Clinical trial NCT02927938
Conditions: Acute Myeloid Leukemia (AML)
Sponsor: Wake Forest University Health Sciences
Start date: 2016-09-01; Completion date: 2018-10-05
Enrollment: 18 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Most patients with acute myeloid leukemia (AML) achieve complete remission (CR) following induction chemotherapy. However, a large majority subsequently relapse and succumb to the disease. Currently, cytogenetics and molecular aberrations are the best prognostic indicators; however, these factors cannot prognosticate accurately for individual patients. Overall, the majority of patients with favorable or intermediate-risk AML will experience relapse. Prognosis after relapse is dismal with a five-year overall survival rate of less than 10%. A leukemia stem cell (LSC) paradigm may explain this failure of CR to reliably translate into cure. This study is undertaken to determine whether the presence of LSCs has prognostic value as well as to determine whether the presence of LSCs has predictive value. This study has an observational component, whereby we intent evaluate whether the presence or absence of LSCs is prognostic. This study also has an interventional component in which it uses LSC status to determine whether favorable and intermediate risk AML patients in CR receive consolidation with chemotherapy or allogeneic HCT.

Detailed description:

Arms: arm 1: Other - Standard of Care Consolidation (HCT)

Enrolled subjects that will contribute to the population of subjects who are evaluable for the primary and secondary objectives. This will not include any subjects who end up in either observational cohort. To be included in the evaluable cohort, the subject must meet the following requirements:

1. Complete remission (CR1) from standard cytarabine or HMA-based induction therapy per standard clinical criteria (Cheson Criteria)
2. Have confirmed presence of CD34+CD38-ALDHint population by flow cytometry at the diagnostic LSC assay (LSC0)
3. Cytarabine-based induction subjects: Are candidates for (as determined by the investigator) and receive consolidation therapy (cytarabine-based chemotherapy or HCT); HMA-based induction subjects: Are candidates for (as determined by the investigator) and receive HCT arm 2: Other - Standard of Care Consolidation (cytarabine-based chemo)

Enrolled subjects that will contribute to the population of subjects who are evaluable for the primary and secondary objectives. This will not include any subjects who end up in either observational cohort. To be included in the evaluable cohort, the subject must meet the following requirements:

1. Complete remission (CR1) from standard cytarabine or HMA-based induction therapy per standard clinical criteria (Cheson Criteria)
2. Have confirmed presence of CD34+CD38-ALDHint population by flow cytometry at the diagnostic LSC assay (LSC0)
3. Cytarabine-based induction subjects: Are candidates for (as determined by the investigator) and receive consolidation therapy (cytarabine-based chemotherapy or HCT) arm 3: Enrolled subjects who do not achieve a CR to induction therapy, regardless of diagnostic phenotype. Following completion of induction therapy and remission bone marrow aspirate, if a subject is determined to not have achieved a complete remission to induction therapy, he or she would be included in observational cohort 1. arm 4: Enrolled subjects who achieve a CR to induction therapy but meet one or more of the following criteria:

* Lack the immunophenotype of interest,
* Cytarabine based induction subjects: Are not candidates for \[as determined by the investigator (e.g. unfit or refusal)\] and do not receive consolidation therapy (cytarabine-based chemotherapy or HCT)
* HMA-based induction subjects: Are not candidates for \[as determined by the investigator (e.g. unfit, lack of donor, refusal)\] and do not receive HCT

Final investigator determination of fit-ness can occur at any time until the start of consolidation therapy.

HMA-based induction subjects will not receive consolidation cytarabine-based chemotherapy as part of the evaluable cohort if they do not receive HCT.
Interventions: intervention 1: Allogeneic HCT intervention 2: Consolidation chemotherapy
Intervention descriptions: intervention 1: Allogeneic HCT intervention 2: Cytarabine-based consolidation chemotherapy

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Number:  LCI-HEM-AML-SCD-001 
Version 2: 2/27//2017   
 
 
 
Protocol #:  LCI-HEM-AML-SCD-001 
TITLE: LEUKEMIA STEM CELL DETECTION IN ACUTE MYELOID LEUKEMIA  
LAY TITLE: STEM CELL DETECTION FOR PATIENTS WITH ACUTE MYELOID 
LEUKEMIA 
Coordinating Site: 
 
Levine Cancer Institute (LCI) 
 
Research and Academic Headquarters 
1021 Morehead Medical Drive 
Charlotte, NC 28204 
 
Sponsor-Investigator: 
Michael R. Grunwald, M.D.  
1021 Morehead Medical Drive, Suite 5300 
Charlotte, NC 28204 
Phone: (980) 442-5125 
Email: Michael.Grunwald@carolinashealthcare.org     
 
 
Statistician: 
James Symanowski, PhD 
1100 Blythe Blvd, Suite 1166 
Charlotte, NC 28203  
Telephone: (980) 442-2348 
Email: James.Symanowski@carolinashealthcare.org  
 
 
Myra Robinson, MSPH 
1100 Blythe Blvd, Suite 1166 
Charlotte, NC 28203  
Telephone: (980) 442-2390 
Email: Myra.Robinson@carolinashealthcare.org  
 
Investigational drug/device: 
N/A 
 
 
 
 


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Version 2: 2/27/2017   
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______________________________________________________________________________ 
The study will be conducted in compliance with the protocol, ICH-GCP and any applicable 
regulatory requirements.  
Confidential 
The information provided in this document is strictly confidential and is intended solely for the 
guidance of the clinical investigation.  Reproduction or disclosure of this document - whether in 
part or in full - to parties not associated with the clinical investigation, or its use for any other 
purpose, without the prior written consent of the Sponsor-Investigator is not permitted. 
Throughout this document, symbols indicating proprietary names (, TM) are not displayed.  
Hence, the appearance of product names without these symbols does not imply that these names 
are not protected. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Protocol Number:  LCI-HEM-AML-SCD-001 
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Original Phase 3 / Version 2 / February 27, 2017 
PROTOCOL SUMMARY 
A. Study Title 
Leukemia stem cell detection in acute myeloid leukemia 
B. Indication 
Acute myeloid leukemia 
C. Clinical Phase 
III 
 
D. Summary of 
Rationale 
 
 
Most patients with AML achieve CR following induction chemotherapy. 
However, a large majority subsequently relapse and succumb to the 
disease. Currently, cytogenetics and molecular aberrations are the best 
prognostic indicators; however, these factors cannot prognosticate 
accurately for individual patients.  
 
Overall, the majority of patients with favorable or intermediate-risk AML 
will experience relapse. Prognosis after relapse is dismal with a five-year 
overall survival rate of less than 10%. A LSC paradigm may explain this 
failure of CR to reliably translate into cure. 
E. Study Objectives 
The primary endpoint is to compare the two-year relapse-free survival 
(RFS) rates of those with and without detectable LSCs at eLSC 
assessment.  
 
The secondary endpoints are to compare the one-year RFS, two-year 
RFS, and overall survival (OS) between different AML groups with and 
without detectable LSCs at LSC1/eLSC. 
 
Safety objectives include the evaluation of graft-versus-host-disease 
timing (acute vs chronic), incidence, and grade/severity in subjects 
undergoing allogeneic hematopoietic cell transplant (HCT). 
 
Exploratory objectives include evaluation of clinical outcomes of subjects 
who are enrolled to the study but do not meet the criteria to be included in 
the evaluable population.  These subjects will be included in one of the 
two following observational cohorts:   
 
Observational cohort 1 (OC1):  enrolled subjects who do not achieve a 
CR to induction therapy 
 
Observational cohort 2 (OC2):  enrolled subjects who achieve a CR to 
induction therapy but meet one or more of the following criteria: 
• Lack the immunophenotype of interest (CD34+CD38-ALDHint ) by 
flow cytometry at the diagnostic LSC assay (LSC0) 
• Cytarabine based induction subjects: Are candidates for (as 
determined by the investigator) and do not receive consolidation 
therapy  


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HMA-based induction subjects: Are not candidates for (as determined 
by the investigator) do not receive HCT 
 
F. Sample 
200 evaluable (for the primary objective) subjects and estimated 300-500 
total including observational subjects. 
 
 
G. Inclusion/ 
Exclusion  
 
 
 
Inclusion: 
• At least 18 years of age 
• New diagnosis of AML, other than APL, confirmed by bone 
marrow aspirate/biopsy  
• Have completed induction therapy and post-induction bone 
marrow biopsy. 
Exclusion: 
• Any illness that would preclude ability to follow study procedures 
• Indeterminate results at LSC0 
I. Statistical Analysis 
The primary endpoint of this study is two-year relapse-free survival 
(RFS) of subjects with acute myeloid leukemia (AML).  Subjects with 
detectable leukemia stem cells (LSCs) in their bone marrow at end of 
consolidation (eLSC+) following the time of achieving first complete 
remission (CR1) will be compared to the two-year RFS of patients 
without detectable LSCs (eLSC-).  RFS is defined as the duration of time 
from the initial CR date (enrollment date) to the date of relapse, as 
determined by the Cheson criteria.  
 


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SCHEMA 
 
 
 


Protocol Number:  LCI-HEM-AML-SCD-001 
Version 2: 2/27/2017   
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Protocol Number:  LCI-HEM-AML-SCD-001 
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LIST OF ABBREVIATIONS 
AE 
Adverse event 
ALDH 
Aldehyde dehydrogenase 
AML 
Acute myeloid leukemia 
APL 
Acute promyelocytic leukemia 
CBF 
Core-binding factor 
CFR 
Code of Federal Regulations 
CML 
Chronic myeloid leukemia 
CR 
Complete response 
CR(1) 
First complete response 
CR(2) 
Second complete response 
CRF 
Case report form 
CSC 
Cancer stem cell 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTMS 
Clinical trial management system 
DSMC 
Data and Safety Monitoring Committee 
DSMP 
Data and Safety Monitoring Plan 
eCRF 
Electronic case report form 
FDA 
Food and Drug Administration 
FISH 
Fluorescence in situ hybridization 
GCP 
Good Clinical Practice 
GVHD 
Graft versus host disease 
HCT 
Hematopoietic cell transplant 
HMA 
Hypomethylating agent   
IRB 
Institutional Review Board 
LCI 
Levine Cancer Institute 
LSC 
Leukemic stem cell 
LSC0 
Leukemic stem cell status pre induction therapy 
LSC1 
Leukemic stem cell status post induction therapy 
eLSC 
Leukemic stem cell status at end of consolidation therapy  
LSC6 
Leukemic stem cell status at six months post day 0 of HCT 
MDS 
Myelodysplastic syndrome 
MRD 
Minimum residual disease 
MPN 
Myeloproliferative neoplasm 
MUD 
Matched unrelated donor 
OS 
Overall survival 
RFS 
Relapse-free survival 
PCR  
Polymerase chain reaction 
SAE 
Serious adverse event 
SI 
Sponsor-Investigator 
SOP 
Standard operating procedure 
NCI 
National Cancer Institute 


Protocol Number:  LCI-HEM-AML-SCD-001 
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t-AML 
Therapy-related acute myeloid leukemia 
VOD 
Veno-occlusive disease 


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GLOSSARY 
LSC0:  Diagnostic bone marrow sample or peripheral blood sample that the leukemia stem cell 
assay is performed on. LSC0 occurs prior to induction therapy for diagnostic purposes.  
LSC1:  Bone marrow sample that the post-induction leukemia stem cell assay is performed on.  
LSC1 occurs following induction chemotherapy at the time of complete remission assessment 
(End of Induction visit). 
eLSC:  Bone marrow sample that the leukemia stem cell assay is performed on, collected on 
evaluable subjects at the end of consolidation therapy. For those undergoing treatment with 
consolidation chemotherapy, eLSC occurs 45-60 days from day 1 of the last consolidation 
chemotherapy cycle.  For those undergoing treatment with consolidation HCT, eLSC occurs 60-
100 days from day 0 (day cells are transplanted). 
LSC6:  This sample only applies to HCT subjects. This is the fourth  sample that the leukemia 
stem cell assay is performed in HCT subjects. For those undergoing treatment with consolidation 
HCT, LSC6 occurs 6 months (180 days +/- 30 days) from day 0 (day cells are transplanted).  
Cytarabine-based induction subjects: Subjects who receive induction therapy that is primarily 
cytarabine-based. Some of these subjects may receive a cycle of hypomethylating agent (HMA)-
based chemotherapy prior to cytarabine-based induction. 
Hypomethylating agent (HMA) based induction subjects: Subjects who receive induction 
therapy that is primarily HMA-based. These subjects will follow the same treatment algorithm as 
the cytarabine-based induction subjects with unfavorable risk except they will not receive 
cytarabine-based consolidation chemotherapy if they are unable to have a HCT. 
Start of treatment:  For those undergoing treatment with consolidation chemotherapy, start of 
treatment is day 1, cycle 1.  For those undergoing treatment with consolidation HCT, start of 
treatment is day 0 (day cells are transplanted). 
End of treatment:  For those undergoing treatment with consolidation chemotherapy, end of 
treatment is defined as 30 days from last administration of consolidation chemotherapy.  For 
those undergoing treatment with consolidation HCT, end of treatment is 30 days from day 0 (day 
cells are transplanted). 
Observational Cohort 1 (OC1): enrolled subjects who do not achieve a CR (CR1) to standard 
cytarabine or HMA-based induction therapy per standard clinical criteria (See Appendix 1).  
Observational Cohort 2 (OC2): enrolled subjects who achieve a CR   
(CR1) from standard cytarabine or HMA-based induction therapy per standard clinical criteria 
(See Appendix 1) and meet one or more of the following criteria:  


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• Lack the immunophenotype of interest (CD34+CD38-ALDHint) by flow cytometry at the 
diagnostic LSC assay (LSC0)  
• Cytarabine based induction subjects: Are not candidates for [as determined by the 
investigator (e.g. unfit or refusal)] and do not receive consolidation therapy (cytarabine-based 
chemotherapy or HCT) 
HMA-based induction subjects: Are not candidates for [as determined by the investigator 
(e.g. unfit, lack of donor, refusal)] and do not receive HCT  
Evaluable Cohort: enrolled subjects who achieve a CR (CR1) from  standard cytarabine or 
HMA-based induction therapy per standard clinical criteria (see Appendix 1) and meet all of the 
following criteria: 
• Have confirmed presence of CD34+CD38-ALDHint population by flow cytometry at the 
diagnostic LSC assay (LSC0) 
• Cytarabine-based induction subjects: Are candidates for (as determined by the investigator) 
and receive consolidation therapy (cytarabine-based chemotherapy or HCT) 
• HMA-based induction subjects:  Are candidates for (as determined by the investigator) and 
receive HCT  
 
 


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TABLE OF CONTENTS 
 
PROTOCOL SUMMARY .......................................................................................................... iii 
SCHEMA ....................................................................................................................................... v 
LIST OF ABBREVIATIONS .................................................................................................... vii 
GLOSSARY................................................................................................................................... x 
TABLE OF CONTENTS ........................................................................................................... xii 
1. 
OBJECTIVES .................................................................................................................... 1 
1.1. 
Primary Objective ................................................................................................. 1 
1.2. 
Secondary Objectives ............................................................................................ 1 
1.3. 
Safety Objective .................................................................................................... 2 
1.4. 
Exploratory Objective ........................................................................................... 2 
2. 
BACKGROUND AND RATIONALE ............................................................................. 2 
2.1. 
Introduction ........................................................................................................... 2 
2.2. 
Leukemia Stem Cells ............................................................................................ 3 
2.3. 
ALDH Activity ...................................................................................................... 4 
2.4. 
Presence of LSCs Following Treatment................................................................ 5 
2.5. 
Leukemic Burden After Chemotherapy ................................................................ 7 
3. 
SUBJECT SELECTION AND ENROLLMENT ............................................................ 9 
3.1. 
Accrual .................................................................................................................. 9 
3.2. 
Eligibility Criteria ................................................................................................. 9 
4. 
INVESTIGATIONAL PLAN ......................................................................................... 10 
4.1. 
Milestone Date Definitions ................................................................................. 10 
4.2. 
Overall Study Design .......................................................................................... 10 
4.3. 
Informed Consent ................................................................................................ 13 
4.4. 
Registration/Study Entry (Pre-Screening) ........................................................... 13 
4.5. 
End of Induction (EOI) ....................................................................................... 13 
4.6. 
End-of-Consolidation (EOC) .............................................................................. 22 
4.7. 
Short and Long-Term Follow-up Visits .............................................................. 22 
4.8. 
Off-Study ............................................................................................................. 23 
4.9. 
Subject Replacements ......................................................................................... 23 


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4.10. 
Subject Withdrawal ............................................................................................. 23 
5. 
STUDY CALENDAR ...................................................................................................... 25 
6. 
TREATMENT EVALUATION ...................................................................................... 26 
7. 
TREATMENT-RELATED ADVERSE EVENTS ........................................................ 26 
8. 
DATA AND SAFETY MONITORING PLAN ............................................................. 26 
8.1. 
Safety Monitoring ............................................................................................... 26 
8.2. 
Data Quality Assurance ....................................................................................... 27 
8.3. 
Communication Between Investigational Sites................................................... 27 
9. 
SAFETY DATA COLLECTION, RECORDING AND REPORTING...................... 27 
9.1. 
Unanticipated Problem Definition ...................................................................... 27 
9.2. 
Study Adverse Event (AE) Definition ................................................................ 28 
9.3. 
“Unexpected” Definition ..................................................................................... 28 
9.4. 
Study Serious Adverse Event (SAE) Definition ................................................. 28 
9.5. 
Event of Clinical Interest – Graft Versus Host Disease (GVHD) ....................... 29 
9.6. 
SAE Reporting Requirements ............................................................................. 30 
9.7. 
Safety Reporting to the IRB ................................................................................ 30 
10. 
STATISTICAL METHODS ........................................................................................... 31 
10.1. 
Sample Size ......................................................................................................... 31 
10.2. 
Endpoint Definitions ........................................................................................... 33 
10.3. 
Analysis Populations ........................................................................................... 33 
10.4. 
Analysis Methods ................................................................................................ 34 
10.5. 
Efficacy Analysis ................................................................................................ 35 
10.6. 
Exploratory Analyses .......................................................................................... 36 
10.7. 
Interim Analyses ................................................................................................. 36 
10.8. 
Safety Analyses ................................................................................................... 36 
11. 
STUDY COMPLETION ................................................................................................. 38 
11.1. 
Completion .......................................................................................................... 38 
12. 
RETENTION OF RECORDS ........................................................................................ 38 
13. 
ETHICAL AND LEGAL ISSUES .................................................................................. 39 
13.1. 
Ethical and Legal Conduct of the Study.............................................................. 39 
13.2. 
Confidentiality ..................................................................................................... 40 


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14. 
CLINICAL TRIAL REGISTRATION AND RESULTS REPORTING .................... 40 
REFERENCES ............................................................................................................................ 41 
APPENDICES ............................................................................................................................. 43 
 
 


Protocol Number:  LCI-HEM-AML-SCD-001 
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1. OBJECTIVES 
 
1.1. 
Primary Objective 
 
The primary objective is to compare the two-year relapse-free survival (RFS) of 
subjects with acute myeloid leukemia (AML) with detectable leukemia stem cells 
(LSCs)  at end of consolidation (eLSC+) to the two-year RFS of subjects without 
detectable LSCs (eLSC-). Before determination of end of consolidation LSC (eLSC) 
status, subjects will have completed either standard cytarabine-based consolidation 
chemotherapy or allogeneic stem cell transplantation.  
 
1.2. 
Secondary Objectives 
 
a. Compare the one-year RFS of AML subjects with detectable LSCs at eLSC to the 
one-year RFS of AML subjects without detectable LSCs at eLSC.   
b. Compare the two-year RFS of favorable risk AML subjects without detectable LSCs 
at the time of achieving CR1 (LSC1 time point) who are treated with standard 
cytarabine-based consolidation to that of favorable risk AML subjects with detectable 
LSCs at LSC1 who are also treated with standard cytarabine-based consolidation. 
c. Compare the two-year RFS of intermediate risk AML subjects with detectable LSCs 
at LSC1 who are treated with allogeneic hematopoietic cell transplant (HCT) to that 
of intermediate risk AML subjects without detectable LSCs at LSC1 who are treated 
with allogeneic HCT. 
d. Compare the two-year RFS of favorable risk AML subjects with detectable LSCs at 
LSC1 who undergo allogeneic HCT to the two-year RFS of favorable risk AML 
subjects with detectable LSCs at LSC1 who receive standard cytarabine-based 
consolidation chemotherapy. 
e. Compare the two-year RFS of intermediate risk AML subjects without detectable 
LSCs at LSC1 who undergo allogeneic HCT to the two-year RFS of intermediate risk 
AML subjects without detectable LSCs at LSC1 who receive cytarabine-based 
consolidation chemotherapy. 
f. Compare the overall survival (OS) of AML subjects with detectable LSCs at eLSC to 
the OS of AML subjects without detectable LSCs at eLSC.  Compare the two-year 
RFS of subjects with AML with detectable LSCs at LSC1 to subjects without 
detectable LSCs at LSC1.   
g. Compare the one-year RFS of subjects with AML with detectable LSCs  at LSC1 to 
subjects without detectable LSCs at LSC1.   
h. Compare the two-year RFS of unfavorable risk AML subjects without detectable 
LSCs at LSC1 who receive standard cytarabine-based consolidation to the two-year 


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RFS of unfavorable risk AML subjects without detectable LSCs at LSC1 who 
undergo allogeneic HCT. 
i. Compare the two-year RFS of subjects with detectable LSCs at LSC6 in HCT 
subjects and detectable LSCs at eLSC in chemotherapy subjects to the two-year RFS 
of HCT/chemotherapy subjects without detectable LSCs at LSC6/eLSC, respectively. 
j. In the subjects that undergo allogeneic HCT, evaluate the change in LSC status from 
eLSC (end of consolidation) to LSC6 (6 months after day 0 of transplant).  
 
1.3. 
Safety Objective 
 
Evaluate the graft-versus-host disease (GVHD) classification (acute vs chronic), 
incidence, and grade/severity in AML subjects undergoing allogeneic HCT. 
 
1.4. 
Exploratory Objective 
 
 
Evaluate the clinical outcomes of the population of enrolled subjects who are not 
included in the evaluable population (as defined in section 8.3).  This will involve the 
following observational cohorts: 
• Observational cohort 1 (OC1):  enrolled subjects who do not achieve a CR 
(CR1) to standard cytarabine or HMA-based induction therapy per standard 
clinical criteria (see Appendix 1). 
• Observational cohort 2 (OC2):  enrolled subjects who achieve a CR (CR1) 
to standard cytarabine or HMA-based induction therapy per standard 
clinical criteria (see Appendix 1) and meet one or more of the following 
criteria:  
• Lack the immunophenotype of interest (CD34+CD38-ALDHint) by flow 
cytometry at the diagnostic LSC assay (LSC0)  
• Cytarabine based induction subjects: Are not candidates for [as 
determined by the investigator (e.g. unfit or refusal)] and do not receive 
consolidation therapy (cytarabine-based chemotherapy or HCT)  
HMA-based induction subjects: Are not candidates for [as determined 
by the investigator (e.g. unfit, lack of donor, refusal)] and do not 
receive HCT 
2. BACKGROUND AND RATIONALE 
 
2.1. 
Introduction 
 
Although most patients with AML achieve CR following standard induction 
chemotherapy, a large majority subsequently relapse and succumb to the disease.1-3 


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Most patients who receive HMA treatment for AML also die of their leukemia.  
Currently, cytogenetic and molecular aberrations are the best prognostic indicators3-5; 
however, these factors predict primarily for groups of patients and cannot 
prognosticate accurately for individual patients. For example, core-binding factor 
(CBF) cytogenetic abnormalities are considered favorable, yet roughly half of these 
patients relapse.6 Overall, the majority of patients with favorable or intermediate-risk 
AML will experience relapse. Prognosis after relapse is dismal with a five-year overall 
survival rate of less than 10%.7 A LSC paradigm may explain this failure of CR to 
reliably translate into cure. Leukemia appears to retain the normal hematopoietic 
hierarchical structure: rare stem cells with self-renewal capacity give rise to partially 
differentiated progeny that comprise the bulk of the leukemia but possess only limited 
proliferative potential.8 Although existing therapies are highly active against the 
leukemic bulk, it appears they spare the hardier LSCs, which are then responsible for 
relapse.9-10 OS and RFS of HCT in CR2 is much worse than in CR1.11  Therefore, 
detection of LSCs in CR1 may provide a more precise method to predict outcome and 
a valuable window of opportunity to guide treatment decisions.  In addition, the 
detection of LSCs at the end of AML treatment may provide prognostic information 
and also an opportunity for intervention. 
 
2.2. 
Leukemia Stem Cells 
 
Since 1994 when Dick and colleagues reported that only rare AML cells, characterized 
by a classical CD34+CD38- normal hematopoietic stem cell (HSC) phenotype, were 
capable of generating leukemia in immunodeficient mice,12 these putative LSCs have 
been the focus of considerable research. Although it is generally accepted that 
CD34+CD38- cells are enriched for LSCs,12 this population is heterogeneous and 
includes both normal and leukemic cells.  Moreover, recent data have challenged the  
CD34+CD38- phenotype of LSCs in AML, leading many investigators to advocate for 
a functional definition of LSCs: those leukemic cells capable of engrafting 
immunodeficient mice.13-16 However, even with this current gold standard, the 
identification of LSCs has proven elusive.8,12 A significant portion of AML patient 
samples will not engraft mice, and the assay is cumbersome and often indeterminate.17 
More importantly, the clinical implications of this assay are unclear.18 Furthermore, 
even in leukemia, where the cancer stem cell (CSC) model is perhaps best established, 
there is a paucity of data demonstrating that LSCs are responsible for disease 
resistance or relapse.  
 
Relapse after CR is due to minimal residual disease (MRD): residual leukemia cells 
persistent in quantities undetectable by conventional assays used to evaluate remission. 
If LSCs are indeed clinically relevant, then their persistence should correlate with 


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recurrence, and MRD should be enriched for these cells. Exploiting the similarities 
between LSCs and their normal counterparts,19 we recently employed strategies 
established for the isolation of normal HSCs20-22 in chronic myeloid leukemia (CML). 
We found that the fraction of the CD34+CD38-  cells with high activity of Aldefluor, 
as determined by aldehyde dehydrogenase (ALDH) levels, was highly enriched for 
leukemic cells capable of engrafting immunodeficient mice.23 Therefore, we assessed 
ALDH activity as a marker for clinically significant MRD in AML. 
 
2.3. 
ALDH Activity 
 
When analyzed for ALDH activity, the normal bone marrow CD34+CD38- cells 
consistently exhibited two, non-overlapping populations: one expressing low ALDH 
activity (CD34+CD38-ALDHlow) and the second expressing high activity ( 
CD34+CD38-ALDHhigh) (Fig 1a). The normal marrow CD34+CD38-ALDHhigh cells 
comprised an average of 10% (range 9-12%) of the total CD34+ cells and 76% (range 
61-85%) of the CD34+CD38- cells. As few as 1000 of these CD34+CD38-ALDHhigh 
cells engrafted NOG mice.  
 
 
Figure 1. Expression of ALDH in the  CD34+CD38- population of bone marrow from a normal donor(a), and three leukemia patients 
with cytogenetic profiles of t(8;21), inv16, and 46xy profiles(b,c,d). Populations with high (boxed) and low ALDH expression are 
apparent in normal marrow(a).  The AML samples contained a discrete third population of ALDH intermediate cells (circled).  In the 
patients with CBF AML this CD34+CD38-ALDHint population was essentially completely leukemic by FISH, as was the  
CD34+CD38-ALDHlow population. In contrast, the small CD34+CD38-ALDHhigh populations from CBF AML patients lacked the 
leukemia-specific FISH marker. 
 
Initial analyses focused on patients with newly diagnosed CBF AML, as the FISH-
detectable abnormalities enabled quantification of leukemic cells in isolated 
populations. In contrast to the normal samples, the CD34+CD38- cells from all CBF 
AML patients exhibited three well-defined populations by ALDH expression. They 
contained a population with intermediate ALDH expression (CD34+CD38-ALDHint) 
(Fig 1b-c), in addition to high and low ALDH populations observed in normal marrow. 
The CD34+CD38-ALDHhigh cells were rare in the newly-diagnosed CBF AML 
patients, constituting an average of only 0.12% of the total CD34+ cells (range 0.005-
0.5%, p<0.001 vs. the normal samples) and 1.24% of the  CD34+CD38- cells (range 
0.03-4.3%, p<0.001 vs. the normal samples). This CD34+CD38-ALDHhigh population 
was essentially devoid of cells with the leukemia-specific cytogenetic abnormality. 


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Similar to those isolated from normal donors, as few as 1000 of these cells yielded 
normal human hematopoietic engraftment of NOG mice. Conversely, the intermediate 
and low ALDH fractions were both virtually entirely leukemic by FISH. As few as 
1000 of the CD34+CD38-ALDHint cells produced leukemic engraftment of NOG mice, 
whereas, the CD34+CD38-ALDHlow cells did not engraft. The CD34+CD38- cells from 
other cytogenetic variants of AML, including those with normal cytogenetics, also 
demonstrated three ALDH populations (Fig 1d) and similar FISH results (when 
applicable). The CD34+CD38-ALDHint population appears to contain a putative LSC 
population, distinguishable from the normal HSC population.  
 
Only four of 20 newly-diagnosed AML patients did not have this characteristic flow 
cytometric pattern containing a CD34+CD38-ALDHint population. In two of these 
patients, most of the CD34+CD38- leukemic cells exhibited high ALDH activity, with 
no discernible separate population of normal CD34+CD38-ALDHhigh cells by FISH or 
PCR. One patient had normal cytogenetics with a FLT3 internal tandem duplication, 
and the second had complex cytogenetics, including deletions of chromosomes 5 and 
7. Notably, both patients had primary refractory disease, which ultimately proved fatal. 
In the two additional patients, the diagnostic leukemic cytogenetic marker was present 
only in the CD34- cells, as has been previously described in a minority of AML 
cases.24 One of these patients had an 11q23 abnormality and has since relapsed within 
a year of diagnosis; the other has remained in CR for more than one year since 
diagnosis and nine months since allogeneic transplantation. 
 
We hypothesized that, because this CD34+CD38-ALDHint population represents 
putative LSCs, if it is present during CR it would correlate with future relapse, and 
therefore define a clinically relevant MRD population. Therefore, we evaluated AML 
patient samples at various stages of treatment to determine if their flow cytometric 
pattern predicted relapse.  
 
2.4. 
Presence of LSCs Following Treatment 
 
Of the 20 AML patients analyzed at diagnosis, three had primary refractory disease, 
one died during induction chemotherapy, one did not receive full induction or 
consolidation chemotherapy, and two others had CD34- leukemia. Of the 13 patients 
with CD34+ leukemia analyzed at diagnosis who achieved morphologic CR after 
induction, follow-up samples were available in nine. An additional seven AML 
patients who achieved CR, but in whom diagnostic samples were not available, were 
also followed: two starting after induction and five after consolidation therapy, for a 
total of 16 evaluable patients. 
 
Of those 16 patients, eight were analyzed in CR1 prior to consolidation. The CR 
samples exhibited two general patterns: two populations, with a predominant 
CD34+CD38-ALDHhigh population and a smaller CD34+CD38-ALDHlow population, as 
seen in normal samples (Figure 2A); three populations including a CD34+CD38-


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ALDHint population (Figure 2B). Five patients exhibited the normal pattern, and both 
of the cell populations were normal by FISH. The three patients who have consistently 
exhibited this normal pattern remain in CR, with an average follow-up of 293 (range 
185-370) days since diagnosis. In the other two patients, the CD34+CD38-ALDHint 
population was detected at follow-up while still in CR after consolidation, and both 
ultimately relapsed. The remaining three patients exhibited an MRD pattern in their 
initial CR1 marrow (Figure 2B) and the CD34+CD38-ALDHint population was ≥85% 
leukemic by FISH. Two of these patients relapsed within 33 days of detection of the 
MRD pattern and subsequently died. The third patient underwent allogeneic HCT in 
CR1 due to adverse risk cytogenetics (complex karyotype, including deletion 7q) and 
has remained in CR for over 17 months.   
 
Figure 2. Aldeflour expression in the CD34+CD38- bone marrow cells. Representative examples of staining patterns in CR1: (A) 
normal, (B) MRD, (C) overt clinical relapse.  
 
Eleven AML patients who achieved morphologic CR with induction chemotherapy 
have been followed since completion of consolidation. Of the seven patients with a 
consistently normal flow pattern after consolidation, none has yet relapsed, with an 
average duration of follow-up of 509 days since diagnosis (range 185-810). However, 
all four of the patients with the MRD pattern after consolidation relapsed at an average 
of 53 days (range 32-81) after detection of the MRD pattern (p=0.003 when compared 
to those patients with a consistently normal pattern). The CD34+CD38-ALDHint 
population was overwhelmingly leukemic (≥95%) by FISH. One of the relapsed 
patients converted to a normal pattern after re-induction and allogeneic transplantation 
and has remained in second CR for over one year. The MRD pattern persisted in two 
other patients, who achieved a second CR after re-induction, both of whom have 
subsequently relapsed. Of note, the CD34+CD38-ALDHint population was not detected 
in an additional two patients who have been in CR for more than three years, but have 
been followed only since their second year post-consolidation. The CD34+CD38-
ALDHint population was present in all six analyzed cases of overt clinical relapse 
(Figure 2C).   
 


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2.5. 
Leukemic Burden After Chemotherapy 
 
According to the LSC paradigm, LSCs should constitute a small fraction of the 
leukemic burden during active disease, but should comprise a substantially larger 
portion of the leukemic population during clinical remission. Although three patients 
in CR after induction who exhibited the MRD pattern had no morphologic, karyotypic, 
or FISH evidence of disease in the unfractionated marrow cells, the cytogenetic 
abnormality was still detectable in an average of 90% of the CD34+CD38-ALDHint 
cells (range 85-95%) and in an average of 22% of the CD34+ cells (range 8-36%). This 
leukemic CD34+CD38-ALDHint population comprised an average of 34% of the total 
leukemic burden by FISH (range 9-51%). In contrast, this population constituted only 
2% of the total leukemic burden at diagnosis (range 0.3-7%, p<0.001 vs. cytogenetic 
CR).  Moreover, although the total leukemic burden decreased by >200-fold from 
diagnosis to CR (p<0.001), the CD34+CD38-ALDHint population decreased by only 
13-fold (p=0.4).  
 
Three different patients who had cytogenetic but no morphologic evidence of disease, 
had an average of 8% leukemic cells (range 4.5-9.5%) in their unfractionated marrow 
as compared to 96% in the CD34+CD38-ALDHint cells (range 94-99%, p<0.001). 
Furthermore, the proportion of the CD34+CD38-ALDHint fraction represented an 
average of 8% of the total leukemic clone (range 2-12%). At overt relapse, as at initial 
diagnosis, this population again comprised only a small fraction (average 1%, range 
0.5-2%) of the total leukemic burden. These data demonstrated that this CD34+CD38-
ALDHint population behaves as a putative LSC population.  
 
Further, these data suggest that the presence of this CD34+CD38-ALDHint population 
predicts relapse, as expected of an LSC population. This group of cells would define a 
clinically relevant MRD population that indicates a high risk of AML relapse. Patients 
with this population, despite lacking the standard poor-risk prognostic markers, would 
be at a high risk of relapse and would likely benefit from more intensive therapy in 
first complete remission, due to the very poor prognosis of AML in relapse: five-year 
overall-survival rates are less than 10% after relapse.7 On the contrary, patients lacking 
the CD34+CD38-ALDHint population in CR1 may not benefit from more intensive 
therapy in first complete remission. 
 
Therefore, we propose a prospective study of AML subjects to evaluate the prognostic 
character of the assay for CD34+CD38-ALDHint LSCs.  In addition, cytarabine-based 
induction subjects with favorable and intermediate risk AML, upon achieving a CR 
after induction therapy, will receive consolidation treatment assignment guided by the 
presence or absence of LSCs in CR1.  This approach contrasts with the present 


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standard strategy using cytogenetic and molecular markers present at diagnosis to 
determine post-remission treatment.   
 
This will be a prospective, randomized, clinical trial, in which favorable and 
intermediate risk evaluable AML subjects who have the CD34+CD38-ALDHint 
population at diagnosis will be assigned (based on LSC1 status and/or randomization) 
to either standard chemotherapy or HCT for consolidation.  
 
 CD34+CD38-ALDHint LSCs must comprise at least 0.001% of total mononuclear 
cells to be considered detectable. 
 
HMA-based induction AML subjects (regardless of risk): 
For HMA-based induction AML subjects who are candidates for transplant, the 
standard of care is allogeneic HCT in CR1.   
• AML subjects who received HMA-based induction who are transplant 
candidates and have an available donor will undergo transplant.  However, not 
all transplant candidates have donors available.   
• An HMA-based induction subject who is unable to receive HCT for any reason 
will be included  in Observational Cohort 2 (OC2). 
 
Favorable risk AML cytarabine-based induction subjects 
• without detectable LSCs in CR1 or who decline Step 2 ICF will receive 
standard consolidation chemotherapy  
• with detectable LSCs in CR1 who accept Step 2 ICF will be randomized to 
receive standard consolidation chemotherapy or allogeneic HCT   
 
Intermediate risk AML cytarabine-based induction subjects  
• with detectable LSCs in CR1 or who decline Step 2 ICF will receive allogeneic 
HCT  
• without detectable LSCs in CR1 who accept Step 2 ICF will be randomized to 
receive standard consolidation chemotherapy or allogeneic HCT 
 
Unfavorable risk AML cytarabine-based induction subjects:  
For unfavorable risk AML subjects who are candidates for transplant, the standard of 
care is allogeneic HCT in CR1.   
• Unfavorable risk subjects on this study who are transplant candidates and have 
an available donor will undergo transplant.  However, not all transplant 
candidates have donors available.   
• An unfavorable risk subject without an available donor will be recommended 
to receive consolidation chemotherapy per standard of care. We will gather 


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information regarding the detectability of LSC in CR1 in this population and 
correlate the detectability of LSCs with outcomes.  
3. SUBJECT SELECTION AND ENROLLMENT 
 
3.1. 
Accrual 
Subjects must meet all of the following criteria to be pre-screened for the study: 
• 18 years and older  
• Newly diagnosed or suspected AML  
• Planned induction therapy (either cytarabine-based or HMA-based) 
Subjects who are consented with the Step 1 consent and meet the eligibility criteria 
after induction chemotherapy will be enrolled on the trial. Subjects will continue to be 
accrued until 200 subjects meeting the criteria for the evaluable cohort as defined in 
Section 4.2.3 with successful and interpretable eLSC evaluations are obtained This 
will constitute the evaluable population (see Section 8.3) for the primary efficacy 
analyses. Subjects who do not meet the criteria for the evaluable cohort will be 
enrolled but followed only in an observational manner for outcomes.  An estimated 
total of 300 to 500 subjects will be enrolled under the treatment and observational 
portion of the study. Subjects that are enrolled, but followed in an exploratory manner 
are not considered to be part of the evaluable population. 
 
The original diagnosis of AML must have been confirmed by bone marrow aspirate 
and/or biopsy review by an institutional hematopathologist.  Subjects are eligible if 
they have AML that is not classified as APL and meet all other eligibility criteria (see 
Section 3.2).  
The study is open to both adult men and women, and to all racial/ethnic subgroups. 
There is no explicit mention of different treatment effects in male and female subjects 
and in different racial/ethnic subgroups in the literature. Therefore, this study will not 
have separate accrual targets for these groups. 
3.2. 
Eligibility Criteria 
 
3.2.1. Inclusion Criteria 
 
Subjects must meet all the following criteria to be enrolled into the study:  
 
a. Must have previously signed the Step 1 informed consent document prior to 
LSC0 (Step 2 informed consent is not required for enrollment) 


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b. Age 18 years and older   
c. New diagnosis of AML, other than APL, confirmed by bone marrow 
aspirate/biopsy and reviewed by an institutional hematopathologist   
d. Have completed induction, as defined by the Investigator and post-induction 
bone marrow biopsy.  
  
3.2.2. Exclusion Criteria 
 
Subjects must not meet the following criterion in order to be enrolled to the study:  
 
a. Any debilitating medical or psychiatric illness that would preclude ability to 
follow study procedures.  
b. Indeterminate results at LSC0 
 
Donor eligibility will be determined per standard HCT criteria.  
4. INVESTIGATIONAL PLAN 
4.1. 
Milestone Date Definitions 
 
Entry Date:  the date the subject signs the Step 1 informed consent document 
Eligibility Date:  the date of the last documented criterion that confirmed post-
induction subject eligibility for enrollment to the study 
Enrollment Date:  the date that the post-induction bone marrow aspirate was 
obtained.  Note: this is the sample that determines post-induction remission status. 
Off Study Date: the date on which the subject is determined to have completed, 
withdrawn, or been withdrawn by the investigator from the study (see section 4.10 Off 
Study). 
 
4.2. 
Overall Study Design 
 
This is a prospective cohort study evaluating the prognostic value of eLSC. This study 
also includes randomization aspects enabling the comparison of the two consolidation 
schemas (standard cytarabine-based consolidation chemotherapy and allogeneic stem 
cell transplantation (HCT)) in AML subjects. Subjects will be enrolled and included in 
one of three cohorts after induction therapy if they meet the post-induction eligibility 
criteria. These cohorts are defined as follows:  
 
4.2.1. Observational Cohort 1 (OC1): enrolled subjects who do not 
achieve a CR to induction therapy, regardless of diagnostic phenotype. 


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Following completion of induction therapy and remission bone 
marrow aspirate, if a subject is determined to not have achieved a 
complete remission to induction therapy, he or she would be included 
in observational cohort 1. 
 
4.2.2. Observational Cohort 2 (OC2): enrolled subjects who achieve a 
CR to induction therapy but meet one or more of the following criteria:  
• Lack the immunophenotype of interest,   
• Cytarabine based induction subjects: Are not candidates for  
[as determined by the investigator (e.g. unfit or refusal)] and do 
not receive consolidation therapy (cytarabine-based 
chemotherapy or HCT)  
• HMA-based induction subjects: Are not candidates for [as 
determined by the investigator (e.g. unfit, lack of donor, 
refusal)] and do not receive HCT 
 
Final investigator determination of fit-ness can occur at any time 
until the start of consolidation therapy. Being unfit for 
consolidation therapy could be a result of any of the following 
reasons:  
 
i. Any debilitating medical or psychiatric illness that would preclude 
ability to receive optimal treatment.  
1. Includes ECOG > 3  
ii. Poor renal function: creatinine > 2.5mg/dL or requiring 
hemodialysis  
iii. Poor liver function: bilirubin > 2.5mg/dL, excluding Gilbert’s 
Syndrome  
iv. Breastfeeding or known pregnancy 
v. Receiving any other investigational agents. 
vi. Uncontrolled intercurrent illness including, but not limited to, 
ongoing or active infection, symptomatic congestive heart failure, 
unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situations that would limit compliance with study 
requirements per the Investigator. 
 
HMA-based induction subjects will not receive consolidation cytarabine-based 
chemotherapy as part of the evaluable cohort if they do not receive HCT. 
 


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Note the subjects enrolled and included in any of the observational cohorts will not 
contribute to the 200 evaluable subjects required for the primary objective. An 
estimated total of 300 to 500 subjects will be enrolled to the study under the 
treatment and observational portions of the study. 
 
4.2.3. Evaluable Cohort: enrolled subjects that will contribute to the 
population of subjects who are evaluable for the primary and 
secondary objectives. This will not include any subjects who end up in 
either observational cohort. To be included in the evaluable cohort, the 
subject must meet the following requirements: 
 
a. Complete remission (CR1) from standard cytarabine or HMA-based 
induction therapy per standard clinical criteria (see Appendix 1) 
b. Have confirmed presence of CD34+CD38-ALDHint population by flow 
cytometry at the diagnostic LSC assay (LSC0) 
c. Cytarabine-based induction subjects: Are candidates for (as determined by 
the investigator) and receive consolidation therapy (cytarabine-based 
chemotherapy or HCT) 
HMA-based induction subjects:  Are candidates for(as determined by the 
investigator) and receive HCT 
 
To be considered a candidate for consolidation therapy, a subject must be 
considered fit. Criteria for fitness may include, but is not limited to the 
following: 
i. Good renal function (creatinine <2.5mg/dL)  
ii. Good liver function (bilirubin <2.5mg/dL)  
iii. ECOG < 3 
iv. Not receiving any other investigational agents 
v. Not breastfeeding or known pregnancy 
vi. No intercurrent illness that would limit compliance with study 
requirements per the Investigator. 
 
Subjects will be assigned to the evaluable cohort based on the criteria above and 
will then be assigned or randomized to either consolidation chemotherapy or HCT. 
Subjects who receive consolidation therapy will be included in the evaluable 
cohort.  
Enrollment to the study will continue until 200 subjects evaluable for the primary 
objective (i.e. with known eLSC status) are enrolled (Section 10.3).  
 
 


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4.3. 
Informed Consent 
 
The applicable informed consent(s) must be obtained and signed prior to any study 
related procedures. 
 
There are multiple informed consents for this study. Patients at Levine Cancer Institute 
with a suspected or confirmed diagnosis of AML will be presented with information 
about this study. All interested subjects will first be consented with the Step 1 consent 
before starting induction chemotherapy for exploratory/observational participation on 
this study.  
 
Once induction chemotherapy is completed, entered subjects must meet the following 
criteria in order to be presented with the Step 2 consent: 
• Received cytarabine-based induction 
• Determined to be a candidate  for consolidation chemotherapy 
• Confirmed presence of CD34+CD38-ALDHint population by flow cytometry at 
diagnosis (LSC0) 
• Favorable or intermediate risk. The risk level is determined prior to induction 
chemotherapy and determines which subjects may have treatment decisions 
informed by their LSC status.  
4.4. 
Registration/Study Entry (Pre-Screening) 
 
Following Step 1 informed consent, subjects will be considered entered into the 
study, and eligibility check per standard operating procedures begins.  Subjects 
will have a bone marrow aspirate/biopsy for diagnostic and clinical care purposes. 
This bone marrow sample may be used for the diagnostic leukemia stem cell 
assay (LSC0). Alternatively, a peripheral blood sample may be used for LSC0. 
Shortly after the diagnostic bone marrow sample, planned induction per standard 
of care will begin. 
 
4.5. 
End of Induction (EOI) 
4.5.1. Procedures and Assessments 
 
At the EOI, eligibility checks will be completed, subject’s remission status will be 
assessed, and other assessments/procedures will occur according to the Study 
Calendar (Section 5.0). A bone marrow aspirate/biopsy evaluating remission status 
will be performed and documented in the electronic medical record. EOI 
assessments/procedures, including bone marrow aspirate/biopsy, may occur on 
different days but must be performed within 30 days prior to the start of 


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consolidation therapy.  An LSC assay (LSC1) will also be performed during this 
time frame.   
4.5.2. Enrollment 
After completion of induction therapy, subjects will be evaluated for any remaining 
eligibility criteria.  All eligible subjects will be considered enrolled to the study as 
of the date of obtaining the post-induction bone marrow aspirate. Subjects meeting 
the requirements described in Section 4.2.3 (achieved CR after induction, fit for 
consolidation chemotherapy) will be included in the evaluable cohort. Subjects not 
meeting the requirements described in section 4.2.3 will fall into one of two 
observational cohorts and will be followed in an exploratory manner evaluating 
LSC status and outcomes as they receive standard of care.   
For evaluable cytarabine-based induction subjects, treatment assignment will be 
informed by the subjects’s clinical risk status (favorable, intermediate, or 
unfavorable) based on cytogenetic and/or molecular abnormalities from the 
diagnostic bone marrow biopsy. If their risk level is intermediate or favorable, 
those subjects will be presented with Step 2 informed consent. Subject’s refusing 
Step 2 consent will be recommended for standard of care treatment according to 
their clinical risk level. Subjects who sign and agree to Step 2 consent will proceed 
through a treatment algorithm by which the subject’s treatment assignment is 
informed by their risk level and LSC1 status (detected/positive, not 
detected/negative, or unknown).   
Evaluable HMA-based induction subjects will be assigned to HCT regardless of 
cytogenetic and/or molecular abnormalities from the diagnostic bone marrow 
biopsy.  
The treatment algorithm assignment will be either deterministic or accomplished by 
randomization in a 1:1 fashion, as described in Section 4.4.3 (Randomization), to 
either the HCT arm or the consolidation chemotherapy arm based on several strata.  
In order to evaluate the putative LSC population, cells from subject bone marrow 
aspirate(s) will be studied. Peripheral blood may be used at the LSC0 timepoint. A 
minimum of 100,000 CD34+ cells is required for complete analysis. The CD34+ 
cells will be isolated by Ficoll density centrifugation separation, followed by 
selection using anti-CD34 magnetic beads.  Cells will be analyzed and sorted based 
on expression of CD34, CD38, and Aldeflour. FISH analysis for cytogenetic 
abnormalities will be performed on sorted cells.  If there are insufficient CD34+ 
cells on remission bone marrow aspirate (up to 2 attempts) and/or the 
hematopathologist is unable to render a definitive LSC status, the LSC status will 
be considered unknown. 


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All enrolled subjects will be assigned a Study ID as determined by the Levine 
Cancer Institute Biostatistics Core.  This will be accomplished by utilizing a list of 
Study IDs and, where applicable, associated treatment arm assignments generated 
prior to study activation.  These will be given by a member of the Levine Cancer 
Institute Biostatistics Core.  The Study ID will be a four digit randomly generated 
ID number, ranging from 0001 to 9999.  
4.5.3. Randomization 
Evaluable subjects, who meet the requirements for certain treatment pathways 
within the treatment algorithm, will be randomized to one of two standard 
treatment groups (HCT versus cytarabine-based consolidation).  
Population eligible for Randomization; 1:1 HCT or consolidation chemotherapy 
• Favorable risk  
o Signed Step 2 ICF 
o Achieved CR with cytarabine-based induction therapy  
o Detectable LSCs at LSC1 (LSC1+)  
o Fit for consolidation chemotherapy.    
• Intermediate risk,  
o Signed Step 2 ICF 
o Achieved CR with cytarabine-based induction therapy  
o No detectable LSCs at LSC1 (LSC1-)  
o Fit for consolidation chemotherapy.   
This randomization will be provided by the Levine Cancer Institute Biostatistics 
Core.  A stratified block randomization will be utilized including the following 
stratification factors to reduce confounding of comparisons between the HCT and 
the conventional therapy arms within each of the selected pathways involving 
randomization:  
• Age (<55 versus > 55) 
• Gender (Female versus Male) 
 
Randomization or assignment to a specific treatment type does not guarantee that 
the subject will ultimately receive that type of therapy. Subject must meet, or 
continue to meet, clinically required criteria to receive that treatment type. Criteria 
includes, but is not limited to, being fit per lab values and vital signs, and 
availability of a donor (for HCT). Cytarabine-based subjects who do not ultimately 


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receive their assigned treatment, but do receive consolidation therapy per the study 
will still be considered part of the evaluable cohort. 
 
Examples of this include, but are not limited to: 
• Cytarabine-based induction, assigned to HCT, donor not available, subject 
fit for and received consolidation chemo 
• Cytarabine-based induction, assigned to, but not fit for HCT, subject fit for 
and received consolidation chemo 
 
4.5.4. Consolidation Therapy 
Consolidation chemotherapy and HCT are both approved procedures and accepted 
clinic standards for treating patients with AML. Any assessments or tests run while 
the subject receives their consolidation therapy will be per investigator discretion 
and are considered to be standard of care. 
 
Procedures required for subjects before and after consolidation therapy are 
described in the Study Calendar (Section 5.0). 
 
Cytarabine-based subjects: 
Subjects who receive cytarabine-based induction will be categorized according to 
risk (from the diagnostic sample) as defined below: 
 
AML Risk Status Definitions 
Subjects with any one or more of the following cytogenetic and/or molecular 
abnormalities will be categorized as having unfavorable risk AML:  
 
• complex karyotype (≥3 clonal chromosomal abnormalities) 
• monosomal karyotype 
• -5 
• 5q- 
• -7 
• 7q- 
• 11q23 [non t(9;11)] 
• inv(3) or t(3;3) 
• t(6;9) 
• t(9;22)  
• normal cytogenetics with the FLT3-ITD mutation. 
 
Subjects will also be categorized as unfavorable risk:  
• Therapy-related AML (t-AML) 


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• AML arising from previously diagnosed myelodysplastic syndrome (MDS) 
or myeloproliferative neoplasm (MPN).   
 
Subjects with any one or more of the following cytogenetic and/or molecular 
abnormalities will be categorized as having favorable risk AML: 
• t(8;21) 
• inv(16) and/or t(16;16) 
• normal cytogenetics with the NPM1 and/or dual CEBPA mutations without 
the FLT3-ITD mutation. 
 
Cytarabine-based induction subjects who cannot be categorized as unfavorable risk 
or favorable risk will be categorized as intermediate risk. 
 
HMA-based induction subjects: 
HMA-based induction subjects will not be categorized according to a risk category 
from the diagnostic sample. 
 
Treatment Algorithms: 
Cytarabine-based induction subjects with favorable risk AML will be treated 
as follows:  
o Subjects without detectable LSCs at LSC1 by flow cytometry will be 
assigned to standard cytarabine-based chemotherapy as per institutional 
standards.  
o Subjects with detectable LSCs at LSC1 by flow cytometry will be 
randomized in a 1:1 ratio to undergo allogenic HCT versus cytarabine-based 
consolidation. Favorable risk subjects who are randomized to HCT but are 
subsequently found to not be fit for HCT and/or who do not have a donor 
(matched related, haploidentical, or matched unrelated donor) will receive 
consolidation chemotherapy. 
Note: Subjects with unknown LSC status at LSC1 or who refuse Step 2 consent for 
entering the treatment algorithm will be assigned to standard cytarabine-based 
chemotherapy as per institutional standards. The study schema for favorable risk 
AML subjects is as follows:  


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Cytarabine-based induction subjects with intermediate risk AML will be 
treated as follows:  
o Subjects with detectable LSCs at LSC1 by flow cytometry will be assigned 
to allogeneic HCT as per institutional standards.  
o Subjects without detectable LSCs at LSC1 by flow cytometry will be randomized in 
a 1:1 ratio to undergo allogeneic HCT versus cytarabine-based consolidation 
therapy.  Intermediate risk subjects who are assigned or randomized to HCT who 
are found to not be candidates for HCT and/or who do not have a donor (matched 
related, haploidentical, or matched unrelated donor) will be treated with standard 
cytarabine-based consolidation.  Note: Subjects with unknown LSC status at LSC1 
or who refuse Step 2 consent for entering the treatment algorithm will be assigned 
to allogeneic HCT (donor and fit-ness for HCT permitting) as per institutional 
standard. The study schema for intermediate risk AML subjects is as follows:  


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o Cytarabine-based induction subjects with unfavorable risk AML will be 
treated as follows:  All unfavorable risk subjects who are candidates for 
HCT and who have an available matched related, matched unrelated, or 
haploidentical donor will be assigned to undergo HCT.  Those subjects who 
lack an available donor or who are not fit for HCT will be treated with 
standard cytarabine-based consolidation chemotherapy.  
Note: The treatment assignment applies even if the subject has unknown LSC 
status.  The study schema for unfavorable risk AML subjects is as follows:  


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HMA-based induction subjects (regardless of risk) will be treated as follows: 
o All HMA-based induction subjects who are candidates for HCT and who have 
an available matched related, matched unrelated, or haploidentical donor will be 
assigned to undergo HCT.  HMA-based induction subjects who do not receive 
HCT (for any reason) will be enrolled and included in the Observational Cohort 
2. Note: The treatment assignment applies even if the subject has unknown LSC 
status. The study schema for HMA-based induction subjects is as follows:  
 


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HCT 
Subjects and their first degree relatives will undergo HLA typing as soon as possible after 
diagnosis according to standard of care.  A matched unrelated donor (MUD) search will 
also be initiated if a related donor is not found.  Umbilical cord transplants will not be 
performed on this trial.  Among multiple suitable donors, selection of the optimal donor 
will use institutional priority extant at the time. Subjects eligible for myeloablative 
conditioning will be prioritized to receive it; subjects not eligible for myeloablative 
conditioning will receive non-myeloablative conditioning. 
 
Subjects undergoing HCT will be permitted to receive up to four cycles of consolidation 
cytarabine or HMA-based therapy prior to undergoing HCT. 
 
4.5.4.1. 
Study Parameters 
Institutional guidelines for consolidation therapy with HCT or standard 
chemotherapy will be followed per routine. 
4.5.4.2. 
Supportive Care 
Supportive care, including infection management and transfusion support, 
will follow good medical practice and institutional standard guidelines. 
 


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4.6. 
End-of-Consolidation (EOC) 
4.6.1. End of treatment 
For evaluable subjects undergoing treatment with consolidation 
chemotherapy, end of treatment is defined as 30 days from day 1 of last 
cycle of chemotherapy.  For subjects undergoing treatment with 
consolidation HCT, end of treatment is defined as 30 days from day 0 of 
HCT.  
4.6.2. End-of-Consolidation Visit 
This visit occurs after completion of consolidation chemotherapy or HCT.  
For evaluable subjects who received consolidation chemotherapy, the 
EOC visit should occur within 60 days of day 1 of the last cycle of 
chemotherapy.  Last cycle of chemotherapy may be cycle 3 or cycle 4.  
For subjects undergoing treatment with consolidation HCT, the EOC visit 
should occur within 100 days from day 0 (day cells are transplanted). 
4.6.3. Procedures and Assessments 
The following procedures and assessments will occur at the end of 
consolidation (EOC) according to the Study Calendar (Section 5.0): bone 
marrow aspirate/biopsy, remission assessment, and maintenance therapy 
status. For subjects undergoing treatment with consolidation HCT, GVHD 
is assessed.  
4.7. 
Short and Long-Term Follow-up Visits  
 
Subjects will be followed for up to five years from enrollment to the study according 
to the Study Calendar (Section 5).  For Year 1, follow-up visits will occur at 6 and 12 
months from the enrollment date.  If a follow-up visit timepoint occurs while the 
subject is still receiving therapy (induction or consolidation), that follow-up visit will 
not be required. For all subjects, the following procedures and/or assessments will be 
recorded during follow-up: remission assessment, maintenance therapy status, 
survival. For subjects who underwent consolidation HCT, GVHD will also be 
assessed.  
 
For subjects who underwent HCT and who have not relapsed, a bone marrow biopsy is 
required at 6 months (± 30 days) and 12 months (± 30 days) from Day 0, and the 6 
month timepoint will include an LSC assay (LSC6) . For subjects who underwent 
consolidation chemotherapy and have not relapsed, bone marrow biopsies are optional 
per investigator discretion during follow-up.   


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During Years 2-5, subjects will complete follow-up annually for relapse and survival 
status from the date of the Year 1, 12-month follow-up visit.  Methods of follow-up 
contact will include standard of care office visits, medical records review, phone calls, 
and/or postal mail.  Bone marrow biopsies during long-term follow-up (years 2-5) are 
optional, per investigator discretion. 
 
4.8. 
Off-Study 
Subjects will be considered Off Study following the final follow-up assessment at year 
5. Subjects may come off-study earlier for the following reasons: 
 
• Study Consent withdrawal (see Section 4.10) 
• Death 
• Lost to follow-up, defined as no successful attempts to contact subject 
over the course of three months 
• Study termination  
• Other, as determined by the Investigator 
 
4.9. Subject Replacements 
 
Enrolled subjects who have discontinued participation in the study after enrollment 
(the time of post-induction bone marrow aspirate) will not be replaced.  
A subject who, for any reason (e.g. failure to satisfy the eligibility criteria or 
withdraws consent), terminates participation in the study before enrollment (the time 
of post-induction bone marrow aspirate) is regarded as a “screen failure”. Screen 
failures may be replaced. 
 
4.10. 
Subject Withdrawal 
 
Subjects are defined as withdrawn from the study if they revoke consent(s) for study 
procedures, or if the Investigator withdraws them from the study. Subjects must be 
withdrawn for the following reasons: 
 
• Subject withdraws consent(s) from study procedures. A subject must be 
removed from the study at his/her own request. At any time during the study 
and without giving reasons, a subject may decline to participate further. The 
subject will not suffer any disadvantage as a result.  
• Subject is lost to follow-up after no successful attempts at contact over three 
consecutive months. 
 


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Subjects may be withdrawn from the study by the Investigator for the following 
reasons:  
 
• The subject is non-compliant with study procedures (as determined by the 
Investigator); including the use of anti-cancer therapy not prescribed by the 
study protocol. 
• Development of an intercurrent illness or situation which would, in the 
judgment of the Investigator, significantly affect assessments of clinical status 
and trial endpoints. 
 
Any subject who withdraws themselves or is withdrawn from the study by the 
Investigator will remain under medical supervision until discharge or transfer is 
medically acceptable. 
 
In all cases, the reason for withdrawal must be recorded in the subject’s medical 
records and/or research chart. Withdrawn subjects are considered to be Off Study.  
 
Details for premature termination of the study as a whole (or components thereof [e.g. 
investigational sites, treatment arms,]) are provided in Section 11.2. 


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5. STUDY CALENDAR 
 
a. 
Required for the purposes of the study. 
b. 
Required at 6 months (± 30 days) and 12 months (± 30 days) from Day 0 for evaluable subjects who received HCT; 
Optional for evaluable subjects who received consolidation chemotherapy, or per investigator discretion. 
c. 
Optional for all evaluable subjects at each long-term follow-up visit, per investigator discretion. 
d. 
Per Cheson Criteria for Leukemia. Also includes reporting first relapse in evaluable and OC2 cohort subjects. 
e. 
For evaluable subjects undergoing consolidation therapy with HCT. 
f. 
For evaluable subjects undergoing HCT, an LSC Assay will be performed 6 months (+/- 30 days) following HCT. 
g. 
Only required for evaluable subjects 
h. 
Required lab tests include at a minimum: Creatinine, bilirubin  
i. 
Favorable and intermediate risk subjects only. 
j. 
If a follow-up visit timepoint occurs while the subject is still receiving therapy (induction or consolidation), that follow-up 
visit will not be required. 
 
 
 
 
Visit Name 
Pre-Screening 
End of 
Induction 
 
End of 
Consolidationg  
Short Term  
(Year 1)  
Follow-Upj 
Long Term (Years 2-5) 
Follow-Up 
 
Visit Occurrence 
Occurs at 
suspected 
AML 
diagnosis 
Occurs at 
completion of 
induction 
therapy 
Occurs at 
completion of 
consolidation 
therapy 
Occurs 6, 12 
months from 
enrollment date 
Occurs annually from 
date of Year 1 Follow-
Up 
 
Visit Window 
Prior to start 
of induction 
chemotherapy 
Within 30 days 
prior to the 
start of 
consolidation 
therapy 
Within 60 
days from day 
1 of last 
chemotherapy 
cycle or within 
100 days from 
Day 0 (HCT) 
+/- 30 days 
+/- 30 days 
 
Step 1 Consent 
X 
 
 
 
 
LSC0 Assay 
X 
 
 
 
 
LSC1 Assay 
 
X 
 
 
 
eLSC Assay 
 
 
X 
 
 
LSC6 Assay 
 
 
 
Xf,g 
 
Step 1 Eligibility 
Check 
 
X 
 
 
 
Step 2 Consenti 
 
X 
 
 
 
 Registration 
 
X 
 
 
 
Physical Exam 
 
Xa 
 
 
 
 
Laboratory Tests 
 
Xa,h 
 
 
 
 
ECOG 
Performance 
Status 
 
Xa 
 
 
 
Bone Marrow 
Aspirate/Biopsy 
X 
X 
X 
Xb,g 
Xc,g 
 
GVHD 
Assessmente 
 
 
X 
Xg 
Xg 
 
Remission 
Evaluationd 
 
X 
X 
X 
X 
 
Maintenance 
Therapy 
Evaluation 
 
 
X 
X 
X 
 
Survival Status 
 
 
 
X 
X 
 


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6. TREATMENT EVALUATION 
Response criteria and measurement of effect will be per the Cheson criteria for AML (see 
Appendix A)1.  
Relapse following complete remission is defined as:  
1. Peripheral Blood Counts: reappearance of blasts in the blood. 
2. Bone Marrow Aspirate and Biopsy: Presence of > 5% blasts, not attributable 
to another cause (e.g., bone marrow regeneration) or dysplasia in greater than 
10% of any lineage with cytopenias in one or more lineages or presence of a 
cytogenetic abnormality consistent with MDS or AML.  
3. Development of extramedullary disease. 
 
7. TREATMENT-RELATED ADVERSE EVENTS 
 
All adverse events will be managed per the Investigator according to standard of care. 
Adverse events can be unpredictable in nature and severity, although all care will be taken to 
minimize them. If subjects suffer any physical injury as a result of participating in this study, 
immediate medical treatment is available at the treatment center. Frequent blood samples will 
be taken to monitor side effects.   
8. DATA AND SAFETY MONITORING PLAN 
 
8.1. 
Safety Monitoring 
 
In addition to the early stopping rules for safety described for GVHD in Section 
10.8.1, this protocol will be monitored according to the processes in effect for all 
Levine Cancer Institute Investigator-Initiated Trials and will abide by standard 
operating procedures set forth by both the Carolinas HealthCare System Office of 
Clinical and Translational Research and the Levine Cancer Institute Clinical Trials 
Office.  It is the responsibility of the Sponsor-Investigator to monitor the safety data 
for this study. The Sponsor-Investigator will monitor the study accrual data every 
month and will evaluate laboratory correlative data on a subject-by-subject basis.  The 
Sponsor-Investigator, Statistician, and other team members as needed will meet 
regularly to monitor subject consents, enrollment and retention, safety data for all 
subjects, and validity/integrity of the data.  Documentation of these meetings will be 
kept with study records.  
 
                                                 
 
 


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SAEs will be reported to the IRB and LCI Data and Safety Monitoring Committee 
(DSMC) per their requirements.  Major protocol deviations that result in a threat to 
subject safety or the integrity of the study will be reported to IRB per their 
requirements. The Sponsor-Investigator will submit data to the DSMC according to 
the overarching LCI Data and Safety Monitoring Plan (DSMP). 
 
8.2. 
Data Quality Assurance 
 
This study will be organized, performed, and reported in compliance with the study 
protocol, standard operating procedures (SOPs) of the Levine Cancer Institute and 
Carolinas HealthCare System Office of Clinical and Translational Research,  and 
other applicable regulations and guidelines (e.g. GCP).  
 
Data will be collected on electronic Case Report Forms (eCRFs). 
A subset of subjects will be monitored by LCI Research Monitors routinely for data 
quality. This monitoring will be done by comparing source documentation to the 
eCRFs.  
 
The study database will be reviewed and checked for omissions, apparent errors, and 
values requiring further clarification using computerized and manual procedures. 
Data queries requiring clarification will be generated and addressed by the 
appropriate research personnel. Only authorized personnel will make corrections to 
the study database and all corrections will be documented in an electronic audit trail. 
 
Any variation between the two data sets will be discussed with the appropriate 
research personnel. 
8.3. 
Communication Between Investigational Sites 
Investigational sites will be required to report any problem that could affect the 
validity/integrity of the study data to the Sponsor-Investigator. Any problem should 
be communicated to the Sponsor-Investigator by email as soon as possible but within 
2 business days of the treating Investigator learning of the event.  
9. SAFETY DATA COLLECTION, RECORDING AND REPORTING 
 
For the purposes of the study, safety variables include instances of GVHD in evaluable 
subjects who have undergone HCT only. Safety assessments should be performed according 
to the study calendar and as clinically necessary.   
 
9.1. 
Unanticipated Problem Definition 
 
A UAP is any incidence, experience or outcome that is unexpected, given the 
information provided in research-related documentation (e.g., Investigator’s brochure, 


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informed consent) and the study population characteristics that is related or possibly 
related to participation in the research study and places the participant at an increased 
risk.   
 
9.2. 
Study Adverse Event (AE) Definition 
 
An adverse event is any untoward medical occurrence associated with the use of a 
drug/device in humans, whether or not considered drug/device related.  An adverse 
event can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of the 
study treatment, whether or not considered related to the study treatment.   
Both forms of treatment on this study (cytarabine-based consolidation chemotherapy 
and HCT) are approved treatments for this disease population, and are considered 
standard of care. Therefore, with the exception of GVHD, which is an event of 
clinical interest, adverse events experienced by the subjects will not be recorded in 
the eCRFs or included in the analysis for the purposes of this study. However, such 
safety events will still be managed according to standard of care and recorded in the 
subject’s medical record per routine clinical standard.  
9.3. 
“Unexpected” Definition 
An AE or SAR is to be considered unexpected if the event is not listed in the current 
Investigator Brochure or is not listed in the severity or specificity observed.  
Investigators should refer to the Safety Information section of the current IB for the 
therapy given, including the DCSI (development core safety information), for the 
expected side effects of the given therapy.  As with any agent, there is always the 
potential for unexpected AEs, including hypersensitivity reactions.   
9.4. 
Study Serious Adverse Event (SAE) Definition 
A serious adverse event is one that results in any of the following:  
• Death 
• Life-threatening illness 
• Inpatient hospitalization or prolongation of existing hospitalization.  
o NOTE: In general, hospitalization means that the subject has been 
detained at the hospital or emergency ward for observation or treatment 
that would not have been appropriate in the physician’s office or 
outpatient setting. For this study, hospitalization that is part of 
planned therapy is not considered an SAE.  
• Persistent or significant disability or incapacity.  


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o NOTE: Disability is defined as a substantial disruption of a person’s 
ability to conduct normal life functions. This definition is not intended 
to include experiences of relatively minor medical significance such as 
uncomplicated headache, nausea, vomiting, diarrhea, influenza, and 
accidental trauma (e.g., sprained ankle) which may interfere or prevent 
everyday life functions but do not constitute a substantial disruption. 
• A congenital anomaly or birth defect 
• An important medical event.  
o NOTE: An event may be considered an important medical event when, 
based upon appropriate medical judgment, it may jeopardize the subject 
and may require medical or surgical intervention to prevent one of the 
outcomes listed in the definition. Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do 
not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
• Pregnancy 
As previously described, each of the treatments in this study (cytarabine-based 
consolidation chemotherapy and HCT) are FDA approved and are considered standard 
of care in this disease population. Therefore, with the exception of GVHD, which is an 
event of clinical interest, safety events experienced by the subjects will not be 
recorded in the eCRF. However, such safety events will still be managed according 
to standard of care and recorded in the subject’s medical record per routine clinical 
standard. 
9.5. 
Event of Clinical Interest – Graft Versus Host Disease (GVHD) 
For the purposes of the study, we will only be collecting and analyzing events of 
GVHD in evaluable subjects who received HCT. This data will be recorded in a study 
eCRF and be analyzed with the study dataset. We will collect data for confirmed 
incidents of GVHD from the time of consolidation HCT through long term follow-up. 
 
The Investigator is responsible for verifying and providing source documentation for 
all incidents of GVHD that occur in evaluable subjects who received HCT, and 
assigning the classification and grade for each incident. 
 
Occurrences of GVHD should be coded using an internationally recognized dictionary. 
Because the CTCAE for toxicity cannot be applied to GVHD, the Investigator will 
classify the toxicity based on the timing as defined below.  
• Acute GVHD: Confirmed GVHD within 100 days of HCT. 


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• Chronic GVHD: Confirmed GVHD 100 days or more after HCT.  
Each occurrence of GVHD in evaluable subjects who received HCT, will be further 
graded by the investigator according to accepted institutional practice which is 
informed by either CIBMTR grading for Acute GVHD or NIH Consensus criteria for 
Chronic GVHD. 
9.6. 
SAE Reporting Requirements 
SAEs meeting each of the following criteria must be reported to the Sponsor-
Investigator within 24 hours of awareness via the CTMS.  
• Subject must be in the evaluable cohort 
• Event meets the definition of SAE per Section 9.4. 
• Event is considered unexpected to the study intervention 
• Event is assigned an attribution of at least possibly related to the study 
intervention 
SAEs will be assigned toxicity scores using the NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.0. 
   
If the SAE is GVHD, the Investigator will quantify the toxicity based on intensity and 
stage per Section 9.5.  
 
Reportable SAEs will be captured from the time of enrollment through 30 days after 
the date of last consolidation chemotherapy administration or 30 days from  
Day 0 of HCT in subjects who receive consolidation HCT. SAEs will be followed 
until clinical recovery is complete, laboratory tests have returned to baseline, or until 
there has been acceptable resolution of the event. This may at times cause the follow-
up period for SAEs to be greater than 30 days. The above referenced 30-day time 
period applies even if the subject begins subsequent anti-cancer therapy during this 
time period. Similarly, the treating investigator is responsible for following the subject 
during the required follow-up period even if the subject lives elsewhere or has been 
released from his or her care and is being treated under another service at LCI. 
 
Confirmed Events of Clinical Interest, as defined in Section 9.5, will be reported in 
evaluable subjects from date of Consolidation HCT (Day 0) through long term follow-
up which is 5 years from enrollment. 
9.7. 
Safety Reporting to the IRB 
All events occurring during the conduct of a protocol and meeting the definition of a 
UAP or SAE will be reported to the IRB per IRB reporting requirements. 


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Protocol deviations will be reported promptly to the IRB per IRB reporting 
requirements.  
10. STATISTICAL METHODS 
 
10.1. 
Sample Size 
The primary analysis will compare two-year RFS probabilities for subjects with 
detectable LSCs at eLSC (eLSC+) and subjects without detectable LSCs at eLSC 
(eLSC-). The targeted sample size is 200 subjects who are evaluable for the primary 
objective (Section 10.3).  Assuming a prevalence of 63% for eLSC+ subjects and 37% 
for eLSC- subjects based on the subject distribution shown in the probability diagram 
(Figure 4), we anticipate that there will be approximately 125 subjects with detectable 
LSCs and 75 subjects without detectable LSCs at end of consolidation. This assumed 
prevalence of having detectable LSCs at end of consolidation (eLSC+) subjects was 
determined using the joint probabilities for each arm shown in the probability tree 
diagram (Figure 4). The probabilities for each of the conditional events (e.g. the 
probability of detectable LSCs post induction given that the subject is determined to be 
intermediate risk is P(LSC1+|IR) = 0.7) are assumed based on previous study data. The 
joint probabilities for each path are determined by multiplying the conditional event 
probabilities along that path. For example, the probability that a favorable risk subject 
who was positive for LSCs post induction (LSC1+) and who underwent HCT will have 
no detectable LSCs at end of consolidation  (eLSC-) is P(FR∩LSC1+∩HCT∩eLSC-) = 
0.1*0.5*0.45*0.5 = 0.01125. The marginal probabilities for eLSC+ and eLSC- are then 
found by summing all the joint probabilities that have the final outcome of eLSC+ and 
eLSC-, respectively. From Figure 4, the resulting probability of having detectable LSCs 
at end of consolidation is P(eLSC+) = 0.62705, while the probability of not having 
detectable LSCs at end of consolidation is P(eLSC-) = 0.37295. This assumed 
distribution of eLSC+ and eLSC- subjects will provide, at minimum, 90% power to 
detect at least a 25% improvement in 2-year recurrence free survival probabilities 
between the eLSC+ and eLSC- groups, assuming two year relapse free survival rates 
ranging from 10% to 40% in the eLSC+ group. These calculations were made to 
preserve a two-sided significance level of alpha = 0.05. Note that, since the assumptions 
about the various pathways may not reflect the subject population accrued for the study, 
the power calculations will be updated according to the true prevalence of having 


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detectable LSCs at end of consolidation.
 
Figure 4. Probability Tree 
 
 


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10.2. 
Endpoint Definitions 
10.2.1. 2-Year and 1-Year Relapse Free Survival 
In this study, 2-year relapse free survival will be calculated from a recorded binary 
variable determined for each subject indicating whether or not the subject experienced 
any disease relapse or death from any cause within two years from CR1. Relapse will be 
classified according to Cheson criteria. The binary variable described above will also be 
calculated at one year for the secondary objective of one-year RFS. 
 
10.2.2. Overall Survival 
 
Overall survival (OS) is defined as the duration of time from the enrollment date (date of 
the post-induction LSC1 assay) to the date of death from any cause. Subjects who are 
alive or lost to follow-up at the time of the analysis will be censored at the last known 
date they were alive. 
 
10.2.3. Relapse Free Survival 
 
Relapse free survival (RFS) is defined as the duration of time from the enrollment date 
(date of the post-induction LSC1 assay) to the date of relapse or death from any cause. 
Relapse will be classified according to Cheson criteria. For surviving subjects who have 
not experienced a relapse or are lost to follow-up at the time of analysis, RFS will be 
censored at the date of last objective disease assessment. 
 
10.2.4. Safety Endpoints 
 
The graft-versus-host disease (GVHD) classification (acute vs chronic), incidence, and 
grade/severity will be the primary safety endpoint.  
 
10.3. 
Analysis Populations 
 
The primary efficacy analyses will be conducted on the subset of subjects who were 
included in the evaluable cohort (defined in Section 4.2) and who also had a 
successful/interpretable end of consolidationeLSC assay. This population of subjects 
will be evaluable for the primary objective. The evaluable populations used for the 
secondary efficacy analyses will be determined by those subjects in the evaluable cohort 
who have successful and interpretable  LSC1, eLSC, and LSC6 assays, depending on the 
objective. Additionally, analyses of the safety endpoints will be conducted on the 
applicable treatment cohort of subjects (Section 4.2). It is noted, however, that the 
evaluation of GVHD will only be applicable to the subjects undergoing HCT. In 


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addition to evaluating outcomes across all enrolled subjects, exploratory assessments 
will be conducted on the following two observational cohorts: 
 
Observational cohort 1 (OC1):  enrolled subjects who do not achieve a CR to induction 
therapy 
 
Observational cohort 2 (OC2):  enrolled subjects who achieve a CR to induction therapy  
but meet one or more of the following criteria:  
• Lack the immunophenotype of interest,   
• Cytarabine based induction subjects: Are not a candidate for [(as determined by the 
investigator (e.g. unfit or refusal)] and do not receive consolidation therapy 
(cytarabine-based chemotherapy or HCT)  
• HMA-based induction subjects: Are not a candidate for [as determined by the 
investigator (e.g. unfit, lack of donor, refusal)] and do not receive HCT 
10.4. 
Analysis Methods 
10.4.1. Timing of Analysis 
The analyses for the primary objective will occur when either all subjects in the study 
have experienced a relapse or else when they have been followed for the 2-year period. 
Subject disposition, baseline characteristics, treatment compliance, will also be assessed 
at that time. Additionally, secondary analyses involving two-year and one-year RFS 
rates will be assessed along with observational cohorts. 
For the purposes of overall survival and relapse free survival, initial analysis will take 
place at the time of the primary analysis. However, subjects will continue to be followed 
until either all subjects have died from any cause or when they have been followed for 5 
years from the date of the post-induction LSC1 assay.  Whenever these criteria are met, 
final survival analysis methods will be implemented for OS and RFS, and the study will 
be closed to IRB. 
10.4.2. Subject Disposition  
 
An accounting of all consented subjects will be provided at the end of the study. 
This will include a breakdown of subjects who consented, enrolled, achieved 
CR1, were included in the evaluable cohort, , received HCT (versus conventional 
consolidation therapy), completed 5 years of follow-up, were discontinued early, 
and those who were lost to follow-up or withdrew consent. 
 
A summary of subject demographics and disease-related characteristics will be 
completed. 


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10.5. 
Efficacy Analysis 
10.5.1. Primary Analysis 
 
The primary analysis will be to compare the relapse free rate at two years between 
the subjects who have detectable LSCs at end of consolidation (eLSC+) and those 
who do not have detectable LSCs at end of consolidation (eLSC-). The relapse 
free survival rates for each group will be calculated and the corresponding 95% 
confidence intervals will be estimated using the Clopper-Pearson method for 
binomial confidence intervals. The relapse free survival rates will also be 
compared between the groups in a stratified logistic regression model. The model 
will be stratified for each of the possible treatment pathways shown in Figure 4 
and will include a covariate for eLSC group. In the event that any pathway is too 
sparse, the stratified design will be collapsed as needed. The group difference will 
be tested at a 2-sided alpha = 0.05 significance level. 
10.5.2. Secondary Analyses 
For each of the secondary objectives, a two-sided alpha = 0.05 level of 
significance will be used for testing. Most of the secondary objectives involve 
comparing RFS rates calculated for selected subgroups (e.g. LSC1+ versus LSC1- 
groups in the subgroup of favorable risk subjects who received consolidation) and 
at the appropriate point in time. The specific group comparisons are described in 
Section 1.2. The RFS rates for the selected subgroups will be analyzed in a similar 
fashion as described above for the 2-year RFS rate primary analysis. 
Overall survival and relapse free survival in the AML subjects will also be 
assessed using Kaplan-Meier techniques. A log-rank test will be used to compare 
the eLSC+ group to the eLSC- group. Cox proportional hazards models will be 
used to evaluate baseline and disease characteristics in addition to the eLSC group 
effect. These methods will also be repeated for all relevant objectives using 
temporally comparable LSC sample measurements (LSC6 samples for HCT 
patients and eLSC samples for the chemo subjects), comparing the LSC6+ (or 
eLSC+, respectively) group versus the LSC6- (or eLSC-) group, as these 
measurements are closer chronologically than the end of consolidation samples 
between the chemotherapy and transplant groups.  
Additional RFS rate comparisons will be explored among other groups. This 
includes comparing the treatment effects (HCT versus consolidation therapy) on 
two-year RFS in unfavorable subjects without detectable LSCs at LSC1, as well 
as broader comparison of the LSC1 groups (LSC1+ versus LSC1-) and their 
impact on the two-year and one-year RFS rates. Additionally, the impact of 


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baseline and disease characteristics on some outcomes will be evaluated. 
Univariate models will be used to identify independent prognostic factors. The 
treatment group term will be added to the models to obtain adjusted group effects. 
In the subjects that undergo allogeneic HCT, descriptive statistics will be used to 
evaluate the change in LSC status from eLSC (end of consolidation) to LSC6 (6 
months after day 0 of transplant). A breakdown will be provided, summarizing the 
frequency and proportion of subjects in each of the following LSC status 
categories: eLSC+/LSC6+; eLSC+/LSC6-; eLSC-/LSC6+; eLSC-/LSC6-. 
10.6. 
Exploratory Analyses 
 
Clinical outcomes of the two observational cohorts (Section 10.3) of subjects who are 
enrolled in the study but do not become a part of the evaluable population will be 
analyzed. Selected comparisons of interest will be evaluated using Fisher’s Exact test. 
Additionally, survival outcomes will be evaluated as necessary using Kaplan Meier 
methods and Cox proportional hazards regression. 
10.7. 
Interim Analyses 
 
A planned interim analysis of the primary endpoint, 2-year relapse free survival, at 50% 
information time (n=100 subjects evaluable for the primary objective with 2 years of 
follow up from CR1) will be conducted. This will be a group sequential analysis 
utilizing the O’Brien-Fleming boundaries shown in Table 1. The corresponding nominal 
2-sided significance level at 50% information time is 0.003, and at 100% information 
time, 0.049, to achieve at total 2-sided alpha of 0.05. If the actual interim analysis does 
not occur at exactly 50% information time, then the O’Brien-Fleming boundaries will be 
updated accordingly. 
Table 1. O’Brien-Fleming boundaries for group sequential interim analysis at 50% 
information time. 
Analysis 
% Information 
Time 
Nominal 
Alpha 
Lower 
Boundary 
Upper 
Boundary 
Interim 
50% 
0.003 
-2.96259 
2.96259 
Final 
100% 
0.049 
-1.96857 
1.96857 
 
 
10.8. 
Safety Analyses 
Incidence rates for GVHD collected from the time of HCT until the off study date 
will be summarized as counts and proportions.  For selected events, 95% 


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confidence intervals will be calculated using the Clopper-Pearson method. Selected 
comparisons of interest will be evaluated using Fisher’s Exact test.  
10.8.1. Early Stopping Rules for Safety 
This study will monitor acute grade III/IV GVHD in the transplant arm in the 
favorable risk cohort.  If it becomes evident that the proportion of acute grade 
III/IV GVHD convincingly exceeds 10%, the study will be halted for a safety 
consultation. The stopping rule will hold enrollment if the posterior probability of 
toxicity risk exceeding 0.10 is 75% or higher in the favorable risk cohort.  The 
prior for this monitoring rule is beta (1,9).  This means that our prior guess at the 
proportion of acute grade III/IV GVHD is 10%, and there is 90% probability that 
this proportion is between 0.57% and 28.3%.  The operating characteristics of the 
stopping rule are given in the Table 3 and are based on 5000 simulations: 
     
Table 2. Early stopping rule cutoffs for numbers of GVHD events. 
Number of 
GVHD 
Events 
Max. Denominator 
Cutoff for Early 
Stopping 
Posterior 
Probability 
Pr(Risk>0.1| 
Data) 
2 
8 
0.7556 
3 
16 
0.7744 
4 
25 
0.7552 
5 
34 
0.7520 
6 
43 
0.7528 
7 
50 
0.7738 
 
 
Table 3. Operating characteristics of stopping rule based on 5000 simulations.  
True 
Tolerability 
Risk 
Prob. Declare 
Treatment Not 
Tolerable 
Average 
Sample Size 
0.02 
1.2% 
39.6 
0.05 
9.4% 
37.3 
0.10 
39.8% 
29.9 
0.15 
72.9% 
21.0 
0.20 
91.1% 
14.4 
0.25 
98.0% 
10.1 
0.30 
99.6% 
7.7 
 
 


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11. STUDY COMPLETION 
 
11.1. 
Completion 
 
The study will be considered complete when one or more of the following conditions 
is met: 
• 
All subjects have completed all study visits. 
• 
All subjects have discontinued from the study. 
• 
The IRB, LCI DSMC, or Sponsor-Investigator discontinues the study. 
• 
The Sponsor-Investigator defines an administrative or clinical cut-off date. 
 
11.2. 
Termination 
 
The study will be terminated when one or more of the following conditions occur: 
If risk-benefit ratio becomes unacceptable owing to, for example: 
• Safety findings from this study (e.g. SAEs) 
• Results of any interim analysis 
• Results of parallel clinical studies 
• Results of parallel animal studies (e.g. toxicity, teratogenicity,  
         carcinogenicity or reproduction toxicity). 
• If the study conduct (e.g. recruitment rate; drop-out rate; data quality; protocol 
compliance) does not suggest a proper completion of the study within a 
reasonable time frame. 
The Sponsor-Investigator has the right to close the study at any site and at any time. 
For any of the above closures, the following applies: 
• Closures should occur only after consultation between involved parties. 
• All affected institutions must be informed as applicable according to local 
law.   
• In case of a partial study closure, ongoing subjects, including those in follow-
up, must be taken care of in an ethical manner. 
 
Details for individual subject's withdrawal can be found in Section 4.11. 
 
12. RETENTION OF RECORDS 
 
Essential documentation including all IRB correspondence, will be retained for at least 2 
years after the investigation is completed.  Documentation will be readily available upon 
request. 
 
 


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13. ETHICAL AND LEGAL ISSUES 
13.1. 
Ethical and Legal Conduct of the Study 
 
The procedures set out in this protocol, pertaining to the conduct, evaluation, and  
documentation of this study, are designed to ensure that the Investigators abide by 
Good Clinical Practice (GCP) guidelines and under the guiding principles detailed in 
the Declaration of Helsinki.  The study will also be carried out in keeping with 
applicable local law(s) and regulation(s). 
 
Documented approval from appropriate agencies (IRB) will be obtained before the 
start of the study, according to GCP, Declaration of Helsinki, local laws, regulations 
and organizations.  When necessary, an extension, amendment, or renewal of IRB 
approval must be obtained and kept on file.   
 
Strict adherence to all specifications laid down in this protocol is required for all 
aspects of study conduct; the Investigators may not modify or alter the procedures 
described in this protocol.   
 
Modifications to the study protocol will not be implemented by the Sponsor-
Investigator without IRB approval.  However, the Investigators may implement a 
deviation from, or a change of, the protocol to eliminate an immediate hazard(s) to the 
trial subjects without prior approval from applicable agencies.  As soon as possible, 
the implemented deviation or change, the reasons for it, and if appropriate, the 
proposed protocol amendment should be submitted to the appropriate agencies.  Any 
deviations from the protocol must be explained and documented by the Investigator. 
 
The Sponsor-Investigator is responsible for the conduct of the clinical trial at the sites 
in accordance with  Good Clinical Practice (GCP) and the Declaration of Helsinki.  
The Sponsor-Investigator is responsible for  overseeing the treatment of all study 
subjects.  The Sponsor-Investigator must assure that all study site personnel, including 
sub-investigators and other study staff members, adhere to the study protocol and all 
applicable regulations and guidelines regarding clinical trials both during and after 
study completion. 
 
The Sponsor-Investigator will be responsible for assuring that all the required data will 
be collected and properly documented. 
 
 
 
 


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13.2. 
Confidentiality 
 
All records identifying the subject will be kept confidential and, to the extent 
permitted by the applicable laws and/or regulations, will not be made publicly 
available. 
 
14. CLINICAL TRIAL REGISTRATION AND RESULTS REPORTING 
The Sponsor-Investigator will ensure that the information and results regarding the 
study will be made publicly available on the internet at www.clinicaltrials.gov. 
 
 
 
 


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clearance of human repopulating cells masks the heterogeneity of leukemia-initiating 
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 15.  Sarry JE, Murphy K, Perry R et al. Human acute myelogenous leukemia stem cells are 
rare and heterogeneous when assayed in NOD/SCID/IL2Rgammac-deficient mice. 
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 16.  Eppert K, Takenaka K, Lechman ER et al. Stem cell gene expression programs influence 
clinical outcome in human leukemia. Nat.Med. 2011;17:1086-1093. 
 17.  Pearce DJ, Taussig D, Zibara K et al. AML engraftment in the NOD/SCID assay reflects 
the outcome of AML: implications for our understanding of the heterogeneity of AML. 
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 18.  Rombouts WJ, Martens AC, Ploemacher RE. Identification of variables determining the 
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 20.  Christ O, Lucke K, Imren S et al. Improved purification of hematopoietic stem cells 
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APPENDICES 
 
Appendix A 
Cheson Criteria for Remission 
 
Response Criteria in AML 
 
Response Criterion                            Time of Assessment               Neutrophils (µL)       Platelets (µL)        Bone Marrow              Other 
 
 
 
 
 
 
 
                               Blasts (%) 
___________________________________________________________________________________________________________________________________ 
Early treatment assessment              7-10 days after therapy      NA                  NA                        <5 
Morphologic leukemia-free state     Varies by protocol             NA                  NA                        <5                    Flow cytometry1; no EMD 
Morphologic CR                              Varies by protocol             1,000               100,000                 < 5                   Transfusion independent; no EMD 
Cytogenetic CR                                Varies by protocol             1,000               100,000                 < 5                  Cytogenetics—normal, no EMD 
Molecular CR                                   Varies by protocol             1,000               100,000                 < 5                  Molecular—negative, no EMD 
Partial remission                              Varies by protocol              1,000              100,000     50 or decrease to 5-25     Blasts< 5% if Auer rod positive 
1 Flow cytometry may also be useful to distinguish between leukemia and a regenerating bone marrow 
Abbreviations: AML, acute myelogenous leukemia; EMD, extramedullary disease; CR, complete remission. 
 
Cheson et al: Revised Recommendations of the International Working Group for Diagnosis, Standardization of Response 
Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia ; J Clin Oncol 2003