Clinical trial NCT02395666
Conditions: Neuroblastoma
Keywords: Neuroblastoma in remission Relapsed Neuroblastoma Refractory Neuroblastoma
Sponsor: Giselle Sholler
Start date: 2015-03-05; Completion date: 2023-08-24
Enrollment: 140 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this research study is to evaluate a new investigational drug to prevent reoccurrence of neuroblastoma that is in remission. This study drug is called DFMO. The objectives of this study will be to monitor for safety and look at efficacy of DFMO.

The safety of the proposed dosing regimen in this trial will be tested by an on-going risk/benefit assessment during the study. A patient benefiting from treatment, not progressing on therapy, and in the absence of any safety issues associated with DFMO may continue on treatment up to 27 cycles with the expectation that there will be an overall clinical benefit.

The procedures involved in this study include Medical history, Physical exam, Vital signs (blood pressure, pulse, temperature), Blood tests, Urine tests, MRI or CT scan of the tumor(s), meta-iodobenzylguanidine (MIBG) scans, and Bone marrow aspirations. All of these tests and procedures are considered standard of care for this population. Drug administration is also part of this protocol, including an investigational new drug called DFMO.

The proposed dosing regimen is an oral dose of DFMO tablets two times a day for each day while on study. There will be 27 cycles. Each cycle will be 28 days in length.

Detailed description:

Arms: arm 1: Subjects will receive twenty-seven (27) cycles of oral DFMO at a dose of 500 to 1000 mg/m2 BID on each day of a 28 day cycle.
Interventions: intervention 1: DFMO
Intervention descriptions: intervention 1: Subjects will receive twenty-seven (27) cycles of oral DFMO at a dose of 500 to 1000 mg/m2 BID on each day of a 28 day cycle.

Protocol documents:
Prot_SAP_001.pdf: 
Clinical Protocol DFMO Phase II 
 
NMTRC 003B Version 5.1 Issued 18 February 2015             Confidential 
 Page 1 of 63 
 
 
 
 
 
 
Protocol DFMO Phase II 
 
NMTRC 003B 
 
 
 
 
 
 
 
A Phase II Preventative Trial of DFMO (eflornithine HCl) as a 
single agent in Patients with High Risk Neuroblastoma in 
Remission. 
 
 
 
 
 
 
Version 5.1  
February 18, 2015 
 
 
 
 
 
 
 
 
 
 
 
 


Clinical Protocol DFMO Phase II 
 
NMTRC 003B Version 5.1 Issued 18 February 2015             Confidential 
 Page 2 of 63 
 
Contact Information 
 
 
 
 
 
 
 
Study Chair:    
 
Giselle Sholler, MD  
NMTRC Chair 
100 Michigan Avenue NE  MC 272          
Grand Rapids, MI 49503 
Tel:  (616) 267-0332  
Fax: 616-391-2785 
giselle.sholler@helendevoschildrens.org 
 
 
 
 
 
 
Safety Officer:        
 
Stephen S. Roberts, MD 
Assistant Attending Physician, Neuroblastoma Program 
Memorial Sloan-Kettering Cancer Center 
1275 York Avenue 
New York, NY 10065 
Tel: 212-639-4034  
robertss@mskcc.org 
 
 
 
 
 
 
 
 
 
56B 
 
 


Clinical Protocol DFMO Phase II 
 
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STUDY COMMITTEE 
 
STUDY CHAIR: 
Giselle Sholler, MD  
NMTRC Chair 
100 Michigan Avenue NE  MC 272         
Grand Rapids, MI 49503 
 
Tel:  (616) 267-0332  
Fax: 616-391-2785 
giselle.sholler@helendevoschildrens.org 
 
STUDY COMMITTEE MEMBERS: 
 
André S. Bachmann, PhD 
Professor and Associate Chair for Research 
Department of Pediatrics and Human 
Development 
College of Human Medicine 
Michigan State University 
Cook-DeVos Center for Health Sciences 
301 Michigan Street, NE, Office 514  
Grand Rapids, Michigan 49503, USA 
 
Phone office: 1-616-331-5982 
E-mail: andre.bachmann@hc.msu.edu  
 
Genevieve Bergendahl, RN 
Clinical Program Director 
NMTRC 
100 Michigan Avenue NE  MC 272         
Grand Rapids, MI 49503 
 
 
Tel:  (616) 267-0335 
Fax: 616-391-2785 
E-Mail:  
Genevieve.bergendahl@helendevoschildrens.org  
 
 
Alyssa VanderWerff 
Clinical Program Coordinator 
NMTRC 
100 Michigan Avenue NE  MC 272         
Grand Rapids, MI 49503 
 
Tel:  (616) 267-0327  
Fax: 616-391-2785 
E-Mail:  
Alyssa.VanderWerff@helendevoschildrens.org  
 
Abhinav Nagulapally, MSc 
Computational Biologist 
NMTRC 
100 Michigan Avenue NE  MC 272         
Grand Rapids, MI 49503 
 
Tel:  (616) 267-0309   
Fax: 616-391-2785 
E-Mail:  
Abhinav.Nagulapally@helendevoschildrens.org 
Takamaru Ashikaga, PhD 
Professor and Director of Medical Biostatistics 
University of Vermont 
Burlington, VT 05405 
 
(P)1-802-656-2526 
(F)1-802-656-3632 
Takamaru.Ashikaga@uvm.edu 
 


Clinical Protocol DFMO Phase II 
 
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William Roberts, MD  
Pediatric Hematology/Oncology  
Rady Children's Hospital San Diego  
3020 Children’s Way MC5035  
San Diego, CA 92123-4282  
Tel: (858) 966-5811  
Fax: (858) 966-8035  
Email: wroberts@chsd.org  
William Ferguson, MD  
Pediatric Hematology-Oncology  
Cardinal Glennon Children’s Medical Center  
Saint Louis University School of Medicine  
1465 S. Grand Blvd.  
Saint Louis, MO 63104  
 
Tel: (314) 577-5638  
Fax: (314) 268-4081  
Email: fergusws@slu.edu  
Javier Oesterheld, MD 
Pediatric Hematology/Oncology 
Blumenthal Cancer Center 
1025 Morehead Medical Drive, Suite 600 
Charlotte, NC 28204 
Phone-704-381-9900 
Fax-704-381-9901 
Pager-704-355-4088  #8553 
E-Mail: 
Javier.oesterheld@carolinashealthcare.org 
Jacqueline M Kraveka, DO 
Assistant Professor of Pediatrics 
Medical University of South Carolina 
Division of Pediatric Hematology-Oncology 
135 Rutledge Avenue, Room 480 
MSC 558 
Charleston, SC 29425 
Office: 843-792-2957 
Pager: 843-792-2123 
Fax: 843-792-8912 
E-Mail: kravekjm@musc.edu 
Deanna Mitchell, MD 
Medical Director, Coagulation Disorders Program 
Assistant Professor, Pediatrics 
Michigan State University 
Helen DeVos Children’s Hospital 
100 Michigan NE 
Grand Rapids, MI  49503 
 
Phone:  616-391-2086 
FAX:  616-391-8873 
E-Mail: 
Deanna.Mitchell@devoschildrens.org 
Don Eslin, MD 
MDACCO 
Pediatric Hematology/Oncology 
50 W. Sturtevant St. 
Orlando, FL 32806 
 
Office: 321-841-8588 
E-Mail: Don.Eslin@orlandohealth.com 


Clinical Protocol DFMO Phase II 
 
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Nehal S. Parikh, MD 
Connecticut Children’s Medical Center 
Pediatric Hematology/Oncology 
282 Washington Street, Suite 2J 
Hartford, CT 06106 
Office: 860-545-9630 
Fax: 860-545-9622 
E-Mail: nparikh@connecticutchildrens.org 
 
Kathleen Neville, MD, MS 
Children's Mercy Kansas City 
Director, Experimental Therapeutics 
    in Pediatric Oncology Program 
2401 Gillham Rd. 
Kansas City, MO  64108 
 
 
Tel: 816-234-3059 
Fax: 816-302-9943 
kaneville@cmh.edu 
 
Francis Eshun, MD 
Center for Cancer and Blood Disorders 
Phoenix Children's Hospital 
1919 Thomas Road 
Phoenix Az 85016 
 
 
feshun@phoenixchildrens.com 
 
BIOANALYSIS LABORATORIES 
 
Spectrum Health Flow Cytometry Laboratory 
Attn: Pamela Kidd, MD 
Lemmen-Holton Cancer Pavilion 
145 Michigan Street NE Suite 6201 
Grand Rapids, MI 49503 
 
 
Dr. Shulin Li 
7777 Knight Rd,  
Smith Research Building, MOD2.009 
Houston, TX-77054 
 
 
 


Clinical Protocol DFMO Phase II 
 
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57BINVESTIGATOR SIGNATURE SHEET 
 
I have read the attached protocol and agree that it contains all the necessary details for 
performing the study. 
 
I will provide copies of the protocol and of the preclinical and clinical information 
(Investigator’s Brochure) on the test article, which was furnished to me by the Neuroblastoma 
and Medulloblastoma Translational Research Consortium (NMTRC), to all members of the study 
team responsible to me who participate in the study.  I will discuss this material with them to 
assure that they are fully informed regarding the test article and the conduct of the study. 
 
Once the protocol has been approved by the Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC), I will not modify this protocol without obtaining the prior approval of 
the NMTRC and of the IRB/IEC.  I will submit the protocol modifications and/or any informed 
consent form (ICF) modifications to the NMTRC and the IRB/IEC, and approval will be 
obtained before any modifications are implemented. 
 
I understand the protocol and will work according to it, the principles of Good Clinical Practice 
(GCP) [current International Conference of Harmonization (ICH) guidelines], and the 
Declaration of Helsinki (1964) including all amendments up to and including the Scotland 
revision (2000) and notes of clarification added in 2002 and 2004. 
 
 
 
 
 
 
Investigator's Signature 
 
Date 
 
 
 
Investigator’s Printed Name 
 
 
 
 
 
Investigational Site Name 
 
 
 
 
 
 


Clinical Protocol DFMO Phase II 
 
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8B 
TABLE OF CONTENTS 
 
 
CONTACT INFORMATION ............................................................................................................................................ 2 
STUDY COMMITTEE ............................................................................................................................................. 3 
INVESTIGATOR SIGNATURE SHEET ................................................................................................................. 6 
TABLE OF CONTENTS ........................................................................................................................................... 7 
PROCEDURES AND ASSESSMENTS TABLE ................................................................................................................. 15 
 
1.0  
 PROTOCOL CONCEPT ..................................................................................................................................... 16 
 
2.0 
BACKGROUND AND PRELIMINARY DATA ....................................................................................................... 16 
 
2.1  PREVIOUS PRECLINICAL AND CLINICAL WORK ....................................................................................... 16 
 
2.2  CLINICAL WORK...................................................................................................................................... 20 
 
3.0 
STUDY OBJECTIVES ........................................................................................................................................ 23 
 
3.1 STRATUM 1 PRIMARY OBJECTIVE: ........................................................................................................... 23 
 
3.2 STRATUM 1 SECONDARY OBJECTIVES: ..................................................................................................... 23 
 
4.0 
STUDY DESIGN ............................................................................................................................................... 23 
4.1 DFMO TREATMENT ................................................................................................................................. 23 
4.2 RESPONSE EVALUATIONS ......................................................................................................................... 24 
4.3 BIOLOGICAL STUDIES ............................................................................................................................... 24 
 
5.0 
PATIENT SELECTION ...................................................................................................................................... 25 
 
5.1 INCLUSION CRITERIA: .............................................................................................................................. 25 
 
6.0 
STUDY DRUG ADMINISTRATION .......................................................................................................... 26 
 
6.1 DFMO ..................................................................................................................................................... 26 
6.1.1 DESCRIPTION, FORMULATION, AND STORAGE OF DFMO ....................................................... 26 
6.1.2 DFMO DOSING TABLE PER WEIGHT GROUP: ......................................................................... 27 
6.1.3 ADMINISTRATION OF DFMO .................................................................................................. 27 
6.1.4 SUBJECTS UNABLE TO SWALLOW TABLETS ............................................................................ 27 
6.1.5 MISSED DOSES ........................................................................................................................ 28 
6.1.6 DFMO CLINICAL PHARMACOLOGY ........................................................................................ 28 
6.2 GUIDELINES FOR DOSE MODIFICATIONS .................................................................................................. 30 
6.3 DOSE MODIFICATION ............................................................................................................................... 31 
6.4 STUDY DRUG ACCOUNTABILITY .............................................................................................................. 31 
6.5 CONCOMITANT MEDICATIONS AND TREATMENTS .................................................................................... 32 
 
7.0 
STUDY PROCEDURES AND ASSESSMENTS ......................................................................................... 32 
7.1 ENROLLMENT OF PATIENTS ...................................................................................................................... 32 
7.2 SCREENING ............................................................................................................................................... 33 
7.3 TREATMENT PHASE – CYCLE 1 ................................................................................................................. 34 
7.4 TREATMENT PHASE – CYCLES 2-27 .......................................................................................................... 35 
7.5 OTHER SCHEDULES: ................................................................................................................................. 37 
7.6 PROTOCOL TREATMENT COMPLETION ..................................................................................................... 37 
7.7 OFF-THERAPY / 30 DAY FOLLOW-UP VISIT .............................................................................................. 38 
7.8 SURVIVAL FOLLOW-UP ............................................................................................................................. 38 
 
 
 


Clinical Protocol DFMO Phase II 
 
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8.0 
ADVERSE EVENT REPORTING ......................................................................................................................... 39 
 
8.1 DEFINITIONS ............................................................................................................................................. 39 
8.1.1 ADVERSE EVENT ..................................................................................................................... 39 
8.1.2 UNEXPECTED ADVERSE EVENT ............................................................................................... 39 
8.1.3 SERIOUS ADVERSE EVENT ...................................................................................................... 39 
8.2 DOCUMENTING ADVERSE EVENTS ........................................................................................................... 40 
8.3 EXPEDITED REPORTING OF ADVERSE EVENTS .......................................................................................... 40 
8.4 GRADING AND RELATEDNESS OF ADVERSE EVENTS ................................................................................ 41 
8.4.1 GRADING OF SEVERITY OF AN ADVERSE EVENT ..................................................................... 41 
 
8.5 RELATEDNESS TO STUDY DRUG ............................................................................................................... 42 
 
9.0 
PATIENT WITHDRAWAL AND TRIAL DISCONTINUATION ................................................................................ 43 
9.1 CRITERIA FOR SUBJECT OFF-THERAPY .................................................................................................... 43 
9.3 TRIAL DISCONTINUATION ........................................................................................................................ 43 
 
10.0 DATA ANALYSIS ....................................................................................................................................... 44 
10.1 DATA QUALITY ASSURANCE .................................................................................................................. 44 
10.2 STATISTICAL ANALYSIS ......................................................................................................................... 44 
10.3 EVENT FREE SURVIVAL .......................................................................................................................... 46 
10.4 OVERALL SURVIVAL .............................................................................................................................. 46 
 
11.0 ADMINISTRATIVE PROCEDURES .......................................................................................................... 46 
11.1 PATIENT INFORMED CONSENT ................................................................................................................ 46 
11.2 ETHICAL CONDUCT OF THE STUDY AND IRB/IEC APPROVAL ................................................................ 47 
11.3 DATA SAFETY MONITORING BOARD (DSMB) ....................................................................................... 47 
11.4 MONITORING AND AUDITING ................................................................................................................. 47 
11.5 PRE-STUDY DOCUMENTATION ............................................................................................................... 49 
11.6 CONFIDENTIALITY .................................................................................................................................. 49 
11.7 SOURCE DOCUMENTS ............................................................................................................................. 49 
11.8 RECORD RETENTION .............................................................................................................................. 50 
 
12.0 BIOLOGICAL EVALUATION ............................................................................................................................. 50 
12.1 BIOLOGICAL CORRELATIVE STUDIES SAMPLE INSTRUCTIONS ................................................................ 53 
12.2 SAMPLE STORAGE AND DESTRUCTION ................................................................................................... 56 
 
 
APPENDIX I: PERFORMANCE STATUS/SCORES .......................................................................................................... 57 
 
 
 
 


Clinical Protocol DFMO Phase II 
 
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A Phase II Preventative Trial of DFMO (eflornithine HCl) as a Single Agent in Patients 
with High Risk Neuroblastoma in Remission. 
 
PROTOCOL SYNOPSIS 
PROTOCOL TITLE 
A Phase II Preventative Trial of DFMO as a Single Agent in 
Patients with High Risk Neuroblastoma in Remission. 
PROTOCOL NUMBER 
NMTRC 003B 
PHASE OF DEVELOPMENT 
Phase II 
OBJECTIVES 
Primary 
• To evaluate the preventative activity of DFMO as a single 
agent in  patients with neuroblastoma who are in remission 
based on: 
 
Event free survival (EFS)  
Secondary 
• To evaluate the preventative activity of DFMO as a single 
agent in  patients with neuroblastoma who are in remission 
based on: 
 
Overall Survival (OS) 
• Correlation of urinary polyamine levels with progression of 
disease in neuroblastoma. 
• To determine the safety and tolerability of DFMO as a single 
agent in pediatric and young adult patients with high risk 
neuroblastoma that is in remission. 
• To evaluate the pharmacokinetics (PK) of new DFMO 
formulation in a minimum of 10 patients 
• Biological Correlates to minimally include: 1) Urine: 
polyamine levels and blood inflammatory markers, 2) Blood: 
microRNA analysis as predictor of DFMO effect, ODC SNP 
analysis in DNA isolated from nucleated cells, circulating 
tumor cell analysis, and explorative biomarker analysis 3) 
Bone Marrow: flow cytometry of minimal residual disease of 
tumor; explorative biomarker analysis 
 
 
STUDY DESIGN 
DFMO will be used in an open label, single agent, multicenter, 
study for patients with neuroblastoma that are in remission. 
Cycle 1-27: DFMO 
In this study subjects will receive twenty-seven (27) cycles of 
oral DFMO at a dose of 500 to 1000 mg/m2 BID (per dosing 
chart in section 6.1.2) on each day of a 28 day cycle. 
 
 
 


Clinical Protocol DFMO Phase II 
 
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Subjects will be evaluated in 2 strata: 
 
• Stratum 1: Subjects that are in remission at the end of 
upfront therapy defined as chemotherapy (5-7 cycles), 
surgery as indicated, consolidation therapy as indicated, 
radiation therapy as indicated, anti-GD2 antibody therapy 
with retinoic acid up to 6 cycles. 
 
• Stratum 2: Subjects that are in remission after any 
previous relapse or refractory therapy 
 
 
Stratum 1- A total of 127-140 subjects in Stratum 1 will be 
enrolled to ensure that there will be 127 evaluable subjects.  
 
Stratum 2- A total of 38 subjects in Stratum 2 will be enrolled to 
ensure that there will be 33 evaluable subjects.  
 
ELIGIBILITY 
Inclusion Criteria 
1. Age: 0-21 years at the time of diagnosis. 
2. Diagnosis: histological verification at either the time of 
original diagnosis or a previous relapse of high risk 
neuroblastoma. 
3. Disease Status: Neuroblastoma that is in Remission 
4. Greater than 30 days from completion of cytotoxic and 
antibody therapy and less than 120 days from previous 
therapy. 
5. A negative serum or urine pregnancy test is required for 
female subjects of child bearing potential (onset of menses 
or ≥13 years of age).  
6. Both male and female post-pubertal study subjects need to 
agree to use one of the more effective birth control methods 
during treatment and for six months after treatment is 
stopped. These methods include total abstinence (no sex), 
oral contraceptives (“the pill”), an intrauterine device (IUD), 
levonorgestrol implants (Norplant), or medroxyprogesterone 
acetate injections (Depo-provera shots). If one of these 
cannot be used, contraceptive foam with a condom is 
recommended. 
7. ANC > 500/μl and platelet count >50,000/μl 
8. Organ Function Requirements 


Clinical Protocol DFMO Phase II 
 
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Subjects must have adequate liver function as defined by 
AST and ALT <10x upper limit of normal  
Serum bilirubin must be ≤ 2.0 mg/dl 
Serum creatinine based on age/gender as follows: 
Age  
Maximum Serum Creatinine 
(mg/dL) 
 
 
  
 
  
 Male  
Female  
 
1 month to < 6 
months  
0.4 
0.4   
6 months to < 1 year  
0.5 
05   
1 to < 2 years  
0.6 
0.6   
2 to < 6 year 
  
0.8 
0.8   
6 to < 10 years  
1 
1   
10 to < 13 years  
1.2 
1.2   
13 to < 16 years  
1.5 
1.4   
≥ 16 years  
1.7 
1.4   
 
9. Informed Consent: All subjects and/or legal guardians must 
sign informed written consent. Assent, when appropriate, 
will be obtained according to institutional guidelines 
 
Exclusion Criteria 
1. Lansky score  < 60% 
2. BSA (m2) of  <0.25 
3. Investigational Drugs: Subjects who are currently receiving 
another investigational drug are excluded from participation. 
4. Anti-cancer Agents: Subjects who are currently receiving 
other anticancer agents are not eligible. Subjects must have 
fully recovered from the effects of prior chemotherapy 
(hematological and bone marrow suppression effects). 
5. Infection: Subjects who have an uncontrolled infection are 
not eligible until the infection is judged to be well controlled 
in the opinion of the investigator. 
6. Subjects who, in the opinion of the investigator, may not be 
able to comply with the safety monitoring requirements of 
the study, or in whom compliance is likely to be suboptimal, 
should be excluded.  
 
 
 
 


Clinical Protocol DFMO Phase II 
 
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Additional criteria: 
Subjects willing to participate in the correlative biologic studies 
will sign an additional consent form to provide bone marrow and 
blood for analysis. 
ESTIMATED NUMBER OF 
SUBJECTS/GEOGRAPHIC 
REGIONS 
A projected total of 160 evaluable subjects (Stratum 1-127 
subjects, Stratum 2-33 subjects).  Up to 25 sites in the United 
States will participate. 
LENGTH OF STUDY 
Accrual to this study is estimated to be at least 2 years, with 
follow up until all patients meet primary and secondary 
endpoints. 
INVESTIGATIONAL PRODUCT 
DOSE/ROUTE/REGIMEN 
DFMO is an oral tablet that will be administered daily on a 28-
day cycle.  
Dose will be 500 to 1000mg/m2 given BID (per dosing chart in 
section 6.1.2) 
STUDY ASSESSMENTS 
Refer to Table of Assessments for timing of study procedures. 
CRITERIA FOR EVALUATION 
Preventative Activity Measures: Primary objectives 
• Event Free Survival (EFS), defined as period from the first 
day of administration of study drug to the first occurrence of 
relapse, progressive disease, secondary cancer, or death or, if 
none of these events occurred, until the last contact with the 
subject. 
Safety Measures and Preventative Activity Measures: 
Secondary objectives 
• Correlation of urinary polyamine levels with progression of 
disease in neuroblastoma. 
 
• Overall Survival (OS) will be defined as the first day of 
administration of study drug until death or will be censored at 
the last contact with the subject if death did not occur during 
the study. 
 
• Safety analysis will be conducted on all subjects who have 
received at least one dose of study drug, and will include the 
frequency of all adverse events and laboratory abnormalities 
as well as frequency of dose interruptions, dose reductions 
and treatment discontinuation. 
 
 
 
 
 


Clinical Protocol DFMO Phase II 
 
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STATISTICS/SAMPLE SIZE 
ESTIMATE 
Statistics: 
All baseline subject characteristics will be summarized in a 
tabular format.  Safety data will be described for all subjects 
receiving at least one dose of DFMO.  Safety data will include 
values for hematology, serum chemistry, vital signs, and adverse 
events. The proportion of subjects experiencing adverse events, 
serious adverse events, and treatment delays will be summarized 
for each dosing cohort. 
 
Sample Size: 
Stratum 1: A 70% 2-year EFS rate was selected as the Phase I 
baseline rate.  The baseline 70% EFS rate at 2-years was based 
upon published data by Yu et al. It is hypothesized that DFMO 
will increase the 2-year EFS rate to 80% which represents an 
approximately 10% increase in the duration of the EFS rate at 2-
years.  Assuming a directional 5% one population binomial test, 
a sample size of n = 127 patients will be required to achieve an 
80% power to detect this 2-year difference in EFS (70% vs 
80%). 
The comparison of the biomarker prevalence rates assumes that 
the Stratum 1 cohort will experience a relapse rate of 30% and a 
70% non-relapse rate at up to two years of follow-up.  It is also 
assumed that the corresponding prevalence of an elevated 
biomarker will be 60% for the relapse group and 30% in the non-
relapse group, respectively.  This hypothesized prevalence 
difference will be detectable with at least an 85% power with an 
overall sample size of N=127 patients (38 relapse and 89 non-
relapse) using a Fisher’s Exact Test and a non-directional Type I 
Error level of 5%.   
 
Stratum 2: Examination of EFS will require a sample size of n = 
33 subjects in Stratum 2  to test whether treatment with DFMO 
can prolong the overall estimated EFS at two years to 30% from 
the 10% at two years estimate  for the known historical data in 
this subject population. A one population binomial test with 
power 80% and a 5% two-tailed Type I error level will require n 
= 33 total patients 
The one population binomial test will reject the null hypothesis 
that the two year EFS rate = 10% in favor of the EFS = 30% if 7 
or more of the n = 33 patients are observed to have at least a two 
year EFS.  
 
 
 


Clinical Protocol DFMO Phase II 
 
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Pharmacokinetics: 
DFMO plasma concentration-time data will be determined for 
the first 10 or more subjects enrolled on the new DFMO 
formulation.  The following PK parameters will be determined 
for the last dose of current formulation DFMO and after 2-5 
days of new formulation DFMO: 
•     Cmax 
•     AUC (0 – 24 hr) 
•     Plasma half-life (t1/2) 
•     Plasma clearance (Cl) 
• 
Vd                                                                   
 


Clinical Protocol DFMO Phase II 
 
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 Procedures and Assessments 
a Voluntary – additional informed consent required for blood 
b End of cycle 3, 6, 9, 13, 20, and at off protocol therapy (end of Cycle 27)- or as 
clinically indicated.; see section 7.4 for specific details) 
c If concern for progression of disease (and/or if hematologic toxicity grade 4) 
d End of cycle 6, 12, and off protocol therapy (and as clinically indicated; see section 7.4 
for specific details) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
e PK’s on last day of tablet formulation and Day 2-5 of starting new  formulation per 
Sec 12. 
f- Cycles 1-12, 14, 17, 21, 24, and off therapy (end of cycle 27) 
g- Cycles 3, 6, 9, 12, 14, 17, 21, 24, and off therapy (end of cycle 27) 
^Standard of Care Bone Marrow. (Separate consent required for additional research 
samples per section 12.0) 
 
Pre 
Cycles 1  
Cycles 2-27 
Follow Up 
 
 
  Day 1 
Day 8 
Day 
15 
Day 
21 
End of 
cycle 
Day 
1 
Day 8 
Day 
15 
Day 
21 
End of 
Cycle 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical & surgical history, demographics
histologic evidence of malignancy 
X 
 
 
 
 
 
 
 
 
 
 
 
Prior therapy for malignancy 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical examination  
X 
X 
 
X 
 
 
X 
 
 
 
 
X 
Vital signs 
X 
X 
 
X 
 
 
X 
 
 
 
 
X 
ECOG performance/Lansky play status 
X 
 
 
 
 
 
X 
 
 
 
 
X 
CBC and differential 
X 
 
 
X 
 
 
Xf 
 
 
 
 
X 
Serum chemistries (as defined in 7.0) 
X 
 
 
X 
 
 
Xf 
 
 
 
 
X 
CRP and ESR 
X 
 
 
 
 
 
Xf 
 
 
 
 
 
Pregnancy test 
X 
 
 
 
 
 
X 
 
 
 
 
 
MIBG/CT/MRI Scan as needed 
X 
 
 
 
 
 
 
 
 
 
Xb 
X 
Concomitant medications 
X 
X 
 
X 
 
 
X 
 
 
 
 
X 
BSA calculation 
X 
 
 
 
 
 
X 
 
 
 
 
 
Administration of DFMO 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense (and collect) drug dosing diary 
 
X 
 
 
 
 
X 
 
 
 
 
X 
AE monitoring 
 
X 
 
X 
 
 
X 
 
 
 
 
X 
PK blood samples (Minimum 10 subjects)
 
 
 
 
 
 
Xe 
 
 
 
 
 
Urine  for biological correlates 
X 
X 
 
X 
 
 
X 
 
 
 
 
 
Blooda for biological correlates 
 
X 
 
X 
 
 
Xf 
 
 
 
 
 
Blood for Circulating tumor cellsa 
 
X 
 
 
 
 
Xg 
 
 
 
 
 
Audiogram 
X 
 
 
 
 
 
 
 
 
 
Xd 
Xd 
Urine VMA/HVA 
X 
 
 
 
 
 
X 
 
 
 
 
 
Bone Marrow^ 
X 
 
 
 
 
 
 
 
 
 
Xc 
Xc 


Clinical Protocol DFMO Phase II 
 
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1.0 
Protocol Concept 
High risk Neuroblastoma (NB) remains a challenge in pediatric oncology, accounting for 15% of 
all pediatric cancer deaths.  While most patients are able to attain remission, approximately 50% 
will relapse.  Once relapsed, there is currently no curative treatment for these children, and for 
these children the 5-year survival rate is <10%.  As such, new therapeutic approaches are needed 
to treat these children. Relapsed patients who are able to obtain a second remission are not 
eligible for relapse therapeutic trials since they have no evidence of disease and yet they are 
likely to relapse within 6 months-1year.  Prevention of relapse is one such approach to improve 
outcome in these patients.  This study will address this concept while using a well-tolerated 
targeted medication for children in remission. 
  
These more aggressive forms of NB respond poorly to hormonal and chemotherapeutic 
approaches, and therefore, there is a great need for antineoplastic agents with novel mechanisms 
of action. The MYCN protein up-regulates ornithine decarboxylase (ODC), a gene encoding for 
the ODC enzyme that is pivotal in polyamine biosynthesis. High polyamine content and elevated 
ODC activities are commonly found in NB as well as many other tumors, and therefore, 
suppression of polyamines in cancer cells is an effective means to reduce tumor cell 
proliferation. Specific polyamine inhibitors such as DFMO have been evaluated in adult clinical 
cancer trials and shown to prevent formation of polyps and colon cancer. DFMO will be 
evaluated in this pediatric NB trial as a preventative single agent therapy. 
 
2.0 
Background and Preliminary Data 
2.1 
Previous Preclinical and Clinical Work  
Neuroblastoma (NB) is a tumor of the autonomous nervous system originating from the adrenal 
medulla and autonomous ganglia in the chest and abdomen. After leukemia and brain tumors, 
NB is the third most frequent malignant tumor of childhood. The incidence in the United States 
is approximately one in 7,000 children (Ater, Gardner et al. 1998). Therapy for NB is very 
intense, especially in advanced stages of the disease with widespread metastases to liver, bone, 
lymph nodes, and bone marrow. Current therapy includes chemotherapy, radiation, and high 
dose chemotherapy with subsequent bone marrow transplantation followed by differentiating 
therapy. More recently, immunotherapy has been added using monoclonal antibodies to the GD2 
glycolipid antigen that is heavily expressed by NB cells (Bosslet, Mennel et al. 1989; Cheung, 
Kushner et al. 1998). Over the last 30 years, significant therapeutic progress has been made with 
an increase in the five-year relative survival rate from approximately 25% to 55%. However, 
almost 50% of patients are estimated to die of their tumor, and over the past decade improvement 
in the five-year survival rate of NB patients has been slow (Harras 1996). NB has a particularly 
poor prognosis in patients older than 2 years at diagnosis, advanced stage disease and/or disease 
characterized by MYCN gene amplification (Seeger, Brodeur et al. 1985; Brodeur 2003). These 
more aggressive forms of NB respond poorly to chemotherapeutic approaches, and therefore, 
there is great need for a better understanding of the cellular regulation of MYCN-amplified NB 
tumors in an effort to search for alternative molecular drug targets. Although a role for the 
MYCN oncoprotein has been established in NB pathogenesis, the mechanism by which MYCN 
contributes to both the development of this disease and its poor prognosis is still unclear. The 


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MYCN oncoprotein functions as a transcriptional regulator (Ben-Yosef, Yanuka et al. 1998) and 
thus may influence tumorigenesis and patient survival by regulating the expression of key genes 
involved in the NB malignant phenotype. MYCN regulates the expression of genes that encode 
ornithine decarboxylase (ODC), the multi-drug resistance-associated protein 1 (MRP1), and 
MDM2 (Slack, Chen et al. 2005). ODC is the rate-limiting enzyme in the production of 
polyamines (Marton and Pegg 1995). Although polyamines, and therefore ODC, are essential for 
normal cell proliferation, increased ODC activity can induce cellular transformation in vitro 
(Auvinen, Paasinen et al. 1992), and high ODC levels are associated with a variety of tumors, 
including those of the brain and prostate (Mohan, Challa et al. 1999; Ernestus, Rohn et al. 2001). 
Several studies have identified ODC as a target gene for the MYCN oncoprotein (Lutz, Stohr et 
al. 1996; Ben-Yosef, Yanuka et al. 1998; Lu, Pearson et al. 2003), and it is possible that ODC, 
and therefore polyamines, play a significant role in NB tumorigenesis. 
 
Role of polyamines in cancer 
The identification of novel inhibitors of enzymes involved in polyamine biosynthesis with 
antitumor activities has recently revived interest in polyamine homeostasis and in designing 
strategies of cancer chemotherapy (Mamont, Duchesne et al. 1978; Porter, Regenass et al. 1992; 
Seiler, Atanassov et al. 1998). Selective pharmacological interference with the synthesis of 
natural polyamines results in tumor cell growth inhibition under both in vitro and in vivo 
conditions (Mamont, Duchesne et al. 1978; McCann and Pegg 1992). Although the precise 
mechanism by which polyamines contribute to cell proliferation is not well known, it has been 
suggested that it may be a result of their ability to bind DNA and affect gene expression by 
bringing about structural changes in chromatin, thereby stimulating cell growth (Marton and 
Morris 1987). Furthermore, the dramatic increases in the activity of ODC in certain tumor cells 
have been linked to G1-S transition (Fuller, Gerner et al. 1977; Kahana and Nathans 1984; 
Kaczmarek, Calabretta et al. 1987). As mentioned above, an apparent molecular basis for this 
derives from the fact that ODC is among those genes, which can be regulated by c-Myc and 
MYCN (Bello-Fernandez, Packham et al. 1993; Pena, Reddy et al. 1993; Wagner, Meyers et al. 
1993; Lutz, Stohr et al. 1996; Lu, Pearson et al. 2003), both of which regulate entry into and exit 
from the cell cycle. Because cell growth is absolutely dependent on polyamines, interference 
with polyamine biosynthesis has long been considered a promising therapeutic approach against 
proliferative diseases, including various malignancies (Heby and Persson 1990; Auvinen, 
Paasinen et al. 1992; McCann and Pegg 1992). α-difluoromethylornithine (DFMO or 
eflornithine), a suicide substrate inhibitor of ODC (Metcalf, Bey et al. 1978; Poulin, Lu et al. 
1992), has been the prototype tool for the study of therapeutic effectiveness of polyamine 
depletion in experimental tumors (McCann and Pegg 1992; Meyskens and Gerner 1999) (Fig. 1). 
 
 
 
 
 
 
 


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DFMO inhibits cell growth of many cancer cells including NB and induces cell differentiation 
(Chapman 1980; Melino, Farrace et al. 1988). These processes are accompanied by an apparent 
depletion of putrescine (Put) and spermidine (Spd) pools (Pegg 1988; Heby and Persson 1990; 
McCann and Pegg 1992). DFMO has also been shown to induce apoptosis and inhibit metastasis 
in a human gastric cancer model (Takahashi, Mai et al. 2000).  
 
Role of polyamines in NB cell differentiation   
The fluctuation in the levels of intracellular polyamines such as Put, Spd, and spermine (Spm) 
has been observed in association with cell differentiation (Heby 1981; Tabor and Tabor 1984; 
Pegg 1986), and inhibition of ODC by DFMO and reduction in polyamine pools stimulates 
various cancer cells including NB cells to differentiate (Chen, Nau et al. 1983; Melino, Farrace et 
al. 1988; Melino, Piacentini et al. 1991).  DFMO treatment of NB cells can change the triangular 
NB morphology by inducing a different phenotype; one which resembles elongated fibroblast-
like cells without typical neuritic processes. By comparison, treatment with retinoic acid (RA) 
induces neural differentiation of NB cells as indicated by the outgrowth of definite neurites 
(Melino, Farrace et al. 1988; Melino, Piacentini et al. 1991; Wainwright, Lasorella et al. 2001). 
The role of polyamines in cell differentiation has been studied for many years, and yet the 
precise role of polyamines at the cellular and molecular level is still not well understood and may 
play a key role in tumor regression. 
 
Effect of polyamine inhibitor DFMO in a transgenic neuroblastoma animal model 
Encouraging data by two groups (Hogarty, Norris et al. 2008; Rounbehler, Li et al. 2009) 
recently emerged and confirmed the effect of DFMO in vivo using the TH-MYCN NB mouse 
model, and DFMO in combination with cisplatin and cyclophosphamide increased the tumor-free 
survival of TH-MYCN homozygous mice (Fig. 2) (Hogarty, Norris et al. 2008). Additional 
studies have revealed that DFMO combined with SAM486A act synergistically and result in a 
significantly reduced tumor burden in TH-MYCN mice (56). 
Figure 1. Pathway of polyamine metabolism showing two 
target enzymes of the polyamine inhibitors DFMO and 
SAM486A. By inhibiting ODC, DFMO depletes putrescine 
(Put) and spermidine (Spd) pools, whereas it only modestly 
affects spermine (Spm) pools. By inhibiting AdoMetDC, 
SAM486A depletes Spd and Spm, whereas it markedly 
increases Put. The inhibitor combination lowers all three 
polyamine pools until cells stop growing and the pools are no 
longer diluted by cell division. SSAT and PAO work in concert 
to acetylate and oxidize polyamines during retro-conversion. 
SMO converts Spm back to Spd without the need for an 
acetylation 
step. 
AdoMetDC, 
S-adenosylmethionine 
decarboxylase; MTA, methyl-thioadenosine; ODC, ornithine 
decarboxylase; 
PAO, 
polyamine 
oxidase; 
SAM, 
S-
adenosylmethionine; 
SMO, 
spermine 
oxidase; 
SSAT, 
spermidine/spermine N1-acetyltransferase 


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Figure 2. Extended tumor-free survival in neuroblastoma-prone mice treated with DFMO. A, tumor-free survival 
curves for homozygous (TH-MYCN +/+) or hemizygous (TH-MYCN +/-) mice stratified by DFMO therapy. DFMO-
treated mice (dashed lines) received DFMO from birth onward (preemptive treatment trial). DFMO therapy was 
stopped at day 70 in tumor-free mice. B, delayed treatment trial: TH-MYCN homozygous and hemizygous mice 
were randomized to DFMO (dashed lines) or control (solid lines) following weaning at day 25. Tumor-free survival 
for TH-MYCN homozygous mice with advanced tumor from the time of treatment with (C) cisplatin alone (black 
line), cisplatin followed by DFMO (gray line), or cisplatin administered simultaneously with DFMO (dashed line) or 
(D) cyclophosphamide alone (solid line) or combined with DFMO (dashed line). P values using the method of 
Kaplan-Meier are shown (75). 
 
Relevance of ODC in patients with neuroblastoma 
Further evidence of the importance of ODC in NB tumorigenesis is available from our recent 
studies with human NB tumors. We analyzed the expression levels of ODC mRNA from 88 NB 
patients and found significant correlations between ODC expression and the overall survival 
probability. High levels of ODC were predictive of low survival probability and vice versa (Fig. 
3A). Most surprisingly, ODC was also predictive in tumors without MYCN amplification (Fig. 
3B), thus suggesting that ODC also plays a role in NB tumorigenesis independent of MYCN 
amplification (57). These findings were independently confirmed by two other groups (Hogarty, 
Norris et al. 2008; Rounbehler, Li et al. 2009). 
 
 
 
 
 
 
 
 
 


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A                                                        B 
 
 
Figure 3. Correlation of ODC gene expression with NB patient survival prognosis. A, Kaplan-Meier graphs 
representing the survival prognosis of 88 NB patients based on high or low expression levels of ODC. Survival 
probability of NB patients (follow-up over 196 months) with low ODC expression is significantly higher than of 
patients with high ODC expression. B, Kaplan-Meier graphs representing the survival prognosis based on high or 
low expression levels of ODC stratified for patients without MYCN amplification. The survival probability of NB 
patients with low ODC expression is significantly higher than of patients with high ODC expression (80). 
2.2 
Clinical Work 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 


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Chemopreventive effects of DFMO and sulindac in sporadic colorectal adenomas 
A recent randomized placebo-controlled double-blind trial was performed by Drs. Meyskens and 
Gerner to test whether the combination of a low dose of DFMO plus a low dose of sulindac 
reduces the recurrence of human colorectal adenomas (59). This study revealed that recurrent 
adenomatous polyps in patients can be markedly reduced by a combination of low oral doses of 
DFMO and sulindac (Table 1). Moreover, the study provided strong evidence that these two 
drugs are safe together with minimal side effects. 
 
 
Cancer Prev Res, 2008, 1:32-8 
 
Specific experience with DFMO and studies in patients with adenomatous polyps are described 
in four clinical chemoprevention studies that have been performed (59, 60, 61, 62) 
Boyle JO et al in a pilot study established that DFMO could lower polyamine content in 
colorectal mucosa. They demonstrated that shed oral extracellular mucosal cells did not provide 
a reliable surrogate estimate of DFMO effects on the colon.  Meyskens et al performed a dose 
de-escalation chemoprevention trial of 2 difluoromethylornithine in patients with colon polyps. 
The short-term (one month) Phase IIa study established that DFMO was both safe and effective 
in reducing colorectal mucosal polyamine contents when administered orally to patients as low 
as .25 gm/m2 for 28 days. No ototoxicity was observed at doses up to twice this amount. They 
Table 1 


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demonstrated that both putrescine content and the ratio of spermidine to spermine and changes in 
these parameters as a function of DFMO treatment decreased as a function of donor age, which 
is an important consideration in the evaluation of DFMO effect.  Meyksens then showed in an 
intermediate term (12 months) Phase IIb clinical chemoprevention trial demonstrated that 
polyamine levels in rectal mucosa can be continually suppressed by daily oral doses of DFMO 
that produced few or no side effects (62). Using a similar design, a smaller study has confirmed 
these results (63); this trial reported some reversible ototoxicity (at a dose of DFMO of 500 
mg/m2/day). 
Usage of DFMO at high doses for therapeutic purposes indicated that hearing loss occurred and 
seemed to be related to the total dose; this toxicity was however reversible (64). The toxicity of 
low dose DFMO has been assessed in both a phase IIa and phase IIb trial and the evaluation of 
hearing changes was described in the phase IIb trial (65).  They were unable to demonstrate an 
effect of DFMO on otoacoustic emissions at any dose of DFMO (0, -75, 200, 400 mg/m2/day) 
administered. They detected a subtle decrease in pure tone threshold that was dose-related. 
However, even at the highest dose the decrease was less than 4 db. Since 1-2 db decreases per 
year of hearing in normal humans in this age range have now been shown (66,67), results should 
not raise undue concern since: (1) the effects of DFMO on hearing loss are known to be rapidly 
reversible after discontinuation of the drug and (2) 10-20 db losses need to occur before the 
effect is clinically evident. Nevertheless, all participants will receive a baseline audiogram, will 
be check throughout the study and if clinically indicated.  
 
 
Summary 
These studies suggest that ODC/polyamines are critical in oncogenesis and therefore present a 
therapeutic target for the treatment and prevention of recurrence of NB and other types of cancer. 
This study will focus on the use of DFMO in high risk neuroblastoma patients that are in 
remission as a strategy to prevent recurrence.  
 
 


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3.0 
Study Objectives 
3.1 
Primary Objective:  
3.1.1 To evaluate the preventative activity of DFMO as a single agent in 
patients that are in remission based on: Event free survival (EFS)  
 
 
3.2 
Secondary Objectives: 
3.2.1 Correlation of urinary polyamine levels with progression of disease in 
neuroblastoma. 
3.2.2 To evaluate the preventative activity of DFMO as a single agent in 
patients with neuroblastoma who are in remission based on: 
 Overall Survival (OS) 
3.2.3 To continue to determine the safety and tolerability of DFMO as a 
single agent and in pediatric and young adult patients with high risk 
neuroblastoma that is in remission. 
 
3.2.4 To evaluate the pharmacokinetics (PK) of new DFMO formulation in a 
minimum of 10 patients. 
 
3.2.5 Biological Correlates to minimally include: 1) Urine: polyamine levels 
and blood inflammatory markers, 2) Blood- microRNA analysis as 
predictor of DFMO effect; ODC SNP analysis in DNA isolated from 
nucleated cells; circulating tumor cell analysis, and explorative 
biomarker analysis 3) Bone Marrow- flow cytometry of minimal 
residual disease of tumor; explorative biomarker analysis 
 
4.0 
Study Design 
 
This study is an open label, single agent, multicenter, study for patients with neuroblastoma that 
is in remission.   
 
4.1 
DFMO Treatment 
 
In this study subjects will receive twenty-seven (27) cycles of oral DFMO at a dose of 500 to 
1000 mg/m2 BID (per dosing chart in section 6.1.2) on each day of a 28 day cycle.  DFMO is 
provided as 250mg tablets.   
 
 
 


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4.2 
 Response Evaluations  
 
At the times indicated in Section 7.0 and the Table of Procedures and Assessments, scans will be 
obtained to evaluate response for subjects enrolled in this study.  Response will be assessed to 
evaluate the potential benefit of DFMO in this patient population.   
 
 
4.3 
Pharmacokinetic Evaluations  
 
Plasma concentrations of DFMO will be determined for a minimum of 10 currently enrolled 
subjects on their last day of taking the DFMO tablet formulation and on Days 2-5 post start of 
taking the new formulation at hours 0 (pre dose), 30min, 1 hour, 3 hours, and 6 hours post-dose. 
Change from old formulation to new formulation may take place on any day of any cycle after 
subject consents to new formulation. 
 
 
4.4 
Biological Studies 
These studies will evaluate the level of polyamines and inflammatory markers in urine.  Subjects 
will have an opportunity to participate in additional correlative biological studies on a voluntary 
basis to evaluate the level of polyamines and inflammatory markers in blood. These voluntary 
studies will evaluate microRNA levels in blood and ODC activity in white blood cells. They will 
also evaluate minimal residual disease in bone marrow. These biologic studies may be able to 
contribute to our knowledge of molecular determinants of response to therapy and/or the 
development of biomarkers to help guide future therapy.  
 
 


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5.0 
Patient Selection  
Eligibility: 
5.1 
Inclusion Criteria: 
 
1. Age: 0-21 years at the time of diagnosis. 
2. Diagnosis: histologic verification at either the time of original diagnosis or a 
previous relapse of high risk neuroblastoma. 
3. Disease Status: Neuroblastoma that is in remission 
4. Greater than 30 days from completion of cytotoxic and antibody therapy and less 
than 120 days from previous therapy  
5. A negative serum or urine pregnancy test is required for female subjects of child 
bearing potential (onset of menses or ≥13 years of age).  
6. Both male and female post-pubertal study subjects need to agree to use one of the 
more effective birth control methods during treatment and for six months after 
treatment is stopped. These methods include total abstinence (no sex), oral 
contraceptives (“the pill”), an intrauterine device (IUD), levonorgestrol implants 
(Norplant), or medroxyprogesterone acetate injections (Depo-provera shots). If one 
of these cannot be used, contraceptive foam with a condom is recommended. 
7. ANC > 500/μl and platelet count >50,000/μl 
8. Organ Function Requirements: Subjects must have adequate liver function as 
defined by: 
a. AST and ALT <10x upper limit of normal  
b.Serum bilirubin must be ≤ 2.0 mg/dl 
c. Serum creatinine based on age/gender as follows: 
Age  
Maximum Serum Creatinine (mg/dL) 
 
 
  
 
  
 Male  
Female  
 
1 month to < 6 months  
0.4 
0.4 
  
6 months to < 1 year  
0.5 
05 
  
1 to < 2 years  
0.6 
0.6 
  
2 to < 6 year 
  
0.8 
0.8 
  
6 to < 10 years  
1 
1 
  
10 to < 13 years  
1.2 
1.2 
  
13 to < 16 years  
1.5 
1.4 
  
≥ 16 years  
1.7 
1.4 
  
 
9. Informed Consent: All subjects and/or legal guardians must sign informed written 
consent. Assent, when appropriate, will be obtained according to institutional 
guidelines 
 
 


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5.2 
Exclusion Criteria: 
 
1. Lansky score  < 60% 
2. BSA (m2) of  <0.25 
3. Investigational Drugs: Subjects who are currently receiving another investigational 
drug are excluded from participation. 
4. Anti-cancer Agents: Subjects who are currently receiving other anticancer agents are 
not eligible. Subjects must have fully recovered from the effects of prior 
chemotherapy (hematological and bone marrow suppression effects). 
5. Infection: Subjects who have an uncontrolled infection are not eligible until the 
infection is judged to be well controlled in the opinion of the investigator. 
6. Subjects who, in the opinion of the investigator, may not be able to comply with the 
safety monitoring requirements of the study, or in whom compliance is likely to be 
suboptimal, should be excluded.  
 
Additional criteria: 
Subjects willing to participate in the correlative biologic studies will sign an additional consent 
form to provide bone marrow and blood for analysis. 
 
• Stratum 1: Subjects that are in remission at the end of upfront therapy defined as 
chemotherapy (5-7 cycles), surgery as indicated, consolidation therapy as indicated, 
radiation therapy as indicated, anti-GD2 antibody therapy with retinoic acid up to 6 
cycles. 
 
• 
Stratum 2: Subjects that are in remission after any previous relapse or refractory therapy 
 
 
6.0 
STUDY DRUG ADMINISTRATION 
6.1 
DFMO 
6.1.1 
Description, Formulation, and Storage of DFMO 
 
   
 
The dosage form to be used in this study are provided as a convex tablet containing 250 mg of 
eflornithine HCl, monohydrate.  The tablets are packaged and sealed in opaque white HDPE 
bottles, and each bottle contains 100 tablets.  
  
 
The tablets are to be stored at room temperature (20-250C). 
 
 


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6.1.2 
DFMO Dosing Table Per Weight Group: 
 
The following table will be used to determine dispensing of DFMO.   
 
 
DFMO Dose: 
 
BSA (m2) 
Tablets to be Dispensed for 
each dose 
Total Tablets 
Per Day 
Actual 
Mg/m2 
>1.5 
Four (4) tablets orally twice a day 
Eight (8) 
625 and down 
per dose 
0.75 to 1.5 
Three (3) tablets orally twice a day 
Six (6) 
500 to 1000 per 
dose 
0.5 to < 0.75  
Two (2) tablets orally twice a day 
Four (4) 
675 to 1000 per 
dose 
 0.25 to < 0.5  
One (1) tablet orally twice a day 
Two (2) 
500 to 1000 per 
dose 
 
<0.25 
Not eligible for trial 
 
 
 
 
6.1.3 
Administration of DFMO 
 
Treatment will be administered on an outpatient basis unless hospitalization is required for 
another reason. 
 
Each entire dose of DFMO tablets should be swallowed.  
 
Subjects will be advised to maintain a low Polyamine diet during the duration of this study. 
A handout will be provided to subjects with foods they should avoid while on this study.  
 
Subjects will be given a ‘patient instruction sheet’ for home administration.   
 
Subjects will be given a ‘dosing diary’ to keep track of doses given at home.  
 
6.1.4 
Subjects Unable to Swallow Tablets 
For subjects unable to swallow tablets, DFMO tablets may be chewed or crushed in a teaspoon 
and administered with a small amount of liquid. Note, tablets may not completely dissolve. 
 
  Recommended liquids for mixing (to cover taste)- Lemonade, Apple, Cranberry, Grape, or 
Pineapple 
 
DFMO may NOT be mixed in high polyamine juices such as orange or grapefruit juice. 


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Crushed DFMO tablets may also be mixed in 1-2 tablespoons of chocolate syrup or apple sauce.  
The DFMO tablets will not dissolve, and this is acceptable.  This technique is simply to mask the 
flavor. 
 
6.1.5 
Missed Doses 
If a subject vomits or misses a dose they should skip the missed dose and continue the drug at the 
next dose.  Do not make up missed doses.  
 
6.1.6 
DFMO Clinical Pharmacology 
DFMO (Eflornithine hydrochloride) is a member of the following drug classes: 1) inhibitor of 
ornithine decarboxylase (ODC), 2) hirsutism (excess hair growth) retardant, and 3) 
antiprotozoals. Eflornithine is FDA approved as a cream for treatment of female hirsutism, and 
in intravenous form for treatment of trypanosomiasis. The oral tablet form is not available 
outside of the clinical trial setting in the U.
 
 
 
Contraindications:  Prior hypersensitivity to eflornithine.  
Common side effects:   
Overall, the most frequently reported AEs in NCI, DCP-sponsored chemoprevention studies 
were diarrhea (9.0%), headache (7.5%), nausea (6.5%), hearing loss (5.6%), tinnitus (4.3%) and 
asthenia (4.7%). Other common toxicities (each accounting for 2 to 3% of all AEs reported) were 
epigastric pain, flatulence, dyspepsia, anemia, dizziness and skin rash. Less common toxicities 
(each accounting for 1 to 2% of all AEs reported) were: stomatitis, rhinitis, insomnia, infections, 
vomiting, vasodilation, dry mouth, constipation, dry skin, menstrual disorders, pharyngitis, 
emotional lability, pruritis, myalgia, and pain (miscellaneous). The most significant adverse 
effects associated with clinical administration of DFMO in chemoprevention trials are loss of 
hearing acuity and tinnitus. These effects have generally been found to be reversible when 
DFMO treatment is stopped. Patients who receive a cumulative oral dose of DFMO below 150 
g/m2 experience minimal ototoxicity. 
 
Infrequent side effects:  Hearing loss/change by audiometry testing has been reported in 8.4% 
of patients on high dose eflornithine.  Rash and alopecia have been reported in 3% of patients.  
Anorexia and abdominal pain have been reported in 2% of patients treated with eflornithine. 
Rare but serious side effects include dizziness (1%), headaches (2%), and seizures (8%), have 
been reported in patients on intravenous eflornithine.  Myelosuppression (including leukopenia, 
[37%], anemia [55%], and thrombocytopenia [14%]) has been reported at high intravenous 
doses, but does not usually occur at the low dose (500 mg) utilized in this study. 
Pregnancy and Lactation:  Pregnancy category C. It is unknown if eflornithine crosses the 
placenta.  Case reports in humans along with animal studies (mice, rats) indicate potential for 
fetotoxicity.  Experiments in rodents indicate that eflornithine blocks yolk sac formation and 
trophoblast differentiation, affecting processes such as vasculogenesis and steriodgnesis (Lopez-


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Garcia, et al., 2008).  The World Health Organization has not determined a breast feeding rating 
for eflornithine due to insufficient data. The Thompson lactation rating is that infant risk cannot 
be ruled out.  No studies investigating the safety of lactation after eflornithine administration 
have been conducted, nor are there data to determine drug levels in breast milk after drug 
administration.  
 
Toxicity for DFMO Potential Risk: 
Likely 
Happens to 10-30  patients out 
of every 100 
Less Likely 
Happens to 3-10 patients out of 
every 100 
Rare  
Happens to fewer than 3 patients 
out of every 100 
• 
Fewer red and white 
blood cells  
o a low number of red 
blood cells can make 
you feel tired and 
weak and may 
require transfusion. 
o a low number of 
white blood cells can 
make it easier to get 
infections 
• Decrease in the number 
of platelets made in the 
bone marrow 
• Nausea  
• Hearing Loss 
• Ringing in ears 
• Diarrhea 
• Headache 
• Weakness 
• Loss of appetite 
• Abdominal Pain 
• Flatulence (gas) 
• Dizziness 
•  
• Skin Rash 
• Seizures 
• Sores in the mouth 
• Runny nose 
• Difficulty sleeping 
• Infections 
• Dry mouth 
• Constipation 
• Dry skin 
• Menstrual disorders 
• Sore throat 
• Vomiting 
• Vasodilation (the 
relaxation of blood 
vessels possibly causing 
low blood pressure) 
• Emotional ups and downs 
• Itchiness 
• Body aches 
• Pain  
 
Four particular areas of concern have been identified with regard to the safety of subjects 
participating in this study and an attempt to address each of them is outlined below: The main 
considerations are: thrombocytopenia, hearing loss, gastrointestinal and non-G.I. side effects. 
a) 
DFMO Specific Adverse Events 
(1) 
Hearing loss. 
Although hearing loss has been a problem at higher doses (see below), clinical changes in 
hearing have been uncommon (one of 123 subjects in the phase IIb trial) and reversible in the 
doses proposed for this trial (62). An extended analysis of these observations is in press (65). 
There was no statistically significant shifts in distortion product otoacoustic emission levels. For 


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auditory pure tone thresholds, there was a subtle 2-3 dB decrease in hearing sensitivity for the 
two higher DFMO doses (0.2 and 0.4 gm/M2/day), but only for the two lowest frequencies at 250 
and 500 HZ. However in two phase I trials using lower doses of DFMO, done by other 
investigators, no audiometric changes were seen after approximately 6 months of DFMO therapy 
at 0.50Gm/m2/d (total dose 90Gm) although some changes were seen at higher doses (79). 
Hearing loss may occur in association with DFMO administration at high doses. In a meta-
analysis of previous studies, (64), it was reported that less than 10% of the subjects who received 
cumulative doses below 150 Gm/m2 developed a demonstrable hearing deficit, while hearing 
losses were observed in up to 75% of subjects who received cumulative doses above 250 Gm/m2. 
This side effect was thought to be totally reversible upon drug discontinuation.  Some study 
participants taking DFMO at doses similar to those used in this study have experienced mild 
decreases in hearing soft sounds. These changes have been uncommon and usually subclinical 
(that is, noticeable on special hearing tests called audiograms, but not in normal conversation or 
daily activities). Although an affected participant's level of hearing usually returns to its usual 
state when drug is stopped, in a small percentage of cases (occurring in less than 5% of 
participants taking the drug) effects have persisted even after drug was stopped. These changes 
are most, likely not reversible in all subjects.  However, in a recently completed phase IIb trial of 
DFMO audiologic changes that were clinically significant were not detected, even in the highest 
dose group (0.4Gm/m2/day) (extended analysis, 65). It is probable that at low doses of DFMO 
ongoing recovery of inner ear polyamines occurs and clinical hearing loss will be rarely, if ever, 
seen. Since hearing loss is usually totally reversible after drug discontinuation, this approach 
appears safe and cost effective. 
(2) 
Thrombocytopenia (low platelets)  
Thrombocytopenia has been reported predominantly in studies using “therapeutic” doses of 
DFMO (>3Gm/m2/day) and primarily in cancer patients who had previously undergone 
chemotherapy or patients with compromised bone marrow.  Other side effects ascribed to DFMO 
have been rare and, to date, seen only at high doses. 
(3)  
Other 
Skin rash, anemia, and neutropenia have also been seen with DFMO administration 
(4) 
GI 
Abdominal pain, loss of appetite, diarrhea have been reported.  
 
6.2 
Guidelines for Dose Modifications 
 Toxicities and dose modifications (refer to section 6.3) will be monitored in all cycles.  
Adjustments to the doses of study drug will be based upon toxicity, graded according to the NCI 
Common Toxicity Criteria (CTC), Version 4.0, if these were normal at baseline. Events that are 
not described in the NCI criteria will be assigned grades according to the criteria provided in 
Section 8.0.  Criteria for determining the relatedness of clinical adverse events to treatment 
(Section 8.0) should be utilized to determine the relationship of adverse events to the treatment.   
 
 
 


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6.3 
Dose Modification  
Patients experiencing any toxicity specified below attributable to DFMO or any intolerable 
toxicity will have their dose of DFMO held until toxicities have reverted to ≤Grade 2 toxicity 
(with the exceptions below for neutropenia, thrombocytopenia and transaminases). Upon 
resolution of the toxicity, subjects will receive a dose reduction of DFMO to one step down on 
the dosing table in section 6.1.2 (one less tablet per dose than their current dose). Subjects that 
are currently only taking one tablet per dose BID will be dose reduced to one tablet per day 
(QD).  Subjects will be allowed to dose reduce for subsequent toxicities defined here as many 
times as they can until they reach the one tablet per day (QD) dosing. At that point if they 
experience another dose reducing toxicity (as defined here) they will be required to go off 
protocol therapy.  
 
• Grade 4 neutropenia or thrombocytopenia that persists for 7 days or longer after holding 
study drug. (DFMO should be held as soon as a Grade 4 related neutropenia is discovered. 
Subject will only dose reduced if toxicity persists at a Grade 4 for longer than 7 days after 
the hold starts. If subject resolves before 7 days of hold they will resume at previous dose. 
If subject does not resolve after 14 days of a hold they will be discontinued from study).  
• >10 X elevation of transaminases that persists for 7 days or longer after holding study 
drug. (DFMO should be held as soon as a 10X related elevation is discovered. Subject will 
only dose reduced if toxicity persists at >10X ULN or higher for longer than 7 days after 
the hold starts. If subject resolves before 7 days of hold they will resume at previous dose. 
If subject does not resolve to <10x ULN after 14 days of a hold they will be discontinued 
from study). 
• Any other Grade 3 non-hematologic toxicity, excluding alopecia, nausea, vomiting, and 
diarrhea that does not adequately respond to treatment. 
If there is no resolution of above toxicity by 14 days, DFMO should be discontinued, and 
subjects should be discontinued from the study.   
 
6.4 
Study Drug Accountability  
Study drug must only be used for subjects enrolled in the trial. The investigational site staff must 
maintain a careful inventory of study drug.  Drugs will be distributed to investigational site staff 
and patients using FDA guidelines for distribution of investigational agents. Study drug use will 
be recorded on a Study Drug Inventory Form.   This form will contain the following information: 
• 
Subject number and initials for each subject receiving study drug 
• 
Date and quantity of DFMO received by the site 
• 
Date and quantity of DFMO dispensed 
• 
Date and quantity of DFMO returned 
At each monitoring visit, the clinical monitor will reconcile with the actual inventory of study 
drug at each site.    
 


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6.5 
Concomitant Medications and Treatments 
All intercurrent medical conditions will be treated at the discretion of the Investigator according 
to acceptable community standards of medical care.  All concomitant medications and treatments 
will be documented on the appropriate case report form. 
The following medications are not permitted during the trial: 
• 
Any cytotoxic chemotherapy 
• 
Any other investigational treatment  
• 
Any other systemic anti-neoplastic therapy including, but not limited to, immunotherapy, 
hormonal therapy, targeted therapies, anti-angiogenic therapies, or monoclonal antibody 
therapy 
• 
Any radiotherapy, including systemically administered radioisotopes, unless administered 
with palliative intent 
Erythropoietin, blood products, anti-emetics, steroids, and transfusions may be administered at 
the discretion of the Investigator based on established criteria  
 
 
7.0 
STUDY PROCEDURES AND ASSESSMENTS 
7.1 
Enrollment of Patients 
Prior to consent of the subject, the research coordinator at the lead site will be contacted (via e-
mail). This coordinator will then reply with study space availability. If the subject fits all 
enrollment criteria, the site will again contact the coordinator at the lead site for official 
enrollment confirmation and unique subject identifier assignment. In addition, a study enrollment 
form will be faxed to the coordinator at the lead site. This determination will be made based on 
subject data (subject qualifies for study) and current subjects enrolled in trial (i.e. how many 
subjects are currently enrolled).  A subject may NOT be enrolled on trial until official approval 
from the lead site is received. 
 
All subjects (or patients’ legal representatives) must provide written informed consent before any 
study specific assessments may be performed. 
 
 


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7.2 
Screening 
The Investigator is responsible for keeping a record of all subjects screened for entry into the 
study and subsequently excluded. 
 
The following screening procedures must be performed within 14 days prior to the first dose of 
study drug (7 days extra may be requested from the study chair in exceptional cases).  Studies must 
be done after last previous treatment for malignancy: 
1. Signed informed consent form.  All subjects (or patients’ legal representatives) must 
provide written informed consent before any study specific assessments may be 
performed.  Signed informed consent form for voluntary participation in correlative 
biologic analysis will also be obtained.  
2. CT or MRI to confirm remission status 
3. MIBG scan to confirm remission status. Consider PET scan for non MIBG avid subjects.   
4. Audiogram 
5. Bone marrow aspirate and biopsy  
6. Additional Optional Bone Marrow-for subjects with additional informed consent bone 
marrow samples for biological correlatives per section 12.0) 
The following screening procedures must be performed up to 5 days prior to the first dose 
of study drug. 
1. Complete medical and surgical history, including documentation of the histologic 
evidence of malignancy and prior treatments for cancer.  Include all other pertinent 
medical conditions and a careful history of all prior medical treatments; 
2. Demographics; 
3. Physical examination (including height and weight), noting all abnormalities including 
baseline dermatologic and neurologic exam, 
4. BSA calculation (from body weight and height); 
5. Vital signs, including temperature, pulse rate, and blood pressure 
6. ECOG Performance status/Lansky Play status (Appendix I);  
7. CBC with differential; 
8. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, AST 
and ferritin; 
9. C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR) 
10. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA) 
11. Urine or serum pregnancy test for female subjects of child bearing potential (onset of 
menses or ≥13 years of age); 
12. Concomitant medications/therapies including documentation of steroid use and dose; 
13. Confirmation of inclusion and exclusion requirements 
14. Urine for Biological Correlates per section 12.0 
 
Following completion of all required screening procedures and certification of all inclusion and 
exclusion criteria by the Investigator, the lead site will be contacted (via e-mail or phone call), at 


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which time the subject will be enrolled in the trial and a unique subject number assigned.  The 
lead site will act as the central coordinating body.  The subject may not start on study until the 
lead site has provided official approval of enrollment.  
 
 
7.3 
Treatment Phase – Cycle 1 
The first cycle will be 28 days in duration.  The following procedures must be completed: 
Cycle 1 Day 1 (may be performed up to 5 days prior to DFMO administration unless 
otherwise indicated) 
1. Medical History and Physical examination (including body weight), including 
documentation of an update of all previous abnormalities, any new abnormalities. 
2. Vital signs, including temperature, pulse rate, blood pressure (sitting) (to be done on 
Cycle 1 Day 1); 
3. Review and recording of concomitant medications; (to be done on Cycle 1 Day 1) 
4. Monitoring and documentation of all AEs and review of concurrent illnesses (to be done 
on Cycle 1 Day 1) 
5. Urine for Biological Correlates per section 12.0 (to be done on Cycle 1 Day 1 in addition 
to screening sample that was sent in section 7.2) 
6. Optional: Blood for Biological Correlates per section 12.0 (additional consent required) 
7. Optional: Blood for Biological Correlates (Circulating Tumor Cells to Houston, TX) per 
section 12.0 (additional consent required). 
8. Dispense drug dosing diary 
 
Cycle 1 Day 15 (+/- 3 day window) 
Subjects will return to the clinic on Cycle 1 Day 15 for evaluations. The following evaluations 
will be conducted: 
 
1. History and Physical exam  
2. Vital signs, including temperature, pulse rate, and blood pressure (sitting) 
3. CBC with differential; 
4. Serum electrolytes, BUN, creatinine, bilirubin, ALT, AST; and LDH 
5. Review and recording of concomitant medications; 
6. Monitoring and documentation of all AEs and review of concurrent illnesses 
7. Urine for Biological Correlates per section 12.0 
8. Optional: Blood for Biological Correlates per section 12.0 (additional consent required) 
 
 
 
 


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7.4 
Treatment Phase – Cycles 2-12 
 
All Cycles will be 28 days in duration without interruption.  The following procedures must be 
completed: 
Cycle 2-12 Day 1  
The following procedures may be performed up to +/- 5 days from Day 1:  
1. Medical History and Physical examination (including body weight), including 
documentation of an update of all previous abnormalities, any new abnormalities. 
2. Vital signs, including temperature, pulse rate, blood pressure (sitting); 
3. Review and recording of concomitant medications; 
4. Monitoring and documentation of all AEs and review of concurrent illnesses 
5. BSA calculation (from body weight and height); 
6. ECOG Performance status/Lansky Play status (Appendix I);  
7. CBC with differential; 
8. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, and 
AST; 
9. C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR) 
10. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA) 
11. Urine for Biological Correlates per section 12.0 
12. Optional: Blood for Biological Correlates per section 12.0 (additional consent required) 
13. Cycles 3, 6, 9, and 12- Optional: Blood for Biological Correlates (Circulating Tumor 
Cells to Houston, TX) per section 12.0 (additional consent required). 
14. Collection of previous cycle drug dosing diary and dispensing of new drug dosing diary 
15. Urine or serum pregnancy test for female subjects of child bearing potential (onset of 
menses or ≥13 years of age); 
 
7.5 
Treatment Phase – Cycles 13-27 
 
All Cycles will be 28 days in duration without interruption.  The following procedures must be 
completed: 
Cycle 13-27 Day 1  
The following procedures may be performed up to +/- 5 days from Day 1:  
1. Medical History and Physical examination (including body weight), including 
documentation of an update of all previous abnormalities, any new abnormalities. 
2. Vital signs, including temperature, pulse rate, blood pressure (sitting); 
3. Review and recording of concomitant medications; 
4. Monitoring and documentation of all AEs and review of concurrent illnesses 
5. BSA calculation (from body weight and height); 


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6. ECOG Performance status/Lansky Play status (Appendix I);  
7. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA) 
8. Urine for Biological Correlates per section 12.0 
9. Urine or serum pregnancy test for female subjects of child bearing potential (onset of 
menses or ≥13 years of age); 
10. Collection of previous cycle drug dosing diary and dispensing of new drug dosing diary 
 
 
Additionally at the start of cycles 14*, 17, 21*, 24, and at off therapy (end of cycle 27)  
Windows- The following should be done within +/-5 days of the start of specified cycle. For 
off-therapy/end of cycle 27 they should be done within 30 days (+7) after stopping study 
drug: 
1. CBC with differential; 
2. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, and 
AST; 
3. C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR) 
4. Optional: Blood for Biological Correlates per section 12.0 (additional consent required) 
5. Optional: Blood for Biological Correlates (Circulating Tumor Cells to Houston, TX) per 
section 12.0 (additional consent required). 
* Start of cycles 14 and 21 bloodwork stated above can be done at the same time as the IV is 
placed for the scans done at the end of cycles 13 and 20 in order to reduce the amount of 
venipuncture the subject experiences.  
 
End of Cycles 3, 6, 9, 13, 20 and off therapy (cycle 27) – scans may be done more often per 
institutional standards.  
Windows- Scans should be done within +/-14 days of the start of next cycle. Off 
therapy/end of cycle 27 scans should be done within 30 days (+7) after stopping study drug. 
1. MIBG scan (for MIBG avid subjects only). Consider PET scan for non MIBG avid 
subjects.   
2. CT/MRI (use same radiologic method  as baseline) 
3. Bone marrow biopsy and aspirate is to be performed if the treating physician has 
concerns for progression   (If bone marrow testing is being performed and the subject has 
signed informed consent for the use of tissue in the correlative biologic study as a post-
treatment sample please send additional samples per section 12.0) 
 
For patients in first remission who require evaluations for previous front line treatments that are 
different than the timing of the above schedule, contact the study chair for approval to accept the 
subjects previously scheduled evaluations instead of repeating the above studies. 
 
 


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End of Cycles 6, 12, and 27  
Windows- The following should be done within +/-14 days of the start of the next cycle. For 
off-therapy/end of cycle 27 it should be done within 30 days (+7) after stopping study drug. 
 
1. Audiogram  
(Audiogram should also be done at any time point for any suspected hearing loss) 
 
7.6 
PK’s for Currently Enrolled Subjects switching to new Formulation DFMO 
(approximately 10 subjects): 
 
1. Last Day of Old DFMO Formulation: Blood for Pharmacokinetic sampling will be 
done on last day of taking old formulation*. Subject should be instructed to hold their 
morning dose of old DFMO.  Pre-dose PK sample will be collected, and then subject will 
take old DFMO in clinic. In total, Samples will be collected pre-dose, (Time 0), as well 
as at 30min, 1 hour, 3 hours, and 6 hours post-dose (exact time should be noted on CRF). 
Plasma will be separated and frozen at –80°C until analysis per section 12.1. 
 
* Change from old formulation to new formulation may take place on any day of any 
cycle after subject consents to new formulation and does not need to be on day 1 of a 
cycle. 
 
2. Start new Formulation of DFMO- Subject should start new formulation at second dose of 
this day in clinic once all PK’s are drawn, this will be Day 1 of new drug formulation.   
3. One day during Days 2-5 of taking on new formulation. Blood for Pharmacokinetic 
sampling on one day during days 2-5 of taking the new formulation. Subject should be 
instructed to hold their morning dose of new DFMO.  Pre-dose PK sample will be 
collected, and then subject will take new DFMO in clinic. In total,  Samples will be 
collected pre-dose, (Time 0), as well as at 30min, 1 hour, 3 hours, and 6 hours post-dose 
(exact time should be noted on CRF). Plasma will be separated and frozen at –80°C until 
analysis per section 12.1. 
 
7.7 
Other Schedules: 
1. Additional imaging or assessments may be done if clinically indicated.  Type of imaging, 
type of assessment and timing should be recorded as well as reason for imaging and/or 
assessment. 
2. Survival will be monitored on an ongoing basis during the study, then every 6 months 
from the time the subject is off-treatment for a period of 2 years, then yearly for up to 
five years off therapy.  
7.8 
Protocol Treatment Completion 
Subjects who receive 27 total 28-day treatment cycles will be considered as having completed 
protocol (total of 756 days).  
 


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7.9 
Off-Therapy / 30 Day Follow-up Visit 
Subjects will return to the clinic 30 (+7) days after the last dose of DFMO, and the following 
evaluations will be conducted: 
1. Medical History and Physical examination (including body weight), including 
documentation of an update of all previous abnormalities, any new abnormalities, and a 
detailed neurological exam; 
2. ECOG Performance status/Lansky Play status (Appendix I); 
3. Vital signs, including temperature, pulse rate, blood pressure (sitting); 
4. Review and recording of concomitant medications; 
5. Monitoring of AEs and review of concurrent illnesses  
6. Collect previous cycles drug dosing diaries (if not collected already) 
 
Any subject with a suspected study drug-related toxicity at the follow-up visit must be 
followed until all current adverse events have resolved to baseline or ≤ grade 2.  This may 
require additional clinical assessments and laboratory tests.  The follow-up results will be 
recorded on the appropriate page of the CRF, as well as in the subject's source 
documentation. Subjects that have started a new anti-cancer treatment since going off DFMO 
will be censored from any further AE collection at the date of starting the new therapy.  
 
 
7.10 
Survival Follow-up 
Subjects will be followed for long term survival by contact with parent or treating institution to 
confirm survival at the following time points (time from last dose of study drug): 
− 6 months 
− 1 year 
-   18 months 
-2 years 
-Yearly after (up to 5 years total) 
 
Follow up will continue for five (5) years off-therapy or until subject death or subject is lost to 
follow up (per definition in section 9.0). The follow-up results including follow-up status, 
remission status, and date of death will be recorded on the appropriate page of the CRF, as well 
as in the subject's source documentation. 
 


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8.0 
Adverse Event Reporting  
8.1 
Definitions 
8.1.1 Adverse Event 
An adverse event is any untoward medical occurrence in a patient or clinical investigation 
subject administered a pharmaceutical product and which does not necessarily have to have a 
causal relationship with this treatment.  An adverse event can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding, for example), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related to 
the medicinal product. 
 
An untoward medical event which occurs outside the period of follow-up as defined in the 
protocol will not be considered an adverse event unless related to study drug.  Worsening of a 
medical condition for which the efficacy of the study drug is being evaluated will not be 
considered an adverse event. 
 
8.1.2 Unexpected Adverse Event 
An unexpected adverse event is one for which the nature or severity of the event is not 
consistent with the applicable product information, e.g., the investigator’s brochure. 
 
8.1.3 Serious Adverse Event 
A serious adverse event is any untoward medical occurrence that at any dose: 
• 
Results in death 
• 
Is life-threatening (an event in which the patient was at risk of death at the time of the 
event; it does not refer to an event which hypothetically might have caused death if it were 
more severe) 
• 
Requires in-patient hospitalization or prolongation of existing hospitalization 
• 
Results in persistent or significant disability/incapacity 
 
• 
Is a congenital anomaly or birth defect 
• 
Other important medical events that may not be immediately life-threatening or result in 
death or hospitalization but may jeopardize the patient or may require intervention to 
prevent one of the other outcomes listed above.  Examples of such events are intensive 
treatment in an emergency room for allergic bronchospasm; blood dyscrasias or 
convulsions that do not result in hospitalization; or development of drug dependency or 
drug abuse. 
 
The term “severe” is often used to describe the intensity (severity) of an event; the event itself 
may be of relatively minor medical significance (such as a severe headache).  This is not the 
same as “serious”, which is based on patient/event outcome or action criteria usually associated 
with events that pose a threat to a patient’s life or functioning. 


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8.2 
Documenting Adverse Events  
The Investigator should elicit information regarding the occurrence of adverse events through 
open-ended questioning of the patient, physical examination and review of laboratory results.   
 
All Grade 2 or higher (per CTCAE version 4.0) adverse events, whether serious or not, will be 
described in the source documents and the adverse event page of the case report form.  All new 
events (Grade 2 or higher), as well as those that worsen in intensity or frequency relative to 
baseline, which occur after administration of study drug through the period of protocol-specified 
follow-up, must be captured. 
 
Information to be reported in the description of each adverse event includes: 
• 
A medical diagnosis of the event (if a medical diagnosis cannot be determined, a 
description of each sign or symptom characterizing the event should be recorded)  
• 
The date of onset of the event  
• 
The date of resolution of the event and whether the event is serious or not 
• 
Action taken; drug treatment required; non-drug treatment required; hospitalization or 
prolongation of hospitalization required; diagnostic procedure performed; patient 
discontinued from the study 
• 
Outcome:  complete recovery or return to baseline; unknown/lost to follow-up; adverse 
event persisting; patient died (notify lead site immediately, and complete the Serious 
Adverse Event page and the Final Visit section of the case report form)   
 
Adverse events, regardless of suspected cause, will be collected for 30 days following the last 
dose of DFMO and until all current adverse events have resolved to baseline or ≤ grade 2. 
 
All adverse events will be reviewed by the safety officer.  
 
8.3 
Expedited Reporting of Adverse Events 
All fatal or life–threatening adverse events must be reported to the lead site immediately by 
telephone, fax, or e-mail within 24 hours of discovery as well as to the appropriate regulatory 
authorities (local IRB and FDA if required and 
 as appropriate.). The lead site will 
then report directly to the safety officer. If full information is not known, additional follow-up by 
the Investigator will be required. 
 
All other serious adverse events must be reported to the lead site by telephone, fax, or e-mail 
within 5 days as well as to the appropriate regulatory authorities (local IRB and FDA and 
 
 as required and appropriate). Sponsor will aid site in determining FDA reporting 
requirements for any SAE. All medwatches must be submitted by the sponsor holding the IND 
and should not be submitted locally by the participating site to the FDA. 
 
The Investigator must report all serious adverse events reported to regulatory authorities in an 
expedited manner to the local IRB or IEC.  All serious adverse events must be followed until 
resolution or stabilization.  
   


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8.4 
Grading and Relatedness of Adverse Events 
8.4.1 Grading of Severity of an Adverse Event 
Each adverse event (Grade 2 or higher) will be graded for severity per the National Cancer 
Institute Common Terminology Criteria for Adverse Events (CTCAE V 4.0), and these criteria 
must be used in grading the severity of adverse events.  The criteria can be found at: 
http://ctep.cancer.gov/reporting/ctc.html. 
 
Grading of Severity of an Adverse Event Not Listed in Published Criteria: 
For those adverse events which are not listed as part of the NCI CTCAE V 4.0, the same grading 
system should be used, where: 
• 
Mild corresponds to an event not resulting in disability or incapacity and which resolves 
without intervention 
• 
Moderate corresponds to an event not resulting in disability or incapacity but which 
requires intervention 
• 
Severe corresponds to an event resulting in temporary disability or incapacity and which 
requires intervention 
• 
Life-threatening corresponds to an event in which the patient was at risk of death at the 
time of the event 
• 
Fatal corresponds to an event that results in the death of the patient 
 
 


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8.5 
Relatedness to Study Drug 
The Investigator must attempt to determine if an adverse event is in some way related to the use 
of the study drug and define an attribution category. This relationship should be described as 
follows: 
 
RELATIONSHIP 
ATTRIBUTION 
DESCRIPTION 
Unrelated to 
investigational 
agent/intervention 
 
Unrelated 
The AE is clearly NOT related to the intervention. 
The event is clearly due to causes distinct from the use 
of the study drug, such as a documented pre-existing 
condition, the effect of a concomitant medication, or a 
new condition which, based on the pathophysiology of 
the condition, and the pharmacology of the study drug, 
would be unrelated to the use of the study drug. 
Unlikely 
The AE is doubtfully related to the intervention. 
Adverse event does not have temporal relationship to 
intervention, could readily have been produced by the 
subject’s clinical state, could have been due to 
environmental or other interventions, does not follow 
known pattern of response to intervention, does not 
reappear or worsen with reintroduction of intervention. 
Related to 
investigational 
agent/intervention 
 
Possible 
The AE may be related to the intervention. 
The event follows a reasonable temporal sequence from 
administration of the study drug and the event follows a 
known response pattern to the study drug BUT the event 
could have been produced by an intercurrent medical 
condition which, based on the pathophysiology of the 
condition, and the pharmacology of the study drug, 
would be unlikely related to the use of the study drug 
OR the event could be the effect of a concomitant 
medication. 
Probable 
The AE is likely related to the intervention. 
The event follows a reasonable temporal sequence from 
administration of the study drug, the event follows a 
known response pattern to the study drug AND the event 
cannot have been reasonably explained by an 
intercurrent medical condition OR the event cannot be 
the effect of a concomitant medication. 
Definite 
The AE is clearly related to the intervention. 
The event follows a reasonable temporal sequence from 
administration of the study drug, the event follows a 
known response pattern to the study drug and based on 
the known pharmacology of the study drug, the event is 
clearly related to the effect of the study drug.  The 
adverse event improves upon discontinuation of the 
study drug and reappears upon repeat exposure. 
 
 


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9.0 
Patient Withdrawal and Trial Discontinuation 
9.1 
Criteria for Subject Off-Therapy 
Subjects may be withdrawn from the study treatment for the following reasons: 
• 
Subject completes all protocol defined therapy- 27 cycles 
• 
Relapsed neoplastic disease  
• 
Subject or guardian withdraws consent to continue study drug 
• 
Subject develops an intercurrent illness that precludes further participation, or requires a 
prohibited concomitant treatment 
• 
The Investigator withdraws the subject in the subject’s best interests 
• 
Subject is lost to follow-up (defined as the inability to contact the subject on 3 separate 
occasions over a period of 2 weeks) 
• 
Administrative reasons (e.g., the subject is transferred to hospice care) 
• 
An adverse event, which in the opinion of the Investigator, precludes further trial 
participation or fulfills the protocol requirements for withdrawal (e.g., the development of 
dose limiting toxicity despite a reduction in protocol therapy for a previous episode of dose 
limiting toxicity) 
• 
Death 
 
9.2 
Criteria for Subject Off-Study 
Subjects may be withdrawn from the study completely which includes survival follow-up 
for the following reasons: 
• 
Subject or guardian withdraws consent to continue in the trial 
• 
Subject is lost to follow-up (defined as the inability to contact the subject on 3 separate 
occasions over a period of 2 weeks) 
• 
Subject completes all protocol defined therapy including all follow-up time points. 
• 
Death 
 
9.3 
Trial Discontinuation 
The lead site may discontinue the trial as a whole or at an individual investigational site at any 
time.  Reasons for early trial discontinuation may include, but are not limited to, unacceptable 
toxicity of study drug, a request to discontinue the trial from a regulatory authority, protocol 
violations at an investigational site, violations of good clinical practice at an investigational site, 
or poor enrollment.  The lead site will promptly inform all Investigators in the event of 
premature study discontinuation and provide all Investigators with instructions regarding the 
disposition of subjects still on study.  Should the study be terminated prematurely, all unused 
study drug, case report forms and any other study material will be returned to the lead site. 
 


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10.0 DATA ANALYSIS 
10.1 
Data Quality Assurance 
Electronic case report forms (CRF) will be checked for correctness against source document data 
by the monitor.  If any entries into the CRF are incorrect, incomplete or illegible, the monitor 
will ask the Investigator or the study site staff to make appropriate corrections.   
10.2 
Statistical Analysis 
 
This is an open label, single agent, multicenter, study for patients with neuroblastoma that are in 
remission. 
 
The following data sets will be used in this study: 
• All enrolled and eligible subjects (ITT) population: All eligible subjects who have a signed 
informed consent form. 
• All treated and eligible subjects (Safety evaluable) population: All subjects who received at 
least one dose of study drug 
• All eligible subjects treated to first evaluable time point with evaluation completed (generally 
3 cycles) (as Treatment Efficacy) population, unless subjects have reached the study endpoint 
of progression of disease at an earlier time point. 
 
Efficacy analyses will be performed on the Treatment evaluable population. Safety analysis will 
be performed on the Safety and Efficacy evaluable population. 
10.3 
All baseline patient characteristics will be summarized in a tabular format.  
Safety data will be described for all subjects receiving at least one dose of 
DFMO.  Safety data will include values for hematology, serum chemistry, 
vital signs, and adverse events. The proportion of subjects experiencing 
adverse events, serious adverse events, dose limiting toxicities and treatment 
delays will be summarized.  Enrollment to study will not pause at interim 
analysis time points. 
 
Sample Size and Analysis: 
 
Stratum 1:  
A 70% 2-year EFS rate was selected as the Phase I baseline rate.  The baseline 70% EFS rate at 
2-years was based upon published data by Yu et al. (82). It is hypothesized that DFMO will 
increase the 2-year EFS rate to 80% which represents an approximately 10% increase in the 
duration of the EFS rate at 2-years.  Assuming a directional 5% one population binomial test, a 
sample size of n = 127 patients will be required to achieve an 80% power to detect this 2-year 
difference in EFS (70% vs 80%). 


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The comparison of the biomarker prevalence rates assumes that the Stratum 1 cohort will 
experience a relapse rate of 30% and thus a 70% non-relapse rate at up to two years of follow-
up.  It is also assumed that the corresponding prevalence of an elevated biomarker will be 60% 
for the relapse group and 30% in the non-relapse group, respectively.  This hypothesized 
prevalence difference will be detectable with 85% power with an overall sample size of N=127 
patients (38 relapse and 89 non-relapse) using a Fisher’s Exact Test and a non-directional Type I 
Error level of 5%.   
 
The sequential biomarker data over the course of treatment for each patient will be classified as 
elevated or not based upon the following a priori study criteria and blinded to patient 
progression status. Elevation of the biomarker is defined as diacetylspermine levels > 
500nmol/gm creatinine on 2 consecutive urine levels. The biomarker prevalence rates for the 
patients who progress (estimated to be n= 38) and those who do not progress (estimated n=89) 
over the two years of the trial treatment will be compared using a Fisher’s exact test of the 
equality of the two EFS prevalence rates implemented with a 5% two-tailed Type I error 
level.  A 95% exact confidence interval for the absolute difference in prevalence rates along with 
a 95% exact confidence interval for the odds ratio for the resulting prevalence rates will be 
obtained to complement the formal hypothesis testing.  Since OS will also be a secondary 
outcome for comparing the utility of the biomarker prevalence, time-to-event analyses will also 
be implemented using the Kaplan-Meier approach initially using the a priori biomarker 
definition.  These initial time-to-event analyses will then be supplemented with a Cox 
proportional hazard modeling effort using the quantitative biomarker assessment to allow for an 
exploratory analysis of other cut-point definitions that can be used to contrast with that of the 
original a priori definition.  Potential confounding clinical measures that are identified for the 
recurrent and non-recurrent patients will be incorporated in a limited fashion using Cox 
proportional hazard models that also include the biomarker effect.  This exploration of 
confounders will need to be limited due to the anticipated small sample sizes. Data from this 
stratum will guide the statistical design for a Phase II study in this patient population.  
 
Stratum 2:  
 
Based upon tabled median EFS times all patients presented by Santana et al. (Cancer, June 15, 
2008) for first and second recurrence times, a median first recurrence time of 8.7 months is 
equivalent to a 14.8% EFS at two years, and the median time to a subsequent second recurrence 
of 3.8 months is equivalent to an EFS rate of 1.3% at two years assuming an exponential time-to-
event model.  Assuming that two thirds of the relapse patient population will have had only a 
single relapse and that the other third of the patients will have had two or more relapses, then a 
weighted average of these two EFS rates (14.8% and 1.3%)  equals 10.3%.  A 10% historical 
EFS 
rate 
for 
two 
years 
was 
assumed 
for 
simplicity. 
Examination of an increase in EFS will require a sample size of n = 33 subjects in Stratum 2 to 
test whether treatment with DFMO can prolong the overall estimated EFS at two years to 30% 
from the  10% two  years estimate for the above historical data in this patient population. The 
sample size is based upon a one sample binomial test of proportions with a   power 80% and a 
5% two -tailed Type I error level.      
 


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An observation of 7 or more patients with at least a two year EFS rate out of the n = 33 patients 
will result in the rejection of the null hypothesis of a 10% EFS in favor of the alternative of a 
30% two year EFS rate.  An exact binomial 95% confidence interval for the two year estimated 
EFS will complement the prior formal two-stage hypothesis test.  Since the actual EFS time-to-
event data is derived from the times to recurrence, a Kaplan-Meier analysis of the time-to-event 
data analysis will be conducted, and the median time to recurrence and a 95% confidence for this 
value will be obtained to supplement this EFS point estimate.  Since this group of Stratum 2 
patients will consist of patients with prior single and multiple recurrence histories, EFS and time 
to event data will be examined by prior recurrence histories.  In particular, the impact of prior 
recurrence history on the time to recurrence will be explored using Kaplan-Meier analysis and 
Cox proportional hazard models.  The response to DFMO therapy and subsequent recurrences 
will also be explored in combination with prior recurrence histories using Cox models. 
 
10.4 
Event Free Survival  
The event free survival (EFS) is defined as the period from the first day of administration of 
study drug until the first occurrence of relapse, progressive disease, secondary cancer, or death 
or, if none of these events occurred, until the last contact with the subject. 
Progression is defined as the appearance of any new lesions >10mm on CT/MRI, any new 
lesions on MIBG or new disease present in the bone marrow. 
 
10.5 
Overall Survival 
Overall Survival (OS) will be defined as the first day of administration of study drug until death 
or will be censored at the last contact with the subject if death did not occur during the study. 
Overall survival will be monitored on an ongoing basis during the study, then every 6 months 
from the time the subject is off-study treatment up to a period of 2 years, then yearly for up to 
five years total. 
 
 
11.0 ADMINISTRATIVE PROCEDURES 
11.1 
Patient Informed Consent 
No study related procedures will be performed until a patient or a patient’s legal representative 
has given written informed consent.  The lead site will provide to the site Investigators a sample 
informed consent document that conforms to all the requirements for informed consent according 
to ICH GCP and US FDA guidelines (21 CFR 50).  However, it is up to each site Investigator to 
provide a final informed consent that may include additional elements required by the 
Investigator’s institution or local regulatory authorities.  The IRB/EC for each investigational site 
must approve the consent form document prior to study activation; changes to the consent form 
during the course of the study may also require IRB/EC approval.  The informed consent 
document must clearly describe the potential risks and benefits of the trial, and each prospective 
participant must be given adequate time to discuss the trial with the Investigator or site staff and 
to decide whether or not to participate.  Each patient who agrees to participate in the trial and 


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who signs the informed consent will be given a copy of the signed dated and witnessed 
document.  The original copy of the signed, dated, and witnessed informed consent document 
will be retained by the Investigator in the study files. 
 
The Investigator must also obtain authorization from the patient to use and/or disclose protected 
health information in compliance with the Health Insurance Portability and Accountability Act 
(HIPAA).  Written HIPAA authorization may be obtained as part of the informed consent 
process.   
11.2 
Ethical Conduct of the Study and IRB/IEC Approval 
The study will be conducted according to the principles of the 2004 version of the Declaration of 
Helsinki, the International Conference on Harmonization Guidance on Good Clinical Practice 
and the requirements of all local regulatory authorities regarding the conduct of clinical trials and 
the protection of human subjects.  
 
The Investigator will submit the protocol, the Investigator’s Brochure, the informed consent and 
any other material used to inform patients about the trial to the local IRB/IEC for approval prior 
to enrolling any patient into the trial.  The IRB/IEC should be duly constituted according to 
applicable regulatory requirements.  Approval must be in the form of a letter signed by the 
Chairperson of the IRB/IEC or the Chairperson’s designee, must be on IRB/IEC stationary and 
must include the protocol by name and/or designated number.  If an Investigator is a member of 
the IRB/IEC, the approval letter must stipulate that the Investigator did not participate in the final 
vote, although the Investigator may participate in the discussion of the trial.  The Investigator 
will also inform the IRB/IEC of any serious adverse events that are reported to regulatory 
authorities and will provide to the IRB/IEC a final summary of the results of the trial at the 
conclusion of the trial. 
 
Any amendments to the protocol will be done at the lead site, and will be submitted to the 
IRB/IEC for review and written approval before implementation. These approved changes will 
then be forwarded to sites for review by their local IRB/IEC.  Written approval from sites will be 
forwarded to the lead site. 
 
11.3 
Data Safety Monitoring Board (DSMB) 
An independent Data Safety and Monitoring Board (DSMB) will oversee the conduct of the 
study.  The members of this Board will receive database summaries, including adverse event 
reports, and will convene either in person or via teleconference every 6 months.  The Board will 
be responsible for decisions regarding possible termination and/or early reporting of the study. 
 
11.4 
Monitoring and Auditing 
A clinical monitor will make regularly scheduled trips to the investigational site to review the 
progress of the trial as defined in the Monitoring Plan.  The actual frequency of monitoring trips 
will depend on the enrollment rate and performance at each site.  At each visit, the monitor will 
review various aspects of the trial including, but not limited to, screening and enrollment logs; 
compliance with the protocol and with the principles of Good Clinical Practice; completion of 


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case report forms; source data verification; study drug accountability and storage; facilities and 
staff data quality; regulatory documentation; and study integrity. In addition the site may be 
audited by 
 government inspectors who must be allowed access to CRFs, source 
documents and other study files.  The site must promptly notify the study chair of any 
inspections scheduled by regulatory authorities, and also forward copies of the inspection reports 
to the study chair. The study chair will promptly forward this information to 
. 
 
 
During scheduled monitoring visits, the Investigator and the investigational site staff must be 
available to meet with the study monitor in order to discuss the progress of the trial, make 
necessary corrections to case report form entries, respond to data clarification requests and 
respond to any other trial-related inquiries of the monitor. 
 
 


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11.5 
Pre-Study Documentation 
Prior to initiating the trial, the Investigators at each site will provide to the Lead site the 
following documents: 
• 
A signed FDA Form 1572 
• 
A current curriculum vitae for the Principal Investigator and each sub-investigator listed on 
the FDA Form 1572 
• 
A copy of the Investigator’s medical license from the state in which the study is being 
conducted 
• 
A letter from the IRB or EC stipulating approval of the protocol, the informed consent 
document and any other material provided to potential trial participants with information 
about the trial (e.g., advertisements) 
• 
A copy of the IRB- or EC-approved informed consent document 
• 
Current IRB membership list for IRB’s without a multiple project assurance number or an 
IRB 
organization 
number 
under 
the 
Federal 
Wide 
Assurance 
program 
(www.ohrp.osophs.dhhs.gov).   
• 
A signed Investigator Protocol Agreement 
• 
A completed financial disclosure form for each person listed in box 6 of the FDA form 
1572. 
• 
Current laboratory certification for the reference laboratory 
• 
A list of current laboratory normal values for the reference laboratory 
 
11.6 
Confidentiality 
It is the responsibility of the investigator to insure that the confidentiality of all subjects 
participating in the trial and all of their medical information is maintained.  Case report forms 
and other documents submitted must never contain the name of a trial participant.  Each subject 
in the trial will be identified by a unique identifier that will be used on all CRF’s and any other 
material submitted to the lead site.  All case report forms and any identifying information must 
be kept in a secure location with access limited to the study staff directly participating in the trial. 
 
The results of the study may be presented in reports, published in scientific journals or presented 
at medical meetings; however, subject names will never be used in any reports about the study. 
 
11.7 
Source Documents 
The Investigator will maintain source records separate from the case report forms in the form of 
clinical charts, medical records, original laboratory, radiology and pathology reports, pharmacy 
records, etc.  The Investigator will document in the clinic chart or medical record the name and 
number of the trial and the date on which the subject signed informed consent prior to the 
subject’s participation in the trial.  Source documents must completely reflect the nature and 
extent of the patient’s medical care, and must be available for source document verification 
against entries in the case report forms when the monitor visits the investigational site.  All 
information obtained from source documents will be kept in strict confidentiality. 


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11.8 
Record Retention 
The Investigator will retain the records of the study for 15 years, or for 2 years following the date 
that a marketing application for the study drug is approved, or if no marketing application is 
filed, or if such an application is not approved, for 2 years after the IND has been closed.  The 
lead site will notify Investigators when retention of study records is no longer required.  All 
study records must be maintained in a safe and secure location that allows for timely retrieval, if 
needed. 
 
Study records that must be retained include copies of case report forms, signed informed 
consents, correspondence with the IRB or IEC, study drug dispensing and inventory records, 
source documents, clinic charts, medical records, laboratory results, radiographic reports) and 
screening/enrollment logs. 
 
Should the Investigator relocate or retire, or should there be any changes in the archival 
arrangements for the study records, the lead site must be notified.  The responsibility for 
maintaining the study records may be transferred to another suitable individual, but the lead site 
must be notified of the identity of the individual assuming responsibility for maintaining the 
study records and the location of their storage.   
 
12.0 Biological Evaluation  
A major challenge in the development of cancer therapeutics is an absence of understanding the 
relationship of the disease to response to therapy, and the ability to predict which subjects are 
most likely to respond to any particular agent. Emerging technologies including ability to 
establish primary tumor cells in culture, evaluate pharmacogenomics and explore biomarkers 
may provide a way to explore relationships between clinical benefit and treatment.  
 
Urine Sample Analysis – 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Blood and Bone Marrow Samples Analysis 
 
ODC SNP analysis –Performed by Pediatric Oncology Translational Research Laboratory 
(Section 12.1.2) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biomarker Analysis –Performed by Pediatric Oncology Translational Research Laboratory 
(Section 12.1.2) 
Subject biomarkers will be evaluated in blood plasma using antibody array analysis in the 
laboratory of Dr. Sholler. Antibody arrays will be generated to target biomarker candidates 
derived from gene expression, proteomic, and glycomic studies of tumor tissue (obtained from 
related biological studies). We will use these arrays to quantify the various protein levels in order 
to identify proteins or panels of proteins that differentiate patients with poor prognosis from 
patients with good prognosis. Specific carbohydrate levels also will be characterized on each 
protein to determine the associations of those measurements with prognosis.  
 
Circulating Tumor Cell Analysis –Performed by Dr. Li (Section 12.1.2) 
 
 
 


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Bone Marrow –Performed by Dr. Kidd (Sections 12.1.3) 
 
 
 
12.1 
  Pharmacokinetic Sample Instructions 
 
Blood samples for the pharmacokinetic assay of DFMO and metabolites will be collected 
from approximately 10 currently enrolled subjects on their last day of taking the old DFMO 
formulation and on one day during Days 2-5 of taking the new formulation. Samples will be 
collected prior to administration of DFMO (Time 0) and at the following times: 30min, 1 hour, 
3 hours, and 6 hours post-dose (exact time should be noted on CRF). 
 
The blood samples will be processed to plasma, aliquoted and stored frozen at  -80ºC until 
assayed.    
 
PK  parameters  (Cmax,  Tmax,  AUC0-24,  AUC0-∞,  t1/2,  Cl,  and  Vd)  will  be  determined  as 
appropriate   using   standard   noncompartmental   procedures   (WINNONLIN   software). 
Individual  subject  measurements,  as  well  as  summary  statistics  (e.g.,  mean,  standard 
deviation, range, %CV) will be reported. 
 
Procedures  for  obtaining,  storing  and  shipping  of  the  pharmacokinetic  samples  are  as 
follows: 
 
1.   One 2cc whole blood specimen will be collected in a purple top tube (EDTA) 
for each time point. 
2.   Place samples immediately in ice water bath until ready to process (no longer 
than 24 hours). 
3.   Specimen will be spun in a centrifuge at 2,000g for 15 minutes 
4.   Following centrifugation; using a clean pipette, the plasma from EDTA tube 
will be aliquoted into a plastic screw capped cryovial. 
5.   Specimens must be labeled with the participants unique study identifier, PK 
specific time point (include cycle, day, and time point) and date and time of 
specimen collection 
6.   Specimens will then be stored in a -80 freezer until ready to be shipped. 
 
 
 


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PK Specimen shipping: 
• All specimens will be shipped on dry ice following the federal IATA hazardous 
Specimen Shipping Guidelines 
 
• Specimens should be batch shipped (after each subject completes all PK time 
points) on a Monday-Thursday. 
 
• PK Specimens will be shipped to the following address: 
 
Ping Zhao  
Pediatric Oncology Translational Research Laboratory  
Coopers Landing 
1345 Monroe Ave, Suite 121 
Grand Rapids, MI  49505 
Ph: 616-486-8645 
Ping.zhao@helendevoschildrens.org 
 
12.2 
  Biological Correlative Studies Sample Instructions 
 
Subjects will have the following required urine samples sent. 
 
12.2.1 Required Urine Polyamines Collection:  
At screening, on Days 1 and 15 of Cycle 1, and on Day 1 of cycles 2-27, 
urine polyamines will be collected. 
A. 
First morning void urines will be collected using a clean catch 
method in containers and placed at 4oC until transported to the 
laboratory. Collection of 15ml is ideal, but lab can accept amounts 
down to a minimum of 1 (one) ml.  
B. 
Specimens will be transferred to 15ml polypropylene conical tubes 
(use multiple 15ml tubes as needed) and stored at -80oC until 
analysis for the dc-SAM and polyamines. Samples should be kept 
at 4oC (either in refrigerator or on ice pack during shipping) until 
frozen and must be frozen within 48 hours from collection time. If 
unable to freeze within 48 hours please note this on the container 
along with time before frozen. 
C. 
Samples will be batch shipped on a Monday (send after 
approximately 20 samples are collected) and sent on dry ice to: 
Ping Zhao  
Pediatric Oncology Translational Research Laboratory  
Coopers Landing 
1345 Monroe Ave, Suite 121 
Grand Rapids, MI 49505  
Ph: 616-486-8645 
Ping.zhao@helendevoschildrens.org 


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Subjects will have the following optional (recommended) blood and bone marrow samples sent. 
12.2.2 Optional Blood Collection 
 
1. Sample One, Two, and Three-  
 
A. 
Sample 1- On Day 1:  2 mls of blood will be collected in an EDTA 
tube (s). At the time of collection, the tube should be gently 
inverted 6-8 times. 
 
B. 
Sample 2- On Days 1 and 15 of Cycle 1, Day 1 of cycles 2 through 
12, and 14, 17, 21, 24 and off -therapy- 5 mls of blood will be 
collected in an EDTA tube(s). At the time of collection, the tube 
should be gently inverted 6-8 times.  
 
C. 
Sample 3- On Days 1 and 15 of Cycle 1, Day 1 of cycles 2 through 
12, and 14, 17, 21, 24 and off-therapy- 5 mls of blood will be 
collected in an EDTA tube(s). At the time of collection, the tube 
should be gently inverted 6-8 times. 
 
 
D. 
Shipment/Storage will be as follows: 
Specimens A-C above should be placed on ice immediately upon 
draw and sent on an ice pack immediately via FED-EX overnight. 
(If Friday collection site may store in refrigerator and ship on 
Monday). Ship to: 
 
Ping Zhao  
Pediatric Oncology Translational Research Laboratory  
Coopers Landing 
1345 Monroe Ave, Suite 121 
Grand Rapids, MI 49505  
Ph: 616-486-8645 
Ping.zhao@helendevoschildrens.org 
 
 
 
 
 
 
 
 
 
 


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2. A 7.5cc whole blood specimen will be collected in a BD CPT Vacutainer 
tube(s) containing the anti-coagulant Sodium Heparin (BD Cat no. 
REF362753, 8mL draw capacity). 
 
A. 
At screening and then start of cycles 3, 6, 12, 14, 17, 21, 24 and 
off-therapy. 
 
 
 
B. 
Specimen Handling: 
1. If BD CPT Vacutainer tube is not available at the site, collect 
blood in a Green top heparin tube (do not use glass tubes). Do not 
follow steps 2-5- Ship without processing in the green top tube.  
Skip to C below for shipping instructions.  
2. After collection, store tube upright at room temperature until 
centrifugation. Blood samples should be centrifuged within two 
hours of blood collection. 
3. Centrifuge tube/blood sample at room temperature (18-25° C) in a 
horizontal rotor for a minimum of 15 minutes 
4. at 1500 to 1800 RCF (Relative Centrifugal Force). 
NOTE: Remix the blood sample immediately prior to 
centrifugation by gently inverting the tube 8 to 10 times. Also, 
check to see that the tube is in the proper centrifuge carrier/adapter. 
5. After centrifugation, mononuclear cells and platelets will be in a 
whitish layer just under the plasma layer. These tubes are now 
ready for transportation. If not transporting immediately, they can 
be stored at room temperature until transportation. Samples should 
be shipped at room temperature.  
 
 
C. 
Ship samples the same day as collection.  Samples will be sent 
ambient temperature to: 
 
 
 
Xueqing Xia 
Dept. Pediatrics—Research, MOD2.016 
UT MD Anderson Cancer Center 
7777 Knight Rd. 
Houston, TX 77054 
713-563-9617 
 
 
 


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12.2.3 Optional Bone Marrow Collection 
 
If subject agrees to optional biology portion of study, additional bone marrow 
samples should be sent at the following time points: 
(Please note that if subject has also signed to protocol NMTRC 00B, these 
samples should be sent under that study instead): 
 
All Subjects- At Enrollment, Any time a bone marrow is done during protocol 
therapy, Early Withdrawal (if indicated), and per institutional standards after that. 
 
Sample Collection-  
 
Send green top (sodium heparin) tube(s) with a minimum of 2cc and preferably 
5cc of bone marrow aspirate in them.    The bone marrow samples should be 
shipped room temperature- but needs to be sent out same day priority overnight 
(must get there within 24 hours of the draw).   Shipments are only accepted 
Monday through Friday, so Bone Marrow draw needs to be done and shipped out 
on Monday through Thursday only please.  
Please contact the lab at the number below to let them know of pending arrival. 
 
Bone marrow 2-5cc in green top (sodium heparin) tube(s) at room temperature 
and will be shipped FED-EX overnight to:  
 
Flow Cytometry Lab of Spectrum Health  
Attn: Pamela Kidd, MD 
Lemmen-Holton Cancer Pavilion   
145 Michigan Street NE Suite 6201 
Grand Rapids, MI 49503 
phone: 616-486- 6270 
 
 
 
 
12.3 
Sample Storage and Destruction 
Samples collected for any studies performed in this protocol may be stored 
indefinitely to research scientific questions related to cancer and/or study drugs. 
The subject retains the right to have the sample material destroyed at any time by 
contacting the principal investigator. 
60B 
 
 


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Appendix I: Performance Status/Scores 
 
 
 
 


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