Clinical trial NCT01824693
Conditions: Juvenile Myelomonocytic Leukemia
Sponsor: Children's Oncology Group
Start date: 2013-06-24; Completion date: 2017-12-31
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This randomized phase II trial studies how well giving busulfan, cyclophosphamide, and melphalan or busulfan and fludarabine phosphate before donor hematopoietic cell transplant works in treating younger patients with juvenile myelomonocytic leukemia. Giving chemotherapy before a donor hematopoietic transplant helps stop the growth of cancer cells. It may also stop the patient's immune system from rejecting the donor's stem cells. When the healthy stem cells from a donor are infused into the patient, they may help the patient's bone marrow make stem cells, red blood cells, white blood cells, and platelets. It is not yet known whether giving busulfan, cyclophosphamide, and melphalan or busulfan and fludarabine phosphate before a donor stem cell transplant is more effective in treating juvenile myelomonocytic leukemia.

Detailed description:
PRIMARY OBJECTIVES:

I. To compare ? in a randomized fashion ? the day 100 treatment related mortality (TRM) incidence for two myeloablative conditioning regimens, busulfan-fludarabine (fludarabine phosphate) (BU-FLU) and busulfan-cyclophosphamide-melphalan (BU-CY-MEL), prior to hematopoietic cell transplant (HCT) for children with juvenile myelomonocytic leukemia (JMML), in order to determine the preferred regimen for future trials.

II. To compare ? in a randomized fashion ? the 18-month event-free survival (EFS) following two different myeloablative conditioning regimens (BU-FLU vs. BU-CY-MEL) prior to HCT for children with JMML, in order to determine the preferred regimen for future trials.

SECONDARY OBJECTIVES:

I. To determine the 18-month relapse incidence (RI) following two different myeloablative conditioning regimens (BU-FLU vs. BU-CY-MEL) prior to HCT for children with JMML.

II. To determine the graft failure rates following two different myeloablative conditioning regimens (BU-FLU vs. BU-CY-MEL) prior to HCT for children with JMML.

TERTIARY OBJECTIVES:

I. To determine the rates of severe toxicities (grade 3/4) at day 100 post-HCT between the two myeloablative conditioning regimens (BU-FLU vs. BU-CY-MEL).

II. To determine the rates of acute and chronic (at 18 months post-HCT) graft-versus-host disease (GVHD) following HCT using two different conditioning regimens (BU-FLU vs. BU-CY-MEL) in children with JMML.

III. To create a JMML-specific pre-HCT index to allow better risk-stratification of future patients.

IV. To determine the feasibility of assessing post-transplant disease burden by donor chimerism measurements and allele-specific polymerase chain reaction (PCR) in mononuclear and sorted cell subsets.

V. To validate gene expression and methylation classifiers predictive of relapse in patients with JMML.

VI. To comprehensively assess genetic and biochemical alterations amongst patients with JMML who are treated on this transplant protocol.

OUTLINE: Patients are randomized to 1 of 2 treatment arms.

ARM I:

CONDITIONING REGIMEN: Patients receive busulfan intravenously (IV) over 2-3 hours once daily (QD), every 12 hours, or every 6 hours on days -8 to -5, cyclophosphamide IV over 60 minutes QD on days -4 and -3, and melphalan IV over 15-30 minutes on day -1.

TRANSPLANT: Patients undergo allogeneic HCT on day 0.

Patients receive tacrolimus IV or orally (PO) on days -1 to 98 (related donor) or 180 (unrelated donor) and mycophenolate mofetil IV over 2 hours or PO every 8 hours on days 1-30 (related donor) or 45 (unrelated donor).

ARM II:

CONDITIONING REGIMEN: Patients receive busulfan as in Arm I and fludarabine phosphate IV over 30-60 minutes on days -5 to -2.

TRANSPLANT: Patients undergo allogeneic HCT as in Arm I.

Patients receive tacrolimus IV or PO on days -1 to 98 (related donor) or 180 (unrelated donor) and mycophenolate mofetil IV over 2 hours or PO every 8 hours on days 1-30 (related donor) or 45 (unrelated donor).

After completion of study treatment, patients are followed up for 5 years.

Arms: arm 1: CONDITIONING REGIMEN: Patients receive busulfan IV QD, every 12 hours, or every 6 hours over 2-3 hours on days -8 to -5, cyclophosphamide IV QD over 60 minutes on days -4 and -3, and melphalan IV over 15-30 minutes on day -1.

TRANSPLANT: Patients undergo allogeneic HCT no sooner than 24 hours after the last dose of chemotherapy.

Patients receive tacrolimus IV or PO on days -1 to 98 (related donor) or 180 (unrelated donor) and mycophenolate mofetil IV over 2 hours or PO every 8 hours on days 1-30 (related donor) or 45 (unrelated donor). arm 2: CONDITIONING REGIMEN: Patients receive busulfan as in Arm I and fludarabine phosphate IV over 1 hour on days -5 to -2.

TRANSPLANT: Patients undergo allogeneic HCT as in Arm I.

Patients receive tacrolimus IV or PO on days -1 to 98 (related donor) or 180 (unrelated donor) and mycophenolate mofetil IV over 2 hours or PO every 8 hours on days 1-30 (related donor) or 45 (unrelated donor).
Interventions: intervention 1: Allogeneic Hematopoietic Stem Cell Transplantation intervention 2: Busulfan intervention 3: Cyclophosphamide intervention 4: Fludarabine Phosphate intervention 5: Laboratory Biomarker Analysis intervention 6: Melphalan intervention 7: Mycophenolate Mofetil intervention 8: Pharmacological Study intervention 9: Tacrolimus
Intervention descriptions: intervention 1: Undergo allogeneic HCT intervention 2: Given IV intervention 3: Given IV intervention 4: Given IV intervention 5: Correlative studies intervention 6: Given IV intervention 7: Given IV or PO intervention 8: Correlative studies intervention 9: Given IV or PO

Protocol documents:
Prot_SAP_000.pdf: 
ASCT1221 
06/05/14 
 
1 
 
Activated: 
06/24/13      
Version Date: 
06/05/14 
Closed: 
02/02/16 
Amendment: 
#1 
 
 
 
CHILDREN'S ONCOLOGY GROUP 
 
 
ASCT1221 
 
A Randomized Phase II Study Comparing Two Different Conditioning Regimens Prior to 
Allogeneic Hematopoietic Cell Transplantation (HCT) for Children with Juvenile Myelomonocytic 
Leukemia (JMML) 
 
 
A Groupwide Phase II Study 
 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, AND SHOULD NOT BE COPIED, REDISTRIBUTED OR 
USED FOR ANY OTHER PURPOSE.  MEDICAL AND SCIENTIFIC INFORMATION CONTAINED WITHIN THIS 
PROTOCOL IS NOT INCLUDED TO AUTHORIZE OR FACILITATE THE PRACTICE OF MEDICINE BY ANY 
PERSON OR ENTITY.  RESEARCH MEANS A SYSTEMATIC INVESTIGATION, INCLUDING RESEARCH 
DEVELOPMENT, 
TESTING 
AND 
EVALUATION, 
DESIGNED 
TO 
DEVELOP 
OR 
CONTRIBUTE 
TO 
GENERALIZABLE KNOWLEDGE.  THIS PROTOCOL IS THE RESEARCH PLAN DEVELOPED BY THE 
CHILDREN’S ONCOLOGY GROUP TO INVESTIGATE A PARTICULAR STUDY QUESTION OR SET OF STUDY 
QUESTIONS AND SHOULD NOT BE USED TO DIRECT THE PRACTICE OF MEDICINE BY ANY PERSON OR TO 
PROVIDE INDIVIDUALIZED MEDICAL CARE, TREATMENT, OR ADVICE TO ANY PATIENT OR STUDY 
SUBJECT.  THE PROCEDURES IN THIS PROTOCOL ARE INTENDED ONLY FOR USE BY CLINICAL 
ONCOLOGISTS IN CAREFULLY STRUCTURED SETTINGS, AND MAY NOT PROVE TO BE MORE EFFECTIVE 
THAN STANDARD TREATMENT.  ANY PERSON WHO REQUIRES MEDICAL CARE IS URGED TO CONSULT WITH 
HIS OR HER PERSONAL PHYSICIAN OR TREATING PHYSICIAN OR VISIT THE NEAREST LOCAL HOSPITAL OR 
HEALTHCARE INSTITUTION. 
 
 
 
STUDY CHAIR 
 
 
  
 
 
 
 
      
     
     
 
 
 
 
 


ASCT1221 
TABLE OF CONTENTS 
SECTION 
PAGE 
STUDY COMMITTEE 
6 
ABSTRACT 
9 
EXPERIMENTAL DESIGN SCHEMA 
10 
1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
11 
1.1 
Primary Objectives 
11 
1.2 
Secondary Objectives 
11 
1.3 
Exploratory Objectives 
11 
2.0 
BACKGROUND 
12 
2.1 
Rationale for Selected Approach and Trial Design 
12 
2.2 
Trial Impact 
13 
2.3 
HCT for JMML 
13 
2.3.1 
“Standard of Care” HCT for JMML 
13 
2.3.2 
Use of Cyclophosphamide vs. Fludarabine 
14 
2.3.3 
Serotherapy 
15 
2.4 
Clinical Implications of Genetic Mutations in JMML 
16 
2.4.1 
Diagnostic and Risk-Stratification Implications 
16 
2.4.2 
Transplant vs. No-Transplant for Patients with Germline Syndromes who     
Develop JMML 
17 
2.4.3 
Correlative Biology Studies 
17 
3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
20 
3.1 
Study Enrollment 
20 
3.1.1 
Patient Registration 
20 
3.1.2 
IRB Approval 
20 
3.1.3 
Study Enrollment 
20 
3.1.4 
Timing 
21 
3.1.5 
Randomization 
22 
3.2 
Patient Eligibility Criteria 
22 
3.2.1 
Age 
22 
3.2.2 
Diagnosis 
22 
3.2.3 
Prior Therapy 
23 
3.2.4 
Mutation Status of PTPN11 (Germline) 
23 
3.2.5 
Neurofibromatosis Type 1 (NF1) Status 
23 
3.2.6 
HIV Status 
23 
3.2.7 
All patients and/or their parents or legal guardians must sign a written informed 
consent. 
23 
3.2.8 
All institutional, FDA, and NCI requirements for human studies must be met. 
23 
4.0 
TREATMENT PROGRAM 
24 
4.1 
Overview of Treatment Plan 
24 
4.2 
Hematopoietic Stem Cell Matching and Cell Dose Requirements 
24 
4.2.1 
Bone Marrow and Peripheral Blood Stem Cell (PBSC) Requirements 
24 
4.2.2 
Matched Related Donor and Cord Blood Unit Requirements 
25 
4.2.3 
Hierarchy of Stem Cell Choices 
25 
4.3 
Requirements to Proceed to HCT 
25 
4.3.1 
Confirmation of JMML Diagnosis 
25 
4.3.2 
Prior Therapy Restrictions 
26 
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4.3.3 
Organ Function Requirements 
26 
4.3.4 
Performance Level 
27 
4.3.5 
Infection Status 
27 
4.3.6 
Pregnancy and Breast Feeding 
27 
4.4 
Hematopoietic Stem Cell Infusion Guidelines 
28 
4.4.1 
Donor Bone Marrow or Peripheral Blood Infusion Guidelines 
28 
4.4.2 
Donor Umbilical Cord Blood Infusion Guidelines 
28 
4.5 
Concomitant Therapy and Study-Specific Supportive Care Guidelines 
29 
4.5.1 
Growth Factors 
29 
4.5.2 
Infectious Prophylaxis 
29 
4.5.3 
Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease 
30 
4.6 
Matched Family Donor HCT for Patients Assigned to Arm A (BU-CY-MEL) 
30 
4.6.1 
HCT Regimen: Matched Family Donor -Arm A 
33 
4.7 
Unrelated Donor HCT for Patients Assigned to Arm A (BU-CY-MEL+ rATG) 
34 
4.7.1  HCT Regimen - Unrelated Donor -Arm A 
38 
4.8 
UCB Donor HCT for Patients Assigned to Arm A (BU-CY-MEL+ rATG) 
39 
4.8.1  HCT Regimen - UCB Donor -Arm A 
42 
4.9 
Matched Family Donor HCT for Patients Assigned to Arm B (BU-FLU) 
43 
4.9.1   HCT Regimen: Matched Family Donor -Arm B 
46 
4.10 
Unrelated Donor HCT for Patients Assigned to Arm B (BU-FLU + rATG) 
47 
4.10.1   HCT Regimen: Unrelated Donor -Arm B 
50 
4.11 
UCB Donor HCT for Patients Assigned to Arm B (BU-FLU + rATG) 
51 
4.11.1   HCT Regimen: UCB Donor -Arm B 
54 
5.0 
DOSE MODIFICATIONS FOR TOXICITIES 
55 
5.1 
Dose Adjustment of Chemotherapy for Patients Whose Weight Exceeds > 125% IBW 55 
5.1.1 
Recommended Ideal Body Weight Calculation for Children Age 1- 17 years 
55 
5.1.2 
Recommended Ideal Body Weight Calculation for Adults (≥ 18 years) 
55 
5.2 
Tacrolimus 
55 
5.3 
Mycophenolate 
56 
6.0 
DRUG INFORMATION 
56 
7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
57 
7.1 
Evaluations at Study Entry and Requirements Prior to Stem Cell Transplant 
57 
7.2 
Required & Optional Clinical, Laboratory and Disease Evaluations Post HCT 
59 
7.2.1  
Suspicion of Relapse 
59 
7.3 
Required Busulfan Pharmacokinetic Studies* (see Section 16). 
60 
7.4 
Follow-up 
60 
8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY     
CRITERIA 
61 
8.1 
Criteria for Removal from Protocol Therapy 
61 
8.2 
Off Study Criteria 
61 
9.0 
STATISTICAL CONSIDERATIONS 
61 
9.1 
Study Design 
62 
9.1.1 
Primary Endpoints: 
62 
9.1.2 
Secondary Endpoints: 
62 
9.1.3 
Stratification 
62 
9.2 
Sample Size and Study Duration 
63 
9.2.1 
Justification for this Design 
63 
9.2.2 
Trial Outcome Probabilities given TRM and Relapse Probabilities 
63 
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ASCT1221 
9.2.3  
TRM and Relapse Probabilities for the Outcome Probability Calculations 
65 
9.3 
Methods of Analysis 
67 
9.3.1 
Primary objectives 
67 
9.3.2 
Secondary objectives 
67 
9.3.3 
Exploratory objectives 
67 
9.3.4 
Safety Monitoring 
69 
9.4 
Evaluability for Response 
70 
9.5 
Evaluability for Toxicity 
70 
9.6 
Gender and Minority Accrual Estimates 
71 
10.0 
EVALUATION CRITERIA 
71 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
71 
10.2 
JMML Response Criteria 
71 
10.2.1 Definition of Complete Remission prior to HCT: 
71 
10.2.2 Definition of Complete Remission after HCT: 
72 
10.2.3 Definition of Relapse: 
72 
10.2.4 Definition of Non-Response: 
73 
10.3 
Graft Failure following Allogeneic HCT 
73 
10.3.1 Primary Graft Failure 
73 
10.3.2 Secondary Graft Failure: 
73 
10.4 
Definition of Engraftment 
73 
10.5 
Evaluation and Diagnosis of GVHD 
74 
10.5.1 Evaluation and Diagnosis of Acute GVHD 
74 
10.5.2 Evaluation and Diagnosis of Chronic GVHD 
75 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS 
76 
11.1 
Purpose 
76 
11.2 
Determination of Reporting Requirements 
76 
11.3 
Reporting of Adverse Events for Commercial Agents – via CTEP-AERS 
77 
11.4 
Routine Adverse Event Reporting 
78 
12.0 
STUDY REPORTING AND MONITORING 
78 
12.1 
CDUS 
78 
12.2 
Data and Safety Monitoring Committee 
78 
13.0 
CYTOGENETICS ANALYSIS GUIDELINES AND REQUIREMENTS 
79 
13.1 
Cytogenetic Analysis Overview 
79 
13.2 
Local Cytogenetic Analysis and Central Review Submission Guidelines 
79 
13.2.1 Specimen Collection for Local Cytogenetics Analysis 
79 
13.2.2 Data Submission for Central Cytogenetics Review at Diagnosis and Relapse 
79 
13.2.3 Data Submission for Central Cytogenetics Review 
80 
13.2.4 Central Cytogenetics Review Submission Guidelines 
80 
14.0 
PATHOLOGY GUIDELINES AND SPECIMEN REQUIREMENTS 
81 
14.1 
Central Pathology Review Submission Guidelines 
81 
15.0 
SPECIAL STUDIES SPECIMEN REQUIREMENTS 
82 
15.1 
NIH-Funded Laboratory for Mutation Analysis at Diagnosis 
82 
15.2 
NRAS, KRAS, CBL, and PTPN11 Mutation Analysis 
82 
15.3 
Minimal Residual Disease (MRD) Analysis 
82 
15.3.1 CLIA/CAP-Approved Laboratory for Mutant Allele Testing after Diagnosis 
82 
15.3.2 Description of MRD Analysis 
82 
15.4 
Specimen Collection Overview 
83 
15.5 
Specimen Details 
83 
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ASCT1221 
15.6 
Specimen Shipping Details 
84 
15.6.1 Shipments to the University of California, San Francisco 
84 
15.6.2 Shipments to the COG Leukemia Reference Laboratory 
85 
15.7 
Description of Correlative Biology Studies 
85 
16.0 
PHARMACOKINETICS FOR BUSULFAN DOSE ADJUSTMENT 
87 
16.1 
1st Dose Pharmacokinetics 
87 
16.2 
Shipping 
88 
16.3 
Guidelines for Adjusting Busulfan Dosing Based on Results of First Dose 
Pharmacokinetic Results 
88 
APPENDIX I:  JMML GENE MUTATIONS 
89 
APPENDIX II:  WHO CRITERIA FOR NEUROFIBROMITOSIS-1 
91 
APPENDIX IIIA: DRUGS KNOWN TO MODULATE THE ACTIVITY OF CYP450 ISOENZYMES 
3A4, CYP2C19 AND CYP1A2 
92 
APPENDIX IIIB: LIST OF ANTICONVULSANTS BASED ON CYP3A4/5 ENZYME INDUCTION 93 
APPENDIX IV: PRE-TRANSPLANT RECOMMENDATIONS 
94 
APPENDIX V:  YOUTH INFORMATION SHEETS 
95 
REFERENCES 
97 
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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
STUDY COMMITTEE 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
  
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
 
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
 
 
 
 
     
 
 
   
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AGENT                             NSC#         IND# 
 
Antithymocyte Globulin  720095      Exempt 
 
Busulfan  
 
750            Exempt 
 
Cyclophosphamide 
26271        Exempt 
 
Fludarabine 
 
312887      Exempt 
 
Melphalan 
 
008806      Exempt 
 
Mesna  
 
113891      Exempt 
 
Mycophenolate mofetil 724229      Exempt 
 
Tacrolimus 
 
717865      Exempt 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SEE SECTION 14-16 FOR SPECIMEN SHIPPING ADDRESSES 
06/05/14 
 
8 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
The Children's Oncology Group has received a Certificate of Confidentiality from the federal 
government, which will help us protect the privacy of our research subjects.  The Certificate protects 
against the involuntary release of information about your subjects collected during the course of our 
covered studies.  The researchers involved in the studies cannot be forced to disclose the identity or any 
information collected in the study in any legal proceedings at the federal, state, or local level, regardless 
of whether they are criminal, administrative, or legislative proceedings.  However, the subject or the 
researcher may choose to voluntarily disclose the protected information under certain circumstances.  For 
example, if the subject or his/her guardian requests the release of information in writing, the Certificate 
does not protect against that voluntary disclosure.  Furthermore, federal agencies may review our records 
under limited circumstances, such as a DHHS request for information for an audit or program evaluation 
or an FDA request under the Food, Drug and Cosmetics Act. 
The Certificate of Confidentiality will not protect against mandatory disclosure by the researchers of 
information on suspected child abuse, reportable communicable diseases, and/or possible threat of harm 
to self or others 
 
 
ABSTRACT 
ASCT1221 is a randomized Phase II trial designed to identify which myeloablative hematopoietic cell 
transplant (HCT) preparative regimen has a lower relapse and treatment related mortality (TRM) rate in 
children with juvenile myelomonocytic leukemia (JMML). Patients with JMML according to Chan et al 
(including diagnostic genotyping from a CLIA/CAP-certified central laboratory) will be randomized to 
one of two myeloablative conditioning regimens within strata defined by donor type and PTPN11 
mutation status. The objective of the study is to identify the better conditioning regimen. It is 
hypothesized that the regimens will have equivalent relapse rates but may differ in TRM, a feature that 
has been exploited in the study design. Our hypothesis is that patients randomized to receive a busulfan-
fludarabine (BU-FLU) conditioning regimen will have less TRM and comparable event-free survival 
(EFS) when compared to a busulfan-cyclophosphamide-melphalan (BU-CY-MEL) conditioning regimen. 
Correlative biologic studies will determine the feasibility of assessing post-transplant disease burden by 
donor chimerism measurements and mutant allele burden. We will also validate gene expression 
classifiers using RNA-based or methylation-based arrays in patients with JMML that may predict relapse, 
and will comprehensively assess genetic and biochemical alterations amongst patients with JMML who 
are treated on this transplant protocol.  
 
 
 
 
 
 
 
06/05/14 
 
9 


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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
1.1 
Primary Objectives 
 
1.1.1 
To compare – in a randomized fashion – the Day 100 TRM incidence for two 
myeloablative conditioning regimens, busulfan-fludarabine (BU-FLU) and 
busulfan-cyclophosphamide-melphalan (BU-CY-MEL), prior to HCT for 
children with JMML, in order to determine the preferred regimen for future trials. 
 
1.1.2 
To compare – in a randomized fashion – the 18-month event-free survival (EFS) 
following two different myeloablative conditioning regimens (BU-FLU vs. BU-
CY-MEL) prior to HCT for children with JMML, in order to determine the 
preferred regimen for future trials. 
 
1.2 
Secondary Objectives 
 
1.2.1 
To determine the 18-month relapse incidence (RI) following two different 
myeloablative conditioning regimens (BU-FLU vs. BU-CY-MEL) prior to HCT 
for children with JMML. 
 
1.2.2 
To determine the graft failure rates following two different myeloablative 
conditioning regimens (BU-FLU vs. BU-CY-MEL) prior to HCT for children 
with JMML. 
 
1.3 
Exploratory Objectives 
 
1.3.1 
To determine the rates of severe toxicities (Grade 3/4) at Day 100 post-HCT 
between the two myeloablative conditioning regimens (BU-FLU vs. BU-CY-
MEL).  
 
1.3.2 
To determine the rates of acute and chronic (at 18 months post-HCT) graft-
versus-host disease (GVHD) following HCT using two different conditioning 
regimens (BU-FLU vs. BU-CY-MEL) in children with JMML. 
 
1.3.3 
To create a JMML-specific pre-HCT index to allow better risk-stratification of 
future patients. 
 
1.3.4 
To determine the feasibility of assessing post-transplant disease burden by donor 
chimerism measurements and allele-specific PCR in mononuclear and sorted cell 
subsets. 
 
1.3.5 
To validate gene expression and methylation classifiers predictive of relapse in 
patients with JMML.  
 
1.3.6 
To comprehensively assess genetic and biochemical alterations amongst patients 
with JMML who are treated on this transplant protocol.  
 
 
 
06/05/14 
 
11 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
 
2.0 
BACKGROUND 
2.1 
Rationale for Selected Approach and Trial Design  
JMML is an uncommon disease occurring exclusively in young children who frequently 
present with a high disease burden and severe clinical symptoms including massive 
hepatosplenomegaly, pulmonary infiltrates, fevers, infections, and rash. Median survival 
of 1-2 years is commonly reported without HCT.1,2 With few exceptions, long-term EFS 
has only been achieved following HCT, with the main cause of treatment failure being 
relapse.3,4 Though not intuitive, there is little evidence that reduction of disease burden 
using chemotherapy prior to the preparative transplant regimen has an effect on post-
transplant outcomes.4 These patients are at high risk for transplant morbidity and 
mortality with conventional myeloablative regimens, which have been dose escalated to 
maximum tolerability in an attempt to prevent the high relapse risk. Reported rates of 
TRM range from 13-33% depending upon stem cell source utilized.4-7 The median age at 
transplant is 2.5 years (0.3 – 15 years),4 and thus, these very young children are also at 
increased risk of late effects when maximally intensive conditioning regimens are 
utilized.  
 
Studies have demonstrated that immunotherapy-based interventions, either rapid 
withdrawal of immunosuppression8,9 or administration of donor lymphocyte infusion 
(DLI),10-12 can result in temporary and rarely long-lasting disappearance of 
residual/relapsing JMML cells following HCT, particularly when introduced in the 
setting of a low disease burden early in relapse. In addition, the use of ex vivo T-cell 
depletion has been shown to result in increased rates of relapse.13 Therefore, we 
hypothesize that the primary mechanism by which HCT results in long-term disease-free 
survival in some patients with JMML is due to the provision of a source of alloreactive 
graft-versus leukemia (GVL) activity. As such, we further hypothesize that the 
myeloablative preparative regimen prior to HCT plays little role per se in the long-term 
disease-control of patients with JMML, provided that it allows donor cell engraftment 
and adequate transfer of alloreactive cells. Thus, in this trial, we will test if a BU/FLU 
conditioning regimen proves to have less TRM compared to BU-CY-MEL with 
equivalent EFS. If BU/FLU is chosen as the preferred regimen to carry forward, we 
believe that the reduced toxicity of this regimen will facilitate future trials incorporating 
novel pre- or post-HCT targeted agents to optimize the outcomes for these children. 
During the course of this trial, we will capture the molecular genotypes of patients and 
we will also validate approaches to measuring minimal residual disease, which we 
hypothesize will provide more sensitive new metrics that will facilitate assessing 
response to therapeutic interventions for JMML.  
 
The study design is a modified randomized phase II trial. Patients will be assigned to BU-
FLU or BU-CY-MEL myeloablative conditioning regimen using a randomized block 
strategy within strata defined by donor type and PTPN11 mutation status. The outcome of 
the trial will be determined after all eligible patients have 18 months of follow-up. If the 
difference in the estimated 18-month EFS is more than 5%, then the regimen with the 
higher EFS will be declared the superior treatment. If the difference in EFS is less than 
5%, then the regimen with the lowest 100 day TRM risk will be declared the superior 
treatment. The randomized phase II approach is appropriate since both arms can be 
considered experimental and requires a much smaller sample size to complete than a 
phase III trial. As discussed in Section 9.2, the modification of the design described 
above exploits the a priori belief that relapse probabilities will not influence the choice of 
regimen, therefore increasing the probability that the trial will pick the better regimen. 
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2.2 
Trial Impact 
Because JMML is a relatively rare malignancy unique to children, advances in the care of 
these children can only be accomplished in the setting of a cooperative group trial. This 
was demonstrated in Europe by the European Working Group on Childhood 
Myelodysplastic Syndromes and Bone Marrow Transplant Working Group (EWOG-
MDS/EBMT). This consortium has used a maximally intense conditioning regimen, BU-
CY-MEL in a limited institution setting.4 However, it is unclear if the toxicity of the 
preparative regimen plays a significant role in the ultimate control of the disease, or if 
less toxic regimens will still accomplish the goal of providing a sustained production of 
alloreactive cells with less treatment-related morbidity and mortality. Since the current 
BU-CY-MEL conditioning regimen cannot be dose-escalated further, we hypothesize that 
superior results will be accomplished by maximizing the alloreactivity of the transplant 
process. In addition, we hypothesize that improving outcome will ultimately require 
therapies to block important cell growth pathways, either pre- or post-HCT with the use 
of targeted agents. Both of these goals may be significantly easier to accomplish if the 
patients have less morbidity and mortality from their HCT. By testing the approach of 
utilizing a fully myeloablative but lower toxicity conditioning regimen needed to achieve 
engraftment and thereby harnessing post-transplant GVL responses, we hope to 
implement a novel paradigm of how to approach HCT for JMML. Such a strategy is 
already being implemented in acute myeloid leukemia (AML); however the disease 
burden in JMML is generally higher at the start of the conditioning regimen and it 
remains untested if this will impede the use of lower doses of chemotherapy using 
BU-FLU.  
 
2.3 
HCT for JMML 
2.3.1 
“Standard of Care” HCT for JMML 
There is no currently agreed upon “standard-of-care” preparative regimen in use 
for patients with JMML. Aricò et al. reviewed the outcome of 91 children with 
JMML treated with HCT in 16 different reports, with an OS of 41%.1  The 
EWOG-MDS/EBMT reported the largest cohort to date (n = 100) in which the 
5-year EFS was 52% (95% CI, 42-62%) and utilized a myeloablative preparative 
regimen with high doses of BU-CY-MEL.4 Importantly, the European triple-
alkylator-based approach was done in a limited-institution fashion. Thus, there is 
the potential that this regimen might be infeasible in a group-wide context. 
Because of the limited-center design of that trial, we cannot estimate a 
meaningful toxicity baseline for this regimen when implemented across the 50+ 
COG-accredited centers. Cyclophosphamide significantly contributes to the 
development of sinusoidal obstruction syndrome post-HCT,14 and thus BU-FLU 
regimens have been noted to have a lower incidence of this toxicity.15 The 
EWOG-MDS/EBMT also reported the results of 42 children with JMML 
undergoing umbilical cord blood transplant (UCBT).6 The cumulative 5-year 
incidence of TRM, relapse, and EFS were 33%, 22%, and 45%, respectively. The 
results of AAML0122, which utilized a TBI-based conditioning regimen, were 
no better, with a 5-year EFS of 41% (95% CI, 30-52%) and a 2-year RI of 40% 
(95% CI, 29-51%) (Robert Gerbing, unpublished data). The lack of apparent 
benefit with a TBI-based conditioning, in conjunction with the increased risk of 
late effects due to TBI, led to the abandonment of a TBI-based approach for this 
trial. Further escalation of the conditioning regimen in JMML appears unlikely to 
produce significant improvement in EFS without unacceptable rates of TRM and 
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we hypothesize that novel strategies will ultimately be needed in order to reduce 
the high rates of relapse seen in these patients. 
 
The COG protocol AAML0122 was closed in October 2006 after the European 
approach was published in January 2005.4 A request to the CIBMTR in April 
2011 revealed that from 2005 to 2009, the majority of children in North America 
with JMML (n = 75) were transplanted with busulfan and cyclophosphamide 
(63%). The COG approach, based on TBI and cyclophosphamide, was utilized in 
16%, and the European approach of BU/CY/MEL was used in only 9% of cases, 
with TBI + Other (1%), BU + FLU +/- MEL (3%), MEL + Other (5%), and 
Unknown (4%) representing the remaining 12% of cases (Mary Horowitz, 
Scientific Director, personal communication). Thus, despite the fact that the 
European approach has been published since 2005, the majority of US transplant 
physicians have not adopted this approach for JMML, but rather utilized a 
preparative regimen considered standard for myeloid malignancies. 
 
Busulfan can be administered on a variety of schedules following HCT. 
Classically, busulfan has been given every 6 hours for 16 doses. More recently, 
many centers have moved to a once daily for 4 doses regimen,16 and this 
approach has been adopted for AAML1031. It has also been shown that every 
12 hour dosing is comparable to every 6 hour dosing.17 Since no head-to-head 
comparison has ever been performed, all three options will be acceptable 
practices on this trial. 
 
The approach to GVHD prophylaxis for this trial was guided by an attempt to 
establish a uniform regimen for all patients despite differing stem cell sources. 
Standard GVHD prophylaxis regimens combine a calcineurin inhibitor 
(tacrolimus or cyclosporine) with short-course methotrexate. Cyclosporine and 
tacrolimus are relatively comparable medications, though tacrolimus has been 
used more often in COG trials (e.g. ASCT0431 and AAML1031). However, 
traditionally, methotrexate use has been avoided in recipients of UCB grafts, due 
to concerns of prolonging count recovery. For many years corticosteroids were 
utilized instead, however, recently mycophenolate mofetil (MMF) has become 
the most common second agent. It is a non-competitive selective inhibitor of 
inosine monophosphate dehydrogenase, and lacks major mucosal, hepatic, or 
renal toxicity compared to other prophylactic aGVHD immunosuppressant drugs. 
Because of this, MMF was utilized in the COG / Clinical Trials Network shared 
trial 0501 studying single vs. double UCB HCT at a dose of 15 mg/kg every 
8 hours, a dose found to be important in children.18 It has also been demonstrated 
that MMF can be successfully utilized in patients receiving non-UCB 
transplants.19,20  
 
2.3.2 
Use of Cyclophosphamide vs. Fludarabine 
One study  demonstrated that 15.75 Gy of TBI is superior to 12 Gy of TBI for 
preventing relapse in patients with AML, albeit with an increased risk of TRM, 
so that EFS and OS were identical.21 Outside of this trial, we are unaware of any 
studies that demonstrate that – within the range of myeloablative preparative 
regimens, and with a comparable serotherapy regimen – there is any difference in 
relapse incidence (RI) for more toxic cyclophosphamide-based regimens versus 
less toxic fludarabine-based regimens. In fact, there are several retrospective 
studies that suggest that the RI for patients with AML/MDS is comparable 
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between the two regimens and potentially favors BU-FLU. Andersson et al. 
demonstrated a 3-year RI of 43% for the BU-CY group vs. 37% for the BU-FLU 
group,22 while Chae et al. demonstrated a 2-year RI of 40% for the BU-CY group 
vs. 23% for the BU-FLU group.15 In children with AML undergoing matched 
related donor HCT, unpublished CIBMTR data demonstrates a one year OS of 
73% in 40 children conditioned with myeloablative doses of BU-FLU, compared 
to 63% in 200 children conditioned with BU-CY (per AAML1031). 
 
Furthermore, as noted above, the introduction of the highly immunosuppressive 
fludarabine into myeloablative transplant preparative regimens for AML has 
demonstrated lower rates of TRM and equivalent relapse rates than 
cyclophosphamide-containing regimens.23,24 The Japanese Childhood MDS 
Study Group reported a pilot trial in 10 children with JMML that demonstrated 
that fludarabine could be safely substituted for cyclophosphamide.25 In addition, 
in a non-randomized cohort of 27 children with JMML in Japan, the 4-year EFS 
following HCT with a wide variety of conditioning regimens was similar for 
patients who received melphalan (67%; 95% CI, 42-85%) versus those who did 
not (58%; 95% CI 32-81%, P = .71).5 
 
Given that Locatelli et al. demonstrated that pre-transplant chemotherapy 
intensity has not impacted outcome post-transplant in JMML,4 it is reasonable to 
question whether highly toxic, dose-maximized myeloablative regimens, 
including BU-CY-MEL, are needed. However, many patients with JMML 
coming to transplant will be naïve to chemotherapy and thus may be at higher 
risk of graft failure if there is insufficient immunosuppression. Pilot studies in 
other patient groups with similar issues (chronic myeloid leukemia and non-
malignant conditions) have demonstrated that approaches including busulfan, 
fludarabine, and anti-thymocyte globulin (ATG)26 or alemtuzumab27,28-based 
regimens can successfully achieve engraftment in the majority of chemotherapy-
naïve patients (89-91%), with low rates of Day 100 TRM (0-9%).  
 
Taken together, since there is no data to suggest that the toxicity of the 
conditioning regimen in JMML is critical  for long-term disease control, we 
therefore propose to determine which fully myeloablative29 regimen (BU-FLU 
vs. BU-CY-MEL, plus/minus rabbit ATG) produces the least amount of TRM, 
while achieving stable donor cell engraftment and equivalent rates of relapse.  
 
2.3.3 
Serotherapy 
Serotherapy (ATG) will only be utilized for patients undergoing unrelated adult 
donor and umbilical cord blood transplants, since a prospective trial showed no 
difference in 2-year RI  in patients undergoing unrelated donor transplant who 
did (28.9%) or did not (23.6%) receive ATG (P = .55), but did decrease rates of 
both acute and chronic GVHD.30 This was confirmed in another recent 
prospective trial.31 Furthermore, Locatelli et al. reported no difference in 5-year 
RI in patients with JMML undergoing unrelated donor HCT who did (36%) or 
did not (33%) receive serotherapy (P = NS), though there was a trend towards 
less non-relapse mortality in the serotherapy group.4 This practice is in 
uniformity with the conditioning regimen on the COG AAML1031 trial, in which 
only unrelated donors receive ATG. This is also similar to the European 
approach in which the use of serotherapy was optional, but was primarily given 
only to unrelated donors.4 
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The timing of serotherapy will vary between patients receiving UCB grafts vs. 
non-UCB grafts. Traditional dosing of ATG with non-UCB grafts occurs on the 
days immediately prior to HCT. However, there is concern that this proximal 
dosing of ATG causes excessive in vivo T-cell depletion in recipients of UCBTs, 
which typically have at least a 10-fold lower T cell amounts than non-UCB stem 
cell sources. In turn, this excessive T cell depletion has been linked to a higher 
incidence of mixed chimerism or frank graft rejection. One approach to solving 
this quandary has been to administer the ATG at an earlier time period before 
UCBT, and preliminary data suggests that this can produce excellent results 
(Boelens JJ, et al. unpublished data). In a European study of pediatric patients 
with either malignancy (n = 40) or inborn errors (n = 63), Bu-Flu recipients from 
2009-11 were compared to a historical population of Bu-Cy+/-Mel recipients 
from 2005-09, with ATG administered to UCB recipients at Day -9 to -6. The 
majority of patients in the study (63%) received UCB, with a slight 
preponderance of UCB donor transplants in the Bu-Flu group (40/55 vs. 25/48; 
P = 0.051). Despite this imbalance, the incidence of graft failure was identical 
between the 2 groups (4%+/-3% in Bu-Flu vs. 5%+/-3% in Bu-Cy+/-Mel; p = 
0.89), and this effect was retained in the minimally-pretreated inborn errors 
group (0%+/-6% in Bu-Flu vs. 7%+/-6% in Bu-Cy+/-Mel; P = 0.34), a group 
more similar to minimally-pretreated JMML patients than a typically heavily-
pretreated patient with AML. We therefore have adopted this “early ATG” 
approach for patients getting UCB donor transplants on this trial in order to try to 
avoid excessive rates of graft failure.    
 
2.4 
Clinical Implications of Genetic Mutations in JMML 
2.4.1 
Diagnostic and Risk-Stratification Implications 
Establishing the diagnosis of JMML can be difficult for many clinicians and is 
currently made by meeting a combination of clinical and laboratory parameters, 
according to expert modifications of the World Health Organization (WHO) 
criteria (see Table 1 in Section 4.3.1).32,33 Taken together, 85% of patients with 
de novo JMML harbor a genetic lesion in molecules that encode proteins 
predicted to activate the Ras/MAPK pathway. Thirty-five percent have PTPN11 
mutations, 25% have NRAS/KRAS mutations, 10-15% have clinical NF1, and 
another 10-15% of patients have been recently described to harbor mutations in 
CBL.34-36 Animal models demonstrate that Nf1, Kras, Ptpn11, and Cbl mutant 
mice all develop fatal myeloproliferative disorders that model JMML.37-42 These 
genetic criteria have been recently added to the WHO criteria to reflect these 
advances in unraveling the molecular genetics of this disorder, with the caveat 
that a minority (15%) of patients will not have a lesion in one of the 
aforementioned genes, nor will they have clinical neurofibromatosis. Funding 
through the Biomarker, Imaging, and Quality of Life Studies Funding Program 
has been obtained for this protocol for the assessment of genetic alterations in 
PTPN11, NRAS, KRAS, and CBL through the UCSF CLIA/CAP approved 
laboratory environment. At the time of enrollment onto ASCT1221, samples will 
be submitted to UCSF for diagnostic genotyping, as well as research studies. 
Pyrosequencing with confirmation using an orthogonal platform (Sanger 
sequencing) will be performed on blood and/or bone marrow with further 
assessment of either buccal or fibroblasts to determine germline origin.   
 
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Considerable controversy remains about the prognostic significance of individual 
genetic lesions. The largest series of transplanted JMML patients did not 
document the presence of NRAS/KRAS, PTPN11, or CBL (first described in 
2009) mutations in their cohort.4 Only patients with clinical evidence of NF1 
were reported, and though it did not reach statistical significance, there was a 
difference in outcomes in those patients with clinical NF1 (EFS 36%, RI 50%) 
compared to those without (EFS 55%, RI 34%).4 A small Japanese study reported 
that a PTPN11 mutation was the only unfavorable factor predicting EFS after 
HCT (30% in the PTPN11 mutants vs. 69%; P = .018).43 Another recently 
published small Korean report also demonstrated a trend towards an unfavorable 
outcome for patients with PTPN11 mutations (59% vs. 86% for those without 
PTPN11, P = .22).44 A trend towards worse EFS for PTPN11 mutated patients, 
versus better EFS for RAS mutated patients was also noted in a European study 
(31% vs. 71%, respectively, vs. 44% for those with no mutation).45 Finally, it is 
known that rare patients have been reported to spontaneously improve or resolve 
their disease.  For example, 5 out of 6 patients with recently documented CBL 
lesions spontaneously resolved while only 1 died of progressive disease.46 
Spontaneous resolution is not uniform among CBL patients, as at least one of the 
patients who received HCT died from relapsed disease—and this patient’s 
affected relatives also died from JMML. In other case reports, rare patients with 
RAS mutations have spontaneously improved.47  
 
Given the genetic heterogeneity of JMML, we feel this is a unique opportunity to 
capture the “genetic demographics” of patients enrolled on this study. While 
randomization provides probabilistic balance of genetic risks for the outcomes 
across the treatment groups, there is good evidence that PTPN11 mutation status 
is strong predictor of relapse so block randomization by PTPN11 status (by donor 
type) will be used to assure absolute balance on this factor.  
 
2.4.2 
Transplant vs. No-Transplant for Patients with Germline Syndromes who 
Develop JMML 
Patients with germline mutations in PTPN11 (Noonan Syndrome) develop a 
myeloproliferative disorder that strongly resembles JMML, though nearly all 
cases appear to spontaneously regress over the first 18 months of life.48,49 As 
such, these patients will not be eligible for treatment on this protocol. Patients 
with germline mutations in CBL develop JMML were discussed in Section 2.4.1, 
and as noted, we believe that patients with a germline CBL mutation are eligible 
for HCT if their clinical course is aggressive. Likewise, we feel that patients with 
NRAS or KRAS mutations are also eligible for HCT. Patients with germline 
mutations in HRAS (Costello Syndrome) are at increased risk of certain 
malignancies, but JMML has not been reported, and as such, there is no a priori 
reason to think that this would spontaneously regress. Therefore, such a patient 
would be eligible for treatment on this protocol. 
 
2.4.3 
Correlative Biology Studies 
While very rare patients have been reported to resolve spontaneously,47 the data 
also support that the best survival outcomes are associated with swift transplant 
at an early age. Thus, clinicians are routinely faced with the clinical dilemma of 
watchful waiting versus moving to swift HCT, a paradoxical approach that is 
understandably anxiety provoking for patients and physicians. Improved risk 
stratification for patients with JMML is warranted, but as we have presented 
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earlier, genotype may or may not be associated with outcome. Recent reports 
indicate that gene expression or methylation profiles may predict which patients 
will do well with currently available therapies45,50 versus those patients who may 
require additional interventions to optimize cure. Furthermore, following these 
patients on therapy and diagnosing relapse has been problematic and has largely 
relied on measuring falling donor chimerism after HCT. We would thus propose 
a series of correlative biology studies to address these management issues for 
newly diagnosed JMML patients who are enrolled on this clinical trial.  
 
A recent publication by Bresolin et al. identified an AML-type gene expression 
signature in JMML patients that was associated with a high risk of relapse45 - 
such assays will be performed in RNA extracted from CD34+ cells selected from 
patients enrolled onto this protocol. The RNA will be extracted upon receipt, 
stored, and analyzed for only those patients who proceed to the HCT so that 
outcome can be assessed.  An additional report by Olk-Batz and colleagues 
indicated that hypermethylation of 4 genes was associated with an inferior 
outcome for patients and raised the possibility of therapeutic interventions using 
DNA methyl-transferase inhibitors. Through additional collaborations, we will be 
assessing genome-wide methylation profiling50 to determine the prognostic 
significance of such signatures and associated novel therapies.  
 
Given the fact that JMML is a difficult disease to follow during therapy, we 
would also propose validation of an allele-specific PCR-based reaction for 
subsets of patients with mutations in PTPN11 and RAS. These measurements will 
be done on diagnostic specimens (prior to receiving significant cytoreductive or 
differentiating therapy), pre-HCT (this will control for individualized pre-HCT 
therapy) and post-HCT at defined time-points in bulk mononuclear cells and in 
sorted cell subsets. We have previously designed and optimized a fluorescently 
based allele-specific PCR reaction for 12 Ras pathway mutations and published 
these results with EWOG-MDS colleagues to validate the assay.51 In the analysis, 
we were able to demonstrate the a) sensitivity and specificity of the assay in 
selectively detecting these point mutations, b) detection of molecular relapse well 
ahead of clinical relapse and frequently ahead of falling donor chimerism 
measurements (currently the standard to detect relapse after HCT), and 
c) concordance of simultaneously obtained peripheral blood and bone marrow 
measurements. As a result of this successful collaboration, we will also be 
conducting these studies for our EWOG-MDS colleagues who will be treating 
their patients on their clinical trial. In this manner, we will be able to assess 
different clinical interventions (EWOG-MDS versus COG) using measurements 
obtained at the same timepoints post-HCT and analyzed in the same lab. Given 
the very recent developments of using sensitive 454 sequencing methodologies to 
detect JMML-specific mutations, we will also compare our allele-specific PCR 
assays against 454 sequencing results. While we predict that the pyrosequencing 
strategies will not be as sensitive as the allele-specific PCR, we know that they 
will be applicable to more patients and thus may have broader use in this 
population. 
 
Because JMML exhibits considerable cellular heterogeneity, it has been difficult 
to elucidate the biologic features of cells that contribute to cancer phenotypes in 
vivo, and of precursor populations that might carry genetic lesions predisposing 
cells to an oncogenic fate. Phosphoflow cytometry is capable of measuring 
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surface antigens to identify subsets of cells with simultaneous assessments of 
intracellular protein phosphorylation to provide a dynamic view of perturbed 
signaling cascades.52 JMML is an ideal disease for assessing the biochemical 
signature of aberrant signaling induced through GM-CSF stimulation and 
presumably via the Ras pathway. We previously identified a reproducible 
phosphoflow signature after exposing JMML samples to low doses of GM-CSF 
that closely mimics the readout of a 2-3 week CFU-GM assay, and will capitalize 
on this technology to design additional studies focused on identifying new drugs 
that could be employed in this disease.53  
 
Finally, recent data indicates that a subset of JMML patients exhibit autoimmune 
features at diagnosis, manifested by high IgG levels and positivity on direct 
antibody tests using Coombs methods. Some patients may further exhibit features 
consistent with autoimmune lymphoproliferative disorders.54 We recognize that 
this protocol will facilitate the capture of these variables in this patient cohort and 
wish to measure IgG and Coombs at the time of enrollment in order to determine 
their effect on patient outcomes. We anticipate that the laboratory assays that we 
and others have developed will allow us to identify patients at risk for poor 
outcomes and ultimately hope to identify novel agents that can be used to treat 
patients with this aggressive myeloid malignancy.  
 
 
 
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3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
3.1 
Study Enrollment 
 
3.1.1 
Patient Registration 
Prior to enrollment on this study, patients must be assigned a COG patient ID 
number. This number is obtained via the COG Registry system once 
authorization for the release of protected health information (PHI) has been 
obtained. The COG patient ID number is used to identify the patient in all future 
interactions with COG. If you have problems with the registration, please refer to 
the online help.   
 
In order for an institution to maintain COG membership requirements, every newly 
diagnosed patient needs to be offered participation in ACCRN07, Protocol for the 
Enrollment on the Official COG Registry, The Childhood Cancer Research Network 
(CCRN).  
 
A Biopathology Center (BPC) number will be assigned as part of the registration 
process. Each patient will be assigned only one BPC number per COG Patient ID. 
For additional information about the labeling of specimens please refer to the 
Pathology and/or Biology Guidelines in this protocol.  
 
3.1.2 
IRB Approval 
Local IRB/REB approval of this study must be obtained by a site prior to 
enrolling patients. Sites must submit IRB/REB approvals to the NCI’s Cancer 
Trials Support Unit (CTSU) Regulatory Office and allow 3 business days for 
processing. The submission must include a fax coversheet (or optional CTSU 
IRB Transmittal Sheet) and the IRB approval document(s). The CTSU IRB 
Certification Form may be submitted in lieu of the signed IRB approval letter. 
All CTSU forms can be located on the CTSU web page (https://www.ctsu.org). 
Any other regulatory documents needed for access to the study enrollment 
screens will be listed for the study on the CTSU Member’s Website under the 
RSS Tab.  
 
IRB/REB approval documents may be faxed (1-215-569-0206), 
E-mailed (CTSURegulatory@ctsu.coccg.org) or mailed to the CTSU Regulatory 
office.  
 
When a site has a pending patient enrollment within the next 24 hours, this is 
considered a “Time of Need” registration. For Time of Need registrations, in 
addition to marking your submissions as ‘URGENT’ and faxing the regulatory 
documents, call the CTSU Regulatory Helpdesk at: 1-866-651-CTSU. For 
general (non-regulatory) questions call the CTSU General Helpdesk at: 1-888-
823-5923. 
 
3.1.3 
Study Enrollment 
Patients may be enrolled on the study once all eligibility requirements for the 
study have been met. Study enrollment is accomplished by going to the CTSU 
OPEN 
(Oncology 
Patient 
Enrollment 
Network) 
https://www.ctsu.org/OPEN_SYSTEM/. For questions, please contact the CTSU 
OPEN helpdesk at ctsucontact@westat.com or call 1-888-823-5923.  
 
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3.1.4 
Timing 
Patients must be enrolled before treatment begins. Guidelines for patient 
management pre-HCT are provided in Appendix IV and may be used per 
physician discretion.  
 
All clinical and laboratory studies to determine eligibility must be performed 
within 7 days prior to enrollment unless otherwise indicated in the eligibility 
section below. 
 
Protocol therapy (HCT conditioning) must begin within 1 year of enrollment.  
 
 
3.1.4.1 Cytogenetics 
Specimens for cytogenetic analysis are required and must be obtained 
prior to therapy initiation (other than hydroxyurea, 6-mercaptopurine, 
cis-retinoic 
acid, 
or 
low-dose 
intravenous 
cytarabine 
[≤ 200 mg/m2/cumulative dose]). Use of a COG-approved institutional 
cytogenetics laboratory is required (see Section 13.2 for specimen 
submission guidelines). A listing of these laboratories may be found on 
the COG website as well as methods of attaining COG approval for local 
cytogenetics laboratories. Results of cytogenetics are not required to be 
completed prior to enrollment; however cytogenetic reports and 
associated materials must be sent for central review within 2 weeks of 
enrollment 
(see 
Section 13.2 
for 
central 
review 
submission 
requirements). 
 
3.1.4.2 Genotyping  
Submission of diagnostic specimens for genotyping analysis at the UCSF 
CLIA/CAP-approved laboratory is required at the time of enrollment. 
See Section 15 for specimen submission and shipping requirements. 
 
3.1.4.3 Pathology 
Submission of central pathology review specimens is required. Specimens 
used for institutional diagnosis must have been obtained prior to therapy 
initiation (other than hydroxyurea, 6-mercaptopurine, cis-retinoic acid, or 
low-dose intravenous cytarabine [≤ 200 mg/m2/cumulative dose]). 
Specimens must be sent for central review within 2 weeks of enrollment. 
See Section 14 for specimen submission guidelines. 
 
3.1.4.4  Initiation of HCT Donor Search 
At the time of diagnosis, the HCT donor search is to be initiated 
according to institutional procedures. Stem cell donor requirements are 
described in Section 4.2. 
 
3.1.4.5 Obtaining Informed Consent 
The consent process for ASCT1221 is divided into 2 stages. The Part 1 
Consent is provided to allow patient enrollment and submission of clinical 
genotyping specimens and correlative biology to UCSF (see Section 15). 
When the requirements to proceed to HCT (as described in Section 4.3) 
are met, the Part 2 Consent is provided to allow HCT conditioning 
regimen randomization. 
 
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3.1.5 
Randomization 
Randomization will occur via OPEN when: 
(1) The diagnosis of JMML according to Chan et al33 is confirmed (see 
Section 4.3.1) 
(2) The HCT donor source has been identified (see Section 4.2) 
(3) The patient has met all requirements to proceed to HCT (see Section 4.3).  
 
Patients will be randomly assigned to either myeloablative allogeneic HCT with a 
busulfan-fludarabine conditioning regimen or a busulfan-cyclophosphamide-
melphalan conditioning regimen. HCT donor source and PTPN11 mutational status 
must be entered into the OPEN system for patient stratification. Stratification groups 
are defined as:  
(1)  Matched related donor, PTPN11 mutation present 
(2)  Matched related donor, PTPN11 mutation absent 
(3)  Closely matched unrelated donor, PTPN11 mutation present 
(4)  Closely matched unrelated donor, PTPN11 mutation absent 
(5)  Umbilical cord blood, PTPN11 mutation present 
(6)  Umbilical cord blood, PTPN11 mutation absent 
 
HCT conditioning regimen must begin within 5 days of randomization.  
 
3.2 
Patient Eligibility Criteria 
Important note: The eligibility criteria listed below are interpreted literally and 
cannot be waived (per COG policy posted 5/11/01). All clinical and laboratory data 
required for determining eligibility of a patient enrolled on this trial must be 
available in the patient's medical/research record which will serve as the source 
document for verification at the time of audit.  
 
All clinical and laboratory studies to determine eligibility must be performed within 
7 days prior to enrollment unless otherwise indicated. See Section 4.3 and Section 7.1 
for required studies to be obtained prior to starting protocol therapy. 
 
INCLUSION CRITERIA 
 
3.2.1 
Age 
 
 
 
Patients must be ≥ 3 months and ≤ 18 years at the time of study enrollment. 
 
3.2.2 
Diagnosis  
a.  Patients must have a strong clinical suspicion of JMML, based on a modified 
 Category 1 of the revised diagnostic criteria, as described by Chan et al.33 
 Specifically, eligible patients must have all of the following:  
• 
Splenomegaly* 
• 
Absolute monocyte count (AMC) > 1000/µL 
• 
Blasts in PB/BM < 20% 
 
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*For the 7-10% of patients without splenomegaly, the diagnostic entry criteria 
must include all other features described above and at least 2 of the following 
criteria (based on a modified Category 3 of Chan et al)33:  
o Circulating myeloid precursors  
o WBC > 10,000/μL 
o Increased fetal hemoglobin (HgbF) for age 
o GM-CSF hypersensitivity 
OR 
b. Patients must have been previously diagnosed with JMML (per Section 4.3.1).  
 
 
3.2.3 
Prior Therapy 
Patients must be previously untreated with HCT.  
 
Please see Section 4.5 for the concomitant therapy restrictions for patients during 
treatment. 
 
 
 
EXCLUSION CRITERIA 
 
3.2.4 
Mutation Status of PTPN11 (Germline) 
Patients with a known germline mutation of PTPN11 (Noonan’s Syndrome) are 
not eligible.  
 
3.2.5 
Neurofibromatosis Type 1 (NF1) Status 
Patients with a known history of NF1 (Neurofibromatosis Type 1) and either  
 
a history of a tumor of the central nervous system (astrocytoma or optic 
glioma), or  
 
a malignant peripheral nerve sheath tumor with a complete remission of 
< 1 year are not eligible.  
 
3.2.6 
HIV Status 
 
HIV positive patients are not eligible. 
 
REGULATORY 
 
3.2.7 
All patients and/or their parents or legal guardians must sign a written informed 
consent. 
 
3.2.8 
All institutional, FDA, and NCI requirements for human studies must be met. 
 
 
 
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4.0 
TREATMENT PROGRAM 
Timing of protocol therapy administration, response assessment studies, and surgical interventions are 
based on schedules derived from the experimental design or on established standards of care. Minor 
unavoidable departures (up to 72 hours) from protocol directed therapy and/or disease evaluations (and up 
to 1 week for surgery) for valid clinical, patient and family logistical, or facility, procedure and/or 
anesthesia scheduling issues are acceptable per COG administrative Policy 5.14 (except where explicitly 
prohibited within the protocol). 
 
All transplants performed on COG trials must occur at FACT-accredited SCT programs. 
 
 
4.1 
Overview of Treatment Plan 
Patients on ASCT1221 will receive an allogeneic stem cell transplant utilizing one of 
2 conditioning regimens:  
 
- 
Arm A: a Busulfan, Cyclophosphamide and Melphalan (BU-CY-MEL) 
regimen, 
or  
- 
Arm B: a Busulfan and Fludarabine (BU-FLU) regimen.  
 
Patients will be randomized to Arm A or Arm B and stratified equally by stem cell source 
and PTPN11 mutational status as described in Section 3.1.5. Randomization will occur 
when: 
(1) The diagnosis of JMML according to Chan et al33 is confirmed (see Section 4.3.1) 
(2) The HCT donor source has been identified (see Section 4.2) 
(3) The patient has met all requirements to proceed to HCT (see Section 4.3).  
 
Note: If the patient was enrolled at a non-COG transplant center, the patient must be 
transferred to a COG-approved transplant center prior to randomization.   
 
For the duration of the study, specimens will be collected and sent to the UCSF CLIA-
certified laboratory and Biopathology Center (BPC; if patient has consented to specimen 
banking) (see Section 7 and 15 for details).  
 
See Appendix IV for pre-transplant therapy recommendations.  
 
“Protocol Therapy” for the purposes of this study encompasses the time from the 
initiation of the conditioning regimen through the end of the GVHD prophylaxis 
(Day +98) for matched family member donors. Day +180 for unrelated and cord blood 
donors). 
 
4.2 
Hematopoietic Stem Cell Matching and Cell Dose Requirements 
 
4.2.1 
Bone Marrow and Peripheral Blood Stem Cell (PBSC) Requirements 
Allele level typing of HLA A, B, C, and DRB1 is required for unrelated or 
related bone marrow and PBSC donors but not matched siblings (i.e. matched 
parent, partially mismatched sibling, etc). Allele level typing of DQB1 is 
recommended.  
 
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4.2.2 
Matched Related Donor and Cord Blood Unit Requirements 
Serological matching of Class I antigens is acceptable, but allele level typing is 
required for DRB1 for matched related donors and cord blood units. Allele level 
typing of Class I antigens and DQB1 is recommended.  
 
4.2.3 
Hierarchy of Stem Cell Choices 
The hierarchy of stem cell choices is as follows: 
 
1. Preferred: Genotypically HLA matched relatives. Syngeneic (identical twin) 
donors are not allowed.   
 
2. If number 1 above not available, choices a and b below are equally 
acceptable:  
a) Unrelated or “other” related (non-genotypically matched) donor: 
High resolution typing of 8 alleles required. Consistent with NMDP 
guidelines, minimum standard is a 5/6 match (antigen level at class I (HLA 
A, B) and allele level class II (DRB1)), but 7/8 or 8/8 including matching at 
HLA C at the allele level is desired. Haploidentical relatives (> 1 HLA 
mismatch) are not allowed. 
 
b) Cord blood: Serologic Class I (A and B) and allele level Class II 
(DRB1) typing is required. Minimum requirement is a 4/6 antigen match 
(HLA A, B, and DRB1). Minimum pre-thaw cell dose of 5 x 107 nucleated 
cells/kg recipient body weight is required. 
 
4.3 
Requirements to Proceed to HCT 
The following are requirements for a patient to proceed to randomization and assignment 
of HCT preparative regimen. HCT preparative regimen must be initiated within 5 days of 
randomization. If a patient fails to meet the requirements described below within one year 
of enrollment, the patient will be Off Protocol Therapy and ASCT1221 Follow up 
Evaluations will be collected.  
 
4.3.1 
Confirmation of JMML Diagnosis 
Patients must have confirmed JMML, ascertained upon completion of genotype 
testing at UCSF and cytogenetic analysis, based on the revised diagnostic 
criteria, as described by Chan et al33 (see table below).  
 
Table 1: JMML Diagnostic Criteria (Chan et al33)  
Category 1 
Category 2 
Category 3 
All of the following: 
• Splenomegaly* 
• Absolute monocyte count 
(AMC) > 1000/µL 
• Blasts in PB/BM < 20% 
• Absence of the t(9;22) 
BCR/ABL fusion gene 
 
At least 1 of the following 
• Somatic mutation in RAS or 
PTPN11  
• Clinical diagnosis of NF1 or 
NF1 gene mutation 
• Homozygous mutation in CBL  
• Monosomy 7 
At least 2 of the following 
• Circulating myeloid precursors 
• WBC > 10,000/µL 
• Increased fetal hemoglobin 
(Hgb F) for age 
• Clonal cytogenetic abnormality 
excluding monosomy 7 
• GM-CSF hypersensitivity 
The diagnosis of JMML is made if a patient meets all of the Category 1 criteria and one of the Category 2 
criteria without needing to meet the Category 3 criteria. If there are no Category 2 criteria met, then the 
Category 3 criteria must be met.  
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* For the 7-10% of patients without splenomegaly, the diagnostic criteria must include all other features 
in Category 1 AND one of the parameters in Category 2 OR no features in Category 2 but two features in 
Category 3. 
 
 
4.3.2 
Prior Therapy Restrictions 
To proceed with HCT randomization and preparative regimen, patients must 
have fully recovered from the acute toxic effects of all prior chemotherapy, 
immunotherapy, or radiotherapy. 
a. Myelosuppressive Chemotherapy and Targeted Agents: Must not have 
received myelosuppressive chemotherapy or targeted agents within 2 weeks of 
the start of HCT conditioning regimen (4 weeks if prior nitrosourea). 
b. Biologic (anti-neoplastic agent): Must not have received a biologic agent 
within 7 days of the start of HCT conditioning regimen. 
c. Radiation therapy (RT): Must not have received local palliative RT (small 
port) within 2 weeks of the start of HCT conditioning regimen; ≥ 6 months 
must have elapsed if prior craniospinal RT or if ≥ 50% radiation of pelvis; 
≥ 6 weeks must have elapsed if other substantial BM radiation. 
 
Please see Section 4.5 for the concomitant therapy restrictions for patients during 
treatment. 
 
4.3.3 
Organ Function Requirements 
To proceed with HCT randomization and preparative regimen, patients must 
meet the following organ function requirements. See Section 7.1 for timing 
requirements for organ function studies.  
 
4.3.3.1 Adequate Renal Function Defined As: 
- 
Creatinine clearance or radioisotope GFR ≥ 70 mL/min/1.73 m2 or 
- 
A serum creatinine based on age/gender as follows: 
 
Age 
 
Maximum Serum 
Creatinine (mg/dL) 
 
Male 
Female 
3 months to < 6 months 
0.4 
0.4 
6 months to < 1 year 
0.5 
0.5 
1 to < 2 years 
0.6 
0.6 
2 to < 6 years 
0.8 
0.8 
6 to < 10 years 
1 
1 
10 to < 13 years 
1.2 
1.2 
13 to < 16 years 
1.5 
1.4 
≥ 16 years 
1.7 
1.4 
The threshold creatinine values in this Table were derived from the Schwartz 
formula for estimating GFR55 utilizing child length and stature data 
published by the CDC. 
 
4.3.3.2 Adequate Liver Function Defined As: 
- Total bilirubin < 2.5 mg/dL unless the increase in bilirubin is 
attributable to Gilbert’s Syndrome, and 
- SGOT (AST) or SGPT (ALT) < 5 x upper limit of normal (ULN) for 
age. 
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4.3.3.3 Adequate Cardiac Function Defined As: 
- 
Shortening fraction of ≥ 27% by echocardiogram, or 
- 
Ejection fraction of ≥ 50% by radionuclide angiogram. 
 
4.3.3.4 Adequate Pulmonary Function Defined As: 
- 
FEV1, FVC, and DLCO corrected for Hgb ≥ 60% by pulmonary 
function tests (PFTs). 
- 
For children who are unable to cooperate for PFTs, the criteria are: 
no evidence of dyspnea at rest, no exercise intolerance, and not 
requiring supplemental oxygen therapy.  
 
4.3.3.5  Central Nervous System Function Defined As: 
- 
Patients with seizure disorder may proceed to HCT if on non-enzyme 
inducing anticonvulsants (see Appendix IIIB) and well controlled. 
- 
CNS toxicity ≤ Grade 2. 
 
4.3.4 
Performance Level 
Patients must have a Lansky or Karnofsky performance status score ≥ 60. Use 
Karnofsky for patients > 16 years of age and Lansky for patients ≤ 16 years of 
age.  
See https://members.childrensoncologygroup.org/prot/reference_materials.asp 
under Standard Sections for Protocols. 
 
Performance status must be assessed within the 14 days prior to the start of the 
HCT conditioning regimen.  
 
4.3.5 
Infection Status  
Patients with HIV or uncontrolled fungal, bacterial or viral infections are not 
permitted to proceed to HCT conditioning regimen. 
-  
Patients must not have begun treatment for or have any documentation of the 
presence of a new fungal infection within 30 days of the start of HCT 
conditioning regimen. 
 
4.3.6 
Pregnancy and Breast Feeding 
 
4.3.6.1  Female patients who are pregnant are not permitted to proceed to HCT 
since fetal toxicities and teratogenic effects have been noted for several 
of the study drugs. 
 
4.3.6.2  Lactating females are not permitted to proceed to HCT unless they have 
agreed not to breastfeed their infants. 
 
4.3.6.3  Female patients of childbearing potential are not permitted to proceed to 
HCT unless a negative pregnancy test result has been obtained. 
 
4.3.6.4  Sexually active patients of reproductive potential are not permitted to 
proceed to HCT unless they have agreed to use an effective contraceptive 
method for the duration of their study participation. 
 
 
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4.4 
Hematopoietic Stem Cell Infusion Guidelines 
Stem cells for allogeneic HCT are infused no sooner than 24 hours after the last dose of 
chemotherapy (not including ATG), according to the institution’s standard operating 
procedure. 
 
4.4.1 
Donor Bone Marrow or Peripheral Blood Infusion Guidelines 
For related donors, bone marrow (or peripheral blood stem cells, if donor is 
unwilling to provide bone marrow) will be collected locally according to the 
institution’s standard operating procedure. 
 
For unrelated donors, bone marrow (or peripheral blood stem cells, if donor is 
unwilling to provide bone marrow) will be collected and transported from the 
NMDP Donor Center (or equivalent country-specific national donor program) 
according to standard operating procedure. A minimum of 2 x 108 nucleated bone 
marrow cells/kg (2 x 106 CD34+ cells/kg if peripheral blood stem cells) of the 
recipients ideal weight must be collected, but 4 x 108 nucleated bone marrow 
cells/kg (4 x 106 CD34+ cells/kg if peripheral blood stem cells) is desirable. 
There will be no manipulation of the bone marrow (or peripheral blood stem 
cells) to deplete T cells prior to marrow infusion. Bone marrow should be 
processed to deplete red blood cells if there is a major ABO mismatch (e.g., 
O type in recipient and A type in donor) to avoid an acute hemolytic transfusion 
reaction, per institutional standard operating practices. Bone marrow (or 
peripheral blood stem cells) will be infused according to the institution’s standard 
operating procedure. Donor bone marrow (or peripheral blood stem cell) grafts 
should be assessed for quantity of total nucleated cells, CD3, and CD34 cells. 
 
4.4.2 
Donor Umbilical Cord Blood Infusion Guidelines 
 
4.4.2.1 UCB Unit Thaw 
The cord blood should be thawed, diluted with or without wash per validated 
institutional or supplying cord blood bank procedures with the exception that 
bedside thawing and direct infusion is not allowed. Bedside thaws are not 
recommended because of the inability to rescue the product if there is loss of 
integrity of the UCB bag on thaw at the bedside and because of the instability of 
the cells in 10% DMSO post thaw. 
 
All transplant centers/cellular therapy laboratories must be familiar with thawing 
of cord blood units. They must have validated procedures and maintain 
competency in the thaw process. The cord blood unit must be thawed in a 
qualified laboratory by trained personnel. Generally the cryopreserved unit is 
removed from the protective cassette, placed in a ziplock bag and thawed rapidly 
in a 37°C waterbath. The ziplock bag allows for recovery of cells if the 
cryopreservation bag cracks or leaks during the thawing process, a rare but 
possible event. Once the contents of the bag reach a slushy consistency, the cells 
can be diluted in dextran/albumin, a hypertonic solution that buffers against the 
intracellular hypertonicity created by DMSO. Cell suspensions can subsequently 
be washed to remove DMSO, free hemoglobin and other cellular debris allowing 
for resuspension in a volume appropriate for the size of the patient to be 
transplanted.  
 
 
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4.4.2.2 UCB Infusion 
Under no circumstances is the cord blood to be irradiated. There must be no in-
line leukocyte filter used with product infusion nor any medications or fluids 
infused in the same line with the cord blood (i.e., no piggyback fluids). Vital 
signs should be monitored before beginning the infusion and periodically during 
administration. Infusion should begin within 2 hours of washing. The infusion 
should take no longer than 1 hour. Pre-medications and hydration prior to cord 
blood 
infusion 
will 
be 
administered 
per 
institutional 
procedure. 
Diphenhydramine, epinephrine, and hydrocortisone should be available at the 
bedside for emergency use if infusion reactions occur. Oxygen with nasal prongs 
for standby use should be present in the room.  
 
4.5 
Concomitant Therapy and Study-Specific Supportive Care Guidelines 
The following supportive care guidelines are provided for institutional consideration. 
Investigator discretion should be used, and individual considerations made for specific 
patient situations and institutional practices. Please see the COG Supportive Care 
Guidelines at: 
https://members.childrensoncologygroup.org/prot/reference_materials.asp under Standard 
Sections for Protocols.  
 
4.5.1 
Growth Factors 
Filgrastim (G-CSF) is not generally recommended for BM or PBSC sources, but 
is usually administered following UCB transplantation. If a center opts to 
administer filgrastim, 5 mcg/kg daily is recommended (with a start time 
according to institutional practice). In patients with evidence of Monosomy 7 by 
cytogenetics, center should consider avoiding filgrastim (by analogy to patients 
with AML).56  
 
4.5.2 
Infectious Prophylaxis 
Co-enrollment on COG Cancer Control and Supportive Care clinical trials 
evaluating anti-infectious prophylaxis strategies is permitted.  
 
Trimethoprim-sulfamethoxazole (TMP-SMX) and Intravenous Immunoglobulin: 
see COG Supportive Care Guidelines. 
 
Fluconazole: ≥ 6 mg/kg dose QD IV or PO for at least the first 28 days 
(prophylaxis against molds is acceptable, according to institutional standards). 
 
Acyclovir: Standard doses for up to one year after transplant.  Acyclovir or 
valacyclovir will be used in patients who have been exposed to HSV, VZV or 
CMV or whose donors have been infected by CMV, as per institutional 
guidelines. 
 
Weekly CMV antigenemia or PCR screening from Day +14 through at least 
Day +100 and pre-emptive treatment with ganciclovir. 
 
Also see the Parenteral Chemotherapy Administration Guidelines (CAG) on the 
COG website at:  
https://members.childrensoncologygroup.org/_files/disc/Pharmacy/ChemoAdmin
Guidelines.pdf for special precautions and suggestions for patient monitoring 
during the infusion. As applicable, see the CAG for suggestions on hydration, or 
hydrate according to institutional guidelines. 
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4.5.3 
Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease 
When availale centers may use defibrotide for either prophylaxis or treatment per 
institutional guidelines 
 
 
4.6 
Matched Family Donor HCT for Patients Assigned to Arm A (BU-CY-MEL)  
The condition regimen described below is for patients with a matched family donor 
randomized to receive the Arm A conditioning regimen. Patients with a matched family 
donor randomized to Arm A will receive an 8-day HCT conditioning regimen that 
consists of Busulfan, Cyclophosphamide and Melphalan (BU-CY-MEL). Tacrolimus and 
mycophenolate mofetil will be administered for GVHD prophylaxis.  
 
Doses should be adjusted for patients > 125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1. 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose  
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
Patients > 4 years: 3.2 mg/kg/dose  
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16)  
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 6 or 12 hours) is permitted per institutional practice.  
 
Q6 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 6 hours  
Dose: Patients < 10 kg: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
Patients ≥ 10 kg but ≤ 4 years old: 1 mg/kg/dose q 6 hours (x 4 doses) 
Patients > 4 years: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
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Days: -7 through -5, every 6 hours (x 12 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 6 hour 
dosing should target an AUC of 900 to 1500 (micromole/liter)*minute per dose. 
 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
 
Cyclophosphamide (CPM): IV over 60 minutes 
Days: -4 through -3 (2 doses) 
Dose: 60 mg/kg/dose, once daily 
 
Mesna: IV over 15 minutes per dose or continuous infusion  
Days: -4 through -3  
Dose: 60 mg/kg/DAY, given as per the recommendations below: 
 
Mesna can be administered in 5 divided doses by short infusion over 15 to 30 minutes. 
The initial bolus dose of mesna may be administered 15 minutes before or at the same 
time as the cyclophosphamide dose; subsequent doses are given 3, 6, 9, and 12 hours 
after the start of cyclophosphamide. This total daily dose of mesna can also be 
administered as IV continuous infusion. The continuous infusion should be started 
15-30 minutes before or at the same time as cyclophosphamide and finished no sooner 
than 12 hours after the end of the cyclophosphamide infusion. 
 
Examples: 
Short infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily mesna 
dose is 1,000 mg; 200 mg of mesna will be given 15 minutes before or with the 
cyclophosphamide dose (Hour 0) and 4 boluses of 200 mg each will be given at Hours 3, 
6, 9 and 12. 
 
Continuous infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily 
mesna dose is 1,000 mg; the 1,000 mg mesna continuous infusion will start 
15-30 minutes before or at the same time as the cyclophosphamide and be completed no 
sooner than 12 hours after the end of the cyclophosphamide infusion. If the 
cyclophosphamide is administered over 2 hours and mesna is started 30 minutes before 
the cyclophosphamide infusion, the total mesna infusion will last at least 14 hours and 
30 minutes. 
 
Melphalan (MEL): IV over 15-30 minutes (at a rate not to exceed 10 mg/minute) 
Days: -1 (1 dose) 
Dose:  < 10 kg: 4.67 mg/kg/dose 
≥ 10 kg: 140 mg/m2/dose 
 
Note: Melphalan infusion must be completed within 60 minutes of preparation.  
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
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IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
 
Tacrolimus levels should be maintained between 5-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO formulations may be used when clinically 
appropriate per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will 
begin no later than Day +60 and will be complete by Day +98. For patients with > Grade 
I acute GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day). The oral 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 5-12 ng/mL. Dose adjustments are 
based on clinical judgment of the treating physician after considering clinical toxicity, 
serum levels, GVHD, concomitant drug use and the rate of rise or decline of the serum 
level. Refer to Section 5.2 for guidance on dose adjustment based on serum trough levels.  
 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +30. For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
 
The therapy delivery maps (TDMs) for this HCT treatment plan are on the next (one) 
page. 
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4.6.1 
HCT Regimen: Matched Family Donor -Arm A 
Treatment for the HCT preparative regimen lasts 8 days. After the HCT, the 
GVHD prophylaxis treatment lasts about 3+ months. 
                    
 _____________________   ____________ 
      Patient COG ID number                    DOB   
Extensive treatment details are in Section 4.1-4.6. This Therapy Delivery Map is on 1 page.   
 
 
 **HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
 
 
Ht __________cm           Wt ___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure prophylaxis 
BUS 
____mg 
CPM 
____mg 
MESNA 
___mg 
 MEL  
___mg 
TAC 
___mg 
MMF  
__mg __mg __mg 
Studies 
Comments 
 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg __mg __mg __mg 
_____mg 
 
 
 
 
 
(a, b, c, d, g) 1 
 
 
 
-7 
__mg __mg __mg __mg 
_____mg 
 
 
 
 
 
 
 
 
 
-6 
__mg __ mg __mg __mg 
_____mg 
 
 
 
 
 
 
 
 
 
-5 
__mg __mg __mg __mg 
_____mg 
 
 
 
 
 
 
 
 
 
-4 
__mg __mg __mg __mg 
 
_____mg 
___mg 
 
 
 
 
 
 
 
-3 
 
 
_____mg 
___mg 
 
 
 
 
 
 
 
-2 
 
 
 
 
 
 
 
 
 
 
 
-1 
 
 
 
 
_____mg 
 ____mg 
 
 
 
 
 
0 
 
 
 
 
 
 
 
 
 
 
 
+1 
 
 
 
 
 
__mg ___mg __mg 
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
+30 
 
 
 
 
 
b, c, d, e, f, g 
 
 
 
... 
 
 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
 
 
c, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
 
 
b, c, d, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+98 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
 
 
c, e, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+180 
 
See Section 7.2 for evaluations after Day +180. 
 
c, e, g 
 
1Prior to HCT conditioning.             SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE.
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis: 
_____________ 
Indicate agent, i.e., 
lorazepam, 
levetiracetam 
 
IV or PO  
 
_____________________ 
(indicate dose) 
Preferred regimen: lorazepam 
(0.02-0.05 mg/kg/dose every 
6 hours) given 30 minutes prior to 
each busulfan dose. Continue until 
≥ 24 hours after last busulfan dose. 
-8 to -4 
 
 
a. History (incl. 
cardiac), PE w/ 
VS,  NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, 
differential, 
HgbF, spleen & 
liver measure., 
concomitant tx 
d. Cytogenetics & 
FISH (not req. 
for all pts, see 
Section 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: 
PB & BM (BM 
only if clinically 
indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS 
REQUIRED FOR 
GOOD PATIENT 
CARE   
Busulfan  
(BUS) 
 
IV over 3 hours  For 
once daily dosing 
only.  
See Section 4.6 for 
alternative dosing 
schedules. 
Age & wt-based (1st dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8 
Submit BUS PK samples 
following 1st dose (Section 16).  
See Section 4.6 for alternative 
dosing schedules. 
Note: Refrain from using 
polycarbonate syringes or filter 
needles during busulfan preparation. 
Dose adjusted per targeted AUC, 
once daily (3 doses) 
-7 to -5 
Cyclophosphamide 
(CPM) 
IV over 60 minutes 
60 mg/kg/dose, once daily  
(2 doses) 
-4 to -3 
 
MESNA 
IV over 15 minutes per 
dose or continuous 
infusion 
60 mg/kg/DAY  
-4 to -3 
See Section 4.6 for administration 
guidelines. 
Melphalan 
(MEL) 
IV  over 15-30 
minutes at a rate not to 
exceed 10 mg/min 
Weight-based dosing: 
< 10 kg: 4.67 mg/kg/dose 
≥ 10 kg: 140 mg/m2/dose  
(1 dose) 
-1 
Melphalan infusion must be 
completed within 60 minutes of 
preparation.  
 
Tacrolimus 
(TAC) 
IV or PO 
See Section 4.6 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to 
+98 
Maintain 5-12 ng/mL. If ≤ Grade I 
aGVHD, begin taper by Day +60.  
Discontinue by Day +98. See 
Section 4.6 for additional 
monitoring.  
Mycophenolate 
Mofetil (MMF) 
IV over 2 hours.  
Convert to oral when 
tolerated. 
15 mg/kg/dose, every 8 hours   
+1 to 
+30 
If ≤ Grade I aGVHD, discontinue 
by Day +30.  
 
06/05/14 
 
33 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
4.7 
Unrelated Donor HCT for Patients Assigned to Arm A (BU-CY-MEL+ rATG)  
The condition regimen described below is for patients with an unrelated donor 
randomized to receive the Arm A conditioning regimen. Patients with an unrelated donor 
and randomized to Arm A will receive an 8-day HCT conditioning regimen that consists 
of Busulfan, Cyclophosphamide and Melphalan (BU-CY-MEL), as well as rabbit Anti-
Thymocyte Globulin (rATG). Tacrolimus and mycophenolate mofetil will be 
administered for GVHD prophylaxis.  
 
Doses should be adjusted for patients >125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose 
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
Patients > 4 years: 3.2 mg/kg/dose 
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16) 
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 6 or 12 hours) is permitted per institutional practice.  
 
Q6 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 6 hours  
Dose: Patients < 10 kg: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
Patients ≥ 10 kg but ≤ 4 years old: 1 mg/kg/dose q 6 hours (x 4 doses) 
Patients > 4 years: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
Days: -7 through -5, every 6 hours (x 12 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 6 hour 
dosing should target an AUC of 900 to 1500 (micromole/liter)*minute per dose. 
 
06/05/14 
 
34 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
 
Cyclophosphamide (CPM): IV over 60 minutes 
Days: -4 through -3 (2 doses) 
Dose: 60 mg/kg/dose, once daily 
 
Mesna: IV over 15 minutes per dose or continuous infusion  
Days: -4 through -3  
Dose: 60 mg/kg/DAY, given as per the recommendations below: 
 
Mesna can be administered in 5 divided doses by short infusion over 15 to 30 minutes. 
The initial bolus dose of mesna may be administered 15 minutes before or at the same 
time as the cyclophosphamide dose; subsequent doses are given 3, 6, 9, and 12 hours 
after the start of cyclophosphamide. This total daily dose of mesna can also be 
administered as IV continuous infusion. The continuous infusion should be started 
15-30 minutes before or at the same time as cyclophosphamide and finished no sooner 
than 12 hours after the end of the cyclophosphamide infusion. 
 
Examples: 
Short infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily mesna 
dose is 1,000 mg; 200 mg of mesna will be given 15 minutes before or with the 
cyclophosphamide dose (Hour 0) and 4 boluses of 200 mg each will be given at Hours 3, 
6, 9 and 12. 
 
Continuous infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily 
mesna dose is 1,000 mg; the 1,000 mg mesna continuous infusion will start 
15-30 minutes before or at the same time as the cyclophosphamide and be completed no 
sooner than 12 hours after the end of the cyclophosphamide infusion. If the 
cyclophosphamide is administered over 2 hours and mesna is started 30 minutes before 
the cyclophosphamide infusion, the total mesna infusion will last at least 14 hours and 
30 minutes. 
 
Rabbit Anti-thymocyte globulin (rATG): IV over 4-6* hours 
Days: -4 through -1 (4 doses) 
Dose: 2 mg/kg/dose, once daily 
 
*The first dose should be infused over at least 6 hours through a high-flow vein. 
Subsequent doses should be administered over at least 4 hours. Administer through an in-
line 0.22 micron filter. 
 
Premedications should be considered prior to rATG per institutional protocols. This may 
include diphenhydramine 1 mg/kg/dose (max 50 mg), or a comparable anti-histamine; 
acetaminophen 
10-15 mg/kg/dose 
(max 
1000 
mg); 
and 
methylprednisolone 
1 mg/kg/dose. Premedication may be repeated or increased as needed to control allergic 
reactions, chills, or fever. Patients who are unable to tolerate the rabbit product may 
receive equine ATG 25 mg/kg/day x 4 days with similar premedication. 
06/05/14 
 
35 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
Melphalan (MEL): IV over 15-30 minutes at a rate not to exceed 10 mg/minute 
Days: -1 (1 dose) 
Dose:   < 10 kg: 4.67 mg/kg/dose 
≥ 10 kg: 140 mg/m2/dose  
 
Note: Melphalan infusion must be completed within 60 minutes of preparation.  
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
 
Tacrolimus levels should be maintained between 8-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO formulations may be used when clinically 
appropriate per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will 
begin no later than Day +100 and will be complete by Day +180. For patients with 
> Grade I acute GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day). The oral 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 8-12 ng/mL for mismatched or 
unrelated donors. Dose adjustments are based on clinical judgment of the treating 
physician after considering clinical toxicity, serum levels, GVHD, concomitant drug use 
and the rate of rise or decline of the serum level. Refer to Section 5.2 for guidance on 
dose adjustment based on serum trough levels.   
 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
06/05/14 
 
36 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +45.  For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
See Section 5.0 for Dose Modifications based on Toxicities. The therapy delivery maps 
(TDMs) for this HCT treatment plan are on the next (one) page. 
06/05/14 
 
37 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.7.1  HCT Regimen - Unrelated Donor -Arm A  
Treatment for the HCT preparative regimen lasts 8 days. After the HCT, the GVHD 
prophylaxis treatment lasts about 6 months. 
                    
 _____________________   ____________ 
        Patient COG ID number                         DOB   
Extensive treatment details are in Section 4.1-4.5 and 4.7. This Therapy Delivery Map is on 1 page.   
 
 **HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
Ht__________cm 
          Wt___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure 
prophylaxis 
BUS 
____mg 
CPM 
____mg 
MESNA 
____mg 
rATG 
___mg  
 MEL  
___mg 
TAC 
___mg 
MMF  
__mg __mg __mg 
Studies 
Comments 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg __mg __mg __mg 
____mg 
 
 
 
 
 
 
(a, b, c, d, g) 1 
 
 
 
-7 
__mg __mg __mg __mg 
____mg 
 
 
 
 
 
 
 
 
 
 
-6 
__mg __mg __mg __mg 
____mg 
 
 
 
 
 
 
 
 
 
 
-5 
__mg __mg __mg __mg 
____mg 
 
 
 
 
 
 
 
 
 
 
-4 
__mg __mg __mg __mg 
 
____mg 
____mg 
_____mg 
 
 
 
 
 
 
 
-3 
 
 
____mg 
____mg 
_____mg 
 
 
 
 
 
 
 
-2 
 
 
 
 
_____mg 
 
 
 
 
 
 
 
-1 
 
 
 
 
_____mg 
____mg 
____mg 
 
 
 
 
 
0 
 
 
 
 
 
 
 
 
 
 
 
 
+1 
 
 
 
 
 
 
__mg __mg __mg  
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+30 
 
 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+45 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
 
 
 
c, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
 
 
 
b, c, d, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
 
 
c, e, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+180 
 
 
 
 
 
 
 
c, e, g 
 
 
 
 
See Section 7.2 for evaluations after Day +180. 
 
 
1Prior to HCT conditioning. SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE.
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis:  
_____________ 
Indicate agent, i.e., 
lorazepam, 
levetiracetam 
IV or PO 
_____________________ 
(indicate dose) 
Preferred regimen: lorazepam 
(0.02-0.05 mg/kg/dose every 6 
hours) given 30 minutes prior to 
each busulfan dose. Continue until 
≥ 24 hours after last busulfan dose. 
-8 to -4 
 
a. History (incl. 
cardiac), PE w/ 
VS,  NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, 
differential, 
HgbF, spleen & 
liver measure., 
concomitant tx 
d. Cytogenetics & 
FISH (not req. 
for all pts, see 
Sect. 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: 
PB & BM (BM 
only if clinically 
indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS 
REQUIRED FOR 
GOOD PATIENT 
CARE 
Busulfan  
(BUS) 
 
IV over 3 hours  
For once daily dosing 
only. See Section 4.7 for 
alternative dosing 
schedules. 
Age & wt-based (1st dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8 
 
 
 
Submit BUS PK samples following 1st dose 
(Section 16).  See Section 4.7  for alternative 
dosing schedules. 
Note: Refrain from using polycarbonate 
syringes or filter needles during busulfan 
preparation. 
Dose adjusted per targeted AUC, 
once daily (3 doses) 
-7 to -5 
 
Cyclophosphamide 
(CPM) 
IV over 60 minutes 
60 mg/kg/dose, once daily 
(2 doses) 
-4 to -3 
 
MESNA 
IV over 15 minutes per 
dose or continuous 
infusion 
60 mg/kg/DAY  
-4 to -3 
See Section 4.7 for administration guidelines. 
Rabbit ATG  
(rATG) 
IV over 4-6* hours  
2 mg/kg/dose, once daily  
(4 doses) 
-4 to -1 
*1st dose: infuse over ≥ 6 hours through a high-
flow vein. Subsequent doses: infuse over 
≥ 4 hours. Administer through an in-line 0.22 
micron filter. Premedications should be 
considered. Equine ATG may be substitutued if 
rabbit product is not tolerated. See Section 4.7 
for details. 
Melphalan 
(MEL) 
IV  over 15-30 minutes 
at a rate not to exceed 
10 mg/min 
Weight based dosing: 
< 10 kg: 4.67 mg/kg/dose 
≥ 10 kg: 140 mg/m2/dose  
(1 dose) 
-1 
Note: Melphalan infusion must be completed 
within 60 minutes of preparation.  
Tacrolimus 
(TAC) 
IV or PO 
See Section 4.7. 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to 
+180 
Maintain 8-12 ng/mL. If ≤ Grade I aGVHD 
begin taper by Day +100. Discontinue by Day 
+180. See Section 4.7 for additional monitoring. 
Mycophenolate 
mofetil (MMF) 
IV over 2 hours.  Convert 
to oral when tolerated. 
15 mg/kg/dose, every 8 hours  
+1 to 
+45 
If ≤ Grade I aGVHD, discontinue by Day +45.   
 
06/05/14 
 
38 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.8 
UCB Donor HCT for Patients Assigned to Arm A (BU-CY-MEL+ rATG)  
The condition regimen described below is for patients with an UCB donor randomized to 
receive the Arm A conditioning regimen. Patients with an UCB donor and randomized to 
Arm A will receive an 8-day HCT conditioning regimen that consists of Busulfan, 
Cyclophosphamide and Melphalan (BU-CY-MEL), as well as rabbit Anti-Thymocyte 
Globulin (rATG). Tacrolimus and mycophenolate mofetil will be administered for 
GVHD prophylaxis.  
 
Doses should be adjusted for patients >125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose 
 
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
 
Patients > 4 years: 3.2 mg/kg/dose 
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16) 
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 12 hours) is permitted per institutional practice. Every 6 hour 
dosing is NOT permitted with UCB donors due to conflicts with ATG timing. 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
 
 
06/05/14 
 
39 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Cyclophosphamide (CPM): IV over 60 minutes 
Days: -4 through -3 (2 doses) 
Dose: 60 mg/kg/dose, once daily 
 
Mesna: IV over 15 minutes per dose or continuous infusion  
Days: -4 through -3  
Dose: 60 mg/kg/DAY, given as per the recommendations below: 
 
Mesna can be administered in 5 divided doses by short infusion over 15 to 30 minutes. 
The initial bolus dose of mesna may be administered 15 minutes before or at the same 
time as the cyclophosphamide dose; subsequent doses are given 3, 6, 9, and 12 hours 
after the start of cyclophosphamide. This total daily dose of mesna can also be 
administered as IV continuous infusion. The continuous infusion should be started 
15-30 minutes before or at the same time as cyclophosphamide and finished no sooner 
than 12 hours after the end of the cyclophosphamide infusion. 
 
Examples: 
Short infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily mesna 
dose is 1,000 mg; 200 mg of mesna will be given 15 minutes before or with the 
cyclophosphamide dose (Hour 0) and 4 boluses of 200 mg each will be given at Hours 3, 
6, 9 and 12. 
 
Continuous infusion: if the cyclophosphamide dose is 1,000 mg, then the total daily 
mesna dose is 1,000 mg; the 1,000 mg mesna continuous infusion will start 
15-30 minutes before or at the same time as the cyclophosphamide and be completed no 
sooner than 12 hours after the end of the cyclophosphamide infusion. If the 
cyclophosphamide is administered over 2 hours and mesna is started 30 minutes before 
the cyclophosphamide infusion, the total mesna infusion will last at least 14 hours and 
30 minutes. 
 
Rabbit Anti-thymocyte globulin (rATG): IV over 4-6* hours 
Days: -8 through -5 (4 doses) 
Dose: 2 mg/kg/dose, once daily 
 
*The first dose should be infused over at least 6 hours through a high-flow vein. 
Subsequent doses should be administered over at least 4 hours. Administer through an 
in-line 0.22 micron filter. 
 
Premedications should be considered prior to rATG per institutional protocols. This may 
include diphenhydramine 1 mg/kg/dose (max 50 mg), or a comparable anti-histamine; 
acetaminophen 
10-15 mg/kg/dose 
(max 
1000 
mg); 
and 
methylprednisolone 
1 mg/kg/dose. Premedication may be repeated or increased as needed to control allergic 
reactions, chills, or fever. Patients who are unable to tolerate the rabbit product may 
receive equine ATG 25 mg/kg/day x 4 days with similar premedication. 
 
Melphalan (MEL): IV over 15-30 minutes at a rate not to exceed 10 mg/minute 
Days: -1 (1 dose) 
Dose:   < 10 kg: 4.67 mg/kg/dose 
 
≥ 10 kg: 140 mg/m2/dose  
 
Note: Melphalan infusion must be completed within 60 minutes of preparation.  
 
 
06/05/14 
 
40 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
Tacrolimus levels should be maintained between 8-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO fomulatios may be used when clinically appropriate 
per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will begin no later 
than Day +100 and will be complete by Day +180. For patients with > Grade I acute 
GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day). The oral 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 8-12 ng/mL for mismatched or 
unrelated donors. Dose adjustments are based on clinical judgment of the treating 
physician after considering clinical toxicity, serum levels, GVHD, concomitant drug use 
and the rate of rise or decline of the serum level. Refer to Section 5.2 for guidance on 
dose adjustment based on serum trough levels.   
 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +45.  For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
See Section 5.0 for Dose Modifications based on Toxicities. The therapy delivery maps 
(TDMs) for this HCT treatment plan are on the next (one) page. 
06/05/14 
 
41 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.8.1  HCT Regimen - UCB Donor -Arm A  
Treatment for the HCT preparative regimen lasts 8 days. After the HCT, the GVHD 
prophylaxis treatment lasts about 6 months. 
                    
 _____________________   ____________ 
        Patient COG ID number                         DOB   
Extensive treatment details are in Section 4.1-4.5 and 4.8. This Therapy Delivery Map is on 1 page.   
 
 **HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
Ht__________cm 
          Wt___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure 
prophylaxis 
BUS 
____mg 
CPM 
____mg 
MESNA 
____mg 
rATG 
___mg  
 MEL  
___mg 
TAC 
___mg 
MMF  
__mg __mg __mg 
Studies 
Comments 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg __mg __mg __mg  
____mg 
 
 
_____mg 
 
 
 
(a, b, c, d, g) 1 
 
 
 
-7 
__mg __mg __mg __mg 
____mg 
 
 
_____mg 
 
 
 
 
 
 
 
-6 
__mg __mg __mg __mg 
____mg 
 
 
_____mg 
 
 
 
 
 
 
 
-5 
__mg __mg __mg __mg 
____mg 
 
 
_____mg 
 
 
 
 
 
 
 
-4 
__mg __mg __mg __mg 
 
____mg 
____mg 
 
 
 
 
 
 
 
 
-3 
 
 
____mg 
____mg 
 
 
 
 
 
 
 
 
-2 
 
 
 
 
 
 
 
 
 
 
 
 
-1 
 
 
 
 
 
____mg 
____mg 
 
 
 
 
 
0 
 
 
 
 
 
 
 
 
 
 
 
 
+1 
 
 
 
 
 
 
__mg __mg __mg  
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+30 
 
 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
+45 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
 
 
 
c, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
 
 
 
b, c, d, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
 
 
c, e, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
+180 
 
 
 
 
 
 
 
c, e, g 
 
 
 
 
See Section 7.2 for evaluations after Day +180. 
 
 
1Prior to HCT conditioning. SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE.
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis:  
_____________ 
Indicate agent, i.e., 
lorazepam, 
levetiracetam 
IV or PO 
_____________________ 
(indicate dose) 
Preferred regimen: lorazepam 
(0.02-0.05 mg/kg/dose every 
6 hours) given 30 minutes prior to 
each busulfan dose. Continue until 
≥ 24 hours after last busulfan dose. 
-8 to -4 
 
a. History (incl. 
cardiac), PE w/ 
VS,  NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, 
differential, 
HgbF, spleen & 
liver measure., 
concomitant tx 
d. Cytogenetics & 
FISH (not req. 
for all pts, see 
Sect. 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: 
PB & BM (BM 
only if clinically 
indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS 
REQUIRED FOR 
GOOD PATIENT 
CARE 
Busulfan  
(BUS) 
 
IV over 3 hours  
For once daily dosing 
only. See Section 4.8 
for alternative dosing 
schedules. 
Age & wt-based (1st dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8 
 
 
 
Submit BUS PK samples following 1st dose 
(Section 16).  See Section 4.8 for alternative 
dosing schedule. 
Note: Refrain from using polycarbonate 
syringes or filter needles during busulfan 
preparation. 
Dose adjusted per targeted AUC, 
once daily (3 doses) 
-7 to -5 
 
Cyclophosphamide 
(CPM) 
IV over 60 minutes 
60 mg/kg/dose, once daily (2 doses) 
-4 to -3 
 
MESNA 
IV over 15 minutes per 
dose or continuous 
infusion 
60 mg/kg/DAY  
-4 to -3 
See Section 4.8 for administration guidelines. 
Rabbit ATG  
(rATG) 
IV over 4-6* hours  
2 mg/kg/dose, once daily  
(4 doses) 
-8 to -5 
*1st dose: infuse over ≥ 6 hours through a high-
flow vein. Subsequent doses: infuse over 
≥ 4 hours. Administer through an in-line 0.22 
micron filter. Premedications should be 
considered Equine ATG may be substitutued if 
rabbit product is not tolerated. See Section 4.8 
for details. 
Melphalan 
(MEL) 
IV  over 15-30 minutes 
at a rate not to exceed 
10 mg/min 
Weight based dosing: 
< 10 kg: 4.67 mg/kg/dose 
≥ 10 kg: 140 mg/m2/dose  
(1 dose) 
-1 
Note: Melphalan infusion must be completed 
within 60 minutes of preparation.  
Tacrolimus 
(TAC) 
IV or PO 
See Section 4.8. 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to 
+180 
Maintain 8-12 ng/mL. If ≤ Grade I aGVHD 
begin taper by Day +100. Discontinue by Day 
+180. See Section 4.8 for additional monitoring. 
Mycophenolate 
mofetil (MMF) 
IV over 2 hours.  Convert 
to oral when tolerated. 
15 mg/kg/dose, every 8 hours  
+1 to 
+45 
If ≤ Grade I aGVHD, discontinue by Day +45.   
 
06/05/14 
 
42 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.9 
Matched Family Donor HCT for Patients Assigned to Arm B (BU-FLU)  
The condition regimen described below is for patients with a matched family donor 
randomized to receive the Arm B conditioning regimen. Patients randomized to Arm B 
will receive an 8-day HCT conditioning regimen that consists of Busulfan and 
Fludarabine (BU-FLU). Tacrolimus and mycophenolate mofetil will be administered for 
GVHD prophylaxis.  
 
Doses should be adjusted for patients > 125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1. 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose 
 
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
 
Patients > 4 years: 3.2 mg/kg/dose 
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16) 
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 6 or 12 hours) is permitted per institutional practice.  
 
Q6 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 6 hours  
Dose: Patients < 10 kg: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 1 mg/kg/dose q 6 hours (x 4 doses) 
 
Patients > 4 years: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
Days: -7 through -5, every 6 hours (x 12 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 6 hour 
dosing should target an AUC of 900 to 1500 (micromole/liter)*minute per dose. 
 
 
06/05/14 
 
43 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
  
Fludarabine (FLUD): IV over 30-60 minutes  
Days: -5 through -2 (4 doses) 
Dose:   < 10 kg:  1.33 mg/kg/dose once daily 
 
≥ 10 kg:  40 mg/m2/dose once daily 
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
 
Tacrolimus levels should be maintained between 5-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO formulations may be used when clinically 
appropriate per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will 
begin no later than Day +60 and will be complete by Day +98. For patients with > Grade 
I acute GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day). The oral 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 5-12 ng/mL. Dose adjustments are 
based on clinical judgment of the treating physician after considering clinical toxicity, 
serum levels, GVHD, concomitant drug use and the rate of rise or decline of the serum 
level. Refer to Section 5.2 for guidance on dose adjustment based on serum trough levels.  
 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
06/05/14 
 
44 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +30. For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
The therapy delivery maps (TDMs) for this HCT treatment plan are on the next (one) 
page. 
06/05/14 
 
45 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.9.1   HCT Regimen: Matched Family Donor -Arm B  
Treatment for the HCT preparative regimen lasts 8 days. 
After the HCT, the GVHD prophylaxis treatment lasts about 3+ months. 
                    
 _____________________   ____________ 
           Patient COG ID number                    DOB   
Extensive treatment details are in Section 4.1-4.5 and 4.9. This Therapy Delivery Map is on 1 page.   
 
 
 **HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
 
 
Ht__________cm 
          Wt___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure prophylaxis 
____mg 
BUS 
____mg 
FLUD 
___mg  
TAC 
___mg 
MMF 
___mg ___mg ___mg 
Studies 
Comments 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
(a, b, c, d, g) 1 
 
 
 
-7 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
 
 
 
 
-6 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
 
 
 
 
-5 
__mg  __mg  __mg  __mg 
____mg 
_____mg 
 
 
 
 
 
 
-4 
__mg  __mg  __mg  __mg 
 
_____mg 
 
 
 
 
 
 
-3 
 
 
_____mg 
 
 
 
 
 
 
-2 
 
 
_____mg 
 
 
 
 
 
 
-1 
 
 
 
____mg 
 
 
 
 
 
0 
 
 
 
____mg 
 
 
 
 
 
+1 
 
 
 
____mg 
___mg ___mg ___mg 
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
+30 
 
 
 
b, c, d, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
c, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
b, c, d, e, f, g 
 
 
 
… 
 
 
 
 
 
 
 
 
+98 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
c, e, g 
 
 
 
… 
 
 
 
 
 
 
 
 
 
+180 
See Section 7.2 for evaluations after Day +180 
c, e, g 
 
1Prior to HCT conditioning.   SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE.
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis 
___________ 
Indicate agent, 
i.e., lorazepam, 
levetiracetam  
 
IV or PO 
____________________ 
(indicate dose) 
Preferred regimen: lorazepam 
(0.02-0.05 mg/kg/dose every 
6 hours) given 30 minutes prior to 
each busulfan dose. Continue until 
≥ 24 hours after last busulfan dose. 
-8 to -4 
 
a. History (incl. 
cardiac), PE w/ VS , 
NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, differential, 
HgbF, spleen & liver 
measure., 
concomitant tx 
d. Cytogenetics & FISH 
(not req. for all pts, 
see Sect. 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: PB 
& BM (BM only if 
clinically indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS REQUIRED 
FOR GOOD PATIENT 
CARE 
  
Busulfan  
(BUS) 
 
 IV over 3 hours  
For once daily 
dosing only. See 
Section 4.9 for 
alternative dosing 
schedules. 
Age & wt-based (1 dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8 
Submit BUS PK samples 
following 1st dose (Section 16).  
See Section 4.9 for alternative 
dosing schedules. 
Note: Refrain from using 
polycarbonate syringes or filter 
needles during busulfan 
preparation. 
Dose adjusted per targeted AUC, 
once daily (3 doses) 
-7 to -5 
Fludarabine  
(FLUD) 
IV over 
30-60 minutes 
Weight-based dosing: 
< 10 kg:  1.33 mg/kg/dose 
≥ 10 kg:  40 mg/m2/dose   
(4 doses) 
-5 to -2 
 
Tacrolimus 
(TAC) 
IV or PO  
See Section 4.9. 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to +98 
Maintain levels between 
5-12 ng/mL. If ≤ Grade I acute 
GVHD, taper will begin no later 
than Day +60 and will be complete 
by Day +98.  See Section 4.9 for 
additional monitoring. 
Mycophenolate 
mofetil (MMF) 
IV over 2 hours. 
Convert to oral 
when tolerated. 
15 mg/kg/dose, every 8 hours   
+1 to +30 
If ≤ Grade I acute GVHD, MMF 
will be discontinued by Day +30. 
 
06/05/14 
 
46 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
4.10 
Unrelated Donor HCT for Patients Assigned to Arm B (BU-FLU + rATG)  
The condition regimen described below is for patients with an unrelated donor 
randomized to receive the Arm B conditioning regimen. Patients randomized to Arm B 
will receive an 8-day HCT conditioning regimen that consists of Busulfan and 
Fludarabine (BU-FLU), as well as rabbit Anti-Thymocyte Globulin (rATG). Tacrolimus 
and mycophenolate mofetil will be administered for GVHD prophylaxis.  
 
Doses should be adjusted for patients > 125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1. 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose 
 
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
 
Patients > 4 years: 3.2 mg/kg/dose 
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16) 
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 6 or 12 hours) is permitted per institutional practice.  
 
Q6 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 6 hours  
Dose: Patients < 10 kg: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 1 mg/kg/dose q 6 hours (x 4 doses) 
 
Patients > 4 years: 0.8 mg/kg/dose q 6 hours (x 4 doses) 
 
Days: -7 through -5, every 6 hours (x 12 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 6 hour 
dosing should target an AUC of 900 to 1500 (micromole/liter)*minute per dose. 
 
 
06/05/14 
 
47 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
 
Fludarabine (FLUD): IV over 30-60 minutes 
Days: -5 through -2 (4 doses) 
Dose:  < 10 kg:  1.33 mg/kg/dose once daily 
 
≥ 10 kg:  40 mg/m2/dose once daily 
 
 
Rabbit Anti-thymocyte globulin (rATG): IV over 4-6* hours 
Days: -4 through -1 (4 doses) 
Dose: 2 mg/kg/dose 
 
*The first dose should be infused over at least 6 hours through a high-flow vein. 
Subsequent doses should be administered over at least 4 hours. Administer through an 
in-line 0.22 micron filter. 
 
Premedications should be considered prior to rATG per institutional protocols. This may 
include diphenhydramine 1 mg/kg/dose (max 50 mg), or a comparable anti-histamine; 
acetaminophen 
10-15 mg/kg/dose 
(max 
1000 
mg); 
and 
methylprednisolone 
1 mg/kg/dose. Premedication may be repeated or increased as needed to control allergic 
reactions, chills, or fever. Patients who are unable to tolerate the rabbit product may 
receive equine ATG 25 mg/kg/day x 4 days with similar premedication. 
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
 
Tacrolimus levels should be maintained between 8-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO formulations may be used when clinically 
appropriate per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will 
begin no later than Day +100 and will be complete by Day +180. For patients with 
> Grade I acute GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day).  The oral 
06/05/14 
 
48 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 8-12 ng/mL for mismatched or 
unrelated donors. Dose adjustments are based on clinical judgment of the treating 
physician after considering clinical toxicity, serum levels, GVHD, concomitant drug use 
and the rate of rise or decline of the serum level. Refer to Section 5.2 for guidance on 
dose adjustment based on serum trough levels.  
 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +45.  For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
The therapy delivery maps (TDMs) for this HCT treatment plan are on the next (one) 
page. 
06/05/14 
 
49 


 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
4.10.1   HCT Regimen: Unrelated Donor -Arm B  
Treatment for the HCT preparative regimen lasts 8 days. 
After the HCT, the GVHD prophylaxis treatment lasts about 6 months. 
                    
 _____________________   ____________ 
           Patient COG ID number                    DOB   
Extensive treatment details are in Section 4.1-4.5 and 4.10. This Therapy Delivery Map is on 1 page.   
 
 
**HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
 
 
Ht__________cm 
          Wt___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure prophylaxis 
____mg 
BUS 
____mg 
FLUD 
___mg 
rATG 
___mg 
TAC 
___mg 
MMF  
__mg __mg __mg 
Studies 
Comments 
 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
 
(a, b, c, d, g) 1  
 
 
 
-7 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
 
 
 
 
 
-6 
__mg  __mg  __mg  __mg 
____mg 
 
 
 
 
 
 
 
 
-5 
__mg  __mg  __mg  __mg 
____mg 
___mg 
 
 
 
 
 
 
 
-4 
__mg  __mg  __mg  __mg 
 
___mg 
___mg 
 
 
 
 
 
 
-3 
 
 
___mg 
___mg 
 
 
 
 
 
 
-2 
 
 
___mg 
___mg 
 
 
 
 
 
 
-1 
 
 
 
___mg 
____mg 
 
 
 
 
 
0 
 
 
 
 
____mg 
 
 
 
 
 
+1 
 
 
 
 
____mg 
____mg ____mg ____mg 
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
+30 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
+45 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
 
c, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
c, e, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+180 
 
 
 
 
 
c, e, g 
 
 
 
 
See Section 7.2 for evaluations after Day +180. 
 
 
1Prior to HCT conditioning.      SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE. 
 
 
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis:  
_____________ 
Indicate agent, i.e., 
lorazepam, 
levetiracetam 
IV or PO 
_____________________ 
(indicate dose) 
Preferred regimen: lorazepam (0.02-
0.05 mg/kg/dose every 6 hours) given 
30 minutes prior to each busulfan 
dose. Continue until ≥ 24 hours after 
last busulfan dose. 
-8 to -4 
 
a. History (incl. 
cardiac), PE w/ 
VS, NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, 
differential, 
HgbF, spleen & 
liver measure., 
concomitant tx 
d. Cytogenetics & 
FISH (not req. 
for all pts, see 
Sect. 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: 
PB & BM (BM 
only if clinically 
indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS 
REQUIRED FOR 
GOOD PATIENT 
CARE 
Busulfan  
(BUS) 
For once daily 
dosing only.  
 
IV over 3 hours  For 
once daily dosing 
only.  See 
Section 4.10 for 
alternative dosing 
schedules. 
Age & wt-based (1 dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8  
Submit BUS PK samples following 1st 
dose (Section 16).   
See Section 4.10 for alternative dosing 
schedules.  
Note: Refrain from using 
polycarbonate syringes or filter needles 
during busulfan preparation. 
Dose adjusted per targeted AUC, once 
daily (3 doses) 
-7 to -5 
 
Fludarabine  
(FLUD) 
IV over 
30-60 minutes 
Weight-based dosing: 
≥ 10 kg:  40 mg/m2/dose  
< 10 kg:  1.33 mg/kg/dose 
(4 doses) 
-5 to -2 
 
Rabbit ATG  
(rATG) 
IV over 4-6* hours 
2 mg/kg/day  
(4 doses) 
 
-4 to -1 
*1st dose: infuse over ≥ 6 hours through 
a high-flow vein. Subsequent doses: 
infuse over ≥ 4 hours. Administer 
through an in-line 0.22 micron filter. 
Premedications should be considered. 
Equine ATG may be substitutued if 
rabbit product is not tolerated. See 
Section 4.10 for details. 
Tacrolimus 
(TAC) 
IVor PO 
See Section 4.10. 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to +180 
Maintain levels between 8-12 ng/mL. If 
≤ Grade I aGVHD, begin taper by Day 
+100. Discontinue by Day +180. See 
Section 4.10 for additional monitoring. 
Mycophenolate 
mofetil (MMF) 
IV over 2 hours. 
Convert to oral when 
tolerated. 
15 mg/kg/dose, every 8 hours  
+1 to +45 
If ≤ Grade I aGVHD, discontinue by 
Day +45.   
 
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50 


 
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ASCT1221 
4.11 
UCB Donor HCT for Patients Assigned to Arm B (BU-FLU + rATG)  
The condition regimen described below is for patients with an UCB donor randomized to 
receive the Arm B conditioning regimen. Patients randomized to Arm B will receive an 
8-day HCT conditioning regimen that consists of Busulfan and Fludarabine (BU-FLU), 
as well as rabbit Anti-Thymocyte Globulin (rATG). Tacrolimus and mycophenolate 
mofetil will be administered for GVHD prophylaxis.  
 
Doses should be adjusted for patients > 125% of Ideal Body Weight (IBW). Adjusted 
weight = 1.25*IBW. See Section 5.1. 
 
Anti-seizure Prophylaxis:   
Days: -8 through at least Day -4 
The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose PO or IV every 6 hours) 
given 30 minutes prior to each busulfan dose and then continuing for at least 24 hours 
after last busulfan dose. An alternative regimen is levetiracetam (10 mg/kg/dose PO or IV 
BID, max dose 1000 mg) beginning 12 hours prior to busulfan and continuing for at least 
24 hours after last busulfan dose.  
 
Busulfan (BUS): IV over 3 hours 
Day: -8, once daily (1 dose) 
Dose:  Patients < 10 kg: 3.2 mg/kg/dose 
 
Patients ≥ 10 kg but ≤ 4 years old: 4 mg/kg/dose  
 
Patients > 4 years: 3.2 mg/kg/dose 
 
Days: -7 through -5, once daily (3 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16) 
 
First-dose busulfan pharmacokinetic studies will be performed in all patients. Daily 
dosing will be adjusted to achieve an overall exposure target area under the curve (AUC) 
of 3600-6000 (micromole/liter)*minute. Refer to Section 16 for additional information.  
 
Acetaminophen should be held for 72 hours before and during busulfan administration, 
but may be given per individual institutional standard policies if clinically necessary. 
 
Note: Refrain from using polycarbonate syringes or filter needles during busulfan 
preparation. 
 
Alternate Busulfan Dosing: 
Divided dosing (every 12 hours) is permitted per institutional practice. Every 6 hour 
dosing is NOT permitted with UCB donors due to conflicts with ATG timing. 
 
Q12 Hour Dosing Schedule: IV over 2 hours 
Day: -8, every 12 hours  
Dose: Patients < 10 kg: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
Patients ≥ 10 kg but ≤ 4 years old: 2 mg/kg/dose q 12 hours (x 2 doses) 
Patients > 4 years: 1.6 mg/kg/dose q 12 hours (x 2 doses) 
 
Days: -7 through -5, every 12 hours (x 6 doses) 
Dose: Targeted dose adjustment based on PK results (see Section 16). Every 12 hour 
dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute per dose.  
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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
 
Fludarabine (FLUD): IV over 30-60 minutes 
Days: -5 through -2 (4 doses) 
Dose:  < 10 kg:  1.33 mg/kg/dose once daily 
 
≥ 10 kg:  40 mg/m2/dose once daily 
 
 
Rabbit Anti-thymocyte globulin (rATG): IV over 4-6* hours 
Days: -8 through -5 (4 doses) 
Dose: 2 mg/kg/dose 
 
*The first dose should be infused over at least 6 hours through a high-flow vein. 
Subsequent doses should be administered over at least 4 hours. Administer through an 
in-line 0.22 micron filter. 
 
Premedications should be considered prior to rATG per institutional protocols. This may 
include diphenhydramine 1 mg/kg/dose (max 50 mg), or a comparable anti-histamine; 
acetaminophen 
10-15 mg/kg/dose 
(max 
1000 
mg); 
and 
methylprednisolone 
1 mg/kg/dose. Premedication may be repeated or increased as needed to control allergic 
reactions, chills, or fever. Patients who are unable to tolerate the rabbit product may 
receive equine ATG 25 mg/kg/day x 4 days with similar premedication. 
 
Tacrolimus (TAC): IV or PO 
Days: -1 onwards (see below) 
Dose is dependent upon route of administration. 
IV administration, Dose: 0.03 mg/kg/DAY 
PO administration, Dose: 0.12 mg/kg/DAY 
 
Tacrolimus levels should be maintained between 8-12 ng/mL. The use of intermittent IV 
dosing, continuous IV dosing, or PO formulations may be used when clinically 
appropriate per institutional protocols. If ≤ Grade I acute GVHD, tacrolimus taper will 
begin no later than Day +100 and will be complete by Day +180. For patients with 
> Grade I acute GVHD, the tacrolimus taper will be per institutional protocols.  
 
Serum tacrolimus troughs and serum magnesium, potassium, and creatinine should be 
drawn at least twice per week while hospitalized, then as per good clinical practice 
thereafter unless a change in medication (e.g. use of concomitant CYP3A4 inhibitors, see 
Appendix IIIA) or renal function might result in an acute change in level. At that point, 
levels will be measured as clinically indicated. Levels sent when dosing by continuous 
infusion are not true trough concentrations, however, the same target range of drug levels 
will be used for both continuous IV and bolus PO routes of administration. When 
converting patients at a therapeutic tacrolimus level from IV to PO formulation, multiply 
total daily IV dose times 4 and administer in 2 divided oral doses per day, every 12 hours 
(e.g., 1 mg of IV tacrolimus per day equates to 4 mg of PO tacrolimus per day).  The oral 
dose should be administered 8-12 hours after the end of the tacrolimus continuous 
infusion.57 
 
The target serum trough level for tacrolimus is 8-12 ng/mL for mismatched or 
unrelated donors. Dose adjustments are based on clinical judgment of the treating 
physician after considering clinical toxicity, serum levels, GVHD, concomitant drug use 
and the rate of rise or decline of the serum level. Refer to Section 5.2 for guidance on 
dose adjustment based on serum trough levels.  
 
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ASCT1221 
Mycophenolate Mofetil (MMF): IV over 2 hours 
Days: +1 onwards (see below) 
Dose: 15 mg/kg/dose, every 8 hours 
 
The FDA has determined that a REMS (Risk Evaluation and Mitigation Strategy) 
program is necessary to ensure the benefits of mycophenolate outweigh the risks of first 
trimester pregnancy loss and congenital malformations associated with mycophenolate 
use during pregnancy. Mycophenolate REMS is a program to tell doctors, nurses, 
pharmacists and patients about the risks of taking mycophenolate during 
pregnancy. Providers and patients (females of child-bearing potential) are required to 
enroll in the program. The program also contains important information about patient 
education and reporting details for pregnancy. Providers and patients are required to sign 
acknowledgement forms prior to initiating therapy. Please refer to the website for 
additional information (https://www.mycophenolaterems.com/).   
 
See Section 2.3.1 for additional information regarding the GVHD prophylaxis approach. 
If ≤ Grade I acute GVHD, MMF will be discontinued by Day +45.  For patients with 
> Grade I acute GVHD, the MMF taper will be per institutional protocols. Patients may 
transition to PO formulations at a 1:1 IV to PO conversion when clinically appropriate. 
Refer to Section 5.3 for dose adjustments based on serum drug levels. 
 
See Section 5.0 for Dose Modifications based on Toxicities.  
The therapy delivery maps (TDMs) for this HCT treatment plan are on the next (one) 
page. 
 
 
 
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THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, SEE PAGE 1 FOR USAGE POLICY 
ASCT1221 
Extensive treatment details are in Section 4.1-4.5 and 4.11. This Therapy Delivery Map is on 1 page.   
 
 
**HEMATOPOIETIC CELL INFUSION:  DAY 0 (see Section 4.4)** 
 
 
Ht__________cm 
          Wt___________kg           BSA____________m2 
Date 
Due 
Date 
Given 
Day 
Anti-seizure prophylaxis 
____mg 
BUS 
____mg 
FLUD 
___mg 
rATG 
___mg 
TAC 
___mg 
MMF  
__mg __mg __mg 
Studies 
Comments 
 
 
 
Enter calculated dose above and actual dose administered below 
 
 
 
 
-8 
__mg  __mg  __mg  __mg 
_____mg 
 
_____mg 
 
 
(a, b, c, d, g) 1  
 
 
 
-7 
__mg  __mg  __mg  __mg 
_____mg 
 
_____mg 
 
 
 
 
 
 
-6 
__mg  __mg  __mg  __mg 
_____mg 
 
_____mg 
 
 
 
 
 
 
-5 
__mg  __mg  __mg  __mg 
_____mg 
____mg 
_____mg 
 
 
 
 
 
 
-4 
__mg  __mg  __mg  __mg 
 
____mg 
 
 
 
 
 
 
 
-3 
 
 
____mg 
 
 
 
 
 
 
 
-2 
 
 
____mg 
 
 
 
 
 
 
 
-1 
 
 
 
 
____mg 
 
 
 
 
 
0 
 
 
 
 
____mg 
 
 
 
 
 
+1 
 
 
 
 
____mg 
____mg ____mg ____mg 
 
 
 
 
+2 
 
 
 
 
 
 
 
 
 
 
+3 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
+30 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
+45 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
+60 
 
 
 
 
 
c, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+90 
 
 
 
 
 
b, c, d, e, f, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+120 
 
 
 
 
 
c, e, g  
 
 
 
… 
 
 
 
 
 
 
 
 
 
+180 
 
 
 
 
 
c, e, g 
 
 
 
 
See Section 7.2 for evaluations after Day +180. 
 
 
1Prior to HCT conditioning.      SEE PROTOCOL SECTION 5 FOR DOSE MODIFICATIONS. SEE SECTION 4.5 FOR SUPPORTIVE CARE.
4.11.1   HCT Regimen: UCB Donor -Arm B  
Treatment for the HCT preparative regimen lasts 8 days. 
After the HCT, the GVHD prophylaxis treatment lasts about 6 months. 
                    
 _____________________   ____________ 
           Patient COG ID number                    DOB   
DRUG 
ROUTE 
DOSAGE 
DAYS 
IMPORTANT NOTES 
OBSERVATIONS 
Anti-Seizure 
Prophylaxis:  
_____________ 
Indicate agent, i.e., 
lorazepam, 
levetiracetam 
IV or PO 
_____________________ 
(indicate dose) 
Preferred regimen: lorazepam (0.02-
0.05 mg/kg/dose every 6 hours) given 
30 minutes prior to each busulfan 
dose. Continue until ≥ 24 hours after 
last busulfan dose. 
-8 to -4 
 
a. History (incl. 
cardiac), PE w/ 
VS, NF1 status, 
pregnancy, PS 
b. BMA 
c. CBC, 
differential, 
HgbF, spleen & 
liver measure., 
concomitant tx 
d. Cytogenetics & 
FISH (not req. 
for all pts, see 
Sect. 7) 
e. GVHD, donor 
chimerism 
f. Complications 
g. Req. specimens: 
PB & BM (BM 
only if clinically 
indicated). 
 
See Section 7 for 
detailed list of 
evaluations.  
 
OBTAIN OTHER 
STUDIES AS 
REQUIRED FOR 
GOOD PATIENT 
CARE 
Busulfan  
(BUS) 
For once daily 
dosing only.  
 
IV over 3 hours  For 
once daily dosing 
only.  See 
Section 4.11 for 
alternative dosing 
schedule. 
Age & wt-based (1 dose): 
< 10 kg: 3.2 mg/kg/dose 
≥ 10 kg but ≤ 4 yr: 4 mg/kg/dose 
> 4 yr: 3.2 mg/kg/dose 
-8  
Submit BUS PK samples following 1st 
dose (Section 16).  See Section 4.11 for 
alternative dosing schedule.  
Note: Refrain from using polycarbonate 
syringes or filter needles during busulfan 
preparation. 
Dose adjusted per targeted AUC, once 
daily (3 doses) 
-7 to -5 
 
Fludarabine  
(FLUD) 
IV over 
30-60 minutes 
Weight-based dosing: 
≥ 10 kg:  40 mg/m2/dose  
< 10 kg:  1.33 mg/kg/dose 
(4 doses) 
-5 to -2 
 
Rabbit ATG  
(rATG) 
IV over 4-6* hours 
2 mg/kg/day  
(4 doses) 
 
-8 to -5 
*1st dose: infuse over ≥ 6 hours through 
a high-flow vein. Subsequent doses: 
infuse over ≥ 4 hours. Administer 
through an in-line 0.22 micron filter. See 
Section 4.11: premedications should be 
considered Equine ATG may be 
substitutued if rabbit product is not 
tolerated. See Section 4.11 for details. 
Tacrolimus 
(TAC) 
IV or PO 
See Section 4.11 
IV Dose: 0.03 mg/kg/DAY 
PO Dose: 0.12 mg/kg/DAY 
-1 to 
+180 
Maintain levels between 8-12 ng/mL. If 
≤ Grade I aGVHD, begin taper by Day 
+100. Discontinue by Day +180. See 
Section 4.11 for add’l monitoring. 
Mycophenolate 
mofetil (MMF) 
IV over 2 hours. 
Convert to oral when 
tolerated. 
15 mg/kg/dose, every 8 hours  
+1 to +45 
If ≤ Grade I aGVHD, discontinue by 
Day +45.   
 
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54 


 
 
ASCT1221 
5.0 
DOSE MODIFICATIONS FOR TOXICITIES 
Notify the Study Chair of any serious adverse events, protocol violations and/or if a patient is 
removed from protocol therapy for toxicity. 
 
5.1 
Dose Adjustment of Chemotherapy for Patients Whose Weight Exceeds > 125% 
IBW 
Chemotherapy given during the preparative regimen (including rATG) will be dosed 
based on actual weight for patients ≤ 125% IBW. Those > 125% IBW will be dosed 
based upon adjusted ideal body weight as follows: 
 
Adjusted ideal body weight = IBW + 0.25 (Actual weight – IBW). 
 
The following formulas for pediatric and adult IBW calculations are recommended, but 
IBW may be calculated according to institutional SOPs. 
 
5.1.1 
Recommended Ideal Body Weight Calculation for Children Age 1- 17 years 
 
IBW = Height (cm)2 x 1.65 
 
 
 
1000 
 
 
5.1.2 
Recommended Ideal Body Weight Calculation for Adults (≥ 18 years) 
 
IBW (females) = (cm ÷ 2.54 – 60) x 2.3 kg + 45.5 kg      
 
IBW (males) = (cm ÷ 2.54 – 60) x 2.3 kg + 50 kg  
 
5.2 
Tacrolimus 
Tacrolimus commonly causes mild/moderate hypertension and alopecia and less 
commonly kidney or liver dysfunction, transplant associated microangiopathy (TAM), 
and neurological changes associated with significant hypertension. When trough levels 
are kept in the therapeutic range and patients receive adequate hydration and magnesium 
replacement, most of these side effects can be minimized. Hypertension should be 
managed with single or combination antihypertensive therapy per institutional standards. 
Tacrolimus should be held for severe toxicities thought to be related to its administration 
(significant neurological changes/malignant hypertension, TAM, kidney failure, etc). 
Other immune suppressive medications may be substituted if tacrolimus is not tolerated 
(cyclosporine, etc). 
 
For levels < 5 ng/mL (or < 8 ng/mL for mismatched or unrelated donors), it is suggested 
that the dose of tacrolimus be increased by approximately 25% increments every 
1-2 days, rounded to the nearest 0.5 milligram (when dosing is oral) until the target range 
is achieved (dose adjustments may vary from the suggested 25% as needed to achieve the 
therapeutic range based upon the measured level and the clinical status of the patient). 
Conversely, for levels > 12 ng/mL, it is suggested that the dose of tacrolimus be 
decreased by approximately 25% every 1-2 days until the target level is achieved. For 
very high levels (> 20 ng/mL), tacrolimus may need to be held entirely for a period of 
time. This may be done as long as serum levels are monitored daily and the drug is 
restarted at an appropriate dosage when the level returns to the therapeutic range. 
 
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55 


 
 
ASCT1221 
5.3 
Mycophenolate 
Mycophenolic Acid (MPA) trough levels should be monitored as clinically indicated and 
dose modified according to the table below (or as per institutional practice). 
 
 
Table 2: Mycophenolate Dose Adjustment Guidelines 
MPA Concentrations (mcg/mL) 
MMF dosing adjustment 
<1-3.5 
No change 
3.6-6 
Decrease dose by 25% 
>6 
Hold one dose, decrease dose by 50%  
 
 
6.0 
DRUG INFORMATION  
See the consent document for toxicities. All other information is available on the COG website in 
the commercial agent monographs manual titled “Drug Information for Commercial Agents used 
by the Children’s Oncology Group.” This manual is provided under Standard Sections for 
Protocols at: https://members.childrensoncologygroup.org/prot/reference_materials.asp. 
 
 
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56 


 
 
ASCT1221 
 
 
7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
Timing of protocol therapy administration, response assessment studies, and surgical interventions are based on schedules derived 
from the experimental design or on established standards of care. Minor unavoidable departures (up to 72 hours) from protocol 
directed therapy and/or disease evaluations (and up to 1 week for surgery) for valid clinical, patient and family logistical, or facility, 
procedure and/or anesthesia scheduling issues are acceptable per COG administrative Policy 5.14 (except where explicitly 
prohibited within the protocol). 
 
7.1 
Evaluations at Study Entry and Requirements Prior to Stem Cell Transplant 
In cases that the patient’s diagnosis occurred prior to study entry, data obtained at the diagnosis of JMML 
prior to study entry must be submitted instead of readings taken at enrollment. 
STUDIES TO BE OBTAINED 
Time of 
Diagnosis 
Study Entry  
Prior to HCT* 
Study Requirements 
Clinical Evaluations: 
 
 
 
 
History (incl. cardiac) 
 
X 
 
 
Physical exam with VS 1 
 
X 
 
 
NF1 status by clinical exam 
 
X 
 
See Appendix II 
Pregnancy Test 2 
 
X 
2 weeks 
Negative  
IgG level 
X 
X^ 
 
 
Direct Coombs Antibody Test 
X 
X^ 
 
 
Performance Status 
 
 
2 weeks 
Lansky or Karnofsky performance status score ≥ 
60. 
Pulmonary Function Tests 
 
 
2 weeks 
FEV1, FVC, & DLCO corrected for Hgb ≥ 60% (or 
if unable to obtain PFTs: no evidence of dyspnea at 
rest, no exercise intolerance, & not requiring 
supplemental oxygen therapy). 
Echo/EKG 
 
 
2 weeks 
SF ≥ 27% or EF ≥ 50% by radionuclide angiogram. 
Liver Function Tests 
 
 
1 week 
Total bilirubin < 2.5 mg/dL (unless due to Gilbert’s 
Syndrome); Transaminases < 5 x ULN 
Creatinine Clearance or GFR  
or Serum Creatinine  
 
 
2 weeks 
≥ 70 mL/minute/1.73m2  
or serum creatinine by age/gender 
CNS Function 
 
 
2 weeks 
CNS toxicity ≤ Grade 2 
CBC, differential, HgbF 
X 
X^ 
2 weeks 
 
BMA (biopsy optional) 3 
X 
X^ 
2 weeks 
 
Cytogenetics & FISH for 
Monosomy 74 
X 
X^ 
X4 
 
BCR/ABL by FISH or PCR 
X 
X^ 
 
 
GM-CSF Hypersensitivity 
X 
X^ 
 
Only for patients without mutations in PTPN11, 
RAS, CBL, or NF1, and who otherwise do not fulfill 
the diagnostic criteria in Section 4.3.1. 
Spleen & liver measurement5 
X 
X^ 
X 
 
Concomitant Therapy 
 
X 
X 
 
HCT Donor Information 
 
 
X 
 
Infection Status 
 
 
X 
See Section 4.3.5 for requirements.  
HSV and CMV titers 
 
 
1 month 
 
Hepatitis C by serology and PCR 
 
 
1 month 
 
Hepatitis B surface antigen 
 
 
1 month 
 
HIV Test 
 
X 
X 
Negative 
 
 
 
 
 
Required Sample Submission: 6 
 
 
 
 
Buccal swab / Oragene Saliva Kit 
 
X 
 
 
Peripheral blood  
 
X 
X 
 
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57 


 
 
ASCT1221 
Bone marrow aspirate 
 
X 
X 
 
 
 
 
 
 
Central Review Submissions: 
 
 
 
 
Cytogenetics Report7 
 
X 
 
 
Pathology Review Materials8 
 
X 
 
 
 
 
 
 
 
Optional Samples: 9 
 
 
 
 
Spleen Tissue 
 
X 
X 
If splenectomy was performed.  
Skin sample  
 
X 
 
Requested only for patients with buccal swabs 
demonstrating suspicious germline mutations in 
PTPN11, RAS, or CBL 
Skin rash biopsy 
 
X 
X 
Only if a rash is biopsied. 
Parent buccal swabs  
 
X 
 
 
Peripheral blood 
 
X 
 
 
Bone marrow aspirate 
 
X 
 
 
1 Including NF-1 status, Noonan syndrome, and assessment of hepatosplenomegaly. 
2 Women of childbearing potential require a negative pregnancy test prior to starting treatment; sexually active 
patients must use an acceptable method of birth control. 
3 Submit pathology materials for central review. 
4 Performed at a COG-approved cytogenetics laboratory. Submit results for central review. Repeat cytogenetic 
analysis pre-HCT if patient received any pre-HCT chemotherapy OR if there was a delay of ≥ 30 days between 
initial diagnosis and HCT. See Section 13.  
5 By ultrasound. 
6 See Section 15 for sample collection & shipping instructions. Samples will be assessed for genotyping in the 
CLIA-certified laboratory at UCSF. Other research studies will be performed on samples in the Loh Laboratory 
(results will not be returned for these research studies).   
7 See Section 13 for central cytogenetics review submission requirements.  
8 See Section 14 for central pathology review submission requirements.  
9 See Section 15 for sample collection & shipping instructions. 
 
^ If performed at diagnosis of JMML prior to study entry, does not need to be repeated at study entry. 
* Please note that pre-HCT observations reflect recommended timing by the study committee. It will not be    
considered a protocol violation if there is a deviation from this recommended timing. 
 
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ASCT1221 
 
 
7.2 
Required & Optional Clinical, Laboratory and Disease Evaluations Post HCT 
Evaluations post-HCT may occur within +/- 3 days of the collection day specified below. 
 
 
STUDIES TO BE 
OBTAINED 
+30 
+60 
+90 
+120 
+180 
+270 
+365 
+540 
Suspected 
of Relapse 
Clinical Evaluations: 
 
 
 
 
 
 
 
 
 
 
CBC, differential, HgbF 
X 
X 
X 
X 
X 
X 
X 
X 
X 
BMA (biopsy optional) 1 
X 
 
X 
 
 
 
 
 
X 
Cytogenetics & FISH 2 
X 
 
X 
 
 
 
 
 
X 
Spleen & liver measurement 3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant therapy 
X 
X 
X 
X 
X 
X 
X 
X 
X 
GVHD assessment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Donor chimerism 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Complications 4 
X 
X 
X 
X 
 
 
 
 
 
 
Required Samples: 5 
Peripheral blood 6 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Bone marrow 6 
X 
X7 
X 
X7 
X7 
X7 
X7 
X7 
X7 
 
Optional Samples: 5 
Spleen Tissue18 
X 
Skin rash biopsy 9 
X 
1 
Submit pathology materials for central review. See Section 14. 
2 
Submit Day +30 and +90 bone marrow for cytogenetic analysis if an abnormal cytogenetic clone was 
identified at initial diagnosis (or pre-HCT). Performed at a COG-approved cytogenetics laboratory. Submit 
results for central review. See Section 13. 
3 
By ultrasound. 
4 
Grade 3-4 non-hematologic toxicities 
5 
See Section 15 for sample collection & shipping instructions.  
6 
Samples will be assessed at each time point for repeat genotyping/454 sequencing (MRD) in the 
CLIA-certified laboratory at UCSF. Qualitative sequencing results will be returned to the treating 
institution. Other research studies will be performed on samples in the Loh Laboratory (results will 
not be returned for these research studies).   
7 
Only if clinically indicated. 
8 
Only if splenectomy was performed.  
9 
Only if a rash is biopsied. 
 
This table only includes evaluations necessary to answer the primary and secondary aims. Obtain 
other studies as indicated for good clinical care.  
 
 
 
7.2.1  Suspicion of Relapse 
Patients may be suspected of relapse (or “non-response,” see Section 10.2 for 
definition details) based on either clinical findings (such as increasing spleen 
size), or laboratory features (such as increasing monocytosis). At the discretion 
of the treating physician, patients should have a bone marrow aspirate performed, 
in addition to the other evaluations listed in the table in Section 7.2. Furthermore, 
some patients may have a “weak-positive” mutation analysis following HCT 
(defined as a 1-14.99% mutant allele burden). It is highly recommended that 
these patients be considered “suspected of relapse,” and that that evaluations 
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59 


 
 
ASCT1221 
listed in the table in Section 7.2 be performed within a month of the test yielding 
the first “weak-positive” result. 
 
 
7.3 
Required Busulfan Pharmacokinetic Studies* (see Section 16).   
For Single (once daily) IV Dosing 
Time Following Start of 1st Dose 
3 hours 
3 hours 15 minutes 
4 hours 
5 hours 
6 hours 
8 hours 
 
* To be done on 1st dose only. 
 
 
For Q6 hour IV Dosing 
Time Following Start of 1st Dose 
2 hours 
2 hours 15 minutes 
2 hours 30 minutes 
3 hours 
4 hours 
5 hours 
6 hours 
* To be done on 1st dose only. 
 
 
 
For Q12 hour IV Dosing 
Time Following Start of 1st Dose 
1 hour 
2 hours  
3 hours  
5 hours  
6 hours 
7 hours 
8 hours 
* To be done on 1st dose only. 
 
7.4 
Follow-up  
See COG Late Effects Guidelines for recommended post treatment follow-up:  
http://www.survivorshipguidelines.org/ 
 
Although the primary end-points of the trial are the 100-day TRM and 18 month EFS, 
patients will be followed until off-therapy for other reasons, or to 5 years from study 
enrollment. 
 
Note: Follow-up data are expected to be submitted per the Case Report Forms (CRFs) 
schedule. 
 
 
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8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY 
CRITERIA 
8.1 
Criteria for Removal from Protocol Therapy 
a) 
Progressive disease (non-response, as described in Section 10.2.4). 
b) 
Refusal of further protocol therapy by patient/parent/guardian. 
c) 
Completion of planned therapy and post-therapy sample collections. 
d) 
Physician determines it is in patient’s best interest. 
e) 
Development of a second malignancy. 
f) 
Criteria to proceed to HCT (Section 4.3) are not met within 1 year of enrollment.  
 
Patients who are off protocol therapy are to be followed until they meet the criteria for Off 
Study (see below). Follow-up data will be required unless consent was withdrawn. 
 
8.2 
Off Study Criteria 
a) 
Death. 
b) 
Lost to follow-up. 
c) 
Patient enrollment onto another COG study with tumor therapeutic intent (eg, at 
recurrence). 
d) 
Withdrawal of consent for any further data submission. 
e) 
The fifth anniversary of the date the patient was enrolled on this study. 
 
 
9.0 
STATISTICAL CONSIDERATIONS 
From June 11, 2001 to October 23, 2006 patients with JMML could enroll on COG AAML0122, 
a single arm trial evaluating a targeted agent following by HCT. This trial enrolled 88 patients 
(~18 cases/year); however a survey of transplant physicians indicated that many eligible patients 
were not enrolled due to concerns regarding trial design that mandated the use of total body 
irradiation in this very young patient population. In addition, diagnostic testing for JMML is now 
more advanced to what was previously available and will likely diagnose a greater number of 
children. We anticipate at least equal, if not superior, accrual to this trial due to the removal of 
TBI from the conditioning regimen and more precise diagnostic procedures.  
 
Enrollment onto COG study AAML0122 averaged 18 patients per year. This study will benefit 
from the support of the JMML Foundation so it is estimated that at least 24 patients per year can 
be enrolled. Enrollment occurs when a clinical diagnosis of JMML is made, but eligibility for the 
HCT randomization is only determined after 1) genetic assessment of the diagnosis is made and 
the complete WHO diagnostic criteria are fulfilled; 2) an eligible donor is found; and 3) the 
patient survives and consents to be randomized. It is estimated that approximately 75% of 
enrolled patients will be eligible (18 eligible patients/year) for the HCT randomization. Thus, we 
calculate that up to 145 patients will need to be enrolled in order attain the target 108 eligible 
patients and, with a 1 year ramp up period, the enrollment period is expected to require 7 years. 
Given the complexities of diagnosing and treating these patients, precise data-driven calculations 
are impossible and the study committee would welcome more rapid accrual. Conversely, as the 
trial progresses, if we find that we are not meeting this metric, we will amend the number of 
children enrolled accordingly, and have secured funding from the BIQSFP mechanism to 
genotype up to 220 children.    
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9.1 
Study Design 
The study design is a modified randomized phase II trial. Patients will be assigned to BU-
FLU or BU-CY-MEL myeloablative conditioning regimen using a randomized block 
strategy within strata defined by donor type and PTPN11 mutation status. The outcome of 
the trial will be determined after all eligible patients have 18 months of follow-up. If the 
difference in the estimated 18 month EFS is more than 5%, then the regimen with the 
higher EFS will be declared the superior treatment. If the difference in EFS is less than 
5%, then the regimen with the lowest 100 day TRM risk will be declared the superior 
treatment. This design was chosen to exploit the a-priori belief that relapse probabilities 
will not influenced by the choice of regimen to increase the probability that the trial will 
pick the better regimen. 
 
9.1.1 
Primary Endpoints:  
Each patient will be evaluated for Day 100 treatment related mortality (TRM) 
and 18 month EFS. A patient who dies prior to relapse or non-response will be 
considered to be a TRM and time to TRM will be defined as time from transplant 
to TRM. Patients who die between the start of the conditioning regimen and 
transplant will be considered a TRM with time to TRM of zero. The estimated 
100 day TRM is defined the cumulative incidence of TRM at 100 days as 
estimated using the method of Aalen and Johannsen.58 An event is either TRM, 
primary or secondary graft failure, or relapse/non-response (as defined in 
Section 10). Time to event will be computed from transplant with patients who 
die between the start of the conditioning regimen and transplant given a time to 
event of zero. Estimated 18-month EFS is defined as the Kaplan-Meier estimator 
of EFS at 18 months. A patient who is in complete remission at transplant 
(Section 10.2.1) is followed for relapse (Section 10.2.3). A patient not in 
complete remission at transplant is followed for either non-response 
(Section 10.2.4) or until the patient meets the complete remission criteria 
(Section 10.2.2). If non-response, the patient has experienced an event so 
statistical follow-up ends. If complete remission, the patient is still “event-free” 
and the patient is further followed for relapse (Section 10.2.3).  
 
 
9.1.2 
Secondary Endpoints:  
For the purpose of Objective 1.2.1, relapse will include “relapse” or “non-
response” as defined by the criteria described in Section 10. Time to 
“relapse/non-response” will be from transplant to when all criteria of 
Section 10.2.3 or 10.2.4 are met. 
 
9.1.3 
Stratification 
The 1:1 randomization will be block stratified within six strata defined by the 
cross classification of stem cell source defined as: (1) matched related donor; 
(2) closely matched unrelated donor; or (3) umbilical cord blood, and mutation in 
the PTPN11 gene. We expect the distribution of donors to be 30:35:35 and the 
prevalence of PTPN11 mutation to be 35% so that the expected number in each 
stratum is given in Table 3 below. 
 
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Table 3: Expected number in each stratum for randomization 
 
 
Donor source 
 
 
Matched Related 
Matched unrelated 
Cord 
Total 
PTPN11 WT 
21 
25 
25 
70 
PTPN11 mutation 
11 
13 
13 
38 
Total 
32 
38 
38 
108 
 
9.2 
Sample Size and Study Duration 
A sample size of 108 (54 in each arm) was chosen so that in the situation in which the 
regimens have the same relapse rate and the difference in true 100 day TRM risk is about 
5% (7%), the modified phase II design will have over 75% (80%) probability of picking 
the superior regimen. 
 
 
9.2.1 
Justification for this Design 
While EFS is the natural measure for comparing treatment regimens, the 
modified randomized phase II design, which puts more weight on 100 day TRM 
risk, was chosen because of an a priori belief that the relapse rates will be similar 
in each. With this design, if there is a large enough difference in observed 
18 month EFS, higher EFS determines the winner, whether that difference is 
because of lower TRM or lower relapse risk. If the difference in EFS is small, 
TRM risk will determine the winner. This design relies primarily on TRM risk 
for picking the winner but provides protection against a situation in which there 
was a large differences in relapse risk that was opposite in direction from the 
difference in TRM risk. As shown in Section 9.4.2, if the true relapse risks are 
similar, then this design has much higher probability of picking the regimen that 
is actually superior to a rule that depends only on the observed EFS.  
 
9.2.2 
Trial Outcome Probabilities given TRM and Relapse Probabilities 
As originally conceived, the proposed study design was a RPII “pick the winner” 
design comparing treatment related mortality (TRM) between the two treatment 
arms. In response to concerns by the COG Scientific Council that the winning 
treatment could have lower event-free survival (EFS), a modification of the RPII 
design was developed that picks the treatment that is EFS superior and exploits 
the Study Committee's a-priori belief that only TRM (and not relapse) rates will 
be influenced by the treatment to increase the probability of picking the regimen 
that is truly better. Specifically, the modified RPII design for this trial chooses 
the winning regimen as the one that has (1) an event rate at least 5% lower than 
the other regimen or (2) a lower TRM rate than the other regimen when the 
difference in event rates is less than 5%. The trial outcome probabilities are a 
function of pkt and pkr, the true TRM and relapse rates for regimen k=A,B; δ, the 
tolerated difference in event rates; and n, the number of patients in each arm of 
the trial. Following the approach of Simon, Wittes, and Ellenberg for the 
standard RPII design,59 let mult(t,r,pt,pr,n) be the multinomial probability of t-
TRM, r-relapse, and n-t-r “no events” patients out of n in the group. The 
probability that the trial result is TA TRM and RA relapse patients from 
Regimen A and TB TRM and RB relapse patients from Regimen B is  
 
p(TA,RA,TB,RB)= mult(TA,RA, pAt, pAr,n)x mult(TB,RB, pBt, pBr,n). 
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The probability that Regimen A is selected using this design is the sum over all 
possible outcomes of TA,RA,TB,RB in which Regimen A meets criteria (1) or (2). 
Letting Δ=nδ, I, the indicator function, and noting that the event rate is given by 
T+R, the probability of Regimen A as winner is  
 
∑[I(TA+RA+ Δ ≤ TB+RB) + I(|(TA+RA) –(TB+RB)| ≤ Δ and TA< TB) + ½ I(|(TA+RA) 
–(TB+RB)| ≤ Δ and TA= TB)]x p(TA,RA,TB,RB)  
 
where the sum is over all possible TA,RA,TB,RB. These probabilities, as well as the 
probabilities of picking Regimen A using standard RPII designs with TRM or 
EFS as the outcome, were computed using a macro written by the Study 
Statistician using the SAS statistical package (Cary, NC) posted on 
http://hydra.usc.edu/timefactors. The program was validated using a computer 
simulation study that generated 10,000 studies with the parameters given in the 
Table below and the winner picked according to the modified RPII design. All 
estimated Regimen A winner probabilities based on the simulation were within 
1% of those calculated analytically. 
 
The table below shows the probability of picking Regimen A (the “superior” 
regimen). For comparison, the probability of picking Regimen A using a standard 
RPII design with just estimated 100 day TRM risk or just estimated 18 month 
EFS probability as the comparison measure. Scenarios 1a to 1d are considered 
“realistic” situations in which Regimen A has lower true TRM probability than 
B, and the same true relapse probability. In this situation, inclusion of relapse in 
the outcome only adds variability to the TRM difference so the standard RPII 
with TRM outcome has the highest probability  of picking Regimen A while the 
standard RPII with EFS has more than 10% lower probability of picking A. The 
modified RPII that will be used in this study suffers only a slight loss of relative 
to the TRM standard design. Under Scenario 2, Regimen A has both lower TRM 
and relapse risk. In this case, the modified design is the most likely to pick 
Regimen A. In Scenario 3a, with TRM equal and Regimen A associated with 
lower relapse risk, the TRM standard design is focused on the wrong outcome 
and picks either regimen with 50% probability. However, the modified design 
still has high probability of picking the Regimen A. In scenarios 3b and 3c, the 
TRM risk from Regimen A is higher than in Regimen B, but relapse risk is 
lower, such that the true EFS is 10% higher in A than in B (i.e., A is the better 
regimen). In this case, the TRM-standard design will be more likely to pick (the 
“wrong” regimen) B, while the modified design picks A with probability close to 
that using a standard design with EFS as the outcome. In Scenario 3d, the true 
EFS difference is 5%, equal to the “EFS tolerance.” In this situation, the 
probability that the observed EFS difference is less than 5% is 50% and the 
probability that the observed TRM risk for A is less than B, conditional on 
observed EFS difference less than 5%, is 56%. Although the probability of 
picking the preferable regimen is not much higher than 50%, the modified design 
is much better in these situations than the TRM standard design which would 
preferentially pick the inferior regimen with high probability. Finally, in Scenario 
3e, there is no difference in EFS so that, arguably, the regimens are equivalent in 
effectiveness. In this case, the modified design favors regimen B.  
 
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Thus, the design focuses on TRM differences but provides some protection if, in 
fact, there is relapse difference in the opposite direction of the TRM difference. 
At the planned sample size of 108 patients (54 in each arm), if there is no 
difference in relapse risk and a true difference of 5% in TRM risk (Scenario 1b, 
1c), there is at least an 75% chance of picking the better regimen.  
 
Probabilities of picking Regimen A under the proposed modified randomized 
Phase II, and standard randomized Phase II designs using estimated 100 day 
TRM and 18 month EFS as the outcomes as a function of true 100 day TRM and 
18 month relapse probabilities with a sample size of 108 (54 in each arm). 
 
 
Table 4: Probability of Picking Regimen A for the Modified Randomized Phase II, Standard 
Randomized Phase II with TRM as outcome, and Standard Randomized Phase II with EFS as 
Outcome for a Range of Hypothetical TRM and Relapse Scenarios. 
 
 
TRM probability 
Relapse probability 
 
Probability of picking Regimen A 
Scenario 
Regimen 
A 
Regimen 
B 
Regimen 
A 
Regimen 
B 
A-B EFS 
difference 
Modified 
RPII 
Standard 
RPII with 
TRM 
outcome 
Standard 
RPII with 
event 
outcome 
Modified 
RPII: 
Simulationa 
1a 
0.08 
0.15 
0.35 
0.35 
0.07 
0.84 
0.87 
0.77 
0.84 
1b 
0.05 
0.12 
0.35 
0.35 
0.07 
0.85 
0.91 
0.77 
0.84 
1c 
0.10 
0.15 
0.35 
0.35 
0.05 
0.76 
0.78 
0.70 
0.76 
1d 
0.05 
0.10 
0.35 
0.35 
0.05 
0.78 
0.84 
0.70 
0.78 
2 
0.05 
0.10 
0.30 
0.40 
0.15 
0.95 
0.84 
0.94 
0.95 
3a 
0.10 
0.10 
0.30 
0.40 
0.10 
0.79 
0.50 
0.85 
0.79 
3b 
0.15 
0.08 
0.33 
0.50 
0.10 
0.73 
0.13 
0.85 
0.73 
3c 
0.15 
0.10 
0.30 
0.45 
0.10 
0.74 
0.22 
0.85 
0.74 
3d 
0.15 
0.10 
0.30 
0.40 
0.05 
0.56 
0.22 
0.70 
0.56 
3e 
0.15 
0.10 
0.30 
0.35 
0.00 
0.39 
0.22 
0.50 
0.38 
aBased on 10,000 trials. 
 
9.2.3  TRM and Relapse Probabilities for the Outcome Probability Calculations 
To calculate the probability of picking a given regimen using the criteria 
described in Section 9.3.1, the probabilities of 100 day TRM and 18 month 
relapsed risk need to be specified for each regimen. These were estimated from 
reports in the literature. The expected Day 100 TRM for patients receiving BU-
CY-MEL can be determined by analysis of the published reports: 
 
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Table 5: Expected Day 100 TRM for Arm A Based on Published Reports. 
 
 
MRD 
URD 
UCB 
BU-CY-MEL4 
5%^ 
8%^ 
14%^ (n = 7) 
BU-CY-MEL6 
NA 
NA 
~25%* (n = 42) 
Summary 
5% 
8% 
~22% 
Expected Proportion of Patients 
on Trial 
0.3 
0.35 
0.35 
Expected Rate of Day 100 TRM 
1.5% 
2.8% 
7.7% 
Total = 12% 
 
 
 
^Day 100 TRM not reported, but extrapolated from reported 5-year TRM, that 
occurred at a median of 2.7 months (range, 1 to 16) from transplant, by dividing 
reported figure in half.  
*Day 100 TRM not reported, but extrapolated from 2-year TRM of 33%, with 
approximately 75% of this expected to occur within 100 days. 
 
Although there is no data on the BU-FLU regimen in patients with JMML, a 
careful review of the literature can provide an expected baseline Day 100 TRM 
in children with non-malignant diseases or myeloid malignancies.  
 
 
Table 6: Expected Day 100 TRM for Arm B Based on Published Reports. 
 
MRD 
URD 
UCB 
BU-FLU26 
0/7 
2/11 
0/1 
BU-FLU27 
1/12 
1/23 
NA 
BU-FLU60 
NA 
NA 
0/30 
BU-FLU61 
0/17 
0/3 
2/20 
BU-FLU^16 
0/60 
1/36 
NA 
Summary 
1/96 = 1% 
4/73 = 5.5% 
2/51 = 3.9% 
Expected Proportion of Patients 
on Trial 
0.3 
0.35 
0.35 
Expected Rate of Day 100 TRM 
0.3% 
1.9% 
1.4% 
Total = 3.6% 
 
 
 
 
 
^Adults with AML/MDS 
Thus, given the expected proportion of stem cell sources used for patients 
enrolling on this trial, we would expect a TRM of approximately 12% in the BU-
CY-MEL arm, and approximately 4% in the BU-FLU arm. Since neither of these 
approaches has been implemented in a truly multicenter approach, we would 
expect slightly higher rates, such as 15% for BU-CY-MEL and 7% for BU-FLU.  
 
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9.3 
Methods of Analysis 
9.3.1 
Primary objectives 
The trial is a modified randomized phase II design. The trial will end when all 
eligible patients have accrued at least 18 months of follow-up time. The analysis 
to determine the superior regimen is as follows: If there is at least a 5% 
difference in 18 month EFS (as estimated using the Kaplan-Meier method), then 
the regimen with the higher 18 month EFS is superior. If the difference in 
estimated 18 month EFS is less than 5%, then the regimen with the lower 
estimated 100 day TRM risk (cumulative incidence as estimated using the Aalen-
Johansen method58) is superior.  
 
 
9.3.2 
Secondary objectives 
9.3.2.1 Relapse / non-response and graft failure rates will be estimated using the 
cumulative incidence via the method of Aalen and Johannsen.58 For relapse, 
death and non-response (per Section 10.2.3) will be treated as competing risks. 
For graft failure, death will be the competing risk. 
 
9.3.2.2  While we have made a reasonable estimate that we will need to screen 
145 patients in order to enroll 108 to the randomized intervention, we 
nonetheless have funding from the BIQSFP agency to perform genotyping on up 
to 220 patients if necessary.  
 
9.3.3 
Exploratory objectives 
9.3.3.1 To determine the rates of severe toxicities (Grade 3/4) at Day 100 post-
HCT between the two myeloablative conditioning regimens (BU-FLU vs. 
BU-CY-MEL).  
 
For Grade 3/4 toxicities (by CTCAEv.4.0), as well as severe SOS (as defined 
below) occurrence will be compared in a number of ways: proportion of patients 
experiencing each toxicity and number of Grade 3/4 toxicities per patient. High 
levels to busulfan, as measured by serum levels, have been associated with 
excessive rates of liver VOD/SOS and TRM.62 Thus, severe toxicities (Grade 
3/4) at Day 100 post-HCT will be assessed as a function of busulfan dose. 
 
Unacceptable (severe) SOS: The CTC does not include SOS as a single toxicity. 
Therefore, this study will utilize a composite definition of SOS used in previous 
studies (NANT N01-02; ANBL0532, ANBL09P1, ANBL12P1) to evaluate SOS:  
Serum total bilirubin > 2.0 mg/dL, with 2 of the following findings within 
21 days of transplantation: hepatomegaly with right upper quadrant pain, ascites, 
or weight gain >5% above baseline. Severe SOS is defined as an episode of SOS 
in addition to a specific organ failure:  
a. Hepatic encephalopathy (CTC Grade 4 hepatic failure), OR  
b. Pulmonary dysfunction: Continuous oxygen support (CTC Grade 
hypoxia) for > 48 hours, ventilatory support not clearly attributable to 
another cause,  
OR 
c. Renal dysfunction: serum creatinine > 3 times the ULN (CTC Grade 3 
creatinine), or the need for dialysis (CTC Grade 4 renal), not clearly 
attributable to another cause. 
 
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9.3.3.2 To determine the rates of acute and chronic (at 18 months post-HCT) 
graft-versus-host disease (GVHD) following HCT using two different 
conditioning regimens (BU-FLU vs. BU-CY-MEL) in children with JMML. 
 
The 18 month risk of acute and chronic GVHD will be estimated using 
cumulative incidence curves with TRM and relapse as the competing risks. 
 
9.3.3.3 To create a JMML-specific pre-HCT co-morbidity index to allow better 
risk-stratification of future patients.   
 
Constellations of potential prognostic factors that can be assessed pre-HCT, that 
identify relapse and mortality risk diversity will be identified using Cox 
regression and semi-parametric survival methods. These factors may include 
patient gender, age at diagnosis, complete blood count values at diagnosis, 
genetic karyotype, genetic mutation status, hemoglobin F percentage, bone 
marrow blast percentage, and spleen size. Standard cross- and other validation 
approaches will be used to estimate the actual relapse risk in the risk-groups. 
 
9.3.3.4  To determine the feasibility of assessing post-transplant disease burden 
by donor chimerism measurements and allele specific PCR in mononuclear and 
sorted cell subsets. 
 
We will perform 454Jr next generation sequencing to assess post-transplant 
disease burden for patients enrolled on the study (as a quantitative value for 
research only) and we plan to release qualitative results for unsorted 
mononuclear cells based on predetermined thresholds (defined and validated in 
the CLIA lab for the BIQSFP application). In other independently funded work, 
we will compare the sensitivity of 454 sequencing performed on subsets of 
sorted/selected cells (CD3, CD14/15, and CD34) against allele specific PCR 
based assays and donor chimerism to identify the optimal methods to diagnose 
relapse. Any patient who is followed on protocol therapy for at least 30 days post 
HCT will be considered in the evaluation of feasibility of post-HCT disease 
burden assessment. The proportion of patients who are successfully assessed will 
be calculated as the number of patients with at least one re-sequencing evaluation 
30 days post-HCT divided by the number of patients who are evaluable for post-
HCT disease burden. This proportion will be compared with 75% using a 1-sided 
exact test of approximate size 0.05. If this test is rejected, the procedures used for 
obtaining this specimen will be examined. For example, we expect that 81/108 
patients will be evaluable for post-HCT disease burden using 454 sequencing. If 
72 or less are successfully evaluated, the procedures for sending the specimens 
will be examined. If the true rate of successful evaluation is only 60%, the post-
HCT disease burden evaluability rate will be considered too low with 95% 
probability. 
 
9.3.3.5 To determine predictors of overall survival using either gene expression 
or methylation classifiers determined at study enrollment  
 
A gene expression profile was previously described by Bresolin et al45 and 
predicted for poorer survival in those patients that displayed an AML-associated 
signature. Similarly, Olk-Batz has identified that the constellation of  
hypermethylation of 4-genes was associated with a higher rate of treatment 
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failure.50  Similar studies will be performed at study enrollment for those patients 
who are ultimately transplanted.  Our analysis strategy will be to compare the 
event (relapse / non-response, and TRM rates) across groups defined by the 
prognostic levels. For the gene expression profile (GEP) studies, prior work  has 
indicated that a cohort of only 44 patients treated on various protocols, revealed 
statistically different groups with either an “AML-type” or a “non-AML” like 
GEP. Those with an AML type GEP had 6% EFS versus 63% EFS for those 
without (P = .0010).45 On that study, 50% of JMML patients were AML-like. 
Cause-specific relapse and TRM rates in AML gene expression positive and 
negative patients will be compared using Cox regression, controlling for other 
prognostic factors and treatment arms to the extent possible. Relapse/non-
response and TRM rates will be estimated using the method of Aalen and 
Johansen. With 108 patients on this study there is over 70% power to detect EFS 
differences of 25% and over 90% power to detect DFS differences of 30% when 
the high-risk group has an EFS rate in the range of 10-50%.  
 
Hypermethylation signatures will be analyzed in a similar manner. For instance, 
methylation signatures will be summarized into a continuous methylation score 
based on the four genes defined in the Olk-Batz work that predicted outcome. On 
this prospective trial there will be at least 65% power to detect DSF differences 
of 20% and over 80% power to detect DFS differences of 30% between the 
lowest quartile and the upper three quartiles of the score. 
 
9.3.3.6   To comprehensively assess genetic and biochemical alterations amongst 
patients with JMML who are treated on this transplant protocol.  
 
This exploratory aim will build on previous and simultaneously conducted work 
to determine the proportion of children with JMML who harbor new mutations 
that may be discovered in parallel whole exome sequencing approaches, as well 
as biochemical perturbations using phosphoflow signaling analyses. In addition, 
biomarkers of autoimmunity (IgG levels and direct Coombs tests) will be 
measured and assessed descriptively to determine the incidence and effect of 
these abnormalities in children with JMML enrolled on this trial.  Comparison of 
event rates by genetic and biomarkers will be done using standard Cox regression 
methods with event risk in patient groups estimated using Kaplan-Meier curves. 
Changes in the genetic or biochemical measures between diagnosis and 
transplant will be correlated with treatments received during this period. 
 
9.3.4 
Safety Monitoring 
Safety monitoring of outcomes will take place two years after the start of the 
study as part of the Study Progress Report cycles. The rules for each outcome to 
be evaluated are constructed such that there is no more than a 10% chance of 
stopping if, in fact, an excess of the event of concern is not elevated above 
expected. 
 
 
9.3.4.1 Graft Failure in the BU-FLU Arm 
Patients on the BU-FLU arm with follow-up of at least 100 days post-HCT will 
be assessed for graft failure. A graft failure (primary or secondary) rate in these 
patients of up to 10% is acceptable. The table below gives the number of graft 
failures (or greater) that would be observed with probability of 10% given a true 
graft failure rate of 10%, given the number of BU-FLU patients assessed. If the 
number of graft failures is greater than or equal to the number shown, the study 
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will be temporarily suspended for DSMC evaluation. If there are 30 patients and 
the true graft failure rate is 30%, then the probability of observing six or more 
graft failures is 92%. 
 
Table 7: Number of BU-FLU Arm Patients with Graft Failure to Trigger 
DSMC Review by Number of BU-FLU Patients with More than 100 Days 
Follow-Up. 
 
BU-FLU patients 
15 
20 
25 
30 
Graft failures for 
temporary suspension 
4 
5 
5 
6 
 
 
 
 
9.3.4.2 Relapse Rate in the BU-FLU Arm  
All patients on the BU-FLU arm will be evaluated for relapse and the relapse rate 
at 18 months, with TRM as a competing risk, will be estimated using the method 
of Aalen and Johansen. If the estimated relapse/non-response is greater than 
40%, the study will be temporarily suspended for DSMC evaluation.  
 
9.3.4.3 TRM Rate in the BU-CY-MEL Arm  
Patients on the BU-CY-MEL arm with follow-up of at least 100 days post-HCT 
will be assessed for TRM. A TRM rate in these patients of up to 20% is 
acceptable. The table below indicates the number of TRMs (or greater) that 
would be observed with probability of 10% given a TRM rate of 20%, given the 
number of BU-CY-MEL patients assessed. If the number of TRMs is greater than 
or equal to the number shown, the study will be temporarily suspended for 
DSMC evaluation. If there are 30 patients and the true graft failure rate is 40%, 
then the probability of observing 10 or TRMs is 82%. 
 
 
Table 8: Number of BU-CY-MEL Arm Patients with TRM to Trigger 
DSMC Review by Number of BU-CY-MEL Patients with More than 100 
Days Follow-Up. 
 
BU-CY-MEL patients 
15 
20 
25 
30 
TRMs for temporary 
suspension 
6 
7 
9 
10 
 
 
9.4 
Evaluability for Response  
An enrolled patient will be evaluable for response (EFS and TRM) if  
1) Diagnosis is confirmed based on criteria in Section 4.3.1, 
2) A stem cell donor source, meeting the criteria in Section 4.2 is identified, 
3) Other patient HCT requirements are met, 
4) The patient provides consent and starts a study preparative regimen. 
 
 
9.5 
Evaluability for Toxicity  
An enrolled patient will be evaluable for toxicity if the patient meets the criteria for 
evaluability for response. 
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9.6 
Gender and Minority Accrual Estimates  
The gender and minority distribution of the study population is expected to be: 
 
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
10 
19 
29 
Not Hispanic or Latino 
63 
128 
191 
Ethnic Category: Total of all subjects 
73 
147 
220 
Racial Category 
 
American Indian or Alaskan Native 
2 
0 
2 
Asian 
4 
11 
15 
Black or African American 
4 
12 
16 
Native Hawaiian or Pacific Islander 
2 
0 
2 
White 
61 
124 
185 
Racial Category: Total of all subjects 
73 
147 
220 
This distribution was derived from the gender/race/ethnicity data from the Statistics & Data 
Center childhood cancer registry grant, ICD-O morphology codes for JMML (9945/3, 
9946/3) between 2005-2012. 
 
 
10.0 
EVALUATION CRITERIA 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
This study will utilize version 4.0 of the CTCAE of the National Cancer Institute (NCI) 
for toxicity and performance reporting. A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP website  
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm). 
Additionally, toxicities are to be reported on the appropriate case report forms. 
 
Please note: ‘CTCAE v4.0’ is understood to represent the most current version of 
CTCAE v4.0 as referenced on the CTEP website (ie, v4.02 and all subsequent iterations 
prior to version 5.0). 
 
 
 
10.2 
JMML Response Criteria 
 
10.2.1 Definition of Complete Remission prior to HCT: 
The majority of patients with JMML will enter HCT with some evidence of 
active disease; however, some patients may enter HCT in complete remission. 
Complete remission will be determined upon entry to HCT (i.e., start of 
conditioning regimen) as those patients that achieve ALL of the following 
criteria: 
 
1. Absence of splenomegaly on exam and imaging, if the spleen is present 
2. Absolute monocyte count < 1000/µL 
3. Absence of myeloid precursors (including blasts) in peripheral blood 
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4. Normal HgbF for age  
5. Blasts in bone marrow of < 5% with normal cytogenetics 
6. For patients with previously identified molecular abnormality, 
disappearance of molecular abnormality in NRAS, KRAS, or PTPN11 
(below a level of 1% on 454Jr sequencing).  
 
 
10.2.2 Definition of Complete Remission after HCT: 
The majority of patients with JMML will enter HCT with some evidence of 
active disease. Complete remission will be defined on Day +90 (for those who 
enter HCT without remission) as those patients that achieve the following 
criteria: 
 
Blasts in bone marrow of < 5%; AND EITHER: 
1. Achievement of donor chimerism of > 95% in unsorted mononuclear 
cells from bone marrow;  
OR 
2. For patients with previously identified cytogenetic or molecular 
abnormality, disappearance of clonal cytogenetic abnormality or 
molecular abnormality in NRAS, KRAS, PTPN11 (below a level of 1% on 
454Jr sequencing)  
OR 
3. For patients without cytogenetic abnormalities or mutations, or with 
mutations in NF1 or CBL at diagnosis, who do not achieve full donor 
chimerism (as defined above), ALL of the following features of clinical 
remission must be achieved: 
a. 
Absence of splenomegaly on exam and imaging, if the spleen is 
present 
b. 
Absolute monocyte count < 1000/µL 
c. 
Absence of myeloid precursors (including blasts) in peripheral 
blood 
d. 
Normal HgbF for age 
 
A small number of patients may not achieve CR at the Day +90 evaluation and 
may convert to full donor chimerism or meet negative cytogenetic/molecular 
criteria at a later point. These patients will initially be scored as a “Non-
Response” however, if they are alive at Day +540 without the initiation of 
additional anti-JMML-directed pharmacotherapy following HCT, then their 
status will be changed to CR. 
 
10.2.3 Definition of Relapse: 
Post-HCT relapse can only be determined in patients who meet the criteria for 
clinical remission prior to or post HCT as defined above, and will require 
EITHER:  
ALL of the following 3 features: 
1. Splenomegaly on exam and/or imaging, if the spleen is present, and had 
previously normalized in size  
2. An increase in host chimerism by ≥ 5% (from the highest achieved 
chimerism) in unsorted mononuclear cells of bone marrow or peripheral 
blood 
3.  Re-emergence of an elevated HgbF for age, if initially elevated at 
diagnosis  
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OR, for patients with previously identified cytogenetic or molecular 
abnormality, the following feature: 
Re-emergence of a clonal cytogenetic abnormality or molecular 
abnormality in NRAS, KRAS, or PTPN11 as measured by two sequential 
molecular tests (within one month of each other) if weakly positive at the 
first time-point followed by another positive (weak or strong) at the 
second sequential measurement OR a single strong positive at any time 
(“strong positive” definition corresponds to ≥ 15% mutant allele burden 
that can be verified by Sanger sequencing, “weakly positive” 
corresponds to 1-14.99% mutant allele burden) 
 
Time to “relapse” will be from transplant date to the date at which all criteria 
have been documented, or the initiation of additional anti-JMML-directed 
pharmacotherapy following HCT, whichever occurs sooner. 
 
10.2.4 Definition of Non-Response:  
Post-HCT “Non-Response” is defined as those patients who never achieved CR 
prior to or after transplant (as measured on Day +90). Time to “Non-Response” 
will be from transplant date to the date at which the Day +90 bone marrow 
evaluation was performed, or the initiation of additional anti-JMML-directed 
pharmacotherapy following HCT, whichever occurs sooner. 
 
10.3 
Graft Failure following Allogeneic HCT 
10.3.1 Primary Graft Failure 
Failure to achieve an ANC ≥ 500/µL after 42 days, determined by three 
consecutive measurements on different days; or < 5% donor cells in blood or 
bone marrow by Day +42 (as demonstrated by a chimerism assay), without 
evidence of JMML.  
 
10.3.2 Secondary Graft Failure:   
Initial engraftment followed by severe neutropenia (ANC ≤ 500/µL) that is not 
caused by recurrent leukemia, or < 5% donor cells in the blood or bone marrow 
(as demonstrated by a chimerism assay) without subsequent improvement 
occurring either spontaneously or after growth factor treatment.  Improvement is 
defined as an ANC > 500/µL consistently. Severe neutropenia with bone marrow 
cellularity ≥ 25% is not secondary graft failure. 
 
Marrow, UCB or peripheral blood stem cell reinfusion carried out any time after 
Day 0 because of inadequate hematopoietic function will be taken as a definitive 
indication of graft failure regardless of ANC values and marrow cellularity. 
Donor lymphocyte infusion is not considered a stem cell reinfusion.  
 
10.4 
Definition of Engraftment 
Neutrophil engraftment is defined as the first of three consecutive days in which the 
absolute neutrophil count exceeded 500 per cubic millimeter. Platelet engraftment is 
defined as the first of seven consecutive days in which the platelet count exceeded 
20,000 per cubic millimeter without platelet transfusions. The presence of donor cells is 
demonstrated by the detection of informative variable-number tandem-repeat 
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polymorphisms or by fluorescent in situ hybridization with a Y-chromosome–specific 
probe in cases of sex-mismatched transplants. 
 
10.5 
Evaluation and Diagnosis of GVHD 
 
10.5.1 Evaluation and Diagnosis of Acute GVHD 
 
Review of systems and physical examinations should be performed daily, while 
in hospital, and weekly thereafter to screen for signs and symptoms of acute graft 
versus host disease (GVHD). Acute GVHD generally develops within the first 
three months after transplantation and appears as a characteristic dermatitis often 
accompanied by hepatitis, cholestasis and enteritis. The onset of acute GVHD 
usually heralds itself as a pruritic macular exanthem of the palms and soles with 
morbilliform lesions on the extremities, trunk, and face.  With progression, the 
lesions become confluent, leading to generalized erythroderma and bulla 
formation complicated by bacterial superinfection and exfoliation. The clinical 
appearance of skin GVHD can be mimicked by toxicity of the hematopoietic cell 
transplant conditioning regimen and by drug reactions. Therefore, documentation 
of the diagnosis by skin biopsy is recommended. Severity of liver GVHD is 
usually described according to the serum bilirubin level.  Hepatic GVHD cannot 
be assessed solely on clinical grounds in patients who have concurrent drug 
toxicity, viral hepatitis, or toxicity caused by pre-transplant chemotherapy 
irradiation. Liver biopsy can be helpful but often cannot be done because of 
clinical contraindications, such as thrombocytopenia or coagulopathy.  
Gastrointestinal GVHD is indicated by watery diarrhea and anorexia, nausea and 
vomiting accompanied by abdominal cramps, gastrointestinal hemorrhage, and 
ileus. Eating often exacerbates symptoms.  The volume of diarrhea has been used 
as an indicator of the severity of gut GVHD, but this can be inaccurate and highly 
variable from day to day. In many cases, it can be difficult to distinguish GVHD 
from infectious enteritis, and endoscopic biopsy is often helpful and should be 
done whenever possible. 
 
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ORGAN STAGING OF GVHD  
Stage 
Skin 
Liver (bilirubin) 
Gut (stool output/day) 
0 
No GVHD rash 
< 2 mg/dL 
(< 34 μmol/L) 
Adult: < 500 mL/day 
Child: < 10 mL/kg/day 
 
1 
Maculopapular rash 
< 25% BSA 
2–3 mg/dL 
(34.1–51 μmol/L) 
Adult: 500–999 mL/day 
Child: 10–19.9 mL/kg/day 
Or persistent nausea, vomiting, 
or anorexia, with a positive 
EGD biopsy. 
 
2 
Maculopapular rash 
25–50% BSA 
3.1–6 mg/dL 
(51–102 μmol/L) 
Adult: 1000–1500 mL/day 
Child: 20–30 mL/kg/day 
 
3 
Maculopapular rash 
> 50% BSA 
6.1–15 mg/dL 
(102.1–255 μmol/L) 
Adult: > 1500 mL/day 
Child: > 30 mL/kg/day 
 
4 
Generalized 
erythroderma with 
bullous formation 
and desquamation 
> 5% BSA 
 
> 15 mg/dL 
(> 255 μmol/L) 
Severe abdominal pain with or 
without ileus, or grossly 
bloody stool (regardless of 
stool volume). 
 
 For GI staging: The “adult” stool output values should be used for patients > 50 kg 
in weight. 
 
 Use 3 day averages for GI staging based on stool output. If stool and urine are 
mixed, stool output is estimated to be 50% of total stool / urine mix.  
 
 For stage 4 GI: the term “severe abdominal pain” will be defined as: 
(a) Pain control requiring institution of opioid use, or an increase in on-going opioid 
use, PLUS  
 (b) Pain that significantly impacts performance status, as determined by the treating 
MD. 
 
 If colonic or rectal biopsy is +, but stool output is < 500 mL/day (< 10 mL/kg/day), 
then consider as GI stage 0. 
 
 There is no modification of liver staging for other causes of hyperbilirubinemia.  
 
Overall Clinical Grade:  
Grade 0 
No Stage 1-4 of any organ 
Grade I  
Stage 1-2 rash and no liver or gut involvement 
Grade II 
Stage 3 rash, or Stage 1 liver involvement, or Stage 1 GI 
Grade III 
Stage 0-3 skin, with Stage 2-3 liver, or Stage 2-3 GI 
Grade IV 
Stage 4 skin, liver or GI involvement 
 
 
10.5.2 Evaluation and Diagnosis of Chronic GVHD 
Review of systems and physical examinations should be performed weekly to 
screen for signs and symptoms of chronic graft versus host disease (cGVHD). 
cGVHD should be described in terms of: 
• 
Extent of skin involvement 
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• 
Liver dysfunction: AST, ALT, total bilirubin levels 
• 
Eye involvement: results of Schirmer’s test 
• 
Involvement of salivary glands or oral mucosa 
• 
Pulmonary involvement 
• 
Presence or absence of joint contractures or limitation on range of 
movement 
• 
other organ involvement 
• 
Results of biopsies (skin, liver, intestine), where applicable 
 
cGVHD should be graded according to the criteria set out by Shulman et al.63 as 
follows: 
 
Limited cGVHD 
Either or both: 
1.) Localized skin involvement 
2.) Hepatic dysfunction due to chronic GVHD 
 
Extensive cGVHD 
Either: 
1.) Generalized skin involvement, or 
2.) Localized skin involvement and/or hepatic dysfunction due to cGVHD 
 
Plus: 
3a.)  liver histology showing chronic aggressive hepatitis, bridging necrosis, 
or cirrhosis, or 
3b.) involvement of eye (Schirmer test with < 5 mm wetting), or 
3c.) involvement of minor salivary glands or oral mucosa demonstrated on 
labial biopsy, or 
3d.) involvement of any other target organ 
 
 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS  
11.1 
Purpose 
Adverse event data collection and reporting, which are required as part of every clinical 
trial, are done to ensure the safety of patients enrolled in the studies as well as those who 
will enroll in future studies using similar agents.  
 
11.2 
Determination of Reporting Requirements 
Reporting requirements may include the following considerations: 1) the characteristics 
of the adverse event including the grade (severity); 2) the relationship to the study 
therapy (attribution); and 3) the prior experience (expectedness) of the adverse event. 
  
Commercial agents are those agents not provided under an IND but obtained instead from 
a commercial source. In some cases an agent obtained commercially may be used for 
indications not included in the package label.  In addition, NCI may on some occasions 
distribute commercial supplies for a trial.  Even in these cases, the agent is still 
considered to be a commercial agent and the procedures described below should be 
followed. 
 
Determine the prior experience Expected events are those that have been previously 
identified as resulting from administration of the agent.  An adverse event is considered 
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unexpected, for reporting purposes only, when either the type of event or the severity of 
the event is not listed in:  
 
• 
the current known toxicities for each commercial agent as provided in 
the Drug Information for Commercial Agents Used by the Children’s 
Oncology Group posted on the COG website; or 
• 
the drug package insert. 
 
Secondary Malignancy 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (eg, 
treatment with investigational agent/intervention, radiation or chemotherapy). A 
metastasis of the initial neoplasm is not considered a secondary malignancy.  
 
All secondary malignancies that occur following treatment need to be reported via CTEP-
AERS. Three options are available to describe the event:  
• 
Leukemia secondary to oncology chemotherapy   
• 
Myelodysplastic syndrome  
• 
Treatment related secondary malignancy 
 
11.3 
Reporting of Adverse Events for Commercial Agents – via CTEP-AERS  
Expedited AE reporting must use CTEP-AERS (Adverse Event Expedited 
Reporting System), accessed via htt;s://eapps-ctep.nci.nih.gov/ctepaers 
 
Commercial reporting requirements are provided in Table B.  The commercial agent(s) 
used in this study are listed in the front of this protocol immediately following the Study 
Committee roster.  
 
• 
COG requires the CTEP-AERS report to be submitted within 7 calendar 
days of learning of the event.   
• 
Use the NCI protocol number and the protocol-specific patient ID provided 
during trial registration on all reports. 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will 
be utilized for AE reporting and are located on the CTEP website at:  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. All 
appropriate treatment areas should have access to a copy of the CTCAE. 
 
 
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Table B  
Reporting requirements for adverse events experienced by patients on study who 
have NOT received any doses of an investigational agent on this study. 
 
CTEP-AERS Reporting Requirements for Adverse Events That Occur During 
Therapy with a Commercial Agent or Within 30 Days1 
Attribution 
Grade 4 
Grade 5 
 
Unexpected 
Expected 
 
Unrelated 
or 
Unlikely 
 
 
CTEP-AERS 
Possible, 
Probable, 
Definite 
CTEP-AERS 
 
CTEP-AERS 
1This includes all deaths within 30 days of the last dose of treatment with a 
commercial agent, regardless of attribution.  Any death that occurs more 
than 30 days after the last dose of treatment with a commercial agent which 
can be attributed (possibly, probably, or definitely) to the agent and is not 
due to cancer recurrence must be reported via CTEP-AERS. 
 
11.4 
Routine Adverse Event Reporting 
Note: The guidelines below are for routine reporting of study specific adverse events on 
the COG case report forms and do not affect the requirements for CTEP-AERS reporting. 
 
The NCI defines both routine and expedited AE reporting. Routine reporting is 
accomplished via the Adverse Event (AE) Case Report Form (CRF) within the study 
database. For this study, routine reporting will include all toxicities reported via CTEP-
AERS and all Grade 3 and higher non-hematological Adverse Events. In addition, routine 
reporting will include unacceptable (severe) SOS as defined in Section 9.3.3. 
 
 
 
12.0 
STUDY REPORTING AND MONITORING  
The Case Report Forms and the submission schedule are posted on the COG web site with each 
protocol under “Data Collection/Specimens”. A submission schedule is included. 
 
12.1 
CDUS 
This study will be monitored by the Clinical Data Update System (CDUS). Cumulative 
CDUS data will be submitted quarterly to CTEP by electronic means. Reports are due 
January 31, April 30, July 31 and October 31. This is not a responsibility of institutions 
participating in this trial. 
 
12.2 
Data and Safety Monitoring Committee 
To protect the interests of patients and the scientific integrity for all clinical trial research 
by the Children’s Oncology Group, the COG Data and Safety Monitoring Committee 
(DSMC) reviews reports of interim analyses of study toxicity and outcomes prepared by 
the study statistician, in conjunction with the study chair’s report. The DSMC may 
recommend the study be modified or terminated based on these analyses.  
 
Toxicity monitoring is also the responsibility of the study committee and any unexpected 
frequency of serious events on the trial are to be brought to the attention of the DSMC. 
The study statistician is responsible for the monitoring of the interim results and is 
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expected to request DSMC review of any protocol issues s/he feels require special 
review. Any COG member may bring specific study concerns to the attention of the 
DSMC.  
 
The DSMC approves major study modifications proposed by the study committee prior to 
implementation (eg, termination, dropping an arm based on toxicity results or other trials 
reported, increasing target sample size, etc.). The DSMC determines whether and to 
whom outcome results may be released prior to the release of study results at the time 
specified in the protocol document. 
 
 
13.0 
CYTOGENETICS ANALYSIS GUIDELINES AND REQUIREMENTS  
13.1 
Cytogenetic Analysis Overview 
It is required that specimens be sent for cytogenetic analysis at the time of initial 
diagnosis and at relapse (if applicable). These require submission of karyotypes, any 
applicable FISH images and corresponding forms for central review. Additionally 
specimens should be sent for cytogenetic analysis pre-HCT if there was a delay of 
30 days or more between initial diagnosis and HCT, or if the patient received any pre-
HCT chemotherapy. Full central review of karyotypes, FISH images and forms from pre-
HCT studies is required ONLY for those cases in which there is a change in cytogenetic 
status from the initial diagnostic study. If pre-HCT cytogenetic studies reveal the same 
results as those of initial diagnosis, only the forms (G-banding and FISH) need to be 
submitted. Day +30 and Day +90 (following HCT infusion date) must be submitted for 
those patients who had an abnormal clone identified by cytogenetics at initial diagnosis. 
Cytogenetic studies must be performed by a COG-approved laboratory.  
 
The institutional CRA must inform the cytogenetics laboratory that the patient is 
anticipated to enroll in a COG JMML study and that the cytogenetics/FISH data must be 
submitted within 2 weeks after enrollment on the ASCT1221 protocol.  
 
13.2 
Local Cytogenetic Analysis and Central Review Submission Guidelines 
 
13.2.1 Specimen Collection for Local Cytogenetics Analysis 
A minimum of 2 mL (optimal volume, 3 mL) of fresh whole bone marrow 
aspirated through a needle into a syringe containing sodium heparin 
(preservative-free is preferable) is recommended in all cases. A first or second 
draw, or a draw from a repositioned needle, is best to ensure a sufficient number 
of leukemic cells in the aspirate. The specimens should be kept at room 
temperature and transported to the institutional cytogenetics laboratory as 
quickly as possible (always within 24 hours of collection). It is critical for the 
laboratory to know that the specimen is for a COG registered patient, the 
registration number of the patient, and the COG study on which the patient is 
enrolled. 
 
If bone marrow cannot be aspirated, a core biopsy, or peripheral blood can be 
submitted. These are much less favorable for diagnosis of a clone than is a bone 
marrow aspirate, and should only be sent in exceptional situations. 
 
13.2.2 Data Submission for Central Cytogenetics Review at Diagnosis and Relapse 
Submit the following for central review following completion of local 
cytogenetic studies: 
 
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- 
The COG G-banding Reporting Form along with two G-banded karyotypes 
(and their associated metaphase spreads) for each clone identified. 
 
- 
The COG FISH Reporting Form along with images of two cells 
demonstrating any abnormal FISH pattern observed. This protocol requires 
only a single FISH assay: a dual probe set (for example, CEP7, D7S486) for 
monosomy 7 that has been validated by the Clinical Laboratory. The probe 
set must include a probe to the centromere of chromosome 7 (CEP 7). 
 
In addition, BCR/ABL1 testing is required, either by PCR or FISH. If PCR has 
not been performed, then the cytogenetics laboratory is required to perform FISH 
testing utilizing a probe set validated in the clinical laboratory. 
 
*NOTE:  Some laboratories will perform additional FISH testing as part of the 
diagnostic work-up. All FISH testing should be recorded on the COG FISH 
Reporting Form. Only abnormal FISH results need be documented with images 
for review. 
 
Use the COG Microarray Reporting Form for results of genomic microarray 
(SNP or array CGH). 
 
* NOTE:  This protocol does NOT require microarray testing by the local 
laboratory. However, if such testing was performed as part of the diagnostic 
work-up, results should be summarized on the Microarray Reporting Form and 
submitted for central review. 
 
13.2.3 Data Submission for Central Cytogenetics Review  
Pre-HCT cytogenetics is performed locally if: 1) the patient received pre-
enrollment chemotherapy, or 2) there was a delay between initial diagnosis and 
HCT of ≥ 30 days. Full central review of karyotypes, FISH images and forms 
from pre-HCT studies is required ONLY for those cases in which there is a 
change in cytogenetic status from the initial diagnostic study. If pre-HCT 
cytogenetic studies reveal the same results as those of initial diagnosis, only the 
forms (G-banding and FISH) need to be submitted.   
 
For patients with an abnormal cytogenetics clone at initial diagnosis (or pre-HCT 
as described above), Day +30 and +90 samples must be submitted for local 
cytogenetics analysis. Central review materials must be submitted if the 
abnormal clone is shown to have evolved.  
 
 
13.2.4 Central Cytogenetics Review Submission Guidelines 
Label all documents with the patient’s COG ID number and the protocol ID 
number. 
 
Please send above materials by e-mail (preferably as a PowerPoint file) to one of 
the following COG Cytogenetics Reviewers:  
 
WEST OF MISSISSIPPI RIVER  
(Include Minnesota and Wisconsin),  
Australia, New Zealand, Western Canada  
EAST OF MISSISSIPPI RIVER  
(Exclude Minnesota and Wisconsin),  
Europe, Eastern Canada 
 
SEND TO: Betsy Hirsch, PhD 
 
SEND TO: Susana C. Raimondi, PhD, FACMG 
Telephone: (612) 273-4952/3171 
Telephone: (901) 595-3537/3536 
E-mail: hirsc003@umn.edu 
E-mail: susana.raimondi@stjude.org 
 
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14.0 
PATHOLOGY GUIDELINES AND SPECIMEN REQUIREMENTS 
This study requires a peripheral blood and bone marrow specimen to be submitted to your local 
institution to make the diagnosis of JMML. See Section 14.1 for central pathology review 
submission requirements.  
 
14.1 
Central Pathology Review Submission Guidelines 
Central review specimens must be submitted at study entry, Day +30, Day +90 and at 
relapse (if applicable). Diagnostic specimens should be submitted within 2 weeks of 
enrollment. Other timepoints should be submitted within 2 weeks of the procedure.  
 
Minimum specimen requirements for central review are as follows:  
1. 
Stained Slides (cover-slipped): 
- 
2 well-stained representative bone marrow aspirate smears 
- 
1 well-stained peripheral blood smear  
- 
1 H&E stained clot section and/or core biopsy (if performed) 
 
2. 
Unstained Slides: 
- 
2 BMA smears 
- 
4 recuts from clot section and/or core biopsy (if performed) 
 
3. 
Other H&E stained and unstained surgical specimen slides (e.g., spleen, 
others) as applicable.  
 
4. 
Copies of the following documents:  
A. local institution’s pathology reports  
B. peripheral blood counts (preferably the most recent) 
C. hemoglobin F level (from a pre-transfusion sample, if the 
patient was blood transfused) 
D. cytogenetics/molecular genetics studies 
E. immunophenotyping by flow cytometry report 
F. Copy of GM-CSF hypersensitivity report (if performed) 
G. Completed COG Specimen Transmittal Form  
 
Please label all materials (slides and reports) for central review with the patient’s surgical 
pathology identification number (SPID) and block number found on the corresponding 
report and the COG patient identification number. 
 
The above material along with contact information of contributing institutions should be 
forwarded to the Biopathology Center for central review by mail or using your 
institutional courier account. 
 
 
 
Biopathology Center – ASCT1221 
Nationwide Children’s Hospital   
700 Children's Drive, WA1340 
Columbus, OH 43205 
Phone: (614) 722-2865 
Email:BPCParaffinTeam@nationwidechildrens.org 
 
The BPC will send central pathology review materials to the study pathologist (Dr Samir 
Kahwash).   
 
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15.0 
SPECIAL STUDIES SPECIMEN REQUIREMENTS 
Specimens for mutation analysis (genotyping) should be obtained at study enrollment. The result 
of the mutation analysis will be used to support the diagnosis of JMML and in the case of a 
positive PTPN11 mutation, will serve as a variable for blocked randomization, in conjunction 
with donor type.  
 
15.1 
NIH-Funded Laboratory for Mutation Analysis at Diagnosis 
In order to provide uniform mutation analysis that is used for risk-based therapy, all 
NRAS, KRAS, CBL, and PTPN11 mutation analysis will be performed as part of the 
protocol therapy at a single site through NIH funding by the Center of Diagnostic 
Excellence for Pediatric Myeloproliferative Neoplasms at the UCSF Benioff Children’s 
Hospital in San Francisco, CA free of charge to patients enrolled onto ASCT1221. If 
samples for mutational analysis were processed at UCSF prior to patient enrollment (as 
fee for service), it is acceptable to use these results to fulfill the requirements in 
Section 4.3 (confirmation of JMML diagnosis according to Chan et al33). 
 
15.2 
NRAS, KRAS, CBL, and PTPN11 Mutation Analysis 
 
As previously described, diagnostic DNA samples from each patient will be used to 
amplify appropriate regions of the genes of interest using pyrosequencing on the Roche 
454Jr platform. The 454Jr utilizes bead-based emulsion PCR target amplification coupled 
with pyrosequencing on a PicoTiterPlate to produce up to 35 million bases of sequencing 
data per run. The JMML test panel is a target-based approach, for which the 454JrJr is 
capable of sequencing up to 70,000 amplicons of approximately 300-400bp in length.   
 
15.3 
Minimal Residual Disease (MRD) Analysis  
Specimen submission for MRD analysis is required and must be collected at the defined 
timepoints in Section 7.2.  All patients that do not have adequate specimens submitted 
will be followed based on their other clinical and laboratory criteria. Qualitative results 
prior to and post HCT will be returned to the institution.   
 
15.3.1 CLIA/CAP-Approved Laboratory for Mutant Allele Testing after Diagnosis 
In order to provide uniform MRD analysis, qualitative MRD testing in 
mononuclear cells will be performed as part of the protocol therapy the Center of 
Diagnostic Excellence for Pediatric Myeloproliferative Neoplasms at the UCSF 
Benioff Children’s Hospital in San Francisco, CA. MRD analysis will be 
performed at the timepoints listed in Section 7.2 free of charge to the patient.  
Investigators are encouraged to call the laboratory (415 514-8488).  
 
 
15.3.2 Description of MRD Analysis  
All post remission specimens will undergo the identical genotype screening as 
performed at initial diagnosis based on the genotype identified. Reporting out of 
weakly positive and strongly positive qualitative results will only be disclosed. 
Weakly positive are those results on the 454Jr sequencing platform that 
correspond to 1-14.99% mutant allele while strongly positive correspond to 
> 15% mutant allele.  Note, due to the germline nature of CBL mutations, the 
interpretation of such results is complex enough that these are not currently 
factored into the response and relapse criteria; however studying these patients 
will be critical for future trials.  
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15.4 
Specimen Collection Overview 
This section describes the sample submission requirements for the genetic 
characterization of JMML cells in the UCSF CLIA/CAP-approved laboratory as well as 
other correlative research studies. 
 
 
Table 9: Study Specimens at Enrollment  
The specimens described below are to be submitted at the time of enrollment.  
Specimen 
Required: UCSF Sample Submissions 
Optional: Banking2 
CLIA-Certified Laboratory 
(genotyping)1 
Loh Laboratory2 
COG Leukemia 
Reference Lab3 
Peripheral blood 
3 mL purple top 
3-5 mL green top5 
3-5 mL purple top5 
Bone marrow aspirate 
3 mL green top 
3-5 mL green top5 
5 mL purple top5 
Buccal swab or Oragene 
saliva kit 
X6 
 
  
Skin biopsy 
X4 
 
  
1 Label with patient name, COG ID, Date of Birth, type of specimen (BMA or PB), draw date and time. 
Samples without a name will be discarded. 
2 Label with BPC number, COG ID, collection date and time, and type of specimen (BMA or PB).   
3 Optional banking specimens will be submitted at diagnosis with appropriate consent. Label with the 
COG ID, BPC number, collection date and time, and type of specimen (BMA or PB). When submitting 
bone marrow also state collection site (L or R).  
4 A skin biopsy will be requested only if the buccal swab or saliva DNA is either inadequate or 
demonstrates an unexpected suspected germline lesion.  
5 Volume of blood or bone marrow may need to be adjusted based on the appropriate weight-based 
volumes described in Section Section 15.5. 
6 Optional parental buccal swabs to be submitted at study entry with appropriate consent. Label with 
Mother/Father (as applicable) of [patient name], COG ID, Date of Birth and type of specimen.  
 
Table 10: Study Specimens pre- and post-HCT 
Specimen 
Required: UCSF Sample Submissions 
CLIA-Certified Laboratory 
(genotyping) 1, 3 
Loh Laboratory2, 3 
Peripheral blood 
3 mL purple top 
5 mL green top4 
Bone marrow aspirate 
3 mL green top 
5 mL green top4 
1 Label with patient name, COG ID, Date of Birth, type of specimen (BMA or PB), draw date and time. 
Samples without a name will be discarded. 
2 Label with BPC number, COG ID, collection date and time, and type of specimen (BMA or PB).   
3  Required within 2 weeks prior to HCT and at defined timepoints in Section 7.2. 
4 Volume of blood or bone marrow may need to be adjusted based on the appropriate weight-based 
volumes described in Section 15.5. 
 
15.5 
Specimen Details  
The total weight-based volume of blood and bone marrow to be collected at each time 
point is provided below.  
 
Peripheral blood: 
10-15 mL for patients weighing > 10 kg  
5-10 mL for patients weighing ≤ 10 kg 
 
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Collected in tubes indicated in Tables 9 and 10 above. Mix well.  
 
Bone marrow:  
10 mL for patients weighing > 10 kg 
5 mL for patients weighing ≤ 10 kg 
 
Collected in tubes indicated in Tables 9 and 10 above. Mix well.  
 
Buccal swab: 
Use swab from local institutional genetics service or obtain from 
the CLIA-approved Diagnostic Laboratory at UCSF (call 
415-514-8488 to request a kit). Gently but firmly swab the inner 
buccal mucosa. Submit 2 swabs (at the time of enrollment only). 
The buccal swab will allow somatic versus germline mutations 
to be distinguished in some cases of JMML or NS/MPN. 
 
Oragene saliva kit:  
Use saliva kit if child is deemed mature enough to collect 
enough saliva (obtained from the CLIA-approved Diagnostic 
Laboratory at UCSF; call 415-514-8488 to request a kit).  
 
Skin biopsy 
A skin biopsy will be requested only if the buccal swab or saliva 
DNA is either inadequate or demonstrates an unexpected 
germline lesion. At the time of bone marrow aspirate/biopsy, a 
small skin sample from the exact spot that the bone marrow 
aspirate or biopsy needle is inserted is encouraged to be 
submitted. Place sample in a tube containing RPMI with fetal 
calf serum. If unavailable, the Loh laboratory will provide the 
media upon request.  
 
The following samples are optional and should be submitted at the time of enrollment, if 
patient has consented to these studies: 
 
Additional Specimens for Patients with Extramedullary Involvement (optional):  
These samples are optional and should be submitted at any point during study 
participation, if the patient has consented to submission of these materials: 
 
Skin rash biopsy 
If a skin rash is biopsied, a sample is encouraged to be 
submitted.  
 
Spleen specimen 
If a splenectomy is performed, investigators are strongly 
encouraged to submit the fresh specimen to the Loh laboratory. 
Please contact the Loh laboratory up to one week prior to 
surgery to allow adequate time to prepare to receive the 
specimen. 
 
15.6 
Specimen Shipping Details 
 
15.6.1 Shipments to the University of California, San Francisco 
Specimens for genotyping at the CLIA-certified laboratory and specimens for 
correlative biology studies at the Loh laboratory should be batched for shipment. 
Specimens must be shipped on the day of collection. Do not ship fresh specimens 
on Friday or during weekends or holidays. Shipments should arrive at the 
laboratory between Tuesday and Friday. 
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Place samples in a sterile inner container and sealed in a light-tight outer 
container. Shipment in small Styrofoam containers is preferred, to insulate 
samples from extreme temperature changes. However, shipment in standard 
biological specimen envelopes (with inner padding) is a suitable alternative. 
 
All specimens should be shipped at room temperature or cooled with a cold pack 
(not frozen) via Federal Express PRIORITY OVERNIGHT using the COG 
Federal Express Account number  
(https://members.childrensoncologygroup.org/_files/reference/FEDEXmemo.pdf). 
Include a COG specimen transmittal form.  
 
Loh laboratory 
HSE 302  
513 Parnassus Ave 
San Francisco, CA 94143 
 
 
Notify the Loh Lab: (415) 514-9389 of the time of specimen shipment to prepare 
to receive research samples. 
 
Leftover specimens will be sent to the Biopathology Center (BPC) if the patient 
has consented to specimen banking.  
 
15.6.2 Shipments to the COG Leukemia Reference Laboratory  
An optional bone marrow and peripheral blood specimen will be collected at the 
time of enrollment for specimen banking, if patient consent if obtained. 
Specimens should be shipped at room temperature or cooled with a cold pack 
(not frozen) priority overnight using the COG courier account number  
(https://members.childrensoncologygroup.org/_files/reference/FEDEXmemo.pdf).  
Include a COG specimen transmittal form with each shipment.  
 
Specimens should be sent to the following address: 
 
COG Leukemia Reference Laboratory 
Nationwide Children’s Hospital 
Protocol ASCT1221 
700 Children’s Drive, C1961 
Columbus, OH 43205 
 
Phone: (614) 722-2866 
Fax: (614) 722-2887  
Email: MGLab@nationwidechildrens.org 
 
15.7 
Description of Correlative Biology Studies 
While correlative biology studies will develop over the course of the clinical trial, a brief 
discussion of some of the intended studies are presented but will be contingent upon 
successful acquisition of independent funding. Importantly, the UCSF CLIA laboratory 
will remove any name, DOB, or COG number prior to distributing the research samples 
to the appropriate laboratories. A unique specimen identifier (USI) generated by COG 
will be assigned to the sample prior to delivery to the appropriate reference lab. In the 
Loh laboratory, aliquots of cells will have DNA and RNA selected from whole 
mononuclear and sorted cell subsets. In addition, one aliquot of cells will have 
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phosphoflow cytometry performed in increasing concentrations of GM-CSF53 and 
potentially exposed to selected drugs for assessment of novel therapies that may be used 
in the future.  
 
RNA from selected cell subsets will be archived for future gene expression studies for 
those patients participating in the HCT randomization. DNA from selected and 
unselected cell subsets will be used for the assessment of mutant allele burden using 
either pyrosequencing methodologies or allele specific PCR.51 At the current time CD3, 
CD14/15, and CD34 selection is planned for post HCT research studies. In addition, 
DNA from diagnosis will also be archived and assessed for genome wide methylation 
studies in those patients participating on the randomized arm. Together, the gene 
expression and methylation assays will be used to define classifiers of relapse, which will 
be a critical step forward in advancing care for children with JMML. DNA from selected 
cell subsets may also be subjected to additional genotyping for novel genes discovered to 
be mutated in JMML. Finally, such strategies including genome wide association studies 
may be performed on specific cell subsets or buccal or fibroblast DNA. In addition to the 
aforementioned studies, other strategies to maximize the use of this precious human 
tissue may be employed in order to gain additional reagents that will ultimately be used 
to improve outcome. Such strategies include, but are not limited to, creation of cell lines 
or induced pluripotent cells that will be used for additional genetic and biochemical 
studies. 
 
 
 
 
 
 
 
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16.0 
PHARMACOKINETICS FOR BUSULFAN DOSE ADJUSTMENT 
 
16.1 
1st Dose Pharmacokinetics 
Please note: Pharmacokinetics studies must be performed in a CLIA or NATA certified 
Laboratory. 
 
Target first dose pharmacokinetics should be performed in all patients regardless of 
busulfan dosing schedule.  
 
Once daily dosing should target an area under the curve (AUC) of 3,600 to 
6,000 (micromole/liter)*minute per dose.  
 
Every 6 hour dosing should target an AUC of 900 to 1500 (micromole/liter)*minute per 
dose. 
 
Every 12 hour dosing should target an AUC of 1800 to 3000 (micromole/liter)*minute 
per dose.  
 
Instructions for Q24 hr dosing: Busulfan concentrations will be determined in plasma by 
collecting blood into a green top sodium heparin tube. Samples should be collected at the 
end of the first 3 hour infusion, 195 minutes after start of infusion (or end of infusion plus 
15 minutes), and 4, 5, 6, and 8 hours after the start of the first infusion. Samples should 
not be drawn from the lumen used to infuse busulfan. See table in Section 7.3. 
 
Institutional pharmacokinetic studies are acceptable and sample submission 
requirements per the local laboratory should be followed. If pharmacokinetics 
cannot be performed at a local laboratory, sites may opt to use the Seattle Cancer 
Care Alliance laboratory (as fee for service) or other regional laboratories 
experienced in this assay. Results are usually available in time for adjusting doses 3 and 
4. 
 
Instructions for Q6hr dosing: Busulfan concentrations will be determined in plasma by 
collecting blood into a green top sodium heparin tube. Samples should be collected at the 
end of the first 2 hour infusion, 135 minutes after start of infusion, 150 minutes after start 
of infusion, and 3, 4, 5, and 6 hours after the start of the first infusion. Samples should 
not be drawn from the lumen used to infuse busulfan. See table in Section 7.3. 
 
In case infusion runs more or less than 2 hours, draw 1 sample immediately when 
infusion ends. Then, draw the next 2 samples 15 minutes apart and continue to draw 
samples at 3, 4, 5, and 6 hours samples by counting from beginning of the infusion.  
 
Instructions for Q12 hr dosing: Busulfan concentrations will be determined in plasma by 
collecting blood into a green top sodium heparin tube. Samples should be collected at 
1 hour following start of infusion, and then at 2 hours (at the end of the busulfan 
infusion), 3, 5, 6, 7, and 8 hours after the START of the infusion. Samples should not be 
drawn from the lumen used to infuse busulfan. See table in Section 7.3. 
 
In case infusion runs more or less than 2 hours, draw 1 sample immediately when 
infusion ends and another 15 minutes later. Then, draw the next samples as scheduled at 
3, 5, 6, 7, and 8 hours after the START of the infusion.  
 
Sample collection instructions for institutions that opt to use the SCCA Pharmacokinetics 
Laboratory are as follows: 
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Collect 1-3 mL of blood into sodium heparin tubes (green top) according to the schedule 
above. Place labeled samples immediately on wet ice and refrigerate. Centrifuge samples 
as soon as possible at 4oC. Separate plasma from RBCs. Store plasma at –20°C. Plasma 
tubes must be labeled with the patient’s name, medical records number, and the date and 
actual clock time that the sample was drawn. If using the SCCA Pharmacokinetics 
Laboratory, sample requisition form, collection schedule, and instructions can also be 
found at: http://www.seattlecca.org/busulfan-lab-samples.cfm. Sites will be required to 
set up an institutional account with the SCCA PK laboratory for billing.  
 
16.2 
Shipping 
For institutions that opt to use the SCCA Pharmacokinetics Laboratory, samples must be 
shipped on a minimum of 3 kg of dry ice the day they were drawn using an urgent 
overnight carrier. Please include IV Busulfex PK requisition form and ship using your 
institutional courier account. 
 
Send samples to: 
Pharmacokinetics Laboratory  
Seattle Cancer Care Alliance 
Room G7-405  
825 Eastlake Ave. East 
Seattle, WA 98109 
Tel: (206) 288-7389 
Fax: (206) 288-7397   
Email: pklab@seattlecca.org 
 
Note: Notify the lab staff of sample shipment at least 48 hours prior to arrival. 
Samples can be received Tuesday through Friday, or on Saturdays with at least 
3 days advance notice. Coordinate the taking of samples so that delivery will occur 
Tuesday through Saturday and there will be time for a possible dose adjustment. 
Thus, the dose must be administered to the patient Monday through Friday. Please 
provide the lab with a tracking number for the package at the email (preferred) or 
phone listed above.   
 
Provide fax and phone numbers for the attending physician or pharmacist so that 
results can be verbally reported and faxed in time for targeted dose adjustment. 
 
16.3 
Guidelines for Adjusting Busulfan Dosing Based on Results of First Dose 
Pharmacokinetic Results 
Once the results of the area under the curve (AUC) analysis of the first busulfan dose are 
available, subsequent doses will be adjusted to achieve an overall exposure target AUC of 
3600-6000 (micromole/liter)*minute (or as listed in Section 16.1 if using q6 hour or q12 
hour dosing). The amount by which the dose is increased or decreased should be decided 
by the patient’s attending physician in conjunction with the institutional toxicology 
laboratory director, or the Seattle Cancer Care Alliance Pharmacokinetics Laboratory 
(see above). 
 
In cases where the pharmacokinetics are performed locally, a second AUC analysis 
should be performed after administration of the first modified dose, and further dose 
adjustments made accordingly, whenever feasible. 
 
 
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APPENDIX I: JMML GENE MUTATIONS 
List of reported mutations in CBL/KRAS/NRAS/PTPN11 for JMML patients 
gene 
chr 
exon 
cDNA coordinates 
nt change 
AA change 
notes/het or hom 
CBL 
11 
7 c. 1096-1G>C 
1096-1G>C 
splice site variant 
Homo 
CBL 
11 
8 c. 1096-1G>C 
1096-1G>C 
splice site variant 
Homo 
CBL 
11 
8 c. 1106_1171del 
1106 del 
splice site variant 
Homo 
CBL 
11 
8 c. 1111T>C 
1111T>C 
Y371H 
Homo 
CBL 
11 
8 c. 1111T>C 
1111T>C 
Y371N 
Homo 
CBL 
11 
8 c. 1111T>G 
1111T>G 
Y371D 
Homo 
CBL 
11 
8 c. 1112A>G 
1112A>G 
Y371C 
Homo 
CBL 
11 
8 c. 1112A>C 
1112A>C 
Y371S 
Homo 
CBL 
11 
8 c. 1139T>C 
1139T>C 
L380P 
Homo 
CBL 
11 
8 c. 1141T>C 
1141T>C 
C381R 
Homo 
CBL 
11 
8 c. 1150T>C 
1150T>C 
C384R 
Homo 
CBL 
11 
8 c. 1150T>G 
1150T>G 
C384G 
Homo 
CBL 
11 
8 c. 1186T>C 
1186T>C 
C396R 
Homo 
CBL 
11 
8 c. 1190_1199del 
1190 del 99  
deletion 
Homo 
CBL 
11 
8 c. 1202G>C 
1202G>C 
C401S 
homo 
CBL 
11 
8 c. 1210T>C 
1210T>C 
C404R 
Homo 
CBL 
11 
8 c. 1222T>C 
1222T>C 
W408R 
Homo 
CBL 
11 
8 c. 1227+4C>T 
1227+4C>T 
splite site variant 
Homo 
CBL 
11 
8 c. 1228-2A>G 
1228-2A>G 
splice site variant 
Homo 
CBL 
11 
9 c. 1244G>T 
1244G>T 
G415V 
Homo 
CBL 
11 
9 c. 1254C>G 
1254C>G 
F418L 
Homo 
PTPN11 
12 
3 c. 155C>G 
155C>G 
T52S 
hetero 
PTPN11 
12 
3 c. 178G>C 
178G>C 
G60R 
hetero 
PTPN11 
12 
3 c. 179G>T 
179G>T 
G60V 
hetero 
PTPN11 
12 
3 c. 179_181delGTG 
179delGTG 
del60G 
hetero 
PTPN11 
12 
3 c. 181G>T 
181G>T 
D61Y 
hetero 
PTPN11 
12 
3 c. 181G>A 
181G>A 
D61N 
hetero 
PTPN11 
12 
3 c. 182A>T 
182A>T 
D61V 
hetero 
PTPN11 
12 
3 c. 182A>G 
182A>G 
D61G 
hetero 
PTPN11 
12 
3 c. 205G>A 
205G>A 
E69K 
hetero 
PTPN11 
12 
3 c. 214G>A 
214G>A 
A72T 
hetero 
PTPN11 
12 
3 c. 215C>T 
215C>T 
A72V 
hetero 
PTPN11 
12 
3 c. 215C>G 
215C>G 
A72G 
hetero 
PTPN11 
12 
3 c. 218C>T 
218C>T 
T73I 
hetero 
PTPN11 
12 
3 c. 226G>A 
226G>A 
E76K 
hetero 
PTPN11 
12 
3 c. 226G>C 
226G>C 
E76Q 
hetero 
PTPN11 
12 
3 c. 227A>T 
227A>T 
E76V 
hetero 
PTPN11 
12 
3 c. 227A>G 
227A>G 
E76G 
hetero 
PTPN11 
12 
3 c. 227A>C 
227A>C 
E76A 
hetero 
PTPN11 
12 
3 c. 226_227delGAinsAT 
226-227GA>AT 
E76M 
hetero 
PTPN11 
12 
4 c. 417G>C 
417G>C 
E139D 
hetero 
PTPN11 
12 
13 c. 1505C>T 
1505C>T 
S502L 
hetero 
PTPN11 
12 
13 c. 1508G>C 
1508G>C 
G503A 
hetero 
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PTPN11 
12 
13 c. 1508G>T 
1508G>T 
G503V 
hetero 
PTPN11 
12 
13 c. 1508G>A 
1508G>A 
G503E 
hetero 
PTPN11 
12 
13 c. 1517A>C 
1517A>C 
Q506P 
hetero 
KRAS 
12 
1 c. 34G>A 
34G>A 
G12S 
hetero 
KRAS 
12 
1 c. 34G>T 
34G>T 
G12C 
hetero 
KRAS 
12 
1 c. 34G>C 
34G>C 
G12R 
 
KRAS 
12 
1 c. 35G>A 
35G>A 
G12D 
hetero 
KRAS 
12 
1 c. 35G>T 
35G>T 
G12V 
hetero 
KRAS 
12 
1 c. 37G>T 
37G>T 
G13C 
hetero 
KRAS 
12 
1 c. 38G>A 
38G>A 
G13D 
hetero 
KRAS 
12 
2 c. 90C>T 
90C>T 
V34L 
synonmous SNP 
KRAS 
12 
2 
c. 112-12C>T  
112-12C>T 
(intronic) 
 
 
KRAS 
12 
2 c. 182A>T 
182A>T 
Q61L 
hetero 
KRAS 
12 
2 c. 182A>C 
182A>C 
Q61P 
hetero 
NRAS 
1 
1 c. 34G>T 
34G>T 
G12C 
hetero 
NRAS 
1 
1 c. 34G>A 
34G>A 
G12S 
hetero 
NRAS 
1 
1 c. 35G>A 
35G>A 
G12D 
hetero 
NRAS 
1 
1 c. 35G>T 
35G>T 
G12V 
hetero 
NRAS 
1 
1 c. 35G>C 
35G>C 
G12A 
hetero 
NRAS 
1 
1 c. 36G>C 
36G>C 
G13R 
hetero 
NRAS 
1 
1 c. 37G>T 
37G>T 
G13C 
hetero 
NRAS 
1 
1 c. 38G>A 
38G>A 
G13D 
hetero 
NRAS 
1 
2 c. 181C>A 
181C>A 
Q61K 
hetero 
NRAS 
1 
2 c. 181A>T 
181A>T 
Q61L 
hetero 
NRAS 
1 
2 c. 182A>G 
182A>G 
Q61R 
hetero 
NRAS 
1 
2 c. 183A>C 
183A>C 
Q61H 
hetero 
NRAS 
1 
2 c. 183A>T 
183A>T 
Q61H 
hetero 
NRAS 
1 
2 c. 183A>G 
183A>G 
Q61P 
hetero 
 
 
 
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APPENDIX II: WHO CRITERIA FOR NEUROFIBROMITOSIS-1  
The World Health Organization (WHO) defines neruofibromitosis-1 (NF-1) as two or more of the 
following:64 
 
1. Six or more café-au-lait spots 1.5 cm or larger in post-pubertal individuals, 0.5 cm or larger in pre-
pubertal individuals 
2. Two or more neurofibromas of any type or one or more plexiform neurofibroma 
3. Freckling in the axilla or groin 
4. Optic glioma (tumor of the optic pathway) 
5. Two or more Lisch nodules (benign iris hamartomas) 
6. A distinctive bony lesion: dysplasia of the sphenoid bone or dysplasia or thinning of long bone cortex 
7. A first-degree relative with NF1 
 
 
 
 
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APPENDIX IIIA: DRUGS KNOWN TO MODULATE THE ACTIVITY OF CYP450 
ISOENZYMES 3A4, CYP2C19 AND CYP1A2 
Note: The use of the agents listed below should be limited due to concerns of drug interactions.  
Avoid using these medications if possible. Every attempt should be made to switch from the medications marked 
with an asterisk (*) to an acceptable alternative. Anticonvulsants that induce CYP3A4/5 are not allowed (**). This 
may not be a complete list; refer to http://medicine.iupui.edu/clinpharm/ddis/ClinicalTable.aspx for additional 
information. 
 
 
CYP 3A4 
Inhibitors 
CYP3A4 
Inducers 
CYP 2C19 
Inhibitors 
CYP 2C19 
Inducers 
CYP 1A2 
Inhibitors 
CYP 1A2 
Inducers 
amiodarone* 
X 
 
 
 
X 
 
aprepitant 
X 
 
 
 
 
 
barbiturates 
 
X 
 
 
 
 
carbamazepine** 
 
X 
 
 
 
 
chloramphenicol 
X 
 
 
 
 
 
cimetidine* 
X 
 
X 
 
X 
 
ciprofloxacin 
 
 
 
 
X 
 
clarithromycin* 
X 
 
 
 
 
 
diethylthiocarbamate 
X 
 
 
 
 
 
diltiazem 
X 
 
 
 
 
 
erythromycin* 
X 
 
 
 
 
 
felbamate** 
 
X 
 
 
 
 
fluconazole 
X 
 
 
 
 
 
fluoroquinolones 
 
 
 
 
X 
 
fluoxetine* 
 
 
X 
 
 
 
fluvoxamine* 
X 
 
X 
 
X 
 
gestodene 
X 
 
 
 
 
 
grapefruit juice* 
X 
 
 
 
 
 
Imatinib 
X 
 
 
 
 
 
itraconazole* 
X 
 
 
 
 
 
ketoconazole* 
X 
 
X 
 
 
 
lansoprazole 
 
 
X 
 
 
 
mibefradil*   
X 
 
 
 
 
 
mifepristone 
X 
 
 
 
 
 
modafinil 
 
X 
 
 
 
 
nefazodone* 
X 
 
 
 
 
 
norfloxacin 
X 
 
 
 
 
 
norfluoxetine 
X 
 
 
 
 
 
omeprazole 
 
 
X 
 
 
 
oxcarbazepine** 
 
X 
X 
 
 
 
pantoprazole 
 
 
X 
 
 
 
pioglitazone 
 
X 
 
 
 
 
phenobarbital** 
 
X 
 
 
 
 
phenytoin** 
 
X 
 
 
 
 
posaconazole* 
X 
 
 
 
 
 
primidone** 
 
X 
 
 
 
 
rifampin* 
 
X 
 
 
 
 
star fruit 
X 
 
 
 
 
 
St. John's wort* 
 
X 
 
 
 
 
telithromycin* 
 
X 
 
 
 
 
ticlopidine 
 
 
X 
 
X 
 
tobacco 
 
 
 
 
 
X 
troglitazone* 
 
X 
 
 
 
 
verapamil*   
X 
 
 
 
 
 
voriconazole 
X 
 
 
 
 
 
 
 
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APPENDIX IIIB: LIST OF ANTICONVULSANTS BASED ON CYP3A4/5 ENZYME 
INDUCTION 
 
 
Anticonvulsant drugs with little or no enzyme induction: ELIGIBLE 
 
Generic Name 
Trade Name 
Gabapentin 
Neurontin 
Lamotrigine 
Lamictal 
Levetiracetam  
Keppra 
Tigabine 
Gabitril 
Topiramate 
Topamax 
Valproic Acid 
Depakote, Depakene 
Zonisamide 
Zonegran 
 
Enzyme inducing anticonvulsant drugs (EIACD): NOT ELIGIBLE 
 
Generic Name 
Trade Name 
Carbamazepine 
Tegretol 
Felbamate 
Felbatol 
Phenobarbital 
Phenobarbital 
Phenytoin 
Dilantin 
Primidone 
Mysoline 
Oxcarbazepine 
Trileptal 
 
 
 
 
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APPENDIX IV: PRE-TRANSPLANT RECOMMENDATIONS 
Pre-transplant chemotherapy for JMML has not demonstrated any benefit upon EFS or OS. AAML0122 
evaluated the efficacy of a pre-transplant window therapy with a farnesyl transferase inhibitor followed 
by two cycles of intensive AML-type chemotherapy. The unpublished 2-year EFS from COG 
AAML0122 was 40% and 2-year OS was 55%. The EWOG-MDS/EBMT JMML trial also demonstrated 
that no matter what type of therapy patients had prior to HCT (no therapy and low-dose chemotherapy vs. 
high-dose chemotherapy) the 5-year EFS (52% vs. 50%), RI (35% vs. 38%), and TRM (13% vs. 13%) 
were nearly identical.4 However, it is difficult to interpret this data, given that patients with higher disease 
burdens were likely more intensively pre-treated. Thus, given the heterogeneity of disease burden at 
diagnosis in children with JMML, it is not feasible to be prescriptive about specific pre-HCT therapies.  
 
Pre-transplant splenectomy is also controversial. The previous COG trial for JMML mandated 
splenectomy for all children prior to HCT. However, the EWOG-MDS/EBMT JMML trial demonstrated 
that a comparison of children with: 1) a spleen size < 5 cm at HCT;  or 2) spleen > 5 cm at HCT; or 3) 
post-splenectomy at HCT, showed no statistical benefit in event-free survival (5-year EFS; 61% vs. 44% 
vs. 48%, respectively), relapse incidence (5-year RI, 24% vs. 45% vs. 39%, respectively), or treatment-
related mortality (5-year TRM; 15% vs. 11% vs. 13%, respectively).4 Therefore, splenectomy is not 
mandated prior to HCT unless clinically indicated for symptomatic relief.  
 
There is no data to suggest that a lifetime or yearly maximum amount of fludarabine is required to safely 
administer without the risk of neurotoxicity, and so pre-HCT fludarabine is allowed regardless of eventual 
treatment assignment. Finally, there may be targeted therapies against JMML available on a clinical trial, 
and their use would be allowed prior to but not following HCT, unless a post-HCT relapse or progression 
was to occur. 
 
The following pre-HCT guidelines are provided for physician consideration. 
 
1. Splenectomy is not mandated but may be performed at the discretion of the clinician for control 
of severe hypersplenism and organomegaly.   
 
2. For asymptomatic patients, no pre-transplant therapy is recommended. 
 
3. For patients with very high WBC counts, pulmonary problems, and/or prominent organomegaly, 
oral 6-mercaptopurine (50 mg/m2/day) +/- cis-retinoic acid (100 mg/m2/day) is recommended. 
 
4. For severely ill children, low-dose intravenous cytarabine (40 mg/m2/day x 5 days) administration 
is recommended. If this fails to improve the patient’s status, the combination of high-dose 
cytarabine (2 g/m2/day x 5 days) plus fludarabine (30 mg/m2/day x 5 days) may also be utilized.  
 
5. Chloromas may be treated with radiotherapy per physician discretion. 
 
Patients must have fully recovered from the acute toxic effects of all prior chemotherapy, immunotherapy, 
or radiotherapy prior to start of protocol therapy (HCT conditioning). Specific prior therapy wash-out 
periods are detailed in Section 4.3.2. 
 
 
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APPENDIX V: YOUTH INFORMATION SHEETS 
 
INFORMATION SHEET REGARDING RESEARCH STUDY 
(for children from 7 through 12 years of age) 
 
ASCT1221 
 
1. We have been talking with you about a condition you may have called Juvenile Myelomonocytic 
Leukemia (JMML). JMML is a type of cancer that grows in the center of your bones. The center 
of your bones is called bone marrow. After doing tests, we think you may have this type of 
cancer. 
 
2. We are asking you to take part in a research study because you may have JMML. A research 
study is when doctors work together to try out new ways to help people who are sick. In this 
study, we are trying to learn more about how to treat JMML.  
 
3. Children who are part of this study will first have some tests to check that JMML is the correct 
condition. If the condition of JMML is confirmed, children will be treated with a stem cell 
transplant. The first part of a stem cell transplant involves treatment with cancer-fighting drugs. 
Then, the patient is given new stem cells from a healthy donor. A donor is a healthy person who 
gives their stem cells to a person who needs a stem cell transplant. Children on this study will be 
treated with one of two types of stem cell transplant. Study doctors want to see which stem cell 
transplant works better in children with JMML. We do not know which one will be better. That is 
why we are doing this study.   
 
4. Sometimes good things can happen to people when they are in a research study. These good 
things are called “benefits.” A benefit to you of being part of this study may be having a 
transplant that works better than another kind of transplant. However, we don’t know for sure if 
there is any benefit of being part of this study. 
 
5. Sometimes bad things can happen to people when they are in a research study. These bad things 
are called “risks.” The risks to you from this study are receiving a stem cell transplant that does 
not work as well or causes more side effects than another type of stem cell transplant. Other 
things may happen to you that we don’t yet know about. 
 
6. Your family can choose to be part of this study or not. Your family can also decide to stop being 
in this study at any time once you start. There may be other treatments for your illness that your 
doctor can tell you about. Make sure to ask your doctors any questions that you have. 
 
7. Children who are part of the study will have extra bone marrow, blood, saliva and possibly other 
samples collected for testing. We want to learn more about JMML and see if there are ways to tell 
how the cancer will respond to treatment. These samples would be taken when other standard 
blood and bone marrow tests are being performed, so there would be no extra procedures. 
 
 
 
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INFORMATION SHEET REGARDING RESEARCH STUDY 
(for teens from 13 through 17 years of age) 
 
ASCT1221 
 
1. We have been talking with you about a condition you may have called Juvenile Myelomonocytic 
Leukemia (JMML). JMML is a type of cancer that grows in the center of your bones. The center 
of your bones is called bone marrow. After doing tests, we think you may have this type of 
cancer. 
 
2. We are asking you to take part in a research study because you may have JMML. A research 
study is when doctors work together to try out new ways to help people who are sick. In this 
study, we are trying to learn more about how to treat JMML.  
 
3. Children and teenagers that are part of this study will first have some tests performed to check the 
JMML diagnosis. If the diagnosis is confirmed, children and teenagers will be treated with a stem 
cell transplant. The first part of a stem cell transplant involves treatment with cancer-fighting 
drugs. Then, the patient is given new stem cells from a healthy donor. A donor is a healthy person 
who gives their stem cells to a person who needs a stem cell transplant. Children and teenagers on 
this study will be treated with one of two types of stem cell transplant. The stem cell transplant 
you get will be decided by chance, like flipping a coin for “heads” or “tails”. Study doctors want 
to see which stem cell transplant works better for children and teenagers with JMML. We do not 
know which one will be better. That is why we are doing this study.   
 
4. Sometimes good things can happen to people when they are in a research study. These good 
things are called “benefits.” A benefit to you of being part of this study may be having a 
transplant that works better than another kind of transplant. However, we don’t know for sure if 
there is any benefit of being part of this study. 
 
5. Sometimes bad things can happen to people when they are in a research study. These bad things 
are called “risks.” The risks to you from this study are receiving a stem cell transplant that does 
not work as well or causes more side effects than another type of stem cell transplant. Other 
things may happen to you that we don’t yet know about. 
 
6. Your family can choose to be part of this study or not. Your family can also decide to stop being 
in this study at any time once you start. There may be other treatments for your illness that your 
doctor can tell you about. Make sure to ask your doctors any questions that you have.  
 
7. Children and teenagers that are part of the study will have extra bone marrow, blood, saliva and 
possibly other samples collected for testing. We want to learn more about JMML and see if there 
are ways to tell how the cancer will respond to treatment. These samples would be taken when 
other standard blood and bone marrow tests are being performed, so there would be no extra 
procedures. 
  
 
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