate gene expression 
levels with survival. Stat Med 2005;24:3077-3088. 
 

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APPENDIX I – DRUG INFORMATION 
 
1. CYTARABINE (Ara-C) (Cytosar-U) 
 
Source and pharmacology: Cytarabine is a deoxycytidine analogue. It must be tri-phosphorylated 
to its active form, ARA-CTP, by deoxycytidine kinase and other nucleotide kinases.  ra-CTP 
inhibits DNA polymerase. In addition, ara-CTP is incorporated into DNA as a false base, causing 
inhibition of DNA synthesis. It is cell cycle, S phase specific. Cytarabine does penetrate the blood 
brain barrier. It is converted to its inactive form, uracil arabinoside, by pyrimidine nucleoside 
deaminase. Approximately 80% of the dose is recovered in the urine, mostly as uracil arabinoside 
(ara-U). 
 
Formulation and stability: Cytarabine is available in multi-dose vials containing 100, 500, 1000 
and 2000mg of lyophilized drug. Intact vials can be stored at room temperature. For IV use, 
either sterile water for injection or bacteriostatic water for injection can be used to reconstitute 
the lyophilized drug. For intrathecal use, only sterile water for injection should be used for 
reconstitution. The 100 and 500 mg vials are reconstituted with 2 and 10 ml respectively 
resulting in a final concentration of 50mg/ml. The 1000 and 2000mg vials are reconstituted with 
20ml and 40 ml respectively resulting in a final concentration of 50mg/ml. After reconstitution, 
the drug is stable for 8 days at room temperature. 
 
Supplier: Commercially available. 
 
Toxicity: Myelosuppression is the dose limiting adverse effect, with leukopenia and 
thrombocytopenia being predominant. Other adverse effects reported commonly include nausea 
and vomiting (may be severe at high doses), diarrhea, mucositis, anorexia, alopecia, skin rash 
and liver dysfunction. A flu-like syndrome characterized by fever, muscle and bone aches is 
common. Less common side effects include allergic reactions and cellulitis at the injection site. 
High doses of cytarabine can cause conjunctivitis, hepatitis, and a group of CNS symptoms 
including somnolence, peripheral neuropathy, ataxia and personality changes. CNS symptoms 
are usually reversible and are more common in patients who have received previous cranial 
irradiation. In addition, a syndrome of sudden respiratory distress progressing to pulmonary 
edema has occurred. 
 
Dosage and route of administration:  See Treatment sections 4.2 – 4.6 and 4.9. 
 
2. CYTARABINE (High Dose Ara-C) 
 
Source and pharmacology: Cytarabine is a deoxycytidine analogue. It must be tri-phosphorylated 
to its active form, ARA-CTP, by deoxycytidine kinase and other nucleotide kinases. Ara-CTP 
inhibits DNA polymerase. In addition, ara-CTP is incorporated into DNA as a false base, causing 
inhibition of DNA synthesis. It is cell cycle, S phase specific. Cytarabine does penetrate the blood 
brain barrier. It is converted to its inactive form, uracil arabinoside, by pyrimidine nucleoside 
deaminase. Approximately 80% of the dose is recovered in the urine, mostly as uracil arabinoside 
(ara-U). 
 

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Formulation and stability: Cytarabine is available in multi-dose vials containing 100, 500, 1000 
and 2000mg of lyophilized drug. Intact vials can be stored at room temperature. For IV use, 
either sterile water for injection or bacteriostatic water for injection can be used to reconstitute 
the lyophilized drug. For intrathecal use, only sterile water for injection should be used for 
reconstitution. The 100 and 500 mg vials are reconstituted with 2 and 10 ml respectively 
resulting in a final concentration of 50mg/ml. The 1000 and 2000mg vials are reconstituted with 
20ml and 40 ml respectively resulting in a final concentration of 50mg/ml. After reconstitution, 
the drug is stable for 8 days at room temperature. 
 
Supplier: Commercially available. 
 
Toxicity: Myelosuppression is the dose limiting adverse effect, with leukopenia and 
thrombocytopenia being predominant. Other adverse effects reported commonly include nausea 
and vomiting (may be severe at high doses), diarrhea, mucositis, anorexia, alopecia, skin rash 
and liver dysfunction. A flu-like syndrome characterized by fever, muscle and bone aches is 
common. Less common side effects include allergic reactions and cellulitis at the injection site.   
High doses of cytarabine can cause conjunctivitis, hepatitis, and a group of CNS symptoms 
including somnolence, peripheral neuropathy, ataxia and personality changes. CNS symptoms 
are usually reversible and are more common in patients who have received previous cranial 
irradiation. In addition, a syndrome of sudden respiratory distress progressing to pulmonary 
edema has occurred. 
 
Dosage and route of administration: See Treatment Sections 4.2 – 4.6. 
 
3. DAUNORUBICIN (Daunomycin, Cerubidine) 
 
Source and pharmacology: Daunorubicin is an anthracycline antibiotic derived from 
Streptomyces coeruleorubidus. Daunorubicin intercalates between base pairs of DNA causing 
steric obstruction, disruption of DNA function and inhibition of RNA synthesis. In addition, 
daunorubicin inhibits topoisomerase II, an enzyme responsible for allowing strands of DNA to 
pass through one another as they unwind. Even though daunorubicin exerts its major effects in 
the S phase, it is considered to be cell cycle phase non-specific. Daunorubicin is widely 
distributed in tissues but does not cross the blood brain barrier. It is metabolized to 
daunorubicinol which is the major active metabolite and aglycones (inactive). The major route of 
elimination is through the bile (40%) with additional elimination through the urine. Dosages 
should be reduced in patients with liver dysfunction (bilirubin > 1/2 mg/dL) or renal dysfunction 
(creatinine > 3 mg/dL).   
 
Formulation and stability: Daunorubicin is supplied in vials containing 20 mg of reddish colored 
lyophilized powder and 100 mg of mannitol. The intact vials should be stored at room 
temperature. Each vial can be reconstituted with 4 ml of sterile water for injection to give a final 
concentration of 5 mg/ml. Reconstituted solutions are stable for 24 hours at room temperature 
and 48 hours if refrigerated. 
 
Supplier: Commercially available. 
 

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Toxicity: Dose-limiting toxicities of daunorubicin include myelosuppression and cardiotoxicity. 
Two forms of cardiac toxicity can occur. Acute toxicity may take the form of arrhythmias, heart 
block or pericarditis and may be fatal. The chronic form of cardiotoxicity is related to total 
cumulative dose and is characterized by heart failure. Mediastinal radiotherapy and/or other 
cardiotoxic drugs may increase the risk of cardiotoxicity. In general, total lifetime dosages of 
450-550mg/m2 should not be exceeded. Other toxicities include nausea and vomiting, mucositis, 
alopecia, diarrhea and red discoloration of the urine and other body fluids. Severe tissue damage 
and necrosis can occur upon extravasation. Radiation recall reactions can occur and can be 
severe. Rarely, allergic reactions have occurred. Typhlitis can occur when combined with 
cytarabine. 
 
Dosage and route of administration: See Treatment Section 4.2. 
 
4. ETOPOSIDE (VP-16) (Vepesid) 
 
Source and pharmacology: Etoposide is an epipodophyllotoxin derived from Podophyllum 
pelatatum. It is thought to act mainly by inhibiting topoisomerase II, causing double and single 
strand DNA breaks. Etoposide is cell cycle, phase-specific, with activity in the G2 and S phases. 
Absorption of etoposide is approximately 30-40% and is highly variable and somewhat dose-
dependent. It is extensively bound to serum proteins and is metabolized in the liver, including 
cytochrome P450 3A metabolism to several moieties that include a reactive oxidized species. 
Etoposide and its metabolites are excreted mainly in the urine with a smaller amount excreted in 
the feces. Dosage adjustments should be considered in patients with liver dysfunction, kidney 
dysfunction or hypoalbuminemia. 
 
Formulation and stability: Etoposide is available in multi-dose vials containing 100mg, 150mg, 
500mg and 1000mg of etoposide as a 20mg/ml solution and 30% alcohol. Etoposide is also 
available as a 50 mg capsule. The intact vials of etoposide solution should be stored at room 
temperature. The capsules should be stored under refrigeration. Etoposide solution should be 
diluted in D5W or 0.9% NaCl prior to administration. Solutions with a final concentration of 0.2 
and 0.4 mg/ml are stable at room temperature for 96 hours and 24 hours respectively. 
 
Supplier: Commercially available. 
 
Toxicity: Dose limiting toxicity is myelosuppression. Nausea and vomiting (usually of low to 
moderate severity), diarrhea, mucositis (particularly with high doses), alopecia and anorexia are 
fairly common. Hypotension can occur with rapid infusions. Other side effects reported less 
commonly include hepatitis, fever and chills, anaphylaxis and peripheral neuropathy. Secondary 
leukemia has been reported. 
 
Dosage and route of administration: See Treatment Sections 4.2 and 4.6. 
 

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5. CLOFARABINE (Clolar™, Clofarex) 
 
Source and pharmacology: Clofarabine is a purine nucleoside analog. It is intracellulary 
metabolized to the active metabolite clofarabine 5’-triphosphate which competes with 
deoxyadenosine triphosphate for binding to ribonucleotide reductase and DNA polymerase. It 
inhibits DNA synthesis, terminates DNA chain elongation and inhibits DNA repair. Clofarabine 
also disrupts the mitochondrial membrane which results in the release of proteins, cytochrome C 
and apoptosis-inducing factor leading to cell death. It is mainly excreted in the urine as 
unchanged drug. 
 
Formulation and stability: Clofarabine is available in the parenteral form as a preservative-free 
solution that is 1 mg/mL. It is available in 20 mL vials. The undiluted drug should be stored at 
room temperature. The diluted solution is stable for 24 hours at room temperature. Clofarabine 
injection should be filtered through sterile 0.2 micrometer syringe filter and then further diluted 
with 5% dextrose or 0.9% NaCl containing solutions.   
 
Supplier: The injection is commercially available, however for this study clofarabine will be 
supplied by Genzyme. 
 
Toxicity: The most common side effects are nausea, vomiting, diarrhea, headache, fever and 
pruritus. Also reported are pericardial effusion, tachycardia, hypotension, left ventricular systolic 
dysfunction, edema, flushing, hypertension, fatigue, anxiety, pain, dizziness, depression, 
irritability. Patients who receive clofarabine are at risk for tumor lysis syndrome and need to be 
monitored closely. Patients may also experience a systemic inflammatory response syndrome 
(SIRS) or capillary leak syndrome. Patients should be monitored for this during the infusion. 
 
Dosage and route of administration: See Treatment Sections 4.2. 
 
6. MITOXANTRONE (Novantrone) 
 
Source and pharmacology: Mitoxantrone is an anthracenedione that is structurally similar to the 
anthracyclines. It is thought to act by intercalating into DNA, causing template disorder, steric 
obstruction and inhibition of DNA and RNA synthesis. In addition, mitoxantrone inhibits the 
action of topoisomerase II. Mitoxantrone is active throughout the cell cycle. Mitoxantrone is 
about 78% protein bound and does cross the blood brain barrier. Mitoxantrone is metabolized in 
the liver to inactive metabolites. The parent drug and metabolites are excreted primarily via 
hepatobiliary excretion with small amounts excreted in the urine. Dosage adjustment is 
recommended for patients with severe hepatic dysfunction (total bilirubin > 3.4 mg/dl). 
 
Formulation and stability: Mitoxantrone is available in multi-dose vials containing 20 mg, 25 mg 
or 30 mg of mitoxantrone as a dark blue, aqueous solution at a concentration of 2 mg/ml. The 
intact vials should be stored at room temperature. Refrigeration may result in precipitation of 
mitoxantrone, which will re-dissolve upon warming to room temperature. The drug should be 
further diluted to at least 50 ml in 5% dextrose or 0.9% NaCl prior to administration. These 
solutions are chemically stable for at least 7 days when stored at room temperature. 
 

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Supplier: Commercially available. 
 
Toxicity: The major dose-limiting toxicity of mitoxantrone is leukopenia with thrombocytopenia 
and anemia occurring much less frequently. Nausea and vomiting are usually moderate in 
severity. Other side effects reported commonly include alopecia, diarrhea, headache, fever and 
stomatitis. Blue to green discoloration of urine and other body fluids occurs. Other side effects 
reported less commonly include elevated liver function tests, allergic reactions, seizures, 
jaundice and renal failure. Congestive heart failure has been reported, but is much less common 
than with doxorubicin. CHF has been reported primarily in patients receiving prior therapy with 
anthracyclines. Patients with an increased risk of cardiotoxicity include those having received 
prior therapy with anthracyclines, those with previous mediastinal radiotherapy and those with 
pre-existing cardiac conditions. 
 
Dosage and route of administration: See Treatment Sections 4.3 and 4.4. 
 
7. THIOGUANINE 
 
Source and pharmacology: Thioguanine is a purine antimetabolite. It is intracellularly converted 
to ribonucleotides which are incorporated into DNA and RNA. Absorption of thioguanine is 
variable and poor and is decreased by food. Thioguanine undergoes extensive metabolism in the 
liver and other tissues to the inactive, methylated derivative and to 6-thiouracil by xanthine 
oxidase. Thioguanine is excreted in the urine almost completely as metabolites.  
 
Formulation and stability: Thioguanine is available as a 40 mg scored tablet. It may be stored at 
room temperature. 
 
Supplier: Tablets are commercially available.  
 
Toxicity: The major dose-limiting toxicity is myelosuppression. Nausea and vomiting are usually 
mild. Other toxicities reported include diarrhea, rash, anorexia, stomatitis, and hyperuricemia. 
Jaundice and elevated liver function tests have been reported rarely 
 
Guidelines for administration: See Treatment Section 4.4. 
 
8. L-ASPARAGINASE (Elspar) 
 
Source and pharmacology: Asparaginase is an enzyme. It is derived from Escherichia coli. 
Asparaginase hydrolyzes serum asparagine (an amino acid required to synthesize proteins) to 
aspartic acid and ammonia, and is therefore lethal to cells that cannot synthesize asparagine. 
Asparaginase is active during all phases of the cell cycle. Asparaginase is not absorbed from the 
GI tract and must be given parenterally. Asparaginase does not cross into the CSF. The plasma 
half-life of L-asparaginase when given IV is ≈ 8-30 hours. When given IM the half-life is ≈30 
hours. Only minimal urinary and biliary excretion occurs. Clearance is unaffected by age, renal 
function or hepatic function. 
 

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IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Formulation and stability: E. Coli asparaginase is available in vials containing 10,000 units of 
lyophilized drug and 80 mg mannitol. Unused vials should be refrigerated. The contents of each 
vial should be diluted with 1 cc of preservative-free normal saline or sterile water, giving a 
resultant solution of 10,000 units/ml. Once in solution, it is recommended that it be used within 8 
hours as no preservative is added. Occasionally a small number of gelatinous-like fibers may 
develop upon standing. If this occurs, the solution can be filtered through a 5 micron filter to 
remove the particles with no change in potency. 
 
Supplier: Commercially available 
 
Toxicity: Acute toxicity includes anaphylactic reactions that occur most commonly when the 
drug is given IV. These can be characterized by laryngeal constriction, hypotension, diaphoresis, 
fever, chills, edema and loss of consciousness. Allergic reactions at the site of IM injection 
include pain, swelling and erythema. Other adverse effects include neutropenia and associated 
immunosuppression, mild nausea and vomiting, malaise, anorexia, elevated LFT’s, pancreatitis 
and hyperglycemia. A decrease in protein synthesis including albumin, fibrinogen and other 
coagulation factors may occur which can result in hemorrhage. Thrombosis and/or pulmonary 
embolism can also occur. Less common side effects include renal dysfunction and CNS 
complications including somnolence, weakness, lethargy, coma and seizures. 
 
Dosage and route of administration: See Treatment Section 4.5. 
 
9. CYCLOPHOSPHAMIDE (Cytoxan) 
 
Source and pharmacology: Cyclophosphamide is a nitrogen mustard derivative. It acts as an 
alkylating agent that causes cross-linking of DNA strands by binding with nucleic acids and other 
intracellular structures, thus interfering with the normal function of DNA. Cyclophosphamide is 
cell-cycle, phase non-specific. Cyclophosphamide is well absorbed from the GI tract with a 
bioavailability of > 75%. Cyclophosphamide is a prodrug that requires activation. It is metabolized 
by mixed-function oxidases in the liver to 4-hydroxycyclophosphamide, which is in equilibrium 
with aldofosfamide. Aldofosfamide spontaneously splits into cyclophosphamide mustard, which is 
considered to be the major active metabolite, and acrolein.  In addition, 4-
hydroxycyclophosphamide may be enzymatically metabolized to 4-ketocyclophosphamide and 
aldofosfamide may be enzymatically metabolized to carboxyphosphamide, which are generally 
considered to be inactive. Cyclophosphamide and its metabolites are excreted mainly in the urine.  
Dosage adjustments should be made in patients with a creatinine clearance of < 50 ml/min. 
 
Formulation and stability: Cyclophosphamide is available in 25 and 50 mg tablets. 
Cyclophosphamide is also available in vials containing 100, 200, 500, 1000 and 2000mg of 
lyophilized drug and 75 mg mannitol per 100 mg of cyclophosphamide. Both forms of the drug 
can be stored at room temperature. The vials are reconstituted with 5, 10, 25, 50 or 100 ml of 
sterile water for injection respectively to yield a final concentration of 20 mg/ml. Reconstituted 
solutions may be further diluted in either 5% dextrose or 0.9% NaCl containing solutions. 
Diluted solutions are physically stable for 24 hours at room temperature and 6 days if 
refrigerated, but contain no preservative, so it is recommended that they be used within 24 hours 
of preparation. 

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Supplier:  Commercially available. 
 
Toxicity: Dose limiting toxicities of cyclophosphamide are bone marrow suppression and cardiac 
toxicity. Cardiac toxicity is typically manifested as congestive heart failure, cardiac necrosis or 
hemorrhagic myocarditis and can be fatal. Hemorrhagic cystitis may occur and necessitates 
withholding therapy. The incidence of hemorrhagic cystitis is related to cyclophosphamide dose 
and duration of therapy. Forced fluid intake and/or the administration of MESNA decreases the 
incidence and severity of hemorrhagic cystitis. Other toxicities reported commonly include 
nausea and vomiting (may be mild to severe depending on dosage), diarrhea, anorexia, alopecia, 
immunosuppression and sterility. Pulmonary fibrosis, SIADH, anaphylaxis and secondary 
neoplasms have been reported rarely. 
 
Dosage and route of administration:  See Treatment Section 4.7. 
 
10. FLUDARABINE (Fludara) 
 
Source and pharmacology: Fludarabine phosphate is a synthetic purine nucleoside analog. It acts 
by inhibiting DNA polymerase, ribonucleotide reductase and DNA primase by competing with 
the physiologic substrate, deoxyadenosine triphosphate, resulting in inhibition of DNA synthesis. 
In addition, fludarabine can be incorporated into growing DNA chains as a false base, thus 
interfering with chain elongation and halting DNA synthesis. Fludarabine is rapidly 
dephosphorylated in the blood and transported intracellularly via a carrier mediated process. It is 
then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate form. 
Approximately 23% of the dose is excreted as the active metabolite in the urine (with dosages of 
18-25 mg/m2/day for 5 days). Renal clearance appears to become more important at higher 
doses, with approximately 41-60% of the dose being excreted as the active metabolite in the 
urine with dosages of 80-260 mg/m2. 
   
Formulation and stability: Fludarabine is supplied in single-dose vials containing 50 mg 
fludarabine as a white lyophilized powder and 50 mg of mannitol. The intact vials should be 
stored under refrigeration. Each vial can be reconstituted by adding 2 ml of sterile water for 
injection resulting in a final concentration of 25mg/ml. Because the reconstituted solution 
contains no antimicrobial preservative, the manufacturer recommends that it should be used 
within 8 hours of preparation. The solution should be further diluted in 5% dextrose or 0.9% 
NaCl prior to administration. 
 
Supplier:  Commercially available. 
 
Toxicity: The major dose-limiting toxicity of fludarabine is myelosuppression. Nausea and 
vomiting are usually mild. Side effects reported commonly include, anorexia, fever and chills, 
alopecia and rash. Neurotoxicity can be manifested by somnolence, fatigue, peripheral 
neuropathy, mental status changes, cortical blindness and coma and is more common at high 
doses. Neurotoxicity is usually delayed, occurring 21-60 days after the completion of a course of 
therapy and may be irreversible. Side effects reported less commonly include diarrhea, 
stomatitis, increased liver function tests, liver failure, chest pain, arrhythmias and seizures. 
Pulmonary toxicity includes allergic pneumonitis characterized by cough, dyspnea, hypoxia and 

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pulmonary infiltrates. Drug induced pneumonitis is a delayed effect, occurring 3-28 days after 
the administration of the third or later course of therapy. Administration of corticosteroids 
usually results in resolution of these symptoms. 
 
Dosage and route of administration: See Treatment Section 4.7. 
 
11. METHOTREXATE 
 
Source and pharmacology: Methotrexate is a folate analogue that acts by inhibiting dihydrofolate 
reductase. Dihydrofolate reductase is an enzyme important in the conversion of folic acid to 
tetrahydrofolic acid, which is necessary in the synthesis of purine nucleotides and thymidylate. 
By inhibiting the production of tetrahydrofolic acid, methotrexate interferes with DNA, RNA 
and protein synthesis. Methotrexate is poorly and variably absorbed orally, with an average of ≈ 
40% for doses of < 30 mg/m2. At higher dosages, the extent of absorption decreases. 
Methotrexate is approximately 50% protein bound. It distributes widely into body tissues and 
fluids with sustained concentrations in the kidney and the liver. Methotrexate undergoes 
metabolism by cytosolic aldehyde oxidase to hydroxy methotrexate. It is excreted mainly in the 
urine as unchanged drug with small amounts being excreted in the bile and feces. The percent 
recovered as unchanged drug in the urine is higher with short infusions than with prolonged 
infusions. Methotrexate has a biphasic elimination with an initial half-life of ≈ 2-3 hours and a 
terminal half-life of 10-12 hours. Methotrexate may be “sequestered” in body fluid collections 
and eliminated slowly from these areas. Patients with effusions or GI obstruction should have 
plasma levels monitored closely for delayed excretion following high-dose methotrexate. 
 
Formulation and stability: Methotrexate is supplied in single-dose vials containing 50mg, 
100mg, 200mg, and 250 mg of methotrexate as a 25 mg/ml preservative-free solution and in 
vials containing 20mg, 50 mg, 100mg, 250 mg and 1000mg of lyophilized drug. It is also 
available in 2.5 mg tablets. Methotrexate preservative-free solution and lyophilized drug should 
be stored at room temperature and protected from light. Methotrexate tablets can also be stored at 
room temperature. The vials containing 20, 50, 100 and 250 mg of lyophilized product can be 
reconstituted by adding sterile water, 0.9% NaCl or D5W to a final concentration not exceeding 
25 mg/ml. The 1000mg vials containing lyophilized product are reconstituted to a final 
concentration of 50 mg/ml. 
 
Supplier:  Commercially available. 
 
Toxicity: The dose limiting toxicities of methotrexate are generally bone marrow suppression, 
ulcerative stomatitis, severe diarrhea or acute nephrotoxicity. Toxicities reported frequently 
include nausea and vomiting, diarrhea, anorexia, alopecia, hepatic toxicity and alopecia. Less 
common side effects include blurred vision, photosensitivity, anaphylaxis, headache, 
pneumonitis, skin depigmentation or hyperpigmentation, rash, vasculitis and encephalopathy. 
During high-dose methotrexate therapy, most patients experience a transient decrease in GFR, 
but renal failure can occur, particularly if the patient does not receive urinary alkalinization and 
aggressive hydration before, during and after receiving high dose methotrexate. Leucovorin 
rescue should be initiated within 48 hours of starting high-dose methotrexate and adjusted based 
on MTX levels to prevent bone marrow toxicity and mucositis. Leucovorin may also be 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
necessary after IT administration, especially if IT methotrexate therapy is given to patients with 
renal dysfunction. Patients with Down syndrome have a tendency to have delayed methotrexate 
clearance and a greater risk of toxicity, despite increased leucovorin rescue. 
 
Dosage and route of administration: Intrathecal, See Treatment Section 4.9. 
 
12. HYDROCORTISONE, Intrathecal (Cortef, Solu-Cortef) 
 
Source and pharmacology: Hydrocortisone is a synthetic steroid akin to the natural adrenal 
hormone cortisol. Hydrocortisone has phase-specific cytotoxicity, killing lymphoblasts primarily 
during S phase. It has catabolic effect on proteins and alters the kinetics of peripheral blood 
leukocytes. It is excreted in the urine and catabolized in the liver. 
 
Formulation and stability: Solu-Cortef sterile powder is supplied in the following package: 100 
mg plain, and 100 mg, 250 mg, 500 mg, and 1000 mg ACT-O-VIAL (MIX-O-VIAL). Store 
unreconstituted product at controlled room temperature 15-30°C (59-86°F). Store reconstituted 
solution in the refrigerator and protect from light. Unused solution should be discarded after 3 
days. Use Solu-Cortef (plain vial) for intrathecal use, and reconstitute with 0.9% sodium 
chloride, USP for injection. 
 
Supplier: Commercially available. 
 
Toxicity: If given intrathecally, sterile arachnoiditis may occur. Headache, seizures, unusual 
feelings or sensations, loss of feeling or ability to move arms or legs, and difficulty with 
urination or bowel movements may also occur. 
 
Dosage and route of administration: Intrathecal, See Treatment Section 4.9 
 
13. MESNA (Mesnex) 
 
Source and pharmacology: Mesna is a synthetic sulfhydryl (thiol) compound. Mesna contains 
free sulfhydryl groups that interact chemically with urotoxic metabolites of oxazaphosphorine 
derivatives such as cyclophosphamide and ifosfamide. Oral bioavailability is ≈50%. Upon 
injection into the blood, mesna is oxidized to mesna disulfide, a totally inert compound. 
Following glomerular filtration, mesna disulfide is rapidly reduced in the renal tubules back to 
Mesna, the active form of the drug. Mesna and mesna disulfide are excreted primarily via the 
urine. 
 
Formulation and stability: Mesna is available in 2 ml, 4 ml and 10 ml amps containing 
100 mg/ml of mesna solution. The intact vials can be stored at room temperature. Mesna may be 
further diluted in 5% dextrose or 0.9% NaCl containing solutions. Diluted solutions are 
physically and chemically stable for at least 24 hours under refrigeration.  
 
Supplier: Commercially available. 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Toxicity: Mesna is generally well tolerated. Nausea and vomiting, headache, diarrhea, rash, 
transient hypotension and allergic reactions have been reported. Patients may complain of a bitter 
taste in their mouth during administration. Mesna may cause false positive urine dipstick 
readings for ketones. 
 
Dosage and administration: See Treatment Section 4.7. 
 
14. SORAFENIB (Nexavar) – Participants with FLT3-ITD 
 
Source and pharmacology: Sorafenib (BAY 54-9085) is a kinase inhibitor (Raf, VEGF-R, and 
PDGF-R). The ras/raf signaling pathway is an important mediator of responses to growth 
signals and angiogenic factors. This pathway is often aberrantly activated in human tumors due 
to presence of activated ras, mutant b-raf, or over expression of growth factor receptors. 
Sorafenib is a potent inhibitor of c-raf, and wild-type and mutant b-raf in vitro. Additionally, 
further characterization of BAY 43-9006 tosylate revealed that this agent inhibits several 
receptor tyrosine kinases (RTKs) that are involved in tumor progression (VEGF-R, PDGF-R, 
Flt3, and c-KIT) and p38α, a member of the MAPK family 
 
Formulation and stability:  Sorafenib is supplied as a salmon-colored 200 mg, round, 
immediate-release film-coated tablet  
 
Sorafenib 200 mg tablets are supplied in bottles of 120 tablets. Sorafenib should be stored at 59 
- 86ºF(15ºC – 30ºC) in a dry place. Tablets are stable until the date of expiration listed on the 
manufacturer’s container. 
 
Supplier: Commercially available. 
 
Dosage and route of administration: Sorafenib is administered orally as tablets BID 
approximately every 12 hours for 21 days after a course of chemotherapy. See Treatment 
Section 4.2.5. 
 
Tablets can be cut into fourths and the dose rounded to the nearest 50 mg. Tablets should be 
taken with clear liquids (approximately 2 to 4 ounces for children < 12 and 4 to 8 ounces for 
patients ≥12 years). It is recommended that sorafenib be taken on an empty stomach. If taken 
with food, sorafenib should be taken with a moderate to low fat meal. 
 
Toxicity: The most frequently occurring side effects (> 20% of patients) include: fatigue 
(asthenia, lethargy, malaise), rash/desquamation, hand-foot syndrome, diarrhea. Hypertension, is 
frequently seen during the first 6 weeks of therapy and should be monitored and treated. Elevated 
lipase and amylase were commonly reported during adult clinical trials, as was 
hypophosphatemia. Patients on sorafenib have also experienced cardiac ischemia or infarction, 
increased risk of hemorrhage, wound healing complications and gastrointestinal perforation. 
Based on animal studies sorafenib is expected to be teratogenic. 
 
CYP3A4 inhibitors have not been demonstrated to alter the metabolism of sorafenib. 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
CYP 450 inducers: Intake of the following cytochrome P450 enzyme-inducing agents is expected 
to increase the metabolism of sorafenib: Antiepileptic drugs (phenytoin, carbamazepine or 
phenobarbital), rifampin, dexamethasone or St. Johns Wort. These agents should be used with 
caution as they are likely to decrease the concentration of sorafenib. 
 
Drug interactions: Sorafenib is metabolized by the P450 CYP3A4 and UGT1A9 enzymes and has 
been shown in preclinical studies to inhibit multiple CYP isoforms. Therefore, it is possible that 
sorafenib may interact with drugs that are metabolized by the P450 CYP isoenzymes or with drugs 
that inhibit CYP 3A. Close monitoring is recommended for patients taking agents with narrow 
therapeutic indices and metabolized by the liver, such as warfarin, quinidine, cyclosporine, and 
digoxin. Drug interactions (increase in AUC) have been noted with doxorubicin, docetaxel, and 
irinotecan.  
 
Sorafenib is 97% to 99% protein bound. 
 
15. VORINOSTAT (ZOLINZA®, SUBEROYLANILIDE HYDROXAMIC ACID, SAHA) 
 
Source and pharmacology: vorinostat is a histone deacetylase (HDAC) inhibitor. Its chemical 
name is N-hydroxy-N’-phenyl-octane-1, 8-diotic acid diamide, N-hydroxy-N’-phenyl (9CI) 
octanediamide. The HDAC enzymes catalyze the removal of acetyl groups from the lysine 
residues of proteins, such as histones and transcription factors. In some cancer cells, there is an 
overexpression of HDACs or an abnormal recruitment of HDACs to oncogenic transcription 
factors causing hypoacetylation of core nucleosomal histones. Hypoacetylation of histones is 
associated with a condensed chromatin structure and repression of gene transcription. 
 
Vorinostat inhibits HDAC by binding directly to the catalytic pocket of HDAC1, HDAC2, and 
HDAC3 (Class I) and HDAC6 (Class II) enzymes. Inhibition of HDAC activity allows for the 
accumulation of acetylated histones. This accumulation influences the regulation of gene 
expression. In vitro, exposure of cultured transformed cell to vorinostat led to G1 or G2 phase 
cell-cycle arrest, apoptosis, or differentiation and demonstrated synergistic and additive activity 
in combination with other cancer therapies (including radiation, kinase inhibitors, cytotoxic 
agents, and differentiating agents). The mechanism of the antineoplastic effect of vorinostat has 
not been fully characterized. 
 
After oral administration, vorinostat is rapidly absorbed, however, administration with a high-fat 
meal resulted in a 33% increase in the extent of absorption and a 2.5-hour delay in the rate of 
absorption compared to the fasted state. Vorinostat is approximately 71% bound to human 
plasma protein. It is extensively metabolized to inactive metabolites, primarily by 
glucuronidation and hydrolysis followed by beta-oxidation. The two metabolites, O-glucuronide 
of vorinostat and 4-anilino-4-oxobutanoic acid are pharmacologically inactive. In vitro studies 
indicate that vorinostat is not metabolized by and does not inhibit the activity of cytochrome P-
450 enzymes. Less than 1% of an administered dose is excreted unchanged in the urine. 
Approximately 35-52% of an oral dose of vorinostat is excreted in the urine as the two major 
metabolites. The mean terminal half-life of vorinostat and the O-glucuronide metabolite is 
approximately 2 hours, while that of the 4-anilino-4-oxobutanoic acid metabolite it is 11 hours. 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Prolongation of prothrombin time (PT) and International Normalized Ratio (INR) were observed 
in patients receiving vorinostat with coumarin-derivative anticoagulants (e.g., warfarin). 
Therefore PT and INR should be monitored when coumarin-dervative anticoagulants are started 
or discontinued. When vorinostat was administered with other HDAC inhibitors (e.g., valproic 
acid), severe thrombocytopenia and gastrointestinal bleeding have been reported. 
 
Formulation and stability: Vorinostat is supplied as a white, opaque gelatin, size 3 capsule, 
containing 100 mg of vorinostat. The inactive ingredients in each capsule include icrocrystalline 
cellulose, sodium croscarmellose, and magnesium stearate. Vorinostat 100 mg capsules are 
supplied in bottles containing 120 capsules. 
 
Store vorinostat capsules at room temperature, 15 to 30ºC (59 to 86ºF). Do not store above 30ºC 
and avoid exposure to excessive moisture. 
 
Guidelines for administration: See Treatment sections of the protocol. Vorinostat should be taken 
with food. The capsules should not be opened or crushed. A suspension can be prepared by the 
pharmacy for patients that cannot swallow pills. If a patient needs less than 120 capsules for a 
treatment cycle, the exact number of capsules needed for treatment can be counted into a 
prescription bottle. 
 
Direct contact of the powder in vorinostat capsules with the skin or mucous membranes should 
be avoided. If such contact occurs, wash thoroughly. Clean powder spills from broken or 
damaged vorinostat capsules carefully minimizing inhalation. Wash spill area at least 3 times 
with ethyl alcohol, followed by water. 
 
Toxicity: Likely (>20%) adverse events include anemia, diarrhea, nausea, vomiting, fatigue, 
thrombocytopenia, anorexia. See package insert for full information. 
 
Supplier: commercially available. 
 
16. 
INTERLEUKIN-2 (IL-2, ALDESLEUKIN, PROLEUKIN) 
 
Source and pharmacology: Aldesleukin is a biosynthetic cytokine (a lymphokine) of recombinant 
DNA origin. It differs from human interleukin-2 by the absence of an N-terminal alanine, the 
replacement of cysteine with serine at position 125 of the sequence, and the absence of 
glycosylation. It is a biologic response modifier with complex antineoplastic and 
immunomodulating activities.  
 
Formulation and stability: Aldesleukin vials contain 22 million units of lyophilized recombinant 
interleukin. Each single use vial is reconstituted with 1.2 ml of sterile water for injection to give 
18 million units/ml. Aldesleukin should be admixed with D5W for infusion, with albumin 0.1% 
added to decrease adsorption. Do not use an in-line filter. Do not mix in saline.  
 
Supplier: Commercially available. 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Toxicities: Aldesleukin is a highly toxic drug. Most adverse effects are dose related and schedule 
dependent, with fewer toxicities associated with low dose, subcutaneous or continuous IV 
infusions as compared to high dose, rapid IV infusions. Most adverse effects are self-limiting and 
reversible within 2 to 3 days of drug discontinuance. Many of the adverse effects of aldesleukin 
are related to capillary leak syndrome, which has been associated with this drug. The most 
frequently reported serious adverse effects include hypotension, renal dysfunction with 
oliguria/anuria, dyspnea or pulmonary congestion, and mental status changes (lethargy, 
somnolence, confusion, agitation). Additional serious adverse effects reported include 
myocardial ischemia, myocarditis, gangrene, respiratory failure leading to intubation, GI 
bleeding, intestinal perforation, ileus, coma, seizures, sepsis, and renal impairment requiring 
dialysis. Most patients receiving aldesleukin develop some degree of a flu-like syndrome that 
may include fever, chills, rigors, fatigue, weakness, malaise, arthralgia and myalgia. 
 
Dosage and administration: NK Cell transplant participants, see Section 4.7. 
 
17. 
LEUCOVORIN (Folinic Acid) -  
 
Source and pharmacology: Leucovorin is a racemic mixture of tetrahydrofolic acid, which is 
involved as a cofactor for 1-carbon transfer reactions in the synthesis of purine and pyrimidines. 
Leucovorin is a potent antidote for both the hematopoietic and reticuloendothelial toxic effects of 
folic acid antagonists by replenishing reduced folate pools. It is postulated that in some cancers, 
leucovorin enters and “rescues” normal cells from the toxic effects of folic acid antagonists, in 
preference to tumor cells, because of differences in membrane transport and affinity for 
polyglutamylation. Leucovorin is converted in the intestinal mucosa and the liver to 5-methyl-
tetrahydrofolate, which is also active as a reduced folate. It is excreted primarily in the urine with 
minor excretion occurring in the feces. 
 
Formulation and stability: Leucovorin is supplied in 5, 15 and 25 mg tablets and vials containing 
50, 100 or 350 mg of leucovorin as a lyophilized powder. The tablets and the lyophilized powder 
can be stored at room temperature. The 50 mg and 100 vials can be reconstituted by adding 5 or 
10 ml of sterile water or bacteriostatic water for injection respectively to yield a final 
concentration of 10 mg/ml. The 350 mg vials can be reconstituted with 17 ml of sterile water or 
bacteriostatic water for injection to yield a final concentration of 20 mg/ml. The reconstituted 
solution is stable for at least 7 days at room temperature. Leucovorin may be further diluted in 
5% dextrose or 0.9% NaCl containing solutions.   
 
Supplier:  Commercially available. 
 
18.  
HYDROXYUREA – ORAL (Hydrea, HU) 
 
Source and pharmacology: Hydroxyurea is well absorbed after oral administration with the peak 
serum concentration achieved in two hours. The drug is excreted primarily in the urine, either as 
urea or as the unchanged compound. 
 
Formulation and stability: capsules, white crystalline powder. Store at room temperature, avoid 
excessive heat and keep bottle tightly closed. 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Administration: PO. 
 
Supplier: Commercially available. 
 
19. 
ERWINIA L-ASPARAGINASE (Erwinaze) 
 
Source and pharmacology: Erwinia asparaginase is an enzyme. It is derived from Erwinia 
chrysanthemi and may be useful in patients with an allergy to the E. coli derived product. 
Asparaginase hydrolyzes serum asparagine (an amino acid required to synthesize proteins) to 
aspartic acid and ammonia, and is therefore lethal to cells that cannot synthesize asparagine.  
Asparaginase is active during all phases of the cell cycle. Asparaginase is not absorbed from the 
GI tract and must be given parenterally. Asparaginase does not cross into the CSF. The plasma 
half-life of Erwinia asparaginase when given IM is approximately 16 hours. Only minimal 
urinary and biliary excretion occurs. Clearance is unaffected by age, renal function or hepatic 
function. 
 
Formulation and stability: Erwinia asparaginase is available in vials containing 10,000 units of 
lyophilized drug. Unused vials should be refrigerated. The contents of each vial should be diluted 
with 1 ml of preservative-free normal saline, giving a resultant solution of 10,000 units/ml. Once 
in solution, it is recommended that it be used within 8 hours as no preservative is added. 
Occasionally a small number of gelatinous-like fibers may develop upon standing. If this occurs, 
the solution can be filtered through a 5 micron filter to remove the particles with no change in 
potency. 
 
Supplier: Commercially available. 
 
Toxicity: Acute toxicity includes anaphylactic reactions that occur most commonly when the 
drug is given IV. These can be characterized by laryngeal constriction, hypotension, diaphoresis, 
fever, chills, edema and loss of consciousness. Allergic reactions at the site of IM injection 
include pain, swelling and erythema. Other adverse effects include neutropenia and associated 
immunosuppression, mild nausea and vomiting, malaise, anorexia, elevated LFTs, pancreatitis 
and hyperglycemia. A decrease in protein synthesis including albumin, fibrinogen and other 
coagulation factors may occur which can result in hemorrhage. Thrombosis and/or pulmonary 
embolism can also occur. Less common side effects include renal dysfunction and CNS 
complications including somnolence, weakness, lethargy, coma and seizures. 
 
For additional information about this drug, please see package insert. 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX II 
Criteria for Acute Graft-vs.-Host Disease 
   Organ staging:  
Organ 
Stage 0 
Stage 1 
Stage 2 
Stage 3 
Stage 4 
Skin 
No rash 
Rash on       
< 25% body 
surface areaa 
Rash  > 25% to 
< 50%  
Rash >50% 
generalized 
erythroderma 
Plus bullae and 
desquamation 
Gastro-
intestinal 
Diarrhea < 
500 mL/day 
Diarrhea 501 
to 1000 
mL/dayc 
or persistent 
nauseac  
Diarrhea 1001 
to 1500 mL/day  
Diarrhea >1500 
mL/day 
Severe 
abdominal pain 
with or without 
ileus 
Liver 
Bilirubin       
< 2.0 mg/dl 
Bilirubin 2.1 
to 3.0 mg/dlb 
Bilirubin 3.1 to 
6.0 mg/dl 
Bilirubin 6.1 to 
15 mg/dl 
Bilirubin 15 
mg/dl 
 
    Overall Grading for acute GVHDe 
Grade 
Skin 
Liver 
Gut 
I 
Stage 1-2 
None 
None 
II 
Stage 3 or 
Stage 1 or 
Stage 1 
III 
- 
Stage 2-3 or 
Stage2-4  
IVf 
Stage 4 or 
Stage 4 
- 
a) Use rule of Nines” or burn chart to determine extent of rash. 
b) Range given as total bilirubin. Downgrade one stage if additional cause of elevated bilirubin 
is documented.  
c) Volume of diarrhea applies to adults. For pediatric patients, the volume of diarrhea should be 
based on body surface area. Downgrade one stage if additional cause of diarrhea has been 
documented.  
d) Persistent nausea with histological evidence of GVHD in stomach or duodenum. 
e) Criteria for grading given as minimum degree of organ involvement required to confer that 
grade.  
f) Grade IV may also include lesser organ involvement but with extreme decrease in 
performance status.  

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX II (continued) 
CRITERIA FOR GRADING CHRONIC GVHD GRADE 
Staging of Chronic GVHD:  
Limited - Localized skin and/or hepatic dysfunction. 
Extensive - One or more of the following (as clinically judged by a physician and deemed as 
chronic GVHD by the PI): 
• 
Generalized skin involvement 
• 
Liver histology showing chronic aggressive hepatitis, bridging necrosis and/or cirrhosis. 
• 
Eye dryness with Schirmer’s test <5 mm wetting 
• 
Oral:  involvement of salivary glands or oral mucosa. 
• 
Other:  another target organ involvement.  
 
 
  

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX III – SPECIMEN SUBMISSION GUIDELINES 
 
Diagnostic studies 
 
 
 Morphology review 
 
As of Amendment 9.0, central review of morphology is no longer required. 
However, Dr. John Choi 
 is available for review or 
assistance if requested by any collaborating site.  
 
Cytogenetic review 
 
Conventional karyotyping and MLL FISH analysis will be performed at local 
institutions and the results will be reviewed by Dr. Susana Raimondi at St. Jude. 
Please submit two karyotypes of each abnormal line (this can be done 
electronically) and a final report to the following: 
 
 
Dr. Susana Raimondi 
 
Department of Pathology 
 
St. Jude Children’s Research Hospital 
 
262 Danny Thomas Place 
 
Memphis, TN 38105 
 
Phone: 
 
 
FAX: 
 
 
Email: 
 
*Please include FedEx tracking number 
 
Immunophenotyping, molecular studies, MRD, and cell banking 
 
Immunophenotyping for diagnostic purposes may be performed at local institutions or 
centrally, as desired by the treating physician. If the local institution prefers 
immunophenotyping to be done at St. Jude, please indicate this at the time the diagnostic 
bone marrow is shipped. Immunophenotyping to determine a leukemic-specific phenotype 
for future MRD studies will be performed at St. Jude.   
 
The Molecular Pathology laboratory (Dr. Sheila Shurtleff, technical director) will carry out 
molecular studies, including testing for the presence of the AML1-ETO, MLL-AF9, and 
MYH11-CBFβ fusion transcripts, FLT3-ITD, and mutations in the FLT3, NPM1, and CEPBA 
genes. Selected cases will also be screened for the presence of MLL gene rearrangements by 
FISH and for the MLL-ENL, MLL-ELL, MLL-AF10, or RBM15/MKL1 fusions as needed.  
 
At diagnosis, please collect a minimum of 10 mL of anti-coagulated bone marrow 
(preservative free heparin). Place in a 15 mL sterile conical centrifuge tube and add 5 mL 
of sterile RPMI-1640 with 20% fetal calf serum (bovine serum albumin). Label the 
tube(s) with patient name, date of birth, and date sample obtained. Seal centrifuge tube 
with parafilm or equivalent. Complete the AML08 Specimen Submission Form and 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
include with specimen(s) for shipping. Clinical specimens with a low probability of 
containing an infectious agent must be “triple packaged” in accordance with Dangerous 
Goods Regulations (DRG), International Air Transport Association (IATA) and OSHA 
guidelines (see http://www.iata.org, http://osha.gov, and AML08 Submission Form for 
additional information). Specimens should be shipped at ambient temperature.  
 
Note: If bone marrow is unattainable (dry tap) or is less than 5 mL, peripheral blood 
containing leukemic blasts should submitted along with any bone marrow obtained. 5 to 
10 mL of peripheral blood collected in preservative free heparin should be added to 
RPMI 1640 with 20% FCS or BSA, processed and packaged for shipment as described 
for bone marrow specimens.  
 
Ship by Federal Express for next day delivery to: 
 
Tissue Resources 
 Department of Pathology, Room C5013B 
 St. Jude Children’s Research Hospital 
 262 Danny Thomas Place 
 Memphis, TN 38105 
 Phone: 
 
On call mobile:  
 
 Email: 
 
 *Please include FedEx tracking number 
 
Please notify the Tissue Resources lab (Matthew Lear, technical director, 
) and Dr. John Choi 
 via email 
when shipping MRD samples. 
 
For Saturday delivery (new patients only), please notify the Tissue Resources Lab by 
email and by phone on Friday before 4:30 PM Central Standard time at 
. 
 
Ship by Federal Express to: 
 
Tissue Resources 
Delivery to Guards desk (PCC)  
Department of Pathology 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place  
Memphis, TN 38105 
Tel: 
 
Fax: 
 
On call mobile:  
 
 
For unantipicated Saturday deliveries (e.g., diagnostic samples from new patients who arrive on 
a Friday evening), please inform the Tissue Resources director (Matthew Lear, 
) to inform him of the Saturday delivery. 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Minimal Residual Disease studies 
 
At days 8 and 22, please send 5 ml of blood for MRD studies. Samples for MRD should 
be sent to the Tissue Resources Lab in preservative-free heparin stored at ambient 
temperature. 
 
At day 22 and all subsequent time points, please send 5 ml of bone marrow for MRD 
studies. Samples for MRD should be sent to the Tissue Resources Lab in preservative-free 
heparin stored at ambient temperature. In addition, for patients with leukemia-specific 
fusion transcripts, all bone marrow aspirates should be sent for RT-PCR analysis. 
 
Note that MRD studies are not performed on weekends. Hence, samples for MRD should 
be drawn Monday through Thursday and sent by overnight delivery. MRD samples that 
arrive on weekends are processed on Monday, but such delay may affect the quality of 
the assay. Only diagnostic specimens will be accepted on weekends and holidays with 
prior notice. 
 
Please notify the Tissue Resources lab and Dr. John Choi 
 
) via email when shipping MRD samples. 
 
*Please include FedEx tracking number 
 
NK cell studies 
 
All samples related to NK cell research and therapy should be sent at room temperature 
by FEDEX priority overnight delivery to: 
 
 Barbara Rooney 
St. Jude Children's Research Hospital 
262 Danny Thomas Place 
Rm D5032 
Memphis, TN 38105 
Phone 
 
Email: 
  
*Please include FedEx tracking number 
 
Please have the blood drawn Mon-Thurs, for delivery Tuesday-Friday.   
 
 
NK cell receptor studies (all patients) 
 
 At the time of diagnosis and prior to consolidation I, all patients should have 
blood sent for NK cell receptor genotyping and phenotyping. Please send 3 ml at 
diagnosis and 8.5 ml at consol I in yellow top (ACD) tubes. 
 
In addition please send 8.5 ml yellow top (ACD) tube for HLA typing or include 
current HLA typing with shipment. 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
NK typing of parents (all patients who are standard risk or high risk who will 
recieve NK cell transplantation) 
 
For NK typing, 8cc blood should be drawn in yellow top (ACD) tubes. If HLA 
typing needs to be performed, include one extra yellow top tube for each donor. 
Tubes must to be clearly labeled with the donor’s full name and date of birth. 
Please include the completed NK HLA shipping form 
 
NK cell studies for patients who have recieved NK infusions 
 
Following the NK cell infusions, NK chimerism and phenotyping will be 
performed on days 7, 14, 21, and 28. Please send 2 yellow top tubes to Barbara 
Rooney at each of those time points. Note: because NK cell studies are performed 
only on weekdays (Monday through Friday), these tests may be sent within 48 
hours of the time they are due (e.g., if day 2 falls on a Sunday, the tests may be 
drawn on day 3). Post-transplant chimerism studies should also be performed 
after day 28 in cases of persistent chimerism. 
 
Pharmacokinetic studies – (completed with LOA #4, 5/29/15) 
 
Sample processing for sorafenib plasma pharmacokinetic studies: after obtaining whole 
blood, the sample should be placed on ice. The sample is centrifuged at 3000 rpm for 10 
minutes, plasma transferred to a pre-labeled cryovial, and then frozen at -20 °C until the 
time of shipment. 
 
Sample shipment: Samples, and copies of completed pharmacokinetic sample collection 
forms and sorafenib dosing diaries for cycle 1 should be shipped on dry ice in batch 
every 4-6 months to Dr. Sharyn Baker. A FedEx account number will be provided to 
individual sites. Please notify Dr. Baker’s laboratory by email before shipment and 
provide a FedEx tracking number. Ship to: 
 
Dr. Sharyn Baker c/o Shelley Orwick 
Pharmaceutical Sciences Department 
St. Jude Children's Research Hospital 
262 Danny Thomas Place  
CCC Room I5504 
Memphis, TN 38105 
Phone (1): 
 
Phone (2): 
 
 
 
*Please include FedEx tracking number 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
Pharmacogenetic studies – (completed with LOA #4, 5/29/15) 
 
Peripheral blood (10 ml) will be collected at the time of diagnosis and at the time of 
count recovery prior to consolidation I and sent to the Tissue Resources laboratory. 
Plasma will be banked for future proteomic studies and DNA will be extracted from 
lymphocytes for genotyping studies. These samples should be sent in purple top tubes 
and may be shipped with the bone marrow specimens. 
 
Histone acetylation studies – (completed with Revision 10.1, 6/2/16) 
 
For patients who receive vorinostat (see section 4.2.6), histone acetylation will be 
assessed in peripheral blood samples (collected in 3 mL of preservative-free heparin) on 
days -2 and 1 of Induction II (section 4.2.6). Samples may be ficolled, frozen and 
batched for shipping to: 
 
Drs. Yubin Ge and Larry Matherly 
Karmanos Cancer Institute 
110 East Warren Avenue 
Detroit, Michigan 48201 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX IV: MOLECULAR DIAGNOSTICS FOR FEVER AND NEUTROPENIA 
 (St. Jude patients only) 
 
Note:  As of Amendment 9.0, this research study will no longer be done. 
 
Background – A significant challenge among patients with neutropenia and fever is 
identification of causative infectious agents. Present technology is limited in this regard, with an 
etiology typically identified in only 30-50% of cases. Detection of bacterial agents continues to 
largely rely upon culture-based methods. In recent years, we have improved our blood culture 
methodologies and have markedly improved the positive culture rate at St. Jude; however, even 
with these enhancements, bacterial or fungal pathogens are isolated from less than 25% of cases. 
While viral diagnostics have made much greater use of molecular diagnostic techniques; most 
assays in routine use target only a single agent or class of agents, again potentially limiting their 
sensitivity. 
 
We propose the application of broad-range molecular diagnostic assays, targeting bacterial, 
fungal, and viral pathogens, in an effort to identify infectious agents in patients with febrile 
neutropenia. Several small studies in the literature provide evidence that such techniques may 
result in a substantial increase in sensitivity over culture; however, these methods have not been 
broadly applied to pediatric oncology patients. 
 
Hypothesis – The use of broad-range molecular diagnostic methods will enable the direct 
detection of infectious agents associated with febrile neutropenia, resulting in an increased speed 
and sensitivity of detection of pathogens currently recovered by culture, and allowing the 
identification of non-cultivable organisms. 
 
Objectives – Determine the performance characteristics of broad-range, molecular diagnostic 
methods for the detection of bacterial, fungal, and viral agents, in comparison to methods 
currently in routine clinical use. 
 
Methods – Concomitant with initial bacterial blood culture for each episode of febrile 
neutropenia, two (2) 4 ml EDTA vacutainer tubes of whole blood will be collected in an aseptic 
manner (patients less than 5 years of age will have only one (1) 4 ml EDTA tube of blood 
collected). Samples will transported to the Clinical Molecular Microbiology Laboratory at room 
temperature. Samples will be held at 4°C and processed within 7 days or frozen at -80° C until 
processing. Samples will be coded and de-identified prior to all processing and analysis. 
Processing will be performed in large batches. Following rapid thawing, nucleic acid extraction 
will be performed on each sample. Broad-range molecular detection methods will be performed 
for the detection of bacterial, fungal, and viral agents. Results of analysis will remain coded and 
unavailable to clinical care providers, as this methodology remains experimental. Results of 
testing will be correlated to the underlying clinical condition and history of patients, clinical 
course, and results of routine laboratory testing, including, but not limited to, culture, antigen 
detection, and viral PCR. Discrepant results will be reconciled based on chart review, together 
with results of tissue pathology and other laboratory results. Comparisons of current methods 
will be made relative to test sensitivity, specificity, clinical predictive value, time required to 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
detect a positive result, and ability to detect novel or non-cultivable organisms. The ability to 
risk-stratify pathogens based on subspecies-level variation will also be examined. 
 
Agreement and discrepancies between the molecular and standard methods will be assessed by 
the study pathologist. Probability (rates) of agreement and discrepancies will be estimated with a 
95% confidence interval. Of particular interest will be the probability (rate) of infections 
immediately following the febrile neutropenia episode among the patients tested positive by the 
molecular method but negative by the standard method; this will be estimated with a 95% 
confidence interval. Rates of infection immediately following the febrile neutropenia episode 
among the patients tested positive vs. negative by the two methods will be estimated and 
compared as well using models of paired categorical (binary) responses.  
 
References 
 
1. Ammann RA, Zucol F, Aebi C, Niggli FK, Kuhne T, Nadal D. Real-time broad-range 
PCR versus blood culture. A prospective pilot study in pediatric cancer patients with 
fever and neutropenia. Support Care Cancer 2007 June;15(6):637-641. 
2. Hakim H, Flynn PM, Knapp KM, Srivastava DK, Gaur AH. Etiology and Clinical Course 
of Febrile Neutropenia in Children With Cancer. J Pediatr Hematol Oncol 2009 July 28. 
3. Seng P, Drancourt M, Gouriet F et al. Ongoing revolution in bacteriology: routine 
identification of bacteria by matrix-assisted laser desorption ionization time-of-flight 
mass spectrometry. Clin Infect Dis 2009 August 15;49(4):543-551. 
4. Sjoholm MI, Dillner J, Carlson J. Multiplex detection of human herpesviruses from 
archival specimens by using matrix-assisted laser desorption ionization-time of flight 
mass spectrometry. J Clin Microbiol 2008 February;46(2):540-545. 
5. Varani S, Stanzani M, Paolucci M et al. Diagnosis of bloodstream infections in 
immunocompromised patients by real-time PCR. J Infect 2009 May;58(5):346-351. 
6. Xu J, Moore JE, Millar BC et al. Improved laboratory diagnosis of bacterial and fungal 
infections in patients with hematological malignancies using PCR and ribosomal RNA 
sequence analysis. Leuk Lymphoma 2004 August;45(8):1637-1641 
 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX V: RANDOMIZATION FORM 
 
AML08 RANDOMIZATION FORM 
 
Please call the St. Jude Pharmacy at 
 to let them know you have enrolled a new patient and for 
randomization. 
Check only one box below and FAX this information to the St. Jude pharmacy at 
. If you are 
unable to send this form by FAX, the information may be given by phone and sent by FAX later. 
 
MRN:                                                           Initials:                                Date:                                                
                                                                                                                                   MM        DD         YY    
                                                                                                                                              
Institution: 
 St. Jude Children’s Research Hospital 
 Stanford University Medical Center 
 
 
 Children’s Hospital of Michigan 
 Cook Children’s Medical Center 
 
 
 Dana Farber Cancer Institute 
 
 Singapore 
 
 
 University of Chicago                               
 Rady Children’s Hospital 
 
       
If Karyotype is available, select one category 
below. If the patient has a high-risk feature listed 
below and one of the favorable translocations, 
please check the box for high-risk only. 
 
 
 inv(16) 
 
 t(8,21) 
 
 11q23 
 
 M7 
 
 Others 
        
If Karyotype is not available, select one category below. 
 
  
 M2 with Auer rods 
  
 M4Eo 
  
 M5 
  
 M7 
  
 Other 
Criteria for Randomization: 
 
 Normal creatinine for age  
 Serum bilirubin ≤ 1.5 x ULN (bili _____ mg/dl) 
 AST and ALT ≤ 2.5 x ULN (AST/SGOT ____ mg/dl AND ALT/SGPT _____ mg/dl) 
 Alkaline phosphatase ≤ 2.5 x ULN (alkaline phosphatase ____ mg/dl) 
 
Patients with hepatic dysfunction who do not meet all of the above criteria WILL NOT BE RANDOMIZED, 
but can be enrolled on AML08 and treated with Induction I (HD-ADE). 
 
 Do NOT randomize this patient. 
 
 
Physician:  
                       Please print. 
 
Contact number for randomization results:                                                                          
           
 
(Be sure to provide your contact number (phone or pager) so that the pharmacist can give you the randomization 
results.) 

AML08 
 
Revision 10.1, dated: 06-02-2016 
 
 
 
 
IRB Approval date: 06-07-2016 
 
Protocol document date: 06-02-2016 
APPENDIX VI:  TREATMENT SCHEMA 
 
 
Note:  collaborating sites may elect to opt out of vorinostat administration during Induction II.  Participants at these sites will receive Induction II 
with LD-ADE alone.