Clinical trial NCT01588951
Conditions: Acute Myeloid Leukemia
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
Start date: 2013-12-01; Completion date: 2017-04-01
Enrollment: 10 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The primary objective of the trial is to compare the two-year relapse-free survival (RFS) of patients with acute myeloid leukemia (AML), presumed to be at high risk for relapse due to the presence of leukemia stem cells (LSCs) in their bone marrow at first complete remission (CR1), who receive either standard cytarabine-based chemotherapy or allogeneic stem cell transplantation (SCT).

Detailed description:

Arms: arm 1: Without LSC, standard cytarabine consolidation arm 2: LSC present, randomized to cytarabine consolidation arm 3: LSC present, randomized to allogeneic transplant
Interventions: intervention 1: Cytarabine consolidation intervention 2: Allogeneic transplant
Intervention descriptions: intervention 1: Cytarabine-based consolidation per institutional standards. intervention 2: Allogeneic stem cell transplant per institutional standards.

Protocol documents:
Prot_SAP_000.pdf: 
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Protocol Title:  J1227 - Consolidation Therapy for Acute Myeloid Leukemia Guided by 
Leukemia Stem Cell Behavior 
 
 
Principal Investigator: B Douglas Smith, MD 
 
Investigators:  
Mark Levis, MD, Ph.D. 
Margaret M. Showel, MD 
 
 
        
Richard J. Jones, MD 
 
 
 
Amy Dezern, MD 
 
 
 
Ivana Gojo, MD 
 
 
 
Keith Pratz, MD 
 
 
 
Gabriel Ghiaur, MD, Ph.D. 
 
 
 
Douglas Gladstone, MD 
 
 
 
Karen Mackey 
 
 
 
Jackie Greer 
 
 
 
Jackie Jung 
 
 
 
Statistician: 
 
Gary Rosner, PhD 
 
 


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Table of Contents 
 
 
 
 
 
 
 
Page 
1. Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3 
1.1. Primary Objective. . . . . . . . . . . . . . . . . . . . .  
3  
1.2. Secondary Objectives. . . . . . . . . . . . . . . . . . . .  
3  
2. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3 
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .  3 
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. . . . . . . . . . . .  6 
3. Patient Selection and Enrollment . . . . . . . . . . . . . . . . . .  6 
3.1. Inclusion Criteria. . . . . . . . . . . . . . . . . . . . . . .  6 
3.2. Inclusion Criteria Definitions. . . . . . . . . . . . . . . . .  6 
3.3. Exclusion Criteria. . . . . . . . . . . . . . . . . . . . . . .  6 
3.4. Inclusion of Women and Minorities . . . . . . . . . . . . .  6 
3.5. Donor eligibility criteria . . . . . . . . . . . . . . . . . . .  7 
3.6. Informed Consent . . . . . . . . . . . . . . . . . . . . . . .  7 
4. Treatment plan . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7 
4.1. Preconsolidation Plan . . . . . . . . . . . . . . . . . . . . .  7 
4.2. Treatment Schedule . . . . . . . . . . . . . . . . . . . . . .  7 
4.3. Duration of Follow-up . . . . . . . . . . . . . . . . . . . .  7 
4.4. Supportive Care Guidelines . . . . . . . . . . . . . . . . .  7 
4.5. Study Parameters . . . . . . . . . . . . . . . . . . . . . . .  7 
5. Treatment Evaluation. . . . . . . . . . . . . . . . . . . . . . . . .  7 
6. Laboratory correlative studies . . . . . . . . . . . . . . . . . . . .  7 
7. Statistical  Methods . . . . . . . . . . . . . . . . . . . . . . . . .  8 
7.1. Study Design and Objectives . . . . . . . . . . . . . . . . .  8 
7.2. Sample size and Power Considerations . . . . . . . . . . .  8 
7.3. Interim Analysis and Stopping Guidelines . . . . . . . . .  10 
7.4. Secondary Objectives . . . . . . . . . . . . . . . . . . . . .  10 
7.5. Early Stopping Rules for Safety . . . . . . . . . . . . . . .  10 
7.6. Early Stopping Guideline for Futility . . . . . . . . . . . .  11 
8. Data Safety and Monitoring Plan . . . . . . . . . . . . . . . . . .  12 
8.1. Adverse Event Definition . . . . . . . . . . . . . . . . . . .  12 
8.2. Relationship to Investigational Product . . . . . . . . . . .  12 
8.3. Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . .  12 
8.4. Serious Adverse Event (SAE) Definition . . . . . . . . . .  13 
8.5. Adverse Drug Reaction and Toxicity Monitoring . . . . .  13 
8.6. Toxicity Reporting . . . . . . . . . . . . . . . . . . . . . .  13 
8.7. Data Handling and Record Keeping . . . . . . . . . . . . .  13 
9. Ethics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14 
9.1. Institutional Review Board . . . . . . . . . . . . . . . . . .  14 
9.2. Ethical Conduct of the Study . . . . . . . . . . . . . . . . .  14 
9.3. Evaluation of Benefits and Risks/Discomforts . . . . . . .  14 
9.4. Financial Disclosure . . . . . . . . . . . . . . . . . . . . . 14 
10. Off Study. . . . . . . . . . . . . . . . . . . . . . . . . . . .  .  15 
11. References. . . . . . . . . . . . . . . . . . . . . . . . . . . .  .  16 
  
 


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Objective  
1.1. Primary Objective  
The primary objective of the trial is to compare the two-year relapse-free survival (RFS) of patients with 
acute myeloid leukemia (AML), presumed to be at high risk for relapse due to the presence of leukemia 
stem cells (LSCs) in their bone marrow at first complete remission (CR1), who receive either standard 
cytarabine-based chemotherapy or allogeneic stem cell transplantation (SCT). 
1.2. Secondary Objectives 
1. Compare the 2-year RFS survival of those without detectable LSCs in CR1 to those patients 
with detectable LSCs in CR1 who are randomized to cytarabine-based consolidation 
chemotherapy. 
2.  Describe the changes in the leukemic population in blood and marrow during and after 
treatment. 
 
2. Background  
2.1. Introduction 
Although most patients with AML achieve CR following standard induction chemotherapy, the majority 
subsequently relapse and succumb to the disease.1-3 Currently, cytogenetic and molecular aberrations are 
the best prognostic indicators for AML patients.3-5 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, between 40-60% of patients with favorable or intermediate-risk AML will experience relapse. 
Prognosis after relapse is poor with 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 some semblance 
of 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 responsible for relapse.9;10 and OS and RFS of SCT in CR2 is much worse 
than in CR1 11 
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 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 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.  


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2.3. ALDH activity distinguishes LSCs from their normal counterparts 
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 AML analyses focused on patients with newly diagnosed CBF leukemias, as the FISH-detectable 
abnormalities enabled quantification of the percentage 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. 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 
from were both virtually entirely leukemic by FISH. As few as 1000 of the ALDHint cells produced 
leukemic engraftment of NOG mice, whereas, the 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. The ALDHint population appears to contain a 
putative LSC population, distinguishable from the normal HSC population.  
This flow cytometric pattern containing an ALDHint population was absent only in four of 20 newly-
diagnosed AML patients. In two of these patients, most of the CD34+CD38- leukemic cells, as defined by 
FISH, exhibited high ALDH activity, with no discernible separate population of normal CD34+CD38-
ALDHhigh cells. 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 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. 


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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 predicts relapse   
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 sixteen evaluable patients. 
Of those 16 patients, 8 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-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 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 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 SCT 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+38- 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 
have 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 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 is enriched for LSCs  
According to the LSC paradigm, LSCs should constitute a small fraction of the leukemic burden during 
active disease, but should comprise the majority 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 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 3-logs from diagnosis to CR (p<0.001), 
the ALDHint population decreased by only 1 log (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 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 ALDHint population predicts relapse, as expected of 
an LSC population. This 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 Therefore, we propose a study of patients without poor-risk AML, as defined by 
current standards, who, upon achieving a CR after induction therapy, will receive consolidation treatment 
guided by the presence of LSCs in CR1.This will be a prospective, randomized, clinical trial, and patients 
who have the CD34+CD38-ALDHint population will be randomized to either standard chemotherapy (Arm 
A) or SCT (Arm B) for consolidation. Patients without LSCs in CR1 will receive standard consolidation 
chemotherapy (Arm C). 
 
3. Patient Selection and Enrollment 
3.1. Inclusion Criteria 
1. Age > 18 years 
2. Ability to give informed consent 
3. New diagnosis of AML other than APL or poor-risk AML as defined in section 3.2 
3.2. Inclusion Criteria Definitions 
The original diagnosis of AML must have been confirmed by bone marrow aspirate and/or biopsy review 
by a JH Hematopathologist.  Patients are eligible if they have AML that is not classified as poor-risk or 
APL. Poor-risk AML is defined as therapy-related, arising from a previous marrow disorder, or de novo 
AML associated with any of the following characteristics: trilineage dysplasia, FLT3/ITD mutation, poor-
risk cytogenetics including: chromosome 3, 5, or 7 abnormalities, t(6;9), and complex karyotype. 
3.3. Exclusion Criteria 
1. Has already had a bone marrow biopsy and aspirate to assess remission status after induction 
therapy 
2. Any debilitating medical or psychiatric illness that would preclude ability to give informed 
consent or receive optimal treatment and follow-up  
3. Pregnancy: Women of childbearing potential who are β- HCG+   
3.4. Inclusion of Women and Minorities  
The proposed study is opened to both men and women, and to all racial/ethnic subgroups. There is no 
explicit mention of different treatment effects in male and female patients and in different racial/ethnic 
subgroups in the literature. Therefore, this study will not have separate accrual targets for these groups. 


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3.5. Donor Eligibility Criteria 
Donor eligibility will be determined per standard BMT criteria.  
3.6. Informed Consent 
All patients eligible for the study must be evaluated by one of the study investigators.  
Informed consent must be obtained and the consent form signed. A Johns Hopkins On-Study Form will 
be completed following fulfillment of the on-study requirements for laboratory work and eligibility 
criteria. For enrollment of non-English-speaking candidates, the JH IRB-mandated policies and 
procedures will be followed as listed at the following website: 
https://irb.jhmi.edu/Guidelines/nonenglishconsent.html  
 
4. Treatment Plan 
4.1. Preconsolidation Plan 
A bone marrow aspirate or biopsy confirming morphologic complete remission (< 5% blasts and normal 
cytogenetics and/or FISH) will be documented in the electronic medical records. Bone marrow aspirate / 
biopsy documenting complete remission and evaluations for consolidation registration (3.2) will be done 
within 2-4 weeks of the start of consolidation therapy. Patients with a bone marrow aspirate that is not 
evaluable for LSCs will be removed from the study. 
4.2. Treatment Schedule 
Patients without detectable LSCs by flow cytometry, will be assigned to standard cytarabine-based 
chemotherapy as per institutional standards. Patients with detectable LSCs by flow cytometry will be 
randomized to either standard cytarabine-based consolidation therapy or allogeneic SCT, per institutional 
standards. Patients will receive the transplant from a related donor who is at least a haploidentical match.  
The choice of the optimal donor will use institutional priority extant at the time. Patients eligible for 
myeloablative conditioning will be prioritized to receive it; patients not eligible for myeloablative 
conditioning will receive non-myeloablative conditioning.  
4.3. Duration of Follow-up 
Patients will be followed for up to five years from start of consolidation. Patients are routinely followed 
for by JH physicians for their post SCT or post-chemotherapy monitoring and patients who complete the 
study and remain with their care at JH will have survival captured at least annually.   
4.4. Supportive Care Guidelines 
Supportive care, including infection management and transfusion support, will follow good medical 
practice and institutional standard guidelines. 
4.5. Study Parameters 
There are institutional guidelines for study parameters for consolidation therapy with SCT or standard 
chemotherapy. Those guidelines will be followed per routine.  
 
5. Treatment Evaluation 
Response criteria  and measurement of effect will be per the NCI criteria for relapse in AML. 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. 
 
6. Laboratory Correlative Studies 
In order to evaluate the putative LSC population, we will study cells from patient bone marrow aspirate. 
A minimum of 100,000 34+ cells is required for complete analysis. The 34+ 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.  


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7. Statistical  Methods 
7.1. Study Design and Objectives 
This is designed as a prospective, randomized, clinical trial comparing two consolidation schema: 
conventional cytarabine-based consolidation and allogeneic blood or marrow transplantation (BMT). 
Patients will be in first complete remission (CR1) after cytarabine-based induction therapy.  The 
randomized cohort of patients will be those with detectable leukemia stem cells (LSCs) in their remission 
marrow, whom we believe to be at high-risk for disease relapse. 
7.1.1. Primary Objective 
The primary objective of the trial is to compare the treatment-specific relapse-free survival (RFS) 
experiences of AML patients in CR1 with detectable LSCs, as described in the previous paragraph.  The 
primary comparison will be with respect to two-year RFS.  We chose two-year RFS, based on historical 
information that indicates that despite crossing hazards, the benefit of BMT to AML patients is realized 
by two years and extends beyond that point.25;26 RFS is defined as the time until relapse/progression or 
death from any cause from randomization. 
7.1.2. Primary Hypothesis 
The primary hypothesis of the study is that allogeneic BMT will improve outcome (RFS) for AML 
patients in CR1 with detectable LSCs. 
7.1.3. Secondary Hypothesis  
A secondary hypothesis is that the presence of detectable LSCs will correlate with a poorer outcome 
for those AML patients randomized to conventional cytarabine-based consolidation therapy. 
7.2. Sample Size and Power Considerations 
The primary analysis will compare two-year RFS probabilities for the different groups.  The comparisons 
are (1) BMT vs. non-BMT among LSC-positive patients and (2) non-BMT in LSC-positive patients vs. 
non-BMT in LSC-negative patients.  The time point of 2 years was chosen based on data that indicate that 
most events with standard consolidation and allogeneic BMT occur by 2 years.  The targeted sample size 
is 80 patients, 40 per treatment arm.  The baseline RFS at 2 years is assumed to be approximately 10% for 
poor-risk AML patients receiving standard cytarabine-based consolidation. In actuality, the survival in 
our series of patients with detectable LSCs who received conventional-dose cytarabine-based 
consolidation was 0, but this was based on only a small number of patients. Furthermore, we assume that 
allogeneic BMT is associated with at least a 50% 2-year RFS in poor-risk AML patients in CR1.  The 
assumed RFS distributions are shown in figure 3. The targeted sample size of 40 patients per group is 
sufficient to provide over 90% power to detect a difference in 2-year RFS (10% versus 40%), based on 
5000 simulations from the assumed RFS curves, testing (at the 2-sided 0.1-level of significance) the 
equality of the Kaplan-Meier estimated treatment-specific 2-year RFS and associated standard errors.  
Even with the logrank test, we will have around 84% power to detect this difference.  If, however, the 2-
year RFS is 50% (the red curve in Fig 3), as we anticipate, we will have more than 90% power to detect 
this difference when testing the 2-year RFS or with a logrank test.     
About 120 new AML patients are seen at Johns Hopkins per year. We anticipate about 1/3 (or 40 patients 
per year) will be poor-risk by standard cytogenetic/molecular criteria, so about 80 will be eligible for this 
study. If 3/4s agree to enroll on the study, 60 patients per year will be accrued. Our data suggest that half 
(30 per year, 15 per arm) will have detectable LSCs. Thus, the study will take about 3 years to accrue 40 
patients to each arm. The 30 patients per year without detectable LSCs will serve as controls for the 
secondary objective. The final patient enrolled will be followed up for a minimum of 2 years, so that the 
targeted total study duration is 5 years (3 years accrual + 2 years follow-up on the last patient).  


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Figure 3. Assumed Relapse-Free Survival Curves 
The study’s secondary objective concerns comparing relapse-free survival among patients without 
detectable LSCs to the group of patients with detectable LSCs randomized to receive cytarabine-based 
consolidation .  We anticipate roughly twice as many patients without detectable LSCs treated with 
cytarabine-based consolidation as with LSC, as shown in the previous paragraph.  Therefore, we assumed 
that 40 LSC-positive patients will be randomized to receive cytarabine-based consolidation and that the 
study will enter 80 patients who are LSC-negative and will receive standard cytarabine-based 
consolidation.  Assuming the same RFS curve for LSC-positive patients receiving cytarabine-based 
consolidation as in the sample size considerations for the primary objective (i.e., the randomized 
comparison), we will have roughly 85% power to detect a 55% reduction in the risk of relapse or death 
when comparing 40 LSC-positive patients getting cytarabine-based consolidation and 80 LSC-negative 
patients receiving cytarabine-based consolidation. 
Table 1 shows various RFS values at 6-month increments up to two years from the start of treatment for 
various hypothetical RFS scenarios for the LSC-negative patients receiving cytarabine-based 
consolidation, relative to the hazard assumed for the LSC-positive patients randomized to receive 
cytarabine-based consolidation (i.e., the lowest curve in the figure 3).  As with the primary objective, 
these calculations are based on 5000 simulations for each scenario with each test two-sided at the 10% 
level.  In this case, the treatment-related piecewise exponential hazard functions are assumed 
proportional, unlike when comparing BMT to standard cytarabine-based consolidation.  Nonetheless, we 
show power for both the logrank test (which is more powerful in this case) and a comparison of 
treatment-specific two-year RFS estimates. 
 


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Table 1. RFS Estimates 
Relative Risk  
For LSC-Negative on cytarabine-
based consolidation,  
RFS Probabilities After Start of Treatment 
Power Compared to LSC-
Positive 
Compared to LSC-Positive 
6 mo. 
12 mo. 
18 mo. 
24 mo. 
Logrank Test 
Test 2-Year 
RFS 
100% 
 0.85  
 0.42  
 0.21  
 0.10  
0.10 
0.10 
75% 
 0.89  
 0.52  
 0.31  
 0.18  
0.41 
0.38 
65% 
 0.90  
 0.57  
 0.36  
 0.23  
0.66 
0.60 
60% 
 0.91  
 0.59  
 0.39  
 0.25  
0.78 
0.71 
55% 
 0.91  
 0.62  
 0.42  
 0.29  
0.88 
0.83 
50% 
 0.92  
 0.65  
 0.46  
 0.32  
0.94 
0.92 
 
7.3. Interim Analysis and Stopping Guidelines 
Interim analysis for efficacy and futility will be conducted annually by the SKCCC-appointed Data and 
Safety Monitoring Board (DSMB).  The stopping guidelines serve as a trigger for consultation with the 
DSMB for additional review and are not formal “stopping rules” that would mandate automatic closure of 
study enrollment.  Toxicity, adverse events, and other safety endpoints will be monitored regularly and 
reported to the DSMB at each interim analysis. 
7.4. Secondary Objectives 
1. The major secondary objective is to compare RFS of those without detectable LSCs in CR1 
to those with detectable LSCs while in CR1 who are randomized to cytarabine-based 
consolidation chemotherapy. The analysis will compare RFS by the logrank test, as discussed 
at the end of section 7.2 
2. The other secondary objective is to describe the changes in the clonogenic population, 
including changes in detectable cytogenetic abnormalities in blood and marrow, during 
consolidation (both cytarabine-based consolidation and allogeneic BMT)  
7.5. Early Stopping Rules for Safety    
This study will monitor acute grade III/IV GVHD in the transplant arm.  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.  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 1 and 
are based on 5000 simulations: 
 
Toxicity stopping rule: 
 
Serious AE in 2 out of 2 patients.  Pr(Risk>0.1| Data) = 0.91 
 
Serious AE in 2 out of 3 patients.  Pr(Risk>0.1| Data) = 0.889 
 
Serious AE in 2 out of 4 patients.  Pr(Risk>0.1| Data) = 0.866 
 
Serious AE in 2 out of 5 patients.  Pr(Risk>0.1| Data) = 0.842 
 
Serious AE in 2 out of 6 patients.  Pr(Risk>0.1| Data) = 0.816 
 
Serious AE in 2 out of 7 patients.  Pr(Risk>0.1| Data) = 0.789 
 
Serious AE in 2 out of 8 patients.  Pr(Risk>0.1| Data) = 0.762 
 
Serious AE in 3 out of 9 patients.  Pr(Risk>0.1| Data) = 0.902 
 
Serious AE in 3 out of 10 patients.  Pr(Risk>0.1| Data) = 0.885 
 
Serious AE in 3 out of 11 patients.  Pr(Risk>0.1| Data) = 0.867 
 
Serious AE in 3 out of 12 patients.  Pr(Risk>0.1| Data) = 0.848 
 
Serious AE in 3 out of 13 patients.  Pr(Risk>0.1| Data) = 0.828 
 
Serious AE in 3 out of 14 patients.  Pr(Risk>0.1| Data) = 0.807 
 
Serious AE in 3 out of 15 patients.  Pr(Risk>0.1| Data) = 0.786 
 
Serious AE in 3 out of 16 patients.  Pr(Risk>0.1| Data) = 0.764 


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 
Serious AE in 4 out of 17 patients.  Pr(Risk>0.1| Data) = 0.888 
 
Serious AE in 4 out of 18 patients.  Pr(Risk>0.1| Data) = 0.873 
 
Serious AE in 4 out of 19 patients.  Pr(Risk>0.1| Data) = 0.858 
 
Serious AE in 4 out of 20 patients.  Pr(Risk>0.1| Data) = 0.842 
 
Serious AE in 4 out of 21 patients.  Pr(Risk>0.1| Data) = 0.825 
 
Serious AE in 4 out of 22 patients.  Pr(Risk>0.1| Data) = 0.807 
 
Serious AE in 4 out of 23 patients.  Pr(Risk>0.1| Data) = 0.789 
 
Serious AE in 4 out of 24 patients.  Pr(Risk>0.1| Data) = 0.77 
 
Serious AE in 4 out of 25 patients.  Pr(Risk>0.1| Data) = 0.75 
 
Serious AE in 5 out of 26 patients.  Pr(Risk>0.1| Data) = 0.868 
 
Serious AE in 5 out of 27 patients.  Pr(Risk>0.1| Data) = 0.855 
 
Serious AE in 5 out of 28 patients.  Pr(Risk>0.1| Data) = 0.84 
 
Serious AE in 5 out of 29 patients.  Pr(Risk>0.1| Data) = 0.825 
 
Serious AE in 5 out of 30 patients.  Pr(Risk>0.1| Data) = 0.81 
 
Serious AE in 5 out of 31 patients.  Pr(Risk>0.1| Data) = 0.794 
 
Serious AE in 5 out of 32 patients.  Pr(Risk>0.1| Data) = 0.777 
 
Serious AE in 5 out of 33 patients.  Pr(Risk>0.1| Data) = 0.76 
 
Serious AE in 6 out of 34 patients.  Pr(Risk>0.1| Data) = 0.867 
 
Serious AE in 6 out of 35 patients.  Pr(Risk>0.1| Data) = 0.854 
 
Serious AE in 6 out of 36 patients.  Pr(Risk>0.1| Data) = 0.841 
 
Serious AE in 6 out of 37 patients.  Pr(Risk>0.1| Data) = 0.828 
 
Serious AE in 6 out of 38 patients.  Pr(Risk>0.1| Data) = 0.814 
 
Serious AE in 6 out of 39 patients.  Pr(Risk>0.1| Data) = 0.8 
 
Serious AE in 6 out of 40 patients.  Pr(Risk>0.1| Data) = 0.785 
Table 2 
True Tolerability 
Risk 
Prob. Declare 
Treatment Not 
Tolerable 
Avg. 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 
 
 
 
7.6. Early Stopping Guideline for Futility and Simulations for Each Cohort  
The simulation studies demonstrate the power for the respective cohort sizes. Since we anticipate 
randomizing 30 patients per year and the primary endpoint of the study is 2-year RFS, 60 patients would 
be accrued by the time the first patient enrolled on study would reach two years of follow-up.  Thus, 
almost all patients will have entered the study by the time we would have enough data to carry out an 
interim futility analysis.  Therefore, the study does not include a formal futility monitoring rule.  If, 
however, accrual is slower than we anticipate, we will consider including an analysis for futility.  
 
Table 1. Operating characteristics of stopping rule based on 5000 simulations. 
Table 2. Operating characteristics of stopping rule based on 5000 simulations.


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8. Data Safety and Monitoring Plan 
This is a DSMP Level I "Medium Risk" study under the SKCCC Data Safety Monitoring Plan 
(9/22/2011). The Clinical Research Office QA Group will perform an audit after the first subject has been 
treated and then periodically depending on the rate of accrual and prior audit results. All trial monitoring 
and reporting will be reviewed annually by the SKCCC Safety Monitoring Committee. The PI is 
responsible for monitoring the study. Data must be reviewed to assure the validity of data, as well as, the 
safety of the subjects. The PI will also monitor the progress of the trial, review safety reports, and clinical 
trial efficacy endpoints and to confirm that the safety outcomes favor continuation of the study.  
8.1. Adverse Event Definition 
An adverse event (AE) is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product, regardless of whether the occurrence is 
considered to have a causal relationship with treatment. An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated 
with the use of a medicinal (investigational) product, allogeneic SCT in this study, whether or not 
considered related to the medicinal (investigational) product. AEs are to be coded using an internationally 
recognized dictionary. This study will utilize the Common Toxicity Criteria (CTC) version 4.0 for 
toxicity where applicable for adverse event reporting. In cases where CTC cannot be applied to the toxic 
event, the investigator will quantify the toxicity based on intensity as defined below.  
1. Mild: The subject is aware of signs or symptoms but they are easily tolerated; usually does 
not require additional therapy or discontinuation of investigational product. 
2. Moderate: The signs and symptoms are sufficient to restrict but do not prevent usual activity; 
possible requires additional therapy but usually does not require discontinuation of 
investigational product 
3. Severe: The Subject is unable to perform usual activities and usually requires discontinuation 
of investigational product. 
8.2. Relationship to Investigational Product (SCT) 
The investigator will classify the relationship of an adverse event to the investigational product  according 
to the following definitions:  
1. None: The time course between the investigational product and the occurrence or worsening 
of the adverse event rules out a causal relationship and or another cause is confirmed and no 
indication of involvement of the study product in the occurrence/worsening of the adverse 
event exists.  
2. Unlikely: the time course between investigational product  and occurrence or worsening of 
AE makes a causal relationship unlikely; the known effects of investigational product  
provide no indication of involvement in AE and another cause adequately explains the AE; 
regarding the AE, a plausible causal chain may be deduced from the known effects of 
investigational product   but another cause is much more probably; or another cause is 
confirmed and involvement of investigational product  in the AE is unlikely. 
3. Possible: Regarding the AE, a plausible causal chain may be deduced, but another cause just 
as likely to be involved is also known; although there is no indication which possible cause is 
involved in the AE.  
4. Probable: the course of the AE suggest involvement of the investigational product  in the AE, 
although another cause cannot be ruled out.  
5. Definite: the course of the AE indicates involvement of the investigational product  in the AE 
and no indication of other causes exists.  
6. Unclassifiable: only used for SAE: the available information is not sufficient for causality 
assessment. 
8.3. Outcome 
The investigator will record the outcome of the AE choosing one of the following categories 
1. Recovered/resolved 
2. Recovering/resolving 
3. Not recovered/ not resolved 


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4. Recovered/resolved with residual effects as specified 
5. Fatal 
6. Unknown  
8.4. Serious Adverse Event (SAE) Definition  
As defined by the FDA CFR 312, a serious adverse event is one, occurring at any dose (including 
overdose), that results in any of the following:  
1. Death 
2. Life-threatening illness 
3. Inpatient hospitalization or prolongation of existing hospitalization 
4. Persistent or significant disability or incapacity 
5. A congenital anomaly or birth defect 
6. An important medical event 
7. Pregnancy 
8.5. Adverse Drug Reaction and Toxicity Monitoring 
The study team (research nurse, study coordinator or attending) will assign toxicity scores using the NCI 
common Toxicity Criteria version 4.0, during consolidation therapy. A copy of the CTC version 4.0 is 
available at the CTEP home page (http://ctep.info,nih.gov). If an unexpected and serious toxicity occurs 
that would result in patients being subjected to unacceptable risk, the trial will be placed on hold while 
this toxicity is investigated.  
 
       8.6. Adverse Event Reporting 
For study purposes, the following will be recorded and reported in accordance with IRB requirements: 
 
All unexpected serious adverse events 
The PI will be responsible for reviewing the clinical course of all patients. All serious events will be 
confirmed as expected or unexpected by an expert panel of leukemia doctors. Reporting of serious 
adverse events will cease for subjects who are not following the protocol arm that they were assigned to. 
In addition, the following will be tracked for study purposes and reported on a yearly basis to the IRB, or 
earlier if warranted:  
 
 
Any adverse events related to grades III-IV GVHD or to chronic GVHD in the first 2 years 
after BMT 
 
All other adverse events will be neither tracked nor reported. 
 
Reporting of subject deaths will cease in the following cases: 
 
 
Subjects on Arm A or Arm C who are greater than 30 days from the completion of 
consolidation chemotherapy 
 
Subjects on Arm B who are greater than 365 days from the date of bone marrow transplant 
 
Subjects who are not following the protocol arm that they were assigned to 
 
8.7. Data Handling and Record Keeping 
8.7.1. Case Report Forms 
The PI and study coordinator will document in the patient files. Data required according to this protocol 
will be recorded on the CRFs developed by the PI. Entries on the CRF must be made with a ball-point pen 
and must be legible. Any documents related to the study must be archived at the study site or in a central 
archive. This includes the careful listing of the identities of the patients involved in the study. This list 
and the signed informed consent statements are key documents in the files to be stored by the PI. Patient 


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files will be archived according to local regulations. All documents related to the study must be retained 
until at least 15 years after the end of the study.  
8.7.2. Patient Registry  
The PI should maintain a patient registry of all patients entered into the study in the event that a safety 
issue arises after study completion. 
 
9. Ethics 
9.1.  Institutional Review Board  
The study protocol and any amendment that is not solely of an administrative nature must be approved by 
an Institutional Review Board (IRB).  
9.2. Ethical Conduct of the Study  
The study will be conducted in accordance with the ethical principles of the Declaration of Helsinki. 
9.3. Evaluation of Benefits and Risks/Discomforts  
9.3.1. Potential Benefits  
Patients will receive evaluation and treatment of their malignancy as a result of participating in this trial. 
Moreover, our preliminary published data suggest that patients with detectable LSCs after induction 
therapy have a poor outcome with standard cytarabine-based consolidation therapy, and allogeneic SCT 
improves outcomes in poor-risk patients, as defined by standard cytogenetic/molecular criteria. Therefore 
in patients with detectable LSCs, SCT may delay or prevent relapse. Alternative approaches to entering 
this trial, standard of care cytarabine-based consolidation or allogeneic SCT, will also be discussed before 
the verbal and written consent. 
9.3.2. Measures for Minimizing Risk 
Side effects can be unpredictable in nature and severity, although all care will be taken to minimize them. 
If patients suffer any physical injury as a result of participating in this study, immediate medical treatment 
is available at the treatment center. Frequent blood work will be taken to monitor side effects. Although 
no compensation is available, any injury will be evaluated and treated in keeping with the benefits or care 
to which patients are entitled under applicable regulations.  
9.3.3. Risks/Benefits Analysis  
Data gathered from both clinical and laboratory evaluations in this trial will be analyzed frequently to 
ensure safety of patients. Any new or significant finding(s) found during the course of the research will be 
shared and explained to each participant since that may affect a patient’s willingness to participate further. 
Patient’s anonymity will be protected to the maximum extent in all publications and presentations that 
result from this research.  
9.3.4. Patient Information and Consent  
The investigator or consent designee will explain the nature of the study, its purpose and associated 
procedures, the expected duration, and the potential benefits and risks of participation to each patient 
prior to his/her entry into the study (i.e., before examinations and procedures associated with selection for 
the study are performed). Each patient will have ample opportunity to ask questions and will be informed 
about the right to withdraw from the study at any time without any disadvantage and without having to 
provide reasons for this decision. Following this informative discussion, a patient will be asked if he/she 
is willing to sign and personally date a statement of informed consent. Only if the patient voluntarily 
agrees to sign the informed consent statement and has done so, may he/she enter the study. The patient 
will receive a copy of the signed and dated informed consent form. The signed informed consent 
statement is to remain in the investigator's files. The informed consent form and any other written 
information provided to patients will be revised whenever important new information becomes available 
that may be relevant to the patient's consent, or there is an amendment to the protocol which necessitates a 
change to the content of the written informed consent form. The investigator will inform the patient of 
changes in a timely manner and will ask the patient to confirm continuation of his/her participation in the 
study by his/her signature on the revised informed consent form. Any revised written informed consent 
form must receive the IRB’s approval/favorable opinion in advance of use. 
9.4. Financial Disclosure  
Each investigator (including the principal investigator and any sub-investigators) who is directly involved 


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in the treatment or evaluation of research subjects must disclose certain financial arrangements. There are 
no financial disclosures for this protocol. 
 
10. Removal of Subjects from Study 
Subjects will be taken off study for the following events: 
1. Death 
2. Completion of 5 years of follow-up 
3. Diagnostic bone marrow aspirate, if available, has no LSCs. (Patients without an available 
diagnostic aspirate will stay on study.) 
4. Recovery marrow after induction is not evaluable for LSCs 
5. Subject is not in CR1 after induction 
6. Prior to randomization, subject is found to have no donor available for bone marrow transplant 
7. Prior to randomization, subject is unable to proceed with study for any other reason 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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